**Anticancer Agents Design, Synthesis and Evaluation**

Printed Edition of the Special Issue Published in *Molecules* Qiao-Hong Chen Edited by

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## **Anticancer Agents: Design, Synthesis and Evaluation**

## **Anticancer Agents: Design, Synthesis and Evaluation**

Editor

**Qiao-Hong Chen**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editor* Qiao-Hong Chen California State University USA

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## **Contents**





## **About the Editor**

**Qiao-Hong Chen** (Associate Professor) Dr. Qiao-Hong Chen received her PhD degree in Medicinal Chemistry from Sichuan University, China. Appointed as a Lecturer in 2001, she was promoted to the position of Full Professor in 2003 at Sichuan University. She was a Postdoctoral Fellow for three years at the University of Alberta in Canada and a Senior Research Fellow for six years at Virginia Tech. She joined Fresno State in 2012 and is currently an Associate Professor in the Chemistry Department. Her research interests focus on natural product-based anticancer agents. She has published 151 peer-reviewed scientific publications, as well as 5 book chapters.

## **Preface to "Anticancer Agents: Design, Synthesis and Evaluation"**

Current cancer therapies still have various limitations, such as multi-drug resistance, undesired off-target effects, and unpredictable efficacies. The cancer-related mortality rate remains high. The development of novel anticancer agents thus continues to be imperative to combat various deadly cancers. The emerging molecular targets and signal pathways enable the development of novel strategies for the rational design of new anticancer agents. Numerous well-established synthetic methods and biological screening assays have paved the way for the discovery and development of new anticancer agents. This Special Issue of Molecules is devoted to all aspects of recent studies searching for new anticancer agents. Both original research and review articles focusing on the rational design, synthesis, and/or biological evaluation of various agents (including small molecules, natural products, intrinsic molecules, antibodies, and vaccines) as potential cancer therapeutics are welcome to be submitted for publication in this Special Issue.

> **Qiao-Hong Chen** *Editor*

### *Review* **Mimicking Strategy for Protein–Protein Interaction Inhibitor Discovery by Virtual Screening**

**Ke-Jia Wu 1,**†**, Pui-Man Lei 1,**†**, Hao Liu 2, Chun Wu 2, Chung-Hang Leung 1,\* and Dik-Lung Ma 2,\***


### Academic Editor: Qiao-Hong Chen

Received: 29 October 2019; Accepted: 28 November 2019; Published: 4 December 2019

**Abstract:** As protein–protein interactions (PPIs) are highly involved in most cellular processes, the discovery of PPI inhibitors that mimic the structure of the natural protein partners is a promising strategy toward the discovery of PPI inhibitors. In this review, we discuss recent advances in the application of virtual screening for identifying mimics of protein partners. The classification and function of the mimicking protein partner inhibitor discovery by virtual screening are described. We anticipate that this review would be of interest to medicinal chemists and chemical biologists working in the field of protein–protein interaction inhibitors or probes.

**Keywords:** protein–protein interactions; virtual screening; mimetics; drug discovery

#### **1. Introduction**

Protein–protein interactions (PPIs) are involved in the regulation of biological processes, including cell proliferation, signal transduction, transcription, and apoptosis [1]. Since numerous ailments are associated with abnormal PPIs, the inhibition of PPIs is an attractive approach for the generation of new therapeutics. However, because of their large and amorphous interfaces, targeting PPIs is a great challenge in pharmaceutical and academic research.

In recent years, computer-aided approaches became useful tools to assist scientists in drug discovery. In particular, virtual screening emerged as a complementary technique to aid high-throughput screening (HTS) in pharmaceutical development. Virtual screening can reduce the number of compounds to be screened in bioassays, leading to a large reduction of time and cost [2,3]. Virtual screening strategies can be traditionally classified into two broad types: ligand-based virtual screening (LBVS) and structure-based virtual screening (SBVS). LBVS strategies include approaches such as pharmacophore-based methods, quantitative structure–activity relationships (QSAR), and three-dimensional shape matching [4]. On the other hand, SBVS techniques mainly revolve around the docking of molecules to three-dimensional (3D) structures of the biological target as determined by X-ray crystallography, nuclear magnetic resonance (NMR), or homology modeling.

Recently, virtual screening found increasing use for identifying inhibitors against various targets. Sun et al. constructed QSAR models of Sirtuin 1 (SIRT1) ligands and discovered 12 compounds as inhibitors of SIRT1 through ligand-based virtual screening [5]. Yang et al. identified a potent and selective KDM5A inhibitor using structure-based virtual screening [6]. Wu et al. reported mitoxantrone as an inhibitor of NAE using virtual screening of an approved drug database [7]. Virtual screening was also used to develop inhibitors of PPIs. Yang et al. described a compound as an inhibitor

of the VHL–HIF1α interaction using structure-based virtual screening [8]. Zhong et al. utilized structure-based virtual screening and identified a cytosine alkaloid compound as an inhibitor that inhibited the menin–MLL interaction and another compound as a potential inhibitor of TLR1–TLR2 heterodimerization [9,10]. However, virtual screening still has many challenges and limitations that need to be overcome, especially its high false positive rate, which limits virtual screening to initial screening only [11].

The rational design of compounds that mimic key interactions at the protein–protein interface is another successful strategy for PPI inhibitor discovery [12]. Compared with low-molecular-weight compounds, mimetics can be more selective and show lower toxicity. Modification and optimization of peptide mimetics can also improve structural stability, resulting in increased oral availability [13]. For example, Groß et al. designed and synthesized a soluble peptide mimicking CXCR4 to interrupt the gp-120 and CXCR4 interaction [14]. In this context, the combined utilization of mimicking strategies and virtual screening has broad application prospects for drug discovery (Figure 1). Based on this strategy, many mimetics targeting cancer-related PPIs were discovered using virtual screening, such as p53 mimetics for the MDM2–p53 interaction [15–20], BH3 mimetics for the Bcl-2–BH3 interaction [21–26], and SMAC mimetics for the IAP–SMAC interaction [1,27–29]. Knowledge of the 3D structures of proteins allows the use of different approaches for mimetics design. For the interruption of PPIs, inhibitors may be larger in size than traditional drug compounds. Peptides and proteins mimetics are increasingly considered to be viable therapeutics for PPI inhibitor discovery [30]. However, there are still some challenges in protein or peptide mimetics design. For example, we still do not fully understand folding and the physical forces that stabilize protein structures. Moreover, sequences with many degrees of freedom can complicate the sequence search, which leads to a requirement for effective methods to find sequences related to a particular structure and measure essential protein folding criteria.

**Figure 1.** Mimicking strategy for inhibitor discovery by virtual screening [31]. (Reprinted with permission from Copyright (2015) Wiley—VCH Verlag GmbH & Co. KGaA.).

In this review, we discuss the recent advances in the application of virtual screening to design protein or peptide mimetics for PPI inhibitor discovery, and we summarize different methods for virtual screening. The classification of mimicking strategies and the function of the mimicking protein partner inhibitors discovered using virtual screening techniques are also described.

#### **2. Integrating Mimicking and Virtual Screening Strategy for Protein–Protein Interaction Inhibitor Discovery**

Recent studies indicated that certain types of PPIs are amenable for targeting by small molecules, which can block the interaction between a protein and its peptide or protein partner via binding at the proteinous interface [7,32–38]. As key residues of the protein or peptide may serve as a beginning point for PPI inhibitor design, the effective mimicking of peptides in their biologically active conformation and the development of mimicking ligands are important goals in science beyond the development of PPI modulators. Meanwhile, virtual screening emerged as a complementary technique to aid mimicking strategy in pharmaceutical development, complementing high-throughput screening (HTS) techniques. Integrating mimicking and virtual screening is a potentially viable strategy for protein–protein interaction inhibitor discovery. In this section, we describe the application of virtual screening and a mimetic strategy for PPI inhibitor discovery and discuss their merits and drawbacks.

#### *2.1. Virtual Screening for PPI Inhibitor Discovery*

Virtual screening is a kind of computer-aided technique that is usually considered as an initial step in the lead discovery process in order to enrich the library with active compounds and predict experimental activity [11]. Usually, based on the information about the target or the ligands of reported compounds, virtual screening is usually classified into two types: ligand-based virtual screening (LBVS) and structure-based virtual screening (SBVS). LBVS strategies depend on the similarity or dissimilarity of the compounds of interest, and they require a large amount of structure–activity data from a large chemical compound library. One of the ligand-based approaches is QSAR modeling, which focuses on achieving a correlation between the physicochemical and structural properties of the ligands and their biological function and potency. QSAR modeling includes two-dimensional (2D-QSAR) and three-dimensional QSAR (3D-QSAR). Scientists use 2D-QSAR and 3D-QSAR properties of ligands to build up a model of biological activity, which can be applied to predict the activity of some new compounds [39]. Compared with 3D-based algorithms, 2D-based algorithms are usually faster but may be less accurate; moreover, 2D-based algorithms cannot find new active compounds with dissimilar chemical structures [40]. Moreover, 2D- and 3D-QSAR do not consider ligand conformations, protein structure and flexibility, or solvation effects. Another LBVS approach is based on the similarity of compounds, which is a simple computational method with low cost, focusing on obtaining compounds that are similar to known ligands. However, this method is easily influenced by human users because it is difficult to objectively select the input molecules [41]. It is usually carried out by using common chemical features from the 3D structures of some known ligands that represent interactions between the ligands and the target. Pharmacophore modeling is another approach for LBVS. Based on analyzing the structures of known inhibitors against a target, a ligand-based pharmacophore can be generated that describes the spatial arrangement of chemical features of active compounds. However, under many circumstances, it is hard to find a library with functionally and structurally diverse molecules with quantitative activity data for a given protein. More importantly, the lack of publications with negative results hinders the identification of inactive molecules, resulting often in the development of qualitative common feature pharmacophores only from active compounds [41,42]. Finally, as LBVS applications are generally based on the properties of the known ligands, the diversity of the hits discovered are generally limited.

In contrast, SBVS techniques do not require knowledge of the biological activity of known compounds. Instead, 3D structures of the protein must be known or inferred. Protein-ligand docking is widely applied to identify compounds that are predicted to bind tightly to the active sites of the target. During the SBVS process, the 3D structure of a target protein and a set of ligands are considered as starting points and screened by virtual filtering, followed by docking and scoring to identify potential lead candidates. Many algorithms were developed to perform SBVS, such as DOCK, GOLD, And AUTODOCK, which can be used for identifying the binding mode and binding affinity between protein and ligand [43]. After docking and scoring, a set of compounds with the highest predicted

binding affinity against the target can be obtained [44]. One relatively new strategy that falls within the purview of SBVS is the binding site comparison approach. This strategy, which can be utilized for drug repurposing and polypharmacology, relies on the recognized fact that many different proteins have similar binding sites [45]. Thus, binding sites in any given protein can be searched and matched with specific chemical structures. Finally, the pharmacophoric approach can also be employed within SBVS. If a high-resolution 3D structure of the target is available, a structure-based pharmacophore of the binding site can be generated based on the structural features of the binding site. In this case, a library of known inhibitors against the target is not needed. Compared to LBVS (Table 1), SBVS is more likely to identify new scaffolds because it is based on physical interactions calculated in silico, rather than relying on the similarity/dissimilarity of known ligand compounds. Thus, SBVS may be able to identify inhibitors with unique mechanisms of action [11,46]. Another difference is that, unlike LBVS, the docking model obtained by SBVS can be used for interaction analysis, in order to further enhance the affinity or selectivity of the compounds.

**Table 1.** The advantages and limitations of structure-based virtual screening (SBVS) and ligand-based virtual screening (LBVS). QSAR—quantitative structure–activity relationships.


Receptor and ligand flexibility is crucial for predicting drug binding and evaluating thermodynamic and kinetic properties. Molecular dynamics (MD) simulation is a technique for investigating atomic and molecular motion, and it is widely and effectively used for analyzing the relationship between the structure and function of molecules [47,48]. The main advantage of MD simulations is that they can thoroughly sample the conformational space around both the protein and the ligand under realistic conditions, accommodating both structural flexibility and entropic effects, thus allowing the thermodynamics and kinetics of the drug–target interaction to be more accurately calculated [47]. Therefore, MD simulation can be combined with SBVS or LBVS to further understand the binding mode of candidate molecules, thus accelerating the process of drug development [49]. Additionally, MD simulations can be used to identify potential pockets and binding hotspots of PPIs [50]. Saez et al. used MD simulations to predict the atomic interactions of the PcTx1–cASIC1 interaction and the hotspot residues of their interface, which could be beneficial for designing therapeutically useful PcTx1 mimetics [51]. By combining structural information, MD, and functional experiments, they obtained detailed insight into the molecular basis of this PPI. The TRAF6–Basigin interaction is implicated in melanoma metastasis. Biswas et al. used MD simulations to study the interactions between individual proteins and TRAF6–Basigin complexes, revealing conformation changes in the PPI and the adoption of a helical conformation [52].

In terms of the chemical library used for SBVS or LBVS, different filters can be used. For fragment-sized compounds, Congreve et al. described a "rule of three" with molecular weight < 300, logP < 3, number of hydrogen bond donors and acceptors < 3, and number of rotatable bonds < 3 [53]. Alternatively, based on physicochemical properties, the "Pfizer's Rule of 3/75" can be applied to predict the toxicology of compounds. Compounds with calculated partition coefficient (ClogP) < 3 and topological polar surface area (TPSA) > 75 are approximately 2.5 times more likely to be safe in in vivo

assays [54]. However, overly strict application of filters may introduce bias, leading to the exclusion of potentially active compounds. To assess the potency of the hits derived from virtual screening, IC50, EC50, Ki, or Kd values can be calculated. Ripphausen considered four subdivisions of potency (<1, >1–10, >10–100, and >100 μM), and suggested that docking hits are generally weakly potent, falling into the 1–100 μM range [55].

As virtual screening is often used as first stage of the primary screening process, false positives are a common problem. Based on six cases, Schierz et al. reported that the average percentage of false positives from the high-throughput primary screen is quite high at 64% [56]. To eliminate false positives, cross-referencing between primary and confirmatory screening assays is required. On the other hand, selectivity is a crucial aspect for developing potent PPI inhibitors. Off-target effects can arise when the compounds bind to other protein targets rather than their intended target, leading to side effects [57]. Virtual screening can be used for resigning, repositioning, and predicting side effects or toxicity of drugs, which can significantly decrease the time and cost of development compared to the traditional drug discovery process [58,59]. In one example, Spahn et al. used a computational simulation to create chemical modifications of fentanyl, an opioid pain killer with severe adverse effects due to off-target effects throughout the body. The newly discovered compound, named NFEPP, possesses a lower pKa and eliminates pain by selectively activating the MOR pathway in the inflamed acidic area without causing side effects [60].

Taken together, virtual screening greatly decreases the time and money costs by processing thousands of compounds in a short time in silico, thereby reducing the number of compounds to be synthesized or purchased [61]. However, because virtual screening relies on analyzing the physicochemical properties of compounds rather than biological activity directly, it has a high rate of false positives or false negatives compared to cellular or phenotypic screens [62].

In order to improve the efficiency of screening for more bioactive PPI lead inhibitors, the mimicking peptide strategy can be used. Virtual screening can be employed to target PPI surfaces or to mimic "hotspot" residues. Peptide mimetics that mimic the bioactivity of the parent peptides can also show improved pharmacokinetic and pharmacodynamic properties, such as bioavailability and stability [63]. It should be noted that the mimicking protein domain can be achieved with protein backbone scaffolds or small molecules [30,31].

#### *2.2. Structure-Based Mimicking Peptide Strategy for PPI Inhibitor Discovery*

Peptides are utilized as feasible molecules to mimic protein binding sites [64]. Peptidomimetics are non-peptide compounds that mimic the conformation and characteristics of peptide molecules to interrupt PPIs [63]. In general, chemical synthetic, screening, and structure design approaches are usually used for designing and extending the diversity of peptide-derived chemical structures, as well as enhancing their metabolic stability [64]. Synthetic strategies can explore and expand the chemical space for peptidomimetics. Screening strategies, including high-throughput screening and fragment screening, are often used to identify hot hits and discover peptidomimetics based on reported compounds [65]. Meanwhile, design strategies can use hotspot residues as starting points to design analogues by mimicking key secondary-structure motifs involved in the PPI interface. Design strategies can be subdivided into sub-structure search, *de novo* design, and bioisostere design [65]. As peptides usually contain secondary structures such as α-helices and β-sheets, peptidomimetics for inhibiting PPIs should be able to mimic these structures in order to be able to displace the natural peptides [66]. Therefore, peptides that mimic α-helices or β-sheets of proteins are attractive targets for drug discovery. α-helix structures are indispensable secondary structural elements which constitute most structured protein domains and contribute greatly to the protein–protein interface. Main strategies for synthesizing α-helix mimetics include (i) cross-linking of peptide side chains and the incorporation of stabilizing caps at the N-terminus, (ii) use of foldamers to modulate backbone variations, and (iii) introduction of projecting rod-like elements that mimic the side chains of an α-helix [31,67]. For example, Ernst et al. designed a polyamide foldamer as an α-helix mimetic, leading to the synthesis

of inhibitors of the Bak BH3/Bcl-xL complex [68]. Meanwhile, a β-strand is an extended structural element between three and 10 amino acids long, and adjacent β-strand structures can be connected laterally backbone hydrogen bonds, forming a twisted or pleated sheet. β-sheets play key roles in maintaining the tertiary and quaternary structures of proteins, as well as PPIs. A number of strategies were utilized to design β-strand and β-sheet mimetics: (i) incorporation of turn mimetics to nucleate β-sheet generation, (ii) macrocyclization via covalent or noncovalent linkages, and (iii) introduction of β-strand-enforcing residues [31]. In addition to α-helix structures and β-strand structure mimetics, mimicking the turn structure of peptides is another potential approach for PPI inhibitor discovery. Turn structures are anomalous secondary structures that differ from α-helix and β-sheet structures due to the non-repetitive dihedral angles of the main chains. Turn motifs allow a peptide chain to fold back, and are important for forming globular proteins [31,69,70]. For instance, Bartfai et al. designed a β-turn mimetic that interrupted the interaction between IL-1RI and MyD88 in the TIR domains [71].

Gimeno and co-workers developed a classification of peptide mimetics depending on the extent of similarity to the native peptide. Class A mimetics contain the parent peptide amino-acid sequence, with the side chains being arranged to closely mimic the active conformation of the native peptide. Class B mimetics possess further modification of the native sequence, including the introduction of non-natural amino-acid residues, other small molecular motifs, or changes of the backbone sequence. Class B mimetics include foldamers, β- and α/β-peptides, and peptoids. Class C mimetics are highly modified structures with small molecular motifs and changes in the main chains of the peptide. Class D mimetics mimic the method of action of the native peptide rather than through structural mimicry of the side chains, and they can be developed via affinity optimization of class C mimetics or, alternatively, they can be identified by virtual screening [31]. An alternative classification of peptide mimetics was also described in the past two decades. Type I mimetics are short peptide sequences that mimic the α-helical motif of a PPI interface. Type II mimetics are functional mimetics that are based on a small molecular scaffold rather than a peptide scaffold. Type III mimetics include non-peptide templates that mimic the topography of the original helix by retaining the spatial arrangement of key binding residues [31,72,73]. In peptide mimetics design, one of the main challenges is that the topological shapes of proteins are complex, leading to variations in the types of interactions, binding pockets, and recognition sites formed [74]. This variability of PPIs is a crucial aspect that has to be mastered for the design of peptide mimetics targeting PPIs.

#### *2.3. Integration of Mimicking Strategies with VS for PPI Inhibitor Discovery*

Effectively mimicking the bioactive conformation of a peptide is a critical part of developing mimics as PPI inhibitors. However, developing mimetics with appropriate pharmacokinetic properties is a key challenge to overcome [31]. To strike a balance for these two properties, applying virtual screening can allow for simultaneous optimization of affinity and pharmacokinetic properties [75–77]. Thus, the integration of mimicking strategies and virtual screening is a complementary strategy for efficiently developing PPI inhibitors. In this section, we introduce and classify mimicking strategies using different virtual screening approaches (Figure 2).

**Figure 2.** Different approaches for mimetics discovery based on structure-based virtual screening (SBVS) and ligand-based virtual screening (LBVS) [78]. (Reprinted with permission from Copyright (2002) Elsevier Science B.V.).

#### 2.3.1. *De Novo* Peptide Design Approach

*De novo* peptide design is an attractive approach for constructing designed peptides with desired structures and functions, including peptide mimetics targeting PPIs. *De novo* design can create novel molecules that do not exist in known compound databases. This method only requires a scaffold library and a few key anchor residues as a starting point. By knowledge of the structural features of the native peptide, new inhibitors with the desired secondary structural characteristics can be built up *de novo* according to the targeted binding site. The virtual *de novo* peptide design method can be considered to comprise the following stages: (i) documentation of key anchor residues and the preparation of the scaffold library, (ii) virtual screening to find scaffold fragments that the anchor residues can be attached to, (iii) sequence design and structure refinement, and (iv) experimental validation [79]. There are several advantages of *de novo* peptide design to developing protein mimics. The first one is that the backbone of the natural protein sequence can be utilized as a template to initiate the design. Another advantage is that knowledge of sequence/structure relationships and/or statistical forcefields from native proteins can be used to guide the sequences of the designed peptides [80]. Therefore, *de novo* peptide design approach is a complementary strategy for peptide mimetics discovery.

Using the joint application of the *de novo* peptide design approach and peptide mimetics design, Li et al. designed PD-1-binding peptides by mimicking five residues (Y56, R113, A121, D122, and Y123) of the ligand PD-L. The most potent peptide Ar5Y\_4 had a KD value of 1.38 ± 0.39 μM, which was comparable to the binding affinity of the PD-L1. Ar5Y\_4 showed the ability to interrupt the binding of PD-L1 to PD-1, providing a potential strategy for further optimization of PD-L1 peptide mimetics [74,81]. Smadbeck et al. used a three-stage *de novo* peptide design approach to design EZH2 inhibitory peptides. The approach comprises a sequence selection stage, a fold specificity calculation stage, and an approximate binding affinity calculation stage. The novel peptide SQ037 showed the highest in vitro response, with an IC50 of 13.5 μM. Compared to the native and K27A mutant control peptides, SQ037 had greater potency as an inhibitor and showed higher specificity to EZH2 [79]. Ruiz-Gómez et al. optimized the *de novo* design approach for small scaffolds mimicking protein recognition epitopes of large, non-structured, and discontinuous PPIs. They applied this novel re-scaffolding approach to the *de novo* design of potent interleukin 10 (IL-10) ligands that mimic the high-affinity receptor IL-10R1 [82]. Overall, these studies demonstrate that computer-aided *de novo* design is an effective strategy for peptide mimetics discovery.

#### 2.3.2. Fragment-Based Design Approach

The fragment-based design approach uses fragments with low molecular weight and small size as starting points for modifying into high-affinity compounds. Compared with HTS, the fragment-based design approach can result in higher hit rates and a higher probability of synthesizing an efficient binding compound [83]. Hence, fragment-based approaches are particularly effective for generating small molecules or peptides targeting PPIs. The most common biophysical screening techniques for the fragment-based design method include differential scanning calorimetry (DSF), ligand- or protein-based nuclear magnetic resonance (NMR), surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or X-ray crystallography [1,74]. The fragment-based design approach is widely applied to mimetics design. For example, Petros et al. discovered a high-affinity ligand for the anti-apoptotic protein Bcl-XL using fragment-based screening. From NMR-based structural studies and parallel synthesis, a potent BH3 mimetic ligand was obtained, which bound to Bcl-xL with an inhibition constant (Ki) of 36 ± 2 nM [22,23]. Using NMR-based screening, parallel synthesis, and structure-based design, further modification of this ligand was achieved by Oltersdorf and co-workers. They discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-XL, and Bcl-w, which showed two to three orders of magnitude more potent affinity than previously reported compounds [24]. Although ABT-737 showed great antitumor activity in murine tumor xenograft models, it lacked oral bioavailability. Park et al. reported that targeted modifications at three positions of ABT-737 led to a 20-fold improvement in the pharmacokinetic/pharmacodynamic relationship (PK/PD). The resulting compound ABT-263 was orally available in a xenograft model of human small-cell lung cancer, and induced complete tumor regressions in all animals [25]. Based on ABT-263, Souers et al. redesigned and reported the first-in-class orally bioavailable Bcl-2-selective inhibitor with potent anticancer activity in vitro and in vivo [26]. Fragment-based design, in concert with computational approaches, show high promise for peptide mimetic design and discovery.

#### 2.3.3. Pharmacophore-Based Design Approach

Pharmacophore-based approaches enables the virtual screening of large numbers of peptide mimetics using a conventional pharmacophore broadly derived either using structure-based or ligand-based methods, depending on whether a 3D structure of the target is available or not [74,84]. The pharmacophore-based approach can be divided into four steps: (i) atom typing, (ii) conformational sampling, (iii) hypothetical pharmacophore construction, and (iv) virtual screening of candidate ligands against the hypothetical pharmacophore [85]. Using the pharmacophore approach, Hansen et al. synthesized small beta-peptidomimetics with anti-staphylococcal activity. Their research showed that small β-peptidomimetics can mimic the antimicrobial activity of much larger antimicrobial peptides (AMPs), making them promising candidates for treating bacterial infections [74,86]. Caporuscio et al. developed compounds that showed micromolar potency against replication of HIV-1 in cells via a target-based pharmacophore model mapping the CD4-binding site on HIV-1 gp-120 [87]. Hall et al. conducted a two-round computational screening of potential peptide mimetic compounds in order to develop inhibitors of the ανβ3 integrin receptor. Biological testing revealed that the peptide mimetic molecules potently inhibited hantavirus with two thousand times more potency than the natural cyclic peptide (cyclo-[CPFVKTQLC]). The second round of screening furnished molecules with improved chemical diversity by building up the pharmacophore models [74,88]. Atatreh et al. started with a 3D pharmacophore and performed virtual screening to discover a series of MDM2–p53 interaction inhibitors with inhibition activity at the submicromolar level, which showed anticancer activities against different breast cancer cell lines [89]. Overall, the pharmacophore-based design approach is a suitable method for peptide mimetics discovery when the target protein structure is unavailable.

#### 2.3.4. Integration of Mimicking Strategies with LBVS for PPI Inhibitor Discovery

In contrast to structure-based approaches, LBVS uses the structures of known binders as templates to discover and identify diverse bioactive compounds with high affinity. In general, LBVS methods depend on the application of computational descriptors of molecular structure, properties, or pharmacophore features, and they analyze relationships between active and database or test compounds in various defined chemical descriptor spaces [90,91]. Three major methods are usually utilized for LBVS: QSAR modeling, pharmacophore modeling, and the efficient similarity method. QSAR modeling can be broadly divided into three steps: (i) collect compound data, (ii) develop and validate QSAR models, and (iii) use the models to identify compounds from a chemical library. QSAR approaches are not computationally intensive, so they can be performed relatively quickly [92]. However, ligand-based 2D- and 3D-QSAR models do not consider ligand conformations, protein structure and flexibility, or solvation effects, which may lead to poor accuracy. When the three-dimensional (3D) structure of a target is unknown, pharmacophore modeling can be utilized to identify biologically active compounds via chemical features such as hydrogen bonding and lipophilicity as the input data for flexible alignment. Ligand-based virtual screening involves two different methods: (i) flexible alignment of molecules by considering only the atomic contributions, and (ii) use of other chemical features that are unrelated to 3D pharmacophore representations, such as hydrogen bonding and lipophilicity [84,93]. In the similarity method, compounds with similar structures are assumed to have similar activities, allowing the topological, steric, electronic, and/or physical properties of compounds to be predicted by comparison with known molecules [94]. Six types of similarity are exploited: chemical similarity, molecular/2D similarity, 3D similarity, biological similarity, global similarity, and local similarity [95]. In recent years, LBVS was increasingly applied to identify active compounds as PPI inhibitors [96]. Švajger et al. used two parallel virtual screening methods targeting the TLR4–MD-2 interface by mimicking interactions with MD-2 to discover novel TLR4 antagonists. They identified a potent hit compound with an IC50 value of 16.6 μM and no cytotoxic properties, which may be a potential agent to treat sepsis and neuropathic pain [97]. Varney et al. previously reported the interaction of Lipid II with defensins, based on the 3D structure of the human defensin peptide HNP1–Lipid II complex. They designed a pharmacophore model and used it for screening for defensin mimetics, leading to the first Lipid II-targeted low-molecular-weight compound, BAS00127538 [98]. Ambaye et al. used the co-crystal structure of a lead peptide antagonist and combined a shape-based similarity search, molecular docking, and 2D-similarity searches to identify nine novel phenylbenzamide-based antagonists of the Grb7 SH2 domain as potential Grb7 anticancer therapeutics [99].

#### **3. Conclusions**

Due to the critical roles of PPIs in disease, targeting PPIs is a potential therapeutic strategy. High-throughput screening is a widely used technique in drug discovery; however, a large investment into compounds and screening assays is required. Virtual screening is an emerging technology for drug discovery because there is no need for physical compounds and bioassays for screening. As we highlighted in this review, virtual screening is used for discovering mimetics of PPIs based on different approaches, such as *de novo* peptide design approach, fragment-based design approach, pharmacophore-based design approach, and ligand-based design approach. We anticipate that the integration of virtual screening with mimicking strategies will become a powerful tool in cancer research and that that this review could arouse the interest of chemical and biological scientists working in the field of PPI inhibitors.

**Author Contributions:** Writing—original draft preparation, K.-J.W. and P.-M.L.; writing—review and editing, C.W. and H.L., writing—review and editing, project administration, D.-L.M. and C.-H.L.

**Funding:** This research was funded by the Hong Kong Baptist University (FRG2/17-18/003), the Health and Medical Research Fund (HMRF/14150561), the National Natural Science Foundation of China (201575121and 21775131, China), the Hong Kong Baptist University Century Club Sponsorship Scheme 2018 (China), the Interdisciplinary Research Matching Scheme (RC-IRMS/16-17/03, China), the Interdisciplinary Research Clusters Matching Scheme (RC-IRCs/17-18/03, China), the Collaborative Research Fund (C5026-16G, China), the SKLEBA and HKBU Strategic Development Fund (SKLP\_1718\_P04, China), the Science and Technology Development Fund, the Macau SAR (File no. 0072/2018/A2 and SKL-QRCM-2017-2019), and the University of Macau (MYRG2018-00187-ICMS, China).

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Second-Generation Androgen Receptor Antagonists as Hormonal Therapeutics for Three Forms of Prostate Cancer**

### **Pravien Rajaram, Alyssa Rivera, Kevin Muthima, Nicholas Olveda, Hubert Muchalski \* and Qiao-Hong Chen \***

Department of Chemistry, California State University, Fresno, CA 93740, USA; pravien@mail.fresnostate.edu (P.R.); alyssamrivera@mail.fresnostate.edu (A.R.); muthima@mail.fresnostate.edu (K.M.); nicolveda08@mail.fresnostate.edu (N.O.) **\*** Correspondence: hmuchalski@csufresno.edu (H.M.); qchen@csufresno.edu (Q.-H.C.); Tel.: +1-559-278-2711 (H.M.); +1-559-278-2394 (Q.-H.C.)

Academic Editor: Laura Cerchia Received: 1 May 2020; Accepted: 21 May 2020; Published: 24 May 2020

**Abstract:** Enzalutamide is the first second-generation nonsteroidal androgen receptor (AR) antagonist with a strong binding affinity to AR. Most significantly, enzalutamide can prolong not only overall survival time and metastatic free survival time for patients with lethal castration-resistant prostate cancer (CRPC), but also castration-resistant free survival time for patients with castration-sensitive prostate cancer (CSPC). Enzalutamide has thus been approved by the US Food and Drug Administration (FDA) for the treatment of both metastatic (in 2012) and non-metastatic (in 2018) CRPC, as well as CSPC (2019). This is an inspiring drug discovery story created by an amazing interdisciplinary collaboration. Equally important, the successful clinical use of enzalutamide proves the notion that the second-generation AR antagonists can serve as hormonal therapeutics for three forms of advanced prostate cancer. This has been further verified by the recent FDA approval of the other two second-generation AR antagonists, apalutamide and darolutamide, for the treatment of prostate cancer. This review focuses on the rational design and discovery of these three second-generation AR antagonists, and then highlights their syntheses, clinical studies, and use. Strategies to overcome the resistance to the second-generation AR antagonists are also reviewed.

**Keywords:** androgen receptor; prostate cancer; enzalutamide; apalutamide; darolutamide

### **1. Introduction**

### *1.1. Prostate Cancer*

Prostate cancer continues to be a main health concern due to the highest incidence and the second highest cancer-related death rate in American men. In 2020, estimates indicate about 21% of all new cancer cases will be attributed to prostate cancer, while over 33,000 deaths caused by prostate cancer are projected to occur in the United States [1]. The critical driving force for prostate cancer is the androgen receptor (AR)-regulated gene expression that is initiated by the binding of androgen to AR [2]. Consequently, the mainstay therapy for castration-sensitive prostate cancer (CSPC) since 1941 is androgen deprivation therapy (ADT). However, after the initial response to ADT for about 18 to 24 months, most CSPC will inevitably shift to castration resistant prostate cancer (CRPC) [3]. In the CRPC stage, prostate cancer continues to grow under extremely low levels of male hormone testosterone in serum. The majority of prostate cancer deaths in the United States are caused by late state (metastatic) CRPC (mCRPC). Within the past decade, several new treatments have been approved for three forms of prostate cancer: metastatic castration-sensitive prostate cancer (mCSPC),

non-metastatic castration-resistant prostate cancer (nmCRPC), and metastatic castration-resistant prostate cancer (mCRPC). Table 1 lists the current treatments that have been approved by the US Food & Drug Administration (FDA) since 2004, according to the information published on the official website of the US FDA. Current treatments for prostate cancer can be classified into taxane-based chemotherapeutics, hormonal therapy, immunotherapy, and radiotherapy. As illustrated in Table 1, far more hormonal therapies than other categories have recently been approved by the US FDA for prostate cancer.


nmCRPC: non-metastatic castration-resistant prostate cancer. mCRPC: metastatic castration-resistant prostate cancer mCSPC: metastatic castration-sensitive prostate cancer.

#### *1.2. Hormonal Therapeutics*

The timeline for the development of hormonal therapeutics for prostate cancer is illustrated in Figure 1. The pioneering hormonal therapeutic for prostate cancer is the well-known androgen deprivation therapy originally reported by Huggins and Hodges in 1941 [4]. At that point, orchiectomy (surgical castration) and administration of high dose of estrogen (non-surgical castration) were established to be two strategies to cut down the circulating testosterone to castrate (or near castrate) levels, resulting in appreciable biochemical response in a cohort of eight patients with metastatic prostate cancer. It was recognized by the Veterans Administration Cooperative Urological Research Group in 1970s that treatment of patients with advanced prostate cancer with high dose of estrogen led to good efficacy, but accompanying with enhanced mortality rate associated with cardiovascular complications [5]. The serious undesired effect of estrogen urged the scientists to search for a safer non-surgical castration strategy in 1980s. Encouraged by the finding that testicular production of testosterone can be indirectly controlled by long-lasting elevation of gonadotropin-releasing hormone (GnRH), GnRH agonists were designed and found to possess potential in suppressing prostate tumor growth in vivo and in clinical settings [6,7]. Synthetic GnRH agonists, e.g., goserelin (Zoladex) [8]

and leuprolide (Lupron) [9], were developed as a replacement for estrogen as a better non-surgical castration strategy by the mid-1980s and have served as the centerpiece of ADT for CSPC since then. To conquer the testosterone surge as well as other side effects caused by GnRH agonists, the US FDA has approved degarelix (a GnRH antagonist) as an alternative medical castration for patients with advanced CSPC in 2008. As compared with GnRH agonists, degarelix can provide rapid suppression of prostate specific antigen (PSA) and testosterone so as to better control testosterone and prolong PSA progression-free survival [10]. Degarelix is, thus, a better non-surgical castration therapy for those CSPC patients at more advanced stages and with more apparent symptoms.

**Figure 1.** Timeline for the development of hormonal therapeutics for prostate cancer.

On the other hand, examination of antiandrogen compounds as another alternative to estrogen castration was initiated in the late 1960s and early 1970s, leading to the development of three first generation nonsteroidal androgen antagonists, flutamide (**1**), nilutamide, (**2**) and bicalutamide (**3**), shown in Figure 2 [11]. These antiandrogen agents were revealed to competitively bind to the ligand-binding domain on androgen receptors. Monotherapy of bicalutamide (the one with the most extensive investigation) cannot offer better clinical benefit to patients with CSPC than ADT. The combination therapy of bicalutamide with ADT is widely used by CSPC patients owing to the greater safety profile than ADT alone, even though it does not grant significant overall survival benefit [12].

**Figure 2.** First generation of nonsteroidal AR antagonists.

The clearer understanding of the structure and function of the androgen receptor revealed that the androgen receptor plays a pivotal role for not only CSPC but also CRPC [13]. This notation stimulated the successful design and discovery of three US FDA-approved second-generation androgen receptor antagonists, enzalutamide (**4**) [14], apalutamide (**5**) [15], and darolutamide (**6**) [16] (Figure 3). As illustrated in Table 2, enzalutamide (**4**) is now the first FDA-approved antiandrogen to treat three forms of advanced prostate cancer after the US FDA approval of enzalutamide (**4**) on 16 December 2019 for the treatment of metastatic castration-sensitive prostate cancer (mCSPC). The intriguing discovery

stories of these three successful second-generation nonsteroidal AR antagonists are reviewed in this article. Their syntheses, clinical studies, and clinical use are highlighted as well. Current strategies to overcome the resistance to these AR antagonists are also summarized.

**Figure 3.** Second generation of nonsteroidal AR antagonists.



nmCRPC: non-metastatic castration-resistant prostate cancer. mCRPC: metastatic castration-resistant prostate cancer. mCSPC: metastatic castration-sensitive prostate cancer.

#### **2. Discovery and Preclinical Studies**

#### *2.1. Enzalutamide* (**4**) *and Apalutamide* (**5**)

Enzalutamide (**4**) and apalutamide (**5**) were discovered by the interdisciplinary collaboration of Sawyers/Jung groups, which was motivated by the notion that "growth of castration-resistant prostate cancer appears to depend upon continued androgen receptor signaling," facilitated by the in vitro AR-overexpressing prostate cancer cell models, and benefited from a complementary collaboration [17]. As illustrated in Figure 4, RU59063 (**7**) was selected as the original lead compound because it is a potent and selective nonsteroidal AR agonist with high affinity for AR [18,19]. Enzalutamide (**4**) and apalutamide (**5**) were eventually identified as two lead candidates for preclinical development on the grounds of in vitro evaluation of their capability of agonistic and antagonistic activity of AR signaling in a castration-resistant LNCaP/AR prostate cancer cell model [20]. The in vitro relative luciferase activity and relative PSA level were measured using bicalutamide as positive control.

**Figure 4.** Discovery of enzalutamide and apalutamide.

The structural modification started with substituting the ω-hydroxybutyl at *N*1 with azidoalkyl and azidoaryl groups in the light of the hypothesis that the small polar azido group might function as a bioisostere of the hydroxyl in RU59063 (**7**). The eastern side of RU59063 (**7**) was the first focus of the chemical manipulation probably due to the fact that the electron-deficient aromatic ring on the western side is a well-established pharmacophore for anti-androgenic activity [21]. Among the first set of analogues, compound **8** was identified as the optimal derivative that had higher binding affinity than bicalutamide. Further modification on compound **8** indicated that the 4-position of the *N*1-phenyl ring can accommodate several different groups without losing the desired activity. Compound **9** with a 4-methyl on the *N*1-phenyl ring was chosen for further structure-activity relationship studies, indicating that the location for the C-4 and C-5 on the thiohydantoin ring cannot be switched and that the geminal dimethyl group on the thiohydantoin ring can be substituted by the cyclobutyl ring in compound **10**. On the basis of abovementioned promising in vitro bioassay data, compounds **9** and **10** were moved forward for in vivo evaluation in a castrate mice model with LAPC4/AR or LNCaP/AR xenografts. Both compounds are more effective than bicalutamide in suppressing PSA secretion, but with a short half-life due to rapid clearance. Considering that electron-rich *N*1-phenyl ring and hydroxylation of the benzylic methyl, directly appending electron-withdrawing groups to the *N*1-phenyl ring led to the discovery of 3-fluoroamide analogues **11** (also called RD162) and enzalutamide (also called MDV3100). Both enzalutamide and RD162 (**11**) have greater (5–8 times) AR binding affinity relative to bicalutamide in the LNCaP/AR cell line with high level expression of wild-type AR. More importantly, their binding is specific to AR because only little or no binding to other nuclear receptors was observed [14].

Both enzalutamide and RD162 (**11**) possess not only excellent in vivo anti-tumor efficacy in the castrate mice model but also superb pharmacokinetic profile [14]. The in vivo pharmacokinetic properties of RD162 (**11**) were first evaluated in mice. The results showed that, after a 24-h oral treatment with a single 20 mg/kg dose, the plasma concentration (~23 μM) of RD162 (**11**) exceeds the concentration (~1–10 μM) necessary to block AR activity. The in vivo pharmacodynamic experiments suggest that RD162 (**11**) can significantly reduce AR transcriptional function and suppress LNCaP/AR tumor cell proliferation. The excellent in vivo efficacy of RD162 (**11**) in castration-resistant prostate tumor models was confirmed to be associated with AR suppression. This is because the effective dose for antitumor efficacy in the LNCaP/AR model is closely correlated with that for AR transcriptional activity as measured by luciferase imaging experiments. The fact that enzalutamide instead of RD162 (**11**) was chosen, at that moment, as the drug candidate for further preclinical studies is simply because enzalutamide can be prepared from an inexpensive starting material. Enzalutamide was successfully approved by FDA, but was found to be associated with seizure side effect caused by antagonizing GABAA receptor in the central nervous system [22]. With the hope to find out a second-generation nonsteroidal AR antagonist with a high therapeutic index, apalutamide was later chosen for further preclinical investigation because of its lower steady-state brain tissue level in mice.

As shown in Figure 4, apalutamide (**5**, also named ARN-509) has very similar chemical structure to RD-162 (**11**) with only difference being the replacement of the *N*3-phenyl ring in RD162 (**11**) with a *N*3-pyridyl ring in apalutamide. Apalutamide possesses comparable in vitro activity to enzalutamide, but with greater anti-tumor efficacy in CRPC xenograft models and lower potential in causing seizure as an adverse effect in the central nervous system [14,23]. Specifically, enzalutamide (**4**) and apalutamide (**5**) were demonstrated to retain full antagonist activity in an AR overexpression setting and have a higher binding affinity of up to 10-fold for AR when compared to bicalutamide (**3**). Both of them compete with bicalutamide (**3**) for the same ligand-binding domain of AR. The selective binding of apalutamide (**5**) for AR over other nuclear hormone receptors was observed. Unlike the first-generation AR antagonists, both enzalutamide (**4**) and apalutamide (**5**) can interrupt multiple steps of the AR-signaling pathway, including the androgen binding to AR, nuclear translocation of AR, DNA binding, and coactivator recruitment. Good in vivo pharmacokinetic profiles, including good oral availability, long plasma half-life, and low systemic clearance, were found for both enzalutamide and apalutamide in mouse and dog models. However, apalutamide (**5**) has less chance than enzalutamide (**4**) to bind to plasma proteins, and 2-fold higher concentration of free apalutamide (**5**) was detected in mouse and human plasma. The in vivo pharmacodynamics studies of enzalutamide (**4**) and apalutamide (**5**) were carried out in a CRPC animal model with LNCaP/AR-luc xenograft tumors. Both of them have potent in vivo anti-tumor efficacy because they can significantly decrease androgen driven luciferase reporter-gene activity and reduce tumor volume compared to vehicle. However, apalutamide (**5**) only needs 10 to 30 mg/kg/d to reach maximum efficacy in the castrate mouse model with the LNCaP/AR xenografts, while enzalutamide (**4**) requires 30 to 100 mg/kg/d. Additionally, apalutamide exhibits antitumor activity in a CSPC xenograft model. The above-mentioned data provide preclinical proofs for further clinical development of apalutamide and enzalutamide for patients with CSPC and CRPC [14,23].

#### *2.2. Darolutamide* (**6**)

An AR transactivation screening of a group of nonsteroidal pyrazole-carboxamide and imidazole-carboxamide derivatives in an AR-HEK293 cell model in combination with a lead optimization process led to the discovery of darolutamide (**6**, also named ODM-201) [24]. Similar to enzalutamide (**4**) and apalutamide (**5**), darolutamide (**6**) is a full antagonist that has high affinity for AR in the AR overexpressing setting and suppresses nuclear translocation of AR. In contrast to enzalutamide (**4**) and apalutamide (**5**), darolutamide (**6**) possesses the following features [24]: (i) a different chemical scaffold that may bypass the side effects caused by enzalutamide (**4**) and apalutamide (**5**); (ii) antagonistic effects towards AR mutants AR(F876L), AR(W741L), and AR(T877A) that facilitate resistance to the first- and second-generation nonsteroidal AR antagonists; (iii) an inability to cross over the brain–blood barrier, suggesting a lower seizure risk than observed with enzalutamide; (iv) not increasing the concentration of serum testosterone in a mouse model; (v) having higher in vivo antitumor efficacy in the mouse model, and (vi) a shorter half-life (1.6 h vs 18.3 h for enzalutamide). Taken together, these promising preclinical results imply that darolutamide (**6**) is complementary to enzalutamide (**4**) and apalutamide (**5**) and that darolutamide (**6**) is an excellent addition to the family of the second generation of AR antagonists. However, higher dose and more frequent administration are recommended for darolutamide (**6**) due to its shorter half-life.

#### **3. Syntheses**

A detailed review of the development of synthetic approaches to enzalutamide, apalutamide, and darolutamide was recently published [25].

#### *3.1. Synthetic Approaches to Enzalutamide* (**4**) *and Apalutamide* (**5**)

Structures of enzalutamide (**4**) and apalutamide (**5**) are highly functionalized and offer multiple disconnection approaches to their synthesis. Each strategy is based on a key transformation of advanced intermediates and the bulk of the synthetic effort is spent on synthesizing those intermediates. The synthesis of the core structure of enzalutamide (**4**) and apalutamide (**5**) has been accomplished using three main strategies, which are presented in Scheme 1. Strategy 1 and Strategy 2 construct the thiohydantoin core toward the end of the synthesis, whereas Strategy 3 begins with the formation of thiohydantoin and the aromatic rings are added later. Regardless of the strategy, the assembly of the final drug begins from similar advanced aryl intermediates. Thus, the majority of process development was focused on preparation of aniline derivatives **D** and **E**, and aryl halides **H** and **I**.

**Scheme 1.** Overview of synthetic approaches to hydantoin-based enzalutamide and apalutamide.

#### 3.1.1. Strategy 1: Cyclization of Isothiocyanate

The first strategy is based on tandem condensation–cyclization cascade initiated by addition of isothiocyanate **B** to the α-amino acid derivative **C**, which delivers the thiourea intermediate **A** (Scheme 1). The compound **A** is not isolated, the nitrogen and the pendant carboxylic acid derivative (Z = CO2H, CO2R, or CN) react to form the thiohydantoin core. Thus, the key intermediates in this approach are isothiocyanate **B** and carboxylic acid intermediate **C**.

Sawyers and Jung disclosed the synthesis of **13** from aniline **12** and thiophosgene (Scheme 2) [17,26–28], to which alternative approaches that avoid use of thiophosgene were later developed [29–32]. The intermediate **12** needed for enzalutamide is commercially available. However, the aniline precursor of isothiocyanate fragment **18** needed for preparation of apalutamide had to be synthesized and proved to be a major challenge typical of heterocyclic amines containing both electron-rich and electron poor substituents. All published approaches to **17** vary in the order and method in which the substituents are added to 2-chloro-3-(trifluoromethyl)pyridine (**14**). The initially disclosed synthesis uses nitration–reduction sequence to install the amine functional group (Scheme 2). This strategy required uneconomical functional group interconversion (conversion **14** to hydroxypyridine **15**, then back to chloride **16** after nitration) as well as protection of the amine. The overall yield of this 7-step sequence is difficult to assess because yields for all steps were not reported. Alternately, cyanation can be accomplished prior to reduction of the nitro group and thus avoiding wasteful protection if a bromide is used instead of the chloride (steps i–k, Scheme 2) [33,34].

**Scheme 2.** Synthesis of isothiocyanate fragments of enzalutamide and apalutamide. Reaction conditions: (a) CSCl2, H2O; (b) AcOH, H2O, reflux; (c) HNO3, H2SO4, 90 ◦C; (d) POCl3, PCl5, 110–120 ◦C; H2, (e) Raney Ni, THF; (f) Boc2O, pyridine, DMAP, rt; (g) KCN, CuCN, phenantroline, DMA, 110 ◦C; (h) TFA, CH2Cl2; (i) POBr3, PBr3, Br2, 90–110 ◦C; (j) CuCN, phenantroline, CH3CONMe2, 160 ◦C; (k) AcOH, Fe.

Highly hazardous nitration at elevated temperature can be avoided by installing the amine via C–*N* cross-coupling reaction, although functional group interconversion (Cl to OH and back to Cl) remains as part of the route (Scheme 3). Initially developed conditions for the cross-coupling delivered **23** in low yield (40%) [33,34], but the reaction conditions were later improved to reliably generate the amine **23** in 71–85% yield [35,36]. Other routes to **17** were also disclosed, but they contain serious inefficiencies are less likely to be adopted on process scale [33,34].

$$\begin{array}{ccccccccc} \text{\textquotedblleft}\_{\text{"\textquotedbl{}}\_{\text{"\textquotedbl{}}\_{\text{N}}}^{\text{\textquotedbl{}}\_{\text{CM}}} & \stackrel{\text{\textquotedbl{}}\_{\text{S}}}{\text{\textquotedbl{}}\_{\text{S}}} & \stackrel{\text{\textquotedbl{}}\_{\text{S}}}{\text{\textquotedbl{}}\_{\text{S}}} & \stackrel{\text{\textquotedbl{}}\_{\text{S}}}{\text{\textquotedbl{}}\_{\text{S}}} & \stackrel{\text{\textquotedbl{}}\_{\text{S}}}{\text{\textquotedbl{}}\_{\text{S}}} & \stackrel{\text{\textquotedbl{}}\_{\text{S}}}{\text{\textquotedbl{}}\_{\text{S}}} & \stackrel{\text{\textquotedbl{}}\_{\text{S}}}{\text{\textquotedbl{}}\_{\text{S}}} & \stackrel{\text{\textquotedbl{}}\_{\text{S}}}{\text{\textquotedbl{}}\_{\text{S}}} & \stackrel{\text{\textquotedbl{}}\_{\text{S}}}{\text{\textquotedbl{}}\_{\text{S}}} & \end{array}$$

**Scheme 3.** Nitration-free synthesis of the substituted pyridine intermediate **17**. Reaction conditions: (a) NIS, DMF, CH3CN, 80 ◦C; (b) POCl3, DMF, microwave 130 ◦C; (c) PMBNH2, Pd(OAc)2, BINAP, Et3N, CsCO3, toluene; (d) Zn(CN)2, Pd2(dba)3, dppf, DMF, 110 ◦C; (e) TFA, CH2Cl2.

Synthesis of the advanced intermediate **C** (R = CN, Scheme 1) can be accomplished in several ways. In their initial route to enzalutamide, Sawyers and Jung reported preparation of **26** in 52% overall yield in a 4-step sequence which begins with oxidation of 2-fluoro-4-nitrotoluene (**24**) to the corresponding carboxylic acid [17], which was converted to an *N-*methyl amide **25** via the acid chloride intermediate. Reduction of the nitro group to an amine and addition to acetone cyanohydrin furnish α-amino nitrile **26**, which upon reaction with isothiocyanate **13** under microwave heating in DMF, gave enzalutamide (**4**). In another approach, amino ester **29** was prepared in 4 steps from 4-bromo-2-fluorobenzoic acid (**27**) in 64% yield by first converting the acid to the *N-*methyl amide followed by a SNAr reaction with 2-aminoisobutyric acid and esterification. Although this route is shorter and the overall yield is slightly higher than the synthesis of nitrile **26**, on a process scale the likely starting material would be 4-bromo-2-fluorotoluene which would add one additional step and likely reduce the overall yield (Scheme 4).

**Scheme 4.** Synthesis of the amino nitrile **26** (top) and amino ester **29** (bottom) for the assembly of enzalutamide. Reaction conditions: (a) CrO3, H5IO6, CH3CN, CH2Cl2; (b) SOCl2, DMF, then MeNH2; (c) Fe, AcOH, EtOAc, reflux; (d) 2-cyano-2-hydroxypropane, MgSO4, EtOAc, 80 ◦C; (e) **13**, DFM, microwave 100 ◦C; (f) SOCl2, DMF (cat), 2-PrOAc, 60 ◦C, then MeNH2; (g) 2-aminoisobutyric acid, CuI, K2CO3, 2-acetylcyclohexanone, DMF, H2O, 105 ◦C; (h) MeI, K2CO3, DMF, H2O, 40 ◦C; **13**, DMSO, 2-PrOAc, 83–83 ◦C.

The amino nitrile fragment needed for assembly of apalutamide was prepared similarly in a 4-step sequence which begins with acylation of methyl amine with 2,4-difluorobenzoyl chloride (**30**) followed by SNAr reaction with 4-methoxybenzyl amine under microwave conditions to give **31**. Acid-mediated deprotection and a Strecker reaction with cyclobutanone in the presence of sodium cyanide give amino nitrile **32** (Scheme 5, top). Although this route is relatively short, it will be difficult to implement at scale. The yield of the SNAr reaction is low (40%) due to poor regioselectivity; the yield of the Strecker reaction was not reported. Additionally, use of cyanide at scale is challenging due to potential release of HCN. The company Hinova developed this route into a 3-step preparation (69%–82% yield) of **32** in which 2-fluoro-4-nitrobenzoic acid (**33**) was converted to a methyl amide followed by reduction of the nitro group and a Strecker reaction with cyclobutanone with TMSCN as cyanide source (Scheme 5, bottom) [37,38].

**Scheme 5.** Two options for synthesis of amino nitrile **32** for the assembly of apalutamide. Reaction conditions: (a) MeNH2, THF; (b) PMBNH2, MeCN, microwave 190 ◦C; (c) TFA, CH2Cl2; (d) cyclobutanone, NaCN; (e) **18**, CSCl2, MeCONMe2, 80 ◦C, then HCl, MeOH; (f) Me2NH–HCl, CDI, Et3N, CH2Cl2, rt; (g) Fe, AcOH, EtOAc, reflux; (h) cyclobutanone, TMSCN, AcOH, 80 ◦C.

Several companies subsequently explored other carboxylic acid derivatives, with the most successful one being ester-based route shown in Scheme 6. Coupling between aryl bromide **36** and cyclobutane amino acid **37** gives amino acid derivative **38** (step a, Scheme 6) [39,40]; alternatively, acid **38** can be prepared from amine **40** and α-bromo acid **41** (step b, Scheme 6) [41]. Alkylation of the carboxylic acid **38** followed by a reaction with isothiocyanide **18** delivered apalutamide in 47% yield from **38**. It is important to note that condensation/cyclization cascade of esters and isothiocyanate delivers thiohydantoin in higher yield than the same reaction with the nitrile, but the higher yield comes with trade-offs. First, one additional step will be needed to convert the ester to amide (MeNH2, heat). Second, the cyclization reaction of isothiocyanate with esters produces alcohol by-product, which is reactive toward the isothiocyanate present in the reaction mixture. Therefore, excess of isothiocyanate has to be used in this step and at least one equivalent is ultimately lost to alcoholysis.

**Scheme 6.** Cyclobutanecarboxylic acid-based approaches to the advanced intermediate **39** needed for assembly of apalutamide. Reaction conditions: (a) DMF, K2CO3, CuCl, H2O, 100–105 ◦C; (b) i-Pr2Net, Et3N, CH2Cl2, reflux; (c) MeI, DMF, K2CO3, rt; (d) **18**, DMSO, 2-PrOAc, 90 ◦C.

Other iterations of Strategy 1 were also used in synthesis of apalutamide where the thiohydantoin core was constructed prior to installation of the methyl benzamide (Scheme 7) [42,43]. Halogenated aniline **42** was subjected to a Strecker reaction with cyclobutanone and the resulting amino nitrile **43** was reacted with isothiocyanate **13** formed in-situ from aniline **17** and 1,1 -thiocarbonylbis(pyridine-2(1*H*)-one (vide infra). Appending the methyl amide functional group can then be accomplished in several ways (Scheme 7, step c): (i) Grignard synthesis of carboxylic acid followed by CDI coupling with methyl amine; (ii) direct Pd-catalyzed amide formation with methylamine and carbon monoxide; or (iii) Pd-catalyzed carbonylative esterification followed by a conversion of the ester to the amide.

**Scheme 7.** Synthesis of thiohydantoin core of apalutamide and late-stage amide formation. Reaction conditions: (a) cyclobutanone, NaCN, AcOH; (b) **17**, 1,1 -thiocarbonylbis (pyridine-2(1*H*)-one, toluene, 100 ◦C then HCl, EtOH, DMA, 70 ◦C; (c) *n-*C5H11MgBr, THF, CO2, then CDI, MeNH2, THF; or Pd(*t-*Bu3P)2, CO (5 bar), *i-*Pr2Net, MeNH2, THF, 60 ◦C; or Pd(OAc)2, dppf, *i-*Pr2NH, CO, MeOH, 60 ◦C, then MeNH2, MeOH.

#### 3.1.2. Strategy 2: Late Stage Cyclization of Amino Amide

The second strategy focuses on the late stage formation of thiohydantoin core through cyclization of the amino amide **M** with thiophosgene or its surrogate (Scheme 1, Strategy 2). Depending on the chosen disconnection, the amino amide **M** can be constructed by condensation of amine **D** with amino acid **N**, or aryl-aryl cross-coupling of the amine **D** with bromide **H**. An application of this strategy was reported by Meng and co-workers in their approach to enzalutamide, which started with a carbodiimide coupling of aniline **12** and protected amino acid **45**, affording amino amide **46** after deprotection (Scheme 8). Copper-catalyzed aryl amination with methyl 4-bromo-2-fluorobenzoate (**47**) affords amino amide **49**, which undergoes the cyclization reaction with thiophosgene in the presence of 8-fold excess of DMAP to provide ester **49** in 28% overall yield [44].

**Scheme 8.** Synthesis of advanced intermediate ester **49** via late-stage formation of thiohydantoin. Reaction conditions: (a) CDI, DBU, *i-*Pr2NEt, THF, 55–65 ◦C; (b) HCl, 2-PrOH; (c) methyl 4-bromo-2-fluorobenzoate (**47**), acetylacetone, CuI, K2CO3, DMF, 120–130 ◦C; CSCl2, DMAP, THF, 40–50 ◦C.

As illustrated in Scheme 9, a modified version of this strategy was used in the synthesis of apalutamide. The key differences include more streamlined synthesis of the starting aniline **12**, use of Boc as protecting group for the amino acid **50**, and use of 1,1 -thiocarbonylbis(pyridine-2(1*H*)-one (or phenylthionochloroformate) as thiophosgene alternative for the formation of thiohydantoin core [45]. This route is a highly developed process that achieves synthesis of apalutamide in seven linear steps with only three purifications. Accounting for two-step synthesis of hydroxypyridine **15** (see Scheme 2), this route achieves preparation of apalutamide in nine linear steps.

**Scheme 9.** Synthesis of apalutamide via late-stage, thiophosgene-free cyclization. Reaction conditions: (a) **12**, CDI, DBU, *i-*Pr2NEt, THF, 60 ◦C; (b) HCl, 2-PrOH, 70 ◦C; (c) **28**, DMA, KOAc, CuBr, TMEDA, 120 ◦C; (d) 1,1 -thiocarbonylbis(pyridine2(1*H*)-one, DMAP, DMA, 90 ◦C.

#### 3.1.3. Strategy 3: Functionalization of Thiohydantoin

The third strategy focuses on the formation of thiohydanotin core first and then functionalization the nitrogens (Strategy 3, Scheme 1). In comparison to the two strategies outlined above, this one is relatively less developed. Nevertheless, it holds the most promise for large-scale synthesis of enzalutamide because it is still highly divergent and, even more importantly, avoids use of thiophosgene. The reported synthesis of enzalutamide using this approach begins with a reaction of methyl 2-chloroisobutyrate (**53**) with thiourea in the presence of triethylamine to give thiohydantoin **54**. Compound **54** then undergoes two SNAr reactions. Deprotonation with NaH in DMF followed by addition of 4-bromo-3-(trifluoromethyl)benzonitrile (**55**) furnished compound **56**, which is deprotonated again and reacted with aryl bromide **28** to provide enzalutamide (Scheme 10) [32]. It should be noted that, at the time of this writing, methyl 2-chloroisobutyrate is not widely available from commercial sources and will have to be synthesized [46].

**Scheme 10.** Synthesis of enzalutamide via functionalization of thiohydantoin. Reaction conditions: (a) thiourea, Et3N, DMF, 80–90 ◦C; (b) 4-bromo-3-(trifluoromethyl)- benzonitrile (**55**), NaH, DMF, rt; (c) **28**, NaH, DMF.

A hybrid variant of this strategy and Strategy 1 were also used in synthesis of apalutamide [47]. The ester **57** was first reacted with potassium isothiocyanate to give thiohydantoin **58**, which was then

coupled with aryl bromide **59** to give apalutamide. Both **57** and **58** can be purified by crystallization, which is a significant advantage for process development (Scheme 11).

**Scheme 11.** Synthesis of apalutamide using atom-economical formation of isothiohydantoin core. Reaction conditions: (a) KSCN, *i-*Pr2NEt, MeOH/H2O, reflux; (b) **59**, K2CO3, CuI, DMF, 100–110 ◦C.

#### *3.2. Synthesis of Darolutamide*

The development of synthesis of darolutamide was mostly focused on process improvements, such as isolation via crystallization, elimination of expensive and hazardous reagents, and the general strategy remained unchanged since the original disclosure (Scheme 12) [48–51]. The synthesis relies on cross-coupling and substitution chemistry with some protection/deprotection and functional group interconversions. First, a pinacol boronic ester is installed on the THP-protected pyrazole **60** by treatment with *n-*BuLi, then with triisopropylborane, and finally with pinacol. Palladium(II) acetate-catalyzed Suzuki cross-coupling of **61** with aryl bromide **62** delivers intermediate **63**. The nitrogen was deprotected with aqueous acid and reacted with *N-*protected (*S*)-(+)-2-amino-1-propanol (**64**) under Mitsunobu conditions followed by deprotection at low pH to give intermediate **65**. The synthesis of darolutamide diastereomers was completed by amide coupling of **65** with carboxylic acid **66** followed by reduction of the methyl ketone with NaBH4 in ethanol.

**Scheme 12.** Synthesis of darolutamide. Reaction conditions: (a) n-BuLi, THF, toluene; (b) (i-PrO)3B; (c) pinacol, AcOH; (d) **62**, Pd(OAc)2, K2CO3, CH3CN/H2O, 70 ◦C; (e) aq HCl, MeOH, 10 ◦C; (f) **64**, PPh3, DIAD, EtOAc; (g) aq HCl, 45 ◦C; (h) **66**, T3P, EtOAc, i-Pr2NEt, 10 ◦C; (i) NaBH4, EtOH.

#### **4. Clinical Studies and Use of Second Generation Nonsteroidal AR Antagonists**

Only those clinical studies directly associated with the US FDA approval of enzalutamide, apalutamide, and darolutamide were highlighted in this review.

#### *4.1. Enzalutamide*

Enzalutamide (also named MDV3100; trade name: Xtandi) has been approved by the FDA in 2012, 2018, and 2019, respectively, for the treatment of mCRPC, nmCRPC, and mCSPC. Enzalutamide possesses a generally good safety profile and is now widely used as a standard-of-care for the treatment of three forms of prostate cancer. The clinical use of enzalutamide not only helps to better manage prostate cancer but also verifies that androgen receptor signaling continues to be one of critical driving forces for CRPC. Enzalutamide can be sequentially used with other therapeutic methods.

The earlier clinical trials of enzalutamide were initiated by its promising efficacy and drug-like properties collected from the preclinical CRPC models. The competitive AR binding capability and clinically effective antitumor activity, together with tolerable safety profile of enzalutamide, were verified in human by a phase I/II study that enrolled 140 patients with CRPC in both pre- and post-chemotherapy settings [52]. The maximal tolerated dose for enzalutamide was determined to be 240 mg/day and the recommended dose for the advanced clinical trials was identified to be 160 mg/day. In view of these inspiring early-stage clinical results, the first phase III trial of enzalutamide, named AFFIRM, has been started to scrutinize enzalutamide versus placebo in patients with mCRPC in a post-chemotherapy setting.

#### 4.1.1. Enzalutamide for mCRPC

With overall survival as its primary end point, AFFIRM aimed to assess whether enzalutamide can prolong the survival time of patients with mCRPC in a post-chemotherapy setting [53]. The clinical benefits derived from AFFIRM as demonstrated by the primary end points and the secondary end points were summarized in Table 3. The primary outcome from this phase III trials established enzalutamide as the first nonsteroidal AR antagonist with significant increase in patient's overall survival time. The secondary outcome suggested that enzalutamide can appreciably slow cancer progression, reduce PSA response, and improve patient's quality of life when compared to placebo. The major adverse effects of enzalutamide observed from AFFIRM are seizures, with 0.6% (five patients out of 800) of patients from the enzalutamide treatment group being reported to have a seizure. This study verified that AR and AR signaling continue to play a pivotal role for the progression of CRPC. Enzalutamide was thus quickly approved by the US FDA on 31 August 2012, for the treatment of late-stage mCRPC due to its capability of prolonging patient's life.


**Table 3.** Major therapeutic benefits brought by enzalutamide based on AFFIRM.

OS: Overall Survival; rPFS: Radiographic Progression-free Survival; SRE: Skeletal-related Event; PSA: Prostate-specific Antigen; AEs: Adverse Events. \* Does not include patients who discontinued or died during the trial.

Inspired by the positive results from AFFIRM and the greater benefit observed in chemotherapy-naïve patients in the phase I-II clinical trial [52], another phase III trial (named PREVAIL, ClinicalTrials.gov number, NCT01212991) of enzalutamide was designed to explore the possibility of extending the application of enzalutamide to patients with mCRPC before chemotherapy [54]. A total of 1717 patients with chemotherapy-naïve mCRPC were enrolled in this trial and randomized into enzalutamide treatment (872; 160 mg daily) group and placebo group (845). Radiographic progression-free survival and overall survival were used as the two primary end points. The clinical benefits brought by enzalutamide treatment in this study with respect to all primary and secondary end points are illustrated in Table 4. It can be concluded from these data that treatment with enzalutamide resulted in a noticeable decrease in risk of radiographic progression and death, as well as a marked

delay in the need of chemotherapy. Due to the promising results from PREVAIL phase III trial, the clinical use of enzalutamide was therefore extended by the US FDA to chemotherapy-naïve patients with mCRPC on 10 September 2014.


**Table 4.** Major therapeutic benefits brought by enzalutamide based on PREVAIL.

OS: Overall Survival; rPFS: Radiographic Progression-free Survival; SRE: Skeletal-related Event; CC: Cytotoxic Chemotherapy; PSA: Prostate-specific Antigen; AEs: Adverse Events. **\*** Does not include patients who discontinued or died during the trial.

#### 4.1.2. Enzalutamide for nmCRPC

The patients with nmCRPC are refractory to ADT treatment and at the onset of metastasis. It has therefore been recognized that new treatment strategies are urgently needed to reduce the risk for metastasis in men with nmCRPC in accompany with a short PSA doubling time. PROSPER phase III trial was designed to investigate whether enzalutamide can meet this need [55]. This study enrolled a total of 1401 patients with nmCRPC and a PSA doubling time no more than 10 months. A total of 933 patients received enzalutamide treatment (160 mg daily) in combination with ADT; while the remaining 468 patients were assigned to the placebo group with continued ADT. Metastasis-free survival was set as the primary end point. As illustrated in Table 5, enzalutamide treatment is superior to placebo with regards to the primary end point and most of the secondary end points. The clinical results from the PROSPER trial highlighted that the risk of progression to mCRPC or death in the enzalutamide treatment group has been lowered by 71% as compared with the placebo group. Because of the promising results from the PROSPER phase III trial, enzalutamide was approved by the US FDA, after Priority Review designation, on 13 July 2018, for the treatment of patients with nmCRPC. This approval extends the enzalutamide treatment to patients with nmCRPC, and makes enzalutamide the first FDA-approved oral medicine for both mCRPC and nmCRPC.


**Table 5.** Major therapeutic benefits brought by enzalutamide based on PROSPER.

MFS: Metastasis Free Survival; rPFS: Radiographic Progression-free Survival; OS: Overall Survival; C-FS: Chemotherapy-Free Survival; PP: Pain Progression; CC: Cytotoxic Chemotherapy; PSA: Prostate-specific Antigen; AEs: Adverse Events; \* Does not include patients who discontinued or died during the trial.

#### 4.1.3. Enzalutamide for mCSPC

Patients with mCSPC is defined as those who have metastatic prostate cancer that still responds to ADT. Up to 5% of annual prostate cancer incidences belong to mCSPC in the United States [56]. With ADT as the original standard of care, most of the patients with mCSPC inevitably progress to high-risk mCRPC in 1–3 years. To help meet the needs of this big group of patients, the ARCHES Phase III trial (ClinicalTrials.gov identifier: NCT02677896) [57] aimed to investigated whether enzalutamide in combination with ADT can prolong radiographic progression-free survival using ADT alone as control. The clinical outcomes from this trial pertaining to the primary end point (radiographic progression-free survival) and key secondary end points are summarized in Table 6. A conclusion can be drawn from these data that enzalutamide, plus ADT, demonstrate clinically significant improvement in efficacy by prolonging the radiographic progression-free survival while maintaining the safety level, as compared with ADT alone. The favorable results encouraged the US FDA to grant an extension of enzalutamide for the treatment of mCSPC on 16 December 2019.


**Table 6.** Major therapeutic benefits brought by enzalutamide based on ARCHES.

rPFS: Radiographic Progression-free Survival; OS: Overall Survival; NAT: New Antineoplastic Therapy; CR: Castration resistance; PSA: Prostate-specific Antigen; AEs: Adverse Events; ‡ Radiographic Progression-Free Survival (rPFS) Based on Independent Central Review (ICR) of Bone Scan According to Prostate Cancer Clinical Trials Working Group 2 (PCWG2) Criteria; † rPFS Based on ICR of Bone Scan According to Protocol Assessment Criteria; \* Does not include patients who discontinued or died during the trial.

#### *4.2. Apalutamide*

The promising preclinical data of apalutamide were well-mirrored by its phase I clinical trial [23,58]. Thirty men with mCRPC were enrolled in its first-in-human study. Apalutamide displayed AR inhibitory ability as evidenced by the 47% PSA response (defined as ≥50% reduction from baseline) at 12 weeks at all tested doses from 30 to 480 mg. The pharmacokinetic profile of oral-administrated apalutamide is linear and dose-dependent in the range of 30–480 mg. The rapid absorption was demonstrated by the fact that it can be measured in the plasma at 30 min after oral intake and that it can reach peak concentrations after 2–3 h. A half-life of 3–4 days was observed in the systematic circulation and most enrolled patients reached a steady-state concentration after 3 weeks of non-interrupted administration of apalutamide. A good safety profile observed from this clinical study further confirmed the high therapeutic index evaluated from the preclinical models. A daily dose of 240 mg was recommended for the follow-up clinical studies of apalutamide considering its dose to maximize the tumor regression in preclinical models, along with peak plasma concentration, safety profile, and efficacy in the phase I clinical study.

#### 4.2.1. Apalutamide for nmCRPC

The SPARTAN trial is a randomized, double blind and multicenter phase III study that evaluated the efficacy of apalutamide in nmCRPC. There were 1207 enrolled men with nmCRPC on ADT with a PSA doubling time of over 10 months randomized to 2:1 to receive apalutamide with ADT or placebo with ADT [59].

The first interim analysis, which concluded in May 2017, determined that there was statistical significance in metastasis free survival (MFS), progression free survival (PFS), time to metastasis and time to symptomatic progression (Table 7). Due to the compelling evidence shown, the safety monitoring committee recommended that the placebo group be allowed to receive the treatment in July 2017. The second interim analysis continued to 2019 to better characterize the effect of apalutamide. It was determined that the median MFS was 40.5 months for the treatment group and 16.2 months in the placebo group with a 95% confidence interval. The four-year survival rate of apalutamide was found to be 72% compared to 65% of the placebo group. When considering the patients that crossed over from placebo to treatment, the four-year survival rate remained at 72% for apalutamide [59].


**Table 7.** Major therapeutic benefits brought by apalutamide based on SPARTAN.

MFS: Metastasis Free Survival; PFS: Progression Free Survival; OS: Overall Survival; CC: Cytotoxic Chemotherapy; AEs: Adverse Events; **\*** Does not include patients who discontinued or died during the trial.

Disease progression was the most frequent indicator for treatment discontinuation. Only 28% of the treatment group compared to 37% of control experienced cancer progression for discontinuation. Apalutamide treatment led to the extension of the second PFS rate by approximately 11.8 months versus placebo. The four-year second PFS rate for the treatment had a 19% difference when compared to the control [59]. Adverse effects were reported in 97% of patients in the treatment group and 94% of patents in the control group [59,60]. Median overall survival (OS) was not reached in either the treatment or placebo group, as the threshold of 427 deaths have not yet been reached as specified in the O'Brien-Fleming boundary [59].

Overall, apalutamide showed a quarter reduction for risk of death when compared to placebo as well as a higher subsequent life-prolongation, despite crossover. The resulting data for SPARTAN included statistical significance in improving MFS and time to symptomatic progression compared to placebo. The observations that apalutamide delays progression and death when combined with ongoing ADT suggests that the drug may be advantageous to high-risk nmCRPC patients. FDA immediately approved apalutamide for high-risk nmCRPC patients on 14 February 2018 [59].

#### 4.2.2. Apalutamide for mCSPC

Apalutamide was approved by the US FDA, after a priority review, on 17 September 2019, to extend its treatment from patients with nmCRPC to those with mCSPC based on the efficacy demonstrated by its TITAN (NCT02489318) phase III trial. This clinical trial was designed as a randomized, double blind and multicenter phase III study and aimed to assess the clinical efficacy of apalutamide in combination with ADT (surgical or medical castration) in patients with mCSPC. A study of 1052 men with mCSPC were randomized 1:1 to treatment and control groups. This involved patients regardless of disease volume and with a history of docetaxel treatment and treatment for localized prostate cancer. At the conclusion of the first interim analysis in November 2018, it was found that 68.2% of patients in the apalutamide and ADT group had a 24-month radiographic progression-free survival compared to 47.5% of the placebo group (Table 8). The overall survival, measured after 200 deaths, for 24-months is 82% for apalutamide group and 73% for the placebo group. The average time for second progression-free survival was also longer in the treatment group compared to that of the placebo group. Analysis of adverse effects between apalutamide and placebo did not differ significantly [61]. In summary, the TITAN trial revealed that apalutamide in combination with ADT resulted in life-prolongation and radiographic progression-free survival relative to placebo with ADT while also preserving quality of life for men with mCSPC [61].


**Table 8.** Major therapeutic benefits brought by apalutamide based on TITAN.

OS: Overall Survival; rPFS: Radiographic Progression-free Survival; PSA: Prostate-specific Antigen; AEs: Adverse Events; \* Does not include patients who discontinued or died during the trial.

#### 4.2.3. Indirect Comparison with Enzalutamide

In the lack of direct comparative studies, Chowdhury et al. conducted a matching-adjusted indirect comparison (MAIC) of the efficacy and quality of life of apalutamide to that of enzalutamide in nmCRPC using data collected from SPARTAN and PROSPER trials [62]. A total of 1171 patients were matched from the SPARTAN trial (n = 1207) to the PROSPER trial (n = 1401). Relative to enzalutamide, apalutamide was better tolerated based on adverse events with an overall decrease in fatigue, low appetite, hypertension, and nausea occurrences. It was also associated with a better health-related qualify of life based on the Functional Assessment of Cancer Therapy-Prostate score [63]. Additionally, the calculated hazard ratios for apalutamide versus enzalutamide were 0.77 for OS and 0.91 for MFS [62]. Based on these MAIC results, apalutamide demonstrates slightly better overall tolerability and is associated with slightly higher efficacy in nmCRPC patients.

#### *4.3. Darolutamide*

It has recently been recognized that early and effective suppression of AR signaling may serve as a good strategy to manage nmCRPC [64], which has been firmly supported by the obvious metastasis-free survival benefits reported in the PROPER trial for enzalutamide and the SPARTAN trial for apalutamide. The clinical phases I and II studies [65,66] suggest that darolutamide provides not only meaningful antitumor efficacy but also a favorable safety profile in clinical settings. ARAMIS phase III trial was thus conducted to further assess the treatment benefits and the possible adverse events of darolutamide in men with nmCRPC. A total of 1509 men with nmCRPC and a PSA doubling time no more than 10 months were enrolled in this trial and were randomized in a 2:1 ratio to receive darolutamide (600 mg twice daily) plus ADT or ADT alone. Metastasis free survival was employed as the primary end point, and the appearance of metastasis was judged by blinded and independent central imaging review. The therapeutic benefits of darolutamide were consistently and affirmatively judged by the primary end point and the entire secondary end points (Table 9). Darolutamide in combination with ADT was demonstrated to prolong metastasis-free survival by 22 months and to reduce the risk of metastasis or death by 59% when compared with ADT alone. The therapeutic benefits brought by darolutamide, enzalutamide, or apalutamide are generally similar in patients with nmCRPC. However, darolutamide exhibited a good safety profile in this phase III trial with approximately similar incidence of adverse events in the darolutamide treatment and placebo groups. The fact that darolutamide has less common adverse effects in the phase III trial relative to enzalutamide and apalutamide is associated with its low penetration of the blood-brain barrier as evidenced in preclinical studies. Darolutamide was approved by the US FDA on 30 July 2019, for the treatment of nmCRPC in line with ARAMIS phase III trial [16].


**Table 9.** Major therapeutic benefits brought by darolutamide based on ARAMIS.

MFS: Metastasis Free Survival; OS: Overall Survival; PP: Pain Progression; SSE: Symptomatic Skeletal Event; AEs: Adverse Events; **\*** Does not include patients who discontinued or died during the trial.

#### **5. Mechanism of Action of the Second-Generation AR Antagonists**

The AR is a nuclear receptor and a ligand-dependent transcription factor that regulates the expression of certain specific genes, including PSA. Its most potent native ligand is 5α-dihydrotestosterone (DHT) that is generated by intracellular metabolism of testosterone, an endogenous androgen synthesized primarily in the testes. The DHT-AR binding drives the AR translocation from cytoplasm to cell nucleus where the AR forms dimer and binds to the androgen response elements in DNA. Co-activators (coregulatory proteins) are then recruited to boost transcription, leading to prostate cancer cell proliferation and survival [2,3]. As shown in Figure 5, castration therapies (both surgical or medical) and abiraterone acetate block the androgen production, while AR antagonists restrain the AR function through competitively binding to the androgen binding site of AR in cell cytoplasm. Intriguingly, enzalutamide, apalutamide, and darolutamide mechanistically distinguish themselves from the first generation nonsteroidal AR antagonists by interfering several stages in the AR signaling pathway [14,23,24]. In addition to competitively suppressing androgen-AR binding, these second-generation nonsteroidal AR antagonists also inhibit the AR translocation from cytoplasm to cell nucleus, the coactivator recruitment, and the AR-DNA binding.

\*First generation nonsteroidal AR antagonist

**Figure 5.** Mechanism of action of different hormonal therapies in AR signaling pathway. \* First generation nonsteroidal AR antagonist.

#### **6. Strategies to Overcome the Resistance to the Second-Generation AR Antagonists**

In spite of the above-mentioned therapeutic benefits of three second-generation AR antagonists in patients with mCRPC, nmCRPC, or mCSPC, a considerable portion of patients are primarily resistant to the treatment. As summarized in Table 10, 63% of the patients with mCRPC after treating with enzalutamide are still at risk of death; 28%–41% of the patients with nmCRPC under the treatment of enzalutamide, apalutamide, or darolutamide, in combination with ADT, are on the line of progression to metastasis or death; 31%–48% of patients of mCSPC under the treatment of enzalutamide or apalutamide are exposed to radiographic progression or death. Additionally, acquired resistance emerges with the time of treatment.


**Table 10.** Hazard Ratios for the primary end points in the phase III trials of three AR antagonists.

#### *6.1. Mechanisms of the Resistance to the Second-Generation AR Antagonists*

The accurate mechanisms of the resistance to the second-generation AR antagonists are still not very clear. Several proposed mechanisms underlying the resistance to the second-generation AR antagonists are summarized in Figure 6. These mechanisms can be classified into two categories: reactivating androgen receptor signaling and bypassing androgen receptor signaling [67]. The reactivation of AR signaling can be achieved by AR amplification and AR overexpression, AR gain-of-function mutations, spliced AR variants (e.g., AR-V7), and intramolecular generation of androgens. The resistance can also be gained through bypassing AR signaling pathway including glucocorticoid receptor takeover, epithelial-mesenchymal transition, neuroendocrine transformation, autophagy, and immune system activation. More details about these proposed mechanisms have been comprehensively reviewed in the literature [67–70].

**Figure 6.** Mechanisms of resistance to the second-generation AR antagonists.

#### *6.2. Strategies to Overcome the Resistance to the Second-Generation AR Antagonists*

#### 6.2.1. Combination Therapy

Combination therapy through targeting multiple complementary mechanisms of action has been recognized in recent years to be a promising strategy to overcome drug resistance [71]. Taking advantage of currently available therapies for CRPC, development of their optimal combinations as multifaceted therapies emerges as one of research hotspots in the field. Additionally, numerous of enzalutamide-based combinations are currently under clinical investigation at different phases, most of which have been tabulated by Tucci et al. in their review article [67]. With the goal to sensitize enzalutamide, these combinations were designed based on the current proposed mechanisms of resistance to the second-generation AR antagonists, as listed in Figure 6. For example, the CORT125281 (glucocorticoid receptor antagonist, NCT03437941), metformin (induces epithelial-mesenchymal transition via suppression of transforming growth factor beta 1/signal transducer and activator of transcription 3 (TGF-β1/STAT3) pathway, NCT02339168 and NCT02640534), galunisertib (TGF-β inhibitor, NCT02452008), AZD5363 (protein kinase B (AKT) inhibitor, NCT0331054), pembrolizumab (an anti-PD-1 checkpoint, NCT02861573 & NCT02787005), and AZD5069 (chemokine receptor antagonist, NCT03177187) are currently in clinical studies in combination with enzalutamide.

#### 6.2.2. Target AR with Other Strategies

AR is a nuclear steroid receptor that comprises three functional domains including the ligand-binding domain (LBD, *C*-terminal end), the DNA-binding domain (DBD, central portion), and the transactivation domain (NTD or TAD, *N*-terminal end) [3]. Enzalutamide, apalutamide, and darolutamide competitively bind to the ligand-binding pocket of the LBD and inhibit the agonistic action of intrinsic ligands. The compounds that still target AR but can overcome the resistance of the second-generation AR antagonists include new LBD-targeted AR antagonists with novel chemical scaffolds, TAD (or NTD)-targeted AR antagonists, DBD-targeted AR antagonists, and AR degraders.

New AR antagonists that still bind to the LBD but with distinct chemical structures can overcome the resistance due to the point mutations, which has been exemplified by the successful story of darolutamide [72]. As abovementioned, darolutamide was developed targeting enzalutamide-resistant prostate cancer. It was revealed to suppress the transcriptional activity of some AR mutants including T878G that was responsible of converting enzalutamide into a partial AR agonist. Recently, halogen-substituted anthranilic acid derivatives have been established as a new chemical scaffold that inhibits the transactivation of both wild-type AR and AR mutants that render treatment resistance to the first-generation and second-generation nonsteroidal AR antagonists [73].

Several compounds that target the TAD or DBD of the AR have been demonstrated to possess potential in treating CRPC, which have been comprehensively summarized in an excellent review article [3]. The EPI compounds that were first isolated from marine sponges and derived from bisphenol A represent the most well-established inhibitors of AR-TAD. This group of compounds down-regulates the expression of full length AR and truncated AR variants (e.g., AR-V7) through suppressing tau-1 (transcriptional activation unit 1) and tau-5 of the TAD [74]. They inhibited AR-positive prostate cancer cell proliferation in both in vitro and in vivo experiments and suppressed the growth of AR-positive prostate cancer cell-derived tumors. The most developed EPI compound, EPI-506, has advanced to a Phase I/II clinical trial (NCT02606123) in patients with mCRPC after enzalutamide and/or abiraterone treatment.

Hairpin polyamide was developed as an AR antagonist that directly inhibits AR binding to DNA and blocks the transcription processes mediated by AR [75]. Hairpin polyamide compounds may be a good strategy to overcome the resistant to the second-generation AR antagonists because they target the transcription driven by both AR and glucocorticoid receptor. Moreover, overexpression of glucocorticoid receptor has been proposed to be one of the critical pathways leading to the resistance of the second-generation AR antagonists.

A new strategy to combat the resistance of the second-generation AR antagonists is to target AR protein for degradation, which is a completely different mechanism compared with those for AR antagonists. Additionally, AR degradation has been reported to be a likely prerequisite for prostate cancer tumor shrinkage based on an in vivo experiment [76]. AR degradation via proteolysis-targeting chimeras (PROTACs) is currently the most intriguing development in this field because this technology replaces the occupancy-driven mechanism for AR antagonist with an event-driven mechanism [77,78]. AR-PROTACs are bifunctional chimeras that can bring AR protein and the E3 ubiquitin ligase in close proximity, resulting in AR ubiquitination and subsequent degradation. AR-PROTACs have been verified in vitro and in vivo to be a better therapeutic strategy than AR antagonists for targeting AR signaling [79]. ARV-110 is the first AR-PROTAC to enter a phase I clinical trial (NCT03888612) in 2019. This clinical study aims to evaluate the pharmacokinetics, safety, and tolerability of ARV-110 in patients with mCRPC who have received more than two systemic therapies.

Additionally, the association between metformin (an antidiabetic drug) and reduced prostate cancer risk in patients with type 2 diabetics prompted a plethora of investigations on the therapeutic effects of metformin. Metformin is undergoing several clinical trials for prostate cancer, which has been summarized in the literature [80]. The mechanism of action underlying its anti-prostate cancer activity has been extensively explored [80], with the crosstalk between adenosine monophosphate-activated protein kinase (AMPK) activation and AR degradation as the most attractive one [81]. It may therefore be a new strategy, especially for prostate cancer patients with diabetics, to use AMPK activators to overcome the resistance to second-generation nonsteroidal AR antagonists. In addition, cyclin-dependent protein kinase 9 (CDK9) is a druggable target for prostate cancer because CDK9 can not only phosphorylate AR and activate AR transcriptional activity but also target anti-apoptotic proteins [82]. Therefore, CDK9 inhibitors may serve as a better therapeutic strategy over the second-generation nonsteroidal AR antagonists for the patients with CRPC.

#### **7. Conclusions**

In conclusion, an interdisciplinary collaboration led the discovery of enzalutamide as the first second-generation nonsteroidal androgen receptor (AR) antagonist with a strong binding affinity to AR. Enzalutamide can significantly prolong not only overall survival time and metastatic free survival time for patients with lethal CRPC, but also castration resistant free survival time for patients with CSPC. Enzalutamide has thus been approved by the US Food and Drug Administration (FDA) for the treatment of both metastatic (in 2012) and non-metastatic (in 2018) CRPC, as well as CSPC (2019) on the basis of the therapeutic benefits observed from AFFIRM, PREVAIL, PROSPER, and ARCHES Phase III clinical trials. Encouraged by the positive clinical results of enzalutamide, two other second-generation AR antagonists, apalutamide, and darolutamide have recently been approved by the FDA for the treatment of prostate cancer. These three second-generation AR antagonists not only offer patients with different stages of prostate cancer with alternative therapeutics, but also verified that AR signaling pathway plays a pivotal role in the progression of both CSPC and CRPC. Several approaches have been developed for the syntheses of these three second-generation AR antagonists, with three main strategies for the syntheses of the core structure of enzalutamide and apalutamide. The drawback of these AR antagonists as therapeutics for prostate cancer is the drug resistance, which can be developed by reactivating or bypassing androgen receptor signaling pathway. Combination therapies taking advantage of multiple complementary mechanisms of action and targeting AR with other mechanisms may serve as good strategies to overcome the resistance.

**Author Contributions:** Q.-H.C. and H.M. conceptualized this review. P.R., H.M., and Q.-H.C. performed the literature search. P.R., A.R., K.M., N.O., H.M., and Q.-H.C. interpreted and summarized the original data from the references. P.R., A.R., H.M., and Q.-H.C. drafted the manuscript. H.M. and Q.-H.C. revised and finalized the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Institutes of Health (National Institute of General Medical Sciences) under Award Number SC2GM121185 (Q. Chen). K. Muthima was supported by the Fresno State NIH Bridges to Doctorate Program; the funding for this program is provided by the National Institutes of Health under Grant Number R25GM115293. N. Olveda was supported by the Fresno State NIH RISE Program; the funding for this program is provided by the National Institutes of Health under Grant Number R25GM131956. The APC was funded in part by the Henry Madden Library at California State University Fresno.

**Acknowledgments:** We thank the Department of Chemistry and College of Science and Mathematics at the California State University Fresno for all administrative support.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **Design, Synthesis, and Biological Evaluation of Proteolysis Targeting Chimeras (PROTACs) for the Dual Degradation of IGF-1R and Src**

### **Sudhakar Manda 1,**†**, Na Keum Lee 1,**†**, Dong-Chan Oh <sup>2</sup> and Jeeyeon Lee 1,\***


#### Academic Editor: Qiao-Hong Chen Received: 30 March 2020; Accepted: 20 April 2020; Published: 23 April 2020

**Abstract:** A focused PROTAC library was developed to degrade both IGF-1R and Src proteins, which are associated with various cancers. PROTACs with IGF-1R and Src degradation potentials were synthesized by tethering different inhibitor warhead units and the E3 ligase (CRBN) recruiting-pomalidomide with various linkers. The designed PROTACs 12a–b inhibited the proliferation and migration of MCF7 and A549 cancer cells with low micromolar potency (1–5 μM) in various cellular assays.

**Keywords:** PROTACs; anticancer activity; protein degradation; IGF-1R; Src

### **1. Introduction**

For the past two decades, targeted protein degradation strategies have been explored widely to develop treatments for various diseases, particularly for cancers [1–4]. Proteolysis targeting chimeras (PROTACs) have emerged as a novel therapeutic strategy in drug discovery for targeted protein degradation [4–6]. PROTACs are heterobifunctional molecules that possess one warhead ligand that binds to the target protein of interest (POI), and a second ligand that recruits an E3 ligase system, which is connected by a chemical linker. The recruitment of the E3 ligase to the target protein results in ubiquitination and the subsequent degradation of the target protein by the proteasome, which is a distinct mechanism from the occupancy-driven modulation of enzyme function by conventional small-molecule inhibitors. Initially described by Crews and Deshaies in 2001, PROTACs have been successfully applied to numerous target proteins [7–12]. As opposed to the stoichiometric binding of drugs that prevent the enzymatic activity of target proteins, PROTACs need to transiently bind to their targets for degradation; this is promising for the potential to address therapeutic challenges in targeting intractable proteins in the current drug discovery [13]. In addition, they can impede the feedback-mediated increased expression of the target proteins that often result in pharmacological inefficacy by small-molecule inhibitors [14,15].

Several E3 ligases have been used in PROTAC technology to degrade recruited target proteins, which include β-TRCP, MDM2 and cIAP [7–9]. In addition, von Hippel-Lindau (VHL) and cereblon (CRBN) have been extensively studied in relation to PROTAC-mediated protein degradation [2,16–18]. Cereblon (CRBN), which is a part of a cullin-RING ubiquitin ligase complex, was identified as the target of thalidomide and its derivatives [19]. These phthalimide immunomodulatory drugs (IMiDs), including lenalidomide, pomalidomide, and thalidomide, play a pivotal role in the treatment of

multiple myeloma and now serve as potent binders of the CRBN. To date, PROTACs have been successfully applied to various target proteins with different cellular locations, including estrogen and androgen receptors, BET proteins, tau protein, FKBP12, and kinases [2,11,12,17,20,21]. In particular, PROTACs, which hijack various cellular kinases, have resulted in effective target degradations [22–26]. Furthermore, the enhanced selectivity of kinase degraders, as compared to their parental kinase inhibitors, were implicated as having potential applications for clinical study [23,27]. Currently, many efforts are underway in medicinal chemistry to convert previously ineffective inhibitors to selective PROTACs for a next-generation drug platform.

The insulin-like growth factor 1 receptor (IGF-1R) is a membrane receptor tyrosine kinase that is implicated in several cancers, including prostate, breast, and lung cancers [28–30]. IGF-1R plays a key role in the proliferation, transformation and survival of various cancer cells. Frequently, its anti-apoptotic properties result in resistance to cytotoxic chemotherapeutic drugs or radiotherapy. For these reasons, the IGF-1R signaling pathway has been a major target for the development of anticancer agents [31,32]. Src, known as proto-oncogene tyrosine-protein kinase, is also associated with cancer cell survival and resistance to targeted anticancer therapies [33]. In particular, Src activation is related to the resistance of many anti-IGF-1R therapeutics. Indeed, a combined inhibition of IGF-1R and the Src family kinases have been shown to enhance antitumor effects in various cancers by decreasing the activated survival pathways [34,35]. We have also shown that the dual inhibition of IGF-1R and Src could be a viable approach to develop effective anticancer therapies to overcome resistance [36].

In this study, we report the design and synthesis of IGF-1R/Src dual degraders using the PROTAC strategy (Figure 1). The (5-cyclopropyl-1*H*-pyrazol-3-yl)pyrimidine-2,4-diamine (A) is known as a potent c-Src inhibitor with an IC50 value of 720 nM from the enzyme inhibition assay [37,38]. RBx10080307 (B) is known as an IGF-1R inhibitor with a cell free IC50 value of 277 nM [39,40]. A common structure, *N*2-phenyl-*N*4-(1*H*-pyrazol-3-yl)pyrimidine-2,4-diamine (C), was derived and was connected with the pomalidomide as a ligand for cereblon for dual IGF-1R/Src degradation. We reasoned that a common chemical structure would target both IGF-1R and Src, bringing both proteins close to the E3 ligase for degradation in PROTACs. We also adopted previously reported 2,4-bis-arylamino-1,3-pyrimidine (D) as an IGF-1R warhead [41], and the aminopyrazolo [3,4-*d*]pyrimidine module (E) as a Src warhead for the PROTAC approach [42,43], and compared their efficacy to those of 12 with the newly designed inhibitor units. Through extensive optimization of the linker region, we have synthesized a series of dual IGF-1R/Src degraders. Among these, PROTACs 12a and 12b were capable of inducing the degradation of both IGF-1R and Src proteins at 1–5 μM in the MCF7 (human breast cancer) and A549 (human lung cancer) cells.

**Figure 1.** Chemical structures of representative Src inhibitor **A**, IGF-1R inhibitors **B**, and a schematic diagram of PROTACs for IGF-1R and Src.

#### **2. Results**

#### *2.1. Design and Synthesis*

We developed a convergent synthetic route to prepare the designed dual IGF-1R/Src PROTAC degraders and their control compounds (Scheme 1). We prepared 4a–b in two steps. An azidation was first carried out from 2-chloropolyethoxy ethanols 1a-b to provide 2a–b [44], which were coupled with 4-flouronitrobenzene 3 to give 4a–b, respectively [45]. 4c was synthesized according to the previous reports, as depicted in the supplementary information [46]. The reduction of azides 4a–c, in the presence of triphenylphosphine, leads to the amine intermediates 5a–c, which were subsequently attached with thalidomide derivative 6 to obtain 7a–c, followed by a reduction with Fe/NH4Cl to provide 8a–c. PROTACs 10a–c were synthesized by coupling 8a–c with 2-chloro-4-arylamino-1,3-pyrimidines 9 in DMSO. The key intermediates 8a–b were also treated with their corresponding reagents 11a or 11b, to produce PROTACs 12a–d (Scheme 1) [36,47].

**Scheme 1.** Reagents and conditions: (a) NaN3, H2O, 80 ◦C, 16 h, 69–82%; (b) K2CO3, DMSO, 80 ◦C, 4 h, 53–61%; (c) Ph3P, THF-H2O, RT, 14 h, 61–85%; (d) DIPEA, DMF, 90 ◦C, 16 h; (e) Fe (powder), NH4Cl, EtOH-H2O, 80 ◦C, 2 h (42–44% over 2 steps); (f) DMSO, 100 ◦C, 4 h (10a–c, 12a–b; 21–57%); (g) p-TsOH, butanol, 100 ◦C, 4 h (12c–d; 22–32%).

The thalidomide-attached linkers 8a–b were also coupled with azidoacetic acid 13 in the presence of EDC·HCl to provide 14a–b, which were further treated with *N*-substituted 4-aminopyrazolo[3–*d*]pyrimidines intermediate (15) in the presence of copper sulfate and sodium ascorbate in THF/H2O/tBuOH to obtain the click products 16a–b via a copper (I)-catalyzed alkyne azide 1,3-dipolar cycloaddition (CuAAC) reaction (Scheme 2). 8a-b reacted with the bromopropyl group-attached pyrazolo[3,4-*d*]pyrimidin-4-amine (17) under the basic condition yielded PROTACs 18a-b. Next, *N*-propagylation of 8a–b under the basic condition afforded 20a-b, which were coupled with azido intermediate (19) via the CuAAC reaction to obtain the desired triazole products 21a–b [36].

**Scheme 2.** Reagents and conditions: (a) EDC·HCl, DIPEA, DMF, RT, 16 h, 40–62%; (b) CuSO4·5H2O, sodium ascorbate, THF, t-Butanol-H2O, RT, 6 h, 26–27%; (c) K2CO3, DMF, 16 h, 50.5–50.8%; (d) Propargyl bromide, K2CO3, DMF, 16 h, 52–55%; (e) CuSO4·5H2O, sodium ascorbate, THF, t-Butanol/H2O, RT, 6 h, 38–40%.

#### *2.2. CPR3 (12a) and CPR4 (12b), Synthesized PROTAC Compounds, Inhibited Cancer Cell Proliferation*

Next, we investigated the cell cytotoxicity of the synthesized PROTAC compounds 10a–c, 12a–d, 16a–d, 18a–b and 21a–b in both MCF7 (human breast cancer) and A549 (human lung cancer) cells. As the cell permeability is more crucial for PROTAC compounds with higher molecular weights than for conventional small-molecule inhibitors, we first screened the synthesized compounds via the MTT assay. Cell growth inhibition was measured at the concentrations of 1 or 10 μM PROTAC compounds, and the measured absorbance values were normalized to DMSO-treated cells. In Figure 2, CPR3 and CPR4 inhibited cell growth dose-dependently in both MCF7 (Figure 2a) and A549 (Figure 2b) cells. Compounds CPR3 and CPR4, with a methyl-1*H*-pyrazole group, showed significant growth inhibitions. In contrast, other compounds did not show significant dose-dependent growth inhibitions. IC50 values were further determined through the growth inhibition curve by varying the concentrations from 0.5 to 80 μM. The IC50 values of CPR3 and CPR4 were measured to be 3.3 and 2.7 μM in MCF7 cells (Figure 2c), whereas they were 4.2 and 7.6 μM in A549 cells (Figure 2d), respectively. Taken together, CPR3 and CPR4 showed anti-cancer potency by effectively inhibiting cancer cell growth.

#### *2.3. CPR3 and CPR4 Degraded Both Src and IGF-1R Proteins*

Cell growth inhibition is closely related to the post translational regulation of gene expression [48]. Therefore, we examined whether CPR3 and CPR4 reduce the expression level of the IGF-1R or Src proteins, serving as a dual degrader. 2-Chloro-*N*-(5-methyl-1*H*-pyrazol-3-yl)pyrimidin-4-amine (NC) was used as a negative control. In MCF7 cells, NC did not degrade Src or the IGF-1R protein, whereas CPR3 and CPR4 all degraded Src and IGF-1R at 5 μM of concentration (Figure 3a). Similarly, CPR3 and CPR4 also degraded Src and IGF-1R in A549 cells (Figure 3b). These results indicated that CPR3 and CPR4 play a key role as a dual degrader for both IGF-1R and Src proteins.

**Figure 2.** Inhibitory effect of PROTAC compounds on the viability of two different cancer cell lines, MCF7 (**a**) and A549 (**b**). PROTAC compounds were treated at 1 or 10 μM of concentration for 3 days in a complete RPMI-1640 medium. At various concentrations of CPR3 (**c**) or CPR4 (**d**), the IC50 value was calculated through the growth curve in both cell lines. Cell viability was determined by the changes in absorbance at 570 nm. \* *P* ≤ 0.05 and \*\* *P* ≤ 0.01, as determined by Student's t-test.

**Figure 3.** Immunoblotting for IGF-1R and Src in both MCF7 (**a**) and A549 (**b**) cells. The cells were treated with CPR3 or CPR4 at 1 and 5 μM of concentration for 24 h. NC was used as a negative control (5 μM). For detection of the protein expression, extracted lysate was incubated with IGF-1R or Src antibody for 1 h.

#### *2.4. Invasion and Migration Ability Were Suppressed by CPR3 and CPR4 Treatment in Both MCF7 and A549 Cells*

IGF-1R or Src mediated cancer cells were correlated with proliferation, differentiation, survival and metastasis, promoting cell-cell signal transduction. The characteristics of cancer cells have resistance to chemotherapy by processing the pathway related to the epithelial to mesenchymal transition (EMT) [49]. Therefore, we tested cell migration and invasiveness through the treatment of CPR3 and CPR4, which can block IGF-1R or Src in both MCF7 and A549 cells. In both cells, CPR3 and CPR4 significantly delayed the ratio of wound closure in a dose dependent manner, 1 or 5 μM (Figure 4), suggesting a reduction of migration by interrupting the pathway of IGF-1R or Src. In addition, Figure 4 shows a decreased invasiveness of CPR3 and CPR4 in both MCF7 (Figure 5a) and A549 (Figure 5b) cells. Cells with the ability to penetrate into the matrix were stained and counted on a bright field microscopy. The number of cells was significantly reduced at 5 μM of concentration of CPR3 and CPR4, as compared to DMSO (Figure 5). These results indicated that CPR3 and CPR4 can block the IGF-1R/Src regulated cancer cell progressions, including migration and invasion.

**Figure 4.** Wound healing images of MCF7 (**a**) and A549 (**b**) cells treated with CPR3 or CPR4 for 24 h. The PROTAC compound-treated cells were scratched with micropipette tip at 0 h. After 24 h, the migration status was monitored by bright optical microscopy. \* *P* ≤0.05 and \*\* *P* ≤0.01, as determined by Student's t-test.

**Figure 5.** Invasiveness images of MCF7 (**a**) and A549 (**b**) cells treated with CPR3 or CPR4 for 24 h. The cells, resuspended in RPMI-1640 without serum, were reseeded on matrigel-coated insert transwell for 24 h. After 24 h, the cells that migrated to the membrane of the transwell were stained with 0.1% crystal violet and were counted with bright optical microscopy. \* *P* ≤ 0.05 and \*\* *P* ≤ 0.01, as determined by Student's t-test.

#### *2.5. PROTAC Compounds Inhibited the Cell Growth of Both MCF7 and A549 Cells in the Soft Agar Colony Formation Assay*

Next, we examined tumorigenesis by treatment with PROTAC compounds in both MCF7 and A549 cells. It is well known that cancer cells differentiate rapidly and proliferate infinitely. In addition, the capability of single cells to form into a colony is a hallmark of cancer cell survival and proliferation. To test cellular anchorage-independent growth in vitro, we performed the soft agar colony formation assay after treatment with PROTAC compounds. In Figure 6, the number of colonies was significantly increased in DMSO or NC in both MCF7 (Figure 6a) and A549 (Figure 6b) cells. In contrast to the control group, the colony forming ability sharply declined with a 5 μM concentration of PROTAC compounds. Moreover, the sizes of the colonies formed from a single cell were much smaller in PROTAC compounds than in DMSO or NC. These results indicated that PROTAC compounds, with the dual degradation of IGF-1R and Src, affected cell survival.

**Figure 6.** Soft agar colony formation images after treatment with CPR3 or CPR4 in both MCF7 (**a**) and A549 (**b**) cells. CPR3 or CPR4 was treated at 5 μM of concentration, followed by an incubation period of 2 weeks. The formed colonies were stained with 0.1% crystal violet and were detected on a bright field microscopy. \* *P* ≤ 0.05, as determined by Student's t-test.

#### **3. Discussion**

In this study, we rapidly synthesized and screened PROTACs for dual degradation of IGF-1R and Src by employing different warhead ligands and varied linker lengths and compositions, which brought target proteins and E3 ligases into proximity for ubiquitination. Our work demonstrated that efficient PROTAC molecules (12a–b), which had single warhead ligands that degraded two target proteins, exhibited low micromolar anticancer activity, measured by different cellular assays, including cancer cell proliferation, immunoblotting, wound healing assay, and soft agar colony formation assays.

Interestingly, the potency of the synthesized compounds obviously varied, depending on the warhead units. Our data revealed that the previously reported Src or IGF-1R modules (D and E) were not sufficient, as individual warheads, for dual PROTACs, whereas the *N*2-phenyl-*N4*-(1*H*-pyrazol-3-yl)pyrimidine-2,4-diamine (C) exhibited better potency for dual degradation. The induced degradation of the protein was found to be intrinsic to target proteins, which are affected by numerous factors, including the linker length and composition, E3 ligase ligand, spatial orientation of the formed ternary complex and their stability, cell permeability, and ubiquitination efficiency. One important factor for PROTACs is a drug-like property with cell permeability, for which atom numbers need to be minimized in the design of the chemical structure. Indeed, the linker size with 7-10 atoms, between the warhead ligand and the E3 ligase ligand in PROTACs 12a–b, appears to be sufficient for proximity-induced ubiquitination of Src or IGF-1R with the E3 ligase.

The possibility of off-target degradation is still likely to happen with small-warhead ligands, due to their promiscuity. However, recent advancements in PROTACs with promiscuous kinase inhibitors have shown that efficiency is not directly correlated with the binding affinity of the ligands [27]. Rather, it is more closely related to the geometry of the ternary complex of the target protein-PROTAC-E3 ligases, as well as to the efficiency of the proximity-induced enzymatic ubiquitin transfer. Further optimization needs to be followed in regard to the optimal CRBN ligand orientation, nano to pico molar potency, etc. However, our work demonstrated a facile preparation of efficient dual degraders, which enrich our knowledge in establishing dual degradation as an alternative therapeutic strategy in cancers.

#### **4. Materials and Methods**

#### *4.1. Chemistry*

#### 4.1.1. General Information

All reagents and solvents were obtained from commercial suppliers and used as received unless otherwise specified. All reactions were performed under nitrogen atmosphere using oven-dried glassware and monitored by thin-layer chromatography (TLC) on a silica-coated plate (60 F254, Merck, Darmstadt, Germany). Separated compounds on the TLC plate were visualized under UV light at 254 nm and 365 nm (VL-4.LC, Vilber Lourmat, Eberhardzell, Germany). Column chromatography was carried out using a silica gel 230-400 mesh (ZEOprep, Zeochem, Lake Zurich, Switzerland) with *n*-hexane, EtOAc, CH2Cl2 and MeOH as eluents. 1H NMR (400 MHz) and 13C NMR (125 MHz) spectra were recorded using a FT-NMR Avance III HD (Bruker, Billerica, MA, USA) at ambient temperature. Chemical shifts were reported in ppm (parts per million) relative to tetramethylsilane and coupling constants (*J*) were expressed in hertz (Hz). The following abbreviations are used for multiplicities: s = singlet; brs = broad singlet; d = doublet; t = triplet; q = quartet; m = multiplet; dd = doublet of doublets. Mass-to-charge ratio (m/z) values were obtained by using high-resolution mass spectrometry (HRMS) under fast atom bombardment (FAB) conditions with a JMS-700 MStation (JEOL, Tokyo, Japan).

#### 4.1.2. Synthesis of 10a–c, 12a–d, 16a–b, 18a–b and 21a–b

General procedure for synthesis of 8a–c: A reaction mixture of 7a–c (11.036 mmol, 1.0 eq), iron powder (10.363 mmol, 10 eq) and ammonium chloride (10.363 mmol, 10 eq) in ethanol-water (8:2) was stirred for 2 h under N2 atmosphere at 80 ◦C. The reaction mixture was cooled down to room temperature, filtered through celite bed, which was washed twice with ethyl acetate. Solvent was removed under reduced pressure and the obtained residue was dissolved in ethyl acetate and washed with water and brine solution. Organic layer was dried over anhydrous sodium sulfate. Upon concentration under reduced pressure, the residue was purified by column chromatography on silica gel to give 8a–c in 42–44% yield.

*4-((2-(2-(4-aminophenoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (8a):* Yellow solid; yield 42.1%; *R*<sup>f</sup> = 0.50 (methanol/dichloromethane = 0.5:9.5); 1H-NMR (400 MHz, CDCl3) δ 8.57 (s, 1H), 7.43 (t, *J* = 8.8 Hz, 1H), 7.06 (d, *J* = 7.2 Hz, 1H), 6.89 (d, *J* = 8.4 Hz, 1H), 6.71 (d, *J* = 9.2 Hz, 2H), 6.58 (d, *J* = 9.2 Hz, 2H), 6.49 (t, *J* = 5.6 Hz, 1H), 4.87 (dd, *J* = 5.6, 12.0 Hz, 1H), 4.03 (t, *J* = 4.8 Hz, 2H), 3.80–3.74 (m, 4H), 3.45 (dd, *J* = 5.6, 11.2 Hz, 2H), 2.84–2.65 (m, 3H), 2.08–2.02 (m, 1H); 13C-NMR (125 MHz, CDCl3) δ 171.39, 169.18, 168.53, 167.59, 151.80, 146.77, 140.14, 135.95, 132.42, 116.75, 116.31 (2C), 115.87 (2C), 111.57, 110.24, 69.89, 69.63, 68.21, 48.78, 42.36, 31.32, 22.67; HR-MS (FAB<sup>+</sup>) calcd for C23H25N406 [M + H]<sup>+</sup> 453.1774, found 453.1777.

*4-((2-(2-(2-(4-aminophenoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoli ne-1,3-dione (8b)*: Yellow solid; yield 44.7%; *R*<sup>f</sup> = 0.50 (methanol/dichloromethane = 1:9); 1H-NMR (400 MHz, CDCl3) δ 8.54 (s, 1H), 7.44 (t, *J* = 7.2 Hz, 1H), 7.05 (d, *J* = 7.2 Hz, 1H), 6.88 (d, *J* = 8.4 Hz, 1H), 6.71 (d, *J* = 8.8 Hz, 2H), 6.58 (d, *J* = 8.8 Hz, 2H), 6.47 (t, *J* = 5.6 Hz, 1H), 4.84 (dd, *J* = 5.2, 12.0 Hz, 1H), 4.02 (t, *J* = 4.8 Hz, 2H), 3.79 (t, *J* = 5.2 Hz, 2H), 3.71–3.65 (m, 6H), 3.43 (dd, *J* = 5.6, 11.2 Hz, 3H), 2.77–2.64 (m, 3H), 2.04-2.00 (m, 1H); 13C-NMR (125 MHz, CDCl3) δ 171.29, 169.18, 168.46, 167.59, 151.85, 146.77, 140.11, 135.95, 132.42, 116.73, 116.30 (2C), 115.79 (2C), 111.53, 110.19, 70.70, 70.66, 69.92, 69.44, 68.07, 48.78, 42.32, 31.31, 22.65; HR-MS (FAB<sup>+</sup>) calcd for C25H29N407 [M + H]<sup>+</sup> 497.2036, found 497.2029.

*4-((2-(2-(2-(2-(4-aminophenoxy)ethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline -1,3-dione (8c):* Yellow solid; yield 42.9%; *R*<sup>f</sup> = 0.50 (methanol/dichloromethane = 1:9); 1H-NMR (400 MHz, CDCl3) δ 8.46 (s, 1H), 8.13 (d, *J* = 9.2 Hz, 2H), 7.44 (t, *J* = 8.4 Hz, 1H), 7.05 (d, *J* = 6.8 Hz, 1H), 6.93 (d, *J* = 9.2 Hz, 2H), 6.87 (d, *J* = 8.4 Hz, 1H), 6.44 (t, *J* = 5.6 Hz, 1H), 4.88 (dd, *J* = 6.0, 12.4 Hz, 1H), 4.17 (t, *J* = 4.4 Hz, 2H), 3.85 (t, *J* = 4.8 Hz, 2H), 3.70–3.64 (m, 10H), 3.42 (dd, *J* = 5.6, 11.1 Hz, 2H), 2.85–2.68 (m, 3H), 2.10–2.02 (m, 1H); 13C-NMR (125 MHz, CDCl3) δ 171.36, 169.18, 168.54, 167.49, 163.75, 146.68, 141.42, 135.92, 132.35, 125.73 (2C), 116.67, 114.48 (2C), 111.50, 110.10, 70.75, 70.54, 70.50, 69.40, 69.22, 68.10, 48.75, 42.26, 31.28, 22.63; HR-MS (FAB<sup>+</sup>) calcd for C27H33N4O8 [M + H]<sup>+</sup> 541.2298, found 541.2299.

General procedure for synthesis of 10a–c and 12a–b: A reaction mixture of 8a–c (0.044 mmol, 1.0 eq) and corresponding pyrimidines 9 or 11a–b (0.044 mmol, 1.0 eq) were dissolved in DMSO (1.0 mL), and the resulting mixture was stirred for 16 h under N2 atmosphere at 90 ◦C. The progress of the reaction was monitored by TLC. After completion of the reaction, 50 mL of cold water was added and extracted with ethyl acetate (3 × 50 mL). The combined organic layer was dried over anhydrous sodium sulfate. Upon concentration under reduced pressure, the residue was purified by column chromatography on silica gel to give 10a–c and 12a–b, respectively. 21–57% yields.

*2-(2,6-dioxopiperidin-3-yl)-4-((2-(2-(4-((4-(quinoline-3-ylamino)pyrimidin-2-yl)amino)pheno xy)ethoxy) ethyl)amino)isoindoline-1,3-dione (10a, CPR-2)*: Yellow solid; yield 52.0%; *R*<sup>f</sup> = 0.45 (methanol/dichloromethane = 1:9); mp 160.2–161.8 ◦C; 1H-NMR (400 MHz, MeOD + CDCl3) δ 8.81 (s, 1H), 8.70 (s, 1H), 7.90 (d, *J* = 5.6 Hz, 1H), 7.85–7.83 (m, 1H), 7.53–7.42 (m, 4H), 7.35 (d, *J* = 9.2 Hz, 2H), 6.98-6.95 (m, 2H), 6.87 (d, *J* = 9.2 Hz, 2H), 6.19 (d, *J* = 6.0 Hz, 1H), 4.85 (dd, *J* = 5.6, 12.4 Hz, 1H), 4.14 (t, *J* = 4.4 Hz, 2H), 3.87 (t, *J* = 4.8 Hz, 2H), 3.79 (t, *J* = 5.6 Hz, 2H), 3.48 (t, *J* = 5.2 Hz, 2H), 2.68–2.53 (m, 3H), 1.99–1.87 (m, 1H); 13C-NMR (125 MHz, MeOD + CDCl3) δ 174.08, 170.92, 170.46, 169.29, 162.30, 161.34, 156.69, 156.32, 147.99, 145.73, 144.42, 137.28, 134.97, 133.92, 133.60, 129.87, 128.86, 128.83 (2C), 128.26, 124.99, 124.87, 118.17, 116.25 (2C), 112,67, 111.33, 99.83, 71.05, 70.95, 69.10, 50.06, 43.45, 32.49, 23.85; HRMS (FAB<sup>+</sup>) calcd for C36H33N8O6 [M + H]<sup>+</sup> 673.2523, found 673.2532.

*2-(2,6-dioxopiperidin-3-yl)-4-((2-(2-(2-(4-((4-(quinolin-3-ylamino)pyrimidin-2-yl)amino)phen oxy)ethoxy) ethoxy)ethyl)amino)isoindoline-1,3-dione (10b, CPR-5)*: Yellow solid; yield 57.7%; *R*<sup>f</sup> = 0.45 (methanol/dichloromethane = 1:9); mp 185.3–186.4 ◦C; 1H-NMR (400 MHz, DMSO-d6) δ 11.06 (s, 1H), 9.82 (s, 1H), 9.10 (s, 1H), 8.85 (s, 2H), 8.02 (d, *J* = 5.6 Hz, 1H), 7.86 (d, *J* = 9.2 Hz, 1H), 7.69 (brs, 1H), 7.54–7.49 (m, 5H), 7.08 (d, *J* = 8.8 Hz, 1H), 6.97 (d, *J* = 6.8 Hz, 1H), 6.83 (d, *J* = 9.2 Hz, 2H), 6.57 (t, *J* = 6.0 Hz, 1H), 6.25 (d, *J* = 5.6 Hz, 1H), 4.99 (dd, *J* = 5.6, 12.8 Hz, 1H), 4.01 (t, *J* = 4.4 Hz, 2H), 3.72 (t, *J* = 4.8 Hz, 2H), 3.60–3.56 (m, 6H), 3.46–3.40 (m, 2H), 2.85–2.76 (m, 1H), 2.53–2.48 (m, 2H), 1.96–1.91 (m, 1H); 13C-NMR (125 MHz, MeOD + CDCl3) δ 173.69, 170.58, 170.29, 169.04, 162.05, 161.17, 156.78, 156.05, 147.74, 145.64, 144.31, 137.07, 134.69, 133.73, 133.39, 129.66, 128.84, 128.64, 128.61, 128.09, 124.68, 124.61 (2C), 117.91, 115.97 (2C), 112.52, 111.07, 99.53, 71.82, 71.67, 70.91, 70.53, 68.82, 49.88, 43.21. 32.35, 23.68; HRMS (FAB<sup>+</sup>) calcd for C38H37N8O7 [M + H]<sup>+</sup> 717.2785, found 717.2770.

*2-(2,6-dioxopiperidin-3-yl)-4-((2-(2-(2-(2-(4-((4-(quinolin-3-ylamino)pyrimidin-2-yl)amino)phenoxy) ethoxy)ethoxy)ethoxy)ethyl)amino)isoindoline-1,3-dione (10c, CPR-1)*: Yellow solid; yield 21.0%; *R*<sup>f</sup> = 0.45 (methanol/dichloromethane = 1:9); mp 151.8–152.9 ◦C; 1H-NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 9.77 (s, 1H), 9.05 (s, 1H), 8.88 (brs, 1H), 8.86 (s, 1H), 8.03 (d, *J* = 5.6 Hz, 1H), 7.87 (d, *J* = 9.6 Hz, 1H), 7.70 (brs, 1H), 7.55–7.48 (m, 5H), 7.07 (d, *J* = 8.4 Hz, 1H), 6.97 (d, *J* = 6.8 Hz, 1H), 6.84 (d, *J* = 9.2 Hz, 2H), 6.55 (t, *J* = 5.6 Hz, 1H), 6.25 (d, *J* = 5.6 Hz, 1H), 5.01 (dd, *J* = 5.0, 12.8 Hz, 1H), 4.01 (t, *J* = 4.4 Hz, 2H), 3.70 (t, *J* = 4.4 Hz, 2H), 3.58–3.52 (m, 10H), 3.40 (dd, *J* = 5.2, 10.8 Hz, 2H), 2.87–2.78 (m, 1H), 2.55–2.49 (m, 2H), 1.99–1.94 (m, 1H); 13C-NMR (125 MHz, CDCl3) δ 172.76, 170.03, 169.22, 167.68, 160.78, 159.85, 156.15, 154.50, 146.63, 145.50, 144.23, 135.89, 132.88, 132.86, 132.32, 128.72, 128.26, 127.63, 127.36, 126.94, 123.82, 122.48 (2C), 116.67, 114.82 (2C), 111.51, 110.08, 97.51, 70.78, 70.68, 70.60 (2C), 69.74, 69.34, 67.72, 48.83, 42.28, 31.44, 22.79; HR-MS (FAB<sup>+</sup>) calcd for C40H41N8O8 [M + H]<sup>+</sup> 761.3047, found 761.3039.

*2-(2,6-dioxopiperidin-3-yl)-4-((2-(2-(4-((4-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl) amino) phenoxy)ethoxy)ethyl)amino)isoindoline-1,3-dione (12a, CPR-3)*: Yellow solid; yield 32.6%; *R*<sup>f</sup> = 0.45 (methanol/dichloromethane = 1:9); mp 158.1–159.3 ◦C; 1H-NMR (400 MHz, MeOD + CDCl3) δ 7.84 (d, *J* = 6.0 Hz, 1H), 7.42 (dd, *J* = 7.2, 8.4 Hz, 1H), 7.34 (d, *J* = 9.2 Hz, 2H), 7.00 (d, *J* = 6.8 Hz, 1H), 6.90 (d, *J* = 8.8 Hz, 1H), 6.83 (d, *J* = 8.8 Hz, 2H), 6.13 (s, 1H), 5.87 (s, 1H), 4.85 (dd, *J* = 5.6, 12.4 Hz, 1H), 4.08 (t, *J* = 4.4 Hz, 2H), 3.80 (t, *J* = 4.8 Hz, 2H), 3.73 (t, *J* = 5.2 Hz, 2H), 3.44 (t, *J* = 5.2 Hz, 2H), 2.77–2.58 (m, 3H), 2.16 (s, 3H), 2.04–1.99 (m, 1H); 13C-NMR (125 MHz, DMSO-d6) δ 172.84, 170.11, 168.94, 167.31, 160.75, 159.39, 157.14, 150.01, 147.37, 146.40, 141.86, 138.84, 136.21, 132.11, 117.41, 115.46 (2C), 115.24 (2C), 110.70, 109.31, 104.99, 95.52, 69.09, 68.93, 67.64, 48.58, 41.71, 31.01, 22.16, 10.63; HR-MS (FAB+) calcd for C31H32N9O6 [M + H]<sup>+</sup> 626.2476, found 626.2477.

*2-(2,6-dioxopiperidin-3-yl)-4-((2-(2-(2-(4-((4-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)amino) phenoxy)ethoxy)ethoxy)ethyl)amino)isoindoline-1,3-dione (12b, CPR-4)*: Yellow solid; yield 44.5%; *R*<sup>f</sup> = 0.45 (methanol/dichloromethane = 1:9); mp 176.2–177.5 ◦C; 1H-NMR (400 MHz, MeOD + CDCl3) δ 7.75 (d, *J* = 6.4 Hz, 1H), 7.43 (dd, *J* = 7.2, 8.4 Hz, 1H), 7.32 (d, *J* = 8.8 Hz, 2H), 7.00 (d, *J* = 7.2 Hz, 1H), 6.91 (d, *J* = 8.8 Hz, 1H), 6.86 (d, *J* = 9.2 Hz, 2H), 6.23 (s, 1H), 5.96 (s, 1H), 4.85 (dd, *J* = 5.2, 11.2 Hz, 1H), 4.08 (t, *J* = 4.4 Hz, 2H), 3.83 (t, *J* = 4.8 Hz, 2H), 3.71–3.65 (m, 6H), 3.42 (t, *J* = 5.2 Hz, 2H), 2.75–2.65 (m, 3H), 2.18 (s, 3H), 2.06–1.98 (m, 1H); 13C-NMR (125 MHz, DMSO-d6) δ 172.80, 170.08, 168.94, 167.29, 160.72, 159.36, 157.06, 150.84, 147.35, 146.40, 140.13, 138.85, 136.20, 132.08, 117.44, 116.17 (2C), 115.31 (2C), 110.66, 109.24, 105.01, 95.47, 69.90, 69.79, 69.19, 68.91, 67.53, 48.55, 41.68, 30.98, 22.14, 10.62; HRMS (FAB<sup>+</sup>) calcd for C33H36N9O7 [M + H]<sup>+</sup> 670.2738, found 670.2745.

*4-((2-(2-(4-((5-chloro-4-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)amino)phenoxy) ethoxy)ethyl) amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dion(12c, CPR-10)*: To the solution of 8a (30 mg, 0.066 mmol) in n-butanol (4.0 mL) were added 11b (16 mg, 0.066 mmol) and p-toluenesulfonic acid monohydrate (13.5 mg, 0.066 mmol). The resulting mixture was stirred for 4 h under N2 atmosphere at 100 ◦C. The progress of reaction was monitored by TLC. After completion of the reaction, the reaction mixture was cooled down to room temperature, saturated NaHCO3 solution was added to adjust pH to 7, then extracted with ethyl acetate (3 x 20 mL). The combined organic layer was dried over anhydrous sodium sulfate. After concentration of the reaction mixture under reduced pressure, the residue was purified by column chromatography on silica gel to give 12c (13.19 mg, 32.5%) as a yellow solid. *R*<sup>f</sup> = 0.45 (methanol/dichloromethane = 1:9); mp 196.2–197.8 ◦C; 1H-NMR (400 MHz, MeOD + CDCl3) δ 7.90 (s, 1H), 7.46 (t, *J* = 8.4 Hz, 1H), 7.32 (d, *J* = 8.8 Hz, 2H), 6.98 (dd, *J* = 7.2, 10.4 Hz, 2H), 6.85 (d, *J* = 8.8 Hz, 2H), 6.20 (s, 1H), 4.92 (dd, *J* = 6.0, 12.4 Hz, 1H), 4.11 (t, *J* = 4.4 Hz, 2H), 3.83 (t, *J* = 4.4 Hz, 2H), 3.76 (t, *J* = 5.2 Hz, 2H), 3.47 (t, *J* = 5.2 Hz, 2H), 2.74–2.63 (m, 3H), 2.20 (s, 3H), 2.04–1.96 (m, 1H); 13C-NMR (125 MHz, DMSO-d6) δ 172.80, 170.07, 168.91, 167.28, 157.04, 156.78, 155.09, 149.72, 146.38, 145.70, 142.45, 138.74, 136.19, 132.09, 117.40, 115.44 (2C), 114.91 (2C), 113.15, 110.67, 109.28, 98.02, 69.08, 68.92, 67.63, 48.56, 41.70, 30.98, 22.14, 10.73; HR-MS (FAB<sup>+</sup>) calcd for C31H31ClN9O6 [M + H]<sup>+</sup> 660.2086, found 660.2077.

*4-((2-(2-(2-(4-((5-chloro-4-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)amino)phenoxy) ethoxy) ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (12d, CPR-12)*: 12d was synthesized according to the procedure for 12c. Yellow solid; yield 22.6%; *R*<sup>f</sup> = 0.55 (EtOAc); mp 180.9–182.1 ◦C; 1H-NMR (400 MHz, CDCl3) δ 8.00 (brs, 1H), 7.94 (s, 1H), 7.60 (brs, 1H), 7.42 (t, *J* = 7.2 Hz, 1H), 7.30 (d, *J* = 9.2 Hz, 2H), 7.01 (d, *J* = 7.2 Hz, 1H), 6.87 (d, *J* = 8.4 Hz, 1H), 6.78–6.71 (m, 2H), 6.63 (brs, 1H), 6.42 (t, *J* = 5.6 Hz, 1H), 6.03 (s, 1H), 4.85 (dd, *J* = 5.6, 12.4 Hz, 1H), 4.11–4.08 (m, 2H), 3.84 (t, *J* = 4.8 Hz, 2H), 3.75–3.69 (m, 6H), 3.46–3.42 (m, 2H), 2.81–2.59 (m, 3H), 2.24 (s, 3H), 2.05–2.02 (m, 1H); 13C-NMR (125 MHz, DMSO-d6) δ 172.79, 170.07, 168.93, 167.28, 157.04, 156.78, 155.10, 149.74, 146.40, 145.69, 142.41, 138.74, 136.19, 132.08, 117.42, 115.30 (2C), 114.92 (2C), 113.16, 110.65, 109.24, 98.01, 69.90, 69.79, 69.24, 68.91, 67.54, 48.56, 41.69, 30.98, 22.14, 10.73; HR-MS (FAB<sup>+</sup>) calcd for C33H35ClN9O7 [M + H]<sup>+</sup> 704.2348, found 704.2340.

General procedure for synthesis of 14a–b: Dried round bottom flask with stir bar was charged with azidoacetic acid 13 (0.06 mmol, 1.0 eq) and *N*-ethyl-*N* -(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) (0.120 mmol, 2.0 eq) followed by slow addition of dry DMF (3 mL). Then substituted alkyl amine 8a–b (0.06 mmol, 1.0 eq) was added, followed by addition of diisopropyl ethylamine (0.018 mmol, 3.0 eq). The resulting mixture was stirred for 16 h at room temperature. After completion of the reaction, cooled water was added to the reaction mixture, which was extracted with ethyl acetate twice. Separated organic layer was dried with anhydrous sodium sulfate and solvent was evaporated under reduced pressure. Crude product was purified by column chromatography on silica gel using EtOAc: Hexane as mobile phase to get desired product 14a–b in 40–63% yields.

*2-azido-N-(4-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethox y)phenyl) acetamide (14a):* Yellow solid; yield 40.6%; *R*<sup>f</sup> = 0.50 (methanol/dichloromethane = 1:9); 1H-NMR (400 MHz, CDCl3) δ 8.23 (s, 1H), 7.97 (s, 1H), 7.46 (t, *J* = 7.2 Hz, 1H), 7.38 (d, *J* = 9.2 Hz, 2H), 7.07 (d, *J* = 7.2 Hz, 1H), 6.88 (t, *J* = 8.8 Hz, 3H), 6.53 (brs, 1H), 4.88 (dd, *J* = 5.2 Hz, *J* = 12.0 Hz 1H), 4.14-4.10 (m, 4H), 3.84 (t, *J* = 4.8 Hz, 2H), 3.78 (t, *J* = 5.2 Hz, 2H), 3.46 (t, *J* = 5.2 Hz, 2H), 2.87–2.70 (m, 3H), 2.11–2.08 (m, 1H); 13C-NMR (125 MHz, CDCl3) δ 171.08, 169.17, 168.41, 167.59, 164.47, 156.01, 146.79, 136.02, 132.46, 130.08, 121.77 (2C), 116.74, 115.19 (2C), 111.69, 110.33, 69.80, 69.72, 67.88, 52.96, 48.84, 42.41, 31.40, 22.76; HR-MS (FAB<sup>+</sup>) calcd for C25H26N7O7 [M + H]<sup>+</sup> 536.1894, found 536.1894.

*2-azido-N-(4-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy) ethoxy)ethoxy) phenyl)acetamide (14b)*: Yellow solid; yield 62.8%; *R*<sup>f</sup> = 0.70 (ethyl acetate); 1H-NMR (400 MHz, CDCl3) δ 8.08 (s, 1H), 7.93 (s, 1H), 7.46 (t, *J* = 8.0 Hz, 1H), 7.38 (d, *J* = 8.8 Hz, 2H), 7.07 (d, *J* = 7.2 Hz, 1H), 6.90–6.84 (m, 3H), 6.46 (t, *J* = 6.4 Hz, 1H), 4.83 (dd, *J* = 6.8, 13.2 Hz, 1H), 4.12-4.07 (m, 4H), 3.84 (t, *J* = 4.8 Hz, 2H), 3.73–3.68 (m, 6H), 3.44 (dd, *J* = 5.6 Hz, 2H), 2.84–2.65 (m, 3H), 2.08–2.03 (m, 1H); 13C-NMR (125 MHz, CDCl3) δ 170.93, 169.20, 168.28, 167.60, 164.42, 156.09, 146.82, 136.04, 132.49, 129.99, 121.83 (2C), 116.76, 115.01 (2C), 111.63, 110.24, 70.86, 70.76, 69.76, 69.49, 67.71, 52.96, 48.84, 42.41, 31.39, 22.71; HR-MS (FAB<sup>+</sup>) calcd for C27H30N7O8 [M + H]<sup>+</sup> 580.2156, found 580.2156.

*3-(4-chlorophenyl)-1-(prop-2-yn-1-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine* (**15**): Compound 15 was synthesized according to the reported procedure [36,50].

*2-(4-((4-amino-3-(4-chlorophenyl)-1H-pyrazol[3,4-d]pyrimidin-1-yl)methyl)-1H-1,2,3-triazol -1-yl)-N-(4- (2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)phen yl)acetamide (16a, CPR-7)*: To a reaction mixture of 8a (12 mg, 0.022 mmol) and 15 (6.3 mg, 0.022 mmol) dissolved in THF (1.0 mL) was added t-butanol (0.5 mL). Then CuSO4·5H2O (1.1 mg, 0.004 mmol) dissolved in 0.2 mL of water was added to it, followed by the addition of sodium ascorbate (3.55 mg, 0.017 mmol) dissolved in 0.2 mL of water. The reaction mixture was stirred at room temperature for 6 h. The progress of reaction was monitored by TLC. After completion of the reaction, solvent was evaporated, and the residue was extracted with ethyl acetate (3 x 20 mL). The combined organic layer was dried over anhydrous sodium sulfate. Upon concentration under reduced pressure, the residue was purified by column chromatography on silica gel to give 16a (5 mg, 27.2%) as a yellow solid. *R*<sup>f</sup> = 0.15 (methanol/ethyl acetate = 1:9); mp 186.3–187.6 ◦C; 1H-NMR (400 MHz, MeOD + CDCl3) δ 8.28 (s, 1H), 7.98 (s, 1H), 7.59 (d, *J* = 8.4 Hz, 2H), 7.48-7.41 (m, 3H), 7.32 (d, *J* = 9.2 Hz, 2H), 6.99 (d, *J* = 6.8 Hz, 1H), 6.93 (d, *J* = 8.4 Hz, 1H), 6.79 (d, *J* = 9.2 Hz, 2H), 5.71 (s, 2H), 5.15 (s, 2H), 4.87 (dd, *J* = 6.0, 12.4 Hz, 1H), 4.07 (t, *J* = 4.4 Hz, 2H), 3.80 (t, *J* = 4.8 Hz, 2H), 3.74 (t, *J* = 5.2 Hz, 2H), 3.44 (t, *J* = 5.2 Hz, 2H), 2.73–2.63 (m, 3H), 2.05–1.96 (m, 1H); 13C-NMR (125 MHz, MeOD + CDCl3) δ 173.62, 170.49, 170.22, 169.03, 164.18, 157.27, 156.78, 154.71, 147.73, 145.77, 143.70, 137.07, 136.73, 133.37, 131.74 (2C), 131.65 (2C), 130.76, 130.61, 126.08, 124.26, 122.55 (2C), 117.90, 115.99 (2C), 112.55, 112.16, 111.12, 70.73, 70.68, 68.78, 53.81, 43.45, 43.22, 32.33, 30.61, 23.68; HR-MS (FAB<sup>+</sup>) calcd for C39H36ClN12O7 [M + H]<sup>+</sup> 819.2518, found 819.2532.

*2-(4-((4-amino-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-(4- (2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy) ethoxy)ethoxy)phenyl)acetamide (16b, CPR-9)*: 16b was synthesized according to the procedure for 16a. Yellow solid; yield 26.8%; *R*<sup>f</sup> = 0.20 (ethyl acetate); mp 192.8–194.1 ◦C; 1H-NMR (400 MHz, MeOD) δ 8.28 (s, 1H), 7.92 (s, 1H), 7.55 (d, *J* = 6.4 Hz, 2H), 7.46-7.38 (m, 4H), 7.31 (d, *J* = 9.2 Hz, 2H), 6.99 (d, *J* = 6.8 Hz, 1H), 6.86 (d, *J* = 8.4 Hz, 1H), 6.76 (d, *J* = 9.2 Hz, 2H), 5.69 (s, 2H), 5.09 (s, 2H), 4.77 (dd, *J* = 5.2, 12.4 Hz, 1H), 3.78 (t, *J* = 4.8 Hz, 2H), 3.68-3.58 (m, 6H), 3.39 (t, *J* = 5.2 Hz, 2H), 3.29-3.27 (m, 2H), 2.73-2.59 (m, 3H), 2.00–1.94 (m, 1H); 13C-NMR (125 MHz, MeOD + CDCl3) δ 173.69, 170.51, 170.29, 169.19, 169.12, 164.28, 157.73, 156.85, 147.78, 137.14, 136.81, 136.43, 133.42, 131.81 (2C), 131.64 (2C), 130.82, 130.67, 129.34, 126.22, 122.61 (2C), 119.64, 117.97, 117.61, 115.87 (2C), 112.53, 111.08, 71.75, 71.65, 70.79, 70.49, 68.66, 53.80, 43.50, 43.23, 32.36, 30.67, 23.70; HR-MS (FAB<sup>+</sup>) calcd for C41H40ClN12O8 [M + H]<sup>+</sup> 863.2781, found 863.2768.

*1-(3-bromopropyl)-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine* (**17**): To a reaction mixture of 22 (400 mg, 1.628 mmol) and 1,3-dibromo propane (394 mg, 1.953 mmol) dissolved in DMF (4.0 mL) was added K2CO3 (562 mg, 4.070 mmol). The resulting mixture was stirred at room temperature for 16 h. The progress of the reaction was monitored by TLC. After completion of the reaction, cold water was added and extracted with ethyl acetate (3 x 20 mL). The combined organic layer was dried over anhydrous sodium sulfate. Upon concentration under reduced pressure, the residue was purified by column chromatography on silica gel to give 17 (388 mg, 65.0%) as a white solid. *R*<sup>f</sup> = 0.80 (methanol/dichloromethane = 1:9); 1H-NMR (400 MHz, CDCl3) δ 8.35 (s, 1H), 7.62 (d, *J* = 8.8 Hz, 2H), 7.50 (d, *J* = 8.8 Hz, 2H), 5.90 (brs, 2H), 4.57 (t, *J* = 6.8 Hz, 2H), 3.42 (t, *J* = 6.8 Hz, 2H), 2.54–2.47 (m, 2H); 13C-NMR (125 MHz, CDCl3) δ 157.84, 155.94, 154.66, 143.32, 135.36, 131.55, 129.68 (2C), 129.60 (2C), 98.38, 45.55, 32.52, 29.79; HR-MS (FAB<sup>+</sup>) calcd for C14H14BrClN5 [M + H]<sup>+</sup> 366.0121, found 366.0121.

*4-((2-(2-(4-((3-(4-amino-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propyl)amino) phenoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione(18a, CPR-8)*: To the solution of 8a (20 mg, 0.044 mmol) in DMF (3.0 mL) were added potassium carbonate (12.21 mg, 0.066 mmol) and 17 (16.2 mg, 0.044 mmol). The reaction mixture was stirred for 16 h under N2 atmosphere at room temperature. The progress of reaction was monitored by TLC. After completion of the reaction, cold water (20 mL) was added and extracted with ethyl acetate (3 x 20 mL). The combined organic layer was dried over anhydrous sodium sulfate. Upon concentration under reduced pressure, the residue was purified by column chromatography on silica gel to give 18a (16.5 mg, 50.6%) as a yellow solid. *R*<sup>f</sup> = 0.50 (methanol/dichloromethane = 1:9); mp 146.3–147.6 ◦C; 1H-NMR (400 MHz, CDCl3) δ 8.35 (s, 1H), 7.63 (d, *J* = 8.4 Hz, 2H), 7.48–7.42 (m, 3H), 7.05 (d, *J* = 6.8 Hz, 1H), 6.89 (d, *J* = 8.4 Hz, 1H), 6.71 (d, *J* = 8.8 Hz, 2H), 6.58 (d, *J* = 8.8 Hz, 2H), 6.46 (t, *J* = 6.0 Hz, 1H), 5.53 (brs, 2H), 4.84 (dd, *J* = 5.2, 12.4 Hz, 1H), 4.45 (td, *J* = 7.2, 2.8 Hz, 2H), 4.03 (t, *J* = 4.4 Hz, 2H), 3.91 (td, *J* = 6.8, 2.4 Hz, 2H), 3.78 (p, 2H), 3.74 (t, *J* = 5.2 Hz, 2H), 3.46 (dd, *J* = 5.6, 11.2 Hz, 2H), 2.88 (dd, *J* = 12.8, 2.8 Hz, 1H), 2.72–2.66 (m, 2H), 2.23 (p, 2H), 2.04–1.99 (m, 1H); 13C-NMR (125 MHz, CDCl3) δ 170.94, 169.29, 168.75, 167.69, 157.69, 155.86, 154.51, 151.75, 146.77, 142.98, 140.30, 135.93, 135.13, 132.49, 131.75, 129.79 (2C), 129.49 (2C), 116.69, 116.23 (2C), 115.91 (2C), 111.57, 110.37, 98.40, 69.94, 69.71, 68.21, 49.56, 44.84, 42.42, 38.29, 31.96, 27.73, 22.06; HR-MS (FAB<sup>+</sup>) calcd for C37H37ClN9O6 [M + H]<sup>+</sup> 738.2555, found 738.2544.

*4-((2-(2-(2-(4-((3-(4-amino-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propyl)amin o)phenoxy) ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (18b, CPR-11)*: 18b was synthesized according to the procedure for 18a. Yellow solid; yield 50.8%; *R*<sup>f</sup> = 0.45 (methanol/dichloromethane = 1:9); mp 148.2–149.9 ◦C; 1H-NMR (400 MHz, CDCl3) δ 8.35 (s, 1H), 7.63 (d, *J* = 8.4 Hz, 2H), 7.49–7.42 (m, 3H), 7.05 (d, *J* = 6.8 Hz, 1H), 6.88 (d, *J* = 8.8 Hz, 1H), 6.70 (d, *J* = 8.8 Hz, 2H), 6.57 (d, *J* = 8.8 Hz, 2H), 6.44 (t, *J* = 5.6 Hz, 1H), 4.82 (dd, *J* = 4.8, 12.0 Hz, 1H), 4.49-4.20 (m, 2H), 4.01 (t, *J* = 4.8 Hz, 2H), 3.93–3.87 (m, 2H), 3.78 (t, *J* = 5.2 Hz, 2H), 3.70–3.63 (m, 6H), 3.43 (dd, *J* = 5.2, 10.8 Hz, 2H), 2.88–2.82 (m, 1H), 2.74–2.62 (m, 2H), 2.23 (p, 2H), 2.06–1.97 (m, 1H); 13C-NMR (125 MHz, CDCl3) δ 170.94, 169.29, 168.75, 167.70, 157.65, 155.80, 154.49, 151.86, 146.77, 143.01, 140.19, 135.93, 135.14, 132.49, 131.74, 129.79 (2C), 129.50 (2C), 116.68, 116.25 (2C), 115.80 (2C), 111.52, 110.31, 98.39, 70.76, 70.71, 69.97, 69.53, 68.08, 49.55, 44.84, 42.37, 38.26, 31.94, 27.74, 22.04; HR-MS (FAB+) calcd for C39H41ClN9O7 [M + H]<sup>+</sup> 782.2817, found 782.2834.

*1-(3-azidopropyl)-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine* (**19**): To the solution of 17 (200 mg, 0.545 mmol) in DMF (2.0 mL) was added sodium azide (46.10 mg, 0.709 mmol) at room temperature. The reaction mixture was stirred for 16 h under N2 atmosphere at room temperature. The progress of reaction was monitored by TLC. After completion of the reaction, cold water (50 mL) was added and extracted with ethyl acetate (3 x 20 mL). The combined organic layer was dried over anhydrous sodium sulfate. Upon concentration under reduced pressure, the residue was purified by column chromatography on silica gel to give 19 (119 mg, 66.1%) as a white solid. *R*<sup>f</sup> = 0.50 (EtOAc/n-Hexane = 1:1); 1H-NMR (400 MHz, CDCl3) δ 8.36 (s, 1H), 7.62 (d, *J* = 8.0 Hz, 2H), 7.50 (d, *J* = 8.4 Hz, 2H), 5.90 (brs, 2H), 4.52 (t, *J* = 6.4 Hz, 2H), 3.36 (t, *J* = 6.4 Hz, 2H), 2.23–2.17 (m, 2H); 13C-NMR (125 MHz, CDCl3) δ 157.84, 155.96, 154.63, 143.31, 135.36, 131.55, 129.67 (2C), 129.60 (2C), 98.34, 48.71, 44.26, 28.93; HR-MS (FAB<sup>+</sup>) calcd for C14H14ClN8 [M + H]<sup>+</sup> 329.1030, found 329.1033.

*2-(2,6-dioxopiperidin-3-yl)-4-((2-(2-(4-(prop-2-yn-1-ylamino)phenoxy)ethoxy)ethyl)amino)iso indoline -1,3-dione (20a):* To the solution of 8a (50 mg, 0.110 mmol) in DMF (3.0 mL) were added potassium carbonate (22.9 mg, 0.165 mmol) and propargyl bromide (14.5 mg, 0.110 mmol) at room temperature. The reaction mixture was stirred for 16 h under N2 atmosphere at room temperature. The progress of reaction was monitored by TLC. After completion of the reaction, cold water (20 mL) was added and extracted with ethyl acetate (3 x 20 mL). The combined organic layer was dried over anhydrous sodium sulfate. Upon concentration under reduced pressure, the residue was purified by column chromatography on silica gel to give 20a (30 mg, 55.4%) as a yellow solid. *R*<sup>f</sup> = 0.60 (ethyl acetate = 100%); 1H-NMR (400 MHz, CDCl3) δ 7.45 (t, *J* = 8.4 Hz, 1H), 7.07 (d, *J* = 7.2 Hz, 1H), 6.91 (d, *J* = 8.4 Hz, 1H), 6.73 (d, *J* = 8.8 Hz, 2H), 6.59 (d, *J* = 8.8 Hz, 2H), 6.49 (t, *J* = 5.6 Hz, 1H), 4.92 (dd, *J* = 7.6, 12.4 Hz, 1H), 4.55 (d, *J* = 2.4 Hz, 2H), 4.04 (t, *J* = 4.4 Hz, 2H), 3.81–3.74 (m, 4H), 3.47 (dd, *J* = 5.2, 10.8 Hz, 2H), 3.00–2.93 (m, 1H), 2.81–2.72 (m, 2H), 2.14 (s, 1H), 2.08–2.04 (m, 1H); 13C-NMR (125 MHz, CDCl3) δ 170.11, 169.23, 167.96, 167.63, 151.77, 146.81, 140.30, 135.97, 132.46, 116.75, 116.23 (2C), 115.92 (2C), 111.61, 110.33, 77.94, 70.76, 69.94, 69.70, 68.22, 49.52, 42.42, 31.87, 29.62, 21.92; HR-MS (FAB+) calcd for C26H27N4O6 [M + H]<sup>+</sup> 491.1931, found 491.1938.

*2-(2,6-dioxopiperidin-3-yl)-4-((2-(2-(2-(4-(prop-2-yn-1-ylamino)phenoxy)ethoxy)ethoxy)ethyl) amino) isoindoline-1,3-dione (20b)*: 20b was synthesized according to the procedure for 20a. White solid; yield 52.5%; *R*<sup>f</sup> = 0.60 (EtOAc/n-Hexane = 1:1); 1H-NMR (400 MHz, CDCl3) δ 7.45 (t, *J* = 8.4 Hz, 1H), 7.07 (d, *J* = 6.8 Hz, 1H), 6.90 (d, *J* = 8.4 Hz, 1H), 6.72 (d, *J* = 8.8 Hz, 2H), 6.59 (d, *J* = 8.8 Hz, 2H), 6.47 (t, *J* = 5.6 Hz, 1H), 4.87 (dd, *J* = 4.8, 7.2 Hz, 1H), 4.55 (d, *J* = 2.4 Hz, 2H), 4.03 (t, *J* = 4.8 Hz, 2H), 3.81 (t, *J* = 5.2 Hz, 2H), 3.72–3.66 (m, 6H), 3.44 (dd, *J* = 5.6, 11.2 Hz, 2H), 2.97–2.86 (m, 1H), 2.76–2.67 (m, 2H), 2.15 (s, 1H), 2.08–2.01 (m, 1H); 13C-NMR (125 MHz, CDCl3) δ 170.10, 169.23, 167.95, 167.66, 151.90, 146.81, 140.20, 135.98, 132.48, 116.74, 116.26 (2C), 115.82 (2C), 111.58, 110.30, 77.97, 70.77, 70.74, 69.99, 69.57, 69.51, 68.11, 49.51, 42.39, 31.85, 29.63, 21.93; HR-MS (FAB<sup>+</sup>) calcd for C28H31N407 [M + H]<sup>+</sup> 535.2193, found 535.2200.

*4-((2-(2-(4-(((1-(3-(4-amino-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propyl)-1H-1,2,3-triazol -4-yl)methyl)amino)phenoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindol ine-1,3-dione (21a, CPR-13)*: 21a was synthesized according to the procedure for 16a. Yellow solid; yield 40.1%; *R*<sup>f</sup> = 0.45 (methanol/dichloromethane = 1:9); mp 159.1–160.6 ◦C; 1H-NMR (400 MHz, CDCl3) δ 8.35 (s, 1H), 7.69 (s, 1H), 7.61 (d, *J* = 8.4 Hz, 2H), 7.48 (d, *J* = 8.4 Hz, 2H), 7.42 (t, *J* = 7.2 Hz, 1H), 7.01 (d, *J* = 7.2 Hz, 1H), 6.88 (d, *J* = 8.4 Hz, 1H), 6.70 (d, *J* = 8.8 Hz, 2H), 6.57 (d, *J* = 8.8 Hz, 2H), 6.47 (t, *J* = 5.6 Hz, 1H), 5.13-5.04 (m, H), 4.92 (dd, *J* = 5.2, 12.4 Hz, 1H), 4.47 (t, *J* = 7.2 Hz, 2H), 4.34 (t, *J* = 6.8 Hz, 2H), 4.03 (t, *J* = 4.8 Hz, 2H), 3.78 (t, *J* = 4.8 Hz, 2H), 3.74 (t, *J* = 5.6 Hz, 2H), 3.44 (dd, *J* = 4.4, 10.0 Hz, 2H), 2.97–2.90 (m, 1H), 2.79–2.68 (m, 2H), 2.53 (p, 2H), 2.07-2.00 (m, 1H); 13C-NMR (125 MHz, CDCl3) δ 170.52, 169.25, 168.70, 167.80, 167.68, 159.35, 157.61, 155.89, 154.58, 146.79, 146.73, 143.55, 143.07, 135.94, 135.40, 132.46, 131.37, 129.69 (2C), 129.62 (2C), 123.83, 116.74, 116.44 (2C), 115.90 (2C), 111.56, 110.33, 69.92, 69.68, 68.23, 49.54, 47.58, 44.12, 42.39, 35.62, 31.91, 29.98, 22.04; HR-MS (FAB<sup>+</sup>) calcd for C40H40ClN12O6 [M + H]<sup>+</sup> 819.2882, found 819.2887.

*4-((2-(2-(2-(4-(((1-(3-(4-amino-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propyl)-1H-1,2,3 triazol-4-yl)methyl)amino)phenoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (21b, CPR-14)*: 21b was synthesized according to the procedure for 16a. Yellow solid; yield 38.3%; *R*<sup>f</sup> = 0.250 (methanol/dichloromethane = 1:9); mp 164.5–165.9 ◦C; 1H-NMR (400 MHz, CDCl3) δ 8.35 (s, 1H),

7.69 (s, 1H), 7.61 (d, *J* = 8.0 Hz, 2H), 7.48 (d, *J* = 8.4 Hz, 2H), 7.41 (t, *J* = 7.6 Hz, 1H), 6.99 (d, *J* = 7.2 Hz, 1H), 6.87 (d, *J* = 8.8 Hz, 1H), 6.69 (d, *J* = 8.4 Hz, 2H), 6.57 (d, *J* = 8.4 Hz, 2H), 6.45 (t, *J* = 6.0 Hz, 1H), 5.12–5.03 (m, 2H), 4.88 (dd, *J* = 5.2, 11.6 Hz, 1H), 4.47 (t, *J* = 6.0 Hz, 2H), 4.36 (t, *J* = 7.2 Hz, 2H), 4.01 (t, *J* = 5.2 Hz, 2H), 3.79 (t, *J* = 5.2 Hz, 2H), 3.71-3.65 (m, 6H), 3.44–3.40 (m, 2H), 2.93–2.87 (m, 1H), 2.75–2.64 (m, 2H), 2.55 (p, 2H), 2.04–1.98 (m, 1H); 13C-NMR (125 MHz, MeOD+CDCl3) 170.75, 169.10, 168.74, 167.77, 155.58, 154.17, 146.67, 146.60, 135.90, 135.39, 135.15, 133.28, 132.35, 132.27, 131.00, 130.89, 129.57 (2C), 129.51 (2C), 128.92, 128.67, 116.72 (2C), 111.40 (2C), 109.99, 109.50, 70.58, 70.52, 69.82, 69.37, 68.01, 47.62, 44.02, 42.09, 35.33, 31.62, 29.80, 29.54, 21.90; HR-MS (FAB<sup>+</sup>) calcd for C42H44ClN12O7 [M + H]<sup>+</sup> 863.3144, found 863.3149.

#### *4.2. Anticancer Activity*

#### 4.2.1. MTT Assay

MCF7 (human breast cancer) and A549 (human lung cancer) cells were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea) and incubated in 5% CO2 at 37 ◦C. The cells were plated in 96-well culture plates (SPL Life Science Co., Gyeonggi-do, Republic of Korea) for 24 h. For cell viability test, the synthesized PROTACs were treated at the various concentrations from 0 to 80 μM for 3 days. After that, 100 μL of 20 mM MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (Thermo Fisher Scientific, Rockford, IL, USA) (5 mg/mL) dissolved in RPMI-1640 medium was added to the incubated well for 3 h. The supernatant was discarded and 100 μL of DMSO was reacted with viable cells. The product of reaction was measured by absorbance at 570 nm using Mithras2 plate reader (Berthold Technologies, Bad Wildbad, Germany).

#### 4.2.2. Western Blot Assay

The PROTAC-treated MCF7 (human breast cancer) and A549 (human lung cancer) cells were lysed with 1X lysis buffer containing protease inhibitor. Extracted protein lysate (20 μg) were loaded on 12% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (ATTO, Tokyo, Japan). The membrane was blocked with 3% Bovine Serum Albumin (BSA) in tris-phosphate buffer containing 0.1% Twin 20, and incubated with primary antibodies for 1 h at room temperature. The following antibodies were IGF1Rβ (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Src (1:000, Cell Signaling Technology, Beverly, MA, USA) and GAPDH (1:200, Santa Cruz Biotechnology) as internal control. The signal was reacted with ECL solution (GenDEPOT, Barker, TX, USA) and detected on an Image Quant LAS 4000 biomolecular imager (GE Healthcare, Chicago, IL, USA).

#### 4.2.3. Wound Healing and Invasion Assay

MCF7 (human breast cancer) and A549 (human lung cancer) cells were treated with 1 or 5 μM of PROTAC compounds for 24 h. After 24 h, the treated cells were collected and re-implanted (3 <sup>×</sup> 105) on 24-well culture plates for 24 h. When cells reached confluence, wound healing assay was performed by scratching with pipette tips. For invasion assay, matrigel (BD Biosciences, San Diego, CA, USA) coated transwell (BD Biosciences, San Diego, CA, USA, 353097, 8 μm pore size) was used to mimic extra cellular matrix in vitro. The PROTAC-treated cells (1 <sup>×</sup> 105) suspended in RPMI-1640 without FBS were seeded into transwell and each of the insert wells was put in 24-well culture plates containing RPMI-1640 with FBS. After incubation, the membrane of insert transwell was stained with 0.1% crystal violet (Sigma Aldrich, St. Louis, USA). The wound closure or invasive images were analyzed based on bright field microscopy.

#### 4.2.4. Soft Agar Colony Formation Assay

To investigate growth ability on solid surface in vitro, a colony forming assay was performed by using top and bottom agarose method. The base of layer was poured with 1.5 mL of 0.5% agarose

(BD Biosciences, San Diego, CA, USA) containing RPMI-1640 with FBS and solidified. 1 mL of PROTAC-treated cells (3 <sup>×</sup> 105) resuspended in complete medium with 0.3% agarose (BD Biosciences, San Diego, CA, USA) was covered on the base of layer. The plates were incubated for approximately 2 weeks in 5% CO2 at 37 ◦C. Formed colony was counted on bright field microscopy.

#### **5. Conclusions**

We developed dual degrader PROTACs to target both IGF-1R and Src, which are associated with various cancer cells. We evaluated the degradation potentials of the synthesized PROTAC compounds by different cellular assays. Interestingly, PROTACs with a dual IGF-1R/Src inhibitor warhead (**C**), which possess a common structure from the reported IGF-1R and Src inhibitors, showed significant degradation potency, whereas previously reported individual modules, with similar sizes for Src or IGF-1R, were not active in PROTACs. Our data support the notion that the binding affinities of warhead ligands are not the sole determinant of PROTAC efficiency. Off-target effects, with promiscuous ligands, may be counteracted with other factors, including the formation of a stable ternary complex with target protein-E3 ligase, as well as efficient ubiquitination. Our dual degrader PROTACs, along with various cell-based assays, indicated that the PROTACs developed in this study serve as valuable tools to understand the mechanism and process of induced degradation of target proteins, which will result in next-generation anticancer therapeutics.

**Supplementary Materials:** The following are available online: Experimental details, synthesis of intermediates (7a–c, 9, 11a–b, 15, 17 and 19), 1H and 13C-NMR spectra of the synthesized compounds.

**Author Contributions:** Conceptualization, J.L.; methodology, S.M. and N.L.; validation, N.L.; formal analysis, S.M. and N.L.; investigation, J.L.; writing—original draft preparation, S.M. and N.L.; writing—review and editing, J.L.; review and editing, D.O.; supervision, J.L.; funding acquisition, J.L. and D.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Research Foundation of Korea (NRF) grants (NRF-2018R1A2B2005535 and 2018R1A4A1021703) funded by the Korean government (MSIT).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Sample Availability:** Samples of the compounds are not available from the authors.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Phenylpyrazalopyrimidines as Tyrosine Kinase Inhibitors: Synthesis, Antiproliferative Activity, and Molecular Simulations**

### **Bhupender S. Chhikara 1,2, Sajda Ashraf 3, Saghar Moza**ff**ari 4, Nicole St. Jeans 2, Dindyal Mandal 2,4, Rakesh Kumar Tiwari 2,4, Zaheer Ul-Haq 3,\* and Keykavous Parang 2,4,\***


#### Academic Editor: Qiao-Hong Chen

Received: 15 April 2020; Accepted: 29 April 2020; Published: 2 May 2020

**Abstract:** *N*1-(α,β-Alkene)-substituted phenylpyrazolopyrimidine derivatives with acetyl and functionalized phenyl groups at α- and β-positions, respectively, were synthesized by the reaction of 3-phenylpyrazolopyrimidine (PhPP) with bromoacetone, followed by a chalcone reaction with differently substituted aromatic aldehydes. The Src kinase enzyme assay revealed modest inhibitory activity (half maximal inhibitory concentration, IC50 = 21.7–192.1 μM) by a number of PhPP derivatives. Antiproliferative activity of the compounds was evaluated on human leukemia (CCRF-CEM), human ovarian adenocarcinoma (SK-OV-3), breast carcinoma (MDA-MB-231), and colon adenocarcinoma (HT-29) cells in vitro. 4-Chlorophenyl carbo-enyl substituted 3-phenylpyrazolopyrimidine (**10**) inhibited the cell proliferation of HT-29 and SK-OV-3 by 90% and 79%, respectively, at a concentration of 50 μM after 96 h incubation. The compound showed modest inhibitory activity against c-Src (IC50 = 60.4 μM), Btk (IC50 = 90.5 μM), and Lck (IC50 = 110 μM), while it showed no activity against Abl1, Akt1, Alk, Braf, Cdk2, and PKCa. In combination with target selection and kinase profiling assay, extensive theoretical studies were carried out to explore the selectivity behavior of compound **10**. Specific interactions were also explored by examining the changing trends of interactions of tyrosine kinases with the phenylpyrazolopyrimidine derivative. The results showed good agreement with the experimental selectivity pattern among c-Src, Btk, and Lck.

**Keywords:** protein kinase; phenylpyrazolopyrimidine; antiproliferative activity; enzyme inhibition; molecular simulation

#### **1. Introduction**

Cancer cells show the overexpression of different protein tyrosine kinases (PTKs). These enzymes catalyze the phosphorylation of many protein substrates by the transfer of the γ-phosphate group from ATP to specific tyrosine residues. PTKs have critical roles in signal transduction pathways.

Src family kinases (SFKs) have nine different PTKs, including c-Src, c-Yes, Fyn, Lck, Lyn, Hck, Frk, Blk, and c-Fgr of which Src is the prototype [1]. SFKs have essential roles in the regulation of a wide variety of normal cellular signal transduction pathways, such as cell division, differentiation, growth factor signaling, survival, adhesion, migration, and invasion [2]. Src tyrosine kinase expression is frequently elevated in several tumors, including breast, colon, prostate, lung, ovary, and pancreas, when it is compared with the adjacent healthy tissues. Src kinase was shown to be the critical modulator of cancer cell metastasis and invasion [3].

In recent years, a number of small cell-permeable inhibitors of protein kinases were investigated for the treatment of different cancers [4]. Different small molecules that were actively investigated as kinase inhibitor include quinazolines [5], quinolinecarbonitriles [6], pyrazolopyrimidines [7,8], imidazo [1,5-a]pyrazines [9], Calixarene [10], benzotriazines [11,12], indoles [13], and ATP- phosphopeptide conjugates [14]. The research effort led to the generation of small molecules for the treatment of different malignancies. Imatinib, a 2-phenylaminopyrimidine, was approved for the treatment of chronic myelogenous leukemia (CML) and gastrointestinal stromal tumors (GISTs) [15]. Dasatinib (BMS-354825), a pyrimidinylthiazole derivative, was approved to use in patients with CML after imatinib treatment [16], and bosutinib (SKI-606), a 3-quinolinecarbonitrile analogue, is known to suppress migration and invasion of human breast cancer cells [17]. Ibrutinib (Figure 1) is a pyrazolopyrimidine-based [18] small-molecule kinase inhibitor that was approved for the treatment of B-cell cancers like mantle cell lymphoma, chronic lymphocytic leukemia, and Waldenström's macroglobulinemia [19,20].

**Figure 1.** Chemical structures of 3-phenylpyrazolopyrimidine (PhPP) derivatives (PP1 and PP2) and Ibrutinib.

A number of crystallographic studies showed that adenosine 5 -(β,γ-imido)triphosphate (AMP-PNP), an ATP mimic, binds to the ATP-binding site of c-Src (Protein Data Bank (PDB) 2SRC) [21]. Furthermore, complexes of 3-phenylpyrazolopyrimidine (PhPP) derivatives (PP1 and PP2) (Figure 1) act as ATP-binding site inhibitors of Hck (PDB 1QCF) and Lck (PDB 1QPE) [22]. Indeed, the pyrazolopyrimidine core of PP1 and PP2 (Figure 1) mimics the adenine base of ATP in binding to the nucleotide-binding site. In another study [8], we showed that the 3-phenyl group interacts with a large hydrophobic pocket in the ATP-binding site, contributing significantly to Src kinase inhibitory activity. There is also another cavity formed from side chains of helix αC and helix αD that is normally occupied by a carbohydrate moiety followed by a triphosphate group of ATP. This cavity is mostly unfilled by the tert-butyl of 3-phenylpyrazolopyrimidines (PP1 and PP2). Furthermore, we investigated the variation of *N*1 substitution in 3-phenylpyrazolopyrimidines with different 1,2,3-triazoles containing hydrophobic residues to interact with amino acids of this cavity and contribute to the enhancement of Src kinase inhibitory potency [8]. The cysteine residue present in the cavity could be a potential target to bind with PhPP molecules irreversibly, leading to a potential increase in kinase inhibition.

Here, we describe the synthesis and evaluation of kinase inhibitory activity of *N*1-substituted PhPP with α-acetyl-β-phenyl-alkene groups (Figure 2). We hypothesized that this substitution at the *N*1 position of PhPP could contribute significantly to the kinase inhibition through generating specific interactions. We further extended our study to investigate the selectivity profile of one of the active compounds (**10**) with a panel of nine different kinases by applying MD and MMGBSA calculations. This study helped to reveal the inhibitory mechanism of PhPP derivatives based on their distinct structural characteristics required for its interaction with a specific kinase.

**Figure 2.** Chemical structures of *N*1-substituted PhPP with α-acetyl-β-phenyl-alkene groups (*E* and *Z* isomers).

#### **2. Results and Discussion**

#### *2.1. Chemical Synthesis*

The products were synthesized by following the chemical pathway shown in Scheme 1. The PhPP (**1**) was synthesized according to a reported procedure [23]. The N1 of **1** was substituted with bromoacetone in the presence of potassium carbonate as a base. Product **2** was confirmed by NMR, which showed the disappearance of the proton peak of NH at 8.07 ppm of **1** and appearance of the peak at 5.24 ppm and 2.21 ppm for CH2 and CH3, respectively, in 1H NMR. Furthermore, 13C NMR showed peaks at 201.34 ppm, 56.15 ppm, and 27.14 ppm representing the carbonyl group, CH2, and CH3, respectively, in product **2**.

**Scheme 1.** Chemical synthesis of PhPP derivatives.

Next, the double bond group was introduced by the chalcone reaction. The anion of **2** generated by sodium hydroxide was reacted with different benzaldehydes at room temperature to give product **3**. The ratio of starting material and amount of solvent defined the formation of product and side products. A higher concentration (i.e., less amount of solvent ethanol) and the presence of higher equivalents of **2** than the aldehyde lead to a second internal Michael reaction where the anion of **2** reacts with product **3**, leading to the formation of side product as indicated by Electrospray Ionization Mass Spectrometry (ESI-MS) at 659 Da (data not shown). In an optimum condition, the aldehyde and reactant **2** should be present in more than 1.4:1 equivalent, and solvent ethanol should be present in approximately 20 mL for 26 mg (0.1 mmol) of **2**. Out of two possible products after the conjugation of **2** with 4-methylbenzaldehyde, only product **3** was observed, suggesting the reaction of the carbanion of methylene (CH2) group between the carbonyl and nitrogen rather than that of methyl (CH3). The formation of product **3** was confirmed by ESI-MS by the presence of a mass peak at 370 Da [M + H]+. This was confirmed by 1H NMR, which showed the absence of a peak at 5.24 ppm for CH*<sup>2</sup>* of **2,** while the three protons for CH*<sup>3</sup>* were present at 2.31 ppm. Correspondingly, in 13C NMR, the peak at 56.15 ppm (assigned to *C*H2) was absent in **3** while the peak at 26.13 ppm (assigned to *C*H3 with slight upfield shift) was present. Furthermore, out of EZ possible isomers for the double bond, both the isomers were present as indicated by NOE NMR spectra, possibly by rotation introduced due to keto-enol tautomerism.

In the case of product **8**, the chalcone reaction was carried with precaution to avoid the formation of side products due to the presence of α-hydrogen in acetaldehyde. The anion of **2** was generated first with 1.1 equivalent of sodium hydroxide over extended time and added to the solution of acetaldehyde (1.2 equivalents). The reaction was quenched with acetic acid after 30 min. The product was confirmed by ESI-MS and NMR. The products were purified by HPLC and showed a purity of 95–99%.

#### *2.2. Src-Kinase Assay*

In vitro Src kinase assay for some of the compounds was conducted, as presented in Table 1. As results indicated, the compounds were moderately active against the Src kinase. Among all the tested compounds, compounds **6** and **4** were found to be the most potent with IC50 values of 21.7 and 24.7 μM, respectively. Compounds **3** and **7** exhibited IC50 values of 32.9 μM and 36.8, respectively. Compound **5** showed the lowest activity with an IC50 of 192.1 μM. Since the compounds showed only moderate Src kinase inhibitory activity, they were evaluated for antiproliferative activity first to find the lead compound for further kinase inhibitory assays.


**Table 1.** In vitro Src kinase inhibition assay for the selected compounds.

<sup>a</sup> All experiments were repeated in triplicate. All the standard deviations were in the range of 6–13%.

#### *2.3. Antiproliferative Activities*

The effect of compounds on the cell proliferation of cancer cells was evaluated in vitro in a human leukemia cell line (CCRF-CEM) according to the previously reported procedures [24,25], breast adenocarcinoma (MDA-MB-231), ovarian adenocarcinoma (SK-OV-3), and colon adenocarcinoma (HT-29) cell lines up to 72 h. Doxorubicin (Dox) was used as a positive control as shown previously [26–29]. These cell lines were selected to determine the antiproliferative activity against diverse cancer cells. Compounds **10** and **11** consistently inhibited the cell proliferation 72–96 h in all cell lines. The percentage of cell proliferation was significantly reduced in HT-29 cells in the presence of **10** and **11** when compared with Dox and other compounds. Compound **10** was more effective in the order of HT-29 (90%) > CCRF-CEM, SK-OV-3 (79%) > MDA-MB-231 (60%), while Dox showed HT-29 (75%) > CCRF-CEM, SK-OV-3 (73%) > MDM-MB-231 (64%) after 96 h. Compounds **2**, **7**, **9**, and **12** were found to be inactive in reducing the cell proliferation in all the cell lines after 24–96 h while other compounds **3** and **13**–**15** exhibited comparable or slightly less antiproliferative activity (Figure 3A–D) than compounds **10** and **11** after 96 h. These data indicate that the presence of the chlorine group at the para position in compounds **10** and **11** were effective in improving the antiproliferative activities. However, the absence of phenyl and/or double bonds in compounds **2** and **8** significantly reduced the antiproliferative activities versus **10** and **11**. The presence of *p*-dimethylamino, *m*-nitro, and *o*-chlorine in **7**, **9**, and **12** were not productive while *o*-fluorine (**13**), *p*-fluorine (**14**), unsubstituted phenyl (**15**), and *p*-Cl (**10** and **11**) enhanced the antiproliferative activity. Compounds **5** and **6** with *p*-ph and *p*-methoxy exhibited antiproliferative activity against some of the cell lines, with **5** being more active in HT-29 and **6** in CCRF-CEM and SK-OV3 cells. However, their activity was generally less than compounds **3**, **10**, **11**, **13**, and **14**. Compounds **10** and **11** exhibited IC50 values of 25.5 μM and 34.3 μM, respectively, after 72 h incubation against SK-OV-3 cells (Figure 4).

**Figure 3.** *Cont*.

**DͲDͲϮϯϭ**

**Figure 3.** Antiproliferative activity of PhPP derivatives (50 μM) and doxorubicin (Dox) (1 μM) against CCRF-CEM (**A**), SK-OV-3 (**B**), HT-29 (**C**), and MDA-MB-231 (**D**) cell lines. All experiments were repeated in triplicate.

**Figure 4.** Dose-response curves of **10** (**left**) and **11** (**right**) after 72 h incubation against SK-OV-3 cells.

Compound **10** was selected for further evaluation against a panel of kinases to determine the mechanism of activity. The results are shown in Table 2 against Abl1, Akt1, Alk, Braf, Btk, Cdk2, cSrc, Lck, and PKCa enzymes. The data indicate modest activity against Btk, c-Src, and Lck while the compound was inactive or showed very low inhibition activity against other kinases.


**Table 2.** The activity of compound **10** against a panel of protein kinases.

<sup>a</sup> Not active (NA) indicates no inhibition or compound activity that could not be fit to an IC50 curve; <sup>b</sup> Staurosporine; <sup>c</sup> GW5074.

#### *2.4. Molecular Modeling*

To rationalize the activity of compound **10**, KEGG pathway and molecular dynamics simulation studies were utilized to explore the specific behavior of the *Z* configuration compound **10** against multiple kinases.

#### 2.4.1. Target Identification

Conventional identification of drug targets is an expensive, time-consuming, and difficult process; only a few drug targets can be identified. In contrast, the computational method permits a great deal of analysis within a short period and brings a large number of potential drug targets from a pool of information [30]. In the present study, an integrated in silico approach was used to identify potential targets [31] for the active compound **10**. Initially, the disease search tool in the KEGG database was used against breast, ovarian, and colorectal cancer to extract the targets that may be involved in these diseases (Figures 5–7) [32]. KEGG uses the knowledge of gene function and linking this information with advanced order functional information by using systematic analysis. The schematic presentation of the KEGG pathway shows genes marked as light-blue color as a drug target and genes marked as pink as associated with the disease, whereas when the gene is linked with both a disease and a drug target, its color is split into light blue and pink. There were several target proteins involved in one pathway; therefore, protein-drug association servers Similarity Ensemble Approach (SEA, http://sea.bkslab.org/) [33], Search Tool for the Retrieval of Interacting Genes (STRING, http://string-db.org) [34], and Search Tool for Interacting Chemicals (STITCH, http://stitch.embl.de/) [35] were used. The STRING database was used to explain the molecular function, biological processes, cellular components, and pathways of the target proteins. The SEA relates target proteins based on set-wise chemical similarity among their compounds. A total of 14 potential targets (Btk, Itk, c-Src, EGFR, Akt1, Fyn, Lyn, Lck, PKC, Abl1, Hck, Cdk2, Braf, and Her2) were selected based on the data obtained from these servers that further proved the reliability of text mining and molecular docking.

**Figure 5.** The KEGG pathway for ovarian cancer.

**Figure 7.** The KEGG pathway for breast cancer.

#### 2.4.2. Docking Studies

The known compounds that were already reported as inhibitors of the target proteins, as well as nature and crucial active site residues, were specified in their accessible complexes, used as a positive control. Prior to docking, validation of the software and docking conditions was performed by retrieving the control compounds from their crystal complexes and then redocking by MOE against their relevant targets. The redocking results are presented in Table 3. After validation, docking of compound **10** was performed with all 14 targets, and their docking scores were compared with the control in order to select a target with the highest docking score. We observed that compound **10** presented good scores against Btk, Itk, c-Src, EGFR, Akt1, Fyn, Lyn, Lck, PKC, and Abl1 kinase as compared to Hck, Cdk2, Braf, and Her2. The docking scores of compound **10** are presented in Table 4.

**Table 3.** Predicted binding affinity (docking scores in kcal/mol) and root-mean-square deviation (RMSD) of control inhibitors against related proteins.


**Table 4.** MM/PBSA derived binding free energies of complexes computed from the MD simulations.


#### 2.4.3. Structure Architecture of Kinases

The structure of all protein kinase domains is composed of a small, typically β-stranded *N*-lobe linked with a small hinge region to a bigger α-helical *C*-lobe. In protein kinases, ATP binds to the cavity that lies between *N*- and C-terminal lobes creating the kinase activation site, where the adenine moiety of ATP is packed like a sandwich between hydrophobic residues, while it is associated with the hinge region by hydrophilic contact. Typical kinase elements include the G-loop (glycine-rich residues forming the active site roof), A-loop, or activation loop crucial for kinase activation and deactivation, C-helix responsible for the selectivity of the active site, and the hinge region that plays a vital role in inhibitor binding. The ATP-binding site categorized into five distinct regions: adenine region where the adenine moiety of ATP forms a hydrogen bond with the hinge, hydrophobic region-I corresponds to the entrance pocket, hydrophobic region-II also known as the selectivity pocket is determined by an extremely conserved DFG (aspartate-phenylalanine-glycine) motif, a hydrophilic region where the ATP sugar binds, and a solvent-exposed region for phosphate binding.

#### 2.4.4. Molecular Dynamic Simulation

Although X-ray crystallography and molecular docking are powerful techniques for biological macromolecules, they are unable to examine the protein's flexibility and they provide a momentary binding mode. Therefore, MD simulation was used to investigate the ligand-protein interactions [36] and the conformational changes which occur in the structure of kinases with respect to motions. Based on the docking results, nine complexes were selected for further MD simulation to understand the binding mechanism of compound **10** and its stability against multiple kinases.

#### **c-Src**

The stereo view of the binding mode of Src (PDB ID: 2SRC) is depicted in Figure 8A. It was found that, initially, compound **10** was bound to the active site of Src kinase similar to a cognate ligand by making a hydrogen bond with the NH group of Met341 and CO group of Glu339, while these bonds were observed to undergo destabilization during the simulation. Comparatively, three new hydrogen bonds were formed, involving the side chain amino group of Lys295 (solvent accessible area) and the hydroxyl group of Ser345, which interact with the pyrazolopyrimidine scaffold located in the adenine region of Src kinase. The spatial position of the compound was found to be different from the observed dock pose because of major conformational changes during simulation.

**Figure 8.** Binding mode of Z configuration of compound **10** (khaki) with the active site residues of (**A**) c-Src, (**B**) Lck, (**C**) Btk, (**D**) Akt, (**E**) Abl1, (**F**) Braf, (**G**) Alk, (**H**) PKC, and (**I**) Cdk2. Amino acid residues involved in crucial interactions are labeled.

The activation loop in the DFG-in conformation provides enough space to accommodate the ring of compound **10**, which may cause a collision with Phe405 of the DFG-out conformation. Moreover, distinct hydrophobic interactions were also observed with Leu273, and Val281 (hydrophobic region I) that maintain the binding mode of complexes. The MD results suggest that the non-flexible part of the compound might be the reason for its moderate activity as compared to the reference compound.

#### **Lck**

The binding elements responsible for the modest activity of Lck kinase are depicted in Figure 8B. Consistent with the results from other reported studies, compound **10** is well accommodated in the ATP site of Lck by forming multiple hydrophobic, π-stacking, π-cation, and Van der Waals interactions with Leu251, Ala271, Lys273, Thr316, Lys318, and Asp382. Due to the different conformation of the DFG motif, compound **10** slightly moves toward the adenine region with its core pyrimidine ring in the adenine region by forming a hydrophilic interaction with Met319. In the meantime, Phe385 of the DFG motif is involved in a weak hydrophobic interaction due to the far distance. The chloro-benzene ring engages the gatekeeper residue Thr316 via hydrophobic interaction, while the non-substituted benzene ring occupies the extended hydrophobic pocket, forming π-stacking and Van der Waals interactions with Leu251, Ala271, and Tyr318. The complex was further stabilized by the formation of a halogen bond between the backbone of the carbonyl oxygen of Ile314 and the fluorine of the compound **10**. In summary, a marked similarity of interaction was observed with the reference compound except for the hydrophilic interaction responsible for the inhibitory activity of Lck kinase.

#### **Btk**

The docked complex of BTK presented a number of hydrophilic and hydrophobic interactions with the active site residues Leu408, Val416, Lys430, Thr474, Glu475, Met477, Leu528, Asp539, and Phe540. The compound occupied the same position consistent with the reference compound. Molecular dynamics studies revealed that compound **10** binding to BTK, is stable and it was found to involve in hydrogen bond interactions with key residues of the glycine-rich P-loop like Leu408, Gly409, and Asp539. Transient hydrogen bond interactions were also detected with crucial active site residues Glu475 and Met477 of the hinge region, catalytic residue Lys430, and the DFG motif that is crucial for kinase activity, indicating that compound **10** probably has the appropriate interaction profile with BTK (Figure 8C).

#### **Akt**

According to the binding mode of the Akt complex [37], compound **10** adopts a Y-shape conformation, where the presence of residues Phe469 and Phe472 that are essential for the regulatory control of Akt1, make the pocket hydrophobic. Initially, the pyrimidine ring occupies the adenosine portion of the binding site, forming hydrogen bonding to the hinge region of the kinase through Ala230 (Figure 8D). During the simulation, this hydrogen bond is replaced by hydrophobic interactions, while the phenyl substituent presents a parallel aromatic interaction with Phe438. Moreover, residues Lys179, Leu181, and Val164 also stabilize the compound by making hydrophobic contact. In general, theoretical studies indicate that compound **10** stabilizes Akt via hydrophobic interactions; however, after losing its interactions with the hinge region, the gatekeeper residue and the DFG motif lead to its low kinase inhibitory properties.

#### **Abl**

The binding mode of the Abl1 complex (Figure 8E) showed that the compound is accommodated in the lipophilic channel slightly away from the hinge region. The bent conformation of the molecule does not allow its tight binding to the ATP site. This resultant compound is unable to mimic interaction with the adenine moiety of ATP. Only a few hydrophobic interactions were observed with Glu305, Val308, Leu473, and Asp400, but these interactions were also not found to be persistent throughout the

md simulation. It is hypothesized that the absence of specific interactions of compound **10** with the residues of the hinge region and DFG motif could be the reason for its inactivity.

#### **Braf**

According to the docking calculations, compound **10** is involved in a number of hydrophilic and hydrophobic interactions with Braf kinase. However, after a short 50 ns production run, the simulated binding mode provided a different picture to understand the selectivity trend. During the simulation, these non-bonded interactions were weakened due to loop flipping of the motif from the DGF-in to the DGF-out conformation. However, in order to accommodate the core pyrimidine ring, the benzene ring was required to tilt out of the plane due to restricted space. As a result of this movement, intermolecular interactions broke, which penalized the binding affinity of compound **10** for Braf (Figure 8F).

#### **Alk**

The binding interactions of the Alk with compound **10** are depicted in Figure 8G. The complex obtained after simulation indicates that the compound partially aligned on the reference compound ceritinib, and it was unable to form any hydrophilic interaction with the active site residues, especially with the hinge region. A similar type of binding mode was observed in the case of PKC (Figure 8H) and Cdk2 (Figure 8I).

#### 2.4.5. Binding Free Energy Calculations

MM/PBSA is considered as the best choice for predicting the binding affinities of a protein-ligand complex with implicit solvent at a low computational cost. It has the potential to recognize the true binding structure of the complex from docking. In the present study, MM/PBSA was used to re-rank the nine complexes after a short production run of 50 ns. The calculated binding free energy of all complexes is presented in Table 4 [38]. The binding position and atomic interaction between protein kinases and ligand are the main factors that play a role in measuring the binding affinity. The predicted binding poses of compound **10** significantly overlaid with the reported inhibitors of c-Src, Lck, Btk, and Akt, but partially aligned in the case of Braf and Alk. According to Table 4, the binding free energy computed for c-Src, Lck, and Akt complexes was −20.63, −15.64, and −18.09, respectively, which were more stable than ALK, CDK2, and Abl1 kinases, agreeing with the selectivity profile observed experimentally. In order to comprehend the effect of the individual energy term in the binding process, total binding free energy was decomposed into the Eele, Evdw, EGB, and ESURF energy components. The values of these components indicated that van der Waals (ΔGvdw) energy contributed significantly to the total binding free energy. The superior contribution of Van der Waals interaction (Table 4) was in agreement with the fact that compound **10** mainly stabilizes via hydrophobic interactions with the kinases.

#### **3. Experimental Protocols**

#### *3.1. Materials and Methods*

Bromoacetone was purchased from ChemService Inc. (West Chester, PA, USA). Anhydrous dichloromethane, anhydrous pyridine, *N*,*N*-dimethylformamide (DMF), and other chemicals and reagents were purchased from Sigma-Aldrich Chemical Co. (Milwaukee, WI, USA). The chemical structures of final products were characterized by nuclear magnetic resonance spectra (1H NMR, 13C NMR) determined on a Bruker (Billerica, MA, USA) NMR spectrometer (400 MHz) or a Varian (Silicon Valley, CA, USA) NMR spectrometer (500 MHz). 13C NMR spectra were fully decoupled. Chemical shifts were reported in parts per millions (ppm) using deuterated solvent peak as the standard. The chemical structures of final products were confirmed by a high-resolution Biosystems QStar Elite (QStar Technologies, Denver, CO, USA) time-of-flight electrospray mass spectrometer. Details of procedures and spectroscopic data of the respective compounds are presented below. Final compounds

were purified on a Phenomenex (Torrance, CA, USA) Prodigy 10 μm ODS reverse-phase column (2.1 cm × 25 cm) with a Hitachi (Tokyo, Japan) HPLC system using a gradient system of acetonitrile or methanol and water (CH3CN/CH3OH/H2O, 0−100%, pH 7.0, 60 min). The purity of final products (>99%) was confirmed by analytical HPLC. The analytical HPLC was performed on the Hitachi analytical HPLC system using a C18 Shimadzu (Kyoto, Japan) Premier 3 μm column (150 cm × 4.6 mm) using two different isocratic systems, and a flow rate of 1 mL/min with UV detection at 265 nm.

#### *3.2. Synthesis*

3-Phenyl-1*H*-pyrazolo[3,4-*d*]pyrimidin-4-amine (**1**): Compound **1** was synthesized according to a previously reported procedure [23].

1-(4-Amino-3-phenyl-1*H*-pyrazolo[3,4-*d*]pyrimidin-1-yl)propan-2-one (**2**): Compound **1** (156 mg, 0.73 mmol) and potassium carbonate (200 mg, 1.4 mmol) were taken in dry DMF (10 mL). Bromoacetone (100 mg, 0.72 mmol) was diluted with dry DMF (5 mL) and was added dropwise to the reaction mixture at room temperature. The reaction mixture was stirred for 4 h at room temperature. After completion of the reaction, the solvent was removed under reduced pressure, added water (50 mL) and extracted with dichloromethane. The organic layer was filtered through filter paper and dried over Na2SO4 (anhydrous). The crude product was further purified on silica gel flash chromatography using dichloromethane and methanol (0–20%) as eluents. 160 mg, 81% yield. 1H-NMR (CDCl3, 500 MHz, δ ppm): 8.3 (s, 1H), 7.7 (d, *J* = 11 Hz, 2H), 7.5–7.5 (m, 3H), 5.2 (s, 2H), 2.2 (s, 3H).13C NMR (CDCl3, 125 MHz, δ ppm): 201.3, 158.1, 156.2, 155.2, 145.4, 132.8, 129.4, 129.3, 128.4, 98.4, 56.1, 27.1. HR-MS (ESI-TOF) (*m*/*z*): calculated for C14H13N5O + H+: 268.1193; found, 268.0852 [M + H]+.

General Procedure for the Synthesis of Pyrazolopyrimidine Chalcones:

Compound **2** (26 mg, 0.1 mmol) was dissolved in ethanol (5 mL). Sodium hydroxide (5 mg, 20 μL of 250 mg/mL NaOH solution in water) was added to the compound **2** solution. The reaction mixture solution was stirred for 15 min. Then, a solution of *p*-methylbenzaldehyde (24 mg, 0.2 mmol) in ethanol (15 mL) was added to the reaction mixture. The reaction mixture was allowed to stir at room temperature for 5 h. On completion of the reaction, the reaction mixture was neutralized with dilute acetic acid in ethanol and purified over reverse phase HPLC using water and methanol as eluents to obtain pure product **3** (22 mg, 61% yield). A similar procedure was adopted for the synthesis of other pyrazolopyrimidine derivatives.

3-(4-Amino-3-phenyl-1*H*-pyrazolo[3,4-*d*]pyrimidin-1-yl)-4-*p*-tolylbut-3-en-2-one (**3**). (Yield: 61%). 1H-NMR (CDCl3, 500 MHz, δ ppm): 8.3 (s, 1H), 7.7 (s, 1H), 7.7 (d, *J* = 7 Hz, 2H), 7.6–7.5 (m, 3H), 7.0 (d, *J* = 8.2 Hz, 2H), 6.9 (d, *J* = 8.2 Hz, 2H), 2.3 (s, 3H), 2.9 (s, 3H). 13C NMR (CDCl3, 125 MHz, δ ppm): 194.3, 157.9, 156.9, 155.8, 146.8, 141.8, 140.5, 132.7, 131.8, 130.7, 129.6, 129.5, 129.4, 128.5, 26.1, 21.5. HR-MS (ESI-TOF) (*m*/*z*): calculated for C22H19N5O + H+: 370.1662; found, 370.1534 [M + H]+.

3-(4-Amino-3-phenyl-1*H*-pyrazolo[3,4-d]pyrimidin-1-yl)-4-*m*-tolylbut-3-en-2-one (**4**). (Yield: 64%). 1H-NMR (CDCl3, 500 MHz, δ ppm): 8.3 (s, 1H), 7.9 (s, 1H), 7.7 (d, *J* = 7 Hz, 2H), 7.6–7.5 (m, 3H), 7.1–7.0 (m, 2H), 6.8 (s, 1H), 6.7 (d, *J* = 10 Hz, 1H), 2.3 (s, 3H), 2.2 (s, 3H). 13C NMR (CDCl3, 125 MHz, δ ppm): 194.3, 157.8, 155.1, 154.9, 146.8, 140.6, 133.9, 131.7, 131.5, 130.2, 129.5, 129.4,128.6, 128.4, 26.2, 21.3. HR-MS (ESI-TOF) (*m*/*z*): calculated for C22H19N5O + H+: 370.1662; found, 370.1454 [M + H]+.

3-(4-Amino-3-phenyl-1*H*-pyrazolo[3,4-d]pyrimidin-1-yl)-4-*p*-biphenylbut-3-en-2-one (**5**). (Yield: 59%). 1H-NMR (CDCl3, 500 MHz, δ ppm): 8.0 (s, 1H), 7.9 (s, 1H), 7.7 (d, *J* = 8.2 Hz, 2H), 7.6–7.6 (m, 3H), 7.6–7.4 (m, 9H), 2.3 (s, 3H). 13C NMR (CDCl3, 125 MHz, δ ppm): 191.9, 147.2, 135.2, 131.1, 130.3, 129.9, 129.9, 129.8, 129.4, 129.0, 128.7, 128.5, 128.5, 127.7, 127.3, 127.3, 127.2, 127.1, 127.0, 126.1, 22.5. HR-MS (ESI-TOF) (*m*/*z*): calculated for C27H21N5O + H+: 432.1819; found, 432.1616 [M + H]+.

3-(4-Amino-3-phenyl-1*H*-pyrazolo[3,4-d]pyrimidin-1-yl)-4-(4-methoxyphenyl)but-3-en-2-one (**6**). (Yield: 63%). 1H-NMR (CDCl3, 500 MHz, δ ppm): 8.3 (s, 1H), 7.9 (s, 1H), 7.7 (d, *J* = 7 Hz, 2H), 7.6–7.5 (m, 3H), 6.9 (d, *J* = 8.9 Hz, 2H), 6.7 (d, *J* = 8.9 Hz, 2H), 3.7 (s, 3H), 2.3 (s, 3H). 13C NMR (CDCl3, 125 MHz, δ ppm): 194.2, 161.9, 158.0, 156.9, 155.7, 146.9, 140.2, 132.8, 132.7, 130.6, 129.5, 129.4, 128.5, 124.3, 114.4, 98.9, 55.3, 26.0. HR-MS (ESI-TOF) (*m*/*z*): calculated for C22H19N5O2 + H+: 386.1612; found, 386.1573 [M + H]+.

3-(4-Amino-3-phenyl-1*H*-pyrazolo[3,4-d]pyrimidin-1-yl)-4-(4-(dimethylamino)phenyl)but-3-en-2-one (**7**). (Yield: 66%). 1H-NMR (CDCl3, 500 MHz, δ ppm): 8.3 (s, 1H), 7.9 (s, 1H), 7.7 (d, *J* = 8 Hz, 2H), 7.6–7.5 (m, 3H), 6.8 (d, *J* = 9.1 Hz, 2H), 6.4 (d, *J* = 9.1 Hz, 2H), 2.9 (s, 6H), 2.3 (s, 3H). 13C NMR (CDCl3, 125 MHz, δ ppm): 194.4, 162.1, 158.2, 157.1, 155.9, 147.1, 140.4, 133.0, 132.9, 130.8, 129.7, 129.6, 128.7, 124.5, 114.6, 99.0, 41.4, 26.3. HR-MS (ESI-TOF) (*m*/*z*): calculated for C23H22N6O + H+: 399.1928; found, 399.1981 [M + H]+.

3-(4-Amino-3-phenyl-1*H*-pyrazolo[3,4-d]pyrimidin-1-yl)-pent-3-en-2-one (**8**). (Yield: 60%). 1H-NMR (CDCl3, 500 MHz, δ ppm): 8.37 (s, 1H), 7.8–7.7 (m, 2H), 7.6–7.5 (m, 3H), 7.4 (q, *J* = 7.1 Hz, 1H), 2.3 (s, 3H), 1.8 (d, *J* = 7 Hz, 3H). 13C NMR (CDCl3, 100 MHz, δ ppm): 157.9, 156.3, 154.8, 141.7, 132.6, 129.6, 129.5, 129.5, 128.5. 128.4, 98.3, 56.2, 26.1, 14.6. HR-MS (ESI-TOF) (*m*/*z*): calcd for C16H15N5O + H+: 294.1349; found, 294.099 [M + H]+.

3-(4-Amino-3-phenyl-1*H*-pyrazolo[3,4-d]pyrimidin-1-yl)-4-(3-nitrophenyl)but-3-en-2-one (**9**). (Yield: 44%). 1H-NMR (CDCl3, 500 MHz, δ ppm): 8.3 (s, 1H), 7.9 (s, 1H), 7.7 (d, *J* = 7 Hz, 2H), 7.5–7.5 (m, 3H), 6.9 (d, *J* = 8.9 Hz, 2H), 6.7 (d, *J* = 8.9 Hz, 1H), 2.3 (s, 3H). 13C NMR (CDCl3, 125 MHz, δ ppm): 194.2, 161.9, 158.0, 156.9, 155.7, 146.9, 140.2, 132.8, 132.7, 130.6, 129.5, 129.4, 128.5, 124.3, 114.4, 98.9, 55.3, 26.0. HR-MS (ESI-TOF) (*m*/*z*): calculated for C21H16N6O3 + H+: 401.1357; found, 401.0957 [M + H]+, 801.1652 [2M + H]+.

3-(4-Amino-3-phenyl-1*H*-pyrazolo[3,4-d]pyrimidin-1-yl)-4-(4-chlorophenyl)but-3-en-2-one (**10**). (Yield: 57%). 1H-NMR (CDCl3, 500 MHz, δ ppm): 8.1 (s, 1H), 7.9 (s, 1H), 7.6 (m, 2H), 7.5–7.5 (m, 3H), 7.2 (d, *J* = 8.6 Hz, 2H), 6.9 (d, *J* = 8.6 Hz, 2H), 2.4 (s, 3H). 13C NMR (CDCl3, 125 MHz, δ ppm): 192.4, 153.7, 153.5, 149.5, 147.2, 140.6, 137.9, 132.5, 131.5, 131.3, 130.9, 130.4, 130.1, 129.5, 128.1, 97.6, 55.3, 25.8. HR-MS (ESI-TOF) (*m*/*z*): calculated for C21H16ClN5O + H+: 390.1116; found, 390.0951 [M + 1]+.

3-(4-Amino-3-phenyl-1*H*-pyrazolo[3,4-d]pyrimidin-1-yl)-4-(2,4-dichlorophenyl)but-3-en-2-one (**11**). (Yield: 58%). 1H-NMR (CDCl3, 500 MHz, δ ppm): 8.3 (s, 1H), 7.9 (s, 1H), 7.7 (d, *J* = 7 Hz, 2H), 7.5–7.5 (m, 3H), 6.9 (s, 1H), 6.7 (d, *J* = 8.6 Hz, 2H), 2.6 (s, 3H). 13C NMR (CDCl3, 125 MHz, δ ppm): 192.2, 153.7, 136.9, 136.2, 130.9, 130.2, 130.1, 129.8, 129.0, 128.2, 128.1, 127.5, 127.5, 97.6, 66.4, 53.4, 25.9. HR-MS (ESI-TOF) (*m*/*z*): calculated for C21H15Cl2N5O + H+: 424.0726; found, 424.1220 [M + H]+.

3-(4-Amino-3-phenyl-1*H*-pyrazolo[3,4-d]pyrimidin-1-yl)-4-(2-chlorophenyl)but-3-en-2-one (**12**). (Yield: 57%). 1H-NMR (CDCl3, 500 MHz, δ ppm): 8.3 (s, 1H), 8.2 (s, 1H), 7.6–7.5 (m, 5H), 7.3–7.2 (m, 4H), 2.6 (s, 3H). 13C-NMR (CDCl3, 125 MHz, δ ppm): 153.6, 153.3, 149.1, 146.5, 138.2, 132.0, 130.8, 130.2, 130.1, 129.3, 128.9, 128.1, 126.9, 52.5, 25.9. HR-MS (ESI-TOF) (*m*/*z*): calcd for C21H16ClN5O + H+: 390.1116; found, 390.1427 [M + H]+.

3-(4-Amino-3-phenyl-1*H*-pyrazolo[3,4-d]pyrimidin-1-yl)-4-(2-fluorophenyl)but-3-en-2-one (**13**). (Yield: 62%). 1H-NMR (CDCl3, 500 MHz, δ ppm): 8.3 (s, 1H), 7.9 (s, 1H), 7.7 (d, *J* = 7 Hz, 2H), 7.6–7.5 (m, 3H), 7.2–6.9 (m, 4H), 2.3 (s, 3H). 13C NMR (CDCl3, 125 MHz, δ ppm): 193.8, 158.1, 155.6, 147.4, 132.8, 132.7, 130.3, 131.7, 129.7, 129.5, 129.5, 129.3, 129.2, 128.4, 128.4, 128.4, 98.7, 26.2. HR-MS (ESI-TOF) (*m*/*z*): calculated for C21H16FN5O + H+: 374.1412; found, 374.1526 [M + H]+.

3-(4-Amino-3-phenyl-1*H*-pyrazolo[3,4-d]pyrimidin-1-yl)-4-(4-fluorophenyl)but-3-en-2-one (**14**). (Yield: 64%). 1H-NMR (CDCl3, 500 MHz, δ ppm): 8.1 (s, 1H), 7.8 (s, 1H), 7.5–7.4 (m, 5H), 6.9 (dd, *J* = 14 Hz, *J* = 5.6 Hz, 2H), 6.8 (t, *J* = 7.6 Hz, 2H), 2.4 (s, 3H). 13C NMR (CDCl3, 125 MHz, δ ppm): 192.5, 153.7, 153.5, 147.2, 140.7, 132.5, 132.4, 130.9, 130.1, 130.0, 128.1, 128.1, 116.7, 116.5, 25.7. HR-MS (ESI-TOF) (*m*/*z*): calculated for C21H16FN5O + H+: 374.1412; found, 374.1679 [M + H]+.

3-(4-Amino-3-phenyl-1*H*-pyrazolo[3,4-d]pyrimidin-1-yl)-4-phenyl but-3-en-2-one (**15**). (Yield: 59%). 1H-NMR (CDCl3, 500 MHz, δ ppm): 8.2 (s, 1H), 8.0 (s, 1H), 7.7–7.6 (m, 5H), 7.4–6.9 (m, 5H), 2.5 (s, 3H). 13C-NMR (CDCl3, 125 MHz, δ ppm): 192.5, 153.7, 153.5, 147.2, 140.7, 132.5, 132.4, 130.9, 130.1, 130.0, 128.1, 128.1, 116.7, 116.5, 25.7. HR-MS (ESI-TOF) (*m*/*z*): calculated for C21H17N5O + H+: 356.1506; found, 356.1703 [M + H]+.

#### *3.3. Kinase Enzyme Assay*

#### 3.3.1. c-Src Kinase Activity Assay

The effect of compounds on the activity of *c*-Src kinase was assessed using a Transcreener® ADP [2,8] fluorescence intensity (FI) Assay, from Bell Brook Labs, Madison, WI, USA, (catalog no. 3013-1K) according to the manufacturer's protocol. A 384-well low-volume black non-binding surface round-bottom microplate was purchased from Corning Inc. (Corning, NY, USA) (#3676). In summary, the kinase reaction was started in the 384-well low volume black microplate with the incubation of 2.5 μL of the reaction cocktail (0.7 nM of His6-Src kinase domain in kinase buffer) with 2.5 μL of prediluted compounds (dissolved in 10% DMSO, 4× target concentration) for 10 min at room temperature using microplate shaker. The reaction cocktail was made using the kinase buffer HEPES (200 mM, pH 7.5), MgCl2 (16 mM), EGTA (8 mM), DMSO (4%), Brij-35 (0.04%), and 2-mercaptoethanol (43 mM). The kinase reaction was started by adding 5 μL of ATP/substrate (40 μM/600 μM) cocktail and incubated for 30 min at room temperature on a microplate shaker. Src optimal peptide (AEEEIYGEFEAKKKK) was used as the substrate for the kinase reaction. The kinase reaction was stopped by adding 10 μL of the 1× ADP Detection Mixture to the enzyme reaction mixture and mixed using a plate shaker. The mixture was incubated at room temperature for 1 h, and the fluorescence intensity was measured. The 1× ADP Detection Mixture was prepared by adding ADP<sup>2</sup> Antibody-IRDyeR QC-1 (10 μg/mL) and ADP Alexa594 Tracer (8 nM) to Stop and Detect Buffer B(1×). The fluorescence Intensity measurements were performed using a fluorescence intensity optical module using the excitation of 580 nm and emission of 630 nm with bandwidths of 10 nm by Optima, BMG Labtech microplate reader (BMG Labtech, Ortenberg, Germany). The IC50 values of the compounds were calculated using ORIGIN 6.0 (Origin Lab, Microcal Software, Inc. Northhampton, MA, USA) software. IC50 is the concentration of the compound that inhibited enzyme activity by 50%. All the experiments were carried out in triplicate.

3.3.2. Evaluation of Compound **10** Against a Panel of Kinases (Abl1, Akt1, Alk, Braf, Btk, Cdk2, cSrc, Lck, and PKCa)

Compound **10** was evaluated for inhibitory activity against Abl1, Akt1, Alk, Braf, Btk, Cdk2, cSrc, Lck, and PKCa, in duplicate by Reaction Biology Corporation. Compounds were tested in a five-dose IC50 mode with four-fold serial dilution starting at 20 μM. Control compounds Staurosporine and GW5074 were tested in a 10-dose IC50 starting at 20 μM with a four-fold or three-fold serial dilution, respectively. All kinase reactions were performed at 200 μM ATP. The compounds were pre-incubated with the enzyme and substrate mixture about 20 min, and then ATP was added to start the reaction. The reaction was 2 h at room temperature. Curve fits were performed when the activities at the highest concentration of compounds were less than 65%. The detailed experimental procedure is provided here. An IC50 value of less than 0.04 nM or higher than 1 μM is estimated based on the best curve fitting available. The substrates for kinases are shown in Table 5.


**Table 5.** Substrates used for kinases.

Base Reaction buffer was prepared from 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT, and 1% DMSO. Required cofactors were added individually to each kinase reaction. Testing compounds were dissolved in 100% DMSO to a specific concentration. The serial dilution was conducted by Integra Viaflo Assist in DMSO. The substrate was prepared in a freshly prepared reaction buffer. The required cofactors for each kinase were added to the substrate solution. The kinase was added into the substrate solution, and the solution was gently mixed. The compounds in 100% DMSO were added into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range) and incubated for 20 min at room temperature. P-ATP (Specific activity 10 μCi/μl) was added into the reaction mixture to initiate the reaction. The mixture was incubated for 2 h at room temperature. The radioactivity was detected using the filter-binding method. The kinase activity data were expressed as the percentage remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions. IC50 values, and curve fits were obtained using GraphPad Prism software (version 7, Informer Technologies, Inc., Los Angeles, CA, USA).

#### *3.4. Cell Culture and Cell Proliferation Assay*

#### 3.4.1. Cell Culture

Human leukemia cell line CCRF-CEM (ATCC no. CCL-119), human ovarian adenocarcinoma cell line SK-OV-3 (ATCC no. HTB-77), human breast carcinoma cell line MDA-MB-231 (ATCC no.HTB-26), and human colon adenocarcinoma cell line HT-29 (ATCC no. HTB-38) were obtained from American Type Culture Collection. Cells were grown on 75 cm<sup>2</sup> cell culture flasks with RPMI-16 medium (for leukemia cells) and EMEM (Eagle's minimum essential medium) (for SK-OV-3, HT-29, MDA-MB-231 cells), supplemented with 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin solution (10,000 units of penicillin and 10 mg of streptomycin in 0.9% NaCl) in a humidified atmosphere of 5% CO2, 95% air at 37 ◦C.

#### 3.4.2. Cell Proliferation Assay

The cell proliferation assay was carried out using a CellTiter 96 aqueous one-solution cell proliferation assay kit (Promega, Madison, WI, USA). Briefly, upon reaching about 75–80% confluence, 5000 cells/well were plated in 96-well microplate in 100 μL of the medium. After seeding for 24 h, the cells were treated with 50 μM compound in triplicate. Doxorubicin (10 μM) was used as a positive control. At the end of the sample exposure period (72 h), 20 μL of CellTiter 96 aqueous solution was added. The plate was returned to the incubator for 1 h in a humidified atmosphere at 37 ◦C. The absorbance of the formazan product was measured at 490 nm using a microplate reader. The blank control was recorded by measuring the absorbance at 490 nm with wells containing medium mixed with CellTiter 96 aqueous solution but no cells. The percentage of cell survival was calculated as the OD value of cells treated with test compound − OD value of culture medium/(OD value of control cells

− OD value of culture medium) × 100%. The same strategy was used for generating a dose-response graph for compound **10**.

#### *3.5. Molecular Modeling*

#### 3.5.1. Target Fishing

Potential targets were identified against the phenylpyrazolopyrimidine scaffold using a combination of information integration, chemometric methods, and data mining approaches. Initially, targets involved in breast, ovarian, and colorectal cancer pathways were extracted from the database of KEGG. In the next step, protein network association servers STRING, STITCH, and SEA were used to characterize the protein-ligand interactions. Similarly, the PhPP scaffold was also searched using different search engines to understand protein-ligand interactions. Based on the above-mentioned information, we selected fourteen different targets, including Btk, Itk, c-Src, EGFR [39], Braf, Fyn, Lyn, Hck, Cdk2/4, Alk, Akt1 Lck, PKC, and Abl1 kinase. To understand the binding mechanism of active compound **10** against all selected targets, molecular docking and MD simulations were performed.

#### 3.5.2. Preparation of Complexes

Crystal structures of the Anaplastic lymphoma kinase [PDB ID: 4MKC], ABL1 kinase [PDB ID: 6HD4], Protein kinase B also known as AKT [PDB ID: 4GV1], bruton tyrosine kinase [PDB ID: 6DIS], BRAF [PDB ID: 6B8U], c-Src kinase [PDB ID: 2SRC], CDK2 [PDB ID: 4KD1], LCK [PDB ID: 2OFU], ITK [PDB ID: 4HUC], EGFR [PDB ID: 4HJO], FYN [PDB ID: 2DQ7], LYN [PDB ID: 2ZV9], HCK [PDB ID: 1QCF], and PKCa [PDB ID: 3IW4] were retrieved from RCSB Protein Databank. The missing part of the starting structures was constructed using MOE software (Molecular Operating Environment, v2016.08, 2016). Compound partial charges were derived with the MMFF94x force field. All stabilizing agents, water molecules, and ions present in the crystal structures were removed. The protonate 3D module in MOE was used for assigning the protonation state to every titratable residue at physiological conditions within the complexes. MOE docking suite was employed for docking studies. The standard default settings were used with the London dG scoring function as a fitness function in all calculations.

#### 3.5.3. MD Simulations

After analysis of docking results, nine complexes were subjected to all-atom MD simulation using the graphics processing unit (GPU) version of the PMEMD.cuda engine provided with Amber18 [40]. The gaff [41] and ff14SB force field [42] were implemented to ligand and protein in all simulations, respectively. Each complex was neutralized by adding counter ions, and the systems were solvated by a cubic box of the TIP3P water model with a 10 Å box size from the solute. Systems were relaxed by adjusting hydrogen position, while 1000 cycles of steepest descent, and 5000 cycles of the conjugate gradient with restraints of 100 kcal·mol−<sup>1</sup> were applied on heavy atoms. Finally, another minimization of 5000 steps was performed to relax the system without any restraint. It was followed by equilibration of each system by gradual heating from 0 K to 300 K in the NVT ensemble. Systems were further equilibrated under the NPT ensemble (Pressure = 1 atm). The production run of 50 ns was performed for each system using the isothermal-isobaric (NPT) ensemble at constant temperature and pressure with periodic boundary conditions. The Particle Mesh Ewald Method (PMEM) [43] was used to calculate long-range electrostatic interactions with cut off value of 8 Å for the non-bonded (electrostatics and Van der Waals) interactions. An integration time step of 2 fs was set to integrate Newton's equations of motion. The SHAKE algorithm [44] was employed to restrain the bonds involving hydrogen atoms. The trajectories were analyzed using CPPTRAJ module incorporated in AMBER suite. All visualization was performed using Chimera and VMD.

#### 3.5.4. MM/PBSA Calculations

The binding free energy calculation for each complex was performed using the MM(GB/PB)SA method based on 1000 frames extracted from the 50 ns trajectory. The equations associated with binding free energy calculation were as follows:

$$
\Delta \mathbf{G} = \mathbf{G}\_{\text{complex}} - (\mathbf{G}\_{\text{receptor}} + \mathbf{G}\_{\text{compound}}) \tag{1}
$$

$$\mathbf{G} = \mathbf{H} - \mathbf{T} \mathbf{S} \tag{2}$$

$$\text{pH} = \text{E}\_{\text{vdw}} + \text{E}\_{\text{ele}} + \text{E}\_{\text{irt}} + \text{G}\_{\text{gb}} + \text{G}\_{\text{surf}} \tag{3}$$

The total binding free energy is ΔG, whereas the free energies for the complex, receptor, and compound are represented as Gcomplex, Greceptor, and Gcompound, respectively. The terms H and TΔS were used for the contribution of enthalpy and entropy. The binding free energy further decomposed into electrostatic (Eele), Van der Waals interaction (Evdw), internal energy (Eint), and polar (Ggb) and non-polar desolvation energy (Gsurf). The value of the dielectric constant was set to 1, and the external was set to 80 for solute protein (εin). The Generalized Born (GB) model established by Onufriev (GBOBC1) with modified Bondi radii (mbondi2) and igb = 2 implemented in AMBER18 was used to calculate the electrostatic contribution to the solvation free energy. The non-polar surface area was calculated using the LCPO model. The offset (β) and surface tension (γ) values were set to 0.92 kcal/mol and 0.00542 kcal/(mol·Å2), respectively.

#### **4. Conclusions**

A number of novel *N*1-(α,β-alkene) substituted phenylpyrazolopyrimidine (PhPP) derivatives with acetyl and functionalized phenyl groups at α- and β-positions were synthesized, and they showed modest inhibitory activity (IC50 = 21.7–192.1 μM) against c-Src kinases. A number of para-chlorophenyl substituted compounds, **10** and **11,** consistently inhibited the cell proliferation 72–96 h in all cell lines and were more effective against HT-29 cells. Compound **10** was also found to be modestly active against c-Src, Lck, and Btk. The target-based study of these compounds was performed by text mining, similarity search, and ligand-protein interaction pattern to identify potential targets. Further MD simulation and binding free energy calculations were carried out to discriminate the selectivity of the investigated compound with a panel of different kinases and provide more in-depth structural insight into the interaction pattern involved in the inhibition process. The structural and energetic features highlighted in this study can provide a theoretical basis for further research on selective protein kinase inhibitors.

**Author Contributions:** Methodology: B.S.C., N.S.J., S.A., S.M., and R.K.T.; writing—original draft preparation: B.S.C., Z.U.-H, and K.P.; supervision: Z.U.-H. and K.P.; funding acquisition: K.P.; performed the synthesis: B.S.C. and N.S.J.; conducted the antiproliferative assays: S.M. and D.M.; performed the kinase assays: R.K.T.; performed molecular modeling: S.A.; supervised, wrote, and edited the manuscript: Z.U.-H. and K.P. All authors contributed to writing the manuscript, editing, and reviewing of the manuscript. All authors read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the American Cancer Society Grant # RSG-07-290-01-CDD.

**Acknowledgments:** We acknowledge the financial support from the American Cancer Society Grant # RSG-07-290-01-CDD and Chapman University School of Pharmacy.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


**Sample Availability:** Samples of the limited number the compounds are available from the authors upon request.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## **Synthesis, Docking Studies and Biological Activity of New Benzimidazole- Triazolothiadiazine Derivatives as Aromatase Inhibitor**

**Ulviye Acar Çevik 1,2, Betül Kaya Çavu¸so ˘glu 3, Begüm Nurpelin Sa ˘glık 1,2, Derya Osmaniye 1,2, Serkan Levent 1,2, Sinem Ilgın 4, Yusuf Özkay 1,2,\* and Zafer Asım Kaplancıklı <sup>1</sup>**


Academic Editor: Filippo Minutolo

Received: 3 March 2020; Accepted: 1 April 2020; Published: 2 April 2020

**Abstract:** In the last step of estrogen biosynthesis, aromatase enzyme catalyzes the conversion of androgens to estrogens. Aromatase inhibition is an important way to control estrogen-related diseases and estrogen levels. In this study, sixteen of benzimidazole-triazolothiadiazine derivatives have been synthesized and studied as potent aromatase inhibitors. First, these compounds were tested for their anti-cancer properties against human breast cancer cell line (MCF-7). The most active compounds **5c**, **5e**, **5k**, and **5m** on MCF-7 cell line were subject to further in vitro aromatase enzyme inhibition assays to determine the possible mechanisms of action underlying their activity. Compound **5e** showed slight less potent aromatase inhibitory activity than that of letrozole with IC50 = 0.032 ± 0.042 μM, compared to IC50 = 0.024 ± 0.001 μM for letrozole. Furthermore, compound **5e** and reference drug letrozole were docked into human placental aromatase enzyme to predict their possible binding modes with the enzyme. Finally, ADME parameters (absorption, distribution, metabolism, and excretion) of synthesized compounds (**5a**–**5p**) were calculated by QikProp 4.8 software.

**Keywords:** aromatase; MCF-7; NIH3T3; benzimidazole; triazolothiadiazine; docking; ADME

#### **1. Introduction**

Aromatase is a member of the cytochrome P450 enzyme that catalyzes the biosynthesis of estrogens from androgens [1,2]. Estrogen levels have been shown to be higher in breast cancer cells. Inhibition of aromatase is one of the effective current therapeutic strategies for controlling estrogen-dependent breast cancer. Therefore, one of the commonly used classes of drugs for the management of estrogen-dependent cancer is aromatase inhibitors (AIs) [1,3,4].

Aromatase inhibitors that have been used clinically can be categorized as first-, second-, and third generations based on their evolution time or steroidal or steroidal or non-steroidal aromatase inhibitors (NSAIs) based on their structural similarity with steroids [5,6].

Aminoglutethimide is the prototype nonsteroidal inhibitor of aromatase. Problems with the side effects and selectivity of aminoglutethimide led to the development of the second-generation of nonsteroidal aromatase inhibitor (Fadrazole bearing imidazole structure) [7]. However, this

compound still has some nonselective inhibitory activity with respect to progesterone, corticosterone, and aldosterone biosynthesis. Competitive nonsteroidal inhibitors can also be constructed with a triazole ring, which is found in the third generation of aromatase inhibitors [8–10]. Most of the nonsteroidal aromatase inhibitors of therapeutic importance act covalently bind to the substrate-binding site of aromatase by coordinate the heme iron [11–13]. Especially, the heterocyclic nitrogen atom of triazole and imidazole plays an important role by coordinating with the heme iron of the aromatase enzyme [12,13]. Some studies have shown that imidazole and triazole derivatives have promising aromatase inhibition [14–17].

Recent clinical studies have shown that aromatase inhibitors, especially the third-generation, are more effective than fulvestrant and tamoxifen because of their lower side effects and higher clinical efficacy. Nevertheless, the development of acquired resistance after prolonged AIs therapy, undesirable side effects (bone loss, cardiac events, increased rash, insomnia, headaches, and arthralgia) limits their use in clinical practice. Thus, the search for new potent molecules that impair cancer growth, strongly inhibit aromatase enzyme, and present fewer side effects is of major importance [18–21].

In the previous study, 3-[4-(5-methyl-1*H*-benzo[*d*]imidazol-2-yl)phenyl]-6- (substituted phenyl) -7H-[1,2,4] triazolo [3,4-b] [1,3,4]thiadiazines derivatives have been synthesized and promising compounds have been obtained that need further development as a new class of aromatase inhibitors [22]. In this study, new benzimidazole-triazolothiadiazine derivatives were synthesized and structure of these compounds was characterized by spectroscopic data. Their antiproliferative activities against MCF-7 were evaluated. To identify the possible modes of action, aromatase inhibition experiments were performed for the most active compounds against the MCF-7 cell line. Finally, in silico prediction of pharmacokinetic profiles (ADME) were calculated for physicochemical properties of these drug candidates.

#### **2. Results and Discussion**

#### *2.1. Chemistry*

Synthesis of the target compounds was accomplished according to the steps illustrated in Scheme 1. Substituents of the synthesized compounds (**5a**–**5p**) were shown in Table 1. The starting intermediated compound (**1a**,**1b**) was prepared by the reaction of 1,2-phenylenediamine and sodium metabisulfite with 4-formylbenzoic acid methyl ester as described in a previous study [23]. The ester derivative (**1a**,**1b**) reacted with hydrazine hydrate producing compounds **2a**, **2b** in a microwave synthesis reactor. Then, the compounds-(5-substitüe-1*H*-benzimidazol-2-yl)benzoic acid hydrazide derivatives (**2a**,**2b**) were treated with carbon disulfide in NaOH solution affording, after acidic treatment, compounds **3a**, **3b**. The reaction of compound **3a, 3b** with hydrazine hydrate in the presence of ethanol produced compounds **4a**, **4b**. Then cyclization of compounds **4a**, **4b** with appropriate phenacyl bromide in the presence of anhydrous ethanol provided the desired final products (**5a**–**5p**). The characterization of these new derivatives was done by their spectroscopic (1H NMR, 13C NMR and Mass) data. 1H NMR and 13C NMR spectra of the synthesized compounds were observed at the expected region. Mass spectra (HRMS) of all the synthesized compounds showed that the molecular ion [M + H]<sup>+</sup> peak is in agreement with their molecular formula (see Supplementary Materials).

**Scheme 1.** The reaction sequence for the synthesis of the compounds (**5a**–**5p**).


**Table 1.** The synthesized compounds (**5a**–**5p**).

#### *2.2. Cytotoxicity Assay*

The newly synthesized compounds were evaluated for their in vitro anticancer potential against MCF-7 breast cancer cell lines by MTT assay [24] using cisplatin as a reference standard. The 50% inhibitory concentration (IC50) values were determined for these compounds. In addition, the cytotoxic activities of compounds **5c**, **5e**, **5k**, and **5m** were assessed against healthy NIH3T3 [25] cells, in order to

express the selectivity toward carcinogenic cells. The IC50 values of test compounds were determined as the mean IC50 of 4 independent experiments. Results are presented in Table 2, as IC50 values are in μM and revealed that some of the tested compounds were remarkably more cytotoxic than the cisplatin against MCF-7 cell lines. Concerning MCF-7 cell line, the most potent compounds were the 4-cyano derivatives **5e** and **5m** with IC50 = 0.016 ± 0.001 and 0.018 ± 0.001 μM, respectively compared to IC50 = 0.020 ± 0.009 μM for the reference drug cisplatin. Furthermore, the most promising activity was observed for the compounds **5c** and **5k** bearing 4-fluorophenyl derivatives with IC50= 0.119 ± 0.005 and 0.110 ± 0.005 μM, respectively compared to IC50 = 0.020 ± 0.009 μM for the reference drug cisplatin.


**Table 2.** IC50 (μM) values of compounds (**5a**–**5p**) (*n* = 4).

Benzimidazole-triazolothiadiazine derivatives led to a promising increase in the anti-proliferative activity. The synthesized compounds can be divided into two groups as nonsubstituted benzimidazole and 5-chlorobenzimidazole. Test compounds showed variable activities against MCF-7. In particular, compounds **5e** and **5m** displayed the highest cytotoxic activity against MCF-7 cell line. The most active compounds **5e** and **5m** carried a 4-cyanophenyl substituent. The chlorine substituent in the fifth position (**5m**) of the benzimidazole ring did not significantly affect the activity. Compounds **5c** and **5k** carrying fluorine substituents in the fourth position of the phenyl ring showed lower activity than compounds **5e** and **5m** bearing cyano group in the fourth position of the phenyl ring.

#### *2.3. Aromatase Inhibition Assay*

The in vitro anti-aromatase activity of the most active compounds **5c**, **5e**, **5k,** and **5m** was valued using commercial fluorometric assay kit (Aromatase-CYP19A Inhibitor Screening kit, Bio Vision) with letrozole as the reference drug. Results are presented in Table 3. The IC50 values of compounds were in the sub-micromolar range (2.276 ± 0.106–0.03 2 ± 0.001 μM). The best value was shown by compound **5e** with IC50 value (0.032 ± 0.001 μM).


**Table 3.** IC50 (μM) values of compounds **5c**, **5e**, **5k**, **5m,** and letrozole.

#### *2.4. Molecular Docking*

After the most active derivative was selected as compound **5e** according to in vitro aromatase enzyme inhibition assay and letrozole, molecular docking studies were performed to explain its binding modes with human aromatase enzyme active site. For this purpose, the crystal structure of human aromatase enzyme (PDB ID: 3EQM) [26] was retrieved from the Protein Data Bank server (www.pdb.org). The 2D and 3D docking poses of compound 5e are presented in Figures 1 and 2. The docking poses of letrozole are presented in Figures 3 and 4.

According to docking poses, it is understood that compound 5e displays compatible settlement with the enzyme active region. The benzimidazole ring in the structure forms two π–π interactions with Arg115 and Phe134. Also, it can be seen from 2D docking pose, this compound is in interaction with Hem molecules and Cys437 via its nitrogen atoms of triazolothiadiazine ring by salt bridge. There is another π–π interaction between 4-cyanophenyl ring and HEM molecule. The nitrogen atom of cyano group at C-4 position of phenyl ring interacts with hydroxyl of Ser314 doing hydrogen bonding. It is thought that this interaction is important for compound **5e** in terms of explaining its inhibitory activity. It is seen that the presence of an electron withdrawing group such as cyano at this position is a positive contribution to the activity.

As seen in the docking poses, letrozole is settled down in the enzyme active site properly. For letrozole, it is seen that benzonitrile ring creates π–π interaction with HEM molecule. Another π–π interaction is observed between 1,2,4-triazole ring and Arg115. Also, this 1,2,4-triazole ring forms two hydrogen bonds via its second and fourth nitrogen atoms with amino groups of Ala438 and Arg115, respectively.

**Figure 1.** Two-dimensional interaction mode of compound **5e** in the enzyme active site (Human aromatase PDB Code: 3EQM).

**Figure 2.** The interacting mode of compound **5e** in the active region of human aromatase. The inhibitor, colored with maroon, and the important residues, colored with purple, in the active site of the enzyme are presented by tube model.

**Figure 3.** Two-dimensional interaction mode of letrozole in the enzyme active site (Human aromatase PDB Code: 3EQM).

**Figure 4.** The interacting mode of letrozole in the active region of human aromatase. The inhibitor, colored with orange, and the important residues, colored with turquoise, in the active site of the enzyme are presented by tube model.

#### *2.5. Theoretical Determination of ADME Properties*

QikProp allows to provide acceptable ranges for comparing the predicted properties of compounds with those of 95% of known drugs and estimate drug-likeness properties. The drug-likeness of a compound was assessed according to Jorgensen's rule of three [27], which regards PCaco (>22 nm/s), logS (>−5.7), primary metabolites (PM) (<7), and Lipinski's rule of five [28], which considers number of hydrogen bond acceptors (≤10) and donors (≤5), molecular weight (<500 Da), and octanol/water partition coefficient (≤5).

Table 4. presents the predicted ADME properties of all compounds. According to Lipinski's rule of five and Jorgensen's rule of three, all compounds (**5a**–**5p**) are in accordance with the rule by causing no more than one violation. Consequently, according to predictions of ADME properties, it can be suggested that the active compounds may have a good pharmacokinetic profile.



<25 poor, >500 great) **PM:** Number of likely metabolic reactions

(active)) **%HOA:** Predicted human oral absorption percent 500, logP < 5, DHB ≤ 5, AHB≤ 10, Positive PSA value. **VRT:** Number of violations of Jorgensen's rule of three. The three rules are: logS > -5.7, PCaco > 22 nm/s, PM < 7.

(recommended

(recommended

 value: >80% is high, <25% is poor) **VRF:** Number of violations of Lipinski's rule of five. The rules are: MW <

 value: 1-8) **CNS:** Predicted central nervous system activity

(recommended

 value: –2 (inactive), +2

#### **3. Materials and Methods**

#### *3.1. Chemistry*

Whole chemicals employed in the synthetic procedure were purchased from Sigma-Aldrich Chemicals (Sigma-Aldrich Corp., St. Louis, MO, USA) or Merck Chemicals (Merck KGaA, Darmstadt, Germany). Melting points of the obtained compounds were determined by MP90 digital melting point apparatus (Mettler Toledo, OH, USA) and were uncorrected. Microwave syntheses were realized by using a Monowave 300 high-performance microwave reactor (Anton-Paar, Austria). 1H NMR and 13C NMR spectra of the synthesized compounds were registered by a Bruker 300 MHz and 75 MHz digital FT-NMR spectrometer (Bruker Bioscience, Billerica, MA, USA) in DMSO-*d6*, respectively. Splitting patterns were designated as follows: s: singlet; d: doublet; t: triplet; m: multiplet in the NMR spectra. Coupling constants (*J*) were reported as Hertz. M+1 peaks were determined by Shimadzu LC/MS ITTOF system (Shimadzu, Tokyo, Japan). All reactions were monitored by thin-layer chromatography (TLC) using Silica Gel 60 F254 TLC plates (Merck KGaA, Darmstadt, Germany).

3.1.1. Synthesis of 4-(5-substitüe-1H-benz[d]imidazol-2-yl)benzoic Acid Methyl Ester (**1a**,**1b**)

Yields: 82–78%. 4-(5-substitüe-1*H*-benz[*d*]imidazol-2-yl)benzoic acid hydrazide derivatives (**2a**,**2b**) (yields: 76–72%) and 5-[4-(5(6)-substituted-1*H*-benz[*d*]imidazol-2-yl)phenyl]-1,3,4-oxadiazole-2-thiol derivatives (**3a**,**3b**) (yields: 78–7 %) were prepared following the reported procedures [23].

3.1.2. 4-Amino-5-(4-(5-substitüe-1H-benzo[d]imidazol-2-yl)phenyl)-4H-1,2,4-triazole-3-thiol (**4a**,**4b**)

Compound **3a** or **3b** (0.02 mol) was dissolved in ethanol. Hydrazine hydrate (5 mL) was added to the mixture. The reaction mixture was heated at 240 ◦C and 10 bar for 10 min under microwave synthesis reactor (Anton-Paar Monowave 300). After the reaction ended, the product was washed with water, dried, and crystallized using ethanol (96%) [22]. Yields: 68–64%.

3.1.3. 3-(4-(5-substitüe-1H-benzo[d]imidazol-2-yl)phenyl)-6-(substitüephenyl)-7H-[1,2,4]triazole [3,4-b][1,3,4]thiadiazine (**5a**–**5p**)

Compound **4a** or **4b** (0.003 mol) and appropriate phenacyl bromide (0.003 mol) in ethanol was heated under reflux for 8 h. After the reaction ended, the product was filtered and dried [22].

3-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-6-phenyl-7H-[1,2,4]triazole[3,4-b][1,3,4] thiadiazine (5a): Yields: 77%. Mp 230.7–232.9 ◦C. 1H-NMR (300 MHz, DMSO-*d6*): δ = 4.52 (2H, s, -CH2-), 7.52-7.55 (2H, m, aromatic CH), 7.59–7.67 (3H, m, Aromatic CH), 7.83–7.86 (2H, m, aromatic CH), 8.06–8.09 (2H, m, aromatic CH), 8.34 (2H, d, *J*=8.67 Hz, 1,4-disubstitutedbenzene), 8.43 (2H, d, *J*=8.64 Hz, 1,4-disubstitutedbenzene). 13C-NMR (75 MHz, DMSO-d6): δ = 23.31, 114.89, 125.84, 126.55, 128.13, 128.50, 129.13, 129.65, 132.64, 133.78, 134.17, 143.96, 149.21, 151.22, 156.82, 163.07. HRMS (*m*/*z*): [M + H]<sup>+</sup> calcd for C23H16N6S: 409.1224; found: 409.1230.

3-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-6-(4-chlorophenyl)-7H-[1,2,4]triazole[3,4-b][1,3,4] thiadiazine (**5b**): Yields: 72%. Mp 190.9–192.6 ◦C. 1H-NMR (300 MHz, DMSO-*d6*): δ = 4.50 (2H, s, -CH2-), 7.56–7.59 (2H, m, aromatic CH), 7.65–7.70 (3H, m, aromatic CH), 7.86–7.89 (2H, m, aromatic CH), 8.06–8.09 (2H, m, aromatic CH), 8.31 (2H, d, *J*=8.61 Hz, 1,4-disubstitutedbenzene), 8.57 (2H, d, *J*=8.61 Hz, 1,4- Disubstitutedbenzene). 13C-NMR (75 MHz, DMSO-*d6*): δ=23.27, 114.67, 125.49, 126.35, 128.87, 129.11, 129.32, 129.45, 129.73, 129.92, 130.11, 130.71, 132.62, 133.01, 137.50, 144.01, 148.52, 151.17, 156.87. HRMS (*m*/*z*): [M + H]<sup>+</sup> calcd for C23H15N6SCl: 443.0843; found: 443.0840.

3-(4-(1*H*-benzo[*d*]imidazol-2-yl)phenyl)-6-(4-fluorophenyl)-7H-[1,2,4]triazole[3,4-b][1,3,4] thiadiazine (**5c**): Yields: 69%. Mp 274.2–277.1 ◦C. 1H-NMR (300 MHz, DMSO-*d6*): δ = 4.50 (2H, s, -CH2-), 7.12 (1H, s, aromatic CH), 7.29 (1H, s, aromatic CH), 7.46–7.52 (4H, m, aromatic CH), 7.81–7.84 (2H, m, aromatic CH), 8.33 (2H, d, *J* = 8.64 Hz, 1,4-disubstitutedbenzene), 8.51 (2H, d, J=8.76 Hz, 1,4-disubstitutedbenzene). 13C-NMR (75 MHz, DMSO-*d6*): δ = 23.27, 114.32, 114.90, 116.76 (d, *J* = 21.98 Hz), 126.40, 127.68, 127.96, 128.43, 128.56, 128.69, 128.87, 128.95, 130.32 (d, *J* = 2.77 Hz), 130.78 (d, *J* = 8.43 Hz), 134.60, 143.66, 151.30, 155.71, 166.36 (d, *J* = 249.53 Hz). HRMS (*m*/*z*): [M + H]<sup>+</sup> calcd for C23H15N6FS: 427.1137; found: 427.1136.

3-(4-(1*H*-benzo[*d*]imidazol-2-yl)phenyl)-6-(4-methylphenyl)-7H-[1,2,4]triazole[3,4-b][1,3,4] thiadiazine (**5d**): Yields: 70%. Mp 207.5–209.1 ◦C. 1H-NMR (300 MHz, DMSO-*d6*): δ = 2.41 (3H, s, CH3), 4.49 (2H, s, -CH2-), 7.41 (2H, d, *J* = 8.13 Hz, 1,4-disubstitutedbenzene), 7.52–7.55 (2H, m, benzimidazole CH), 7.83–7.86 (2H, m, benzimidazole CH), 7.97 (2H, d, *J* = 8.25 Hz, 1,4-disubstitutedbenzene), 8.33 (2H, d, *J* = 8.58 Hz, 1,4-disubstitutedbenzene), 8.43 (2H, d, *J* = 8.61 Hz, 1,4-disubstitutedbenzene). 13C-NMR (75 MHz, DMSO-*d6*): δ=21.55, 23.18, 114.86, 125.84, 126.42, 128.08, 128.24, 128.47, 129.02, 129.22, 129.71, 130.20, 130.90, 134.11, 142.96, 144.00, 147.35, 149.13, 151.07, 156.69. HRMS (*m*/*z*): [M + H]<sup>+</sup> calcd for C24H18N6S: 423.1381; found: 423.1386.

3-(4-(1*H*-benzo[*d*]imidazol-2-yl)phenyl)-6-(4-cyanophenyl)-7H-[1,2,4]triazole[3,4-b][1,3,4] thiadiazine (**5e**): Yields: 68%. Mp 259.3–263.1 ◦C. 1H-NMR (300 MHz, DMSO-*d6*): δ = 4.53 (2H, s, -CH2-), 7.56–7.58 (2H, m, benzimidazole CH), 7.85–7.88 (2H, m, benzimidazole CH), 8.06 (2H, d, *J* = 8.37 Hz, 1,4-disubstitutedbenzene), 8.21 (2H, d, *J* = 8.46 Hz, 1,4-disubstitutedbenzene), 8.30 (2H, d, *J* = 8.49 Hz, 1,4-disubstitutedbenzene), 8.42 (2H, d, *J* = 8.49 Hz, 1,4-disubstitutedbenzene). 13C-NMR (75 MHz, DMSO-*d6*): δ = 23.35, 114.58, 114.71, 118.68, 125.51, 126.39, 127.20, 128.48, 128.74, 128.87, 129.19, 129.43, 129.84, 133.03, 133.44, 137.97, 143.97, 148.72, 151.25, 155.45. HRMS (*m*/*z*): [M + H]<sup>+</sup> calcd for C24H15N7S: 434.1186; found: 434.1182.

3-(4-(1*H*-benzo[*d*]imidazol-2-yl)phenyl)-6-(4-bromophenyl)-7H-[1,2,4]triazole[3,4-b][1,3,4] thiadiazine (**5f**): Yields: 74%. Mp 284.8–286.2 ◦C. 1H-NMR (300 MHz, DMSO-*d6*): δ = 4.50 (2H, s, -CH2-), 7.53–7.56 (3H, m, aromatic CH), 7.81–7.85 (3H, m, aromatic CH), 7.99–8.02 (2H, m, aromatic CH), 8.32 (2H, d, *J* = 8.64 Hz, 1,4-disubstitutedbenzene), 8.42 (2H, d, *J* = 8.70 Hz, 1,4-disubstitutedbenzene). 13C-NMR (75 MHz, DMSO-*d6*): δ = 23.20, 114.91, 125.04, 125.96, 126.50, 127.03, 128.56, 129.19, 130.09, 130.90, 132.37, 132.67, 133.00, 142.94, 143.96, 147.34, 149.17, 151.22, 155.95. HRMS (*m*/*z*): [M + H]<sup>+</sup> calcd for C23H15N6SBr: 487.0311; found: 487.0335.

3-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-6-(2,4-difluorohenyl)-7H-[1,2,4]triazole [3,4-b][1,3,4] thiadiazine (**5g**): Yields: 72%. Mp 208.5–210.7 ◦C. 1H-NMR (300 MHz, DMSO-*d6*): δ = 4.42 (2H, s, -CH2-), 7.46–7.49 (4H, m, aromatic CH), 7.79–7.82 (3H, m, aromatic CH), 7.29–7.35 (2H, m, aromatic CH), 8.45–8.51 (2H, m, aromatic CH). 13C-NMR (75 MHz, DMSO-*d6*): δ = 25.37, 113.01 (dd, *J1* = 3.08 Hz, *J2* = 21.45 Hz), 106.08 (d, *J* = 26.01 Hz), 114.05, 114.45, 119.72 (dd, *J1* = 3.02 Hz, *J2* = 11.51 Hz), 126.56, 127.17, 128.25, 128.75, 129.15, 132.37, 132.67 (dd, *J1* = 3.12 Hz, *J2* = 10.36 Hz), 134.23, 135.55, 143.74, 148.22, 151.06, 153.70, 164.66 (d, *J* = 251.18 Hz), 164.83 (d, *J* = 250.91 Hz). HRMS (*m*/*z*): [M + H]<sup>+</sup> calcd for C23H14N6SF2: 445.1053; found: 445.1041.

3-(4-(1*H*-benzo[*d*]imidazol-2-yl)phenyl)-6-(2,4-dichlorophenyl)-7H-[1,2,4]triazole[3,4-b] [1,3,4] thiadiazine (**5h**): Yields: 77%. Mp 190.7–193.4 ◦C. 1H-NMR (300 MHz, DMSO-*d6*): δ = 4.36 (2H, s, -CH2-), 7.34–7.37 (3H, m, aromatic CH), 7.65 (1H, dd, *J1* = 2.04 Hz, *J2* = 8.34 Hz, aromatic CH), 7.70–7.73 (2H, m, aromatic CH), 7.78–7.81 (1H, m, aromatic CH), 8.20 (2H, d, *J* = 8.64 Hz, 1,4-disubstitutedbenzene), 8.40 (2H, d, *J* = 8.61 Hz, 1,4-disubstitutedbenzene). 13C-NMR (75 MHz, DMSO-*d6*): δ = 26.23, 115.05, 115.32, 124.22, 124.72, 126.99, 127.78, 128.08, 128.29, 128.57, 128.96, 129.59, 130.38, 132.93, 133.05, 133.74, 136.86, 143.65, 149.92, 151.62, 156.53. HRMS (*m*/*z*): [M + H]<sup>+</sup> calcd for C23H14N6SCl2: 477.0457; found: 477.0450.

3-(4-(5-chloro-1*H*-benzo[*d*]imidazol-2-yl)phenyl)-6-phenyl-7H-[1,2,4]triazole[3,4-b][1,3,4] thiadiazine (**5i**): Yields: 80%. Mp: 247.3–249.6 ◦C. 1H-NMR (300 MHz, DMSO-*d6*): δ = 4.50 (2H, s, -CH2-), 7.49 (1H, dd, *J1* = 1.89 Hz, *J2*=8.67 Hz, aromatic CH), 7.59–7.62 (3H, m, aromatic CH), 7.78–7.84 (2H, m, aromatic

CH), 8.03–8.06 (2H, m, aromatic CH), 8.27 (2H, d, *J* = 8.64 Hz, 1,4-disubstitutedbenzene), 8.37 (2H, d, *J* = 8.64 Hz, 1,4-disubstitutedbenzene). 13C-NMR (75 MHz, DMSO-*d6*): δ = 23.32, 114.60, 116.29, 125.95, 126.40, 128.11, 128.47, 128.97, 129.20, 129.63, 129.94, 132.62, 133.27, 133.71, 135.39, 143.97, 150.42, 151.04, 156.76. HRMS (*m*/*z*): [M + H]<sup>+</sup> calcd for C23H15N6SCl: 443.0826; found: 443.0840.

3-(4-(5-chloro-1*H*-benzo[*d*]imidazol-2-yl)phenyl)-6-(4-chlorophenyl)-7H-[1,2,4]triazole [3,4-b][1,3,4] thiadiazine (**5j**): Yields: 70%. Mp: 226.0–228.9 ◦C. 1H-NMR (300 MHz, DMSO-*d6*): δ = 4.46 (2H, s, -CH2-), 7.33–7.37 (1H, m, benzimidazole CH), 7.63 (2H, d, *J* = 8.61 Hz, 1,4-disubstitutedbenzene), 7.68–7.73 (2H, m, benzimidazole CH), 8.04 (2H, d, *J* = 8.61 Hz, 1,4-disubstitutedbenzene), 8.19 (2H, d, *J* = 8.46 Hz, 1,4-disubstitutedbenzene), 8.39 (2H, d, *J* = 8.43 Hz, 1,4-disubstitutedbenzene). 13C-NMR (75 MHz, DMSO-*d6*): δ = 23.13, 115.14, 116.51, 124.18, 127.79, 128.20, 128.33, 128.83, 128.98, 129.19, 129.68, 129.87, 130.51, 131.57, 132.60, 137.42, 143.58, 151.40, 155.59. HRMS (*m*/*z*): [M + H]<sup>+</sup> calcd for C23H14N6SCl2: 477.0427; found: 477.0450.

3-(4-(5-chloro-1*H*-benzo[*d*]imidazol-2-yl)phenyl)-6-(4-fluorophenyl)-7H-[1,2,4]triazole [3,4-b][1,3,4] thiadiazine (**5k**): Yields: 71%. Mp: 249.1–250.9 ◦C. 1H-NMR (300 MHz, DMSO-*d6*): δ = 4.48 (2H, s, -CH2-), 7.38 (1H, dd, *J1* = 1.98 Hz, *J2* = 8.58 Hz, aromatic CH), 7.46–7.48 (2H, m, aromatic CH), 7.71–7.74 (1H, m, aromatic CH), 7.77 (1H, s, aromatic CH), 8.12–8.14 (2H, m, aromatic CH), 8.25 (2H, d, *J* = 8.64 Hz, 1,4-disubstitutedbenzene), 8.40 (2H, d, *J* = 8.61 Hz, 1,4-disubstitutedbenzene). 13C-NMR (75 MHz, DMSO-*d6*): δ = 23.45, 114.95, 116.45, 116.77 (d, *J* = 22.09 Hz), 124.83, 128.07, 128.21, 128.39, 128.84, 128.89, 130.30 (d, *J* = 2.99 Hz), 130.85 (d, *J* = 8.93 Hz), 135.26, 137.58, 143.69, 150.99, 151.27, 155.73, 164.82 (d, *J* = 249.42 Hz). HRMS (*m*/*z*): [M + H]<sup>+</sup> calcd for C23H14N6FSCl: 461.0741; found: 461.0746.

3-(4-(5-chloro-1*H*-benzo[*d*]imidazol-2-yl)phenyl)-6-(4-methylphenyl)-7H-[1,2,4]triazole [3,4-b][1,3,4] thiadiazine (**5l**): Yields: 65%. Mp: 250.7–252.3 ◦C. 1H-NMR (300 MHz, DMSO-*d6*): δ = 4.46 (2H, s, -CH2-), 7.40 (2H, d, *J* = 8.07 Hz, 1,4-disubstitutedbenzene), 7.45 (1H, dd, *J1* = 1.95 Hz, *J2* = 8.67 Hz, benzimidazole CH), 7.76–7.79 (1H, m, benzimidazole CH), 7.82–7.83 (1H, m, benzimidazole CH), 7.95 (2H, d, *J* = 8.31 Hz, 1,4-disubstitutedbenzene), 8.28 (2H, d, *J* = 8.67 Hz, 1,4-disubstitutedbenzene), 8.37 (2H, d, *J* = 8.67 Hz, 1,4-disubstitutedbenzene). 13C-NMR (75 MHz, DMSO-*d6*): δ = 21.54, 23.15, 114.88, 116.44, 125.26, 127.74, 128.08, 128.21, 128.95, 129.24, 129.72, 130.21, 130.90, 134.62, 136.89, 142.95, 143.90, 150.99, 151.15, 156.66. HRMS (*m*/*z*): [M + H]<sup>+</sup> calcd for C24H17N6SCl: 457.0994; found: 457.0997.

3-(4-(5-chloro-1*H*-benzo[*d*]imidazol-2-yl)phenyl)-6-(4-cyanophenyl)-7H-[1,2,4]triazole [3,4-b][1,3,4] thiadiazine (**5m**): Yields: 62%. Mp: 254.0–255.7 ◦C. 1H-NMR (300 MHz, DMSO-*d6*): δ = 4.53 (2H, s, -CH2-), 7.50 (1H, dd, *J1* = 1.95 Hz, *J2* = 8.70 Hz, benzimidazole CH), 7.85–7.86 (1H, m, aromatic CH), 8.06–8.08 (3H, m, aromatic CH), 8.21 (2H, d, *J* = 8.61 Hz, 1,4-disubstitutedbenzene), 8.26 (2H, d, *J* = 8.64 Hz, 1,4-disubstitutedbenzene), 8.38 (2H, d, *J* = 8.64 Hz, 1,4-disubstitutedbenzene). 13C-NMR (75 MHz, DMSO-*d6*): δ = 23.30, 114.57, 114.71, 118.70, 125.83, 126.85, 128.48, 128.86, 129.11, 129.37, 129.81, 133.46, 133.58, 135.73, 137.98, 143.88, 150.56, 151.33, 155.41. HRMS (*m*/*z*): [M + H]<sup>+</sup> calcd for C24H14N7SCl: 468.0782; found: 468.0793.

3-(4-(5-chloro-1*H*-benzo[*d*]imidazol-2-yl)phenyl)-6-(4-bromophenyl)-7H-[1,2,4]triazole [3,4-b][1,3,4] thiadiazine (**5n**): Yields: 76%. Mp: 243.4–247.3 ◦C. 1H-NMR (300 MHz, DMSO-*d6*): δ = 4.48 (2H, s, -CH2-), 7.43 (1H, dd, *J1* = 1.98 Hz, *J2* = 8.67 Hz, benzimidazole CH), 7.75 (1H, s, aromatic CH), 7.80–7.82 (3H, m, aromatic CH), 7.99 (2H, d, *J* = 8.70 Hz, 1,4-disubstitutedbenzene), 8.26 (2H, d, *J* = 8.58 Hz, 1,4-disubstitutedbenzene), 8.37 (2H, d, *J* = 8.64 Hz, 1,4-disubstitutedbenzene). 13C-NMR (75 MHz, DMSO-*d6*): δ=23.14, 115.01, 116.52, 124.92, 126.45, 128.10, 129.05, 130.07, 131.76, 132.00, 132.15, 132.67, 133.00, 141.45, 143.77, 148.89, 149.69, 151.39, 155.91. HRMS (*m*/*z*): [M + H]<sup>+</sup> calcd for C23H14N6SClBr: 520.9930; found: 520.9945.

3-(4-(5-chloro-1*H*-benzo[*d*]imidazol-2-yl)phenyl)-6-(2,4-difluorohenyl)-7H-[1,2,4]triazole [3,4-b][1,3,4] thiadiazine (**5o**): Yields: 66%. Mp: 182.2–185.0 ◦C. 1H-NMR (300 MHz, DMSO-*d6*): δ = 4.39 (2H, s, -CH2-), 7.28–7.34 (1H, m, aromatic CH), 7.42–7.48 (1H, m, aromatic CH), 7.48–7.52 (2H, m, aromatic CH), 7.77–7.83 (2H, m, aromatic CH), 7.93–8.01 (1H, m, aromatic CH), 8.25 (2H, d, *J* = 8.64 Hz, 1,4-disubstitutedbenzene), 8.44 (2H, d, *J* = 8.58 Hz, 1,4-disubstitutedbenzene). 13C-NMR (75 MHz, DMSO-*d6*): δ = 25.31, 105.76 (d, *J* = 26.01 Hz), 106.28, 113.19 (dd, *J1* = 2.63 Hz, *J2* = 20.86 Hz), 114.74, 116.39, 119.76 (dd, *J*<sup>1</sup> = 3.73 Hz, *J2* = 7.83 Hz), 125.45, 127.49, 128.32, 128.94, 129.14, 129.40, 132.71 (dd, *J1* = 3.21 Hz, *J2* = 9.80 Hz), 134.22, 143.80, 151.60, 151.26, 153.78, 161.28 (d, *J* = 252.62 Hz), 164.46 (d, *J* = 256.22 Hz). HRMS (*m*/*z*): [M + H]<sup>+</sup> calcd for C23H13N6F2SCl: 479.0632; found: 479.0652.

3-(4-(5-chloro-1*H*-benzo[*d*]imidazol-2-yl)phenyl)-6-(2,4-dichlorophenyl)-7H-[1,2,4]triazole [3,4-b][1,3,4] thiadiazine (**5p**): Yields: 68%. Mp: 114.4–116.5 ◦C. 1H-NMR (300 MHz, DMSO-*d6*): δ = 4.35 (2H, s, -CH2-), 7.25 (1H, dd, *J1* = 1.74 Hz, *J2* = 8.52 Hz, aromatic CH), 7.65 (2H, dd, *J1* = 2.07 Hz, *J2* = 8.34 Hz, aromatic CH), 7.88 (2H, d, *J* = 8.34 Hz, aromatic CH), 7.90 (1H, m, aromatic CH), 8.16 (2H, d, *J* = 8.61 Hz, 1,4-disubstitutedbenzene), 8.32 (2H, d, *J* = 8.58 Hz, 1,4-disubstitutedbenzene). 13C-NMR (75 MHz, DMSO-*d6*): δ = 26.33, 106.76, 125.55, 127.29, 127.35, 128.58, 128.90, 129.64, 130.37, 131.63, 132.35, 132.96, 133.06, 133.44, 133.77, 136.56, 136.84, 143.51, 149.25, 151.77, 156.48. HRMS (*m*/*z*): [M + H]<sup>+</sup> calcd for C23H13N6SCl3: 511.0037; found: 511.0061.

#### *3.2. Cytotoxicity Assay*

The anticancer activity of compounds **5a**–**5p** were screened according to the MTT assays. The MTT assays were performed as previously described [24,25]. Cisplatin was used as the reference drug for the MCF7 cell line in the MTT assays.

#### *3.3. Aromatase Inhibition Assay*

This method was carried out according to the kit procedure (Bio Vision, Aromatase (CYP19A) Inhibitor Screening Kit (Fluorometric)). The compounds were dissolved in dimethyl sulfoxide (DMSO) and added to the assay in at least seven concentrations ranging from 10<sup>−</sup>3–10−<sup>9</sup> M. The recombinant human aromatase stock was prepared by reconstituting with 1 mL of aromatase assay buffer. The contents were mixed thoroughly by vortexing to obtain a homogeneous solution and the solution was transferred to a 15-mL conical tube. The volume was brought to 2450 μL with the aromatase assay buffer and 50 μl of NADPH production system (100X) was added for a final total volume of 2.5 mL. Letrozole was used as a positive inhibition control. For solvent control, a small aliquot of aromatase assay buffer containing the organic solvent was used to dissolve the test compounds that were prepared. Reaction wells containing test compounds and the corresponding no inhibitor controls (which may also serve as a solvent control), as well as a background control (containing no fluorogenic Aromatase Substrate) were prepared. The plate was incubated for at least 10 min at 37 ◦C to allow test ligands to interact with the aromatase. After incubation, 30 μl of the aromatase substrate/NADP+ mixture was added to each well. Immediately (within 1 min), the fluorescence at Ex/Em = 488/527 nm was measured.

#### *3.4. Molecular Docking*

A structure-based in silico procedure was applied to discover the binding modes of compound 5e to human aromatase enzyme active site. The crystal structures of human aromatase (PDB ID: 3EQM) [26] was retrieved from the Protein Data Bank server (www.pdb.org).

The structures of ligands were built using the Schrödinger Maestro [29] interface and then were submitted to the Protein Preparation Wizard protocol of the Schrödinger Suite 2016 Update 2 [30]. The ligands were prepared by the LigPrep 3.8 [31] to assign the protonation states at pH 7.4 ± 1.0 and the atom types, correctly. Bond orders were assigned, and hydrogen atoms were added to the structures. The grid generation was formed using Glide 7.1 [32]. Flexible docking runs were performed with single precision docking mode (SP).

#### *3.5. Theoretical Determination of ADME Properties*

Physicochemical parameters of obtained compounds (**5a**–**5p**) were evaluated by using QikProp 4.8 [33].

#### **4. Conclusions**

Inhibition of aromatase has proved to be an effective approach for the treatment of hormone-dependent breast cancer in the postmenopausal women. Imidazole and triazole groups are important rings for the development new potent aromatase inhibitors with high affinity for the enzyme. A series of benzimidazole-triazolothiadiazine derivatives have been synthesized with different substituents at benzimidazole and phenyl rings. The newly synthesized compounds were tested for their anti-cancer properties against human breast cancer cell line (MCF-7). The synthesized compounds were then tested in in vitro aromatase assay and two compounds (**5e** and **5m**) exhibited activity similar to letrozole.

**Supplementary Materials:** The following are available online.

**Author Contributions:** Z.A.K., Y.Ö., conceived and designed the experiments; D.O., U.A.Ç. performed the synthesis; S.L. performed analysis studies; B.N.S., U.A.Ç., and S.I. performed activity tests; D.O., U.A.Ç., B.N.S., B.K.Ç. wrote the paper. All authors have read and agreed to the published version of the manuscript.

**Acknowledgments:** This study was financially supported by Anadolu University Scientific Projects Fund, Project No: 1905S032.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Sample Availability:** Samples of the compounds **5a**–**5p** are available from the authors.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **Synthesis, Characterization and Biological Evaluation of Novel Dihydropyranoindoles Improving the Anticancer E**ff**ects of HDAC Inhibitors**

**Murat Bingul 1,2,3, Greg M. Arndt 2,4, Glenn M. Marshall 2,5, Belamy B. Cheung 2,6,\*, Naresh Kumar 1,\* and David StC. Black 1,\***


Academic Editor: Qiao-Hong Chen Received: 28 February 2020; Accepted: 16 March 2020; Published: 18 March 2020

**Abstract:** The dihydropyranoindole scaffold was identified as a promising target for improving the anti-cancer activity of HDAC inhibitors from the preliminary screening of a library of compounds. A suitable methodology has been developed for the preparation of novel dihydropyranoindoles via the Hemetsberger indole synthesis using azido-phenylacrylates, derived from the reaction of corresponding alkynyl-benzaldehydes with methyl azidoacetate, followed by thermal cyclization in high boiling solvents. Anti-cancer activity of all the newly synthesized compounds was evaluated against the SH-SY5Y and Kelly neuroblastoma cells as well as the MDA-MB-231 and MCF-7 breast adenocarcinoma cell lines. Biological studies showed that the tetracyclic systems had significant cytotoxic activity at higher concentration against the neuroblastoma cancer cells. More importantly, these systems, at the lower concentration, considerably enhanced the SAHA toxicity. In addition to that, the toxicity of designated systems on the healthy human cells was found to be significantly less than the cancer cells.

**Keywords:** dihydropyranoindole; anticancer; HDAC inhibitors; neuroblastoma; breast cancer

#### **1. Introduction**

Chemotherapy is one of the most widely used treatments for high-risk cancer patients [1,2]. A range of well-known agents, namely Doxorubicin, Cyclophosphamide, Etoposide have been used to combat various cancers in modern chemotherapeutic therapy [3,4]. However, a major problem faced in chemotherapy is resistance to commonly-used anti-cancer drugs [5]. Therefore, development of novel anti-cancer chemotherapeutic agents is of utmost importance in the area of drug discovery and development.

The histone deacetylase (HDAC) inhibitors are a class of chemotherapeutic agents that hold promise in cancer therapy [6,7]. HDAC inhibitors have been reported to suppress cell proliferation and angiogenesis, induce cell differentiation and promote apoptosis in a number of cancer cell types [8,9]. Suberoylanilide hydroxamic acid (SAHA) is the first HDAC inhibitor to obtain meet FDA approval [10],

and has been considered to be a highly promising anticancer therapeutic agent due to its potent cytotoxic effect on a number of tumor cell types as well as low toxicity towards healthy normal cells [11–13]. However, single agent treatment with SAHA has been found to be ineffective against several cancers [14,15]. On the other hand, the combination of SAHA with other chemotherapeutic agents with different mechanisms of action has been considered to be more promising as the drug resistance caused by single agent therapies may be overcome [14,16]. Due to the significant cytotoxic effects observed during clinical trials of SAHA in combination with a variety of chemotherapeutic agents, many synthetic compounds have been produced and screened to identify molecules capable of enhancing the cytotoxic activity of SAHA, while producing fewer side effects.

In order to provide a basis for this study, a subset (10,560 compounds) of the Walter & Eliza Hall Institute (WEHI) compound library was screened to identify molecules that can act synergistically with a clinical dose of SAHA to overcome drug resistance in SAHA-resistant MDA-MB-231 breast cancer cell lines [17]. The compounds that reduced viability to <40% in the presence of SAHA but to >70% in the absence of SAHA, with a difference of at least 55% between the two conditions have been identified as hit molecules for their SAHA enhancing capability [17]. A structural analysis of the hit molecules demonstrated that the main structural feature to be the presence of 5- or 6-membered fused heterocyclic systems, most commonly containing one or more nitrogen atoms. Furthermore, these fused systems typically involved 3 or 4 conjoined rings. Due to the ongoing experiments regarding the hit molecules, the structures of these compounds are not discussed in this manuscript.

#### *Identification of New Target Molecules*

The starting point of the current work was the selection of a range of compounds exhibiting structural similarity to the hits from compound library in order to expand the structural diversity of the screening set. The six related monomeric dihydropyranoindoles **1**–**3** and dimeric furoquinolines **4**–**6** were selected in order to understand the effect of the indole and quinoline structures, as well as the fused dihydropyran and furan rings, on biological activities (Figure 1). The molecules were tested against SH-SY5Y neuroblastoma and MDA-MB-231 breast cancer cell lines, using the Alamar blue (Resazurin) assay [18] to measure the cell viability. The same screening conditions were used with WEHI screening. Briefly, the designated cells were exposed to 1 μM SAHA, 10 μM compound and the combination of SAHA and compound for 72 h.

**Figure 1.** Six compounds from the NK library.

The in vitro assays revealed that the MDA-MB-231 were the more resistant cell lines than the SH-SY5Y cell lines for the single and combination treatments of all compounds. The compounds **5** and **6**, analogues of quinolines, displayed the lowest reduction on SH-SY5Y cell viability and also no SAHA enhancement was found by the combination with SAHA (Figure S1A, see Supplementary Materials). In the case of compound **6**, the single and combination treatments showed no inhibition of MDA-MB-231 cell viability, while the 20% and 5% of inhibitions were obtained with the treatment of compound **5** alone and combination with 1 μM SAHA (Figure S1B, see Supplementary Materials). The compound **4** was the most active ligand among the quinoline candidates, with 30% inhibition of SH-SY5Y cell viability in the absence of SAHA and 20% additional cytotoxic effect in the presence of SAHA (Figure S1A, see Supplementary Materials). The designated compound displayed 25% reduction on MDA-MB-231 cells, while no SAHA enhancement effect was obtained against the same cell line (Figure S1B, see Supplementary Materials).

Overall, **1**, **3** and **4** were determined as the most promising compounds as SAHA enhancer and it was concluded that the indole heterocyclic systems **1** and **3** were found to be a potential target for the SAHA enhancement with the higher differential values between in the absence and presence of SAHA In order to further validate these compounds **1**, **3** and **4** as viable lead structures, it was essential to determine their toxicity towards normal and non-malignant cells. The most effective ligands were screened against the MRC-5 normal human lung fibroblast cells, as described for cytotoxic assay. This screen showed that the toxicity of **3** was found to be greater than **1** and **4** proposing that specificity of **3** against the normal and cancer cells was not different (Figure S2, see Supplementary Materials). The single treatment of compound **3** showed the cytotoxic activity with the values of 33% and 28% reduction against the SH-SY5Y and MDA-MB-231 cells respectively, while the toxicity of this compound against the MRC-5 cells was 26% and 30% greater than the SH-SY5Y and MDA-MB-231 cancer cells. However, compounds **1** and **4** displayed non-toxic behavior on the MRC-5 healthy human cells with values of 103% and 98% viable cells.

Based on the results of screening against the cancer cells and toxicity study on normal cells, the indole heterocyclic system **1** was identified as promising leads for further development on the enhancement of SAHA activity and compound **1** was also determined to be non-toxic across the healthy human cells. The main work described in this manuscript focused on the synthesis, characterization and in vitro biological evaluation of a series of targeted compounds based on tricyclic and tetracyclic dihydropyrano derivatives. The effectiveness of the novel compounds as single agents and in combination with a clinical dose of SAHA was determined against neuroblastoma and breast cancer cells.

#### **2. Results and Discussion**

The preparation of dihydropyranoindole systems was achieved by two synthetic methods, and the generality of these pathways were discussed in this paper. 3,4-Dihydroxybenzaldehyde **7** has been given as an example for the representation of two possible methods which could be used for the preparation of the desired pyranoindole methyl 5,10-dihydro-7*H*-dipyrano [3,2-*e*:2 ,3 -*g*]indole-6-carboxylate **10** (Figure 2). The first method was to prepare the compound **10** via methyl hydroxyindole-2-carboxylate **8** which could be prepared by the Hemetsberger indole synthesis [19]. Methyl hydroxyindole-2-carboxylate **8** on reacting with haloalkynes would give the alkyne indole-ether **9** which upon Claisen cyclization would afford the desired dihydropyranoindole **10**. As an alternative pathway, aryl ether benzaldehyde **11** would be prepared via simple alkylation of phenol **7** with haloalkynes and the Hemetsberger indole synthesis would then be applied to build the indole moiety. It is anticipated that thermal cyclization would yield the desired compound **10** in one step.

**Figure 2.** Two methods for the preparation of pyranoindole systems.

#### *2.1. Preparation of Tetracyclic Dihydropyranoindole 10 Via (Method 1)*

In the first synthetic method, methyl 5,6-dihydroxyindole-2-carboxylate **8** was generated via the Hemetsberger indole synthesis by benzyl-protected benzaldehydes and the subsequent deprotection afforded the corresponding hydroxyindoles. The 3,4-dihydroxybenzaldehyde **7** was reacted with benzyl bromide in the presence of potassium carbonate in acetone to afford the protected carbaldehydes **12** [20] in 88% yield (Scheme 1). Treatment of 3,4-dibenzyloxy benzaldehyde **12** with methyl azidoacetate in methanol, in the presence of strong basic environment (sodium methoxide) gave the vinyl azido intermediate **13** in 59% yield. Thermal decomposition of the arylazido **13** was performed by heating at reflux in xylene, generating the methyl 5,6-dibenzyloxyindole-2-carboxylate **14** in 71% yield. Hydrogenolysis of the benzyl group was carried out by treating the compound **14** with 5% *w*/*w* palladium on carbon under hydrogen atmosphere at room temperature for 2 h, to yield the desired methyl 5,6-dihydroxyindole-2-carboxylate **8** [21] in 77% yield. The dihydroxyindole **8** was reacted with propargyl bromide in the presence of potassium carbonate in acetone (Scheme 1). The desired dipropargyloxyindole intermediate **9** was prepared in 61% yield. The Claisen cyclization of **9** was explored in xylene, 1,2-dichlorobenzene and toluene. It was found that heating at reflux in chlorobenzene gave the optimum result in terms of the completion of the reaction as well as the yield and purity of the product. Using this approach, the desired tetracyclic dihydropyranoindole **10** was isolated in 51% yield.

**Scheme 1.** Reagents and conditions: (i) Benzyl bromide, K2CO3, DMF, reflux, overnight; (ii) Methyl azidoacetate, NaOMe, anhyd. MeOH, <−10 ◦C, 4 h; (iii) Xylene, reflux, 2 h, (iv) 5% Pd/C, H2, MeOH/THF, 2h, rt, (v) Propargyl bromide, K2CO3, acetone, reflux, 4 h, 61%, (vi) Chlorobenzene, reflux 2 h, 52%.

The possible reaction mechanism could be explained as in Figure 3. Methyl 5,6-bis(prop-2-yn-1-yloxy)-1*H*-indole-2-carboxylate **9** undergoes an initial Claisen rearrangement to generate intermediate **A**, which subsequently enolizes to produce 5,6-dihydroxyindole derivative (intermediate **B**). A double hydride shift in intermediate **B** gives the keto intermediate **C**, which undergoes an electrocyclic ring closing reaction to form methyl 5,10-dihydro-2*H*-dipyrano[3,2-*e*:2 ,3 -*g*]indole-6-carboxylate **10**.

**Figure 3.** Possible reaction mechanism.

The 1H-NMR spectrum of compound **10** in CDCl3 showed a singlet at 3.96 ppm corresponding to the methyl ester protons, and two doublets of doublets at 4.91 and 4.96 ppm assigned to the two CH2 groups. A multiplet at 5.87–5.97 ppm corresponded to H3 and H9, while another multiplet at 6.72–6.80 ppm corresponded to H4 and H8. Furthermore, H3 appeared as a doublet at 7.19 ppm and the NH proton appeared as a broad singlet at δ 8.85 ppm. The DEPT 135 spectrum of compound **10** confirmed the structure of the molecule, displaying the loss of two CH carbon signals as a result of cyclization of the alkyne group as well as the appearance of two negative signals at 64.68 and 64.72 ppm corresponding to the methylene carbon atoms in the product.

#### *2.2. Alternative Pathway for the Preparation of Tetracyclic Dihydropyranoindole 10 (Method 2)*

The alternative approach began with the reaction of 3,4-dihydroxybenzaldehyde **7** with propargyl bromide under basic conditions (potassium carbonate), which afforded the propargyloxy benzaldehyde **11** [22] in 87% yield. At this point, two synthetic strategies using benzaldehyde **11** as a key intermediate were envisioned (Scheme 2). The first route involved Claisen cyclization to build the dihydropyrano rings **15**, followed by the application of the Hemetsberger indole synthesis to construct the indole scaffold via the unsaturated azido intermediate **16** (Route 1). The cyclization of the aryl ether intermediate **11** was attempted by refluxing in high-boiling solvents such as xylene, toluene, 1,2-dichlorobenzene and chlorobenzene (Scheme 2). In all cases, the novel compound **15** was afforded in low yields, with unreacted starting material being recovered as the major product. The highest yield (35%) was obtained by the use of chlorobenzene. In order to synthesize the desired tetracyclic dihydropyranoindole **10**, the standard Hemetsberger indole synthesis was applied to new intermediate **15**. The condensation of benzaldehyde **15** with methyl azidoacetate was carried out at low temperature (ice-salt bath) in the presence of sodium methoxide. The addition of a small amount of crushed ice to the reaction mixture resulted in the isolation of azido ester **16** as an oily compound which was found to be unstable at room temperature, and was hence used in the next step without further purification. It was postulated that the presence of the dihydropyran ring caused the generation of an unstable azido ester intermediate. Thermal cyclization of the azido ester **16** was performed by heating at reflux in xylene, which afforded the desired dihydropyranoindole **10** in 62% yield.

**Scheme 2.** Reagents and conditions: (a) Propargyl bromide, K2CO3, acetone, reflux, 4 h, (b) Methyl azidoacetate, metallic Na, anhyd. MeOH, <−10 ◦C, 2 h (c) Chlorobenzene, reflux, 2 h, (d) Xylene, reflux, 4 h.

In the second route, it was anticipated that the indole heterocyclic system could be constructed first, followed by the installation of dihydropyrano rings onto the indole moiety. Since the cyclization of both aryl ether and unsaturated azide ester moieties require refluxing conditions in a high-boiling solvent, it was predicted that the indole and dihydropyran rings could be prepared in one step from the key azide intermediate **17** (Route 2). The benzaldehyde **11** was condensed with methyl azidoacetate under strongly basic conditions at low temperature to generate the novel unsaturated stable azide intermediate **17** in 63% yield (Scheme 2). Since xylene was the common solvent for the generation of the indole moiety from the azido ester intermediate, the cyclization reaction was first carried out in refluxing xylene. As anticipated, the thermal cyclization of intermediate **17** resulted in both the decomposition of the azidocinnamate moiety and formation of the indole heterocyclic system, as well as the cyclization of the propargyloxy moiety to furnish the fused pyran ring in a single step. Hence, the desired tetracyclic dihydropyranoindole **10** was obtained in a yield of 72%.

Taken altogether, it was concluded that the first method is a less favorable synthetic pathway consisting of six steps to prepare the desired dihydropyranoindole and also generating the lowest of yield (51%) at the final step. In the case of the second method, route 1 was also found to be an unfavorable synthetic method for further synthesis due to the low-yielding cyclization step to generate the new tricyclic aldehyde **15**, the formation of the unstable azido intermediate **16** from the Hemestberger indole synthesis and the four step pathway for the desired dihydropyranoindole **10**. On the other hand, route 2 of the second strategy was deemed to be the most favorable synthetic route towards dihydropyranoindoles, as it contained three steps to afford the desired dihydropyranoindole and gave the highest yield of 72%. Thus, route 2 of the second method was chosen for further studies in this study.

#### *2.3. The Preparation of Monomeric Hydropyrano System*

The synthetic strategy (Route 2) was extended to the synthesis of indole systems containing a single fused dihydropyran ring. Hence, 4-hydroxy-3-methoxy benzaldehyde **18** was reacted with propargyl bromide in the presence of potassium carbonate to give aryl ether **19a** [23] in 84% yield, which was then reacted with methyl azidoacetate under basic conditions to give azidocinnamate **20a** in 76% yield (Scheme 3). Interestingly, thermal cyclization of the **20a** in xylene afforded the corresponding propargyloxy indole **21a** in the yield of 68% instead of the expected dihydropyranoindole system **22a**. Further heating of indole **21a** in refluxing chlorobenzene afforded the desired dihydropyranoindole **22a** in 77% yield.

**Scheme 3.** Reagents and conditions: (a) Propargyl bromide or 3-chloro-3-methylbut-1-yne or 1-bromobut-2-yne, K2CO3, acetone, reflux, 4 h, (b) Methyl azidoacetate, metallic Na, anhyd. MeOH, <−10 ◦C, 2 h (c) Xylene, reflux, 4 h, (d) Chlorobenzene, reflux, 2 h.

The 1H-NMR spectrum of compound **22a** in CDCl3 showed the presence of two singlets at 3.94 ppm and 3.96 ppm corresponding to the methoxy and methyl ester protons, and doublets of doublets at 4.96 ppm corresponding to the CH2 protons. Protons H8 and H9 appeared as multiplets in a range of 5.89–5.95 ppm and 6.73–6.79 ppm respectively. Furthermore, H4 appeared as a singlet at 7.02 ppm, while H3 was appeared as a doublet at 7.13 ppm (*J* = 2.1 Hz) due to its coupling with the NH proton, which resonated as a broad singlet at 8.94 ppm. The DEPT 135 spectrum showed the presence of a negative peak at 65.79 ppm corresponding to a methylene carbon atom. Furthermore, the spectrum revealed the absence of the alkyne CH signal due to cyclization of the alkyne moiety in indole **21a**.

The synthesis of tricyclic dihydropyranoindole systems containing methyl substituents on the fused dihydropyran ring at different positions was also investigated. To achieve this, 4-hydroxy-3-methoxybenzaldehyde **18** was reacted with 1-bromobut-2-yne and 3-chloro-3-methylbut-1-yne in the presence of potassium carbonate to give the aryl ether aldehydes **19b** [24] and **19c** [25] in 92% and 84% yields, respectively (Scheme 3). The aldehydes **19b** and **19c** were treated with methyl azidoacetate in the presence of sodium methoxide to give azidocinnamates **20b** and **20c** in yields of 61% and 62%. Finally, the thermal cyclization of the unsaturated azide **20b** and **20c** in refluxing xylene generated the desired product **22c** in 74% yields, while the indole ether **21b** was isolated in 67% yield. The desired dihydropyranoindole **22b** was obtained in 49% yield, by the further cyclization of **21b** in refluxing chlorobenzene.

In the 1H-NMR spectrum of methyl 5-methoxy-7,7-dimethyl-1,7-dihydropyrano[2,3-*g*]indole-2 carboxylate **22c**,the two methyl groups appeared as a singlet at 1.55 ppm, while H8 and H9 appeared as doublet signals at 5.71 and 6.66 ppm (*J* = 9.7 Hz). Furthermore, H4 appeared as a singlet at 7.01 ppm and H3 appeared as a doublet at 7.13 ppm (*J* = 2.1 Hz), confirming that cyclization occurred at C7. In the 1H-NMR spectrum of the cyclization product **22b**, the olefinic proton on the dihydropyran ring appeared as multiplet signals in the ranges of 5.65–5.68 ppm, due to coupling with the neighboring

CH2 and CH3 groups. For compound **22b**, the O-CH2 protons resonated as a multiplet at 4.77 ppm and the methyl protons of the dihydropyran ring appeared as a quartet at 1.71 ppm.

The same synthetic route was applied to 3-hydroxy-4-methoxy benzaldehyde **23** in order to generate an analogue with the dihydropyran ring fused at a different position. Thus, hydroxybenzaldehyde **23** was reacted with propargyl bromide in the presence of potassium carbonate to generate the intermediate ether **24a** [26] in 87% yield, which upon reaction with methyl azidoacetate in strongly basic conditions gave the azidocinnamate **25a** in 72% yield (Scheme 4). Heating the azidocinnamate **25a** in refluxing xylene gave the propargyloxy indole **26a** in 73% yield, which was then cyclized in chlorobenzene to afford the desired dihydropyranoindole **27a** in 74% yield.

The 1H-NMR spectrum of compound **27a** in CDCl3 showed two singlet signals at 3.92 and 3.93 ppm corresponding to the methoxy and methyl ester protons, and doublets of doublets at 4.90 ppm corresponding to the CH2 protons. H8 and H9 appeared as multiplets at 5.89–5.95 and 6.76–6.79 ppm respectively. The H4 appeared as a singlet at 6.77 ppm, while H1 appeared as a doublet at 7.17 ppm (*J* = 2.1 Hz). Moreover, the NH proton resonated as a broad singlet at δ 8.96 ppm. The formation of the desired dihydropyranoindole was further confirmed by DEPT 135 spectroscopy which revealed the presence of a negative peak at 65.71 ppm corresponding to the CH2 group accompanied by the disappearance of the signal corresponding to the CH group of the starting alkyne **26a**.

3-Hydroxy-4-methoxybenzaldehyde **23** was further treated with 1-bromobut-2-yne and 3-chloro-3-methylbut-1-yne under basic conditions to generate the corresponding intermediates **24b** [24] and **24c** [27] 89% and 82% yields, respectively (Scheme 4). The aryl ethers **24b** and **24c** were condensed with methyl azidoacetate in the presence of sodium methoxide to generate the azidoacrylates **25b** and **25c** in 64% and 62% yields respectively. Thermal decomposition of the unsaturated azides **25b** gave the indole ethers **26b** in 71%, while the desired hydropyrano compound **27c** was directly isolated in 68% yield. The desired dihydropyranoindoles **27b** was obtained in 54% yields respectively, by the cyclization of **26b** and in refluxing chlorobenzene. It was also of interest to construct new dihydropyranoindole systems containing substituents other than a methyl group on a pyran ring. The haloalkyne, 3-phenylprop-2-yn-1-ol, was treated with 3-hydroxy-4-methoxybenzaldehyde **23** in the presence of potassium carbonate to produce the alkylated carbaldehyde **24d** in 92% yield (Scheme 4). The reaction between the aryl ether **24d** with methyl azidoacetate afforded the unsaturated azido ester **25d** in 64% yield. Finally, heating compound **25d** in refluxing xylene afforded the corresponding indole ether **26d** in 74% yield, which further underwent thermal cyclization in chlorobenzene to furnish the desired dihydropyranoindole **27d** in 81% yield.

In the 1H-NMR spectrum of methyl 5-methoxy-7,7-dimethyl-3,7-dihydropyrano[3,2-*e*]indole-2 carboxylate **27c**, the two methyl groups appeared as a sharp singlet at 1.53 ppm. The H8 and H9 of the dihydropyran ring appeared as two doublets at 5.73 and 6.68 ppm that coupled to each other with a coupling constant of 9.7 Hz. H1 appeared as a doublet (*J* = 2.1 Hz) at 7.20 ppm while H4 was assigned as a singlet at 6.79 ppm. In the 1H-NMR spectrum of the cyclization product **27b** the olefinic protons on the dihydropyran ring appeared as multiplet signals in range of 5.69–5.72, due to coupling with the neighboring CH2 and CH3 groups. The characteristic O-CH2 protons resonated as a multiplet at 4.77 ppm, while the methyl group of the dihydropyran ring appeared at as a quartet 1.63 ppm. The 1H-NMR spectrum of compound **27d** displayed the O-CH2 protons as a doublet at 4.85 ppm with a coupling constant of 4.4 Hz, and the olefinic proton as a multiplet at 5.94–5.97 ppm. The aromatic protons of phenyl ring appeared as multiplet in the range of 7.34–7.44 ppm. The H4 and H1 appeared as two singlets at 5.98 and 6.88 ppm.

Similar attempts were made to the prepare methyl substituted tetracyclic dihydropyranoindole compounds from the 3,4-dihydroxybenzaldehyde **7**, 1-bromobut-2-yne and 3-chloro-3-methylbut-1-yne. The benzaldehyde **7** was alkylated with 2 equivalents of 1-bromobut-2-yne in the presence of potassium carbonate to generate the novel aryl ether **29** in 87% yield (Scheme 5). The benzaldehyde **29** was then treated with methyl azidoacetate under strongly basic conditions to generate the azido compound **30** in 63% yield, which subsequently underwent thermal decomposition in refluxing xylene to afford

the indole ether **31** in 65% yield. However, the cyclization of **31** could not be achieved in a number of solvents, including chlorobenzene, toluene, xylene and 1,2-dichlorobenzene which resulted in the formation of decomposed reaction mixture. The reaction between 3,4-dihydroxybenzaldehyde **7** and 3-chloro-3-methylbut-1-yne was investigated in an attempt to produce the dipropargyloxybenzaldehyde intermediate **28** (Scheme 5). However, this reaction resulted in a black reaction mixture, presumably due to decomposition.

**Scheme 4.** Reagents and conditions: (a) Propargyl bromide or 3-chloro-3-methylbut-1-yne or1-bromobut-2-yne or 3-phenylprop-2-yn-1-ol, K2CO3, acetone, reflux, 4 h, (b) Methyl azidoacetate, metallic Na, anhyd. MeOH, <−10 ◦C, 2 h (c) Xylene, reflux, 4 h, (d) Chlorobenzene, reflux, 2 h.

**Scheme 5.** Reagents and conditions: (a) 1-Bromobut-2-yne or 3-Chloro-3-methyl-1-butyne, K2CO3, acetone, reflux, 4 h, (b) methyl azidoacetate, metallic Na, anhdyrous MeOH, −10 ◦C, 2 h (c) Xylene, reflux, 2 h, (d) Xylene, chlorobenzene, toluene, 1,2-dichlorobenzene reflux, overnight.

#### *2.4. Biological Study*

#### 2.4.1. Preliminary Biological Screening of Dihydropyranoindoles for SAHA Enhancement Activity

Novel dihydropyranoindole analogues were screened to determine the levels of SAHA enhancement activity as well as their own cytotoxic profile against the SH-SY5Y and Kelly neuroblastoma cells and the MDA-MB-231 and MCF-7 breast adenocarcinoma cell lines using the Alamar blue (Resazurin) assay [18]. Briefly, the cells were allowed to attach for 24 h in a 96-well culture plate before being exposed to the ligands at a concentration of 10 μM in DMSO for 72 h, either in the presence or absence of SAHA. Comparative values for cell viability in each well were determined by a Wallac 1420 Victor III spectrophotometer, which measured light absorbance in each well at 570 nm. The mean and standard deviation (SD) values for each compound were calculated from at least three replicate experiments. The anticancer activity of the compounds was evaluated by comparison to a negative (DMSO) control. Figure 4 shows the tested novel dihydropyranoindoles synthesized in this study and the compound **33** was selected from the NK library. In this assay, the cell lines were exposed to SAHA, 10 μM of the compounds as well as the combination of SAHA with the compounds for 72 h.

**Figure 4.** Synthesized and selected compounds.

According to the results from the screening, the neuroblastoma cells were found to be the more sensitive cells towards the treatment of new pyranoindoles, while these compounds showed a moderate effect on the breast cancer cell lines (Figures S3A,B and S4A,B, see Supplementary Materials). MDA-MB-231 cells were determined as the most resistant cell line for the single as well as the combination treatments (Figure S3A, see Supplementary Materials). The dihydropyranoindole **27c** exhibited the greatest cell viability inhibition with the values of 34% and 35% in the absence and presence of SAHA against the MDA-MB-231 cells. The SAHA enhancement was achieved by the additional reduction value of 20% across MDA-MB-231 viability. MCF-7 cells showed an identical pattern of sensitivity. However, most of the dihydropyranoindoles were able to reduce the viability by the value of 20% (Figure S3, see Supplementary Materials).

Compound **33** displayed the highest reduction with the 35% inhibition, while compounds **27c** and **22c** generated similar reductions with the values of 20% and 22% on the same cell line. Unfortunately the combination of pyranoindoles with 1 μM SAHA had very low SAHA enhancement by the average values of 5% across the MCF-7 cell lines.

It was observed that dihydropyranoindoles had a variety of impact on the viability of neuroblastoma cells (Kelly and SH-SY5Y) with the single and combination treatments (Figure S4A,B, see Supplementary Materials). Out of all the novel ligands synthesized, ligand **33** had the strongest cytotoxic activity with the reduction value of 50% at 10 μM, while ligands **27c** and **10** showed similar

inhibition behaviors by the average reduction value of 32% against Kelly cells for the single treatment. The dihydropyranoindoles **27c** and **33** displayed the enhancement of SAHA cytotoxicity with the values of 24 and 22% respectively (Figure S4A, see Supplementary Materials). The value of 40% SAHA enhancement was obtained by the combination of compound **33**. However, the ligand **33** was considered as self-toxic compound due to the reduction value for the single treatment. The levels of individual cytotoxic efficiency against the SH-SY5Y were found to be higher than those observed on the Kelly cells (Figure S4B, see Supplementary Materials). The compounds **27d** and **10** showed the greatest cytotoxic activity reducing 44% and 56% of the cells respectively. Most importantly, the enhancement of SAHA activity was achieved by the treatment of almost all of the novel dihydropyranoindole analogues showing at least 20% additional reduction to SAHA cytotoxicity. Although the highest SAHA enhancement was obtained in the case of compound **10** with the value of 55%, it was not considered as a promising enhancer since the single treatment also generated the greatest reduction (56%) on the SH-SY5Y cell. The best enhancers were assigned as compounds **22a**, **27a**, **27c** and **33** with the reduction values of 25%, 29%, 30% and 31%respectively due to the lower toxicity but the higher enhancement effects on SAHA cytotoxicity.

2.4.2. Determination of SAHA Enhancement Activity of Selected Dihydropyranoindoles at Lower Concentrations

Since the levels of SAHA enhancement were found to be promising, further investigations were carried out using five different concentrations (0.01, 0.1, 1, 10, 20 μM), using the Alamar blue (Resazurin) assay [18] to determine whether the lower concentrations would provide higher enhancement of the SAHA cytotoxicity or the cytotoxic manner would be dependent on the dose usage. The initial screening revealed that the single treatment of dihydropyranoindoles **33**, **27c** and **10** at a concentration of 10 μM showed the highest cytotoxic efficiency against the Kelly cells. The designated compounds were treated with the Kelly cells by the combinations of 0.5 μM SAHA with the five different concentrations (0.01, 0.1, 1, 10, 20 μM) and the results are shown at Figure S5A,C (see Supplementary Materials). In general, the single treatment of these compounds provided lower cytotoxic activity at the concentrations of 0.01, 0.1 and 1 μM compared with the values at 10 μM and the cell viability differentials between the absence and presence of SAHA were greater than those at 10 μM concentrations, suggesting that the designated compounds behave as suitable SAHA enhancers (Figure S5A,C, see Supplementary Materials). Similar SAHA enhancement pattern was observed at the concentrations of 0.01, 0.1 and 1 μM for all three compounds, while the combination of 1 μM compound **27c** or **10** with the 0.5 μM SAHA revealed the best SAHA enhancement with the values of 24% and 25% additional inhibition (Figure S5B,C, see Supplementary Materials).

In addition to those two compounds, compound **33** showed the greatest viability differentials with the values of 61%, 63% and 65% at the concentrations of 0.01, 0.1 and 1 μM and the desired SAHA enhancement was observed for all the concentrations. The lowest concentration of compound **33** (0.01 μM) revealed a remarkable reduction with the value of 25%, while the best combination for the highest SAHA enhancement was determined as a 10:0.5 ratio of compound and SAHA with the additional reduction value of 55% (Figure S5 A, see Supplementary Materials). It was concluded that the highest cell viability differentials was achieved by the use of nanomolar concentrations in the case of all three compounds and the best SAHA enhancer was determined as compound **33**.

Similarly, in the case of the SH-SY5Y cell line, further investigations were undertaken with **27a**, **33** and **27c** which were chosen due to their highest additional effects on the SAHA cytotoxicity with the lower toxicity behavior in the absence of SAHA. Compound **10** was also explored to determine its cytotoxic behavior as well as the SAHA enhancement potential at lower concentrations. The combinations of 1 μM SAHA with the five different concentrations (0.01, 0.1, 1, 10, 20 μM) of designated compounds were treated with the SH-SY5Y cells and the results are shown at Figure S6A–D (see Supplementary Materials).

The SAHA enhancement pattern was found to be identical on SH-SY5Y value for all cases of combinations of **27a** with SAHA. The highest viability differential was obtained in the ratio 1:10 of SAHA to compound with the reduction value of 35% (Figure S6A, see Supplementary Materials). The dose-dependent pattern of single treatments of **33** and **27c** suggested that these compounds displayed higher toxicity at 20 μM with the values of 45% and 55% reductions. Furthermore, the combinations of these compounds at the concentrations of 0.01, 0.1 and 1 μM with SAHA enhanced the SAHA cytotoxicity in a similar manner and the best combinations were found to be as the ratio 1:10 of SAHA to compound with the additional reduction values of 28% and 33% (Figure S6B,C, see Supplementary Materials). The greatest cytotoxicity was obtained at a concentration of 20 μM of **10** alone with the reduction values of 88% on SH-SY5Y cell viability and the ratio of 1:20 SAHA to compound combination provided 94% cell death (Figure S6D, see Supplementary Materials).

More importantly, at the lower concentrations (0.01, 0.1 and 1 μM) of **10**, the cytotoxicity was found to be lower with the average reduction value of 25%. Encouragingly, the biggest cell viability differential was obtained at the ratio of 1:10 SAHA to compound with the value of 44%, and the greatest SAHA enhancement was also found as 37% at the same combination. It was concluded that **10** was the most cytotoxic compound at higher concentration (20 μM, 88% reduction) and also it was found to be the best SAHA enhancer at the ratio of 1:10 SAHA to compound combination.

#### 2.4.3. SAR Study of Selected Dihydropyranoindoles

These observations suggested an important structure-activity relationship among **27a**, **33**, **27c** and **10**. That is, the incorporation of tetracyclic dihydropyranoindoles **33** and **10** resulted in lower cell viability reductions at higher concentrations (20 μM) and also this system was found to offer the best enhancement with the ratio of 1:10 SAHA to compound combinations against both neuroblastoma cancer cells (Kelly and SH-SY5Y). The SAR analysis revealed that the location of dihydropyran ring on the benzene ring was important for the cytotoxic efficiency of tricyclic dihydropyranoindoles, The compounds **27c**, an example of dihydropyrano[3,2-*e*]indole system, was found to be more potent than the dihydropyranoindole **22c** which is a member of dihydropyrano[2,3-*g*]indole scaffold against both neuroblastoma cells (Kelly and SH-SY5Y). In comparison of two tricyclic dihydropyranoindoles **27c** and **22c** at 20 μM, it was found that the compound **27c** reduced 20% more SH-SY5Y cell with the combination of 1 μM SAHA than compound **22c**, while the 20% SAHA enhancement was achieved using the combination of compound **27c** and SAHA but no SAHA enhancement was found with the compound **22c** in the case of Kelly cells. Furthermore, the dihydropyrano[2,3-*g*]indole analogues **22a** and **22b** displayed the lowest reduction on Kelly cells in the presence and absence of SAHA. Similar results were observed in the case of SH-SY5Y cells with the exception of 21% additional reduction on SAHA enhancement obtained by the use of **22a**.

#### 2.4.4. Toxicity Study Against Normal Cells

While selected dihydropyranoindoles showed potent SAHA enhancement activity against Kelly and SH-SY5Y neuroblastoma and MCF-7 and MDA-MB-231 breast cancer cells, they must be markedly less toxic against the healthy cells in order to be considered as potential SAHA enhancers. Thus, the dihydropyranoindoles **27a**, **33**, **27c** and **10** were also examined for their toxicity effects on the MRC-5 and WI-38 lung fibroblasts in order to determine whether these compounds exhibited selectivity for tumor cells (Figure S7A–D, see Supplementary Materials). Comparison of the cytotoxic activity and toxicity levels of the dihydropyranoindoles against cancer cells and the healthy cells revealed that the cancer cells were more sensitive to the dihydropyranoindoles compared to the normal cells. The toxicity of 1 μM of SAHA reduced the viability of MRC-5 and WI-38 cells with the value of 9% and 24% respectively, while **27a** alone displayed a similar pattern of toxicity against normal cells with values of 10% for MRC-5 and 22% for WI-38 at a concentration of 10 μM (Figure S7A, see Supplementary Materials). The viability of MRC-5 and WI-38 were reduced 21% and 16%, 3% and 26% by the use of **33** and **10** alone (Figure S7B,C, see Supplementary Materials), while the highest toxicity levels were

obtained in the case of **27c** with the reduction values of 20% and 43% (Figure S7D, see Supplementary Materials). The combination treatments were found to be quite identical to the reduction values of single treatments of each compound. These observations demonstrated that **27a**, **33** and **10** were either less toxic or slightly less toxic than SAHA, while **27c** displayed more toxicity than SAHA for both normal cells.

#### **3. Materials and Methods**

#### *3.1. General Information*

Commercially available reagents were purchased from Fluka (Sydney, NSW, Australia), Aldrich (Sydney, NSW, Australia), Acros Organics (Morris Plains, NJ, USA), Alfa Aesar (Lancashire, UK) and Lancaster (Lancashire, UK) and purified if necessary. The synthetic procedures have been reported for all compounds as general methods and appropriate references have been given for known compounds. 1H (300 MHz) and 13C-NMR (75 MHz) spectra were obtained in the designated solvents on a DPX 300 spectrometer (Bruker, Sydney, NSW, Australia). Melting points were measured using a Mel-Temp melting point apparatus and are uncorrected. Infrared spectra were recorded on Avatar Series FT-IR spectrophotometer as KBr disks (Thermo Nicolet, Waltham, MA, USA). Ultraviolet spectra were measured using a Cary 100 spectrophotometer (Varian, Santa Clara, CA, USA) in the designated solvents and data reported as wavelength (λ) in nm and adsorption coefficient (ε) in cm−1M−1. High-resolution [ESI] mass spectra were recorded by the UNSW Bioanalytical Mass Spectrometry Facility, on an Orbitrap LTQ XL (Thermo Scientific, Waltham, MA, USA) ion trap mass spectrometer using a nanospray (nano-electrospray) ionization source.

#### 3.1.1. GP-1: General Procedure for the Benzylation of Dihydroxybenzaldehydes

A solution of hydroxybenzaldehyde (1 equiv.), anhydrous potassium carbonate (per hydroxyl group, 1 equiv.) and benzyl bromide (per hydroxyl group, 1 equiv.) in anhydrous DMF (100 mL) was heated at reflux until TLC analysis showed consumption of the starting aldehyde (9 h). Upon cooling to room temperature, the reaction mixture was diluted with water (300 mL) and the resulting precipitate was collected via filtration and washed with water (2 × 250 mL). Upon drying, the residue was recrystallized from dichloromethane and *n*-hexane to give the title compound.

#### 3.1.2. GP-2: General Procedure for the Preparation of Vinyl-Azido Intermediates

A solution of sodium methoxide was prepared via the portion-wise addition of metallic sodium (17 equiv.) to anhydrous methanol (30 mL) with stirring under nitrogen. A dropping funnel was attached to the reaction flask and charged with the corresponding aldehyde (1 equiv.) and methyl azidoacetate (10 equiv.) in methanol (15 mL). The contents of the funnel were added dropwise to the sodium methoxide solution over 1.5 h under a nitrogen atmosphere. Once the addition was completed, the reaction mixture was warmed to 5 ◦C, where it remained for 4 h. The heterogeneous mixture was poured into crushed ice. The resulting precipitate was filtered, washed with water and dried to give the title compound. The crude product was used in the next step without further purification.

#### 3.1.3. GP-3: General Procedure for the Preparation of Methyl Benzyloxyindole-2-carboxylates

The vinyl azido intermediate (6.66 mmol) was dissolved in xylene (50 mL) and the reaction mixture was heated under reflux for 6 h. After refluxing, the solvent was evaporated under reduced pressure and the remaining residue was extracted with boiling hexane. Upon cooling, the resulting solid was filtered to give crude product which was recrystallized from dichloromethane and *n*-hexane to give the title compound.

3.1.4. GP-4: General Procedure for the Hydrogenolysis of Methyl Dibenzyloxyindole-2-carboxylate

After vacuum/H2 cycles to remove air from the reaction flask, a stirred mixture of benzyloxyindole (1 mmol) and 5% Pd/C (10% *w*/*w*) in THF/MeOH (1:1, 15 mL) was exposed to a hydrogen atmosphere (1 atm) and stirred at room temperature for 2 h. The reaction mixture was filtered through a pad of Celite® and the filtrate was then concentrated under reduced pressure. The crude product was purified by flash column chromatography (SiO2), eluted with dichloromethane to give the title compounds.

#### 3.1.5. GP-5: General Procedure for the Synthesis of Propargyloxybenzaldehydes:

Propargyl bromide (per hydroxyl group, 1.2 equiv.) was added to a mixture of potassium carbonate (per hydroxyl group, 1 equiv.) and hydroxybenzaldehyde (1 equiv.) in acetone. The reaction mixture was heated under reflux with stirring until no more starting material remained (~30 h). The reaction mixture was cooled to room temperature and Et2O (100 mL) was added. The ethereal solution was washed with NaOH (1 N, 3 × 50 mL). The organic layer was dried over MgSO4, concentrated under reduced pressure and recrystallized from dichloromethane/*n*-hexane to give the compound.

3.1.6. GP-6 General Procedure for the Synthesis of Dihydropyranoindoles:

A solution of alkyne indole ethers (1.04 mmol) in chlorobenzene (20 mL) was heated under reflux until TLC analysis showed consumption of the starting indole (12 h). The heating was discontinued and the solvent was evaporated under reduced pressure. The crude product was purified using flash column chromatography (SiO2), eluted with 30% dichloromethane/*n*-hexane, to give the dihydropyranoindole.

*Methyl 2-azido-3-(3,4-dibenzyloxyphenyl)-propenoate* (**13**) The *title* compound was prepared as described in GP-2 from 3,4-dibenzyloxybenzaldehyde (**12**) (2.95 g, 9.3 mmol) and methyl azidoacetate (10.69 g, 93 mmol) in anhydrous methanol (30 mL) to give the product (2.27 g, 59%) as a pale yellow granular solid; m.p. 114–116 ◦C; IR (KBr): *v*max 2917, 2119, 1701, 1683, 1590, 1508, 1432, 1379, 1233, 1201, 1130, 999, 802, 728 cm<sup>−</sup>1; UV-vis (CH3CN): λmax325 (24,500); 1H-NMR: (300 MHz, CDCl3): δ 3.91 (s, 3H, CO2Me), 5.25 (s, 4H, 2 × O-CH2), 6.83 (s, 1H, CH=C), 7.35–7.52 (m, 12H, ArH), 7.64 (d, *J* = 2.2 Hz, 1H, H2 ); 13C-NMR: (75.6 MHz, CDCl3): δ 52.7 (CO2Me), 70.9 (CH2), 71.3 (CH2), 113.8 (C2 ), 116.6 (C5 ), 123.3 (CH=C), 125.4 (C6 ), 125.7 (ArC), 126.6 (ArC), 127.1 (2 × ArC), 127.2 (2 × ArC), 127.8 (ArC), 127.9 (ArC), 128.5 (2 × ArC), 128.6 (2 × ArC), 136.8 (C1 ), 137.1 (CH=C), 148.3 (C4 ), 150.1 (C3 ), 164.1 (CO2Me); HRMS (+ESI): Found *m*/*z* 438.1439 [M + Na]<sup>+</sup>, C24H21N3O4Na requires 438.1439.

*Methyl 5,6-dibenzyloxyindole-2-carboxylate* (**14**) The *title* compound was prepared as described in GP-3 from methyl 2-azido-3-(3 ,4 -dibenzyloxyphenyl)propenoate (**13**) (2.76 g, 6.66 mmol) in xylene (50 mL) to give the product (1.84 g, 71%) as a yellow granular solid; m.p. 148–150 ◦C; IR (KBr): *v*max 3309, 2942, 2871, 2113, 1680, 1627, 1519, 1488, 1452, 1353, 1288, 1246, 1208, 1144, 1000, 918, 905, 839, 794 cm−1; UV-vis (CH3CN): λmax 208 nm (ε 72,400 cm−<sup>1</sup> M<sup>−</sup>1), 320 (31,700); 1H-NMR (300 MHz, CDCl3): δ 3.93 (s, 3H, CO2Me), 5.21 (s, 2H, O-CH2), 5.24 (s, 2H, O-CH2), 7.00 (s, 1H, H4), 7.01 (s, 1H, H7), 7.23 (d, *J* = 2.1 Hz, 1H, H3), 7.33–7.51 (m, 10H, ArH), 8.73 (bs, 1H, NH); 13C-NMR (75.6 MHz, CDCl3): 51.8 (CO2Me), 71.3 (CH2), 72.0 (CH2), 97.0 (C7), 107.0 (C4), 108.8 (C3), 121.0 (ArC), 126.0 (C2), 127.0 (ArC), 127.2 (2 × ArC), 127.3 (ArC), 127.7 (2 × ArC), 127.8 (ArC), 128.4 (ArC), 128.5 (ArC), 132.4 (ArC), 137.0 (ArC), 137.4 (2 <sup>×</sup> ArC), 145.8 (C5), 149.9 (C6), 162.1 (CO2Me); HRMS (+ESI): Found *m*/*z* 410.1364 [M + Na]+, C24H21NO4Na requires 410.1363.

*Methyl 5,6-dihydroxyindole-2-carboxylate* (**8**) The *title* compound was prepared as described in GP-4 from methyl 5,6-dibenzyloxyindole-2-carboxylate (**14**) (0.387 g, 1.0 mmol) and 5% Pd/C catalyst (40 mg) in methanol/THF mixture (15 mL) to give the product (159 mg, 77%) as yellow solid; m.p. 256–258 ◦C; IR (KBr): *v*max 3437, 3315, 2953, 2107, 1653, 1632, 1531, 1506, 1437, 1311, 1283, 1230, 1198, 1139, 992, 937, 849, 825, 767 cm−1; UV-vis (CH3CN): λmax 203 nm (ε 42,100 cm−<sup>1</sup> M−1), 318 (27,000);1H-NMR (300MHz, *d*6-DMSO): δ 3.82 (s, 3H, CO2Me), 6.79 (d, *J* = 0.8 Hz, 1H, H4), 6.88 (s, 1H, H3), 6.90 (d, *J* = 0.8 Hz, 1H, H7), 8.84 and 9.17 (bs, each 1H, OH), 11.28 (bs, 1H, NH); 13C-NMR (75.6 MHz, CDCl3): 52.0

(CO2Me), 97.3 (C7), 105.4 (C4), 108.4 (C3), 120.3 (ArC), 125.0 (C2), 133.2 (ArC), 142.3 (C5), 146.7 (C6), 162.5 (CO2Me); HRMS (+ESI): Found *m*/*z* 230.0423 [M + Na]<sup>+</sup>, C10H9NO4Na requires 230.0424.

*Methyl 5,6-bis(prop-2-yn-1-yloxy)-1H-indole-2-carboxylate* (**9**) The *title* compound was prepared as described in GP-5 from methyl 5,6-dihydroxyindole-2-carboxylate (**8**) (993 mg, 4.8 mmol), potassium carbonate (1.32 g, 9.6 mmol) and propargyl bromide (1.36 g, 10.6 mmol) in acetone to give the product (828 mg, 61%) as a yellow solid;m.p. 170–172 ◦C; IR (KBr): νmax 3332, 3285, 2939, 2925, 2865, 2292, 2108, 1720, 1684, 1625, 1520, 1480, 1435, 1380, 1365, 1280, 1247, 1203, 1150 1024, 986, 892, 819, 805, 759, 720, 672 cm<sup>−</sup>1; UV-vis (CH3CN): λmax 209 nm (ε 28,300 cm−<sup>1</sup> M<sup>−</sup>1), 313 (18,000); 1H-NMR (300 MHz, CDCl3): δ 2.55 (t, *J* = 1.6 Hz, 1H, CH≡C), 2.57 (t, *J* = 1.6 Hz, 1H, CH≡C), 3.95 (s, 3H, CO2Me), 4.82 (d, *J* = 2.4 Hz, 2H, O-CH2), 4.85 (d, *J* = 2.4 Hz, 2H, O-CH2), 7.10 (d, *J* = 0.7 Hz, 1H, H7), 7.16 (q, *J* = 0.9 Hz, 1H, H3), 7.31 (s, 1H, H4), 8.86 (bs, 1H, NH), 13C-NMR (75.6 MHz, CDCl3): δ51.8 (CO2Me), 57.0 (O-CH2), 57.5 (O-CH2), 75.7 (C≡CH), 75.7 (C≡CH), 78.6 (C≡CH), 79.1 (C≡CH), 97.2 (C7), 107.2 (C4), 108.9 (C3), 121.3 (aryl C), 126.4 (C2), 132.4 (aryl C), 144.2 (C4), 148.2 (C5), 162.1 (CO2Me); HRMS (+ESI): Found *m*/*z* 306.0736 [M + Na]<sup>+</sup>, C16H13NO4Na requires 306.0737.

*2,9-Dihydropyrano[3,2-h]chromene-5-carbaldehyde* (**15**) The *title* compound was prepared as described in GP-6 from 3,4-bis(prop-2-yn-1-yloxy)benzaldehyde (**11**) (214 mg, 1.04 mmol) in chlorobenzene (20 mL) to give the product (77 mg, 35%) as a white solid; m.p. 102–104 ◦C; IR (KBr): νmax 1680, 1570, 1490, 1440, 1340, 1301, 1205, 1102, 995, 945, 900, 898, 861, 745 cm<sup>−</sup>1; UV-vis (CH3CN): λmax 225 nm (ε 27,400 cm−<sup>1</sup> M−1), 268 (18,800), 305 (17,500); 1H-NMR (300 MHz, CDCl3): δ 4.40 (dd, *J* = 3.0, 1.4 Hz, 2H, O-CH2), 4.79 (dd, *J* = 3.0, 1.4 Hz, 2H, O-CH2), 6.97 (d, *J* = 8.7 Hz, 2H, H4 and H7), 7.37 (s, 1H, H6), 7.40–7.42 (m, 2H, H3 and H8), 9.80 (s, 1H, CHO); 13C-NMR (75.6 MHz, CDCl3): δ71.3 (O-CH2), 71.5 (O-CH2), 119.6 (C6), 120.6 (aryl C), 121.6 (C4), 121.8 (C7), 125.3 (aryl C), 126.5 (C3), 126.9 (C8), 127.1 (C5), 147.9 (aryl C), 154.3 (aryl C), 190.7 (CHO); HRMS (+ESI): Found *m*/*z* 237.0533 [M + Na]+, C13H10O3Na requires 237.0528.

*3,4-Bis(but-2-yn-1-yloxy)benzaldehyde* (**29**) The *title* compound was prepared as described in GP-5 from 3,4-dihydroxybenzaldehyde (7) (660 mg, 4.8 mmol), potassium carbonate (1.32 g, 9.6 mmol) and 1-bromobut-2-yne (1.40 g, 10.6 mmol) in acetone to give the product (1.01 g, 87%) as a white solid; m.p. 86–88 ◦C; IR (KBr): νmax 1688, 1582, 1499, 1431, 1375, 1248, 1201, 1125, 987, 920, 857, 797, 729 cm−1; UV-vis (CH3CN): λmax 228 nm (ε 31,500 cm−<sup>1</sup> M−1), 272 (22,200), 303 (14,800); 1H-NMR (300 MHz, CDCl3): δ 1.85–1.87 (m, 6H, 2 × C-CH3), 4.79 (q, *J* = 2.3 Hz, 2H, O-CH2), 4.82 (q, *J* = 2.3 Hz, 2H, O-CH2), 7.17 (d, *J* = 8.2 Hz, 1H, H5), 7.52 (dd, *J* = 8.2, 1.9 Hz, 1H, H6), 7.57 (d, *J* = 1.9 Hz, 1H, H2), 9.89 (s, 1H, CHO); 13C-NMR (75.6 MHz, CDCl3): <sup>δ</sup> 3.6 (C-CH3), 3.7 (C-CH3), 57.2 (2 <sup>×</sup> O-CH2), 73.1 (C-CH3), 73.3 (C-CH3), 84.6 (C≡C(CH3)), 84.9 (C≡C(CH3)), 112.0 (C5), 112.7 (C2), 126.4 (C6), 130.3 (aryl C), 147.9 (C4 ), 152.9 (C3 ), 190.8 (CHO); HRMS (+ESI): Found *m*/*z* 265.0840 [M + Na]<sup>+</sup>, C15H14O3Na requires 265.0835.

*4-Methoxy-3-((3-phenylprop-2-yn-1-yl)oxy)benzaldehyde* (**24d**) The *title* compound was prepared as described in GP-5 from 3-hydroxy-4-methoxybenzaldehyde **23** (730 mg, 4.8 mmol), potassium carbonate (662 mg, 4.8 mmol) and 3-phenylprop-2-yn-1-yl-4-methylbenzenesulfonate (1.66 g, 5.8 mmol) in acetone to give the product (1.17 g, 92%) as a white solid; m.p. 112–114 ◦C; IR (KBr): νmax 1673, 1579, 1506, 1430, 1378, 1256, 1221, 1165, 1130, 1012, 931, 874, 810, 754, 686 cm<sup>−</sup>1; UV-vis (CH3CN): λmax 232 nm (ε 45,500 cm−<sup>1</sup> M<sup>−</sup>1), 272 (21,500), 302 (14,800); 1H-NMR (300 MHz, CDCl3): δ 4.00 (s, 3H, OMe), 5.08 (s, 2H, O-CH2), 7.03 (d, *J* = 8.2 Hz, 1H, H5), 7.30–7.33 (m, 3H, ArH), 7.34–7.45 (m, 2H, ArH), 7.54 (dd, *J* = 8.2, 1.9 Hz, 1H, H6), 7.68 (d, *J* = 1.9 Hz, 1H, H2), 9.90 (s, 1H, CHO); 13C-NMR (75.6 MHz, CDCl3): δ 56.2 (OMe), 57.5 (O-CH2), 83.0 (C≡C(Ph)), 88.0 (C-Ph), 110.9 (C5), 112.1 (C2), 122.1 (aryl C), 127.1 (2 × aryl CH), 127.8 (aryl CH), 128.8 (C6), 130.0 (aryl C), 131.9 (2 × aryl CH), 147.5 (C4), 155.0 (C3), 190.7 (CHO); HRMS (+ESI): Found *m*/*z* 289.0837 [M + Na]<sup>+</sup>, C17H14O3Na requires 289.0835.

*Methyl (Z)-2-azido-3-(3,4-bis(prop-2-yn-1-yloxy)phenyl) acrylate* (**17**) The *title* compound was prepared as described in GP-2 from 3,4-bis(prop-2-yn-1-yloxy)benzaldehyde (**11**) (558 mg, 2.6 mmol) and methyl azidoacetate (2.99 g, 26.0 mmol) in anhydrous methanol (30 mL) to give the product (582 mg, 63%) as a pale yellow granular solid; m.p. 114–116 ◦C; IR (KBr): νmax 2954, 2123, 1709, 1594, 1506, 1433, 1378, 1239, 1133, 1081, 1009, 793, 745, 683 cm−1; UV-vis (CH3CN): λmax 327 nm (ε 19,100 cm−<sup>1</sup> M−1); 1H-NMR (300 MHz,CDCl3): <sup>δ</sup> 2.57 (t, *<sup>J</sup>* <sup>=</sup> 2.4 Hz, 1H, CH≡C), 2.59 (t, *<sup>J</sup>* <sup>=</sup> 2.4 Hz, 1H, CH≡C), 3.92 (s, 3H, CO2Me), 4.83 (d, *J* = 2.4 Hz, 4H, 2 × O-CH2), 6.91 (s, 1H, CH=C), 7.09 (d, *J* = 8.5 Hz, 1H, H5 ), 7.44 (dd, *J* = 8.5, 2.0 Hz, 1H, H6 ), 7.77 (d, *J* = 2.0 Hz, 1H, H2 ), 13C-NMR (75.6 MHz, CDCl3): δ 52.8 (CO2Me), 56.6 (OMe), 57.0 (2 × O-CH2), 76.1 (C≡CH), 76.2 (C≡CH), 77.2 (C≡CH), 77.4 (C≡CH), 113.8 (C5 ), 116.6 (C2 ), 123.9 (CH=C), 125.3 (C6 ), 125.6 (aryl C), 146.9 (aryl C), 148.5 (aryl C), 164.1 (CO2Me); HRMS could not be determined due to the unstable properties of the compound

*Methyl (Z)-2-azido-3-(4-methoxy-3-(prop-2-yn-1-yloxy)phenyl)acrylate* (**25a**) The *title* compound was prepared as described in GP-2 from 4-methoxy-3-(prop-2-yn-1-yloxy)benzaldehyde (24a) (494 mg, 2.6 mmol) and methyl azidoacetate (2.99 g, 26.0 mmol) in anhydrous methanol (30 mL) to give the product (537 mg, 72%) as a pale yellow granular solid; m.p. 116–118 ◦C; IR (KBr): νmax 2951, 2101, 1698, 1592, 1507, 1432, 1379, 1254, 1214, 1139, 1084, 1016, 809, 745 cm<sup>−</sup>1; UV-vis (CH3CN): λmax 235 nm (<sup>ε</sup> 16,600 cm−<sup>1</sup> <sup>M</sup><sup>−</sup>1), 330 (27,100); 1H-NMR (300 MHz, CDCl3): <sup>δ</sup> 2.58 (t, *<sup>J</sup>* <sup>=</sup> 2.4 Hz, 1H, CH≡C), 3.94 (s, 3H, OMe), 3.94 (s, 3H, CO2Me), 4.83 (d, *J* = 2.4 Hz, 2H, O-CH2), 6.90 (s, 1H, CH=C), 6.93 (d, *J* = 8.5 Hz, 1H, H5 ), 7.43 (dd, *J* = 8.5, 2.0 Hz, 1H, H6 ), 7.76 (d, *J* = 2.0 Hz, 1H, H2 ); 13C-NMR (75.6 MHz, CDCl3): δ 52.8 (CO2Me), 55.9 (OMe), 56.8 (O-CH2), 76.0 (C≡CH), 77.4 (C≡CH), 111.2 (C5 ), 116.1 (C2 ), 123.4 (CH=C), 125.6 (C6 ), 125.0 (aryl C), 126.1 (CH=C), 146.3 (C4 ), 150.8 (C3 ), 164.1 (CO2Me); HRMS (+ESI): Found *m*/*z* 310.0800 [M + Na]<sup>+</sup>, C14H13N3O4Na requires 310.0798.

*Methyl (Z)-2-azido-3-(3-methoxy-4-(prop-2-yn-1-yloxy) phenyl)acrylate* (**20a**) The *title* compound was prepared as described in GP-2 from 3-methoxy-4-(prop-2-yn-1-yloxy)benzaldehyde (**19a**) (494 mg, 2.6 mmol) and methyl azidoacetate (2.99 g, 26.0 mmol) in anhydrous methanol (30 mL) to give the product (567 mg, 76%) as a pale yellow granular solid; m.p. 120–122 ◦C; IR (KBr): νmax 2920, 2122, 1704, 1592, 1509, 1450, 1345, 1243, 1206, 1141, 1008, 922, 804, 764 cm−1; UV-vis (CH3CN): λmax 323 nm (ε 17,500 cm−<sup>1</sup> <sup>M</sup><sup>−</sup>1); 1H-NMR (300 MHz, CDCl3): <sup>δ</sup> 2.56 (t, *<sup>J</sup>* <sup>=</sup> 2.4 Hz, 1H, CH≡C), 3.94 (s, 3H, OMe), 3.95 (s, 3H, CO2Me), 4.83 (d, *J* = 2.4 Hz, 2H, O-CH2), 6.91 ( s, 1H, CH=C), 7.06 (d, *J* = 8.5 Hz, 1H, H5 ), 7.43 (dd, *J* = 8.5, 1.9 Hz, 1H, H6 ), 7.56 (d, *J* = 1.9 Hz, 1H, H2 ); 13C-NMR (75.6 MHz, CDCl3): δ 52.8 (CO2Me), 55.9 (OMe), 56.5 (O-CH2), 76.2 (C≡CH), 77.4 (C≡CH), 113.3 (C5 ), 113.5 (C2 ), 123.7 (CH=C), 124.4 (C6 ), 125.5 (aryl C), 127.3 (CH=C), 147.9 (C3 ), 149.1 (C4 ), 164.1 (CO2Me); HRMS could not be determined due to the unstable properties of the compound.

*Methyl (Z)-2-azido-3-(3,4-bis(but-2-yn-1-yl)oxy)phenyl) acrylate* (**30**) The *title* compound was prepared as described in GP-2 from 3,4-bis(but-2-yn-1-yloxy)benzaldehyde (**29**) (629 mg, 2.6 mmol) and methyl azidoacetate (2.99 g, 26.0 mmol) in anhydrous methanol (30 mL) to give the product (555 mg, 63%) as a pale yellow granular solid; m.p. 124–126 ◦C; IR (KBr): νmax 2918, 2119, 1712, 1687, 1590, 1506, 1432, 1373, 1314, 1243, 1128, 992, 804, 745 cm<sup>−</sup>1; UV-vis (CH3CN): λmax 307 nm (ε 14,500 cm−<sup>1</sup> M<sup>−</sup>1); 1H-NMR (300 MHz, CDCl3): δ 1.87 (t, *J* = 2.3 Hz, 6H, 2 × C-CH3), 3.93 (s, 3H, CO2Me), 4.79 (q, *J* = 2.3 Hz, 2H, O-CH2), 4.82 (q, *J* = 2.3 Hz, 2H, O-CH2), 6.91 (s, 1H CH=C), 7.05 (d, *J* = 8.5 Hz, 1H, H5 ), 7.44 (dd, *J* = 8.5, 1.8 Hz, 1H, H6 ), 7.73 (d, *J* = 2.0 Hz, 1H, H2 ); 13C-NMR (75.6 MHz, CDCl3): <sup>δ</sup> 3.7 (2×C-CH3), 52.8 (CO2Me), 57.1 (OMe), 57.2 (O-CH2), 57.5 (O-CH2), 73.9 (2×C-CH3), 84.2 (C≡C(CH3)), 84.9 (C≡C(CH3)), 113.2 (C5 ), 116.0 (C2 ), 125.3 (CH=C), 125.8 (C6 ), 126.5 (aryl C), 126.6 (CH=C), 147.9 (C4 ), 148.8 (C3), 164.2 (CO2Me); HRMS could not be determined due to the unstable properties of the compound.

*Methyl (Z)-2-azido-3-(3-(but-2-yn-1-yloxy)-4-methoxyphenyl) acrylate* (**25b**) The *title* compound was prepared as described in GP-2 from 3-(but-2-yn-1-yloxy)-4-methoxybenzaldehyde (**24b**) (530 mg, 2.6 mmol) and methyl azidoacetate (2.9 g, 26.0 mmol) in anhydrous methanol (30 mL) to give the product (500 mg, 64%) as a pale yellow granular solid; m.p. 96–98 ◦C; IR (KBr): νmax 2951, 2101, 1698, 1592, 1507, 1431, 1379, 1254, 1139, 1016, 810, 745 cm<sup>−</sup>1; UV-vis (CH3CN): λmax 327 nm (ε 25,700 cm−<sup>1</sup> M<sup>−</sup>1); 1H-NMR (300 MHz, CDCl3): δ 1.89 (t, *J* = 2.3 Hz, 3H, CH3), 3.93 (s, 3H, OMe), 3.94 (s, 3H, CO2Me), 4.78 (q, *J* = 2.3 Hz, 2H, O-CH2), 6.91 (d, *J* = 8.5 Hz, 1H, H5 ), 6.92 (s, 1H, CH=C), 7.41 (dd, *J* = 8.5, 2.0 Hz, 1H, H6 ), 7.73 (d, *J* = 2.0 Hz, 1H, H2 ); 13C-NMR (75.6 MHz, CDCl3): δ 3.7 (C-CH3), 52.8 (CO2Me), 55.9 (OMe), 57.4 (O-CH2), 73.7 (C-CH3), 84.3 (C≡C(CH3)), 111.0 (C5 ), 115.6 (C2 ), 123.3 (CH=C), 125.6 (C6 ), 125.9 (aryl C), 126.0 (CH=C), 146.7 (C4 ), 150.7 (C3 ), 164.2 (CO2Me); HRMS (+ESI): Found *m*/*z* 324.0944 [M + Na]<sup>+</sup>, C15H15N3O4Na requires 324.0955.

*Methyl (Z)-2-azido-3-(4-(but-2-yn-1-yloxy)-3-methoxyphenyl)acrylate* (**20b**) The *title* compound was prepared as described in GP-2 from 4-(but-2-yn-1-yloxy)-3-methoxybenzaldehyde (19b)(530 mg, 2.6 mmol) and methyl azidoacetate (2.99 g, 26.0 mmol) in anhydrous methanol (30 mL) to give the product (477 mg, 61%) as a pale yellow granular solid; m.p. 94–96 ◦C; IR (KBr): νmax 2920, 2120, 1702, 1592, 1509, 1434, 1377, 1244, 1140, 1011, 801, 763 cm<sup>−</sup>1; UV-vis (CH3CN): λmax 324 nm (ε 14,900 cm−<sup>1</sup> M−1); 1H-NMR (300 MHz, CDCl3): δ 1.87 (t, *J* = 2.3 Hz, 3H, CH3), 3.94 (s, 3H, OMe), 3.94 (s, 3H, CO2Me), 4.77 (q, *J* = 2.3 Hz, 2H, O-CH2), 6.90 (s, 1H, CH=C), 7.05 (d, *J* = 8.5 Hz, 1H, H5 ), 7.38 (dd, *J* = 8.5, 1.8, Hz, 1H, H6 ), 7.53 (d, *J* = 1.8 Hz, 1H, H2 ); 13CNMR (75.6 MHz, CDCl3): δ 3.7 (C-CH3), 52.8 (CO2Me), 55.9 (OMe), 57.1 (O-CH2), 73.5 (C-CH3), 84.4 (C≡C(CH3)), 112.9 (C5 ), 113.3 (C2 ), 123.4 (aryl C), 124.5 (CH=C), 125.8 (C6 ), 126.8 (CH=C), 14.34 (C3 ), 149.0 (C4 ), 164.2 (CO2Me); HRMS (+ESI): Found *m*/*z* 324.0955 [M + Na]<sup>+</sup>, C15H15N3O4Na requires 324.0955.

*Methyl (Z)-2-azido-3-(4-methoxy-3-((3-phenylprop-2-yn-1-yl) oxy)phenyl)acrylate* (**25d**) The *title* compound was prepared as described in GP-2 from 4-methoxy-3-((3-phenylprop-2-yn-1-yl)oxy)benzaldehyde (**24d**) (692 mg, 2.6 mmol) and methyl azidoacetate (2.99 g, 26.0 mmol) in anhydrous methanol (30 mL) to give the product (632 mg, 67%) as a pale yellow granular solid; m.p. 118–120 ◦C; IR (KBr): νmax 2921, 2827, 2119, 1674, 1612, 1580, 1506, 1431, 1378, 1275, 1232, 1129, 1008, 966. 873, 809, 753, 686 cm<sup>−</sup>1; UV-vis (CH3CN): λmax 234 nm (ε 57,700 cm−<sup>1</sup> M−1), 272 (22,600), 306 (21,800); 1H-NMR (300 MHz, CDCl3): δ 3.93 (s, 3H, OMe), 3.96 (s, 3H, CO2Me), 5.04 (d, *J* = 6.0 Hz, 2H, O-CH2), 6.91 (s, 1H, CH≡C), 6.92 (d, *J* = 8.0 Hz, 1H, H5 ), 7.09 (dd, *J* = 8.0, 1.8 Hz, 1H, H6 ), 7.27–7.33 (m, 3H, ArH), 7.43–7.46 (m, 2H, ArH), 7.85 (d, *J* = 1.8 Hz, 1H, H2 ); 13C-NMR (75.6 MHz, CDCl3): δ 52.6 (CO2Me), 55.9 (OMe), 57.5 (O-CH2), 83.9 (C-Ph), 87.5 (C≡C(Ph)), 111.1 (C5 ), 116.0 (C2 ), 120.4 (aryl C), 125.7 (CH=C), 126.0 (C6 ), 126.1 (aryl C), 128.3 (2×aryl CH), 128.6 (CH=C), 130.6 (aryl CH), 131.9 (2×aryl CH), 146.7 (C4 ), 150.9 (C3 ), 164.2 (CO2Me); HRMS (+ESI): Found *m*/*z* 386.1099 [M + Na]<sup>+</sup>, C20H17N3O4Na requires 386.1117.

*Methyl (Z)-2-azido-3-(4-methoxy-3-((2-methylbut-3-yn-2-yl) oxy)phenyl)acrylate* (**25c**) The *title* compound was prepared as described in GP-2 from 4-methoxy-3-((2-methylbut-3-yn-2-yl)oxy)benzaldehyde (**24c**) (567 mg, 2.6 mmol) and methyl azidoacetate (2.99 g, 26.0 mmol) in anhydrous methanol (30 mL) to give the product (507 mg, 62%) as a pale yellow granular solid; m.p. 122–124 ◦C; IR (KBr): νmax 2988, 2835, 2094, 1691, 1619, 1565, 1506, 1425, 1374, 1253, 1138, 1084, 1027, 962. 891, 799, 756, 687 cm−1; UV-vis (CH3CN): λmax 238 nm (ε 16,800 cm−<sup>1</sup> M<sup>−</sup>1), 328 (35,400); 1H-NMR (300 MHz, CDCl3): δ 1.70 (s, 6H, 2 × C-CH3), 2.59 (d, *J* = 0.9 Hz, 1H, CH≡C), 3.87 (s, 3H, OMe), 3.92 (s, 3H, CO2Me), 6.91 (s, 1H, CH=C), 6.90 (d, *J* = 8.5 Hz, 1H, H5 ), 7.45 (dd, *J* = 1.9, 8.5 Hz, 1H, H6 ), 8.16 (d, *J* = 2.0 Hz, 1H, H2 );13C-NMR (75.6 MHz, CDCl3): δ 29.3 (2×C-CH3), 52.7 (CO2Me), 55.7 (OMe), 73.6 (C≡CH), 74.1 (C-(CH3)2), 86.1 (C≡CH), 111.5 (C5 ), 123.2 (C2 ), 124.8 (CH=C), 125.7 (C6 ), 125.9 (aryl C), 127.4 (CH=C), 144.2 (C4 ), 153.9 (C3 ), 164.2 (CO2Me); HRMS (+ESI): Found *m*/*z* 338.1099 [M + Na]<sup>+</sup>, C16H17N3O4Na requires 338.1117.

*Methyl (Z)-2-azido-3-(3-methoxy-4-((2-methylbut-3-yn-2-yl)oxy)phenyl)acrylate* (**20c**) The *title* compound was prepared as described in GP-2 from 3-methoxy-4-((2-methylbut-3-yn-2-yl)oxy)benzaldehyde (**19c**) (567 mg, 2.6 mmol) and methyl azidoacetate (2.1 g, 26. mmol) in anhydrous methanol (30 mL) to give the product (524 mg, 62%) as a pale yellow granular solid; m.p. 116–118 ◦C; IR (KBr): νmax 2975, 2802, 2112, 1703, 1635, 1565, 1512, 1400, 1387, 1212, 1100, 1052, 998, 957, 888, 765, 745, 677 cm−1; UV-vis (CH3CN): <sup>λ</sup>max 328 nm (<sup>ε</sup> 17,100 cm−<sup>1</sup> M−1); 1H-NMR (300 MHz, CDCl3): <sup>δ</sup> 1.72 (s, 6H, 2 <sup>×</sup> C-CH3), 2.60 (d, *J* = 0.9 Hz, 1H, CH≡C), 3.89 (s, 3H, OMe), 3.93 (s, 3H, CO2Me), 6.91 (s, 1H, CH=C), 7.33 (dd, *J* = 8.5, 1.9 Hz, 1H, H6 ), 7.45 (d, *J* = 8.5 Hz, 1H, H5 ), 7.52 (d, *J* = 1.9 Hz, 1H, H2 ); 13C-NMR (75.6 MHz, CDCl3): δ 29.4 (2 × C-CH3), 52.8 (CO2Me), 55.9 (OMe), 73.9 (C≡CH), 73.9 (C-(CH3)2) 85.8 (C≡CH), 113.9 (C5 ), 121.5 (C2 ), 123.8 (CH=C), 123.9 (C6 ), 125.7 (aryl C), 128.5 (CH=C), 146.1 (C3 ), 152.0 (C4 ), 164.1 (CO2Me); HRMS (+ESI): Found *m*/*z* 338.1104 [M + Na]<sup>+</sup>, C16H17N3O4Na requires 338.1117.

*Methyl 6-methoxy-5-(prop-2-yn-1-yloxy)-1H-indole-2-carboxylate* (**26a**) The *title* compound was prepared as described in GP-3 from methyl (*Z*)-2-azido-3-(3-(but-2-yn-1-yloxy)-4-methoxyphenyl)acrylate (**25a**) (596 mg, 2.08 mmol) in xylene (20 mL) to give the product (196 mg, 73%) as a yellow solid; m.p. 164–166 ◦C; IR (KBr): νmax 3325, 3259, 2953, 2925, 2123, 1671, 1624, 1520, 1480, 1440, 1367, 1320, 1249, 1208, 1185, 1146, 1005, 890, 833, 766, 694 cm−1; UV-vis (CH3CN): λmax 208 nm (ε 40,600 cm−<sup>1</sup> M−1), 318 (28,700); 1H-NMR (300 MHz, CDCl3): <sup>δ</sup> 2.55 (t, *J* = 2.4 Hz, 1H, CH≡C), 3.95 (s, 3H, OMe), 3.96 (s, 3H, CO2Me), 4.82 (d, *J* = 2.4 Hz, 2H, O-CH2), 6.90 (d, *J* = 0.8 Hz, 1H, H4), 7.16 (q, *J* = 0.9 Hz, 1H, H3), 7.28 (s, 1H, H7), 8.91 (bs, 1H, NH), 13C-NMR (75.6 MHz, CDCl3): δ 51.8 (CO2Me), 56.0 (OMe), 57.3 (O-CH2), 75.7 (C≡CH), 78.7 (C≡CH), 94.0 (C7), 106.6 (C4), 109.0 (C3), 120.2 (aryl C), 125.9 (C2), 132.9 (aryl C), 143.7 (C6), 150.6 (C5), 162.3 (CO2Me); HRMS (+ESI): Found *m*/*z* 282.0725 [M + Na]<sup>+</sup>, C14H13NO3Na requires 282.0742.

*Methyl 5-methoxy-6-(prop-2-yn-1-yloxy)-1H-indole-2-carboxylate* (**21a**) The *title* compound was prepared as described in GP-3 from methyl (*Z*)-2-azido-3-(4-(but-2-yn-1-yloxy)-3-methoxyphenyl)acrylate (**20a**) (596 mg, 2.08 mmol) in xylene (20 mL) to give the product (183 mg, 68%) as a yellow solid; m.p. 152–154 ◦C; IR (KBr): νmax 3325, 3241, 2999, 2937, 2113, 1681, 1638, 1522, 1475, 1452, 1360, 1280, 1237, 1211, 1195, 1143, 1003, 924, 843, 819, 761, 675 cm<sup>−</sup>1; UV-vis (CH3CN): λmax 209 nm (ε 35,800 cm−<sup>1</sup> M<sup>−</sup>1), 308 (21,400); 1H-NMR (300 MHz, CDCl3): <sup>δ</sup> 2.57 (t, *J* = 2.4 Hz, 1H, CH≡C), 3.95 (s, 3H, OMe), 3.96 (s, 3H, CO2Me), 4.85 (d, *J* = 2.4 Hz, 2H, O-CH2), 7.08 (d, *J* = 0.8 Hz, 1H, H4), 7.11 (s, 1H, H7), 7.15 (q, *J* = 0.9 Hz, 1H, H3), 8.87 (bs, 1H, NH); 13C-NMR (75.6 MHz, CDCl3): δ 51.8 (CO2Me), 56.2 (OMe), 56.9 (O-CH2), 76.1 (C≡CH), 77.4 (C≡CH), 96.8 (C7), 103.0 (C4), 108.7 (C3), 121.4 (aryl C), 126.1 (C2), 131.6 (aryl C), 146.6 (C5), 147.6 (C6), 162.2 (CO2Me); HRMS (+ESI): Found *m*/*z* 282.0726 [M + Na]<sup>+</sup>, C14H13NO3Na requires 282.0742.

*Methyl 5,6-bis(but-2-yn-1-yloxy)-1H-indole-2-carboxylate* (**31**) The *title* compound was prepared as described in GP-3 from methyl (*Z*)-2-azido-3-(3,4-bis(but-2-yn-1-yloxy)phenyl)acrylate (**30**) (704 mg, 2.08 mmol) in xylene (20 mL) to give the product 210 mg, 65%) as a yellow solid; m.p. 146–148 ◦C; IR (KBr): νmax 3332, 2912, 2294, 2120, 1682, 1644, 1519, 1434, 1379, 1244, 1204, 1145, 984, 891, 819, 765 cm<sup>−</sup>1; UV-vis (CH3CN): λmax 210 nm (ε 39,600 cm−<sup>1</sup> M<sup>−</sup>1), 317 (24,900); 1H-NMR (300 MHz, CDCl3): δ 1.87 (q, *J* = 2.4 Hz, 6H, 2 × C-CH3), 3.96 (s, 3H, CO2Me), 4.76 (q, *J* = 2.4 Hz, 2H, O-CH2), 4.79 (q, *J* = 2.4 Hz, 2H, O-CH2), 7.08 (d, *J* = 0.7 Hz, 1H, H4), 7.16 (q, *J* = 0.9 Hz, 1H, H3), 7.25 (s, 1H, H7), 9.03 (bs, 1H, NH); 13C-NMR (75.6 MHz, CDCl3): <sup>δ</sup> 3.7 (2 <sup>×</sup> C-CH3), 51.8 (CO2Me), 57.4 and 57.8 (O-CH2), 73.9 (C-CH3), 74.2 (C-CH3), 83.8 (C≡C(CH3)), 84.2 (C≡C(CH3)), 96.5 (C7), 106.1 (C4), 108.9 (C3), 120.9 (aryl C), 126.0 (C2), 132.5 (aryl C), 144.4 (C5), 148.5 (C6), 162.3 (CO2Me); HRMS (+ESI): Found *m*/*z* 334.1046 [M + Na]<sup>+</sup>, C18H17NO4Na requires 334.1050.

*Methyl 5-(but-2-yn-1-yloxy)-6-methoxy-1H-indole-2-carboxylate* (**26b**) The *title* compound was prepared as described in GP-3 from methyl (*Z*)-2-azido-3-(3-(but-2-yn-1-yloxy)-4-methoxyphenyl)acrylate (**25b**) (626 mg, 2.08 mmol) in xylene (20 mL) to give the product (201 mg, 71%) as a yellow solid; m.p. 160–162 ◦C; IR (KBr): νmax 3324, 3258, 3009, 2918, 2795, 2123, 2108, 1672, 1600, 1520, 1490, 1425, 1400, 1385, 1346, 1249, 1208, 1185, 1146, 1005, 980, 890, 833, 766 cm−1; UV-vis (CH3CN): λmax 208 nm (ε 27,700 cm−<sup>1</sup> M−1), 319 (18,900);1H-NMR (300 MHz, CDCl3): δ 1.88 (q, *J* = 2.4 Hz, 3H, C-CH3), 3.95 (s, 3H, OMe), 3.96 (s, 3H, CO2Me), 4.77 (q, *J* = 2.4 Hz, 2H, O-CH2), 6.88 (d, *J* = 0.7 Hz, 1H, H4), 7.16 (q, *J* = 0.9 Hz, 1H, H3), 7.23 (s, 1H, H7), 8.82 (bs, 1H, NH); 13C-NMR (75.6 MHz, CDCl3): δ 5.2 (C-CH3), 53.2 (CO2Me), 57.4 (OMe), 59.1 (O-CH2), 75.5 (C-CH3), 85.3 (C≡C), 95.2 (C7), 107.1 (C4), 110.4 (C3), 121.7 (aryl C), 127.1 (C2), 134.0 (aryl C), 145.5 (C6), 151.9 (C5), 163.7 (CO2Me); HRMS (+ESI): Found *m*/*z* 296.0884 [M + Na]<sup>+</sup>, C15H15NO4Na requires 296.0899.

*Methyl 6-(but-2-yn-1-yloxy)-5-methoxy-1H-indole-2-carboxylate* (**21b**) The *title* compound was prepared as described in GP-3 from methyl (*Z*)-2-azido-3-(4-(but-2-yn-1-yloxy)-3-methoxyphenyl)acrylate (**20b**) (626 mg, 2.08 mmol) in xylene (20 mL) to give the product (190 mg, 67%) as a yellow solid; m.p. 164–166 ◦C; IR (KBr): νmax 3324, 3224, 3005, 2936, 2835, 2215, 2108, 1680, 1584, 1495, 1455, 1433, 1362, 1285, 1243, 1228, 1146, 1045, 988, 946, 846, 819, 760, 672 cm<sup>−</sup>1; UV-vis (CH3CN): λmax 210 nm (ε 37,100 cm−<sup>1</sup> M<sup>−</sup>1), 319 (22,300); 1H-NMR (300 MHz, CDCl3): δ 1.87 (q, *J* = 2.4 Hz, 3H, C-CH3), 3.94 (s, 3H, OMe), 3.96 (s, 3H, CO2Me), 4.80 (q, *J* = 2.4 Hz, 2H, O-CH2), 7.06 (d, *J* = 0.7 Hz, 1H, H4), 7.08 (s, 1H, H7), 7.14 (q, *J* = 0.9 Hz, 1H, H3), 9.01 (bs, 1H, NH); 13C-NMR (75.6 MHz, CDCl3): δ 3.7 (C-CH3), 51.8 (CO2Me), 56.1 (OMe), 57.4 (O-CH2), 73.8 (C-CH3), 84.3 (C≡C(CH3)), 96.2 (C7), 102.7 (C4), 108.7 (C3), 121.0 (aryl C), 125.8 (C2), 131.8 (aryl C), 146.1 (C5), 148.0 (C6), 162.3 (CO2Me); HRMS (+ESI): Found *m*/*z* 296.0883 [M + Na]<sup>+</sup>, C15H15NO4Na requires 296.0889.

*Methyl 6-methoxy-5-((3-phenylprop-2-yn-1-yl)oxy)-1H-indole-2-carboxylate* (**26d**) The *title* compound was prepared as described in GP-3 from methyl (Z)-2-azido-3-(4-methoxy-3-((3-phenylprop-2-yn-1-yl)oxy)phenyl)acrylate (**25d**) (754 mg, 2.08 mmol) in xylene (20 mL) to give the product (257 mg, 74%) as a yellow solid; m.p. 138–140 ◦C; IR (KBr): νmax 3326, 2984, 2924, 2740, 2113, 1675, 1645, 1520, 1480, 1439, 1381, 1247, 1195, 1145, 996, 845, 823, 761, 672 cm−1; UV-vis (CH3CN): λmax 203 nm (ε 58,900 cm−<sup>1</sup> M−1), 318 (22,200); 1H-NMR (300 MHz, CDCl3): δ 3.95 (s, 3H, OMe), 3.98 (s, 3H, CO2Me), 5.04 (s, 2H, O-CH2), 6.90 (s, 1H, H4), 7.17 (q, *J* = 0.9 Hz, 1H, H3), 7.31 (s, 1H, H7), 7.32–7.36 (m, 3H, ArCH), 7.44–7.47 (m, 2H, ArCH), 8.84 (bs, 1H, NH); 13C-NMR (75.6 MHz, CDCl3): <sup>δ</sup> 51.8 (CO2Me), 56.0 (OMe), 58.2 (O-CH2), 84.1 (C≡C(Ph)), 87.4 (C-Ph), 93.9 (C7), 106.6 (C4), 109.0 (C3), 120.3 (aryl C), 122.4 (aryl C), 125.8 (C2), 128.2 (2 × aryl CH), 128.5 (aryl CH), 131.8 (2 × aryl CH), 132.8 (aryl C), 144.0 (C6), 150.7 (C5), 162.2 (CO2Me); HRMS (+ESI): Found *m*/*z* 336.1221 [M + H]<sup>+</sup>, C20H18NO4 requires 336.1236.

*Methyl 5,10-dihydro-7H-dipyrano[3,2-e:2 ,3 -g]indole-6-carboxylate* (**10**) *Method 2: Route 2* The title compound was prepared as described in GP-3 from methyl (Z)-2-azido-3-(3,4-bis(prop-2-yn-1-yloxy)phenyl) acrylate (**9**) (646 mg, 2.08 mmol) in xylene (20 mL) to give the product (61.4 mg, 72%) as a yellow solid; m.p. 168–170 ◦C; IR (KBr): νmax 3415, 3328, 2947, 2831, 2341, 2116, 1674, 1640, 1575, 1524, 1486, 1441, 1400, 1274, 1213, 1158, 1135, 1025, 1000, 920, 813, 755 cm−1; UV-vis (CH3CN): λmax 225 nm (ε 24,100 cm−<sup>1</sup> M−1), 269 (13,800), 360 (13,000); 1H-NMR (300 MHz, CDCl3): δ 3.96 (s, 3H, CO2Me), 4.91 (dd, J = 3.8, 1.8 Hz, 2H, O-CH2), 4.94 (dd, *J* = 3.8, 1.8 Hz, 2H, O-CH2), 5.87–5.97 (m, 2H, H3 and H9), 6.72–6.80 (m, 2H, H4 and H8), 7.19 (d, *J* = 2.1 Hz, 1H, H5), 8.85 (s, 1H, NH); 13C-NMR (75.6 MHz, CDCl3): δ52.0 (CO2Me), 65.7 (O-CH2), 65.8 (O-CH2), 106.5 (C5), 107.0 (aryl C), 115.3 (aryl C), 118.8 (C4), 119.3 (C8), 120.8 (aryl C), 121.8 (C3), 121.9 (C9), 126.5 (C6), 128.8 (aryl C), 138.1 (aryl C), 141.7 (aryl C), 162.3 (CO2Me); HRMS (+ESI): Found *m*/*z* 306.0736 [M + Na]<sup>+</sup>, C16H13NO4Na requires 306.0737.

This compound was also prepared by the methods described below. Method 1: The title compound was prepared as described in GP-6 from methyl 5,6-bis(prop-2-yn-1-yloxy)-1*H*-indole-2-carboxylate (**16**) (118 mg, 0.42 mmol) in chlorobenzene (30 mL) to give the product (43.5 mg, 51%) as a yellow solid.

Method 2: Route 1 The title compound was prepared as described in GP-6 from methyl (*Z*)-2-azido-3-(2*H*,10*H*-pyrano[4,3-h]chromen-5-yl)acrylate (**17**) (646 mg, 2.08 mmol) in xylene (20 mL) to give the product (52.9 mg, 62%) as a yellow solid.

*Methyl 5-methoxy-3,7-dihydropyrano[3,2-e]indole-2-carboxylate* (**27a**) The title compound was prepared as described in GP-6 from methyl 6-methoxy-5-(prop-2-yn-1-yloxy)-1H-indole-2-carboxylate (**26a**) (108 mg, 0.42 mmol) in chlorobenzene (30 mL) to give the product (80 mg, 74%) as a yellow solid; m.p. 166–168 ◦C; IR (KBr): νmax 3325, 2948, 2839, 2340, 2110, 1677, 1637, 1515, 1439, 1271, 1193, 1143, 1098,

998, 931, 883, 808, 752 cm−1; UV-vis (CH3CN): λmax 212 nm (ε 37,400 cm−<sup>1</sup> M−1), 271 (13,300), 322 (30,600); 1H-NMR (300 MHz, CDCl3): δ 3.92 (s, 3H, OMe), 3.93 (s, 3H, CO2Me), 4.90 (dd, *J* = 3.7, 1.7 Hz, 2H, O-CH2), 5.89–5.95 (m, 1H, H8), 6.76–6.79 (m, 1H, H9), 6.79 (s, 1H, H4), 7.17 (d, *J* = 2.1 Hz, 1H, H1), 8.96 (s, 1H, NH); 13C-NMR (75.6 MHz, CDCl3): δ 51.8 (CO2Me), 56.0 (OMe), 65.7 (O-CH2), 93.8 (C4), 106.1 (C1), 114.9 (aryl C), 118.3 (aryl C), 121.6 (C9), 121.8 (C8), 126.0 (C2), 132.2 (aryl C), 139.3 (aryl C), 148.8 (C5), 162.2 (CO2Me); HRMS (+ESI): Found *m*/*z* 282.0731 [M + Na]<sup>+</sup>, C14H13NO4Na requires 282.0737.

*Methyl 5-methoxy-1,7-dihydropyrano[2,3-g]indole-2-carboxylate* (**22a**) The *title* compound was prepared as described in GP-6 from methyl 5-methoxy-6-(prop-2-yn-1-yloxy)-1*H*-indole-2-carboxylate (**21a**) (108 mg, 0.42 mmol) in chlorobenzene (30 mL) to give the product (83 mg, 77%) as a yellow solid; m.p. 188–190 ◦C; IR (KBr): νmax 3327, 2944, 2827, 2366, 2106, 1677, 1528, 1444, 1306, 1221, 1148, 1101, 989, 955, 831, 755 cm<sup>−</sup>1; UV-vis (CH3CN): λmax 226 nm (ε 28,100 cm−<sup>1</sup> M<sup>−</sup>1), 290 (16,700), 339 (18,400); 1H-NMR (300 MHz, CDCl3): δ 3.94 (s, 3H, OMe), 3.96 (s, 3H, CO2Me), 4.96 (dd, *J* = 3.8, 1.8 Hz, 2H, O CH2), 5.89–5.95 (m, 1H, H8), 6.75–6.79 (m, 1H, H9), 7.02 (s, 1H, H4), 7.13 (d, *J* = 2.1 Hz, 1H, H3), 8.94 (s, 1H NH); 13C-NMR (75.6 MHz, CDCl3): δ51.9 (CO2Me), 56.2 (OMe), 65.7 (O-CH2), 103.1 (C4), 107.1 (aryl C), 109.2 (C3), 119.3 (aryl C), 120.8 (C8), 121.0 (C9), 126.0 (C2), 128.7 (aryl C), 137.6 (aryl C), 145.2 (C5), 162.3 (CO2Me); HRMS (+ESI): Found *m*/*z* 282.0738 [M + Na]<sup>+</sup>, C14H13NO4Na requires 282.0737.

*Methyl 5-methoxy-9-methyl-3,7-dihydropyrano[3,2-e]indole-2-carboxylate* (**27b**) The *title* compound was prepared as described in GP-6 from methyl 5-(but-2-yn-1-yloxy)-6-methoxy-1*H*-indole-2-carboxylate (**26b**) (114 mg, 0.42 mmol) in chlorobenzene (30 mL) to give the product (62 mg, 54%) as a yellow solid; m.p. 196–198 ◦C; IR (KBr): νmax 3310, 2948, 2925, 2833, 2106, 1675, 1640, 1517, 1495, 1439, 1366, 1274, 1265, 1194, 1146, 1080, 1004, 960, 880, 815, 765, 672 cm<sup>−</sup>1; UV-vis (CH3CN): λmax 213 nm (ε 28,900 cm−<sup>1</sup> M<sup>−</sup>1), 267 (9,500), 322 (24,200); 1H-NMR (300 MHz, CDCl3): δ 1.63 (s, 3H, C-CH3), 3.94 (s, 3H, OMe), 3.95 (s, 3H, CO2Me), 4.77 (q, *J* = 2.4 Hz, 2H, O-CH2), 5.69–5.72 (m, 1H, H8), 6.87 (s, 1H, H1), 7.23 (s, 1H, H4), 8.82 (bs, 1H, NH); 13C-NMR (75.6 MHz, CDCl3): δ 22.6 (C-CH3), 51.8 (CO2Me), 56.1 (OMe), 65.2 (O-CH2), 93.8 (C4), 109.3 (C1), 117.4 (aryl C), 117.9 (aryl C), 118.3 (C8), 126.5 (C2), 132.0 (C-CH3), 132.9 (aryl C), 140.4 (aryl C), 149.0 (C5), 162.2 (CO2Me); HRMS (+ESI): Found *m*/*z* 296.0887 [M + Na]+, C15H15NO4Na requires 296.0893.

*Methyl 5-methoxy-9-methyl-1,7-dihydropyrano[2,3-g]indole-2-carboxylate* (**22b**) The *title* compound was prepared as described in GP-6 from methyl 6-(but-2-yn-1-yloxy)-5-methoxy-1*H*-indole-2-carboxylate (**21b**) (114 mg, 0.42 mmol) in chlorobenzene (30 mL) to give the product (56 mg, 49%) as a yellow solid; m.p. 176–178 ◦C; IR (KBr): νmax 3410, 2980, 2930, 2845, 1695, 1645, 1606, 1534, 1445, 1430, 1385, 1375, 1232, 1189, 1139, 1096, 1040, 998, 981, 927, 857, 744, 712cm<sup>−</sup>1; UV-vis (CH3CN): λmax 224 nm (ε 32,500 cm−<sup>1</sup> M<sup>−</sup>1), 286 (22,200), 337 (23,500); 1H-NMR (300 MHz, CDCl3): δ 1.71 (s, 3H, C-CH3), 3.94 (s, 3H, OMe), 3.96 (s, 3H, CO2Me), 4.77 (q, *J* = 1.6 Hz, 2H, O-CH2), 5.65–5.68 (m, 1H, H8), 7.05 (s, 1H, H4), 7.14 (d, *J* = 2.1 Hz, 1H, H3), 8.81 (bs, 1H, NH), 13C-NMR (75.6 MHz, CDCl3): δ 20.8 (C-CH3), 51.8 (CO2Me), 56.2 (OMe), 65.4 (O-CH2), 103.2 (C4), 108.9 (C3), 109.9 (aryl C), 117.7 (C8), 121.9 (aryl C), 126.0 (C2), 128.7 (C-CH3), 129.3 (aryl C), 144.2 (aryl C), 145.4 (C5), 162.1 (CO2Me); HRMS (+ESI): Found *m*/*z* 296.0886 [M + Na]<sup>+</sup>, C15H15NO4Na requires 296.0893.

*Methyl 5-methoxy-9-phenyl-3,7-dihydropyrano[3,2-e]indole-2-carboxylate* (**27d**) The *title* compound was prepared as described in GP-6 from methyl 6-methoxy-5-((3-phenylprop-2-yn-1-yl)oxy)-1*H*-indole-2-carboxylate (**26d**) (140 mg, 0.42 mmol) in chlorobenzene (30 mL) to give the product (113 mg, 81%) as a yellow solid; m.p. 188–190 ◦C; IR (KBr): νmax 3311, 2949, 2816, 2114, 1684, 1639, 1598, 1515, 1500, 1438, 1400, 1360, 1260, 1241, 1193, 1141, 1042, 1011, 995, 920, 823, 759, 703 cm−1; UV-vis (CH3CN): λmax 274 nm (ε 11,600 cm−<sup>1</sup> M−1), 326 (26,400); 1H-NMR (300 MHz, CDCl3): δ 3.82 (s, 3H, OMe), 4.00 (s, 3H, CO2Me), 4.85 (d, *J* = 4.4 Hz, 2H, O-CH2), 5.94–5.97 (m, 1H, H8), 5.98 (s, 1H, H4), 6.88 (s, 1H, H1), 7.30–7.34 (m, 2H, ArH), 7.41–7.44 (m, 3H, ArH), 8.76 (bs, 1H, NH), 13C-NMR (75.6 MHz, CDCl3): δ 51.7 (CO2Me), 58.1 (OMe), 65.1 (O-CH2), 94.2 (C4), 109.4 (C1), 117.0 (aryl C), 117.5 (aryl C), 125.0 (C2), 120.4 (C8), 127.9 (aryl CH), 128.3 (2 × arylCH), 128.5 (2 × aryl CH), 132.8 (aryl C), 139.4 (C-Ph), 139.4 (aryl C), 141.1 (aryl C), 149.0 (C5), 162.1 (CO2Me); HRMS (+ESI): Found *m*/*z* 358.1057 [M + Na]<sup>+</sup>, C20H17NO4Na requires 358.1055.

*Methyl 5-methoxy-7,7-dimethyl-3,7-dihydropyrano[3,2-e] indole-2-carboxylate* (**27c**) The title compound was prepared as described in GP-3 from methyl (*Z*)-2-azido-3-(4-methoxy-3-((2-methylbut-3-yn-2-yl)oxy)phenyl)acrylate (**25c**) (654 mg, 2.08 mmol) in xylene (20 mL) to give the product (220 mg, 68%) as a yellow solid; m.p. 144–146 ◦C; IR (KBr): νmax 3316, 2962, 2975, 2111, 1681, 1625, 1600, 1510, 1439, 1400, 1366, 1270, 1230, 1192, 1131, 1075, 999, 934, 887, 818, 754, 728 cm<sup>−</sup>1; UV-vis (CH3CN): λmax 219 nm (ε 40,000 cm−<sup>1</sup> M<sup>−</sup>1), 271 (13,500), 323 (31,100); 1H-NMR (300 MHz, CDCl3): <sup>δ</sup> 1.53 (s, 6H, 2 <sup>×</sup> CH3), 3.92 (s, 3H, OMe), 3.95 (s, 3H, CO2Me), 5.73 (d, *J* = 9.7 Hz, 1H, H8), 6.68 (d, *J* = 9.7 Hz, 1H, H9), 6.79 (s, 1H, H4), 7.19 (d, *J* = 2.1 Hz, 1H, H1), 9.00 (bs, 1H, NH); 13C-NMR (75.6 MHz, CDCl3): <sup>δ</sup> 27.3 (2 <sup>×</sup> C-CH3), 51.7 (CO2Me), 58.1 (OMe), 76.2 (C-(CH3)2), 93.1 (C4), 106.1 (C1), 113.4 (aryl C), 118.2 (aryl C), 119.3 (C9), 125.7 (C2), 130.6 (C8), 132.0 (aryl C), 138.1 (aryl C), 149.5 (C5), 162.3 (CO2Me); HRMS (+ESI): Found *m*/*z* 310.1044 [M + Na]+, C16H17NO4Na requires 310.1050.

*Methyl 5-methoxy-7,7-dimethyl-1,7-dihydropyrano[2,3-g] indole-2-carboxylate* (**22c**) The title compound was prepared as described in GP-3 from methyl (*Z*)-2-azido-3-(3-methoxy-4-((2-methylbut-3-yn-2-yl)oxy)phenyl)acrylate (**20c**) (654 mg, 2.08 mmol) in xylene (20 mL) to give the product (203 mg, 74%) as a yellow solid; m.p. 158–160 ◦C; IR (KBr): νmax 3338, 2955, 2919, 2845, 2111, 1676, 1640, 1595, 1534, 1444, 1370, 1350, 1310, 1265, 1217, 1195, 1134, 1099, 1050, 985, 960, 880, 826, 760, 714 cm<sup>−</sup>1; UV-vis (CH3CN): λmax 226 nm (ε 29,600 cm−<sup>1</sup> M<sup>−</sup>1), 262 (15,200), 341 (19,000); 1H-NMR (300 MHz, CDCl3): <sup>δ</sup> 1.55 (s, 6H, 2 <sup>×</sup> CH3), 3.93 (s, 3H, OMe), 3.96 (s, 3H, CO2Me), 5.71 (d, *J* = 9.7 Hz, 1H, H8), 6.66 (d, *J* = 9.7 Hz, 1H, H9), 7.02 (s, 1H, H4), 7.13 (d, *J* = 2.1 Hz, 1H, H3), 8.95 (bs, 1H, NH); 13C-NMR (75.6 MHz, CDCl3): <sup>δ</sup> 27.4 (2 <sup>×</sup> C-CH3), 51.8 (CO2Me), 56.5 (OMe), 76.6 (C-(CH3)2), 103.3 (C4), 105.8 (aryl C), 109.3 (C3), 116.8 (C9), 120.6 (aryl C), 125.7 (C2), 128.9 (aryl C), 129.8 (C8), 142.3 (aryl C), 149.9 (C5), 162.4 (CO2Me); HRMS (+ESI): Found *m*/*z* 288.1228 [M + H]<sup>+</sup>, C16H18NO4 requires 288.1236.

#### *3.2. Cell Biology Techniques*

The SH-SY5Y and Kelly human neuroblastoma cell lines were generously donated by Dr. J. Biedler (Memorial Sloan-Kettering Cancer Center, New York, NY, USA). The MDA-MB-231 and MCF-7 breast cancer cell lines were purchased from the American Type Culture Collection. All cell lines were cultured under standard conditions at 37 ◦C in 5% CO2 as an adherent monolayer in Dulbecco's modified Eagle's medium supplemented with l-glutamine (DMEM) (Invitrogen, Waltham, MA, USA) and 10% fetal calf serum (FCS) (Thermo Fisher Scientific, Waltham, MA, USA).

#### Method for Cell Viability Assays

Cell viability was measured by the standard Alamar blue assay, as previously described [18]. Briefly, cells were allowed to attach for 24 h in 96-well culture plates. The cells were then continuously exposed to serial dilutions of the hydrazide-hydrazone derivatives for 72 h, either in the presence or absence of SAHA (0.5 or 1 μM), with five replicate wells for each determination. Cell viability was determined by the addition of 22 μL of Alamar blue reagent, recorded at comparative 0 h and 5 h values, using a Wallac 1420 Victor III spectrophotometer (GMI, Ramsey, MN, USA), which measured light absorbance in each well at 570 nm. The cell viability of each plate was calculated as a percentage compared to matched DMSO controls (0.5%). The mean (+/'SEM) is shown for three independent experiments.

#### *3.3. Statistical Analysis*

Results of the cell viability studies were statistically analyzed using the two-tailed, unpaired Student's t-test. Results are expressed as mean values with 95% confidence intervals.

#### **4. Conclusions**

The studies presented in this study have contributed to the investigation of the potential of dihydropyranoindoles to act as SAHA enhancers for the treatment of neuroblastoma and breast cancer cells. To the best of our knowledge, this is the first study which was carried out on the combination of SAHA with dihydropyranoindoles. The desired tricyclic and tetracyclic dihydropyranoindoles was achieved by the reaction of corresponding hydroxybenzaldehydes with haloalkynes followed by the application of the Hemetsberger indole synthesis to yield the related indoles. Claisen cyclization in the presence of chlorobenzene afforded the desired compounds in good yields. Some of the dihydropyranoindoles were successfully synthesized from the corresponding azido intermediates in one step by the thermal decomposition step of the Hemetsberger indole synthesis. The biological studies revealed that the breast cancer cells displayed significant resistance towards the combination treatments of the dihydropyranoindoles compared to the neurobalstoma cells. It was also found that tetracyclic analogues of dihydropyranoindoles **33** and **10** were found to be more favorable for the enhancement of SAHA activity, while only dihydropyrano[3,2-*e*]indole tricyclic systems **27a** and **27c** resulted in the additional reduction of the SAHA cytotoxicity against the neuroblastoma cell lines. Taken altogether, the tetracyclic dihydropyranoindole **10** was determined as the most cytotoxic compound at the concentration 20 μM, while the lower concentration of the designated compound displayed the best enhancement on SAHA activity with the value of 44% additional reduction at the ratio of 1:10 SAHA to compound.

**Supplementary Materials:** The following figures are available online: **Figure S1** Cell viability of A) SH-SY5Y, B) MDA-MB-231 cancer cells treated with the selected six compounds **1**–**6** (10 μM) over 72 h. Error bars represent mean values (±S.D.) for three independent determinations. **Figure S2**. Comparative toxicity of compounds **1, 3** and **4** (10 μM) against SH-SY5Y, MDA-MB-231 and MRC-5 cell lines after 72 h exposure. Error bars represent mean values (±S.D.) for three independent determinations. **Figure S3**. Cell viability of A) MCF-7 *p* < 0.01 B) MDA-MB-231 *p* < 0.05 breast cancer cells treated with 10 μM compounds over 72 h, in the presence or absence of 1 μM of SAHA. Error bars represent mean values (±S.D.) for three independent determinations **Figure S4**. Cell viability of A) Kelly *p* ≥ 0.05 (ns) B) SH-SY5Y *p* < 0.0005 neuroblastoma cancer cellstreated with 10 μM compounds over 72 h, in the presence or absence of 1 μM of SAHA. Error bars represent mean values (±S.D.) for three independent determinations. **Figure S5**. Cell viability of KELLY neuroblastoma cancer cells treated with compounds A) **33** *p* < 0.01 B) **27c** *p* < 0.05 and C) **10** *p* < 0.05 at different concentrations (0.01, 0.1, 1, 10, 20 μM) over 72 h in the absence and presence of SAHA. Error bars represent mean values (±S.D.) for three independent determinations. **Figure S6**. Cell viability of SH-SY5Y neuroblastoma cancer cells treated with compounds A) **27a**, *p* < 0.0005 B) **33**, *p* < 0.0005 C) **10**, *p* < 0.01 and D) **27c**, *p* < 0.01 at different concentrations (0.01, 0.1, 1, 10, 20 μM) over 72 h in the absence and presence of SAHA. Error bars represent mean values (±S.D.) for three independent determinations. **Figure S7**. Comparative toxicity of compounds A) **27a**, *p* < 0.05 B) **33**, *p* < 0.05 C) **10**, *p* < 0.05 and D) **27c**, *p* < 0.0005 (10 μM) against cancer cells (Kelly, SH-SY5Y, MCF-7 and MDA-MB-231) and human lung fibroblasts (WI-38 and MRC-5) cell lines after 72 h exposure. Error bars represent mean values (±S.D.) for three independent determinations.

**Author Contributions:** N.K., D.S.B., G.M.M. and B.B.C. conceived and designed the experiments, G.M.A. provided the data from library screening, M.B. performed the experiments, analyzed, interpreted the data and wrote the paper, N.K. and G.M.M. supported financially. All authors have read and agree to the published version of the manuscript.

**Funding:** This research was funded by Australian Research Council Discovery Project, grant number DP180100845.

**Acknowledgments:** We thank the University of New South Wales, and the Australian Research Council (ARC) for funding to N.K. and D.S.B. Mass spectrometric analysis for this work was carried out at the Bioanalytical Mass Spectrometry Facility, UNSW and was supported in part by infrastructure funding from the New South Wales Government as part of its co-investment in the National Collaborative Research Infrastructure Strategy. This research was supported by Australian Postgraduate Research Scholarships, University of NSW, Program Grants from the NHMRC Australia, Cancer Institute NSW Australia and Cancer Council NSW Australia. The Children's Cancer Institute Australia for Medical Research is affiliated with the University of NSW and Sydney Children's Hospital.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


**Sample Availability:** Samples of the compound are not available from the authors.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Design, Synthesis, and In Vitro Evaluation of the Photoactivatable Prodrug of the PARP Inhibitor Talazoparib**

**Jiaguo Li 1,2, Dian Xiao 2, Lianqi Liu 2, Fei Xie 2, Wei Li 2, Wei Sun 1,\*, Xiaohong Yang 1,\* and Xinbo Zhou 2,\***


Academic Editor: Qiao-Hong Chen Received: 26 November 2019; Accepted: 16 January 2020; Published: 18 January 2020

**Abstract:** In this article, we report the design, synthesis, photodynamic properties, and in vitro evaluation of photoactivatable prodrug for the poly (ADP-ribose) polymerase 1 (PARP-1) inhibitor Talazoparib. In order to yield a photoactivatable, inactive prodrug, photoactivatable protecting groups (PPGs) were employed to mask the key pharmacophore of Talazoparib. Our study confirmed the good stability and photolytic effect of prodrugs. A PARP-1 enzyme inhibition assay and PARylation experiment showed that the inhibitory activity of the prodrug was reduced 380 times and more than 658 times, respectively, which proved that the prodrug's expected activity was lost after PPG protection. In *BRCA1*- and *BRCA2*-deficient cell lines, the inhibitory activity of the compound was significantly restored after ultraviolet (UV) irradiation. The results indicate that the photoactivatable prodrug strategy is an interesting approach for studying PARP inhibitors. Meanwhile, the described photoactivatable prodrug also provided a new biological tool for the mechanism research of PARP.

**Keywords:** Talazoparib; PARP inhibitor; prodrug; o-nitro-benzyl; photoactivatable protecting groups

### **1. Introduction**

DNA repair in normal cells mainly relies on the base excision repair (BER) pathway for single-strand DNA breaks and the homologous recombination (HR) pathway for double-strand DNA breaks [1]. Poly (ADP-ribose) polymerase (PARP) plays a key role in the repair of single-strand DNA breaks, through its ability to bind to DNA gaps and recruit other DNA repair enzymes in the BER pathway [1–4]. The tumor suppressor proteins encoded by the breast cancer genes *BRCA1* and *BRCA2* participate in the HR of double-strand DNA breaks [5]. Usually, in tumor cells with *BRCA1* and *BRCA2* mutations or defects, the inhibition of the PARP enzyme can lead to the accumulation of single-strand DNA breaks, owing to the HR repair pathway being blocked, which causes further double-strand DNA breaks and finally results in the death of tumor cells [6–8]. Currently, PARP inhibitors have been a hot topic in the tumor research area. Among them, Olaparib, Rucaparib, Niraparib, and Talazoparib have been approved for the treatment of *BRCA*-deficient breast, ovarian, fallopian tube, and primary peritoneal cancers [9]. In addition, PARP inhibitors are also used in combination with some kinase inhibitors, and show significant synergistic antitumor effects on prostate cancer, pancreatic cancer, gastric cancer, acute leukemia, and non-small-cell lung cancer [10–13]. The number of diseases illustrate the huge potential for therapeutic agents and for biological probes in PARP research.

Talazoparib (trade name: Talzenna), an oral PARP inhibitor developed by Pfizer, was approved by the U.S. Food and Drug Administration (FDA) on 16 October 2018 [14,15]. It is currently the most active compound among the PARP inhibitors, with an IC50 of 0.57 nM, which is 4–10 times lower than that of the other PARP inhibitors [16]. The clinical dose of Talazoparib is only 1 mg (once a day). In contrast, to achieve similar effects as Talazoparib, the dosage of Olaparib [17] and Niraparib [18] are 400 mg (twice a day) and 300 mg (once a day), respectively. In addition, while Talazoparib is highly effective in killing tumor cells, there may still be serious side effects, such as myelodysplastic syndrome or acute myeloid leukemia [14,19]. In light of the immense significance of PARP inhibitors, we aimed to develop a relevant, photoactivatable Talazoparib prodrug.

Photoactivatable prodrugs are usually designed by blocking a key pharmacophore moiety of the inhibitor using photoactivatable protecting groups (PPGs), which have been widely used in biology and medicine in recent years as a non-invasive approach [20–22]. This method can provide temporal and spatial control for the release of bioactive substance by UV irradiation. Thus, highly active inhibitors can be generated in irradiated areas of interest at defined points in time. The photoactivatable prodrugs can serve as a novel biological tool for kinetic or mechanistic studies. Moreover, blocked inhibitors may minimize the systemic side effects of Talazoparib, in order to enable a higher dosage of inactive prodrugs.

The o-nitrobenzyl system and its derivatives are one of the most commonly used PPGs [23–26]. The photolysis mechanism is intramolecular rearrangement. As shown in Figure 1, under 365 nm UV irradiation, carbonyl compounds containing o-nitrobenzyl can form a highly active bi-radical, which will undergo hydrogen abstraction on the carbon atom at the γ position and release the parent drugs [26–29]. The advantages of such PPG technology are a high release speed and easy chemical synthesis. At the same time, the UV release wavelength of 365 nm involves less DNA damage than wavelengths shorter than 300 nm, and is harmless to tissues and cells at low doses [29,30]. This method has potential clinical application value [25,26]. For example, irradiation can be temporarily applied to the lesion site during or after surgery to release active substances for killing residual cancer cells, thereby preventing postoperative recurrence. Optical fibres and endoscopic probes can also be used to transmit the required light to the action site to treat some superficial tumors.

**Figure 1.** Photolysis mechanism of o-nitrobenzyl upon 365 nm UV irradiation and the inferred drug release process of the Talazoparib prodrug.

In this study, we report the design, synthesis, photodynamic properties, and in vitro evaluation of a photoactivatable prodrug for the PARP-1 inhibitor Talazoparib. We used the concept of PPGs to covalently bond o-nitrobenzyl derivatives (herein designated as compound **2**) to the lactam pharmacophore of Talazoparib, under the guidance of molecular docking; this disrupts its key hydrogen bonding interaction with the PARP protein residue to yield an inactive photoactivatable

prodrug, i.e., compound **3**. Our research proved that newly designed prodrug **3** showed good stability and could be rapidly deprotected after UV radiation. A PARP-1 enzyme inhibition assay and PARylation experiment showed that inhibitory activity of the PPG-protected prodrug was greatly reduced by 380 and more than 658 times, respectively. In addition, in *BRCA1*- and *BRCA2*-deficient cell lines, the inhibitory activity of the prodrug **3** was significantly restored after transitory UV irradiation, and the data implies that inhibitory activity could increase with prolonged irradiation time. The results of this preliminary study indicate that the photoactivatable prodrug strategy was an interesting approach for studying PARP inhibitors. Meanwhile, the described photoactivatable prodrugs also provide a novel biological tool for the signal transduction research.

#### **2. Results and Discussion**

#### *2.1. Molecular Modelling*

Molecular docking of the Tazaloparib into the catalytic domain of PARP-1 (catPARP-1) revealed that its lactam moiety was the key pharmacodynamic group (PDB ID 4PJT). N and O atoms on the lactam moiety could form hydrogen bonds with the Gly863 and Ser904 residues of PARP-1, respectively (Figure 2A) [31,32]. To block its pharmacophoric features, we linked PPGs (namely, compound **2**) to the lactam moiety to form compound **3**, and simulated the action mode of this compound in the same activity pocket. Consistent with our hypothesis, no proper binding of compound **3** to the enzyme active sites were found during the docking; that is, the PPGs blocked the interaction of the lactam moiety with the Gly863 and Ser904 residues (Figure 2B). In addition, the introduction of PPGs led to spatial conflict, which rendered compound **3** unable to maintain the same binding mode as Talazoparib, and the whole molecule was turned over by a certain angle degree (Figure 2C). Inspired by this docking result, we synthesised compound **3** and carried out the following series of photochemical characterisations and biological evaluations.

**Figure 2.** Predicted ligand binding models relative to the catalytic domain of poly (ADP-ribose) polymerase 1 (catPARP-1). (**A**) The co-crystallized binding modes of Talazoparib and the catPARP-1 protein were taken from PDB ID 4PJT. (**B**) Predicted co-crystallized binding poses of compound **3** in a complex with catPARP-1. (**C**) Superimposition of the binding models of Tazaloparib (blue) and compound **3** (red) with catPARP-1 protein (green).

#### *2.2. Chemistry*

As shown in Scheme 1, Talazoparib could generate the intermediate compound **4** under the action of n-Butyllithium. Compound **4** subsequently underwent a nucleophilic substitution reaction with compound **2** to generate the photoactivatable target compound **3**.

**Scheme 1.** Synthesis of compound **3**. Reagents: (a) n-BuLi, tetrahydrofuran (THF), N2, –80 ◦C, 10 min; (b) THF, N2, 0 ◦C, 1 h.

#### *2.3. Stability Assays and UV Cleavage Test In Vitro*

#### 2.3.1. UV Stability of Talazoparib

Talazoparib should have sufficient stability at the light radiation wavelength used; otherwise, it will be degraded immediately after release or even before the breaking of the PPG covalent bond. Therefore, we first evaluated the UV stability of Talazoparib at a wavelength of 365 nm. To this end, we used a UV cross-linker with an emission wavelength of 365 nm (4.1 mWs/cm2) to irradiate 2.63 mM Talazoparib in methanol, and took aliquots of the sample at different times for high-performance liquid chromatography (HPLC) analysis. The result indicated that there was no significant loss of Talazoparib in the peak area within 10 min, showing good UV stability (Figure 3A). In addition, the UV/vis absorption spectra of Talazoparib and compound **3** was shown in supplementary materials (See Figure S1).

**Figure 3.** Stability assays and UV cleavage test in vitro. Compounds were irradiated at 365 nm (4.1 mW/cm2). Samples were determined on a C18 column using a water/acetonitrile (ACN) gradient. The detection wavelength for high-performance liquid chromatography (HPLC) analysis was 220 nm. The peak area or peak area percentage was plotted against the irradiation time. (**A**) UV stability of Talazoparib: 2.63 mM Talazoparib in methanol were irradiated up to 10 min, and aliquot samples at different time points were analyzed by HPLC. Talazoparib was stable under the described UV irradiation. (**B**) Phosphate-buffered saline (PBS) stability of the photoactivatable prodrug: 20 μM compound **3** in pH = 7.4 PBS (containing 10% DMSO) were incubated at 37 ◦C for 48 h and analyzed by HPLC. The peak area of compound **3** did not show significant changes. (**C**) UV cleavage test on compound **3**: 20 μM of the compound in methanol was irradiated at 365 nm for 5 min and analyzed by HPLC. By progressing irradiation, compound **3** was converted into Talazoparib.

#### 2.3.2. Stability of the Photoactivatable Prodrug in Phosphate-Buffered Saline

The synthesised photoactivatable prodrug should be sufficiently stable. If a high dose of inactive prodrugs were converted to the parent drug in advance, this might produce some undesirable toxic and side effects. Therefore, we tested the stability of compound **3** in phosphate-buffered saline (PBS) with the same pH as blood. Then 20 μM of compound **3** were added to pH = 7.4 PBS containing 10% dimethyl sulfoxide, which was incubated in a 37 ◦C thermostatic incubator for 48 h under normal brightness, during which samples of equal concentration were taken at different time points for HPLC analysis. The results were plotted according to the sampling time points and the peak areas of the compound (Figure 3B). The peak area of compound **3** did not show significant changes within 48 h, indicating that the compound **3** had good stability in PBS.

#### 2.3.3. UV Release of the Photoactivatable Prodrug

A photoactivatable prodrug needs to not only have good stability, but also be rapidly and effectively released at the irradiated site. Therefore, we tested the photolysis of compound **3**. Figure 3C shows the release situation of compound **3** (20 μM in methanol) after UV irradiation. Under the current experimental conditions, compound **3** could rapidly and completely release Talazoparib in 3 min. In addition, the release experiment also provided a reference for the irradiation duration to be used for the subsequent cell experiments.

#### *2.4. Enzymatic Experiments In Vitro*

#### 2.4.1. Inhibition of the PARP-1 Enzyme

According to the guidance of molecular docking, after the PPG was introduced at the lactam pharmacophore of Talazoparib, the prodrug **3** should have had a reduced inhibitory effect on the PARP-1 enzyme. Therefore, we evaluated the inhibitory activities of Talazoparib and compound **3** on PARP-1. As shown in the dose-dependent curve in Figure 4A, the inhibitory activity of the PPG-inactivated compound **3** was significantly lower than that of Talazoparib. The IC50 values of Talazoparib and compound **3** were 0.005 and 1.919 μM, respectively, indicating that the inhibitory activity was reduced by 380 times (Table 2). This was consistent with the prediction from the molecular docking. The test results strongly proved that the interaction between the inhibitor and PARP-1 enzyme could be significantly blocked after introducing PPG to the lactam pharmacophore of Talazoparib.

**Table 1.** Inhibitory activity of Talazoparib and compound **3** on poly (ADP-ribose) (PAR) polymerization.

**Figure 4.** (**A**) Dose–response curve of Talazoparib and compound **3** on PARP-1 enzyme inhibition. Compound **3** showed a significant decrease in the inhibitory activity compared to the active Talazoparib to PARP-1 enzyme. Error bars represent standard deviation from duplicate determinations, and results are shown as mean ± standard deviation (SD). The IC50 value is presented in Table 2. (**B**) Dose–response curve of Talazoparib and compound **3** on PARylation assay. The ability of compound **3** to inhibit the

polymerization of PAR significant decreased compared to the active Talazoparib in Hela cells. Error bars represented standard deviation from duplicate determinations, and results are shown as mean ± SD. The IC50 value is presented in Table 1.

**Table 2.** Poly (ADP-ribose) polymerase 1 (PARP-1) enzyme inhibitory activity of Talazoparib and compound **3**.


#### 2.4.2. PARylation Assay

The inhibitory activity of compounds on the PARP-1 enzyme could also be evaluated at the cellular level by using a hydrogen peroxide solution to damage the DNA of cells. Under normal circumstances, PARP-1 would be activated immediately, and the poly (ADP-ribose) polymer would be synthesised with NAD+ as the substrate. In the presence of a PARP-1 inhibitor, however, the biological function of PARP-1 would be inhibited. Therefore, the inhibitory activity on PARP-1 can be determined by detecting the level of poly (ADP-ribose) [16,33]. From the data in Figure 4B and Table 1, it can be seen that Talazoparib showed a strong inhibitory effect on poly ADP-ribosylation in HeLa cells, with an IC50 value of less than 0.0005 μM, whereas compound **3** showed a weak inhibitory effect, with an IC50 value of 0.329 μM, a decrease of 658 times at least.

#### *2.5. Compound's Cytotoxicity Assay in the Absence and Presence of UV Irradiation*

After confirming that the introduction of PPG could block the inhibitory activity of Talazoparib on PARP-1, we speculated that compound **3** should also have less cytotoxicity than Talazoparib. To verify this idea, cell proliferation inhibition experiments were conducted. As Talazoparib should only be applied to *BRCA1* or BRCA*2*-defective cell lines, the principle is simultaneous inhibition of the BER pathway and HR repair pathway of DNA to produce a synthetic lethality effect and consequently, the apoptosis of the tumour cells. Therefore, we selected the well-recognized *BRCA1* and *BRCA2*-defective cell lines MX-1 and Capan-1 for the following tests. The results indicate that Talazoparib exhibited effective cytotoxicity in the MX-1 and Capan-1 cell lines (IC50 values: 0.015 μM and 0.003 μM, respectively). In contrast, the PPG-inactivated compound **3** showed no significant cytotoxicity, with IC50 values of 1.873 μM and 0.863 μM, respectively (Figure 5 and Table 3).

**Figure 5.** Dose–response curves of compound **3** in the absence and presence of ultraviolet irradiation. Cells were incubated for 1 h with the compounds and were then irradiated at 365 nm (4.1 mW/cm2). Cell growth was determined 10 days after incubation with the compounds. Irradiated compounds showed left-shifted dose–response curve compared to that of the unirradiated compounds. Error bars represent the standard deviation from duplicate determinations, and results are shown as mean ± SD. The IC50 value is presented in Table 3.


**Table 3.** Capan-1 and MX-1 cell inhibitory activities of Talazoparib and compound **3** in the absence and presence ultraviolet radiation.

Next, we tested whether the inhibitory activity of compound **3** could be restored after UV irradiation. In order to negate the influence of UV irradiation on cells, to avoid interfering with the experiment results, we first measured the maximum tolerance of cells to UV light. As shown in Figure 6, the MX-1 cell line was relatively sensitive to UV irradiation, and could tolerate an irradiation duration of 1 min, whereas further extension of the duration would affect cell proliferation. In contrast, the Capan-1 cell line could tolerate UV irradiation for a relatively long time, and approximately 85% of the cells were still alive after irradiation for 5 min. On the basis of the principle of maximising drug release, while ensuring normal cell proliferation as much as possible, we determined the UV irradiation durations for the MX-1 and Capan-1 cells to be 1 min and 3 min, respectively.

**Figure 6.** Cellular ultraviolet light tolerance test. Cells were irradiated at 365 nm (4.1 mW/cm2) for indicated periods of time, and cell growth was determined after 10 days. The MX-1 cell line was relatively sensitive to UV irradiation. Radiation over 1 min affected cell proliferation. In contrast, the Capan-1 cell line tolerated UV irradiation for a relatively long time.

As shown in Table 3, the inhibitory activity of compound **3** on MX-1 cells after UV irradiation was approximately three times higher than that on the non-irradiated group (IC50 values: 1.873 μM vs. 0.577 μM, respectively). However, it did not reach the same level of inhibition as the parent drug Talazoparib, likely because the irradiation duration of 1 min was not enough to allow the release of all the compound **3**. Therefore, we tested the Capan-1 cells that could tolerate a longer UV irradiation duration. To our delight, the IC50 value of compound *3* for Capan-1 cells was 0.092 μM after 3 min of irradiation, which was nine times higher than that of the non-irradiated group (IC50 value: 0.863 μM). Further analysis was needed to determine the reason why the inhibitory activity of compound **3** still did not reach the same level as Talazoparib after UV irradiation. We simulated the same cytotoxicity experimental conditions to evaluate the photolysis of 10 μM compound **3** (10 μM is the starting concentration of compound **3** in cytotoxicity experiments) in a 96-well plate. The results showed that compound **3** was not released completely after 3 min UV irradiation (see Figure S2).

The above cytotoxicity experiment data indicated that compound **3** could significantly restore the effective activity of Talazoparib after being irradiated by UV light, and the data from both cells imply that inhibitory activity could increase with prolonged irradiation time. However, owing to the limited types of *BRCA*-deficient cells that could be selected for study and their restrictive UV light tolerance, compound **3** did not reach the same level of inhibitory activity as Talazoparib. The experimental results could provide inspiration for future research.

#### *2.6. Verification of the Toxicity of Leaving Photoactivatable Protecting Groups*

Subsequently, the leaving PPG after irradiation was also evaluated for its cytotoxicity. Compound **3** was obviously not suitable for verifying this, since it released active Talazoparib after its photocleavage. Therefore, we used an indirect test method by synthesising compound **6** via the nucleophilic substitution reaction between the non-cytotoxic Boc-protected amino acid L-alanine (Boc-Ala), as reported in the literature, and compound **2** under the catalysis of potassium carbonate (Scheme 2), and examined compound **6** s effect on cell proliferation before and after irradiation [25,26].

**Scheme 2.** Synthesis of compound **6**. Reagents: (c) K2CO3, reflux, 5 h.

We first evaluated the photolysis of compound **6**, and the experimental results showed that 20 μM of compound **6** in methanol solution were almost completely released within 20 min (see Figure S3). Next, we used the same initial concentration as that of compound **3** for the measurement. To fully release the drug, we UV-irradiated compound **6** at different concentrations for 20 min, and then added each sample to cells for co-culture. As a result, compound **6** showed no significant cytotoxicity in MX-1 and Capan-1 cells both before and after irradiation (Figure 7 and Table 4). Since the Boc–Ala released after irradiation was not cytotoxic, the results verified that the leaving PPG at the indicated concentrations did not cause any cytotoxicity. This therefore proved that the cytotoxicity of compound **3** after irradiation could be attributed entirely to the released Talazoparib.

**Figure 7.** Dose–response curves of compound **6** in the absence and presence of ultraviolet radiation in the MX-1 and Capan-1 cells. After 20 min of irradiation at 365 nm (4.1 mW/cm2), the compounds were transferred to 96-well plates and incubated with the cells. Cell growth was determined 10 days after incubation with the compounds. Irradiated compounds showed similar dose–response curves as that of unirradiated compounds. Error bars represent standard deviation from duplicate determinations, and results are shown as mean ± SD. The IC50 value is presented in Table 4.


**Table 4.** MX-1 and Capan-1 cell inhibitory activities of compound **6** in the absence and presence ultraviolet radiation.

#### **3. Materials and Methods**

#### *3.1. Molecular Docking*

The catPARP-1 crystal structure (PDB ID 4PJT) was downloaded from the Protein Data Bank. Molecular docking was carried out using Gold (The Cambridge Crystallographic Data Centre). Before docking calculations, water molecules of the crystal structure were deleted, and hydrogen atoms were added. The whole 4PJT was defined as a receptor, and the site sphere was selected based on the active site of 4PJT. Compound **3** was placed during the molecular docking procedure. After the end of molecular modelling, types of interactions of the docked protein with ligand were analyzed.

#### *3.2. Chemistry*

All of the reagents and solvents were purchased from Innochem (Beijing, China), Aladdin (Shanghai, China), Energy Chemical (Shanghai, China), TCI (Tokyo, Japan), Ark Pharm (Libertyville, IL, USA), and used without additional purification. Anhydrous solvents were stored in sure-seal bottles under dry nitrogen. Analytical thin-layer chromatography was conducted on pre-coated silica gel plates (Yantai Dexin Biotechnology Co., Ltd., Yantai, China). Visualization was accomplished with 254 nm and 365 nm UV light. Column chromatography was performed using silica gel (200–300 mesh; Qingdao Ocean Chemical Engineering Co., Ltd., Qingdao, China).

Nuclear magnetic resonance (NMR) spectra were obtained on a JNM-ECA-400 400 MHz spectrometer (JEOL Ltd., Tokyo, Japan). Chemical shifts were reported in ppm and TMS was used as the internal standard. Coupling constants (*J*) are given in Hertz. Spin multiplicities were reported as the following abbreviations: s (singulet), d (doublet), dd (doublet doublet), t (triplet), q (quadruplet), m (multiplet). The mass spectrometry (MS) systems were the API 3000 triple-quadrupole mass spectrometer equipped with a Turbo Ion Spray electrospray ionization (ESI) source (AB Sciex, Concord, ON, Canada). HPLC analysis was performed using an Agilent 1260 Series (California, CA, USA).

3.2.1. Synthesis of (8*S*,9*R*)-5-Fluoro-8-(4-fluorophenyl)-9-(1-methyl-1*H*-1,2,4-triazol-5-yl)-2- ((6-nitrobenzo[d][1,3]dioxol-5-yl)methyl)-2,7,8,9-tetrahydro-3*H* pyrido[4,3,2-de]phthalazin-3-one (**3**)

To a solution of Talazoparib (70 mg, 0.18 mmol) in anhydrous tetrahydrofuran (THF) (30 mL) was added to a 100 mL, two-necked flask with a magnetic stir bar. The flask was evacuated and backfilled with argon three times. Then 2.5 M n-Butyllithium (108 μL, 0.27 mmol) in hexanes were added the reaction solution, and the mixture was cooled at −80 ◦C. After stirring for 10 min, a solution of 5-(bromomethyl)-6-nitrobenzo[d][1,3] dioxole (93.9 mg, 0.27 mmol) in anhydrous THF was slowly added to the mixture, was warmed to 0 ◦C, and stirred overnight. When the reaction was completed, the mixture was carefully quenched by ammonium chloride saturated solution, and was extracted with ethyl acetate (50 mL × 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by column chromatography (dichloromethane/methanol = 50:1) to give compound **3** (46 mg, yield 45.7%) as a light yellow solid; 1H NMR (400 MHz, DMSO-*d*6) δ 7.79 (s, 1H); 7.73 (s, 1H); 7.63 (s, 1H); 7.53–7.45 (m, 2H); 7.20–7.13 (m, 2H); 7.10 (dd, *J* = 9.0, 2.4 Hz, 1H); 6.93 (dd, *J* = 11.1, 2.5 Hz, 1H); 6.61 (s, 1H); 6.22–6.21 (m, 2H); 5.40–5.29 (m, 2H); 5.08–5.00 (m, 2H); 3.56 (s, 3H). ESI m/z (M + H)<sup>+</sup> calculated for C27H20F2N7O5 + 560.15 found 560.15.

### 3.2.2. (6-Nitrobenzo[d][1,3]dioxol-5-yl)methyl (tert-butoxycarbonyl)-l-alaninate (**6**)

To a solution of (tert-butoxycarbonyl)-l-alanine (190 mg, 1.0 mmol) in acetone, potassium carbonate (207.31 mg, 1.50 mmol) and compound **3** were added. Then the mixture was stirred and heated at reflux for 5 h. After the reaction completed, the mixture was cooled to room temperature and removed under reduced pressure. The residue was dissolved in water and then the aqueous solution was extracted with dichloromethane (50 mL × 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated to give the crude product, which was purified by column chromatography (ethyl acetate/petroleum ether = 5:1) to give compound **6** (264 mg, yield 71.4%) as a light yellow solid; 1H NMR (400 MHz, DMSO-*d*6) δ 7.74 (s, 1H); 7.46 (d, *J* = 7.3 Hz, 1H); 7.22 (s, 1H), 6.26 (s, 2H); 5.43–5.32 (m, 2H); 4.15–4.08 (m, 1H); 1.38 (s, 9H); 1.27 (d, *J* = 7.4 Hz, 3H). ESI m/z (M + Na)<sup>+</sup> calculated for C16H20N2NaO8 <sup>+</sup> 391.11 found 391.11.

#### *3.3. Talazoparib UV Stability Assays*

Talazoparib was dissolved in methanol (2.63 mM), and the solution was divided into six equal portions for UV irradiation at 365 nm by using an UV crosslinker (CL-1000L UVP Crosslinker, 5 × 8 W; Analytikjena, IL, USA). The irradiation times were 1 min, 2 min, 3 min, 4 min, 5 min, and 10 min. The irradiated solution was transferred to a volumetric flask and diluted to the same volume with methanol. Then HPLC analysis was performed to detect the compound peak area. Samples were determined on an Agilent Eclipse Plus C18 column (4.6 × 150 mm, 5 μm particle size; California, CA, USA) using a mobile phase A (water) and mobile phase B (acetonitrile (ACN)) to elute. The gradient changed from 70% A to 30% A in 10 min, then continuing to elute with 30% A for 5 min. The flow rate was 1 mL/min, and the detection wavelength was 220 nm.

#### *3.4. Phosphate-Bu*ff*ered Saline Stability of the Photoactivatable Prodrug*

Twenty μM of the test compound in pH = 7.4 PBS, containing 10% DMSO, were transferred to 13 1 mL volumetric flasks in equal volumes, incubated at 37 ◦C. The volumetric flasks were taken out at 0 min, 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, and 7 days, and were frozen at −80 ◦C. The samples were thawed in sequence and diluted to the same volume with methanol. Then HPLC analysis was performed to detect the compound peak area. Samples were determined on an Agilent Eclipse Plus C18 column (4.6 × 150 mm, 5 μm particle size; California, CA, USA) using a mobile phase A (water) and mobile phase B (ACN) to elute. The gradient changed from 70% A to 30% A in 10 min, continuing to elute with 30% A for 5 min. The flow rate was 1 mL/min, and the detection wavelength was 220 nm.

#### *3.5. UV Cleavage Test for Photoactivatable Prodrug*

The compound was dissolved in methanol (20 μM), and the solution were irradiated at 365 nm by using an UV crosslinker. Every minute from 0 to 10 min, aliquot samples (200 μL) were determined on an Agilent Eclipse Plus C18 column (4.6 × 150 mm, 5 μm particle size) using mobile phase A (water) and mobile phase B (ACN) to elute. The gradient changed from 70% A to 30% A in 10 min, continuing to elute with 30% A for 5 min. The flow rate was 1 mL/min and the detection wavelength was 220 nm. The peak areas percentage of the compounds at each time point were plotted against the sampling time.

#### *3.6. PARP-1 Enzyme Inhibition Assay*

A PARP-1 Chemiluminescent Assay Kit (Cat# 80569, BPS, San Diego, CA, USA) was used to detect the ability of compounds to inhibit PARP-1 enzyme activity. All experimental steps followed the operating manual. Firstly, each well was precoated with 25 μL 1× histone mixture that was diluted in PBS by incubation at 4 ◦C overnight. The wells were blocked by adding 100 μL of blocking buffer, then incubated at room temperature for 90 min. Then 12.5 μL of the prepared 1× PARP assay mixture and 1× activated DNA in 1× PARP buffer were added into each well of the assay plate, and 2.5 μL of

compound or solvent control were added at varying concentrations. The reaction was initiated by the addition of 10 μL 1 ng/μL PARP-1 enzyme in 1× PARP buffer at room temperature for 60 min. Next, the reaction solution was added with 25 μL of diluted Streptavidin-HRP (1:50 in blocking buffer) to each well at room temperature for an additional 30 min. Finally, 25 μL of Horseradish Peroxidase (HRP) chemiluminescent substrate A and 25 μL of HRP chemiluminescent substrate B were mixed on ice, and the mixture was added by 50 μL per well. The luminescent signal was measured using a multi-function microplate reader, and IC50 values were calculated using GraphPad Prism 8.0 software.

#### *3.7. PARylation Assay*

The cellular PARylation assay evaluated the ability of test compounds to inhibit polymerization of PAR. Hela cells were seeded in 96-well plates (10,000 cells/well) in 200 μL of cell complete medium, and were allowed to adhere for 4 h at 37 ◦C in a humidified atmosphere of 5% CO2. The medium was then removed from the plates, and the cells were treated with varying concentrations of test compounds for 18 h. 100 μL serum-free medium with 500 μM H2O2 solution was added, and the plate was kept at 37 ◦C for 5 min. Next, cells were fixed for 20 min with prechilled methanol at −20 ◦C and were incubated with 60 μL Poly(ADP-ribose) monoclonal antibody (1:2000) (prepared at 1x PBS containing 0.05% Tween-20 with 1% bovine serum albumin) at 37 ◦C for 2 h, followed by incubation with 60 μL IRDye 800CW goat anti-mouse IgG (diluted to 1:5000) and DNA stain DRAQ5 (1:5000) (prepared at 1× PBS containing 0.05% Tween-20 with 1% bovine serum albumin) at 37 ◦C for 2 h. The fluorescent signal was normalized with the DRAQ5 signal, and IC50 values were calculated using GraphPad Prism 8.0 software.

#### *3.8. Cell Proliferation Assay*

To assess the cytotoxicity of test compounds on *BRCA*-deficient cells using a CellTiter-Glo assay. MX-1 cells (*BRCA1*-deficient) and Capan-1 cells (*BRCA2*-deficient) were seeded in 96-well plates at densities that allowed linear growth for 10 days, and adhered overnight at 37 ◦C in a humidified atmosphere of 5% CO2. Cells were treated in their recommended growth medium containing varying concentrations of test compounds.When the cells were incubated with the test compounds for 1 h, the cell culture plates were irradiated to the corresponding time with an ultraviolet crosslinker (for compounds that did not require UV irradiation, this step could be omitted). After 10 days of incubation, CellTiter-Glo Reagent (Promega, Madison, WI, USA) was added to the plates, which continued to be incubated for 30 min at room temperature. Chemiluminescence values were recorded by a multiplate reader, and IC50 values were calculated using GraphPad Prism 8.0 software.

#### **4. Conclusions**

In summary, this paper reports, for the first time, a photoactivatable Talazoparib prodrug. During the research process, we blocked the key lactam pharmacophore of Talazoparib with PPGs under the guidance of computer molecular docking. The in vitro enzyme inhibition assay and PARylation experiment proved that the inhibitory activity of the PPG-inactivated compound was greatly reduced (by 380 and more than 658 times, respectively). In *BRCA1*- and *BRCA2*- deficient cell lines, the prodrug could significantly restore the effective activity of Talazoparib after being irradiated by UV light, and the data imply that inhibitory activity could increase with prolonged irradiation time. However, because of the limitations of the relatively few types of *BRCA*-deficient cells that could be selected for study, as well as their restricted tolerance to UV light, we could not obtain the same level of inhibitory activity from the irradiated prodrug as from the parent drug. Further structural optimisation of the compound and appropriate cell selection would be helpful for further research on this photoactivatable Talazoparib prodrug strategy.

In conclusion, the results of this preliminary study prove that the photoactivatable prodrug strategy was an interesting approach for studying PARP inhibitors. On the one hand, the described photoactivatable prodrug could provide a new biological tool for the mechanism research of PARP. On the other hand, photoactivatable prodrug also create new possibilities for therapeutic applications. However, this requires profound research on the prodrug' stability, toxicity, and bioavailability, and further cell and animal studies will be planned to address these questions.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1420-3049/25/2/407/s1, Figure S1: UV/Vis absorption spectra of compound 3 and Talazoparib in methanol solution, Figure S2: UV-cleavage test on the compound 3 in culture medium DMEM, Figure S3: UV-cleavage test on the compound 6, Figure S4 and S6: The 1H-NMR spectrometry of compound 3 and 6, Figure S5 and S7: Mass spectrometry of compound 3 and 6.

**Author Contributions:** Conceptualization, J.L.; methodology, J.L.; software, L.L.; validation, W.S., X.Y. and X.Z.; formal analysis, D.X. and F.X.; investigation, D.X. and W.L.; resources, X.Z.; data curation, J.L. and L.L.; writing—original draft preparation, J.L.; writing—review and editing, W.S., X.Y., and X.Z.; supervision, X.Y. and X.Z.; project administration, X.Z.; funding acquisition, X.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Science and Technology Major Project for Major New Drugs Innovation and Development, under grant no. 2018ZX09711003-009, and the Chinese National Natural Science Foundation, under grant no. 81872736.

**Acknowledgments:** The authors gratefully acknowledge research support from the Dan Jiang laboratory for structural confirmation.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Sample Availability:** Samples of the compounds are not available from the authors.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Article*

## **P-MAPA and Interleukin-12 Reduce Cell Migration**/**Invasion and Attenuate the Toll-Like Receptor-Mediated Inflammatory Response in Ovarian Cancer SKOV-3 Cells: A Preliminary Study**

**Luiz Antonio Lupi 1, Flávia Karina Delella 2, Maira Smaniotto Cucielo 1, Graziela Gorete Romagnoli 3, Ramon Kaneno 3, Iseu da Silva Nunes 4, Raquel Fantin Domeniconi 1, Marcelo Martinez 5, Francisco Eduardo Martinez 1, Wagner José Fávaro <sup>6</sup> and Luiz Gustavo de Almeida Chu**ff**a 1,\***


### Academic Editor: Qiao-Hong Chen

Received: 16 October 2019; Accepted: 9 December 2019; Published: 18 December 2019

**Abstract:** Immunotherapies have emerged as promising complementary treatments for ovarian cancer (OC), but its effective and direct role on OC cells is unclear. This study examined the combinatory effects of the protein aggregate magnesium–ammonium phospholinoleate–palmitoleate anhydride, known as P-MAPA, and the human recombinant interleukin-12 (hrIL-12) on cell migration/invasion, apoptosis, toll-like receptor (TLR)-mediated inflammation, and cytokine/chemokine profile in human OC cell line SKOV-3. P-MAPA and IL-12 showed cancer cell toxicity under low doses after 48 h. Although apoptosis/necrosis and the cell cycle were unchanged by the treatments, P-MAPA enhanced the sensitivity to paclitaxel (PTX) and P-MAPA associated with IL-12 significantly reduced the migratory potential and invasion capacity of SKOV-3 cells. P-MAPA therapy reduced TLR2 immunostaining and the myeloid differentiation factor 88 (MyD88), but not the TLR4 levels. Moreover, the combination of P-MAPA with IL-12 attenuated the levels of MyD88, interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-kB p65). The IL-12 levels were increased and P-MAPA stimulated the secretion of cytokines IL-3, IL-9, IL-10, and chemokines MDC/CCL22 and, regulated on activation, normal T cells expressed and secreted (RANTES)/CCL5. Conversely, combination therapy reduced the levels of IL-3, IL-9, IL-10, MDC/CCL22, and RANTES/CCL5. Collectively, P-MAPA and IL-12 reduce cell dynamics and effectively target the TLR-related downstream molecules, eliciting a protective effect against chemoresistance. P-MAPA also stimulates the secretion of anti-inflammatory molecules, possibly having an immune response in the OC microenvironment.

**Keywords:** ovarian cancer; P-MAPA; IL-12; TLR signaling; inflammation; chemoresistance

#### **1. Introduction**

Ovarian cancer (OC) is the fifth largest cause of cancer-related death in the United States and is the most lethal of all gynecological malignancies [1]. OC originates from the ovarian surface epithelium, fallopian tube (fimbriae region) or endometriosis-related tissue, and often exhibits a late diagnosis due to the lack of clear signs or symptoms in the early stages of development [2–5]. Unfortunately, there is no screening method for achieving the best curative result, and traditional chemotherapy and surgery are limited for patients with advanced OC [6]. After cisplatin and paclitaxel (PTX) resistance, several patients become susceptible to developing recurrent OC and metastasis [7]. Therefore, new therapeutic options that overcome chemoresistance and enhance drug sensitivity are promising for the treatment of OC.

Immunotherapies have demonstrated great efficiency by activating host immune responses into the OC microenvironment [8]; however, what is happening with the cancer cells as a direct result of immunostimulation requires further investigation. We recently reported the effect of the immunomodulatory agent termed protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride (P-MAPA), a natural biopolymer extracted from the *Aspergillus oryzae*, which exhibits a number of antitumor responses in different experimental models of cancer [9–11]. In association with cisplatin, P-MAPA showed a greater survival rate and a reduced OC volume in addition to the increased expression of proteins involved in toll-like receptor (TLR)-mediated inflammatory response (canonical and non-canonical pathways) in OC-bearing animals [11]. Recent studies also supported the immunological mechanism of action of P-MAPA through the activation of TLR2 and TLR4 signaling in both cancer and infections, in addition to regulating the activity of T cells (especially CD4+T and CD8+T cells) and natural killer (NK) cells [9,10]. Importantly, P-MAPA did not show toxicity in preclinical in vitro (V-79 Chinese hamster cell line) and in vivo models (Swiss mice, Wistar rats, and monkeys) nor in human clinical trial phase I [9]; its effects in cancer treatment have been tested in non-muscle invasive urinary bladder cancer [9,10] and in OC [11]. The directive role of P-MAPA on human OC cells, considering its feasibility, sensibility, resistance, and toxicity, has not been explored yet.

TLRs are transmembrane molecules that signal via myeloid differentiation factor 88 (MyD88) or TLR-associated activator of interferon (TRIF) to induce cell proliferation, chemoresistance, and cytokine/chemokine production [12]. Most importantly, TLRs can trigger a different response depending on the cell type (e.g., cancer cell or immune cell), and particularly, TLR4 has been reported to be a precursor of the immune escape of OC cells [13,14]. The crosstalk between the immunoadjuvant and the OC cells targeting the TLR signaling and related cytokine secretion has never been proposed as to their impact and specific response.

Interleukin-12 (IL-12) is a cytokine related to innate and adaptive immunity, being mainly produced by the antigen-presenting cells (APCs), such as B lymphocytes, dendritic cells (DCs), and others [15,16]. It acts on T and NK cells stimulating a cytotoxic CD8+ response and inducing cytokine production, especially interferon-γ (IFN-γ) [15,17]; IL-12 is involved in the differentiation of naïve T cells into a polarized T helper 1 (Th1) immune response. Patients with recurrent OC who underwent IL-12 treatment, in a well-established dosage regimen, showed significant tumor regression [18]; the major challenge involving the treatment with IL-12 is related to the adverse effects due to its high diffusion and toxicity (e.g., lymphopenia and irreversible elevation of transaminases at 600 ng/kg and neutropenia, fatigue, and headache at 300 ng/kg) [19], and a more precise and direct administration may limit their undesirable effects. Notably, a phase II study involving intraperitoneal infusions of recombinant IL-12 in patients with residual disease ≤1 cm showed to be well-tolerated after fist-line therapy for OC-related peritoneal carcinomatosis [19]. Recently, Cohen et al. [16] studied the relationship between membrane-bound IL-12 in tumor cells and the potential to disrupt protumorigenic signaling and tumor outgrowth within peritoneal cavities; IL-12 significantly led to a tumor refractory state, thereby delaying the onset of metastatic disease.

Because P-MAPA is thought to increase IFN-γ levels, which may potentiate the Thelper (Th1)-mediated immune response, and adjuvant therapies with IL-12 have long been proposed as beneficial for patients with OC, this study investigates the effects of P-MAPA and IL-12, alone or in combination, on cancer cell activities with focus to the TLR-mediated inflammatory process and cytokines/chemokines profiling in human SKOV-3 cell line.

#### **2. Results**

#### *2.1. P-MAPA and IL-12 Reduce Cell Viability and Induce Apoptosis in the Presence of PTX in SKOV-3 Cells*

An MTT assay was carried out using three biological and technical replicates to unravel the most suitable dose and period of treatment. Based on previous results, the SKOV-3 cells were challenged with P-MAPA at doses of 25 μg/mL, 50 μg/mL, and 100 μg/mL, and rhIL-12 at doses of 0.5 ng/mL, 1 ng/mL, and 2 ng/mL for 24 h, 48 h, and 72 h. Then, we tested three different cell concentrations to achieve the better treatment response (1 <sup>×</sup> 10<sup>3</sup> cells, 1 <sup>×</sup> 10<sup>4</sup> cells, and 5 <sup>×</sup> 104 cells; Supplementary Figure S1). The SKOV-3 cell viability was efficiently reduced after the low-dose P-MAPA treatment (25 μg/mL for 48 h; viability was reduced by ~27%), and then started to increase after 72 h exposure (Figure 1A). Treatment with rhIL-12 also showed reduction in cell viability at an intermediate dose of 1 ng/mL mainly after 48-h exposure (Figure 1B; viability reduction by ~25% after 48 h). Combinatory therapy with P-MAPA and IL-12 also reduced the cell viability by about 33% (Figure 1C), and the treatment doses were set at 25 μg/mL of P-MAPA and 1 ng/mL of IL-12. Annexin V- fluorescein isothiocyanate (FITC)/Propidium iodide (PI) staining was used to effectively determine the apoptosis/necrosis rate induced by P-MAPA and rhIL-12 immunotherapies. In this study, we set early apoptosis (Annexin V-FITC+/PI−) as apoptosis and late apoptosis (Annexin V+/PI+) and necrosis (Annexin V−/PI+) as necrosis. The apoptosis index was not influenced by P-MAPA, IL-12, or P-MAPA+IL-12 (Figure 1D,E), which pointed out that these agents are unable to induce cell death at this concentration. We also observed that P-MAPA and IL-12 did not alter the cell cycle significantly (Figure 1F,G). These data indicate that these immunotherapeutic agents do not have a direct and high toxic effect to the cell, which is, in fact, expected from immunotherapies. Because the treatments did not promote cell death or cell proliferation, the reduction of cell viability might have been a result of decreased cell metabolism, because the MTT test indirectly reflects the mitochondrial activity.

To better understand whether P-MAPA and IL-12 therapies potentially increase the effect of standard chemotherapy, they were tested in association with PTX (Figure 2A,B). After 48 h, P-MAPA decreased cell viability in association with different doses of PTX compared with cells treated with PTX only (~30% reduction with 5 μM PTX; ~30% reduction with 2.5 μM PTX; ~24% reduction with 1.25 μM PTX and ~34% reduction with 0.625 μM PTX). Because IL-12 had no effect on cell viability when associated with PTX, the decreased cell viability observed after combining P-MAPA with IL-12 in association with doses of PTX is probably due to the P-MAPA effect (~19% reduction with 5 μM PTX, ~30% reduction with 2.5 μM PTX, ~30% reduction with 1.25 μM PTX, and ~38% reduction with 0.625 μM PTX). To confirm these effects on SKOV-3 cell death, an Annexin V-FITC/PI assay was performed using the lowest dose of PTX (0.625 μM). Concordantly, with the MTT results, P-MAPA and P-MAPA+IL-12 increased cell death in association with PTX at dose of 0.625 μM. The apoptosis/necrosis ratio of PTX was 0.64 and became higher when associated with P-MAPA (1.18) or P-MAPA+IL-12 (0.88), thus enhancing the P-MAPA effect as apoptotic inductor. Finally, PTX significantly increased the number of cells in G0/G1 and decreased cells in G2/M phase of the cell cycle, thus inducing cell cycle arrest. In this case, the addition of P-MAPA and IL-12 did not potentiate the PTX effects in cell cycle (Figure 2D,E).

**Figure 1.** Protein aggregate magnesium–ammonium phospholinoleate–palmitoleate anhydride (P-MAPA) and interleukin-12 (IL-12) suppress cell viability without changing the apoptosis rate and cell cycle. SKOV-3 cells were treated with various concentrations of P-MAPA (**A**) and IL-12 (**B**) for 24, 48, and 72 h, and the cytotoxicity (expressed as percentage) was assayed by MTT; (**C**) Cell viability (%) after standardization of treatments (25 <sup>μ</sup>g P-MAPA and 1 ng IL-12) after 48 h exposure; 1 <sup>×</sup> 103 SKOV-3 cells showed the best reproducibility. (**D**) Representative apoptotic index in SKOV-3 cells treated with P-MAPA and IL-12 for 48 h detected by annexin V/PI flow cytometry. (**E**) Percentage of cells in apoptosis and necrosis. (**F**) Representative cell cycle analysis in SKOV-3 cells treated with P-MAPA and IL-12 for 48 h detected by PI and RNAase flow cytometry. (**G**) Percentage of PI+ cells in G0/G1, S, and G2/M phases. The samples were assayed in three technical and biological replicates. Results are expressed as the mean ± SD and described as column chart. \* *p* < 0.05 vs. different doses at 48 h; # *p* < 0.05 vs. control group.

**Figure 2.** P-MAPA and P-MAPA+IL-12 reduce cell viability and apoptosis/necrosis rate in the presence of PTX. (**A**) Cell viability was assessed by an MTT assay; SKOV-3 cells were treated with various concentrations of PTX alone or in association with P-MAPA and IL-12 for 48 h. \* *p* < 0.05 vs. PTX and PTX+IL-12 groups. (**B**) Percentage of cells in apoptosis and necrosis after exposure to 0.625 μM PTX plus P-MAPA, IL-12, or P-MAPA+IL-12. \* *p* < 0.05. (**C**) Representative apoptotic index in SKOV-3 cells detected by Annexin V/PI flow cytometry. (**D**) Representative cell cycle analysis in SKOV-3 cells treated with PTX, P-MAPA and IL-12 for 48 h detected by PI and RNase flow cytometry. (**E**) Percentage of PI+ cells in the G0/G1, S, and G2/M phases. The samples were assayed in three technical and biological replicates.\* *p* < 0.05 vs. control group. Results are expressed as the mean ± SD.

#### *2.2. Combination of P-MAPA with IL-12 Is Essential to Reduce Cell Migration Whereas P-MAPA Alone Decreased The Invasive Potential of SKOV-3 Cells*

To investigate the inhibitory effect of P-MAPA and IL-12 as a single or combinatory treatment on SKOV-3 cells, a wound-healing assay was performed in different periods. Although the treatment with IL-12 showed a reduced migration rate (~ 17%) after 36 h and 48 h exposure, cells treated with P-MAPA migrated significantly less (~ 13%) only at 48 h exposure (Figure 3A,B). Combination of P-MAPA and IL-12 reduced the migratory potential of cells after 36 h and 48 h treatment (>20%), being the most efficient after 36 h (Figure 3A,B); this overall analysis suggests that IL-12 was more effective than P-MAPA in delaying wound closure. SKOV-3 cells that were treated with 0.9% saline solution (control group) had an accelerated growth and migration rate when compared with all the treatments. Because the wound-healing assay might be biased by cell proliferation, transwell migration and invasion assays were performed and effectively showed that P-MAPA reduced cell migration when administered alone or in association with IL-12 (Figure 4A). When Geltrex® was added to the chambers, the number of invasive cells was reduced after P-MAPA treatment in comparison to IL-12 treatment or its association (Figure 4B).

#### *2.3. Immunotherapy with P-MAPA and IL-12 Significantly Reduced the TLR-Mediated Downstream Molecules Involved in the Inflammatory Process of SKOV-3 Cells*

One of the most important factors responsible for the acquisition of malignant phenotypes of OC cells is associated with chemoresistance to treatments and uncovering new agents that downregulate the inflammatory pathway(s) may be of significant interest. To evaluate whether P-MAPA or IL-12 play a role on OC-related inflammation, TLR2- and TLR4-mediated pathways were evaluated through canonical and non-canonical signaling. Although the expressions of TLR2 and TLR4 did not vary significantly, the downstream target molecules were affected by the treatments (Figure 5A,B). P-MAPA and IL-12 alone or in combination led to a profound reduction in the MyD88 levels (0.53-, 0.48-, and 0.61-fold reduction, respectively vs. the control group; Figure 5A,B). We also evaluated the NF-kB p65 expression in the extracts of SKOV-3 cells, and notably, IL-12 alone or combined with P-MAPA induced a significant reduction in the p65 levels (0.66- and 0.63-fold reduction, respectively vs. control group; Figure 5A,B). To explore the non-canonical pathway, the TRIF and IRF3 levels were measured. Although the TRIF levels were unchanged after the treatments (*p* > 0.05), P-MAPA, IL-12, and the combination of P-MAPA with IL-12 induced a significant decrease in the IRF3 levels (0.70-, 0.43-, and 0.71-fold reduction, respectively vs. the control group; Figure 5A,B). These findings provide evidence that P-MAPA and IL-12 potentially act on MyD88-dependent and MyD88-independent pathways, which also encourages the possibility that these immunomodulatory agents could even increase the chemosensitivity of other therapeutics.

**Figure 3.** Migratory potential of SKOV-3 cells determined by wound-healing assay. (**A**) Percentage of wound closure after 0, 6, 12, 24, 36, and 48 h. \* *p* < 0.05, # *p* < 0.01 vs. control group. (**B**) Photographs of each wound-healing analysis at 0, 24, 36, and 48 h which were representative for specific closing area; vertical black bars were used to show the incision edges (10× magnification). The samples were assayed in three technical and biological replicates.

**Figure 4.** Effects of P-MAPA and IL-12 alone or in combination on the migration and invasion capacity of SKOV-3 cells. (**A**) Cell migration was measured by the amount of cells located in the lower part of the insert. (**B**) Cell invasion was determined by the amount of cells located in the lower part of the insert previously covered by Geltrex®. Representative images of the assay were obtained at 20<sup>×</sup> magnification. The samples were assayed in three technical and biological replicates. Results are expressed as the mean ± SD; \* *p* < 0.05; CT, control group.

To further investigate and elucidate the location and the relative expression levels of the TLR2 and TLR4 receptors, an immunofluorescence assay was performed on SKOV-3 cells. As evidenced by the cellular fluorescence level, the P-MAPA treatment significantly decreased the expression level of cytoplasmic and nuclear TLR2 (48% fluorescence reduced vs. the control group; Figure 5C). On the contrary, IL-12 and the combination of P-MAPA and IL-12 promoted the highest TLR2 immunofluorescence intensity (135% and 151% fluorescence level, respectively vs. P-MAPA; Figure 5C), which suggests that IL-12 is responsible for restoring TLR2 activation after P-MAPA therapy in SKOV-3 cells. Lastly, the relative immunofluorescence of the TLR4 was unchanged after the treatments (Figure 5D).

#### *2.4. P-MAPA Stimulates the Secretion of Pro- and Anti-Inflammatory Molecules, Whereas Its Association with IL-12 Induced the Synthesis of Inflammatory Cytokines in SKOV-3 Cells*

To determine which cytokines/chemokines are secreted by the SKOV-3 cells and how P-MAPA and IL-12 act to regulate its production, a wide range of these molecules were evaluated in both supernatants (Figure 6A) and cellular extracts (Figure 6B). As expected, the IL-12 levels were higher in the cells treated with IL-12 and P-MAPA+IL-12 (244% and 243% fold-increased, respectively vs. the control group, and 154% and 155% fold-increased, respectively vs. the P-MAPA group; Figure 6A); IL-12 was slightly elevated with P-MAPA, even at low concentrations.

**Figure 5.** TLR2- and TLR4-mediated signaling pathways are involved with inflammatory process and chemoresistance in human OC. (**A**) Representative protein profiles of TLR2, TLR4, MyD88, TRIF, IRF3, and NF-kB; fractions containing 50 μg protein were pooled from 5 samples per group. (**B**) Individual blots were used for densitometric analysis of the TLR2 and TLR4 levels and related downstream molecules (MyD88, TRIF, IRF3, and NF-kB) after normalization to the β-actin. Data are expressed as the mean ± SD. \* *p* < 0.05 vs. control group. Relative fluorescence intensity of TLR2 (**C**) and TLR4 (**D**) receptors. The values are expressed as the mean ± SD. The samples were assayed in three technical and biological replicates. \* *p* < 0.05. Confocal imaging of TLR2 (**E**) and TLR4 (**F**) immunostaining using fluorescein isothiocyanate (FITC)-conjugated antibodies anti-TLR2 and anti-TLR4 was obtained in SKOV-3 cells (Alexafluor®488, bar = 50 μm). DAPI was used for nuclear staining and merged images were performed using Image J software. Negative controls were used. CT, control; DAPI, 4 ,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate.

**Figure 6.** Multiplex analysis of cytokines and chemokines produced by SKOV-3 cells in response to P-MAPA and IL-12 treatments. (**A**) Concentrations of IFN-γ, IL-3, IL-9, IL-10, IL-12, RANTES, and MDC were altered in the supernatants of cell culture. (**B**) Levels of IL-4, IL-8, and MIP-1α were altered in SKOV-3 cell extracts. All data are expressed as the mean ± SD. \* *p* < 0.05 as compared with the corresponding group; One-way ANOVA complemented by the Tukey test. The samples were assayed in three technical and biological replicates. CT, control; IFN-γ, interferon gamma; RANTES, regulated on activation, normal T cell expressed and secreted; MDC, macrophage-derived chemokine; MIP-1α, macrophage inflammatory protein 1-alpha.

Comparing the treatments, P-MAPA significantly stimulated the secretion of IFN-γ (24% vs. the control, IL-12 and P-MAPA+IL-12 groups; Figure 6A), which is probably related to the activation of the Th1 response. P-MAPA also stimulated IL-10 (21% vs. control, 23% vs. IL-12 and 32% vs. P-MAPA+IL-12 groups), MDC/CCL22 (31% vs. control and IL-12 groups, and 45% vs. P-MAPA+IL-12), and, regulated on activation, normal T cells expressed and secreted (RANTES)/CCL5 (25% vs. the control, 27% vs. the IL-12, and 30% vs. the P-MAPA+IL-12 groups). Furthermore, P-MAPA increased IL-3 and treatment with IL-12 or P-MAPA+IL-12 promoted a reduction in its levels (13% and 17% reduction, respectively vs. P-MAPA; Figure 6A). Treatment with P-MAPA also increased the IL-9 levels (25% increased vs. the control and P-MAPA+IL-12 groups; Figure 6A). A part of these results suggests the secretion of molecules involved in the Th2 response (e.g., IL-10, CCL22), but their regulatory signaling combined with other molecules released by immune cells into the OC microenvironment could not be proved to have a favorable or unfavorable effect. The IL-4 levels were internally elevated in SKOV-3 cells (Figure 6B) after therapies with IL-12 alone or combined with P-MAPA (169% and 254% increased vs. the control group and 198% and 293% increased vs. the P-MAPA group, respectively). Interestingly, the association of P-MAPA with IL-12 induced the highest production of intracellular IL-8 (37% vs. the control group) and MIP-1α (11% vs. control group; Figure 6B). In brief, cytokines that were not significantly influenced by P-MAPA and IL-12 included IL-1β, IL-2, IL-6, IL-7, IL-13, IL-15, IL-17, IP-10, MCP-1, and MIP-1β (Supplementary Tables S1 and S2).

#### **3. Discussion**

We reported that lower doses of P-MAPA efficiently reduce cell invasion, whereas P-MAPA in combination with IL-12 reduces cell migration in addition to attenuating the TLR-mediated inflammatory response in human SKOV-3 cells (Figure 7). Although these compounds have individually showed important effects in reducing OC volume and mass while enhancing overall survival and immunostimulation in animals and humans [11,16], this study is the first to describe a therapeutic rationale against human OC, thus revealing the mechanisms underlying cancer cell-related inflammatory aspects rather than those of immune cells.

P-MAPA and IL-12 elicited a decrease in cell metabolism, but no effect on cell apoptosis/necrosis and cell cycle was observed. Although an MTT assay is largely used as the viability/toxicity assay, it could be biased by decreased metabolism activity. This utilizes mitochondrial machinery to convert a colorless tetrazolium salt solution into purple formazan crystals [20]. Once we had pursued a treatment dose with low cell toxicity but considerable modulatory potential, we believed that either P-MAPA or IL-12 may be changing OC cell metabolism to a reduced activity (metabolomics could be of significant value to find the targets more precisely). The impact of this reduction was indeed confirmed by the migration and invasion assays. P-MAPA was able to reduce the migration and invasion capacity, and the delayed wound closure in IL-12-treated cells may be likely due to its decreased metabolic activity. Given that lower doses of P-MAPA and IL-12 are recommended to be well-tolerated, OC cell apoptosis could be prevented by the low-level toxicity of chemicals; conversely, these concentrations are safely used to activate immune responses and modulate cell metabolism. When associated with PTX, P-MAPA was able to increase cell death and apoptosis/necrosis ratio without potentiating the PTX effects on cell cycle. In accordance with a previous study in which approximately 50% of SKOV-3

cells died after 48 h exposure to 5 μM of PTX [21], we observed that P-MAPA not only was effective to increase cell death in the presence of PTX but also to maintain cell toxicity even after considerably lower levels of PTX. This increase in cell death might be a result of apoptosis induction, since the apoptosis/necrosis ratio was significantly higher in animals treated with the combination of P-MAPA and PTX. Because apoptosis lead to the release of the damage-associated molecular pattern (DAMP), this higher ratio may facilitate immunogenic cell death (ICD) [22].

**Figure 7.** Schematic view of the effects of P-MAPA and IL-12 on human SKOV-3 cells. The effect of each treatment is shown by its representative color. Up and down arrows indicate whether the treatment increased or decreased, respectively, such as cellular function or molecule at these levels. The treatments reduced the downstream molecules (MyD88, NF-kB, and IRF3, with the exception of P-MAPA in reducing NF-kB) of both canonical and non-canonical toll-like receptor (TLR) signaling pathways, and only P-MAPA significantly downregulated the TLR2 levels. The intracellular cytokine/chemokine levels were increased after treatment with IL-12 (IL-4) or P-MAPA+IL-12 (IL-4, IL-8 and MIP-1α); P-MAPA was more effective in increasing the secretion of the following cytokines/chemokines: IFN-γ, IL-3, IL-9, IL-10, RANTES/CCL5, MDC/CCL22). As expected, IL-12 was used to treat SKOV-3 cells and appeared increased after IL-12 and P-MAPA+IL-12 treatments. Although IL-12 only reduced the SKOV-3 cells' wound closure, P-MAPA efficiently regulated the cellular dynamics by reducing the wound closure, cell migration and invasion. The combination of P-MAPA with IL-12 decreased the period of wound closure and cell migration rate. MyD88, myeloid differentiation primary response 88; NF-kB, nuclear factor kappa B; TRIF, TIR-domain-containing adapter-inducing interferon-β; IRF-3, interferon regulatory factor 3; TLR2, toll-like receptor 2; TLR4, toll-like receptor 4; P-MAPA, protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride; IL, interleukin; MIP-1α, macrophage inflammatory protein 1α; IFN-γ, interferon gamma; RANTES, regulated on activation, normal T cell expressed and secreted; MDC, macrophage-derived chemokine; CCL, C-C motif chemokine ligand.

The pro-inflammatory actions of TLR signaling in cancer cells and immune cells may severely affect tumor progression [23], and, particularly, the immunosuppressive OC microenvironment needs to be continuously immunostimulated [23,24]. Although TLR4 was unchanged by the treatments, P-MAPA induces a reduction in the TLR2 levels together with downstream molecules in SKOV-3 cells. Additionally, we found TLR2 nuclear expression in SKOV-3 cells treated with P-MAPA. Although we are not the first to observe nuclear staining of TLR2 and TLR4 [25], the effect of the switch of cytoplasmic to nuclear expression in SKOV-3 cells remains unclear. Based on our results, this nuclear expression seems not to have any negative effect favoring cancer development. Although the TLR2-induced MyD88-dependent pathway is related to an increase in IL-12 secretion [26], the immunoregulatory mechanisms whereby IL-12 restores TLR2 to levels close to control remain to be elucidated. In immune cells, P-MAPA has reportedly been described as a TLR4 agonist [9–11], thereby enhancing the synthesis of cytokines and activating a Th1-polarized response. Our data evidenced the protective effect of P-MAPA in attenuating TLR2-mediated signaling in OC cells. The activation of TLR, especially via MyD88, is associated with increased tumor growth, chemoresistance, and the early recurrence of ovarian epithelial tumors [7,27]. In addition, OC expressing high levels of MyD88 presents a higher proliferative index and increased production of pro-inflammatory cytokines and chemokines [7,28,29]. Importantly, the association of P-MAPA and IL-12 decreased the levels of MyD88 in SKOV-3 cells compared with their respective control; because P-MAPA and P-MAPA + IL-12 also significantly decreased the migratory and invasive potential of the cells, it can be suggested that the MyD88-dependent signaling pathway may be one of the mechanisms by which SKOV-3 cells promote migration and invasion. It has also been reported that increased expression of MyD88 is related to a decreased sensitivity to chemotherapy (e.g., PTX) in the MyD88-negative OC A2780 cell line [30]; moreover, the activation of MyD88 and NF-kB in MyD88-positive SKOV-3 cells promoted cell proliferation and tumor growth, likely due to the increased secretion of pro-inflammatory cytokines, thus rendering the cells PTX resistant [13]. In this line, the downregulation of MyD88 and NF-kB may be a possible mechanism by which P-MAPA improves the chemosensitivity of PTX in SKOV-3 cells.

The activation of TLR4/MyD88/NF-kB signaling by ligands is strongly related to an inflammatory microenvironment, thereby contributing to a more aggressive OC phenotype and poorer clinical outcomes in women [31]. In SKOV-3 cells, silencing the membrane-associated RING-CH (MARCH), an ubiquitin ligase that downregulates MHC class II expression, resulted in reduced cell migration and invasion in addition to inhibition of NF-kB signaling [32], thus reinforcing that NF-kB may play a role in the regulation of numerous cellular dynamics. Taken together, the combination of P-MAPA and IL-12 was efficient to induce the downregulation of both MyD88 and NF-kB, which can be of great value to enhance the overall survival of patients while attenuating OC progression and metastasis.

TRIF activation is responsible for triggering the TLR non-canonical pathway, thus activating several transcriptional factors, such as NF-kB, IRF3 and AP-1, and resulting in cytokines and type I IFN production [33,34]. Although few studies have explored the role of TRIF/IRF3 in OC cells, they seem to be critical for therapy [35]. Recently, Chuffa et al. [23] reported the downregulation of TRIF and IRF3 in OC-induced rats, whereby the immunomodulatory agent dramatically reduced the volume and mass of ovarian tumors. Importantly, P-MAPA and IL-12, either alone or in association, significantly downregulated IRF3 levels, evidencing the potential of immunotherapies to act by both canonical and non-canonical TLR pathways.

Immunostimulatory and immunosuppressive molecules can contribute to either inhibiting or enhancing anti-tumor immune activity of such a response by immunotherapy. We screened a number of molecules displaying important effects in OC, but most of them displayed no changes in SKOV-3 cells after P-MAPA and IL-12 treatments. Treatment of SKOV-3 with rhIL-12 showed its availability in both IL-12 and P-MAPA+IL-12 groups, thus proving that the agent was present in the supernatants; whether IL-12 therapy was still capable of stimulating more IL-12 production by the cells is uncertain. IL-3 levels are stimulated by P-MAPA therapy, and this increase might be protective for patients during OC chemotherapy; administration of rhIL-3 to patients with platelet count < 75,000/mm3 is effective to

fight thrombocytopenia and neutropenia after chemotherapy [36]. Moreover, IL-3 is able to intensify the dose of carboplatin for primary advanced OC [37]. P-MAPA also enhanced IL-9 secretion by SKOV-3 cells, and this effect seems to have a dual impact on the immune system. IL-9 was initially recognized as a T-cell growth factor with oncogenic potential. However, Th9 cell-secreted IL-9 has been revisited for the immunity of some tumors [38]. IL-9 activates innate immune cells like mast cells, contributing to tumor growth prevention [38], and in addition to Th9 cell-derived IL-3, induction of adaptive anti-cancer responses that favor DCs survival has been reported in various cancers [39].

Regulatory T (Treg) cells can infiltrate into solid OC or ascitic fluid, contributing to an immunosuppressive microenvironment by secreting IL-10 and TGFB-1 while reducing the IFN-γ levels [40]. Because these cytokines participate either in stimulating or inhibiting the activities of other immune cells, P-MAPA seems to work as a double-edge sword regarding the SKOV-3 cells. Although mechanistically unclear, P-MAPA contributes partially to reducing the immunosuppression, likely due to the increased levels of IFN-γ rather than IL-10. In fact, high IFN-γ secretion meets our proposal by enhancing OC-cell immunogenicity through the recruitment of CD8+T and natural killer (NK) cells, in addition to increasing the anti-tumor activity of macrophages; we recently found that P-MAPA reduces Treg cells and stimulates CD8+T effector cells in OC-bearing animals (unpublished data). Recently, the expression of programmed death-ligand 1 (PD-L1) on tumor cells represents an important pathway by which malignant cells evade the immune system. In SKOV-3 cells, PD-L1 was variably found in the surface and cytoplasm [41], and its expression was correlated with high levels of TNF-α, IL-10, and IL-6 released from tumor-associated macrophages (TAMs). Because P-MAPA and IL-12 attenuated the downstream mediators of the TLR signaling, the altered expression of some cytokines may be independent of TLR-mediated inflammatory response.

A complex network represented by chemokines and its receptors, growth factors, inflammatory products, and other molecules (e.g., NF-kB), is responsible for tumor progression or rejection. Chemokines signal not only for tumor cells but may control tumor development through activation of specific receptors expressed in a variety of cells, thereby regulating the traffic of infiltrating macrophages, lymphocytes, DCs, and neutrophils [42]. The secretion of CCL5, which binds to the CCR4 receptor, is strongly associated with the presence of tumor-infiltrating CD8+T cells; the upregulation of its receptor in activated vaccine-primed T cells improved tumor homing in OC [43]. In SKOV-3 cells, the levels of CCL22/MDC and CCL5/RANTES were higher after P-MAPA therapy. Although these chemokines appear to be associated with a poor prognosis when released by immune cells, the functional meaning of their production by OC cells as to their significance in the OC microenvironment remains to be investigated. CCL22 is secreted by DCs and macrophages and acts on target cells by interacting with the CCR4 receptor located in the cell surface [44]. CCL22 is correlated to the chemoattraction of Treg cells in advanced stages of OC, and its expression was increased in response to IFN-γ signaling [45]. This finding partially corroborates our results in which both CCL22 and IFN-γ were higher in SKOV-3 cells after P-MAPA therapy; in contrast, P-MAPA does not promote elevation in the number of Treg cells (data not shown). A previous study by Giuntoli et al. [46] showed that ascites specimens originating from patients with malignant OC were accomplished by elevated levels of IL-6, IL-8, IL-10, IL-15, IP-10, MCP-1, MIP-1β, and VEGF, in contrast to significantly reduced levels of IL-2, IL-5, IL-7, IL-17, and CCL5/RANTES. Although controversial, IL-8 production by human OC cells plays a role in controlling tumor growth [47], and MIP-1α is involved in the recruitment of Th1 and cytotoxic effector T cells [48]. We found a higher secretion IL-8 and MIP-1α after combinatory treatment of P-MAPA and IL-12. Partially supporting our results, IL-8 levels have already been reported to be augmented in OC, even after the downregulation of NF-kB [49]; however, the alternative mechanism involved with this production remains unclear. Additionally, we observed increased levels of RANTES after P-MAPA treatment. RANTES is responsible for recruiting T cells, macrophages, eosinophils, and basophils into the inflammatory sites. In addition to IL-2 and IFN-γ, RANTES is thought to induce the activation and proliferation of NK cells while enhancing the anti-tumor response in animal models [46]. In addition to the overexpression of VEGF and MCP-1, a reduction in RANTES levels is associated with activating

pathways for tumor growth. Normally, OCs produce large amounts of RANTES, and this production is correlated with the infiltration of TAMs, CD8+T cells, and tumor progression [50,51]; these chemokines are further related to the acquisition of polarized immune responses (Th1 versus Th2). Although some negative effects are associated with the efficacy of chemotherapy related to RANTES, therapy with P-MAPA may be helpful against tumor expansion.

#### **4. Materials and Methods**

#### *4.1. Cell Line and Cell Culture*

The human OC cell line SKOV-3 was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). The SKOV-3 cells were routinely incubated in RPMI 1640 (Life Technologies, Grand Island, NY, USA) supplemented with 10% Fetal Bovine Serum (FBS) and 1% anti-anti solution (100 mg/mL penicillin G, and 100 μg/mL streptomycin (Merck, Darmstadt, Germany). The culture medium was changed every 2 to 3 days. All cells were maintained at 37 ◦C in a humidified atmosphere of 5% CO2.

#### *4.2. Treatments with P-MAPA and IL-12*

To evaluate the in vitro effect of the treatments, different doses of P-MAPA (25 μg/mL, 50 μg/mL, and 100 μg/mL) were tested in accordance with Favaro et al. [9]. Initially, 5 mg P-MAPA was diluted in 1 mL of saline solution to achieve the desired stock solution of 5 mg/mL, which were then diluted in the cell culture medium to obtain the testing doses. For the treatment with recombinant (rh)IL-12, concentrations of 0.5 ng/mL, 1 ng/mL, and 2 ng/mL were diluted in the culture medium based on the previous report by Su et al. [52]. For the combination of P-MAPA and IL-12, the most effective dose and incubation period were determined after performing an MTT assay. The saline solution was used as a solvent vehicle control and prepared in the same volume and dilution for both treatments. All experiments were performed at 0, 24, 48, and 72 h time exposure and assayed in three technical and biological replicates.

#### *4.3. Cell Cytotoxicity (MTT Assay)*

The SKOV-3 cells were seeded in a 96-well plate at a density of 1 <sup>×</sup> 103 cells/well. The cellular activity and/or toxicity were evaluated in different concentrations of P-MAPA and IL-12 and periods (0, 24, and 48 h) to define the best treatment protocol. For this experiment, the choice of dose and period of treatment was the combination of high cellular viability with low toxicity and efficient signaling regulation. Thereafter, different doses of PTX were associated with P-MAPA and IL-12 to verify the sensitivity effects. An MTT solution (5 mg/mL) was added to the wells for 4 h, and the crystals were diluted with DMSO under agitation. The concentration was determined by an Epoch microplate reader (BioTek Instruments, Highland Park, PO, USA) at 540 nm, being the reference curve fixed at 650 nm. The percentage of crystal formation was calculated by fixing the control group crystal formation as 100%.

#### *4.4. Apoptosis Rate by Annexin V-FITC*/*PI Staining*

During the apoptosis process, cells normally externalize the phospholipid phosphatidylserine (PS), which binds with high affinity to Annexin V in the presence of calcium. Herein, we used the Annexin V assay with the BD PharmingenTM Annexin V-FITC Apoptosis Detection Kit (ApoAlert Annexin V, Clontech, CA, USA). The SKOV-3 cells (1 <sup>×</sup> 10<sup>5</sup> cells) were placed in a 6-well plate and left for 6 h to attach. The cells were then treated with 25 μg/mL of P-MAPA, 1 ng/mL of rhIL-12 or their association for 48 h. In addition, P-MAPA and IL-12 were added to the lowest dose of PTX (0.625 μM) to confirm chemosensitivity. After the treatment period, cells were trypsinized and centrifuged (Centrifuge 5804 R, Eppendorf, Hamburg, Germany)) for 10 min at 1200 rpm for culture medium removal. The cell pellet was washed twice with phosphate-buffered saline (PBS) and centrifuged at 10,000 rpm for 30 s, followed by resuspension with 100 μL Annexin V binding buffer and incubation with Annexin V and propidium iodide (PI) for 15 min in the dark at room temperature. After the incubation, the prepared cells were then analyzed by flow cytometry in a FACSCantoTMII with FACSDiva (BD Biosciences, Clontech, CA, USA) software. The flow cytometric results were analyzed by the FlowJo software (vX.10.6 version, Tree Stars Inc., Ashland, OR, USA).

#### *4.5. Cell Cycle Determination by PI Staining*

The cell cycle stages (G0/G1, S, and G2/M) were performed by flow cytometry analysis through the DNA content measurement of nuclei stained with PI dye. After all the treatments, the SKOV-3 cells (1 <sup>×</sup> 105) were trypsinized, washed with PBS, and centrifuged for 5 min at 1500 rpm. After the cells were fixed in 70% cold ethanol at 4 ◦C for 1 h, they were incubated with PI staining solution of 50 μg/mL PI and 10 mg/mL RNase A for 1 h at room temperature in a dark room. Flow cytometry was performed in a FACSCantoTMII with FACSDiva (BD Biosciences, Clontech, CA, USA) software to analyze DNA content. The relative ratios of cells in the G0/G1, S, and G2/M phases were calculated using FlowJo software (vX.10.6 version, Tree Stars Inc.).

#### *4.6. Wound-Healing Assay*

The wound-healing method was performed to verify the effects of P-MAPA and rhIL-12 alone or in combination on cell migration capacity. Briefly, 3 <sup>×</sup> 10<sup>5</sup> SKOV-3 cells were placed in 6-well plates with a serum-free culture medium for cell starvation. When the cells reached high confluence, a wound was created through the confluent cell monolayer using an angled tip at 45◦, and the SKOV-3 cells were then immediately treated with the pre-determined doses of P-MAPA, rhIL-12, or both, diluted in 2 mL of complete RPMI 1640 medium. Images were obtained at 0 h, 6 h, 12 h, 24 h, 36 h, and 48 h until the wound closure. Migration area (%) was measured using Image-J software. All experiments were analyzed in biological and technical triplicate.

#### *4.7. Cell Migration Using Transwell Insert*

To evaluate the migratory potential of the cells, 1 <sup>×</sup> <sup>10</sup><sup>4</sup> SKOV-3 cells were seeded in triplicates into the upper chambers of 24-well ThinCert™ cell culture inserts (GBO, Americana, SP, Brazil) with PVDF filters (8.0 μm pore size) containing the corresponding treatment diluted in serum free RPMI 1640 medium. In the lower chamber, RPMI supplemented with 10% FBS was added as a chemotactic factor. After the plates were incubated at 37 ◦C and 5% CO2 for 24 h, the cells in the upper chamber were gently removed with a cotton swab. The cells that had migrated to the lower surface of the insert through the 8.0 μm pore were fixed in methanol for 8 min and stained with hematoxylin for 45 s. The migrated cells were photographed with 20<sup>×</sup> objectives under an inverted microscope (ZeissAxiovert®, Germany). Four non-overlapped images were randomly analyzed for each well and the migrated cells were counted using Image J software.

#### *4.8. Invasion Assay*

The invasion assay was performed using a 24-well ThinCert™ cell culture insert (GBO, Americana, SP, Brazil) with PVDF filters (8.0 μm pore size). Briefly, 24-well plates were previously coated with Geltrex® (ThermoFisher, Waltham, MA, USA), which mimics the biological basement membrane matrix, and 1 <sup>×</sup> <sup>10</sup><sup>4</sup> cells were placed in the upper chamber with a serum-free culture medium containing the treatments. After 24 h, the invasive potential was determined by the amount of cells capable of crossing the barrier when chemotactically attracted by the RPMI 1640 medium supplemented with 10% FBS in the lower chamber. The protocol of cell fixing and staining was the same described for cell migration. Finally, four non-overlapped images were randomly analyzed per well and the invasive cells were counted using Image J software.

#### *4.9. Immunofluorescence Assay*

After the SKOV-3 cells were seeded in coverslips and treated with P-MAPA, rhIL-12, or their association for 48 h, they were fixed in methanol for 8 min at room temperature and then washed in sterile ice-cold PBS. To avoid nonspecific bindings, a blocking solution containing 3% (*v*/*v*) bovine serum albumin (BSA) was added for 1 h. Then, cells were incubated overnight with rabbit polyclonal anti-TLR2 and anti-TLR4 antibodies diluted 1:200 (Abcam, Cambridge, MA) for 4 h, followed by post incubation with secondary polyclonal anti-IgG conjugated to FITC (1:200 dilution in 1% BSA, Santa Cruz Biotechnology, Inc., CA) for 1 h. After the reactions, 4,6-diamidino-2-phenylindole (DAPI; Sigma, St Louis, MO) was used for nuclei staining. Positive staining was analyzed using a confocal fluorescence microscope Zeiss Axiophot II (Carl Zeiss, Oberköchen, Germany) at different magnification (excitation filter 590 nm, emission filter 650 nm) and for DAPI staining (excitation filter 365 nm, emission filter 485 nm). The relative fluorescence in FITC images was calculated using Image J software.

#### *4.10. Western Blot Analysis*

After the treatments, five replicates of each experiment were used. After being washed with cold PBS, 1 <sup>×</sup> 106 SKOV-3 cells were added to radioimmunoprecipitation assay buffer (RIPA) lysis buffer containing protease inhibitors and rapidly frozen for 24 h. Under constant agitation for 30 min at 4 ◦C, the cells were resuspended and transferred to 1.5 mL tubes and centrifuged for 20 min at 12,000 rpm. Protein quantification was performed using a Bradford assay, and the same amount of protein (40 μg) was solubilized in 1.5×Laemmli buffer and then used for 4–20% SDS-PAGE (Bio-Rad Laboratories, Hercules, CA, USA). After electrophoresis was carried out on tris-glycine running buffer system (120 V for 2 h), the proteins were electro-transferred (350 mA) to nitrocellulose membranes, and then blocked with 3% BSA in tris-buffered saline plus tween 20 (TBS-T) solution for 1 h. Afterward, the proteins were incubated with respective primary antibodies (1:500; Abcam, Cambridge, UK) at 4 ◦C overnight: anti-TLR2, anti-TLR4, anti-MyD88, anti-NF-kB p65, anti-TRIF, anti-IRF3. Subsequently, the membranes were washed three times and then incubated for 90 min with specific secondary antibodies (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:20,000 in 1% BSA. After sequential washes, positive reactions were performed using ECL kit (Thermo Fisher Scientific, MA). All of the blots were calculated using individual samples obtained from three replicates/group and were represented as the mean optical density (band intensity/housekeeping protein). β-actin was used as the endogenous control.

#### *4.11. Cytokine and Chemokine Assay*

Levels of different cytokines and chemokines were determined in both supernatants and cell homogenates using a MilliPlex® Map Kit (EMD Millipore, Darmstadt, Germany) with a standard 20-plex detection kit according to the manufacturer's protocols. The human cytokine/chemokine Panel I kit (cat. no. HCYTOMAG-60K) included the following analytes: interferon-gamma (IFN-γ), interleukin (IL)-1β, IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-9, IL- 10, IL-12, IL-13, IL-15, IL-17, IP-10, macrophage-derived chemokine (MDC/CCL22), monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1 (MIP)-1α, MIP-1β, and regulated upon activation normal T cell expressed and secreted (RANTES/CCL5). The concentrations were ranged between 0 and the lowest detectable level in each assay before log transformation. The analyses combined fluorescent cytometry and ELISA technology, so that each magnetic bead was added to a specific anti-cytokine to achieve a specific binding. For this experiment, the levels of analytes varied from 0.4 to 3500 pg/mL and the intra-assay CV was < 10%, inter-assay CV < 15%. No cross-reactivity was observed among cytokines and other molecules. The fluorescence intensity was measured using the MAGPIX system (Luminex Corporation, Austin, TX, USA).

#### *4.12. Statistical Analysis*

All data were evaluated using the analysis of variance (ANOVA) and presented as the mean ± standard deviation (SD). Significant results were then compared by Tukey or Newman-Keuls *post hoc* tests, and statistical significance was set at *p* < 0.05 for all analyses. Data were analyzed and constructed using GraphPad Prism 5.0 scientific graphing software (GraphPad Software, San Diego, CA, USA).

#### **5. Conclusions**

In summary, we demonstrated that P-MAPA in association with IL-12, both considered potent immunomodulatory agents, exhibited anti-cancer activities on human SKOV-3 cells. Although P-MAPA combined with IL-12 promotes a reduction in cell viability and cell migration, P-MAPA alone reduces the invasion capacity and enhances apoptosis in the presence of PTX. Similarly to either P-MAPA or IL-12 alone, the combinatory therapy induced the downregulation of TLR-downstream molecules involved with inflammation, which may result in protection against chemoresistance; these effects seem to be associated with TLR2 suppression rather than TLR4 signaling. P-MAPA alone stimulated the secretion of pro- and anti-inflammatory mediators, and its association with IL-12 increased the production of IL-4, -8, and MIP-1α by the SKOV-3 cells; this may promote changes in the immune responsiveness of the OC microenvironment. In addition to the effect on OC-infiltrated immune cells, these immunotherapies might provide a sustained opportunity for a novel combined strategy against malignant OC cells.

**Supplementary Materials:** The following are available online. Figure S1: MTT assay was tested with different cell densities to find the most appropriate strategy for cell counting. SKOV-3 cells at density of 1 <sup>×</sup> 10<sup>3</sup> were representative to determine cell viability; Table S1: Multiplex assay of the cytokines and chemokines (pg/mL) in the supernatant of cell culture; Table S2: Multiplex assay of the cytokines and chemokines (pg/mL) in the SKOV-3 cells.

**Author Contributions:** L.A.L., L.G.d.A.C., F.K.D.: conceived the hypothesis of the study, collected and analyzed the data, and drafted the manuscript. W.J.F., F.E.M., M.M., G.G.R., R.K., M.S.C., R.F.D., I.d.S.N.: participated in its design, intellectual conception of the study, and in the acquisition of data. All authors have read and agreed to the published version of the manuscript.

**Funding:** We would like to give a special thanks to Farmabrasilis-Brazil, FAPESP (*Fundação de Amparo à Pesquisa do Estado de São Paulo*, grant numbers: 2019/00906-6 and 2016/03993-9), CAPES (grant number: 0708/2018), and CNPq (grant number: 401040/2016-0) by providing financial support.

**Acknowledgments:** We are grateful to HélioKushima and Valeria Sandrim from Department of Pharmacology, IBB/UNESP, Botucatu, SP, Brazil, for excellent technical assistance and support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**


#### **References**


**Sample Availability:** Samples of the compounds are not available from the authors.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **Design, Synthesis, and Biological Evaluation of Pyridineamide Derivatives Containing a 1,2,3-Triazole Fragment as Type II c-Met Inhibitors**

### **Hehua Xiong** †**, Jianxin Cheng** †**, Jianqing Zhang, Qian Zhang, Zhen Xiao, Han Zhang, Qidong Tang \* and Pengwu Zheng \***

School of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang 330013, China; 18296154955@163.com (H.X.); cjx3159@163.com (J.C.); zhang1405474771@163.com (J.Z.); 15797966937@163.com (Q.Z.); xz950420@163.com (Z.X.); zbl1045762244@163.com (H.Z.)

**\*** Correspondence: tangqidongcn@126.com (Q.T.); zhengpw@jxstnu.edu.cn (P.Z.); Tel.: +86-791-8380-2393 (P.Z.)

† These authors contribute equally to this work.

Academic Editor: Qiao-Hong Chen

Received: 21 November 2019; Accepted: 10 December 2019; Published: 18 December 2019

**Abstract:** A series of 4-(pyridin-4-yloxy)benzamide derivatives containing a 1,2,3-triazole fragment were designed, synthesized, and their inhibitory activity against A549, HeLa, and MCF-7 cancer cell lines was evaluated. Most compounds exhibited moderate to potent antitumor activity against the three cell lines. Among them, the promising compound **B26** showed stronger inhibitory activity than Golvatinib, with IC50 values of 3.22, 4.33, and 5.82 μM against A549, HeLa, and MCF-7 cell lines, respectively. The structure–activity relationships (SARs) demonstrated that the modification of the terminal benzene ring with a single electron-withdrawing substituent (fluorine atom) and the introduction of a pyridine amide chain with a strong hydrophilic group (morpholine) to the hinge region greatly improved the antitumor activity. Meanwhile, the optimal compound **B26** showed potent biological activity in some pharmacological experiments in vitro, such as cell morphology study, dose-dependent test, kinase activity assay, and cell cycle experiment. Finally, the molecular docking simulation was performed to further explore the binding mode of compound **B26** with c-Met.

**Keywords:** 4-(pyridin-4-yloxy)benzamide; 1,2,3-triazole; c-Met; inhibitor

#### **1. Introduction**

Cancer has become a serious threat to human life and health [1,2]. c-Met tyrosine kinase is a kind of type III tyrosine kinase, which is closely related to cellular activities [3] such as growth, reproduction, metastasis, etc. However, abnormal expression of c-Met kinases in cells usually triggers the occurrence, invasion, and metastasis of various cancer diseases [4]. c-Met kinase has been found overexpressed in various cancer cells and has become an attractive target for cancer treatment [5,6]. Nowadays, developing small molecule c-Met kinase inhibitors has become a hotspot in the treatment of human cancer [7].

c-Met inhibitors are classified as type I and type II inhibitors according to the binding mode of inhibitors with c-Met. Usually, c-Met inhibitors of type I are single-target inhibitors that bind to the hinge region of the ATP pocket, and c-Met inhibitors of type II are multitarget inhibitors that bind to the hinge region and an extra hydrophobic pocket [8,9]. With the development of biology and pharmacology, the intracellular mechanisms have been elucidated, and research in small molecule inhibitors has made significant progress. Type II c-Met inhibitors possess more potent inhibitory activity and better tolerance to drug resistance through binding to kinases of multiple types. Cabozantinib, a c-Met inhibitor of type II, was approved by the FDA for treatment of prostate cancer in 2012. Some representative c-Met inhibitors (type II) in the clinical trial phase are listed in Figure 1, including

Golvatinib, BMS-777607, Altriatinib, and TAS-115 [10–12]. Furthermore, according to the structure characteristics of inhibitors, the skeleton of c-Met inhibitor (type II) was summarized into blocks A, B, C, and D by our research group, as shown in Figure 1 [13].

**Figure 1.** Some representative c-Met inhibitors of type II and the summarized skeleton.

We explored the SARs of c-Met inhibitors (type II) bearing pyridinamide structure using Golvatinib as a lead compound in this study. In order to guide our modification of the lead compound, a molecular docking simulation of Golvatinib and c-Met protein was performed, as shown in Figure 2. The docking results indicated that blocks A and C, which possessed hydrogen bonding interactions with residues Met1160, Asp1222, and Lys1110, played a key role in maintaining the inhibitory activity against c-Met. Therefore, the modification was mainly concentrated on blocks A and C. Firstly, a pyridylamide structure in block A, including hydrogen bond donor and hydrogen bond acceptor atoms, was introduced into the meta position of pyridine in order to enhance the interaction with the hinge region residues, and then some alkyl chains bearing morpholine or thiophene groups were connected to the terminal of the pyridine amide structure for better water solubility of compounds. Then, a 1,2,3-triazole fragment with good biological activity and two hydrogen bond acceptor atoms was introduced to block C for more tight combination between the compounds and c-Met. In addition, considering the steric hindrance problem [14], H/F atoms and different substituents were introduced in blocks B and D, respectively, to explore their effects on biological activity of target compounds. Finally, a series of small molecule inhibitors containing the 1,2,3-triazole fragment were designed, synthesized, and biologically evaluated.

**Figure 2.** Design strategy of target compounds based on the molecular simulation and structure–activity relationships (SARs).

#### **2. Results and Discussion**

#### *2.1. Chemistry*

The synthesis route of target compounds **B1–B27** is shown in Scheme 1. Firstly, the commercially available pyridinecarboxylic acid **6** was reacted with SOCl2 using NaBr as catalyst to yield 4-chloropicolinoyl chloride **7**, which was then converted into ethyl 4-chloropicolinate **8** with ethanol. Next, 4-chloropicolinate **8** was subjected to nucleophilic substitute with *p*-nitrophenol or 2-fluoro-4-nitrophenol using chlorobenzene as solvent to give picolinate analogues **9a–9b**, which were then dissolved in 1,4-dioxane and hydrolyzed with NaOH aqueous solution to obtain picolinic acid analogues **10a–10b**. Intermediates **10a–10b** were reacted with SOCl2 to get the corresponding acyl chlorides and then connected with amines (including propan-1-amine, 2-(thiophen-2-yl)ethan-1-amine, pyrrolidine and 3-morpholinopropan-1-amine) to obtain picolinamide analogues **11a–11e**. Finally, the key intermediates **12a–12e** were produced by the reduction of **11a–11e** with hydrazine hydrate as reducing agent. Other intermediates **13a–13i**, which have been reported in our previous research [15], were combined with intermediates **12a**–**12e** by a nucleophilic substitute reaction to get the target compounds **B1–B27**.

The structural information of the target compounds was confirmed by 1H-NMR, 13C-NMR, and TOF MS (ES+), which were consistent with the structures depicted.

**Scheme 1.** The synthetic route for target compounds **B1–B27**. Reagents and conditions: (i) NaBr, SOCl2, 85 ◦C, 18 h; (ii) triethylamine, dichloromethane (DCM), EtOH, 0 ◦C, 1 h; (iii) *p*-nitrophenol or 2-fluoro-4-nitrophenol, chlorobenzene, 135 ◦C, 4 h; (iv) 1,4-dioxane, sodium hydroxide (10%), r.t., 0.5 h; (v) SOCl2, amines, Et3N, DCM, 0–85 ◦C, 1 h; (vi) EtOH, activated carbon, FeCl3·6H2O, hydrazine hydrate (80%), 90 ◦C, 4 h. (vii) EtOH, activated carbon, FeCl3·6H2O, hydrazine hydrate (80%), 90 ◦C, 4 h; (vii) DCM, NaHCO3, 0 ◦C, 1 h.

#### *2.2. Antitumor Activity of Compounds B1–B27 Against A549, HeLa, and MCF-7 Cell Lines and SAR Analysis*

The antitumor activity of target compounds against A549, HeLa, and MCF-7 cell lines was evaluated to investigate their inhibitory activity against cancer cells [16]. The antitumor activities of compounds were displayed as the IC50 (half-inhibitory concentration) values, as shown in Tables 1 and 2. Most compounds exhibited moderate to potent antitumor activity against three cell lines. Compounds **B25–B27** possessed more potent inhibitory activity than Golvatinib, as shown in Table 2, with IC50 values in the range of 3.22–13.60 μM. The optimal compound **B26** showed excellent inhibitory activity against A549, HeLa, and MCF-7 cell lines with IC50 values of 3.22 ± 0.12, 4.33 ± 0.09, and 5.82 ± 0.09 μM, respectively, which were more efficient than Golvatinib (8.14 ± 0.45, 15.17 ± 0.17, and 16.91 ± 0.29 μM). **Table 1.** Antitumor activities of the target compounds **B1–B18** against A549, HeLa, and MCF-7 cell lines.


<sup>a</sup> NA: Low inhibitory activity, <sup>b</sup> Used as the positive control.

**Table 2.** Antitumor activities of the target compounds **B10–B12** and **B19–B27** against A549, HeLa, and MCF-7 cell lines.

<sup>a</sup> Bold values show the IC50 values of the target compounds lower than the values of the positive control; <sup>b</sup> Used as the positive control.

According to the antitumor activity of compounds **B1–B27** listed in Tables 1 and 2, the SARs were summarized as follows. Overall, the F atom on the meta-central benzene ring provided stronger inhibitory activity against cancer cells than H atom, for example, compounds **B1–B8** displayed less inhibitory activity than compounds **B9–B18**. A single substitution (such as H/F/OCF3/Cl/CF3) linked to the terminal benzene ring was favorable for maintaining the biological activity, such as compounds **B1–B5** and **B10–B15** that possessed moderate antitumor activity with IC50 values less than 60 μM against A549 and HeLa cell lines. However, the double substituents connected to the terminal benzene ring obviously impaired the inhibitory activity of compounds **B6–B9** and **B15–B18,** whose IC50 values were more than 120 μM against three cell lines. A possible explanation would be that the introduction of two substituents to the terminal benzene ring may create steric hindrance, which made it difficult for compounds to extend into the hydrophobic pocket and to bind tightly to c-Met kinase.

Inspired by the inhibitory activity of compounds **B1–B3** and **B10–B12**, as shown in Table 1, the F atom of the central benzene ring and the single group (H, F, or OCF3) of the terminal benzene ring were reserved to perform a SAR study of the pyridine amide moiety. Hydrophilic groups, including morpholine or thiophene groups attached to the terminal of pyridine amide, have a great contribution to inhibitory activity of the compounds **B22–B27** (with IC50 values in the range of 3.22–45.18 μM).

In summary, introducing morpholino groups and F atoms to the pyridylpropyl amide and the terminal benzene ring, respectively, made significant advances in the inhibitory activity.

#### *2.3. Dose-Dependent Test of Compound B26 Against A549 Cells*

To explore the relationship between concentrations and antitumor activity of compound **B26**, we carried out a dose-dependent test based on the MTT method [16], in which A549 cells were treated with seven different concentrations of compound **B26** (0.137, 0.411, 1.234, 3.704, 11.11, 33.33, and 100 μM), using Golvatinib as a positive control, as seen in Figure 3. The results revealed that compound **B26** could effectively inhibit cancer cells in a dose-dependent manner. Notably, compound **B26** inhibited A549 cells more than 50% at the concentration of 3.704 μM, while the inhibitory rate of Golvatinib was less than 40% at the same concentration.

**Figure 3.** Relationship between concentration and inhibition rate of compound **B26** against A549 cells.

#### *2.4. Cell Cycle Study of A549 Cells Treated with Compound B26*

To further investigate the inhibitory pattern of compound **B26** on the growth of cancer cells, cell cycle distribution analysis [17] was carried out on A549 cells, as shown in Figure 4. Compound **B26** at concentrations of 5, 10, and 15 μM and Golvatinib at a concentration of 5 μM were delivered to A549 cells for 24 h, as shown in Figure 4. With the increased concentration of compound **B26**, the percentage of cells blocked in G0/G1 phase was increased from 65.44% to 79.18%, while the percentage in the S phase decreased from 21.91% to 16.56%. There was no significant change in G2/M phase. The result showed that compound **B26** (74.41%) and Golvatinib (74.77%) had the same inhibitory effect on A549 cells at the concentration of 5 μM, both of which could block cells in the G0/G1 phase.

**Figure 4.** Cell cycle study of A549 cells treated with compound **B26** and Golvatinib.

#### *2.5. c-Met Enzyme Assay of Compounds B25–B27 and Staurosporine*

Compounds **B25**, **B26**, and **B27**, possessing potent antitumor activity against A549, HeLa, and MCF-7 cell lines, were selected to investigate their c-Met kinase assay by mobility shift assay method [18] with staurosporine as a positive control, which was used to insure the reliability of the experimental data. Compound **B26** exhibited potent inhibitory activity against c-Met with an inhibition rate of 36% at the concentration of 0.625 μM, as shown in Table 3.

**Table 3.** c-Met kinase activity of selected compounds **B25**, **B26**, **B27**, and staurosporine.


#### *2.6. Molecular Docking Simulation of Compounds (B26 and Golvatinib) and c-Met*

Next, we further explored the binding mode of compound **B26** and Golvatinib with c-Met kinase (PDB: 3LQ8, extracted from the PDB database) through molecule docking simulation using AutoDock 4.2 software [19], as shown in Figure 5. The docking results indicated that compound **B26** and Golvatinib in an extended conformation formed strong hydrogen bonding interactions with residues Met1160, Asp1222, and Lys1110, as shown in Figure 5a–d. Interestingly, the pyridylamide structure of compound **B26** formed a bidentate hydrogen bond with residue Met1160, as shown in Figure 5a,b, which may offer a stronger ability of compound **B26** to bind with c-Met. Compound **B26** formed more hydrogen bonding interactions with Met1160 than Golvatinib, which may explain the better antitumor activity of compound **B26**.

**Figure 5.** Figure 5a, 5b, and 5c are partial views, two-dimensional partial views, and integral cavity views of the docking results of c-Met (PDB code: 3LQ8) and compound **B26**, respectively. Figure 5d shows partial views of c-Met and Golvatinib. The c-Met protein and compounds are displayed by cartoons and sticks, respectively. Hydrogen bonding interactions between compounds and c-Met are indicated with dashed lines or arrows in red.

#### **3. Experimental Section**

#### *3.1. Chemistry*

Unless specifically required, all reagents used in the experiments were purchased as commercial analytical grade and used without further purification. Frequently used solvents (dichloromethane, ethyl acetate, tetrahydrofuran, 1,4-dioxane, methanol, ethanol, etc.) were absolutely anhydrous. Flash column chromatography was performed on silica gel (300 mesh) using a mixture of petroleum ether (PE) and ethyl acetate (EA). The reaction process was monitored by thin-layer chromatography (TLC, SH-GH254) and spots were visualized with ultraviolet analyzer (light in 254 or 365 nm). 1H-NMR and 13C-NMR spectra analysis of compounds was implemented in Bruker 400 MHz spectrometer (Bruker Bioscience, Billerica, MA, USA) using tetramethylsilane (TMS) as an internal standard at room temperature. Mass spectrometry (MS) of target compounds was carried out Waters High Resolution Quadrupole Time of Flight Tandem Mass Spectrometry (Waters, Xevo G2-XS Tof). The melting point of the compounds was measured by SGW X-4 micro melting point instrument. The purity of compounds was determined by an Agilent 1260 liquid chromatograph equipped with an Inertex-C18 column, and all were more than 95%.

#### 3.1.1. Preparation of 4-Chloropicolinoyl Chloride (**7**)

Pyridinecarboxylic acid **6** (10.00 g, 0.081 mol), NaBr (0.10 g, 0.001 mol), and a drop of *N,N*-dimethylformamide (DMF) were added in thionyl chloride (SOCl2, 50 mL) and stirred at 85 ◦C for 18 h. The reaction was monitored by TLC. After completion of the reaction, the solvent was removed under vacuum to obtain a yellow liquid, which was dissolved in DCM for further use.

#### 3.1.2. Preparation of Ethyl 4-Chloropicolinate (**8**)

Dichloromethane (30 mL), ethanol (16.43 g, 0.170 mol), and triethylamine (17.17 g, 0.170 mol) were successively added to a beaker and stirred at 0 ◦C for 0.5 h. Subsequently, a mixture of

4-chloropicolinoyl chloride **7** (16.43 g, 0.110 mol) and DCM (5 mL) was added dropwise, stirred at 0 ◦C for 0.5 h, and monitored by TLC. After completion of the reaction, NaOH solution was added to the mixture to adjust the pH value 9–10. Ultimately, the organic layers were combined and dried to obtain a brown liquid.

3.1.3. Preparation of Ethyl 4-(4-Nitrophenoxy)picolinate (**9a**) and Ethyl4-(2-Fluoro-4-Nitrophenoxy) picolinate (**9b**)

Intermediate **8** (13.22 g, 0.071 mol) and *p*-nitrophenol or 2-fluoro-4-nitrophenol (0.026 mol) were dissolved in chlorobenzene (40 mL) and then stirred at 130 ◦C for 4 h. The completion of the reaction was monitored by TLC. After completion of the reaction, the cold petroleum ether (100 mL) was poured into the reaction solvent and stirred at room temperature for 0.5 h. Next, petroleum ether (100 mL) was poured off to give a viscous liquid, which was then dissolved in DCM (300 mL) and extracted with aqueous solution of NaOH. The organic layer was combined, dried, and concentrated under vacuum to get a pale yellow solid.

3.1.4. Preparation of 4-(4-Nitrophenoxy)picolinic Acid (**10a**) and 4-(2-Fluoro-4-Nitrophenoxy)picolinic Acid (**10b**)

Intermediates **9a–9b** (0.035 mol) were dissolved in 1,4-dioxane (50 mL) and stirred at room temperature for 0.5 h. A solution of 10% NaOH (2.28 mL, 0.057 mol) was then added dropwise to the above mixture, stirred at room temperature for 0.5 h, and monitored by TLC. After completion of the reaction, the solvent was evaporated under vacuum to give a white solid. The white solid was then dissolved in saturated NaCl solution (500 mL) and stirred at room temperature for 10 h. Subsequently, after the pH of the solution was adjusted to 2–3 by 75% hydrochloric acid (HCl), a yellow solid was precipitated, filtered, and dried.

3.1.5. Preparation of 4-(4-Nitrophenoxy)picolinamide or 4-(2-Fluoro-4-Nitrophenoxy)picolinamide Analogues (**11a–11e**)

Intermediates **10a–10b** (0.020 mol) and one drop of DMF were successively dissolved in SOCl2 (50 mL) and stirred at 85 ◦C for 0.5 h. Then reaction solvent was concentrated in vacuum, dissolved in DCM (8 mL), and added dropwise to a mixture of DCM (50 mL), ammonia analogs (0.020 mol), and triethylamine (0.020 mol). The reaction solution was stirred at 0 ◦C for 0.5 h, and monitored by TLC. Finally, a gray slid was obtained by the same post-treatment method as that used for preparation of ethyl 4-chloropicolinate **8**.

3.1.6. Preparation of 4-(4-Aminophenoxy)picolinamide or 4-(2-Fluoro-4-aminophenoxy)picolinamide Analogues (**12a–12e**)

Intermediates **11a–11e** (0.016 mol), activated carbon (1.92 g, 0.160 mol), and FeCl3·6H2O (0.43 g, 0.016 mol) were dissolved in ethanol (30 mL) and stirred at 90 ◦C for 0.5 h. Then, 80% hydrazine hydrate (8.00 g, 0.128 mol) was added dropwise and stirred for 3.5 h. The reaction was monitored by TLC. After completion of the reaction, the solution was filtered, concentrated, and recrystallized to give a green solid.

3.1.7. Preparation of 5-Methyl-1-Phenyl-1H-1,2,3-Triazole-4-Carbonyl Chloride or 1-Phenyl-5- (trifluoromethyl)-1H-1,2,3-Triazole-4-Carbonyl Chloride Analogues (**13i–13e**)

The preparation of intermediates **13a–13i** was carried out according to our previous research [15].

#### 3.1.8. Preparation of Target Compounds **B1–B27**

Intermediates **12a–12e** (0.002 mol) and sodium bicarbonate (4.20 g, 0.005 mol) were dissolved in DCM and stirred at 0 ◦C for 0.5 h. The solution of intermediates **13a–13i** (0.002 mol) and DCM were added dropwise to the above mixture, and stirred at 0 ◦C for 0.5 h. The reaction was monitored by TLC. After completion of the reaction, the solution was extracted with a mixture of DCM/NaOH (500 mL) three times and then the organic layers were combined, dried over sodium sulfate, evaporated, and purified by chromatographic column to possess a light yellow or white solid.

*4-(4-(5-methyl-1-phenyl-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-propylpicolinamide* (**B1**) Light yellow solid; Yield: 78.5%; m.p.: 116.8–117.1 ◦C; 1H-NMR (400 MHz, DMSO-*d*6, ppm) δ 10.75 (s, 1H), 8.82 (s, 1H), 8.52 (d, *J* = 5.5 Hz, 1H), 8.03 (s, 1H), 8.01 (s, 1H), 7.67 (s, 2H), 7.64 (d, *J* = 6.4 Hz, 2H), 7.43 (s, 1H), 7.23 (d, *J* = 8.4 Hz, 2H), 7.18 (d, *J* = 4.5 Hz, 1H), 3.22 (dd, *J* = 12.8, 6.3 Hz, 2H), 2.59 (s, 3H), 1.50 (dt, *J* = 14.1, 7.0 Hz, 2H), 0.84 (t, *J* = 7.2 Hz, 3H). TOF MS ES+ (*m*/*z*): [M + H]<sup>+</sup>, calcd for C25H24N6O3: 457.1988, found, 457.1986.

*4-(4-(1-(4-fluorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-propylpicolinamide* (**B2**) Light yellow solid; Yield: 59.3%; m.p.: 110.4–110.8 ◦C; 1H-NMR (400 MHz, DMSO-*d*6, ppm) δ 10.64 (s, 1H), 8.71 (s, 1H), 8.40 (d, *J* = 5.6 Hz, 1H), 7.91 (s, 1H), 7.89 (s, 1H), 7.65 (d, *J* = 4.8 Hz, 1H), 7.63 (d, *J* = 4.6 Hz, 1H), 7.39 (t, *J* = 8.7 Hz, 2H), 7.31 (d, *J* = 2.1 Hz, 1H), 7.11 (d, *J* = 8.8 Hz, 2H), 7.06 (d, *J* = 3.1 Hz, 1H), 3.10 (dd, *J* = 13.3, 6.6 Hz, 2H), 2.46 (s, 3H), 1.38 (dt, *J* = 14.2, 7.1 Hz, 2H), 0.72 (t, *J* = 7.3 Hz, 3H). TOF MS ES+ (*m*/*z*): [M + H]<sup>+</sup>, calcd for C25H23FN6O3: 475.1894, found, 475.1896.

*4-(4-(5-methyl-1-(2-(trifluoromethoxy)phenyl)-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-propylpicolinamide* (**B3**) White solid; Yield: 78.2%; m.p.: 97.7–98.0 ◦C; 1H-NMR (400 MHz, DMSO-*d*6, ppm) δ 10.81 (s, 1H), 8.81 (s, 1H), 8.52 (d, *J* = 5.3 Hz, 1H), 8.03 (s, 1H), 8.01 (s, 1H), 7.88 (d, *J* = 7.7 Hz, 1H), 7.84 (d, *J* = 7.6 Hz, 1H), 7.78 (d, *J* = 7.7 Hz, 1H), 7.72 (t, *J* = 7.2 Hz, 1H), 7.42 (s, 1H), 7.23 (d, *J* = 8.6 Hz, 2H), 7.18 (d, *J* = 0.8 Hz, 1H), 3.22 (d, *J* = 6.2 Hz, 2H), 2.46 (s, 3H), 1.51 (dd, *J* = 14.0, 7.0 Hz, 2H), 0.84 (t, *J* = 7.2 Hz, 3H). TOF MS ES+ (*m*/*z*): [M + H]<sup>+</sup>, calcd for C26H23F3N6O4: 541.1811, found, 541.1813.

*4-(4-(1-(4-chlorophenyl)-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-propylpicolinamide* (**B4**) White solid; Yield: 66.4%; m.p.: 159.0–159.2 ◦C; 1H-NMR (400 MHz, DMSO-*d*6, ppm) δ 11.19 (s, 1H), 8.80 (s, 1H), 8.52 (d, *J* = 5.5 Hz, 1H), 7.99 (s, 1H), 7.97 (d, *J* = 1.2 Hz, 1H), 7.79 (d, *J* = 4.4 Hz, 3H), 7.75 (d, *J* = 8.7 Hz, 1H), 7.42 (s, 1H), 7.27 (d, *J* = 8.7 Hz, 2H), 7.19 (d, *J* = 5.5 Hz, 1H), 3.23 (dd, *J* = 13.2, 6.7 Hz, 2H), 1.57–1.47 (m, 2H), 0.84 (t, *J* = 7.4 Hz, 3H). 13C-NMR (100 MHz, DMSO-*d*6, ppm) δ 165.73, 163.13, 156.58, 152.54, 150.36(2,C), 149.46, 142.31, 136.19, 135.84, 134.09, 129.70(2,C), 128.22(2,C), 122.40(2,C), 121.33(2,C), 114.18(2,C), 108.95, 40.58, 22.34, 11.27. TOF MS ES+ (*m*/*z*): [M + H]+, calcd for C25H20ClF3N6O3: 545.1316, found, 545.1317.

*N-propyl-4-(4-(5-(trifluoromethyl)-1-(2-(trifluoromethyl)phenyl)-1H-1,2,3-triazole-4-carboxamido)phenoxy) picolinamide* (**B5**) White yellow solid; Yield: 69.4%; m.p.: 163.4–163.7 ◦C; 1H-NMR (400 MHz, DMSO-*d*6, ppm) δ 11.26 (s, 1H), 8.80 (s, 1H), 8.53 (d, *J* = 5.2 Hz, 1H), 8.15–8.09 (m, 2H), 8.05 (d, *J* = 7.2 Hz, 1H), 8.00 (d, *J* = 7.7 Hz, 3H), 7.43 (s, 1H), 7.27 (d, *J* = 8.5 Hz, 2H), 7.18 (d, *J* = 2.7 Hz, 1H), 3.23 (dd, *J* = 12.4, 6.0 Hz, 2H), 1.57–1.47 (m, 2H), 0.84 (t, *J* = 7.3 Hz, 3H). 13C-NMR (100 MHz, DMSO-*d*6, ppm) δ 165.72, 163.14, 156.20, 152.53, 150.36(2,C), 149.53, 141.89, 135.72, 134.28(2,C), 132.86, 132.02, 130.02(2,C), 127.71, 122.60(2,C), 121.29(2,C), 114.17(2,C), 108.96, 40.57, 22.33, 11.24. TOF MS ES+ (*m*/*z*): [M + H]+, calcd for C26H20F6N6O3: 579.1580, found, 579.1573.

*4-(4-(1-(3,4-difluorophenyl)-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-propylpicolinamide* (**B6**) Light yellow solid; Yield: 85.0%; m.p.: 97.6–97.8 ◦C; 1H-NMR (400 MHz, DMSO-*d*6, ppm) δ 11.21 (s, 1H), 8.80 (s, 1H), 8.52 (d, *J* = 4.9 Hz, 1H), 8.12 (s, 1H), 7.98 (d, *J* = 6.9 Hz, 2H), 7.81 (d, *J* = 8.9 Hz, 1H), 7.74 (s, 1H), 7.41 (s, 1H), 7.27 (d, *J* = 6.9 Hz, 2H), 7.20 (d, *J* = 2.3 Hz, 1H), 3.26–3.19 (m, 2H), 1.52 (dd, *J* = 13.7, 6.8 Hz, 2H), 0.84 (t, *J* = 7.1 Hz, 3H). 13C-NMR (100 MHz, DMSO-*d*6, ppm) δ 165.73, 163.13, 156.48, 152.54, 150.36(2,C), 149.48, 142.09, 135.81, 131.59, 124.44, 122.46(2,C), 121.32(2,C), 118.63, 118.44, 117.06, 116.86, 114.19(2,C), 108.93, 40.58, 22.34, 11.26. TOF MS ES+ (*m*/*z*): [M + H]+, calcd for C25H19F5N6O3: 547.1517, found, 547.1517.

*4-(4-(1-(3-chloro-4-fluorophenyl)-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-propylpicolinamide* (**B7**) White solid; Yield: 81.6%; m.p.: 153.6–153.9 ◦C; 1H-NMR (400 MHz, DMSO-*d*6, ppm) δ 11.23 (s, 1H), 8.80 (t, *J* = 5.9 Hz, 1H), 8.52 (d, *J* = 5.6 Hz, 1H), 7.99 (s, 1H), 7.97 (s, 1H), 7.79 (d, *J* = 4.0 Hz, 3H), 7.42 (s, 1H), 7.27 (d, *J* = 8.6 Hz, 2H), 7.18 (d, *J* = 3.8 Hz, 1H), 3.23 (dd, *J* = 13.3, 6.5 Hz, 2H), 1.57–1.47 (m, 2H), 0.84 (t, *J* = 7.3 Hz, 3H). 13C-NMR (100 MHz, DMSO-*d*6, ppm) δ 165.73, 163.15, 156.61, 152.52, 150.33(2,C), 149.45, 142.38, 136.20, 135.87, 134.06, 129.69(2,C), 128.18(2,C), 122.39(2,C), 121.29(2,C), 114.13(2,C), 108.99, 40.58, 22.34, 11.25. TOF MS ES+ (*m*/*z*): [M + H]<sup>+</sup>, calcd for C25H19ClF4N6O3: 563.1222, found, 563.1216.

*4-(4-(1-(2-chloro-5-(trifluoromethyl)phenyl)-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamido)phenoxy)-Npropylpicolinamide* (**B8**) White solid; Yield: 56.7%; m.p.: 143.3–143.7 ◦C; 1H-NMR (400 MHz, DMSO-*d*6, ppm) δ 11.30 (s, 1H), 8.81 (s, 1H), 8.65 (s, 1H), 8.53 (d, *J* = 4.9 Hz, 1H), 8.21 (d, *J* = 8.0 Hz, 1H), 8.15 (d, *J* = 7.9 Hz, 1H), 8.00 (d, *J* = 8.4 Hz, 2H), 7.42 (s, 1H), 7.27 (d, *J* = 8.5 Hz, 2H), 7.19 (d, *J* = 2.1 Hz, 1H), 3.22 (dd, *J* = 12.2, 6.0 Hz, 2H), 1.52 (dq, *J* = 13.9, 7.1 Hz, 2H), 0.84 (t, *J* = 7.1 Hz, 3H). 13C-NMR (100 MHz, DMSO-*d*6, ppm) δ 165.72, 163.14, 156.13, 152.54, 150.37(2,C), 149.56, 141.97, 135.71, 135.05, 133.58, 131.78(2,C), 131.49, 130.44, 127.03, 122.63(2,C), 121.28(2,C), 114.18(2,C), 108.96, 40.58, 22.33, 11.25. TOF MS ES+ (*m*/*z*): [M + H]<sup>+</sup>, calcd for C26H19ClF6N6O3: 613.1190, found, 613.1189.

*4-(4-(1-(4-chloro-3-(trifluoromethyl)phenyl)-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamido)phenoxy)-Npropylpicolinamide* (**B9**) Light yellow solid; Yield: 66.3%; m.p.: 155.5–155.8 ◦C; 1H-NMR (400 MHz, DMSO-*d*6, ppm) δ 11.21 (s, 1H), 8.81 (s, 1H), 8.53 (d, *J* = 5.3 Hz, 1H), 8.44 (s, 1H), 8.17 (d, *J* = 8.1 Hz, 1H), 8.10 (d, *J* = 8.4 Hz, 1H), 7.99 (d, *J* = 8.7 Hz, 2H), 7.41 (s, 1H), 7.27 (d, *J* = 8.6 Hz, 2H), 7.19 (d, *J* = 2.9 Hz, 1H), 3.22 (dd, *J* = 12.8, 6.2 Hz, 2H), 1.52 (dq, *J* = 14.8, 7.4 Hz, 2H), 0.84 (t, *J* = 7.3 Hz, 3H). TOF MS ES+ (*m*/*z*): [M + H]<sup>+</sup>, calcd for C26H19ClF6N6O3: 613.1190, found, 613.1188.

*4-(2-fluoro-4-(5-methyl-1-phenyl-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-propylpicolinamide* (**B10**) White solid; Yield: 76.8%; m.p.: 143.3–143.7 ◦C; 1H-NMR (400 MHz, DMSO-*d*6, ppm) δ 10.96 (s, 1H), 8.83 (s, 1H), 8.54 (d, *J* = 5.4 Hz, 1H), 8.11 (d, *J* = 13.2 Hz, 1H), 7.86 (d, *J* = 8.7 Hz, 1H), 7.67 (d, *J* = 2.6 Hz, 5H), 7.48–7.39 (m, 2H), 7.23 (d, *J* = 4.7 Hz, 1H), 3.23 (dd, *J* = 12.9, 6.3 Hz, 2H), 2.59 (s, 3H), 1.57–1.47 (m, 2H), 0.84 (t, *J* = 7.3 Hz, 3H). 13C-NMR (100 MHz, DMSO-*d*6, ppm) δ 165.66, 163.43, 160.15, 153.09, 150.87(2,C), 138.46, 138.29, 135.61, 130.48, 130.09(3,C), 125.84(3,C), 124.07, 117.64, 113.82, 109.37, 108.46, 41.02, 22.73, 11.67, 9.86. TOF MS ES+ (*m*/*z*): [M + H]<sup>+</sup>, calcd for C25H23FN6O3: 475.1894, found, 475.1894.

*4-(2-fluoro-4-(1-(4-fluorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-propylpicolinamide* (**B11**) White solid; Yield: 65.1%; m.p.: 143.5–143.8 ◦C; 1H-NMR (400 MHz, DMSO-*d*6, ppm) δ 10.95 (s, 1H), 8.82 (s, 1H), 8.54 (d, *J* = 5.1 Hz, 1H), 8.10 (d, *J* = 12.7 Hz, 1H), 7.86 (d, *J* = 8.6 Hz, 1H), 7.76 (s, 2H), 7.51 (t, *J* = 8.4 Hz, 2H), 7.44 (d, *J* = 12.9 Hz, 2H), 7.22 (d, *J* = 2.5 Hz, 1H), 3.22 (d, *J* = 6.0 Hz, 2H), 2.58 (s, 3H), 1.51 (dd, *J* = 13.9, 6.9 Hz, 2H), 0.84 (t, *J* = 7.2 Hz, 3H). 13C-NMR (100 MHz, DMSO-*d*6, ppm) δ 165.65, 164.20, 163.42, 161.74, 160.11, 153.08, 150.88, 138.75, 138.22, 131.99, 128.44, 128.35, 124.08, 117.65, 117.18(2,C), 116.95, 113.83, 109.60, 109.38, 108.44, 41.01, 22.73, 11.67, 9.78. TOF MS ES+ (*m*/*z*): [M + H]+, calcd for C25H22F2N6O3: 493.1800, found, 493.1797.

*4-(2-fluoro-4-(5-methyl-1-(2-(trifluoromethoxy)phenyl)-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-propylpicolinamide* (**B12**) Light yellow solid; Yield: 63.2%; m.p.: 132.6–132.9 ◦C; 1H-NMR (400 MHz, DMSO-*d*6, ppm) δ 10.99 (s, 1H), 8.82 (s, 1H), 8.55 (d, *J* = 5.6 Hz, 1H), 8.10 (d, *J* = 13.2 Hz, 1H), 7.88 (d, *J* = 7.5 Hz, 1H), 7.85 (d, *J* = 8.0 Hz, 2H), 7.79 (d, *J* = 8.4 Hz, 1H), 7.73 (t, *J* = 7.5 Hz, 1H), 7.46 (d, *J* = 9.0 Hz, 1H), 7.41 (d, *J* = 2.5 Hz, 1H), 7.23 (dd, *J* = 5.5, 2.6 Hz, 1H), 3.26–3.20 (m, 2H), 2.46 (s, 3H), 1.58–1.47 (m, 2H), 0.85 (t, *J* = 7.4 Hz, 3H). TOF MS ES+ (*m*/*z*): [M + H]<sup>+</sup>, calcd for C26H22F4N6O4: 559.1717, found, 559.1717.

*4-(4-(1-(4-chlorophenyl)-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamido)-2-fluorophenoxy)-N-propylpicolinamide* (**B13**) Light yellow solid; Yield: 76.3%; m.p.: 176.5–176.8 ◦C; 1H-NMR (400 MHz, DMSO-*d*6, ppm) δ 11.37 (s, 1H), 8.81 (s, 1H), 8.55 (d, *J* = 5.3 Hz, 1H), 8.06 (d, *J* = 12.8 Hz, 1H), 7.79 (d, *J* = 7.4 Hz, 5H), 7.48 (d, *J* = 8.9 Hz, 1H), 7.44 (s, 1H), 7.27–7.20 (m, 1H), 3.23 (d, *J* = 6.1 Hz, 2H), 1.52 (dd, *J* = 14.1, 7.1 Hz, 2H), 0.84 (t, *J* = 7.3 Hz, 3H). TOF MS ES+ (*m*/*z*): [M + H]<sup>+</sup>, calcd for C25H19ClF4N6O3: 563.1222, found, 563.1216.

*4-(2-fluoro-4-(5-(trifluoromethyl)-1-(2-(trifluoromethyl)phenyl)-1H-1,2,3-triazole-4-carboxamido)phenoxy)-Npropylpicolinamide* (**B14**) Light yellow solid; Yield: 79.6%; m.p.: 172.8–173.1 ◦C; 1H-NMR (400 MHz, DMSO-*d*6, ppm) δ 11.43 (s, 1H), 8.82 (s, 1H), 8.55 (d, *J* = 5.3 Hz, 1H), 8.12 (s, 1H), 8.09 (d, *J* = 4.7 Hz, 1H), 8.05 (s, 1H), 8.00 (d, *J* = 9.1 Hz, 2H), 7.84 (d, *J* = 8.3 Hz, 1H), 7.49 (d, *J* = 8.9 Hz, 1H), 7.44 (s, 1H), 7.24 (d, *J* = 2.5 Hz, 1H), 3.23 (d, *J* = 5.6 Hz, 2H), 1.55–1.49 (m, 2H), 0.84 (t, *J* = 7.2 Hz, 3H). 13C-NMR (100 MHz, DMSO-*d*6, ppm) δ 165.15, 163.01, 156.39, 152.70, 150.48(2,C), 141.52, 134.25(2,C), 134.18, 132.86, 132.00, 129.99(2,C), 127.65, 123.86(2,C), 117.59, 113.43(2,C), 109.61, 109.39, 108.12, 40.60, 22.31, 11.21. TOF MS ES+ (*m*/*z*): [M + H]<sup>+</sup>, calcd for C26H22F4N6O3: 543.1614, found, 543.1616.

*4-(4-(1-(3,4-difluorophenyl)-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamido)-2-fluorophenoxy)-N-propylpicolinamide* (**B15**) White solid; Yield: 55.4%; m.p.: 134.4–134.8 ◦C; 1H-NMR (400 MHz, DMSO-*d*6, ppm) δ 11.40 (s, 1H), 8.83 (s, 1H), 8.55 (d, *J* = 5.4 Hz, 1H), 8.13 (d, *J* = 9.0 Hz, 1H), 8.06 (d, *J* = 13.0 Hz, 1H), 7.82 (d, *J* = 7.0 Hz, 2H), 7.74 (s, 1H), 7.48 (t, *J* = 8.8 Hz, 1H), 7.42 (s, 1H), 7.25 (d, *J* = 2.0 Hz, 1H), 3.22 (d, *J* = 6.2 Hz, 2H), 1.52 (d, *J* = 7.1 Hz, 2H), 0.84 (t, *J* = 7.1 Hz, 3H). TOF MS ES+ (*m*/*z*): [M + H]+, calcd for C26H19F7N6O3: 597.1485, found, 597.1486.

*4-(4-(1-(3-chloro-4-fluorophenyl)-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamido)-2-fluorophenoxy)-Npropylpicolinamide* (**B16**) White solid; Yield: 59.7%; m.p.: 163.3–163.5 ◦C; 1H-NMR (400 MHz, DMSO-*d*6, ppm) δ 11.39 (s, 1H), 8.82 (t, *J* = 5.8 Hz, 1H), 8.56 (d, *J* = 5.6 Hz, 1H), 8.23 (d, *J* = 4.8 Hz, 1H), 8.06 (d, *J* = 12.9 Hz, 1H), 7.88 (d, *J* = 8.5 Hz, 1H), 7.83 (d, *J* = 8.8 Hz, 1H), 7.77 (t, *J* = 8.9 Hz, 1H), 7.48 (t, *J* = 9.0 Hz, 1H), 7.42 (s, 1H), 7.25 (d, *J* = 5.4 Hz, 1H), 3.23 (dd, *J* = 13.3, 6.5 Hz, 2H), 1.53 (dt, *J* = 14.5, 7.4 Hz, 2H), 0.85 (t, *J* = 7.4 Hz, 3H). TOF MS ES+ (*m*/*z*): [M + H]<sup>+</sup>, calcd for C25H18ClF5N6O3: 581.1127, found, 581.1128.

4-(4-(1-(2-chloro-5-(trifluoromethyl)phenyl)-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamido)-2 fluorophenoxy)-N-propylpicolinamide (**B17**) White solid; Yield: 64.5%; m.p.: 154.1–154.4 ◦C; 1H-NMR (400 MHz, DMSO-d6, ppm) δ 11.48 (s, 1H), 8.83 (s, 1H), 8.65 (s, 1H), 8.55 (d, *J* = 5.0 Hz, 1H), 8.21 (d, *J* = 7.8 Hz, 1H), 8.15 (d, *J* = 8.2 Hz, 1H), 8.07 (d, *J* = 12.9 Hz, 1H), 7.84 (d, *J* = 8.6 Hz, 1H), 7.49 (t, *J* = 8.8 Hz, 1H), 7.43 (s, 1H), 7.25 (d, *J* = 1.4 Hz, 1H), 3.23 (d, *J* = 6.0 Hz, 2H), 1.52 (dd, *J* = 13.9, 6.9 Hz, 2H), 0.84 (t, *J* = 7.3 Hz, 3H). TOF MS ES+ (m/z): [M + H]<sup>+</sup>, calcd for C26H18ClF7N6O3: 631.1089, found, 631.1089.

4-(4-(1-(4-chloro-3-(trifluoromethyl)phenyl)-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamido)-2 fluorophenoxy)-N-propylpicolinamide (**B18**) White solid; Yield: 55.9%; m.p.: 153.4–153.7 ◦C; 1H-NMR (400 MHz, DMSO-d6, ppm) δ 11.41 (s, 1H), 8.83 (s, 1H), 8.56 (d, *J* = 5.3 Hz, 1H), 8.45 (s, 1H), 8.18 (d, *J* = 8.4 Hz, 1H), 8.12–8.01 (m, 2H), 7.84 (d, *J* = 8.4 Hz, 1H), 7.48 (t, *J* = 8.9 Hz, 1H), 7.42 (s, 1H), 7.25 (s, 1H), 3.26–3.19 (m, 2H), 1.52 (dq, *J* = 14.4, 7.0 Hz, 2H), 0.84 (t, *J* = 7.2 Hz, 3H). TOF MS ES+ (m/z): [M + H]<sup>+</sup>, calcd for C26H18ClF7N6O3: 631.1089, found, 631.1096.

*N-(3-fluoro-4-((2-(pyrrolidine-1-carbonyl)pyridin-4-yl)oxy)phenyl)-5-methyl-1-phenyl-1H-1,2,3-triazole-4 carboxamide* (**B19**) Light yellow solid; Yield: 63.2%; m.p.: 169.7–170.0 ◦C; 1H-NMR (400 MHz, DMSO-*d*6, ppm) δ 10.94 (s, 1H), 8.50 (d, *J* = 4.4 Hz, 1H), 8.09 (d, *J* = 12.9 Hz, 1H), 7.84 (d, *J* = 8.4 Hz, 1H), 7.65 (d, *J* = 14.4 Hz, 5H), 7.42 (t, *J* = 8.6 Hz, 1H), 7.13 (d, *J* = 14.2 Hz, 2H), 3.59 (s, 2H), 3.45 (s, 2H), 2.59 (s, 3H), 1.82 (s, 4H). TOF MS ES+ (*m*/*z*): [M + H]<sup>+</sup>, calcd for C26H23FN6O3: 487.1897, found, 487.1902.

*N-(3-fluoro-4-((2-(pyrrolidine-1-carbonyl)pyridin-4-yl)oxy)phenyl)-1-(4-fluorophenyl)-5-methyl-1H-1,2,3 triazole-4-carboxamide* (**B20**) Light yellow solid; Yield: 66.4%; m.p.: 164.3–164.7 ◦C; 1H-NMR (400 MHz, DMSO-*d*6, ppm) δ 10.94 (s, 1H), 8.51 (d, *J* = 5.3 Hz, 1H), 8.09 (d, *J* = 13.2 Hz, 1H), 7.84 (d, *J* = 8.4 Hz, 1H), 7.75 (d, *J* = 3.2 Hz, 2H), 7.51 (t, *J* = 8.4 Hz, 2H), 7.42 (t, *J* = 8.9 Hz, 1H), 7.18 (s, 1H), 7.12 (d, *J* = 4.6 Hz, 1H), 3.59 (s, 2H), 3.45 (s, 2H), 2.57 (s, 3H), 1.81 (s, 4H). TOF MS ES+ (*m*/*z*): [M + H]+, calcd for C26H22F2N6O3: 505.1800, found, 505.1807.

*N-(3-fluoro-4-((2-(pyrrolidine-1-carbonyl)pyridin-4-yl)oxy) phenyl)-5-methyl-1-(2-(trifluoromethoxy)phenyl)- 1H-1,2,3-triazole-4-carboxamide*(**B21**) White solid; Yield: 66.5%; m.p.: 176.3–176.6 ◦C; 1H-NMR (400 MHz,

DMSO-d6, ppm) δ 11.00 (s, 1H), 8.51 (d, *J* = 4.9 Hz, 1H), 8.09 (d, *J* = 12.8 Hz, 1H), 7.89 (d, *J* = 7.3 Hz, 1H), 7.85 (d, *J* = 8.0 Hz, 2H), 7.79 (d, *J* = 8.2 Hz, 1H), 7.75–7.70 (m, 1H), 7.43 (t, *J* = 8.6 Hz, 1H), 7.16 (s, 1H), 7.11 (d, *J* = 1.9 Hz, 1H), 3.60 (s, 2H), 3.46 (s, 2H), 2.46 (s, 3H), 1.82 (s, 4H). TOF MS ES+ (m/z): [M + H]<sup>+</sup>, calcd for C27H22F4N6O4: 571.1717, found, 571.1722.

*4-(2-fluoro-4-(5-methyl-1-phenyl-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-(2-(thiophen-2-yl)ethyl)picolinamide* (**B22**) Light yellow solid; Yield: 61.9%; m.p.: 124.6–125.0 ◦C; 1H NMR (400 MHz, DMSO-*d*6, ppm) δ 10.97 (s, 1H), 8.98 (t, *J* = 5.9 Hz, 1H), 8.55 (d, *J* = 5.6 Hz, 1H), 8.11 (d, *J* = 13.2 Hz, 1H), 7.86 (d, *J* = 8.8 Hz, 1H), 7.67 (d, *J* = 2.3 Hz, 5H), 7.48–7.41 (m, 2H), 7.32 (d, *J* = 4.9 Hz, 1H), 7.24 (d, *J* = 3.0 Hz, 1H), 6.96-6.91 (m, 1H), 6.89 (d, *J* = 2.6 Hz, 1H), 3.54 (dd, *J* = 13.4, 6.8 Hz, 2H), 3.06 (t, *J* = 7.1 Hz, 2H), 2.59 (s, 3H). TOF MS ES+ (*m*/*z*): [M + H]<sup>+</sup>, calcd for C28H23FN6O3S: 543.1614, found, 543.1616.

*4-(2-fluoro-4-(1-(4-fluorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-(2-(thiophen-2-yl) ethyl)picolinamide* (**B23**) White solid; Yield: 51.7%; m.p.: 110.4–110.8 ◦C; 1H-NMR (400 MHz, DMSO-*d*6, ppm) δ 10.99 (s, 1H), 9.00 (t, *J* = 5.7 Hz, 1H), 8.57 (d, *J* = 5.6 Hz, 1H), 8.13 (d, *J* = 13.2 Hz, 1H), 7.88 (d, *J* = 8.8 Hz, 1H), 7.79 (d, *J* = 4.8 Hz, 1H), 7.77 (d, *J* = 4.8 Hz, 1H), 7.54 (t, *J* = 8.7 Hz, 2H), 7.47 (d, *J* = 9.1 Hz, 1H), 7.43 (d, *J* = 2.1 Hz, 1H), 7.34 (d, *J* = 4.6 Hz, 1H), 7.26 (d, *J* = 2.4 Hz, 1H), 6.98–6.94 (m, 1H), 6.91 (s, 1H), 3.55 (dd, *J* = 13.3, 6.8 Hz, 2H), 3.08 (t, *J* = 7.1 Hz, 2H), 2.60 (s, 3H). TOF MS ES+ (*m*/*z*): [M + H]<sup>+</sup>, calcd for C28H22F2N6O3S: 561.1520, found, 561.1529.

*4-(2-fluoro-4-(5-methyl-1-(2-(trifluoromethoxy)phenyl)-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-(2-(thiophen-2-yl)ethyl)picolinamide* (**B24**) White solid; Yield: 56.6%; m.p.: 103.4–103.7 ◦C; 1H-NMR (400 MHz, DMSO-d6, ppm) δ 10.98 (s, 1H), 8.94 (s, 1H), 8.55 (d, *J* = 5.3 Hz, 1H), 8.10 (d, *J* = 13.2 Hz, 1H), 7.88 (d, *J* = 6.4 Hz, 2H), 7.84 (d, *J* = 7.0 Hz, 1H), 7.78 (d, *J* = 8.1 Hz, 1H), 7.72 (t, *J* = 7.4 Hz, 1H), 7.44 (t, *J* = 8.8 Hz, 2H), 7.31 (d, *J* = 4.1 Hz, 1H), 7.23 (d, *J* = 2.3 Hz, 1H), 6.96–6.92 (m, 1H), 6.90 (s, 1H), 3.55 (d, *J* = 6.2 Hz, 2H), 3.07 (t, *J* = 6.9 Hz, 2H), 2.47 (s, 3H). TOF MS ES+ (m/z): [M + H]<sup>+</sup>, calcd for C29H22F4N6O4S: 627.1437, found, 627.1439.

*4-(2-fluoro-4-(5-methyl-1-phenyl-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-(3-morpholinopropyl)picolinamide* (**B25**) White solid; Yield: 42.6%; m.p.: 108.3–108.6 ◦C; 1H-NMR (400 MHz, DMSO-*d*6, ppm) δ 10.67 (s, 1H), 9.05 (s, 1H), 8.53 (s, 1H), 8.03 (d, *J* = 7.5 Hz, 2H), 7.67 (s, 5H), 7.45 (s, 1H), 7.23 (d, *J* = 7.6 Hz, 1H), 7.17 (s, 1H), 3.64 (s, 4H), 3.36 (s, 2H), 2.60 (s, 3H), 2.44 (s, 6H), 1.72 (s, 2H). 13C-NMR (100 MHz, DMSO-*d*6, ppm) δ 166.25, 163.53, 159.92, 152.85, 150.72, 149.29, 138.56, 138.11, 136.82, 135.67, 130.44, 130.10(2,C), 125.83(2,C), 122.70(2,C), 121.51(2,C), 114.53, 109.30, 66.25(2,C), 56.81, 53.53(2,C), 38.36, 25.59, 9.85. TOF MS ES+ (*m*/*z*): [M + H]<sup>+</sup>, calcd for C29H30FN7O4: 560.2421, found, 560.2416.

*4-(2-fluoro-4-(1-(4-fluorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-(3-morpholinopropyl) picolinamide* (**B26**) White solid; Yield: 39.3%; m.p.: 108.5–108.9 ◦C; 1H-NMR (400 MHz, DMSO-*d*6, ppm) δ 10.73 (s, 1H), 9.10 (s, 1H), 8.52 (d, *J* = 3.7 Hz, 1H), 8.03 (d, *J* = 7.9 Hz, 2H), 7.76 (s, 2H), 7.52 (d, *J* = 6.9 Hz, 2H), 7.43 (s, 1H), 7.23 (d, *J* = 7.9 Hz, 2H), 3.61 (s, 4H), 3.35 (s, 2H), 2.59 (s, 3H), 2.34 (s, 6H), 1.68 (s, 2H). 13C-NMR (100 MHz, DMSO-*d*6, ppm) δ 166.25, 163.48, 159.87, 152.90, 150.69, 149.33, 138.38, 136.78, 132.06, 128.39, 128.30, 122.71(2,C), 121.46(2,C), 117.16(2,C), 116.93(2,C), 114.50, 109.32, 66.54(2,C), 57.08, 53.77(2,C), 38.55, 25.80, 9.74. TOF MS ES+ (*m*/*z*): [M + H]<sup>+</sup>, calcd for C29H29F2N7O4: 578.2327, found, 578.2347.

*4-(2-fluoro-4-(5-methyl-1-(2-(trifluoromethoxy)phenyl)-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-(3 morpholinopropyl)picolinamide* (**B27**) White solid; Yield: 41.0%; m.p.: 97.6–97.9 ◦C; 1H-NMR (400 MHz, DMSO-d6, ppm) δ 10.78 (s, 1H), 9.05 (s, 1H), 8.54 (s, 1H), 7.99 (d, *J* = 5.2 Hz, 2H), 7.86 (d, *J* = 3.8 Hz, 2H), 7.79 (s, 1H), 7.73 (s, 1H), 7.41 (s, 1H), 7.23 (d, *J* = 6.1 Hz, 2H), 3.91 (d, *J* = 8.3 Hz, 2H), 3.85 (d, *J* = 11.1 Hz, 2H), 3.36 (d, *J* = 5.0 Hz, 2H), 3.06 (s, 4H), 2.45 (s, 2H), 1.98 (s, 2H). TOF MS ES+ (m/z): [M + H]<sup>+</sup>, calcd for C30H29F4N7O5: 643.1059, found, 643.1094.

1H-NMR spectra of representative target compounds (**B1**, **B6**, **B12**, **B13**, **B20**, **B25**, and **B26**), 13C-NMR spectra of representative target compounds (**B6**, **B7**, **B10**, **B11**, **B14**, **B25**, and **B26**), and TOF-MS spectra of representative target compounds (**B13**, **B17**, **B19**, **B21**, **B22**, **B25**, and **B26**) can be seen in the Supplementary Materials.

#### *3.2. Antitumority Assay*

The antitumor activities of target compounds were determined by the MTT method using Golvatinib as a positive control. All cancer cell lines (A549, HeLa, and MCF-7) were cultured with Dulbecco Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute (1640) containing 10% fetal bovine serum and 0.1% penicillin–streptomycin, under ambient conditions of 5% CO2 and 37 ◦C. Cells were digested with an appropriate amount of Trypsin–EDTA solution to obtain the cell suspension, which was diluted with medium and inoculated into 96-well plates at 5\*104 cells per well. After cells were incubated for 24 h, the target compounds diluted by medium to suitable concentrations were added into 96-well plates, and the cells were cultured continue for 72 h. Next, the medium was removed, and then thiazolyl blue tetrazolium bromide (MTT) was added to each well to treat cells for 3.5 h. Ultimately, dimethyl sulfoxide (DMSO) was added to each well after removal of MTT and the absorbance values were measured with the ELISA (enzyme-linked immunosorbent assay) reader. All antitumor activities were tested for three times. The IC50 values were the average of three measurements and were calculated using the Bacus Laboratories Incorporated Slide Scanner (Bliss) software.

#### *3.3. Dose-Dependent Test*

The dose-dependent effect of compound **B26** on A549 cells was tested by the MTT method using Golvatinib as a positive control. The experimental procedure was identical to that of the cytotoxic activity, wherein the concentration of the test compound was configured to be seven and the cytotoxic activity was five. Seven different concentrations of compound **B26** treated on A549 cells to obtain the corresponding inhibition rates. Experimental data was obtained by Orange (2018 64 bit) software based on the inhibition rate.

#### *3.4. Cell Morphology Studies*

Cell morphology studies aimed to explore the morphological changes of A549 cells with and without treatment with compound **B26**, and the cell morphology was visualized by acridine orange (AO) staining. The culture environment of A549 cells was consistent with that in the antitumor activity experiments. Cells were digested with Trypsin–EDTA solution to get the cell suspension, which was diluted with 1640 medium and inoculated into a 24-well plate at 2\*10<sup>4</sup> cells per well. After incubating for 12 h, compound **B26** diluted to suitable concentrations by 1640 medium was added into a 24-well plate, and cell culture was continued for 12 h. Then, the 1640 medium in the well plate was removed and every well were washed three times with phosphate buffer saline (PBS). After washing with PBS three times, A549 cells were treated with AO for 15 min. A549 cells were washed three times with PBS again, and the cell morphology distribution was observed directly by a fluorescence microscope. Ultimately, the picture is exported via computer.

#### *3.5. Cell Cycle Study*

A549 cells were seeded into six-well plates at 1\*10<sup>5</sup> cells per well and incubated for 24 h. Then, the medium containing DMEM (control, without compound) or compound **B26** with different concentrations was added to the culture plate, and A549 cells were further cultured for 24 h. Then, A549 cells were collected into a centrifuge tube and fixed with a small amount of ice-cold 70% ethanol at 4 ◦C for 6–12 h. Cells washed three times with PBS were incubated with Rnase (1 mg/mL, diluted with PBS) for 30 min at room temperature. Finally, propidium iodide (PI) was added to staining without light for 30 min at room temperature, and the DNA content was measured by flow cytometry within 1 h. Experimental data was obtained by Modify software.

#### *3.6. c-Met Kinase Assay*

The kinase assay was implemented through Mobility shift assay. The Mixture the configured available Brij-35 (0.0015%) and 50 mM HEPES (pH = 7.5) to get a kinase buffer base. Then, compounds **B25**, **B26**, and **B27** were configured to five concentrations using dimethyl sulfoxide. The kinase buffer containing the compounds or dimethyl sulfoxide was added to 96-well plates and mixed on the shaker for fifteen minutes. Next, the solution was transferred in duplicate from a 96-well plate to a 384-well plate, and then an enzyme solution (c-Met kinase mixed with kinase buffer) was added to each well. After incubation of the 384-well plate for 10 min at room temperature, a 2.5\*peptide solution (formed by the addition of FAM-labeled peptide and ATP to the kinase base buffer) was added to each well. After incubation at 28 ◦C for a certain period of time, a stop buffer was added to each well for stop the reaction. The inhibition value is obtained by converting the conversion data on the caliper. The formula is that percent inhibition = (max − conversion) / (max − min) \* 100. Among them, "max" stands for DMSO control, "min" stands for low control. Finally, the IC50 values were obtained by fitting the inhibition rate data using the XLFit excel add-in version software.

#### *3.7. Molecular Docking Study*

All the molecular docking simulations were performed by the AutoDock 4.2 software. The crystal structure of c-Met (PDB code: 3LQ8) used in the docking was downloaded from http://www.rcsb.org/. The preparation process of the protein for docking mainly involves the addition of hydrogen atoms and charges, elimination of unrelated water molecules, immobilization of exact residues, and removal of endogenous ligands (Foretinib), etc. The preparation process of the molecules for docking mainly includes the addition of hydrogen atoms and charges. Then the prepared molecules (target compounds) were docked to the certified binding site of c-Met protein. Subsequently, the genetic algorithm was used for energy optimization. All the docking results were modified and processed by PyMOL 1.8.x software (https://pymol.org).

#### **4. Conclusions**

In conclusion, a series of 4-(pyridin-4-yloxy)benzamide derivatives bearing a triazole fragment were designed and synthesized. In addition, we evaluated them for antitumor activity against three cancer cell lines and c-Met kinase activity (only for compounds **B25**–**B27**) in vitro. The pharmacological results indicated that most compounds showed moderate antitumor activity against the three cancer cell lines. In particular, compound **B26** showed excellent inhibitory activity with IC50 values of 3.22, 4.33, and 5.82 μM against A549, HeLa, and MCF-7 cell lines, which were more potent than Golvatinib, respectively. The SARs study indicated that the introduction of a morpholino group in the hydrophilic region was more favorable than an alkane chain in terms of antitumor activity, and a single electron-withdrawing substituent (such as a fluorine atom) on the terminal phenyl ring improved the inhibitory activity of the target compounds. Further studies will be carried out in the near future.

#### **Supplementary Materials:** The Supplementary Materials are available online at http://www.mdpi.com/1420-3049/ 25/1/10/s1.

**Author Contributions:** P.Z., Q.T. and J.C. conceived and designed the experiments; H.X., J.Z., Q.Z., Z.X. and H.Z. performed the experiments and statistics analysis; J.C. and H.X. completed the molecular docking simulation and H.X. started the project and wrote the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** We gratefully acknowledge the generous support provided by Natural Science Foundation of National Natural Science Foundation of China (NSFC No. 81660572), Jiangxi Province (20171BAB215071, 20192ACBL21009) and Top-notch talent project of Jiangxi Science & Technology Normal University (2016QNBJRC002).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Sample Availability:** Samples of all target compounds are available from the authors.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Synthesis and Evaluation of Novel 2***H***-Benzo[***e***]-[1,2,4]thiadiazine 1,1-Dioxide Derivatives as PI3K**δ **Inhibitors**

### **Ya-Ping Gong** †**, Long-Qian Tang** †**, Tong-Shen Liu and Zhao-Peng Liu \***

Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China; 201614474@mail.sdu.edu.cn (Y.-P.G.); lqtang.student@sina.com (L.-Q.T.); jshrlts@163.com (T.-S.L.)

**\*** Correspondence: liuzhaop@sdu.edu.cn; Tel.: +86-531-88382006; Fax: +86-531-88382548

† Ya-Ping Gong and Long-Qian Tang contributed equally to this work.

Academic Editor: Qiao-Hong Chen

Received: 8 November 2019; Accepted: 22 November 2019; Published: 25 November 2019

**Abstract:** In previous work, we applied the rotation-limiting strategy and introduced a substituent at the 3-position of the pyrazolo [3,4-*d*]pyrimidin-4-amine as the affinity element to interact with the deeper hydrophobic pocket, discovered a series of novel quinazolinones as potent PI3Kδ inhibitors. Among them, the indole derivative **3** is one of the most selective PI3Kδ inhibitors and the 3,4-dimethoxyphenyl derivative **4** is a potent and selective dual PI3Kδ/γ inhibitor. In this study, we replaced the carbonyl group in the quinazolinone core with a sulfonyl group, designed a series of novel 2*H*-benzo[*e*][1,2,4]thiadiazine 1,1-dioxide derivatives as PI3Kδ inhibitors. After the reduction of nitro group in *N*-(2,6-dimethylphenyl)-2-nitrobenzenesulfonamide **5** and *N*-(2,6-dimethylphenyl)-2-nitro-5-fluorobenzenesulfonamide **6**, the resulting 2-aminobenzenesulfonamides were reacted with trimethyl orthoacetate to give the 3-methyl-2*H*-benzo[*e*][1,2,4]thiadiazine 1,1-dioxide derivatives. After bromination of the 3-methyl group, the nucleophilic substitution with the 3-iodo-1*H*-pyrazolo[3,4-*d*]pyrimidin-4-amine provided the respective iodide derivatives, which were further reacted with a series of arylboronic acids via Suzuki coupling to furnish the 2*H*-benzo[*e*][1,2,4]thiadiazine 1,1-dioxide derivatives **15a**–**J** and **16a**–**d**. In agreement with the quinazolinone derivatives, the introduction of a 5-indolyl or 3,4-dimethoxyphenyl at the affinity pocket generated the most potent analogues **15a** and **15b** with the IC50 values of 217 to 266 nM, respectively. In comparison with the quinazolinone lead compounds **3** and **4**, these 2*H*-benzo[*e*][1,2,4]thiadiazine 1,1-dioxide derivatives exhibited much decreased PI3Kδ inhibitory potency, but maintained the high selectivity over other PI3K isoforms. Unlike the quinazolinone lead compound **4** that was a dual PI3Kδ/γ inhibitor, the benzthiadiazine 1,1-dioxide **15b** with the same 3,4-dimethoxyphenyl moiety was more than 21-fold selective over PI3Kγ. Moreover, the introducing of a fluorine atom at the 7-position of the 2*H*-benzo[*e*][1,2,4]thiadiazine 1,1-dioxide core, in general, was not favored for the PI3Kδ inhibitory activity. In agreement with their high PI3Kδ selectivity, **15a** and **15b** significantly inhibited the SU-DHL-6 cell proliferation.

**Keywords:** PI3Ks; PI3Kδ inhibitors; 2*H*-benzo[*e*][1,2,4]thiadiazine 1,1-dioxide; anticancer; anticancer agents

#### **1. Introduction**

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that regulate numerous biological functions, including cell growth, proliferation, differentiation, motility, and intracellular trafficking, through the phosphorylation of the phosphatidylinositol 4,5-bisphosphate (PIP2) to generate the lipid

second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3) [1–3]. There are three classes of the PI3K enzyme, of which class I PI3Ks are the mostly studied and further divided into subgroups IA (PI3Kα, PI3Kβ, and PI3Kδ) and IB (PI3Kγ) based on the signaling pathways and the regulatory proteins to which they bind [4,5]. The class IA PI3K isoforms mediate the signal transduction from receptor tyrosine kinases [6], while the IB isoform PI3Kγ is principally activated by G-protein coupled receptors [7]. PI3Kα and PI3Kβ are ubiquitously expressed, while PI3Kδ and PI3Kγ are dominantly expressed in leukocytes [8–10]. All the class I PI3K isoforms are implicated in cancer [11–16]. There are also mounting evidences that support a therapeutic role for inhibition of PI3Kα in diabetes [17,18], PI3Kβ in thrombosis [19,20], PI3Kδ and PI3Kγ in both rheumatoid arthritis and asthma [21–24], PI3Kδ in activated PI3Kδ syndrome (APDS) [25–27], and PI3Kγ in idiopathic pulmonary fibrosis [28]. Therefore, the development of PI3K isoform selective inhibitors is a promising therapeutic strategy for the treatment of these PI3Ks-related diseases. The selective PI3Kδ inhibitor idelalisib (Figure 1) is approved by FDA for follicular lymphoma (FL) and small lymphocytic lymphoma (SLL) and for chronic lymphocytic leukemia (CLL) in combination with rituximab [29,30]. The dual PI3Kδ/γ inhibitor duvelisib (Figure 1) is approved for adult patients with relapsed or refractory CLL or SLL, and relapsed or refractory FL after at least two prior systemic therapies [31]. However, in the clinical application of idelalisib, infectious and autoimmune toxicities were observed, and the unique toxicities are associated with inhibition of different isoforms of the PI3K enzyme [32]. To improve the isoform selectivity of the quinazolinone-based PI3Kδ inhibitors, in previous work, we introduced a pyrazolo [3,4-*d*]pyrimidin-4-amine moiety as the hinge region binding group, a substituent at the 3-position of the pyrazolo[3,4-*d*]pyrimidine core as the affinity element to interact with the deeper hydrophobic pocket, and a 2,6-dimethylphenyl to limit the free rotation of the 3-phenyl in idelalisib, discovered the indole derivative **3** as one of the most selective PI3Kδ inhibitors (IC50 = 8.6 nM) with more than 3630-fold, 390-fold and 40-fold selective for PI3Kδ over PI3Kα, β and γ, and the 3,4-dimethoxyphenyl derivative **4** as a potent and selective dual PI3Kδ/γ inhibitor (IC50 = 8.4 nM for PI3Kδ, IC50 = 62 nM for PI3Kγ) with more than 1400-fold, 820-fold selective for PI3Kδ over PI3Kα and PI3Kβ [33]. Considering the importance of sulfonamides in drug discovery [34–36], we replaced the carbonyl group in the quinazolinone core, and reported here the synthesis and preliminary evaluation of 2*H*-benzo[*e*][1,2,4]thiadiazine 1,1-dioxide derivatives as PI3Kδ inhibitors.

**Figure 1.** Selective PI3Kδ and dual PI3Kδ/γ inhibitors.

#### **2. Results and Discussion**

#### *2.1. Chemistry*

All the new 2*H*-benzo[*e*][1,2,4]thiadiazine 1,1-dioxide derivatives were prepared following a general synthetic route shown in Scheme 1. The 2-nitrobenzene-1-sulfonamides **5** and **6** were readily prepared according to the reported method by the reaction of 2-nitrobenzene-1-sulfonyl chloride or 5-fluoro-2-nitrobenzene-1-sulfonyl chloride with 2,6-dimethylbenzenamine in methanol and water solution in the presence of CH3COONa under refluxing conditions [37]. Reduction of the nitro

group to amine was carried out using hydrazine monohydrate in the presence of ferric chloride and activated charcoal in methanol under reflux conditions in excellent yields (95% and 99%). The resulting 2-aminobenzenesulfonamides **7** and **8** were reacted with trimethyl orthoacetate to give the corresponding 3-methyl-2*H*-benzo[*e*][1,2,4]thiadiazine 1,1-dioxide derivatives **9** and **10** in 51% and 40%, respectively. In the bromination of the allyl methyl group using *N*-bromosuccinimide (NBS), the main compounds were found to be the dibrominated products. Therefore, compounds **9** and **10** were reacted with only 0.5 equivalent of NBS in glacial acetic acid to give the monobrominated derivatives **11** and **12** in moderate yields (79% and 72% based on NBS). Nucleophilic substitution of **11** and **12** with 3-iodo-1*H*-pyrazolo[3,4-*d*]pyrimidin-4-amine, which was readily prepared from 5-amino-1*H*-pyrazole-4-carbonitrile in two steps by the known procedures [38], resulted in the iodides **13** and **14** in 86 and 58% yields, respectively. Finally, the incorporation of the affinity elements was achieved through the Suzuki coupling of **13** and **14** with the appropriate boronic acid in dioxane and water catalyzed by Pd(PPh3)4 under refluxing conditions, and the target 2*H*-benzo[*e*][1,2,4]thiadiazine 1,1-dioxide derivatives **15a**–**J** and **16a**–**d** were obtained in 34–91% and 51–84% yields. The structures of these 2*H*-benzo[*e*][1,2,4]thiadiazine 1,1-dioxide derivatives were characterized by 1H-nuclear magnetic resonance (NMR) and 13C-NMR (please refer to the Supplementary Materials).

**Scheme 1.** Synthesis of 2*H*-benzo[*e*][1,2,4]thiadiazine 1,1-dioxide derivatives.

#### *2.2. PI3K*δ *Inhibitory Activity and Isoform Selectivity*

Compounds **15a**–**J** were first tested for their inhibitory activity against PI3Kδ using the ADP-Glo luminescent assay [39], using a pan-PI3K inhibitor PI-103 as a positive control [40]. As shown in Table 1, the substitution at the 3-position of the pyrazolo[3,4-d]pyrimidine with 5-indolyl or 3,4-dimethoxyphenyl led to the relative potent analogues **15a** and **15b** with IC50 values of 217 to 266 nM, respectively. The 6-methoxypyridin-3-yl derivative **15d** exhibited moderate PI3Kδ inhibitory activity (IC50 = 498 nM), whereas the 3-fluoro-4-methoxyphenyl analogue **15c** only had marginal activity. In comparison with **15b**, the substitution of the 3,4-dimethoxypheny group for 2,3-dihydrobenzo[*b*][1,4]dioxin-6-yl (**15e**), benzo[*d*][1,3]dioxol-5-yl (**15f**), 4-methoxyphenyl (**15g**), 3-methoxyphenyl (**15h**), 4-(trifluoromethoxy)phenyl (**15i**), and phenyl (**15j**) was not tolerated, indicating the subtle requirements at the affinity pocket of PI3Kδ.


**Table 1.** PI3Kδ inhibitory activity of **15a**–**j** and **16a**–**d**.

<sup>a</sup> The IC50 values are shown as the mean ± SD from two separate experiments.

In order to increase the inhibitory activity of compound **15a**–**d**, a fluorine atom was introduced at the 7-position of the 2*H*-benzo[*e*][1,2,4]thiadiazine 1,1-dioxide, and compounds **16a**–**d** were prepared and evaluated for their PI3Kδ inhibitory activity. In comparison with **15a**, the fluorinated compound **16a** lost its activity (Table 1), and compounds **16b** and **16d** showed almost 2-fold decrease in potency. In contrast, compound **16c** with a 3-fluoro-4-methoxyphenyl moiety at the affinity pocket showed a slight increase in potency.

Compared with the leading quinazolinone derivatives **3** and **4**, these 2*H*-benzo[*e*][1,2,4]-thiadiazine 1,1-dioxide derivatives **15a**–**j** and **16a**–**d** showed much decreased PI3Kδ inhibitory activity. However, the most potent derivatives **15a** and **15b** proved to be selective PI3Kδ inhibitors (Table 2). The indole derivative **15a** showed significantly lower potency against other three isoforms of class I PI3K and was more than 140-fold selective for PI3Kδ over PI3Kα, β and γ. The 3,4-dimethoxyphenyl derivative **15b** was more than 60-fold, 90-fold and 20-fold selective for PI3Kδ over PI3Kα, PI3Kβ, and PI3Kγ, respectively. In comparison with the lead **4**, **15b** was more selective over PI3Kγ (21-fold vs. 7-fold).



<sup>a</sup> The IC50 or GI50 values are shown as the mean ± SD from two separate experiments; <sup>b</sup> CAL-101 was the positive control.

#### *2.3. SU-DHL-6 Cell Growth Inhibitory Activity*

The selective PI3Kδ inhibitors **15a** and **15b** were further evaluated for their antiproliferative activity against human B-cell SU-DHL-6. **15a** and **15b** significantly inhibited SU-DHL-6 cell proliferation with the GI50 of 2.13 and 2.50 μM, respectively (Table 2), which were in consistent with their PI3Kδ inhibitory potency.

#### *2.4. Molecular Modeling Study*

Molecular docking studies were conducted on the new discovered selective PI3Kδ inhibitors **15a** and **15b**. As shown in Figure 2, the pyrazolo[3,4-d]pyrimidine portion in both compounds **15a** and **15b** forms hydrogen bonds with Glu826 and Val828 in the hinge region.

**Figure 2.** Molecular docking studies of **15a** (left) and **15b** (right).

Both inhibitors bind to the PI3Kδ isoform in an 'induced fit' conformation in which the 2*H*-benzo[*e*][1,2,4]thiadiazine 1,1-dioxide moiety is sandwiched between Trp760 and Met752 as these residues move apart to create the specificity pocket. The indol-5-yl (**15a**) in the specificity pocket forms an additional hydrogen bond with Asp787. Like the carbonyl oxygen in **3** and **4**, one sulfonyl oxygen in both **15a** and **15b** acts as hydrogen bond acceptor from the H2O2278. In compound **15b**, only the 3-methoxy group forms hydrogen bonding with Lys779, while in lead **4**, the 3-methoxy interacts with Tyr813 and Asp911, the 4-methoxy interacts with Lys779 [33]. These differences may contribute its less potency against PI3Kδ than **4**. In comparison with **3** and **4**, the lack of a 8-fluorine at the 2*H*-benzo[*e*][1,2,4]thiadiazine 1,1-dioxide core in **15a** and **15b** may also be related to their lower PI3Kδ inhibitory activity.

#### **3. Materials and Methods**

#### *3.1. General Chemical Experimental Procedures*

1H- and 13C-NMR spectra were recorded on a Bruker-600 NMR spectrometer (Brucker Co., Ltd., Zurich, Switzerland). All spectra were recorded at room temperature for DMSO or CDCl3 solutions. High resolution mass spectra (HRMS) were obtained on a 6520 QTOF instrument (Agilent Technologies Inc.*,* Santa Clara, CA, USA by electrospray ionization (ESI). Melting points were determined on an X-6 micromelting point apparatus (Beijing Tech. Co., Ltd., Beijing, China) without corrections. Column chromatography was performed on silica gel (200–300 mesh). All reactions involving oxygen- or moisture sensitive compounds were carried out under a dry N2 atmosphere using anhydrous solvents. Unless otherwise noted, reagents were added by syringe.

#### *2-Amino-N-(2,6-dimethylphenyl)benzenesulfonamide* (**7**)

To a stirred solution of *N*-(2,6-dimethylphenyl)-2-nitrobenzenesulfonamide (**5**, 19.4 g, 63.3 mmol) in methanol (200 mL), ferric chloride (5.1 g, 19 mmol) and activated charcoal (6.5 g) was added and refluxed for 30 min. 80% Hydrazine monohydrate (31.7 g, 633 mmol) was then added dropwise and refluxed for 5 h. After filtration, the filtrate was concentrated and the residue was dissolved in EtOAc (200 mL), washed with brine, dried over anhydrous Na2SO4. After filtration and evaporation, the residue was purified by silica gel chromatography (EtOAc/hexane = 1:3) to give **7** (16.5 g, 95%) as a white solid, m.p. 144–146 ◦C; lit. [41] m.p. 144–145 ◦C.

#### *2-Amino-N-(2,6-dimethylphenyl)-5-fluorobenzenesulfonamide* (**8**)

According to the procedures described for the synthesis of **7**, compound **8** were obtained as a colorless solid (16 g) in 99% yield, m.p. 185–186 ◦C; 1H-NMR (DMSO-d6) δ 9.44 (s, 1H, SO2NH), 7.21 (td, *J* = 9.0, 3.0 Hz, 1H, Ar-H), 7.07 (td, *J* = 8.4, 1.5 Hz, 1H, Ar-H), 7.01 (d, *J* = 7.8 Hz, 2H, Ar-H), 6.97 (dd, *J* = 8.4, 3.0 Hz, 1H, Ar-H), 6.85 (dd, *J* = 9.0, 4.8 Hz, 1H, Ar-H), 5.85 (s, 2H, NH2), 2.04 (s, 6H, 2,6-(CH3)2); MS (ESI) calcd. for C14H14FN2O2S [M − H]−: 293.1, found: 293.3.

#### *2-(2,6-Dimethylphenyl)-3-methy-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide* (**9**)

A mixture of **7** (5 g, 18.1 mmol), trimethyl orthoacetate (50 mL) and 4Ă molecular sieve (10 g) was refluxed for 10 h. After cooling to room temp., the mixture was concentrated and the residue was dissolved in EtOAc (200 mL), washed with brine, dried over anhydrous Na2SO4. After filtration and evaporation, the residue was purified by silica gel chromatography (EtOAc/hexane = 1:5) to give **9** (2.8 g, 51%) as a white solid, m.p. 162–163 ◦C; 1H-NMR (CDCl3) δ 7.89 (d, *J* = 7.8 Hz, 1H, Ar-H), 7.70 (td, *J* = 8.4, 1.2 Hz, 1H, Ar-H), 7.60 (d, *J* = 8.4 Hz, 1H, Ar-H), 7.47 (td, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.21 (t, *J* = 7.2 Hz, 1H, Ar-H), 7.18 (d, *J* = 7.8 Hz, 2H, Ar-H), 2.22 (s, 6H, 2,6-(CH3)2), 2.11 (s, 3H, 3-CH3); 13C-NMR (CDCl3) δ 154.34, 142.55, 138.58, 133.51, 132.59, 129.99, 129.27, 127.65, 127.23, 126.90, 121.01, 23.38, 18.62; MS (ESI) *m*/*z* calcd. for C16H17N2O2S [M + H]<sup>+</sup> 301.1, found 301.0.

#### *2-(2,6-Dimethylphenyl)-7-fluoro-3-methy-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide* (**10**)

According to the procedures described for the synthesis of **9**, compound **10** were obtained as a colorless solid (6.9 g) in 40% yield, m.p. 150–151 ◦C; 1H-NMR (DMSO-d6) δ 7.89 (dt, *J* = 7.2, 1.5 Hz, 1H, Ar-H), 7.73 (dd, *J* = 7.2, 1.2 Hz, 2H, Ar-H), 7.36 (t, *J* = 7.2 Hz, 1H, Ar-H), 7.29 (d, *J* = 7.8 Hz, 2H, Ar-H), 2.13 (s, 6H, 2,6-(CH3)2), 2.06 (s, 3H, 3-CH3); 13C-NMR (DMSO-d6) δ 160.28 (d, *J*C–F = 249.2 Hz), 154.02, 139.26 (d, *J*C–F = 3.0 Hz), 138.59, 132.62, 130.88 (d, *J*C–F = 7.6 Hz), 130.66, 129.79, 127.96 (d, *J*C–F = 9.1 Hz), 122.58 (d, *J*C–F = 24.2 Hz), 107.95 (d, *J*C–F = 27.2 Hz), 23.25, 18.47; MS (ESI) *m*/*z* calcd. for C16H16FN2O2S [M + H]<sup>+</sup> 319.1, found 319.0.

#### *3-Bromomethyl-2-(2,6-dimethylphenyl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide* (**11**)

Compound **9** (1.0 g, 3.3 mmol) was dissolved in glacial acetic acid (10 mL), and then NBS (0.3 g, 1.65 mmol) was added. After the mixture was stirred at room temperature for 0.5 h, distilled water (50 mL) was added. The mixture was extracted by dichloromethane, washed with brine, dried over anhydrous Na2SO4. After filtration and evaporation, the residue was purified by silica gel chromatography (EtOAc/hexane = 1:10) to give **11** (0.5 g) with a conversion yield of 79% as a white solid, m.p. 150–151◦C; 1H-NMR (CDCl3) δ 7.90 (dd, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.75 (td, *J* = 8.4, 1.2 Hz, 1H, Ar-H), 7.69 (dd, *J* = 7.8, 0.6 Hz, 1H, Ar-H), 7.55 (td, *J* = 8.4, 1.2 Hz, 1H, Ar-H), 7.29 (t, *J* = 7.8 Hz, 1H, Ar-H), 7.19 (d, *J* = 7.8 Hz, 2H, Ar-H), 3.97 (s, 2H, CH2Br), 2.24 (s, 6H, 2,6-(CH3)2); 13C-NMR (CDCl3) δ 151.86, 142.16, 138.89, 133.69, 132.14, 130.36, 129.51, 128.37, 128.25, 127.79, 121.00, 28.91, 18.95; MS (ESI) *m*/*z* calcd. for C16H16BrN2O2S [M + H]<sup>+</sup> 379.0 and 381.0, found 381.3 and 383.4.

#### *3-Bromomethyl-2-(2,6-dimethylphenyl)-7-fluoro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide* (**12**)

According to the procedures described for the synthesis of **11**, compound **12** were obtained as a colorless solid (0.65 g) in 72% conversion yield, m.p. 185–186 ◦C; 1H-NMR (DMSO-d6) δ 7.95 (dd, *J* = 8.4, 3.0 Hz, 1H, Ar-H), 7.84 (dd, *J* = 10.8, 5.4 Hz, 1H, Ar-H), 7.79 (td, *J* = 10.8, 3.0 Hz, 1H, Ar-H), 7.37 (t, *J* = 8.4 Hz, 1H, Ar-H), 7.29 (d, *J* = 9.0 Hz, 2H, Ar-H), 4.10 (s, 2H, CH2Br), 2.14 (s, 6H, 2,6-(CH3)2); 13C-NMR (DMSO-d6) δ 161.21 (d, *J*C–F = 250.6 Hz), 151.77, 138.82, 132.20, 131.54 (d, *J*C–F = 9.1 Hz), 130.94, 129.97, 129.28, 128.60 (d, *J*C–F = 9.1 Hz), 122.87 (d, *J*C–F = 24.2 Hz), 108.32 (d, *J*C–F = 25.7 Hz), 30.02, 18.72; MS (ESI) *m*/*z* calcd.for C16H15BrFN2O2S [M + H]<sup>+</sup> 397.0 and 399.0, found 397.2 and 399.1.

#### *3-((4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-(2,6-dimethylphenyl)-2Hbenzo[e]-[1,2,4]thiadiazine 1,1-dioxide* (**13**)

To a solution of **11** (1.1 g, 2.9 mmol) and 3-iodo-1*H*-pyrazolo[3,4-d]pyrimidin-4-amine (1.1 g, 4.4 mmol) in DMF (8 mL), K2CO3 (0.8 g, 5.8 mmol) was added. After stirring at 60 ◦C for 5 h, the mixture was poured into water (100 mL), extracted by EtOAc. The organic layer was washed with brine, dried over anhydrous Na2SO4. After filtration and evaporation, the residue was purified by silica gel chromatography (EtOAc/hexane = 1:10) to give **13** (1.4 g, 86%) as a white solid, m.p. 242–243 ◦C; 1H-NMR (DMSO-d6) δ 8.07 (s, 1H, Ar-H), 7.94 (dd, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.80 (td, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.65 (td, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.43 (d, *J* = 7.8 Hz, 1H, Ar-H), 7.25 (t, *J* = 7.2 Hz, 1H, Ar-H), 7.17 (d, *J*= 7.2 Hz, 2H, Ar-H), 5.12 (s, 2H, NCH2), 2.07 (s, 6H, 2,6-(CH3)2); 13C-NMR (CDCl3) δ 162.77, 161.35, 159.35, 156.17, 146.35, 143.45, 139.66, 136.44, 135.32, 134.48, 133.91, 133.26, 132.44, 108.30, 95.82, 60.13, 54.33, 23.17; MS (ESI) *m*/*z* calcd. for C21H19IN7O2S [M + H]<sup>+</sup> 560.0, found 560.2.

#### *3-((4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-(2,6-dimethylphenyl)-7-fluoro-2H-benzo- [e][1,2,4]thiadiazine 1,1-dioxide* (**14**)

Following the procedures described for the synthesis of **13**, compound **14** were obtained as a white solid (1.6 g) in 58% yield, m.p. 243–244 ◦C; 1H-NMR (DMSO-d6) δ 8.08 (s, 1H, Ar-H), 7.92 (dd, *J* = 11.4, 4.2 Hz, 1H, Ar-H), 7.68 (td, *J* = 13.2, 4.2 Hz, 1H, Ar-H), 7.54 (dd, *J* = 13.2, 7.2 Hz, 1H, Ar-H), 7.26 (dd, *J* = 13.2, 10.2 Hz, 1H, Ar-H), 7.17 (d, *J* = 11.4 Hz, 2H, Ar-H), 5.13 (s, 2H, NCH2), 2.06 (s, 6H, 2,6-(CH3)2); 13C-NMR (DMSO-d6) δ 160.89 (d, *J*C–F = 250.7 Hz), 158.02, 156.61, 154.57, 150.89, 138.69, 131.55, 131.46 (d, *J*C–F = 7.6 Hz), 130.64, 129.76, 128.54 (d, *J*C–F = 7.6 Hz), 122.71 (d, *J*C–F = 22.7 Hz), 108.28 (d, *J*C–F = 25.7 Hz), 103.55, 91.13, 79.59, 49.55, 18.40; MS (ESI) *m*/*z* calcd. for C21H18FIN7O2S [M + H]<sup>+</sup> 578.0, found 578.0.

#### *3-((4-Amino-3-(1H-indol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-(2,6-dimethylphenyl)- 2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide* (**15a**)

To a solution of **13** (180 mg, 0.30 mmol) in dioxane (4 mL) and distilled water (1.5 mL) was added 1*H*-indol-5-ylboronic acid (88 mg, 0.55 mmol), sodium carbonate anhydrous (103 mg, 0.97 mmol) and Pd(PPh3)4 (12 mg, 0.03 mmol). The mixture was degassed with N2, and refluxed for 4 h. After cooling to room temperature, EtOAc (50 mL) and distilled water (10 mL) were added, and the organic layer was washed brine, dried over Na2SO4. After filtration and evaporation, the residue was purified by silica gel chromatography (EtOAc/hexane = 1:1) to give **15a** (161 mg, 91%) as a white solid, m.p. 216–217 ◦C; 1H-NMR (CDCl3) δ 11.32 (s, 1H, NH), 8.09 (s, 1H, Ar-H), 7.95 (dd, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.81 (td, *J* = 7.8, 1.2 Hz, 2H, Ar-H), 7.65 (td, *J* = 7.8, 0.6 Hz, 1H, Ar-H), 7.57 (d, *J* = 8.4 Hz, 1H, Ar-H), 7.52 (d, *J* = 7.8 Hz, 1H, Ar-H), 7.45 (t, *J* = 3.0 Hz, 1H, Ar-H), 7.38 (dd, *J* = 8.4, 1.8 Hz, 1H, Ar-H), 7.25 (t, *J* = 7.8 Hz, 1H, Ar-H), 7.15 (d, *J* = 7.8 Hz, 2H, Ar-H), 6.55 (t, *J* = 8.4, 1.8 Hz, 1H, Ar-H), 5.20 (s, 2H, NCH2), 2.04 (s, 6H, 2,6-(CH3)2); 13C-NMR (CDCl3) δ 158.53, 156.22, 155.43, 152.04, 146.79, 141.79, 138.70, 136.53, 134.86, 131.96, 130.48, 129.70, 129.09, 128.51, 128.47, 127.83, 127.04, 123.90, 121.76, 121.20, 120.52, 112.68, 102.22, 97.86, 49.61, 18.35; HRMS (ESI) *m*/*z* calcd. for C29H25N8O2S [M + H]<sup>+</sup> 549.1816, found 549.1829.

#### *3-((4-Amino-3-(3,4-dimethoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-(2,6-dimethylphenyl)- 2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide* (**15b**)

According to the procedures described for the synthesis of **15a**, compound **15b** were obtained as a white solid (61 mg) in 75% yield, m.p. 220–222 ◦C; 1H-NMR (DMSO-d6) δ 8.31 (s, 1H, NH), 8.09 (s, 1H, Ar-H), 7.94 (dd, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.81 (td, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.65 (td, *J* = 7.2, 1.2 Hz, 1H, Ar-H), 7.50 (d, *J* = 8.4 Hz, 1H, Ar-H), 7.26 (t, *J* = 7.8 Hz, 1H, Ar-H), 7.15–7.19 (m, 4H, Ar-H), 7.12 (d, *J* = 8.4 Hz, 1H, Ar-H), 5.18 (s, 2H, NCH2), 3.81 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 2.02 (s, 6H, 2,6-(CH3)2); 13C-NMR (DMSO-d6) δ 158.50, 156.26, 155.50, 151.98, 149.83, 149.51, 145.20, 141.74, 138.71, 134.86, 131.98, 130.50, 129.70, 129.13, 128.47, 127.84, 125.53, 121.19, 121.03, 112.71, 111.99, 97.70, 79.63, 56.04, 55.89, 49.64, 18.32; HRMS (ESI) *m*/*z* calcd. for C29H28N7O4S [M + H]<sup>+</sup> 570.1918, found 570.1921.

#### *3-((4-Amino-3-(3-fluoro-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2- (2,6-dimethyl-phenyl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide* (**15c**)

According to the procedures described for the synthesis of **15a**, compound **15c** were obtained as a white solid (59 mg) in 66% yield, m.p. 226–227 ◦C; 1H-NMR (DMSO-d6) δ 8.31 (s, 1H, NH), 8.10 (s, 1H, Ar-H), 7.94 (d, *J* = 7.8 Hz, 1H, Ar-H), 7.81 (t, *J* = 7.8 Hz, 1H, Ar-H), 7.65 (t, *J* = 7.8 Hz, 1H, Ar-H), 7.49 (d, *J* = 8.4 Hz, 1H, Ar-H), 7.43–7.38 (m, 2H, Ar-H), 7.33 (t, *J* = 8.4 Hz, 1H, Ar-H), 7.25 (t, *J* = 7.8 Hz, 1H, Ar-H), 7.15 (d, *J* = 7.8 Hz, 2H, Ar-H), 5.18 (s, 2H, NCH2), 3.90 (s, 3H, OCH3), 2.02 (s, 6H, 2,6-(CH3)2); 13C-NMR (DMSO-d6) δ 158.50, 156.33, 155.58, 152.84, 152.03 (d, *J*C–F = 244.6 Hz), 151.85, 148.13 (d, *J*C–F = 9.1 Hz), 144.02, 141.72, 138.69, 134.86, 131.93, 130.51, 129.71, 128.82 (d, *J*C–F = 95.1 Hz), 127.82, 125.78 (d, *J*C–F = 6.0 Hz), 125.21, 121.20, 116.11 (d, *J*C–F = 19.6 Hz), 114.91, 97.67, 79.65, 56.57, 49.64, 18.32; HRMS (ESI) *m*/*z* calcd. for C28H25FN7O3S [M + H]<sup>+</sup> 558.1718, found 558.1733.

#### *3-((4-Amino-3-(6-methoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-(2,6-dimethylphenyl)- 2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide* (**15d**)

According to the procedures described for the synthesis of **15a**, compound **15d** were obtained as a white solid (41 mg) in 47% yield, m.p. 248–249 ◦C; 1H-NMR (DMSO-d6) δ 8.39 (d, *J* = 1.8 Hz, 1H, Ar-H), 8.31 (s, 1H, NH), 8.11 (s, 1H, Ar-H), 7.94 (dd, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.92 (dd, *J* = 8.4, 2.4 Hz, 1H, Ar-H), 7.81 (td, *J* = 8.4, 1.2 Hz, 1H, Ar-H), 7.65 (td, *J* =7.8, 1.2 Hz, 1H, Ar-H), 7.49 (d, *J* = 7.8 Hz, 1H, Ar-H), 7.26 (t, *J* = 7.8 Hz, 1H, Ar-H), 7.16 (d, *J* = 7.8 Hz, 2H, Ar-H), 6.98 (d, *J* = 8.4 Hz, 1H, Ar-H), 5.20 (s, 2H, NCH2), 3.92 (s, 3H, OCH3), 2.03 (s, 6H, 2,6-(CH3)2); 13C-NMR (DMSO-d6) δ 164.14, 158.60, 156.38, 155.61, 151.81, 146.69, 142.36, 141.71, 139.29, 138.69, 134.87, 131.91, 130.51, 129.70, 129.13, 128.50, 127.80, 122.70, 121.19, 111.48, 97.92, 79.64, 53.89, 49.66, 18.33; HRMS (ESI) *m*/*z* calcd. for C27H25N8O4S [M + H]<sup>+</sup> 541.1765, found 541.1781.

#### *3-((4-Amino-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2- (2,6-dimethylphenyl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide* (**15e**)

According to the procedures described for the synthesis of **15a**, compound **15e** were obtained as a white solid (71 mg) in 64% yield, m.p. 225–226 ◦C; 1H-NMR (DMSO-d6) δ 8.31 (s, 1H, NH), 8.08 (s, 1H, Ar-H), 7.94 (dd, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.82 (td, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.65 (td, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.51 (d, *J* = 8.4 Hz, 1H, Ar-H), 7.24 (t, *J* = 8.4 Hz, 1H, Ar-H), 7.14 (d, *J* = 7.8 Hz, 2H, Ar-H), 7.11–7.09 (m, 2H, Ar-H), 7.02 (d, *J* = 7.8 Hz, 1H, Ar-H), 5.18 (s, 2H, NCH2), 4.30 (s, 4H, OCH2CH2O), 2.01 (s, 6H, 2,6-(CH3)2); 13C-NMR (DMSO-d6) δ 158.45, 156.25, 155.47, 151.92, 144.79, 144.60, 144.23, 141.74, 138.66, 134.86, 131.94, 130.47, 129.68, 129.12, 128.48, 127.83, 126.18, 121.59, 121.19, 118.30, 117.23, 97.63, 79.64, 64.67, 64.59, 49.62, 18.30; HRMS (ESI) *m*/*z* calcd. for C29H26N7O4S [M + H]<sup>+</sup> 568.1761, found 568.1775.

#### *3-((4-Amino-3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2- (2,6-dimethyl-phenyl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide* (**15f**)

According to the procedures described for the synthesis of **15a**, compound **15f** were obtained as a white solid (29 mg) in 34% yield, m.p. > 250 ◦C; 1H-NMR (DMSO-d6) δ 8.31 (s, 1H, NH), 8.08 (s, 1H, Ar-H), 7.94 (dd, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.82 (td, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.65 (td, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.50 (d, *J* = 7.8 Hz, 1H, Ar-H), 7.25 (t, *J* = 7.8 Hz, 1H, Ar-H), 7.15 (d, *J* = 7.2 Hz, 2H, Ar-H), 7.12–7.09 (m, 2H, Ar-H), 7.07 (d, *J* = 8.4, 1H, Ar-H), 6.10 (s, 2H, OCH2O), 5.17 (s, 2H, NCH2), 2.01 (s, 6H, 2,6-(CH3)2); 13C-NMR (DMSO-d6) δ 158.44, 156.28, 155.47, 151.89, 148.36, 144.98, 141.73, 138.67, 134.87, 131.93, 130.49, 129.70, 129.14, 128.50, 127.82, 126.85, 122.65, 121.19, 109.44, 108.80, 101.89, 97.65, 79.64, 49.62, 18.30; HRMS (ESI) *m*/*z* calcd. for C28H24N7O4S [M + H]<sup>+</sup> 554.1605, found 554.1614.

#### *3-((4-Amino-3-(4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-(2,6-dimethylphenyl)-2Hbenzo[e][1,2,4]thiadiazine 1,1-dioxide* (**15g**)

According to the procedures described for the synthesis of **15a**, compound **15g** were obtained as a white solid (45 mg) in 74% yield, m.p. 248–249 ◦C; 1H-NMR (DMSO-d6) δ 8.32 (s, 1H, NH), 8.09 (s, 1H, Ar-H), 7.94 (dd, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.82 (td, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.65 (td, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.57 (d, *J* = 8.4 Hz, 2H, Ar-H), 7.50 (d, *J* = 8.4 Hz, 1H, Ar-H), 7.25 (t, *J* = 7.8 Hz, 1H, Ar-H), 7.15 (d, *J* = 7.8 Hz, 2H, Ar-H), 7.11 (d, *J* = 8.4 Hz, 2H, Ar-H), 5.19 (s, 2H, NCH2), 3.82 (s, 3H, OCH3), 2.02 (s, 6H, 2,6-(CH3)2); 13C-NMR (DMSO-d6) δ160.18, 158.51, 156.26, 155.51, 151.92, 145.03, 141.75, 138.69, 134.85, 131.93, 130.48, 129.95, 129.69, 129.10, 128.49, 127.82, 125.43, 121.19, 115.12, 97.70, 79.63, 55.72, 49.60, 18.34; HRMS (ESI) *m*/*z* calcd. for C28H26N7O3S [M + H]<sup>+</sup> 540.1812, found 540.1829.

#### *3-((4-Amino-3-(3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-(2,6-dimethylphenyl)-2Hbenzo[e][1,2,4]thiadiazine 1,1-dioxide* (**15h**)

According to the procedures described for the synthesis of **15a**, compound **15h** were obtained as a white solid (46 mg) in 59% yield, m.p. 221–222 ◦C; 1H-NMR (DMSO-d6) δ 8.31 (s, 1H), 8.10 (s, 1H, Ar-H), 7.94 (dd, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.82 (td, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.63 (td, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.50 (d, *J* = 7.8 Hz, 1H, Ar-H), 7.47 (t, *J* = 7.8 Hz, 1H, Ar-H), 7.26 (t, *J* = 7.8 Hz, 1H, Ar-H), 7.23 (d, *J* = 7.8 Hz, 1H, Ar-H), 7.20–7.11 (m, 3H, Ar-H), 7.06 (dd, *J* = 8.4, 2.4 Hz, 1H, Ar-H), 5.21 (s, 2H, NCH2), 3.82 (s, 3H, OCH3), 2.02 (s, 6H, 2,6-(CH3)2); 13C-NMR (DMSO-d6) δ 160.11, 158.43, 156.31, 155.56, 151.88, 145.02, 141.72, 138.70, 134.87, 134.36, 131.95, 130.86, 130.51, 129.71, 129.14, 128.48, 127.83, 121.20, 120.76, 115.19, 113.85, 97.74, 79.64, 55.63, 49.68, 18.32; HRMS (ESI) *m*/*z* calcd. for C28H26N7O3S [M + H]<sup>+</sup> 540.1812, found 540.1825.

#### *3-((4-Amino-3-(4-(trifluoromethoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)- 2-(2,6-dimethyl-phenyl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide* (**15i**)

According to the procedures described for the synthesis of **15a**, compound **15i** were obtained as a white solid (61 mg) in 61% yield, m.p. 206–207 ◦C; 1H-NMR (DMSO-d6) δ 8.12 (s, 1H, Ar-H), 7.94 (dd, *J* = 8.4, 1.2 Hz, 1H, Ar-H), 7.81 (dt, *J* = 8.4, 1.2 Hz, 1H, Ar-H), 7.76 (d, *J* =8.4 Hz, 2H, Ar-H), 7.65 (dt, *J* = 8.4, 0.6 Hz, 1H, Ar-H), 7.52 (d, *J* = 7.8 Hz, 2H, Ar-H), 7.48 (d, *J* = 7.8 Hz, 1H, Ar-H), 7.26 (t, *J* = 7.8 Hz, 1H, Ar-H), 7.16 (d, *J* = 7.8 Hz, 2 H, Ar-H), 5.21 (s, 2H), 2.03 (s, 6H); 13C-NMR (DMSO-d6) δ 158.52, 156.38, 155.72, 151.77, 149.08, 143.91, 141.70, 138.70, 134.87, 132.28, 131.90, 130.59, 130.53, 129.72, 129.14, 128.50, 127.80, 122.10, 121.21, 120.57 (q, *J*C–F = 256.70 Hz), 97.73, 49.68, 18.34; HRMS (ESI) *m*/*z* calcd. for C28H23F3N7O3S [M + H]<sup>+</sup> 594.1530, found 594.1547.

#### *3-((4-Amino-3-phenyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-(2,6-dimethylphenyl)-2Hbenzo[e]-[1,2,4]thiadiazine 1,1-dioxide* (**15j**)

According to the procedures described for the synthesis of **15a**, compound **15j** were obtained as a white solid (40 mg) in 73% yield, m.p. 193–194 ◦C; 1H-NMR (DMSO-d6) δ 8.30 (s, 1H, NH), 8.09 (s, 1H, Ar-H), 7.92 (dd, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.80 (td, *J* = 7.8, 1.2 Hz, 1H, Ar-H), 7.66–7.62 (m, 3H, Ar-H), 7.54 (t, *J* = 7.8 Hz, 2H, Ar-H), 7.52–7.32 (m, 2H, Ar-H), 7.24 (t, *J* = 7.8 Hz, 2H, Ar-H), 7.14 (d, *J* = 7.8 Hz, 2H, Ar-H), 5.19 (s, 2H, NCH2), 2.01 (s, 6H, 2,6-(CH3)2); 13C-NMR (DMSO-d6) δ 1158.46, 156.31, 155.60, 151.86, 145.16, 141.72, 138.69, 134.54, 133.53, 132.51, 131.99, 131.92, 130.51, 130.47, 129.71, 129.67, 129.27, 129.20, 128.60, 127.81, 121.20, 97.73, 79.65, 49.65, 18.34; HRMS (ESI) *m*/*z* calcd. for C27H24N7O2S [M + H]<sup>+</sup> 510.1707, found 510.1722.

#### *3-((4-Amino-3-(1H-indol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-(2,6-dimethylphenyl)-7-fluoro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide* (**16a**)

According to the procedures described for the synthesis of **15a**, compound **16a** were obtained as a white solid (65 mg) in 73% yield, m.p. 132–133 ◦C; 1H-NMR (DMSO-d6) δ 8.30 (s, 1H, NH), 8.08 (s, 1H, Ar-H), 7.91 (dd, *J* = 7.2, 2.4 Hz, 1H, Ar-H), 7.81 (brs, 1H, Ar-H), 7.69 (td, *J* = 9.6, 3.0 Hz, 1H, Ar-H), 7.61 (dd, *J* = 9.0, 4.8 Hz, 1H, Ar-H), 7.56 (d, *J* = 8.4 Hz, 1H, Ar-H), 7.44 (t, *J* = 3.0 Hz, 1H, Ar-H), 7.36 (dd, *J* = 8.4, 1.8 Hz, 1H, Ar-H), 7.25 (t, *J* = 7.28Hz, 1H, Ar-H), 7.16 (t, *J* = 7.8 Hz, 2H, Ar-H), 6.54 (s, 1H, 3-CH-indolyl), 5.20 (s, 2H, NCH2), 2.01 (s, 6H, 2,6-(CH3)2); 13C-NMR (DMSO-d6) δ 160.08 (d, *J*C–F = 250.7 Hz), 158.52, 156.23, 155.41, 151.51, 146.84, 138.68, 138.61 (d, *J*C–F = 3.0 Hz), 136.53, 131.81, 131.44 (d, *J*C–F = 9.1 Hz), 130.55, 129.74, 128.66 (d, *J*C–F = 9.1 Hz), 128.46, 127.05, 123.87, 122.67 (d, *J*C–F = 24.2 Hz), 121.74, 120.52, 112.67, 108.19 (d, *J*C–F = 27.2 Hz), 102.21, 97.85, 79.65, 49.57, 18.32; HRMS (ESI) *m*/*z* calcd. for C29H24FN8O2S [M + H]<sup>+</sup> 567.1721, found 567.1739.

#### *3-((A-amino-3-(3,4-dimethoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-(2,6-dimethylphenyl)-7 fluoro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide* (**16b**)

According to the procedures described for the synthesis of **15a**, compound **16b** were obtained as a white solid (47 mg) in 51% yield, m.p. 229–230 ◦C; 1H-NMR (DMSO-d6) δ 8.31 (s, 1H, NH), 8.09 (s, 1H, Ar-H), 7.91 (dd, *J* = 7.2, 2.4 Hz, 1H, Ar-H), 7.69 (td, J = 9.0, 2.4 Hz, 1H, Ar-H), 7.59 (dd, *J* = 9.0, 4.8 Hz, 1H, Ar-H), 7.26 (t, *J* = 7.8 Hz, 1H, Ar-H), 7.21–7.10 (m, 5H, Ar-H), 5.19 (s, 2H, NCH2), 3.82 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 2.01 (s, 6H, 2,6-(CH3)2); 13C-NMR (DMSO-d6) δ 160.78 (d, *J*C–F = 250.7 Hz), 158.51, 156.27, 155.49, 151.46, 149.84, 149.52, 145.24, 138.70, 138.57 (d, *J*C–F = 3.0 Hz), 131.84, 131.41 (d, *J*C–F = 7.6 Hz), 130.57, 129.74, 128.69 (d, *J*C–F = 7.6 Hz), 125.52, 122.66 (d, *J*C–F = 24.2 Hz), 121.03, 112.72, 112.02, 108.20 (d, *J*C–F = 27.2 Hz), 97.71, 79.65, 56.04, 55.89, 49.60, 18.31; HRMS (ESI) *m*/*z* calcd. for C29H27FN7O4S [M + H]<sup>+</sup> 588.1824, found 588.1838.

#### *3-((4-Amino-3-(3-fluoro-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2- (2,6-dimethyl-phenyl)-7-fluoro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide* (**16c**)

According to the procedures described for the synthesis of **15a**, compound **16c** were obtained as a white solid (76 mg) in 84% yield, m.p. 201–202 ◦C; 1H-NMR (DMSO-d6) δ 8.31 (s, 1H, NH), 8.09 (s, 1H, Ar-H), 7.91 (dd, *J* = 7.2, 3.0 Hz, 1H, Ar-H), 7.69 (td, *J* = 7.4, 2.4 Hz, 1H, Ar-H), 7.59 (dd, *J* = 9.0, 4.2 Hz, 1H, Ar-H), 7.44–7.38 (m, 2H, Ar-H), 7.33 (t, *J* = 9.0 Hz, 1H, Ar-H), 7.26 (t, *J* = 7.2 Hz, 1H, Ar-H), 7.15 (d, *J* = 7.8 Hz, 2H, Ar-H), 5.19 (s, 2H, NCH2), 3.90 (s, 3H, OCH3), 2.01 (s, 6H, 2,6-(CH3)2); 13C-NMR (DMSO-d6) δ 160.89 (d, *J*C–F = 250.7 Hz), 158.50, 156.34, 155.56, 152.02 (d, *J*C–F = 246.1 Hz), 151.32, 148.14 (d, *J*C–F = 10.6 Hz), 144.06, 138.68, 138.62 (d, *J*C–F = 19.6 Hz), 131.78, 131.44 (d, *J*C–F = 7.6 Hz), 130.59, 129.74, 128.66 (d, *J*C–F = 7.6 Hz), 125.76 (d, *J*C–F = 7.6 Hz), 125.21, 122.68 (d, *J*C–F = 22.7 Hz), 116.11 (d, *J*C–F = 19.6 Hz), 114.92, 108.21 (d, *J*C–F = 25.7 Hz), 97.67, 79.63, 56.59, 49.61, 18.30; HRMS (ESI) *m*/*z* calcd. for C28H24F2N7O3S [M + H]<sup>+</sup> 576.1624, found 576.1637.

#### *3-((4-Amino-3-(6-methoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-(2,6-dimethylphenyl)- 7-fluoro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide* (**16d**)

According to the procedures described for the synthesis of **15a**, compound **16d** were obtained as a white solid (51 mg) in 58% yield, m.p. 225–226 ◦C; 1H-NMR (DMSO-d6) δ 8.38 (s, 1H, NH), 8.31 (s, 1H, Ar-H), 8.10 (s, 1H, Ar-H), 7.90 (d, *J* = 8.4 Hz, 2H, Ar-H), 7.69 (t, *J* = 7.2 Hz, 1H, Ar-H), 7.59 (dd, *J* = 9.0, 4.2 Hz, 1H, Ar-H), 7.25 (t, *J* = 7.2 Hz, 1H, Ar-H), 7.15 (d, *J* = 8.4 Hz, 2H, Ar-H), 6.97 (d, *J* = 8.4 Hz, 1H, Ar-H), 5.20 (s, 2H, NCH2), 3.92 (s, 3H, OCH3), 2.01 (s, 6H, 2,6-(CH3)2); 13C-NMR (DMSO-d6) δ 164.15, 160.89 (d, *J*C–F = 250.7 Hz), 158.60, 156.39, 155.59, 151.29, 146.69, 142.41, 139.29, 138.69, 138.54 (d, *J*C–F = 3.0 Hz), 131.77, 131.45 (d, *J*C–F = 7.6 Hz), 130.59, 129.74, 128.66 (d, *J*C–F = 9.1 Hz), 122.75, 122.68, 122.67 (d, *J*C–F = 22.7 Hz), 111.48, 108.20 (d, *J*C–F = 27.2 Hz), 97.93, 79.64, 53.89, 49.63, 18.32; HRMS (ESI) *m*/*z* calcd. for C27H24FN8O3S [M + H]<sup>+</sup> 559.1671, found 559.1678.

#### *3.2. PI3K Kinase Assay*

The ADP-Glo luminescent assay was used for PI3Kδ, PI3Kβ and PI3Kγ isoforms and the kinase-Glo luminescent assay was used for PI3Kα according to the standard protocols of Promega [40]. PI-103 was used as a positive control. The compounds were tested from 1 μM or 10 μM, 3-fold dilution, in duplicate for 10 concentrations. The kinase reaction was done in 384-well plate (Corning, Los Altos, MA, USA). Each well was loaded with test compounds (in 100% DMSO) and reaction buffer containing PI substrate. After the PI3K proteins were then added, the reaction was started by the addition of PIP2 and ATP prepared in the reaction buffer and ran for either 60 (for PI3Kα, PI3Kβ, and PI3Kγ) or 120 min (for PI3Kδ). ADP-Glo reagent was then added to terminate the reavtion. The plates were then read in a Synergy 2 reader (BioTek, Shanghai, China) for luminescence detection.

#### *3.3. Cell Proliferation Assay*

Cell proliferation was evaluated by The CellTiter-Glo luminescent cell viability assay (Promega, Shanghai, China) was used to evaluate the inhibitory activity of compounds **15a** and **15b** following the manufacturer's protocol. In brief, SU-DHL-6 (ATCC) cells were seeded in 96-well plates (Corning, Los Altos, MA, USA) at density of 1 <sup>×</sup> 104 cells per well, and incubated with medium alone or with the tested compounds at the indicated concentrations (50 μM in DMSO, 3-fold dilution, in duplicate for 10 concentrations). 50 μL CellTiter-Glo (Promega, Shanghai, China) reagent was added to each well to induce cell lysis, and the plate was incubated at room temperature for 10 min to stabilize luminescent signal. After 100 μL of the mixture from each well was transferred to a new 96-well black plate, the fluorescence signal was read on EnVision (Shanghai, China) and the data were analysized by XLFit 4 software (IDBS, Berlin, Germany).

#### *3.4. Molecular Docking*

X-ray cocrystal structure of PI3Kδ enzyme was downloaded from RCSB Protein Date Bank (PDB ID: 2WXH) [42]. The molecular docking of **15a** and **15b** was carried out following the same procedures as reported for compounds **3** and **4** [33].

#### **4. Conclusions**

In a continuous study to find more potent and selective PI3Kδ inhibitors based on the rotation-limiting strategy, we substituted the carbonyl in the quinazolinone core for the sulfonyl group, designed and synthesized a series of novel 2*H*-benzo[*e*][1,2,4]thiadiazine 1,1-dioxide derivatives **15a**–**J** and **16a**–**d**. In agreement with the quinazolinone derivatives, the introduction of a 5-indolyl or 3,4-dimethoxyphenyl at the affinity pocket generated the most potent analogues **15a** and **15b** with the IC50 values of 217 to 266 nM, respectively. In comparison with the quinazolinone lead compounds **3** and **4**, the 2*H*-benzo[*e*][1,2,4]thiadiazine 1,1-dioxide derivatives exhibited much reduced PI3Kδ inhibitory activity, but maintained high selectivity over other PI3K isoforms. Unlike the quinazolinone lead compound **4** that was a dual PI3Kδ/γ inhibitor, the 2*H*-benzo[*e*][1,2,4]thiadiazine 1,1-dioxide **15b** was more than 21-fold selective over PI3Kγ. This may provide a structural base for the further design of more potent and selective PI3Kδ inhibitors. In agreement with their high PI3Kδ inhibitory activity, **15a** and **15b** exhibited high antiproliferative potency against B-cell leukemia SU-DHL-6 cells.

**Supplementary Materials:** Supplementary materials are available online.

**Author Contributions:** Conceptualization, Y.-P.G. and Z.-P.L.; methodology, Y.-P.G., L.-Q.T. and T.-S.L.; validation, Y.-P.G., L.-Q.T. and T.-S.L.; formal analysis, L.-Q.T. and Y.-P.G.; investigation, Y.-P.G., L.-Q.T. and T.-S.L.; resources, Z.-P.L.; data curation, L.-Q.T. and Y.-P.G.; writing—original draft preparation, L.-Q.T.; writing—review and editing, Z.-P.L.; visualization, Y.-P.G. and L.-Q.T.; supervision, Z.-P.L.; project administration, Z.-P.L.; funding acquisition, Z.-P.L.

**Funding:** This work was partially supported by the key research and development program of Shandong province (2017CXGC1401).

**Conflicts of Interest:** The authors declare no conflict of interest.

### **Abbreviations**


#### **References**


**Sample Availability:** Samples of the compounds **15a**–**j** and **16a**–**d** are available from the authors.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Design, Synthesis, and Biological Evaluation of Novel Thienopyrimidine Derivatives as PI3K**α **Inhibitors**

### **Lide Yu 1,2,**†**, Qinqin Wang 1,**†**, Caolin Wang 1, Binliang Zhang 1, Zunhua Yang 2, Yuanying Fang 2, Wufu Zhu 1,\* and Pengwu Zheng 1,\***


Received: 23 August 2019; Accepted: 19 September 2019; Published: 20 September 2019

**Abstract:** Three series of novel thienopyrimidine derivatives **9a**–**l**, **15a**–**l**, and **18a**–**h** were designed and synthesized, and their IC50 values against four cancer cell lines HepG-2, A549, PC-3, and MCF-7 were evaluated. Most compounds show moderate cytotoxicity against the tested cancer cell lines. The most promising compound **9a** showed moderate activity with IC50 values of 12.32 ± 0.96, 11.30 ± 1.19, 14.69 ± 1.32, and 9.80 ± 0.93 μM, respectively. The inhibitory activities of compounds **9a** and **15a** against PI3Kα and mTOR kinase were further evaluated. Compound **9a** exhibited PI3Kα kinase inhibitory activity with IC50 of 9.47 ± 0.63 μM. In addition, docking studies of compounds **9a** and **15a** were also investigated.

**Keywords:** Thienopyrimidine; Pyrazole; PI3Kα inhibitor

#### **1. Introduction**

The PI3K-Akt-mTOR signaling pathway plays an important role in tumorigenesis and development [1,2]. The activation of related proteins in this pathway is closely related to the occurrence and development of tumors. In recent years, the development of small molecule drugs to effectively inhibit the overexpression of this pathway has become a research hotspot in cancer therapy [3]. Moreover, the study of the complex co-crystals structure of protein and small molecular Ligand also promoted the development of new drugs [4–6]. Many small molecule inhibitors targeting PI3K-Akt–mTOR signaling pathway have entered clinical studies, and some of them were even approved by FDA, such as PI3K inhibitor GDC-0941 [7]; AKT inhibitor GSK2110183 [8]; mTOR inhibitor AZD2014 (Figure 1) [9], Rapamycin, Deforolimus. PI3Kα is the most important isoform in cell proliferation in response to growth factor-tyrosine kinase pathway activation. There are currently more than ten PI3Kα inhibitors undergoing clinical trials. Many research groups are attempting to develop some more PI3Kα inhibitors.

**Figure 1.** Structures of representative clinical PI3K/mTOR dual inhibitors and target compounds.

The thienopyrimidine core is widely used in small molecule inhibitors of the PI3K-Akt-mTOR signaling pathway. Among them, thienopyrimidine derivative GDC-0941 developed by Genentech, is the first PI3K inhibitor entering the clinical stage. Right after, several analogues of GDC-0941 were obtained. Two of them, GNE-477 and GDC-0980 (Figure 1), exhibited excellent activities and were regarded as potent PI3K/mTOR inhibitors [10,11]. Research showed that thienopyrimidines core were very important to the activity of these compounds, and it was considered to be an active pharmacophore.

In our previous research, several series of thienopyrimidine-containing compounds were designed and synthesized [12–14], one representative compound EJMC20159364-16i (Figure 1) exhibited the best in vitro cytotoxic activity and kinase inhibitory activity (PI3K, mTOR). SARs of this series of compounds were summarized. The results showed that the introduction of the flexible hydrazinyl linker at the pyrimidine 2 position helps to increase the cytotoxic activity of the target compounds. Continuous to this work, the hydrazinyl linker was kept unchanged in the new designed compounds, and we constructed a pyrazole ring on the hydrazinyl as a new flexible linker to connect with the aryl group, hoping to increase the compounds' inhibitory activity by improving the interaction of the compounds with the receptor (Figure 2). As a result, two series of thieno[2,3-*d*]pyrimidines and thieno[3,2-*d*]pyrimidines containing pyrazole unit (**9a**–**l**, **15a**–**l**) were designed and synthesized. Inspired by GDC-0084, in order to investigate the influence of the thienopyrimidine core to the activity, a flexible cyclohexane was introduced to the target compounds, resulting in a series of tricyclic thienopyrimidine compounds **21a**–**h**. It is expected to increase the cytotoxic activity and increase the interaction of the compounds with the enzyme to enhance the activity and selectivity. Finally, three series of thienopyrimidine derivatives containing pyrazole structure (**9a**–**l**, **15a**–**l, 21a**–**h**) were designed, synthesized and evaluated for their cytotoxic activity.

**Figure 2.** The design concept based on the co-crystal structure of GDC-0941 with protein.

#### **2. Results and Discussion**

#### *2.1. Chemistry*

The synthetic routes for thienopyrimidine derivatives **9a**–**l**, **15a**–**l,** and **21a**–**h** are outlined in Schemes 1 and 2. Condensation of the 4-substituted benzaldehydes (**1a**–**d**) with the 4-substituted acetophenones (**2a**–**e**) produced the corresponding chalcones **3a**–**l** (Scheme 1). Treatment of **5** with POCl <sup>3</sup> afforded 2,4-dichlorothieno[2,3-*d*]pyrimidine **6**, which was then treated with morpholine to give 4-(2-chlorothieno[2,3-*d*]pyrimidin-4-yl)morpholine **7**. Treatment of **7** with hydrazine gave the key intermediate **8**. Intermediate **8** condensed with the corresponding chalcones **3a**–**l** to afford the target compounds **9a**–**l**. The synthesis of compounds **15a**–**l** and **21a**–**h** was similar to that of compounds **9a**–**l** (Scheme 2). Generated by condensation reaction between **8** chalcone **3a**–**l,** there appears a chiral carbon atom at C-5 position of pyrazole of all of the compounds. The similar reaction was reported in previous research and the structures of target compounds can be easily identified. The structures of target compounds were confirmed by 1H-NMR, 13C-NMR, and TOF MS (ESI+), which were in agreement with the structures depicted.

**Scheme 1.** Synthetic routes of chalcones **3a**–**l**. Reagents and conditions: **a** 10% NaOH, EtOH, r.t., 24 h.

**Scheme 2.** Synthetic routes of target compounds **9a**–**l**, **15a**–**l**, and **21a**–**h**. Reagents and conditions: **a** 5 eq urea, 180 ◦C, 2h; **b** POCl3, DMF (cat.), reflux, 8 h; **c** 2.1 eq morpholine, MeOH, 0 ◦C, 30 min, r.t., 2h; **d** 80% hydrazine monohydrate, reflux, 8h; **e** Glacial acetic acid, H2SO4 (78%), 100 ◦C.

#### *2.2. Biological Discussion*

All target compounds were evaluated for their cytotoxic activities against A549 (human lung cancer), PC-3 (human prostate cancer), MCF-7 (human breast cancer), HepG2 (human hepatoma) cell lines. In addition, the activity against PI3Kα/mTOR kinase of the most promising compounds **9a** and **15a** was further evaluated. The results expressed as IC50 were summarized in Tables 1–3.


**Table 1.** In vitro cytotoxic activities against different cancer cell lines of **9a**–**l** and **15a**–**l**.


The value ">50" indicates that no inhibitory effect at 50 μM compound concentration. <sup>a</sup> The values are an average of two separate determinations; <sup>b</sup> Calculated by ChemBioDraw Ultra 15.0; <sup>c</sup> used as a positive control.

**Table 2.** In vitro cytotoxic activities against different cancer cell lines of **21a**–**h**.


The value ">50" indicates that no inhibitory effect at 50 μM compound concentration. <sup>a</sup> The values are an average of two separate determinations; <sup>b</sup> Calculated by ChemBioDraw Ultra 15.0; <sup>c</sup> used as a positive control.


**Table 3.** Enzymatic activities of compounds **9a** and **15a** against PI3Kα and mTOR (IC50, μM).

<sup>a</sup> The values are an average of two separate determinations. <sup>b</sup> used as a positive control.

As illustrated in Tables 1 and 2, most of the target compounds exhibited moderate cytotoxic activities. The compounds whose skeleton are thieno[2,3-*d*]pyrimidine (**9a**–**1**) has slightly stronger inhibitory activity against the tested cancer cell lines than the compounds whose skeleton are thieno[3,2-*d*]pyrimidine (**15a**–**l** and **21a**–**h**). The most promising compound **9a** showed moderate to well inhibitory activity with IC50 values of 12.32 ± 0.96, 11.30 ± 1.19, 14.69 ± 1.32, and 9.80 ± 0.93 μM, respectively.

It is worth noting that when R1 of the aryl group is an electron-withdrawing group (EWG) and R2 is a H atom, the inhibitory activity of the compounds (**9g**–**h**, **15g**–**h,** and **21a**–**h**) against cancer cells is decreased, especially for MCF-7 and A549 cells. The effect is greater than that of HepG2 and PC-3 cancer cell lines. Among them, the inhibitory activity of the compound **15h** against MCF-7 cells was IC50 > 50 μM, which means that the inhibitory activity was almost lost. When the C-3 and C-4 positions of the aryl group were both replaced by chlorine, the activity of the compounds (**9h**–**l**, **15h**–**l**) is slightly lower than that of the compounds (**9b**–**g**, **15b**–**g**) substituted by F or Br atoms.

When R1 of the aryl group was hydrogen atom and R2 was an electron-donating group, the inhibitory activities of the compounds **9b** and **15b** against the tested cancer cells decreased. The compound **15b** which contained the core skeleton thieno[3,2-*d*]pyrimidine was more obvious in this respect, and its IC <sup>50</sup> values for A549 and MCF-7 were both higher than 50 μM. When R <sup>1</sup> of the aryl group was an electron withdrawing group, the inhibitory activity against cancer cells of the compounds **9c**–**l** and **15c**–**l** tended to decrease irrespective whether the substituent of R2 was an electron withdrawing group or an electron donating group. Comparing the difference in activity between **9e**–**f** and **15e**–**f**, it is easily to find that the effect of the electron withdrawing group on the activity is slightly less than that of the electron-donating group. As shown in Table 3, compounds **21a**–**h** exhibited poor cytotoxic activity against the tested cell lines. This may attributed to the introduction of the tricyclic thieno-pyrimidine structure, resulting in an increase in steric hindrance which reduces the solubility of the compound at physiological pH and the relative molecular mass of the compound exceeds 500.

Finally, the inhibitory activity against PI3Kα kinase and mTOR kinase of selected compounds **9a** and **15a** were further examined. As shown in Table 3, enzymatic activity results of compounds **9a** and **15a** exhibited a moderate to excellent inhibitory activity against PI3Kα kinase. The inhibitory activity of the compounds against PI3Kα kinase is better than that of mTOR kinase.

#### *2.3. Molecular Docking Study of Compounds 9a and 15a.*

To further explore the binding modes of target compounds (**9a** and **15a**) with the active site of PI3Kα, molecular docking simulation studies were carried out by the AutoDock 4.2 software. Based on in vitro inhibition results, we selected compounds **9a** and **15a** as ligand examples, and the structures of PI3Kα (PDB code: 3TL5) were selected as the docking models. The best-scoring ligand–protein complex (Figure 3) was used for binding site analysis.

**Figure 3.** Docking modes of **9a** and **15a**. (**A**) Overview of the binding site of compound **9a** with PI3Kα (3TL5) kinase. (**B**) Binding model of compound **9a** with PI3Kα (3TL5) kinase. (**C**) Overview of the binding site of compound **15a** with PI3Kα (3TL5) kinase. (**D**) Binding model of compound **15a** with PI3Kα (3TL5) kinase.

The binding models of compounds **9a** and **15a** to the 3TL5 protein are approximately the same as our previously assumed binding model (Figure 3A,C). The detailed binding models of compounds **9a** and **15a** with the active site of PI3Kα kinase are shown in Figure 3. In the docking model of compound **9a** with PI3Kα (Figure 3B), we can easily see that the oxygen atom on the morphine ring formed a hydrogen bond with the hinge region residue VAL882. After careful observation of the binding model of compound **15a** with 3TL5 protein kinase (Figure 3D), it is not difficult to find that the N atom on the pyrimidine ring of thieno[3,2-*d*]pyrimidine formed a hydrogen bond interaction with residue SER806. Compared with the original ligand GDC-0980, the hydrogen bonding interaction between compounds **9a** and **15a** with protein is reduced, which may be one of the reasons why this series of compounds failed to achieve excellent cytotoxic activity.

#### **3. Experimental Section**

#### *3.1. General Information*

Unless otherwise required, all reagents used in the experiment were purchased as commercial analytical grade and used without further purification. Frequently used solvents (Ethanol, petroleum ether, ethyl acetate, dichloromethane, etc.) were absolutely anhydrous. All actions were monitored through GF254 thin-layer chromatography plate (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) and spots were visualized with iodine or light (in 254 nm or 365 nm). The structure of the target compound was confirmed by 1H-NMR and 13C-NMR spectra at room temperature on Bruker 400 MHz spectrometer (Bruker Bioscience, Billerica, MA, USA) with tetramethylsilane (TMS) as an internal standard. Mass spectrometry (MS) was performed on Waters High Resolution Quadrupole Time of Flight Tandem Mass Spectrometry (QTOF) (Waters Corporation, Milford, MA, USA). The purity of the

compound was determined by Agilent 1260 liquid chromatograph (Agilent Technologies Inc., Palo Alto, CA, USA) fitted with an Inertex-C18 column. All target compounds had the purity of ≥95%.

#### *3.2. Chemistry*

3.2.1. General Procedure for the Preparation of Compounds **3a**–**l**

Compounds **3a**–**l** were synthesized according to the reported procedures by our research group [15].

#### 3.2.2. General Procedure for the Preparation of Compounds **8**, **14** and **20**

Compound **5** was obtained by a cyclization reaction starting from commercially available methyl 2-aminothiophene-3-carboxylate (**4**) and urea. Subsequently, compound **5** is subjected to a chlorination reaction with POCl3 to give compound **6**. Compound **6** was substituted by morphine and hydrazine hydrate respectively to obtain key intermediate **8**. Similarly, we used commercially available methyl 2-aminothiophene-3-carboxylate (**10**) or commercial available methyl 2-amino-4,5,6,7-tetrahydrobenzo[*b*]thiophene-3-carboxylate (**16**) as the starting material to obtain intermediates **14** or **20** through a similar reaction condition. Detailed synthesis of key intermediates **8**, **14**, and **20**, and their precursors **7**, **11**, **12**, and **13**. can be found in the article reported by our research group [13].

#### 3.2.3. General Procedure for the Preparation of Target Compounds **9a**–**l**, **15a**–**l**, and **21a**–**h**.

A mixture of different substituted chalcone **3a**–**l** (1.5 mmol) and 4-(2-mercaptothieno[2,3-*d*] pyrimidin-4-yl) morpholine (**8**) or 4-(2-mercaptothieno[3,2-*d*]pyrimidin -4-yl)morpholine (**14**) or 4-(2-hydrazinyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-*d*]pyrimidin-4-yl)morpholine (**20**) were dissolved (1.5 mmol, **8**/**14**/**20**) in 25 mL of glacial acetic acid. Subsequently, the reaction solution was stirred at 100–110 ◦C for 1.5 h under the action of 2–3 drops concentrated H2SO4 (78%) as a catalyst and monitored by thin-layer chromatography (TLC). The reaction mixture was concentrated under reduced pressure. After the completion of concentrated, the mixture was filtered and the precipitate was washed with ethanol. If the purify of the compound is not high enough, recrystallization by 95% EtOH or column chromatography (EtOAc:PE = 1:3) are needed. The obtained solids were then dried to give the target compounds **9a**–**l**, **15a**–**l**, and **18a**–**h** with the yield ranging from 30%–60%.

*4-(2-(3-(3,4-dichlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]pyrimidin-4-yl) morpholine* (**9a**). Light yellow solid, mp 202–205 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 8.42 (d, *J* = 5.3 Hz, 1H), 7.95 (d, *J* = 8.7 Hz, 2H), 7.73 (s, 1H), 7.62 (m, *J* = 16.2, 7.0 Hz, 2H), 7.33 (d, *J* = 8.1 Hz, 1H), 7.12 (d, *J* = 8.4 Hz, 2H), 5.85–5.75 (m, 1H), 4.08 (m, *J* = 17.7, 11.6 Hz, 1H), 3.85 (s, 3H), 3.84–3.75 (m, 3H), 3.67 (s, 3H), 3.60–3.54 (m, 1H), 3.50–3.46 (m, 2H). HRMS (ESI): *m*/*z* calcd for (C27H25N3OS + H)+: 440.1797; found: 440.1834.

*4-(2-(5-(4-bromophenyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]pyrimidin-4-yl) morpholine* (**9b**). Light yellow solid, m.p. 280–282 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 8.18 (d, *J* = 5.5 Hz, 1H), 7.90 (dd, *J* = 8.4, 5.5 Hz, 2H), 7.63 (dd, *J* = 8.4, 5.0 Hz, 2H), 7.37 (dd, *J* = 13.9, 7.0 Hz, 3H), 7.30–7.26 (m, 1H), 5.79 (dd, *J* = 12.1, 6.1 Hz, 1H), 3.95 (dd, *J* = 17.8, 12.2 Hz, 1H), 3.82–3.65 (m, 6H), 3.59 (d, *J* = 7.7 Hz, 3H). HRMS (ESI): *m*/*z* calcd for (C28H27N3O2S + H)+: 470.1902; found: 470.1946.

*4-(2-(3,5-bis(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]pyrimidin-4-yl)Morpholine* (**9c**).Yellow solid, m.p. 225–227 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 8.47 (d, *J* = 5.6 Hz, 1H), 8.13 (s, 2H), 7.63 (s, 1H), 7.50–7.42 (m, 4H), 7.25 (t, *J* = 8.5 Hz, 2H), 5.88 (s, 1H), 4.13 (d, *J* = 12.1 Hz, 1H), 3.87 (s, 4H), 3.70 (s, 3H), 3.49 (s, 1H), 3.22 (s, 1H). HRMS (ESI): *m*/*z* calcd for (C27H23F2N3OS + H)+: 476.1608; found: 476.1598.

*4-(2-(3-(3,4-dichlorophenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]pyrimidin-4-yl) morpholine* (**9d**).Yellow solid, m.p. 286–289 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 7.73 (d, *J* = 7.5 Hz, 2H), 7.54 (d, *J* = 7.9 Hz, 2H), 7.31 (d, *J* = 6.6 Hz, 3H), 7.25 (d, *J* = 7.8 Hz, 2H), 5.74 (d, *J* = 6.6 Hz, 1H), 3.95–3.84 (m, 1H), 3.76–3.63 (m, 6H), 3.59 (s, 2H), 3.49 (d, *J* = 5.9 Hz, 1H). HRMS (ESI): *m*/*z* calcd for (C28H26FN3O2S + H)+: 488.1808; found: 488.1853.

*4-(2-(3-(3,4-Dichlorophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]pyrimidine-4-yl)morpholine* (**9e**). Yellow solid, m.p. 228.3~229.1 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 8.07 (d, *J* = 5.4 Hz, 1H), 7.77 (d, *J* = 7.1 Hz, 2H), 7.55 (d, *J* = 8.4 Hz, 2H), 7.44 (d, *J* = 7.6 Hz, 3H), 7.25 (d, *J* = 5.5 Hz, 2H), 5.72 (dd, *J* = 12.1, 5.7 Hz, 1H), 3.87 (dd, *J* = 18.0, 12.3 Hz, 1H), 3.71 (s, 2H), 3.66–3.58 (m, 4H), 3.54 (s, 2H), 3.18 (dd, *J* = 18.1, 5.8 Hz, 1H). HRMS (ESI): *m*/*z* calcd for (C28H26BrN3OS + H)+: 534.1041; found: 534.0991.

*4-(2-(5-(4-methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]pyrimidine-4-yl)morpholine* (**9f**). Orange solid, m.p. 237.6~239.1 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 8.17 (d, *J* = 4.9 Hz, 1H), 7.79 (d, *J* = 8.1 Hz, 2H), 7.35 (d, *J* = 4.7 Hz, 1H), 7.29 (d, *J* = 7.3 Hz, 2H), 7.24 (d, *J* = 7.7 Hz, 3H), 7.03 (d, *J* = 8.3 Hz, 2H), 5.69 (d, *J* = 6.4 Hz, 1H), 3.98–3.87 (m, 2H), 3.80 (s, 3H), 3.68 (s, 4H), 3.57 (s, 4H). HRMS (ESI): *m*/*z* calcd for (C27H23BrFN3OS + H)+: 538.0790; found: 538.0882.

*4-(2-(3-(3,4-dichlorophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]pyrimidin-4-yl)morpholine* (**9g**). Yellow solid, m.p. 207.4~208.2 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 8.43 (d, *J* = 5.4 Hz, 1H), 7.90 (d, *J* = 7.6 Hz, 2H), 7.74 (s, 1H), 7.62 (dd, *J* = 15.4, 6.7 Hz, 2H), 7.41–7.29 (m, 3H), 5.80 (dd, *J* = 11.6, 6.2 Hz, 1H), 4.09 (dd, *J* = 18.2, 11.5 Hz, 1H), 3.82 (s, 4H), 3.67 (s, 3H), 3.48 (s, 2H), 2.40 (s, 3H). HRMS (ESI): *m*/*z* calcd for (C27H24BrN3OS + H)+: 520.0884; found: 520.0865.

*4-(2-(3-(4-bromophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]pyrimidine-4-yl)morpholine* (**9h**). Light yellow solid, m.p. 222.8~223.9 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 8.11 (d, *J* = 5.0 Hz, 1H), 7.73 (d, *J* = 7.5 Hz, 2H), 7.54 (d, *J* = 7.9 Hz, 2H), 7.31 (d, *J* = 6.6 Hz, 3H), 7.25 (d, *J* = 7.8 Hz, 2H), 5.74 (d, *J* = 6.6 Hz, 1H), 3.95–3.84 (m, 1H), 3.76–3.63 (m, 6H), 3.59 (s, 2H), 3.13 (d, *J* = 13.7 Hz, 1H), 2.40 (s, 3H). 13C NMR (101 MHz, DMSO-*d6*) δ 163.89, 157.95, 156.29, 150.82, 145.81, 134.03, 132.46, 131.45, 131.41, 129.81, 129.71(2,C), 129.17, 128.43, 126.68(2,C), 126.29, 124.43, 106.60, 66.29(2,C), 61.90, 46.26(2,C).HRMS (ESI): *m*/*z* calcd for (C27H23Cl2N3OS + H)+: 508.1017; found: 508.1059.

4-(2-(3,5-diphenyl-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]pyrimidin-4-yl)morpholine (**9i**). Light yellow solid, m.p. 231.7~233.2 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 8.04 (d, *J* = 5.5 Hz, 1H), 7.77 (d, *J* = 7.6 Hz, 2H), 7.48–7.37 (m, 3H), 7.30–7.17 (m, 6H), 5.71 (dd, *J* = 12.0, 5.3 Hz, 1H), 3.87 (dd, *J* = 17.4, 12.2 Hz, 1H), 3.63 (dt, *J* = 19.1, 10.7 Hz, 6H), 3.49 (d, *J* = 13.7 Hz, 2H), 3.45–3.40 (m, 1H). HRMS (ESI): *m*/*z* calcd for (C27H22Cl2FN3OS + H)+: 528.0898; found: 528.0891.

*4-(2-(3-(4-fluorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]pyrimidin-4-yl) morpholine* (**9j**). Light yellow solid, m.p. 215.8~216.8 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 7.91–7.86 (m, 2H), 7.50 (t, *J* = 5.0 Hz, 1H), 7.39–7.24 (m, 4H), 7.16 (t, *J* = 8.8 Hz, 1H), 7.06 (d, *J* = 8.6 Hz, 2H), 5.72 (dd, *J* = 11.0, 5.3 Hz, 1H), 4.07–3.96 (m, 1H), 3.82 (s, 3H), 3.74 (s, 4H), 3.65–3.56 (m, 3H), 3.48 (s, 2H). HRMS (ESI): *m*/*z* calcd for (C27H22BrCl2N3OS + H)+: 588.0100; found: 588.0110.

*4-(2-(3-(4-bromophenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]Pyrimidin-4-yl) morpholine* (**9k**). Yellow solid, m.p. 221.2~222.0 ◦C; 1H -NMR (400 MHz, DMSO-*d6*) δ (ppm): 8.14 (d, *J* = 5.6 Hz, 1H), 7.93–7.85 (m, 2H), 7.55 (d, *J* = 8.2 Hz, 2H), 7.39–7.30 (m, 3H), 7.27 (d, *J* = 8.3 Hz, 2H), 5.77 (dd, *J* = 11.9, 5.4 Hz, 1H), 3.93 (dd, *J* = 17.7, 12.3 Hz, 1H), 3.80–3.65 (m, 6H), 3.59 (d, *J* = 10.2 Hz, 2H), 3.19 (dd, *J* = 17.9, 5.8 Hz, 1H). HRMS (ESI): *m*/*z* calcd for (C28H25Cl2N3O2S + H)+: 538.1123; found: 538.1199.

*4-(2-(3-(4-bromophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thieno[2,3-d]pyrimidine-4-Morpholine* (**9l**). Light yellow solid, m.p. 221.2~222.0 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 8.05 (d, *J* = 5.5 Hz, 1H), 7.76 (d, *J* = 7.3 Hz, 2H), 7.50 7.39 (m, 5H), 7.21 (dd, *J* = 17.0, 6.8 Hz, 3H), 5.70 (d, *J* = 6.7 Hz, 1H), 3.86 (d, *J* = 5.2 Hz, 1H), 3.69 (t, *J* = 10.6 Hz, 2H), 3.64–3.57 (m, 4H), 3.53 (t, *J* = 9.8 Hz, 2H), 3.12 (d, *J* = 5.5 Hz, 1H). HRMS (ESI): *m*/*z* calcd for (C28H25Cl2N3OS + H)+: 522.1174; found: 522.1203.

*4-(2-(3-(3,4-dichlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]pyrimidin-4-yl) morpholine* (**15a**). Light yellow solid, m.p. 131.2~132.5 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 7.73 (d, *J* = 8.4 Hz, 2H), 7.57 (d, *J* = 8.1 Hz, 2H), 7.40 (d, *J* = 6.2 Hz, 1H), 7.19 (d, *J* = 6.0 Hz, 2H), 7.02 (d, *J* = 8.5 Hz, 2H), 5.69 (dd, *J* = 12.0, 5.5 Hz, 1H), 3.92–3.84 (m, 1H), 3.81 (s, 3H), 3.62 (dd, *J* = 22.9, 8.6 Hz, 6H), 3.51 (d, *J* = 8.6 Hz, 2H), 3.17 (dd, *J* = 17.7, 5.6 Hz, 1H). HRMS (ESI): *m*/*z* calcd for(C27H25N3OS + H)+: 440.1797; found: 440.1808.

*4-(2-(5-(4-bromophenyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]pyrimidin-4-yl) morpholine* (**15b**). Light yellow solid, m.p. 265.3~266.3 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 7.69 (d, *J* = 7.8 Hz, 2H), 7.50 (d, *J* = 8.1 Hz, 2H), 7.40 (d, *J* = 6.2 Hz, 1H), 7.27 (d, *J* = 7.8 Hz, 2H), 7.20 (d, *J* = 7.8 Hz, 2H), 5.69 (dd, *J* = 12.2, 5.1 Hz, 1H), 3.93–3.82 (m, 1H), 3.62 (dd, *J* = 23.2, 8.0 Hz, 6H), 3.51 (d, *J* = 7.4 Hz, 2H), 3.12 (d, *J* = 18.1 Hz, 1H). HRMS (ESI): *m*/*z* calcd for (C28H27N3O2S + H)+: 470.1902; found: 470.1939.

*4-(2-(3,5-bis(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]pyrimidin-4-yl)Morpholine* (**15c**). Yellow solid, m.p. 254.7~255.4 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 7.88–7.82 (m, 2H), 7.39 (d, *J* = 6.1 Hz, 1H), 7.29 (dd, *J* = 16.3, 8.0 Hz, 4H), 7.19 (d, *J* = 6.1 Hz, 1H), 7.14 (d, *J* = 8.5 Hz, 2H), 5.73 (dd, *J* = 12.2, 5.2 Hz, 1H), 3.92–3.83 (m, 1H), 3.62 (dd, *J* = 20.8, 8.0 Hz, 6H), 3.51 (d, *J* = 8.0 Hz, 2H), 3.13 (dd, *J* = 18.0, 5.0 Hz, 1H). HRMS (ESI): *m*/*z* calcd for (C27H23F2N3OS + H)+: 476.1608; found: 476.1640.

*4-(2-(3-(3,4-dichlorophenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]pyrmidin-4-yl) morpholine* (**15d**). Yellow solid, m.p. 237.7~238.8 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 7.60 (d, *J* = 7.9 Hz, 2H), 7.48 (d, *J* = 6.8 Hz, 2H), 7.31 (d, *J* = 5.8 Hz, 1H), 7.19 (d, *J* = 7.8 Hz, 2H), 7.11 (d, *J* = 6.4 Hz, 2H), 5.62 (d, *J* = 6.1 Hz, 1H), 3.82–3.73 (m, 1H), 3.55 (s, 2H), 3.51 (d, *J* = 7.4 Hz, 4H), 3.43 (d, *J* = 8.8 Hz, 2H), 3.08 (d, *J* = 12.8 Hz, 1H). 13C NMR (101 MHz, DMSO-*d6*) δ 162.70, 162.39, 161.13, 160.78, 157.90, 156.38, 140.04, 128.77, 128.03, 121.75, 115.91(2,C), 115.77(2,C), 114.63(3,C), 110.46, 66.28, 62.03, 55.83, 47.11, 42.77. HRMS (ESI): *m*/*z* calcd for (C28H26FN3O2S + H)+: 488.1808; found: 488.1842.

*4-(2-(3-(3,4-Dichlorophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]pyrimidine-4-yl)morpholine* (**15e**). Yellow solid, m.p. 263–265 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 7.80 (d, *J* = 6.9 Hz, 2H), 7.61–7.55 (m, 2H), 7.49–7.44 (m, 3H), 7.43–7.39 (m, 1H), 7.21 (d, *J* = 6.2 Hz, 2H), 5.73 (dd, *J* = 12.2, 5.8 Hz, 1H), 3.89 (dd, *J* = 17.7, 12.2 Hz, 1H), 3.69–3.56 (m, 6H), 3.53–3.48 (m, 2H), 3.20 (dd, *J* = 17.8, 5.8 Hz, 1H). HRMS (ESI): *m*/*z* calcd for (C28H26BrN3OS + H)+: 534.1041; found: 534.1008.

*4-(2-(5-(4-methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]pyrimidine-4-y)morpholine* (**15f**). Yellow solid, m.p. 235–237 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 7.78 (d, *J* = 8.5 Hz, 2H), 7.42 (d, *J* = 6.1 Hz, 1H), 7.30 (d, *J* = 7.2 Hz, 2H), 7.25 (d, *J* = 6.2 Hz, 4H), 7.04 (d, *J* = 8.6 Hz, 2H), 5.73–5.65 (m, 1H), 3.91 (dd, *J* = 18.0, 12.1 Hz, 1H), 3.82 (s, 3H), 3.66–3.53 (m, 6H), 3.46 (s, 2H), 3.16 (d, *J* = 17.8 Hz, 1H). HRMS (ESI): *m*/*z* calcd for (C27H23BrFN3OS + H)+: 538.0790; found: 538.0832.

*4-(2-(3-(3,4-dichlorophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]pyrimidin-4-yl)morpholine* (**15g**). Yellow solid, m.p. 267–268 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 7.69 (d, *J* = 7.7 Hz, 2H), 7.57 (d, *J* = 8.2 Hz, 2H), 7.40 (d, *J* = 6.2 Hz, 1H), 7.28 (d, *J* = 8.0 Hz, 2H), 7.20 (d, *J* = 6.3 Hz, 2H), 5.70 (dd, *J* = 11.9, 5.6 Hz, 1H), 3.91–3.82 (m, 1H), 3.65–3.56 (m, 6H), 3.51 (d, *J* = 8.4 Hz, 2H), 3.21–3.13 (m, 1H), 2.35 (s, 3H). HRMS (ESI): *m*/*z* calcd for (C27H24BrN3OS + H)+: 517.0823; found: 517.0798.

*4-(2-(3-(4-bromophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]pyrimidine-4-yl)morpholine* (**15h**). Yellow solid, m.p. 255–257 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 7.68 (d, *J* = 8.1 Hz, 2H), 7.50 (d, *J* = 8.3 Hz, 2H), 7.40 (t, *J* = 6.6 Hz, 1H), 7.29 (d, *J* = 10.6 Hz, 2H), 7.22 – 7.17 (m, 3H), 5.69 (dd, *J* = 11.7, 4.7 Hz, 1H), 3.92–3.83 (m, 1H), 3.63–3.55 (m, 6H), 3.50 (d, *J* = 11.1 Hz, 2H), 3.09 (dd, *J* = 17.6, 5.7 Hz, 1H), 2.35 (s, 3H). 13C NMR (101 MHz, DMSO-*d6*) δ 171.77, 158.31, 154.66, 151.51, 145.64, 132.28, 131.46, 131.41, 129.95, 129.76, 129.17(2,C), 128.46, 126.76(2,C), 126.26, 121.64, 117.97, 110.41, 66.32(2,C), 61.88, 46.96(2,C), 42.01.HRMS (ESI): *m*/*z* calcd for (C27H23Cl2N3OS + H)+: 508.1017; found: 508.1044.

*4-(2-(3,5-diphenyl-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]pyrimidin-4-yl)morpholine* (**15i**). Yellow solid, m.p. 266–267 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 8.04 (d, *J* = 5.5 Hz, 1H), 7.77 (d, *J* = 7.6 Hz, 2H), 7.56–7.35 (m, 3H), 7.32–7.15 (m, 6H), 5.71 (dd, *J* = 12.0, 5.3 Hz, 1H), 3.87 (dd, *J* = 17.4, 12.2 Hz, 1H), 3.70–3.54 (m, 6H), 3.50 (s, 2H), 3.09 (dd, *J* = 17.6, 5.3 Hz, 1H). HRMS (ESI): *m*/*z* calcd for (C27H22Cl2FN3OS + H)+: 526.0923; found: 526.0889.

*4-(2-(3-(4-fluorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]pyrimidin-4-yl) morpholine* (**15j**). Yellow solid, m.p. 271–272 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 7.74 (d, *J* = 7.9 Hz, 2H), 7.35 (d, *J* = 6.0 Hz, 1H), 7.26–7.17 (m, 3H), 7.07 (d, *J* = 8.8 Hz, 2H), 6.95 (d, *J* = 8.6 Hz, 2H), 5.63 (dd, *J* = 11.8, 5.6 Hz, 1H), 3.85 (d, *J* = 17.8 Hz, 1H), 3.73 (s, 3H), 3.55 (dd, *J* = 25.4, 8.6 Hz, 7H), 3.09 (d, *J* = 16.8 Hz, 2H). HRMS (ESI): *m*/*z* calcd for (C27H22BrCl2N3OS + H)+: 588.0100; found: 588.0132.

*4-(2-(3-(4-bromophenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]Pyrimidin-4-yl)morpholine* (**15k**). Yellow solid, m.p. 251–252 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 7.75 (s, 2H), 7.41 (d, *J* = 7.6 Hz, 2H), 7.31 (d, *J* = 5.5 Hz, 1H), 7.21 (t, *J* = 8.0 Hz, 2H), 7.12 (d, *J* = 7.9 Hz, 3H), 5.62 (d, *J* = 6.8 Hz, 1H), 3.85–3.74 (m, 1H), 3.53 (d, *J* = 13.2 Hz, 6H), 3.44 (d, *J* = 8.9 Hz, 2H), 3.05 (d, *J* = 17.7 Hz, 1H). HRMS (ESI): *m*/*z* calcd for (C28H25Cl2N3O2S + H)+: 538.1123; found: 538.1161.

*4-(2-(3-(4-bromophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)thieno[3,2-d]pyrimidine-4-Morpholine* (**15l**). Yellow solid, m.p. 244–245 ◦C; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 7.79 (d, *J* = 7.3 Hz, 2H), 7.50 (d, *J* = 8.3 Hz, 2H), 7.44 (t, *J* = 8.0 Hz, 3H), 7.40 (d, *J* = 6.2 Hz, 1H), 7.24–7.18 (m, 3H), 5.71 (dd, *J* = 12.1, 5.7 Hz, 1H), 3.89 (dd, *J* = 17.5, 12.6 Hz, 1H), 3.62 (dd, *J* = 23.5, 8.5 Hz, 6H), 3.51 (d, *J* = 8.0 Hz, 2H), 3.13 (dd, *J* = 17.7, 5.6 Hz, 1H). HRMS (ESI): *m*/*z* calcd for (C28H25Cl2N3OS + H)+: 522.1174; found: 522.1201.

4-(2-(3-(3,4-dichlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-5,6,7,8-tetrahydrobenzo [4,5]thieno[2,3-d]pyrimidin-4-yl)morpholine (**21a**). Dark blue solid, m.p. 252–253 ◦C; ESI-MS [M + H]<sup>+</sup> *m*/*z*: 594.5; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 7.73 (d, *J* = 8.7 Hz, 2H), 7.56 (d, *J* = 7.0 Hz, 2H), 7.20 (d, *J* = 8.4 Hz, 1H), 7.02 (d, *J* = 7.0 Hz, 2H), 5.67 (d, *J* = 5.7 Hz, 1H), 3.88 (d, *J* = 17.1 Hz, 1H), 3.81 (s, 3H), 3.62 (d, *J* = 5.4 Hz, 2H), 3.58 (s, 2H), 3.18 (d, *J* = 12.1 Hz, 3H), 2.99 (s, 2H), 2.73 (s, 4H), 1.81 (s, 4H).

4-(2-(3-(4-bromophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-5,6,7,8-tetrahdrobenzo[4,5]thieno [2,3-d]pyrimidin-4-yl)morpholine (**21b**). Dark blue solid, m.p. 243–244 ◦C; ESI-MS [M + H]<sup>+</sup> *m*/*z*: 588.5; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 7.69 (d, *J* = 7.9 Hz, 2H), 7.50 (d, *J* = 8.3 Hz, 2H), 7.27 (d, *J* = 7.7 Hz, 2H), 7.19 (d, *J* = 8.3 Hz, 2H), 5.70 (dd, *J* = 12.3, 5.4 Hz, 1H), 3.88 (dd, *J* = 17.7, 12.3 Hz, 1H), 3.64 (s, 2H), 3.57 (s, 2H), 3.15 (d, *J* = 16.2 Hz, 3H), 2.99 (s, 2H), 2.74 (s, 4H), 2.35 (s, 3H), 1.78 (d, *J* = 13.4 Hz, 4H).

4-(2-(3,5-diphenyl-4,5-dihydro-1H-pyrazol-1-yl)-5,6,7,8-tetrahydrobenzo[4,5]thiophene[2,3-d]pyramidin -4-yl)morpholine (**21c**). Dark blue solid, m.p. 212–213 ◦C; ESI-MS [M + H]<sup>+</sup> *m*/*z*: 495.6; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 7.82–7.79 (m, 2H), 7.46 (d, *J* = 7.6 Hz, 3H), 7.29 (d, *J* = 7.3 Hz, 2H), 7.23 (dd, *J* = 9.2, 1.9 Hz, 3H), 5.73 (dd, *J* = 12.1, 5.5 Hz, 1H), 3.92 (dd, *J* = 17.7, 12.1 Hz, 1H), 3.65–3.60 (m, 2H), 3.55 (d, *J* = 6.5 Hz, 2H), 3.14 (dd, *J* = 17.7, 5.7 Hz, 3H), 3.02–2.94 (m, 2H), 2.74 (s, 4H), 1.80 (d, *J* = 28.8 Hz, 4H).

4-(2-(3-(4-fluorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-5,6,7,8-tetrahydrobenzo [4,5]thieno[2,3-d]pyrimidin-4-yl)morpholine (**21d**). Dark blue solid, m.p. 189-193 ◦C; ESI-MS [M + H]<sup>+</sup> *m*/*z*: 543.6; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 7.74 (d, *J* = 8.7 Hz, 2H), 7.28–7.23 (m, 2H), 7.12 (t, *J* = 8.8 Hz, 2H), 7.02 (d, *J* = 8.8 Hz, 2H), 5.71 (dd, *J* = 12.0, 5.4 Hz, 1H), 3.87 (d, *J* = 5.0 Hz, 1H), 3.81 (s, 3H), 3.68–3.61 (m, 2H), 3.57 (d, *J* = 6.4 Hz, 2H), 3.20–3.12 (m, 3H), 3.00 (s, 2H), 2.73 (s, 4H), 1.79 (d, *J* = 29.8 Hz, 4H).

4-(2-(3-(4-bromophenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-5,6,7,8-tetrahydrobenzo[4,5] thieno[2,3-d]pyrimidin-4-yl)morpholine (**21e**). Dark blue solid, m.p. 272–273 ◦C; ESI-MS [M + H]<sup>+</sup> *m*/*z*: 592.5; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 7.85 (d, *J* = 6.4 Hz, 2H), 7.50 (d, *J* = 7.3 Hz, 2H), 7.30 (s, 2H), 7.20 (d, *J* = 8.0 Hz, 2H), 5.77–5.66 (m, 1H), 3.91 (t, *J* = 16.3 Hz, 1H), 3.63 (s, 2H), 3.58 (s, 2H), 3.18 (s, 3H), 3.01 (s, 2H), 2.74 (s, 4H), 1.79 (d, *J* = 18.0 Hz, 4H).

4-(2-(3-(4-bromophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno [2,3-d]pyrimidin-4-yl)morpholine (**21f**). Dark blue solid, m.p. 257–259 ◦C; ESI-MS [M + H]<sup>+</sup> *m*/*z*: 574.5; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 7.80 (d, *J* = 7.8 Hz, 2H), 7.48 (dd, *J* = 17.6, 7.9 Hz, 5H), 7.20 (d, *J* = 8.4 Hz, 2H), 5.73 (dd, *J* = 11.9, 5.5 Hz, 1H), 3.91 (dd, *J* = 17.9, 12.1 Hz, 1H), 3.64 (d, *J* = 6.0 Hz, 2H), 3.57 (s, 2H), 3.15 (dd, *J* = 17.8, 5.5 Hz, 3H), 3.02 (s, 2H), 2.74 (s, 4H), 1.78 (d, *J* = 18.3 Hz, 4H).

4-(2-(5-(4-bromophenyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-5,6,7,8-tetrahydrobenzo [4,5]thieno[2,3-d]pyrimidin-4-yl)morpholine (**21g**). Dark blue solid, m.p. 231–234 ◦C; ESI-MS [M + H]<sup>+</sup> *m*/*z*: 643.4; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 7.71 (d, *J* = 8.3 Hz, 2H), 7.63 (d, *J* = 8.3 Hz, 2H), 7.57 (d, *J* = 11.3 Hz, 2H), 7.22 (d, *J* = 8.8 Hz, 1H), 5.71 (dd, *J* = 11.9, 5.9 Hz, 1H), 3.89 (dd, *J* = 18.0, 12.5 Hz, 1H), 3.62 (s, 2H), 3.57 (s, 2H), 3.21 (dd, *J* = 17.9, 6.2 Hz, 3H), 2.99 (s, 2H), 2.73 (s, 4H), 1.78 (d, *J* = 37.6 Hz, 4H).

4-(2-(3-(3,4-dichlorophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno [2,3-d]pyrimidin-4-yl)morpholine (**21h**). Dark blue solid, m.p. 293–294 ◦C; ESI-MS [M + H]<sup>+</sup> *m*/*z*: 563.1; 1H NMR (400 MHz, DMSO-*d6*) δ (ppm): 7.80 (d, *J* = 6.5 Hz, 2H), 7.58 –7.55 (m, 2H), 7.46 (d, *J* = 7.6 Hz, 3H), 7.23–7.19(m, 1H), 5.73 (dd, *J* = 12.1, 5.9 Hz, 1H), 3.92 (dd, *J* = 17.8, 12.2 Hz, 1H), 3.63 (d, *J* = 6.2Hz, 2H), 3.58 (d, *J* = 6.4Hz, 2H), 3.21 (dd, *J* = 18.1, 6.3 Hz, 3H), 3.01 (s, 2H), 2.74 (s, 4H), 1.79 (d, *J* = 23.9 Hz, 4H).

#### *3.3. Cytotoxicity Assay In Vitro*

The *in vitro* cytotoxic activities of Compounds **9a**–**l**, **15a**–**l,** and **21a**–**h** were evaluated with A549, PC-3, HepG2, and MCF-7 cell lines by the standard MTT assay, with GDC-0941 as a positive control. The cancer cell lines were cultured in minimum essential medium (MEM) supplement with 10% fetal bovine serum (FBS). Approximately, 4 <sup>×</sup> 10 <sup>3</sup> cells, suspended in MEM medium, were plated onto each well of a 96-well plate and incubated in 5% CO2 at 37 ◦C for 24 h. The test compounds at the indicated final concentrations were added to the culture medium, and cell cultures continued for 72 h. Fresh MTT was added to each well at a terminal concentration of 5 μg/mL and incubated with cells at 37 ◦C for 4 h. The formazan crystals were dissolved in 100 μL of DMSO in each well, and the absorbency at 492 nm (for absorbance of MTT formazan) and 630 nm (for the reference wavelength) was measured with an enzyme linked immunosorbent assay (ELISA) reader (MR-96A Mindray Elisa Microplate Reader, Guangzhou, China). All compounds were tested three times in each of the cell lines. The results expressed as inhibition rates or IC50 was the averages of two determinations and calculated using the Bacus Laboratories Inc. Slide Scanner (Bliss) software (the Bacus Laboratories Inc. Slide Scanner (BLISS) system, Lombard, IL, USA).

#### *3.4. mTOR Kinase Assay*

The potent compounds **9a** and **15a** were tested for their activities against mTOR enzyme using Kinase-Glo® Luminescent Kinase Assay (Promega, Madison, WI, USA), with NVPBEZ-235 and PI103 as positive controls. The kinase reaction was done in a 384-well black plate. Each well was loaded with 50 μL of test items (in 90% DMSO) and 5 μL reaction buffer containing 10 μg/mL PI substrate (l-α-phosphatidylinositol); Avanti Polar Lipids (Avanti Polar Lipids, Inc., Alabaster, AL, USA); prepared in 3% octyl-glucoside) and the mTOR protein 2.5 nM was then added to it. The reaction was started by the addition of 5 μL of 10 μM ATP prepared in the reaction buffer (50 mM HEPES pH 7.5, 1 mM EGTA, 3 mM MnCl2, 10 mM MgCl2, 2 mM DTT and 0.01% Tween-20) and was incubated for 60 min. It was terminated by the addition of 10 μL Kinase-Glo buffer. The plates were then read in Synergy 2 readers (BioTek, Winooski, VT, USA) for luminescence detection. The assay was repeated two times and the results expressed as IC50 (inhibitory concentration 50%) were the averages of two determinations.

#### *3.5. PI3K*α *Kinase Assay*

The potent compounds **9a** and **15a** were tested for their activity against PI3Kα using a Kinase-Glo® Luminescent Kinase Assay (Promega, Madison, WI, USA), with GDC-0941 and PI103 as positive controls. The kinase reaction occurred in a 384-well black plate. Each well was loaded with 50 μL of test items (in 90% DMSO) and 5 μL of reaction buffer containing 10μg/mL PI substrate (l-α-phosphatidylinositol; Avanti Polar Lipids (Avanti Polar Lipids, Inc.); prepared in 3% octyl-glucoside), and the PI3Kα protein (10 nM) was then added to it. The reaction was started by the addition of 5 μL of 1 μM Adenosine triphosphate (ATP) prepared in the reaction buffer and incubated for 60 min for p110α. It was terminated by the addition of 10 μL of Kinase-Glo buffer. The plates were then read in a Synergy 2 reader (BioTek, Winooski, VT, USA) for luminescence detection. The assay was repeated two times and the results expressed as IC50 (inhibitory concentration 50%) were the averages of two determinations.

#### *3.6. Docking Studies*

Molecular docking simulation studies were carried out by the AutoDock 4.2 software (The Scripps Research Institute, San Diego, CA, USA). The docking tutorial we used and the detailed AutoDock basic operational methods can be found at: http://autodock.scripps.edu/faqs-help/tutorial. The protein preparation process of flexible docking mainly includes fixing the exact residues, adding hydrogen atoms, removing irrelevant water molecules, adding charges, etc. The potent compounds were selected as ligand examples, and the structures of PI3Kα (PDB code: 3TL5, http://www.pdb.org/) were selected as the docking models. Only the best-scoring ligand–protein complexes were used for the binding site analyses. All the docking results were processed and modified in Open-Source PyMOL 1.8.x software (https://pymol.org).

#### **4. Conclusions**

Three series of thienopyrimidine derivatives containing pyrazole structure were designed and synthesized. In addition, we evaluated their cytotoxic activities against four cancer cell lines *in vitro*. The pharmacological indicated that most of the compounds showed moderate cytotoxic activity against the tested cancer cell lines. What is more, the inhibitory activities of the compounds **9a** and **15a** against PI3Kα and mTOR kinase were further investigated. Compound **9a** exhibited moderate levels of inhibition activity against four cancer cell lines and PI3Kα kinase. Although compound **9a** did not reach the same level of inhibition as the positive control drug, it also gave us new direction for developing novel thienopyrimidines containing the pyrazole linker group as PI3Kα inhibitor.

**Author Contributions:** W.Z., Z.Y., and P.Z. provided and managed the project and supervise the progress of the project; L.Y., Q.W., B.Z., and Y.F. designed the chemical synthesis route and performed the experiments; L.Y., Q.W., and C.W. conducted experimental data analysis and software processing; L.Y., W.Z., and P.Z. started the project, and wrote and revised the paper.

**Funding:** We gratefully acknowledge the generous support provided by The National Natural Science Funds (No. 21967009); The Project Supported by Natural Science Foundation of Jiangxi, China (20171ACB21052 and 2018BBG70003); Natural Science Funds for Distinguished Young Scholar of Jiangxi Province, China (20171BCB23078); Key projects of the youth fund, Natural Science Funds of Jiangxi Province (20171ACB21052).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Sample Availability:** Samples of the compounds **9a**–**l**, **15a**–**l**, and **21a**–**h** are available from the authors.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **Potent Anti-Ovarian Cancer with Inhibitor Activities on Both Topoisomerase II and V600EBRAF of Synthesized Substituted Estrone Candidates**

**Mohamed El-Naggar 1, Abd El-Galil E. Amr 2,3,\*, Ahmed A. Fayed 3,4, Elsayed A. Elsayed 5,6, Mohamed A. Al-Omar <sup>2</sup> and Mohamed M. Abdalla <sup>7</sup>**


Received: 25 April 2019; Accepted: 28 May 2019; Published: 29 May 2019

**Abstract:** A series of 16-(α-alkoxyalkane)-17-hydrazino-estra-1(10),2,4-trien[17,16-c]-3-ol (**3a**–**l**) and estra-1(10),2,4-trien-[17,16-c]pyrazoline-3-ol derivatives (**4a**–**d**) were synthesized from corresponding arylidines **2a,b** which was prepared from estrone **1** as starting material. Condensation of 1 with aldehydes gave the corresponding arylidine derivatives **2a,b** which were treated with hydrazine derivatives in alcohols to give the corresponding derivatives **3a**–**l**, respectively. Additionally, treatment of **2a,b** with methyl- or phenylhydrazine in ethanolic potassium hydroxide afforded the corresponding N-substituted pyrazoline derivatives **4a**–**d**, respectively. All these derivatives showed potent anti-ovarian cancer both in vitro and in vivo. The mechanism of anti-ovarian cancer was suggested to process via topoisomerase II and V600EBRAF inhibition.

**Keywords:** estrone derivatives; hydrazine; N-substituted pyrazoline; anti-ovarian cancer; topoisomerase II inhibitor; kinase inhibitor

#### **1. Introduction**

Synthetic alterations of estrone lead to discovering of compounds with diverse biological activities, for example with antitumor effect [1], as anti-breast cancer agent [2] and with antioxidant activity [3]. Estrone derivatives with antitumor activities must be devoid completely of the estrogenic activities [4–6]. The inversion of the configuration at C-13 lead to estrone derivatives with antitumor activities devoid from hormonal actions due to conformational change for the overall molecule resulting from the cis junction of rings C and D [4,7]. Some recent publications report on the syntheses and in vitro biological evaluation of several 13α-estrone derivatives [8–11]. These derivatives exhibited biological activities with substantial anti-proliferative or enzyme inhibitory potentials. Most literature data are mainly focusing on 13α-estrone substitution in ring D, while modified derivatives with ring A substitutions are rarely reported [12].

Cancer is considered one of the most significant causes of death worldwide. Ovarian cancer is one of the major causes for death among adult females [13]. Cancer is characterized by abnormal cell proliferation bypassing normal cell growth and death mechanisms. Recently, DNA topoisomerase II inhibitors have been researched as a potential target, which interferes with cancer growth and development [14]. Topoisomerase II inhibition leads finally to cell cycle inhibition and apoptosis in abnormal cancer cells [15]. BRAF kinase inhibition is another possible control mechanism, which was also developed to interfere with cancer cell growth [16]. BRAF kinase constitutes a major signaling process integrated in the RAS-MEK proliferation process. The activation of MEK by BRAF signals finally induces cell proliferation and survival [17]. Inhibiting BRAF activity results in inhibition of the proliferation signals, and thus inhibiting cancer cell development and growth [18].

In view of these observations andin continuation of our previousworkin heterocyclic chemistry [19–22], the current work aimed at evaluating the effect or remote long cage distortion caused by introducing some moieties in both 16 and 17 locations of ring D. We screened some of synthesized estrone candidates for their anti-ovarian cancer potential both in vitro and in vivo. Furthermore, the inhibitory effects of the synthesized compounds were investigated against topoisomerase II and V600EBRAF kinase inhibitors.

#### **2. Results and Discussion**

#### *2.1. Results*

#### 2.1.1. Chenical Synthesis

A series of 17-hydrazino-estratrienol (**3a–l**) and pyrazolinestratrienol (**4a–d**) were synthesized from corresponding arylidines **2a,b** which was prepared from estrone **1** as starting material.

Treatment of aryledine derivatives **2a,b** with hydrazine hydrate, in refluxing methanol or ethanol afforded the corresponding 17-hydrazino-16-α-methoxymethane estrone derivatives **3a,b** and 17-hydrazino-16-α-ethoxyethane estrone derivatives **3c,d**, respectively.

Also, reaction of compounds **2a,b** with N-methyl hydrazine, in refluxing methanol or ethanol afford 17-N-methyl hydrazino-16-α-methoxymethane estrone derivatives **3e,f** and 17-*N*-methyl hydrazino-16-α-ethoxyethane estrone derivatives **3g,h** respectively.

Additionally, when compounds **2a,b** reacted with *N*-phenyl hydrazine, under the same conditions afford the corresponding 17-*N*-phenylhydrazino- 16-α-methoxymethane estrone derivatives **3i,j** and 17-*N*-phenylhydrazino- 16-α-ethoxyethane estrone derivatives **3k,l**, respectively. Finally, condensation of **2a,b** with CH3NHNH2 or PhNHNH2 in refluxing dioxane afforded the corresponding N-substituted pyrazoline derivatives **4a–d**, respectively (Scheme 1).

**Scheme 1.** Synthetic route for compounds **2**–**4**.

#### 2.1.2. Biological Screening

#### In Vitro Cytotoxic Activities

The cytotoxicity of some synthesized derivatives against SKOV-3 cancer cell line in vitro was performed with the MTT assay. The obtained results showed that all synthesized compounds showed potent in vitro cytotoxic activities against SKOV-3 cells at the nanomolar level. Moreover, the effect of the synthesized compounds was dose-dependent. From the obtained IC50 values (Figure 1), prepared compounds can be arranged discerningly in the following order: **3a**, **3b**, **3c**, **3d**, **3e**, **3f**, **3g**, **3h**, **3i**, **3j**, **3k**, **3l**, **4a**, **4b**, **4c**, **4d** (Figure 1). Compounds **3a–l** are characterized with the presence of hydrazine group rather than the pyrazoline group present in compounds **4a**–**d**. Furthermore, the alkyl group is found to be more active than the aromatic group. Generally Cpd. **3a** showed highest cytotoxic activity with IC50 value of 4.23 ± 0.12 nM, while Cpd. **4d** was the least active, where its IC50 value was about 9.5% of that obtained for Cpd. **3a**.

The effect of resveratrol (RES) and doxorubicin (DOX) on SKOV-3 showed IC50 of 55 and 250 nM, respectively.

The cytotoxicity of the newly synthesized compounds against fibroblast were as follow **IC50 3a** (1.2 μM), **3b** (2.45 μM), **3c** (5.67 μM), **3d** (7.44 μM), **3e** (8.11 μM), **3f** (9.20 μM), **3g** (11.18 μM), **3h**(16.22 μM), **3i** (15.13 μM), **3j** (16.20 μM), **3k** (17.78 μM), **3l** (20.21 μM), **4a** (23.34 μM), **4b** (24.55 μM), **4c** (25.55 μM), **4d** (30.34 μM).

**Figure 1.** In vitro Cytotoxicity activities of the tested compounds against SKOV-3 cells.

#### In Vivo Anti-Ovarian Cancer

The in vivo anti-ovarian cancer activities of the tested compounds were evaluated using mouse xenograft model. Figure 2 represents the percentages of decrease in tumor growth upon treatment with different synthesized compounds. The obtained results showed that all compounds potentially reduced tumor growth over a period of 40 days of treatment, in comparison to control treatments, which showed normal tumor growth development. Moreover, it can be seen that the reduction in tumor size started directly from the 5th day of treatment, where the reduction percentage ranged from 45.7 ± 0.05% to 54.71 ± 0.01%. The reduction in tumor growth increased significantly reaching its maximal by the end of treatment period (40 days). The highest reduction in tumor growth was recorded for occurred Cpd. **3a**, which significantly decreased tumor growth by about 93.61 ± 0.7%. It can also be seen that Cpd. **3a**, was the most potent for in vivo treatment, where its effect was more obvious starting from day15 of treatment, where the reduction percentage in tumor size due to other compounds was more or less within the same range. We also can see that the in vivo results are coincided with those obtained for *in vitro* results, where the reduction in tumor size followed the same descending order: **3a**, **3b**, **3c**, **3d**, **3e**, **3f**, **3g**, **3h**, **3i**, **3j**, **3k**, **3l**, **4a**, **4b**, **4c**, and **4d**.

**Figure 2.** Percentages of decrease in tumor volume in nude mice in response to different synthesized derivatives.

#### 2.1.3. Inhibition of Topoisomerase II Activities

In order to further investigate the possible mechanism of action of the newly synthesized derivatives, we investigated their inhibitory effects on topoisomerase II. Results obtained (Figure 3) showed that all synthesized compounds showed potential inhibitory effects against topoisomerase II. Furthermore, the inhibitory effect was also found to follow the same descending order obtained before during in vitro and in vivo investigation: **3a**, **3b**, **3c**, **3d**, **3e**, **3f**, **3g**, **3h**, **3i**, **3j**, **3k**, **3l**, **4a**, **4b**, **4c**, and **4d**. Cpd. **3a** showed the most inhibitory action for topoisomerase II with IC50 value of 3.45 ± 0.13 nM, which was about 7,9% of that obtained for the lest potent compound (IC50 for Cpd. **4d**: 43.56 ± 0.98 nM).

**Figure 3.** IC50 of Topoisomerase II Inhibitor activities of the tested compounds.

#### 2.1.4. In Vitro Kinase Assay

The in vitro kinase assay of synthesized derivatives was investigated against both WTBRAF (BRAF kinase wild type) and V600EBRAF (mutant BRAF kinase). Results obtained in Figure 4 showed that all synthesized compounds were highly active inhibitors for V600EBRAF compared with moderate activities against WTBRAF. Again, the descending order of activities was as follow **3a**, **3b**, **3c**, **3d**, **3e**, **3f**, **3g**, **3h**, **3i**, **3j**, **3k**, **3l**, **4a**, **4b**, **4c**, and **4d**. Compound **3a** recorded the most active inhibition (IC50: 0.041 ± 0.0016 and 4.23 ± 0.12 μM for mutant and wild type BRAF kinase, respectively. Furthermore, it can be seen that the inhibitory effect of Cpd. **3a** was much more superior to that obtained for different positive control drugs (0.48, 3.87 and 0.97 μM for Sorafenib, Dabrafenib and Vemurafenib, respectively.

**Figure 4.** IC50 values of synthesized for both WTBRAF and V600EBRAF.

#### *2.2. Discussion*

Within the framework of the current work we synthesized new estrone derivatives from their corresponding arylidines. The newly synthesized derivatives showed potential cytotoxic activities against SKOV-3 cells. Moreover, in vivo investigations revealed that the synthesized compounds were able to potentially reduce tumor volume growth over a treatment period of 45 days.

The newly introduced 1-alkoxy benzyl moiety alone or combined with the 17- hydrazino- moities make remote destortion of the structure cage that completely elminate any hormonal activities of te estrone molceules and deviate the biological activities towards the aniticancer ones, so we investigated this property and tried to find the mechnisms of anticancer actions of these newly synthesized compounds.

In search for the possible mechanism of action of the anticancer activities of the tested compounds, we investigated the possibility of the compounds to inhibit both topoisomerase II and kinase enzymes. Results showed that the prepared compounds can potentially serve as inhibitors for these enzymes. The cytotoxic activities of the prepared estrone derivatives can be due to the inhibition of 17-hydroxysteroid dehydrogenases. We have previously reported on the anti-breast cancer activities of different estrone derivatives [23], and explained that their anticancer activities can be attributed to the inhibition of 17-hydroxysteroid dehydrogenase [24].

Concerning topoisomerase II and V600EBRAF kinase inhibition, our results suggest that the newly synthesized compounds exert their cytotoxic action against SKOV-3 cancer cells by interfering with the metabolic activity of these enzymes, thus preventing cancer cells from obtaining proliferation signaling molecules essential for their growth and survival [16,17]. Different activities of the prepared compounds may be attributed to the structure activity relationship of these compounds.

Hydrazine derivatives are generally potent than pyrazoline ones, due to their open chain structure as well as their higher electron density. Furthermore, it can be suggested that *N*-methyl derivatives are more active than *N*-phenyl ones. On the other hand, hydrazine derivatives can be divided into more active α-methoxyl derivatives and the less active α-ethoxyl, and then the least active *N*-phenyl derivatives. This can be explained due to the fact that phenyl group act as a withdrawing group. On the other hand, the weaker effect of α-ethoxyl derivatives than the α-methoxyl ones can be attributed to their bulky nature. This effect can be observed among different series of compound **3**. As the bulky nature of the substituted groups increase, accordingly more steric hindrance can be observed, leading to decreased cytotoxic activities.

#### **3. Materials and Methods**

#### *3.1. Chemical Synthesis*

"Melting points reported are inaccurate. IR spectra were registered on Shimadzu FT-IR 8300 E (Shimadzu Corporation, Kyoto, Japan) spectrophotometer using the (KBr) disk technique. The 1H-NMR spectra were determined with bruker 600 MHz NMR spectrometer. The chemical shifts are expressed on the δ (ppm) scale using TMS as the standard reference. Mass spectra were recorded on Finnigan SSQ operating at 70 ev. Elemental analysis determined on a Perkin Elmer 240 (Microanalysis), Microanalysis Center, Cairo University, Cairo, Egypt".

#### 3.1.1. Synthesis of 3-Hydroxy-16-[substituted]-estra-1(10),2,4-trien-17-ones (**2a**,**b**)

"A mixture of (**1**) (0.54 g, 20 mmol) and formaldehyde or acetaldehyde (20 mmol) in EtOH (50 mL) and aqueous KOH (10 mL, 30%) was stirred for 24 h at room temperature. The formed solid was collected by filtration, crystallized from ethanol to give compounds **2a** [25] and **2b**, respectively".

#### *3-Hydroxy-16-[ethylene]-estra-1(10),2,4-trien-17-one* (**2b**)

Yield 97%, mp 198–200 ◦C, [α] 25 *<sup>D</sup>* = + 71 (c 1, MeOH). IR (KBr, cm–1): 3340 (OH), 2944 (CH, aliphatic), 1743 (C=O), 1642 (C=C). 1H NMR: (600 MHz, CDCl3): δ ppm 0.61 (1H, m, H-8β), 0.90 (3H, s, CH3-18), 1.01 (1H, m, H-11β), 1.12 (1H, m, H-7α), 1.15 (1H, m, H-12α), 1.25 (1H, m, H-14α), 1.44 (1H, m, H-15β), 1.63 (1H, m, H-15α), 1.72 (1H, m, H-7β), 2.01 (1H, m, H-9α), 2.13 (1H, m, H-11α), 2.50 (1H, m, H-12β), 2.56 (1H, m, H-6α), 2.66 (1H, m, H-6β), 4.90 (1H, s, OH, disappeared with D2O), 5.75 (1H, dd, H-2), 6.67 (1H, d, H-4), 2.08 (3H, d, CH3, enone), 6.41 (1H, s, enone proton), 7.12 (1H, d, H-1). 13C NMR: (150 MHz, CDCl3): δ 13.52, 21.73, 25.51, 26.64, 27.48, 29.75, 36.35, 38.61, 43.81, 48.92, 50.43, 112.79, 115.53, 126.56, 128.48, 132.50, 137.02, 138.44, 153.82, 210.02 (20 C). MS (EI): *m*/*z* 296 (100%) [M+]. Anal. Calcd for C20H24O2 (296.40): Calcd C, 81.04; H, 8.16. Found C, 81.00; H, 8.10.

3.1.2. Synthesis of 16-(α-alkoxy-alkane)-17-hydrazino-estra-1(10),2,4-trien[17,16-c]-3-ol and their *N*-substituted derivatives (**3a–l**)

"A mixture of **2a,b** (14 mmol) and hydrazine derivatives, namely, hydrazine hydrate, methyl hydrazine or phenyl hydrazine (8 mmol) in absolute methanol or ethanol (30 mL) was refluxed for 5–10 h. The solvent was concentrated under reduced pressure, the formed precipitate was filtered off, washed with water, dried and crystallized from ethanol to give the corresponding derivatives **3a–l**, respectively".

#### *16-(*α*-Methoxy-methane)-17-hydrazino-estra-1(10),2,4-trien-[17,16-c]-3-ol* (**3a**)

Yield 80%, mp 199–201 ◦C, [α] 25 *<sup>D</sup>* = + 81 (c 1, MeOH). IR (KBr, cm–1): 3444–3377 (NH, NH2), 3345 (OH), 2951 (CH, aliphatic), 1638 (C=C). 1H NMR: (600 MHz, CDCl3): δ ppm 0.61 (1H, m, H-8β), 0.90 (3H, s, CH3-18), 1.00 (1H, m, H-11β), 1.11 (1H, m, H-7α), 1.14 (1H, m, H-12α), 1.26 (1H, m, H-14 α), 1.39 (1H, m, H-15β), 1.58 (1H, m, H-15α), 1.68 (1H, m, H-7β), 2.01(1H, m, H-9α), 1.84 (1H, m, CH, 16-Hα), 2.11 (1H, m, H-11α), 2.49 (1H, m, H-12β), 2.61 (1H, m, H-6α), 2.66 (1H, m, H-6β), 2.83 (1H, d, H-17), 3.48 (3H, s, OCH3), 4.65 (2H, m, NH2, disappeared with D2O), 4.80 (2H, d, CH2-O), 4.87 (1H, s, OH, disappeared with D2O), 5.70 (1H, dd, H-2), 6.61 (1H, d, H-4), 7.10 (1H, d, H-1), 7.70 (1H, br.s, NH, disappeared with D2O). 13C NMR: (150 MHz, CDCl3): δ 13.58, 21.76, 25.57, 26.76, 28.00, 29.78, 36.38, 38.65, 43.89, 48.40, 50.45, 59.45, 69.11, 112.37, 115.56, 126.53, 132.81, 138.46, 153.84, 167.02 (20 C). MS (EI): *m*/*z* 330 (90%) [M<sup>+</sup>]. Anal. Calcd for C20H30N2O2 (330.46): Calcd C, 72.69; H, 9.15; N, 8.48. Found C, 72.60; H, 9.10; N, 8.40.

#### *16-(*α*-Methoxy-ethane)-17-hydrazino-estra-1(10),2,4-trien[17,16-c]-3-ol* (**3b**)

Yield 57%, mp. 308–310 ◦C, [α] 25 *<sup>D</sup>* = + 112 (c 1, MeOH). IR (KBr, cm–1): 3448-3377 (NH, NH2), 3348 (OH), 2959 (CH, aliphatic), 1634 (C=C). 1H NMR: (600 MHz, CDCl3): δ ppm 0.59 (1H, m, H-8β), 0.91 (3H, s, CH3-18), 1.01 (1H, m, H-11β), 1.12 (1H, m, H-7α), 1.14 (1H, m, H-12 α), 1.26 (1H, m, H-14 α), 1.39 (1H, m, H -15 β), 1.58 (1H, m, H-15α), 1.42 (3H, m, CH3,C-16 ethane), 1.69 (1H, m, H-7β), 2.02(1H, m, H-9α), 1.83 (1H, m, CH, H-16α), 2.11 (1H, m, H-11α), 2.49 (1H, m, H-12β), 2.61 (1H, m, H-6α), 2.66 (1H, m, H-6β), 2.83 (1H, d, H-17), 3.48 (3H, s, OCH3), 4.65 (2H, s, NH2, disappeared with D2O), 4.84 (1H, d, CH-O), 4.99 (1H, s, OH, disappeared with D2O), 5.72 (1H, dd, H-2), 6.61 (1H, d, H-4), 7.11 (1H, d, H-1), 7.78 (1H, bs, NH, disappeared with D2O). 13C NMR: (150 MHz, CDCl3): δ 13.58, 21.77, 22.22, 25.57, 26.47, 28.00, 29.78, 36.68, 38.47, 43.89, 48.69, 50.45, 59.45, 69.11, 112.37, 115.56, 126.55, 132.48, 138.46, 153.54, 167.76 (21 C). MS (EI): *m*/*z* 344 (11%) [M<sup>+</sup>]. Anal. Calcd for C21H32N2O2 (344.49): Calcd C, 73.22; H, 9.36; N, 8.13. Found C, 73.18; H, 9.30; N, 8.10.

*16-(*α*-Ethoxy-methane)-17-hydrazino-estra-1(10),2,4-trien-[17,16-c]-3-ol* (**3c**)

Yield 56%, mp 219–221 ◦C, [α] 25 *<sup>D</sup>* = + 77 (c 1, MeOH). IR (KBr, cm–1): 3445–3376 (NH, NH2), 3346 (OH), 2951 (CH, aliphatic), 1638 (C=C). 1H NMR: (600 MHz, CDCl3): δ ppm 0.61 (1H, m, H-8β), 0.90 (3H, s, CH3-18), 1.00 (1H, m, H-11β), 1.11 (1H, m, H-7α), 1.15 (1H, m, H-12α), 1.21 (3H, m, CH3, ethoxyl), 1.25 (1H, m, H-14α), 1.36 (1H, m, H-15β), 1.57 (1H, m, H-15α), 1.68 (1H, m, H-7β), 2.01 (1H, m, H-9α), 1.84 (1H, m, CH, H-16α), 2.10 (1H, m, H-11α), 2.50 (1H, m, H-12β), 2.60 (1H, m, H-6α), 2.70 (1H, m, H-6β), 6.61 (1H, d, H-4), 2.80 (1H, d, H-17), 3.50 (2H, m, OCH2), 4.70 (2H, m, NH2, disappeared with D2O), 4.80 (2H, d, CH-O), 4.87 (1H, s, OH, disappeared with D2O), 5.70 (1H, dd, H-2), 7.10 (1H, d, H-1), 7.70 (1H, br.s, NH, disappeared with D2O). 13C NMR: (150 MHz, CDCl3): δ 13.65, 18.74, 21.57, 25.17, 26.38, 28.04, 29.64, 36.54, 38.15, 44.11, 48.44, 50.54, 68.45, 69.11, 112.44, 115.67, 126.34, 131.11, 138.46, 153.84, 161.47 (21 C). MS (EI): *m*/*z* 344 (35%) [M<sup>+</sup>]. Anal. Calcd for C21H32N2O2 (344.49): Calcd C, 73.22; H, 9.36; N, 8.13. Found C, 73.16; H, 9.30; N, 8.10.

#### *16-(*α*-Ethoxy-ethane)-17-hydrazino-estra-1(10),2,4-trien-[17,16-c]-3-ol* (**3d**)

Yield 90%, mp 223–225 ◦C, [α] 25 *<sup>D</sup>* = + 95 (c 1, MeOH). IR (KBr, cm–1): 3440-3370 (NH, NH2), 3350 (OH), 2951 (CH, aliphatic), 1631 (C=C). 1H NMR: (600 MHz, CDCl3): δ ppm 0.57 (1H, m, H-8β), 0.90 (3H, s, CH3-18), 1.00 (1H, m, H-11β), 1.10 (1H, m, H-7α), 1.15 (1H, m, H-12α), 1.20 (3H, m, CH3, ethoxyl), 1.24 (1H, m, H-14α), 1.36 (1H, m, H-15β), 1.45 (3H, m, CH3,C-16 ethane), 1.59 (1H, m, H-15α), 1.71 (1H, m, H-7β), 1.96 (1H, m, H-9α), 1.82 (1H, m, CH, H-16α), 2.10 (1H, m, H-11α), 2.50 (1H, m, H-12β), 2.62 (1H, m, H-6α), 2.67 (1H, m, H-6β), 2.86 (1H, d, H-17), 3.48 (2H, m, OCH2), 4.64 (2H, s, NH2, disappeared with D2O), 4.83 (1H, d, CH-O), 4.88 (1H, s, OH, disappeared with D2O), 5.74 (1H, dd, H-2), 6.65 (1H, d, H-4), 7.12 (1H, d, H-1), 7.78 (1H, br.s, NH, disappeared with D2O). 13C NMR: (150 MHz, CDCl3): δ 13.55, 18.79, 22.07, 22.28, 25.47, 26.52, 27.77, 28.09, 36.68, 37.97, 44.09, 48.11, 51.41, 68.43, 69.13, 112.56, 115.57, 126.37, 133.18, 137.87, 155.56, 162.74 (22 C). MS (EI): *m*/*z* 358 (45%) [M+]. Anal. Calcd for C22H34N2O2 (358.51): Calcd C, 73.70; H, 9.56; N, 7.81. Found C, 73.64; H, 9.52; N, 7.75.

#### *16-(*α*-Methoxy-methane)-17-[N-methyl-hydrazino]estra-1(10),2,4-trien[17,16-c]-3-ol* (**3e**)

Yield 76%, mp 222–224 ◦C, [α] 25 *<sup>D</sup>* = + 66 (c 1, MeOH). IR (KBr, cm–1): 3443–3371 (NH), 3344 (OH), 2953 (CH, aliphatic), 1634 (C=C). 1H NMR: (600 MHz, CDCl3): δ ppm 0.61 (1H, m, H-8β), 0.92 (3H, s, CH3-18), 1.03 (1H, m, H-11β), 1.10 (1H, m, H-7α), 1.14 (1H, m, H-12α), 1.25 (1H, m, H-14α), 1.37 (1H, m, H-15β), 1.57 (1H, m, H-15α), 1.66 (1H, m, H-7β), 2.00 (1H, m, H-9α), 1.84 (1H, m, CH, H-16α), 2.13 (1H, m, H-11α), 2.32 (3H, s, NCH3), 2.48 (1H, m, H-12β), 2.63 (1H, m, H-6α), 2.68 (1H, m, H-6β), 2.84 (1H, d, H-17), 3.49 (3H, s, OCH3), 4.86 (2H, d, CH-O), 4.87 (1H, s, OH, disappeared with D2O), 5.72 (1H, dd, H-2), 6.63 (1H, d, H-4), 7.11 (1H, d, H-1), 7.76 (1H, brs, NH, disappeared with D2O), 7.99 (1H, bs, NH, disappeared with D2O). 13C NMR: (150 MHz, CDCl3): δ 13.53, 21.44, 25.67, 26.36, 28.03, 29.23, 36.33, 38.43, 43.83, 47.47, 48.43, 50.34, 59.35, 69.31, 112.36, 115.67, 126.52, 132.32, 138.21, 153.45, 162.32 (21 C). MS (EI): *m*/*z* 344 (45%) [M<sup>+</sup>]. Anal. Calcd for C21H32N2O2 (344.50): Calcd C, 73.22; H, 9.36; N, 8.13. Found C, 73.15; H, 9.31; N, 8.09.

#### *16-(*α*-Methoxy-ethane)-17-[N-methyl-hydrazino]-estra-1(10),2,4-trien-[17,16-c]-3-ol* (**3f**)

Yield 57%, mp 310–312 ◦C, [α] 25 *<sup>D</sup>* = + 112 (c 1, MeOH). IR (KBr, cm–1): 3443–3374 (NH), 3344 (OH), 2955 (CH, aliphatic), 1628 (C=C). 1H NMR: (600 MHz, CDCl3): δ ppm 0.58 (1H, m, H-8β), 0.90 (3H, s, CH3-18), 1.00 (1H, m, H-11β), 1.10 (1H, m, H-7α), 1.14 (1H, m, H-12α), 1.26 (1H, m, H-14 α), 1.39 (1H, m, H-15 β), 1.60 (1H, m, H-15α), 1.44 (3H, m, CH3, C-16 ethane), 1.70 (1H, m, H-7β), 2.00 (1H, m, H-9α), 1.82 (1H, m, CH, H-16α), 2.11 (1H, m, H-11α), 2.36 (3H, s, NCH3), 2.51 (1H, m, H-12β), 2.62 (1H, m, H-6α), 2.65 (1H, m, H-6β), 2.85 (1H, d, H-17), 3.44 (3H, s, OCH3), 4.83 (1H, d, CH-O), 4.99 (1H, s, OH, disappeared with D2O), 5.74 (1H, dd, H-2), 6.65 (1H, d, H-4), 7.13 (1H, d, H-1), 7.73 (1H, br.s, NH, disappeared with D2O), 7.99 (1H, m, NH, disappeared with D2O). 13C NMR: (150 MHz, CDCl3): δ 13.78, 21.54, 22.27, 25.97, 26.69, 28.03, 29.43, 36.45, 38.54, 43.68, 47.88, 48.57, 50.86, 59.46, 69.15, 112.54, 115.69, 126.46, 132.90, 138.75, 153.45, 163.60 (22 C). MS (EI): *m*/*z* 358 (100%) [M+]. Anal. Calcd for C22H34N2O2 (358.50): Calcd C, 73.70; H, 9.56; N, 7.81. Found C, 73.60; H, 9.49; N, 7.75.

#### *16-(*α*-Ethoxy-methane)-17-[N-methyl-hydrazino]-estra-1(10),2,4-trien-[17,16-c]-3-ol* (**3g**)

Yield 90%, mp 286–288 ◦C, [α] 25 *<sup>D</sup>* = + 121 (c 1, MeOH). IR (KBr, cm–1): 3441–3379 (NH), 3348 (OH), 2957 (CH, aliphatic), 1636 (C=C). 1H NMR: (600 MHz, CDCl3): δ ppm 0.61 (1H, m, H-8β), 0.91 (3H, s, CH3-18), 1.01 (1H, m, H-11β), 1.13 (1H, m, H-7α), 1.15 (1H, m, H-12α), 1.22 (3H, m, CH3, ethoxyl), 1.25 (1H, m, H-14α), 1.37 (1H, m, H-15β), 1.58 (1H, m, H-15α), 1.69 (1H, m, H-7β), 2.00 (1H, m, H-9 α), 1.81 (1H, m, CH, H-16α), 2.12 (1H, m, H-11α), 2.33 (3H, s, NCH3), 2.54 (1H, m, H-12β), 2.65 (1H, m, H-6α), 2.76 (1H, m, H-6β), 2.87 (1H, d, H-17), 3.50 (2H, m, OCH2), 4.85 (2H, d, CH2-O), 4.98 (1H, s, OH, disappeared with D2O), 5.74 (1H, dd, H-2), 6.65 (1H, d, H-4), 7.10 (1H, d, H-1), 7.76 (1H, br.s, NH, disappeared with D2O), 7.95 (1H, m, NH, disappeared with D2O). 13C NMR: (150 MHz, CDCl3): δ 13.35, 18.45, 21.43, 25.78, 26.80, 28.14, 29.88, 36.47, 38.90, 44.41, 47.37, 48.34, 50.54, 68.66, 69.67, 113.34, 115.55, 125.54, 131.53, 137.99, 154.44, 162.27 (22 C). MS (EI): *m*/*z* 358 (100%) [M+]. Anal. Calcd for C22H34N2O2 (358.50): Calcd C, 73.70; H, 9.56; N, 7.81. Found C, 73.62; H, 9.50; N, 7.74.

#### *16-(*α*-Ethoxy-ethane)-17-[N-methyl-hydrazino]-estra-1(10),2,4-trien-[17,16-c]-3-ol* (**3h**)

Yield 78%, mp 280–282 ◦C, [α] 25 *<sup>D</sup>* = + 135 (c 1, MeOH). IR (KBr, cm–1): 3441–3371 (NH), 3351 (OH), 2951 (CH, aliphatic), 1631 (C=C). 1H NMR: (600 MHz, CDCl3): δ ppm 0.60 (1H, m, H-8β), 0.93 (3H, s, CH3-18), 1.04 (1H, m, H-11β), 1.11 (1H, m, H-7α), 1.16 (1H, m, H-12α), 1.19 (3H, m, CH3,ethoxyl), 1.23 (1H, m, H-14α), 1.37 (1H, m, H-15β), 1.46 (3H, m, CH3,C-16 ethane), 1.61 (1H, m, H-15α), 1.72 (1H, m, H-7β), 1.82 (1H, m, CH, H-16α), 1.93 (1H, m, H-9α), 2.10 (1H, m, H-11α), 2.50 (1H, m, H-12β), 2.34 (3H, s, NCH3), 2.63 (1H, m, H-6α), 2.62 (1H, m, H-6β), 2.85 (1H, d, H-17), 3.43 (2H, m, OCH2), 4.64 (1H, s, OH, disappeared with D2O), 4.83 (1H, d, CH-O), 5.72 (1H, dd, H-2), 6.64 (1H, d, H-4), 7.10 (1H, d, H-1), 7.75 (1H, br.s, NH, disappeared with D2O), 7.88 (1H, s, NH, disappeared with D2O). 13C NMR: (150 MHz, CDCl3): δ 13.53, 18.67, 22.43, 22.90, 25.67, 26.78, 27.45, 28.29, 36.89, 37.65, 44.65, 47.35, 48.68, 51.90, 68.78, 69.13, 112.56, 115.56, 126.34, 133.55, 137.43, 155.34, 163.34 (23 C). MS (EI): *m*/*z* 372 (100%) [M<sup>+</sup>]. Anal. Calcd for C23H36N2O2 (372.54): Calcd C, 74.15; H, 9.74; N, 7.52. Found C, 74.05; H, 9.68; N, 7.48.

#### *16-(*α*-Methoxy-methane)-17-[N-phenyl-hydrazino]-estra-1(10),2,4-trien-[17,16-c]-3-ol* (**3i**)

Yield 67%, mp 312–314 ◦C, [α] 25 *<sup>D</sup>* = + 95 (c 1, MeOH). IR (KBr, cm–1): 3430–3367 (NH), 3345 (OH), 3068 (CH, aromatic), 2951 (CH, aliphatic), 1638 (C=C). 1H NMR: (600 MHz, CDCl3): δ ppm 0.61(1H, m, H-8β), 0.91 (3H, s, CH3-18), 1.00 (1H, m, H-11β), 1.12 (1H, m, H-7α), 1.15 (1H, m, H-12α), 1.28 (1H, m, H-14α), 1.40 (1H, m, H-15β), 1.60 (1H, m, H-15α), 1.68 (1H, m, H-7β), 2.02 (1H, m, H-9α), 1.86 (1H, m, CH, H-16α), 2.12 (1H, m, H-11α), 2.49 (1H, m, H-12 β), 2.59 (1H, m, H-6α), 2.65 (1H, m, H-6β), 2.84 (1H, d, H-17), 3.47 (3H, s, OCH3), 4.80 (2H, d, CH-O), 4.87 (1H, s, OH, disappeared with D2O), 5.70 (1H, dd, H-2), 6.61 (1H, d, H-4), 7.10 (1H, d, H-1), 7.28-7.48 (5H, m, Ar-H), 7.76 (1H, br.s, NH, disappeared with D2O), 7.99 (1H, s, NH, disappeared with D2O). 13C NMR: (150 MHz, CDCl3): δ 13.45, 21.76, 25.76, 26.67, 28.45, 29.66, 36.43, 38.65, 43.45, 48.40, 50.87, 59.45, 69.67, 112.43, 112.76, 115.66, 119.11, 126.68, 129.45, 132.67, 138.53, 152.55, 153.79, 161.02 (26 C). MS (EI): *m*/*z* 406 (77%) [M<sup>+</sup>]. Anal. Calcd for C26H34N2O2 (406.56): Calcd C, 76.81; H, 8.43; N, 6.89. Found C, 76.75; H, 8.40; N, 6.80.

#### *16-(*α*-Methoxy-ethane)-17-[N-phenyl-hydrazino]-estra-1(10),2,4-trien-[17,16-c]-3-ol* (**3j**)

Yield 66%, mp 229–231 ◦C, [α] 25 *<sup>D</sup>* = + 147 (c 1, MeOH). IR (KBr, cm–1): 3433–3363 (NH), 3346 (OH), 3066 (CH, aromatic), 2955 (CH, aliphatic), 1635 (C=C). 1H NMR: (600 MHz, CDCl3): δ ppm 0.62 (1H, m, H-8β), 0.92 (3H, s, CH3-18), 1.02 (1H, m, H-11β), 1.13 (1H, m, H-7α), 1.18 (1H, m, H-12α), 1.26 (1H, m, H-14α), 1.39 (1H, m, H-15β), 1.42 (3H, m, CH3, C-16 ethane), 1.58 (1H, m, H-15α), 1.69 (1H, m, H-7β), 1.83 (1H, m, CH, H-16α), 2.03 (1H, m, H-9α), 2.10 (1H, m, H-11α), 2.52 (1H, m, H-12β), 2.62 (1H, m, H-6α), 2.65 (1H, m, H-6β), 2.85 (1H, d, H-17), 3.49 (3H, s, OCH3), 4.86 (1H, d, CH-O), 4.89 (1H, s, OH, disappeared with D2O), 5.71 (1H, dd, H-2), 6.61 (1H, d, H-4), 7.12 (1H, d, H-1), 7.27-7.49 (5H, m, Ar-H), 7.77 (1H, br.s, NH, disappeared with D2O), 7.97 (2H, s, NH, disappeared with D2O). 13C NMR: (150 MHz, CDCl3): δ 13.52, 21.78, 22.92, 25.67, 26.45, 28.10, 29.90, 36.17, 38.47, 43.80, 48.69, 50.55, 59.25, 69.11, 112.12, 112.78, 115.54, 119.17, 126.46, 129.53, 132.79, 138.78, 152.42, 153.76, 163.11 (27 C). MS (EI): *m*/*z* 420 (81%) [M<sup>+</sup>]. Anal. Calcd for C27H36N2O2 (420.58): Calcd C, 77.10; H, 8.63; N, 6.66. Found C, 77.00; H, 8.58; N, 6.60.

#### *16-(*α*-Ethoxy-methane)-17-[N-phenyl-hydrazino]-estra-1(10),2,4-trien-[17,16-c]-3-ol* (**3k**)

Yield 60%, mp 338–340 ◦C, [α] 25 *<sup>D</sup>* = + 155 (c 1, MeOH). IR (KBr, cm–1): 3431–3362 (NH), 3343 (OH), 3064 (CH, aromatic), 2956 (CH, aliphatic), 1637 (C=C). 1H NMR: (600 MHz, CDCl3): δ ppm 0.63 (1H, m, H-8β), 0.90 (3H, s, CH3-18), 1.01 (1H, m, H-11β), 1.11 (1H, m, H-7α), 1.16 (1H, m, H-12α), 1.21 (3H, m, CH3, ethoxyl), 1.24 (1H, m, H-14α), 1.36 (1H, m, H-15β), 1.59 (1H, m, H-15α), 1.68 (1H, m, H-7β), 2.00 (1H, m, H-9α), 1.84 (1H, m, CH, H-16α), 2.11 (1H, m, H-11α), 2.50 (1H, m, H-12β), 2.61 (1H, m, H-6α), 2.70 (1H, m, H-6β), 6. disappeared (1H, d, H-4), 2.80 (1H, d, H-17), 3.53 (2H, m, OCH2), 4.80 (2H, d, CH-O), 4.84 (1H, s, OH, exchangeable with D2O), 5.70 (1H, dd, H-2), 7.15 (1H, d, H-1), 7.26-7.47 (5H, m, Ar-H), 7.76 (1H, br.s, NH, disappeared with D2O), 7.98 (1H, m, NH, disappeared with D2O). 13C NMR: (150 MHz, CDCl3): δ 13.64, 18.74, 21.57, 25.17, 26.30, 28.05, 29.64, 36.54, 38.15, 44.18, 48.46, 50.55, 68.43, 69.13, 112.49, 112.72, 115.60, 119.11, 126.34, 129.50, 131.11, 138.49, 152.40, 153.84, 162.47 (27 C). MS (EI): *m*/*z* 420 (88%) [M<sup>+</sup>]. Anal. Calcd for C27H36N2O2 (420.58): Calcd C, 77.10; H, 8.63; N, 6.66. Found C, 77.02; H, 8.57; N, 6.61.

#### *16-(*α*-Ethoxy-ethane)-17-[N-phenyl-hydrazino]-estra-1(10),2,4-trien-[17,16-c]-3-ol* (**3l**)

Yield 96%, mp 288–300 ◦C, [α] 25 *<sup>D</sup>* = + 95 (c 1, MeOH). IR (KBr, cm–1): 3431–3367 (NH), 3343 (OH), 3068 (CH, aromatic), 2951 (CH, aliphatic), 1631 (C=C). 1H NMR: (600 MHz, CDCl3): δ ppm 0.58 (1H, m, H-8β), 0.91 (3H, s, CH3-18), 1.00 (1H, m, H-11β), 1.10 (1H, m, H-7α), 1.14 (1H, m, H-12α), 1.21 (3H, m, CH3, ethoxyl), 1.24 (1H, m, H-14α), 1.36 (1H, m, H-15β), 1.44 (3H, m, CH3, C-16 ethane), 1.60 (1H, m, H-15α), 1.71 (1H, m, H-7β), 1.97 (1H, m, H-9 α), 1.83 (1H, m, CH, H-16α), 2.11 (1H, m, H-11α), 2.51 (1H, m, H-12β), 2.62 (1H, m, H-6α), 2.67 (1H, m, H-6 β), 2.88 (1H, d, H-17), 3.49 (2H, m, OCH2), 4.84 (1H, d, CH-O), 4.87 (1H, s, OH, disappeared with D2O), 5.73 (1H, dd, H-2), 6.62 (1H, d, H-4), 7.13 (1H, d, H-1), 7.23-7.47 (5H, m, Ar-H), 7.77 (1H, br.s, NH, disappeared with D2O), 7.87 (1H, m, NH, disappeared with D2O). 13C NMR: (150 MHz, CDCl3): δ 13.56, 18.77, 22.27, 22.32, 25.76, 26.78, 27.34, 28.29, 36.54, 37.90, 44.21, 48.23, 51.32, 68.34, 69.56, 112.67, 112.77, 115.56, 119.10, 126.45, 129.50, 133.47, 137.23, 152.55, 155.51, 163.73 (28 C). MS (EI): *m*/*z* 434 (93%) [M<sup>+</sup>]. Anal. Calcd for C28H38N2O2 (434.61): Calcd C, 77.38; H, 8.81; N, 6.45. Found C, 77.30; H, 8.75; N, 6.40.

#### 3.1.3. Synthesis of pyrazoline-3-ol derivatives (**4a–d**)

A mixture of **2a,b** (4 mmol) and hydrazine derivatives, namely, methy hydrazine or phenyl hydrazine (16 mmol) in dioxane (25 mL) was refluxed for 5 h. The mixture was evaporated under vacuum, the residue formed was triturated with water, filtered off, washed with water, dried and crystallized from MeOH to give derivatives **4a–d**, respectively.

#### *1'-Methyl-1'H-estra-1(10),2,4-trien-[17,16-c]pyrazoline-3-ol* (**4a**)

Yield 96%, mp 117–119 ◦C, [α] 25 *<sup>D</sup>* = +109 (c 1, MeOH). IR (KBr, cm–1): 3351 (OH), 2946 (CH, aliphatic), 1627 (C=C), 1610 (C=N). 1H NMR: (600 MHz, CDCl3): δ ppm 0.61 (1H, m, H-8β), 0.94 (3H, s, CH3-18), 1.08 (1H, m, H-11β), 1.13 (1H, m, H-7α), 1.18 (1H, m, H-12α), 1.27 (1H, m, H-14α), 1.41 (1H, m, H-15β), 1.66 (1H, m, H-15α), 1.74 (1H, m, H-7β), 1.84 (1H, m, CH, H-16α), 2.01 (1H, m, H-9α), 2.10 (3H, s, NCH3), 2.22 (1H, m, H-11α), 2.42 (1H, m, H-12β), 2.52 (1H, m, H-6α), 2.66 (1H, m, H-6β), 2.84 (2H, d, pyrazoline-5 ), 4.95 (1H, s, OH, disappeared with D2O), 5.75 (1H, dd, H-2), 6.65 (1H, d, H-4), 7.15 (1H, d, H-1). 13C NMR: (150 MHz, CDCl3): δ 14.60, 22.56, 25.35, 26.46, 28.77, 29.56, 36.68, 38.68, 43.34, 45.36, 48.21, 50.24, 64.45, 112.46, 115.57, 126.44, 132.56, 138.45, 152.67, 156.46 (20 C). MS (EI): *m*/*z* 310 (100%) [M<sup>+</sup>]. Anal. Calcd for C20H26N2O (310.43): Calcd C, 77.38; H, 8.44; N, 9.02. Found C, 77.29; H, 8.36; N, 8.98.

#### *1'-Methyl -1'H-5'-methyl-estra-1(10),2,4-trien-[17,16-c]pyrazoline-3-ol* (**4b**)

Yield 88%, mp 216–218 ◦C, [α] 25 *<sup>D</sup>* = +111 (c 1, MeOH). IR (KBr, cm–1): 3351 (OH), 2946 (CH, aliphatic), 1627 (C=C), 1617 (C=N). 1H NMR: (600 MHz, CDCl3): δ ppm 0.62 (1H, m, H-8β), 0.92 (3H, s, CH3-18), 1.02 (1H, m, H-11β), 1.05 (3H, s, CH3-5 -methyl), 1.10 (1H, m, H-7α), 1.14 (1H, m, H-12α), 1.25 (1H, m, H-14α), 1.40 (1H, m, H-15β), 1.66 (1H, m, H -15α), 1.77 (1H, m, H-7β), 1.88 (1H, m, CH, H-16α), 2.00 (1H, m, H-9α), 2.17 (3H, s, N-CH3), 2.20 (1H, m, H-11α), 2.47 (1H, m, H-12β), 2.57 (1H, m, H-6α), 2.66 (1H, m, H-6β), 2.84 (1H, m, pyrazoline-5 ), 4.88 (1H, s, OH, disappeared with D2O), 5.79 (1H, dd, H-2), 6.68 (1H, d, H-4), 7.19 (1H, d, H-1). 13C NMR: (150 MHz, CDCl3): δ 13.35, 21.80, 24.27, 25.30, 26.07, 28.20, 29.77, 36.76, 38.70, 43.67, 45.67, 48.50, 50.70, 64.45, 112.37, 115.54, 126.46, 132.40, 138.77, 153.44, 155.42 (21 C). MS (EI): *m*/*z* 324 (100%) [M<sup>+</sup>]. Anal. Calcd for C21H28N2O (324.45): Calcd C, 77.74; H, 8.70; N, 8.63. Found C, 77.66; H, 8.64; N, 8.58.

#### *1'-Phenyl-1'H-estra-1(10),2,4-trien-[17,16-c]pyrazoline-3-ol* (**4c**)

Yield 70%, mp 255–257 ◦C, [α] 25 *<sup>D</sup>* = + 128 (c 1, MeOH). IR (KBr, cm–1): 3347 (OH), 3071 and 3061 (CH, aromatic), 2931 (CH, aliphatic), 1633 (C=C), 1614 (C=N). 1H NMR: (600 MHz, CDCl3): δ ppm 0.60 (1H, m, H-8β), 0.90 (3H, s, CH3-18), 1.00 (1H, m, H-11β), 1.10 (1H, m, H-7α), 1.20 (1H, m, H-12α), 1.28 (1H, m, H-14α), 1.49 (1H, m, H-15β), 1.68 (1H, m, H-15α), 1.77 (1H, m, H-7β), 1.86 (1H, m, CH, H-16α), 2.04 (1H, m, H-9α), 2.26 (1H, m, H-11α), 2.48 (1H, m, H -12β), 2.54 (1H, m, H-6α), 2.64 (1H, m, H-6β), 2.84 (2H, d, pyrazoline-5'), 5.00 (1H, s, OH, disappeared with D2O), 5.71 (1H, dd, H-2), 6.70 (1H, d, H-4), 7.10 (1H, d, H-1), 7.28-7.40 (5H, m, Ar-H). 13C NMR: (150 MHz, CDCl3): δ 13.49, 21.24, 25.55, 26.46, 28.51, 29.67, 36.64, 38.45, 43.35, 50.73, 58.26, 64.45, 112.10, 112.48, 115.46, 119.31, 126.10, 129.20, 132.52, 138.79, 152.20, 153.13, 163.48 (25 C). MS (EI): *m*/*z* 372 (81%) [M<sup>+</sup>]. Anal. Calcd for C25H28N2O (372.50): Calcd C, 80.61; H, 7.58; N, 7.52. Found C, 80.52; H, 7.50; N, 7.48.

#### *1'-Phenyl-1'H-5'-methyl-estra-1(10),2,4-trien-[17,16-c]pyrazoline-3-ol* (**4d**)

Yield 75%, mp 214–216 ◦C, [α] 25 *<sup>D</sup>* = +127 (c 1, MeOH). IR (KBr, cm–1): 3345 (OH), 3068 (CH, aromatic), 2949 (CH, aliphatic), 1625 (C=C), 1614 (C=N). 1H NMR: (600 MHz, CDCl3): δ ppm 0.62 (1H, m, H-8β), 0.93 (3H, s, CH3-18), 1.02 (1H, m, H-11β), 1.05 (3H, s, CH3-5'-methyl), 1.12 (1H, m, H-7α), 1.17 (1H, m, H-12α), 1.27 (1H, m, H-14α), 1.41 (1H, m, H-15β), 1.71 (1H, m, H -15α), 1.77 (1H, m, H-7β), 1.87 (1H, m, CH, H-16α), 2.02 (1H, m, H-9α), 2.22 (1H, m, H-11α), 2.46 (1H, m, H -12β), 2.54 (1H, m, H-6α), 2.65 (1H, m, H-6β), 3.83 (1H, s, pyrazoline-5'), 4.91 (1H, s, OH, disappeared with D2O), 5.81 (1H, dd, H-2), 6.73 (1H, d, H-4), 7.21 (1H, d, H-1), 7.28-7.48 (5H, m, Ar-H). 13C NMR: (150 MHz, CDCl3): δ 13.81, 21.89, 24.25, 25.80, 26.35, 28.34, 29.23, 36.56, 38.67, 43.34, 48.55, 50.67, 64.44, 112.70, 115.34, 119.10, 126.76, 129.50, 132.45, 138.56, 152.40, 154.36, 163.56, 164.55 (26 C). MS (EI): *m*/*z* 386 (23%) [M+]. Anal. Calcd for C26H30N2O (386.52): Calcd C, 80.79; H, 7.82; N, 7.25. Found C, 80.70; H, 7.76; N, 7.20.

#### *3.2. Biological Screening*

#### 3.2.1. In Vitro Cytotoxic Activities

The cytotoxic activities of the synthesized derivatives against ovarian cancer cell lines (SKOV-3) were evaluated using standard MTT assay [26,27]. SKOV-3 cells cultivated on DMEM medium supplemented with 10% FBS, 100×, 1% antibiotic/antimycotic solution and 3.6 g/L NaHCO3 were used. Cells were incubated under standard laboratory conditions [28,29]. Before testing compounds, cells were trypsinized, centrifuged and prepared as per our developed protocol [30,31]. MTT assay relies on the enzymatic conversion of the MTT substrate into purple formazan by living cells mitochondrial enzymes. Shortly, 96 well plates were seeded (10,000 cells/100 μL/well), then incubated for 24 h at standard conditions. Afterwards, cells were exposed to different concentrations from prepared

compounds and incubated for another 24 h. Thereafter, MTT (10 μL, 5 mg/mL, PBS) were added to each well, and plates were incubated for 4 h. Supernatants were then replaced discarded with 200 μL of DMSO to dissolve the precipitated formazan crystals. Formazan absorbance, proportional to living cell number, was read at 550 nm using a microplate reader. Viability percentages were calculated and the corresponding IC50 values were obtained from the linear regression of the calibration curve. For comparison, doxorubicin (DOX) and resveratrol (RES) were used as positive controls. Furthermore, the effect of the newly synthesized derivatives was tested against normal fibroblast cell line to assess their toxicities.

#### 3.2.2. In Vitro Anti-Ovarian Xenograft Model

Anti-ovarian cancer xenograft animal model was developed in nude mouse using SKOV-3 cells according to McCauley et al. [32]. Treatment protocol was approved by the University of South Dakota, Institutional Animal Care and Use Committee (study protocol 50-01-05-08B). Immunodeficient (athymic nude-Foxn1nu) female mice were inoculated (1 <sup>×</sup> 106 cells/site). Upon tumor development, (av. volume ~50 mm3), 10-mice groups were implanted s.c. with constant release pellets containing tested compound (1 μM/gm) or placebo. Animal and tumor development were monitored measured every 5 days after pellet implantation, using standardized Caliper in two perpendicular diameters of the implant according to our previous work [2]. Due to large tumor volumes, (5-fold difference between largest and smallest tumor), relative tumor volumes were used for comparison. Relative tumor volume = Vtvolume at time t/V0initial volume.

#### 3.2.3. Topoisomerase II Inhibition

The inhibitory effects of the synthesized compounds against topoisomerase II were investigated using relaxation assay according to Goyeneche, et al. [33]. Topoisomerase II was purified from P388 cells, where enzyme unit corresponds to the activity required complete relaxation of 0.125 g of supercoiled pBR-322 DNA at 30 ◦C for 1 h. The assay was performed as per protocol and samples were subjected to electrophoresis (0.7% agarose gels, TBE buffer. Ethidium bromide-stained DNA was examined under UV light.

#### 3.2.4. In Vitro Kinase Inhibition

#### Protein Expression and Purification

BRAF wild-type and V600E mutant kinase domains were expressed and purified according to Nakamura et al. [34]. Shortly, Sf 9 cells infected with BRAF kinase domain containing baculovirus were resuspended, sonicated, and then cleared using ultracentrifugation. Afterwards, the cleared lysate with mixed with equilibrated Talon resin, and the resin then washed with 10 column volumes of wash buffer and then eluted with buffer (25 mM Tris, pH 7.0, 250 mM NaCl, 160 mM imidazole, 10% glycerol). The eluant was then diluted, concentrated using Superdex 200 gel filtration column, purified as previously described and finally stored at –80 ◦C until use.

#### In Vitro ELISA-Based Kinase Assay

"This method was adopted from Nakamura et al. [34] and Qin et al. [35]. The previously prepared GST MEK-His protein was diluted in TTBS buffer (100 μL, 50 μg/mL), and then bound to 96-plate wells coated with glutathione (Pierce Biotechnology). Two DMSO-dilutions of each compound (1 μL) were added to 50 μL (50mM HEPES, pH7.0, 0.7 pmol of BRAFV600E kinase). Tubes were incubated at ambient temperature for 1 h, then added to GST-MEK-His-plate wells. Afterwards, 50 μL of phosphorylation buffer were added to start kinase reaction (37 ◦C/30 min/shaking). Reaction was stopped, then substrate was added, and the signal was recorded. The high throughput inhibitor screening was carried out according to standard assay conditions at different concentrations, to generate a sigmoidal dose response curve, a four-parameter logistic model using GraphPad Prism, for BRAF proteins, which was used to obtain corresponding IC50 values".

#### 3.2.5. Statistical Analysis

"Measurements were carried out in thrice and data are represented as means ± SEM. Significance was calculated by Student's t-test using SPSS software (SPSS Inc., Paris, France). \* *p* <0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001. IC50 values were calculated using GraphPad Prism software, GraphPad Software, San Diego, CA, USA [36]".

#### **4. Conclusions**

During the current investigation, we synthesized a series of 17-hydrazino- and 17,16-c-pyrazoline estrone derivatives. Prepared compounds exhibited potential in vitro and in vivo anti-ovarian cancer activity against SKOV-3 cells. Compound **3a** was found to be the most potent with an IC50 value of 4.23 ± 0.12 nM. Additionally, in vivo xenograft ovarian cancer model showed that Cpd. **3a** was able to reduce tumor growth by 93.61 ± 0.7% after 40 days of treatment. Furthermore, the newly synthesized compounds were able to interfere with tumor proliferation through inhibiting the activity of topoisomerase II and V600EBRAF, where the obtained IC50 values for both enzymes were 3.45 <sup>±</sup> 0.13 nM and 0.041 ± 0.0016 μM, respectively.

**Author Contributions:** M.E.-N., A.E.-G.E.A., A.A.F. performed most of the experiments; A.E.-G.E.A., M.A.A.-O., A.A.F. analyzed the data; E.A.E. and M.M.A. the contributed to the anticancer activity assays; All authors read and approved the final manuscript.

**Funding:** The authors are grateful to the Deanship of Scientific Research, king Saud University for funding through Vice Deanship of Scientific Research Chairs.

**Acknowledgments:** The authors are grateful to the Deanship of Scientific Research, king Saud University for funding through Vice Deanship of Scientific Research Chairs.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


carboxamides on D-secoestrone scaffolds. *J. Enzyme Inhib. Med. Chem.* **2016**, *31*, 574–579. [CrossRef] [PubMed]


**Sample Availability:** Samples of the compounds are available from the authors.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Synthesis, Biological Evaluation, and In Silico Studies of Novel Aminated Xanthones as Potential p53-Activating Agents**

**Agostinho Lemos 1, Ana Sara Gomes 2, Joana B. Loureiro 2, Pedro Brandão 1, Andreia Palmeira 1,3, Madalena M. M. Pinto 1,3, Lucília Saraiva 2,\* and Maria Emília Sousa 1,3,\***


Academic Editor: Qiao-Hong Chen

Received: 7 May 2019; Accepted: 21 May 2019; Published: 22 May 2019

**Abstract:** Xanthone scaffold has been regarded as an attractive chemical tool in the search for bioactive molecules with antitumor activity, and in particular two xanthone derivatives, 12-hydroxy-2,2-dimethyl-3,4-dihydro-2*H*,6*H*-pyrano [3,2-*b*]xanthen-6-one (**4**) and 3,4-dimethoxy-9 oxo-9*H*-xanthene-1-carbaldehyde (**5**), were described as a murine double minute 2 (MDM2)-p53 inhibitor and a TAp73 activator, respectively. The xanthone **5** was used as a starting point for the construction of a library of 3,4-dioxygenated xanthones bearing chemical moieties of described MDM2-p53 inhibitors. Eleven aminated xanthones were successfully synthesized and initially screened for their ability to disrupt the MDM2-p53 interaction using a yeast cell-based assay. With this approach, xanthone **37** was identified as a putative p53-activating agent through inhibition of interaction with MDM2. Xanthone **37** inhibited the growth of human colon adenocarcinoma HCT116 cell lines in a p53-dependent manner. The growth inhibitory effect of xanthone **37** was associated with the induction of G1-phase cell cycle arrest and increased protein expression levels of p53 transcriptional targets. These results demonstrated the potential usefulness of coupling amine-containing structural motifs of known MDM2-p53 disruptors into a 3,4-dioxygenated xanthone scaffold in the design of novel and potent p53 activators with antitumor activity and favorable drug-like properties. Moreover, in silico docking studies were performed in order to predict the binding poses and residues involved in the potential MDM2-p53 interaction.

**Keywords:** antitumor activity; computational docking; MDM2-p53 interaction; xanthones; yeast-based assays

#### **1. Introduction**

The tumor suppressor p53 acts as a transcription factor, triggering the expression of multiple downstream target genes that play a pivotal role in the regulation of cell cycle arrest, apoptosis, senescence, and DNA repair [1–6]. Upon cellular stress stimuli, the activation of the p53 pathway may compromise the tumorigenesis, circumventing the proliferation of damaged cells with oncogenic potential. Inactivation of the p53 function represents one of the most common events in human cancers [7]. In approximately half of all human cancer cells, the p53 inactivation is caused by mutations within the p53 gene (*TP53*) or by post-translational modifications of its gene product [8]. In those tumors retaining the wild type (wt) p53 function, the tumor suppressor activity of p53 can be abolished by multiple mechanisms [9], mainly by direct interaction between p53 and its primary endogenous inhibitor, the murine double minute 2 (MDM2) protein [10,11]. The oncoprotein MDM2 physically interacts with the amino-terminal domain of p53 and negatively regulates its activity by blocking p53-mediated transactivation [12,13], by inducing the export of p53 from nucleus to the cytoplasm [14], and by promoting E3 ubiquitin ligase-mediated p53 degradation via the proteasomal pathway [15,16]. Targeting the MDM2-p53 interaction with small molecules has been regarded an attractive approach for the reactivation of the p53 pathway in wt p53-expressing tumors [17,18]. Since the seminal work of Vassilev et al. reporting the discovery of nutlin-3A (**1**, Figure 1) as the first inhibitor of MDM2-p53 interaction [19], an impressive number of MDM2-p53 disruptors from a huge diversity of chemical families have been identified in recent years, entering some of them into clinical trials [20,21].

**Figure 1.** Chemical structures of nutlin-3A (**1**), gambogic acid (**2**), α-mangostin (**3**), 12-hydroxy-2,2 dimethyl-3,4-dihydro-2*H*,6*H*-pyrano[3,2-*b*]xanthen-6-one (**4**), and 3,4-dimethoxy-9-oxo-9*H*-xanthene-1-carbaldehyde (**5**).

Xanthones represent a privileged class of compounds with a multiplicity of biological activities, in particular with antitumor activity against several human tumor cell lines [22–36]. Intriguingly, our research group has demonstrated that the antitumor activity of some derivatives can be correlated to the disruption of the MDM2-p53 complex and the activation of a p53-dependent pathway [30,31]. In fact, implementing a yeast-based assay, the naturally occurring xanthones gambogic acid (**2**, Figure 1) and α-mangostin (**3**, Figure 1) were found to revert the inhibitory effect of MDM2 on p53-induced growth inhibition and on p53 transcriptional activity [30]. The synthetic xanthone 12-hydroxy-2,2-dimethyl-3,4-dihydro-2*H*,6*H*-pyrano [3,2-*b*]xanthen-6-one (**4**, Figure 1) was identified as a potential MDM2-p53 disruptor, using an in silico virtual screening strategy against MDM2 protein and a yeast-based assay [31]. Its molecular mechanism was corroborated in MDM2-overexpressing human tumor cell lines with wt p53. In fact, xanthone **4** selectively upregulated the p53 transcriptional activity, increasing the protein expression levels of p53-target genes in HCT116 human colon adenocarcinoma cells [31]. Nevertheless, xanthone **4** is highly hydrophobic and poorly water soluble, which could limit its in vivo use. Interestingly, Chen and co-workers explored the antitumor activity of xanthone **4** either in combination or hybridized with 5,6-dimethylxanthone-4-acetic acid (DMXAA) [37], a xanthone derivative with tumor-vascular disrupting activity. A very recent study reported the discovery of

3,4-dimethoxy-9-oxo-9*H*-xanthene-1-carbaldehyde (**5**, Figure 1) as a new TAp73 activator via disruption of its interaction with both MDM2 and mutant p53 [36].

Herein, based on the 3,4-oxygenated pattern of substitution of xanthones **4** and **5** (Figure 2A), and on chemical moieties derived from known MDM2-p53 disruptors, namely the molecules **1** [19], **6** [38], **7** [39], and **8** [40] (Figure 2B), we followed a molecular hybridization approach for the construction of novel 3,4-oxygenated xanthone derivatives (Figure 2D). In the present work, we opted to synthesize a library of aminated xanthones (Figure 2D) via a strategy of reductive amination of the xanthone **5** with a set of commercially available amine precursors (**9**–**19**) containing structural motifs of MDM2-p53 disruptors (Figure 2C). The introduction of amine moieties in the xanthone scaffold may contribute to a potential enhancement of drug-like properties, a critical parameter for the identification of promising drug candidates. The ability of the synthesized ligands to disrupt the MDM2-p53 interaction and to activate a p53-dependent pathway was further investigated using the previously established yeast-based assay and in human cancer cell lines. In silico docking studies were performed in order to predict the binding poses and residues involved in the potential MDM2-p53 interaction.

#### **2. Results and Discussion**

#### *2.1. Chemistry*

#### 2.1.1. Synthesis of 3,4-dimethoxy-9-oxo-9*H*-xanthene-1-carbaldehyde (**5**)

In the present work, xanthone **5** was used as a starting precursor for the synthesis of a set of aminated xanthones with a 3,4-dioxygenated pattern of substitution. Recently, the multi-step synthesis of compound **5** has been reported [36] as consisting in: (i) a Friedel-Crafts acylation of 1,2,3-trimethoxybenzene (**20**) with the commercially available reagent 2-methoxybenzoyl chloride (**21**); (ii) an intramolecular nucleophilic aromatic substitution of (2-hydroxy-3,4-dimethoxyphenyl) (2-methoxyphenyl)methanone (**22**) and consecutive regioselective demethylation of the methoxy group at position C-4 under conventional heating; (iii) a Duff formylation at position C-1 of 4-hydroxy-3 methoxy-9*H*-xanthen-9-one (**23**); and (iv) a methylation of the hydroxyl group at position C-4 of 4-hydroxy-3-methoxy-9-oxo-9*H*-xanthene-1-carbaldehyde (**24**) (Scheme 1A). The difficulty in achieving a regioselective demethylation at position C-4 necessary for Duff formylation at position C-1 demanded the planning of an optimized synthetic route more appropriate for scaling-up. We hypothesized that the implementation of an analog precursor of **20** with a suitable substituent at position C-1 might provide a useful alternative for the synthesis of **5**, avoiding the troublesome demethylation and Duff formylation processes. Herein, the commercially available 1,2,3-trimethoxy-5-methylbenzene (**25**) was selected for the Friedel-Crafts acylation with the acyl chloride **21** and the Lewis acid aluminum chloride to furnish the intermediate (2-hydroxy-3,4-dimethoxy-6-methylphenyl)(2-methoxyphenyl)methanone (**26**). Subsequent intramolecular nucleophilic aromatic substitution followed by elimination of methanol under basic conditions and microwave (MW) irradiation provided the 3,4-dimethoxy-1-methyl-9*H*-xanthen-9-one (**27**) in good yield quantity. This two-step methodology for the construction of xanthone analogues of **27** has been previously described in the literature [32,41–43]. With the application of MW methodology in the basic cyclization of **26** instead of the conventional heating employed in the synthesis of **23**, the reaction time was reduced from 48 h to 6 h. Furthermore, xanthone **27** was obtained in higher reaction yield under MW irradiation (63% yield (**27**) vs. 38% yield (**23**)). Subsequently, the Vohl-Ziegler bromination of **27**, using dibenzoyl peroxide (DBP) and *N*-bromosuccinimide (NBS) as a radical initiator and a brominating agent, respectively, afforded the 1-(dibromomethyl)-3,4-dimethoxy-9*H*-xanthen-9-one (**28**) as the major product. A solvolytic displacement of bromine atoms of the xanthone **28** was effectively attained using 1-butyl-3 methylimidazolium tetrafluoroborate and water, under conventional heating, and furnished xanthone **5** in 64% yield quantity (Scheme 1B). Of note, the two-step procedure of Vohl-Ziegler bromination and subsequent solvolytic displacement required lower reaction times (4.5 h (2 steps from **27** to **5**) vs. 54 h (2 steps from **23** to **5**) and allowed the preparation of xanthone **5** in higher reaction yields (51% yield (2 steps from **27** to **5**) vs. 28% yield (2 steps from **23** to **5**)), compared to the Duff formylation/methylation

pathway. Overall, the replacement of starting precursor **20** by the methylated derivative **25** at position C-1 of the aromatic ring was critical for the successful optimization of the preparation of xanthone **5**.

**Figure 2.** Strategy of molecular hybridization used for the construction of the library of aminated xanthones (**D**) by conjugation of the 3,4-dioxygenated substitution pattern of xanthones **4** and **5** (**A**) with the chemical moieties of known murine double minute 2 (MDM2)-p53 inhibitors (**C**), namely the molecules **1** (IC50 = 0.09 μM)**, 6** (IC50 = 2.6 μM)**, 7** (KI = 0.656 μM), and **8** (KI = 0.023 μM) (**B**).

**Scheme 1.** (**A**) Synthetic route for the preparation of 3,4-dimethoxy-9-oxo-9*H*-xanthene-1-carbaldehyde (**5**) from 1,2,3-trimethoxybenzene (**20**). (**B**) Optimized synthetic route for the preparation of the xanthone **5** using the starting precursor 1,2,3-trimethoxy-5-methylbenzene (**25**). The compound numbering used concerns the nuclear magnetic resonance (NMR) assignments. Note: [BmIm]BF4 = 1-butyl-3-methylimidazolium tetrafluoroborate; DBP = dibenzoyl peroxide; HMTA = hexamethylenetetramine; NBS = *N*-bromosuccinimide. <sup>a</sup> Isolated yields.

#### 2.1.2. Synthesis of a Library of Aminated Xanthones (**30**–**40**)

A library of aminated xanthones **30**–**40** with a 3,4-dioxygenated pattern of substitution was obtained through a one-pot reductive amination of xanthone **5** with the appropriate amine precursors **9**–**19** (Scheme 2). In one-pot reductive aminations, the selection of the reducing agent is critical for their efficiency, since a suitable agent must reduce the Schiff base intermediate selectively over the carbonylated compounds. As an attempt to investigate the best reaction conditions, a solid-supported reactant containing borohydride (MP-BH4) combined with methanol were initially employed, since it was successfully used in the reductive amination of a xanthone derivative (data not shown). When these conditions were applied to the reductive amination of **5**, the starting precursor was not completely consumed and the alcohol derivative 1-(hydroxymethyl)-3,4-dimethoxy-9*H*-xanthen-9-one (**29**) was the major product formed. The replacement of the above-mentioned conditions by the milder reducing agent sodium triacetoxyborohydride (STAB) and the solvent tetrahydrofuran allowed a total consumption of the xanthone **5**, favoring the one-pot amination/reduction pathway. Under the reaction conditions described in the Scheme 2, eleven aminated xanthones **30**–**40** were successfully synthesized in moderate to good yields (11 examples, 39–73% yields).

**Scheme 2.** Reductive amination of **5** with appropriate amine precursors **9**–**19** for the synthesis of aminated xanthones **30**–**40** with a 3,4-dioxygenated substitution pattern. The compound numbering used concerns the NMR assignments. <sup>a</sup> Isolated yields.

The structure elucidation of the xanthones **5** and **27**–**40** was established on the basis of highresolution mass spectrometry (HRMS) (Figures S1–S15) and nuclear magnetic resonance (NMR) techniques (Figures S16–S45). The 13C-NMR assignments were determined by bidimensional heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) experiments (Supplementary Materials).

#### *2.2. Biological Activity Evaluation*

2.2.1. Effect of Synthesized Xanthone Derivatives **30**–**40** on the MDM2-p53 Interaction Using A Yeast-Based Screening Assay

Our group has previously developed a yeast-based screening assay to search for potential inhibitors of the MDM2-p53 interaction [30,31,44–46]. In this yeast cell system, the expression of human wt p53 induces a growth inhibition proportional to the degree of its activity (Figure 3, p53, dimethyl sulfoxide (DMSO)). On the other hand, this p53 inhibitory effect is reversed by yeast co-expression of human MDM2 (Figure 3, MDM2-p53, DMSO). This established yeast screening assay was used to study the potential inhibitory effect of 1-20 μM of xanthone derivatives **30**–**40** on the MDM2-p53 interaction. Figure 3 represents the maximal effect achieved with xanthone derivatives **30**–**40** at 10 μM. The results showed that only the xanthone **37** caused a significant reversion of the inhibitory effect of MDM2 on p53 (77.01 ± 7.35% of reversion, *n* = 3), with an almost complete reestablishment of the p53 growth inhibitory effect (63.55 ± 4.26% vs. 69.22 ± 2.23%; Figure 3, DMSO-treated p53-expressing yeast vs. xanthone **37**-treated MDM2-p53-expressing yeast). It should be noted that xanthone **37** did not interfere with the growth of control yeast (transformed with empty vectors) or yeast expressing human p53 alone (data not shown). As such, these results indicated that the xanthone **37** behaved as a potential p53-activating agent through inhibition of interaction with MDM2, and it was, therefore, selected for further investigation in human cancer cells.

**Figure 3.** Effect of xanthones **30**–**40** on the MDM2-p53 interaction using a yeast-based assay. Effect of 10 μM xanthone derivatives **30**–**40** and nutlin-3A on the growth of yeast co-expressing p53 and MDM2 after 42 h treatment; results were plotted setting as 100% the growth of yeast transformed with the empty vector (control yeast); nutlin-3A (inhibitor of the MDM2-p53 interaction) was used as positive control. Data are mean ± standard error of the mean (SEM) of three independent experiments; mean values were statistically analyzed by one-way analysis of variance (ANOVA) with Tukey's multiple comparisons test, with significant differences from yeast co-expressing MDM2-p53 treated with DMSO. Note: \* *p* < 0.05; \*\* *p* < 0.01.

#### 2.2.2. Evaluation of the Antitumor Activity of Xanthone 37 in Human Cancer Cell Lines

Using the sulforhodamine B (SRB) assay, it was confirmed that xanthone **37** inhibited the growth of human HCT116 p53+/+ colon cancer cells, with an IC50 (concentration that causes 50% growth inhibition) value of 8.67 ± 0.59 μM (*n* = 4), and of the MDM2-overexpressing human HepG2 liver carcinoma cells, with an IC50 value of 18.95 ± 0.39 μM (*n* = 4), after 48 h treatment.

To evaluate the dependency of the antitumor activity of xanthone **37** on the p53 pathway, we next determined the impact of xanthone **37** on the colony forming ability of HCT116 p53+/+ cells and on the respective p53-knockout (HCT116 p53−/−), using a colony formation assay (Figure 4A). The results showed a significant reduction of the growth inhibitory activity of xanthone **37**, at 3–5 μM in HCT116 p53+/+ cells, demonstrating a p53-dependent antitumor effect of xanthone **37**. Accordingly, 10 and 20 μM of xanthone **37** caused a G1-phase cell cycle arrest in HCT116 p53+/+ cells, but not in HCT116 p53−/<sup>−</sup> cells (Figure 4B) after 24 h treatment. It is of note that apoptosis analysis was also investigated by Annexin-V assay; nevertheless, apoptotic events were not detected at 10 and 20 μM of xanthone **37**, after 24 and 48 h treatment. In accordance with these results, we also observed that xanthone **37** upregulated the protein expression levels of MDM2, p53, and p21, in HCT116 p53+/+ cells for 24 h treatment (Figure 4C).

**Figure 4.** Xanthone **37** inhibits the colony forming capability of HCT116 cells through induction of cell cycle arrest and in a p53-dependent manner. (**A**) Evaluation of colony forming ability in HCT116 p53+/+ and HCT116 p53−/<sup>−</sup> colon cancer cells after 11 days treatment with 3-10 μM of xanthone **37**; results were plotted setting as 100% the colonies formed after treatment with dimethyl sulfoxide (DMSO). (**B**) Effect of 10 and 20 μM of xanthone **37** on cell cycle progression of HCT116 p53+/+ and HCT116 p53−/<sup>−</sup> cells, after 24 h treatment. (**C**) Effect of 10 and 20 μM of xanthone **37** on the expression levels of MDM2, p53, and p21 in HCT116 p53+/+ cells analyzed by Western Blot after 24 h treatment. Immunoblots represent one of three independent experiments; Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. (**A**,**B**) Data are mean ± SEM of three independent experiments; values significantly different from HCT116 p53−/<sup>−</sup> cells using two-way ANOVA with Sidak's multiple comparisons test (**A**) or DMSO using Student's *t*-test (**B**) are indicated: \* *p* < 0.05; \*\* *p* < 0.01.

Altogether, these results indicated that xanthone **37** had an in vitro p53-dependent antitumor activity mediated by induction of cell cycle arrest.

#### *2.3. In Slico Studies*

It is common knowledge that the amino-terminal (residues **18**–**26**) p53 α-helical peptide interacts with a deep hydrophobic cleft within the amino-terminal domain of MDM2 [47]. The MDM2 binding site consists of a large and a small pocket that interact with Phe19/Trp23 and Leu26 of p53, respectively (Figure 5A) [48]. The crystal structure of the MDM2 complexed with the transactivation domain of p53 (Protein Data Bank (PDB) id 1YCR [49]) allowed the visual inspection of the interactions between the Phe19, Trp23, and Leu26 residues of p53 and their respective pockets. Hydrogen-bonding interactions are established between the indole group of Trp23 and carbonyl backbone of Leu54, and between the NH backbone of Phe19 and the carbamoyl group of Gln72. Additional interactions are established between p53 and Leu57, Gly58, Ile61, Met62, Val75, Val93, and His96 residues of MDM2 (Figure 5B).

**Figure 5.** (**A**) Crystallographic p53 (for simplification, only Phe19, Trp23, and Leu26 residues are represented as purple sticks) bound to the three pockets of MDM2 (solid surface). (**B**) Binding mode of crystallographic p53 (for simplification, only residues Phe19, Trp23, and Leu26, are represented as purple sticks) in the hydrophobic cleft of MDM2 (transparent surface); MDM2 amino acid residues involved in interactions with p53 are represented as green sticks. Structural data were obtained from the X-ray crystal structure of MDM2-p53 complex (Protein Data Bank (PDB) id 1YCR). MDM2 is represented as surface, where carbon, oxygen, nitrogen, and sulfur are represented in green, red, blue, and yellow, respectively. Hydrogen-bonding interactions are depicted with a dashed blue line. Other types of interactions are represented as a dashed red line.

The 1.6 Å X-ray crystal structure of the small-molecule inhibitor of MDM2-p53 interaction **1** in complex with MDM2 (PDB id 4HG7 [50]) was selected for further molecular docking studies of the aminated xanthone derivatives **30**–**40** onto MDM2 protein, using the MDM2-p53 disruptors **1**–**4** as positive controls. In this work, AutoDock Vina [51] was the software chosen to predict docking conformations, as it has been described as being the best software in predicting crystallographic MDM2-p53 inhibitor poses (root-mean-square deviation (RMSD) < 1.0 Å) by redocking tests [52]. Docking scores for the most stable binding poses of xanthones **30**–**40** on MDM2 were determined using the AutoDock Vina software [51]. The predicted docking scores for known MDM2-p53 inhibitors (positive controls **<sup>1</sup>**–**4**) ranged from <sup>−</sup>7.7 to <sup>−</sup>8.0 kcal·mol−<sup>1</sup> (Table 1). The most stable binding conformation of the synthesized xanthones **30**–**40** exhibited docking scores from −5.9 to <sup>−</sup>7.4 kcal· mol<sup>−</sup>1, therefore being in the same range of binding affinity as the positive controls (Table 1).

Amongst the tested compounds, the xanthone **37** was identified as the most active compound in the yeast-based screening assay. In addition, the predicted free energy values for the most stable binding pose of xanthone **37** were more negative than the majority of the remaining ligands. Therefore, the xanthone **37** was further analyzed in terms of docking poses and residues potentially involved in the interaction with MDM2. Figure 6A shows the most stable conformation of xanthone **37** as suggested by the docking protocol. The xanthone **37** was predicted to interact within the p53-binding site, mimicking the key hydrophobic residues of p53 when bound to MDM2 (docking score of <sup>−</sup>7.3 kcal·mol−1). Similarly to the binding mode of **1** within the p53-binding site (Figure 6B), no hydrogen-bonding interactions between the xanthone **37** and the MDM2 residues are predicted to be established. The *p*-fluorobenzylamino group of xanthone **37** occupies the Trp23 pocket and makes π-stacking interactions with Phe91 residue, amide-π interactions with the Leu57 and Gly58 residues, and CH-π interactions with the Leu54, Leu57, Gly58, Ile61, Val93, and Ile99 residues. Interestingly, the docking conformation of *p*-fluorobenzylamino group and the *p*-chlorophenyl ring of **1** are identical and superimposable (Figure 6A,B). The methoxy groups of the xanthone scaffold are oriented to the Leu26 pocket and make CH-CH interactions with the Leu54 residue. The adopted scaffold orientation of the xanthone **37** in the hydrophobic cleft where p53 is supposed to bind allows additional interactions to be formed, strengthening the binding of xanthone **37** to MDM2. For example, apart from the Gly58 residue of MDM2, the non-substituted aromatic ring of the xanthone scaffold is predicted to establish π-stacking

interactions with the Phe55 residue, and amide-π and CH-π interactions with the Gln59 residue. The establishment of additional interactions within the MDM2 protein that are not observed in the MDM2-p53 binding have been described for a considerable number of high-affinity p53-activating agents [17]. The visual inspection of the xanthone **37** also suggests that an enhancement of its binding affinity towards MDM2 protein can be achieved by molecular modifications in the xanthone scaffold of **37** in order to optimize its interaction with the amino acid residues surrounding the Phe19 pocket.


**Table 1.** Docking scores (kcal·mol<sup>−</sup>1) of xanthones **<sup>30</sup>**–**<sup>40</sup>** and positive controls **<sup>1</sup>**–**<sup>4</sup>** onto MDM2 target.

**Figure 6.** (**A**) Predicted binding pose of the xanthone **37** (purple sticks) in the binding site of MDM2. (**B**) MDM2 (transparent surface) in complex with the crystallographic nutlin-3A (**1**, purple sticks). MDM2 is represented as a transparent surface, where carbon, oxygen, nitrogen, and sulfur are represented in green, red, blue, and yellow, respectively. Small molecule-MDM2 interactions are depicted with a dashed red line. MDM2 residues involved in interactions with the ligands are represented as green sticks and labeled.

#### **3. Materials and Methods**

#### *3.1. Chemistry*

All reagents and solvents were purchased from Sigma Aldrich (Sigma-Aldrich Co. Ltd., Gillinghan, UK) and no further purification process was implemented. Solvents were evaporated using a rotary evaporator under reduced pressure, Buchi Waterchath B-480. Microwave (MW) reactions were

performed using an Ethos MicroSYNTH 1600 Microwave Labstation from Milestone (Thermo Unicam, Portugal). The internal reaction temperature was controlled by a fiber optic probe sensor. All reactions were monitored by thin-layer chromatography (TLC) carried out on precoated plates with 0.2 mm of thickness using Merck silica gel 60 (GF254) with appropriate mobile phases. Compounds **30**–**40** were easily detectable at 254 nm or 365 nm and after revelation with a solution of ninhydrin in ethanol 3 mg·mL−<sup>1</sup> (activated by heat).

Flash column chromatography using silica gel 60 (0.040–0.063 mm, Merck, Darmstadt, Germany), flash cartridge chromatography (GraceResolv®, Grace Company, Deerfield, IL, USA), and Discovery® DSC-SCX SPE cationic exchange cartridge (Grace Company, Deerfield, IL, USA) were used in the purification of the synthesized compounds. Melting points (m.p.) were measured in a Köfler microscope (Wagner and Munz, Munich, Germany) and are uncorrected. 1H- and 13C-nuclear magnetic resonance (NMR) spectra were recorded at the University of Aveiro, Department of Chemistry in CDCl3 or DMSO-*d*<sup>6</sup> (Deutero GmbH, Ely, UK) at room temperature on a Bruker Avance 300 spectrometer (300.13 MHz for 1H and 75.47 MHz for 13C, Bruker Biosciences Corporation, Billerica, MA, USA). Chemical shifts are expressed in δ (ppm) values relative to tetramethylsilane (TMS) as an internal reference. Coupling constants are reported in hertz (Hz). 13C-NMR assignments were made by bidimensional heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) NMR experiments (long-range C, H coupling constants were optimized to 7 Hz) or by comparison with the assignments of similar molecules. High-resolution mass spectroscopy (HRMS) spectra were measured on a Bruker FTMS APEX III mass spectrometer (Bruker Corporation, Billerica, MA, USA) and recorded as electrospray ionization (ESI) mode in Centro de Apoio Cientifico e Tecnolóxico á Investigación (CACTI, University of Vigo, Pontevedra, Spain). The following compounds were synthesized and purified by the described procedures.

#### 3.1.1. Synthesis of 3,4-dimethoxy-1-methyl-9*H*-xanthen-9-one (**27**)

Xanthone **27** (9.34 g, 63% yield) was synthesized from 1,2,3-trimethoxy-5-methylbenzene (**25**) and characterized according to the previously described procedure [42].

#### 3.1.2. Synthesis of 1-(dibromomethyl)-3,4-dimethoxy-9*H*-xanthen-9-one (**28**)

A mixture of **27** (2.52 g, 9.32 mmol), *N*-bromosuccinimide (3.32 g, 18.7 mmol), and dibenzoyl peroxide (0.68 g, 2.8 mmol) in carbon tetrachloride (25 mL) was refluxed (85 ◦C) for 2 h. The reaction was monitored using *n*-hexane/ethyl acetate in a proportion of 8:2. Once the reaction was completed, the resulting orange suspension was cooled at 0 ◦C and stirred for 30 min in an ice bath. The solid was filtered and washed with cold carbon tetrachloride. The filtrate was evaporated under reduced pressure and further purified by flash column chromatography (SiO2, *n*-hexane/ethyl acetate in gradient) to obtain the **28** as a white powder.

For 1-(dibromomethyl)-3,4-dimethoxy-9*H*-xanthen-9-one (**28**): White powder (3.14 g, 80% yield); melting point (m.p.) 125–127 ◦C. 1H-NMR (CDCl3, 300.13 MHz): δ = 8.91 (1H, s, H-1), 8.30 (1H, dd, *J* = 8.0 and 1.6 Hz, H-8), 7.77 (1H, s, H-2), 7.73 (1H, ddd, *J* = 8.4, 7.0, and 1.6 Hz, H-6), 7.54 (1H, d, *J* = 8.4 Hz, H-5), 7.39 (1H, ddd, *J* = 7.5, H-7), 4.11 (3H, s, 4-OCH3), 4.05 (3H, s, 3-OCH3) ppm; 13C-NMR (CDCl3, 75.47 MHz): δ = 177.8 (C-9), 156.5 (C-3), 155.0 (C-10a), 150.4 (C-4a), 139.5 (C-4), 137.6 (C-1), 135.0 (C-6), 126.9 (C-8), 124.3 (C-7), 122.0 (C-8a), 117.6 (C-5), 111.9 (C-2), 110.8 (C-9a), 61.9 (3-OCH3), 56.6 (4-OCH3), 39.2 (C-1 ) ppm. HRMS (ESI+): *m*/*z* [C16H12Br2O5 + H]<sup>+</sup> calcd. for [C16H13Br2O5]: 426.91751; found: 426.91709.

3.1.3. Synthesis of 3,4-dimethoxy-9-oxo-9*H*-xanthene-1-carbaldehyde (**5**)

Xanthone **28** (3.14 g, 7.34 mmol) was added to a solution of 1-butyl-3-methylimidazolium tetrafluoroborate and water (6 mL, 5:1). The mixture was heated at 100 ◦C with stirring for 2.5 h. The reaction was monitored using *n*-hexane/ethyl acetate in a proportion of 7:3. After completion of the reaction, the resulting suspension was cooled at room temperature, diluted with water (10 mL), and extracted with ethyl acetate (5 × 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, and after filtration of the solution, the solvent was evaporated under reduced pressure. The crude product was then purified by flash column chromatography (SiO2, *n*-hexane/ethyl acetate in gradient). A light yellow powder corresponding to **5** was obtained.

For 3,4-dimethoxy-9-oxo-9*H*-xanthene-1-carbaldehyde (**5**): Light yellow powder (1.33 g, 64% yield); m.p. > 330 ◦C. 1H-NMR (CDCl3, 300.13 MHz): δ = 11.21 (1H, s, H-1 ), 8.31 (1H, dd, *J* = 8.0 and 1.7 Hz, H-8), 7.77 (1H, ddd, *J* = 8.5, 7.0, and 1.7 Hz, H-6), 7.60 (1H, dd, *J* = 8.5 and 0.8 Hz, H-5), 7.56 (1H, s, H-2), 7.43 (1H, ddd, *J* = 8.0, 7.0, and 0.8 Hz, H-7), 4.11 (3H, s, 4-OCH3), 4.07 (3H, s, 3-OCH3) ppm; 13C-NMR (CDCl3, 75.47 MHz): δ = 192.8 (C-1 ), 177.9 (C-9), 156.3 (C-3), 155.5 (C-10a), 150.9 (C-4a), 140.7 (C-4), 135.1 (C-6), 133.5 (C-1), 126.7 (C-8), 124.5 (C-7), 121.9 (C-8a), 117.8 (C-5), 116.1 (C-9a), 108.5 (C-2), 61.8 (4-OCH3), 56.6 (3-OCH3) ppm. HRMS (ESI<sup>+</sup>): *m*/*z* [C16H12O5 + H]<sup>+</sup> calcd. for [C16H13O5]: 285.07575; found: 285.07598; [C16H12O5 + Na]<sup>−</sup> calcd. for [C16H12NaO5]: 307.05769; found 307.05750.

#### 3.1.4. Synthesis of 1-(hydroxymethyl)-3,4-dimethoxy-9*H*-xanthen-9-one (**29**)

Xanthone **29** (52 mg, 52% yield) was synthesized from xanthone **5** and characterized according to the previously described procedure [36].

#### 3.1.5. General Procedure for the Synthesis of Xanthones (**30**–**40**)

Xanthone **5** (40 mg, 0.141 mmol) was dissolved in tetrahydrofuran (3 mL) and the appropriate amine precursors **9**–**19** (0.197 mmol) were added to the solution under N2 gas. After the addition of a 4.0 equimolar quantity of sodium triacetoxyborohydride (119 mg, 0.563 mmol), the resulting mixture was stirred at room temperature for 30 min. Subsequently, a 3.0 equimolar quantity of acetic acid (24 μL 0.423 mmol) was added to the solution, The reaction was carried out at room temperature for 12 h under N2 gas. For monitoring the synthesis of xanthones **30**–**40** by TLC, two chromatographic systems were employed: (i) *n*-hexane/ethyl acetate (7:3) and methanol/triethylamine (10:0.1) for xanthones **30**–**36**; (ii) *n*-hexane/ethyl acetate (7:3) and chloroform/acetone/triethylamine (5:5:0.1) for xanthones **37**–**40**. Once completed of the reaction, three distinct work-up approaches were employed.

(a) For xanthones **30**–**32**: the solvent was evaporated under reduced pressure and the resulting crude product was basified with sodium hydroxide (10 mL, 5%, m/v) and extracted with chloroform (3 × 10 mL). The combined organic phases were then acidified and extracted with hydrochloric acid 5 M (3 × 30 mL). Afterwards, the aqueous phases were gathered and then basified with sodium hydroxide (100 mL, 20%, *m*/*v*). The crude product was extracted successively with chloroform (3 × 100 mL). The organic layers were gathered and dried over anhydrous sodium sulfate. After filtration, the solution was concentrated under reduced pressure, and the resulting crude product was purified by flash cartridge chromatography (SiO2, chloroform/acetone/ammonium hydroxide, 90:10:1) to afford the xanthones 30, 31, and 32 as pure compounds.

For 3,4-dimethoxy-1-(((2-morpholinoethyl)amino)methyl)-9*H*-xanthen-9-one (**30**): Green powder (35 mg, 62% yield); m.p. 125–126 ◦C. 1H-NMR (CDCl3, 300.13 MHz): δ = 8.25 (1H, dd, *J* = 8.0 and 1.6 Hz, H-8), 7.72 (1H, ddd, *J* = 8.1, 7.0, and 1.6 Hz, H-6), 7.57 (1H, dd, *J* = 8.4 and 0.8 Hz, H-5), 7.38 (1H, ddd, *J* = 8.0, 7.1, and 0.9 Hz, H-7), 7.09 (1H, s, H-2), 4.34 (2H, s, H-1 ); 4.04 (3H, s, 4-OCH3), 4.01 (3H, s, 3-OCH3), 3.66 (4H, t, *J* = 4.6 Hz, H-5 ), 2.90 (2H, t, *J* = 6.1 Hz, H-2 ), 2.60 (2H, t, *J* = 6.1 Hz, H-3 ), 2.47 (4H, t, *J* = 4.3 Hz, H-4 ) ppm; 13C-NMR (CDCl3, 75.47 MHz): δ = 178.0 (C-9), 156.5 (C-3), 155.3 (C-10a), 152.1 (C-4a), 137.0 (C-1), 136.0 (C-4), 134.6 (C-6), 126.5 (C-8), 124.1 (C-7), 122.1 (C-8a), 117.7 (C-5), 114.6 (C-9a), 111.6 (C-2), 67.0 (C-5 ), 61.5 (4-OCH3), 57.4 (C-2 ), 56.4 (3-OCH3), 53.7 (C-1 ), 53.5 (C-4 ), 45.3 (C-3 ) ppm. HRMS (ESI+): *m*/*z* [C22H26N2O5 + H]<sup>+</sup> calcd. for [C22H27N2O5]: 399.19145; found: 399.19060.

For 1-(((3-(dimethylamino)propyl)(methyl)amino)methyl)-3,4-dimethoxy-9*H*-xanthen-9-one (**31**): White powder (35 mg, 65% yield); m.p. 84–85 ◦C. 1H-NMR (CDCl3, 300.13 MHz): δ = 8.25 (1H, dd, *J* = 8.0 and 1.6 Hz, H-8), 7.69 (1H, ddd, *J* = 8.5, 7.0, and 1.6 Hz, H-6), 7.54 (1H, dd, *J*= 7.4 and 0.7, H-5), 7.53 (1H, s, H-2), 7.35 (1H, ddd, *J* = 8.0, 7.1, and 1.0 Hz, H-7), 4.36 (2H, s, H-1 ); 4.05 (3H, s, 4-OCH3), 4.02 (3H, s, 3-OCH3), 2.65 (2H, t, *J* = 7.2 Hz, H-3 ), 2.44 (2H, t, *J* = 7.5 Hz, H-5 ), 2.39 (3H, s, H-2 ), 2.28 (6H, s, H-6 ), 1.82 (2H, m, *J* = 7.3 Hz, H-4 ) ppm; 13C-NMR (CDCl3, 75.47 MHz): δ = 178.1 (C-9), 156.6 (C-3), 155.2 (C-10a), 151.9 (C-4a), 135.2 (C-1), 135.0 (C-4), 134.2 (C-6), 126.6 (C-8), 123.9 (C-7), 122.4 (C-8a), 117.6 (C-5), 114.5 (C-9a), 109.0 (C-2), 61.5 (4-OCH3), 60.1 (C-1 ), 57.8 (C-5 ), 56.4 (3-OCH3), 56.1 (C-3 ), 45.3 (C-6 ), 42.5 (C-2 ), 25.3 (C-4 ) ppm. HRMS (ESI+): *m*/*z* [C22H28N2O4 + H]<sup>+</sup> calcd. for [C22H29N2O4]: 385.21218; found: 385.21139.

For 1-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-3,4-dimethoxy-9*H*-xanthen-9-one (**32**): Yellow powder (40 mg, 71% yield); m.p. 129–130 ◦C. 1H-NMR (CDCl3, 300.13 MHz): δ = 8.25 (1H, dd, *J* = 8.0 and 1.6 Hz, H-8), 7.68 (1H, ddd, *J* = 8.5, 7.0, and 1.6 Hz, H-6), 7.53 (1H, dd, *J* = 8.4 and 0.7 Hz, H-5), 7.48 (1H, s, H-2), 7.35 (1H, ddd, *J* = 8.0, 7.1, and 1.0 Hz, H-7), 4.32 (2H, s, H-1 ), 4.03 (3H, s, 4-OCH3), 4.01 (3H, s, 3-OCH3), 3.63 (2H, t, *J* = 5.4 Hz), 2.58 (2H, t, *J* = 5.4 Hz) ppm; 13C-NMR (CDCl3, 75.47 MHz): δ = 178.1 (C-9), 156.5 (C-3), 155.2 (C-10a), 152.0 (C-4a), 138.8 (C-1), 134.8 (C-4), 134.2 (C-6), 126.6 (C-8), 123.9 (C-7), 122.5 (C-8a), 117.5 (C-5), 114.6 (C-9a), 108.2 (C-2), 61.5 (4-OCH3), 60.2 (C-1 ), 59.3 (C-5 ), 57.7 (C-4 ), 56.2 (3-OCH3), 53.5 (C-3 ), 53.2 (C-2 ) ppm. HRMS (ESI+): *m*/*z* [C22H26N2O5 + H]<sup>+</sup> calcd. for [C22H27N2O5]: 399.19145; found: 399.19059.

(b) For xanthones **33**–**36**: the solvent was evaporated under reduced pressure and the crude product was extracted with chloroform (3 × 10 mL). The organic phases were gathered, dried over anhydrous sodium sulfate, and after filtration, the resulting solution was concentrated under reduced pressure. Then, a solid phase extraction using a cation exchange cartridge Discovery® DSC-SCX was applied to further purify the crude product. Initially, an activation of the cartridge with methanol (100 mL) was carried out followed by loading the cartridge with the sample. Then, the elution was performed with the following solvents/solutions: (i) dichloromethane/methanol (5:5), (ii) methanol, and (iii) ammonium hydroxide/methanol (2%, *v*/*v*). The fractions obtained from the elution with ammonium hydroxide/methanol (2%, *v*/*v*) were gathered and the solvent was evaporated under reduced pressure. A flash cartridge chromatography with chloroform/acetone/ammonium hydroxide (90:10:1) was also performed to obtain the xanthones **33**, **34**, **35**, and **36** as pure compounds.

For 1-(((2-(diethylamino)ethyl)amino)methyl)-3,4-dimethoxy-9*H*-xanthen-9-one (**33**): Green powder (28 mg, 52% yield); m.p. 68–69 ◦C. 1H-NMR (CDCl3, 300.13 MHz): δ = 8.27 (1H, dd, *J* = 8.0 and 1.4 Hz, H-8), 7.71 (1H, ddd, *J* = 8.5, 7.0, and 1.6 Hz, H-6), 7.56 (1H, dd, *J* = 8.4 and 0.7 Hz, H-5), 7.37 (1H, ddd, *J* = 8.0, 7.1, and 1.0 Hz, H-7), 7.14 (1H, s, H-2), 4.39 (2H, s, H-1 ); 4.05 (3H, s, 4-OCH3), 4.01 (3H, s, 3-OCH3), 2.87 (2H, t, *J* = 6.4 Hz, H-2 ), 2.68 (2H, t, *J* = 6.4 Hz, H-3 ), 2.55 (4H, m, *J* = 7.1 Hz, H-4 ), 1.01 (6H, t, *J* = 7.1 Hz, H-5 ) ppm; 13C-NMR (CDCl3, 75.47 MHz): δ = 178.1 (C-9), 156.5 (C-3), 155.3 (C-10a), 152.1 (C-4a), 137.4 (C-1), 135.8 (C-4), 134.5 (C-6), 126.6 (C-8), 124.0 (C-7), 122.2 (C-8a), 117.7 (C-5), 114.5 (C-9a), 111.1 (C-2), 61.5 (4-OCH3), 56.4 (3-OCH3), 53.4 (C-3 ), 52.4 (C-1 ), 46.9 (C-4 ), 46.8 (C-2 ), 11.6 (C-5 ) ppm. HRMS (ESI+): *m*/*z* [C22H28N2O4 + H]<sup>+</sup> calcd. for [C22H29N2O4]: 385.21218; found: 385.21150.

For 4-((3,4-dimethoxy-9-oxo-9*H*-xanthen-1-yl)methyl)piperazin-2-one (**34**): Orange powder (20 mg, 39% yield); m.p. 245–247 ◦C. 1H-NMR (DMSO-*d*6, 300.13 MHz): δ = 8.13 (1H, dd, *J* = 8.0 and 1.5 Hz, H-8), 7.82 (1H, ddd, *J* = 8.5, 7.0, and 1.6 Hz, H-6), 7.65 (1H, dd, *J* = 8.4 and 0.6, H-5), 7.45 (1H, ddd, *J* = 8.0, 7.1, and 1.0 Hz, H-7), 7.36 (1H, s, H-2), 4.24 (2H, s, H-1 ); 3.98 (3H, s, 4-OCH3), 3.90 (3H, s, 3-OCH3), 3.20 (2H, t, *J* = 5.3 Hz, H-5 ), 3.13 (2H, s, H-2 ), 2.66 (2H, t, *J* = 5.3 Hz, H-4 ) ppm; 13C-NMR (DMSO-*d*6, 75.47 MHz): δ = 176.7 (C-9), 168.0 (C-3 ), 156.2 (C-3), 154.6 (C-10a), 151.8 (C-4a), 136.9 (C-1), 135.0 (C-4), 134.7 (C-6), 126.1 (C-8), 124.3 (C-7), 121.8 (C-8a), 117.7 (C-5), 113.7 (C-9a), 109.2 (C-2), 60.9 (4-OCH3), 58.5 (C-1 ), 57.2 (C-5 ), 56.3 (3-OCH3), 48.7 (C-2 ), 30.7 (C-4 ) ppm. HRMS (ESI+): *m*/*z* [C20H20N2O5 + H]<sup>+</sup> calcd. for [C20H21N2O5]: 369.14450; found: 369.14369.

For 1-((5-amino-3,4-dihydroisoquinolin-2(1*H*)-yl)methyl)-3,4-dimethoxy-9*H*-xanthen-9-one (**35**): White powder (23 mg, 39% yield); m.p. 171–172 ◦C. 1H-NMR (CDCl3, 300.13 MHz): δ = 8.26 (1H, dd, *J* = 8.0 and 1.4 Hz, H-8), 7.69 (1H, ddd, *J* = 8.5, 7.0, and 1.6 Hz, H-6), 7.57 (1H, s, H-2), 7.54 (1H, dd, *J* = 8.4 and 0.7 Hz, H-5), 7.35 (1H, ddd, *J* = 8.0, 7.1, and 1.0 Hz, H-7), 6.99 (1H, t, *J* = 7.7 Hz, H-7 ), 6.57 (1H, d, *J* = 7.8 Hz, H-6 ), 6.53 (1H, d, *J* = 7.6 Hz, H-8 ), 4.48 (2H, s, H-1 ); 4.02 (3H, s, 4-OCH3), 3.97

(3H, s, 3-OCH3), 3.81 (2H, s, H-10 ), 2.93 (2H, t, *J* = 5.9 Hz, H-2 ), 2.66 (2H, t, *J* = 5.9 Hz, H-3 ) ppm; 13C-NMR (CDCl3, 75.47 MHz): δ = 178.2 (C-9), 156.7 (C-3), 155.2 (C-10a), 151.9 (C-4a), 144.1 (C-5 ), 139.3 (C-1), 136.3 (C-9 ), 134.8 (C-4), 134.2 (C-6), 126.6 (C-8), 126.4 (C-7 ), 123.8 (C-7), 122.5 (C-8a), 119.5 (C-4 ), 117.6 (C-5), 117.1 (C-8 ), 114.5 (C-9a), 112.7 (C-6 ), 108.0 (C-2), 61.5 (4-OCH3), 60.1 (C-1 ), 56.7 (C-10 ), 56.3 (3-OCH3), 51.0 (C-2 ), 24.9 (C-3 ) ppm. HRMS (ESI+): *m*/*z* [C25H24N2O4 + H]<sup>+</sup> calcd. for [C25H25N2O4]: 417.18088; found: 417.18018.

For 3,4-dimethoxy-1-(piperidin-1-ylmethyl)-9*H*-xanthen-9-one (**36**): Yellow powder (31 mg, 62% yield); m.p. 108-109 ◦C. 1H-NMR (CDCl3, 300.13 MHz): δ = 8.25 (1H, dd, *J* = 8.0 and 1.6 Hz, H-8), 7.66 (1H, ddd, *J* = 8.5, 8.0, and 1.6 Hz, H-6), 7.56 (1H, s, H-2), 7.52 (1H, dd, *J* = 8.3 and 0.7 Hz, H-5), 7.33 (1H, ddd, *J* = 7.9, 7.1, and 0.9 Hz, H-7), 4.26 (2H, s, H-1 ); 4.03 (3H, s, 4-OCH3), 4.01 (3H, s, 3-OCH3), 2.57 (4H, t, *J* = 4.6 Hz, H-2 ), 1.65 (4H, m, *J* = 5.5 Hz, H-3 ), 1,50 (2H, m, *J* = 5.5 Hz, H-4 ) ppm; 13C-NMR (CDCl3, 75.47 MHz) ppm: δ = 177.5 (C-9), 156.1 (C-3), 154.7 (C-10a), 151.3 (C-4a), 138.9 (C-4), 134.2 (C-1), 133.8 (C-6), 126.0 (C-8), 123.3 (C-7), 121.9 (C-8a), 117.1 (C-5), 113.9 (C-9a), 107.8 (C-2), 60.8 (4-OCH3), 56.1 (3-OCH3), 55.7 (C-2 ), 54.3 (C-1 ), 25.7 (C-3 ), 23.8 (C-4 ) ppm. HRMS (ESI+): *m*/*z* [C21H23NO4 + H]<sup>+</sup> calcd. for [C21H24NO4]: 354.16998; found: 354.16939.

(c) For xanthones **37**–**40**: the solvent was evaporated under reduced pressure and the resulting crude product was basified with sodium hydroxide (10 mL, 5%, *m*/*v*) and extracted with chloroform (3 × 10 mL). The combined organic phases were then acidified and extracted with hydrochloric acid 5 M (3 × 30 mL). The resulting organic phase was dried over anhydrous sodium sulfate and, after filtration, the solution was concentrated under reduced pressure. Then, a solid phase extraction using a cation exchange cartridge Discovery® DSC-SCX was applied to further purify the crude product. Initially, an activation of the cartridge with methanol (100 mL) was carried out followed by loading the cartridge with the sample. Then, the elution was performed with the following solvents/solutions: (i) dichloromethane/methanol (5:5), (ii) methanol, and (iii) ammonium hydroxide/methanol (2%, *v*/*v*). The fractions obtained from the elution with ammonium hydroxide/methanol (2%, *v*/*v*) were gathered and the solvent was evaporated under reduced pressure to furnish the xanthones **37**, **38**, **39**, and **40** as pure compounds.

For 1-(((4-fluorobenzyl)amino)methyl)-3,4-dimethoxy-9*H*-xanthen-9-one (**37**): White powder, (34 mg, 61% yield); m.p. 163–165 ◦C. 1H-NMR (CDCl3, 300.13 MHz): δ = 8.28 (1H, dd, *J* = 8.0 and 1.6 Hz, H-8), 7.71 (1H, ddd, *J* = 8.5, 7.0, and 1.6 Hz, H-6), 7.55 (1H, dd, *J* = 8.5 and 0.8 Hz, H-5), 7.40 (1H, ddd, *J* = 8.0, 7.0, and 0.8 Hz, H-7), 7.36 (2H, m, H-5 ), 7.01 (2H, m, H-4 ), 6.96 (1H, s, H-2), 4.30 (2H, s, H-1 ), 4.01 (3H, s, 4-OCH3), 4.01 (3H, s, 3-OCH3), 3.85 (2H, s, H-2 ) ppm; 13C-NMR (CDCl3, 75.47 MHz): δ = 178.1 (C-9), 160.3 (C-6 ), 156.3 (C-3), 155.3 (C-10a), 152.2 (C-4a), 138.6 (C-4), 135.9 (C-3 ), 135.7 (C-1), 134.5 (C-6), 129.8 (C-5 ), 126.7 (C-8), 124.0 (C-7), 122.2 (C-8a), 117.7 (C-5), 115.3 (C-4 ), 114.7 (C-9a), 110.9 (C-2), 61.5 (4-OCH3), 56.3 (3-OCH3), 53.3 (C-1 ), 52.5 (C-2 ) ppm. HRMS (ESI+): *m*/*z* [C23H20FNO4 + H]<sup>+</sup> calcd. for [C23H21FNO4]: 394.14491; found: 394.14410.

For 1-(((4-chlorobenzyl)amino)methyl)-3,4-dimethoxy-9*H*-xanthen-9-one (**38**): Light yellow powder (35 mg, 61% yield); m.p. 162–163 ◦C. 1H-NMR (CDCl3, 300.13 MHz): δ = 8.25 (1H, dd, *J* = 8.0 and 1.6 Hz, H-8), 7.79 (1H, ddd, *J* = 8.5, 7.0, and 1.6 Hz, H-6), 7.66 (2H, m, H-5 ), 7.60 (1H, dd, *J* = 8.5 and 0.9 Hz, H-5), 7.43 (1H, ddd, *J* = 8.0, 7.0, and 0.9 Hz, H-7), 7.39 (1H, s, H-2), 7.38 (2H, m, H-4 ), 4.53 (2H, s, H-2 ); 4.33 (2H, s, H-1 ), 4.07 (3H, s, 4-OCH3), 4.01 (3H, s, 3-OCH3) ppm; 13C-NMR (CDCl3, 75.47 MHz): δ = 179.6 (C-9), 157.0 (C-3), 155.5 (C-10a), 152.0 (C-4a), 137.7 (C-4), 135.8 (C-1), 135.6 (C-6), 131.7 (C-5 ), 129.5 (C-4 ), 129.4 (C-3 ), 127.3 (C-6 ), 126.6 (C-8), 124.6 (C-7), 121.3 (C-8a), 117.9 (C-5), 115.5 (C-2), 114.8 (C-9a), 61.6 (4-OCH3), 57.1 (3-OCH3), 51.3 (C-1 ), 50.7 (C-2 ) ppm. HRMS (ESI+): *m*/*z* [C23H20ClNO4 + H]<sup>+</sup> calcd. for [C23H21ClNO4]: 410.11536; found: 410.11466.

For (*R*)-1-(((1-(4-chlorophenyl)ethyl)amino)methyl)-3,4-dimethoxy-9*H*-xanthen-9-one (**39**): White powder (39 mg, 65% yield); m.p. 163–164 ◦C. 1H-NMR (CDCl3, 300.13 MHz): δ = 8.26 (1H, dd, *J* = 8.0 and 1.6 Hz, H-8), 7.79 (1H, ddd, *J* = 8.5, 7.0, and 1.6 Hz, H-6), 7.70 (2H, m, H-6 ), 7.59 (1H, dd, *J* = 8.5 and 0.8 Hz, H-5), 7.43 (1H, ddd, *J* = 8.0, 7.0, and 0.8 Hz, H-7), 7.42 (2H, m, H-5 ), 7.25 (1H, s, H-2), 4.54 (2H, s, H-1 ); 4.27 (1H, q, *J* = 6.8 Hz, H-1 ), 4.04 (3H, s, 4-OCH3), 4.01 (3H, s, 3-OCH3), 1.83 (3H, d, *J* = 6.8 Hz, H-3 ) ppm; 13C-NMR (CDCl3, 75.47 MHz): δ = 179.6 (C-9), 156.8 (C-3), 155.4 (C-10a), 151.9 (C-4a), 137.6 (C-4), 135.5 (C-6), 134.5 (C-1), 131.8 (C-7 ), 129.8 (C-6 ), 129.5 (C-5 ), 127.3 (C-4 ), 126.6 (C-8), 124.6 (C-7), 121.3 (C-8a), 117.9 (C-5), 115.6 (C-2), 114.9 (C-9a), 61.6 (4-OCH3), 58.9 (C-2 ), 57.0 (3-OCH3), 50.2 (C-1 ), 20.2 (C-3 ) ppm. HRMS (ESI+): *m*/*z* [C24H22ClNO4 + H]<sup>+</sup> calcd. for [C24H23ClNO4]: 424.13101; found: 424.13018.

For 1-(((4-bromobenzyl)amino)methyl)-3,4-dimethoxy-9*H*-xanthen-9-one (**40**): White powder (47 mg, 73% yield); m.p. 161–162 ◦C. 1H-NMR (CDCl3, 300.13 MHz): δ = 8.27 (1H, dd, *J* = 8.0 and 1.6 Hz, H-8), 7.73 (1H, ddd, *J* = 8.5, 7.0, and 1.6 Hz, H-6), 7.58 (1H, dd, *J* = 8.5 and 0.8 Hz, H-5), 7.48 (2H, m, H-5 ), 7.39 (2H, m, H-4 ), 7.38 (1H, ddd, *J* = 8.0, 7.0, and 0.8 Hz, H-7), 7.08 (1H, s, H-2), 4.31 (2H, s, H-1 ); 4.01 (3H, s, 4-OCH3), 4.01 (3H, s, 3-OCH3), 3.88 (2H, s, H-2 ) ppm; 13C-NMR (CDCl3, 75.47 MHz): δ = 178.6 (C-9), 156.5 (C-3), 155.3 (C-10a), 152.2 (C-4a), 136.4 (C-4), 136.2 (C-1), 134.9 (C-6), 131.8 (C-5 ), 130.7 (C-4 ), 128.4 (C-3 ), 126.6 (C-8), 124.2 (C-7), 121.9 (C-8a), 121.8 (C-6 ), 117.7 (C-5), 114.7 (C-9a), 112.5 (C-2), 61.6 (4-OCH3), 56.7 (3-OCH3), 52.7 (C-1 ), 52.0 (C-2 ) ppm. HRMS (ESI+): *m*/*z* [C23H20BrNO4 + H]<sup>+</sup> calcd. for [C23H21BrNO4]: 454.06485; found: 454.06385.

#### *3.2. Biological Evaluation*

#### 3.2.1. Yeast Strains and Growth Conditions

For the yeast assay, the previously obtained *Saccharomyces cerevisiae* cells co-expressing human p53 or human MDM2, as well as yeast control (transformed with empty vectors) [30,31], were used. To induce expression of human proteins, yeast cells were grown in selective induction medium with 2% (*w*/*w*) galactose and 2% (*w*/*w*) raffinose, in the presence of 1–20 μM of xanthones **30**–**40** or 0.1% DMSO, at 30 ◦C under continuous orbital shaking (200 rpm) for approximately 42 h, as described [30,31]. Yeast cell growth was analyzed by counting the number of colony-forming units (CFU) after 2 days incubation at 30 ◦C on Sabouraud Dextrose Agar plates from Liofilchem (Frilabo, Porto, Portugal). Results were estimated considering 100% growth as the number of CFU obtained with untreated yeast co-expressing p53 and MDM2.

#### 3.2.2. Human Cancer Cell Lines and Culture Conditions

Human colon adenocarcinoma HCT116 cell lines expressing wt p53 (HCT116 p53+/+) and its p53-null isogenic derivative (HCT116 p53−/−) were provided by B. Vogelstein (The Johns Hopkins Kimmel Cancer Center, Baltimore, MD, USA). Human liver HepG2 carcinoma cell lines were purchased from ATCC (Rockville, MD, USA). Cancer cells were cultured in RPMI-1640 medium with ultraglutamine (Lonza, VWR, Carnaxide, Portugal), and supplemented with 10% fetal bovine serum (FBS; Merck Millipore, VWR). Cells were maintained at 37 ◦C in a humidified atmosphere of 5% CO2.

#### 3.2.3. Cell Proliferation Assay

Cell proliferation was determined using the SRB assay, as described [30,31]. Briefly, cells were seeded in 96-well plates at 5.0 <sup>×</sup> 103 cells/well for HCT116 p53+/+ cells and 4.0 <sup>×</sup> 103 cells/well for HepG2 and 24 h later treated with serial dilutions (3.13–50 μM) of xanthone **37** or 0.25% DMSO for 48 h. IC50 values were determined from the concentration-response curves.

#### 3.2.4. Colony Formation Assay

HCT116 p53+/+ and HCT116 p53−/<sup>−</sup> cells were seeded in a 6-well plate at 1.0 <sup>×</sup> 10<sup>3</sup> cells/well and treated with 3, 4, 5, 7, and 10 μM of xanthone **37** or DMSO at the time of seeding. After 11 days incubation, colonies were fixed using 10% of methanol and 10% acetic acid for 10 min and stained with 0.5% crystal violet (Sigma-Aldrich) in 1:1 methanol/water for 15 min. Colonies with more than 20 cells were counted.

#### 3.2.5. Cell Cycle Analysis

HCT116 p53+/+ and HCT116 p53−/<sup>−</sup> cells were seeded in a 6-well plate at 1.5 <sup>×</sup> 105 cells/well for 24 h and treated with 10 and 20 μM of xanthone **37** or DMSO for 24 h. Cells were collected, fixed, and stained with propidium iodide (Fluka, Sigma-Aldrich). Cell cycle was analyzed by flow cytometry, as described [45].

#### 3.2.6. Western Blot Analysis

HCT116 p53+/+ were seeded in a 6-well plate at 1.5 <sup>×</sup> 105 cells/well for 24 h and treated with 10 and 20 μM of xanthone **37** or DMSO for 24 h. Total protein extracts of cancer cells were obtained and analyzed by Western blot, as described [45]. Membranes were probed with a mouse monoclonal anti-p53 (DO-1), anti-MDM2 (D-12), or with a rabbit polyclonal anti-p21 (C-19), followed by an anti-mouse or anti-rabbit horseradish-peroxidase (HRP)-conjugated secondary antibody. For loading control, membranes were stripped and re-probed with mouse monoclonal anti-GAPDH (6C5). All antibodies were purchased from Santa Cruz Biotechnology (Frilabo, Porto, Portugal). The signal was detected with enhanced chemiluminescence (ECL) Amersham Kit from GE Healthcare (VWR) and with Molecular Imager® ChemiDoc™ XRS+ System, using Image Lab™ software.

#### 3.2.7. Statistical Analysis

Data's statistical analysis was performed using the Prism 7 program (GraphPad Software Inc., California, USA). Differences between means were tested for significance using one-way ANOVA with Tukey's multiple comparisons test, two-way ANOVA with Sidak's multiple comparisons test, and Student's *t*-test (\* *p* < 0.05; \*\* *p* < 0.01).

#### *3.3. In Silico Studies onto MDM2*

The chemical structures of gambogic acid (**2**), α-mangostin (**3**), 12-hydroxy-2,2-dimethyl-3,4 dihydro-2*H*,6*H*-pyrano[3,2-*b*]xanthen-6-one (**4**), and the xanthones **30**–**40** were drawn using ChemSketch (Advanced Chemistry Development, Inc. (ACD/Labs), Canada); the structure of known MDM2-p53 inhibitor nutlin-3A (**1**) was obtained from PubChem [53]. The three-dimensional (3D) structures of the xanthones and **1** were minimized using the ArgusLab version 4.0.1 software for Windows by Hamiltonian (quantum mechanics) using the Parameterized Model number 3 (PM3) semi-empirical method [54]. The 3D structure of MDM2 was extracted from Protein Data Bank (PDB id 4HG7) [50]. Docking simulations involving MDM2 and the small molecules were undertaken in AutoDock Vina (Scripps Research Institute, USA) [51]. AutoDock Vina considered the macromolecular target conformation as a rigid unit, while the ligands were allowed to be flexible and adaptable to the target. Vina explored the lowest binding affinity conformations and returned nine different conformations for each ligand. AutoDock Vina was run using an exhaustiveness of 8 and a grid box with the dimensions of 19.7 Å, 26.1 Å, and 14.1 Å, engulfing the binding cavity occupied by the crystallographic nutlin-3A (PDB id 4HG7). Ligand conformations and interactions with the binding cavity were visualized using PyMOL version 1.3 [55]. In order to validate the docking approach for the macromolecular target structure used, nutlin-3A was docked onto the binding cavity of MDM2 using AutoDock Vina, and the obtained docking conformation was compared to crystallographic nutlin-3A (PDB id 4HG7) using RMSD (not shown). Docking scores of xanthones and positive controls were compared.

#### **4. Conclusions**

An alternative and efficient strategy for the preparation of carbaldehydic xanthones was successfully developed. The use of a precursor with a methyl substituent at position C-1 revealed to be crucial for the optimization of the synthesis of **5**. The newly described approach required lower reaction times and enabled the preparation of xanthone **5** in a higher global yield. In this work, a series of eleven aminated xanthones (**30**–**40**) possessing a 3,4-dioxygenated pattern of substitution was efficiently constructed in moderate to good yields. From this group of compounds, xanthone **37** was identified for the first time as a putative p53-activating agent, using a yeast-based screening assay. Xanthone **37** was revealed to inhibit the growth of human HCT116 p53+/+ colon cancer cells, being that this effect is associated with cell cycle arrest through activation of the p53 pathway. Nevertheless, further studies are required to confirm the mechanism of action of **37**, which may lead to the identification of a novel xanthone derivative with promising antitumor activity.

These results demonstrated the potential usefulness of coupling amine-containing structural motifs of known MDM2-p53 disruptors into the 3,4-dioxygenated xanthone scaffold as a starting point for the design of novel and improved p53-activating agents with antitumor activity and drug-like properties.

**Supplementary Materials:** The following are available online. Figure S1–S15: HRMS spectra of the xanthone derivatives **5** and **27**–**40**. Figure S16–S45: 1H- and 13C-NMR spectra of the xanthone derivatives **5** and **27**–**40**.

**Author Contributions:** A.L. performed the in silico studies and the synthesis of xanthones and wrote the manuscript. A.L., A.S.G., and J.B.L. performed biological experiments and analyzed the data. P.B. contributed to the experimental synthetic work. M.E.S. and L.S. conceived and designed the study, coordinated the experimental work, and wrote the manuscript. A.P. coordinated the in silico studies. M.M.M.P., M.E.S., and L.S. provided financial support for the study, analyzed the data, and wrote the manuscript.

**Funding:** The authors thank to national funds provided by FCT—Foundation for Science and Technology and European Regional Development Fund (ERDF) and COMPETE under the Strategic Funding UID/Multi/ 04423/2019, UID/QUI/50006/2019 (UCIBIO/REQUIMTE) the projects (3599-PPCDT) PTDC/DTP-FTO/1981/2014— POCI-01-0145-FEDER-016581, POCI-01-0145-FEDER-028736, and PTDC/MAR-BIO/4694/2014 (POCI-01-0145- FEDER-016790; 3599-PPCDT). FCT fellowships: PD/BD/114046/2015 (A.S. Gomes), SFRH/BD/128673/2017 (J. B. Loureiro), and the Programa Operacional Capital Humano (POCH), specifically the BiotechHealth Programme (PD/00016/2012).

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


**Sample Availability:** Samples of the compounds **5**, **16**–**40** are available from the authors.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Design and Synthesis of Indoleamine 2,3-Dioxygenase 1 Inhibitors and Evaluation of Their Use as Anti-Tumor Agents**

### **Hui Wen** †**, Yuke Liu** †**, Shufang Wang, Ting Wang, Gang Zhang, Xiaoguang Chen, Yan Li, Huaqing Cui, Fangfang Lai \* and Li Sheng \***

State Key Laboratory of Bioactive Substances and Function of Natural Medicine, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100050, China; wenhui@imm.ac.cn (H.W.); liuyuke@imm.ac.cn (Y.L.); wangshufang@imm.ac.cn (S.W.); wangtingdlf@imm.ac.cn (T.W.); gzhang@imm.ac.cn (G.Z.); chxg@imm.ac.cn (X.C.); yanli@imm.ac.cn (Y.L.); hcui@imm.ac.cn (H.C.)


Academic Editor: Qiao-Hong Chen

Received: 14 May 2019; Accepted: 4 June 2019; Published: 5 June 2019

**Abstract:** Indoleamine 2,3-dioxygenase (IDO) 1 is the key enzyme for regulating tryptophan metabolism and is an important target for interrupting tumor immune escape. In this study, we designed four series of compounds as potential IDO1 inhibitors by attaching various fragments or ligands to indole or phenylimidazole scaffolds to improve binding to IDO1. The compounds were synthesized and their inhibitory activities against IDO1 and tryptophan 2,3-dioxygenase were evaluated. The cytotoxicities of the compounds against two tumor cell lines were also determined. Two compounds with a phenylimidazole scaffold (DX-03-12 and DX-03-13) showed potent IDO1 inhibition with IC50 values of 0.3–0.5 μM. These two IDO1 inhibitors showed low cell cytotoxicity, which indicated that they may exert their anti-tumor effect via immune modulation. Compound DX-03-12 was investigated further by determining the in vivo pharmacokinetic profile and anti-tumor efficacy. The pharmacokinetic study revealed that DX-03-12 had satisfactory properties in mice, with rapid absorption, moderate plasma clearance (∼36% of hepatic blood flow), acceptable half-life (∼4.6 h), and high oral bioavailability (∼96%). Daily oral administration of 60 mg/kg of compound DX-03-12 decreased tumor growth by 72.2% after 19 days in a mouse melanoma cell B16-F10 xenograft model compared with the untreated control. Moreover, there was no obvious weight loss in DX-03-12-treated mice. In conclusion, compound DX-03-12 is a potent lead compound for developing IDO1 inhibitors and anti-tumor agents.

**Keywords:** indoleamine 2,3-dioxygenase; inhibitor; anti-tumor; immune modulation; tryptophan metabolism

#### **1. Introduction**

Tryptophan metabolism is an important pathway for cancer immunotherapy [1–3]. The accumulation of tryptophan can activate T cells, and a decrease in tryptophan concentration affects the regular function of T cells and interferes with the differentiation of naïve T cells. The accumulation of tryptophan metabolites, such as l-kynurenine and other downstream kynurenines (3-hydroxykynurenine and 3-hydroxyanthranilic acid), is toxic to T cells and induces T cell apoptosis. Thus, increasing tryptophan degradation inhibits the immune response [4–6].

Indoleamine 2,3-dioxygenase (IDO; E.C. 1.12.11.17) 1 is a monomeric 41 kDa enzyme that contains heme and converts l-tryptophan to *N*-formylkynurenine in non-hepatic tissues [7]. Tryptophan 2,3-dioxygenase (TDO; E.C. 1.13.11.11) also catalyzes the degradation of l-tryptophan to *N*-formylkynurenine [8]. However, these two enzymes are genetically distinct and have low sequence homology [8].

IDO1 is an important enzyme in tryptophan metabolism and contributes to creating peripheral immune tolerance by degrading l-tryptophan, which suppresses the function of T cells, and by producing *N*-formylkynurenine, which inhibits T cells [8]. In addition, IDO1 can also trigger other activators of anti-tumor immunity. Notably, IDO1 is overexpressed in a range of cancer tissues [4–6]. Thus, blocking the activity of IDO1 is a potential strategy for tumor immunotherapy.

The crystal structures of recombinant human IDO1 have been resolved [9,10], and biochemical studies have revealed that the heme iron in the active site of IDO1 binding and forms adducts with exogenous ligands including O2 <sup>−</sup>, NO, CO, H2S, and CN<sup>−</sup> [9,11–14]. IDO1 tolerates various substrates, whereas TDO has strict substrate specificity. This difference can be used to develop selective IDO1 inhibitors [8].

During the development of anti-tumor drugs, potent IDO1 inhibitors were discovered, and intensive efforts have been made to develop potent IDO1/TDO inhibitors with various structural scaffolds. Some typical examples of IDO1/TDO inhibitors are shown in Figure 1 [1,15–21]. The indole and phenylimidazole scaffolds have produced several potent IDO1 inhibitors. For example, IDO1 inhibitors indoximod (pathway IC50 = 450 nM) and PF-0684003 (IDO1 IC50 = 410 nM) are indole derivatives [13], and GDC-0919 (IDO1 IC50 = 13 nM) and AMCL-17g (IDO1 IC50 = 77 nM) are phenylimidazole derivatives [20].

**Figure 1.** Reported IDO1 inhibitors. Indoximod and PF-0684003 have an indole scaffold and GDC-0919 and AMCL-17g have a phenylimidazole scaffold.

In this study, in the early stages of searching for new IDO1 inhibitors, we used indole and phenylimidazole as the core scaffolds. Four series of compounds were designed with different strategies. The compounds were synthesized and the IDO1/TDO inhibitory activities were determined. The in vivo pharmacokinetic profile and anti-tumor efficacy of a potent IDO1 inhibitor were evaluated to explore its potential as an anti-tumor agent.

#### **2. Results and Discussion**

#### *2.1. Design Strategy for the Four Compound Series*

Four series of compounds were designed (Figure 2) to explore the structure–activity relationship (SAR). The SAR information was used for further optimization. Indole is derived from tryptophan and phenylimidazole is an IDO1 inhibitor [18]. Thus, tryptophan and phenylimidazole fit into the active site of IDO1, and they were selected as the core scaffolds for designing the inhibitors. As we mentioned in the introduction, several exogenous ligands including hydroxylamine, O2 −, NO, CO, H2S, and CN<sup>−</sup> were able to bind with the heme iron in the active site of IDO1 [9,11–14]. We attached various fragments and ligands to these scaffolds to strengthen the binding to the active site of IDO1.

In series 1, several ligands, such as hydroxylamine and SO, that interact strongly with heme iron were linked to the double bond of the indole scaffold. We hoped to observe a synergistic effect between the ligand and the indole ring in the IDO1 binding. Series 2 was based on compound PF-0684003 (Figure 1), which is an IDO1 inhibitor developed by Pfizer. The structure of PF-0684003 co-crystalized with IDO1 revealed that there was no interaction between PF-0684003 and the heme iron, which is usually observed between other IDO1 inhibitors and IDO1 [13]. Thus, several potential heme binding ligands, such as hydroxylamine and SO, were added to the structure of PF-0684003 to enhance the inhibitory activity.

Phenylimidazole is a weak IDO1 inhibitor. Crystal structures have revealed that the imidazole ring binds with the heme iron [18,19]. Thus, in series 3 and 4, we used phenylimidazole as the scaffold, and we attached fragments to either the imidazole ring (series 3) or the phenyl ring (series 4). These fragments were intended to improve the binding in the active site of IDO1 through various interactions, such as hydrogen bonds and π–π interactions.

**Figure 2.** Design strategies of the four series of inhibitors. Because the parent compounds of series 1 and 2, indole and PF-0684003, respectively, do not interact with the heme iron, various heme binding ligands were attached to the indole scaffold. The imidazole ring interacts with the heme iron, and thus various fragments were attached to the phenyl ring in series 3 and the imidazole ring in series 4 to strengthen the binding to the active site of IDO1.

#### *2.2. Synthesis of Selected Compounds*

Although the compounds were classified into four series for examining the SAR, the structural diversity meant that they required a variety of synthetic routes. We describe representative synthetic schemes for four compounds. Syntheses for all the compounds are included in the Experimental section.

Scheme 1 shows the synthesis of DX-02-05. In the presence of a Lewis acid, ethyl 5-fluoro-1*H*-indole-2-carboxylate underwent a condensation reaction with 1*H*-pyrrole-2,5-dione to yield compound DX-02-03. Alcohol **1** was obtained by reducing DX-02-03, and then **1** was directly oxidized with MnO2 to produce aldehyde **2**. Aldehyde **2** was condensed with hydroxylamine hydrochloride to obtain compound DX-02-04. Finally, the double bond in DX-02-04 was reduced with NaBH3CN to obtain compound DX-02-05.

**Scheme 1.** Synthesis of DX-02-05. (a) BF3-Et2O, ClCH2CH2Cl, yield = 55%; (b) LiAlH4, CH3OH, yield = 86%; (c) MnO2, dry dichloromethane, yield = 91%; (d) hydroxylamine hydrochloride, DIPEA, CH3OH, yield = 66%; (e) NaBH3CN, 12 N HCl, CH3OH, yield = 43%.

Scheme 2 shows the synthesis of DX-02-07. Sulfur substitution of 5-fluoroindolin-2-one was performed with P2S5 to produce compound **3**. Under basic conditions, compound **4** was obtained through an SN2 substitution reaction with CH3I. A condensation reaction between **4** and 1*H*-pyrrole-2,5-dione was performed at 120 ◦C to obtain sulfide DX-02-06, which was oxidized to racemic sulfoxide DX-02-07.

**Scheme 2.** Synthesis of DX-02-07. (a) P2S5, NaHCO3, dry tetrahydrofuran, yield = 75%; (b) CH3I, K2CO3, acetone, yield = 83%; (c) 1*H*-pyrrole-2,5-dione, CH3CO2H, yield = 61%; (d) m-CPBA, dry dichloromethane, yield = 35%.

Scheme 3 shows the synthesis of DX-03-12. First, the imidazole starting material was protected with trityl chloride to avoid various side reactions. Compound **6** was obtained by the Suzuki–Miyaura reaction with a boronic acid [22]. The methyl group was removed by BBr3 to expose the hydroxyl group in compound **7**. Compound **8** was prepared in parallel by melting 3-chloropropylamine and isobenzofuran-1,3-dione together. Compounds **7** and **8** were subjected to an SN2 nucleophilic reaction to obtain compound **9** under strong basic conditions. This step had a low yield, which was explained by the high steric hindrance of the large molecules and the weak nucleophilicity of compound **7**. The amine protecting group was removed with hydrazine hydrate to obtain compound **10**. Compound **10** was reacted with trifluoromethanesulfonylchloride under basic conditions to give compound **11**. Finally, DX-03-12 was obtained by removing the protecting group on the imidazole with acetic acid.

**Scheme 3.** Synthesis of DX-03-12. (a) Trityl Chloride, DIPEA, dry DMF, yield = 78%; (b) (2-methoxyphenyl)boronic acid, K3PO4, 14% Pd(PPh3)4, DMF, inert atmosphere, yield = 63%; (c) BBr3, inert atmosphere, dry dichloromethane, yield = 65%; (d) solvent-free melt, yield = 55%; (e) KOH, dry DMF, yield = 71%; (f) hydrazine hydrate, CH3OH; (g) trifluoromethanesulfonylchloride, DIPEA, dry dichloromethane, yield = 67%; (h) AcOH, CH3OH, yield = 86%.

Scheme 4 shows the synthesis of DX-04-02. 2-Iodoimidazole was substituted with trifluoromethylbenzyl bromide to give compound **12**. Compound **12** underwent a Suzuki–Miyaura coupling reaction with (5-chloro-2-methoxyphenyl)boronic acid to obtain DX-04-01. Finally, the methyl group was removed with BBr3 to obtain DX-04-02.

**Scheme 4.** Synthesis of DX-04-02. (a) DIPEA, dry DMF; (b) (5-chloro-2-methoxyphenyl)boronic acid, K3PO4, Pd(PPh3)4, DMF, inert atmosphere, yield = 72%; (c) BBr3, inert atmosphere, yield = 53%.

#### *2.3. In Vitro Biological Evaluation*

We prepared the four series of compounds using the synthetic schemes in Section 2.2. Twelve commercially available compounds were purchased (see Section 3.1). In total, we screened 50 compounds in vitro for their IDO1 and TDO inhibitory activities and cell cytotoxicities against tumor cell lines H460 and MCF7 (Table 1). NLG919 was the reported potent IDO1/TDO inhibitor, and Taxol are known commercial anti-cancer drug. They were used as the reference compounds in this study.


**Table 1.** In vitro IDO1/TDO inhibitory activities and cell cytotoxicities of series 1.


**Table 1.** *Cont.*

<sup>a</sup> When the calculated IC50 of a compound is >50 μM, we show the inhibition rate (%) at 50 μM. -: no detectable inhibition. NA: not tested.

The IDO1 and TDO inhibitory activities of the 22 compounds in series 1 were measured (Table 1). The series 1 compounds contained heme binding fragments including CO, NO, CN, H2S, imidazole, and hydroxylamine; however, the inhibition data showed that most compounds in series 1 exhibited marginal IDO1/TDO inhibition. However, four compounds (DX-01-14, DX-01-15, DX-01-16, and DX-01-17) containing hydroxylamine had moderate IDO1 inhibitory activities. The most active IDO1 inhibitor in series 1 was compound DX-01-14 with an IC50 of 11.1 μM. In addition, cell cytotoxicity screening showed that most of the compounds were not toxic to mammalian cancer cell lines.

Generally speaking, these molecules have a rather low molecular weight, and the rather small moiety causes them hard to bind tightly inside the active center of IDO1. Thus, a poor SAR was observed for this series of compounds. Interestingly, for these four active compounds, they all contain the fragment of hydroxylamine. This may suggest that hydroxylamine is the ideal ligand to attach to the double bond of the indole scaffold, while still maintaining the binding with the heme iron.

Series 2 included seven compounds, which were all PF-0684003 derivatives (Table 2). However, the in vitro IDO1/TDO inhibitory activities of all these PF-0684003 derivatives were lower than that of the parent compound, and none were IDO1/TDO inhibitors. The cytotoxicity study demonstrated that these PF-0684003 derivatives were not toxic to cancer cell lines.


**Table 2.** In vitro IDO1/TDO inhibitory activities and cell cytotoxicities of series 2.

<sup>a</sup> When the calculated IC50 of a compound is >50 μM, we show the inhibition rate (%) at 50 μM. -: no detectable inhibition. NA: not tested.

The structure of PF-0684003 co-crystallized with IDO1 shows that PF-0684003 binds tightly in the active site of IDO1 but does not interact with the heme iron [18]. In the present study, the addition of the heme binding ligands [14] (SO and hydroxylamine in DX-02-07 and DX-02-05, respectively) to the double bond of the indole ring abrogated the IDO1 inhibition, similar to the optimization reported in a previous study.

Series 3 contains 13 phenylimidazole derivatives (Table 3). Compounds DX-03-01 to DX-03-10 showed marginal IDO1/TDO inhibition and weak cell cytotoxicities. However, compounds DX-03-11, DX-03-12, and DX-03-13 are potent IDO1 inhibitors with IC50 values of 0.3–2.4 μM. Interestingly, these three compounds show marginal TDO inhibition activity, and thus are IDO1/TDO selective inhibitors. In addition, these compounds are not toxic to mammalian cancer cell lines.

Compound DX-03-01 is a known compound [18], which binds to the active site in IDO1. The imidazole ring interacts with the heme iron, and the hydroxyl group binds to the nearby Ser167 in the active site [7]. However, the substitution of the hydroxyl group with hydroxylamine (DX-03-01 to DX-03-10) did not produce IDO1 inhibitors with higher activity. In DX-03-10 to DX-03-13, the hydroxyl group was used to attach an alkyl chain bearing terminal groups used in other reported IDO1 inhibitors [17,19]. The terminal fragments, such as sulfamide and urea, are important to maintain the binding [17,19].


**Table 3.** In vitro IDO1/TDO inhibitory activities and cell cytotoxicity of series 3.

<sup>a</sup> When the calculated IC50 of a compound is >50 μM, we show the inhibition rate (%) at 50 μM. -: no detectable inhibition. NA: not tested.

Series 4 contains eight phenylimidazole derivatives (Table 4). Most compounds in this series showed marginal IDO1 inhibition, but compound DX-04-05 showed moderate IDO1 inhibition. However, compound DX-04-06, in which the hydroxyl group was methylated, showed decreased IDO1 inhibition. Most compounds in this series were toxic to the cancer cell lines H460 and MCF7 with an IC50 of around 10−<sup>5</sup> M, which indicates the compounds also interrupt the function of other targets in the cells.


**Table 4.** In vitro IDO1/TDO inhibitory activities and cell cytotoxicities of series 4.

<sup>a</sup> When the calculated IC50 of a compound is >50 μM, we show the inhibition rate (%) at 50 μM. -: no detectable inhibition. NA: not tested.

In series 4, the design strategy was to attach fragments to the imidazole ring instead of the phenyl ring in series 3. However, this strategy does not maintain the IDO1 inhibitory activity. In addition, the free hydroxyl group in series 4 appears to be important for forming a hydrogen bond with an amino acid residue, such as Ser167 [7], in the active site; methylating this hydroxyl group decreased IDO1 inhibition (DX-04-05 versus DX-04-06).

In summary, based on the SAR study, compounds DX-03-12 and DX-03-13 showed potent IDO1 inhibition activity, and compound DX-03-13 has some structure similarity as previous published patent (WO2011/056652Al) [23]. Thus, the most potent IDO1 inhibitor DX-03-12 was used as the lead compound for further study.

#### *2.4. Pharmacokinetic Study of DX-03-12*

The in vivo pharmacokinetic properties of compound DX-03-12 were evaluated in male ICR mice. DX-03-12 was administered orally either as a bolus at a dose of 30 mg/kg or intravenously at a dose of 3 mg/kg. The plasma concentration-time profiles are shown in Figure 3. The pharmacokinetic parameters were determined by non-compartmental analysis (Table 5). After intravenous administration (3 mg/kg), compound DX-03-12 showed a moderate clearance of 33.3 mL/min/kg, which was approximately 36% of mouse hepatic blood flow (90 mL/min/kg). The volume of distribution in the steady state was 10 L/kg, suggesting that there was an extensive distribution of compound DX-03-12 in tissues. Compound DX-03-12 was rapidly absorbed after oral administration of 30 mg/kg. The plasma concentration was quantifiable at the first sampling time point (5 min) and remained detectable at 24 h. The maximum plasma concentration of compound DX-03-12 of 2963 ng/mL was observed 1 h post-dose. Compound DX-03-12 was eliminated relatively slowly with a half-life of 4.6 h. The oral bioavailability of compound DX-03-12 was calculated to be 96%.

The pharmacokinetic study revealed that compound DX-03-12 showed rapid absorption and almost complete oral bioavailability in mice. After oral administration of 30 mg/kg in mice, it exhibited a long half-life (4.6 h) due to moderate plasma clearance. The pharmacokinetic properties of compound DX-03-12 in mice indicate that it has the potential to be a once-a-day drug.

**Table 5.** Pharmacokinetic parameters of DX-03-12 after a single oral or intravenous dose in male ICR mice.

**Figure 3.** Mean plasma concentration-time profile in male ICR mice after administration of a single oral (30 mg/kg) or intravenous (3 mg/kg) dose of compound DX-03-12. PO stands for per os, which means oral administration of drug; iv stands for intravenous administration of drug.

#### *2.5. In Vivo Anti-Tumor E*ffi*cacy Study of Compound DX-03-12*

To determine whether the compound was effective against tumors in vivo, the anti-tumor effect of DX-03-12 was evaluated in a B16F10 subcutaneous xenograft model in syngeneic mice (Figure 4). Oral and intraperitoneal (i.p.) administration (30 or 60 mg/kg/day) of DX-03-12 significantly decreased the growth of melanoma B16F10 xenograft tumors in a dose-dependent manner. DX-03-12 oral treatment resulted in a 72.2% decrease in tumor weight and i.p. treatment resulted in a 72.3% decrease at a dose of 60 mg/kg compared with the control tumor after 19 days treatment. DX-03-12 had no obvious effect on the body weight of the mice and the peripheral white blood cells, which indicated that DX-03-12 had low toxicity in mice. In this study, the classic first line anti-cancer drug cyclophosphamide (CTX) was used as the reference drug.

**Figure 4.** In vivo anti-tumor activity of DX-03-12 in B16F10 xenograft mice. (**A**,**B**) Tumors from each group after 19 days of treatment. Mice bearing B16F10 xenografts were dosed orally or i.p. with vehicle (0.5% methylcellulose), CTX (Cyclophosphamide, orally, 60 mg/kg twice a week), DX-03-12 (i.p., 30 mg/kg/day), DX-03-12 (i.p., 60 mg/kg/day), DX-03-12 (orally, 30 mg/kg/day), or DX-03-12 (orally, 60 mg/kg/day). Results are expressed as the mean ± SD (*n* = 5–7 for each group). \* *p* < 0.05 and \*\*\* *p* < 0.001 versus vehicle. (**C**) The body weight from each group after the treatment. There is no obvious body weight difference among all of the groups. (**D**) The white blood cells detected in the blood of all groups of animals. There is also no obvious WBC difference among all of the groups.

#### **3. Experiment Section**

#### *3.1. Chemistry*

Reagents and solvents were obtained from commercial suppliers and used as received. 1H-NMR spectra were obtained on an NMR spectrometer (Mercury, Varian, San Diego, CA, USA; 400 MHz). Electrospray ionization (ESI) mass spectra and high-resolution mass spectroscopy (HRMS) were performed with a liquid chromatograph/mass selective detector time-of-flight mass spectrometer

(LC/MSD TOF, Agilent Technologies, Santa Clara, CA, USA). silica gel column chromatography was performed with silica gel 60G (Qingdao Haiyang Chemical, Qingdao, China). Purity was determined using HPLC, LC/MS and NMR spectroscopy. All of the synthesized compounds have the purity over than 95%.

Several commercial available compounds were purchased from Beijing innochem Co. Ltd. (Beijing, China). They are DX-01-01, DX-01-08, DX-01-09, DX-01-10, DX-01-11, DX-01-12, DX-01-18, DX-01-19, DX-01-20, DX-01-21, DX-01-22, DX-03-01.

3.1.1. Preparation of (*R*/*S*) 3-(5-fluoro-2-((hydroxyamino)methyl)-1*H*-indol-3-yl)pyrrolidine-2,5-dione (DX-02-05)

Preparation of (*R*/*S*) Ethyl 3-(2,5-dioxopyrrolidin-3-yl)-5-fluoro-1*H*-indole-2-carboxylate (DX-02-03)

Ethyl 5-fluoro-1*H*-indole-2-carboxylate (2 g, 9.7 mmol) and 1*H*-pyrrole-2,5-dione (1.40 g, 14.4 mmol) were added to dry 1,2-dichloroethane (50 mL), and 46.5% BF3-Et2O (3.54 g, 11.6 mmol) was added to reaction mixture at 20 ◦C. After the reaction mixture was stirred at 90 ◦C, until the starting material disappeared in thin layer chromatography (TLC), the reaction mixture was distilled in vacuo, and dichloromethane (50 mL) was added to the mixture. The organic extract was washed twice with saturated aqueous NaHCO3 and NaCl (20 mL) respectively, and the organic extract was dried over Na2SO4. After solvent was removed under vacuum, the product was purified as a white solid by silica gel column chromatography, 1.62 g, yield 55%. 1H-NMR (400 MHz, DMSO-*d*6): δ = 12.00 (s, 1H, NH-indolyl), 11.31 (s, 1H, NH), 7.49–7.45 (m, 2H, H-indolyl), 7.18 (td, *J* = 9.2, 2.6 Hz, 1H, H-indolyl), 4.82 (dd, *J* = 9.6, 6.6 Hz, 1H, CH), 4.33–4.27 (m, 2H, CH2), 3.08 (dd, *J* = 17.7, 9.6 Hz, 1H, CH'H"), 2.70 (dd, *J* = 17.7, 6.6 Hz, 1H, CH'H"), 1.30 (t, *J* = 7.1 Hz, 3H, CH3). 13C-NMR (101 MHz, CD3OD): δ =182.17, 180.15, 162.66, 160.66, 134.24, 129.01, 126.54, 119.11, 115.66, 114.82, 104.78, 62.19, 39.68, 39.37, 14.63. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C15H14O4N2F: 305.09321; found: 305.09290.

Preparation of (*R*/*S*) 3-(5-fluoro-2-(hydroxymethyl)-1*H*-indol-3-yl) pyrrolidine-2,5-dione (**1**)

Ethyl 3-(2,5-dioxopyrrolidin-3-yl)-5-fluoro-1*H*-indole-2-carboxylate (DX-02-03, 1.3 g, 4.3 mmol) were added to dry CH3OH (20 mL), LiAlH4 (0.13 g, 4.3 mmol) was added to reaction mixture gradually at 20 ◦C. The reaction mixture was stirred at 20 ◦C until the starting material disappeared in TLC. The reaction mixture was distilled in vacuo, and ethyl acetate (50 mL) was added to the mixture. The organic extract was washed twice with saturated aqueous NaCl (20 mL), and the organic extract was dried over Na2SO4. After solvent was removed under vacuum, the product was purified as a white solid by silica gel column chromatography, 0.97 g, yield 86%. 1H-NMR (400 MHz, CD3OD): δ = 7.30 (dd, *J* = 8.8, 4.4 Hz, 1H, H-phenyl), 6.93 (dd, *J* = 9.9, 2.4 Hz, 1H, H-phenyl), 6.89–6.83 (m, 1H, H-phenyl), 4.72 (d, *J* = 1.3 Hz, 2H, CH2), 4.46 (dd, *J* = 9.7, 5.6 Hz, 1H, CH), 3.20 (dd, *J* = 18.4, 9.7 Hz, 1H, CH'H"), 2.81 (dd, *J* = 18.4, 5.6 Hz, 1H, CH'H"). 13C-NMR (101 MHz, CD3OD): δ = 182.67, 180.16, 160.22, 157.85, 139.57, 133.85, 127.82, 113.46, 110.70, 103.45, 56.67, 39.93, 39.00. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C13H12N2O3F: 263.08265; found: 263.08160.

Preparation of (*R*/*S*) 3-(2,5-dioxopyrrolidin-3-yl)-5-fluoro-1*H*-indole-2-carbaldehyde (**2**)

3-(5-Fluoro-2-(hydroxymethyl)-1*H*-indol-3-yl)pyrrolidine-2,5-dione (**1**, 0.8 g, 3.1 mmol) was added to dry dichloridemethane (20 mL), MnO2 (0.36 g, 4.1 mmol) was added to reaction mixture gradually at 20 ◦C. The reaction mixture was stirred at 20 ◦C until the starting material disappeared in TLC. The reaction mixture was filtered, the solvent was removed under vacuum, and the product was purified as a white solid by silica gel column chromatography, 0.73g, yield 91%. 1H-NMR (400 MHz, CD3OD): δ = 10.00 (s, 1H, CHO), 7.50 (dd, *J* = 9.1, 4.3 Hz, 1H, H-phenyl), 7.27 (dd, *J* = 9.5, 2.3 Hz, 1H, H-phenyl), 7.19 (td, *J* = 9.1, 2.4 Hz, 1H, H-phenyl), 4.91 (dd, *J* = 9.7, 6.0 Hz, 1H, CH), 3.29–3.21 (m, 1H, CH'H"), 2.84 (dd, *J* = 18.1, 6.0 Hz, 1H, CH'H"). 13C-NMR (101 MHz, CD3OD): δ = 183.66,

181.23, 179.66, 160.59, 158.30, 135.75, 135.36, 127.61, 117.26, 115.66, 105.59, 39.77, 39.35. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C13H10N2O3F: 261.06700; found: 261.06638.

Preparation of (*R*/*S*) (*E*)-3-(2,5-dioxopyrrolidin-3-yl)-5-fluoro-1*H*-indole-2-carbaldehyde oxime (DX-02-04)

3-(2,5-Dioxopyrrolidin-3-yl)-5-fluoro-1*H*-indole-2-carbaldehyde (0.5 g, 1.92 mmol) was added to dry CH3OH (30 mL), then *N,N*-Diisopropylethylamine (0.5 g, 3.84 mmol) and hydroxylamine hydro -chloride (0.13 g, 1.92 mmol) were added respectively at 20 ◦C. The reaction mixture was stirred at 60 ◦C until the starting material disappeared in TLC. After solvent was removed under vacuum, the product was purified as a white solid by silica gel column chromatography, 0.35 g, yield 66%. 1H-NMR (400 MHz, CD3OD): δ = 8.22 (s, 1H, =CH), 7.32 (dd, *J* = 8.8, 4.4 Hz, 1H, H-indolyl), 7.06–6.86 (m, 2H, H-indolyl), 4.63 (dd, *J* = 9.7, 5.9 Hz, 1H, CH), 3.19 (dd, *J* = 18.3, 9.7 Hz, 1H, CH'H"), 2.81 (dd, *J* = 18.7, 6.2 Hz, 1H, CH'H"). 13C-NMR (101 MHz, CD3OD): δ =181.92, 179.89, 160.31, 157.98, 141.03, 136.51, 134.81, 132.30, 113.78, 112.58, 103.95, 39.83, 38.66. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C13H11N3O3F: 276.07790; found: 276.07718.

Preparation of (*R*/*S*) 3-(5-fluoro-2-((hydroxyamino)methyl)-1*H*-indol-3-yl) pyrrolidine-2,5-dione (DX-02-05)

3-(2,5-Dioxopyrrolidin-3-yl)-5-fluoro-1*H*-indole-2-carbaldehyde oxime (DX-02-04, 0.3 g, 1.1 mmol) was added to CH3OH (20 mL), then NaBH3CN (0.07 g, 1.1 mmol) was added at 20 ◦C. 12N HCl was added to reaction mixture continuously to keep acidic. The reaction mixture was stirred at 20 ◦C until the starting material disappeared in TLC. After solvent was removed under vacuum, the product was purified as a white solid by silica gel column chromatography, 0.13 g, yield 43%. 1H-NMR (400 MHz, CD3OD): δ = 7.30 (dd, *J* = 8.8, 4.5 Hz, 1H, H-indolyl), 6.93–6.88 (m, 1H, H-indolyl), 6.88–6.82 (m, 1H, H-indolyl), 4.50 (dd, *J* = 9.7, 5.5 Hz, 1H, CH), 4.12 (d, *J* = 3.6 Hz, 2H, CH2NH), 3.21 (dd, *J* = 18.5, 9.7 Hz, 1H, CH'H"), 2.85 (dd, *J* = 18.5, 5.5 Hz, 1H, CH'H"). 13C-NMR (101 MHz, CD3OD): δ = 160.27, 157.92, 135.20, 130.85, 130.02, 113.00, 110.85, 110.54, 104.55, 104.31, 50.48, 38.34, 30.37. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C13H13N3O3F: 278.09355; found: 278.09291.

3.1.2. Preparation of (*R*/*S*) 3-(5-fluoro-2-(methylsulfinyl)-1*H*-indol-3-yl)pyrrolidine-2,5-dione (DX-02-07)

#### Preparation of 5-fluoroindoline-2-thione (**3**)

5-fluoroindolin-2-one (2 g, 13 mmol) and P2S5 (2.92 g, 13 mmol) were added to dry tetrahydrofuran (30 mL). After the reaction mixture was stirred at room temperature for 1 h. Until the starting material disappeared in thin layer chromatography (TLC), the NaHCO3 (3.43 g, 39 mmol) was added gradually. After stirring at room temperature for 1 h, the reaction mixture was distilled in vacuo, dichloromethane (50 mL) was added to the mixture. The organic extract was washed twice with saturated aqueous NaCl (20 mL), and the organic extract was dried over Na2SO4. After solvent was removed under vacuum, the product was obtained as yellow crystals by re-crystallization using ethanol, 1.62 g, yield 75%. 1H-NMR (400 MHz, CDCl3): δ = 10.14 (s, 1H, NH), 7.05–6.95 (m, 1H, H-phenyl), 6.94–6.89 (m, 1H, H-phenyl), 4.08 (s, 1H, CH2). 13C-NMR (101 MHz, CDCl3)): δ = 203.39, 159.10, 140.24, 132.15, 114.87, 112.47, 110.47, 49.15. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C8H7NFS: 168.02777; found: 168.02773.

Preparation of 5-fluoro-2-(methylthio)-1*H*-indole (**4**)

5-fluoroindoline-2-thione (**3**, 1 g, 6 mmol), CH3I (0.99 g, 7 mmol) and K2CO3 (0.97 g, 7 mmol) were added to acetone (30 mL), and the reaction mixture was stirred at room temperature until the starting material disappeared in TLC. The reaction mixture was distilled in vacuo, ethyl acetate (50 mL) was added to the mixture. The organic extract was washed twice with saturated aqueous NaCl (20 mL), and the organic extract was dried over Na2SO4. After solvent was removed under vacuum, the product was purified as a white solid by silica gel column chromatography, 0.90 g, yield 83%. 1H-NMR (400 MHz, CDCl3): δ = 8.04 (s, 1H, NH), 7.23–7.13 (m, 2H, H-indolyl), 6.91 (s, 1H, H-indolyl), 6.49 (s, 1H, H-indolyl), 2.52 (s, 3H, CH3). 13C-NMR (101 MHz, CDCl3): δ = 159.18, 156.85, 133.57, 129.02, 110.94, 110.47, 105.31, 104.73, 18.86. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C9H9NFS: 182.04342; found: 182.04425.

Preparation of (*R*/*S*) 3-(5-fluoro-2-(methylthio)-1*H*-indol-3-yl)pyrrolidine-2,5-dione (DX-02-06)

5-Fluoro-2-(methylthio)-1*H*-indole (**4**, 0.5 g, 2.7 mmol) and 1*H*-pyrrole-2,5-dione (0.54 g, 5.4 mmol) were added to dry CH3CO2H (30 mL), and the reaction mixture was stirred at 120 ◦C until the starting material disappeared in TLC. The reaction mixture was distilled in vacuo, ethyl acetate (100 mL) was added to the mixture. The organic extract was washed twice with saturated aqueous NaCl (30 mL), and the organic extract was dried over Na2SO4. After solvent was removed under vacuum, the product was purified as a white solid by silica gel column chromatography, 0.47 g, yield 61%. 1H-NMR (400MHz, CD3OD): δ = 7.28 (dd, *J* = 8.9, 4.5 Hz, 1H, H-phenyl), 7.00 (dd, *J* = 9.7, 2.4 Hz, 1H, H-phenyl), 6.91 (td, *J* = 9.2, 2.5 Hz, 1H, H-phenyl), 4.56 (dd, *J* = 9.8, 5.6 Hz, 1H, CH), 3.22 (dd, *J* = 18.3, 9.8 Hz, 1H, CH'H"), 2.77 (dd, *J* = 18.3, 5.6 Hz, 1H, CH'H"), 2.41 (s, 3H, CH3). 13C-NMR (101 MHz, CD3OD): δ = 182.38, 180.11, 160.26, 135.05, 132.13, 127.67, 116.34, 113.18, 111.70, 103.25, 40.35, 39.10, 19.59. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C13H12O2N2FS: 279.05980; found: 279.05930.

Preparation of (*R*/*S*) 3-(5-fluoro-2-(methylsulfinyl)-1*H*-indol-3-yl)pyrrolidine-2,5-dione (DX-02-07)

3-(5-Fluoro-2-(methylthio)-1*H*-indol-3-yl)pyrrolidine-2,5-dione (DX-02-06, 0.2 g, 0.7 mmol) was added to dry dichloridemethane (20 mL), and 3-Chloroperoxybenzoic acid (0.15 g, 0.9 mmol) was added gradually at 0 ◦C. The reaction mixture was stirred at 0 ◦C until the starting material disappeared in TLC, and Na2S2O3 (0.11 g, 0.7 mmol) was added to the mixture. After reaction mixture was stirred for 20 min, the reaction mixture was washed twice with saturated aqueous NaCl (20 mL), and the organic extract was dried over Na2SO4. After solvent was removed under vacuum, the product was purified as a white solid by silica gel column chromatography, 0.074 g, yield 35%. 1H-NMR (400MHz, DMSO-*d*6): δ = 12.28 (s, 1H, NH), 11.51 (s, 1H, NH), 7.51–7.48 (m, 1H, H-phenyl), 7.19–7.13 (m, 2H, H-phenyl), 4.61 (ddd, *J* = 26.8, 9.7, 5.8 Hz, 1H, CH), 3.18 (ddd, *J* = 18.0, 9.7, 2.3 Hz, 1H, CH'H"), 2.98 (d, *J* = 16.7 Hz, 3H, CH3), 2.76 (ddd, *J* = 41.8, 18.1, 5.8 Hz, 1H, CH'H"). 13C-NMR (101 MHz, DMSO-d6): δ = 179.74, 177.94, 158.72, 138.29, 134.14, 125.83, 114.50, 113.93, 113.53, 104.46, 41.67, 38.62, 38.11. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C13H12O3N2FS: 295.05472; found: 295.05508.

3.1.3. Preparation of *N*-(3-(2-(1*H*-imidazol-5-yl)phenoxy)propyl)-1,1,1-trifluoromethane sulfonamide (DX-03-12)

Preparation of 4-iodo-1-trityl-1*H*-imidazole (**5**)

4-Iodo-1*H*-imidazole (5 g, 25.8 mmol) and *N,N*-diisopropylethylamine (6.65 g, 51.5 mmol) were added to dry dimethylformamide (30 mL), and (chloromethanetriyl)tribenzene (7.89 g, 28.4 mmol) was added to reaction mixture gradually at 20 ◦C. After the reaction mixture was stirred at 20 ◦C until the starting material disappeared in thin layer chromatography (TLC), the reaction mixture was distilled in vacuo, ethyl acetate (200 mL) was added to the mixture. The organic extract was washed twice with saturated aqueous NaCl (40 mL), and the organic extract was dried over Na2SO4. After solvent was removed under vacuum, the product was purified as a white solid by silica gel column chromatography, 8.77 g, yield 78%. 1H-NMR (400 MHz, CDCl3): δ = 7.38–7.33 (m, 10H, H-phenyl, H-imidazolyl), 7.13–7.09 (m, 6H, H-phenyl), 6.92 (d, *J* = 1.4 Hz, 1H, H-imidazolyl). 13C-NMR (101 MHz, CDCl3): δ = 141.83, 140.56, 129.72 (6C), 128.29 (3C), 128.18 (6C), 127.91, 126.89 (3C), 75.84. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C22H18N2I: 437.05092; found: 437.04934.

#### Preparation of 4-(2-methoxyphenyl)-1-trityl-1*H*-imidazole (**6**)

4-Iodo-1-trityl-1*H*-imidazole (**5**, 2 g, 4.58 mmol), (5-chloro-2-methoxyphenyl)boronic acid (0.852 g, 4.58 mmol), K3PO4 (2.9 g, 13.7 mmol), Pd(PPh3)4 (0.74 g, 0.64 mmol) were added to dry dimethylformamide (30 mL). The reaction mixture was stirred at 100 ◦C under inert atmosphere, until the starting material disappeared in TLC. The reaction mixture was distilled in vacuo, and ethyl acetate (100 mL) was added to the mixture. The organic extract was washed twice with saturated aqueous NaCl (20 mL), and the organic extract was dried over Na2SO4. After solvent was removed under vacuum, the product was purified as a white solid by silica gel column chromatography, 1.2 g, yield 63%. 1H-NMR (400 MHz, CDCl3): δ = 7.51–7.32 (m, 12H, H-phenyl, H-imidazolyl), 7.15–7.04 (m, 8H, H-phenyl, H-imidazolyl), 6.94 (d, *J* = 8.1 Hz, 1H, H-phenyl), 3.99 (s, 3H, CH3). HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C29H25N2O: 417.19614; found: 417.19647.

#### Preparation of 2-(1-trityl-1*H*-imidazol-4-yl)phenol (**7**)

4-(2-Methoxyphenyl)-1-trityl-1*H*-imidazole (**6**, 1.0 g, 2.4 mmol) was added to dry dichloridemethane (20 mL), BBr3 (0.71 g, 2.9 mmol) was added to reaction mixture gradually at −70 ◦C. The reaction mixture was stirred under inert atmosphere until the starting material disappeared in TLC. The reaction mixture was diluted with dichloridemethane (50 mL), and was washed twice with saturated aqueous NaCl (20 mL), then the organic extract was dried over Na2SO4. After solvent was removed under vacuum, the product was purified as a white solid by silica gel column chromatography, 0.63 g, yield 65%. 1H-NMR (400 MHz, DMSO-*d*6): δ = 11.19 (s, 1H, OH), 7.49–7.36 (m, 2H, H-phenyl), 7.33–7.10 (m, 16H, H-phenyl, H-imidazolyl), 6.84–6.70 (m, 2H, H-phenyl), 6.45 (s, 1H, H-phenyl). 13C-NMR (101 MHz, DMSO-*d*6): δ = 147.24, 141.47, 137.70, 136.55, 128.71 (6C), 127.80 (6C), 127.56, 127.24 (3C), 126.99 (3C), 126.10, 118.39, 117.87, 115.68, 74.61. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C28H23N2O: 403.18049; found: 403.18134.

#### Preparation of 2-(3-chloropropyl)isoindoline-1,3-dione (**8**)

3-Chloropropan-1-amine (3.77 g, 40.5 mmol) and isobenzofuran-1,3-dione (2 g, 13.5 mmol) were mixed without solvent, and the reaction mixture was stirred at 140 ◦C (molten condition), until the starting material disappeared in TLC. The reaction mixture was distilled in vacuo, ethyl acetate (100 mL) was added to the mixture. The organic extract was washed twice with saturated aqueous NaCl (20 mL), and the organic extract was dried over Na2SO4. After solvent was removed under vacuum, the product was purified as a white solid by silica gel column chromatography, 1.66 g, yield 55%. 1H-NMR (400 MHz, DMSO-*d*6): δ = 7.85–7.78 (m, 4H, H-phenyl), 3.73–3.62 (m, 4H, CH2, CH2), 2.08–2.01(m, 2H, CH2). 13C-NMR (101 MHz, DMSO-*d*6): δ = 167.38 (2C), 133.75 (2C), 131.19 (2C), 122.43 (2C), 42.31, 34.60, 30.39. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C11H11ClNO2: 224.04728; found: 224.04683.

#### Preparation of 2-(3-(2-(1-trityl-1*H*-imidazol-5-yl)phenoxy)propyl)isoindoline-1,3-dione (**9**)

2-(1-Trityl-1*H*-imidazol-4-yl)phenol (**7**, 0.5 g, 1.2 mmol) and KOH (0.14 g, 2.5 mmol) were added to dry dimethylformamide (20 mL), after the reaction mixture was stirred at 20 ◦C for 15 min, 2-(3-chloropropyl)isoindoline-1,3-dione (**8**, 0.294 g, 1.32 mmol) was added to reaction mixture at 20 ◦C. After the reaction mixture was stirred at 60 ◦C until the starting material disappeared in thin layer chromatography (TLC), the reaction mixture was distilled in vacuo, ethyl acetate (100 mL) was added to the mixture. The organic extract was washed twice with saturated aqueous NaCl (20 mL), and the organic extract was dried over Na2SO4. After solvent was removed under vacuum, the product was purified as a white solid by silica gel column chromatography, 0.5 g, yield 71%. 1H-NMR (400 MHz, DMSO-*d*6): δ = 8.08 (dd, *J* = 7.7, 1.6 Hz, 1H, H-phenyl), 7.88–7.81 (m, 4H, H-phenyl, H-imidazolyl), 7.44–7.35 (m, 10H, H-phenyl), 7.17–7.13 (m, 7H, H-phenyl, H-imidazolyl), 7.12–7.08 (m, 1H, H-phenyl), 6.97 (t, *J* = 7.5 Hz, 1H, H-phenyl), 6.92 (d, *J* = 8.2 Hz, 1H, H-phenyl), 3.93 (t, *J* = 6.4 Hz, 2H, CH2), 3.43 (t, *J* = 6.5 Hz, 2H, CH2), 1.82–1.69 (m, 2H, CH2). 13C-NMR (101 MHz, DMSO-*d*6): δ = 167.25, 154.09,

141.77, 137.17, 134.72, 133.82, 131.11, 128.71 (6C), 128.65 (3C), 127.69, 127.66 (6C), 127.46, 127.36 (3C), 126.71, 125.92, 122.47, 121.86, 121.01, 120.60, 119.93, 111.33, 74.14, 64.42, 33.61, 27.54. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C39H32N3O3: 590.24382; found: 590.24541.

Preparation of 3-(2-(1-trityl-1*H*-imidazol-4-yl)phenoxy)propan-1-amine (**10**)

2-(3-(2-(1-Trityl-1*H*-imidazol-4-yl)phenoxy)propyl)isoindoline-1,3-dione (**9**, 0.4 g, 0.68 mmol) and 35% hydrazine hydrate (0.12 g, 1.35 mmol) were added to CH3OH, and the reaction mixture was stirred at 50 ◦C under inert atmosphere. After the starting material disappeared in thin layer chromatography (TLC), the reaction mixture was distilled in vacuo, ethyl acetate (100 mL) was added to the mixture. The organic extract was washed twice with saturated aqueous NaHCO3 and NaCl (20 mL) respectively, and the organic extract was dried over Na2SO4. After solvent was removed under vacuum, the unpurified product was added to the next step directly.

Preparation of 1,1,1-trifluoro-*N*-(3-(2-(1-trityl-1*H*-imidazol-4-yl)phenoxy)propyl) Methane Sulfonamide (**11**)

3-(2-(1-Trityl-1*H*-imidazol-4-yl)phenoxy)propan-1-amine (crude **10**, 0.3 g, 0.65 mmol) and *N,N*-diisopropylethylamine (0.168 g, 1.3 mmol) were added to dry dichloridemethane (20 mL), and trifluoromethanesulfonylchloride (0.12 g, 0.71 mmol) was added to reaction mixture gradually at 20 ◦C. After the starting material disappeared in thin layer chromatography (TLC), the reaction mixture was distilled in vacuo, the reaction mixture was diluted with dichloridemethane (50 mL), and the organic extract was washed twice with saturated aqueous NaCl (20 mL), and the organic extract was dried over Na2SO4. After solvent was removed under vacuum, the product **11** was purified as a white solid by silica gel column chromatography, 0.257 g, yield 67%. 1H-NMR (400 MHz, CDCl3): δ = 7.80 (s, 1H, NH), 7.43–7.32 (m, 10H, H-phenyl, H-imidazolyl), 7.23–7.16 (m, 8H, H-phenyl, H-imidazol), 7.07 (d, *J* = 1.5 Hz, 1H, H-phenyl), 6.97–6.86 (m, 2H, H-phenyl), 4.16 (t, *J* = 5.2 Hz, 2H, CH2), 3.57–3.46 (m, 2H, CH2), 2.19–2.06 (m, 2H, CH2). 13C-NMR (101 MHz, CDCl3): δ= 154.95, 142.08, 139.92, 138.22, 129.77 (6C), 128.56 (3C), 128.36, 128.15 (3C), 128.09 (6C), 121.68, 120.87, 119.04, 118.47, 111.86, 75.75, 67.83, 43.24, 29.80. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C32H29N3O3F3S: 592.18762; found: 592.18616.

Preparation of *N*-(3-(2-(1*H*-imidazol-5-yl)phenoxy)propyl)-1,1,1-trifluoromethane Sulfonamide (DX-03-12)

1,1,1-Trifluoro-*N*-(3-(2-(1-trityl-1*H*-imidazol-4-yl)phenoxy)propyl)methanesulfonamide (**11**, 0.2 g, 0.34 mmol) and AcOH (0.5 mL) were added to CH3OH (10 mL), and the reaction mixture was stirred at 20 ◦C. After the starting material disappeared in thin layer chromatography (TLC), the reaction mixture was distilled in vacuo, the product was purified as a white solid by silica gel column chromatography, 0.101 g, yield 86%. 1H-NMR (400 MHz, DMSO-*d*6): δ =7.99 (d, *J* = 7.4 Hz, 1H, H-phenyl), 7.72 (s, 1H, H-imidazolyl), 7.49 (s, 1H, H-imidazolyl), 7.18 (t, *J* = 7.8 Hz, 1H, H-phenyl), 7.03 (d, *J* = 8.1 Hz, 1H, H-phenyl), 6.98 (t, *J* = 7.5 Hz, 1H, H-phenyl), 4.15 (t, *J* = 6.0 Hz, 2H, CH2), 3.40 (t, *J* = 6.8 Hz, 2H, CH2), 2.06–2.09 (m, 2H, CH2). 13C-NMR (101 MHz, DMSO-*d*6): δ = 154.86, 135.47, 134.49, 127.46, 127.25, 124.99, 122.69, 120.97, 118.57, 112.34, 65.09, 41.39, 30.14. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C13H15O3N3F3S: 350.07807; found: 350.07782.

3.1.4. Preparation of 4-chloro-2-(1-(4-(trifluoromethyl)benzyl)-1*H*-imidazol-4-yl) Phenol (DX-04-02)

Preparation of 4-iodo-1-(4-(trifluoromethyl)benzyl)-1*H*-imidazole (**12**)

4-Iodo-1*H*-imidazole (1 g, 5.2 mmol) and *N,N*-diisopropylethylamine (1.34 g, 10.4 mmol) were added to dry dimethylformamide (20 mL), and 1-(bromomethyl)-4-(trifluoromethyl)benzene (1.48 g, 6.2 mmol) was added to reaction mixture at 20 ◦C. After the reaction mixture was stirred at 20 ◦C until the starting material disappeared in thin layer chromatography (TLC), the reaction mixture was distilled in vacuo, dichloromethane (50 mL) was added to the mixture. The organic extract was washed

twice with saturated aqueous NaHCO3 and NaCl (20 mL) respectively, and the organic extract was dried over Na2SO4. After solvent was removed under vacuum, the unpurified product was added to the next step directly.

#### Preparation of 4-(5-chloro-2-methoxyphenyl)-1-(4-(trifluoromethyl)benzyl)-1*H*-imidazole (DX-04-01)

4-Iodo-1-(4-(trifluoromethyl)benzyl)-1*H*-imidazole (crude **12**, 0.5 g, 1.4 mmol), (5-chloro-2 methoxyphenyl)boronic acid (0.27 g, 1.4 mmol), K3PO4 (0.9 g, 4.3 mmol), Pd(PPh3)4 (0.23 g, 0.2 mmol) were added to dry dimethylformamide (10 mL). The reaction mixture was stirred at 100 ◦C under inert atmosphere, until the starting material disappeared in TLC. The reaction mixture was distilled in vacuo, ethyl acetate (50 mL) was added to the mixture. The organic extract was washed twice with saturated aqueous NaCl (20 mL), and the organic extract was dried over Na2SO4. After solvent was removed under vacuum, DX-04-01 was purified as a white solid by silica gel column chromatography, 0.37 g, yield 72%. 1H-NMR (400 MHz, CD3OD): δ = 7.94 (d, *J* = 2.5 Hz, 1H, H-phenyl), 7.81 (s, 1H, H-imidazolyl), 7.65–7.63 (m, 3H, H-imidazolyl, H-phenyl), 7.40 (d, *J* = 8.0 Hz, 2H, H-phenyl), 7.15 (dd, *J* = 8.7, 2.5 Hz, 1H, H-phenyl), 6.96 (d, *J* = 8.8 Hz, 1H, H-phenyl), 5.34 (s, 2H, CH2), 3.86 (s, 3H, CH3). 13C-NMR (101 MHz, CD3OD): δ = 156.09, 142.76, 138.77, 137.13, 131.46, 129.05 (2C), 128.22, 127.44, 126.92 (2C), 124.85, 124.18, 121.78, 113.52, 112.48, 56.17, 51.22. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated forC18H15ON2ClF3: 367.08195; found: 367.08374.

#### Preparation of 4-chloro-2-(1-(4-(trifluoromethyl)benzyl)-1*H*-imidazol-4-yl)phenol (DX-04-02)

4-(5-Chloro-2-methoxyphenyl)-1-(4-(trifluoromethyl)benzyl)-1*H*-imidazole (DX-04-01, 0.3 g, 0.82 mmol) was added to dry dichloridemethane (20 mL), BBr3 (0.24 g, 0.98 mmol) was added to reaction mixture gradually at −70 ◦C. The reaction mixture was stirred under inert atmosphere until the starting material disappeared in TLC. The reaction mixture was diluted with dichloridemethane (50 mL), and was washed twice with saturated aqueous NaCl (20 mL), then the organic extract was dried over Na2SO4. After solvent was removed under vacuum, DX-04-02 was purified as a white solid by silica gel column chromatography, 0.15 g, yield 53%. 1H-NMR (400 MHz, CD3OD): δ = 7.85 (s, 1H, H-imidazolyl), 7.70–7.65 (m, 3H, H-imidazolyl, H-phenyl), 7.63 (d, *J* = 2.6 Hz, 1H, H-phenyl), 7.45 (d, *J* = 8.1 Hz, 2H, H-phenyl), 7.02 (dd, *J* = 8.7, 2.6 Hz, 1H, H-phenyl), 6.80 (d, *J* = 8.7 Hz, 1H, H-phenyl), 5.37 (s, 2H, CH2). 13C-NMR (101 MHz, CD3OD): δ = 155.21, 142.66, 140.49, 137.67, 129.87, 129.60, 129.12 (2C), 128.42, 126.89 (2C), 126.06, 125.01, 121.01, 118.79, 118.28, 51.17. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated forC17H13ON2ClF3: 353.06630; found: 353.06635.

#### 3.1.5. Preparation of 4-methyl-1*H*-indole-3-carboxylic Acid (DX-01-02)

Methyl 4-methyl-1*H*-indole-3-carboxylate (200 mg, 1.06 mmol) used hydrolysis reaction (2 N NaOH water solution) to afford DX-01-02 (white solid, 151 mg, yield 82%). 1H-NMR (400 MHz, CD3OD): δ = 8.18 (dd, *J* = 3.9, 1.9 Hz, 1H, H-indolyl), 7.30 (d, *J* = 8.0 Hz, 1H, H-indolyl), 7.17 (t, *J* = 7.7 Hz, 1H, H-indolyl), 7.01 (dd, *J* = 7.3, 0.7 Hz, 1H, H-indolyl), 2.77 (s, 3H, CH3). 13C-NMR (101 MHz, CD3OD): δ = 139.17, 133.89, 126.39, 125.96, 125.68, 120.61, 117.71, 112.19, 111.02, 23.30. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C10H10NO2: 176.07061; found: 176.07129.

#### 3.1.6. Preparation of 5-methyl-1*H*-indole-3-carboxylic Acid (DX-01-03)

Methyl 5-methyl-1*H*-indole-3-carboxylate (200 mg, 1.06 mmol) used hydrolysis reaction (2 N NaOH water solution) to afford DX-01-03 (white solid, 150 mg, yield 81%). 1H-NMR (400 MHz, CD3OD): δ = 8.15 (d, *J* = 1.8 Hz, 1H, H-indolyl), 8.07 (s, 1H, H-indolyl), 7.39 (d, *J* = 8.3 Hz, 1H, H-indolyl), 7.15 (dd, *J* = 8.3, 1.1 Hz, 1H, H-indolyl), 2.46 (s, 3H, CH3). 13C-NMR (101 MHz, CD3OD): δ = 137.41, 134.35, 127.80, 127.00, 122.51, 120.09, 117.20, 113.08, 110.78, 21.77. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C10H10NO2: 176.07061; found: 176.07094.

#### 3.1.7. Preparation of 5-nitro-1*H*-indole-3-carboxylic Acid (DX-01-04)

Methyl 5-nitro-1*H*-indole-3-carboxylate (200 mg, 0.91 mmol) used hydrolysis reaction (2 N NaOH water solution) to afford DX-01-04 (white solid, 172 mg, yield 92%). 1H-NMR (400 MHz, CD3OD): δ = 8.99 (d, *J* = 2.2 Hz, 1H, H-indolyl), 8.14 (s, 1H, H-indolyl), 8.12 (dd, *J* = 9.0, 2.3 Hz, 1H, H-indolyl), 7.58 (d, *J* = 9.0 Hz, 1H, H-indolyl). 13C-NMR (101 MHz, CD3OD): δ = 167.81, 144.32, 141.16, 136.51, 127.01, 118.93, 118.80, 113.45, 111.10, 49.00. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C9H7N2O4: 207.04003; found: 207.03940.

#### 3.1.8. Preparation of 6-nitro-1*H*-indole-3-carboxylic Acid (DX-01-05)

Methyl 6-nitro-1*H*-indole-3-carboxylate (200 mg, 0.91 mmol) used hydrolysis reaction (2 N NaOH water solution) to afford DX-01-05 (white solid, 168 mg, yield 90%). 1H-NMR (400 MHz, CD3OD): δ = 8.38 (d, *J* = 1.5 Hz, 1H, H-indolyl), 8.22 (s, 1H, H-indolyl), 8.18 (d, *J* = 8.9 Hz, 1H, H-indolyl), 8.05 (dd, *J* = 8.8, 1.7 Hz, 1H, H-indolyl). 13C-NMR (101 MHz, CD3OD): δ = 167.87, 144.97, 138.31, 136.78, 132.29, 122.19, 117.33, 109.89, 109.69. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C9H7N2O4: 207.04003; found: 207.03949.

#### 3.1.9. Preparation of 5-methoxy-1*H*-indole-3-carboxylic Acid (DX-01-06)

Methyl 5-methoxy-1*H*-indole-3-carboxylate (200 mg, 0.97 mmol) used hydrolysis reaction (2 N NaOH water solution) to afford DX-01-06 (white solid, 140 mg, yield 75%). 1H-NMR (400 MHz, CD3OD): δ = 7.88 (s, 1H, H-indolyl), 7.57 (d, *J* = 2.4 Hz, 1H, H-indolyl), 7.31 (d, *J* = 8.8 Hz, 1H, H-indolyl), 6.83 (dd, *J* = 8.8, 2.5 Hz, 1H, H-indolyl), 3.83 (s, 3H, CH3). 13C-NMR (101 MHz, CD3OD): δ = 156.99, 133.58, 128.36, 115.37, 114.23, 113.88, 113.61, 104.51, 103.64, 56.06. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C10H10NO3: 192.06552; found: 192.06645.

#### 3.1.10. Preparation of 7-bromo-1*H*-indole-3-carboxylic Acid (DX-01-07)

Methyl 7-bromo-1*H*-indole-3-carboxylate (200 mg, 0.79 mmol) used hydrolysis reaction (2 N NaOH water solution) to afford DX-01-07 (white solid, 160 mg, yield 85%). 1H-NMR (400 MHz, CD3OD): δ = 8.06 (dd, *J* = 8.0, 0.9 Hz, 1H, H-indolyl), 7.98 (s, 1H, H-indolyl), 7.36 (dd, *J* = 7.6, 0.7 Hz, 1H, H-indolyl), 7.08 (t, *J* = 7.8 Hz, 1H, H-indolyl). 13C-NMR (101 MHz, CD3OD): δ = 168.56, 138.29, 134.03, 129.07, 126.24, 123.59, 121.46, 117.02, 106.02. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C9H7NO2Br: 239.96547; found: 239.96452.

#### 3.1.11. Preparation of (*E*) 1*H*-indole-3-carbaldehyde Oxime (DX-01-13)

1*H*-indole-3-carbaldehyde (200 mg, 1.38 mmol) following the similar procedure described for the preparation of DX-02-04 afforded DX-01-13 (white solid, 158 mg, yield 72%). 1H-NMR (400 MHz, DMSO-*d*6): δ = 11.56 (s, 1H, OH), 11.17 (s, 1H, NH), 8.26 (s, 1H, =CH), 8.22 (d, *J* = 1.3 Hz, 1H, H-indolyl), 7.97 (d, *J* = 7.9 Hz, 1H, H-indolyl), 7.44 (d, *J* = 8.1 Hz, 1H, H-indolyl), 7.16 (t, *J* = 7.5 Hz, 2H, H-indolyl). 13C-NMR (101 MHz, CD3OD): δ = 146.89, 138.66, 132.19, 127.98, 123.30, 121.40, 118.79, 112.59, 107.56. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C9H9ON2: 161.07094; found: 161.07076.

#### 3.1.12. Preparation of *N*-((1*H*-indol-3-yl)methyl)hydroxylamine (DX-01-14)

DX-01-13 (100 mg, 0.62 mmol) following the similar procedure described for the preparation of DX-02-05 afforded DX-01-14 (white solid, 70 mg, yield 69%). 1H-NMR (400 MHz, DMSO-*d*6): δ = 10.87 (s, 1H, NH-indolyl), 7.58 (d, *J* = 7.8 Hz, 1H, H-indolyl), 7.34 (d, *J* = 8.1 Hz, 1H, H-indolyl), 7.29 (br, 1H, NH), 7.24 (s, 1H, H-indolyl), 7.06 (t, *J* = 8.0 Hz, 1H, H-indolyl), 6.99–6.95 (m, 1H, H-indolyl), 5.66 (br, 1H, OH), 4.04 (s, 2H, CH2). 13C-NMR (101 MHz, CD3OD): δ = 138.02, 128.61, 125.94, 122.58, 120.11, 119.41, 112.28, 110.24, 55.80. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C9H11ON2: 163.08659; found: 163.08611.

#### 3.1.13. Preparation of *N*-((5-(o-tolyl)-1*H*-indol-3-yl)methyl)hydroxylamine (DX-01-15)

5-Bromo-1*H*-indole-3-carbaldehyde (300 mg, 1.34 mmol) and o-tolylboronic acid (183 mg, 1.34 mmol) following the similar procedure described for the preparation of DX-03-12 and DX-02-05 afforded DX-01-15 (white solid, 145 mg, yield 43% from 5-bromo-1*H*-indole-3-carbaldehyde). 1H-NMR (400 MHz, CD3OD): δ = 7.57–7.39 (m, 3H, H-phenyl, H-indolyl), 7.26–7.04 (m, 5H, H-phenyl, H-indolyl), 5.13 (s, 2H, CH2), 2.24 (s, 3H, CH3). 13C-NMR (101 MHz, CD3OD): δ = 144.63, 137.29, 136.60, 135.38, 131.19, 131.12, 128.56, 127.79, 126.62, 125.69, 124.87, 119.47, 112.33, 107.61, 62.12, 29.31. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C16H17N2O: 253.13354; found: 253.13471.

3.1.14. Preparation of (*E*)-1*H*-indole-2-carbaldehyde Oxime (DX-01-16)

1*H*-indole-2-carbaldehyde (200 mg, 1.38 mmol) following the similar procedure described for the preparation of DX-02-04 afforded DX-01-16 (white solid, 117 mg, yield 53%). 1H-NMR (400 MHz, DMSO-*d*6): δ = 11.29 (s, 1H, OH), 11.17 (s, 1H, NH), 8.14 (s, 1H, =CH), 7.52 (d, *J* = 8.0 Hz, 1H, H-indolyl), 7.37 (d, *J* = 8.2 Hz, 1H, H-indolyl), 7.12 (t, *J* = 7.6 Hz, 1H, H-indolyl), 6.98 (t, *J* = 7.5 Hz, 1H, H-indolyl), 6.56 (s, 1H, H-indolyl). 13C-NMR (101 MHz, CD3OD): δ = 142.67, 138.93, 132.77, 129.48, 124.71, 122.20, 120.89, 112.84, 108.61. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C9H9ON2: 161.07094; found: 161.07077.

3.1.15. Preparation of *N*-((1*H*-indol-2-yl)methyl)hydroxylamine (DX-01-17)

DX-01-16 (60 mg, 0.37 mmol) following the similar procedure described for the preparation of DX-02-05 afforded DX-01-17 (white solid, 39 mg, yield 65%). 1H-NMR (400 MHz, DMSO-d6): δ = 10.88 (s, 1H, NH-indolyl), 7.46–7.39 (m, 2H, H-indolyl, NH), 7.32 (d, *J* = 8.3 Hz, 1H, H-indolyl), 7.03–6.97 (m, 1H, H-indolyl), 6.93 (t, *J* = 8.1 Hz, 1H), 6.28 (s, 1H, H-indolyl), 6.17 (br, 1H, OH), 4.00 (s, 2H, CH2). 13C-NMR (101 MHz, CD3OD): δ = 137.98, 136.56, 122.09, 120.86, 120.84, 120.01, 111.84, 101.86, 51.98. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C9H11ON2: 163.08659; found: 163.08636.

#### 3.1.16. Preparation of (*E*)-3-((1*H*-indol-3-yl)methylene)pyrrolidine-2,5-dione (DX-02-01)

1*H*-indole-3-carbaldehyde (500 mg, 3.45 mmol) and 1*H*-pyrrole-2,5-dione (401 mg, 4.14 mmol) following the similar procedure described for the preparation of DX-02-04 afforded DX-02-01 (white solid, 398 mg, yield 51% from 1*H*-indole-3-carbaldehyde). 1H-NMR (400 MHz, DMSO-*d*6): δ = 11.94 (s, 1H, NH-indolyl), 11.18 (s, 1H, NH), 7.81 (dd, *J* = 12.1, 5.1 Hz, 2H, H-indolyl, =CH), 7.70 (t, *J* = 2.1 Hz, 1H, H-indolyl), 7.54–7.41 (m, 1H, H-indolyl), 7.21–7.16 (m, 2H, H-indolyl), 3.52 (d, *J* = 2.2 Hz, 2H). HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C13H11O2N2: 227.08150; found: 227.08134.

#### 3.1.17. Preparation of (*R*/*S*) 3-((1*H*-indol-3-yl)methyl)pyrrolidine-2,5-dione (DX-02-02)

DX-02-01 (200 mg, 3.45 mmol) following the similar procedure described for the preparation of DX-02-04 afforded DX-02-02 (white solid, 65 mg, yield 32%). 1H-NMR (400 MHz, DMSO-*d*6): δ = 11.06 (s, 1H, NH-indolyl), 10.91 (s, 1H, NH), 7.51 (d, *J* = 7.8 Hz, 1H, H-indolyl), 7.33 (d, *J* = 8.0 Hz, 1H, H-indolyl), 7.16 (s, 1H, H-indolyl), 7.06 (t, *J* = 7.5 Hz, 1H, H-indolyl), 6.97 (t, *J* = 7.5 Hz, 1H, H-indolyl), 3.20–3.12 (m, 1H, CH'H"), 2.95 (dd, *J* = 14.0, 8.5 Hz, 1H, CH'H"), 2.70–2.61 (m, 2H, H-pyrrolidine-2,5-dione), 2.34 (dd, *J* = 18.0, 3.5 Hz, 1H, H-pyrrolidine-2,5-dione). HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C13H13N2O2: 229.09715; found: 229.09663

3.1.18. Preparation of (*E*) 2-(1*H*-imidazol-4-yl)benzaldehyde Oxime (DX-03-02)

Compound **5** (400 mg, 0.92 mmol) and (2-formylphenyl)boronic acid (138 mg, 0.92 mmol) following the similar procedure described for the preparation of DX-03-12 and DX-02-04 afforded DX-03-02 (white solid, 98 mg, yield 57% from (2-formylphenyl)boronic acid). 1H-NMR (400 MHz, CD3OD): δ = 8.32 (s, 1H, =CH), 7.85–7.84 (m, 1H, H-phenyl), 7.79 (s, 1H, H-imidazolyl), 7.52 (d, *J* = 7.7 Hz, 1H, H-phenyl), 7.42–7.38 (m, 1H, H-phenyl), 7.34–7.30 (m, 1H, H-phenyl), 7.12 (s, 1H, H-imidazolyl). 13C-NMR (100 MHz, DMSO-*d*6): δ = 148.70, 136.36, 133.83, 130.10, 129.50, 129.27, 128.72, 128.34, 127.09, 126.20. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C10H10ON3: 188.08184; found: 188.08191.

3.1.19. Preparation of *N*-(2-(1*H*-imidazol-4-yl)benzyl)hydroxylamine (DX-03-03)

Compound DX-03-02 (50 mg, 0.27 mmol) following the similar procedure described for the preparation of DX-02-04 afforded DX-03-03 (white solid, 32 mg, yield 63%). 1H-NMR (400 MHz, CD3OD): δ = 7.75 (s, 1H, H-imidazolyl), 7.53 (d, *J* = 1.7 Hz, 1H, H-phenyl), 7.47–7.44 (m, 1H, H-phenyl), 7.35–7.27 (m, 3H, H-imidazolyl, H-phenyl), 4.04 (s, 2H). HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C10H12ON3: 190.09749; found: 190.09737.

3.1.20. Preparation of (*E*) 2-(2*H*-tetrazol-5-yl)benzaldehyde Oxime (DX-03-04)

2-(2H-tetrazol-5-yl)benzaldehyde (200 mg, 1.15 mmol) following the similar procedure described for the preparation of DX-03-12 and DX-02-04 afforded DX-03-04 (white solid, 78 mg, yield 36% from 2-(2*H*-tetrazol-5-yl)benzaldehyde). 1H-NMR (400 MHz, DMSO-*d*6): δ = 11.23 (s, 1H, OH), 11.07 (s, 1H, H-tetrazolyl), 8.29 (s, 1H, =CH), 7.73–7.56 (m, 4H, H-phenyl). 13C-NMR (101 MHz, DMSO-*d*6): δ = 156.17, 137.81, 132.14, 130.64, 129.27, 127.64, 116.84, 110.69. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C8H8N5O: 190.07234; found: 190.07158.

3.1.21. Preparation of (*E*) 5-chloro-2-(1*H*-imidazol-4-yl)benzaldehyde Oxime (DX-03-05)

Compound **5** (300 mg, 0.69 mmol) and (4-chloro-2-formylphenyl)boronic acid (127 mg, 0.69 mmol) following the similar procedure described for the preparation of DX-03-12 and DX-02-04 afforded DX-03-05 (white solid, 70 mg, yield 46% from (4-chloro-2-formylphenyl)boronic acid). 1H-NMR (400 MHz, DMSO-*d*6): δ = 12.38 (s, 1H, OH), 11.32 (s, 1H, NH-imidazolyl), 8.77 (s, 1H, =CH), 7.80 (s, 1H, H-imidazolyl), 7.74 (s, 1H, H-imidazolyl), 7.63 (d, *J* = 2.4 Hz, 1H, H-phenyl), 7.44–7.42 (m, 2H, H-phenyl). 13C-NMR (101 MHz, DMSO-*d*6): δ = 147.77, 138.56, 136.60, 133.12, 131.72, 131.48, 131.05, 129.25, 125.42, 116.28. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C10H9ClN3O: 222.04287; found: 222.04330.

3.1.22. Preparation of (*E*) 2-fluoro-6-(1*H*-imidazol-4-yl)benzaldehyde Oxime (DX-03-06)

Compound **5** (300 mg, 0.69 mmol) and (3-fluoro-2-formylphenyl)boronic acid (116 mg, 0.69 mmol) following the similar procedure described for the preparation of DX-03-12 and DX-02-04 afforded DX-03-06 (white solid, 75 mg, yield 53% from (3-fluoro-2-formylphenyl)boronic acid). 1H-NMR (400 MHz, DMSO-*d*6): δ = 12.37 (s, 1H, OH), 11.24 (s, 1H, NH-imidazolyl), 8.37 (s, 1H, =CH), 7.76 (s, 1H, H-imidazolyl), 7.52 (s, 1H, H-imidazolyl), 7.36–7.13 (m, 3H, H-phenyl). 13C-NMR (101 MHz, DMSO-*d*6): δ = 161.48, 159.00, 144.68, 136.29, 130.27, 124.36, 122.95, 117.89, 116.35, 113.98. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C10H9FN3O: 206.07242; found: 206.07250.

3.1.23. Preparation of (*E*) 2-(1*H*-imidazol-4-yl)-5-methoxybenzaldehyde Oxime (DX-03-07)

Compound **5** (300 mg, 0.69 mmol) and (2-formyl-4-methoxyphenyl)boronic acid (124 mg, 0.69 mmol) following the similar procedure described for the preparation of DX-03-12 and DX-02-04 afforded DX-03-07 (white solid, 113 mg, yield 76% from (2-formyl-4-methoxyphenyl)boronic acid). 1H-NMR (400 MHz, CD3OD): δ = 8.73 (s, 1H, H-imidazolyl), 7.78 (s, 1H, H-imidazolyl), 7.52–7.38 (m, 3H, H-phenyl), 7.15 (s, 1H, H-phenyl), 7.10–6.96 (m, 4H, H-phenyl), 3.85 (s, 3H, CH3). 13C-NMR (101 MHz, CD3OD): δ = 141.55, 137.87, 133.07, 132.61, 121.44, 118.60, 117.79, 116.37, 113.90, 110.95, 55.90. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C11H12N3O2: 218.09240; found: 218.09206.

3.1.24. Preparation of (*E*) 4-(1*H*-imidazol-4-yl)-2 -(trifluoromethyl)-[1,1 -biphenyl]-3-carbaldehyde Oxime (DX-03-08)

Compound **5** (300 mg, 0.69 mmol) and (3-formyl-2 -(trifluoromethyl)-[1,1 -biphenyl]-4-yl) boronic acid (202 mg, 0.69 mmol) following the similar procedure described for the preparation of DX-03-12 and DX-02-04 afforded DX-03-08 (white solid, 82mg, yield 36% from (3-formyl-2 -(trifluoromethyl)-[1,1 -biphenyl]-4-yl)boronic acid). 1H-NMR (400 MHz, DMSO-*d*6): δ = 12.38 (s, 1H, OH), 11.14 (s, 1H, NH-imidazolyl), 8.85(s, 1H, H-imidazolyl), 7.86–7.79 (m, 2H, H-imidazolyl, H-phenyl), 7.73–7.63 (m, 4H, H-phenyl), 7.46 (br, 2H, H-phenyl), 7.33 (s, 1H, H-phenyl). 13C-NMR (101 MHz, DMSO-*d*6): δ = 148.54, 140.58, 137.83, 136.56, 132.89, 132.57, 130.11, 129.84, 129.53, 128.94, 128.64, 126.58, 126.32, 126.23, 126.04, 123.22, 116.04. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C17H13F3N3O: 332.10052; found: 332.10123.

3.1.25. Preparation of (*E*) 3-(1*H*-imidazol-4-yl)-[1,1 -biphenyl]-4-carbaldehyde Oxime (DX-03-09)

Compound **5** (300 mg, 0.69 mmol) and (4-formyl-[1,1 -biphenyl]-3-yl)boronic acid (156 mg, 0.69 mmol) following the similar procedure described for the preparation of DX-03-12 and DX-02-04 afforded DX-03-09 (white solid, 81 mg, yield 45% from (4-formyl-[1,1 -biphenyl]-3-yl)boronic acid). 1H-NMR (400 MHz, DMSO-*d*6): δ = 11.01 (s, 1H, OH), 10.34 (s, 1H, NH-imidazolyl), 8.55 (s, 1H, =CH), 8.39 (s, 1H, H-imidazolyl), 8.12 (s, 1H, H-imidazolyl), 7.58 (br, 1H, H-phenyl), 7.48–7.22 (m, 2H, H-phenyl), 7.11–7.02 (m, 2H, H-phenyl), 6.87–6,81 (m, 2H, H-phenyl), 6.50 (s, 1H, H-phenyl). HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C16H14N3O: 264.11314; found: 264.11346.

3.1.26. Preparation of 4-(2-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,3-thiadiazole (DX-03-10)

2-(1,2,3-Thiadiazol-4-yl)phenol (100 mg, 0.56 mmol) and 1-(bromomethyl)-4-(trifluoromethyl)benzene (134 mg, 0.56 mmol) following the similar procedure described for the preparation of **12** afforded DX-03-10 (white solid, 128 mg, yield 68%). 1H-NMR (400 MHz, DMSO-*d*6): δ = 9.39 (s, 1H, H-thiadiazolyl), 8.26 (d, *J* = 7.5 Hz, 1H, H-phenyl), 7.76 (d, *J* = 8.2 Hz, 2H, H-phenyl), 7.70 (d, *J* = 8.5 Hz, 2H, H-phenyl), 7.48–7.43 (m, 1H, H-phenyl), 7.31 (d, *J* = 7.7 Hz, 1H, H-phenyl), 7.17 (t, *J* = 7.5 Hz, 1H, H-phenyl), 5.44 (s, 2H, CH2). HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C16H12F3N2OS: 337.06169; found: 337.06116.

3.1.27. Preparation of *N*-(3-(2-(1*H*-imidazol-5-yl)phenoxy)propyl)-aminesulfonamide (DX-03-11)

Compound **9** (200 mg, 0.34 mmol) following the similar procedure described for the preparation of DX-03-12 afforded DX-03-11 (white solid, 45 mg, yield 45% from compound **9**). 1H-NMR (400 MHz, CD3OD): δ = 8.67 (s, 1H, NH-imidazolyl), 8.08 (s, 1H, H-imidazolyl), 7.83 (s, 1H, H-imidazolyl), 7.69 (d, *J* = 7.7 Hz, 1H, H-phenyl), 7.40 (t, *J* = 7.1 Hz, 1H, H-phenyl), 7.16 (d, *J* = 8.2 Hz, 1H, H-phenyl), 7.08 (t, *J* = 7.4 Hz, 1H, H-phenyl), 4.20 (t, *J* = 6.0 Hz, 2H, CH2), 3.44 (t, *J* = 6.9 Hz, 2H, CH2), 2.11–2.03 (m, 2H, CH2). HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C12H17O3N4S: 297.10159; found: 297.10115.

3.1.28. Preparation of 1-(3-(2-(1*H*-imidazol-5-yl)phenoxy)propyl)-3-(4-(trifluoromethyl) phenyl)urea (DX-03-13)

Compound **9** (200 mg, 0.34 mmol) following the similar procedure described for the preparation of DX-03-12 afforded DX-03-13 (white solid, 77 mg, yield 56% from compound **9**). 1H-NMR (400 MHz, DMSO-*d*6): δ = 12.24 (s, 1H, NH-imidazolyl), 8.90 (s, 1H, H-imidazolyl), 8.01 (d, *J* = 7.4 Hz, 1H, H-phenyl), 7.75 (s, 1H, H-imidazolyl), 7.61–7.54 (m, 5H, H-phenyl), 7.21–7.15 (m, 1H, H-phenyl), 7.06 (d, *J* = 7.8 Hz, 1H, H-phenyl), 7.02–6.94 (m, 1H, H-phenyl), 6.48 (t, *J* = 5.7 Hz, 1H, H-phenyl), 4.14 (t, *J* = 6.1 Hz, 2H, CH2), 3.38–3.32 (m, 2H, CH2), 2.08–1.97 (m, 2H, CH2). 13C-NMR (101 MHz, DMSO-*d*6): δ = 155.42, 155.08, 144.71, 135.42, 129.17, 127.38, 127.13, 126.38 (2C), 123.78, 122.81, 121.53, 121.21, 120.86, 117.71 (2C), 112.33, 65.82, 36.89, 29.98. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C20H20O2N4F3: 405.15329; found: 405.15302.

3.1.29. Preparation of 1-(3-bromo-4-fluorobenzyl)-4-(5-chloro-2-methoxyphenyl)-1*H*-imidazole (DX-04-03)

2-Bromo-4-(bromomethyl)-1-fluorobenzene (500 mg, 1.88 mmol) following the similar procedure described for the preparation of DX-04-02 afforded DX-04-03 (white solid, 480 mg, yield 65% from 2-bromo-4-(bromomethyl)-1-fluorobenzene). 1H-NMR (400 MHz, CD3OD): δ = 7.95 (d, *J* = 2.7 Hz, 1H, H-phenyl), 7.78 (d, *J* = 1.3 Hz, 1H, H-imidazolyl), 7.64 (d, *J* = 1.3 Hz, 1H, H-imidazolyl), 7.57 (dd, *J* = 6.5, 2.2 Hz, 1H, H-phenyl), 7.30–7.24 (m, 1H, H-phenyl), 7.20 (d, *J* = 8.5 Hz, 1H, H-phenyl), 7.16 (dd, *J* = 8.8, 2.7 Hz, 1H, H-phenyl), 6.98 (d, *J* = 8.8 Hz, 1H, H-phenyl), 5.22 (s, 2H, CH2), 3.88 (s, 3H, CH3). 13C-NMR (101 MHz, CD3OD): δ = 161.35, 158.96, 156.10, 138.33, 137.40, 133.91, 129.70, 128.04, 127.36, 126.81, 125.19, 121.53, 117.84, 113.47, 110.01, 56.13, 50.30. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C17H14ON2BrClF: 394.99566; found: 394.99728.

3.1.30. Preparation of 2-(1-(3-bromo-4-fluorobenzyl)-1*H*-imidazol-4-yl)-4-chlorophenol (DX-04-04)

DX-04-03 (200 mg, 0.51 mmol) following the similar procedure described for the preparation of DX-04-02 afforded DX-04-04 (white solid, 116 mg, yield 60%). 1H-NMR (400 MHz, DMSO-*d*6): δ = 9.12 (s, 1H, H-imidazolyl), 8.40 (s, 1H, H-imidazolyl), 8.01 (s, 1H, H-imidazolyl), 7.89 (dd, *J* = 6.5, 1.7 Hz, 1H, H-phenyl), 7.56–7.49 (m, 1H, H-phenyl), 7.45 (t, *J* = 8.6 Hz, 1H, H-phenyl), 7.34 (dd, *J* = 8.8, 2.5 Hz, 1H, H-phenyl), 7.00 (d, *J* = 8.8 Hz, 1H, H-phenyl), 5.42 (s, 2H, CH2). 13C-NMR (101 MHz, CD3OD): δ = 161.85, 159.35, 154.93, 136.62, 134.79, 130.61, 130.34, 126.90, 125.53, 119.70, 118.77, 118.32, 118.09, 117.92, 110.27, 51.71. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C16H12ON2BrClF: 380.98001; found: 380.98065.

3.1.31. Preparation of 4-chloro-2-(1-(4-(trifluoromethyl)benzyl)-1*H*-1,2,3-triazol-4-yl)phenol (DX-04-05)

DX-04-06 (300 mg, 0.81 mmol) following the similar procedure described for the preparation of DX-04-02 afforded DX-04-05 (white solid, 176 mg, yield 61%). 1H-NMR (400 MHz, DMSO-*d*6): δ = 10.49 (s, 1H, OH), 7.85 (s, 1H, H-triazolyl), 7.64 (d, *J* = 8.1 Hz, 2H, H-phenyl), 7.34 (dd, *J* = 8.8, 2.7 Hz, 1H, H-phenyl), 7.20 (d, *J* = 8.1 Hz, 2H, H-phenyl), 7.16 (d, *J* = 2.7 Hz, 1H, H-phenyl), 6.98 (d, *J* = 8.8 Hz, 1H, H-phenyl), 5.62 (s, 2H, CH2). 13C-NMR (101 MHz, CD3OD): δ = 155.39, 140.99, 136.49, 134.75, 132.46, 131.76, 131.11, 129.36 (2C), 126.49 (2C), 125.38, 124.09, 118.30, 116.15, 53.27. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C16H12ON3ClF3: 354.06155; found: 354.06015.

3.1.32. Preparation of 4-(5-chloro-2-methoxyphenyl)-1-(4-(trifluoromethyl)benzyl)-1*H*-1,2,3-triazole (DX-04-06)

4-chloro-2-ethynyl-1-methoxybenzene (500 mg, 3.01 mmol) and 1-(bromomethyl)-4- (trifluoromethyl)benzene (717 mg, 3.01 mmol) following the similar procedure described for the preparation of DX-04-02 afforded DX-04-06 (white solid, 675 mg, yield 61% from 4-chloro-2-ethynyl-1-methoxybenzene). 1H-NMR (400 MHz, CD3OD): δ = 7.71 (s, 1H, H-triazolyl), 7.50 (d, *J* = 8.1 Hz, 2H, H-phenyl), 7.40 (dd, *J* = 8.9, 2.7 Hz, 1H, H-phenyl), 7.14 (d, *J* = 8.1 Hz, 2H, H-phenyl), 7.09 (d, *J* = 2.6 Hz, 1H, H-phenyl), 7.03 (d, *J* = 8.9 Hz, 1H, H-phenyl), 5.55 (s, 2H, CH2), 3.64 (s, 3H, CH3). 13C-NMR (101 MHz, CD3OD): δ = 154.74, 143.61, 131.20, 130.91, 129.93 (2C), 129.81, 129.39, 127.59, 127.02 (2C), 125.98, 125.64, 118.63, 117.43, 55.31, 55.04. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C17H14ON3ClF3: 368.07720; found: 368.07599.

3.1.33. Preparation of 4-chloro-2-(5-(4-(trifluoromethyl)phenyl)-1*H*-1,2,3-triazol-4-yl)phenol (DX-04-07)

DX-04-08 (200 mg, 0.57 mmol) following the similar procedure described for the preparation of DX-04-02 afforded DX-04-07 (white solid, 144 mg, yield 75%). 1H-NMR (400 MHz, DMSO-*d*6): δ = 15.28 (s, 1H, H-triazolyl), 9.91 (s, 1H, NH-triazolyl), 7.75–7.68 (m, 4H, H-phenyl), 7.37 (br, 2H, H-phenyl), 6.94 (s, 1H, H-phenyl). 13C-NMR (101 MHz, CD3OD): δ = 155.58, 142.95, 137.77, 136.14, 131.62, 131.27, 130.86, 128.63 (2C), 126.43 (2C), 125.31, 124.28, 119.14, 118.53. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C15H10ON3ClF3: 340.04590; found: 340.04453.

#### 3.1.34. Preparation of 4-(5-chloro-2-methoxyphenyl)-5-(4-(trifluoromethyl)phenyl)-1*H*-1,2,3-triazole (DX-04-08)

4-chloro-2-ethynyl-1-methoxybenzene (300 mg, 1.81 mmol) and 1-iodo-4-(trifluoromethyl)benzene (491 mg, 1.81 mmol) following the similar procedure described for the preparation of DX-04-02 afforded DX-04-08 (white solid, 338 mg, yield 53% from 4-chloro-2-ethynyl-1-methoxybenzene). 1H-NMR (400 MHz, DMSO-*d*6): δ = 15.41 (s, 1H, NH-triazolyl), 7.74 (d, *J* = 8.1 Hz, 2H, H-phenyl), 7.68–7.59 (m, 2H, H-phenyl), 7.54 (d, *J* = 8.9 Hz, 1H, H-phenyl), 7.48 (d, *J* = 2.7 Hz, 1H, H-phenyl), 7.15 (s, 1H, H-phenyl), 3.42 (s, 3H, CH3). 13C-NMR (101 MHz, CD3OD): δ = 157.20, 136.42, 131.71, 131.59, 131.14, 129.65, 128.37 (2C), 126.96, 126.75, 126.40 (2C), 124.27, 121.57, 114.17, 55.99. HRMS (ESI): *m*/*z* [M + H]<sup>+</sup> calculated for C16H12ON3ClF3: 354.06155; found: 354.06079.

#### *3.2. Pharmacological Method*

#### 3.2.1. Enzyme-Based IDO1s or TDOs Activity Assay

The compounds on IDO1 or TDO activity were determined as follows [16,24,25]: A standard reaction mixture (100 μL) containing 100 mM potassium phosphate buffer (pH 6.5), 40 mM ascorbic acid (neutralized with NaOH), 200 μg/mL catalase, 20 μM methylene blue and 0.05 μM rhIDO1 or rhTDO was added to the solution containing the substrate l-tryptophan and the test sample at a determined concentration. The reaction was carried out at 37 ◦C for 45 min and stopped by adding 20 μL of 30% (*w*/*v*) trichloroacetic acid. After heating at 65 ◦C for 15 min, 100 μL of 2% (*w*/*v*) p-dimethylaminobenzaldehyde in acetic acid was added to each well. The yellow pigment derived from kynurenine was measured at 492 nm using a SYNERGY-H1 microplate reader (Biotek Instruments, Inc., Winooski, VT, USA). IC50 was analyzed using the GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA).

#### 3.2.2. Cell Culture

B16-F10 mouse melanoma cells, NCI-H460 human lung cancer cell line and MCF7 were obtained from American Type Culture Collection (ATCC). All the cell lines were cultured with Dulbecco' modified Eagle's medium (DMEM) (Invitrogen Corporation, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Waltham, MA, USA) and 1% penicillin streptomycin (Invitrogen) at 37 ◦C in 5% CO2.

#### 3.2.3. Cell Viability Assay

The in vitro inhibitory effects of the compounds were measured by MTT assay [26]. Briefly, different kinds of cells were plated in 96-well plates at a density of 2000/well. After attachment for overnight at 37 ◦C, the cells were treated with the compounds at various concentrations. After 72 h culture, the MTT reagent was added to each well, and plates were incubated for another 4 h at 37 ◦C. Then, the blue-purple crystal formamidine was dissolved in DMSO after discarding the supernatant. Samples were measured using microplate reader at a wavelength of 570 nm (Biotek Instruments, Inc., Winooski, VT, USA). IC50 values were calculated with GraphPad Prism 8.0 software.

#### 3.2.4. Mice

C57BL/6 mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing Vital River Laboratory Animal Technology Co., Ltd, Beijing, China). Studies involving mice were approved by the Experimental Animal Management and Welfare Committee at the Institute of Materia Medica, Peking Union Medical College.

#### 3.2.5. Pharmacokinetic Studies

The animal Care and Welfare Committee of Institute of Materia Medica, Chinese Academy of Medical Sciences approved all animal care, housing, and laboratory procedures. Male ICR mice were used in the single dose pharmacokinetic studies. Compound DX-03-12 was prepared as 3 mg/mL suspension with 0.5% CMC (containing 0.3% Tween 80) for oral use and was formulated as 3 mg/mL solution with 5% DMSO in saline for intravenous injection. Twenty mice were divided into two groups, 10 in each group. After fasting 12 h with free access to water, mice were treated with a 3 mg/kg i.v. or 30 mg/kg oral dose of compound DX-03-12. Blood samples (50 μL) were collected at 5, 15, 30 min, 1, 2, 4, 6, 8, 12 and 24 h after oral administration and 2, 5, 15, 30 min, 1, 2, 4, 6, 8, 12 and 24 h after intravenous injection. After centrifugation, the plasma samples (15 μL) were precipitated by five volumes of acetonitrile. The supernatant were analyzed by liquid chromatography/tandem mass spectrometry (Agilent Technologies, Santa Clara, CA, USA) with a Zobax C18 column (50 mm × 2.1 mm, 3.5 μm). Compound detection was performed with the mass spectrometer in MRM (multiple reaction monitoring, Agilent Technologies, Santa Clara, CA, USA) negative ionization mode. The selected reaction monitoring transitions were *m*/*z* 348.5→160 for compound DX-03-12. The pharmacokinetic parameters were calculated with WinNonlin software V6.3 using non-compartmental analysis (Pharsight Corporation, Mountain View, CA, USA).

#### 3.2.6. In Vivo Studies

The mouse melanoma cells B16F10 were cultured and harvested in saline. At day 0 of the experiment, 1 <sup>×</sup> 106 cells were injected subcutaneously into mice, and treatment was initiated at day 1 following the mice enrolled randomly in control and experimental groups [27]. For control group, 0.5% CMCNa was orally administered every day. Compound DX-03-12 was dissolved in 0.5% CMC for oral treatment or dissolved in saline for intraperitoneal treatment. When the mice were sacrificed, the tumors were stripped and weighted. The tumor growth inhibition (TGI) was calculated as TGI = (1 – tumor weight treatment/tumor weight vehicle) × 100%. The statistical analysis was performed with GraphPad Prism 8.0 software and the significance level was evaluated with a one-way ANOVA model.

#### **4. Conclusions**

We designed four series of IDO1 inhibitors using indole and phenylimidazole as the scaffolds. The design strategies were based on the mechanism of IDO1, focusing on the interaction between the inhibitor and the heme iron. The four series of compounds were synthesized and their IDO1 and TDO inhibition was evaluated. Most compounds showed marginal IDO1 inhibition, but eight compounds showed moderate IDO1 inhibition. In particular, DX-03-12 and DX-03-13 showed IDO1 inhibition with IC50s of 0.3 and 0.5 μM and low cell cytotoxicities against two cancer cell lines. A pharmacokinetic study showed that DX-03-12 had satisfactory properties, with rapid absorption, moderate plasma clearance, acceptable half-life, and high oral bioavailability. The in vivo anti-tumor efficacy in a B16F10 subcutaneous xenograft mouse model showed that DX-03-12 orally administered at a dose of 60 mg/kg inhibited tumor growth by 72.2% compared with the control tumors. In summary, IDO1 inhibitor DX-03-12 exhibits promising drug like properties, good pharmacokinetic properties, low cellular toxicity, and impressive in vivo anti-tumor efficacy.

**Author Contributions:** L.S., F.L., and H.C. conceived and designed the experiments; H.W., Y.L. (Yuke Liu), S.W., T.W., and G.Z. performed the experiments, analyzed the data; Y.L. (Yan Li), and X.C. checked the data; L.S., F.L., and H.C. wrote the paper. Please contact L.S. for pharmacokinetic study, F.L. for pharmacology study and H.C. for medicinal chemistry.

**Funding:** The research leading to these results has received funding from The CAMS Innovation Fund for Medical Sciences (2017-I2M-1-010), The Drug Innovation Major Project (2018ZX09711001), National Natural Science Foundation of China (81603190).

**Acknowledgments:** We would like to express our appreciation to Jie Li of Beijing Chao-Yang Hospital for the clinical issue discussion.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Sample Availability:** Samples of the compounds are available from the authors.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Synthesis and Cytotoxicity of 7,9-***O***-Linked Macrocyclic C-Seco Taxoids**

### **Yu Zhao 1, Tian-En Wang 1, Alberto Mills 2, Federico Gago <sup>2</sup> and Wei-Shuo Fang 1,\***


#### Academic Editor: Qiao-Hong Chen Received: 23 May 2019; Accepted: 6 June 2019; Published: 8 June 2019

**Abstract:** A series of novel 7,9-*O*-linked macrocyclic taxoids together with modification at the C2 position were synthesized, and their cytotoxicities against drug-sensitive and P-glycoprotein and βIII-tubulin overexpressed drug-resistant cancer cell lines were evaluated. It is demonstrated that C-seco taxoids conformationally constrained via carbonate containing-linked macrocyclization display increased cytotoxicity on drug-resistant tumors overexpressing both βIII and P-gp, among which compound **22b**, bearing a 2-m-methoxybenzoyl group together with a five-atom linker, was identified as the most potent. Molecular modeling suggested the improved cytotoxicity of **22b** results from enhanced favorable interactions with the T7 loop region of βIII.

**Keywords:** taxoids; βIII-tubulin; P-glycoprotein; drug resistance

#### **1. Introduction**

Antitubulin agent paclitaxel (**1a**, Figure 1) and its semi-synthetic derivative docetaxel (**1b**, Figure 1) are successfully used in the clinic for the treatment of ovarian, breast, and non-small cell lung and prostate cancers and Kaposi's sarcoma.

**Figure 1.** Structures of paclitaxel (**1a**), docetaxel (**1b**), and IDN5390 (**2**).

However, their clinical uses are severely restricted by drug resistance. Various resistance mechanisms toward taxane-based anticancer drugs have been revealed, among which only overexpression of P-glycoprotein (P-gp) and βIII-tubulin have been confirmed clinically [1–4].

A great deal of effort has been made to overcome multi-drug resistance (MDR) mediated by overexpression of P-gp. Nevertheless, many taxane-based drug candidates targeting P-gp overexpression did not achieve the expected efficacy in clinical trials [5,6].

A correlation between βIII-tubulin overexpression and poor prognosis has been reported in several human tumors, including various advanced malignancies upon treatment of taxanes, such as breast, non-small cell lung, and ovarian cancers [7–10]. It was reported that a C-seco taxane IDN5390 (**2**, Figure 1) is more active than paclitaxel in several paclitaxel-resistant human ovarian adenocarcinoma cell lines (e.g., A2780TC1, A2780TC3, and OVCAR-3), expressing high βIII-tubulin and P-gp levels. However, IDN5390 was nearly 10-fold less active than paclitaxel in paclitaxel-sensitive cells [11].

To find a modified taxane effective against both paclitaxel-sensitive and -resistant tumors, the 7,9-*O*-linked C-seco taxoid **3** (Figure 2) was synthesized in our lab and shown to exhibit significant activity enhancement against drug-sensitive Hela andβIII-tubulin overexpressing HeLa-βIII tumor cells compared to the C-seco paclitaxel derivative **4** (an analog of IDN5390, Figure 2) [12]. Those macrocyclic taxoids provided a successful example for the compromise between structural pre-organization and sufficient flexibility in the C ring of C-seco taxoids to improve the cytotoxicity against tumor cells that overexpress βIII tubulin [12].

**Figure 2.** Structures of the 7,9-*O*-linked C-seco taxoid **3** and C-seco paclitaxel derivative **4**.

It has long been observed that modification of taxane at the C2 position could play a critical role in its interaction with tubulin. Consistent with this observation, the 7,9-*O*-linked C-seco taxoid with C2 modification **5** (Figure 3) was much more cytotoxic than the C-seco taxoid **6** (Figure 3) in drug-resistant A2780AD and KB-V1 cells (P-gp overexpression) and Hela-βIII (βIII overexpression) [12]. It was demonstrated that the *meta*-substitution (i.e., -OMe, -F, -Cl) of the C2-benzoate moiety of C-seco taxoids derivatives **7a**–**c** (Figure 3) could increase the interaction of C-seco-taxoids with βIII-tubulin to overcome paclitaxel-resistance [13].

**Figure 3.** Structures of the 7,9-*O*-linked C-seco taxoid **5** and C-seco taxoids **6, 7a**–**c**.

In order to explore the effect of macrocyclization in a different way from that employed in our previous study (i.e., via the carbonate formation to restrict the conformation), the synthesis and biological activity assessment of a series of 7,9-*O*-linked macrocyclic taxoids together with modification at the C2 position were performed and are reported here.

#### **2. Results and Discussion**

#### *2.1. Chemistry*

Our synthesis began with the preparation of 2 -*tert*-butyldimethylsilyl-paclitaxel **12a**, which was then converted to 10-deacetylpaclitaxel **13a** upon treatment with hydrazine hydrate in methanol. Synthesis of the analogues **12b**–**e** of 2 -*tert*-butyldimethylsilyl-paclitaxel with modifications at the C2 positions was then realized by selective C2 debenzoylation and then elaboration with various *m*-substituted benzoyl group. C2-modified 10-deacetylpaclitaxel **13b**–**e** were afforded in the same manner as compound **13a**. Then the compounds **13a**–**e** were oxidized by copper (II) diacetate to furnish **14a**–**e** as a mixture of two epimers. Reductive trapping of the ring C-seco tautomers **15a**–**e** were obtained by treatment with l-selectride. Subsequent deprotection with pyridine hydrofluoride (HF-pyridine) afforded **16a**–**e**. The 9-OH in **15a**–**e** was hydroxylalkylated by 2-bromoethanol or 3-bromo-1-propanol in the presence of potassium carbonate and potassium iodide in *N*,*N*-dimethylformamide (DMF). Cyclization of compounds **17a**–**e** could be accomplished by treatment with a slight excess of triphosgene, affording the cyclic carbonate analogues **19a**–**e**. Desilylation of **19a**–**e** with HF-pyridine afforded macrocyclic analogues **21a**–**e**. Accordingly, macrocyclic taxoids **22a**–**e** bearing a linkage with one more carbon atom were synthesized from **18a**–**e** in a similar manner (Scheme 1).

**Scheme 1.** (a) tert-ButyldiMethylsilyl chloride (TBDMSCl), imidazole, DMF, 2 h, 60 ◦C, and; triethylchlorosilane (TESCl), imidazole, DMF, overnight, r.t., 90.1%; (b) Triton-B, tetrahydrofuran (THF), 15 min, −40 ◦C, 47.7%; (c) (**10a**) m-anisic acid, N, N -diisopropylcarbodiimide (DIC), 4-dimethylaminopyridine (DMAP), toluene, overnight, 60 ◦C, 72.5%; (**10b**) m-fluorobenzoic acid, DIC, DMAP, toluene, overnight, 60 ◦C, 91.4%; (**10c**) m-chlorobenzoic acid, DIC, DMAP, toluene, overnight, 60 ◦C, 84.2%; (**10d**) m-trifluoromethyl acid, DIC, DMAP, toluene, overnight, 60 ◦C, 84.2%; (d) HF -pyridine, THF, overnight, r.t.; (**11a**) 81.8%; (**11b**) 91.9%; (**11c**) 85.1%; (**11d**) 91.0%; (e) TBDMSCl, imidazole, DMF, 4 h, r.t.; (f) NH2NH2·H2O, C2H5OH, 2 h, r.t. (e and f two steps), (**13a**) 87.9%, (**13b**) 99.0%; (**13c**) 93.5%; (**13d**) 83.0%; (**13e**) 90.1%; (g) Cu(OAc)2·H2O, CH3OH, overnight, r.t., (**14a**) 85.8%; (**14b**) 69.5%; (**14c**) 79.0%; (**14d**) 84.9%; (**14e**) 81.9%; (h) <sup>l</sup>-selectride, THF, 15 min, <sup>−</sup><sup>20</sup> ◦C, (**15a**) 81.2%; (**15b**) 88.7%; (**15c**) 79.2%; (**15d**) 58.8%; (**15e**) 76.5%; (i) HF/Py, THF, overnight, r.t., (**16a**) 77.4%; (**16b**) 65.2%; (**16c**) 59.3%; (**16d**) 60.3%; (**16e**) 55.5%; (j) 2-bromoethanol, K2CO3, KI, DMF, 5–9 h, r.t., (**17a**) 52.4%; (**17b**) 56.1%; (**17c**) 53.2%; (**17d**) 60.9%; (**17e**) 49.9%; (k) 3-bromo-1-propanol, K2CO3, KI, DMF, 4–9 h, r.t., (**18a**) 72.4%; (**18b**) 75.3%; (**18c**) 69.7%; (**18d**) 52.6%; (**18e**) 71.8%; (l) triphosgene, pyridine, DCM, 3–5 h, 0 ◦C to r.t., (**19a**) 15.9%; (**19b**) 32.7%; (**19c**) 32.1%; (**19d**) 36.1%; (**19e**) 30.5%; (**20a**) 24.9%; (**20b**) 28.6%; (**20c**) 27.1%; (**20d**) 23.6%; (**20e**) 26.7%; (m) HF/Py, THF, 30 h, r.t., (**21a**) 46.2%; (**21b**) 54.1%; (**21c**) 52.2%; (**21d**) 48.7%; (**21e**) 63.7%; (**22a**) 51.0%; (**22b**) 59.2%; (**22c**) 51.5%; (**22d**) 65.4%; (**22e**) 59.5%.

#### *2.2. Bioactivity Evaluation of Taxoids* **21a**–**e** *and* **22a**–**e**

Taxoids **21a**–**e**, **22a**–**e**, and **16a**–**e** were evaluated for their in vitro cytotoxicities against cervical carcinoma HeLa cells and their corresponding drug-resistant cell lines (HeLa-βIII).

As shown in Table 1, all the 7,9-*O*-linked macrocyclic analogues (**21a**–**e, 22a**–**e**) possessed a remarkably higher potency against HeLa and HeLa-βIII cells than their corresponding C-seco taxoids analogues **16a**–**e**. The R/S values (IC50 in drug resistant cells/IC50 in drug sensitive cells) of all macrocyclic analogues were lower than those of their corresponding C-seco taxoids analogues **16a**–**e**. These findings demonstrate that conformational restraint via carbonate-containing linked macrocyclization can improve the cytotoxicity against human carcinoma cell lines overexpressing βIII tubulin.

Furthermore, all of the 7,9-*O*-linked macrocyclic analogues bearing the four-atom linker (**21a**–**e**) turned out to be generally more potent against sensitive HeLa cells and HeLa-βIII cells than the corresponding macrocyclic analogues bearing the five-atom linker (**22a**–**e**). Since the cytotoxicity of **21a**–**d** was approximately equal to that of paclitaxel, these results suggest that a four-atom tether is optimal in these macrocyclic C-seco taxoids.

For the effect of C2 modifications in these macrocyclic C-seco taxoids, we found similar results as previously reported [12,13]. Pepe et al. studied the cytotoxicity of a series of C-seco taxoids, whereby the introduction of a substituent (-OMe, -F, -Cl) to the *meta*-position of the C2 benzoyl moiety increased potency against the βIII-tubulin-overexpressing, paclitaxel-resistant cell line A2780TC3 [13]. Taxoid **6** bearing a 2-(*m*-azido) benzyloxy group and synthesized in our lab exhibited higher binding affinity for microtubulin (MT) than its corresponding taxoids bearing a 2-benzyloxy group [12]. The 7,9-*O*-linked C-seco taxoid bearing a 2-(*m*-azido) benzyloxy group **5** was significantly more active than **6** on drug-resistant A2780AD, HeLa-βIII, and KB-V1 cells [12].

We investigated this effect in the 7,9-*O*-linked C-seco taxoids **21b**–**e** and **22b**–**e** reported here. Thus, introduction of a *meta* substituent (-OMe, -F, -Cl) on derivatives bearing the four-atom linker showed similar cytotoxicity against HeLa sensitive cell lines and Hela-βIII cells lines as their corresponding C-seco taxoid **21a** bearing a 2-benzyloxy group. In contrast, the *meta*-substituted (-OMe, -F, -Cl) derivatives bearing five-atom linker possessed higher potency against HeLa-βIII cells lines than its corresponding C-seco taxoid **22a** (by a factor of 3.1–8.9). Nonetheless, the 2-*m*-CF3 analogues **21e** and **22e** were considerably less cytotoxic against both drug sensitive and resistant cells by one to two orders of magnitude.

The growth inhibition effects of the 7,9-*O*-linked macrocyclic analogues were also measured on human breast cancer MCF-7 cells and their corresponding P-gp-overexpressing drug-resistant MCF-7/R cells. As shown in Table 1, the 7,9-*O*-linked macrocyclic analogues possessed remarkable potency against MCF-7/R cells compared to their C-seco counterparts, except for the 2-*m*-CF3 derivatives. Strikingly, macrocyclic taxoids bearing the five-atom linker were almost inactive. The cytotoxicity against MCF-7/R cells of the 7,9-*O*-linked macrocyclic derivatives (-OMe, -F, -Cl) bearing the five-atom linker were slightly higher than that of the compouds bearing the four-atom linker. Compounds **22b**–**d** (-OMe, -F, -Cl) bearing the five-atom linker and **21a**, **21d** (-H, -Cl) bearing the four-atom linker displayed lower R/S values than that of paclitaxel. All in all, **22b** turned out to be the best compound in the whole series.


**22a**–**e**.


#### *2.3. Binding Mode of* **22b** *and Rationale for the Increased A*ffi*nity for the* β*III Isotype*

Inspection of the paclitaxel-β-tubulin complex in high-resolution cryo-electron microscopy (cryo-EM) structures of mammalian microtubules reveals a very tight fit between the bulky drug and the taxane-binding site, in which the M-loop is structured as an α-helix [14]. The oxetane ring oxygen of paclitaxel (**1a**) accepts a hydrogen bond from the backbone NH of Thr276, whereas the C-3 benzamide carbonyl oxygen acts as a hydrogen bond acceptor for N<sup>ε</sup> of His229. These two hydrogen bonds are likely to be present in the **22b**:β-tubulin complex as well (Figure 4), and definitely contribute to the binding affinity, but these two residues are common to both βIIB and βIII isotypes. In the search for sequence differences that can account for the resistance to taxanes, a lot of emphasis has been placed on the Ser→Ala replacement at position 277, but we believe that the most important replacement in relation to drug resistance possibly involves Cys241→Ser. The reason is that, as a consequence of this substitution, disulfide bond formation with Cys356 is precluded in the βIII isotype and the T7 loop region, which closes over the bound taxane, is likely to be more flexible. By directing the macrocyclic region of **22b** towards this loop, favorable interactions within the taxane-binding site are enhanced and the affinity towards the βIII isotype is improved. We hypothesize that the improved cytotoxicity of **22b** relative to **1a**, and the other taxanes described here, results from enhanced favorable interactions with the T7 loop region of βIII, an isotype in which Cys356 cannot engage in a disulfide bond with the amino acid present at position 241 (Ser).

**Figure 4. 22b** docked into homology models of human tubulins IIb and III bound to guanosine diphosphate (GDP)-Mg2+. Colored in magenta and labeled are those residues close to the taxane-binding site that differ between these two isotypes.

As shown in this modelling study, the binding of these taxoids to microtubules are similar to that of paclitaxel. Although cyclization does increase the cytotoxicity either in drug-sensitive and -resistant cells in most cases, the 7,9-*O*-linked macrocyclic taxoids only showed comparable cytotoxicity to that of paclitaxel. The enhanced activity is not only arisen from the known C-2 modifications [15,16], but also from the cyclization of the cleaved C-ring of taxane.

#### **3. Experimental**

#### *3.1. Chemical Synthesis*

#### 3.1.1. General Methods

All chemicals and reagents were purchased from Beijing Innochem Science and Technology Co. Ltd. (Beijing, China), Sinopharm Chemical Reagent Co. Ltd. (Beijing, China), and thin-layer chromatography (TCI). The 200–300 mesh silica gel used for flash column chromatography was purchased from Rushanshi Shuangbang Xincailiao Co. Ltd. (Rushan, Shandong, China). Visualization on TLC (analytical thin layer chromatography) was achieved by the use of UV light (254 nm) and treatment with phosphomolybdic acid or KMnO4 followed by heating. All solvents were purified and dried according to the standard procedures. The purification was performed on flash column chromatography. The high performance liquid chromatography (HPLC)- electrospray ionization (ESI)-mass spectrometry (MS) analysis was carried out in an Agilent 1260 Infinity HPLC system (Agilent Technologies, Waldbronn, Germany) equipped with a reversed phase 4.68 × 50 mm (1.8 um) XDB-C18 Column and consisted of a binary solvent delivery system, an auto sampler, a column temperature controller, and an UV detector. The mass spectra were acquired by a 6120 Quadrupole LC-MS mass spectrometer (Agilent Technologies, Waldbronn, Germany) connected to the HPLC system via an ESI interface. Proton and carbon magnetic resonance spectra (1H-NMR and 13C-NMR) were recorded on a Bruker BioSpin AG 300 or 400 MHz spectrometer or Varian 300, 400, or 600 MHz spectrometer.1H-NMR data were reported as follows: Chemical shifts, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m=multiplet), coupling constant(s) in Hz, integration. All tested compounds **16a**–**e**, **21a**–**e**, and **22a**–**e** were ≥95% pure by HPLC (column XDB C18 4.6 × 50 mm 1.8 μm, mobile phase: acetonitrile-water (10:90–100:0 gradient in 4.5 min), flow rate 1.0 mL/min, detected at 220 nm).

#### 3.1.2. General Experimental Procedure for Compounds **14a**–**e**

*2 -O-(tert-Butyldimethylsilyl)-10-dehydro-10-deacetylpaclitaxel* (**14a**). To a solution of **13a** (261.3 mg, 0.282 mmol) in methanol (8 mL) was added Cu(OAc)2·H2O (282 mg, 1.41 mmol). The suspension was stirred at room temperature overnight (open air) and then diluted with ethyl acetate. The organic phase was washed sequentially with saturated aqueous NaCl (50 mL), 2 N NH3 to remove copper salts and saturated aqueous NH4Cl. After drying (Na2SO4) and evaporation under reduced pressure, the crude product was purified by silica gel chromatography (ethyl acetate: petroleum ether = 1:2) to give a 4:1 mixture of epimers **14a** (14-β-OH and 14-α-OH, 223.8 mg, 85.8% total yield). 1H-NMR (CDCl3, 600 MHz) δ: 8.19 (d, *J* = 7.8 Hz, 1H, Ar-H), 7.73 (d, *J* = 7.8 Hz, 2H, Ar-H), 7.48 (m, 11H, Ar-H), 7.06 (d, *J* = 9.0 Hz, 1H, NH), 6.28 (t, *J* = 8.7 Hz, 1H, H-13), 5.87 (d, *J* = 7.2 Hz, 1H, H-2), 5.79 (dd, *J* = 1.5, 8.7 Hz, 1H, H-3 ), 4.93 (m, 1H, H-5), 4.67 (d, *J* = 1.8 Hz, 1H, H-2 ), 4.50 (d, *J* = 11.4 Hz, 1H, H-7), 4.44 (d, *J* = 8.4 Hz, 1H, H- 20a), 4.38 (d, *J* = 9.0 Hz, 1H, H-20b), 4.04 (d, *J* = 7.2 Hz, 1H, H-3), 3.88 (m, 1H, OH-7), 2.67 (s, 3H, CH3), 2.48 (dd, *J* = 9.6, 15.6 Hz, 1H, H-6a), 2.29 (m, 2H, H-14b, H-14a), 2.19 (m, 1H, H-6b), 1.86 (s, 3H, CH3), 1.75 (s, 3H, CH3), 1.21 (s, 3H, CH3), 1.13 (s, 3H, CH3), 0.79 (s, 9H, SiC(CH3)3), −0.04 (s, 3H, Si(CH3)), −0.30 (s, 3H, Si(CH3)). (For the chemical characterization refer to http://dx.doi.org/10.1016/j.ejmech.2017.06.001)

*2 -O-(tert-Butyldimethylsilyl)-2-debenzoyl-2-(3-methoxybenzoyl)-7-epi-10-dehydro-10-deacetylpaclitaxel* (**14b**). 69.5% yield as a 4:1 mixture of two epimers; 1H-NMR (CDCl3, 400 MHz) δ: 7.80 (d, *J* = 7.7 Hz, 1H, Ar-H), 7.74 (d, *J* = 7.2 Hz, 2H, Ar-H), 7.41 (m, 10H, Ar-H), 7.21 (dd, *J* = 2.0, 8.0 Hz, Ar-H), 7.09 (d, *J* = 9.1 Hz, 1H, NH), 6.27 (t, *J* = 8.9 Hz, 1H, H-13), 5.89 (d, *J* = 7.2 Hz, 1H, H-2), 5.79 (dd, *J* = 2.2, 9.4 Hz, 1H, H-3 ), 4.94 (m, 1H, H-5), 4.67 (d, *J* = 2.0 Hz, 1H, H-2 ), 4.50 (d, *J* = 11.2 Hz, 1H, H-7), 4.47 (d, *J* = 8.8 Hz, 1H, H- 20a), 4.38 (d, *J* = 8.6 Hz, 1H, H-20b), 4.05 (d, *J* = 7.1 Hz, 1H, H-3), 3.90 (s, 3H, OCH3), 3.88 (m, 1H, OH-7), 2.67 (s, 3H, CH3), 2.49 (dd, *J* = 9.8, 15.4 Hz, 1H, H-6a), 2.29 (m, 2H, H-14b, H-14a), 1.86 (s, 3H, CH3), 2.25 (m, 1H, H-6b), 1.75 (s, 3H, CH3), 1.21(s, 3H, CH3), 1.13 (s, 3H, CH3), 0.81 (s, 9H, SiC(CH3)3), <sup>−</sup>0.02 (s, 3H, Si(CH3)), <sup>−</sup>0.26 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) <sup>δ</sup>: 207.8, 188.7,

172.2, 170.9, 167.0, 166.9, 159.8, 143.3, 141.1, 138.2, 134.1, 131.9, 130.3, 130.0, 128.8 (2C), 128.1, 127.0, 126.4, 122.7, 120.5, 114.4, 82.7, 81.8, 79.2, 77.4, 75.3, 75.1, 70.7, 57.2, 55.5 (2C), 40.3, 39.4, 36.3, 35.4, 26.9, 26.1, 25.5, 22.9, 18.2, 15.0, 14.4, −5.2, −5.8; LC-MS (ESI, *m*/*z*): [M + 1], found 954.4, [M + 23], found 976.4, C52H63NO14Si: 953.40 (see supplementary materials).

*2 -O-(tert-Butyldimethylsilyl)-2-debenzoyl-2-(3-fluorobenzoyl)-7-epi-10-dehydro-10-deacetylpaclitaxel* (**14c**). 79.0% yield as a 4:1 mixture of two epimers; 1H-NMR (CDCl3, 400 MHz) δ: 8.02 (d, *J* = 8.0 Hz, 1H, Ar-H), 7.89–7.86 (m, 1H, Ar-H), 7.73 (d, *J* = 7.2 Hz, 2H, Ar-H), 7.59–7.33 (m, 10H, Ar-H), 7.07 (d, *J* = 9.2 Hz, 1H, BzNH), 6.27 (t, *J* = 8.8 Hz, 1H, H-13), 5.86 (d, *J* = 7.2 Hz, 1H, H-2), 5.80 (dd, *J* = 2.0, 9.2 Hz, 1H, H-3 ), 4.95 (dd, *J* = 4.0, 8.0 Hz, 1H, H-5), 4.68 (d, *J* = 2.4 Hz, 1H, H-2 ), 4.48 (d, *J* = 11.2 Hz, 1H, H-7), 4.44 (d, *J* = 8.8 Hz, 1H, H- 20a), 4.36 (d, *J* = 8.8 Hz, 1H, H-20b), 4.05 (d, *J* = 7.2 Hz, 1H, H-3), 3.92–3.88 (m, 1H, OH-7), 2.68 (s, 3H, COCH3), 2.48 (dd, *J* = 9.6, 15.2 Hz, 1H, H-6a), 2.31–2.29 (m, 2H, H-14b, H-14a), 2.22 (dd, *J* = 8.4, 15.6 Hz, 1H, H-6b), 1.99 (br, 1H, OH), 1.86 (s, 3H, CH3), 1.76 (s, 3H, CH3), 1.22 (s, 3H, CH3), 1.13 (s, 3H, CH3), 0.81 (s, 9H, SiC(CH3)), <sup>−</sup>0.02 (s, 3H, Si(CH3)), <sup>−</sup>0.26 (s, 3H, Si(CH3)2); 13C-NMR (CDCl3, 100 MHz) <sup>δ</sup>: 207.8, 188.6, 172.3, 171.2, 170.9, 167.0, 165.9 (d, <sup>4</sup>*J*C-F <sup>=</sup> 2.9 Hz), 162.7 (d, <sup>1</sup>*J*C-F = 246.5 Hz), 143.5, 140.9, 138.2, 134.1, 131.9, 131.3 (d, <sup>3</sup>*J*C-F = 7.3 Hz), 130.7 (d, <sup>3</sup>*J*C-F = 7.6 Hz), 128.83 (2C), 128.77 (2C), 128.1, 127.0 (2C), 126.4 (2C), 126.1 (d, <sup>4</sup>*J*C-F = 3.1 Hz), 120.0 (d, <sup>2</sup>*J*C-F = 21.1 Hz), 117.1 (d, <sup>2</sup>*J*C-F = 23.1 Hz), 82.8, 81.7, 79.4, 75.5, 75.3, 70.6, 57.2, 55.6, 40.3, 39.5, 36.3, 35.4, 26.1, 25.5 (3C), 22.9 (2C), 18.2, 15.0, 14.5, 14.2, −5.2, −5.8; LC-MS (ESI, *m*/*z*): [M + 1], found 942.4, [M + 23], found 964.3, C51H60FNO13Si: 941.38.

*2 -O-(tert-Butyldimethylsilyl)-2-debenzoyl-2-(3-chlorobenzoyl)-7-epi-10-dehydro-10-deacetylpaclitaxel* (**14d**). 84.9% yield as a 4:1 mixture of two epimers; 1H-NMR (CDCl3, 400 MHz) δ: 8.18 (s, 1H, Ar-H), 8.11 (d, *J* = 7.6 Hz, 1H, Ar-H), 7.73 (d, *J* = 7.6 Hz, 2H, Ar-H), 7.62 (d, *J* = 7.6 Hz, 1H, Ar-H), 7.55–7.50 (m, 2H, Ar-H), 7.44–7.35 (m, 7H, Ar-H), 7.08 (d, *J* = 8.8 Hz, 1H, BzNH), 6.25 (t, *J* = 8.8 Hz, 1H, H-13), 5.84 (d, *J* = 7.2 Hz, 1H, H-2), 5.79 (dd, *J* = 8.8 Hz, 1H, H-3 ), 4.96 (m, 1H, H-5), 4.67 (d, *J* = 2.0 Hz, 1H, H-2 ), 4.47 (d, *J* = 11.2 Hz, 1H, H-7), 4.44 (d, *J* = 8.4 Hz, 1H, H- 20a), 4.35 (d, *J* = 8.4 Hz, 1H, H-20b), 4.05 (d, *J* = 7.2 Hz, 1H, H-3), 3.89 (m, 1H, OH-7), 2.68 (s, 3H, CH3), 2.48 (dd, *J* = 10.0, 15.4 Hz, 1H, H-6a), 2.29 (m, 2H, H-14b, H-14a), 2.23 (m, 1H, H-6b), 1.86 (s, 3H, CH3), 1.76 (s, 3H, CH3), 1.22 (s, 3H, CH3), 1.13 (s, 3H, CH3), 0.82 (s, 9H, SiC(CH3)3), <sup>−</sup>0.02 (s, 3H, Si(CH3)), <sup>−</sup>0.26 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) δ: 207.8, 188.6, 172.2, 170.9, 165.7, 143.5, 140.9, 138.2, 134.9, 134.1, 133.9, 131.9, 130.9, 130.4, 130.3, 128.83 (2C), 128.77 (2C), 128.5, 128.1, 127.0 (2C), 126.5 (2C), 82.7, 81.7, 79.4, 75.4, 75.3, 70.7, 60.4, 57.2, 55.6, 40.2, 39.4, 36.3, 35.3, 29.7, 26.1, 25.5 (2C), 22.9, 22.8, 18.2, 14.9, 14.5, −5.2, −5.8; LC-MS (ESI, *m*/*z*): [M + 1], found 958.4, [M + 23], found 980.4, C51H60ClNO13Si: 957.35.

*2 -O-(tert-Butyldimethylsilyl)-2-debenzoyl-2-(3-trifluoromethyl)-7-epi-10-dehydro-10-deacetylpaclitaxel* (**14e**). 81.9% yield as a 4:1 mixture of two epimers; 1H-NMR (CDCl3, 400 MHz) δ: 8.50 (s, 1H, Ar-H), 8.43 (d, *J* = 7.6 Hz, 1H, Ar-H), 7.91 (d, *J* = 8.0 Hz, 1H, Ar-H), 7.72 (d, *J* = 7.8 Hz, 3H, Ar-H), 7.51 (m, 1H, Ar-H), 7.41 (m, 7H, Ar-H), 7.06 (d, *J* = 9.2 Hz, 1H, BzNH), 6.24 (t, *J* = 8.6 Hz, 1H, H-13), 5.88 (d, *J* = 7.6 Hz, 1H, H-2), 5.78 (dd, *J* = 1.6, 9.2 Hz, 1H, H-3 ), 4.97 (m, 1H, H-5), 4.67 (d, *J* = 2.0 Hz, 1H, H-2 ), 4.45 (d, *J* = 11.2 Hz, 1H, H-7), 4.42 (d, *J* = 8.5 Hz, 1H, H-20a), 4.37 (d, *J* = 8.6 Hz, 1H, H-20b), 4.09 (d, *J* = 7.6 Hz, 1H, H-3), 3.90 (m, 1H, OH-7), 2.66 (s, 3H, CH3), 2.51 (dd, *J* = 9.6, 15.5 Hz, 1H, H-6a), 2.30 (m, 2H, H-14b, H-14a), 2.26 (m, 1H, H-6b), 1.87 (s, 3H, CH3), 1.77 (s, 3H, CH3), 1.23 (s, 3H, CH3), 1.15 (s, 3H, CH3), 0.83 (s, 9H, SiC(CH3)3), <sup>−</sup>0.02 (s, 3H, Si(CH3)), <sup>−</sup>0.24 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) <sup>δ</sup>: 207.7, 188.6, 172.1, 171.0, 167.1, 165.6, 143.6, 140.9, 138.3, 134.2, 133.6, 131.9, 131.4 (q, <sup>2</sup>*J*C-F = 33.2 Hz), 130.3 (q, <sup>3</sup>*J* = 3.4 Hz), 130.3, 130.1, 129.9, 128.9, 128.8, 128.1, 127.1 (q, <sup>3</sup>*J*C-F = 4.0 Hz), 127.0, 126.5, 123.7 (q, <sup>1</sup>*J*C-F = 273.6 Hz), 82.8, 81.7, 79.5, 75.6, 75.3, 70.7, 57.2, 55.6, 40.3, 39.5, 36.3, 35.4, 26.1, 25.6, 22.9, 22.7, 18.2, 14.9, 14.5, −5.2, −5.8; LC-MS (ESI, *m*/*z*): [M + 1], found 992.0, C52H60F3NO13Si: 991.38.

#### 3.1.3. General Experimental Procedure for Compounds **15a**–**e**

*2 -tert-Butyldimethylsilyl-10-dehydro-7-8-seco-10-deacetylpaclitaxel* (**15a**). l-selectride (1M solution in THF, 0.8 mL) was added, dropwise, to a solution of **14a** (171.5 mg, 0.186 mmol) in THF (4.1 mL) at −20 ◦C. After 15 min, the reaction was quenched by addition of cold ethyl acetate (50.0 mL) and 2 N H2SO4 (4.0 mL). The reaction mixture was extracted with ethyl acetate and the combined organic phases were washed with a saturated aqueous NaHCO3 and saturated aqueous NH4Cl. The resulting solution was dried over anhydrous MgSO4 and the solvent evaporated under reduced pressure to give a crude product. The crude product was purified by silica gel chromatography (ethyl acetate: petroleum ether = 1:1) which gave product **15a** (139.5 mg, 81.2% yield) as a white solid

*2 -tert-Butyldimethylsilyl-2-debenzoyl-2-(3-methoxybenzoyl)-10-dehydro-7,8-seco-10-deacetylpaclitaxel* (**15b**). White solid; yield 88.7%; 1H-NMR (CDCl3, 400 MHz) δ: 7.73–7.70 (m, 3H, Ar-H), 7.55 (s, 1H, Ar-H), 7.42 (m, 10H, Ar-H), 7.14 (dd, *J* = 2.3, 8.2 Hz, 1H, BzNH), 6.47 (s, 1H, OH-9), 6.29 (t, *J* = 8.0 Hz, 1H, H-13), 5.91 (d, *J* = 8.8 Hz, 1H, H-3 ), 5.60 (d, *J* = 9.2 Hz, 1H, H-2), 5.24 (brs, 2H, H-5, H-20a), 4.72 (d, *J* = 1.6 Hz, 1H, H-2 ), 4.29 (d, *J* = 6.8 Hz, 1H, H-3), 4.20 (brs, 1H, H-7a), 3.96 (brs, 1H, OH), 3.85 (s, 4H, OCH3, H-7b), 2.82 (brs, 1H, H-6a), 2.71 (brs, 1H, OH), 2.26 (dd, *J* = 9.6, 15.6 Hz, 2H, H-14a, H-14b), 2.09–2.02 (m, 4H, CH3, H-6b), 1.87 (brs, 6H, 2 × CH3), 1.28 (s, 3H, CH3), 1.10 (s, 3H, CH3), 0.79 (s, 9H, SiC(CH3)3), <sup>−</sup>0.11 (s, 3H, Si(CH3)), <sup>−</sup>0.28 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) <sup>δ</sup>: 191.2, 171.2, 171.1, 168.9, 167.4, 167.2, 159.8, 148.9, 142.1, 138.2, 133.9, 131.9, 130.5, 130.3, 128.7 (4C), 128.0, 127.1 (2C), 126.7 (2C), 124.6, 122.2, 119.5, 115.0, 86.3, 76.1, 75.1, 70.6, 59.2, 55.8, 55.5, 43.1, 36.6, 25.5 (3C), 23.4, 22.2, 21.5, 18.3, 14.8, 14.4, −5.6, −6.0; LC-MS (ESI, *m*/*z*): [M + 1], found 956.4, [M + 23], found 978.4, C52H65NO14Si: 955.42.

*2 -tert-Butyldimethylsilyl-2-debenzoyl-2-(3-fluorobenzoyl)-10-dehydro-7,8-seco-10-deacetylpaclitaxel* (**15c**). White solid; yield 79.2%; 1H-NMR (CDCl3, 400 MHz) δ: 7.94 (d, *J* = 7.8 Hz, 1H, Ar-H), 7.76 (d, *J* = 8.7 Hz, 1H, Ar-H), 7.68 (d, *J* = 7.4 Hz, 2H, Ar-H), 7.53–7.44 (m, 3H, Ar-H), 7.40–7.30 (m, 8H, NH, Ar-H), 6.48 (s, 1H, OH-9), 6.30 (t, *J* = 8.0 Hz, 1H, H-13), 5.92 (d, *J* = 9.0 Hz, 1H, H-3 ), 5.60 (d, *J* = 9.5 Hz, 1H, H-2), 5.25 (brs, 2H, H-5, H-20a), 4.71 (d, *J* = 2.1 Hz, 1H, H-2 ), 4.30 (d, *J* = 7.8 Hz, 1H, H-3), 4.19 (brs, 1H, H-20b), 3.96 (brs, 1H, H-7a), 3.80 (brs, 1H, H-7b), 2.78 (brs, 1H, H-6a), 2.69 (brs, 1H, H-14a), 2.26 (dd, *J* = 9.5, 15.6 Hz, 1H, H-14b), 2.10–2.00 (ovelap, 1H, H-6b), 1.90 (s, 3H, CH3), 1.86 (overlap and brs, 6H, 2 × CH3), 1.27 (s, 3H, CH3), 1.10 (s, 3H, CH3), 0.78 (s, 9H, SiC(CH3)3), −0.09 (s, 3H, Si(CH3)), <sup>−</sup>0.29 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) <sup>δ</sup>: 191.1, 171.2, 171.0, 168.8, 167.4, 166.4, 131.0 (d, <sup>1</sup>*J*C-F <sup>=</sup> 248.1 Hz), 128.8 (2C), 128.1, 127.1, 126.7, 125.8 (d, <sup>4</sup>*J*C-F <sup>=</sup> 2.6 Hz), 124.6, 121.1 (d, <sup>2</sup>*J*C-F <sup>=</sup> 21.3 Hz), 116.7 (d, <sup>2</sup>*J*C-F = 22.6 Hz), 86.5, 80.7, 76.1, 75.5, 70.5, 59.0, 55.8, 44.5, 43.2, 36.7, 25.6, 22.3, 21.6, 18.3, 14.9, 14.5, −5.4, −5.9; LC-MS (ESI, *m*/*z*): [M + 1], found 944.4, [M + 23], found 966.4, C51H62FNO13Si: 943.40.

*2 -tert-Butyldimethylsilyl-2-debenzoyl-2-(3-chlorobenzoyl)-10-dehydro-7,8-seco-10-deacetylpaclitaxel* (**15d**). White solid; yield 58.8%; 1H-NMR (CDCl3, 400 MHz) δ: 8.05 (d, *J* = 7.6 Hz, 2H, Ar-H), 8.01 (s, 1H, Ar-H), 7.68 (d, *J* = 7.6 Hz, 2H, Ar-H), 7.57 (d, *J* = 8.0 Hz, 1H, Ar-H), 7.41–7.31 (m, 9H, NH, Ar-H), 6.49 (s, 1H, OH-9), 6.29 (t, *J* = 8.4 Hz, 1H, H-13), 5.92 (d, *J* = 9.3 Hz, 1H, H-3 ), 5.59 (d, *J* = 9.5 Hz, 1H, H-2), 5.23 (brs, 2H, H-5, H-20a), 4.71 (d, *J* = 1.6 Hz, 1H, H-2 ), 4.30 (d, *J* = 6.9 Hz, 1H, H-3), 4.18 (brs, 1H, H- 20b), 3.98 (brs, 1H, H-7a), 3.83 (brs, 1H, H-7b), 2.78 (brs, 1H, H-6a), 2.67 (brs, 1H, H-14a), 2.25 (dd, *J* = 9.6, 15.6 Hz, 1H, H-14b), 2.05 (m, 1H, H-6b), 1.89 (s, 3H, CH3), 1.85 (brs, 6H, 2 × CH3), 1.26 (s, 3H, CH3), 1.10 (s, 3H, CH3), 0.78 (s, 9H, SiC(CH3)3), <sup>−</sup>0.11 (s, 3H, Si(CH3)), <sup>−</sup>0.28 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) <sup>δ</sup>: 191.1, 171.0, 168.7, 167.4, 166.3, 148.9, 142.0, 138.0, 137.1, 135.0, 133.9, 131.9, 131.0, 130.6, 129.6, 128.7 (2C), 128.1, 128.0, 127.0 (2C), 126.7 (2C), 124.5, 86.4, 87.0, 80.5, 76.0, 75.4, 70.5, 55.7, 43.1, 36.6, 29.7, 26.9, 25.5 (3C), 22.2, 21.5, 18.3, 14.8, 14.5, −5.5, −6.0; LC-MS (ESI, *m*/*z*): [M + 1], found 960.0, [M + 23], found 981.9, C51H62ClNO13Si: 959.37.

*2 -tert-Butyldimethylsilyl-2-debenzoyl-2-(3-trifluoromethylbenzoyl)-10-dehydro-7,8-seco-10-deacetylpaclitaxel* (**15e**). White solid; yield 76.5%; 1H-NMR (CDCl3, 400 MHz) δ: 8.38 (d, *J* = 7.9 Hz, 2H, Ar-H), 7.84 (d, *J* = 7.9 Hz, 2H, Ar-H), 7.48 (m, 11H, NH, Ar-H), 6.51 (s, 1H, OH-9), 6.29 (t, *J* = 8.4 Hz, 1H, H-13), 5.89 (d, *J* = 9.3 Hz, 1H, H-3 ), 5.62 (d, *J* = 9.4 Hz, 1H, H-2), 5.25 (brs, 2H, H-5, H-20a), 4.72 (brs, 1H, H-2 ), 4.32 (d, *J* = 6.8 Hz, 1H, H-3), 4.24 (brs, 1H, H- 20b), 3.94 (brs, 1H, H-7a), 3.76 (brs, 1H, H-7b), 2.78 (brs, 1H, H-6a), 2.66 (brs, 1H, H-14a), 2.28 (dd, *J* = 9.5, 15.6 Hz, 1H, H-14b), 2.05 (m, 1H, H-6b), 1.92 (s, 3H, CH3), 1.85 (brs, 6H, 2 × CH3), 1.27 (s, 3H, CH3), 1.11 (s, 3H, CH3), 0.78 (s, 9H, SiC(CH3)3), −0.08 (s, 3H, Si(CH3)), <sup>−</sup>0.29 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) <sup>δ</sup>: 191.0, 170.9, 167.5, 166.2, 148.9, 142.1, 137.9, 133.9, 133.1, 131.9, 131.4 (q, <sup>2</sup>*J*C-F = 33.3 Hz), 130.2, 128.7, 128.0, 126.9, 126.7 (q, <sup>3</sup>*J*C-F = 4.1 Hz), 126.5, 123.5 (q, <sup>1</sup>*J*C-F = 273.7 Hz), 87.2, 86.3, 80.6, 75.8, 75.6, 74.5, 70.4, 67.6, 59.0, 55.7, 44.4, 43.2, 38.3, 36.6, 29.7, 29.1, 25.5, 22.1, 21.5, 18.2, 14.8, 14.4, −5.5, −6.0; LC-MS (ESI, *m*/*z*): [M + 1], found 994.3, [M + 23], found 1016.3, C52H62F3NO13Si: 993.39.

#### 3.1.4. General Experimental Procedure for Compounds **16a**–**e**

*10-Dehydro-7,8-seco-10-deacetylpaclitaxel* (**16a**). To a solution of of **15a** (19.8 mg, 0.0214 mmol) in THF (1.4 mL) was added, dropwise, 0.3 mL of HF-pyridine (*v*/*v* = 1:2) at 0 ◦C, and the reaction mixture was stirred at room temperature for 30 h. The reaction was quenched with saturated aqueous NaHCO3, diluted with ethyl acetate, washed with saturated NH4Cl. The organic layer dried over anhydrous Na2SO4 and then concentrated under reduced pressure. Purification of the crude product by silica gel chromatography (ethyl acetate: petroleum ether = 2:1) gave product **16a** as a white solid (13.4 mg, 77.4% yield). 1H-NMR (CDCl3, 400 MHz) δ: 8.04 (d, *J* = 7.6 Hz, 2H, Ar-H), 7.69 (d, *J* = 7.2 Hz, 2H, Ar-H), 7.57 (t, *J* = 7.4 Hz, 1H, Ar-H), 7.48–7.44 (m, 5H, Ar-H), 7.38–7.27 (m, 6H, NH, Ar-H), 6.47 (s, 1H, OH-9), 6.16 (t, *J* = 7.5 Hz, 1H, H-13), 5.86 (dd, *J* = 2.8, 9.4 Hz, 1H, H-3 ), 5.60 (d, *J* = 9.5 Hz, 1H, H-2), 5.20 (brs, 1H, H-5), 5.16 (d, *J* = 11.4 Hz, 1H, H-20a), 4.78 (d, *J* = 3.0 Hz, 1H, H-2 ), 4.27 (d, *J* = 8.1 Hz, 1H, H-3), 4.20 (brs, 1H, H-20b), 3.89 (brs, 1H, H-7a), 3.68 (dt, *J* = 6.2, 10.1 Hz, 1H, H-7b), 2.77 (brs, 1H, H-6a), 2.61 (brs, 1H, H-14a), 2.38 (dd, *J* = 9.7, 15.9 Hz, 1H, H-14b), 2.29 (brs, 1H, OH), 2.10–2.07 (m, 1H, H-6b), 1.83 (brs, 6H, 2 <sup>×</sup> CH3), 1.75 (s, 3H, CH3), 1.20 (s, 3H, CH3), 1.07 (s, 3H, CH3); 13C-NMR (CDCl3, 100 MHz) δ: 191.0, 172.4, 169.0, 167.3, 167.2, 148.8, 142.2, 138.0, 136.7, 133.8, 133.6, 132.0, 129.7, 129.3, 129.0, 128.8, 128.7, 128.1, 127.1, 127.0, 124.6, 86.9, 86.2, 80.5, 74.8, 73.7, 70.7, 59.6, 54.8, 43.0, 36.6, 29.7, 24.9, 22.1, 21.2, 18.4, 14.6, 14.5; LC-MS (ESI, *m*/*z*): [M + 1], found 812.0, [M + 23], found 834.0, C45H49NO13: 811.32.

*2-Debenzoyl-2-(3-methoxybenzoyl)-10-dehydro-7,8-seco-10-deacetylpaclitaxel* (**16b**). White solid; yield 65.2%; 1H-NMR (CDCl3, 400 MHz) δ: 7.72 (d, *J* = 7.6 Hz, 2H, Ar-H), 7.60 (d, *J* = 7.1 Hz, 1H, Ar-H), 7.47–7.43 (m, 5H, Ar-H), 7.36–7.31 (m, 5H, Ar-H), 7.29–7.25 (m, 1H, Ar-H), 7.10 (d, *J* = 8.1 Hz, 1H, NH), 6.49 (brs, 1H, OH-9), 6.09 (t, *J* = 7.4 Hz, 1H, H-13), 5.81 (d, *J* = 9.2 Hz, 1H, H-3 ), 5.58 (d, *J* = 9.5 Hz, 1H, H-2), 5.20 (brs, 1H, H-5), 5.14 (d, *J* = 11.3 Hz, 1H, H-20a), 4.73 (brs, 1H, H-2 ), 4.24 (d, *J* = 7.2 Hz, 1H, H-3), 4.19 (brs, 1H, H-20b), 3.87 (brs, 1H, H-7a), 3.79 (s, 3H, OCH3), 3.65 (brs, 1H, H-7b), 2.74 (brs, 1H, H-6a), 2.56 (brs, 1H, H-14a), 2.40 (dd, *J* = 9.9, 15.9 Hz, 1H, H-14b), 2.35 (brs, 1H, OH-1), 2.09 (m, 1H, H-6b), 1.83 (brs, 6H, 2 <sup>×</sup> CH3), 1.69 (s, 3H, CH3), 1.17 (s, 3H, CH3), 1.06 (s, 3H, CH3); 13C-NMR (CDCl3, 100 MHz) δ: 191.1, 172.4, 169.0, 167.1, 159.8, 148.8, 142.1, 138.1, 136.7, 133.6, 131.9, 130.7, 130.1, 129.0, 128.7, 128.6, 128.2, 128.1, 127.1, 124.5, 121.8, 119.5, 114.8, 86.9, 86.1, 80.4, 74.8, 73.7, 70.5, 59.7, 55.5, 55.0, 44.5, 42.9, 36.6, 29.8, 29.7, 24.9, 22.2, 21.1, 14.6, 14.5; LC-MS (ESI, *m*/*z*): [M + 1], found 842.4, [M + 23], found 864.3, C46H51NO14: 841.33.

*2-Debenzoyl-2-(3-fluorobenzoyl)-10-dehydro-7, 8-seco-10-deacetylpaclitaxel* (**16c**). White solid; yield 59.3%, 1H-NMR (CDCl3, 300 MHz) δ: 7.90 (d, *J* = 7.7 Hz, 1H, Ar-H), 7.75–7.68 (m, 3H, Ar-H), 7.48–7.44 (m, 4H, Ar-H), 7.41–7.31 (m, 6H, Ar-H), 7.24 (d, *J* = 8.0 Hz, 1H, NH), 6.46 (brs, 1H, OH-9), 6.20 (t, *J* = 7.9Hz, 1H, H-13), 5.88 (d, *J* = 7.6 Hz, 1H, H-3 ), 5.59 (d, *J* =9.5 Hz, 1H, H-2), 5.19 (overlap, *J* = 11.5 Hz, 2H, H-20a, H-5), 4.81 (brs, 1H, H-2 ), 4.46 (brs, 1H, OH), 4.29 (d, *J* = 7.9Hz, 1H, H-3), 4.22 (brs, 1H, H-20b), 3.92 (brs, 1H, H-7a), 3.70 (brs, 1H, H-7b), 3.39 (brs, 1H, OH), 2.77 (brs, 1H, H-6a), 2.60 (brs, 1H, H-14a), 2.37 (dd, *J* = 9.7, 15.8 Hz, 1H, H-14b), 2.09 (m, 1H, H-6b), 1.85 (brs, 6H, 2 × CH3), 1.78 (s, 3H, CH3), 1.64 (brs, 1H, OH-1), 1.22 (s, 3H, CH3), 1.09 (s, 3H, CH3); 13C-NMR (CDCl3, 75 MHz) δ: 190.9, 172.4, 167.2, 166.2, 162.7 (d, <sup>1</sup>*J*C-F = 248.7 Hz), 148.9, 142.1, 137.9, 133.6, 132.0, 131.4 (d, <sup>2</sup>*J*C-F = 7.4 Hz), 130.8 (d, <sup>3</sup>*J*C-F = 7.9 Hz), 128.9, 128.7, 128.2, 127.0, 126.9, 125.5, 124.4, 121.0 (d, <sup>2</sup>*J*C-F = 21.2 Hz), 116.5 (d, <sup>2</sup>*J*C-F = 23.5 Hz), 86.8, 86.2, 80.5, 75.2, 74.7, 73.6, 70.7, 59.5, 54.6, 42.9, 36.5, 29.7, 29.3, 24.9, 22.1, 21.2, 14.7, 14.5; LC-MS (ESI, *m*/*z*): [M + 1], found 830.3, [M + 23], found 852.3, C45H48FNO13: 829.31.

*2-Debenzoyl-2-(3-chlorobenzoyl)-10-dehydro-7, 8-seco-10-deacetylpaclitaxel* (**16d**). White solid; yield 60.3%; 1H-NMR (CDCl3, 400 MHz) δ: 7.98 (d, *J* = 7.5 Hz, 2H, Ar-H), 7.72 (d, *J* = 7.0 Hz, 2H, Ar-H), 7.56 (d, *J* = 8.4 Hz, 1H, Ar-H), 7.47–7.42 (m, 4H, Ar-H), 7.40–7.29 (m, 6H, NH, Ar-H), 6.49 (brs, 1H, OH-9), 6.15 (t, *J* = 7.8 Hz, 1H, H-13), 5.86 (d, *J* = 8.1 Hz, 1H, H-3 ), 5.58 (d, *J* = 9.5 Hz, 1H, H-2), 5.21 (brs, 1H, H-5), 5.14 (d, *J* =11.0 Hz, 1H, H-20a), 4.79 (d, *J* = 2.8 Hz, 1H, H-2 ), 4.27 (d, *J* =7.8 Hz, 1H, H-3), 4.20 (brs, 1H, H-20b), 3.89 (brs, 1H, H-7a), 3.71–3.67 (m, 1H, H-7b), 2.75 (brs, 1H, H-6a), 2.59 (brs, 1H, H-14a), 2.38 (dd, *J* = 9.7, 15.8 Hz, 1H, H-14b), 2.25 (brs, 1H, OH), 2.09 (m, 1H, H-6b), 2.00 (m, 1H, OH), 1.84 (brs, 6H, 2 <sup>×</sup> CH3), 1.74 (s, 3H, CH3), 1.20 (s, 3H, CH3), 1.07 (s, 3H, CH3); 13C-NMR (CDCl3, 100 MHz) δ: 191.0, 172.5, 167.2, 166.0, 148.9, 142.1, 138.1, 135.0, 133.8, 133.7, 132.0, 131.1, 130.4, 129.5, 128.8, 128.7, 128.1, 127.9, 127.0, 124.3, 86.9, 86.2, 80.4, 75.3, 74.7, 73.6, 70.6, 59.6, 54.8, 50.8, 42.9, 36.6, 29.7, 29.5, 29.3, 24.9, 22.2, 21.2, 14.6, 14.5; LC-MS (ESI, *m*/*z*): [M + 1], found 845.9, [M + 23], found 867.8, C45H48ClNO13: 845.28.

*2-Debenzoyl-2-(3-trifluoromethylbenzoyl)-10-dehydro-7,8-seco-10-deacetylpaclitaxel* (**16e**). White solid; yield 55.5%; 1H-NMR (CDCl3, 400 MHz) δ: 8.33 (d, *J* = 7.7 Hz, 1H, Ar-H), 8.25 (brs, 1H, NH), 7.85 (d, *J* = 7.4 Hz, 1H, Ar-H), 7.68–7.62 (m, 3H, Ar-H), 7.48–7.45 (m, 3H, Ar-H), 7.40–7.29 (m, 6H, Ar-H), 6.47 (brs, 1H, OH-9), 6.18 (t, *J* = 7.9 Hz, 1H, H-13), 5.87 (d, *J* = 7.6 Hz, 1H, H-3 ), 5.62 (d, *J* = 9.4 Hz, 1H, H-2), 5.18 (overlap, *J* = 11.5 Hz, 2H, H-20a, H-5), 4.80 (d, *J* = 2.8 Hz, 1H, H-2 ), 4.62 (brs, 1H, OH), 4.28 (d, *J* = 7.6 Hz, 1H, H-3), 4.24 (brs, 1H, H-20b), 3.90 (brs, 1H, H-7a), 3.68 (brs, 1H, H-7b), 3.38 (brs, 1H, OH), 2.77 (brs, 1H, H-6a), 2.58 (brs, 1H, H-14a), 2.37 (dd, *J* = 9.7, 15.7 Hz, 1H, H-14b), 2.05 (brs, 1H, OH), 2.03–1.98 (m, 1H, H-6b), 1.84 (brs, 6H, 2 <sup>×</sup> CH3), 1.78 (s, 3H, CH3), 1.22 (s, 3H, CH3), 1.09 (s, 3H, CH3); 13C-NMR (CDCl3, 100 MHz) <sup>δ</sup>: 190.9, 172.4, 167.2, 166.0, 148.9, 142.1, 138.0, 133.7, 133.0, 132.0, 131.7, 130.3, 130.0, 129.7, 128.9, 128.7, 128.2, 127.0 (2C), 126.4, 124.8, 124.2, 122.1, 86.9, 86.2, 80.5, 75.4, 74.7, 73.5, 70.7, 59.6, 54.7, 43.0, 36.6, 31.9, 29.7, 25.5, 22.1, 21.2, 14.6, 14.4; LC-MS (ESI, *m*/*z*): [M + 1], found 880.0, [M + 23], found 901.9, C46H48F3NO13: 879.31.

3.1.5. General Experimental Procedure for Compounds **17a**–**e**

*2 -O-(tert-Butyldimethylsilyl)-9-O-(2-hydroxyethyl)-10-dehydro-7, 8-seco-10-deacetylpaclitaxel* (**17a**). To a mixture of **15a** (50 mg, 0.0540 mmol), potassium carbonate (104 mg, 0.754 mmol), and potassium iodide (18 mg, 0.108 mmol) in DMF (0.9 mL) was added 2-bromoethanol (54 μL 0.762 mmol), dropwise, at 0 ◦C. The reaction mixture was stirred for 9 h at room temperature and then was diluted with ethyl acetate. The organic phase was washed with saturated aqueous NH4Cl. The organic layer was dried over anhydrous Na2SO4 and then concentrated under reduced pressure. Purification of the crude product by silica gel chromatography (ethyl acetate: petroleum ether = 1:1) gave product **17a** as a white solid (19 mg, 52.4% yield). 1H-NMR (CDCl3, 300 MHz) δ: 8.09 (d, *J* = 7.2 Hz, 2H, Ar-H), 7.62 (d, *J* = 7.2 Hz, 2H, Ar-H), 7.57 (d, *J* = 7.5 Hz, 1H, Ar-H), 7.49 (t, *J* = 7.5 Hz, 2H, Ar-H), 7.36 (m, 8H, Ar-H), 6.30 (t, *J* = 8.4 Hz, 1H, H-13), 5.88 (d, *J* = 9.0 Hz, 1H, H-3 ), 5.57 (d, *J* = 9.6 Hz, 1H, H-2), 5.28 (overlap, 2H, H-5, H-20a), 4.69 (d, *J* = 1.8 Hz, 1H, H-2 ), 4.26 (d, *J* = 7.8 Hz, 1H, H-3), 4.20 (m, 2H, H-20b, OCH2CH2OCO), 3.94 (m, 1H, OCH2CH2OCO), 3.87 (m, *J* = 1.8, 6.3, 8.7, 12.0 Hz, 1H, H-7a), 3.74 (overlap, 2H, H-7b, OCH2CH2OCO), 3.62 (m, 1H, OCH2CH2OCO), 2.71 (m, 2H, H-6a, H-14a), 2.25 (dd, *J* = 9.3, 15.9 Hz, 2H, H-6b, H-14b), 1.97 (s, 3H, CH3), 1.92 (s, 3H, CH3), 1.82 (s, 3H, CH3), 1.27 (s, 3H, CH3), 1.16 (s, 3H, CH3), 0.76 (s, 9H, SiC(CH3)3), <sup>−</sup>0.08 (s, 3H, Si(CH3)), <sup>−</sup>0.33 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) <sup>δ</sup>: 192.3, 170.7, 168.7, 167.3, 153.8, 144.0, 139.4, 138.0, 133.7 (2C), 131.8, 129.9 (2C), 128.9 (2C), 128.6 (4C), 127.9, 126.9 (2C), 126.4 (2C), 87.1, 87.7, 85.7, 79.9, 75.6, 75.4, 74.6, 73.8, 70.5, 61.8, 58.7, 55.6, 42.9, 36.6, 25.4 (3C), 22.9, 22.0, 18.1, 15.3, 14.6, −5.5, −6.1; LC-MS (ESI, *m*/*z*): [M + 1], found 970.5, [M + 23], found 992.4, C53H67NO14Si: 969.43.

*2 -O-(tert-Butyldimethylsilyl)-2-debenzoyl-2-(3-methoxybenzoyl)-9-O-(2-hydroxyethyl)-10-dehydro-7,8-seco-10-deacetylpaclitaxel* (**17b**). White solid; yield 56.1%; 1H-NMR (CDCl3, 400 MHz) δ: 7.72–7.66 (m, 3H, Ar-H), 7.54 (s, 1H, Ar-H), 7.47–7.43 (m, 2H, Ar-H), 7.42–7.29 (m, 8H, Ar-H), 7.12 (dd, *J* = 2.0, 8.4 Hz, 1H, BzNH), 6.29 (t, *J* = 8.5 Hz, 1H, H-13), 5.88 (d, *J* = 9.3 Hz, 1H, H-3 ), 5.59 (d, *J* = 9.6 Hz, 1H, H-2), 5.26 (overlap, 2H, H-5, H-20a), 4.72 (s, 1H, H-2 ), 4.26 (d, *J* = 6.4 Hz, 1H, H-3), 4.19–4.15 (m, 1H, H-20b), 3.94–3.64 (m, 9H, OCH3, OCH2CH2OH, H-7a, H-7b), 2.61 (br, 2H, H-6b, H-14b), 2.25 (dd, *J* = 9.6, 15.6 Hz, 2H, H-6a, H-14a), 2.03 (s, 3H, OAc), 1.93 (s, 6H, 2 × CH3), 1.26 (s, 3H, CH3), 1.16 (s, 3H, CH3), 0.77 (s, 9H, SiC(CH3)3), <sup>−</sup>0.10 (s, 3H, Si(CH3)), <sup>−</sup>0.30 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) <sup>δ</sup>: 192.4, 171.2, 170.9, 168.8, 167.3, 167.0, 162.6, 159.7, 144.1, 138.1, 133.8, 131.8, 130.4, 130.2, 128.6 (2C), 127.9, 127.0 (2C), 126.6 (2C), 122.2, 119.5, 114.9, 85.7, 79.8, 75.8, 74.8, 61.8, 58.9, 55.7, 55.5, 42.9, 36.6, 36.5, 31.9, 31.4, 29.7, 25.5 (3C), 22.9, 22.1, 18.2, 15.2, 14.6, −5.5, −6.1; LC-MS (ESI, *m*/*z*): [M + 1], found 1000.5, [M + 23], found 1022.5, C54H69NO16Si: 999.44.

*2 -O-(tert-Butyldimethylsilyl)-2-debenzoyl-2-(3-fluorobenzoyl)-9-O-(2-hydroxyethyl)-10-dehydro-7,8-seco-10-deacetylpaclitaxel* (**17c**). White solid; yield 53.2%; 1H-NMR (CDCl3, 400 MHz) δ: 7.93 (d, *J* = 7.8 Hz, 1H, Ar-H), 7.76 (d, *J* = 8.8 Hz, 1H, Ar-H), 7.63 (d, *J* = 7.6 Hz, 2H, Ar-H), 7.52–7.28 (m, 11H, NH, Ar-H), 6.29 (t, *J* = 8.1 Hz, 1H, H-13), 5.89 (d, *J* = 9.2 Hz, 1H, H-3 ), 5.58 (d, *J* = 9.6 Hz, 1H, H-2), 5.35–5.15 (overlap, 2H, H-5, H-20a), 4.70 (d, *J* = 1.9 Hz, 1H, H-2 ), 4.26 (d, *J* = 7.6 Hz, 1H, H-3), 4.20–4.16 (m, 2H, H-20b, OCH2CH2OCO), 3.95 (brs, 1H, OCH2CH2OCO), 3.89–3.84 (ddd, *J* = 2.3, 6.8, 8.7, 12.4 Hz, 1H, H-7a), 3.80–3.70 (overlap, 2H, H-7b, OCH2CH2OCO), 3.60–3.58 (m, 1H, OCH2CH2OCO), 2.71–2.63 (m, 2H, H-6a, H-14a), 2.25 (dd, *J* = 9.6, 15.8 Hz, 2H, H-6b, H-14b), 2.03 (s, 3H, CH3), 1.91(s, 3H, CH3), 1.87 (brs, 3H, CH3), 1.83 (brs, 2H, 2 × OH), 1.26 (s, 3H, CH3), 1.15 (s, 3H, CH3), 0.73 (s, 9H, SiC(CH3)3), <sup>−</sup>0.08 (s, 3H, Si(CH3)), <sup>−</sup>0.32 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) <sup>δ</sup>: 192.3, 171.2, 170.8, 168.6, 167.4, 166.2, 162.7 (d, <sup>1</sup>*J*C-F = 248.3 Hz), 144.0, 137.9, 133.9, 131.9, 131.2 (d, <sup>3</sup>*J*C-F = 7.4 Hz), 131.0 (d, <sup>3</sup>*J*C-F = 7.7 Hz), 128.7 (2C), 128.0, 127.0, 126.5, 125.8 (d, <sup>4</sup>*J*C-F = 2.6 Hz), 121.0 (d, <sup>2</sup>*J*C-F = 21.2 Hz), 116.3 (d, <sup>2</sup>*J*C-F = 22.9 Hz), 85.8, 80.0, 75.7, 75.2, 70.5, 61.9, 58.7, 55.6, 45.7, 42.9, 36.7, 25.5, 22.9, 22.1, 18.2, 15.3, 14.6, 13.7, −5.4, −6.0; LC-MS (ESI, *m*/*z*): [M + 1], found 988.0, C53H66FNO14Si: 987.42.

*2 -O-(tert-Butyldimethylsilyl)-2-debenzoyl-2-(3-chlorobenzoyl)-9-O-(2-hydroxyethyl)-10-dehydro-7,8-seco-10-deacetylpaclitaxel* (**17d**). White solid; yield 60.9%; 1H-NMR (CDCl3, 400 MHz) δ: 8.06 (d, *J* = 8.8 Hz, 2H, Ar-H), 7.63 (d, *J* = 7.5 Hz, 2H, Ar-H), 7.57 (d, *J* = 8.2 Hz, 1H, Ar-H), 7.50–7.43 (m, 3H, Ar-H), 7.43–7.33 (m, 8H, Ar-H, BzNH), 6.31 (t, *J* = 8.0 Hz, 1H, H-13), 5.90 (d, *J* = 9.2 Hz, 1H, H-3 ), 5.59 (d, *J* = 9.6 Hz, 1H, H-2), 5.32–5.28 (overlap, 2H, H-5, H-20b), 4.71 (d, *J* = 1.9 Hz, 1H, H-2 ), 4.28 (d, *J* = 7.6 Hz, 1H, H-3), 4.24–4.20 (m, 2H, H-20a, OCH2CH2OH), 3.98–3.95 (m, 1H, OCH2CH2OH), 3.93–3.89 (m, 1H, H-7b), 3.78–3.72 (overlap, 2H, H-7a, OCH2CH2OH), 3.65 (br, 1H, OCH2CH2OH), 2.72–2.68 (m, 2H, H-6b, H-14b), 2.26 (dd, *J* = 7.2, 13.4 Hz, 2H, H-6a, H-14a), 1.98 (s, 3H, CH3), 1.93 (brs, 1H, OH), 1.85 (brs, 2H, OH), 1.29 (brs, 6H, CH3), 1.18 (s, 3H, CH3), 0.78 (s, 9H, SiC(CH3)3), −0.08 (s, 3H, Si(CH3)), <sup>−</sup>0.30 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) <sup>δ</sup>: 192.3, 170.8, 167.4, 166.3, 144.0, 137.9, 135.0, 133.9 (2C), 132.0, 130.8, 130.7, 129.7, 128.8 (2C), 128.7 (2C), 128.2, 128.1, 127.0 (2C), 126.6 (2C), 85.9, 75.8, 75.3, 70.5, 62.0, 55.6, 43.0, 36.7, 32.0, 29.7, 29.4, 27.3, 25.5 (3C), 23.0, 22.7, 22.2, 18.3, 15.3, 14.7, 14.2, −5.4, −6.0; LC-MS (ESI, *m*/*z*): [M + 1], found 1004.4, [M + 23], found 1026.4, C53H66ClNO14Si: 1003.39.

*2 -O-(tert-Butyldimethylsilyl)-2-debenzoyl-2-(3-trifluoromethylbenzoyl)-9-O-(2-hydroxyethyl)-10-dehydro-7, 8-seco-10-deacetylpaclitaxel* (**17e**). White solid; yield 49.9%; 1H-NMR (CDCl3, 400 MHz) δ: 7.83 (d, *J* = 7.7 Hz, 2H, Ar-H), 7.68 (t, *J* = 7.8 Hz, 1H, Ar-H), 7.58 (d, *J* = 7.3 Hz, 1H, Ar-H), 7.45 (t, *J* = 7.4 Hz, 2H, Ar-H), 7.35 (m, 8H, Ar-H), 6.29 (t, *J* = 8.2 Hz, 1H, H-13), 5.87 (d, *J* = 8.7 Hz, 1H, H-3 ), 5.60 (d, *J* = 9.65 Hz, 1H, H-2), 5.26 (overlap, 2H, H-5, H-20a), 4.70 (d, *J* = 1.3 Hz, 1H, H-2 ), 4.28 (d, *J* = 6.8 Hz, 1H, H-3), 4.19 (m, 2H, H-20b, OCH2CH2OCO), 3.98 (m, 1H, OCH2CH2OCO), 3.87 (m, *J* = 1.8, 6.3, 8.7, 12.0 Hz, 1H, H-7a), 3.75 (overlap, 2H, H-7b, OCH2CH2OCO), 3.66 (m, 1H, OCH2CH2OCO), 2.65 (m, 2H, H-6a, H-14a), 2.24 (dd, *J* = 6.7, 16.3 Hz, 2H, H-6b, H-14b), 1.97 (s, 3H, CH3), 1.92 (s, 3H, CH3), 1.82 (s, 3H, CH3), 1.26 (s, 3H, CH3), 1.17 (s, 3H, CH3), 0.77 (s, 9H, SiC(CH3)3), −0.08 (s, 3H, Si(CH3)), −0.32 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) δ: 192.3, 170.8, 167.6, 166.1, 144.1, 137.8, 133.9, 133.2, 132.0, 131.4 (q, 2JC-F = 33.2 Hz), 130.3, 130.2 (q, 3JC-F = 3.5 Hz), 130.0, 128.8, 128.7, 128.1, 126.9, 126.8 (q, 3JC-F = 3.6 Hz), 126.5, 123.6 (q, 1JC-F = 273.8 Hz), 85.8, 75.6, 75.4, 70.5, 62.0, 58.4, 55.6, 43.0, 36.7, 31.9, 29.8, 29.7, 29.6 (2C), 29.3 (2C), 27.2, 25.5, 23.0, 22.7, 22.1, 18.4, 18.2, 15.3, 14.6, 14.1, −5.4, −6.0; LC-MS (ESI, *m*/*z*): [M + 1], found 1038.4, [M + 23], found 1060.4, C54H66F3NO14Si: 1037.42.

3.1.6. General Experimental Procedure for Compounds **19a**–**e**

*2 -O-(tert-Butyldimethylsilyl)-macrocyclic taxoid* (**19a**). To a solution of **17a** (19 mg, 0.0196 mmol) in dichloromethane (4.2 mL) was added, dropwise, pyridine (22 μL, 0.266 mmol) and triphosgene (8.7 mg, 0.0293 mmol) at 0 ◦C, then the reaction mixture was stirred at room temperature for 5 h. The reaction was diluted with ethyl acetate, washed with saturated NaCl. The organic layer was dried over anhydrous Na2SO4 and then concentrated under reduced pressure. Purification of the crude product by silica gel chromatography (ethyl acetate: petroleum ether = 1:3) gave product **19a** as a white solid (3.1 mg, 15.9% yield). 1H-NMR (CDCl3, 400 MHz) δ: 8.17 (d, *J* = 7.2 Hz, 2H, Ar-H), 7.75 (d, *J* = 7.2 Hz, 2H, Ar-H), 7.63 (t, *J* = 7.2 Hz, 1H, Ar-H), 7.54 (t, *J* = 7.6 Hz, 2H, Ar-H), 7.50 (d, *J* = 7.6 Hz, 1H, Ar-H), 7.43 (d, *J* = 7.6 Hz, 2H, Ar-H), 7.39 (t, *J* = 7.2 Hz, 2H, Ar-H), 7.34–7.29 (m, 3H, Ar-H), 7.08 (d, *J* = 9.2 Hz, 1H, NH), 6.27 (t, *J* = 8.8 Hz, 1H, H-13), 5.76 (d, *J* = 8.4 Hz, 1H, H-3 ), 5.60 (d, *J* = 7.6 Hz, 1H, H-2), 4.84 (overlap, 2H, H-5, OCH2CH2OCO), 4.79 (d, *J* = 7.6 Hz, 1H, H-3), 4.67 (d, *J* = 2.0 Hz, 1H, H-2 ), 4.57 (dd, *J* = 6.8, 12.0 Hz, 1H, OCH2CH2OCO), 4.49 (dd, *J* = 6.8, 12.0 Hz, 1H, OCH2CH2OCO), 4.46 (d, *J* = 8.4 Hz, 1H, H-20a), 4.35 (d, *J* = 8.4 Hz, 1H, H-20b), 4.32 (dd, *J* = 5.2, 13.6 Hz, 1H, OCH2CH2OCO), 4.13 (ddd, *J* = 6.4, 10.8, 17.2 Hz, 1H, H-7a), 4.02 (ddd, *J* = 8.8, 11.6, 17.2 Hz, 1H, H-7b), 2.55 (s, 3H, 4-OAc), 2.35 (m, 3H, H-6a, H-6b, H-14a), 2.18 (dd, *J* = 8.8, 15.2 Hz, 1H, H-14b), 1.84 (s, 3H, CH3), 1.25 (s, 3H, CH3), 1.24 (s, 3H, CH3), 1.19 (s, 3H, CH3), 0.79 (s, 9H, SiC(CH3)3), <sup>−</sup>0.03 (s, 3H, Si(CH3)), <sup>−</sup>0.30 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 150 MHz) <sup>δ</sup>: 192.1, 171.0, 169.4, 166.9, 166.8, 152.4, 151.5, 144.5, 139.7, 138.3, 134.6, 134.1, 133.8, 131.8, 130.2 (2C), 129.0, 128.8 (2C), 128.7, 127.9, 127.0 (2C), 126.3 (2C), 89.6, 84.7, 78.6, 76.1, 75.3, 75.2, 71.4, 70.6, 68.9, 65.9, 55.6, 42.9, 42.4, 36.5, 31.0, 26.2, 25.5 (3C), 22.9, 21.6, 18.1, 14.4, 13.6, −5.3, −5.9; LC-MS (ESI, *m*/*z*): [M + 1], found 996.4, [M + 23], found 1018.4, C54H65NO15Si: 995.41.

*2 -O-(tert-Butyldimethylsilyl)-2-debenzoyl-2-(3-methoxybenzoyl)-macrocyclic taxoid* (**19b**). White solid; yield 32.7%; 1H-NMR (CDCl3, 400 MHz) δ: 7.80 (d, *J* = 1.2, 7.6 Hz, 1H, Ar-H), 7.77 (d, *J* = 6.8 Hz, 2H, Ar-H), 7.67 (dd, *J* = 1.5, 2.5 Hz, 1H, Ar-H), 7.55–7.51 (m, 1H, Ar-H), 7.49–7.39 (m, 3H, Ar-H), 7.40–7.31 (m, 5H, Ar-H), 7.20–7.17 (m, 1H, Ar-H), 7.08 (d, *J* = 9.2 Hz, 1H, BzNH), 6.28 (t, 1H, H-13), 5.76 (dd, *J* = 1.6, 8.9 Hz, H-3 ), 5.62 (d, *J* = 7.5 Hz, 1H, H-2), 4.87 (d, *J* = 7.6 Hz, 1H, H-20b), 4.85 (d, *J* = 4.8 Hz, 1H, H-5), 4.81 (d, *J* = 7.5 Hz, 1H, H-20a), 4.68 (d, *J* = 2.0 Hz, 1H, H-2 ), 4.60–4.32 (m, 5H, H-7a, H-7b, H-16a, H-16b, H-3), 4.18–4.03 (m, H-15a, H-15b), 3.91 (s, 3H, OCH3), 2.55 (d, *J* = 1.3 Hz, 6H, 2 × CH3), 2.41–2.35 (m, 3H, H-14b, H-6a, H-6b), 2.24–2.20 (m, 1H, H-14a), 1.84 (d, *J* = 1.2 Hz, 3H, COCH3), 1.26 (s, 3H, CH3), 1.21 (s, 3H, CH3), 0.81 (s, 9H, SiC(CH3)3), <sup>−</sup>0.008 (s, 3H, Si(CH3)), <sup>−</sup>0.28 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) δ: 192.2, 171.1, 169.4, 166.9, 166.8, 159.8, 152.5, 151.6, 144.6, 139.7, 138.4, 134.7, 134.2, 131.9, 130.4, 130.0, 128.82 (2C), 128.78 (2C), 128.0, 127.0 (2C), 126.4 (2C), 122.6, 120.3, 114.7, 89.6, 84.8, 78.5, 75.4, 75.3, 71.5, 70.7, 69.0, 65.9, 55.6, 43.0, 42.5, 36.5, 31.1, 29.7, 26.3, 25.6 (3C), 22.9, 21.6, 18.2, 14.4, 13.7, −5.2,−5.8; LC-MS (ESI, *m*/*z*): [M + 1], found 1026.4, [M + 23], found 1048.4, C55H67NO16Si: 1025.42.

*2 -O-(tert-Butyldimethylsilyl)-2-debenzoyl-2-(3-fluorobenzoyl)-macrocyclic taxoid* (**19c**). White solid; yield 32.1%; 1H-NMR (CDCl3, 400 MHz) δ: 8.01(d, *J* = 7.8 Hz, 1H, Ar-H), 7.84–7.87 (m, 1H, Ar-H), 7.75–7.73 (m, 2 H, Ar-H), 7. 56–7.49 (m, 2H, Ar-H), 7.44–7.31 (m, 8H, Ar-H), 7.09 (d, *J* = 9.0 Hz, 1H, NH), 6.28 (t, *J* = 8.8 Hz,1H, H-13), 5.78 (dd, *J* = 1.4, 9.1 Hz, 1H, H-3 ), 5.61 (d, *J* = 7.6 Hz, 1H, H-2), 4.87–4.82 (overlap, 2H, OCH2CH2OCO, H-5), 4.83 (d, *J* = 7.5 Hz, 1H, H-3), 4.69 (d, *J* = 2.0 Hz, 1H, H-2 ), 4.58 (dd, *J* = 6.4, 11.9 Hz, 1H, OCH2CH2OCO), 4.51 (dd, *J* = 6.4, 13.7 Hz, 1H, OCH2CH2OCO), 4.46 (d, *J* = 8.2 Hz, 1H, H-20a), 4.36–4.30 (overlap, 2H, OCH2CH2OCO, H-20b), 4.16–4.09 (m, 1H, H-7a), 4.06–3.99 (m, 17.2 Hz, 1H, H-7b), 2.57 (s, 3H, COCH3), 2.56 (s, 3H, 4-OAc), 2.39–2.31 (m, 3H, H-6a, H-6b, H-14a), 2.18 (dd, *J* = 8.4, 15.4 Hz, 1H, H-14b), 1.90 (brs, 1H, OH), 1.84 (s, 3H, CH3), 1.23 (s, 3H, CH3), 1.19 (s, 3H, CH3), 0.80 (s, 9H, SiC(CH3)3), <sup>−</sup>0.29 (s, 3H, Si(CH3)), <sup>−</sup>0.03 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) δ: 192.1, 171.1, 169.4, 167.0, 165.7, 162.7 (d, <sup>1</sup>*J*C-F = 247.8 Hz), 152.5, 151.6, 144.5, 139.8, 138.4, 134.5, 134.2, 131.9, 131.3 (d, <sup>3</sup>*J*C-F = 7.6Hz), 130.6 (d, <sup>3</sup>*J*C-F = 7.6 Hz), 128.8 (2C), 128.0, 127.0, 126.5, 126.0 (d, <sup>4</sup>*J*C-F = 3.0 Hz), 121.0 (d, <sup>2</sup>*J*C-F = 21.4 Hz), 117.1 (d, <sup>2</sup>*J*C-F = 23.3 Hz), 89.6, 84.7, 78.6, 76.1, 75.7, 75.3, 71.5, 70.7, 68.9, 65.9, 55.6, 43.0, 42.5, 36.4, 31.0, 26.3, 25.6, 22.8, 21.6, 18.2, 14.4, 13.6, −5.2, −5.8; LC-MS (ESI, *m*/*z*): [M + 1], found 1014.4, [M + 23], found 1036.4, C54H64FNO15Si: 1013.40.

*2 -O-(tert-Butyldimethylsilyl)-2-debenzoyl-2-(3-chlorobenzoyl)-macrocyclic taxoid* (**19d**). White solid; yield 36.1%; 1H-NMR (CDCl3, 400 MHz) δ: 8.19 (t, *J* = 1.6 Hz, 1H, Ar-H), 8.10–8.07 (m, 1H, Ar-H), 7.77–7.75 (m, 2H, Ar-H), 7.64–7.61 (m, 1H, Ar-H), 7.55–7.49 (m, 2H, Ar-H), 7.46–7.33 (m, 7H, Ar-H), 7.08 (d, *J* = 9.2 Hz, 1H, BzNH), 6.25 (m, 1H, H-13), 5.76 (dd, *J* = 1.6, 9.0 Hz, 1H, H-3 ), 5.59 (d, *J* = 7.6 Hz, 1H, H-2), 4.89–4.81 (m, 3H, OCH2CH2OCO, H-5, H-3), 4.68 (d, *J* = 2.0 Hz, 1H, H-2 ), 4.57 (dd, *J* = 6.6, 11.9 Hz, 1H, OCH2CH2OCO), 4.50 (dd, *J* = 6.5, 11.8 Hz, 1H, OCH2CH2OCO), 4.44 (d, *J* = 8.0 Hz, 1H, H-20a), 4.38–4.32 (m, 2H, OCH2CH2OCO, H-20b), 4.15–4.10 (m, 1H, H-7a), 4.07–4.01 (m, *J* = 17.2 Hz, 1H, H-7b), 2.56 (s, 3H, CH3), 2.55 (s, 3H, CH3), 2.41–2.30 (m, 3H, H-6a, H-6b, H-14a), 2.25–2.18 (m, 1H, H-14b), 1.84 (d, *J* = 1.2 Hz, 3H, CH3), 1.25 (s, 3H, CH3), 1.20 (s, 3H, CH3), 0.82 (s, 9H, SiC(CH3)3), −0.01 (s, 3H, Si(CH3)), <sup>−</sup>0.26 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) <sup>δ</sup>: 192.1, 171.1, 169.4, 165.6, 152.5, 151.7, 144.5, 139.8, 138.4, 134.9, 134.5, 133.6, 131.8, 131.0, 130.3, 128.81 (2C), 128.79 (2C), 128.4, 128.0, 127.0 (2C), 126.5 (2C), 89.6, 84.7, 78.7, 77.3, 75.6, 75.3, 70.8, 69.0, 55.7, 43.0, 42.5, 36.4, 29.7, 26.3, 25.6 (3C), 22.8, 21.6, 18.2, 14.4, 13.6, −5.2, −5.8; LC-MS (ESI, *m*/*z*): [M + 1], found 1030.4, [M + 23], found 1052.4, C54H64ClNO15Si: 1029.37.

*2 -O-(tert-Butyldimethylsilyl)-2-debenzoyl-2-(3-trifluoromethylbenzoyl)-macrocyclic taxoid* (**19e**). White solid; yield 30.5%; 1H-NMR (CDCl3, 400 MHz) δ: 8.51 (s, 1H, Ar-H), 8.41 (d, *J* = 7.8 Hz, 1H, Ar-H), 7.92 (d, *J* = 7.8 Hz, 1H, Ar-H), 7.76–7.72 (m, 3H, Ar-H), 7.55–7.51 (m, 1H, Ar-H), 7.47–7.40 (m, 4H, Ar-H), 7.37–7.33 (m, 3H, Ar-H), 7.09 (d, *J* = 9.1 Hz, 1H, NH), 6.24 (t, *J* = 8.8 Hz, 1H, H-13), 5.76 (dd, *J* = 1.0, 9.0 Hz, 1H, H-3 ), 5.63 (d, *J* = 7.6 Hz, 1H, H-2), 4.91–4.84 (m, 3H, H-3, H-5, OCH2CH2OCO), 4.67 (d, *J* = 1.8 Hz, 1H, H-2 ), 4.58 (dd, *J* = 6.8, 11.8 Hz, 1H, OCH2CH2OCO), 4.52 (dd, *J* = 6.8, 13.7 Hz, 1H, OCH2CH2OCO), 4.44 (d, *J* = 8.1 Hz, 1H, H-20a), 4.38–4.33 (m, 2H, OCH2CH2OCO, H-20b), 4.11–4.18 (m, 1H, H-7a), 4.09–4.02 (m, *J* = 17.2 Hz, 1H, H-7b), 2.55 (s, 3H, 4-OAc), 2.42–2.36 (m, 3H, H-6a, H-6b, H-14a), 2.29–2.20 (m,1H, H-14b), 1.94 (s, 1H, OH), 1.86 (d, *J* = 1.04 Hz, 3H, CH3), 1.29 (s, 3H, CH3), 1.26 (s, 3H, CH3), 1.22 (s, 3H, CH3), 0.84 (s, 9H, SiC(CH3)3), <sup>−</sup>0.004 (s, 3H, Si(CH3)), <sup>−</sup>0.24 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) <sup>δ</sup>: 192.0, 171.1, 169.3, 167.0, 165.4, 152.5, 151.6, 144.5, 139.8, 138.4, 134.4, 134.3, 133.6, 131.8, 131.4 (q, <sup>2</sup>*J*C-F = 33.2 Hz), 130.3 (q, <sup>3</sup>*J*C-F = 3.4 Hz), 130.2, 129.7, 128.8, 128.0, 127.0, 126.9 (q, <sup>3</sup>*J*C-F = 4.3 Hz), 126.5, 123.7 (q, <sup>1</sup>*J*C-F = 273.8 Hz), 89.6, 84.7, 78.7, 76.0, 75.8, 75.2, 71.5, 70.8, 69.0, 65.9, 55.7, 43.0, 42.4, 36.4, 31.0, 29.0, 26.3, 25.6, 22.7, 21.6, 18.2, 14.4, 13.6, −5.2,−5.8; LC-MS (ESI, *m*/*z*): [M + 1], found 1064.4, [M + 23], found 1086.4, C55H64F3NO15Si: 1063.40.

3.1.7. General Procedure for the Syntheses of Compounds **21a**–**e**

#### Macrocyclic taxoid (**21a**)

To a solution of of **19a** (32.3 mg, 0.0464 mmol) in THF (8 mL) was added, dropwise, 1.53 mL of HF-pyridine (*v*/*v* = 1:2) at 0 ◦C, and the reaction mixture was stirred at room temperature for 30 h. The reaction was quenched with saturated aqueous NaHCO3, diluted with ethyl acetate, and washed with saturated NH4Cl. The organic layer was dried over anhydrous Na2SO4 and then concentrated under reduced pressure. Purification of the crude product by silica gel chromatography (ethyl acetate: petroleum ether = 2:1) gave product **21a** as a white solid (13.2 mg, 46.2% yield). 1H-NMR (CDCl3, 400 MHz) δ: 8.14 (d, *J* = 7.0 Hz, 2H, Ar-H), 7.73 (d, *J* = 8.6 Hz, 2H, Ar-H), 7.62 (t, *J* = 7.5 Hz, 1H, Ar-H), 7.52–7.45 (m, 5H, Ar-H), 7.40–7.30 (m, 5H, Ar-H), 7.22 (d, *J* = 9.1 Hz, 1H, NH), 6.20 (t, *J* = 8.5 Hz, 1H, H-13), 5.78 (dd, *J* = 2.5, 9.1 Hz, 1H, H-3 ), 5.58 (d, *J* = 7.5 Hz, 1H, H-2), 4.80–4.74 (overlap, 4H, H-5, H-2 , H-3, OCH2CH2OCO), 4.52 (dd, *J* = 6.4, 12.0 Hz, 1H, OCH2CH2OCO), 4.45–4.44 (overlap, 2H, H-20a, OCH2CH2OCO), 4.31-4.25 (overlap, 2H, H-20b, OCH2CH2OCO), 4.07–3.91 (overlap, 2H, H-7b, H-7a),

2.56 (br, 1H, OH-2 ), 2.51 (s, 3H, CH3), 2.35 (s, 3H, CH3), 2.33–2.25 (overlap, 4H, H-14a, H-14b, H-6a, H-6b), 1.68 (s, 3H, CH3), 1.18 (s, 3H, CH3), 1.16 (s, 3H, CH3); 13C-NMR (CDCl3, 150 MHz) δ: 191.8, 172.4, 169.7, 166.9, 166.8, 152.4, 151.5, 144.8, 138.6, 137.9, 135.0, 133.9, 133.6, 132.0, 130.1 (2C), 129.0 (2C), 128.8 (4C), 128.3, 127.0 (2C), 89.4, 84.7, 78.4, 76.1, 75.1, 73.1, 71.8, 71.5, 68.9, 65.8, 54.8, 42.8, 42.4, 36.4, 30.9, 29.7, 26.3, 22.5, 21.2, 14.0, 13.7; LC-MS (ESI, *m*/*z*): [M + 1], found 882.8, [M + 23], found 904.4, C48H51NO15: 881.33.

*2-Debenzoyl-2-(3-methoxybenzoyl)-macrocyclic taxoid* (**21b**). White solid; yield 54.1%; 1H-NMR (CDCl3, 400 MHz) δ: 7.76–7.74 (m, 3H, Ar-H), 7.66-7.65 (m, 1H, Ar-H), 7.53–7.46 (m, 8H, Ar-H), 7.36–7.32 (m, 1H, Ar-H), 7.03 (d, *J* = 8.9 Hz, 1H, NH), 6.17 (t, *J* = 8.2 Hz, 1H, H-13), 5.79 (dd, *J* = 2.3, 9.0 Hz, 1H, H-3 ), 5.59 (d, *J* = 7.5 Hz, 1H, H-2), 4.84–4.77 (overlap, 4H, H-5, H-2 , H-3, OCH2CH2OCO), 4.55 (dd, *J* = 6.5, 11.9 Hz, 1H, OCH2CH2OCO), 4.45 (dd, *J* = 6.6, 13.2 Hz, 1H, OCH2CH2OCO), 4.43 (d, *J* = 7.5 Hz, 1H, H-20a), 4.36 (d, *J* = 8.29 Hz, 1H, H-20b), 4.32 (dd, *J* = 5.2, 13.8 Hz, 1H, OCH2CH2OCO), 4.12–3.96 (m, 2H, H-7b, H-7a), 3.88 (s, 3H, OAc), 3.62 (brs, 1H, OH-2 ), 2.53 (s, 3H, CH3), 2.36 (s, 3H, CH3), 2.40–2.27 (overlap, 4H, H-14a, H-14b, H-6a, H-6b), 1.93 (brs, 1H, OH-1), 1.68 (d, 3H, CH3), 1.23 (s, 3H, CH3), 1.18 (s, 3H, CH3); 13C-NMR (CDCl3, 100 MHz) δ: 191.9, 172.4, 169.7, 167.0, 166.7, 159.8, 152.4, 151.5, 144.8, 138.6, 138.0, 135.1, 133.7, 132.0, 130.3 (2C), 130.0, 129.8, 129.7, 129.0 (2C), 128.7 (4C), 128.3, 127.0 (2C), 122.5, 120.0, 115.0, 89.4, 84.7, 78.4, 76.2, 75.3, 73.2, 71.7, 71.5, 68.9, 65.8, 55.5, 54.9, 42.8, 42.4, 36.4, 31.9, 30.9, 29.7, 26.3, 22.5, 21.2, 14.0, 13.7; LC-MS (ESI, *m*/*z*): [M + 1], found 912.3, [M + 23], found 934.3, C49H53NO16: 911.34.

*2-Debenzoyl-2-(3-fluorobenzoyl)-macrocyclic taxoid* (**21c**). White solid; yield 52.2%; 1H-NMR (CDCl3, 400 MHz) δ: 7.97–7.92 (m, 1H, Ar-H), 7.86–7.83 (ddd, *J* = 1.5, 2.5, 9.2 Hz, 1H, Ar-H), 7.75–7.72 (m, 2H, Ar-H), 7.54–7.47 (m, 4H, Ar-H), 7.43–7.39 (m, 4 H, Ar-H), 7.37–7.32 (m, 2H, Ar-H), 7.02 (d, *J* = 9.1 Hz, 1H, NH), 6.17 (dd, *J* = 7.5, 8.8 Hz, 1H, H-13), 5.79 (dd, *J* = 2.4, 9.0 Hz, 1H, H-3 ), 5.57 (d, *J* = 7.6 Hz, 1H, H-2), 4.84–4.78 (overlap, 4H, H-5, H-2 , H-3, OCH2CH2OCO), 4.55 (dd, *J* = 6.4, 11.9 Hz, 1H, OCH2CH2OCO), 4.47–4.40 (m, 2H, H-20b, H-20a), 4.34–4.29 (m, 2H, OCH2CH2OCO), 4.12–3.96 (m, 2H, H-7b, H-7a), 3.62 (d, *J* = 4.9 Hz, 1H, OH-2 ), 2.53 (s, 3H, CH3), 2.36 (s, 3H, CH3), 2.35–2.25 (m, 4H, H-14a, H-14b, H-6a, H-6b), 1.93 (brs, 1H, OH-1), 1.68 (d, *J* = 1.2 Hz, 3H, CH3), 1.23 (s, 3H, CH3), 1.18 (s, 3H, CH3); 13C-NMR (CDCl3, 100 MHz) δ: 191.8, 172.4, 169.6, 167.1, 165.6 (d, *J*C-F = 3.2 Hz, CO), 162.4 (d, <sup>1</sup>*J*C-F = 247.8 Hz), 152.4, 151.6, 144.7, 138.7, 138.0, 134.8, 133.7, 132.0, 131.3 (d, <sup>3</sup>*J*C-F = 7.6 Hz), 130.5 (d, <sup>3</sup>*J*C-F = 7.8 Hz), 129.9 (d, <sup>4</sup>*J*C-F = 3.9 Hz), 129.7, 129.0, 128.7, 128.4, 127.0, 126.0, 121.0 (d, <sup>2</sup>*J*C-F = 21.3 Hz), 117.0 (d, <sup>2</sup>*J*C-F = 23.3 Hz), 89.5, 84.7, 78.5, 76.0, 75.5, 73.2, 71.7, 71.5, 68.9, 65.7, 55.0, 42.8, 42.4, 36.3, 30.9, 26.3, 25.5, 22.7, 22.4, 21.2, 14.1, 14.0, 13.6; LC-MS (ESI, *m*/*z*): [M + 1], found 900.0, [M + 23], found 922.0, C48H50FNO15: 899.32.

*2-Debenzoyl-2-(3-chlorobenzoyl)-macrocyclic taxoid* (**21d**). White solid; yield 48.7%; 1H-NMR (CDCl3, 400 MHz) δ: 8.16 (t, 1H, Ar-H), 8.06–8.03 (m, 1H, Ar-H), 7.75–7.72 (m, 2H, Ar-H), 7.61 (ddd, *J* = 1.1, 2.1, 8.0 Hz, 1H, Ar-H), 7.53–7.45 (m, 4H, Ar-H), 7.43–7.39 (m, 4 H, Ar-H), 7.36–7.32 (m, 1H, Ar-H), 7.01 (d, *J* = 9.1 Hz, 1H, NH), 6.17 (td, *J* =1.3, 8.7 Hz, 1H, H-13), 5.78 (dd, *J* = 2.3, 9.0 Hz, 1H, H-3 ), 5.56 (d, *J* = 7.6 Hz, 1H, H-2), 4.84–4.78 (overlap,4H, H-5, H-2 , H-3, OCH2CH2OCO), 4.55 (dd, *J* = 6.4, 11.9 Hz, 1H, OCH2CH2OCO), 4.47–4.38 (m, 2 H, H-20b, H20a), 4.34–4.29 (m, 2H, OCH2CH2OCO), 4.11–3.96 (m, 2H, H-7b, H-7a), 3.62 (brs, 1H, OH-2 ), 2.52 (s, 3H, CH3), 2.37 (s, 3H, CH3), 2.42–2.27 (overlap, 4H, H-14a, H-14b, H-6a, H-6b), 2.0 (brs, 1H, OH-1), 1.68 (d, *J* = 1.2 Hz, 3H, CH3), 1.23 (s, 3H, CH3), 1.18 (s, 3H, CH3); 13C-NMR (CDCl3, 100 MHz) δ: 191.8, 172.4, 169.6, 167.1, 165.5, 152.4, 151.6, 144.7, 138.7, 138.0, 134.8 (2C), 133.8, 133.7, 132.0, 130.9, 130.2 (2C), 129.0, 128.7, 128.4 (2C), 127.1, 127.0, 89.4, 84.7, 78.4, 76.0, 75.5, 73.1, 71.7, 71.5, 68.9, 65.7, 55.0, 42.8, 42.4, 36.3, 30.9, 26.2, 22.4, 21.2, 14.0, 13.6; LC-MS (ESI, *m*/*z*): [M + 1], found 916.3, [M + 23], found 938.3, C48H50ClNO15: 915.29.

*2-Debenzoyl-2-(3-trifluoromethylbenzoyl)-macrocyclic taxoid* (**21e**). White solid; yield 63.7%; 1H-NMR (CDCl3, 400 MHz) δ: 8.47 (s, 1H, Ar-H), 8.36 (d, *J* = 7.9 Hz, 1H, Ar-H), 7.90 (d, *J* = 7.8 Hz, 1H, Ar-H), 7.73–7.67 (m, 3H, Ar-H), 7.46–7.52 (m, 3 H, Ar-H), 7.43–7.32 (m, 5H, Ar-H), 6.94 (d, *J* = 9.0 Hz, 1H, NH), 6.16 (dd, *J* = 7.5, 8.8 Hz, 1H, H-13), 5.77 (dd, *J* = 2.0, 8.9 Hz, 1H, H-3 ), 5.60 (d, *J* = 7.5 Hz, 1H, H-2), 4.85–4.78 (overlap, 4H, H-5, H-2 , H-3, OCH2CH2OCO), 4.55 (dd, *J* = 6.6, 12.0 Hz, 1H, OCH2CH2OCO), 4.45 (dd, *J* = 6.7, 13.6 Hz, 1H, OCH2CH2OCO), 4.39 (d, *J* = 8.0 Hz, 1 H, H-20b), 4.33 (dd, 1H, OCH2CH2OCO), 4.31 (d, *J* = 7.7 Hz, 1H, H-20a),4.12-3.97 (m, 2H, H-7b, H-7a), 3.53 (d, *J* = 6.9 Hz, 1H, OH-2 ), 2.53 (s, 3H, CH3), 2.34 (s, 3H, CH3), 2.46–2.28 (m, 4H, H-14a, H-14b, H-6a, H-6b), 1.93 (brs, 1H, OH-1), 1.70 (d, *J* = 1.2 Hz, 3H, CH3), 1.24 (s, 3H, CH3),1.15 (s, 3H, CH3); 13C-NMR (CDCl3, 100 MHz) δ: 192.0, 171.1, 169.3, 167.0, 165.4, 152.5, 151.6, 144.5, 139.8, 138.4, 134.4, 134.3, 133.6, 131.8, 131.4 (q, <sup>2</sup>*J*C-F = 33.2 Hz), 130.3 (q, <sup>3</sup>*J*C-F = 3.4 Hz), 130.2, 129.7, 128.8, 128.0, 127.0, 126.9 (q, <sup>3</sup>*J*C-F = 4.3 Hz), 126.5, 123.7 (q, <sup>1</sup>*J*C-F = 273.8 Hz), 89.6, 84.7, 78.7, 76.0, 75.8, 75.2, 71.5, 70.8, 69.0, 65.9, 55.7, 43.0, 42.4, 36.4, 31.0, 29.0, 26.3, 25.6, 22.7, 21.6, 18.2, 14.4, 13.6, −5.2, −5.8; LC-MS (ESI, *m*/*z*): [M + 1], found 950.3, [M + 23], found 972.3, C49H50F3NO15: 949.31.

#### 3.1.8. General Experimental Procedure for Compounds **18a**–**e**

*2 -O-(tert-Butyldimethylsilyl)-9-O-(3-hydroxyethyl)-10-dehydro-7, 8-seco-10-deacetylpaclitaxel* (**18a**). To a mixture of **15a** (65 mg, 0.0703 mmol), potassium carbonate (67.8 mg, 0.491 mmol) and potassium iodide (11.7 mg, 0.0705 mmol) in DMF (0.65 mL) was added 3-bromo-1-propanol (44 μL 0.487 mmol), dropwise, at 0 ◦C. The reaction mixture was stirred for 4 h at room temperature and then was diluted with ethyl acetate. The organic phase was washed with saturated aqueous NH4Cl. The organic layer was dried over anhydrous Na2SO4 and then concentrated under reduced pressure. Purification of the crude product by silica gel chromatography (ethyl acetate: petroleum ether = 1:1) gave product **18a** as a white solid (50 mg, 72.4% yield). 1H-NMR (CDCl3, 400 MHz) δ: 8.10 (d, *J* = 7.6 Hz, 2H, Ar-H), 7.62 (d, *J* = 7.6 Hz, 2H, Ar-H), 7.58 (t, *J* = 7.2 Hz, 1H, Ar-H), 7.47 (dd, *J* = 7.6, 14.8 Hz, 2H, Ar-H), 7.43–7.40 (m, 1H, Ar-H), 7.38–7.36 (m, 4H, Ar-H), 7.30–7.29 (m, 3H, Ar-H), 6.30 (t, *J* = 8.4 Hz, 1H, H-13), 5.88 (d, *J* = 9.6 Hz, 1H, H-3 ), 5.57 (d, *J* = 9.6 Hz, 1H, H-2), 5.30–5.27 (overlap, 2H, H-5, H-20a), 4.69 (d, *J* = 2.0 Hz, 1H, H-2 ), 4.26 (d, *J* = 7.6 Hz, 1H, H-3), 4.20 (brs, 1H, H-20b), 4.00-3.95 (m, 2H, OCH2CH2CH2OCO), 3.91–3.83 (m, 3H, OCH2CH2CH2OCO, H-7a), 3.76 (brs, 1H, H-7b), 2.75–2.64 (m, 2H, H-6a, H-14a), 2.27–2.21 (m, 2H, H-6b, H-14b), 1.92–1.90 (m, 5H, CH3, OCH2CH2CH2OCO), 1.96 (s, 3H, CH3), 1.81 (brs, 3H, CH3), 1.27 (s, 3H, CH3), 1.18 (s, 3H, CH3), 0.76 (s, 9H, SiC(CH3)3), −0.08 (s, 3H, Si(CH3)), <sup>−</sup>0.33 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) <sup>δ</sup>: 191.6, 170.7, 168.7, 167.3 (2C), 153.1, 152.8, 144.1, 137.9, 133.8, 133.7, 131.9, 129.9 (2C), 128.9 (2C), 128.7 (4C), 128.6, 127.9 (2C), 126.5 (2C), 82.1, 79.9, 75.7, 74.7, 70.5, 68.8, 60.4, 59.3, 58.8, 55.6, 42.8, 36.6, 32.2, 25.4 (3C), 23.2, 22.1, 18.2, 14.9, 14.2, −5.5, −6.1; LC-MS (ESI, *m*/*z*): [M + 23], found 1006.0, C54H69NO14Si: 983.45.

*2 -O-(tert-Butyldimethylsilyl)-2-debenzoyl-2-(3-methoxybenzoyl)-9-O-(3-hydroxyethyl)-10-dehydro-7,8-seco-10-deacetylpaclitaxel* (**18b**). White solid; yield 75.3%; 1H-NMR (CDCl3, 400 MHz) δ: 7.71–7.65 (m, 3H, Ar-H), 7.53 (m, 1H, Ar-H), 7.46–7.41 (m, 2H, Ar-H), 7.11 (dd, *J* = 2.4, 8.0 Hz, 1H, BzNH), 6.28 (t, *J* = 8.6 Hz, 1H, H-13), 5.87 (d, *J* = 9.2 Hz, 1H, H-3 ), 5.58 (d, *J* = 9.5 Hz, 1H, H-2), 5.29–5.26 (overlap, 2H, H-5, H-20a), 4.72 (d, *J* = 2.1 Hz, 1H, H-2 ), 4.26 (d, *J* = 6.6 Hz, 1H, H-3), 4.21 (brs, 1H, H-20b), 4.00-3.95 (m, 2H, OCH2CH2CH2O), 3.98–3.86 (m, 3H, OCH2CH2CH2O, H-7b), 3.83 (overlap, 4H, OCH3, H-7a), 2.77–2.66 (m, 2H, H-6b, H-14b), 2.27–2.21 (m, 2H, H-6a, H-14a), 1.96 (s, 3H, CH3), 1.92 (brs, 2H, OCH2CH2CH2O), 1.83 (m, 2H, OH), 1.84 (brs, 3H, CH3), 1.26 (s, 3H, CH3), 1.19 (s, 3H, CH3), 0.78 (s, 9H, SiC(CH3)3), <sup>−</sup>0.09 (s, 3H, Si(CH3)), <sup>−</sup>0.29 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) <sup>δ</sup>: 191.6, 171.2, 170.9, 168.9, 167.3, 167.2, 159.8, 144.4, 138.1, 133.8, 131.9, 130.4, 130.3, 128.7 (2C), 128.7 (2C), 128.0, 127.0 (2C), 126.7 (2C), 122.3, 119.6, 114.9, 85.8, 79.9, 75.8, 74.9, 70.7, 68.8, 59.3, 59.0, 55.7, 55.5, 42.8, 36.6, 32.3, 25.5 (3C), 25.2, 23.2, 22.2, 18.2, 14.9, 14.5, −5.5, −6.0; LC-MS (ESI, *m*/*z*): [M + 1], found 1014.4, [M + 23], found 1036.4, C55H71NO16Si: 1013.46.

*2 -O-(tert-Butyldimethylsilyl)-2-debenzoyl-2-(3-fluorobenzoyl)-9-O-(3-hydroxyethyl)-10-dehydro-7, 8-seco-10-deacetylpaclitaxel* (**18c**). White solid; yield 69.7%; 1H-NMR (CDCl3, 400 MHz) δ: 7.93 (d, *J* = 7.6 Hz, 1H, Ar-H), 7.75 (d, *J* = 8.9 Hz, 1H, Ar-H), 7.63 (d, *J* = 7.5 Hz, 1H, Ar-H), 7.52–7.28 (m, 11H, NH, Ar-H), 6.29 (t, *J* = 8.1 Hz, 1H, H-13), 5.89 (d, *J* = 9.2 Hz, 1H, H-3 ), 5.57 (d, *J* = 9.6 Hz, 1H, H-2), 5.28–5.26

(overlap, 2H, H-5, H-20a), 4.70 (d, *J* = 2.0 Hz, 1H, H-2 ), 4.27 (d, *J* = 8.1 Hz, 1H, H-3), 4.19 (brs, 1H, H-20b), 3.99–3.94 (m, 2H, OCH2CH2CH2OH), 3.88–3.81 (m, 3H, OCH2CH2CH2OH, H-7a), 3.76 (brs, 1H, H-7b), 2.72–2.63 (m, 2H, H-6a, H-14a), 2.24 (dd, 1H, *J* = 9.5, 15.8 Hz, H-14b), 2.06– 1.98 (overlap, 1 H, H-6b), 1.95 (s, 3H, CH3), 1.93–1.87 (m, 4H, CH3, OCH2CH2CH2OH), 1.83 (brs, 3H, CH3), 1.26 (brs, 3H, CH3), 1.17 (s, 3H, CH3), 0.77 (s, 9H, SiC(CH3)3), <sup>−</sup>0.09 (s, 3H, Si(CH3)), <sup>−</sup>0.32 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) <sup>δ</sup>: 191.5, 171.2, 170.8, 168.6, 167.4, 166.3, 162.7 (d, 1JC-F <sup>=</sup> 248.2 Hz), 144.3, 137.9, 133.9, 131.9, 131.2 (d, 2JC-F = 7.3 Hz), 131.0 (d, 3JC-F = 7.7 Hz), 128.8, 128.7, 128.0, 127.0, 126.5, 125.8 (d, 4JC-F = 2.6 Hz), 121.0 (d, 2JC-F = 21.2 Hz), 116.6 (d, 2JC-F = 22.8 Hz), 85.9, 80.0, 75.8, 75.2, 70.5, 68.9, 59.3, 55.6, 42.8, 36.7, 32.3, 25.5, 23.2, 22.1, 18.2, 15.0, 14.5, −5.4, −6.0; LC-MS (ESI, *m*/*z*): [M + 1], found 1002.0, [M + 23], found 1025.0, C54H68FNO14Si: 1001.44.

*2 -O-(tert-Butyldimethylsilyl)-2-debenzoyl-2-(3-chlorobenzoyl)-9-O-(3-hydroxyethyl)-10-dehydro-7,8-seco-10-deacetylpaclitaxel* (**18d**). White solid; yield 52.6%; 1H-NMR (CDCl3, 400 MHz) δ: 8.03 (d, *J* = 7.6 Hz, 1H, Ar-H), 8.00–7.96 (m, *J* = 7.6 Hz, 1H, Ar-H), 7.65 (d, *J* = 7.6 Hz, 2H, Ar-H), 7.54 (t, *J* = 7.2 Hz, 1H, Ar-H), 7.48–7.44 (m, 2H, Ar-H), 7.43–7.40 (m, 1H, Ar-H), 7.38–7.36 (m, 4H, Ar-H), 7.39–7.28 (m, 7H, Ar-H), 6.30 (t, *J* = 8.4 Hz, 1H, H-13), 5.88 (d, *J* = 9.6 Hz, 1H, H-3 ), 5.57 (d, *J* = 9.6 Hz, 1H, H-2), 5.30–5.27 (overlap, 2H, H-5, H-20a), 4.71 (s, 1H, H-2 ), 4.26 (d, *J* = 7.6 Hz, 1H, H-3), 4.18 (brs, 1H, H-20b), 3.99–3.93 (m, 2H, OCH2CH2CH2O), 3.79–3.40 (m, 3H, OCH2CH2CH2O, H-7a), 3.73–3.64 (brs, 1H, H-7b), 2.94 (s, 3H, CH3), 2.84 (s, 3H, CH3), 2.66 (br, 2H, H-6a, H-14a), 2.29–2.22 (m, 2H, H-6b, H-14b), 1.93 (s, 3H, CH3), 1.93–1.86 (m, 5H, CH3, OCH2CH2CH2O), 1.82 (brs, 3H, CH3), 1.24 (s, 3H, CH3), 1.16 (s, 3H, CH3), 0.76 (s, 9H, SiC(CH3)3), <sup>−</sup>0.11 (s, 3H, Si(CH3)), <sup>−</sup>0.31 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) δ: 191.5, 171.1, 170.8, 167.4, 165.9, 162.5, 144.3, 137.9, 134.8, 133.9, 133.7, 131.8, 131.0, 130.6, 129.5, 128.7 (2C), 128.6 (2C), 128.1, 127.9, 126.9 (2C), 126.5 (2C), 85.8, 75.7, 75.2, 70.5, 68.8, 67.9, 60.4, 59.2, 55.6, 42.7, 36.6, 36.4, 32.3, 31.4, 25.4 (3C), 23.2, 22.1, 21.0, 18.2, 14.5, 14.2, −5.5, −6.1; LC-MS (ESI, *m*/*z*): [M + 1], found 1018.4, [M + 23], found 1040.4, C54H68ClNO14Si: 1017.41.

*2 -O-(tert-Butyldimethylsilyl)-2-debenzoyl-2-(3-trifluoromethylbenzoyl)-9-O-(3-hydroxyethyl)-10-dehydro-7, 8-seco-10-deacetylpaclitaxel* (**18e**). White solid; yield 71.8%; 1H-NMR (CDCl3, 400 MHz): δ 8.38 (d, *J* = 7.8 Hz, 2H, Ar-H), 7.83 (d, *J* = 7.7 Hz, 2H, Ar-H), 7.68 (t, *J* = 7.8 Hz, 1H, Ar-H), 7.44 (dd, *J* = 15.4, 8.0 Hz, 2H, Ar-H), 7.47–7.41 (m, 1H, Ar-H), 7.40–7.36 (m, 4H, Ar-H), 7.30–7.29 (m, 3H, Ar-H), 6.29 (t, *J* = 8.5 Hz, 1H, H-13), 5.86 (d, *J* = 8.6 Hz, 1H, H-3 ), 5.59 (d, *J* = 9.5 Hz, 1H, H-2), 5.26 (overlap, 2H, H-5, H-20a), 4.70 (d, *J* = 2.0 Hz, 1H, H-2 ), 4.29 (d, *J* = 7.3 Hz, 1H, H-3), 4.21 (brs, 1H, H-20b), 4.00–3.96 (m, 2H, OCH2CH2CH2OH), 3.90–3.87 (m, 3H, OCH2CH2CH2OH, H-7a), 3.75 (brs, 1H, H-7b), 2.70–2.60 (m, 2H, H-6a, H-14a), 2.28–2.22 (m, 2H, H-6b, H-14b), 1.97 (s, 3H, CH3), 1.94–1.92 (m, 5H, CH3, OCH2CH2CH2OH), 1.81 (brs, 3H, CH3), 1.27 (s, 3H, CH3), 1.19 (s, 3H, CH3), 0.76 (s, 9H, SiC(CH3)3), <sup>−</sup>0.08 (s, 3H, Si(CH3)), <sup>−</sup>0.32 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) <sup>δ</sup>: 191.5, 170.8, 167.5, 144.3, 137.9, 133.9, 133.2, 132.0, 131.4 (q, 2JC-F = 33.3 Hz), 130.2 (q, 3JC-F = 4.0 Hz), 130.0, 128.8, 128.1, 126.9, 126.8 (q, 3JC-F = 3.9 Hz), 126.5, 123.6 (q, 1JC-F = 274.2 Hz), 85.9, 75.6, 75.4, 70.5, 59.4, 55.6, 42.9, 36.7, 32.3, 25.5, 18.2, 14.6, −5.4, −6.0; LC-MS (ESI, *m*/*z*): [M + 1], found 1052.4, [M + 23], found 1074.4, C55H68NO14F3Si: 1051.44.

#### 3.1.9. General Experimental Procedure for Compounds **20a**–**e**

*2 -O-(tert-Butyldimethylsilyl)-macrocyclic taxoid* (**20a**). To a solution of **18a** (26.2 mg, 0.0266 mmol) in dichloromethane (5.7 mL) was added, dropwise, pyridine (11 μL, 0.133 mmol) and triphosgene (8.7 mg, 0.0293 mmol) at 0 ◦C, then the reaction mixture was stirred at room temperature for 3.5 h. The reaction was diluted with ethyl acetate, then washed with saturated NaCl. The organic layer was dried over anhydrous Na2SO4 and then concentrated under reduced pressure. Purification of the crude product by silica gel chromatography (ethyl acetate: petroleum ether = 1:3) gave product **20a** as a white solid (6.7 mg, 24.9% yield). 1H-NMR (CDCl3, 400 MHz) δ: 8.17 (d, *J* = 8.0 Hz, 2H, Ar-H), 7.75 (d, *J* = 7.6 Hz, 2H, Ar-H), 7.63 (t, *J* = 7.2 Hz, 1H, Ar-H), 7.56–7.49 (m, 3H, Ar-H), 7.44–7.29 (m, 7H, Ar-H), 7.09 (d, *J* = 9.2 Hz, 1H, NH), 6.27 (t, *J* = 8.8 Hz, 1H, H-13), 5.76 (d, *J* = 8.8 Hz, 1H, H-3 ), 5.60 (d, *J* = 8.0 Hz, 1H, H-2), 4.80 (d, *J* = 7.6 Hz, 2H, H-5, H-3), 4.67 (brs, 1H, H-2 ), 4.63 (m, 1H, OCH2CH2CH2OCO), 4.53 (dd, *J* = 6.4, 11.6 Hz, 1H, OCH2CH2CH2OCO), 4.47 (d, *J* = 8.4 Hz, 1H, H-20a), 4.42 (dd, *J* = 4.0, 7.2 Hz, 1H, OCH2CH2CH2OCO), 4.34 (d, *J* = 8.4 Hz, 1H, H-20b), 4.11 (overlap, 2H, H-7a, OCH2CH2CH2OCO), 3.98 (dt, *J* = 6.0, 11.6 Hz, 1H, H-7b), 2.66 (m, 1H, H-6a), 2.56 (s, 3H, CH3), 2.52 (s, 3H, CH3), 2.42–2.11 (m, 5H, H-6b, H-14a, H-14b, OCH2CH2CH2O), 1.82 (s, 3H, CH3), 1.24 (s, 3H, CH3), 1.17 (s, 3H, CH3), 0.79 (s, 9H, SiC(CH3)3), <sup>−</sup>0.04 (s, 3H, Si(CH3)), <sup>−</sup>0.31 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 150 MHz) δ: 191.6, 170.7, 169.7, 169.3, 166.7, 166.6, 153.6, 152.4, 144.1, 138.8, 138.1, 135.5, 133.6, 131.6, 130.2 (2C), 130.0, 128.7, 128.6, 128.5, 127.7, 126.8 (2C), 126.1 (2C), 90.3, 84.4, 78.4, 76.1, 75.2, 75.0, 70.4, 68.9, 65.8, 64.2, 55.4, 42.6 (2C), 36.4, 30.8, 28.8, 25.9, 25.3 (3C), 22.5, 21.5, 17.9, 14.5, 14.1, −5.5, −6.1; LC-MS (ESI, *m*/*z*): [M + 23], found 1032.0, C55H67NO15Si: 1009.43.

*2 -O-(tert-Butyldimethylsilyl)-2-debenzoyl-2-(3-methoxybenzoyl)-macrocyclic taxoid* (**20b**). White solid; yield 28.6%; 1H-NMR (CDCl3, 400MHz) δ: 7.79 (d, *J* = 7.6 Hz, 1H, Ar-H), 7.76 (d, *J* = 7.2 Hz, 2H, Ar-H), 7.67–7.66 (m, 1H, Ar-H), 7.55–7.50 (m, 1H, Ar-H), 7.48–7.42 (m, 3H, Ar-H), 7.40–7.29 (m, 5H, Ar-H), 7.19–7.16 (m, 1H, Ar-H), 7.09 (d, *J* = 8.8 Hz, 1H, BzNH), 6.27 (t, *J* = 8.8 Hz, 1H, H-13), 5.76 (dd, *J* = 1.5, 9.0 Hz, 1H, H-3 ), 5.62 (d, *J* = 7.64 Hz, 1H, H-2), 4.85–4.82 (m, 2H, H-5, H-3), 4.69–4.63 (m, 2H, H-2 , OCH2CH2CH2OCO), 4.56–4.51 (m, 1H, OCH2CH2CH2OCO), 4.48–4.38 (m, 3H, H-20a, H-20b, OCH2CH2CH2OCO), 4.16–4.07 (m, 2H, H-7a, OCH2CH2CH2OCO), 4.01–3.96 (m, 1H, H-7b), 3.90 (s, 3H, OCH3), 2.72–2.62 (m, 1H, H-6a), 2.57 (s, 3H, CH3), 2.53 (s, 3H, CH3), 2.43–2.36 (m, 1H, H-6b), 2.35–2.28 (m, 1H, H-14a), 2.16–2.14 (m, 2H, H-14b, OCH2CH2CH2O), 1.82 (d, *J* = 1.1 Hz, 3H, CH3), 1.25 (s, 3H, CH3), 1.18 (s, 3H, CH3), 0.88 (s, 9H, SiC(CH3)3), <sup>−</sup>0.014 (s, 3H, Si(CH3)), <sup>−</sup>0.29 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) <sup>δ</sup>: 190.8, 171.3, 171.2, 169.2, 166.9, 166.8, 159.8, 144.8, 138.3, 134.2, 131.8, 130.6, 130.1, 128.8 (2C), 128.7 (2C), 128.0, 127.0 (2C), 126.6 (2C), 122.3, 120.0, 114.5, 85.1, 77.4, 77.3, 76.8, 75.3, 74.9, 70.8, 67.8, 60.4, 55.8, 55.5, 42.8, 41.6, 40.4, 36.6, 33.0, 29.7, 25.5 (3C), 23.2, 22.4, 21.0, 18.2, 14.5, 14.2, −5.3,−5.8; LC-MS (ESI, *m*/*z*): [M + 1], found 1040.5, [M + 23], found 1062.5, C56H69NO16Si: 1039.44.

*2 -O-(tert-Butyldimethylsilyl)-2-debenzoyl-2-(3-fluorobenzoyl)-macrocyclic taxoid* (**20c**). White solid; yield 27.1%; 1H-NMR (CDCl3, 400 MHz) δ: 8.00 (d, *J* = 7.7 Hz, 1H, Ar-H), 7.87 (d, *J* = 8.8 Hz, 1H, Ar-H), 7.77 (d, *J* = 7.32 Hz, 1H, Ar-H), 7.57–7.51 (m, 2H, Ar-H), 7.46–7.33 (m, 8H, Ar-H), 7.10 (d, *J* = 9.0 Hz, 1H, NH), 6.26 (t, *J* = 8.5 Hz, 1H, H-13), 5.78 (d, *J* = 8.5 Hz, 1H, H-3 ), 5.60 (d, *J* = 7.7 Hz, 1H, H-2), 4.85–4.82 (overlap, 2H, H-5, H-3), 4.72–4.63 (overlap, 2H, OCH2CH2CH2OCO, H-2 ), 4.55 (dt, *J* = 5.5, 10.3 Hz, 1H, OCH2CH2CH2OCO), 4.47–4.42 (overlap, 2H, OCH2CH2CH2OCO, H-20a), 4.37 (d, *J* = 8.2 Hz, 1H, H-20b), 4.14–4.10 (overlap, 2H, H-7a, OCH2CH2CH2OCO), 3.99 (dt, *J* = 6.4, 11.3 Hz, 1H, H-7b), 2.73–2.64 (m, 1H, H-6a), 2.57 (s, 3H, CH3), 2.54 (s, 3H, CH3), 2.42–2.18 (m, 5H, H-6b, H-14a, H-14b, OCH2CH2CH2O), 2.10 (brs, 1H, OH), 1.83 (s, 3H, CH3), 1.25 (s, 3H, CH3), 1.18 (s, 3H, CH3), 0.82 (s, 9H, SiC(CH3)3), <sup>−</sup>0.008 (s, 3H, Si(CH3)), <sup>−</sup>0.27 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) <sup>δ</sup>: 191.8, 171.0, 169.5, 166.9, 165.7 (d, <sup>5</sup>*J*C-F = 3.0 Hz, ArCO), 162.6 (d, <sup>1</sup>*J*C-F = 247.8 Hz), 153.8, 152.6, 144.3, 139.1, 138.4, 135.5, 134.2, 131.8, 131.3 (d, <sup>3</sup>*J*C-F = 7.5 Hz), 130.6 (d, <sup>3</sup>*J*C-F = 7.7 Hz), 128.8, 128.7, 127.9, 127.0, 126.4, 126.0 (d, <sup>4</sup>*J*C-F = 2.6 Hz), 120.9 (d, <sup>2</sup>*J*C-F = 21.1 Hz), 117.0 (d, <sup>2</sup>*J*C-F = 23.2 Hz), 90.6, 84.6, 78.6, 77.4, 77.1, 76.7, 76.2, 75.8, 75.3, 70.6, 69.2, 66.0, 64.4, 55.6, 42.8 (2C), 36.5, 31.0, 29.0, 26.1, 25.5 (3C), 22.7, 21.7, 18.2, 14.7, 14.4, −5.2, −5.8; LC-MS (ESI, *m*/*z*): [M + 1], found 1028.4, [M + 23], found 1050.4, C55H66FNO15Si: 1027.42.

*2 -O-(tert-Butyldimethylsilyl)-2-debenzoyl-2-(3-chlorobenzoyl)-macrocyclic taxoid* (**20d**). White solid; yield 23.6%; 1H-NMR (CDCl3, 400 MHz) δ: 8.20 (t, *J* = 1.6 Hz, 1H, Ar-H), 8.12–8.09 (m, 1H, Ar-H), 7.77 (d, *J* = 7.2 Hz, 1H, Ar-H), 7.65–7.62 (m, 1H, Ar-H), 7.56–7.51 (m, 2H, Ar-H), 7.47–7.33 (m, 7H, Ar-H), 7.09 (d, *J* = 9.0 Hz, 1H, BzNH), 6.26 (t, *J* = 8.8 Hz, 1H, H-13), 5.77 (dd, *J* = 1.5, 8.98 Hz, 1H, H-3 ), 5.60 (d, *J* = 7.8 Hz, 1H, H-2), 4.85–4.82 (dd, *J* = 3.5, 11.0 Hz, 2H, H-5, H-17b), 4.72–4.64 (m, 2H, OCH2CH2CH2OCO, H-2 ), 4.59–4.53 (m, 1H, OCH2CH2CH2OCO), 4.49–4.43 (m, 2H, OCH2CH2CH2OCO, H-20a), 4.38 (d, *J* = 8.4 Hz, 1H, H-20b), 4.15–4.10 (m, 2H, OCH2CH2CH2OCO, H-20a), 4.02–3.96 (m, 1H, H-7b), 2.74–2.65 (m, 1H, H-6a), 2.57 (s, 3H, CH3), 2.55 (s, 3H, CH3), 2.42–2.20 (m, 5H, H-6b, H-14a, H-14b, OCH2CH2CH2O), 1.84 (d, *J* = 1.0 Hz, 3H, CH3), 1.26 (s, 3H, CH3), 1.19 (s, 3H, CH3), 0.83 (s, 9H, SiC(CH3)3), <sup>−</sup>0.005 (s, 3H, Si(CH3)), <sup>−</sup>0.26 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) <sup>δ</sup>: 190.7, 171.2, 169.1, 167.0, 165.7, 144.8, 138.3, 135.5, 135.0, 134.3, 133.9, 131.8, 131.1, 130.5, 129.9, 128.8 (2C), 128.7 (2C), 128.2, 128.0, 127.0 (2C), 126.7 (2C), 90.6, 84.6, 78.7, 76.2, 75.8, 75.3, 70.7, 67.8, 55.7, 42.8, 41.6, 36.5, 34.7, 31.6, 29.7, 25.6 (3C), 22.7, 22.4, 18.2, 14.5, 14.1, −5.3, −5.8; LC-MS (ESI, *m*/*z*): [M + 1], found 1044.0, [M + 23], found 1065.9, C55H66ClNO15Si: 1043.39.

*2 -O-(tert-Butyldimethylsilyl)-2-debenzoyl-2-(3-trifluoromethylbenzoyl)-macrocyclic taxoid* (**20e**). White solid; yield 26.7%; 1H-NMR (CDCl3, 400MHz) δ: 8.50 (s, 1H, Ar-H), 8.41 (d, *J* = 7.8 Hz, 1H, Ar-H), 7.92 (d, *J* = 7.9 Hz, 1H, Ar-H), 7.55–7.51 (m, 1H, Ar-H), 7.48–7.33 (m, 8H, Ar-H), 7.09 (d, *J* = 9.0 Hz, 1H, NH), 6.26–6.21 (m, 1H, H-13), 5.76 (dd, *J* =1.4, 9.1 Hz, 1H, H-3 ), 5.64 (d, *J* = 7.8 Hz, 1H, H-2), 4.89–4.83 (m, 2H, H-5, H-3), 4.69–4.63 (m, 2H, OCH2CH2CH2OCO, H-2 ), 4.60–4.55 (m, 1H, OCH2CH2CH2OCO), 4.49–4.43 (m, 2H, OCH2CH2CH2OCO, H-20a), 4.37 (d, *J* = 8.0 Hz,1H, H-20b), 4.13 (t, *J* = 7.7 Hz, 2H, H-7a, OCH2CH2CH2OCO), 2.75–2.67 (m, 1H, H-6a), 4.02–3.96 (m, 1H, H-7b), 2.58 (s, 3H, CH3), 2.53 (s, 3H, CH3), 2.45–2.18 (m, 5H, H-6b, H-14a, H-14b, OCH2CH2CH2O), 1.84 (d, *J* =1.0 Hz, 1H, CH3), 1.26 (s, 3H, CH3), 1.25 (s, 3H, CH3), 1.20 (s, 3H, CH3), 0.83 (s, 9H, SiC(CH3)3), −0.006 (s, 3H, Si(CH3)), −0.25 (s, 3H, Si(CH3)); 13C-NMR (CDCl3, 100 MHz) δ: 191.7, 171.1, 167.0, 165.5, 153.9, 152.7, 135.4, 134.3, 133.6, 131.8, 131.4 (q, <sup>2</sup>*J* = 33.3 Hz), 130.9, 130.3 (q, <sup>3</sup>*J* = 3.9 Hz), 130.2, 129.7, 128.8, 128.0, 127.0, 126.9 (q, <sup>3</sup>*J* = 3.8 Hz), 126.5, 123.7 (q, <sup>1</sup>*J* = 273.8 Hz), 90.6, 84.7, 78.7, 76.2, 75.8, 75.3, 70.8, 69.2, 66.0, 64.5, 55.7, 42.9, 42.8, 36.5, 31.0, 29.0, 26.1, 22.6, 21.6, 18.2, 14.6, 14.4, −5.2, −5.8; LC-MS (ESI, *m*/*z*): [M + 1], found 1078.4, [M + 23], found 1100.4, C56H66F3NO15Si: 1077.42.

3.1.10. General Procedure for the Syntheses of Compounds **22a**–**e**

#### Macrocyclic taxoid (**22a**)

To a solution of of **20a** (29.6 mg, 0.0293 mmol) in THF (8 mL) was added, dropwise, 0.47 mL of HF-pyridine (*v*/*v* = 1:2) at 0 ◦C, and the reaction mixture was stirred at room temperature for 30 h. The reaction was quenched with saturated aqueous NaHCO3, diluted with ethyl acetate, and washed with saturated NH4Cl. The organic layer was dried over anhydrous Na2SO4 and then concentrated under reduced pressure. Purification of the crude product by silica gel chromatography (ethyl acetate: petroleum ether = 2:1) gave product **22a** as a white solid (13.4 mg, 51.0% yield). 1H-NMR (CDCl3, 400 MHz) δ: 7.16 (d, *J* = 7.1 Hz, 2H, Ar-H), 7.75 (d, *J* = 7.0 Hz, 2H, Ar-H), 7.64 (t, *J* = 7.5 Hz, 1H, Ar-H), 7.54–7.50 (m, 3H, Ar-H), 7.49–7.47 (m, 2H, Ar-H), 7.43–7.39 (m, 4H, Ar-H), 7.36–7.32 (m,1H, Ar-H), 7.04 (d, *J* = 9.1 Hz, 1H, NH), 6.18 (dd, *J* = 8.7 Hz, 1H, H-13), 5.81 (dd, *J* = 2.4, 9.1 Hz, 1H, H-3 ), 5.59 (d, *J* = 7.7 Hz, 1H, H-2), 4.81–4.79 (overlap, 3H, H-5, H-3, H-2 ), 4.61 (dt, *J* = 5.4, 11.0 Hz, 1H, OCH2CH2CH2OCO), 4.52 (dt, *J* = 6.9, 12.0 Hz, 1H, OCH2CH2CH2OCO), 4.44 (overlap, 2H, H-20a, OCH2CH2CH2OCO), 4.41–4.33 (m, 1H, H-20b), 4.34–4.28 (overlap, *J* = 6.7 Hz, H-7a), 4.08 (overlap, 2H, H-7a, OCH2CH2CH2OCO), 3.95 (m, 1H, H-7b), 3.58 (brs, 1H, OH-2 ), 2.66–2.61 (m, 1H, H-6a), 2.55 (s, 3H, CH3), 2.37–2.33 (brs, 3H, CH3), 2.30–2.12 (overlap, 3H, H-6b, H-14a, H-14b), 1.67 (brs, 2H, OCH2CH2CH2OCO), 1.23 (s, 3H, CH3), 1.16 (s, 3H, CH3); 13C-NMR (CDCl3, 100 MHz) δ: 191.6, 172.4, 169.8, 167.0, 166.9, 153.8, 152.6, 144.6, 138.0 (2C), 136.1, 133.9, 133.7, 130.2 (2C), 129.1, 129.0 (2C), 128.9, 128.8 (2C), 128.7 (2C), 128.3, 127.0 (2C), 90.5, 84.7, 78.5, 76.3, 75.3, 73.2, 71.7, 69.2, 65.6, 64.4, 54.8, 42.9, 42.7, 36.5, 31.0, 29.0, 26.2, 22.4, 21.3, 14.7, 14.0; LC-MS (ESI, *m*/*z*): [M + 1], found 896.3, [M + 23], found 918.3, C49H53NO15: 895.34.

*2-Debenzoyl-2-(3-methoxybenzoyl)-macrocyclic taxoid* (**22b**). White solid; yield 59.2%; 1H-NMR (CDCl3, 400 MHz) δ: 7.75 (d, *J* = 7.4 Hz, 3H, Ar-H), 7.65 (dd, *J* =1.42, 2.3 Hz, 1H, Ar-H), 7.52–7.38 (m, 9H, Ar-H), 7.17 (dd, *J* = 2.1, 8.2 Hz, 1H, Ar-H), 7.06 (d, *J* = 9.0 Hz, 1H, NH), 6.15 (t, *J* = 8.2 Hz, 1H, H-13), 5.78 (dd, *J* = 2.1, 8.9 Hz, 1H, H-3 ), 5.59 (d, *J* = 7.6 Hz, 1H, H-2), 4.80–4.76 (overlap, 3H, H-5, H-3, H-2 ), 4.60 (dt, *J* = 5.6, 11.2 Hz, 1H, OCH2CH2CH2OCO), 4.51 (dt, *J* = 5.7, 11.2 Hz, 1H, OCH2CH2CH2OCO), 4.44–4.37 (overlap, *J* = 5.6 Hz, 2H, OCH2CH2CH2OCO, H-20a), 4.36 (d, *J* = 8.3 Hz, 1H, H-20b), 4.07 (overlap, 2H, H-7a, OCH2CH2CH2OCO), 3.97–3.91 (m, 1H, H-7b), 3.87 (s, 3H, OCH3), 3.66 (d, *J* = 4.1 Hz, 1H, OH-2 ), 2.67–2.56 (m, 1H, H-6a), 2.55 (s, 3H, CH3), 2.34 (s, 3H, CH3), 2.40–2.30 (m, 2H, OCH2CH2CH2OCO), 2.28–2.12 (m, 3H, H-6b, H-14a, H-14b), 2.02 (brs, 1H, OH-1), 1.65 (s, 3H, CH3), 1.22 (s, 3H, CH3), 1.15 (s, 3H, CH3); 13C-NMR (CDCl3, 100 MHz) δ: 191.6, 172.3, 169.8, 167.0, 166.8, 159.8, 153.7, 152.6, 144.6, 138.1, 137.9, 136.1, 133.7, 132.0, 130.3, 129.8, 129.0, 128.7, 128.3, 127.1, 127.0, 122.4, 112.0, 115.0, 90.4, 84.7, 78.4, 76.3, 75.3, 73.3, 71.7, 69.2, 66.0, 64.4, 55.5, 54.9, 42.8, 42.7, 36.5, 31.0, 29.0, 26.1, 22.4, 21.2, 14.7, 14.0; LC-MS (ESI, *m*/*z*): [M + 1], found 926.3, [M + 23], found 948.3, C50H55NO16: 925.35.

*2-Debenzoyl-2-(3-fluorobenzoyl)-macrocyclic taxoid* (**22c**). White solid; yield 51.5%; 1H-NMR (CDCl3, 400 MHz) δ: 7.96 (d, *J* = 7.69 Hz, 1 H, Ar-H), 7.84 (d, *J* = 9.1 Hz, 1H, Ar-H), 7.74 (d, *J* = 7.7 Hz, 2H, Ar-H), 7.53–7.46 (m, 4 H, Ar-H), 7.40 (t, *J* = 7.5 Hz, 4H, Ar-H), 7.36–7.32 (m, 2H, Ar-H), 7.05 (d, *J* = 9.0 Hz, 1H, NH), 6.15 (t, *J* = 8.6 Hz, 1H, H-13), 5.78 (dd, *J* = 2.0, 9.0 Hz, 1H, H-3 ), 5.57 (d, *J* = 7.7 Hz, 1H, H-2), 4.81–4.79 (overlap, 3H, H-5, H-3, H-2 ), 4.60 (dt, *J* = 5.3, 10.7 Hz, 1H, OCH2CH2CH2OCO), 4.52 (dt, *J* = 5.4, 10.8 Hz, 1H, OCH2CH2CH2OCO), 4.44–4.40 (ovelap, 2H, H-7a, OCH2CH2CH2OCO), 4.40 (d, *J* = 8.0 Hz, 1H, H-20a), 4.32 (d, *J* = 8.1 Hz, 1H, H-20b), 4.07 (t, *J* = 7.5 Hz, 2H, OCH2CH2CH2OCO), 3.93 (dt, *J* = 5.9, 11.9 Hz, 1H, H-7b), 3.69 (brs, 1H, OH-2 ), 2.68–2.58 (m, 1H, H-6a), 2.54 (s, 3H, CH3), 2.34 (s, 3H, CH3), 2.33–2.26 (m, 2H, OCH2CH2CH2OCO), 2.24–2.16 (m, 3H, H-6b, H-14a, H-14b), 1.97 (brs, 1H, OH-1), 1.65 (br, 3H, CH3), 1.22 (s, 3H, CH3), 1.15 (s, 3H, CH3); 13C-NMR (CDCl3, 100 MHz) δ: 191.5, 172.4, 169.7, 167.0, 165.7 (d, *J*C-F = 2.8 Hz, CO), 162.6 (d, <sup>1</sup>*J*C-F =247.9 Hz), 153.8, 152.6, 144.5, 138.1 (2C), 135.9, 133.8, 131.9, 131.3 (d, <sup>3</sup>*J*C-F = 7.5 Hz), 130.5 (d, <sup>3</sup>*J*C-F = 7.6 Hz), 129.0, 128.7, 128.3, 127.0 (2C), 126.0 (d, <sup>4</sup>*J*C-F = 2.9 Hz), 121.0 (d, <sup>2</sup>*J*C-F = 21.3 Hz), 116.9 (d, <sup>2</sup>*J*C-F = 23.3 Hz), 90.5, 84.6, 78.5, 76.2, 75.6, 73.3, 71.6, 69.2, 66.0, 64.4, 55.0, 42.8, 42.7, 36.4, 30.9, 29.3, 29.0, 26.1, 22.3, 21.3, 14.6, 14.0; LC-MS (ESI, *m*/*z*): [M + 1], found 914.3, [M + 23], found 936.3, C49H52FNO15: 913.33.

*2-Debenzoyl-2-(3-chlorobenzoyl)-macrocyclic taxoid* (**22d**). White solid; yield 65.4%; 1H-NMR (CDCl3, 400 MHz) δ: 8.16 (t, *J* = 1.8 Hz, 1H, Ar), 8.06–8.04 (m, 1 H, Ar-H), 7.75–7.73 (m, 2H, Ar-H), 7.61 (ddd, *J* =1.0, 2.0, 8.0 Hz, 1H, Ar-H), 7.43–7.39 (m, 4H, Ar-H), 7.52–7.45 (m, 4 H, Ar-H), 7.37–7.32 (m, 1H, Ar-H), 7.01 (d, *J* = 9.0 Hz, 1H, NH), 6.15 (dd, *J* = 7.4, 8.7 Hz, 1H, H-13), 5.78 (dd, *J* = 2.3, 9.0 Hz, 1H, H-3 ), 5.57 (d, *J* = 7.7 Hz, 1H, H-2), 4.81–4.79 (overlap, 3H, H-5, H-3, H-2 ), 4.60 (dt, *J* = 5.3, 10.7 Hz, 1H, OCH2CH2CH2OCO), 4.53 (dt, *J* = 5.5, 11.3 Hz, 1H, OCH2CH2CH2OCO), 4.45–4.44 (m, 1H, OCH2CH2CH2OCO), 4.39 (d, *J* = 8.2 Hz, 1H, H-20a), 4.33 (d, *J* = 8.2 Hz, 1H, H-20b), 4.08 (overlap, *J* = 7.62 Hz, 2H, H-7a, OCH2CH2CH2OCO), 3.96–3.90 (m, 1H, H-7b), 3.60 (brs, 1H, OH-2 ), 2.68–2.58 (m, 1H, H-6a), 2.54 (s, 3H, CH3), 2.35 (s, 3H, CH3), 2.44–2.30 (m, 2H, OCH2CH2CH2OCO), 2.28–2.16 (m, 3H, H-6b, H-14a, H-14b), 1.97 (brs, 1H, OH-1), 1.65 (d, *J* =1.7 Hz, 3H, CH3), 1.23 (s, 3H, CH3), 1.16 (s, 3H, CH3); 13C-NMR (CDCl3, 100 MHz) δ: 191.5, 172.4, 169.7, 167.0, 165.5, 153.8, 152.6, 144.5, 138.0, 135.9, 134.8, 133.8, 133.7, 132.0, 130.9, 130.2 (2C), 129.0, 128.7, 128.4, 127.1, 127.0, 90.4, 84.6, 78.5, 76.1, 75.5, 73.2, 71.7, 69.2, 65.9, 64.4, 55.0, 42.8, 42.7, 36.4, 30.9, 29.0, 26.1, 22.3, 21.2, 14.6, 14.0; LC-MS (ESI, *m*/*z*): [M + 1], found 930.3, [M + 23], found 952.3, C49H52ClNO15: 929.30.

*2-Debenzoyl-2-(3-trifluoromethylbenzoyl)-macrocyclic taxoid* (**22e**). White solid; yield 59.5%; 1H-NMR (CDCl3, 400 MHz) δ: 8.46 (s, 1 H, Ar), 8.36 (d, *J* = 7.8 Hz, 1H, Ar-H), 7.90 (d, *J* =7.7 Hz, 1H, Ar-H), 7.73–7.67 (m, 3 H, Ar-H), 7.51–7.47 (m, 3H, Ar-H), 7.42–7.32 (m, 5H, Ar-H), 6.98 (d, *J* = 9.0 Hz, 1H, NH), 6.15 (t, *J* = 8.1 Hz, 1H, H-13), 5.77 (dd, *J* = 2.0, 9.0 Hz, 1H, H-3 ), 5.61 (d, *J* = 7.7 Hz, 1H, H-2), 4.84–4.78 (overlap, 3H, H-5, H-3, H-2 ), 4.62–4.51 (m, 2H, OCH2CH2CH2OCO), 4.45–4.41 (m, 2H, H-7a, OCH2CH2CH2OCO), 4.39 (d, *J* = 8.0 Hz, 1H, H-20a), 4.31 (d, *J* = 8.2 Hz, 1H, H-20b), 4.07 (t, *J* = 7.6 Hz, 1H, OCH2CH2CH2OCO), 3.93 (dt, *J* = 6.9, 11.6 Hz, 1H, H-7b), 3.62 (brs, 1H, OH-2 ), 2.66–2.58 (m, 1H, H-6a), 2.54 (s, 3H, CH3), 2.44–2.35 (m, 2H, OCH2CH2CH2OCO), 2.32 (s, 3H, CH3), 2.28–2.16 (m, 3H, H-6b, H-14a, H-14b), 1.97 (brs, 1H, OH-1), 1.67 (br, 3H, CH3), 1.23 (s, 3H, CH3), 1.17 (s, 3H, CH3); 13C-NMR (CDCl3, 75 MHz) δ: 191.6, 172.4, 169.7, 167.0, 165.4, 153.7, 152.6, 144.5, 138.1 (2C), 135.8, 133.7, 133.6, 131.9, 131.3 (d, <sup>2</sup>*J*C-F = 33.2 Hz), 130.3 (d, <sup>3</sup>*J*C-F = 3.7 Hz), 130.1, 129.6, 129.0, 128.7, 128.4, 127.1, 127.0, 126.9 (d, <sup>3</sup>*J*C-F = 3.2 Hz), 125.5, 90.5, 84.6, 78.5, 76.1, 75.6, 73.1, 71.7, 69.2, 66.0, 64.4, 55.0, 42.8, 42.7, 36.3, 30.9, 29.3, 28.9, 26.1, 22.2, 21.2, 14.6, 14.0; LC-MS (ESI, *m*/*z*): [M + 1], found 964.4, [M + 23], found 986.3, C50H52F3NO15: 963.33.

#### *3.2. Cell Assays*

Cell viability after taxoids treatment was evaluated using CCK-8 assay. Briefly, 3000 cells per well were seeded in 96-well plates and incubated under normal conditions for 24 h. Cells were treated with different concentrations of the test agent for 72 h, then the medium was removed, 100 μL of CCK-8 working solution was added to each well for 1 h at 37 ◦C. The absorbance was measured at 450 nm with a microplate reader (Tecan Trading, AG, Switzerland). Vehicle-only treated cells served as the indicator of 100% cell viability. The 50% inhibitory concentration (IC50) was defined as the concentration that reduced the absorbance of the vehicle-only treated wells by 50% in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

#### *3.3. Model Building and Molecular Dynamics Simulations*

The X-ray crystal structure of paclitaxel provided a template for the construction of **18b** and **22b** using the molecular editing tools implemented in PyMOL [17]. Geometry optimization of **18b** and **22b** was achieved by means of the updated AM1 hamiltonian implemented in the *sqm* program, which also produced atomic charge distributions for both ligands that can reproduce the molecular electrostatic potential. The ff14SB AMBER force field was used to assign bonded and non-bonded parameters to protein and ligand atoms [18,19]. **18b** was immersed in a cubic box containing TIP3P water molecules and simulated under periodic boundary conditions for 50 ns at 300 K. Subsequent gradual cooling, from 300 to 273 K over 1 ns, of snapshots taken regularly every 2.5 ns, followed by energy minimization until the root-mean-square of the Cartesian elements of the gradient was less than 0.1 kcal·mol−1·Å−1, provided representative structures of this molecule; those displaying the shortest distance between the free hydroxyl groups were chosen to build **22b** by means of a C=O linkage. The simulated macromolecular ensemble representing a short piece of a microtubule with bound **22b** was constructed as previously reported for D-seco taxol derivatives [20].

#### **4. Conclusions**

In summary, this study demonstrated that C-seco taxoids conformationally constrained via carbonate-containing linked macrocyclization display increased cytotoxicity on drug-resistant tumors overexpressing both βIII and P-gp, compared to the non-cyclized C-seco taxoid counterparts Among them, compound **22b**, bearing a 2-*m*-methoxybenzoyl group together with a five-atom linker, was identified as the most potent compound in the series.

**Supplementary Materials:** The supplementary materials are available online, cell lines and culture [21], establishment of model multidrug-resistant breast cancer cell line MCF-7/ADR. Copies of ESI-MS, HPLC, 1H-NMR and 13C-NMR spectra for representative compounds.

**Author Contributions:** Y.Z. performed most of the experiments; T.-N.W. carried out the bioassays; A.M. and F.G. carried out the molecular modeling studies; writing—original draft preparation, Y.Z., T.-N.W. and A.M.; writing—review and editing, W.-S.F. and F.G.; W.-S.F. planned, designed, and organized the whole research of this study. All authors read and approved the final version of the manuscript.

**Funding:** CAMS Innovation Fund for Medical Sciences (CIFMS Grant No. 2016-I2M-1-01) and the Drug Innovation Major Project (Grant Nos. 2018ZX09711001-001-001 and 2018ZX09711001-001-003) by MOST of China (W.-S.F.) and Spanish MEC/MICINN for grant SAF2015-64629-C2-2 (F.G.) are acknowledged.

**Conflicts of Interest:** The authors declare no conflicts of interest regarding the publication of this paper.

#### **References**


**Sample Availability:** Not available.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **New Zampanolide Mimics: Design, Synthesis, and Antiproliferative Evaluation**

### **Guanglin Chen 1, Ziran Jiang 1, Qiang Zhang 2, Guangdi Wang <sup>2</sup> and Qiao-Hong Chen 1,\***


Received: 26 December 2019; Accepted: 14 January 2020; Published: 15 January 2020

**Abstract:** Zampanolide is a promising microtubule-stabilizing agent (MSA) with a unique chemical structure. It is superior to the current clinically used MSAs due to the covalent nature of its binding to β-tubulin and high cytotoxic potency toward multidrug-resistant cancer cells. However, its further development as a viable drug candidate is hindered by its limited availability. More importantly, conversion of its chemically fragile side chain into a stabilized bioisostere is envisioned to enable zampanolide to possess more drug-like properties. As part of our ongoing project aiming to develop its mimics with a stable side chain using straightforward synthetic approaches, 2-fluorobenzyl alcohol was designed as a bioisosteric surrogate for the side chain based on its binding conformation as confirmed by the X-ray structure of tubulin complexed with zampanolide. Two new zampanolide mimics with the newly designed side chain have been successfully synthesized through a 25-step chemical transformation for each. Yamaguchi esterification and intramolecular Horner–Wadsworth–Emmons condensation were used as key reactions to construct the lactone core. The chiral centers at C17 and C18 were introduced by the Sharpless asymmetric dihydroxylation. Our WST-1 cell proliferation assay data in both docetaxel-resistant and docetaxel-naive prostate cancer cell lines revealed that compound **6** is the optimal mimic and the newly designed side chain can serve as a bioisostere for the chemically fragile *N*-acetyl hemiaminal side chain in zampanolide.

**Keywords:** natural product; anticancer agent; synthesis; zampanolide

#### **1. Introduction**

Prostate cancer is still one of the big health concerns, as evidenced by its high incidence and mortality rate in American men. In 2019, 174,650 new prostate cancer cases that account for nearly 20% of all cancer cases and 31,620 prostate cancer deaths are projected to occur in the United States [1]. Prostate cancer is driven by the androgen receptor (AR)-regulated gene expression that is initiated by the binding of androgen to the AR [2]. Androgen deprivation therapy (ADT) has thus been the main treatment for prostate cancer over 70 years. However, the median duration of ADT response is merely 18–24 months due to inevitable progression to castration-resistant prostate cancer (CRPC) [3]. Consequently, approximately 30,000 prostate cancer deaths in the United State each year are caused by CRPC as well as resistance to the current treatments. The critical driving force for the continued progression of lethal CRPC is the reactivation of AR transcriptional activity [4,5]. The current first-line and second-line chemotherapeutics for CRPC are two microtubule-stabilizing cytotoxic agents (MSA), docetaxel and cabazitaxel [6–8]. Promotion of mitotic arrest is the initially well-established mechanism underlying the action of MSAs as anticancer agents [9]. The MSAs were recently revealed to impair AR transcriptional activity by suppressing nuclear importation of AR as downstream of microtubule

stabilization [10]. This alternative mechanism is independent of MSA-induced mitotic arrest and can explain in part the efficacy of MSA in AR-driven CRPC.

The marine macrolide (−)-zampanolide (**1**, Figure 1) [11] has been demonstrated as a unique and promising MSA, which distinguishes it from the current clinically used MSAs due to (1) the covalent nature of its binding to the taxane pocket in β-tubulin as confirmed by a high-resolution X-ray structure of zampanolide in complex with α,β-tubulin, and (2) the very high cytotoxic potency as evidenced by low nanomolar IC50 values in suppressing multidrug-resistant cancer cell proliferation [12].

**Figure 1.** Zampanolide, desTHP-(−)-zampanolide, desTHPzampanolide mimics **3** and **4**.

Although (−)-zampanolide has shown great potential in becoming a more effective chemotherapeutic for CRPC, no in vivo assessment of its antitumor efficacy, pharmacokinetics, and toxicity in animal models has so far been reported yet mainly because of its limited supply. Unfortunately, the supply issue of (−)-zampanolide cannot be addressed either by isolation from marine sponge sources or by current reported total syntheses. The best yield for isolation of (−)-zampanolide from marine sponge sources is merely 0.001% [12,13], and the highest yield for its syntheses is only 0.9% [14–18]. More importantly, the chemically fragile *N*-acyl hemiaminal side chain of zampanolide can compromise its pharmacokinetic profiles as well as the feasibility as a viable drug candidate. The easy cleavage of the *N*-acyl hemiaminal side chain has been confirmed by a thermolysis reaction carried out by Professor Smith and his coworkers [16], and the instability of zampanolide in CDCl3 has been reported by Higa and coworkers when they first identified its structure [13]. The *N*-acyl hemiaminal side chain of zampanolide is, however, very vital to retain its excellent cytotoxic potency, and the derivative without the side chain has 10- to 1000-fold less potency in certain cell models [12,19,20]. Our previous study has confirmed that a suitably designed side chain (e.g., the side chain in desTHPzampanoide mimics **3** and **4**) can function as a bioisostere for the chemically unstable *N*-acyl hemiaminal side chain in desTHPzampanolide (**2**) [21]. However, the bioisosteric side chains that we reported previously do not contain a hydroxyl substituent, corresponding to the C20 alcohol of zampanolide that is hydrogen-bonded to the carbonyl oxygen of Thr276 in β-tubulin. As part of our ongoing project to develop simplified and stabilized zampanolide mimics, this paper presents the design, total synthesis, and antiproliferative evaluation of zampanolide mimics in which the *N*-acyl hemiaminal side chain is substituted by more stable 2-fluorobenzyl alcohol appendages.

#### **2. Results and Discussion**

#### *2.1. Design of New Zampanolide Mimics*

The first group of side chains in the zampanolide mimics (e.g., **3**–**4**) previously published by us [21] do not contain a hydroxyl group in order to render a more simplified synthesis. However, the binding conformation confirmed by the high-resolution crystal structure of zampanolide tubulin complex [22] indicates the hydroxyl group at the C20 position in zampanolide is hydrogen-bonded to the carbonyl oxygen of Thr276 in β-tubulin. With this point in mind, a new side chain (S-2) was designed as a bioisosteric substitution for the N-acyl hemiaminal side chain, an unstable moiety in zampanolide but important for its potent cytotoxic properties. The stabilized side chain (S-2) was

designed to replace the chemically fragile N-acylhemiaminal moiety (blue circle in Figure 2) with a benzyl alcohol. The 20OH group is retained in the target side chain so that it can be hydrogen-bonded to the main-chain carbonyl oxygen of Thr276 in β-tubulin. The 1 O atom of zampanolide's side chain is replaced with fluorine atom in the designed side chain so that it can be hydrogen-bonded to the NH group of Thr<sup>276</sup> in β-tubulin. The E,Z-hexadienoyl amide in zampanolide side chain is replaced with a planar conjugate phenyl ring to preserve the hydrophobic contacts between the proposed side chain and residues of β-tubulin. The Almann's desTHP-dactylolide and its enantiomer [23] were further adopted as the core structure of the new zampanolide mimics (**5** and **6**) in the current study because of the acceptable in vitro potency of desTHP-(−)-zampanolide (**2**) and the practical synthesis of the core. Consequently, we set **5** and **6** as our new target mimics of zampanolide in this study.

**Figure 2.** Design of a new stabilized side chain for zampanolide mimics.

#### *2.2. Synthesis*

Our retrosynthetic analysis of the zampanolide mimics **5** and **6**, using Yamaguchi esterification and intramolecular Horner–Wadsworth–Emmons condensation as key reactions, is illustrated in Scheme 1. The Yamaguchi esterification was employed to connect Fragment C1–C8 (carboxylic acid **9**) with Fragment C9–C18 (alcohol **7** or **8**) via the ester bond between C1 and C17. The intramolecular Horner–Wadsworth–Emmons condensation was used to close the macrolactone ring at C8–C9. These two key reactions were successfully applied to our syntheses of desTHPdactylolide and the mimics of desTHPzampanolides [21,23]. The known Fragment C1–C8 (**9**) was readily synthesized via 10 steps from commercially available 2-butyn-1-ol according to the reported procedure [24].

**Scheme 1.** Retrosynthetic analysis of zampanolide mimics **5** and **6.**

The preparation of Fragment C9–C18 (**7** and **8**) with the desired side chain is shown in Scheme 2. The chiral centers at C17 and C18 in the Fragment C9–C18 (**7** and **8**) were introduced by the Sharpless asymmetric dihydroxylation using the commercially available AD-mix formulations (AD-mix-α and AD-mix-β, respectively) with the ligand and the osmium salt as the critical trace component (0.6%

by wt) and ferricyanide and carbonate as the bulk ingredient (99.4% by wt.) [25]. Compound (*E*)-ethyl 3-(2-fluorophenyl)acrylate was synthesized by the Wittig reaction of the commercially available 2-fluorobenzaldehyde (**10**) with ethyl 2-(triphenyl- phosphoranylidene)acetate (**11**) according to the procedure described in the literature [26]. (*E*)-3-(2-Fluorophenyl)prop-2-en-1-ol (**13**) was synthesized by reducing (*E*)-ethyl 3-(2-fluorophenyl)acrylate (**12**) with diisobutylaluminium hydride (DIBAL) based on the procedure reported in the literature [27]. The Sharpless asymmetric dihydroxylation of the allylic alcohol (**13**) with AD-mix-α or AD-mix-β was performed following the standard experimental procedure reported in the literature and sulfonamide was used to accelerate the hydrolysis of osmate ester [25]. Epoxy alcohols (**18** with *R*,*R* configuration, **19** with *S*,*S* configuration) have been synthesized by sequential selective mesylation of primary alcohol (**14** or **15**) and intermolecular Williamson ether synthesis [28]. The hydroxy group in epoxy alcohols (**18** with *R*,*R* configuration, **19** with *S*,*S* configuration) was then protected as the corresponding methoxymethyl (MOM) ethers (**20** with *R*,*R* configuration, **21** with *S*,*S* configuration). Iodo ether **22** was prepared using the optimized protocol previously developed in our laboratory [23]. Its lithiation with *tert*-butyllithium followed by nucleophilic reaction with epoxy MOM ether (**20** with *R*,*R* configuration, **21** with *S*,*S* configuration) in toluene at −90 ◦C mediated by boron trifluoride etherate furnished Fragment C9–C18 (**7** with *R*,*R* configuration, **8** with *S*,*S* configuration) in 22–23% overall yield from **22**.

As illustrated in Scheme 3, the union of Fragment C1–C8 (alcohol **9**) and Fragment C9–C18 (carboxylic acid, **7** with *R*,*R* configuration, **8** with *S*,*S* configuration) has been accomplished by the Yamaguchi esterification to yield bis-TBS ethers (**23** with *R*,*R* configuration, **24** with *S*,*S* configuration). Removal of the protecting TBS groups at C7 and C9 with HF-pyridine complex followed by double oxidation of the corresponding diol (**25** with *R*,*R* configuration and **26** with *S*,*S* configuration) with Dess–Martin periodinane afforded the keto aldehydes (**27** with *R*,*R* configuration, **28** with *S*,*S* configuration). Without further chromatographic purification, the crude keto aldehydes were directly subjected to the synthesis of zampanolide mimics (**29** with *R*,*R* configuration, **30** with *S*,*S* configuration) through the intramolecular HWE condensation catalyzed by Ba(OH)2. Since extractive work-up resulted in significant hydrolysis of the ester [21], the reaction was quenched by filtration of the reaction mixture through a pad of silica gel and the crude product was subjected to preparative TLC purification. The MOM group in **29** was initially planned to be removed using methanolic HCl, but the methanol participated in a Michael addition to the enone, which resulted in the byproduct **31** (Scheme 4). The removal of the MOM with HCl was, therefore, carried out using THF rather than methanol as solvent, leading to the desired zampanolide mimics (**5** with *R*,*R* configuration at C17 and C18, **6** with *S*,*S* configuration at C17 and C18).

**Scheme 4.** Deprotection of MOM ether 29 with methanolic HCl.

#### *2.3. Antiproliferative Activity toward Prostate Cancer Cell Lines*

The in vitro antiproliferative activity of two pairs of enantiomeric macrolides, **5** and **6**, **29** and **30**, has been assessed against a panel of prostate cancer cell lines, including three docetaxel-sensitive prostate cancer cell models (PC-3 DU145, and LNCaP) and two docetaxel-resistant prostate cancer

cell lines (PC-3/DTX and DU145/DTX). WST-1 cell proliferation assay was used for the in vitro evaluation according to the procedure described in the Experimental Section. Docetaxel was used as a positive control while DMSO as a negative control. The half-maximal inhibitory concentrations (IC50 values) were measured by WST-1 cell proliferation assay after 3 days of exposure, calculated from the dose–response curves, and presented as the mean ± standard deviation of the mean in Table 1. The IC50 values for these four new zampanolide mimics are in the range of 1.00–9.36 μM. Mimic **6** has a set of smallest IC50 values (1.00–3.91 μM) among the four test macrolides. As summarized in Figure 3, the IC50 values of new mimic **6** are very close to those for mimic **3** (0.86–2.97 μM) and mimic **32** (1.92–3.16 μM). The mimic **3** has been evidenced to have similar potency as desTHPzampanolide **2** with the *N*-acetyl hemiaminal side chain of zampanolide against prostate cancer cells [21]. The side chain in mimic **6** was thus identified as a new bioisostere for the chemically unstable *N*-acetyl hemiaminal side chain of zampanolide. The relative resistance for Mimic **6** of PC-3/DTX over PC-3 and DU145/DTX over DU145 is 0.59 and 0.44, respectively.

**Table 1.** Antiproliferative potency of zampanolide mimics against docetaxel-sensitive and docetaxel-resistant prostate cancer cells.


*<sup>a</sup>* The relative resistance of the two cell lines obtained by dividing the IC50 value of the resistance cell line by that of the parental cell line. *<sup>b</sup>* These data were reported in our previous paper [21].

**Figure 3.** Comparison of antiproliferative potency among different zampanolide mimics.

#### **3. Materials and Methods**

#### *3.1. General Procedures*

Optical rotations were measured on a RUDOLPH Research Analytical Autopol III Automatic Polarimeter (RUDOLPH Research Analytical, Hackettstown, NJ, USA). IR spectra were recorded on a Nicolet Nexus 470 FTIR spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). High-Resolution MS were obtained on an Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with electrospray ionization (ESI). NMR spectra (see Supplementary Materials) were obtained on a Bruker Fourier 300 spectrometer (Billerica,MA, USA) in CDCl3. The chemical shifts are given in ppm referenced to the respective solvent peak, and coupling constants are reported in Hz. Anhydrous THF and dichloromethane were purified by PureSolv MD 7 Solvent Purification System from Innovative Technologies (MB-SPS-800) (Herndon, VA, USA). All other reagents and solvents were purchased from commercial sources (Fisher Scientific, Portland, OR, USA) and were used without further purification. Silica gel column chromatography was performed using silica gel (32–63 μM). Preparative thin-layer

chromatography (PTLC) separations were carried out on thin-layer chromatography plates loaded with silica gel 60 GF254 (EMD Millipore Corporation, Berlington, MA, USA). Starting materials **22** and **9** were synthesized using the procedure previously described by us [23]. (*E*)-ethyl 3-(2-fluorophenyl)acrylate (**12**) was prepared from commercially available 2-fluorobenzaldehyde (**10**, CAS 446-52-6) according to the reported procedure [26]. (*E*)-3-(2-Fluorophenyl)prop-2-en-1-ol (**13**) was synthesized by reducing (*E*)-ethyl 3-(2-fluorophenyl)acrylate (**12**) with DIBAL based on the procedure reported in the literature [27]. Its structure was confirmed by 1H NMR and high resolution mass spectrometry (HRMS) data (calculated for C9H10FO (M + H): 153.0716; Found: 153.0710).

#### *3.2. Synthesis of (1R,2R)-1-(2-Fluorophenyl)propane-1,2,3-triol [(17R,18R) triol* **14***]*

AD-mix-α (5.9 g) was dissolved in a mixture of *tert*-butyl alcohol (21 mL) and water (21 mL). The solution was stirred vigorously at room temperature until two clear phases were observed. At this point, the lower aqueous phase emerged as bright yellow. Methanesulfonamide (399 mg, 4.2 mmol, 1 equiv) was added and the reaction mixture was cooled down to 0 ◦C. A solution of 13 (640 mg, 4.2 mmol, 1 equiv) in 1 mL of *tert*-butyl alcohol was then added. The resulting mixture was stirred vigorously at 0 ◦C overnight and the reaction progress was monitored with TLC. The reaction was quenched by adding solid sodium sulfate (6.3 g) at 0 ◦C. The mixture was warmed to room temperature and stirred for 30–60 min. The *tert*-butyl alcohol was removed under vacuum, and saturated sodium bicarbonate (30 mL) and water (300 mL) were added to the residue. The mixture was stirred for additional 30 min and then extracted with ethyl acetate (150 mL × 3). The combined extracts were rinsed with brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified through column chromatography eluting with ethyl acetate. The product after purification, with quantitative yield, still contained a trace amount of methanesulfonamide, which is good enough to be used for the next-step reaction. 1H NMR (300 MHz, CDCl3) δ. 7.38 (t, *J* = 7.2 Hz, 1H, aromatic H), 7.21 (dd, *J* = 13.5, 5.7 Hz, 1H, aromatic H), 7.06 (t, *J* = 7.5 Hz, 1H, aromatic H), 6.95 (t, *J* = 8.4 Hz, 1H, aromatic H), 4.88 (d, *J* = 6.3 Hz, 1H, H-18), 4.23 (br.s, 3H, 3 × OH), 3.77 (q, *J* = 5.1 Hz, 1H, H-17), 3.45 (d, *J* = 4.8 Hz, 2H, H2-16). 13C NMR (75 MHz, CDCl3) δ 159.9 (d, *J*CF = 243.8 Hz), 129.6, 128.5, 127.9 (d, *J*CF = 12.8 Hz), 124.6, 115.4 (d, *J*CF = 21.8 Hz), 75.3, 68.8, 63.5. IR (film) νmax: 3384, 2988, 1617, 1490, 1455, 1320, 1222, 1054 cm<sup>−</sup>1. HRMS (ESI): *m*/*z* calculated for C9H12FO3 [M + H]+: 187.0770. Found: 187.0766.

#### *3.3. Synthesis of (2R,3R)-3-(2-Fluorophenyl)-2,3-dihydroxypropyl methanesulfonate [(17R,18R) mesylate* **16***]*

To a solution of triol **14** (1.37 g, 7.39 mmol) in pyridine (15 mL, 0.5 M), methane sulfonyl chloride (0.57 mL, 7.39 mmol) was added dropwise at 0 ◦C under argon. The reaction mixture was stirred at room temperature for 20 h and the reaction progress was monitored by TLC (Hexane/EtOAc, 1:1, *v*/*v*). The reaction was quenched by diluting with ethyl acetate (800 mL), and the resulting solution was rinsed with brine (50 mL × 3) and dried over anhydrous sodium sulfate. After removing the organic solvent, the residue was subjected to column chromatography, using 40% ethyl acetate in hexane as eluent, to give the desired product. Colorless oil; 81% yield. 1H NMR (300 MHz, CDCl3) δ 7.47 (dt, *J* = 7.5, 1.8 Hz, 1H, aromatic H), 7.34–7.27 (m, 1H, aromatic H), 7.17 (dt, *J* = 7.5, 0.9 Hz, 1H, aromatic H), 7.04 (ddd, *J* = 10.5, 8.1, 0.9 Hz, 1H, aromatic H), 5.00 (d, *J* = 5.7 Hz, 1H, H-18), 4.27–4.16 (m, 2H, H2-16), 4.06–4.01 (m, 1H, H-17), 3.18 (br.s, 2H, 2 <sup>×</sup> OH), 3.03 (s, 3H, SO2*CH3*). 13C NMR (75 MHz, CDCl3) δ 159.9 (d, *J*CF = 244.3 Hz), 130.1 (d, *J*CF = 8.3 Hz), 128.4 (d, *J*CF = 3.8 Hz), 127.0 (d, *J*CF = 12.8 Hz), 124.8 (d, *J*CF = 3.8 Hz), 115.7 (d, *J*CF = 21.8 Hz), 72.9, 70.4, 67.9 (d, *J*CF = 2.3 Hz), 37.6. IR (film) νmax: 3502, 3029, 2940, 1617, 1587, 1490, 1456, 1346, 1171 cm<sup>−</sup>1. HRMS (ESI): *m*/*z* calculated for C10H14FO5S [M + H]+: 265.0546. Found: 265.0547.

#### *3.4. Synthesis of (R)-(2-Fluorophenyl)((R)-oxiran-2-yl)methanol [(17R,18R) epoxide* **18***]*

To a suspension of sodium hydride (275 mg, 60%, 6.87 mmol, 1.5 equiv) in anhydrous THF (36 mL), a solution of **16** (1.21 g, 4.58 mmol, 1 equiv) in anhydrous THF was added at about −30 ◦C under argon. The reaction mixture was stirred at 0 ◦C overnight and the reaction progress was monitored

with TLC (hexane:EtOAc, 3:1). The mixture was filtered through a silica gel pad eluting with ethyl acetate. After concentration to remove the solvent, the residue was subjected to PTLC purification eluting with toluene:EtOAc (3:1, *v*/*v*) to furnish the desired epoxide. Colorless oil; 57% yield. 1H NMR (300 MHz, CDCl3) δ 7.57 (dd, *J* = 7.8, 1.8 Hz, 1H, aromatic H), 7.35–7.28 (m, 1H, aromatic H), 7.20 (dt, *J* = 7.5, 1.2 Hz, 1H, aromatic H), 7.06 (ddd, *J* = 10.5, 8.1, 1.2 Hz, 1H, aromatic H), 4.84 (t, *J* = 6.6 Hz, 1H, H-18), 3.26–3.22 (m, 1H, H-17), 2.92–2.85 (overlapped, 2H, H2-16), 2.47 (d, *J* = 5.7 Hz, 1H, OH). 13C NMR (75 MHz, CDCl3) δ 159.9 (d, *J*CF = 244.5 Hz), 129.6 (d, *J*CF = 8.3 Hz), 127.9 (d, *J*CF = 4.5 Hz), 127.3 (d, *J*CF = 13.5 Hz), 124.5 (d, *J*CF = 3.8 Hz), 115.4 (d, *J*CF = 21.8 Hz), 68.7 (d, *J*CF = 2.3 Hz), 55.6, 45.5 (d, *J* = 2.3 Hz). IR (film) νmax: 3420, 3067, 3002, 2928, 1617, 1586 1488, 1456, 1224 cm<sup>−</sup>1. HRMS (ESI): *m*/*z* calculated for C9H10FO2 [M + H]+: 169.0665. Found: 169.0657.

#### *3.5. Synthesis of (R)-2-((R)-(2-Fluorophenyl)(methoxymethoxy)methyl)oxirane [(17R,18R)MOM ether* **20***]*

To a solution of alcohol **18** (716 mg, 4.3 mmol) in DMF (4.2 mL), sodium hydride (204 mg, 60%, 5.1 mmol) was added at 4 ◦C, and the mixture was stirred at 4 ◦C for 30 min. Methoxymethyl chloride (0.39 mL, 5.1 mmol) followed by tetrabutylammonium iodide (157 mg, 0.43 mmol) was added to the reaction mixture. The reaction was proceeded with stirring at room temperature for 20 h. The reaction mixture was diluted with ethyl acetate (300 mL) and diethyl ether (300 mL) and then rinsed with brine (50 mL × 3). The organic layer was dried over anhydrous sodium sulfate and concentrated to give a crude mass, which was purified by column chromatography eluting with hexane/ethyl acetate (3:1, *v*/*v*) to furnish the MOM ether **20** as a pale-yellow oil in 67% yield. 1H NMR (300 MHz, CDCl3) δ 7.51 (dt, *J* = 7.2, 1.8 Hz, 1H, aromatic H), 7.33–7.25 (m, 1H, aromatic H), 7.17 (dt, *J* = 7.5, 1.2 Hz, 1H, aromatic H), 7.04 (ddd, *J* = 9.9, 8.1, 1.2 Hz, 1H, aromatic H), 4.75 (d, *J* = 6.9 Hz, 1H, O*CH2*OCH3), 4.67 (d, *J* = 6.9 Hz, 1H, O*CH2*OCH3), 4.62 (d, *J* = 6.6 Hz, 1H, H-18), 3.36 (s, 3H, O*CH3*), 3.26–3.22 (m, 1H, H-17), 2.73–2.72 (overlapped, 2H, H2-16). 13C NMR (75 MHz, CDCl3) δ 160.3 (d, *J* = 244.5 Hz), 129.8 (d, *J* = 8.3 Hz), 128.6 (d, *J* = 4.5 Hz), 125.2 (d, *J* = 14.3 Hz), 124.4 (d, *J* = 3.0 Hz), 115.5 (d, *J* = 21.8 Hz), 94.6, 73.0, 55.6, 54.4, 44.2 (d, *J* = 2.3 Hz). IR (film) νmax: 2891, 1615, 1587, 1488, 1455, 1022 cm<sup>−</sup>1. HRMS (ESI): *m*/*z* calculated for C11H14FO3 [M + H]+: 213.0927. Found: 213.0921.

#### *3.6. Synthesis of (5R,6R,E)-5-(2-Fluorophenyl)-8,16,16,17,17-pentamethyl-2,4,11,15-tetraoxa-16 silaoctadec-8-en-6-ol [(17R,18R) Fragment C9–C18 (***7***)]*

To a solution of vinyliodide **22** (164 mg, 0.44 mmol; co-evaporated twice with pentane) in toluene (3 mL) at −78 ◦C, *tert*-butyllithium (0.46 mL, 1.9 M, 0.89 mmol) was added, and the mixture was stirred at −78 ◦C for 45 min prior to being cooled down to −90 ◦C. Epoxide **20** (188 mg, 0.89 mmol; co-evaporated twice with pentane) in toluene (1.5 mL) was added dropwise to make sure that interior temperature lower than −78 ◦C. The reaction solution was re-cooled to −90 ◦C before BF3•OEt2 (0.11 mL, 0.89 mmoL) was added dropwise. The resulting solution was then stirred at −78 ◦C overnight prior to being quenched with ethyl acetate (15 mL) and saturated aqueous sodium bicarbonate (35 mL) was added. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (60 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated *in Vacuum* to remove ethyl acetate. The crude mass was subjected to PTLC purification over silica gel using hexane/ethyl acetate (4:1, *v*/*v*) as eluent to yield secondary alcohol **7** as a pale-yellow oil in 23% yield. 1H NMR (300 MHz, CDCl3) δ. 7.41 (dt, *J* = 7.2, 1.5 Hz, 1H, aromatic H), 7.32–7.24 (m, 1H, aromatic H), 7.15 (dt, *J* = 7.5, 0.9 Hz, 1H, aromatic H), 7.53 (ddd, *J* = 9.9, 8.1, 0.9 Hz, 1H, aromatic H), 5.41 (t, *J* = 6.6 Hz, 1H, H-14), 4.81 (d, *J* = 6.3 Hz, 1H, H-17), 4.62 (d, *J* = 6.9 Hz, 1H, O*CH2*OCH3), 4.58 (d, *J* = 6.6 Hz, 1H, O*CH2*OCH3), 3.94 (d, *J* = 6.6 Hz, 2H, H2-13), 3.67 (t, *J* = 6.3 Hz, 2H, H2-11), 3.46 (t, *J* = 6.3 Hz, 2H, H2-9), 3.36 (s, 3H), O*CH3*), 1.75 (quin, *J* = 6.3 Hz, 2H, H2-10), 1.64 (s, 3H, 15-*CH3*), 0.87 (s, 9H, TBS), 0.03 (s, 6H, TBS). 13C NMR (75 MHz, CDCl3) δ 160.9 (d, *J*CF = 244.8 Hz), 136.4, 129.8, 129.1, 128.7, 126.1 (d, *J*CF = 13.5 Hz), 124.5 (d, *J*CF = 3.8 Hz), 115.6 (d, *J*CF = 21.8 Hz), 95.3, 75.6, 72.5 (d, *J*CF = 12.1 Hz), 67.3, 67.0, 60.2, 56.2, 43.0, 33.2, 26.1, 26.07, 18.5, 16.6, 16.5. IR (film) νmax: 3420, 2929, 1715, 1647, 1615, 1488, 1029 cm−1. HRMS (ESI): *m*/*z* calculated for C24H42FO5Si [M + H]+: 457.2786. Found: 457.2780.

#### *3.7. Synthesis of*

#### *(2E,4Z)-(5R,6R,E)-5-(2-Fluorophenyl)-8,16,16,17,17-pentamethyl-2,4,11,15-tetraoxa-16-silaoctadec-8-en-6-yl 7-((tert-butyldimethylsilyl)oxy)-8-(diethoxyphosphoryl)-5- methylocta-2,4-dienoate [(17R,18R) ester* **23***]*

To a solution of Fragment C1–C8 (**9**, 37 mg, 0.09 mmol; co-evaporated with pentane twice) in toluene (0.5 mL) at room temperature, trimethylamine (28 μL, 0.20 mmol) and 2,4,6-trichlorobenzoyl chloride (18 μL, 0.12 mmol) was sequentially added, and the reaction mixture was stirred at room temperature for 1.5 h. A solution of alcohol **7** (35 mg, 0.08 mmol) and DMAP (9.3 mg, 0.08 mmol) in toluene (0.5 mL) was then added to the reaction mixture. The reaction was allowed to proceed with stirring at room temperature for 19 h prior to being quenched with water (5 mL) and saturated aqueous sodium bicarbonate (5 mL). The mixture was extracted with ethyl acetate (5 mL × 3), the combined organic extracts were dried over anhydrous sodium sulfate and concentrated *in vacuum*. PTLC purification of the crude product over silica gel eluting with hexane/ethyl acetate (60:40, *v*/*v*) yielded the ester **23** as a mixture of diastereoisomers in a 1:1 ratio as a colorless oil in 77% yield. 1H NMR (300 MHz, CDCl3) δ 7.50 (dd, *J* = 15.0, 11.7 Hz, H-3), 7.42 (t, *J* = 7.5 Hz, 1H, aromatic H), 7.25–7.20 (m, 1H, aromatic H), 7.10 (t, *J* = 7.5 Hz, 1H, aromatic H), 7.00 (t, *J* = 8.4 Hz, 1H, aromatic H), 6.03 (d, *J* = 11.4 Hz, H-4), 5.75 (d, *J* = 15.3 Hz, 1H, H-2), 5.43–5.37 (m, 1H, H-17), 5.33 (t, *J* = 6.6 Hz, 1H, H-14), 5.08 (d, *J* = 5.4 Hz, 1H, H-18), 4.58 (d, *J* = 6.6 Hz, 1H, -O*CH2*OCH3), 4.51 (d, *J* = 6.6 Hz, 1H, O*CH2*OCH3), 4.18–4.04 (overlapped, 5H, 2 x O*CH*2CH3, H-7), 3.93–3.79 (m, 2H, H2-13), 3.64 (t, *J* = 6.3 Hz, 2H, H2-11), 3.40 (t, *J* = 6.3 Hz, 2H, H2-9), 3.32 (s, 3H, -OCH2O*CH3*), 2.58–2.48 (m, 2H, H2-16), 2.33–2.24 (m, 2H, H2-8), 2.00–1.92 (m, 2H, H2-6), 1.87 (s, 3H, 5-*CH3*), 1.72 (quin, *J* = 6.3 Hz, 2H, H2-10), 1.64 (1.63) (s, 3H, 15-*CH3*), 1.31 (t, *J* = 7.2 Hz, 6H, 2 × *O*CH2*CH3*), 0.86 (s, 9H, TBS), 0.81 (0.80) (s, 9H, TBS), 0.04 (0.03) (s, 3H, TBS), 0.01 (s, 6H, TBS), <sup>−</sup>0.04(−0.05) (s, 3H, TBS). 13C NMR (75 MHz, CDCl3) <sup>δ</sup> 166.8 (166.7), 160.8 (d, *J* = 244.5 Hz), 146.4 (d, *J* = 9.8 Hz), 141.6, 135.2 (d, *J* = 3.8 Hz), 129.7, 129.0, 126.8, 125.7 (d, *J* = 13.5 Hz), 125.1, 124.3, 119.3, 115.6 (115.3), 95.0, 72.9, 72.3, 67.3, 66.8, 61.7(61.6), 60.5, 60.1, 56.1, 41.8 (41.2), 36.1, 34.3, 33.1, 27.0, 26.11, 26.05, 25.90, 25.87, 25.2, 25.0, 18.5, 18.0, 16.6 (16.5), −4.6, −5.2. IR (film) νmax: 2929, 2856, 1716, 1489, 1251, 1020 cm−1. HRMS (ESI): *m*/*z* calculated for C43H77FO10PSi2 [M + H]+: 859.4777. Found: 859.4756.

#### *3.8. Synthesis of (2E,4Z)-(1R,2R,E)-1-(2-Fluorophenyl)-6-(3-hydroxypropoxy)-1-(methoxymethoxy) -4-methylhex-4-en-2-yl 8-(diethoxyphosphoryl)-7-hydroxy-5-methylocta-2,4-dienoate [(17R,18R) diol* **25***]*

To a solution of di-TBS ether **23** (90 mg, 0.105 mmol) in THF (4.2 mL) at 0 ◦C in a plastic bottle, hydrogen fluoride-pyridine complex (70%, 1.0 mL) was added dropwise. The solution was stirred at 0 ◦C for 5 min and then at room temperature for 16 h. Saturated sodium bicarbonate (30 mL) was added to quench the reaction and the suspension was extracted with ethyl acetate (30 mL × 3). The combined organic extracts were dried over anhydrous sodium sulfate and the dried extract was concentrated under vacuum. The crude product was purified by PTLC, eluting with dichloromethane/methanol (93:7. *v*/*v*), and the pure diol was washed out from the PTLC silica gel with acetone as a pale-yellow oil in 92% yield. 1H NMR (300 MHz, CDCl3) δ 7.50 (7.49) (dd, *J* = 15.3, 11.7 Hz, 1H, H-3), 7.43 (t, *J* = 9.0 Hz, 1H, aromatic H), 7.30–7.23 (m, 1H, aromatic H), 7.13 (7.12) (t, *J* = 7.5 Hz, 1H, aromatic H), 7.031 (7.028) (dd, *J* = 10.2, 9.0 Hz, 1H, aromatic H), 6.11 (d, *J* = 11.4 Hz, 1H, H-4), 5.80 (d, *J* = 15.3 Hz, 1H, H-2), 5.47–5.40 (m, 1H, H-17), 5.32 (t, *J* = 5.7 Hz, 1H, H-14), 5.09 (d, *J* = 5.7 Hz, 1H, H-18), 4.58 (d, *J* = 6.9 Hz, 1H, -O*CH2*OCH3), 4.51 (d, *J* = 6.9 Hz, 1H, O*CH2*OCH3), 4.18–4.06 (overlapped, 5H, 2 × O*CH2*CH3, H-7), 3.90 (t, *J* = 5.7 Hz, 2H, H2-13), 3.69 (3.68) (t, *J* = 5.7 Hz, 2H, H2-11), 3.48 (3.47) (t, *J* = 5.7 Hz, 2H, H2-9), 3.33 (3.32) (s, 3H, -OCH2O*CH3*), 3.03 (s, 2H, 2 × OH), 2.66–2.53 (m, 1H, H-16), 2.46–2.19 (m, 3H, H2-8 & H-16), 1.92 (S, 3H, 5-*CH3*), 1.96–1.88 (m, 2H, H2-6), 1.75 (quin, *J* = 5.7 Hz, 2H, H2-10), 1.65 (s, 3H, 15-*CH3*), 1.33 (t, *J* = 7.2 Hz, 6H, 2 <sup>×</sup> OCH2*CH3*). 13C NMR (75 MHz, CDCl3) δ 167.0 (166.8), 160.9 (d, *J* = 247.5 Hz), 146.0, 140.8 (d, *J* = 17.3 Hz), 135.7 (d, *J* = 9.0 Hz), 129.8, 129.0, 126.8, 125.6 (d, *J* = 12.8 Hz), 124.8 (d, *J* = 11.3 Hz), 124.4, 119.7 (d, *J* = 11.3 Hz), 115.7 (115.4), 94.9, 73.0, 72.4, 68.8, 67.4, 65.3, 62.2, 61.8, 56.1, 41.1, 34.6 (34.5), 32.7, 32.3, 25.3, 25.0, 16.6 (16.5). IR (film) νmax: 3366, 2929, 1711, 1633, 1488, 1222, 1019 cm<sup>−</sup>1. HRMS (ESI): *m*/*z* calculated for C31H49FO10P [M + H]+: 631.3048. Found: 631.3041.

#### *3.9. Synthesis of (R,3E,9E,11Z,15E)-6-((R)-(2-Fluorophenyl)(methoxymethoxy)methyl)-4,12 dimethyl-1,7-dioxacyclooctadeca-3,9,11,15-tetraene-8,14-dione [(17R,18R) macrolactone* **29***]*

To a solution of diol **25** (65 mg, 0.10 mmol) in dichloromethane (6.2 mL) at room temperature, Dess–Martin periodinane (DMP, 131 mg, 0.30 mmol) was added. The reaction mixture was stirred for 30 min before a second portion of DMP (131 mg, 0.30 mmol) was added. The reaction was allowed to proceed with stirring at room temperature for an additional 1 h. The reaction mixture was poured into a stirred mixture of saturated sodium bicarbonate (30 mL) and saturated sodium thiosulfate (30 mL), and the resulting suspension was stirred for 30 min before being extracted with dichloromethane (30 mL × 3). The combined organic extracts were dried over anhydrous sodium sulfate and the dried extract was concentrated under vacuum. The crude product was used for the next reaction without further purification. To a solution of the crude ketoaldehyde (0.10 mmol) obtained above in THF (103 mL) at 0 ◦C was added water (2.2 mL) followed by activated barium hydroxide (15 mg, 0.08 mmol). The reaction mixture was stirred at 0 ◦C for 30 min and at room temperature for 2.5 h, then was filtered through a pad of sodium sulfate and a pad of silica gel that was rinsed with ethyl acetate. After evaporation of the solvent, the crude product was subjected to PTLC purification, using hexane/ethyl acetate (73:37) as eluent, to yield the pure macrolactone **29**. Colorless oil, 46% yield for two steps. [α]: <sup>−</sup>15.6 (*c* = 0.23, MeOH). 1H NMR (300 MHz, CDCl3) <sup>δ</sup> 7.60 (dd, *J* = 15.0, 11.4 Hz, 1H, H-3), 7.46 (dt, *J* = 7.5, 1.8 Hz, 1H, aromatic H), 7.33–7.25 (m, 1H, aromatic H), 7.16 (dt, *J* = 7.5, 1.2 Hz, 1H, aromatic H), 7.05 (ddd, *J* = 9.9, 8.4, 1.2 Hz, 1H, aromatic H), 6.82 (dt, *J* = 16.2, 6.6 Hz, 1H, H-9), 6.12 (d, *J* = 11.4 Hz, 1H, H-4), 6.01 (d, *J* = 16.2 Hz, 1H, H-8), 5.91 (d, *J* = 15.0 Hz, 1H, H-2), 5.51 (ddd, *J* = 11.4, 6.3, 1.2 Hz, 1H, H-17), 5.27 (dd, *J* = 7.8, 4.8 Hz, 1H, H-14), 5.12 (d, *J* = 6.3 Hz, 1H, H-18), 4.60 (d, *J* = 6.9 Hz, 1H, *O*-*CH2*OCH3), 4.53 (d, *J* = 6.9 Hz, 1H, O*CH2*OCH3), 3.99 (dd, *J* = 12.0, 8.1 Hz, 1H, H-16), 3.88–3.81 (m, 2H, H2-13), 3.47–3.28 (m, 2H, H2-11), 3.34 (s, 3H, OCH2O*CH3*), 3.14 (d, *J* = 12.6 Hz, 1H, H-16), 2.43–2.36 (m, 2H, H2-10), 2.33 (d, *J* = 12.0 Hz, 1H, H-6), 2.05 (d, *J* = 12.1 Hz, 1H, H-6), 1.83 (s, 3H, 5-CH3), 1.63 (S, 3H, 15-CH3). 13C NMR (75 MHz, CDCl3) δ 197.2, 166.6, 160.9 (d, *J* = 244.5 Hz), 146.8, 142.5, 139.7, 138.9, 134.3, 130.3, 128.9 125.9 (d, *J* = 7.5 Hz), 125.6 (d, *J* = 13.5 Hz), 125.2 (d, *J* = 5.6 Hz), 124.5, 120.9 (d, *J* = 5.3 Hz), 115.6 (d, *J* = 21.8 Hz), 94.9, 72.9, 72.6, 67.9, 67.8, 56.0, 46.0, 41.4, 33.1, 24.09, 16.7. IR (film) νmax: 2931, 1713, 1633, 1489, 1358, 1226, 1030 cm<sup>−</sup>1. HRMS (ESI): *m*/*z* calculated for C27H34FO6 [M + H]+: 473.2339. Found: 473.2333.

#### *3.10. Synthesis of (R,3E,9E,11Z,15E)-6-((R)-(2-Fluorophenyl)(hydroxy)methyl)-4,12-dimethyl-1,7-dioxacyclooctadeca-3,9,11,15-tetraene-8,14-dione [(17R,18R) macrolactone* **5***]*

To a solution of **29** (30 mg, 0.064 mmol) in a mixture of tetrahydrofuran/water (0.92 mL, 1:1, *v*/*v*), concentrated hydrochloric acid (130 μL) was added. The two more portions of concentrated hydrochloric acid (130 μL × 2) were added sequentially after each one-hour stirring at room temperature. The reaction mixture was stirred at room temperature overnight before the reaction was quenched by adding saturated ammonium chloride (60 mL). The subsequent mixture was extracted with ethyl acetate (40 mL × 3). The combined extracts were dried over anhydrous sodium sulfate and the solvents were evaporated *in vacuum*. The crude product was purified over preparative thin layer chromatography eluting with toluene/ethyl acetate (3:1, *v*/*v*) to furnish the desired product (11 mg). Colorless syrup, 41% yield. 1H NMR (300 MHz, CDCl3) δ 7.62 (dd, *J* = 15.0, 11.7 Hz, 1H, H-3), 7.49 (dt, *J* = 7.5, 1.8 Hz, 1H, aromatic H), 7.31–7.27 (m, 1H, aromatic H), 7.17 (dt, *J* = 7.5, 1.2 Hz, 1H), 7.05 (ddd, *J* = 10.2, 8.1, 0.9 Hz, 1H), 6.83 (dt, *J* = 15.9, 6.9 Hz, 1H, H-9), 6.12 (d, *J* = 11.4 Hz, 1H, H-4), 6.02 (d, *J* = 16.2 Hz, 1H, H-8), 5.91 (d, *J* = 15.0 Hz, 1H, H-2), 5.48–5.38 (m, 1H, H-17), 5.27 (t, *J* = 6.0 Hz, 1H, H-14), 5.10 (d, *J* = 6.3 Hz, 1H, H-18), 3.99 (dd, *J* = 12.0, 8.1 Hz, 1H, H-16), 3.86 (br.s, 1H, H-13), 3.82 (br.s, 1H, H-13),

3.51–3.36 (m, 2H, H2-11), 3.18 (d, *J* = 12.9 Hz, 1H, H-16), 2.44–2.34 (m, 3H, H2-10, H-6), 2.06 (d, *J* = 14.4 Hz, 1H, H-6), 1.83 (s, 3H, 5-*CH3*), 1.62 (s, 3H, 15-*CH3*). 13C NMR (75 MHz, CDCl3) δ 197.2, 167.0, 160.1 (d, *J*CF = 244.5 Hz), 146.9, 143.0, 140.1 (d, *J*CF = 12.8 Hz), 134.2, 130.2, 129.9, 128.8, 127.6 (d, *J*CF = 12.8 Hz), 126.0, 125.2, 124.6, 120.7, 115.6 (d, *J*CF = 21.8 Hz), 79.4, 74.4, 70.3, 68.0, 45.9, 41.4, 33.0, 24.1, 16.7. [α]: −15.2 (*c* = 0.16, MeOH). IR (film) νmax: 3428, 2924, 2854, 1706, 1633, 1558, 1488, 1360, 1221, 1036 cm<sup>−</sup>1. HRMS (ESI): *m*/*z* calculated for C25H30FO5 [M + H]+: 429.2077. Found: 429.2074.

#### *3.11. Synthesis of (1S,2S)-1-(2-Fluorophenyl)propane-1,2,3-triol [(17S,18S) triol* **15***]*

Triol **15** was prepared in 68% yield from 13 catalyzed by AD-mix-β using a similar procedure used to synthesize its antipode **14**. 1H NMR (300 MHz, CDCl3) δ 7.39 (t, *J* = 7.5 Hz, 1H, aromatic H), 7.22 (dd, *J* = 13.8, 7.2 Hz, 1H, aromatic H), 7.07 (t, *J* = 7.5 Hz, 1H, aromatic H), 6.96 (t, *J* = 9.3 Hz, 1H, aromatic H), 4.90 (d, *J* = 6.3 Hz, 1H, H-18), 4.21 (br.s, 3H, 3 × OH), 3.78 (dd, *J* = 10.2, 4.8 Hz, 1H, H-17), 3.48 (d, *J* = 4.8 Hz, 2H, H2-16). 13C NMR (75 MHz, CDCl3) δ 160.0 (d, *J*CF = 244.5 Hz), 129.6, 128.5 (d, *J*CF = 16.5 Hz), 127.8 (d, *J*CF = 12.8 Hz), 124.6, 115.5 (d, *J*CF = 21.8 Hz), 75.4, 68.8, 63.4. IR (film) νmax: 3375, 2930, 1781, 1489, 1456, 1319 cm<sup>−</sup>1. HRMS (ESI): *m*/*z* calculated for C9H12FO3 [M + H]+: 187.0770. Found: 187.0765.

#### *3.12. Synthesis of (2S,3S)-3-(2-Fluorophenyl)-2,3-dihydroxypropyl methanesulfonate [(17S,18S) mesylate* **17***]*

Mesylate **17** was prepared from triol **15** as a colorless oil in 72% yield, employing a procedure similar to that used for the mesylation of triol **14**. 1H NMR (300 MHz, CDCl3) δ 7.48 (dt, *J* = 7.5, 1.8 Hz, 1H, Aromatic H), 7.32–7.29 (m, 1H, aromatic H), 7.18 (dt, *J* = 8.4, 0.9 Hz, 1H, aromatic H), 7.05 (ddd, *J* = 9.3, 8.1, 0.9 Hz, 1H, aromatic H), 5.01 (d, *J* = 5.7 Hz, 1H, H-18), 4.28–4.15 (m, 2H, H2-16), 4.08–4.03 (m, 1H, H-17), 3.04 (s, 3H, SO2*CH3*). 13C NMR (75 MHz, CDCl3) δ 159.9 (d, *J*CF = 244.5 Hz, 1H), 130.1, 128.4, 127.0 (d, *J*CF = 12.8 Hz, 1H), 124.8, 115.7 (d, *J*CF = 21.8 Hz, 1H), 72.9, 70.3, 67.9, 37.6. IR (film) νmax: 3482, 3029, 2939, 1616, 1587, 1489, 1456, 1332, 1168 cm<sup>−</sup>1. HRMS (ESI): *m*/*z* calculated for C10H14FO5S [M + H]+: 265.0546. Found: 265.0542.

#### *3.13. Synthesis of (S)-(2-Fluorophenyl)((S)-oxiran-2-yl)methanol [(17S,18S) epoxide* **19***]*

Epoxide **19** (52%, pale yellow oil) was obtained from mesylate **17** according to the internal Williamson ether synthesis procedure employed for the conversion of mesylate **16** to epoxide **18**. 1H NMR (300 MHz, CDCl3) δ 7.57 (dt, *J* = 7.5, 1.8 Hz, 1H, aromatic H), 7.35–7.25 (m, 1H, aromatic H), 7.20 (dt, *J* = 7.5, 1.2 Hz, 1H, aromatic H), 7.06 (ddd, *J* = 10.5, 8.1, 1.2 Hz, 1H, aromatic H), 4.83 (d, *J* = 5.1 Hz, 1H, H-18), 3.26–3.21 (m, 1H, H-17), 2.92–2.84 (m, 2H, H2-16). 13C NMR (75 MHz, CDCl3) δ 160.0 (d, *J*CF = 244.5 Hz), 129.8, 127.6 (d, *J*CF = 9.0 Hz), 127.4 (d, *J*CF = 13.5 Hz), 124.7, 115.6 (d, *J*CF = 21.0 Hz), 68.6, 55.4, 45.6. IR (film) νmax: 3482, 3029, 2939, 1617, 1587, 1489, 1456, 1331, 1168 cm−1. HRMS (ESI): *m*/*z* calculated for C9H10FO2 [M + H]+: 169.0665. Found: 169.0659.

#### *3.14. Synthesis of (S)-2-((S)-(2-Fluorophenyl)(methoxymethoxy)methyl)oxirane [(17S,18S) MOM ether* **21***]*

MOM ether **21** was prepared from epoxide **19** as a pale-yellow oil in 73% yield employing a procedure similar to that used for conversion of **18**. 1H NMR (300 MHz, CDCl3) δ 7.44 (dt, *J* = 7.5, 1.8 Hz, 1H, aromatic H), 7.32–7.27 (m, 1H, aromatic H), 7.17 (dt, *J* = 7.5, 0.9 Hz, 1H, aromatic H), 7.05 (ddd, *J* = 10.2, 8.4, 1.2 Hz, 1H, aromatic H), 4.76 (d, *J* = 6.6 Hz, 1H, -O*CH2*OCH3), 4.68 (d, *J* = 6.9 Hz, 1H, H-18), 4.63 (d, *J* = 6.6 Hz, 1H, -O*CH2*OCH3), 3.37 (s, 3H, -OCH2O*CH3*), 3.27 – 3.23 (m, 1H, H-17), 2.75–2.73 (m, 2H, H2-16). 13C NMR (75 MHz, CDCl3) δ 160 (d, *J*CF = 245.3 Hz), 129.9 (d, *J*CF = 8.3 Hz), 128.7 (d, *J*CF = 3.8 Hz), 125.3 (d, *J*CF = 14.3 Hz), 124.5 (d, *J*CF = 3.8 Hz), 115.7 (d, *J*CF = 21.8 Hz), 94.8, 73.1, 55.8, 54.6, 44.4. IR (film) νmax: 2891, 2825, 1616, 1587, 1488, 1455, 1149, 1022 cm−1. HRMS (ESI): *m*/*z* calculated for C11H14FO3 [M + H]+: 213.0927. Found: 213.0920.

*3.15. Synthesis of (5S,6S,E)-5-(2-Fluorophenyl)-8,16,16,17,17-pentamethyl-2,4,11,15-tetraoxa-16 silaoctadec-8-en-6-ol [(17S, 18S) Fragment C9–C18 (***8***)]*

(17*S*,18*S*) Fragment C9–C18 (**8**) was synthesized as a colorless oil in 22% yield from MOM ether **21** in a similar way to the synthesis of its antipode (17*R*,18*R*) Fragment C9–C18 (7). 1H NMR (300 MHz, CDCl3) δ 7.41 (dt, *J* = 7.5, 1.8 Hz, 1H, aromatic H), 7.32–7.24 (m, 1H, aromatic H), 7.15 (dt, *J* = 7.5, 1.2 Hz, 1H, aromatic H), 7.03 (ddd, *J* = 10.2, 8.4, 1.2 Hz, 1H, aromatic H), 5.41 (t, *J* = 6.6 Hz, 1H, H-14), 4.81 (d, *J* = 6.6 Hz, 1H, H-18), 4.63 (d, *J* = 6.6 Hz, 1H, O*CH2*OCH3), 4.58 (d, *J* = 6.6 Hz, 1H, O*CH2*OCH3), 3.95 (d, *J* = 6.6 Hz, 2H, H2-13), 3.82 (dt, *J* = 10.2, 5.7 Hz, 1H, H-17), 3.67 (t, *J* = 6.0 Hz, 2H, H2-11), 3.46 (t, *J* = 6.6 Hz, 2H, H2-9), 3.36 (s, 3H, OCH3), 2.23 (dd, *J* = 12.6, 9.6 Hz, 1H, H-16), 2.05 (dd, *J* = 12.6, 2.7 Hz, 1H, H-16), 1.76 (quin, *J* = 6.3 Hz, 2H, H2-10), 1.64 (s, 3H, 15-CH3), 0.87 (s, 9H, TBS), 0.03 (s, 6H, TBS). 13C NMR (75 MHz, CDCl3) δ 160.9 (d, *J*CF = 244.7 Hz), 136.4, 129.2, 128.9, 128.6, 126.1 (d, *J*CF = 12.8 Hz), 124.5, 115.5, 95.3, 75.6, 72.7, 67.3, 67.0, 60.2, 56.2, 43.0, 33.2, 26.0, 18.5, 16.6, 16.5. IR (film) νmax: 3461, 2928, 2855, 1616, 1586, 1252, 1090, 1031 cm<sup>−</sup>1. HRMS (ESI): *m*/*z* calculated for C24H42FO5Si [M + H]+: 457.2786. Found: 457.2782.

#### *3.16. Synthesis of (2E,4Z)-(5S,6S,E)-5-(2-Fluorophenyl)-8,16,16,17,17-pentamethyl-2,4,11,15 tetraoxa-16-silaoctadec-8-en-6-yl 7-((tert-butyldimethylsilyl)oxy)-8-(diethoxyphosphoryl)- 5-methylocta-2,4-dienoate [(17S,18S) ester* **24***]*

(17*S*,18*S*) Ester **24** was obtained as a pale-yellow oil in 91% yield from (17*S*,18*S*) Fragment C9–C18 (**8**) and Fragment C1–C8 (**9**), according to the esterification procedure employed for the preparation of ester **23**. 1H NMR (300 MHz, CDCl3) δ 7.50 (dd, *J* = 14.4, 12.0 Hz, 1H, H-3), 7.42 (t, *J* = 7.2 Hz, 1H, aromatic H), 7.34–7.21 (m, 1H, aromatic H), 7.10 (t, *J* = 7.5 Hz, 1H, aromatic H), 7.01 (t, *J* = 9.3 Hz, 1H, aromatic H), 6.03 (d, *J* = 11.7 Hz, 1H, H-4), 5.76 (d, *J* = 15.0 Hz, 1H, H-2), 5.45–5.37 (m, 1H, H-17), 5.33 (t, *J* = 6.0 Hz, 1H, H-14), 5.08 (d, *J* = 5.1 Hz, 1H, H-18), 4.58 (d, *J* = 6.9 Hz, 1H, O*CH2*OCH3), 4.51 (d, *J* = 6.6 Hz, 1H, O*CH2*OCH3), 4.23–4.04 (m, 5H, 2 × O*CH2*CH3, H-7), 3.93–3.80 (m, 2H, H2-13), 3.65 (t, *J* = 6.6 Hz, 2H, H2-11), 3.41 (t, *J* = 6.3 Hz, H2-9), 3.32 (s, 3H, OCH2O*CH3*), 2.58–2.54 (m, 2H, H2-16), 2.33–2.28 (m, 2H, H2-8), 2.03–1.93 (m, 2H, H2-6), 1.87 (s, 3H, 5-*CH3*), 1.77–1.70 (m, 2H. H2-10), 1.64 (s, 3H, 15-CH3), 1.31 (t, *J* = 6.9 Hz, 6H, 2 × OCH2*CH3*), 0.90 (0.88) (s, 3H, TBS), 0.86 (s, 6H, TBS), 0.81 (0.80) (s, 9H, TBS), 0.08 (0.04) (s, 6H, TBS), 0.02 (-0.04) (s, 6H, TBS). 13C NMR (75 MHz, CDCl3) δ 166.7, 160.8 (d, *J*CF = 245.3 Hz), 146.5 (146.4), 146.3 (146.2), 135.7 (135.2), 129.8, 129.0, 126.8, 125.8 (125.6),125.1 (124.7), 124.3 119.2, 115.5 (d, *J*CF = 27.5 Hz), 95.0, 72.9, 72.3, 68.9, 66.8, 61.8, 61.7, 60.1, 56.1, 41.7 (41.2), 36.0 (34.2), 33.1, 32.3, 29.8, 27.0, 26.0, 25.9, 25.8, 24.9, 18.5, 18.1, 18.0, 16.6, 16.5. IR (film) νmax: 2928, 2856, 1714, 1636, 1250, 1020 cm<sup>−</sup>1. HRMS (ESI): *m*/*z* calculated for C43H77FO10PSi2 [M + H]+: 859.4777. Found: 859.4774.

#### *3.17. Synthesis of (2E,4Z)-(1S,2S,E)-1-(2-Fluorophenyl)-6-(3-hydroxypropoxy)-1-(methoxymethoxy)- 4-methylhex-4-en-2-yl 8-(diethoxyphosphoryl)-7-hydroxy-5-methylocta-2,4-dienoate [(17S,18S) diol* **26***]*

(17*S*,18*S*) Diol **26** was obtained as a colorless syrup in 65% yield according to the TBS deprotection procedure used for the conversion of (17*R*,18*R*) ester **23** to (17*R*,18*R*) diol **25**. 1H NMR (300 MHz, CD3COCD3) δ 7.63–7.55 (m, 1H, H-3), 7.52 (t, *J* = 7.8 Hz, 1H, aromatic H), 7.41–7.33 (m, 1H, aromatic H), 7.23 (t, *J* = 7.2 Hz, 1H, aromatic H), 7.14 (t, *J* = 9.0 Hz, 1H, aromatic H), 6.16 (d, *J* = 11.4 Hz, 1H, H-4), 5.80 (d, *J* = 15.0 Hz, 1H, H-2), 5.46 (quin, *J* = 5.1 Hz, 1H, H-17), 5.31 (t, *J* = 6.6 Hz, 1H, H-14), 5.10 (d, *J* = 5.7 Hz, 1H, H-18), 4.61 (d, *J* = 6.6 Hz, 1H, -O*CH2*OCH3), 4.51 (d, *J* = 6.6 Hz, 1H, -O*CH2*OCH3), 4.22–4.04 (overlapped, 5H, 2 × –O*CH2*CH3, H-7), 3.88 (d, *J* = 6.0 Hz, 2H, H2-13), 3.57 (t, *J* = 4.8 Hz, 2H, H2-11), 3.40 (t, *J* = 6.3 Hz, 2H, H2-9), 3.30 (s, 3H, -OCH2O*CH3*), 2.92 (br.s, 2H, 2 × OH), 2.60–2.57(overlapped, 2H, H2-16), 2.29 (d, *J* = 9.3 Hz, 2H, H2-8), 2.07–2.04 (m, 2H, H2-6), 1.95 (s, 3H, 5-CH3), 1.68 (quin, *J* = 6.0 Hz, H2-10), 1.65 (s, 3H, 15-CH3), 1.29 (t, *J* = 7.2 Hz, 6H, 2 <sup>×</sup> –OCH2*CH3*). 13C NMR (75 MHz, CDCl3) δ 166.0, 160.7 (d, *J*CF = 243.8 Hz), 147.0 (d, *J*CF = 9.8 Hz), 140.8, 134.2, 129.8, 129.3, 125.8 (d, *J*CF = 9.8 Hz), 125.6, 125.4 (d, *J*CF = 4.5 Hz), 124.3, 119.1, 115.3 (115.0), 94.7, 72.3, 66.8, 66.7, 65.5, 61.4(61.3), 61.2 (61.1), 59.1, 55.1, 41.2, 40.7, 34.6, 33.02(32.98), 32.0, 24.4, 15.91(15.86), 15.7(15.6). IR (film) νmax: 3381, 2927, 1709, 1633, 1488, 1019 cm−1. HRMS (ESI): *m*/*z* calculated for C31H49FO10P [M + H]+: 631.3048. Found: 631.3045.

#### *3.18. Synthesis of (2E,4Z)-(1S,2S,E)-1-(2-Fluorophenyl)-1-(methoxymethoxy)-4-methyl-6-(3-oxopropoxy) hex-4-en-2-yl 8-(diethoxyphosphoryl)-5-methyl-7-oxoocta-2,4-dienoate [ketoaldehyde (17S,18S)* **28***]*

The crude product was obtained from (17*S*,18S) diol **26** employing the oxidation procedure used for the synthesis of its antipode **27**. After being confirmed by its 1H NMR data, the crude product was directly used for the next step reaction without further purification. 1H NMR (300 MHz, CDCl3) δ 9.72 (t, *J* = 1.8 Hz, 1H, CHO), 7.41 (dt, *J* = 7.5, 1.8 Hz, 1H, aromatic H), 7.31 (dd, *J* = 15.0, 11.7 Hz, 1H, H-3), 7.29–7.22 (m, 1H), 7.11 (dt, *J* = 7.5, 0.9 Hz, 1H), 7.51 (ddd, *J* = 10.2, 8.1, 0.9 Hz, 1H), 6.17 (d, *J* = 11.1 Hz, 1H, H-4), 5.83 (d, *J* = 15.0 Hz, 1H, H-2), 5.45–5.38 (m, 1H), 5.31 (t, *J* = 5.7 Hz, 1H, H-14), 5.07 (d, *J* = 5.4 Hz, 1H), 4.57 (d, *J* = 6.9 Hz, 1H, O*CH2*OCH3), 4.50 (d, *J* = 6.9 Hz, 1 H, O*CH2*OCH3), 4.20–4.11 (m, 5H, 2 × O*CH2*CH3, H-7), 3.90 (t, *J* = 7.2 Hz, 2H, H2-13), 3.65 (t, *J* = 6.0 Hz, 2H, H2-11), 3.31 (s, 3H, OCH3), 3.10 (d, *J* = 22.8 Hz, 2H, H2-8), 2.39–2.19 (m, 2H, H2-10), 1.87 (s, 3H, 5-CH3), 1.63 (s, 3H, 15-CH3).

#### *3.19. Synthesis of (S,3E,9E,11Z,15E)-6-((S)-(2-Fluorophenyl)(methoxymethoxy)methyl)-4,12 dimethyl-1,7-dioxacyclooctadeca-3,9,11,15-tetraene-8,14-dione [(17S,18S) macrolide* **30***]*

(17*S*,18*S*) Macrolide **30** was synthesized according to the HWE ring closing procedure employed for the conversion of **27** to **29**. Colorless syrup, 68% yield for two steps. [α]: +8.1 (*c* = 0.22, MeOH). 1H NMR (300 MHz, CDCl3) δ 7.59 (dd, *J* = 15.0, 11.4 Hz, 1H, H-3), 7.46 (dt, *J* = 7.5, 1.8 Hz, 1H, aromatic H), 7.36–7.25 (m, 1H, aromatic H), 7.15 (dt, *J* = 7.5, 1.2 Hz, 1H, aromatic H), 7.04 (ddd, *J* = 9.9, 8.1, 1.2 Hz, 1H, aromatic H), 6.81 (dt, *J* = 15.9, 6.9 Hz, 1H, H-9), 6.11 (d, *J* = 11.4 Hz, 1H, H-4), 6.00 (d, *J* = 16.2 Hz, 1H, H-8), 5.90 (d, *J* = 15.0 Hz, 1H, H-2), 5.50 (dd, *J* = 11.4, 6.3 Hz, 1H, H-17), 5.26 (dd, *J* = 7.5, 4.5 Hz, 1H, H-14), 5.11 (d, *J* = 6.3 Hz, 1H, H-18), 4.59 (d, *J* = 6.9 Hz, 1H,–O*CH2*OCH3), 4.52 (d, *J* = 6.6 Hz, 1H, –O*CH2*OCH3), 3.98 (dd, *J* = 12.0, 8.1 Hz, 1H, H-16), 3.88 – 3.81 (overlapped, 2H, H2-13), 3.46 – 3.31 (overlapped, 2H, H2-11), 3.33 (s, 3H, O*CH3*), 3.14 (d, *J* = 12.6 Hz, 1H, H-16), 2.42–2.35 (m, 2H, H2-10), 2.33 (d, *J* = 11.7 Hz, 1H, H-6), 2.04 (d, *J* = 11.7 Hz, 1H, H-6), 1.82 (s, 3H, 5-*CH3*), 1.63 (s, 3H, 15-*CH3*). 13C NMR (75 MHz, CDCl3) δ 197.1, 166.6, 160.9 (d, *J*CF = 244.5 Hz), 146.8, 142.5, 139.8, 134.3, 130.2, 129.9, 129.0, 125.9 (d, *J*CF = 7.5 Hz), 125.5 (d, *J* = 13.5 Hz), 125.2 (d, *J*CF = 4.5 Hz), 124.5, 120.9 (d, *J*CF = 3.8 Hz), 115.6 (d, *J*CF = 21.0 Hz), 94.9, 72.9, 72.6, 67.9, 67.8, 56.0, 45.9, 41.4, 33.1, 24.1, 16.7. IR (film) νmax: 2925, 2854, 1714, 1669, 1634, 1489, 1359, 1280 cm<sup>−</sup>1. HRMS (ESI): *m*/*z* calculated for C27H34FO6 [M + H]+: 473.2339. Found: 473.2332.

#### *3.20. Synthesis of (S,3E,9E,11Z,15E)-6-((S)-(2-Fluorophenyl)(hydroxy)methyl)-4,12-dimethyl-1,7-dioxacyclooctadeca-3,9,11,15-tetraene-8,14-dione [(17S,18S) zampanolide mimic* **6***]*

(17*S*,18*S*) Zampanolide mimic **6** was obtained according to the MOM deprotection procedure used for the conversion of (17*R*,18*R*) macrolide **29** to (17S,18S) zampanolide mimic **5**. Colorless syrup, 45% yield. [α]: +15.6 (*c* = 0.15, MeOH). 1H NMR (300 MHz, CD3COCD3) δ 7.61 (dd, *J* = 15.0, 11.1 Hz, 1H, H-3), 7.34 (dd, *J* = 14.4, 7.2 Hz, 1H, aromatic H), 7.21 (t, *J* = 7.5 Hz, 1H, aromatic H), 7.10 (dd, *J* = 10.5, 8.1 Hz, 1H, aromatic H), 6.83 (dt, *J* = 16.2, 6.3 Hz, 1H, H-9), 6.20 (d, *J* = 11.1 Hz, 1H, H-4), 6.06 (d, *J* = 15.9 Hz, H-8), 5.88 (d, *J* = 12.6 Hz, 1H, H-2), 5.44 (dd, *J* = 11.1, 5.4 Hz, 1H, H-17), 5.27 (t, *J* = 5.4 Hz, 1H, H-14), 5.17 (t, *J* = 5.4 Hz, 1H, H-18), 4.81 (d, *J* = 5.4 Hz, 1H, OH), 4.07–3.97 (m, 1H, H-16), 3.90 (d, *J* = 12.9 Hz, 1H, H-13), 3.83 (dd, *J* = 12.3, 4.5 Hz, 1H, H-13), 3.48– 3.32 (m, 2H, H2-11), 3.17 (d, *J* = 12.6 Hz, 1H, H-16), 2.42–2.33 (overlapped, 3H, H2-10, H-6), 2.19 (d, *J* = 13.8 Hz, 1H, H-6), 1.81 (s, 3H, 5-*CH3*), 1.64 (s, 3H, 15-*CH3*). 13C NMR (75 MHz, CD3COCD3) δ 196.5, 166.5, 160.5 (d, *J*CF = 242.3 Hz), 146.6, 142.7, 139.8, 134.7, 130.6, 129.7, 129.5, 126.3, 126.2, 125.7, 124.8, 121.7, 115.6, 73.8, 69.0, 68.9, 68.2, 45.8, 41.6, 33.2, 23.8, 16.3. IR (film) νmax: 3447, 2916, 2855, 1710, 1669, 1633, 1489, 1456, 1359, 1281, 1148 cm<sup>−</sup>1. HRMS (ESI): *m*/*z* calculated for C25H30FO5 [M + H]+: 429.2077. Found: 429.2073.

#### *3.21. Cell Culture*

All cell lines were initially purchased from American Type Culture Collection (ATCC). The PC-3, PC-3/DTX, and LNCaP prostate cancer cell lines were routinely cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. Cultures were maintained in a high humidity environment supplemented with 5% carbon dioxide at a temperature of 37 ◦C. The DU145 and DU145/DTX prostate cancer cells were routinely cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% FBS and 1% penicillin/streptomycin.

The procedure illustrated in the literature [29,30] was adapted to establish docetaxel-resistant prostate cancer cell lines. Specifically, docetaxel-resistant DU145 and PC-3 cell lines (DU145/DTX and PC-3/DTX) were developed over a period of one year by stepwise increased concentrations of docetaxel. Cells were repeatedly conserved in an appropriate concentration of docetaxel, starting with IC50 value of the respective parent cell lines. Docetaxel-containing media will be replaced every 2–3 days. The concentration of docetaxel was increased when cells exhibited resistance to treatments.

#### *3.22. WST-1 Cell Proliferation Assay*

PC-3, PC-3/DTX, DU145, DU145/DTX, or LNCaP cells were placed in 96-well plates at a density of 3200 cells each well in 200 μL of culture medium. The cells were then treated with synthesized mimics, or positive reference separately at different doses for 3 days, while equal treatment volumes of DMSO were used as vehicle control. After the cells were cultured in a CO2 incubator at 37 ◦C for three days, the premixed WST-1 cell proliferation reagent (10 μL, Clontech) was added to each well. The cells were incubated for additional 3 h at 37 ◦C before mixing gently for one minute on an orbital shaker to ensure homogeneous distribution of color. A microplate-reader (Synergy HT, BioTek) was used to measure the absorbance of each well at a wavelength of 430 nm. The half-maximal inhibitory concentration (IC50 value) is the concentration of each compound that inhibits cell proliferation by 50% under the experimental conditions. Each of IC50 value is represented as the average from triplicate determinations that were reproducible and statistically significant. The IC50 values were calculated based on dose–response curves from at least five dosages for each compound.

#### *3.23. Statistical Analysis*

All data are represented as the mean ± standard deviation (S.D.) for the number of experiments indicated. Other differences between treated and control groups were analyzed using the Student's t-test. A *p*-value < 0.05 was considered statistically significant.

#### **4. Conclusions**

In summary, two new zampanolide mimics have been designed and each of them has been achieved through a 25-step chemical transformation starting from commercially available 2-butyn-1-ol. Yamaguchi esterification and intramolecular Horner–Wadsworth–Emmons condensation were employed as crucial reactions to build up the C-17 and C-1 ester moiety and to close the lactone ring. The chiral centers at C17 and C18 in the Fragment C9–C18 (**7** and **8**) were introduced by the Sharpless asymmetric dihydroxylation using the commercially available AD-mix formulations. Our WST-1 cell proliferation assay data in both docetaxel-resistant and docetaxel-sensitive prostate cancer cell lines revealed that compound **6** is the optimal mimic and the newly designed side chain can act as a bioisostere for the chemically fragile *N*-acetyl hemiminal side chain in zampanolide.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1420-3049/25/2/362/s1, NMR spectra (1H and 13C) of the zampanolide mimics **5** and **6**, as well as the macrolides **30** and **31,** and intermediates **7–8, 14–21,** and **23–26**.

**Author Contributions:** Conceptualization and design, Q.C.; experimentation and data analysis, G.C., Z.J., Q.Z., G.W., and Q.-H.C.; funding acquisition, Q.-H.C. and G.W.; all authors finally reviewed and approved the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Institutes of Health (National Institute of General Medical Sciences) under Award Number SC2GM121185 (Q. Chen). HRMS were supported by the National Institutes of Health RCMI program at Xavier University of Louisiana through Grant 2G12MD007595 (G. Wang).

**Acknowledgments:** We thank the Department of Chemistry and College of Science and Mathematics at CSU-Fresno for all administrative support.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

#### **References**


**Sample Availability:** Samples of the compounds **5, 6, 29 and 30** are available from the authors.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Synthesis and Antiproliferative Screening Of Novel Analogs of Regioselectively Demethylated Colchicine and Thiocolchicine**

**Dominika Czerwonka 1, Szymon Sobczak 2, Ewa Maj 3, Joanna Wietrzyk 3, Andrzej Katrusiak <sup>2</sup> and Adam Huczy ´nski 1,\***


Academic Editor: Qiao-Hong Chen Received: 29 January 2020; Accepted: 3 March 2020; Published: 5 March 2020

**Abstract:** Colchicine, a pseudoalkaloid isolated from *Colchicum autumnale*, has been identified as a potent anticancer agent because of its strong antimitotic activity. It was shown that colchicine modifications by regioselective demethylation affected its biological properties. For demethylated colchicine analogs, 10-demethylcolchicine (colchiceine, **1**) and 1-demethylthiocolchicine (**3**), a series of 12 colchicine derivatives including 5 novel esters (**2b**–**c** and **4b**–**d**) and 4 carbonates (**2e**–**f** and **4e**–**f**) were synthesized. The antiproliferative activity assay, together with in silico evaluation of physicochemical properties, confirmed attractive biological profiles for all obtained compounds. The substitutions of H-donor and H-acceptor sites at C1 in thiocolchicine position provide an efficient control of the hydration affinity and solubility, as demonstrated for anhydrate **3**, hemihydrate **4e** and monohydrate **4a**.

**Keywords:** colchicine analogs; thiocolchicine; colchiceine; antimitotic agents; antiproliferative activity; hydrates

#### **1. Introduction**

Colchicine (Figure 1) is a pseudoalkaloid produced by *Colchicum autumnale* [1]. Its beneficial applications have been known for centuries, as colchicine was used by ancient Greeks and Egyptians to treat swelling and inflammation [2]. Presently, colchicine is considered as the first-line therapy for gout, pericarditis and familial Mediterranean fever [1,3–5]. The most interesting property of colchicine is its high affinity for binding to the tubulin in cancer cells, leading to the inhibition of microtubule polymerization and mitosis arrest [6–8]. These unique properties have attracted great interest to colchicine as an anticancer-drug candidate, however its use is limited by its toxicity towards normal cells [9–11]. For this reason, new derivatives obtained through chemical modifications guided by structure–activity relations are intensively investigated. This research is aimed at reducing the colchicine toxicity and preserving the antimitotic and anticancer properties [12–24]. As it turns out, tubulin interacts with trimethoxyphenyl ring A and tropolone ring C, making the methoxy groups at C1, C2 and C10, as well as the C9-keto group, crucial for colchicine's antimitotic activity [25–27]. Unfortunately, colchicine is also prone to isomerization, resulting in isocolchicine (Figure 1) with 500 times lower affinity for binding to tubulin than colchicine itself [28].

**Figure 1.** Structures of colchicine, lumicolchicine, isocolchicine, colchiceine, isocolchiceine and thiocolchicine.

Colchiceine (Figure 1) is a C10 demethylated colchicine analog characterized by much lower toxicity [29,30]. It is, next to lumicolchicine (Figure 1), the main product of colchicine degradation and can naturally occur in plant extracts together with colchicine [31]. Elguero et al. [32] have established that colchiceine (as well as its C10-acylated analog) can occur in two tautomeric forms, from which the isoform is slightly more dominant. This equilibrium between colchiceine and isocolchiceine (Figure 1) exists also in the solid state, which has been confirmed by X-ray diffraction studies [33]. The tendency of colchiceine towards tautomerization, in combination with the loss of the C10-methoxy group, may be responsible for its poor antimitotic activity. In contrast, colchiceine shows better bacteriostatic and antifungal activity than colchicine, because its free hydroxyl group may increase its binding affinity towards the cell walls of pathogens [29,33].Interestingly, the replacement of the C10-methoxy group with thiomethyl in a colchicine analog thiocolchicine (Figure 1) decreases its isomerization. Thiocolchicine is more stable, exhibits lower toxicity and has a higher binding affinity to tubulin [34–36]. As could be expected, demethylation of thiocolchicine at C1/C2/C3 positions leads to a lower binding ability to tubulin and lower toxicity [37–39]. As the removal of the methoxy groups from colchicine's structure reduces its toxicity but also lowers its binding affinity to tubulin, we decided to replace these groups with a bigger substituent, such as an ester or carbonate moiety, to investigate an effect of this modification on biological activity [40].

For this reason, we carried out a series of regioselective demethylation reactions of colchicine and thiocolchicine, in order to to obtain colchiceine (**1**) and 1-demethylthiocolchicine (**3**). On the basis of **1** and **3**, we synthesized a series of 12 mono- and double-modified colchicine analogs. Within these analogs, we present 5 novel esters (**2b**–**c** and **4b**–**d**) and 4 novel carbonates (**2e**–**f** and **4e**–**f**). The pathways used for these syntheses are depicted in Scheme 1.

We also evaluated the antiproliferative activity of these derivatives using three human cancer cell lines, human lung adenocarcinoma (A549), human breast adenocarcinoma (MCF-7) and human colon adenocarcinoma cell line (LoVo), and one normal murine embryonic fibroblast cell line (BALB/3T3). Moreover, on the basis of the in silico calculations, we were able to predict the physicochemical properties of the obtained compounds and check their compliance with Lipi ´nski's rule of 5, permitting evaluation of its drug-likeness.

#### **2. Results and Discussion**

#### *2.1. Chemistry*

A series of colchicine esters and carbonates substituted at C1 and C10 positions in colchicine were synthesized by performing regioselective demethylation reactions of C1 and C10 methoxy groups, respectively. At first, we performed regioselective demethylation of the colchicine C10 methoxy group by treating colchicine with a mixture of glacial acetic acid and 0.1N hydrochloric acid (i). Reaction resulted in precipitation of yellow solid [41], which was further recrystallized in acetone, leading to growth of good quality, large single crystals. Performed X-ray diffraction analysis proved them to be a colchiceine (**1**, Scheme 1) hemihydrate of the same structure as that reported by Mackay et al. [33], in which the unit-cell accommodates two tautomeric forms: colchiceine and isocolchiceine.

**Scheme 1.** Synthesis of novel colchicine analogs. The obtained crystal structure of **1** was identical to the one proposed by Mackey et al. [33]. Reagents and conditions: (i) glacial acetic acid, hydrochloric acid, 100 ◦C, 6h; (iii) MeOH/H2O, CH3SNa, RT, 24h; (iv) DCM, acetyl chloride, SnCl4, 0 ◦C → RT, 48h; (v) MeOH/H2O, LiOH, RT, 1h; (ii,vi) DCM, Et3N, respective acyl chloride/chloroformate, 0 ◦C → RT, 24h.

Colchicine was also treated with sodium methanethiolate (**iii**) to obtain thiocolchicine [42]. By using a slightly modified method proposed by Bladé-Font [43] (**iv**,**v**), thiocolchicine was regioselectively demethylated at the C1 methoxy group, leading to 1-demethylthiocolchicine (**3**) with 40% yield. This regioselective demethylation was confirmed by X-ray analysis (Figure 2). Finally, compounds **1** and **3** were treated with respective acyl chlorides or chloroformates (**ii**, **vi**) to give single modified derivatives **2a**–**f** and double modified derivatives **4a**–**f** with 38%–58% yields (Scheme 1).

**Figure 2.** (**a**) Molecule of 1-demethylthiocolchicine (**3**) and (**b**) its crystal structure projected along the crystal direction [100].

The purity and structure of synthesized colchicine derivatives **1**, **2a**–**f**, **3** and **4a**–**f** were determined on the basis of ESI-MS, NMR and X-ray analysis. The specific data can be found in Section 3 and in Supplementary Materials. The disappearance of the signal at about 56.5 ppm in the 13C NMR spectrum of colchicine, assigned to the C10 methoxy group, is the evidence of the formation of colchiceine (**1**). Moreover, in the spectra of derivatives **2a**–**2f**, an additional signal from the carbonyl group can be observed in the range 164.1–174.3 ppm for esters **2a**–**2d**, and 151.9–152.5 ppm for carbonates **2e**–**f**. A shift of the signal corresponding to the C10 methoxy group from 56.5 to 15.1 ppm is evidence for the introduction of the thiomethyl group at position C10, and the absence of one of the signals around 61 ppm assigned to the C1 methoxy group is evidence of 1-demethylthiocolchicine (**3**) formation. An additional signal corresponding to the carbonyl carbon atom can be observed in the range 168.3–174.7 ppm for esters **4a**–**4d** and in the range 152.8–152.9 ppm for carbonates **4e**–**f**.

#### *2.2. In Silico Calculations of the Physicochemical Properties*

We used the Molinspiration online database (http://www.molinspiration.com, free of charge) to predict the physicochemical properties of all synthesized compounds and collected them in Table 1 [44].

**Table 1.** Physicochemical properties of the synthesized compounds based on the Molinspiration database [44].


MW: Molecular weight; clog*P*: calculated log octanol/water partition coefficient; tPSA: total polar surface area; n(O,N): number of hydrogen acceptors; n(OH,NH): number of hydrogen donors; Rotb: rotatable bonds; MV: molecular volume.

According to Lipinski's rule of 5, most "drug-like" molecules should have log*P* ≤ 5, molecular weight ≤ 500, number of hydrogen bond acceptors ≤ 10 and number of hydrogen bond donors ≤ 5. Additionally, it was demonstrated that a total polar surface area (tPSA) < 140 and number of rotatable bonds < 10, in combination with log*P* < 5, are crucial for both good bioavailability and permeability through biological membranes [45].

The demethylation of the C1 and C10 position of colchicine, both in **1** and **3**, increases the number of H-donor sites while the substitution of colchicine's C10 methoxy group with thiomethyl results in a decreased number of H-acceptor sites in **3**. Additionally, the demethylation of the C1 and C10 position of colchicine reduces the number of rotatable bonds. Substitution of a hydroxyl group, both in **1** and **3**, with an ester or carbonate substituent, increases the number of H-acceptor sites and reduces the number of H-donor sites to that of colchicine. According to the performed calculations, all synthesized compounds **2a**–**e** and **4a**–**e** have a rather lipophilic character, with the clog*P* values between 0.60 and 3.98. Conducted syntheses allowed us to obtain compounds with less lipophilic character than that of colchicine for compounds **1**, **2a** and **2e**, with clog*P* values 0.83, 060 and 0.95, respectively. Interestingly, esters and carbonates in the C10 position (**2a**–**e**) are in general less lipophilic than corresponding esters and carbonates in the C1 position (**4a**–**e**). Except for **4d**, whose molecular weight is above 500, the calculations for all colchicine analogs are in agreement with Lipinski's descriptors. Importantly, in order to be active, a drug should not have more than one violation [46]. This, in combination with the number of rotatable bonds between 4 and 8, as well as tPSA between 84.86 and 100.18, confirm good physicochemical profiles of all obtained compounds.

#### *2.3. X-ray Analysis*

The structures of compounds **3**, **4a** and **4e** were additionally confirmed by X-ray diffraction. Selected crystallographic data are collected in Table 2, and more detailed information can be found in Supplementary Information. The single crystals of **3**, **4a**, **4e** were obtained by slow evaporation from an ethyl acetate mixture. The 1-Demethylthiocolchicine (**3**) crystallizes in the orthorhombic space group *P*212121 (Figure 2) while the crystals of **4a** and **4e** are monoclinic, space group *P*21. The substitution

at the C1 atom with acetyl ester (**4a**) and methyl carbonate (**4e**), and the following change in the ratio between H-bond acceptors and donors, increases the tendency of these colchicine derivatives to co-crystallize with water; hence, monohydrate **4a**·H2O and hemihydrate **4e**·**1/2**H2O were obtained.

Molecules **4a** and **4e** significantly differ in conformation (Figure 3).The arrangement of the C2 methoxy group, which can be described by the C1-C2-O2-C18 torsion angle, changed from −122.36◦ to 108.75◦ in **4a** and **4e**, respectively. Together with increasing mass of the C1 moiety, the twist between the planar phenyl and tropolone rings around the C13-C16 bond increases, altering the C1-C16-C13-C12 angle from 45.43◦ in **3**, through 58.54◦ in **4a**, to 60.11◦ in **4e**.

**Figure 3.** Comparison of the molecular conformation from the X-ray analysis studies between (**a**) molecule **4a** and (**b**) **4e**, superimposed with respect to the benzene ring on the 1-demethylthiocolchicine (molecule **3**), highlighted with a light-green color.


**Table 2.** Selected crystallographic data for compounds **3**, **4a**·**H2O** and **4e**·**1/2H2O**.

The presence of water significantly alters the pattern of H-bonds between **3** and **4a**·**H2O** and **4e**·**1/2H2O**. Although in all structures amide nitrogen participates in the formation of intermolecular contacts, only in **3** are the NH···S, of 2.793(2) Å, and NH···O4, of 2.370(3) Å,bonds formed. In a monohydrate crystal **4a**·**H2O**, water interrupts the NH···O4 interaction observed in the crystal structure of **3**, accepting NH amide proton, of 2.086(1) Å and further donating its H-atoms to the HO-H···O4 , of 2.007(3) Å, and HO-H···O5 , of 1.994(3) Å bond. Much weaker H-bonds are present in the crystal structure of **4e**·**1/2H2O**. Despite the presence of a water molecule in the crystal structure of **4e**·**1/2H2O**, the strong NH···O5 bond, of 2.033(2) Å is present again. The disordered by location at half-occupied sites water molecule is involved in formation of a weak HO-H···O5 ' interaction of 2.538(1) Åand the H-bond at the disordered terminal carbonyl at C9. Due to the HO-H···O4 of 2.241(1) Å bond, the occupational disorder of H2O is passed onto the orientation of **4e** molecules as illustrated in Figure 3. Depending on the presence or absence of this water molecule, its adjacent molecule **4e** is slightly shifted in the crystal structure. This disorder is most apparent for the terminal carbonyl at C9 (dislocated by 0.358 Å) and thiomethyl at C10 (dislocated by 0.625 Å).

#### *2.4. Antiproliferative Activity*

The antiproliferative activity of all synthesized colchicine analogs **1**, **2a**–**f**, **3**, **4a**–**f** was *tested in vitro* against three human cancer cell lines: human lung adenocarcinoma (A549), human breast adenocarcinoma (MCF-7) and human colon adenocarcinoma cell line (LoVo). For a more accurate evaluation of cytotoxic activity, the effect on normal murine embryonic fibroblast cell line (BALB/3T3) was also tested according to the previously published procedure [12]. Detailed information concerning antiproliferation assay can be found in Supplementary Materials. The mean values of IC50 ± SD of the tested compounds are collected in Table 3.

**Table 3.** Antiproliferative activity (IC50) of 10-demethylcolchicine (colchiceine, **1**), 1-demethylthiocolchicine (**3**) and their derivatives(**2a**–**f**, **4a**–**f**) compared to the antiproliferative activity of colchicine and standard anticancer drugs doxorubicin and cisplatin and the calculated values of selectivity index (SI).


The IC50 value is defined as the concentration of a compound at which 50% growth inhibition is observed. Human lung adenocarcinoma (A549), human breast adenocarcinoma (MCF-7), human colon adenocarcinoma cell line (LoVo) and normal murine embryonic fibroblast cell line (BALB/3T3). The SI (Selectivity Index) was calculated for each compound, using the formula SI = IC50 for normal cell line BALB/3T3/IC50 for the respective cancerous cell line. A beneficial SI > 1.0 indicates a drug with efficacy against tumor cells greater than the toxicity against normal cells.

All colchicine analogs showed stronger antiproliferative activity against all tested cancer cell lines than the conventional chemotherapeutic, cisplatin. In general, the IC50 values are better for colchicines doubly modified at C1 and C10 positions (**3**, **4a**–**f**) than for the analogs singly modified at position C10 only (**1**, **2a**–**f**). Compound **4a**, with a thiomethyl group at position C10 and an acetyl ester substituent at C1, showed the highest activity against all tested cell lines (IC50 = 0.48 ± 0.15, 0.10 ± 0.02, 0.11 ± 0.02 μM for A549, MCF-7, LoVo cancer cell lines, respectively).

All tested colchicine analogs exhibit higher SI values than doxorubicin, which indicates the therapeutic potential of the synthesized compounds. Compound **3**, 1-demethylthiocolchicine, as well as its acylated analog **4a**, showed high SI values against MCF-7 (SI = 4.92, SI = 6.88) as well as LoVo cell lines (SI = 5.36, SI = 5.95). From among analogs modified exclusively at position C10, compound **2b** with propionyl ester substituent revealed a higher SI value towards LoVo cell line (SI = 3.13).

#### **3. Materials and Methods**

#### *3.1. General*

Information concerning reagents and solvents, as well as equipment used for measurements, can be found in the Supplementary Materials.

3.1.1. Synthesis and Characterization of 10-Demethylcolchicine (colchiceine, 1), Thiocolchicine and 1-Demethylthiocolchicine (3)

Information concerning the synthesis of 10-demethylcolchicine (colchiceine, **1**), thiocolchicine and 1-demethylthiocolchicine (**3**) can be found in Supplementary Materials.

#### 3.1.2. General Route for Synthesis of Compounds 2a–f and 4a–f

To a solution of **1** or **3** (100 mg, 0.25 mmol) in dichloromethane (DCM, 10 mL) cooled to 0 ◦C, Et3N (1 mL, 7 mmol) and respective acyl chloride or chloroformate were added (excess). The mixture was first stirred at 0 ◦C for 30 min and then for the next 24h at RT. Reaction time was determined by TLC. After that time the solution was filtered to remove triethylamine hydrochloride, the DCM was evaporated under reduced pressure and the residue was purified by CombiFlash® (chloroform/acetone, increasing concentration gradient) to give respective compounds as amorphous yellow solids.

#### 3.1.3. Characterization of Acetyl Ester of Colchiceine 2a

Amorphous yellow solid, yield 49%, m.p 120–122 ◦C; 1H NMR (CDCl3, 400 MHz) δ 7.50 (1H, s), 7.31 (1H, d, *J* = 11.3 Hz), 7.22 (1H, d, *J* = 11.3 Hz), 6.53 (1H, s), 4.60 (1H, dt, *J* = 12.7, 6.5 Hz), 3.91 (3H, s), 3.88 (3H, s), 3.66 (3H, s, 2.50 (1H, dd, J = 13.6, 6.3 Hz), 2.35 (3H, s), 2.26–2.17 (1H, m), 1.96 (3H, s), 1.87 (1H, td, *J* = 12.1, 6.9 Hz). 13C NMR (CDCl3, 101 MHz) δ 169.9, 168.2, 153.9, 151.0, 148.6, 141.8, 141.5, 137.0, 134.5, 131.2, 130.3, 125.2, 107.4, 61.6, 61.3, 56.0, 52.1, 36.8, 29.7, 22.7, 20.7. ESI-MS (m/z): [M + H]<sup>+</sup> 428, [M + Na]<sup>+</sup> 450, [M + K]<sup>+</sup> 466, [2M + Na]<sup>+</sup> 877.

#### 3.1.4. Characterization of Propionyl Ester of Colchiceine 2b

Amorphous yellow solid, yield 52%, m.p 133-135 ◦C; 1H NMR (CDCl3, 400 MHz) δ 7.50 (1H, s), 7.30 (1H, d, *J* = 11.3 Hz), 7.22 (1H, d, *J* = 11.2 Hz), 6.52 (1H, s), 4.66–4.57 (1H, m), 3.91 (3H, s), 3.88 (3H, s), 3.66 (3H, s), 2.66 (2H, q, *J* = 7.4 Hz), 2.50 (1H, dd, *J* = 13.5, 6.3 Hz), 2.35 (1H, ddd, *J* = 12.7, 10.3, 6.0 Hz), 2.24–2.16 (1H, m), 1.97 (3H, s), 1.87 (2H, td, *J* = 12.0, 6.7 Hz), 1.26 (3H, t, *J* = 7.6 Hz). 13C NMR (CDCl3, 101 MHz) δ 171.8, 169.9, 153.9, 151.1, 148.9, 141.7, 141.6, 136.7, 134.5, 130.9, 130.5, 125.2, 107.4, 61.7, 61.4, 56.1, 52.1, 36.9, 29.8, 27.4, 22.8, 9.0. ESI-MS (m/z): [M + Na]<sup>+</sup> 464, [2M + Na]<sup>+</sup> 905.

#### 3.1.5. Characterization of Isobutyryl Ester of Colchiceine 2c

Yellowish brown oil, yield 55%; 1H NMR (CDCl3, 400 MHz) δ 7.56 (1H, s), 7.28 (1H, d, *J* = 11.0 Hz), 7.23 (1H, d, *J* = 10.6 Hz), 6.52 (1H, s), 4.64 (1H, dt, *J* = 12.2, 6.8 Hz), 3.91 (3H, s), 3.88 (3H, s), 3.65 (3H, s), 2.90–2.82 (1H, m), 2.54–2.47 (1H, m), 2.36 (1H, td, *J* = 13.2, 6.9 Hz), 2.24–2.12 (2H, m), 1.96 (3H, s), 1.92–1.85 (1H, m), 1.35 (3H, d, *J* = 1.4 Hz), 1.33 (3H, d, *J* = 1.3 Hz). 13C NMR (CDCl3, 101 MHz) δ 174.3, 170.0, 153.9, 151.1, 150.3, 142.1, 141.5, 136.0, 134.5, 131.6, 130.1, 125.2, 107.4, 61.7, 61.3, 56.0, 52.1, 36.6, 34.0, 29.8, 26.4, 22.7, 19.0. ESI-MS (m/z): [M + Na]<sup>+</sup> 478, [2M + Na]<sup>+</sup> 933.

### 3.1.6. Characterization of Benzyl Ester of Colchiceine 2d

Amorphous yellow solid, yield 41%, m.p 123–125 ◦C; 1H NMR (CDCl3, 400 MHz) δ 8.18 (2H, d, *J* = 7.4 Hz), 7.65–7.60 (2H, m), 7.49 (1H, ddd, *J* = 4.8, 3.1, 1.1 Hz), 7.34 (2H, s), 6.53 (1H, s), 4.66–4.58 (1H, m), 3.93 (3H, d, *J* = 0.9 Hz,), 3.90 (3H, d, *J* = 0.7 Hz), 3.68 (3H, d, *J* = 1.1 Hz), 2.49 (1H, dd, *J* = 13.4, 4.9 Hz), 2.42–2.32 (1H, m), 2.17–2.06 (1H, m), 1.86 (3H, d, *J* = 4.0 Hz), 1.84–1.78 (1H, m). 13C NMR

(CDCl3, 101 MHz) δ 169.9, 164.1, 153.9, 151.1, 142.0, 141.6, 134.5, 133.8, 130.4, 128.9, 128.5, 125.3, 107.4, 61.7, 61.4, 56.1, 52.0, 36.9, 29.8, 22.7. ESI-MS (m/z): [M + H]<sup>+</sup> 490, [M + Na]<sup>+</sup> 512, [M + K]<sup>+</sup> 528.

#### 3.1.7. Characterization of Methyl Carbonate of Colchiceine 2e

Amorphous yellowish brown solid, yield 58%, m.p 115–118 ◦C; 1H NMR (CDCl3, 400 MHz) δ 7.57 (1H, s), 7.32 (1H, d, *J* = 20.5 Hz), 6.54 (1H, s), 4.62 (1H, dt, *J* = 11.8, 6.0 Hz), 3.92 (3H, s)\*, 3.92 (3H, s)\*, 3.89 (3H, s), 3.68 (3H, s), 2.52 (1H, dd, *J* = 13.4, 6.1 Hz), 2.37 (1H, td, *J* = 13.0, 6.7 Hz), 2.29–2.18 (1H, m), 1.99 (3H, s), 1.91 (1H, dd, *J* = 19.0, 12.2 Hz). 13C NMR (CDCl3, 101 MHz) δ 169.9, 154.1, 152.5, 151.1, 141.6, 134.5, 125.1, 107.4, 61.7, 61.3, 56.1, 55.8, 52.2, 37.2, 29.8, 22.8. ESI-MS (m/z): [M + H]<sup>+</sup> 444, [M + Na]<sup>+</sup> 466, [M + K]<sup>+</sup> 482, [2M + Na]<sup>+</sup> 909.

#### 3.1.8. Characterization of Ethyl Carbonate of Colchiceine 2f

Yellowish brown oil, yield 55%; 1H NMR (CDCl3, 400 MHz) δ 7.46 (1H, s), 7.40–7.32 (1H, m, *J* = 6.2 Hz), 7.30–7.21 (1H, m), 6.55 (1H, s), 4.62 (1H, dt, *J* = 12.6, 6.4 Hz), 4.36 (2H, q, *J* = 7.1 Hz), 3.93 (3H, s, *J* = 2.9 Hz), 3.90 (3H, s), 3.68 (3H, s), 2.54 (1H, dd, *J* = 13.6, 6.4 Hz), 2.41 (1H, td, *J* = 13.1, 7.1 Hz), 2.32–2.21 (1H, m), 2.01 (3H, s), 1.94–1.84 (1H, m), 1.41 (3H, t, *J* = 7.1 Hz). 13C NMR (CDCl3, 101 MHz) δ 169.9, 154.0, 151.9, 151.1, 141.6, 134.5, 125.1, 107.4, 65.4, 61.7, 61.3, 56.1, 52.1, 37.2, 29.8, 22.8, 14.1. ESI-MS (*m*/*z*): [M + H]<sup>+</sup> 458, [M + Na]<sup>+</sup> 480, [M + K]<sup>+</sup> 496, [2M + Na]<sup>+</sup> 937.

#### 3.1.9. Characterization of Acetyl Ester of 1-demethylthiocolchicine 4a

Amorphous yellow solid, yield 45%, m.p 168-172 ◦C; 1H NMR (DMSO-d6, 400 MHz) δ 8.51 (1H, d, *J* = 7.7 Hz), 7.23 (1H, d, *J* = 10.8 Hz), 7.02 (1H, s), 6.97 (1H, s), 6.92 (1H, d, *J* = 10.3 Hz), 3.89 (3H, s), 3.74 (3H, s), 2.65 (1H, dd, *J* = 13.5, 6.0 Hz), 2.41 (3H, s), 2.32–2.24 (1H, m), 2.22 (3H, s), 2.12–2.01 (1H, m), 1.85 (3H, s, *J* = 6.3 Hz), 1.84–1.77 (1H, m, *J* = 12.1, 6.8 Hz). 13C NMR (DMSO-d6, 101 MHz) δ 181.2, 168.9, 168.2, 157.7, 152.8, 150.4, 140.8, 139.3, 136.4, 134.4, 132.7, 128.2, 126.4, 124.6, 109.9, 60.1, 56.0, 50.7, 36.2, 29.1, 22.5, 20.3, 14. ESI-MS (m/z): [M + Na]<sup>+</sup> 466, [2M + Na]<sup>+</sup> 909.

3.1.10. Characterization of Propionyl Ester of 1-demethylthiocolchicine 4b

Amorphous yellow solid, yield 47%, m.p 136-140 ◦C; 1H NMR (DMSO-d6, 400 MHz) δ 8.52 (1H, d, *J* = 7.7 Hz), 7.23 (1H, d, *J* = 10.7 Hz), 7.03 (1H, s), 6.98 (1H, s), 6.92 (1H, d, *J* = 10.2 Hz), 4.34 (1H, dt, *J* = 11.8, 7.1 Hz), 3.90 (3H, s), 3.76–3.71 (3H, m), 2.66 (1H, dd, *J* = 13.2, 6.4 Hz), 2.41 (3H, s), 2.33–2.19 (2H, m), 2.08 (2H, ddd, *J* = 11.4, 9.8, 5.4 Hz), 1.84–1.78 (1H, m), 1.11 (3H, t, *J* = 7.5 Hz). 13C NMR (DMSO-d6, 101 MHz) δ 181.2, 172.3, 168.3, 157.7, 152.8, 150.5, 140.9, 139.2, 136.4, 134.4, 132.9, 128.3, 126.4, 124.7, 109.9, 60.1, 56.0, 50.7, 36.2, 29.1, 26.7, 22.6, 14.5, 9.2. ESI-MS (m/z): [M + H]<sup>+</sup> 458, [M + Na]<sup>+</sup> 480, [M + K]<sup>+</sup> 496, [2M + Na]<sup>+</sup> 937.

#### 3.1.11. Characterization of Isobutyryl Ester of 1-demethylthiocolchicine 4c

Amorphous yellow solid, yield 49%, m.p 165-167 ◦C; 1H NMR (DMSO-d6, 101 MHz) δ 8.50 (1H, d, *J* = 7.8 Hz), 7.22 (1H, d, *J* = 10.6 Hz), 7.03 (1H, s), 6.98 (1H, s), 6.91 (1H, d, *J* = 8.8 Hz), 4.35 (1H, dt, *J* = 11.7, 7.3 Hz), 3.90 (3H, s), 3.72 (3H, s), 2.74 (1H, dt, *J* = 13.9, 6.9 Hz), 2.66 (1H, dd, *J* = 13.6, 6.1 Hz), 2.39 (3H, s), 2.34–2.25 (1H, m), 2.09 (1H, td, *J* = 12.7, 5.9 Hz), 1.86 (3H, s), 1.81 (1H, dd, *J* = 12.0, 6.9 Hz, 1.16 (3H, d, *J* = 3.5 Hz), 1.14 (3H, d, *J* = 3.5 Hz). 13C NMR (DMSO-d6, 101 MHz) δ 181.2, 174.7, 168.3, 157.7, 152.7, 150.4, 140.8, 139.2, 136.4, 134.4, 132.9, 128.3, 126.3, 124.8, 109.9, 60.1, 56.1, 50.7, 36.2, 33.3, 29.1, 22.6, 18.8, 14.6. ESI-MS (m/z): [M + Na]<sup>+</sup> 494, [2M + Na]<sup>+</sup> 965.

3.1.12. Characterization of Benzyl Ester of 1-demethylthiocolchicine 4d

Amorphous yellow solid, yield 38%, m.p 166-170 ◦C; 1H NMR (DMSO-d6, 101 MHz) δ 8.52 (1H, s), 8.07 (2H, d, *J* = 7.3 Hz), 7.77–7.71 (1H, m), 7.63–7.56 (2H, m), 7.22–7.12 (1H, m), 7.06 (1H, s), 7.02 (1H, s), 4.49–4.40 (1H, m), 3.94 (3H, s), 3.74 (3H, s), 2.72 (1H, dd, *J* = 13.0, 5.9 Hz), 2.36 (1H, dd, *J* = 7.4, 5.5 Hz,), 2.31 (3H, s), 2.13 (2H, ddd, *J* = 19.4, 12.8, 6.9 Hz,), 1.87 (3H, s).13C NMR (DMSO-d6, 101 MHz) δ 181.1, 168.3, 157.7, 152.9, 150.6, 141.0, 139.4, 136.3, 134.6, 134.2, 129.8, 129.1, 128.4, 128.3, 126.2, 124.9, 110.2, 60.3, 56.1, 50.8, 36.3, 29.2, 22.6, 14.5. ESI-MS (m/z): [M + Na]<sup>+</sup> 528, [2M + Na]<sup>+</sup> 1033.

#### 3.1.13. Characterization of Methyl Carbonate of 1-demethylthiocolchicine 4e

Amorphous yellow solid, yield 53%, m.p 158-162 ◦C; 1H NMR (DMSO-d6, 101 MHz) δ 8.55 (1H d, *J* = 7.6 Hz), 7.26 (1H, d, *J* = 10.6 Hz), 7.05–6.99 (3H, m), 4.34 (1H, dt, *J* = 11.8, 7.0 Hz), 3.91 (3H, s), 3.82 (3H, s), 3.79 (3H, s), 2.69 (1H, dd, *J* = 13.5, 6.2 Hz), 2.43 (3H, s), 2.29 (1H, ddd, *J* = 18.6, 12.3, 5.4 Hz), 2.09 (1H, ddd, *J* = 18.7, 12.5, 6.2 Hz), 1.88 (3H, s), 1.86–1.80 (1H, m). 13C NMR (DMSO-d6, 101 MHz) δ 181.2, 168.4, 157.9, 152.9, 152.8, 150.5, 140.8, 139.3, 136.0, 134.5, 133.2, 128.2, 126.4, 124.3, 110.2, 60.4, 56.14, 56.07, 50.8, 36.1, 29.1, 22.6, 14.4. ESI-MS (m/z): [M + H]<sup>+</sup> 460, [M + Na]<sup>+</sup> 482, [M + K]<sup>+</sup> 498, [2M + Na]<sup>+</sup> 941.

#### 3.1.14. Characterization of Ethyl Carbonate of 1-demethylthiocolchicine 4f

Amorphous yellow solid, yield 48%, m.p 165-167 ◦C; 1H NMR (DMSO-d6, 400 MHz) δ 8.52 (1H, d, *J* = 7.6 Hz), 7.23 (1H, d, *J* = 10.7 Hz), 7.01 (3H, t, *J* = 10.3 Hz), 4.33 (1H, dt, *J* = 12.6, 7.5 Hz), 4.27–4.20 (2H, m), 3.90 (3H, s), 3.78 (3H, s), 2.67 (1H, dd, *J* = 13.2, 6.6 Hz), 2.41 (3H, s), 2.34–2.23 (1H, m), 2.08 (3H, s), 1.87 (3H, s), 1.85–1.78 (1H, m), 1.22 (3H, t, *J* = 7.1 Hz). 13C NMR (DMSO-d6, 101 MHz) δ 181.2, 168.4, 157.9, 152.8, 152.4, 150.5, 140.8, 139.3, 136.2, 134.5, 133.1, 128.2, 126.3, 124.4, 110.2, 65.1, 60.3, 56.1, 50.8, 36.1, 29.1, 22.6, 14.4, 14.0. ESI-MS (m/z): [M + Na]<sup>+</sup> 496, [M + K]<sup>+</sup> 512, [2M + Na]<sup>+</sup> 969.

#### *3.2. Antiproliferative Activity*

Detailed information concerning antiproliferative activity assay is given in Supplementary Materials.

#### *3.3. X-ray Measurements*

X-ray measurements and detailed crystallographic data are given in Supplementary Materials.

#### **4. Conclusions**

On the basis of regioselectively demethylated colchicine analogs, colchiceine (**1**) and 1-demethylthiocolchicine (**3**), we have designed and synthesized a series of **12** colchicine derivatives bearing ester and carbonate substituents, including **9** entirely novel derivatives (**2b**,**c**,**e**,**f** and **4b**,**c**,**d**,**e**,**f**). Derivatives **4a**·**H2O** and **4e**·**1/2H2O**, with acetyl ester and methyl carbonate moieties, display reduced hydrophilic properties and crystallize as hydrates, which affects their biological activity and increases their solubility. The control over water co-crystallization from monohydrate to hemihydrate has been achieved through the number of H-donor and acceptor sites. These features can be invaluable for the regulation of the bioaccessibility and pharmaceutical processing of this group of compounds [47]. The synthesized derivatives exhibit a considerable in vitro antiproliferative activity against three human cancer cell lines. Compound 4a, carrying the thiomethyl group at position C10 and acetyl ester substituent at C1, showed the highest activity and selectivity index values. The biological evaluation has been supported by prediction of physicochemical properties, which are consistent with Lipinski's rule of five for all synthesized analogs.

**Supplementary Materials:** The following are available online: General procedures, experimental details, as well as an elaborate description of the X-ray diffraction sudies and in vitro methods used.

**Author Contributions:** Conceptualization, D.C. and A.H.; methodology, D.C., A.H., S.S. and E.M. validation, A.H., A.K. and J.W.; investigation, D.C, S.S. and E.M. resources, A.H., A.K. and J.W. data curation, D.C., S.S and E.M writing—original draft preparation, D.C. and S.S writing—review and editing, A.H., A.K. and J.W. visualization, D.C.; supervision, A.H.; project administration, A.H.; funding acquisition, A.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** Financial support with a grant from the Polish National Science Centre (NCN)—No. 2016/21/B/ST5/00111 is gratefully acknowledged.

**Acknowledgments:** D.C. and S.S. acknowledge a scholarship (No. POWR. 03.02.00-00-I026/16 and POWR. 03.02.00-00-I023/17, respectively) co-financed by the European Union through the European Social Fund under the Operational Program Knowledge Education Development.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.

#### **References**


**Sample Availability:** Samples of the compounds **1**, **2a**–**f**, **3** and **4a**–**f** are available from the authors.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **Synthesis, Antiproliferative Activity and Molecular Docking Studies of Novel Doubly Modified Colchicine Amides and Sulfonamides as Anticancer Agents**

**Julia Krzywik 1,2, Witold Mozga 2, Maral Aminpour 3, Jan Janczak 4, Ewa Maj 5, Joanna Wietrzyk 5, Jack A. Tuszy ´nski 3,6 and Adam Huczy ´nski 1,\***


Academic Editor: Qiao-Hong Chen Received: 16 March 2020; Accepted: 11 April 2020; Published: 14 April 2020

**Abstract:** Colchicine is a well-known compound with strong antiproliferative activity that has had limited use in chemotherapy because of its toxicity. In order to create more potent anticancer agents, a series of novel colchicine derivatives have been obtained by simultaneous modification at C7 (amides and sulfonamides) and at C10 (methylamino group) positions and characterized by spectroscopic methods. All the synthesized compounds have been tested in vitro to evaluate their cytotoxicity toward A549, MCF-7, LoVo, LoVo/DX and BALB/3T3 cell lines. Additionally, the activity of the studied compounds was investigated using computational methods involving molecular docking of the colchicine derivatives to β-tubulin. The majority of the obtained derivatives exhibited higher cytotoxicity than colchicine, doxorubicin or cisplatin against tested cancer cell lines. Furthermore, molecular modeling studies of the obtained compounds revealed their possible binding modes into the colchicine binding site of tubulin.

**Keywords:** anticancer agents; colchicine amide; colchicine sulfonamide; tubulin inhibitors; docking studies; crystal structure

#### **1. Introduction**

Microtubules, which are composed of α- and β-tubulin heterodimers, are involved in a large number of processes, such as intracellular transport, cell shape development, cell division and cell motility. During cell division microtubules form the mitotic spindle that in normal cells correctly separates the chromosomes into two daughter cells. In cancer cells the rate of mitosis is typically increased but chromosome segregation is imperfect leading to aneuploidy. Microtubules formed during mitosis are considered as an ideal target for anticancer drugs since no cell can divide without the force generated by microtubules. Therefore, many inhibitors of microtubule dynamics have been investigated for their potential use as cancer chemotherapy drugs [1–7]. One of these compounds is

colchicine **1** (see Scheme 1), a well–known tropolone alkaloid isolated from *Colchicum autumnale*, which has been shown to exhibit very high cytotoxic effects. It binds to tubulin at the colchicine binding site and induces conformational change in the tubulin dimer making it incompetent for microtubule assembly. As a result, the cell cycle is blocked and apoptosis is induced [8–14]. Unfortunately, colchicine is too toxic to be useful as an antitumor agent [8,15–21]. Nevertheless, it has found use in therapy, e.g., for the treatment of familial Mediterranean fever, Behcet's disease or acute gout [22–27].

**Scheme 1.** Synthesis of doubly modified colchicine derivatives (**2**–**21),** changes at C7 and C10 positions are highlighted in red. Reagents and conditions: (**a**) NHCH3/EtOH, reflux; (**b**) 2M HCl, reflux; (**c**) RC(O)Cl or RSO2Cl, Et3N, DCM, 0 ◦C to RT for **4**, **8**, **13**, **15**–**16, 18** and **21**; (**d**) RCOOH, EDCI, DCM, RT for **5**–**7, 9**–**12** and **14**; (**e**) (1) *N*-(*tert*-butoxycarbonyl)-*N*-[4-(dimethylazaniumylidene)- 1,4-dihydropyridin-1-ylsulfonyl]azanide, DCM, RT, (2) 4M HCl/EtOAc, RT for **17**; (**f**) ClCH2CH2SO2Cl, Et3N, DCM, 0 ◦C to RT; (**g**) morpholine, DCM, RT.

Therefore, over the past few decades much interest has been focused on structural modifications of **1** in the hope of improving its therapeutic index [28–31]. Numerous double-modified colchicine derivatives have been synthesized with the group at position C7 substituted by various amide and sulfonamide moieties as well as with replacement of the methoxy group at position C10 by a group containing either a nitrogen or a sulfur atom and their biological activities have been determined [28,29,32–36]. These results have shown that new derivatives may have lower toxicity with respect to normal cells while maintaining high antitumor activity. In addition, from the chemical point of view, such a change at position C10 allows obtaining compounds more resistant to acid hydrolysis [29,30,33]. We have decided to check various amide and sulfonamide moieties because they have long been valued for their rich biological and chemical profiles and have emerged as a promising class of compounds in drug discovery [37–41].

Herein, we report the synthesis, crystallographic and spectroscopic analysis of a series of structurally different derivatives of colchicine obtained by its modification at position C7 (various amide, sulfonamide or sulfamide moieties) and at position C10 (methylamino group). We also describe the results of in vitro antiproliferative activity evaluation of colchicine (**1**) and the obtained colchicine derivatives (**2**–**21**) against four human cancer cell lines and one normal murine embryonic fibroblast cell line. To acquire more knowledge about the molecular mechanism of action of the investigated compounds (**1**–**21**), we also present results of *in silico* molecular docking study of the colchicine binding site (CBS) of β-tubulin.

#### **2. Results and Discussion**

#### *2.1. Chemistry*

To investigate the effect of methylamino group at position C10 and, at the same time, various amide, sulfonamide and sulfamide moieties at position C7 of colchicine **1** on its antiproliferative activity, eighteen new derivatives (**4**–**21**) were synthesized. To facilitate the structure-activity relationship analysis (SAR) we designed compounds with different side chains at position C7: alkyl chains of various length, straight and branched (**4**–**8**), unsaturated alkyl chain (**19**), alkyl chains of various lengths containing halogen atoms (**9**–**11**), an aromatic group without or with substituents (**12**–**16**, **21**), and compounds containing an amino group **17**–**18** and **20**.

The general route for the synthesis of colchicine derivatives **2**–**21** is depicted in Scheme 1. Colchicine (**1**) was treated with methylamine solution in ethanol to give 10-methylamino-10-demethoxycolchicine (**2**) with 80% yield, according to the method described earlier [42]. The replacement of water solution of methylamine by ethanol solution eliminated the work up after the reaction and permitted obtaining comparable final yields. Next, hydrolysis of **2** with 2M HCl yielded *N*-deacetyl-10-methylamino-10-demethoxycolchicine (**3**). Compounds **4**–**16**, **18**–**19** and **21** were readily available from **3** by treatment with respective acid/ sulfonamide/ sulfamide chloride in the presence of triethylamine or with the corresponding carboxylic acid and carbodiimide as a condensing agent. Compound **17** was prepared using a *N*-(*tert*-butoxycarbonyl)-*N*- [4-(dimethylazaniumylidene)-1,4-dihydropyridin-1-ylsulfonyl]azanide and further modified by removal of the *tert*-butoxycarbonyl group from amine with HCl [43]. Vinylsulfonamide **19** was synthesized from 2-chloroethanesulfonyl chloride and **3** through sulfonylation and in situ β-elimination of HCl. Compound **19** was used as the Michael acceptor with an electron-deficient double bond for the reaction with morpholine to produce compound **20 [44,45]**. All synthesized compounds were isolated in pure form after column chromatography.

The purity and structures of the obtained compounds **2**–**21** were determined using the LC-MS, 1H and 13C NMR methods and are shown in the Supplementary Materials and discussed below. The characteristic signals of -OCH3 group at position C10 of **1** in the 1H NMR and 13C NMR spectra were observed as a singlet at 4.0 ppm and at 56.5 ppm, respectively. These signals vanish after the reaction of colchicine with methylamine proving the substitution of the -OCH3 group in the tropolone

ring of **1**. After the introduction of -NHCH3 at position C10 the signals of this group in the obtained derivatives (**2**–**21**) were visible approx. at 3.1 ppm as a doublet and approx. at 7.4 ppm as a quartet in 1H NMR and approx. at 29.5 ppm in 13C NMR. The chemical shifts of the amide moiety can be found in the range 7.9–9.7 ppm in 1H NMR and in the range 164.2–175.2 ppm in 13C NMR, depending on the substituent used. The ESI mass spectrometry confirmed the structure of the obtained compounds by the presence of an *m*/*z* signals assigned to the corresponding pseudomolecular ions of these compounds.

#### *2.2. X-ray Crystal Analysis*

Structural characterization of the colchicine derivatives is very important in order to understand their anticancer properties stemming from their interaction with tubulin as well as to enable structure–activity relationship analysis (SAR) and related investigation. Therefore, structural analyses of all crystals that were suitable for X-ray analysis of single crystals were performed. Crystals of **6**, **11**, **12**, **14**, **18** and **19** suitable for the X-ray single crystal analysis were obtained by recrystallization of the respective colchicine derivatives from acetonitrile, whereas crystals **15** and **16** from ethyl acetate solutions. All crystals were measured at room (295 K) and low (100 K) temperature. Details of the data collection parameters, crystallographic data and the final agreement parameters are listed in Supplementary Table S1. In the temperature range from 295 K to 100 K, no structural phase transitions were observed in the crystals studied, although for colchicine derivative **11** at low temperature some disorder of the -CF3 group in the -CH2-CH2-CF3 group at atom C21 could be observed. Colchicine derivatives **6**, **11**, **12**, **14**, **15,** and **16** crystallize in the P3221 space group of the trigonal system while derivative **18** crystallizes in the P212121 space group of the orthorhombic system and derivative **19** crystallizes in the P21 space group of the monoclinic system. These space groups are chiral since the compounds contain an asymmetric carbon (C7) atom. The absolute configuration at the C7 atom is *S* in all structures. The molecular structures of all colchicine derivatives (**6**, **11**, **12**, **14**, **15**, **16**, **18** and **19**) are illustrated in Supplementary Figure S60. The planar phenyl A and tropolone C rings in all colchicine derivatives (**6**, **11**, **12**, **14**, **15**, **16**, **18** and **19**) are twisted around the C13–C16 bond with the torsion angle describing the twisting conformation C1–C16–C13–C12 between ~53◦ and ~56◦ at 100 K and they do not differ significantly from the values at room temperature (Table 1). Ring B in all colchicine derivatives exhibits a similar puckering pattern and the extent of its non-planarity is such that it adopts a conformation, which is close to the twist-boat with a flattening caused by the fusion of rings A and C (see Supplementary Figure S60). So the conformation of the fused A, B and C rings of colchicine skeleton in the investigated derivatives is quite similar to that in colchicine itself [46].


**Table 1.** Selected torsion angles (◦) of colchicine derivatives **6**, **11**, **12**, **14**, **15**, **16**, **18** and **19** obtained by X-ray analysis and DFT computation for a comparison.

The methoxy group -OCH3 linked to the phenyl ring at C3 is almost coplanar with the ring in all structures of colchicine derivatives, whereas the other two methoxy groups linked at C1 and C2 atoms of the phenyl ring have different orientations in some colchicine derivatives (Table 1). The *N*-methylamino group (-NHCH3) linked to tropolone C ring at C10 atom is almost coplanar with the ring. The differences between the conformations of the investigated colchicine derivatives are illustrated in Figure 1 and Table 1. For clarity, the colchicine derivatives have been divided into two groups, according to the substituent in ring B at position C7; one group comprises the derivatives with a chain substituent (Figure 1a) and the other group comprises the derivatives with a substituent containing an aromatic ring (Figure 1b). Analysis of these results reveals a significant difference in the torsion angle C17-O1-C1-C2 in compounds 18 (~−109◦) and 19 (~66◦) or the difference between the calculated (~−103◦) and measured (~−77◦) torsion angle C18-O2 -C2-C3 in compound 19. These differences result from different approach to the description of the molecule conformations, the X-ray analysis values refer to the conformation of molecules in crystals, in which the intermolecular interactions play a significant role and leads crystallization and specific crystal packing, while the DFT values refer to a single isolated molecule in the gas state with the intermolecular interactions not taken into account.

**Figure 1.** Comparison of the X-ray structure of (**a**) derivatives with a chain substituent at position C7: **6** (blue), **11** (red), **18** (pink) and **19** (green) and (**b**) derivatives with a substituent containing an aromatic ring at position C7: **12** (pink), **14** (blue), **15** (green) and **16** (red) showing the conformation of the colchicine skeleton. The molecules are overlapped one over another so that the ring C1-C2-C3-C4-C15-C16 is at the same position.

#### *2.3. Molecular Electrostatic Potential Map Analysis*

The role of the trimethoxyphenyl ring A of colchicine as well as that of the tropolone ring C in tubulin binding have been studied in great details. The tropolonoic ring C of the colchicine skeleton is found to be crucial for the interaction with tubulin [47,48]. The molecular electrostatic potential map (MESP) correlated with the electronic density in a molecule and is a powerful tool for analyzing interactions [49–51]. It was, therefore, calculated for all structurally characterized colchicine derivatives. Additionally, the gas-phase structures of all molecules were determined using the DFT optimization with the Gaussian09 program package [52]. All calculations were carried out by the DFT method using the Becke3-Lee–Yang–Parr correlation functional (B3LYP) [53–56] with the 6–31 + G basis set, starting from the X-ray geometry of molecules. The gas-phase optimized conformations of all colchicine derivatives are, in general, in good agreement with those obtained from the X-ray single crystal investigation, however the optimized torsion angle C18–O2–C2–C3 describing the orientation of the methoxy group is significantly smaller than that provided by the X-ray analysis (Table 1 and for more details see Supplementary Table S2).

The region of tubulin that interacts with colchicine is near the αβ-tubulin/dimer interface [57]. In order to better understand the interaction of the colchicine derivatives with tubulin, the molecular electrostatic potential was calculated for all structurally characterized colchicine derivatives as well as, for comparison, for colchicine itself. The three-dimensional MESP maps for colchicine derivatives and for colchicine itself were calculated on the basis of the DFT (B3LYP) optimized geometries of molecules and mapped onto the total electron density isosurface (0.008 eÅ<sup>−</sup>3) for both molecules using the GaussView 5.0 program (Figure 2). The color coding of MESP is in the range of −0.05 (red) to 0.05 eÅ-1 (blue). For all colchicine derivatives, the regions of negative MESP are usually associated with the lone pair of electronegative atoms (O and N), whereas the regions of positive MESP are associated with the electropositive atoms (Figure 2).

**Figure 2.** *Cont*.

$$\mathfrak{c}(\mathbf{12})$$

**Figure 2.** *Cont*.

**Figure 2.** Optimized conformation (left) and three-dimensional molecular electrostatic potential map (right) for colchicine derivatives (**a**–**h**) and colchicine itself (**i**), for comparison. Color code: <sup>−</sup>0.05 eÅ−<sup>1</sup> (red) to +0.05 eÅ−<sup>1</sup> (blue).

The nucleophilic regions in colchicine derivatives are observed near oxygen atoms of all methoxy and carbonyl groups. In addition, significantly less negative value of MESP than that near the oxygen atoms spreads across the aromatic phenyl rings. The planar conformation of tropolone ring C, showing the alternating single and double C-C bonds, is manifested as partial delocalization of the π electrons resulting in a slightly negative value of MESP on both sides of the planar fragment of colchicine derivatives. The molecular electrostatic potential for colchicine itself was also calculated for comparison (Figure 2i). In all colchicine derivatives the methoxy (-OCH3) substituent in the tropolone C ring is replaced by *N*-methylamino substituent (-NHCH3), therefore, MESP maps show a less negative area near this group (Figure 2a–h) compared to the MESP map of colchicine itself (Figure 2i). Additionally, the replacement in colchicine molecule of the -NHC(O)CH3 group in ring B at C7 atom with various substituents modified the size of the molecule and the maps of electrostatic potential. This is particularly visible for derivatives **11**, **14** and **18**, **19** in which the -NHC(O)CH3 group is replaced by -NHC(O)C2H4CF3, -NHC(O)C6H4F in **11** and **14**, respectively, and in **18** and **19** by the substituents containing sulfonyl group (-SO2R).

The DFT results, especially the three-dimensional molecular electrostatic potential map (MESP) providing information on the distribution of the electron density of molecules are useful for predictions of interactions between the tested compounds and homology modeled tubulin βI. The formation of guest-host complexes (colchicine derivatives as guest and tubulin as host) depends on the guest's fit into the host cavity and their interactions that result from the mutual matching of electrostatic interactions.

#### *2.4. In Vitro Determination of Drug-Induced Inhibition of Human Cancer Cell Line Growth*

The synthesized colchicine derivatives **2**–**21** and starting material **1** were evaluated for their in vitro antiproliferative effect on four human cancer cell lines, including one cell line displaying various levels of drug resistance and additionally one normal murine embryonic fibroblast cells.

The majority of new derivatives of **1** showed antiproliferative activity in the nanomolar range and were characterized by lower IC50 values than unmodified colchicine **1**, as well as doxorubicin and cisplatin, commonly used as antitumor agents in cancer chemotherapy (Table 2). From the set of tested compounds, the ones most active against A549 tumor cell line were **2**, **4**–**6**, **8**–**14** and **18**–**19** (IC50 ≤ 15 nM), against MCF-7 tumor cell line-were **2**, **9**–**17** and **19** (IC50 ≤ 13 nM), against LoVo tumor cell line-were **2**, **4**–**6**, **8**–**16**, **18**–**19** (IC50 ≤ 11 nM) of which the lowest IC50 values were shown by compounds **9**–**10** and **13** (IC50 = 0.7–1.8 nM). Moreover, compound **13** was observed to be most active towards the LoVo/DX line (IC50 = 9.6 nM), approx. 170 times more potent than unmodified colchicine **1** (IC50 = 1646.6 nM). Compound **7** from amides and compound **20** from sulfonamides showed the weakest activity (the highest IC50 values) against all cancer cell lines tested. The decrease in cytotoxicity of compound **7** could be related to an increase in hydrophobicity (high calculated octanol/water partition coefficient clog*P* = 8.7, see Table 3). It is well known that high clog*P* value and therefore low hydrophilicity are responsible for poor absorption and permeation to the colchicine binding pocket in β-tubulin. The high IC50 value for compound **20** may be due to the presence of a morpholine ring which is a large volume substituent and can adopt different conformations. Although these compounds were the least potent out of the whole series of tested derivatives (**1**–**21**), their IC50 values were in the micromolar range (see Table 2).


**Table 2.** Antiproliferative activity (IC50) of colchicine (**1**) and its derivatives (**2**–**21**) compared with antiproliferative activity of standard anticancer drugs doxorubicin and cisplatin and the calculated values of the resistance index (RI) of tested compounds.

**Table 2.** *Cont*.


The IC50 value is defined as the concentration of a compound at which 50% growth inhibition is observed. The IC50 values shown are mean ± SD. Human lung adenocarcinoma (A549), human breast adenocarcinoma (MCF-7), human colon adenocarcinoma cell line (LoVo) and doxorubicin-resistant subline (LoVo/DX), normal murine embryonic fibroblast cell line (BALB/3T3). The RI (Resistance Index) indicates how many times more chemoresistant is a resistant subline relative to its parental cell line. The RI was calculated for each compound using the formula: RI = (IC50 for LoVo/DX cell line)/(IC50 for LoVo cell line). When RI is 0–2, the cells are sensitive to the compound tested, RI in the range 2–10 means that the cells shows moderate sensitivity to the drug tested, RI above 10 indicates strong drug resistance.

**Table 3.** Computational predictions of interactions between tested compounds (**1**–**21**) and homology modeled tubulin βI. 3D representation and 2D layout of colchicine derivatives–tubulin protein complex, binding energy (BE), calculated octanol/water partition coefficient (clog*P*) and active residues are tabulated.



**Table 3.** *Cont*.


**Table 3.** *Cont*.


**Table 3.** *Cont*.

Although the synthesized compounds are effective toward cancer cells, their potential is limited against cells with developed drug resistance. The data presented in Table 2 show that unmodified colchicine **1** and all of the colchicine derivatives **2**–**21** less effectively inhibited the proliferation of the doxorubicin-resistant subline LoVo/DX than the sensitive LoVo cell line. The calculated values of RI clearly confirmed that none of the tested amides and sulfonamides was able to overcome the drug resistance of LoVo/DX cell line (RI ranges from 11.8 to 306.0). It can be explained by the upregulated expression of efflux transporters in these cells, playing an important role in drug transport in many organs and determining the drug resistance of cancer cells. Because this type of resistance is one of the mechanisms of cancer resistance [58], compounds **1**–**2** and **4**–**21** are probably good substrates for such pumps. Increased efflux of compounds makes it impossible to reach their adequate concentrations in the cell and consequently to exert efficient cytotoxic effect. The only compound which showed RI < 10 was derivative **3**, having at position C7 a free amino group. Keeping in mind its good activity (IC50 < 20 nM for three cancer cells, see Table 2) it can be still considered as a good starting point for the chase after antitumor agents active against drug resistant lines.

The selectivity index (SI) was calculated to evaluate the toxicity of the compounds studied against normal cells and to predict their therapeutic potential (see Figure 3). High SI values result from large differences between the cytotoxicity against cancer and normal cells and this means that cancer cells will be killed at a higher rate than normal ones. From the set of tested compounds with methylamino group at position C10 and alkyl chains at position C7 (**2**, **4**–**8, 19**) the best selectivity index for A549, MCF-7 and LoVo cells (SI = 1.5–4.0) showed **8** with short and branched substituent (isobutyric acid derivative). The 4,4,4-trifluorobutyric acid derivative **11** (from compounds with alkyl chains of various lengths containing halogen atoms **9**–**11**) showed outstanding selectivity for three out of four cancer cells (SI = 5.6–7.8). It should be emphasized that compound **11** was the only compound with SI values greater than that of unmodified colchicine **1** for all tumor cell lines. Despite the fact that compounds **9**, **10,** and **13** stood out from the synthesized compounds in terms of IC50 values, their selectivity indices were high only for LoVo lines (SI = 6.5–9.1), they were very cytotoxic also to non-cancerous BALB/3T3 cells (IC50 = 6.2–11.6 nM). Among aryl amides and sulfonamides **12**–**16** and **21**, distinctive SI values were derived for colchicine derivatives containingnicotinic and isonicotinic amide residue (SI ranges 3.0 to 4.8). These results are noteworthy and suggest that extended and more detailed research of similar derivatives is necessary to determine the importance of ring aromaticity, its size or heteroatom type. Especially high SI values for MCF-7 were obtained for sulfamide **17** (SI = 9.8) and sulfonamide containing a morpholine ring **20** (SI = 6.6). These results deserve special attention and require further studies to determine whether the conversion of an amide bond to a sulfonamide bond with an appropriate substituent would allow obtaining compounds highly selective towards MCF7 cells, compared to colchicine **1**. As many as thirteen of the obtained derivatives exhibited SI ≥ 2 for LoVo cell line (see Figure 3). The results indicated that properly designed doubly modified (at C7 and C10 positions) colchicine derivatives can have greater selectivity towards cancer cells than the parental compound.

**Figure 3.** Comparison of selectivity index (SI) values of the tested compounds. The SI was calculated for each compound using the formula: SI = (IC50 for normal cell line BALB/3T3)/(IC50 for respective cancerous cell line). A favorable SI > 1.0 indicates a drug with efficacy against tumor cells greater than the toxicity against normal cells.

#### *2.5. In Silico Determination of the Molecular Mode of Action*

In the present study, computational investigation including molecular docking, molecular dynamics (MD) simulations, molecular mechanics generalized Born/surface area (MM/GBSA) binding free energy calculations and decomposition of pair-wise free energy on a per-residue basis were conducted to deeply explore the molecular basis for the binding of twenty novel double modified colchicine amides and sulfonamides to β-tubulin. The latter is one of the subunits of microtubules in the cytoskeleton structure of every eukaryotic cell, which is the target of many anticancer drugs. The twenty structures of colchicine derivatives described above were docked into the βI-tubulin (the most abundant isotype in most cancer tumors) colchicine binding site.

On the basis of our computational predictions, according to increasing binding energy, the compounds are ordered as follows: **20** (−59.6), **18** (−54.6), **7** (−53.7), **9** (−44.0), **15** (−43.9), **4** (−43.4), **5** (−43.1), **10** (−41.8), **1** (−41.1), **13** (−40.1), **2** (−39.3), **16** (−38.3), **6** (−37.2), **12** (−35.2), **17** (−34.9), **11** (−34.7), **14** (−34.3), **8** (−32.3), **21** (−29.8), **19** (−23.4), **3** (−4.0) kcal/mol with the binding energies in the parenthesis given in units of kcal/mol. Binding energies of these compounds are shown in Figure 4 and Table 3.

**Figure 4.** Comparison of binding energies of the tested compounds complexed with tubulin βI. Binding energies have been estimated using the MM/GBSA method.

In view of the calculated binding energies, we can conclude that there is no strong correlation (the lower the binding energy, the more biologically active the chemical compound) between *in silico* computer calculations (BE values, Figure 4) and in vitro activity results (IC50 values, Table 2).

The lowest binding energies, −59.6 and −53.7 kcal/mol, were shown by sulfonamide **20** (derivative with the morpholine ring) and amide **7** (derivative of palmitic acid), respectively. However, these compounds showed the weakest antiproliferative activity (highest IC50 values) among all compounds tested (**1**–**21**).

Compound **18** has the third lowest energy (−54.6 kcal/mol) and good cytotoxicity with IC50 from 9.3 to 15.5 nM for LoVo, A549 and MCF-7 cancer cells. The molecular-level computations indicate that **18** fits well to βI-tubulin and probably uses this binding site as a target. The majority of the other derivatives **2**, **4**–**6**, **8**–**17** exhibited binding energy less than −30.0 kcal/mol, so close to the energy of unmodified colchicine **1** (BE = −41.1 kcal/mol), which is a compound known to inhibit tubulin polymerization. These compounds bind to the active sites of βI-tubulin isotype and may therefore

have a mechanism of action similar to **1** and may be colchicine binding site inhibitors, although their in vitro activities towards various cells characterized by an IC50 value (Table 2) were lower than that of colchicine **1**.

Derivative **3** showed the highest binding energy value (BE = −4.0 kcal/mol), therefore its antiproliferative activity is not exclusively the result of interaction with tubulin. This is an important finding because compound **3** was the only one which showed any possibility of breaking the drug resistance of the LoVo/DX line (RI = 9.2, see Table 2).

Therefore, we can see that the results only partly correlate with in vitro determined biological activity of these compounds as indicated though the corresponding IC50 values. This may be explained by several additional effects taking place in living cells compared to the computational simulations that focus only on the binding mode of the compounds to the target. Primarily off-target interactions involving efflux transporters with different affinities for the individual compounds and differences in solubility of these colchicine derivatives or their membrane permeability. Additionally, the lack of significant correlation may be due to the fact that in various cancer cells the expression of specific β-tubulin isotypes significantly varies [59]. We propose the inclusion of binding affinity calculations for these compounds with regard to not only other tubulin isotypes but also with respect to most important efflux transporters in order to minimize them and hence increase the activity of the drug in vitro and *in vivo*.

Schematic interactions of the compounds with CBS of βI-tubulin residues are shown in Table 3.

In 3D representation, the interacting residues predicted from pairwise per-residue binding free energy decomposition calculations (E < −2 kcal/mol) are shown in stick presentation and their carbons and the ribbon are colored as green. Tubulin is shown in cartoon representation. Hydrogen bonds and their directionality are represented as black dashed arrows. The structures are color-coded as follows: tubulin αI, brown; tubulin βI, beige. Compounds are displayed with sticks and the atoms are colored as O (red), C (gray), N (blue), S (yellow), Cl (green) and F (pink). Binding energy defines the affinity of binding for colchicine derivatives complexed with tubulin βI. Binding energies are predicted by MM/GBSA method. The last column contains information about the active residues with the binding free energy decomposition (Edecomp) less than −2 kcal/mol (the residues with Edecomp < −3 kcal/mol are highlighted in boldface). The last line contains the graphical key to help interpret the 2D part of the ligand interactions panel.

Derivative compounds are designed with different side chains at C7 position of colchicine **1**. Compounds **2**, **4**–**8** have alkyl chains of various lengths, straight and branched and compound **19** has an unsaturated alkyl chain. The second lowest binding energy compound **7** within all the compounds is in this group (BE = −53.6 kcal/mol). Compound **7** appears to be engaged in some interactions with α-tubulin (−3 < Edecomp < −2 kcal/mol). Most of the compounds in this group are bound through Lys785, Lys687 and Leu688 residues.

Compounds **9**–**11** were designed with alkyl chains of various length containing halogen atoms side chains at position C7 of colchicine **1**. In this group, the common binding residues with (−3 < Edecomp < −2 kcal/mol) are Ala179, Cys674, Asn691, while the strongest binding residues (Edecomp < −3 kcal/mol) are Leu688 and Lys785.

Compounds **12**–**16, 21** were designed with an aromatic group side chain without or with substituents at C7 position of colchicine **1**. The same trend of strong binding of Leu688 and Lys785 residues with the compounds is seen in this group. The binding energies in this group are on the higher side compared to the other compounds.

As the last group, compounds **17**–**18** and **20** contain the sulfonamide moieties at position C7 of colchicine **1**. The strongest binding residues in this group belong to both α-tubulin (Gln10 and Ser177) in compound **17**, (Gln10, Gly142, Gly143, Thr144 and Ser177) in compound **18** and (Asp68 and Asn100) in compound **20** with (Edecomp < −3 kcal/mol) and β-tubulin (Asp684, Asn691, Lys785) in compound **17** and Lys687 in compound **20**. The lowest binding energy between all the compounds characterizes compounds **20** and **18**, which mainly bind to α-tubulin.

In the experimental part of the study, the highest IC50 values (the weakest activity) were found for compound **7** from the amides and compound **20** from the sulfonamides studied against all cancer cell lines tested. According to our computational analysis, although these compounds show the lowest binding energies, they do not bind to the CBS, which is located in β-tubulin and has the tendency to go beyond and also bind to α-tubulin.

#### **3. Materials and Methods**

#### *3.1. General*

All solvents, substrates and reagents were obtained from TriMen Chemicals (Poland) or Sigma Aldrich and were used without further purification. CDCl3 and CD2Cl2 spectral grade solvents were stored over 3 Å molecular sieves for several days. TLC analysis was performed using pre-coated glass plates (0.2 mm thickness, GF-254, pore size 60 Å) from Agela Technologies and spots were visualized by UV-light. Products were purified by flash chromatography using high-purity grade silica gel (pore size 60 Å, 230–400 mesh particle size, 40–63 μm particle size) from SiliCycle Inc. Preparative HPLC was performed on LC-20AP Shimadzu with ELSD-LTII detector equipped with Phenomenex Luna C18 250 × 21 mm, 5 μm column eluted with 20 mL/min flow over 20 min of acetonitrile in water. Solvents were removed using a rotary evaporator.

#### *3.2. Spectroscopic Measurements*

NMR spectra were recorded on Bruker Avance DRX 500 (1H NMR at 500 MHz and 13C NMR at 126 MHz) magnetic resonance spectrometers. 1H NMR spectra are reported in chemical shifts downfield from TMS using the respective residual solvent peak as internal standard (CDCl3 δ 7.26 ppm, CD2Cl2 δ 5.32 ppm, (CD3)2SO δ 2.50 ppm). 1H NMR spectra are described as follows: chemical shift (δ, ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, dt = doublet of triplets, dq = doublet of quartets, m = multiplet), coupling constant (*J*) in Hz, and integration. 13C NMR spectra are described in chemical shifts downfield from TMS using the respective residual solvent peak as internal standard (CDCl3 δ 77.16 ppm, CD2Cl2 δ 53.84 ppm and (CD3)2SO δ 39.52 ppm).

Electrospray ionization (ESI) mass spectra were obtained on a Waters Alliance 2695 separation module with a PDA 2996 UV detector and Waters Micromass ZQ 2000 mass detector equipped with Restek Ultra Biphenyl 50 × 3 mm, 3 μm column eluted with 0.3 mL/min flow of 3–100% gradient (over 6 min) of acetonitrile in water.

#### *3.3. Synthesis*

#### 3.3.1. Synthesis of **2**

To a solution of **1** (1.0 equiv.) in EtOH, a methylamine (solution 33% in EtOH, 10.0 equiv.) was added. The mixture was stirred at reflux for 24 h and then concentrated under reduced pressure to dryness. The residue was purified using column flash chromatography (silica gel; DCM/MeOH) and next lyophilized from dioxane to give the pure product **2** as a yellow solid with a yield of 80%.

ESI-MS for C22H26N2O5 (*m*/*z*): [M + H]<sup>+</sup> 399, [M + Na]<sup>+</sup> 421, [2M + H]<sup>+</sup> 797, [2M + Na]<sup>+</sup> 819, [M – H]− 397, [M + HCOO−] − 443.

1H NMR (500 MHz, CDCl3) δ 8.70 (d, *J* = 6.4 Hz, 1H), 7.58 (s, 1H), 7.46 (d, *J* = 11.1 Hz, 1H), 7.28–7.25 (m, 1H), 6.58 (d, *J* = 11.3 Hz, 1H), 6.52 (s, 1H), 4.73–4.64 (m, 1H), 3.93 (s, 3H), 3.88 (s, 3H), 3.61 (s, 3H), 3.08 (d, *J* = 5.4 Hz, 3H), 2.47–2.43 (m, 1H), 2.37–2.31 (m, 1H), 2.29–2.22 (m, 1H), 2.02–1.96 (m, 1H), 1.94 (s, 3H).

13C NMR (126 MHz, CDCl3) δ 175.11, 170.19, 155.23, 152.91, 151.61, 151.10, 141.51, 139.37, 134.66, 130.42, 126.93, 122.81, 108.28, 107.20, 61.46, 61.40, 56.16, 52.72, 37.06, 30.15, 29.53, 22.74.

#### 3.3.2. Synthesis of **3**

To a solution of compound **2** (1.0 equiv.) in dioxane, 2M HCl (10.0 equiv.) was added and the mixture was stirred at reflux. Reaction progress was monitored by LC-MS. Then the reaction mixture was neutralized with 4M NaOH to pH~10 and extracted four times with EtOAc. The organic layers were combined, washed with brine, dried over Na2SO4, filtered and evaporated under reduced pressure. The residue was purified using column flash chromatography (silica gel; DCM/MeOH) and next lyophilized from dioxane to give the pure product **3** as a yellow solid with a yield of 73%.

ESI-MS for C20H24N2O4 (*m*/*z*): [M + H]<sup>+</sup> 357, [M + Na]<sup>+</sup> 379, [2M + Na]<sup>+</sup> 735.

1H NMR (500 MHz, CDCl3) δ 7.61 (s, 1H), 7.33 (d, *J* = 11.1 Hz, 1H), 7.23–7.21 (m, 1H), 6.50 (s, 1H), 6.50 (d, *J* = 11.4 Hz, 2H), 3.87 (s, 3H), 3.87 (s, 3H), 3.75–3.72 (m, 1H), 3.59 (s, 3H), 3.05 (d, *J* = 5.5 Hz, 3H), 2.41–2.37 (m, 1H), 2.33–2.31 (m, 2H), 2.25 (s, 2H), 1.71–1.61 (m, 1H).

13C NMR (126 MHz, CDCl3) δ 175.57, 154.99, 153.25, 152.76, 150.59, 141.06, 138.73, 135.41, 129.74, 126.63, 123.68, 107.35, 106.94, 61.19, 60.84, 56.06, 54.01, 40.90, 30.69, 29.48.

#### 3.3.3. General Procedure for the Synthesis of Colchicine Derivatives **4**, **8**, **13**, **15**–**16**, **18** and **21**

Compounds **4**, **8**, **13**, **15**–**16**, **18** and **21** were obtained directly from compound **3**. To a solution of compound **3** (1.0 equiv.) and Et3N (3.0 equiv.) in DCM in an ice bath, the corresponding acid/sulfonyl/sulfamide chloride (1.1 equiv.) diluted with DCM was added slowly. Next the ice bath was removed and the reaction mixture was stirred at RT. Reaction progress was monitored by LC-MS. Then the reaction mixture was diluted with EtOAc, washed with H2O, 1M K2CO3, brine and dried over Na2SO4. The residue was purified using column flash chromatography (silica gel; DCM/MeOH) and next lyophilized from dioxane to give respective compound.

#### Compound **4**

Yellow solid, yield 86%.

ESI-MS for C23H28N2O5 (*m*/*z*): [M + H]<sup>+</sup> 413, [M + Na]<sup>+</sup> 435, [2M + H]<sup>+</sup> 825, [2M + Na]<sup>+</sup> 847, [M−H]<sup>−</sup> 411, [M + HCOO−] − 457.

1H NMR (500 MHz, CDCl3) δ 8.46 (s, 1H), 7.53 (s, 1H), 7.44 (d, *J* = 11.1 Hz, 1H), 7.25–7.21 (m, 1H), 6.55 (d, *J* = 11.4 Hz, 1H), 6.50 (s, 1H), 4.72–4.65 (m, 1H), 3.91 (s, 3H), 3.86 (s, 3H), 3.61 (s, 3H), 3.06 (d, *J* = 5.4 Hz, 3H), 2.44–2.40 (m, 1H), 2.33–2.30 (m, 1H), 2.26–2.17 (m, 3H), 1.94–1.88 (m, 1H), 1.05 (t, *J* = 7.6 Hz, 3H).

13C NMR (126 MHz, CDCl3) δ 175.09, 173.89, 155.20, 152.86, 151.64, 151.09, 141.49, 139.23, 134.67, 130.33, 126.95, 122.94, 108.14, 107.20, 61.44, 56.14, 52.37, 37.20, 30.18, 29.53, 29.11, 9.72.

#### Compound **8**

Yellow solid, yield 96%.

ESI-MS for C24H30N2O5 (*m*/*z*): [M + H]<sup>+</sup> 427, [M + Na]<sup>+</sup> 449, [2M + H]<sup>+</sup> 853, [2M + Na]<sup>+</sup> 875, [M−H]<sup>−</sup> 425, [M + HCOO−] − 471.

1H NMR (500 MHz, CDCl3) δ 7.48 (d, *J* = 11.2 Hz, 1H), 7.45 (s, 1H), 7.31–7.28 (m, 1H), 6.67 (d, *J* = 7.3 Hz, 1H), 6.59 (d, *J* = 11.3 Hz, 1H), 6.53 (s, 1H), 4.75–4.63 (m, 1H), 3.94 (s, 3H), 3.89 (s, 3H), 3.63 (s, 3H), 3.10 (d, *J* = 5.4 Hz, 3H), 2.53–2.43 (m, 2H), 2.38–2.32 (m, 1H), 2.28–2.20 (m, 1H), 1.93–1.82 (m, 1H), 1.14 (t, *J* = 6.7 Hz, 6H).

13C NMR (126 MHz, CDCl3) δ 176.86, 175.21, 155.19, 152.85, 151.15, 141.56, 139.08, 134.55, 130.09, 126.97, 123.07, 107.91, 107.22, 61.51, 61.45, 56.13, 51.96, 37.56, 35.21, 30.19, 29.54, 19.70, 19.60.

#### Compound **13**

Yellow solid, yield 86%.

ESI-MS for C27H27ClN2O5 (*m*/*z*): [M + H]<sup>+</sup> 495/497, [M + Na]<sup>+</sup> 517, [2M + H]<sup>+</sup> 989/991, [M-H]<sup>−</sup> 493/495, [M + HCOO−] − 540.

1H NMR (500 MHz, CD2Cl2) δ 8.12 (d, *J* = 7.5 Hz, 1H), 7.60 (s, 1H), 7.42–7.39 (m, 2H), 7.29–7.22 (m, 2H), 7.19 (t, *J* = 7.5 Hz, 1H), 7.06 (t, *J* = 7.4 Hz, 1H), 6.60 (s, 1H), 6.53 (d, *J* = 11.3 Hz, 1H), 4.88–4.83 (m, 1H), 3.91 (s, 3H), 3.89 (s, 3H), 3.71 (s, 3H), 3.03 (d, *J* = 5.2 Hz, 3H), 2.52–2,48 (m, 1H), 2.42–2.31 (m, 2H), 2.14–2.04 (m, 1H).

13C NMR (126 MHz, CD2Cl2) δ 174.51, 165.99, 155.24, 153.11, 150.94, 150.51, 141.52, 139.19, 135.71, 134.71, 130.64, 130.43, 130.01, 129.60, 129.58, 126.83, 126.79, 123.26, 107.90, 107.46, 61.08, 61.07, 56.02, 52.86, 37.83, 30.11, 29.35.

#### Compound **15**

Yellow solid, yield 23%.

ESI-MS for C26H27N3O5 (*m*/*z*): [M + H]<sup>+</sup> 462, [M + Na]<sup>+</sup> 484, [2M + H]<sup>+</sup> 923, [2M + Na]<sup>+</sup> 945, [M-H]− 460, [M + HCOO−] − 506.

1H NMR (500 MHz, CDCl3) δ 9.35 (d, *J* = 7.0 Hz, 1H), 9.07 (d, *J* = 1.8 Hz, 1H), 8.48 (dd, *J* = 4.8, 1.5 Hz, 1H), 8.13–8.06 (m, 1H), 7.65 (s, 1H), 7.51 (d, *J* = 11.2 Hz, 1H), 7.31 (q, *J* = 5.1 Hz, 1H), 7.05 (dd, *J* = 7.9, 4.9 Hz, 1H), 6.61 (d, *J* = 11.4 Hz, 1H), 6.54 (s, 1H), 4.99–4.86 (m, 1H), 3.95 (s, 3H), 3.89 (s, 3H), 3.70 (s, 3H), 3.08 (d, *J* = 5.4 Hz, 3H), 2.51–2.45 (m, 1H), 2.40–2.34 (m, 1H), 2.33–2.27 (m, 1H), 2.17–2.09 (m, 1H).

13C NMR (126 MHz, CDCl3) δ 174.69, 165.09, 155.37, 153.01, 151.82, 151.42, 151.15, 148.87, 141.59, 139.62, 134.89, 134.61, 130.41, 129.34, 126.86, 123.08, 122.79, 108.49, 107.27, 61.53, 61.48, 56.15, 53.22, 36.83, 30.28, 29.58.

#### Compound **16**

Yellow solid, yield 39%.

ESI-MS for C26H27N3O5 (*m*/*z*): [M + H]<sup>+</sup> 462, [M + Na]<sup>+</sup> 484, [2M + H]<sup>+</sup> 923, [2M + Na]<sup>+</sup> 945, [M-H]− 460, [M + HCOO−] − 506.

1H NMR (500 MHz, CDCl3) δ 9.71 (d, *J* = 6.6 Hz, 1H), 8.37 (d, *J* = 5.8 Hz, 2H), 7.69 (s, 1H), 7.64 (d, *J* = 5.8 Hz, 2H), 7.54 (d, *J* = 11.2 Hz, 1H), 7.32–7.28 (m, 1H), 6.65 (d, *J* = 11.4 Hz, 1H), 6.53 (s, 1H), 4.98–4.81 (m, 1H), 3.95 (s, 3H), 3.87 (s, 3H), 3.71 (s, 3H), 3.10 (d, *J* = 5.4 Hz, 3H), 2.50–2.41 (m, 1H), 2.41–2.25 (m, 2H), 2.16–2.11 (m, 1H).

13C NMR (126 MHz, CDCl3) δ 174.51, 164.91, 155.37, 153.05, 151.64, 151.17, 150.18, 141.59, 140.46, 139.81, 134.59, 130.64, 126.78, 122.68, 120.99, 108.68, 107.27, 61.55, 61.47, 56.14, 53.46, 36.50, 30.29, 29.60.

#### Compound **18**

Yellow solid, yield 38%.

ESI-MS for C22H29N3O6S (*m*/*z*): [M + H]<sup>+</sup> 464, [M + Na]<sup>+</sup> 486, [2M + H]<sup>+</sup> 927, [2M + Na]<sup>+</sup> 949, [M-H]− 462.

1H NMR (500 MHz, CDCl3) δ 7.85 (s, 1H), 7.45–7.41 (m, 2H), 6.58 (d, *J* = 11.3 Hz, 1H), 6.54 (s, 1H), 6.49–6.46 (m, 1H), 4.33–4.19 (m, 1H), 3.93–3.89 (m, 6H), 3.57 (s, 3H), 3.10 (d, *J* = 5.1 Hz, 3H), 2.58 (s, 6H), 2.49–2.39 (m, 2H), 2.35–2.26 (m, 1H), 2.01–1.90 (m, 1H).

13C NMR (126 MHz, CDCl3) δ 174.73, 155.46, 153.08, 150.77, 150.08, 141.39, 139.41, 134.59, 129.64, 126.34, 124.53, 108.22, 107.43, 61.34, 60.80, 56.48, 56.05, 39.91, 37.95, 30.50, 29.57.

#### Compound **21**

Yellow solid, yield 83%.

ESI-MS for C27H27F3N2O6S (*m*/*z*): [M + H]<sup>+</sup> 565, [M + Na]<sup>+</sup> 587, [2M + H]<sup>+</sup> 1129, [2M + Na]<sup>+</sup> 1151, [M-H]− 563.

1H NMR (500 MHz, CDCl3) δ 8.72 (d, *J* = 7.5 Hz, 1H), 8.07 (s, 1H), 7.92 (s, 1H), 7.84 (d, *J* = 8.2 Hz, 2H), 7.53 (d, *J* = 8.3 Hz, 2H), 7.48 (d, *J* = 11.5 Hz, 1H), 6.66 (d, *J* = 11.8 Hz, 1H), 6.46 (s, 1H), 4.16 (q, *J* = 8.8 Hz, 1H), 3.91 (s, 3H), 3.86 (s, 3H), 3.58 (s, 3H), 3.08 (d, *J* = 5.2 Hz, 3H), 2.45–2.31 (m, 1H), 2.31–2.18 (m, 2H), 2.16–2.10 (m, 1H).

13C NMR (126 MHz, CDCl3) δ 170.13, 155.43, 153.53, 150.65, 150.15, 144.53, 141.35, 134.75, 133.34, 131.89, 127.57, 125.82, 125.79, 125.49, 124.38, 123.51, 122.21, 111.26, 107.42, 61.29, 60.95, 56.63, 56.04, 38.98, 30.26, 29.78.

3.3.4. General Procedure for the Synthesis of Colchicine Derivatives **5**–**7**, **9**–**12** and **14**

Compounds **5**–**7**, **9**–**12** and **14** were obtained directly from compound **3**. To a solution of compound **3** (1.0 equiv.) in DCM corresponding carboxylic acid (1.1 equiv.) and 1-(3-dimethylaminopropyl)- 3-ethylcarbodiimide hydrochloride (EDCI, 1.1 equiv.) were added. Reaction progress was monitored by LC-MS. Then the reaction mixture was diluted with EtOAc, washed with H2O, 1M K2CO3, brine and dried over Na2SO4. The residue was purified using column flash chromatography (silica gel; DCM/MeOH) and next lyophilized from dioxane to give respective compound.

#### Compound **5**

Yellow solid, yield 93%.

ESI-MS for C24H30N2O5 (*m*/*z*): [M + H]<sup>+</sup> 427, [M + Na]<sup>+</sup> 449, [2M + H]<sup>+</sup> 853, [2M + Na]<sup>+</sup> 875, [M-H]− 425, [M + HCOO−] − 471.

1H NMR (500 MHz, CDCl3) δ 7.88 (s, 1H), 7.51 (s, 1H), 7.45 (d, *J* = 11.1 Hz, 1H), 7.27–7.25 (m, 1H), 6.56 (d, *J* = 11.3 Hz, 1H), 6.51 (s, 1H), 4.76–4.68 (m, 1H), 3.92 (s, 3H), 3.87 (s, 3H), 3.61 (s, 3H), 3.08 (d, *J* = 5.4 Hz, 3H), 2.45–2.41 (m, 1H), 2.35–2.27 (m, 1H), 2.25–2.17 (m, 3H), 1.91 –1.85 (m, 1H), 1.65–1.55 (m, 2H), 0.87 (t, *J* = 7.4 Hz, 3H).

13C NMR (126 MHz, CDCl3) δ 175.10, 172.96, 155.22, 152.87, 151.43, 151.13, 141.52, 139.20, 134.61, 130.31, 126.94, 123.10, 108.13, 107.22, 61.45, 56.13, 52.21, 38.22, 37.45, 30.18, 29.54, 19.09, 13.93.

#### Compound **6**

Yellow solid, yield 73%.

ESI-MS for C25H32N2O5 (*m*/*z*): [M + H]<sup>+</sup> 441, [M + Na]<sup>+</sup> 463, [2M + H]<sup>+</sup> 881, [2M + Na]<sup>+</sup> 903, [M-H]− 439, [M + HCOO−] − 485.

1H NMR (500 MHz, CDCl3) δ 7.86 (s, 1H), 7.50 (s, 1H), 7.44 (d, *J* = 11.1 Hz, 1H), 7.26–7.21 (m, 1H), 6.55 (d, *J* = 11.3 Hz, 1H), 6.51 (s, 1H), 4.76–4.68 (m, 1H), 3.93 (s, 3H), 3.88 (s, 3H), 3.61 (s, 3H), 3.08 (d, *J* = 5.4 Hz, 3H), 2.45–2.41 (m, 1H), 2.36–2.30 (m, 1H), 2.25–2.18 (m, 3H), 1.93–1.84 (m, 1H), 1.58–1.51 (m, 2H), 1.30–1.22 (m, 2H), 0.81 (t, *J* = 7.3 Hz, 3H).

13C NMR (126 MHz, CDCl3) δ 175.19, 173.02, 155.19, 152.85, 151.32, 151.15, 141.54, 139.11, 134.59, 130.19, 126.98, 123.13, 107.97, 107.20, 61.44, 56.13, 52.20, 37.46, 36.05, 30.19, 29.53, 27.70, 22.46, 13.76.

#### Compound **7**

Yellow solid, yield 24%.

ESI-MS for C36H54N2O5 (*m*/*z*): [M + H]<sup>+</sup> 595, [2M + H]<sup>+</sup> 1189, [M-H]<sup>−</sup> 593, [M + HCOO−] − 639. 1H NMR (500 MHz, CDCl3) δ 8.18–7.90 (m, 2H), 7.73–7.53 (m, 2H), 6.88 (d, *J* = 10.1 Hz, 1H), 6.55 (s, 1H), 4.74 (s, 1H), 3.93 (s, 3H), 3.89 (s, 3H), 3.62 (s, 3H), 3.19 (s, 3H), 2.47 (d, *J* = 7.4 Hz, 1H), 2.39–2.08 (m, 5H), 1.54–1.49 (m, 2H), 1.28–1.16 (m, 26H), 0.86 (t, *J* = 7.0 Hz, 3H).

13C NMR (126 MHz, CDCl3) δ 175.09, 173.07, 155.23, 152.89, 151.32, 151.14, 141.55, 139.25, 134.58, 130.35, 126.91, 123.12, 108.24, 107.23, 61.45, 56.12, 52.16, 37.56, 36.39, 31.95, 30.18, 29.73, 29.69, 29.54, 29.53, 29.43, 29.39, 25.64, 22.72, 14.16.

#### Compound **9**

Yellow solid, yield 83%.

ESI-MS for C22H25ClN2O5 (*m*/*z*): [M + H]<sup>+</sup> 433/435, [M + Na]<sup>+</sup> 455/457, [2M + H]<sup>+</sup> 865, [2M + Na]<sup>+</sup> 887, [M-H]<sup>−</sup> 431.

1H NMR (500 MHz, CDCl3) δ 8.60–8.38 (m, 1H), 7.48–7.39 (m, 2H), 7.29 (q, *J* = 5.2 Hz, 1H), 6.56 (d, *J* = 11.4 Hz, 1H), 6.52 (s, 1H), 4.75–4.63 (m, 1H), 4.08–3.95 (m, 2H), 3.92 (s, 3H), 3.87 (s, 3H), 3.60 (s, 3H), 3.07 (d, *J* = 5.3 Hz, 3H), 2.50–2.43 (m, 1H), 2.40–2.31 (m, 1H), 2.28–2.21 (m, 1H), 2.04–1.93 (m, 1H).

13C NMR (126 MHz, CDCl3) δ 175.04, 166.12, 155.33, 152.97, 151.10, 150.18,141.57, 139.37, 134.41, 129.97, 126.80, 122.73, 108.18, 107.27, 61.45, 61.33, 56.14, 52.91, 42.63, 37.17, 30.02, 29.55.

#### Compound **10**

Yellow solid, yield 46%.

ESI-MS for C22H24Cl2N2O5 (*m*/*z*): [M + H]<sup>+</sup> 467/469, [M + Na]<sup>+</sup> 489/491, [2M + H]<sup>+</sup> 933/935, [2M + Na]<sup>+</sup> 957/959, [M-H]<sup>−</sup> 465/467.

1H NMR (500 MHz, CDCl3) δ 9.53 (d, *J* = 7.2 Hz, 1H), 7.56 (s, 1H), 7.53 (d, *J* = 11.2 Hz, 1H), 7.34 (q, *J* = 5.2 Hz, 1H), 6.64 (d, *J* = 11.4 Hz, 1H), 6.52 (s, 1H), 6.20 (s, 1H), 4.77–4.69 (m, 1H), 3.94 (s, 3H), 3.89 (s, 3H), 3.63 (s, 3H), 3.12 (d, *J* = 5.4 Hz, 3H), 2.46–2.44 (m, 1H), 2.38–2.23 (m, 2H), 1.97–1.91 (m, 1H).

13C NMR (126 MHz, CDCl3) δ 174.73, 164.19, 155.51, 153.12, 151.17, 150.51, 141.69, 139.81, 134.31, 130.45, 126.66, 122.71, 108.80, 107.36, 66.56, 61.52, 61.48, 56.17, 53.28, 37.17, 30.06, 29.66.

#### Compound **11**

Yellow solid, yield 89%.

ESI-MS for C24H27F3N2O5 (*m*/*z*): [M + H]<sup>+</sup> 481, [M + Na]<sup>+</sup> 503, [2M + H]<sup>+</sup> 961, [2M + Na]<sup>+</sup> 983, [M-H]− 479, [M + HCOO−] − 525.

1H NMR (500 MHz, CDCl3) δ 8.77–8.65 (m, 1H), 7.56 (s, 1H), 7.49 (d, *J* = 11.2 Hz, 1H), 7.35–7.28 (m, 1H), 6.61 (d, *J* = 11.4 Hz, 1H), 6.51 (s, 1H), 4.75–4.70 (m, 1H), 3.94 (s, 3H), 3.88 (s, 3H), 3.62 (s, 3H), 3.10 (d, *J* = 5.4 Hz, 3H), 2.56–2.46 (m, 2H), 2.45–2.27 (m, 4H), 2.25–2.20 (m, 1H), 1.90–1.84 (m, 1H).

13C NMR (126 MHz, CDCl3) δ 174.99, 169.79, 155.36, 152.97, 151.33, 151.12, 141.57, 139.53, 134.54, 130.36, 127.98, 126.81, 122.89, 108.41, 107.25, 61.46, 61.34, 56.13, 52.52, 37.37, 30.16, 29.56, 29.33, 28.17.

#### Compound **12**

Yellow solid, yield 89%.

ESI-MS for C27H28N2O5 (*m*/*z*): [M + H]<sup>+</sup> 461, [M + Na]<sup>+</sup> 483, [2M + H]<sup>+</sup> 921, [2M + Na]<sup>+</sup> 943, [M-H]− 459, [M + HCOO−] − 505.

1H NMR (500 MHz, CDCl3) δ 8.36 (d, *J* = 7.3 Hz, 1H), 7.81 (d, *J* = 7.2 Hz, 2H), 7.68 (s, 1H), 7.49 (d, *J* = 11.2 Hz, 1H), 7.29–7.23 (m, 2H), 7.16 (t, *J* = 7.7 Hz, 2H), 6.57 (d, *J* = 11.4 Hz, 1H), 6.53 (s, 1H), 4.99–4.93 (m, 1H), 3.96 (s, 3H), 3.89 (s, 3H), 3.70 (s, 3H), 3.06 (d, *J* = 5.4 Hz, 3H), 2.50–2.45 (m, 1H), 2.41–2.37 (m, 1H), 2.34–2.26 (m, 1H), 2.11–2.06 (m, 1H).

13C NMR (126 MHz, CDCl3) δ 175.02, 167.10, 155.26, 152.91, 151.30, 151.20, 141.57, 139.18, 134.65, 133.84, 131.31, 130.17, 128.28, 127.17, 127.01, 123.23, 108.01, 107.28, 61.53, 61.49, 56.14, 52.85, 37.19, 30.30, 29.53.

#### Compound **14**

Yellow solid, yield 57%.

ESI-MS for C27H27FN2O5 (*m*/*z*): [M + H]<sup>+</sup> 479, [M + Na]<sup>+</sup> 501, [2M + H]<sup>+</sup> 957, [2M + Na]<sup>+</sup> 979, [M-H]− 477, [M + HCOO−] − 523.

1H NMR (500 MHz, CDCl3) δ 8.75 (s, 1H), 7.68 (s, 1H), 7.59 (d, *J* = 7.5 Hz, 1H), 7.53–7.47 (m, 2H), 7.31–7.26 (m, 1H), 7.10–7.05 (m, 1H), 6.97–6.92 (m, 1H), 6.61 (d, *J* = 11.4 Hz, 1H), 6.55 (s, 1H), 4.99–4.90 (m, 1H), 3.96 (s, 3H), 3.90 (s, 3H), 3.70 (s, 3H), 3.09 (d, *J* = 5.4 Hz, 3H), 2.53–2.47 (m, 1H), 2.43–2.37 (m, 1H), 2.35–2.27 (m, 1H), 2.18–2.10 (m, 1H).

13C NMR (126 MHz, CDCl3) δ 174.84, 165.56, 155.31, 152.98, 151.38, 151.20, 141.59, 139.42, 134.63, 130.36, 129.84, 126.92, 123.06, 122.53, 118.29, 118.12, 114.70, 114.52, 108.29, 107.27, 61.52, 61.50, 56.15, 53.09, 37.00, 30.31, 29.56.

#### 3.3.5. Synthesis of **17**

To a solution ofcompound**3**(1.0equiv.) inDCM,*N*-(*tert*-Butoxycarbonyl)-*N*-[4-(dimethylazaniumylidene)- 1,4-dihydropyridin-1-ylsulfonyl]azanide (4.0 equiv.) was added and the mixture was stirred at RT. Reaction progress was monitored by LC-MS. Then the reaction mixture was diluted with EtOAc, washed with H2O, 10% citric acid, 1M K2CO3, brine and dried over Na2SO4. The residue was purified using column flash chromatography (silica gel; EtOAc/hexanes). To the obtained compound, 4M HCl/EtOAc was added and the reaction mixture was stirred at RT overnight. Next the solvent was removed under reduced pressure and the residue was purified using preparative HPLC (MeCN/H2O) and lyophilized from dioxane to give the pure hydrochloride product **17** as a yellow solid with a yield of 21%.

ESI-MS for C20H25N3O6S (*m*/*z*): [M + H]<sup>+</sup> 436, [M + Na]<sup>+</sup> 458, [2M + H]<sup>+</sup> 871, [2M + Na]<sup>+</sup> 893, [M-H]− 434.

1H NMR (500 MHz, CDCl3) δ 8.19 (s, 2H), 7.50–7.39 (m, 2H), 6.66 (s, 1H), 6.57 (s, 1H), 4.41–4.32 (m, 1H), 3.94 (m, 6H), 3.56 (s, 3H), 3.11 (s, 3H), 2.59–2.46 (m, 2H), 2.56–2.49 (s, 1H), 2.21 (s, 1H).

#### 3.3.6. Synthesis of **19**

To a solution of compound **3** (1.0 equiv.) and Et3N (3.0 equiv.) in DCM in an ice bath, 2-chloroethanesulfonyl chloride (1.2 equiv.) diluted with DCM was slowly added. Next, the ice bath was removed and the reaction mixture was stirred at RT. Reaction progress was monitored by LC-MS. Then the reaction mixture was diluted with EtOAc, washed with 1M K2CO3, brine and dried over Na2SO4. The residue was purified using column flash chromatography (silica gel; EtOAc/MeOH) and lyophilized from dioxane to give the pure product **19** as a yellow solid with a yield of 64%.

ESI-MS for C22H26N2O6S (*m*/*z*): [M + H]<sup>+</sup> 447, [2M + H]<sup>+</sup> 893, [2M + Na]<sup>+</sup> 915, [M-H]<sup>−</sup> 445.

1H NMR (500 MHz, (CD3)2SO) δ 8.26 (d, *J* = 7.5 Hz, 1H), 8.04 (s, 1H), 7.41 (s, 1H), 7.23 (d, *J* = 11.2 Hz, 1H), 6.71 (s, 1H), 6.64 (d, *J* = 11.5 Hz, 1H), 6.48 (dd, *J* = 16.4, 9.9 Hz, 1H), 5.71 (dd, *J* = 21.1, 13.1 Hz, 2H), 3.84–3.76 (m, 4H), 3.73 (s, 3H), 3.37 (s, 3H), 2.95 (s, 3H), 2.49 (s, 1H), 2.11–2.01 (m, 2H), 1.88–1.82 (m, 1H).

13C NMR (126 MHz, (CD3)2SO) δ 173.10, 155.58, 153.04, 150.65, 149.28, 141.12, 139.36, 137.66, 134.78, 128.80, 126.48, 125.64, 123.98, 108.88, 108.07, 61.19, 60.73, 56.26, 55.99, 38.70, 29.82.

#### 3.3.7. Synthesis of **20**

Compound **20** was obtained directly from compound **19**. To **19** in DCM, morpholine was added in excess. Reaction progress was monitored by LC-MS. Then the mixture was diluted with EtOAc, washed with H2O, brine and dried over Na2SO4. The residue was purified using column flash chromatography (silica gel; DCM/MeOH) and subsequently lyophilized from dioxane to give the pure product **20** as a yellow solid with a yield of 63%.

ESI-MS for C26H35N3O7S (*m*/*z*): [M + H]<sup>+</sup> 534, [2M + H]<sup>+</sup> 1067, [M-H]<sup>−</sup> 532.

1H NMR (500 MHz, CDCl3) δ 7.72 (s, 1H), 7.43 (d, *J* = 11.2 Hz, 1H), 7.40–7.38 (m, 1H), 6.84–6.50 (m, 1H), 6.56 (d, *J* = 11.3 Hz, 1H), 6.53 (s, 1H), 4.48–4.35 (m, 1H), 3.93 (s, 3H), 3.90 (s, 3H), 3.63 (t, *J* = 4.5 Hz, 4H), 3.58 (s, 3H), 3.17–3.11 (m, 1H), 3.10 (d, *J* = 5.4 Hz, 3H), 3.07–2.99 (m, 1H), 2.83–2.76 (m, 1H), 2.75–2.69 (m, 1H), 2.51–2.33 (m, 7H), 1.99–1.91 (m, 1H).

13C NMR (126 MHz, CDCl3) δ 175.05, 155.45, 153.09, 150.89, 149.61, 141.54, 139.38, 134.35, 129.35, 129.06, 128.25, 126.38, 124.07, 108.00, 107.38, 61.37, 61.02, 56.09, 55.65, 53.37, 52.68, 51.08, 39.95, 30.38, 29.56.

#### *3.4. Single Crystal X-ray Measurement*

The single crystals of colchicine derivatives (**6**, **11**, **12**, **14**, **15**, **16**, **18** and **19**) were used for data collection on a four-circle KUMA KM4 diffractometer equipped with two-dimensional CCD area detector at room (295(1) K) and low temperature (100(1) K. The graphite monochromatized Mo-Kα radiation (λ = 0.71073 Å) and the ω-scan technique (Δω = 1◦) were used for data collection. Lattice parameters were refined by the least-squares methods at all reflection positions. One image was monitored as a standard after every 40 images for a control of the crystal stability. Data collection and

reduction along with absorption correction were performed using the CrysAlis software package [60]. The structures were solved by direct methods using SHELXT [61] giving positions of almost all non-hydrogen atoms. The structures were refined using SHELXL–2018 [62] with the anisotropic thermal displacement parameters. Hydrogen atoms were refined as rigid. Visualizations of the structures were made with the Diamond 3.0 program [63]. Details of the data collection parameters, crystallographic data and final agreement parameters are collected in Supplementary Table S1. The structures have been deposited with the Cambridge Crystallographic Data Center in the CIF format, no. CCDC 1980349–1980354 for **6**, **11**, **12**, CCDC 1980341–1980344 for **14** and **15**, CCDC 1980339–1980340 for **16** and CCDC 1980345–1980348 for **18** and **19** at 100 K and RT, respectively. Copies of this information can be obtained free of charge from The Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: +44 1223 336 033); email: deposit@ccdc.cam.ac.uk or www:http://www.ccdc.cam.ac.uk).

#### *3.5. DFT Molecular Modeling*

Molecular orbital calculations with full geometry optimization of colchicine derivatives (**6**, **11**, **12**, **14**, **15**, **16**, **18** and **19**) were performed with the Gaussian09 program package [52]. All calculations were carried out with the DFT level using the Becke3-Lee–Yang–Parr correlation functional (B3LYP) [53–56] with the 6–31+G basis set assuming the geometry resulting from the X-ray diffraction study as the starting structure. As convergence criteria, the threshold limits of 0.00025 and 0.0012 a.u. were applied for the maximum force and the displacement, respectively. The three-dimensional molecular electrostatic potential (3D MESP) maps are obtained on the basis of the DFT (B3LYP/6–31G) optimized. The calculated 3D MESP is mapped onto the total electron density isosurface (0.008 eÅ-3) for each molecule. The color code of MESP maps is in the range of <sup>−</sup>0.05 eÅ−<sup>1</sup> (red) to <sup>+</sup>0.05 eÅ−<sup>1</sup> (blue).

#### *3.6. In Vitro Antiproliferative Activity*

#### 3.6.1. Cell Lines and Culturing Conditions

Four human cancer cell lines and one murine normal cell line were used to evaluate antiproliferative activity of colchicine and its derivatives **1**–**21**: human lung adenocarcinoma (A549), human breast adenocarcinoma (MCF-7), human colon adenocarcinoma cell lines sensitive and resistant to doxorubicin (LoVo) and (LoVo/DX) respectively, and normal murine embryonic fibroblast cell line (BALB/3T3). The A549 cell line was purchased from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK). The MCF-7, LoVo and LoVo/DX cell lines was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). All the cell lines are maintained in the Institute of Immunology and Experimental Therapy (IIET), Wroclaw, Poland.

Human lung adenocarcinoma cell line was cultured in a mixture of OptiMEM and RPMI 1640 (1:1) medium (IIET, Wroclaw, Poland), supplemented with 5% fetal bovine serum HyClone (GE Healthcare, USA) and 2 mM L-glutamine (Sigma-Aldrich, Germany). Human breast adenocarcinoma cell line was cultured in a mixture of Eagle medium (IIET, Wroclaw, Poland), supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 8 μg/mL insulin and 1% amino acids (Sigma-Aldrich, Germany). Human colon adenocarcinoma cell lines were cultured in a mixture of OptiMEM and RPMI 1640 (1:1) medium (IIET, Wroclaw, Poland), supplemented with 5% fetal bovine serum HyClone (GE Healthcare, USA), 2 mM l-glutamine, 1 mM sodium pyruvate (Sigma-Aldrich, Germany) and 10 μg/100 mL doxorubicin (Accord) for LoVo/DX. Murine embryonic fibroblast cells were cultured in Dulbecco medium (Gibco), supplemented with 10% fetal bovine serum (GE Healthcare, USA) and 2 mM L-glutamine (Sigma-Aldrich, Germany). All culture media contained antibiotics: 100 U/mL penicillin (Polfa-Tarchomin, Poland) and 0.1 mg/mL streptomycin (Sigma Aldrich, Germany). All cell lines were cultured during entire experiment in humid atmosphere at 37 ◦C and 5% CO2.

#### 3.6.2. Cell Viability Assays

Twenty-four hours before adding the tested compounds, all cell lines were seeded in 96-well plates (Sarstedt, Germany) in appropriate media with 0.5 <sup>×</sup> 104 cells per well for A549 cell line, 0.75 <sup>×</sup> <sup>10</sup><sup>4</sup> cells per well for MCF-7 cell line and 1.0 <sup>×</sup> 104 cells per well for LoVo, LoVo/DX and BALB/3T3 cell lines. All cell lines were exposed to each tested agent at different concentrations in the range 100–0.001 μg/mL for 72 h. The cells were also exposed to the reference drug cisplatin (Teva Pharmaceuticals, Poland) and doxorubicin (Accord Healthcare Limited, UK). Additionally, all cell lines were exposed to DMSO (solvent used for tested compounds) (POCh, Poland) at concentrations corresponding to those present in dilutions of tested agents. After 72 h sulforhodamine B assay (SRB) was performed [64].

#### SRB

After 72 h of incubation with the tested compounds, the cells were fixed in situ by gently adding of 50 μL per well of cold 50% trichloroacetic acid TCA (POCh, Poland) and were incubated at room temperature for one hour. Then the wells were washed four times with water and air dried. Next, 50 μL of 0.1% solution of sulforhodamine B (Sigma-Aldrich, Germany) in 1% acetic acid (POCh, Poland) were added to each well and were incubated at room temperature for 0.5 h. After incubation time, unbound dye was removed by washing plates four times with 1% acetic acid, whereas the stain bound to cells was solubilized with 150 μL of 10 mM Tris base (Sigma-Aldrich, Germany). Absorbance of each solution was read from a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, USA) at the 540 nm wavelength.

Results are presented as mean IC50 (concentration of the tested compound that inhibits cell proliferation by 50%) ± standard deviation. IC50 values were calculated in Cheburator 0.4, Dmitry Nevozhay software (version 1.2.0 software by Dmitry Nevozhay, 2004–2014, http://www.cheburator. nevozhay.com, freely available for each experiment [65]. Compounds at each concentration were tested in triplicates in a single experiment and each experiment was repeated at least three times independently. Results are summarized in Table 2.

#### *3.7. Molecular Docking Studies*

#### 3.7.1. Ligand Preparation

The ligand structures were prepared using Ligprep from the Schrödinger suite [66]. Conformations and tautomeric states were assigned to the ligands by following the ligand preparation protocol implemented in Schrödinger suite with default settings. LigPrep generates variants of the same ligand with different tautomeric, stereochemical, and ionization properties.

#### 3.7.2. Tubulin Model

The tubulin crystal structures available in the PDB are those for bovine protein. The bovine tubulin structure of tubulin (PDB ID: 1SA0) [67] was used as a template to construct the homology model of human αβ-tubulin isotypes (βI (UniProtKb: P07437), which is the most abundant isotype in most tumors using the Molecular Operating Environment (MOE) software package [68]. The sequence corresponding to the gene TUBA1A (UniProt ID: Q71U36) was chosen as a reference sequence for human tubulin, whereas gene TUBB associated to I isoform (UniProt ID: P07437) was chosen for human tubulin. Homology modeling was performed using MOE by setting the number of generated models to 10 and by selecting the final model based on MOE's generalized Born/volume integral (GB/VI) scoring function. The models used in the simulations performed for the present study were developed earlier and described in our previous publications [69]. These earlier investigations can also be considered to be validations of the computational model employed here since they were directly compared to the corresponding experimental data.

#### 3.7.3. Molecular Dynamics Simulations

The missing hydrogens for heavy atoms were added using the tLEAP module of AMBER 14 with the AMBER14SB force field [70]. The protonation states of all ionizable residues were determined at pH = 7 using the MOE program [68]. Each protein model was solvated in a 12 Å box of TIP3P water. In order to bring the salt concentration to the physiological value of 0.15 M, 93 Na<sup>+</sup> ions and 57 Cl<sup>−</sup> ions were added. Minimization of the structure was carried out in two steps, using the steepest descent and conjugate gradient methods successively. At first, minimization was made in 2 ps on solvent atoms only, by restraining the protein-ligand complex. Next, minimization was run without the restraint in 10 ps. After minimization, the molecular dynamics (MD) simulations were carried out in three steps: heating, density equilibration, and production. At first, each solvated system was heated to 298 K for 50 ps, with weak restraints on all backbone atoms. Next, density equilibration was carried out for 50 ps of constant pressure equilibration at 298 K, with weak restraints. Finally, MD production runs were performed for all systems for 70 ns. The root-mean-square deviation (RMSD) of both the entire tubulin structure and the colchicine binding site were found to reach a plateau after 40 ns. Clustering analysis of the last 30 ns of the generated MD trajectory was carried out using the Amber's CPPTRAJ program [71] to identify representative conformations of the tubulin dimer. Clustering was made via the hierarchical agglomerative approach using the RMSD of atoms in the colchicine binding site as a metric. An RMSD cutoff of 1.0 Å was set to differentiate the clusters. On the basis of the clustering analysis, three representative structures of the tubulin dimer were found. The docking was performed on all the three representative structures and the one with the highest docking score was selected, which was the largest cluster (about 70% of the simulation) conformation of the tubulin structure. During the modeling, the cofactors including GTP, GDP, colchicine, and the magnesium ion located at the interface between α–and β–monomers were kept as part of the environment and included in the refinement step.

#### 3.7.4. Docking Simulations

We used the AutoDock Vina [72] program to predict the binding pose of the ligands under flexible ligand and rigid receptor conditions. Dockbox package was used to facilitate preparing docking inputs and post-processing of the docking results [73]. Docking simulations performed with a cubic box (size 30.0 Å) were centered at the center of binding pockets and the docking was run separately on tubulin structure. Every generated pose was energy-minimized using Amber14 by keeping the protein fixed and was re-scored using the MOE's GBVI/WSA dG scoring function [68]. No constraints were applied in the docking studies. For each compound/protein-structure pair, the pose with the best score was identified and used as an initial configuration for molecular mechanics Gibbs–Boltzmann surface area MM/GBSA computations.

#### 3.7.5. Binding Energy and Pairwise Per-Residue Free Energy Decomposition Calculations Using MM/GBSA Method

The MM/GBSA technique is used to calculate the free energy associated with the binding of double modified colchicine amides and sulfonamides [74]. This method combines molecular mechanics with continuum solvation models. We performed MM-GBSA integrated in Amber. The binding free energy is estimated as:

$$
\Delta G\_{\text{bind}} = \overline{G}\_{\text{complex}} - \left[ \overline{G}\_{\text{protein}} + \overline{G}\_{\text{ligand}} \right] \tag{1}
$$

where G is the average free energy of the complex, protein, and ligand, are calculated according to the equation:

$$
\overline{G} = \overline{E}\_{M+} \overline{G}\_{\text{solution}} - T\overline{S} \tag{2}
$$

where EMM are determined with the SANDER program and represent the internal energy (bond, angle, and dihedral), van der Waals and electrostatic interactions (see Equation (3)). TS is the entropy contribution estimated using normal mode (nmode) analysis.

$$
\overline{\mathbf{E}}\_{\text{M}} = \overline{\mathbf{E}}\_{\text{int}} + \overline{\mathbf{E}}\_{\text{elec}} + \overline{\mathbf{E}}\_{\text{vdW}} \tag{3}
$$

The solvation free energy can be calculated as the sum of polar and nonpolar contributions. The polar parts are obtained by using the generalized-born (GB) model—resulting in the MM/GBSA method, whereas the nonpolar terms are estimated from a linear relation (Equation (4)) to the solvent accessible surface area (SASA).

$$G\_{non-polar} = \gamma \, SASA + b \tag{4}$$

In the present study, a 2 ns-duration MD trajectory was run in TIP3P water using Amber14, for every top pose generated at the end of the docking step. It is worth noting that to assess the performance of MM/GBSA methodology [75], we evaluated the prediction accuracy of this method by various simulation protocols including 1 ns MD production calculations using PDBbind data set. Too long an MD simulation could be prejudicious for the overall success of the MM/GBSA method. According to this study and the common practice to calculate binding energies using MM/GBSA, we have decided to run MD production simulation for 2 ns. The MM/GBSA calculations were performed on a subset of 200 frames collected at regular time intervals from the trajectory. For PB calculations, an ionic strength of 0.0 nM (istrng = 0.0) and a solvent probe radius of 1.6 Å (prbrad = 1.6) were used. For GB calculations, the igb parameter was set to 5 that corresponds to a modified GB model equivalent to model II in reference. Pairwise free energy decomposition analysis was performed using the MM/GBSA decomposition process by the MM/GBSA program in AMBER 14 to compute the interaction between the inhibitors and each residue. For each of the tested compounds, the active residues estimated from MM/GBSA decomposition process and the best GB score out of the trajectories associated with the representative structures of the tubulin dimer were collected and are reported in Table 3 and Figure 4.

#### *3.8. Calculation clogP*

We used the Molinspiration online database (http://www.molinspiration.com, free of charge) to predict the *clogP* values for all synthesized compounds and collected them in Table 3 [76].

#### **4. Conclusions**

In an attempt to discover novel potent inhibitors of microtubule dynamics, eighteen novel double modified colchicine analogs comprising methylamine substituent at position C10 of ring C and amide or sulfonamide substituents at position C7 of ring B were successfully synthesized, purified and their structures were confirmed by spectroscopic analyses as well as X-ray measurements. Four human cancer cell lines (A549, MCF-7, LoVo, LoVo/DX) were used to evaluate the anticancer potency of all synthesized compounds.

Compared with unmodified colchicine, the majority of its studied derivatives exhibited excellent potency against A549, MCF-7, and LoVo cell lines. The antiproliferative activity of colchicine derivatives was in the nanomolar range and they also were characterized by lower IC50 values also than doxorubicin and cisplatin, commonly used as antitumor agents in cancer chemotherapy. The preliminary SAR revealed that the type of substituent at position C7 in ring B and the presence of –NHCH3 group at position C10 in ring C of colchicine **1** were of cardinal importance to the compounds' cytotoxicity.

The calculated values of the selectivity index (SI) clearly show that it is possible to design more selective compounds than colchicine **1**. The most active double-modified colchicine derivatives were the compounds that included chloroacetamide and dichloroacetamide (**9**, **10**) and 2–chlorobenzamide (**13**) moiety (Table 2). However, in vitro tests showed that they did not have high SI values (except towards the LoVo cells) and *in vivo* testing should be carried out to determine their therapeutic potential. But of all tested compounds, 4,4,4-trifluorobutyric amide of 10-*N*-methylaminocolchicine **11** was the most prominent. Compound **11** showed high activity towards A549, MCF7 and LoVo cell lines, with antiproliferative IC50 values ranging between 10.4 and 14.6 nM as well as SI values higher than 5 for these three cell lines. The most selective towards MCF-7 cell line was compound **17** (SI was almost 10) and towards LoVo cell line the most selective were compounds **7**, **9**, **10**, and **13** (SI were over 6). So we conclude that the appropriate modification of colchicine molecule and synthesis of its analogs might overcome the toxicity, which is a major challenge in designing a potential colchicine-based drug candidate.

Compound **11** was subsequently identified as the that with potentially the best therapeutic index and therefore the most promising candidate for further development, as it showed strong in vitro anticancer activities with high SI against three human cancer cell lines. However, further in vivo studies should be conducted for the successful development of this compound.

Molecular docking study showed the ability of the obtained colchicine derivatives to bind into the active sites of βI-tubulin isotype with the well-defined binding modes. It is worthy to mention that 10-*N*-methylaminocolchicine based compounds serve as useful templates for the further development of the anticancer and antimitotic agents. Further evaluation should help to find more detailed structure-activity relationships of microtubule-targeting drugs and CBS inhibitors, which can help in rational drug design in future.

**Supplementary Materials:** The following are available online, Table S1: Details of the data collection parameters, crystallographic data and the final agreement parameters. (for compounds **6**, **11**, **12**, **14**, **15**, **16**, **18** and **19**); Table S2: Optimized parameters for colchicine derivatives (**6**, **11**, **12**, **14**, **15**, **16**, **18** and **19**), Figure S1: The LC-MS chromatogram and mass spectra of **2**; Figure S2: The 1H NMR spectrum of **2** in CDCl3; Figure S3: The 13C NMR spectrum of **2** in CDCl3; Figure S4: The LC-MS chromatogram and mass spectra of **3**; Figure S5: The 1H NMR spectrum of **3** in CDCl3; Figure S6: The 13C NMR spectrum of **3** in CDCl3; Figure S7: The LC-MS chromatogram and mass spectra of **4**; Figure S8: The 1H NMR spectrum of **4** in CDCl3, Figure S9: The 13C NMR spectrum of **4** in CDCl3, Figure S10: The LC-MS chromatogram and mass spectra of **5**; Figure S11: The 1H NMR spectrum of **5** in CDCl3, Figure S12: The 13C NMR spectrum of **5** in CDCl3; Figure S13: The LC-MS chromatogram and mass spectra of **6**; Figure S14: The 1H NMR spectrum of **6** in CDCl3; Figure S15: The 13C NMR spectrum of **6** in CDCl3; Figure S16: The LC-MS chromatogram and mass spectra of **7**; Figure S17: The 1H NMR spectrum of **7** in CDCl3; Figure S18: The 13C NMR spectrum of **7** in CDCl3; Figure S19: The LC-MS chromatogram and mass spectra of **8**; Figure S20: The 1H NMR spectrum of **8** in CDCl3; Figure S21:. The 13C NMR spectrum of **8** in CDCl3; Figure S22: The LC-MS chromatogram and mass spectra of **9**; Figure S23: The 1H NMR spectrum of **9** in CDCl3; Figure S24: The 13C NMR spectrum of **9** in CDCl3; Figure S25: The LC-MS chromatogram and mass spectra of **10**; Figure S26: The 1H NMR spectrum of **10** in CDCl3; Figure S27: The 13C NMR spectrum of **10** in CDCl3; Figure S28. The LC-MS chromatogram and mass spectra of **11**; Figure S29. The 1H NMR spectrum of **11** in CDCl3; Figure S30: The 13C NMR spectrum of **<sup>11</sup>** in CDCl3; Figure S31. The LC-MS chromatogram and mass spectra of **<sup>12</sup>**; Figure S32: The 1H NMR spectrum of **<sup>12</sup>** in CDCl3; Figure S33: The 13C NMR spectrum of **<sup>12</sup>** in CDCl3; Figure S34: The LC-MS chromatogram and mass spectra of **13**; Figure S35: The 1H NMR spectrum of **13** in CD2Cl2; Figure S36: The 13C NMR spectrum of **<sup>13</sup>** in CD2Cl2. Figure S37: The LC-MS chromatogram and mass spectra of **<sup>14</sup>**; Figure S38: The 1H NMR spectrum of **<sup>14</sup>** in CDCl3; Figure S39: The 13C NMR spectrum of **<sup>14</sup>** in CDCl3; Figure S40: The LC-MS chromatogram and mass spectra of **15**; Figure S41: The 1H NMR spectrum of **15** in CDCl3; Figure S42: The 13C NMR spectrum of **15** in CDCl3; Figure S43: The LC-MS chromatogram and mass spectra of **16**; Figure S44: The 1H NMR spectrum of **16** in CDCl3. Figure S45: The 13C NMR spectrum of **16** in CDCl3; Figure S46: The LC-MS chromatogram and mass spectra of **17**; Figure S47: The 1H NMR spectrum of **17** in CDCl3; Figure S48: The LC-MS chromatogram and mass spectra of **18**;Figure S49: The 1H NMR spectrum of **18** in CDCl3; Figure S50: The 13C NMR spectrum of **18** in CDCl3; Figure S51: The LC-MS chromatogram and mass spectra of **19**; Figure S52: The 1H NMR spectrum of **19** in (CD3)2SO; Figure S53: The 13C NMR spectrum of **19** in (CD3)2SO; Figure S54: The LC-MS chromatogram and mass spectra of **20**; Figure S55**:** The 1H NMR spectrum of **20** in CDCl3. Figure S56: The 13C NMR spectrum of **<sup>20</sup>** in CDCl3; Figure S57: The LC-MS chromatogram and mass spectra of **<sup>21</sup>**; Figure S58: The 1H NMR spectrum of **<sup>21</sup>** in CDCl3. Figure S59: The 13C NMR spectrum of **<sup>21</sup>** in CDCl3; Figure S60: Molecular structure of colchicine derivatives (**6**, **11**, **12**, **14**, **15**, **16**, **18** and **19**) at 295 K and 100 K.

**Author Contributions:** Conceptualization, J.K., and A.H.; methodology, J.K., A.H., J.J., J.W. and J.A.T.; software, J.J.; M.A.; validation, A.H., J.A.T. and J.J.; formal analysis, A.H. and J.K.; investigation, J.K., E.M., J.J. and M.A; resources, A.H.; J.K. and W.M.; data curation, J.K., M.A. and E.M.; writing—original draft preparation, J.K., J.J. and M.A.; writing—review and editing, W.M., A.H. J.W. and J.A.T.; visualization, J.K., J.J. and M.A.; supervision, A.H.; project administration, A.H.; funding acquisition, A.H. and W.M. All authors have read and agreed to the published version of the manuscript.

**Acknowledgments:** Financial support by a grant No. 2016/21/B/ST5/00111 of the Polish National Science Centre (NCN) –is gratefully acknowledged. JAT gratefully acknowledges research support from NSERC and the Allard Foundation for his research.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


#### **Sample Availability:** Not available

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Overcoming Resistance to Platinum-Based Drugs in Ovarian Cancer by Salinomycin and Its Derivatives—An In Vitro Study**

**Marcin Michalak 1, Michał Stefan Lach 2,3,4,\*, Michał Antoszczak 5, Adam Huczy ´nski <sup>5</sup> and Wiktoria Maria Suchorska 2,4,\***


Received: 12 November 2019; Accepted: 24 January 2020; Published: 26 January 2020

**Abstract:** Polyether ionophore salinomycin (SAL) and its semi-synthetic derivatives are recognized as very promising anticancer drug candidates due to their activity against various types of cancer cells, including multidrug-resistant populations. Ovarian cancer is the deadliest among gynecologic malignancies, which is connected with the development of chemoresistant forms of the disease in over 70% of patients after initial treatment regimen. Thus, we decided to examine the anticancer properties of SAL and selected SAL derivatives against a series of drug-sensitive (A2780, SK-OV-3) and derived drug-resistant (A2780 CDDP, SK-OV-3 CDDP) ovarian cancer cell lines. Although SAL analogs showed less promising IC50 values than SAL, they were identified as the antitumor agents that significantly overcome the resistance to platinum-based drugs in ovarian cancer, more potent than unmodified SAL and commonly used anticancer drugs—5-fluorouracil, gemcitabine, and cisplatin. Moreover, when compared with SAL used alone, our experiments proved for the first time increased selectivity of SAL-based dual therapy with 5-fluorouracil or gemcitabine, especially towards A2780 cell line. Looking closer at the results, SAL acted synergistically with 5-fluorouracil towards the drug-resistant A2780 cell line. Our results suggest that combinations of SAL with other antineoplastics may become a new therapeutic option for patients with ovarian cancer.

**Keywords:** salinomycin; anticancer activity; overcoming drug resistance; tumor specificity; synergy; 5-fluorouracil; gemcitabine; amides/esters; ovarian cancer

#### **1. Introduction**

Despite developing new therapeutic strategies and extensive knowledge about tumor biology, ovarian cancer (OvCa) remains a leading cause of mortality among gynecologic malignancies; asymptomatic early stages and lack of ambiguous biomarkers enabling detection of the disease lead to late diagnosis, mostly at stage III and IV [1,2]. Five-year overall survival for advanced OvCa is approximately 30% [3]. Most of the patients respond well to radical surgery and either neoadjuvant or adjuvant chemotherapy. However, 75% of patients develop recurrence [4]. Prognosis for patients with the recurrent disease is poor, especially for those diagnosed with the recurrence earlier than six months after completion of the initial platinum-based therapy [5]. This state is called

platinum-resistance in opposition to platinum-sensitive patients who develop recurrence more than six months after completion of the initial therapy. The intrinsic and acquired resistance to platinum-based chemotherapy is the main reason for OvCa treatment failure [6,7].

A few theories have been proposed to explain the development of chemoresistance in human malignancies. The most likely one is related to cancer stem cell theory [8]. According to this concept, a malignant tumor consists of two populations of cells, namely cancer stem cells (CSCs) and differentiated cells. Self-renewal, repopulation, and resistance to irradiation and cytotoxic drugs are typical of the first narrow population. Contrary to that, differentiated cancer cells are sensitive to treatment and form a bulk population of the tumor cells. CSCs, similarly to pluripotent stem cells, have active developmental signaling pathways, such as Hedgehog, Notch, and Wnt/β-catenin. The latter two seem to be particularly responsible for OvCa platinum resistance [9,10]. Unfortunately, there is no single universal marker capable of distinguishing CSCs from the differentiated cancer cells. Such a marker could help to develop a highly specific targeted therapy against CSCs, which would improve OvCa patient outcome. Therefore, the main focus of OvCa therapies should be the elimination of CSCs. One of the molecules exhibiting anticancer potential and selective properties against CSCs is salinomycin (SAL, **1**, Figure 1).


**Figure 1.** Structure of salinomycin and its derivatives studied in this work.

This is a monocarboxylic polyether antibiotic naturally synthesized by *Streptomyces albus* (strain no. 80614) [11]. SAL was identified in 2009 as the most active agent among 16,000 compounds tested towards breast CSCs [12]. Since then, SAL has been found effective against many other types of cancer cells and CSCs, including those displaying multidrug resistance (MDR) and has been used in a small group of patients with advanced carcinoma of the head, neck, breast, and ovary [13]. SAL acts as a sensitizer of malignant cells to radiotherapy or chemotherapy, i.e., colchicine, doxorubicin, and etoposide [14–17].

#### **2. Results**

#### *2.1. Derivation of Cisplatin-Resistant Cell Lines*

To test the usefulness of SAL and its derivatives in overcoming cisplatin-resistance, chemoresistant OvCa sub-cell lines were established. MTT and RT-qPCR followed the cell exposure to cisplatin to confirm derivation of stable phenotype of the resistant cell lines. A2780 CDDP and SK-OV-3 CDDP lines responded with morphological changes and increased IC50 against cisplatin as compared with their parental population (Figure 2A,B). Both resistant cell lines showed also enhanced expression of ABCB1, ABCG2, and ABCC2 versus control (Figure 2C,D). ABCB4 expression boosted significantly in SK-OV-3 CDDP cell line but only slightly in A2780 CDDP cell line.

**Figure 2.** Overview of cisplatin-resistant ovarian cancer cell lines (A2780, SK-OV-3). (**A**,**B**) Morphological changes of both drug-resistant cancer sub-lines represent enlargement and slight spindle-like shape. Survival curves indicate increased IC50 for both resistant variants (RI = 18.08 for A2780; RI = 1.56 for SK-OV-3). The pictures were taken under 200× magnification. (**C**,**D**) RT-qPCR analysis of A2780 and SK-OV-3 revealed significantly increased expression of ABC drug transporters in derived resistant variants.

#### *2.2. In Vitro Activity of Cytotoxic Drugs, Salinomycin, and Its Derivatives Against OvCa Cells*

It was clearly proven that chemical modification of SAL and other polyether ionophores may not only increase the biological activity of resulting derivatives but also reduce their general toxicity [18–21]. Furthermore, SAL with a modified C1 carboxyl group (amides or esters) transports cations by a biomimetic mechanism, while chemically unmodified SAL transports cations through biological membranes via an electroneutral mechanism [22,23]. This change in ionophoretic properties may result in better biological properties of **SAL** analogs than of those with a native structure.

We devised a library of SAL derivatives based on the most active SAL amides and esters obtained in our previous studies by a chemical modification of C1 carboxyl group, i.e., amides **2** and **3**, as well as esters **5** and **6**, respectively (Figure 1) [18–20]. To expand structural diversity at C1 position and to better determine the structure-activity relationship (SAR), we additionally analyzed propargyl amide **4** and propargyl ester **7** (Figure 1), as these structures had shown promising bioactivity [19].

Data gathered in Table 1 indicate that all tested compounds exhibited biological activity against malignant cells. The effect towards ovarian A2780 cell line was distinctly better than that against metastatic ovarian SK-OV-3 cell line. Briefly, the most effective was chemically unmodified SAL, the activity of which was higher against A2780 cell line and comparable against SK-OV-3 cell line than that of reference anticancer drug—cisplatin (CDDP) (Table 1). In OvCa cell lines A2780, SK-OV-3 as well as their platinum-resistant sub-lines, all semi-synthetic derivatives of SAL (both from amide and ester series) needed significantly higher IC50 values to induce comparable biological effects than SAL itself (Table 1). The most active SAL analog was 4-fluorophenethyl amide **3** (Figure 1) but still its activity was one order of magnitude lower than that of unmodified SAL (Table 1). As expected, cisplatin-resistant sub-lines were more resistant to CDDP than both cisplatin-sensitive variants; thus, the anticancer activity of compounds **3** and **5** (Figure 1) was higher than that exhibited by CDDP towards A2780 CDDP cell line (Table 1).

However, to the best of our knowledge, there are no reports describing the effects of a dual therapy using SAL and 5-fluorouracil (5FU) or gemcitabine (GEM) towards OvCa cells. Therefore, we decided to check if SAL shows desired results when combined with these commonly used anticancer drugs. For this purpose, we prepared 1:1 molar mixtures of SAL and 5FU/GEM (**1** + 5FU and **1** + GEM, respectively) and tested their activity towards OvCa cell lines (Table 1). Interestingly, in both cases we witnessed a strong interaction between SAL and either 5FU or GEM. IC50 values for **1** + 5FU and **1** + GEM against all OvCa cell lines reached a low micromolar concentration range and were significantly lower than those exhibited by individual components (**1** and 5FU) and reference anticancer drug—CDDP (Table 1). More promising (lower) IC50 values were only obtained for GEM (Table 1), which is recommended for treatment of recurrent OvCa.

To determine the real therapeutic potential of novel anticancer drug candidates, it is necessary to check their effects (selectivity) towards normal cells. Judging by IC50 values, the tests performed in normal diploid human MRC-5 cell line suggested that all SAL derivatives were significantly less toxic towards non-malignant human cells than the chemically unmodified SAL (Table 1).


*Molecules* **2020** , *25*, 537

**Table 1.** The IC50 values estimated for ovarian cancer cell lines (A2780, SK-OV-3, both

drug-sensitive

 and

drug-resistant

 variants) and normal diploid human MRC-5

#### *2.3. Salinomycin and Its Derivatives Overcome Cisplatin Resistance and are More Selective Against Cisplatin-Resistant OvCa Cells*

To assess the effectiveness of the studied drugs in overcoming acquired cisplatin resistance, we calculated the resistance index (RI), based on the IC50 of derived cell lines (Figure 3A). The RI indicates how many more times a resistant sub-line (either A2780 CDDP or SK-OV-3 CDDP) is chemoresistant as compared with its parental cell line (either A2780 or SK-OV-3, respectively). An RI between 0 and 2 indicates that the cells are sensitive to the tested compound. An RI in the range from 2 to 10 means that the cells show moderate sensitivity to a drug. An RI above 10 indicates strong drug resistance [24]. None of the cancer cell lines developed strong resistance to the tested agents under the combinatory treatment of SAL with either 5FU or GEM (Figure 3A). Contrary to GEM, both **1** + 5FU and **1** + GEM co-treatments were capable of efficiently overcoming the drug resistance of OvCa cells, as manifested by considerably lower values of RI (Figure 3A).

Deeper analysis of RI parameters revealed that cancer cell line A2780, in opposition to SK-OV-3, developed some resistance to SAL (Figure 3A). This finding may indicate possible treatment failure on SAL clinical application. On the other hand, almost no SAL derivatives (except for **6** and **7**) developed even mild resistance during our experiments in both OvCa cell lines (Figure 3A). Generally, A2780 and SK-OV-3 cell lines turned out more sensitive to amide analogs of SAL (compounds **2**–**4**) than the corresponding ester derivatives (compounds **6**–**7**). The only exception to this rule was ester **5** (RI = 0.30 and RI = 0.50 for A2780 and SK-OV-3 cell line, respectively) (Figure 3A).

**Figure 3.** Calculated values of (**A**) the resistance indexes (RI), and (**B**) selectivity indexes (SI) of the tested compounds.

Further, to establish therapeutic potential of the tested anticancer agents, we used the difference in the antiproliferative activity towards the OvCa cell lines and the corresponding normal cell line to calculate the values of the selectivity index (SI) (Figure 3B). The SI is an important pharmaceutical parameter that facilitates the estimation of possible future clinical development; higher values of SI indicate greater anticancer specificity, and the compounds displaying an SI above 3.0 are considered highly selective agents [21,25]. Therefore, our study clearly proved that normal cells were less sensitive to SAL used alone or its combinations with other cytotoxic agents (5FU or GEM) than to amide and ester derivatives of SAL (Figure 3B).

Selectivity of SAL as well as its amide and ester analogs was very promising, especially in non-resistant and resistant to cisplatin A2780 cell lines (Figure 3B). However, the highest values of SI were noted for SAL and its 1:1 molar mixture with GEM (**1** + GEM, Figure 3B), and this effect decreased with time of exposure to the anticancer agents. On the other hand, SAL turned out more selective against SK-OV-3 cell line and its platinum-resistant variant than all analyzed amide and ester derivatives (Figure 3B). A combinatory treatment of OvCa cells involving SAL and GEM (**1** + GEM, Figure 3B) yielded very high SI values. The combination of the cytotoxic agents (regardless of their mechanism of anticancer action), either 5FU or GEM, with SAL was more effective than SAL or its derivatives used alone (Table 1 and Figure 3).

As OvCa cells seemed highly sensitive to the action of both **1** + 5FU and **1** + GEM (Table 1 and Figure 3), we decided to determine the exact effect (addictive, synergistic, or antagonistic) of the specific compound combinations. Using the 1:1 molar mixtures of SAL and 5FU or GEM, we performed standard viability assays and subjected the results to the analysis and determination of values of the combination indexes (CI) (Table 2). As previously described for a pair of drugs [26,27], CI > 1.3 indicates antagonism, CI = 1.1–1.3 indicates moderate antagonism, CI = 0.9–1.1 indicates additive effect, CI = 0.8–0.9 indicates slight synergism, CI = 0.6–0.8 indicates moderate synergism, CI = 0.4–0.6 indicates synergism, and CI = 0.2–0.4 indicates strong synergism. Interestingly, the results presented in Table 2 indicated that SAL in combination with 5FU acted synergistically (CI = 0.56) in A2780 CDDP cell line, whereas in SK-OV-3 and SK-OV-3 CDDP cell lines, they only showed an additive effect (CI = 1.09 and CI = 1.02, respectively).

**Table 2.** Calculated combination index (CI) values of simultaneously delivered salinomycin (**SAL**, **1**) and cytotoxic drugs (5-fluorouracil **5FU**, gemcitabine **GEM**) in the 1:1 molar mixtures.


After a primary screening of the compounds, SAL and its amides indicated in both OvCa cell lines variants the most promising response. To confirm their effect, Western blot analysis for B-cell lymphoma 2 (Bcl2), Bcl2 associated X protein (Bax), and caspase-3 (CASP3) was performed (Figure 4).


**Figure 4.** Western blot analysis of OvCa cell lines exposed to IC50 values of selected compounds for 72 h (**A**–**D**). The expression of anti-apoptotic (Bcl2) and apoptotic proteins (Bax and CASP3) was evaluated. The numbers describe the quantified level of band intensity normalized to expression of reference protein GAPDH and the control population.

In A2780 cells, the Bcl2 decreased in all groups in comparison with the control (Figure 4A). The lowest expression was observed in the GEM and 1 + GEM variant, but their effect was similar. The proapoptotic protein Bax also represented similar trends of expression as Bcl2. Interestingly, full form of caspase-3 (CASP3) was decreased in cells treated with SAL, 5FU, and their combination, but GEM and its combination with SAL, as well as amide derivatives of SAL, caused the enhanced expression of that protein. The antagonistic effect of combined SAL with 5FU or GEM was observed by decreasing expression of CASP3. In case of the A2780 CDDP variant, Bcl2 expression in cells treated with SAL, 1 + 5FU, GEM, and 1 + GEM was decreased (Figure 4B). Surprisingly, the SAL amides (**3**–**5**) caused increased Bcl2 expression. It is worth mentioning that a combination of 5FU with SAL caused downregulation of Bcl2. In all tested compounds the expression of Bax was inhibited in comparison with the control. Among them, a combination of SAL with 5FU caused decreased expression of Bax compared to 5FU alone. A similar effect onto Bax expression was observed in the GEM and its combination with SAL. In A2780 CDDP cell line, CASP3 expression in SAL and 5FU was decreased, and their combination caused a similar effect in comparison to control. However, the expression of that protein in GEM, 1 + GEM, 3, 4, and 5 variants was enhanced in comparison with the control. The combination of SAL with 5FU caused the enhanced expression of the full form of CASP3, which confirmed synergistic action of these compounds. On the other hand, the antagonistic effect was observed in GEM and its combination with SAL, where the cytotoxic effect was enhanced in only GEM-treated cells.

In SK-OV-3 cell line, Bcl2 expression was decreased in all studied compounds, except amide **4**, compared to control (Figure 4C). Among them, SAL, 5FU, and **1**+GEM showed the lowest expression. All tested compounds indicated decreased expression of Bax in comparison with control. The combination of SAL and 5FU did not cause the upregulation of these proteins exciding the level of compounds tested alone. The GEM indicated the highest expression level of Bax among tested molecules. Amide derivatives of SAL did not indicate increased expression of Bax in comparison with SAL. The similar tendencies of CASP3 expression were observed, excluding compound **2** and **3**, which exhibited its higher expression in comparison with SAL. These observations confirmed the antagonistic effect in studied combinations of antineoplastic agents with SAL. In the case of SK-OV-3 CDDP cell line exposed to distinct molecules, the expression of Bcl2 only in SAL and SAL combined with 5FU was upregulated in comparison with the control (Figure 4D). Only the combination of SAL with GEM caused enhanced downregulation of Bcl-2 compared to exposition to them alone. Bax expression in those cells was decreased in comparison with the control. Among tested variants, 1 + 5FU exceeded the level of its expression compared to 5FU, but not to SAL alone. Again, GEM used separately induced its highest expression among all exposed compounds. The amide derivatives of SAL indicated lower expression of Bax in comparison with SAL. The presence of Bax downregulation in cells exposed to a combination of antineoplastics with SAL confirmed their antagonism. The expression of CASP3 and the action of compounds co-cultured with SK-OV-3 CDDP cells was similar to that of Bax. These results confirmed earlier estimated effects of combined SAL with 5FU or GEM in studied cell lines.

#### **3. Discussion**

One of the major problems in ovarian cancer (OvCa) treatment is development of chemoresistant residual cancer cells. Cancer stem cell (CSC)-targeted therapies should thus be developed [5]. One of the promising compounds in this respect is salinomycin (SAL). Its short-lasting side effects and low solubility in aqueous solutions may be avoided by some chemical modifications. These modifications provide new SAL molecules with improved stability and unaffected selective properties against CSCs, particularly effective in overcoming chemoresistant forms of the disease [28,29]. However, the synthesis of selective SAL derivatives is complicated due to the presence of multiple functional groups and a sensitive tricyclic 6-6-5 bis-spiroketal ring system in SAL structure [20,29,30].

In this study, we derived cisplatin-resistant cell lines A2780 CDDP and SK-OV-3 CDDP and evaluated the antiproliferative activity of selected amides and esters of SAL, as well as SAL used alone or in combination with other commonly used cytostatic agents, such as 5-fluorouracil (5FU) and gemcitabine (GEM), against these OvCa cells. To confirm the observed effect, Western blot analysis of proteins related to apoptosis (Bcl2, Bax and CASP3) was also performed.

We obtained resistant variants of cancer cell lines, as confirmed by increased IC50 against cisplatin estimated by MTT assay. These variants also showed elevated expression of genes related to drug transporters. ABCB1, ABCB4, ABCG2, and ABCC2 are well-described markers of drug resistance development in many cancers [31–35]. These proteins are characteristic of side populations of cells that represent stem-cell-like features, and in some cancers, they are recognized as markers of CSCs. They are responsible for active efflux of many anticancer agents causing treatment failure [35–37].

Our experiments provided interesting data regarding the activity of SAL and its derivatives against platinum resistance developed in OvCa cell lines in vitro. Creation of platinum-resistant OvCa cell line (SK-OV-3) did not induce the resistance of cancer cells to the action of SAL and its derivatives. This indicated that different molecular mechanisms are responsible for either platinum or SAL resistance, and additionally SAL could be identified as an effective agent in overcoming the platinum resistance of OvCa cells. However, A2780 CDDP variant exhibited moderate sensitivity to SAL and its 2,2,2-trifluoroethyl ester derivative (compound **6**, Figure 1). This phenomenon could be related to the increased expression of ABCB1 and ABCG2 in resistant variants of A2780 cancer cell line. A study by the Boesch group revealed that in OvCa cell lines (A2780, IGROV1), selected ABCB1 and ABCG2 positive cells exhibited protective mechanisms against ionophore antibiotics, including SAL [38]. Contrary to that, a study in 2010 demonstrated that SAL treatments restored doxorubicin sensitivity in human doxorubicin-resistant epithelial OvCa cell line (A2780/ADR) exposed to doxorubicin either alone or in combination with SAL by inhibition of ABCB1 functionality [39]. Disparate results of these studies might be related to distinct co-activation of a group of genes in the same chromosomal region, where ABCB1 activation occurs by various cytotoxic agents [31].

Our experiments did not confirm the high anticancer activity of SAL derivatives reported recently in primary acute lymphoblastic leukemia (ALL) cells [20,40], which may be caused by a different type of malignancies and their adverse biology; ALL represents a hematological malignancy, while OvCa represents a malignant solid tumor. Until now, there have been only limited data showing the effects of SAL and its derivatives on OvCa cells. The recent data indicated that SAL itself affects a wide spectrum of mechanisms against OvCa biology, such as inhibition of the epithelial-mesenchymal transition (EMT) process (responsible for the development of metastatic disease), eradication of the CSC population (CD44+CD117+), and inhibition of the NF-kB signaling pathway (upregulation of proteins related to that pathway correspond with poor clinical prognosis in OvCa) [41–44]. Our study, for the first time, demonstrates that the combination of SAL with cytostatic agents (5FU and GEM) is more effective than SAL used alone or its amide and ester derivatives. 5FU is not commonly used in OvCa treatment—a few clinical trials revealed no significant improvement in clinical outcomes in advanced OvCa patients receiving 5FU combined with cisplatin or leucovorin [45–47]. Of note is that SAL acted synergistically with 5FU towards drug-resistant A2780 OvCa cell line. A similar effect was presented in studies concerning colorectal and hepatocellular carcinoma, which indicated the neutral or synergistic effect of the combination of SAL and 5FU [48,49]. In case of GEM, which is mostly used as a secondary line of chemotherapy after the development of resistant disease in OvCa, we did not observe the synergistic effect of combined SAL and GEM in both OvCa cell lines, as was found over their action in pancreatic carcinoma cell lines [50,51]. We think that one of the causes was their high molar ratio 1:1 used in our study versus 5 <sup>μ</sup>M concentration of SAL and 5 <sup>μ</sup>g·mL−<sup>1</sup> applied in the pancreatic cancer cell lines [51]. The differences between the observed effects of these two nucleoside analogs in combination with SAL might be related to a disturbance of their cellular uptake by an ion imbalance caused by SAL. The concentrative nucleoside transporters (CNT) and equilibrative nucleoside transporters (ENT) are mostly engaged in the transport of GEM, while for 5FU, only ENT proteins are involved [52–55]. The mechanism of nucleoside transport by CNT proteins is Na+-dependent, whereas in the case of ENT, they are mediated by facilitated diffusion [53,54]. SAL is responsible for the transport of potassium

and sodium cations, which could lead to disturbance of the GEM uptake through CNT transporters (indicating high affinity to GEM), decreasing its activity against cancer cells [29,54,55].

The effect of combination of SAL with GEM or 5FU was confirmed by Western blot analysis of proteins related to apoptosis (Bcl2, Bax, and CASP3). Besides the same IC50 effect at distinct concentrations of studied compounds, SAL amide derivatives (3–5) caused enhanced expression of CASP3 in A2780 and A2780 CDDP cell lines, which could suggest different or more effective induction of apoptosis by these molecules. We did not observe a similar effect in SK-OV-3 cells, which could be related to intrinsic resistance mechanisms due to their metastatic origin. However, there is a lack of detailed mechanistic studies explaining the differences between SAL and amide derivatives against OvCa cell biology; thus, their further detailed characterization of action should be performed. On the other hand, for SAL used alone, we obtained similar findings as the Parajuli group, where the A2780 CDDP variant was tested, and a decrease of the Bcl2, Bax, and CASP3 was noted at similar doses that we used in our study; however, in their study, only after 1 μM of SAL, caspases were significantly increased [44,56]. They also indicated that the cause of apoptosis is related to activation of death receptor 5 (DR5) pathway, which was observed even at low doses of SAL (0.5 μM) [56]. In contrast, the Kaplan group observed induction of apoptosis by upregulation of CASP3 in OVCAR3 cell line even at 0.1 μM of SAL [57].

In summary, all findings mentioned above corresponded well with our results. Potent anticancer activity of SAL, lack of SAL resistance in platinum-resistant OvCa cell lines, and reversible SAL resistance imply the possible application of SAL in overcoming either primary or acquired platinum resistance (in platinum-resistant cell lines/patients). Further analyses of the SAL treatment used alone or in combination with other anticancer drugs, such as 5FU and GEM, will require identification of the most effective combination of SAL and the cytotoxic agent, and the mode of their administration (synchronous or sequential). Our results may contribute to the development of anticancer therapy based on SAL, which may give hope for heavily treated OvCa patients. Further studies should focus on discovering the mechanisms of action for SAL and its derivatives.

#### **4. Materials and Methods**

#### *4.1. Chemical Part*

#### 4.1.1. Isolation of Salinomycin

Salinomycin sodium salt was isolated from commercially available veterinary premix SACOX® following acidic extraction, using the previously described procedure [18,19]. Briefly, isolated sodium salt of salinomycin was dissolved in CH2Cl2 and stirred vigorously with a layer of aqueous sulfuric acid (pH = 1.0). The organic layer containing salinomycin (SAL, **1**, Figure 1) was washed with distilled water. Then, CH2Cl2 was evaporated under reduced pressure to dryness giving SAL as clear oil. After three cycles of evaporation with *n*-pentane, this oil was transformed into white amorphous solid. Spectroscopic data for SAL were closely matched previously published data [22].

#### 4.1.2. Synthesis of Salinomycin Derivatives

All SAL amides and esters (compounds **2**–**7**, Figure 1) obtained by a chemical modification of the C1 carboxyl group were prepared according to the procedures we described previously [18,19]. Spectroscopic data of all the compounds matched those found in the reference literature [18,19].

#### *4.2. Biological Part*

#### 4.2.1. Cell Culture and Derivation of Cisplatin-Resistant Cell Lines

In this study, OvCa cell lines A2780 and SK-OV-3 (ATCC, Manassas, VA, USA) and human fetal lung fibroblasts cell line (MRC-5 pd19; ECACC, Salisbury, United Kingdom) were used to evaluate the antiproliferative activity of the tested compounds. OvCa cell lines were cultivated in RPMI 1640 containing 25 mM HEPES and 5 mM l-glutamine with 10% fetal bovine serum (FBS) (all provided from Biowest, Nuaillé, France) and 1% penicillin-streptomycin (Merck KGaA, Darmstadt, Germany). MRC-5 pd19 was cultured in DMEM supplemented with 10% FBS, 2 mM l-glutamine (all provided from Biowest, Nuaillé, France), 1% penicillin-streptomycin and 1% non-essential amino acids (NEAA) (both provided from Merck KGa, Darmstadt, Germany). To generate the cisplatin-resistant cell lines (A2780 CDDP; SK-OV-3 CDDP), increasing doses of cisplatin (CDDP) (Teva Pharmaceutical Industries Ltd. Petach Tikwa, Israel) were added to the culture medium, starting from the concentration of 100 ng mL−1. Then, the cells were exposed to CDDP (3 cycles of 3 days each). After that, the cell culture medium was replaced with the fresh one without drugs for the next 3 days or one week until the cells recovered. After the 3 cycles, the dose of cisplatin was doubled until the concentration of 1000 ng· mL−<sup>1</sup> was achieved. Then, to maintain a resistant phenotype, 1000 ng·mL−<sup>1</sup> of **CDDP** was added once per 2 weeks for 3 days.

#### 4.2.2. Isolation of RNA and RT-qPCR

The cells (2.5 <sup>×</sup> 105) were washed twice in Dulbecco's phosphate buffered saline (DPBS, Biowest, Nuaillé, France) and suspended in TRI reagent (Sigma-Aldrich, St. Louis, MO, USA). Next, RNA was isolated using Direct-zol RNA MiniPrep (Zymoresearch, Irvine, CA, USA) according to the manufacturer's instructions. Then 1 μg of RNA was collected to synthesize cDNA using iScript kit (BioRAD, Hercules, CA, USA). The cDNA was diluted 20 times, and 2.5 μL was added to the reaction mix composed of FastStart Essential DNA Probes Mix and specific probes (both provided by Roche Molecular Systems, Inc, Basel, Switzerland). The RT-qPCR reaction was performed as previously described [58]. The tested genes related to drug resistance included: ATP-binding cassette subfamily B member 1 (ABCB1), ATP-binding cassette subfamily B member 4 (ABCB4), ATP-binding cassette subfamily G member 2 (ABCG2), and ATP-binding cassette subfamily C member 2 (ABCC2). Relative gene expression level was determined using reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primers and molecular probes used in this study are listed in Table S1 (Supplementary Material).

#### 4.2.3. Cell Viability Assay

For the evaluation of cell proliferation inhibition, we used the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay as previously described [59]. Briefly, A2780 WT and CDDP (2000 cells/well), SK-OV-3 WT and CDDP (800 cells/well), and MRC-5 pd19 (3000 cells/well) were seeded onto a 96-well plate for overnight until the cells attached. Next, the cells were exposed to the estimated concentrations of SAL, its derivatives, and anticancer drugs for 72 h (Table S2, Supplementary Material). Then the cell culture medium was replaced with the fresh one containing 0.5 mg·mL−<sup>1</sup> of MTT (Affymetrix, Santa Clara, MA, USA) and left for 2 h at 37 ◦C. After that, the medium was replaced with DMSO (VWR, Darmstadt, Germany) and left at 37 ◦C for 10 min until the crystals were dissolved. The measurement was performed with a plate reader, Multiskan FC (Thermofisher, San Jose CA, USA) at 570 and 690 nm. Then, IC50 and mean 95% CI were determined using GraphPad Prism 6 (Graph Pad Software, San Diego, CA, USA). The values of resistance index (RI), selectivity index (SI), and combination index (CI) were calculated as described previously [20,60].

#### 4.2.4. Western Blot Analysis

After 72 h of exposure to IC50 concentration of selected compounds, the OvCa cell lines were lysed using RIPA lysing buffer (Sigma Aldrich, St. Louis, MO, USA). For analysis, 10 μg of protein was used, calculated using Pierce™ BCA Protein Assay Kit (Thermofisher, San Jose, CA, USA) according to the manufacturer's instructions. The procedure was performed as previously described [52]. Briefly, after electrophoresis, proteins were transferred onto polyvinylidene difluoride (PVDF) membrane. After 2 h of blocking with non-fat milk (Sigma Aldrich, St. Louis, MO, USA), the membrane was incubated overnight at 4 ◦C with primary antibodies: anti-Bcl-2 (dilution 1:500; sc-509, Santa Cruz, Dallas, TX, USA), anti-Bax (dilution 1:500; sc-7480, Santa Cruz, Dallas, TX, USA), anti-CASP3 (full-form)(dilution 1:1000; no. ab49822, Abcam, Cambridge, UK), and reference protein GAPDH (dilution 1:500; no. sc-47724, Santa Cruz, Dallas, TX, USA). On the next day, after washes, the membranes were incubated with appropriate secondary antibodies conjugated with horseradish peroxidase (HRP) (dilution 1:1000; no. 7076 and 7074, Cell Signaling Technology, Leiden, Netherlands). Then the protein bands were visualized using WesternBright™ Quantum kit (Advansta, San Jose, CA, USA) and documented using the ChemiDoc Touch Imaging System (Bio-Rad Laboratories Ltd., Hercules, CA, USA). The intensity was measured using Image Lab Software (ver 6.0.1, Bio-Rad Laboratories Ltd., CA, USA). All buffers and equipment used during Western blot analysis were provided from Bio-Rad Laboratories Ltd., CA, USA.

#### 4.2.5. Statistical Analysis

The statistical analysis of genes expression (Student's *t*-test) was performed using GraphPadPrism 6 package (Graph Pad Software, San Diego, CA, USA). The data were deemed significant at *p* < 0.05. All experiments were repeated at least three times.

**Supplementary Materials:** The following are available online, Table S1: List of primers used in this study, Table S2: Concentration of drugs, dilution range, and serial dilution factors used in this study.

**Author Contributions:** Conceptualization, A.H., M.A. and M.M.; methodology, M.A., M.S.L; validation, A.H., M.S.L, M.A., M.M. and W.M.S.; formal analysis, M.M., M.A., A.H, M.S.L., W.M.S.; investigation, M.A, M.S.L and M.M.; resources, A.H., M.A. and M.M., M.S.L.; writing—original draft preparation, M.M, M.A. and M.S.L.; writing—review and editing, M.A, A.H, M.M, M.S.L., W.M.S; supervision, M.M., A.H., W.M.S.; funding acquisition, M.A., M.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** M.A. wishes to acknowledge the National Science Centre (NCN) for financial support under SONATA grant (2016/23/D/ST5/00242). M.M. wishes to acknowledge the Greater Poland Cancer Center for financial support under the internal WCO grant 5/2018/183.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**



#### **References**


**Sample Availability:** Samples of the all compounds are available from the authors.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article*

## **Organosilicon Compounds, SILA-409 and SILA-421, as Doxorubicin Resistance-Reversing Agents in Human Colon Cancer Cells**

### **Olga Wesołowska 1,\*, Krystyna Michalak 1, Maria Błaszczyk 1, Joseph Molnár <sup>2</sup> and Kamila Sroda-Pomianek ´ <sup>1</sup>**


Received: 18 March 2020; Accepted: 1 April 2020; Published: 3 April 2020

**Abstract:** Multidrug resistance (MDR) that occurs in cancer cells constitutes one of the major reasons for chemotherapy failure. The main molecular mechanism of MDR is overexpression of protein transporters from the ATP-binding cassette (ABC) superfamily, such as ABCB1 (multidrug resistance protein 1 (MDR1), P-glycoprotein). At the expense of ATP hydrolysis, ABCB1 pumps a diverse range of substrates (including anticancer drugs) out of the cell, thereby reducing their intracellular concentration. In the present study, the ability of two patented disiloxanes (SILA-409 and SILA-421) to reverse drug resistance in human colon adenocarcinoma cell lines LoVo and LoVo/Dx was investigated. It was demonstrated that both compounds in concentrations of 0.5–1 μM strongly increased the sensitivity of LoVo/Dx cells to doxorubicin. By means of an accumulation test in which rhodamine 123 was used as an ABCB1 substrate analogue, both organosilicon compounds were also shown to inhibit ABCB1 transport activity. The intracellular accumulation of doxorubicin was also increased, and more drug entered the cellular nuclei of resistant cells in the presence of the studied compounds. In conclusion, both SILA-409 and SILA-421 were demonstrated to be effective MDR reversal agents in resistant human colon cancer cells.

**Keywords:** organosilicon compounds; SILA-409 (Alis-409); SILA-421 (Alis-421); multidrug resistance (MDR) reversal; ABCB1 (P-glycoprotein); colon cancer

#### **1. Introduction**

Since chemotherapy continues to be a method of choice for the treatment of various types of cancer, any factors that undermine its effectiveness constitute a serious therapeutic issue. In the majority of patients, the initial response to chemotherapy is satisfactory; however, the occurrence of multidrug resistance (MDR) during treatment results in a development of progressive disease [1,2]. Cells displaying the MDR phenotype are no longer vulnerable to cytotoxic actions of many functionally and structurally dissimilar anti-cancer drugs.

Among several mechanisms that may lead to the development of MDR, the overexpression of ATP-binding cassette (ABC) transporters such as ABCB1 (P-glycoprotein, MDR1: multidrug resistance protein 1), ABCC1 (MRP1: multidrug resistance-associated protein 1), and ABCG2 (BCRP: breast cancer resistance protein) proteins seems to prevail [3,4]. It was recently reported that non-ABC transporters such as Hedgehog receptor Patched were also engaged in doxorubicin (Dox) efflux and conferred Dox resistance to cancer cells [5,6]. ABCB1 is a transporter that utilizes the energy gained from the hydrolysis of ATP to pump many structurally variable substrates (including xenobiotics and chemotherapeutics) out of the cell [7]. Among the numerous strategies proposed to overcome MDR, the most basic approach is the idea to use the inhibitor of a multidrug transporter (MDR modulator) along with chemotherapy in hopes to increase the intracellular accumulation of an anti-cancer drug and to improve the outcome of the treatment. Although this approach is yet to result in any clinical success [8–10], the search for new substances that could serve as clinically applicable MDR modulators is continuously ongoing.

Silicon is a metalloid, which is a group of chemical elements also called semi-metals [11]. It can form chemical bonds with carbon and oxygen, and silicon-containing compounds are widely used in medicine and engineering. Silicone is a synthetic polymer composed of repeating siloxane units. Due to its high biocompatibility and favorable mechanical properties, silicone is used for the production of various medical implants (e.g., breast or testicle), as well as contact lenses [11]. On the other hand, silica materials are applied as controlled drug delivery systems and three-dimensional scaffolds for tissue engineering [11]. The introduction of a silicon atom to a structure usually yields a molecule of higher lipophilicity than its carbon analogue. The increased anti-cancer activity of silicon–indomethacin derivatives was recorded, and it was claimed that the higher lipophilicity of silicon derivatives resulted in their increased uptake by cancer cells [12]. New sila-organosulfur compounds were recently synthesized that were reported to be effective anti-cancer and antibacterial agents [13].

SILA-409 and SILA-421 are water-soluble disiloxanes that were synthesized and patented as putative MDR modulators [14]. They were previously demonstrated to increase fluorescent dye accumulation in human *ABCB1* gene-transfected mouse lymphoma cells and, to much lesser extent, in colon carcinoma Colo320/MDR1-LRP cells, but not in ABCC1 expressing breast cancer (HTB-26/MRP1) and stomach cancer (257P/MDR) cells, which was attributed to their specific interactions with the ABCB1 transporter [15]. The specificity of disiloxanes toward ABCB1 was corroborated in a study by Kars et al. conducted in a model system of breast cancer cells [16]. Recently, the synergism between disiloxanes and amyloid β-protein in the inhibition of ABCB1 transporter activity was reported [17]. In an in vivo study on mice bearing human pancreatic cancer xenografts, the application of SILA-409 resulted in the reduction of tumor growth that was accompanied by increased apoptosis and a reduced level of ABCB1 protein in cancer cells [18]. SILA-421 was found to cause cell-cycle arrest and apoptosis in several non-MDR cancer cell lines [19]. The analysis of the global gene expression profile of HL-60 leukemia cells treated with this compound revealed multiple cellular pathways affected by SILA-421, including DNA replication and transcription processes. Interference with these processes was also attributed to the antimicrobial activity of SILA-409 and SILA-421 compounds [20–22], as well as their ability to eliminate resistance-bearing plasmids from *Escherichia coli* strains [23]. Additionally, the weak chemopreventive activity of disiloxanes was reported both in vitro and in vivo [24].

In the present work, the ability of SILA-409 and SILA-421 to reverse Dox resistance in human adenocarcinoma cells LoVo/Dx was demonstrated. The disiloxanes inhibited the transport function of ABCB1 protein both in LoVo/Dx cells and in Madin-Darby Canine Kidney (MDCK) cells expressing human ABCB1 (MDCK-MDR1). The amount of Dox accumulated inside LoVo/Dx cells was also elevated in the presence of studied compounds, and its distribution pattern was changed.

#### **2. Results and Discussion**

#### *2.1. Cytotoxicity of Disiloxanes*

A human adenocarcinoma cell line sensitive to Dox (LoVo) and its Dox-resistant counterpart (LoVo/Dx) were employed as a model system. It was previously demonstrated that the increased expression of ABCB1 transporter is mainly responsible for the resistance of LoVo/Dx cells [25]. The cytotoxicity of both SILA-409 and SILA-421 to LoVo and LoVo/Dx cells was comparable (Figure 1). Both compounds were strongly cytotoxic to the cells in concentrations above 10 μM. The half maximal inhibitory concentration (IC50) values of SILA-409 were 15.6 μM and 24.6 μM for LoVo and LoVo/Dx

cells, respectively. For SILA-421, IC50 values were 8.4 μM in LoVo cells and 9.2 μM in LoVo/Dx cells. SILA-421 was more toxic than SILA-409, which might be the result of the higher lipophilicity of this compound.

**Figure 1.** Cytotoxicity of SILA-409 (**A**) and SILA-421 (**B**) to LoVo (open symbols) and LoVo/Dx cells (full symbols). Means of three experiments ± SD are presented.

The cytotoxicity of both disiloxanes to MDCK cells and MDCK transfected with human *ABCB1* gene (MDCK-MDR1) was similar (Figure S1, Supplementary Materials). Both compounds in concentrations below 5 μM slightly stimulated cell growth, whereas, in concentrations of 25 μM and higher, <10% of cells survived. IC50 values of SILA-409 in MDCK and MDCK-MDR1 cells were 14.2 μM and 13.9 μM, respectively. For SILA-421, the values of 11.6 μM and 12.1 μM were analogously recorded. Similarly to the findings in colon cancer cells, IC50 values for SILA-421 were slightly lower than those for SILA-409.

The cytotoxicity of the studied disiloxanes to various cancer cell lines reported previously [15,19] was similar to the results of the present study. The IC50 values of SILA-421 analyzed in several various cancer cell lines, as well as in normal cells HEK-293, lay within the range 5–35 μM [19]. In another study, the cytotoxicity of both SILA-409 and SILA-421 was assessed in a panel of multidrug resistant cancer cell lines of different patterns of expression of MDR-associated transporters [15]. IC50 values varied from 8–80 μM depending on cell line. In three out of five cell lines studied, SILA-421 was found to be slightly more cytotoxic than SILA-409.

The analysis of the disiloxanes' chemical structures (Figure S3, Supplementary Materials) and the calculation of their theoretical logP values, performed using the free web tool Molinspiration (https://www.molinspiration.com/cgi-bin/properties), demonstrated that SILA-421 was more lipophilic than SILA-409 (logP values were 5.83 and 9.60 for SILA-409 and SILA-421, respectively). Lipophilicity was observed to positively correlate with the antiproliferative activity of various compounds toward cancer cells [26,27]. However, in other experimental settings, molecular parameters other than lipophilicity were found to affect cytotoxicity to the highest extent [28,29]. Therefore, a larger series of disiloxanes and a characterization of more of their molecular descriptors would be required to determine the effect of lipophilicity on the biological activity of these compounds.

#### *2.2. Influence of Disiloxanes on Doxorubicin Cytotoxicity*

Next, the influence of the studied modulators on Dox cytotoxicity was investigated (Figure 2). SILA-409 and SILA-421 were applied in concentrations in which they killed <10% of LoVo/Dx cells. The treatment of cells with a combination of either SILA-409 or SILA-421 with Dox significantly reduced the survival rate of resistant colon cancer cells, i.e., the disiloxanes partially reversed Dox resistance. Non-toxic concentrations of SILA-409 and SILA-421 were applied together with doxorubicin. Sensitive LoVo cells were more vulnerable to this anticancer drug (IC50 Dox = 4.0 μM) than ABCB1-overexpressing LoVo/Dx cells (IC50 Dox = 30.0 μM) [30]. It was demonstrated that both compounds strongly increased the sensitivity of LoVo/Dx cells to doxorubicin without changing the sensitivity of LoVo cells to this drug. The value of IC50 for Dox was reduced to 5.7 μM in the presence of SILA-409 at 0.5 μM concentration, and to 6.1 μM in the presence of SILA-421 (1 μM). The isobolographic analysis applied to the obtained results revealed the existence of synergism between Dox and each of the studied disiloxanes (Table 1).


**Table 1.** Combination of disiloxanes with Dox against LoVo/Dx cell growth.

Dose and effect data were obtained from the sulforhodamine B (SRB) assay (mean values of three experiments) and subjected to CompuSyn analysis. CI (combination index) values were generated by CompuSyn software. CI = 1 indicates additive effect, CI < 1 indicates synergism, and CI > 1 indicates antagonism.

**Figure 2.** The changes in doxorubicin (Dox) cytotoxicity in LoVo (circles) and LoVo/Dx cells (squares) caused by SILA-409 at 0.5 μM (**A**) and SILA-421 at 1 μM (**B**). Open symbols represent cells treated with Dox only, whereas full symbols represent cells treated with Dox and the modulator. Means of three experiments ± SD are presented.

The reduction of Dox cytotoxicity by SILA-409 and SILA 421 was previously observed in human *ABCB1* gene-transfected mouse lymphoma cells (L5718/MDR) and the resistant colon cancer cell line (Colo320/MDR1-LRP) [15]. Synergism between disiloxanes and Dox was observed in these cell lines but not in breast cancer cell lines MCF-7, T47-D, and HTB-26/MRP1. The authors concluded that the presence of a functional ABCB1 transporter was essential for disiloxanes to be able to revert Dox resistance [15]. This pointed to the specific interactions between SILA-409 and SILA-421 and ABCB1 protein. Similar synergistic interactions with appropriate anticancer drugs were also observed for SILA-409 and SILA-421 in MCF-7 cell lines resistant to paclitaxel and docetaxel, but not in the sublines resistant to Dox and vincristine [16].

Dox cytotoxicity was significantly lower (*p* < 0.05) in LoVo/Dx than in LoVo cells as determined by Student's *t*-test. Significant enhancement (*p* < 0.05) of Dox cytotoxicity was recorded for both SILA-409 and SILA-421in LoVo/Dx cells in the whole concentration range

#### *2.3. Intracellular Accumulation of Rhodamine 123 (R123)*

Rhodamine 123 (R123) is popularly used as a fluorescent reporter substrate of the ABCB1 transporter. Both SILA-409 and SILA-421 caused an increase in R123 accumulation in LoVo/Dx and MDCK-MDR1 cells (Figure 3) in a concentration-dependent manner, which suggested that both compounds were inhibitors of ABCB1 transport activity. SILA-421 seemed to exert its inhibitory action in lower concentrations than SILA-409, which significantly elevated fluorescence intensity ratio (FIR) values only in concentrations above 10 μM in Dox-resistant colon cancer cells.

**Figure 3.** The influence of SILA-409 (gray) and SILA-421 (white) on R123 intracellular accumulation in LoVo/Dx (**A**) and Madin-Darby Canine Kidney-multidrug resistance protein 1 (MDCK-MDR1) cells (**B**). Means ± SD of three experiments are presented. The statistically significant differences from the untreated cells were determined using Student's *t*-test (\* *p* < 0.05).

Molnar et al. [15] also observed increased R123 accumulation in human *ABCB1* gene-transfected mouse lymphoma cells (L5718/MDR) treated with SILA-409 and SILA-421 in concentrations up to 2 μg/mL. Both compounds demonstrated similar activity in this respect. They were, however, unable to affect R123 accumulation in sensitive breast and prostate cancer cells and it was again concluded that the activity of disiloxanes can only be observed in cell lines that express a functional ABCB1 transporter [15]. In the study of Kars et al. [16], both disiloxanes were demonstrated to increase R123 accumulation in a series of breast cancer cell lines resistant to paclitaxel, docetaxel, Dox, and vincristine. In contrast to our findings, SILA-409 was observed to be more active then SILA-421 in this experimental setting.

#### *2.4. Intracellular Accumulation of Doxorubicin*

Accumulation of Dox itself by human colon cancer cells was also investigated. As presented in Figure 4, drug-sensitive LoVo cells accumulated more drug than Dox-resistant LoVo/Dx cells. The treatment of cells by disiloxanes at a concentration of 5 μM resulted in a significant increase in Dox accumulation by LoVo/Dx cells with apparently no effect observed in LoVo cells.

**Figure 4.** Intracellular doxorubicin (Dox) accumulation in LoVo (gray bars) and LoVo/Dx cells (white bars) treated with SILA-409 and SILA-421 at 5 μM concentration. Means ± SD of three experiments are presented. The statistically significant differences between untreated and disiloxane-treated cells were determined using Student's *t*-test (\* *p* < 0.05).

Since Dox is characterized by strong intrinsic fluorescence, its intracellular accumulation may also be examined directly by means of fluorescence microscopy. Again, LoVo cells accumulated more Dox than LoVo/Dx cells (Figure 5 and Figure S2, Supplementary Materials), and the application of disiloxanes at a concentration of 5 μM resulted in an increase in accumulation of the drug in Dox-resistant cells but not in the sensitive ones. SILA-421 was more effective in this respect than SILA-409. The intracellular distribution of Dox was also affected by organosilicon compounds. Dox that was excluded from the nuclei of untreated resistant cells was demonstrated to localize within these organelles in modulator-treated LoVo/Dx cells.

**Figure 5.** Intensity of intracellular fluorescence of Dox, measured by ImageJ software, in LoVo (gray bars) and LoVo/Dx cells (white bars) treated with 5 μM of SILA-409 and SILA-421 is presented as the mean fluorescence values ± SD measured in 20 representative cells. The statistically significant differences from the untreated cells were determined using Student's *t*-test (\* *p* < 0.05).

According to our best knowledge, the influence of SILA-409 and SILA-421 on Dox accumulation and cellular localization was not previously studied. The pattern of changes in Dox accumulation caused by disiloxanes was similar to the changes caused by verapamil (a well-known inhibitor of ABCB1 protein) observed previously in the same colon cancer cell lines as those used in the present work [25].

#### *2.5. Expression of ABCB1 Transporter*

Additionally, the influence of disiloxanes on ABCB1 transporter expression was analyzed by Western blotting (Figure 6). Both SILA-409 and SILA-421 significantly decreased ABCB1 protein level in LoVo/Dx cells during 48 h of treatment. SILA-421 turned out to be more active in this respect in comparison with SILA-409. No effect of organosilicon compounds on ABCB1 protein level was noted during 60 min of treatment with modulators (data not shown).

**Figure 6.** Western blot analysis of ATP-binding cassette B1 (ABCB1) protein level (**A**) in LoVo/Dx cells cultured with 0.5 μM SILA-409 and SILA-421 for 48 h. The molecular masses of the proteins are indicated on the left side of the gel. β-Glucuronidase (β-GUS) was used as a reference protein. The relative level of ABCB1 expression (**B**) was normalized to the control derived from non-treated LoVo/Dx cells. The results of three experiments ± SD are presented. The statistically significant differences from the untreated controls were determined using Student's *t*-test (\* *p* < 0.05).

The downregulation of ABCB1 expression was previously demonstrated using immunohistochemical methods in pancreatic tumor samples isolated from mice treated with SILA-409 [18]. On the other hand, no influence of disiloxanes (tested in concentrations of 380–500 μg/mL) on *ABCB1* expression level was detected in mouse T-lymphoma cells transfected with human *ABCB1* [15].

#### **3. Materials and Methods**

#### *3.1. Chemicals*

Orgasnosilicon compounds 1,3-dimethyl-1,3-bis(4-fluorophenyl)-1,3-bis(3-morpholino-propyl)disiloxan dihydrochloride (SILA-409) and 1,3-dimethyl-1,3-bis(4-fluorophenyl)-1,3 bis{3-[1(4-butylpiperazinyl)]-propyl}-disiloxan tetrahydrochloride (SILA-421) were synthesized and patented [14]. Their chemical structures are presented in Figure S3 (Supplementary Materials). Stock solutions of organosilicon compounds were prepared in dimethyl sulfoxide (DMSO). Sulforhodamine B (SRB), rhodamine 123 (R123), and doxorubicin (Dox) were obtained from Sigma-Aldrich (Poznan, Poland) and dissolved in water.

#### *3.2. Cell Culture*

Human colorectal adenocarcinoma cell line, LoVo, and its resistant subline, LoVo/Dx, obtained by prolonged exposure to Dox [31], were obtained from the Institute of Immunology and Experimental Therapy of Polish Academy of Science (Wroclaw, Poland). Cultivation conditions were Ham's F12 medium (with the addition of 10% fetal bovine serum, l-glutamine, antibiotics, and, in the case of LoVo/Dx cells, Dox at 100 ng/mL), at 37 ◦C and 5% CO2.

Madin-Darby Canine Kidney cells (MDCK) and MDCK cells expressing human ABCB1 (MDCK-MDR1) [32] were purchased from the Netherlands Cancer Institute (NKI-AVL, Amsterdam, the Netherlands). The cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, l-glutamine, and antibiotics at 37 ◦C and 5% CO2.

#### *3.3. Cell Viability Assay*

The SRB assay [33] with minor modifications was used for the estimation of the effect of the studied compounds on cell growth. Cells were seeded in 96-well flat-bottom microtiter plates in 75 μL of medium and allowed to attach (60 min, 37 ◦C). Then, 75 μL of medium containing an amount of the studied compounds (such that a desired compound concentration was obtained in a final sample volume) was added to each well, with the exception of the control wells, which contained medium only. The culture plates were then incubated for 48 h at 37 ◦C. The further procedure was carried out as previously described [34]. Cytotoxicity of DMSO to LoVo and LoVo/Dx cells was negligible.

#### *3.4. Isobolographic Analysis*

Combination index (CI) values were calculated using the CompuSyn software (www.combosyn. com, ComboSyn, Inc., Paramus, USA) according to the classic median-effect equation as described by Chou and Martin [35].

$$CI = \frac{(D)\_1}{(Dx)\_1} + \frac{(D)\_2}{(Dx)\_2},\tag{1}$$

where (Dx)1 is the dose of drug 1 alone that inhibits a system by x%, (Dx)2 is the dose of drug 2 alone that inhibits a system by x%, and (D)1 + (D)2 are doses of drugs 1 and 2 in combination that also inhibit a system by x%.

#### *3.5. Accumulation of Rhodamine 123 in Cancer Cells*

In order to determine R123 accumulation, the cells were harvested and incubated (300,000 cells/mL) with the appropriate concentration of the studied compound (15 min; 25 ◦C). Next, R123 (10 μM) was added, and the cells were incubated for 60 min at 37 ◦C. After centrifugation, the samples were washed twice with ice-cold phosphate-buffered saline (PBS) and dissolved in lysis buffer (20 mM Tris-HCl, 0.2% SDS, pH = 7.7). Intracellular fluorescence (λex = 485 nm, λem = 538 nm) was collected with the use of an Infinite M200Pro plate reader (Tecan Instruments, Maennedorf, Switzerland). Based on measured

the fluorescence intensity (FL) of the treated and control samples (without modulator), the fluorescence intensity ratio (FIR) was calculated according to the following equation:

$$\text{FIR} = \frac{\left(\text{FL}\_{\text{LoVoDx}} \text{or MDCK} - \text{MDR1 treated}\right) / \left(\text{FL}\_{\text{LoVoDx}} \text{or MDCK} - \text{MDR1 control}\right)}{\left(\text{FL}\_{\text{LoVo}} \text{or MDCK treated}\right) / \left(\text{FL}\_{\text{LoVo}} \text{or MDCK control}\right)}. \tag{2}$$

#### *3.6. Intracellular Accumulation of Doxorubicin*

Intracellular Dox accumulation was detected with a fluorimetry based assay as described previously [36]. Briefly, cells were seeded (800,000/well) onto a six-well plate and incubated for 24 h at 37 ◦C. Then, the cells were incubated in PBS containing Dox (4 μM) and treated with the modulators. After 48 h of incubation, cells were washed twice in ice-cold PBS and detached. Next, cells were centrifuged and lysed. The cellular protein content was determined using the standard method of Bradford reaction [37]. Dox content was measured using an LS-5 spectrofluorimeter (Perkin-Elmer, Beaconsfield, UK). Excitation and emission wavelengths were 475 and 553 nm, respectively. Fluorescence was expressed in ng of Dox per mg of cellular protein with the use of the calibration curve prepared previously.

For fluorescence microscopic experiments, LoVo and LoVo/Dx cells were cultivated on eight-well μ-Slide microscopy chambers (Ibidi, Munich, Germany) for 48 h. For the experiment, a fresh portion of F12 medium was added containing 50 μM Dox (plus 5 μM of the studied compounds in treated samples), and cells were incubated for 60 min at 37 ◦C. After incubation, the chambers were washed with PBS and with serum- and phenol red-free F12 medium. The images were collected with a Nikon Eclipse TE2000-E microscope. Fluorescence was excited in the range 528–553 nm and collected in the range 578–633 nm.

#### *3.7. Expression of ABCB1 Protein*

Cell lysates were prepared in ice-cold lysis buffer (1% Triton X-100, 50 mM Hepes, 150 mM NaCl, 1.5 mM MgCl2, 1 mM ethylene glycol-bis(β-aminoethyl ether)-*N,N*,*N* ,*N* -tetraacetic acid (EGTA), 1 mM phenylmethylsulfonyl fluoride (PMSF), 100 mM NaF, 10 mM sodium pyrophosphate, 10 μg/mL aprotinin, and 10% glycerol, pH 7.4). After centrifugation of whole-cell lysates (13,000× *g*, 10 min, 4 ◦C), the supernatants were taken for analysis. The standard method of Bradford reaction was used to measure protein content (Bradford, 1976). The proteins were subjected to SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, and detected using primary antibodies in tris-buffered saline with Tween (TBS-T) buffer (0.1% Triton X-100, 50 mM Tris-HCl, 150 mM NaCl, 1, pH 7.4) with 5% bovine serum albumin (BSA). The anti-ABCB1 mouse monoclonal primary antibodies (C494) (Alexis) were used at dilution 1:1000. The level of β-glucuronidase (β-GUS) was also determined as a reference protein (anti-glucuronidase mouse monoclonal antibody, diluted 1:1000, Thermo Scientific). After incubation (overnight at 4 ◦C), the membranes were washed in TBS-T and incubated with rabbit anti-mouse immunoglobulin G (IgG) secondary antibody conjugated to horseradish peroxidase (HRP) (dilution 1:1000, Thermo Scientific) for 30 min at 4 ◦C. The membranes were then washed with TBS-T, and the proteins were visualized. The relative amount of protein normalized to the control (non-treated cells) was determined. The optical density of the bands on the electrophoregram was detected with the Image J softwar version 1.43m.

#### *3.8. Statistical Analysis*

Data represent the means ± standard deviation (SD) of at least three replications. Student's *t*-test was applied, and *p*-values less than 0.05 were considered to be statistically significant.

#### **4. Conclusions**

Organosilicon compounds, SILA-409 and SILA-421, were demonstrated to reverse doxorubicin resistance in a human colon cancer cell line. Both compounds were inhibitors of the transport function of ABCB1 protein judging from the increased accumulation of R123 by modulator-treated cells. Intracellular accumulation of Dox was also increased, and more drug entered cellular nuclei in the presence of the studied compounds. SILA-421 demonstrated slightly higher activity than SILA-409. Additionally, the decreased expression of ABCB1 protein after treatment with disiloxanes was recorded. Therefore, the studied compounds acted as resistance-reversing agents via two mechanisms. They were effective inhibitors of the ABCB1 transport function, and its reduction was easily observed after 60 min of disiloxane treatment (e.g., in fluorescence microscopy and R123 accumulation experiments). Moreover, after prolonged treatment with SILA-409 and SILA-421, the reduction of ABCB1 protein level that occurred certainly increased the MDR-reversing potency of these compounds. In conclusion, both SILA-409 and SILA-421 were demonstrated to be effective anti-MDR agents in resistant human colon cancer cells.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1420-3049/25/7/1654/s1: Figure S1: Cytotoxicity of SILA-409 (A) and SILA-421 (B) to MDCK (full symbols) and MDCK-MDR1 cells (open symbols). Means of three experiments ± SD are presented; Figure S2: Fluorescence microscopy images illustrating doxorubicin accumulation in LoVo (A) and LoVo/Dx (B) cells treated with 5 μM SILA-409 (C and D for LoVo and LoVo/Dx, respectively) and with 5 μM SILA-421 (E and F). Scale bar is 50 μm. Illumination conditions were the same for all images; Figure S3: Chemical structures of SILA-409 and SILA-421.

**Author Contributions:** Conceptualization, O.W. and K.M.; Validation, O.W.; Formal analysis, O.W. and K.S.-P.; ´ Investigation, O.W., K.S.-P., and M.B.; Resources, J.M.; Writing—Original draft preparation, O.W.; Writing—Review ´ and editing, O.W., K.S.-P., J.M. and K.M.; Visualization, O.W.; Supervision, K.M.; Funding acquisition, K.M. All ´ authors have read and approved the final version of the manuscript.

**Funding:** This work was financed by Wroclaw Medical University, subject Simple No. SUB.A050.20.018. J.M. was supported by the Szeged Foundation for Cancer Research.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


**Sample Availability:** Samples of the compounds are not available from the authors.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Synthesis, Anti-Cancer and Anti-Migratory Evaluation of 3,6-Dibromocarbazole and 5-Bromoindole Derivatives**

**Krystal M. Butler-Fernández 1, Zulma Ramos 1, Adela M. Francis-Malavé 2, Joseph Bloom 1, Suranganie Dharmawardhane <sup>3</sup> and Eliud Hernández 1,\***


Academic Editor: Qiao-Hong Chen

Received: 2 July 2019; Accepted: 19 July 2019; Published: 24 July 2019

**Abstract:** In this study, a new series of *N*-alkyl-3,6-dibromocarbazole and *N*-alkyl-5-bromoindole derivatives have been synthesized and evaluated in vitro as anti-cancer and anti-migration agents. Cytotoxic and anti-migratory effects of these compounds were evaluated in MCF-7 and MDA-MB-231 breast cancer cell lines and an insight on the structure-activity relationship was developed. Preliminary investigations of their anti-cancer activity demonstrated that several compounds have moderate antiproliferative effects on cancer cell lines with GI50 values in the range of 4.7–32.2 μM. Moreover, carbazole derivatives **10**, **14**, **15**, **23**, and **24** inhibit migration activity of metastatic cell line MDA-MB-231 in the range of 18–20%. The effect of compounds **10**, **14**, and **15** in extension of invadopodia and filopodia was evaluated by fluorescence microscopy and results demonstrated a reduction in actin-based cell extensions by compounds **10** and **15**.

**Keywords:** 3,6-dibromocarbazole; 5-bromoindole; carbazole; actin; breast cancer; migration

#### **1. Introduction**

In women, breast cancer is the leading cause of death, mainly due to metastasis [1]. If breast cancer is detected and treated prior to metastasis, the patient has a higher probability of being cured of their disease. Cancer cell invasion involves cell migration through the extracellular matrix (ECM) and the accompanying degradation of the ECM [2]. Several proteins play a key role in this process, by the extension of structures known as invadopodia. Invadopodia are actin-rich protrusive structures with associated matrix degradation activity and are believed to be important for tumor cells to penetrate the basement membrane of epithelia and blood vessels [3]. In cell migration, the reorganization of the actin cytoskeleton produces the force necessary for cell migration [4]. The Rho GTPases, Rac, and Cdc42 are key molecular switches activated by a myriad of cell surface receptors to promote breast cancer cell migration/invasion, proliferation, and survival [5]. Unlike Ras, Rac and Cdc42 are not mutated in breast cancer, but activated via the deregulation of expression and/or activity of their upstream regulators, guanine nucleotide exchange factors (GEFs) [6]. The WASP family proteins are key regulators of the actin cytoskeleton and cell migration through induction of membrane protrusions at the leading edge [7]. In cancer cells, when N-WASP interacts and activates the Arp2/3 complex, it catalyzes actin polymerization and assembly into filopodia and invadopodia [7,8]. To initiate this process, the Rho GTPase Cdc42, in its GTP-activated form, binds and activates N-WASP by inducing a conformational change that liberates the autoinhibited structure, thereby interacting with Arp2/3

complex, and regulating the protrusive formation in membrane structures promoting extracellular matrix (ECM) degradation [8]. Therefore, inhibition of these processes decreases cell motility and invasion, and may greatly improve the potential therapeutic applications of such inhibitors against cancer metastasis.

Natural and synthetic carbazole derivatives comprise a wide variety of biologically active agents with diverse pharmacological activities, including antitumoral, antioxidant, anti-inflammatory, antibacterial, anticonvulsant, antipsychotic, antidiabetic, and larvicidal properties [9–28]. Carbazoles are tricyclic aromatic compounds with a benzene ring fused to the 2,3-positions of an indole ring [29]. The antitumor properties of carbazole derivatives have been correlated to their polycyclic, planar aromatic structure, and large π-conjugated backbone that noncovalently bind with DNA base pairs, hydrophobic pockets, and forms electrostatic interactions to intercalate into DNA [9,30]. In particular, among a wide variety of carbazoles, a series of *N*-alkyl-3,6-disubstituted carbazole derivatives has been discovered and evaluated for their potential as neuroprotective agents [31], antimalarial [32], antitumoral [33,34], anti-apoptotic [35], and antibacterial activities [36]. Selected examples of bioactive *N*-alkyl-3,6-disubstituted carbazole derivatives are represented in Figure 1.

**Figure 1.** Structure of representative *N*-alkyl-3,6-dihalogencarbazole derivatives.

The compound P7C3 (Figure 1) was discovered from a library of 200,000 drug-like molecules, and showed proneurogenic and neuroprotective properties, stabilized mitochondrial membrane potential, and inhibits neuronal apoptosis [37]. Several derivatives of P7C3 have been synthesized with modifications at the linker chain. Replacement of the hydroxyl group with a fluorine atom, and an additional methylene group between the hydroxyl group and the aniline, increases activity [37]. The aromatic ring was replaced with heteroaromatic groups, but activity was found to be less effective. The commercially available TDR30137 (Figure 1) was discovered and characterized as an inhibitor of P. *falciparum* K1 (Pf-K1) in human red blood cells with an IC50 of 57 nM [32]. However, TDR30137 was not active in in vivo studies with the *Plasmodium berghei* mouse model. In structure-activity relationship (SAR) studies, the importance of 3,6-halogen substitution, hydroxyl group, and the tertiary amine correlated with improved activity on Pf-K1 strains [32]. A carbazole derivative named Wiskostatin (Figure 1) was identified to bind within a pocket in the GBD regulatory module that maintains N-WASP in an inactive, autoinhibited conformation [38]. In a pyrene-actin polymerization assay, using purified proteins, it was demonstrated that Wiskostatin inhibited full-length N-WASP activation of the Arp2/3 complex at IC50 = 10 μM [38]. The specific binding site of Wiskostatin was determined to be within the GBD of the autoinhibited conformation of N-WASP after performing the experiment with activated Cdc42-GTP. Unfortunately, a recent report described that Wiskostatin inhibited other cellular functions that are not believed to be N-WASP dependent [39]. These studies revealed that Wiskostatin caused an irreversible decrease in cellular ATP levels, and that it does not function as a selective inhibitor of N-WASP dependent functions in intact cells, and caused an overall change in the energy status of cells; thus, inhibiting normal transport processes [39]. Herein, we designed and synthesized a new series of *N*-alkyl-3,6-dibromocarbazole and 5-bromoindole derivatives, tested for their antiproliferative and antimigratory activities in MCF-7 and MDA-MB-231 breast cancer cell lines, and analyzed the effect of the most active migration inhibitor on actin dynamics and actin cytoskeleton rearrangement.

#### **2. Results and Discussion**

The aim of this study is to design and synthesize a new series of *N*-alkyl-3,6-dibromocarbazole and *N*-alkyl-5-bromoindole derivatives and analyze their cytotoxic effect and potential to inhibit actin cytoskeleton rearrangement and cancer cell migration. The structural elements of Wiskostatin and derivatives identified as pharmacophoric unit are a 3,6-dihalogen carbazole and a dialkylamino-2-propanol chain. Our strategy was to design and synthesize a new series of compounds with a 3,6-dibromocarbazole or 5-bromoindole ring connected, via a three-carbon atom aliphatic chain, to an amide group. The influence of different N-alkyl or aromatic substituents at the amide group was examined.

We screened all compounds to determine their cytotoxic effect against MCF-7 and MDA-MB-231 breast cancer cells using the Sulforhodamine B (SRB) assay [40] (Figure 2). In addition, anti-migratory activity was determined using the wound healing assay (scratch method) [41] on the metastatic MDA-MB-231 cancer cells. In this assay, the relative migration of MDA-MB-231 breast cancer cells in the presence of carbazole or indole derivatives at a concentration of 10 μM (or at concentrations that do not affect cell viability) was compared to the migration in the presence of vehicle (0.02% DMSO). Representative photomicrographs of the migration inhibition of compounds **10**, **14**, **15**, and Wiskostatin are represented in Figure 3. Results show that in the vehicle-treated control experiment, wound healing is progressing considerably, and after 24 h, the wound is completely healed. When cells are incubated with compounds **10**, **14**, and **15** after 24 h, the wound healing is inhibited. However, Wiskostatin did not elicit an inhibitory effect on wound healing when incubated with MDA-MB-231 cells after 24 h at a concentration of 2 μM. Actin and Arp2/3 regulation by active WASP induce de novo actin polymerization and assembly to generate the F-actin structures filopodia and invadopodia used for cell migration [42]. Therefore, to investigate the effect of compounds **10**, **14**, **15**, and Wiskostatin on actin dynamics, we performed immunofluorescence microscopy to detect polymerized actin on MDA-MB-231 cancer cells (Figure 4). The structure and biological activities of new compounds are summarized in Tables 1 and 2.

#### *2.1. N-Alkyl-3,6-Dibromocarbazole Derivatives*

The synthetic method to construct the 3,6-dibromocarbazole derivatives library is described in Scheme 1 (see the Supplementary Materials for representative 1H and 13C NMR spectral data). The *N*-alkyl-3,6-dibromocarbazole derivatives were generated in a three-step synthesis using 3,6-dibromocarbazole **1** as origin of the carbazole derivatives core. Compound **1** was reacted with ethyl 4-bromobutyrate to introduce the aliphatic side chain by nucleophilic substitution (Scheme 1), followed by hydrolysis to afford the corresponding 3,6-diromocarbazole-4-butyric acid **2**. For the generation of 3,6-dibromocarbazole-4-butyramide derivatives **3**, compound **2** was therefore used as starting material, which reacted with different amines via an amide coupling reaction using N-(3-Dimethylaminopropyl)-N'-ethylcarbonate (EDAC) with Hydroxybenzotriazole (HOBt) as an additive dissolved in methylene chloride (CH2Cl2).

**Scheme 1.** General synthetic procedure of 3,6-dibromocarbazole-4-butyramide derivatives **3**. Reagents and conditions: (**a**) (i) Ethyl 4-bromobutyrate, K2CO3, DMF, 80 ◦C, 2h; (ii) KOH, DMF/water, 80 ◦C, 2-6 h, 86%; (**b**) HOBt, EDAC, CH2Cl2, Et3N, rt, amine: R-NH2 or HNR1R2, 2-8 h.

The in vitro anti-proliferative and anti-migratory activities of compounds **4**–**27**, and Wiskostatin are represented in Table 1. From the twenty-four compound derivatives of 3,6-dibromocarbazole, it can be observed that in the MCF-7 (ER+) cancer cell line, compounds **6–10**, **14**, **16–21**, and **32** showed good to moderate antiproliferative activity with a GI50 in the range of 6.8–32.2 μM. In the MDA-MB-231 cell line, compounds **6–10**, **14**, **16**, **18**, **20–21**, and **23** inhibited cell proliferation with a GI50 in the range of 4.7–23 μM. The remaining compounds in that series had a GI50 above 50 μM in both breast cancer cell lines. Compound **8** with a 2-piperazinyl ethyl butyramide chain showed very good anticancer activity against both cancer cell lines MCF-7 and MDA-MB-231 with GI50 values of 8 and 4.7 μM, respectively. Also, compound **18** with a piperazinyl amide showed significant anticancer activity against both cancer cell lines with GI50 values of 7.5 and 6.7 μM, respectively. Similarly, compound **21**, another piperazinyl amide derivative, showed very good in vitro anticancer activity against both cancer cell lines with GI50 values of 6.5 and 8 μM, respectively. Shortening the aliphatic chain between the N-atom of the amide group and the morpholine from C3 (**9**), over C2 (**4**), to C0 (**12**) led to a complete loss of antiproliferative activity on both cancer cell lines. On the other hand, introduction of an aromatic ring or aromatic heterocycle (**22–27**, Table 1) in the amide group led to lack of antiproliferative activity with GI50 above 50 μM on both cancer cell lines. Thus, in general, compounds with a piperazinyl butyramide group attached to the N-atom of the carbazole appear to be more cytotoxic than compounds with other butyramide group in this series of compounds. In addition, three compounds—**8**, **18**, and **21**—were found to be more cytotoxic against both cancer cell lines MCF-7 and MDA-MB-231 than Wiskostatin, which in this assay showed GI50 values of 9.7 and 8.3 μM, respectively (Table 1, Figure 2).

To further assess the anti-migratory activity of carbazole derivatives in vitro, we examined its inhibitory effects on the migration of the metastatic breast cancer cell line MDA-MB-231 using the wound-healing assay at concentrations that do not affect cell viability. We chose the MDA-MB-231 breast cancer cells over MCF-7 due to its enhanced metastatic and migratory properties, with concomitant Rac and Cdc42 expression, compared to the non-metastatic and poorly migrating MCF-7 cells. The relative migration of treated cells with 3,6-dibromocarbazole derivatives compared with control (MDA-MB-231 cells) are summarized in Table 1.


**Table 1.** Cell growth inhibition and anti-migration activity of 3,6-dibromocarbazole derivatives.


**Table 1.** *Cont.*

<sup>a</sup> GI50 = compound concentration required to inhibit MDA-MB-231 proliferation by 50% after 48 h treatment. Values are expressed as the mean of triplicate experiments, and standard deviation (SD) is <10%. <sup>b</sup> After 24 h, MDA-MB-231 cellular migration was determined by measuring the distance traveled from the edge of the scratch toward the center of the scratch, relative to control. <sup>c</sup> Percent relative migration values at 10 μM (or at concentrations that do not affect cell viability). Results are presented as means ± SD of three independent experiments.

**Figure 2.** Log-dose response curve for compounds **7**, **8**, **10**, **18**, **21**, and Wiskostatin on MCF-7 and MDA-MB-231 breast cancer cell lines. Each data point represents the mean of three replicates and error bars represent ± SD. Each GI50 value was calculated based on sigmoidal curve fitting to the respective data set.

Among the twenty-four compounds tested for their anti-migratory effect, five compounds (**10**, **14**, **15**, **23**, **24**) inhibited migration in the range of 10–20%. While compounds **7**, **8**, **18**, and **21** were among the most cytotoxic compounds tested against MDA-MB-231 and MCF-7 cancer cell lines, they did not show significant anti-migratory effect. In contrast, compounds **14–16** and **24** inhibited migration in the range of 18–20%, compared to Wiskostatin that inhibit 5% of migration at 2 μM. Compound **14**, a carbazole derivative with a piperidine-4-carbonitrile amide group that showed moderate anti-proliferative activity on both MCF-7 and MDA-MB-231 cell lines, inhibit 19% of migration. Both compounds **15** and **16**, with phenyl- and acetyl-piperazine amide group, respectively, showed comparable anti-migratory activity with 20% and 18%, respectively. The anti-proliferative effect of **16** on both cancer cell lines was moderate, compared to **15**, which lacks cytotoxic activity. On the other hand, the 3-aminoquinoline amide **24**, showed anti-migratory activity of 18% with GI50 above 50 μM on MDA-MB-231 cell line. When comparing Wiskostatin and carbazole derivatives from Table 1, compound **10** exhibited anti-proliferative activity with GI50 values comparable with Wiskostatin on both cancer cell lines. However, compound **10** showed higher anti-migratory potency with 13% at 2.1 μM, compared with 5% anti-migratory effect of Wiskostatin at 2 μM on MDA-MB-231 breast cancer cells. Therefore, compound **10** exhibits similar cytotoxicity with improved anti-migratory potential compared to Wiskostatin.

**Figure 3.** Inhibitory effect of compounds **10**, **14**, **15**, and Wiskostatin on MDA-MB-231 cells migration detected by wound-healing assay. MDA-MB-231 cells were treated with vehicle or with compounds **10**, **14**, **15**, and Wiskostatin. The photomicrographs were obtained at 0 and 24 h. Percent relative migration values are the average of three independent experiments. Dotted lines show the area occupied by the initial scraping for 0 h, and the wound edge for 24 h.

The Rho GTPases are believed to stimulate plasma membrane protrusion by inducing actin filament nucleation and polymerization on or close to membranes [43]. In particular, the Rho GTPase protein Cdc42, through activation of N-WASP/Arp2/3 pathway, is an important mediator of actin polymerization and filopodium extension [44,45]. Therefore, compounds that interfere with this process might be potentially useful molecular probes for the study of cell migration and invasion. To determine changes in actin cytoskeletal structures, we treated MDA-MB-231 breast cancer cells with vehicle or compounds **10**, **14**, **15**, and Wiskostatin at 10 μM for 24 h (Figure 4).

**Figure 4.** Effect of Wiskostatin and compounds **10**, **14**, and **15** on actin cytoskeleton of metastatic cancer cell MDA-MB-231. MDA-MB-231 metastatic breast cancer cells were treated with vehicle or Wiskostatin (2 μM) and compounds **10**, **14**, or **15** at 10 μM for 24 h to determine changes in actin cytoskeletal structures. Cells were fixed, permeabilized, and stained with rhodamine phalloidin to visualize F-actin. Arrows, lamellipodia; arrowheads, filopodia.

To identify F-actin based cell surface extensions, cells were stained with rhodamine phalloidin to localize F-actin. The results showed that untreated cells (control) demonstrated a strong formation of lamellipodia extensions, membrane ruffles, and stress fibers, with few filopodia. Wiskostatin and compounds **14** and **15** moderately reduced lamellipodia extensions when compared with control cells at concentrations that do not affect cell viability. In addition, compound **10**, at concentrations that inhibit 13% cell migration (2 μM), exhibited a marked reduction in lamellipodia formation when compared to vehicle, and in particular with Wiskostatin, which inhibits cell migration in 5% at 2 μM (Table 1). Compound **15** demonstrates a marked reduction in polymerized actin indicating inhibition of Arp2/3 mediated actin nucleation. Specific inhibition of filopodia, as would be predicted by WASP inhibition, could not be determined since the vehicle control cells exhibited more invadopodia and lamellipodia than filopodia. In general, these results suggest that new N-alkyl-3,6-dibromocarbazoles and compounds derivatives of Wiskostatin can be explored as new probes to study actin dynamics in cancer cells, or to further develop new anti-cancer and anti-metastatic drugs.

#### *2.2. N-alkyl-5-Bromoindole Derivatives*

To further explore the N-alkyl-5-bromoindole butyramide derivative series, we synthesized several compounds in which the carbazole core was replaced by a 5-bromoindole ring (Scheme 2). The strategy is to analyze the effect of using a smaller ring system as a core connected to the C3 linker side chain and the amide group. The synthesis of N-alkyl-5-bromoindole derivatives is described in Scheme 2. The 5-bromoindole **28** was reacted with ethyl 4-bromobutyrate, followed by hydrolysis to yield the corresponding 5-bromoindole-4-butyric acid **29**. Since N-alkylation in indoles is more difficult than in carbazoles, the reaction rate to obtain **29** was improved by using Cs2CO3 as a base over K2CO3, where Cs2CO3 solubility is ten times higher in organic solvents than K2CO3. For the generation of 5-bromoindole-4-butyramide derivative **30**, carboxylic acid **29** was therefore used as starting material, which reacted with different amines via an amide coupling reaction using reaction conditions similar as described in Scheme 1 (see the Supplementary Materials for representative 1H and 13C NMR spectral data).

**Scheme 2.** General synthetic procedure of 5-bromoindole-4-butyramide derivative **30**. Reagents and conditions: (**a**) (i) Ethyl 4-bromobutyrate, Cs2CO3, DMF, 100 ◦C, 16 h; (ii) KOH, DMF/water, 80 ◦C, 2–6 h, 80%; (**b**) HOBt, EDAC, CH2Cl2, Et3N, rt, amine: R-NH2 or HNR1R2, 2-8 h.

The in vitro anti-proliferative and anti-migratory activities of compounds **31–34** are represented in Table 2. In this assay, the replacement of the rigid carbazole ring core by an indole resulted in loss of cytotoxic activity on both MCF-7 and MDA-MB-231 cell lines. For example, from the four representative 5-bromoindole derivatives synthesized, it can be observed that only compound **34** showed moderate cytotoxic effect on MCF-7 cells with GI50 of 18.4 μM, while GI50 value on MDA-MB-231 breast cancer cells was above 50 μM. Furthermore, the remaining compounds in that series had GI50s above 50 μM in both breast cancer cell lines. Additionally, after 24 h treatment at 10 μM using the wound-healing assay in the MDA-MB-231 cell line, no migration inhibition could be observed. Hence, the absence of any activity of 5-bromoindole derivative series, together with the fact that several carbazole derivatives show promising activity, a structure-activity relationship (SAR) can be established. For example, it appears that the presence of a carbazole ring core, the C3 linker region, and amide group improve both anti-proliferative and anti-migratory activity.


**Table 2.** Cell growth inhibition and anti-migration activity of 5-bromoindole derivatives.

<sup>a</sup> GI50 = compound concentration required to inhibit MDA-MB-231 proliferation by 50% after 48 h treatment. Values are expressed as the mean of triplicate experiments, and standard deviation (SD) is <10%. <sup>b</sup> After 24 h, MDA-MB-231 cellular migration was determined by measuring the distance traveled from the edge of the scratch toward the center of the scratch, relative to control. <sup>c</sup> Percent relative migration values at 10 μM. Results are presented as means ± SD of three independent experiments.

#### **3. Materials and Methods**

#### *3.1. General Methods*

All experiments were carried out in pre-dried glassware (≥1 h, 80–90 ◦C) under a nitrogen atmosphere. Nuclear magnetic resonance (NMR) spectra were obtained using a 400 MHz Bruker Avance UltraShield™ spectrometer. 1H (400 MHz) and 13C (100 MHz) NMR were recorded in CDCl3 or DMSO-*d*6, unless otherwise used, and the chemical shift was expressed in parts per million (ppm) relative to CDCl3 (δ 7.26 for 1H and δ 77.0 for 13C) or DMSO-*d*<sup>6</sup> (δ 2.50 for 1H and δ 39.5 for 13C) as the internal standard. 1H NMR data is reported as position (δ), relative integral, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; dt, doublet of triplets; dd, doublet of doublets; dq, doublet of quartets; m, multiplet; br, broad peak), coupling constant (*J*) in hertz (Hz), and the assignment of the atom. The shift in ppm for multiplets correspond to the centermost value of the entire splitting pattern. 13C NMR data are reported as position (δ) and assignment of the atom. Microwave reactions were conducted in a CEM Discovery Microwave for Drug Discovery, SP- 1445. High resolution electrospray ionization mass spectrometry (ESI-HRMS) data were obtained on a Thermo Scientific™ Q Exactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer with high performance liquid chromatography (HPLC) Agilent 1200 utilizing a Zorbax SB-C18 column (2.1 mm × 50 mm, 1.8μm) at 40 ◦C, and a mobile phase of acetonitrile containing 5% Milli Q water at a flow rate of 0.35 mL/min (run time: 6.5 min), and 1 μL injection volume.

#### *3.2. Synthesis Methods*

Progress of the reaction was monitored via TLC analysis using general purpose silica gel on glass 5 × 20 cm with UV indicator, 250 μm, 60 Å medium pore diameter, UV indicator, and visualized by UV fluorescent Spectroline E Series Ultraviolet lamps, in most cases followed by staining with I2. The compounds were purified via column chromatography over silica gel (70–230 mesh, 60 Å) with the appropriate size column (24/40, 12 in. × 0.5 in.) or (24/40, 12 in. × 0.72 in.). Wiskostatin compound was obtained from MilliporeSigma.

#### 3.2.1. General Procedure for the Synthesis of 4-(3,6-Dibromo-Carbazol-9-yl)-Butyric Acid (**2**)

A 50 mL three-neck round-bottom flask, equipped with a reflux condenser, was charged with 3,6-dibromocarbazole **1** (0.325 g, 1.0 mmol), K2CO3 (0.1382 g, 1 mmol), and ethyl 4-bromobutyrate 2 (0.4436 mL, 3.1 mmol), dissolved in DMF (5 mL). After 15 min of stirring at room temperature, the reaction mixture was refluxed at 80 ◦C for 2 h. After completion of the reaction (analyzed by TLC), water (1 mL) and KOH (1.0 mmol) was added and the reaction mixture, refluxed at 80 ◦C for 2 h. After the reaction was completed (analyzed by TLC), the mixture was allowed to reach room temperature. The mixture was washed with water (20 mL) and the product was extracted using dichloromethane (3 × 10 mL). The organic layer was washed with brine and dried with Na2SO4, and filtered and concentrated under reduced pressure. The crude oil was purified via column chromatography over silica gel and 50% ethyl acetate in hexane, and the product obtained as a white solid for the precursor 4-(3,6-Dibromo-carbazol-9-yl)-butyric acid **2** (0.3535 g, 0.86 mmol, 86%). TLC analysis in ethyl acetate-hexane (1:1), *Rf* = 0.19. 1H NMR (400 MHz, CDCl3) δ 1.98 (2H, m), 2.27 (2H, t, *J* = 7.2 Hz), 4.41 (2H, t, *J* = 7.2 Hz), 7.30 (2H, d, *J* = 8.9 Hz), 7.56 (2H, dd, *J* = 2.0, 8.8 Hz), 8.14 (1H, d, *J* = 2.0 Hz); 13C NMR (100 MHz, CDCl3) 24.2, 31.1, 42.2, 111.8, 112.0, 123.4, 123.9, 139.5, 174.3. HR-FTMS (ESI) *m*/*z* calcd. for C16H13Br2NO2, [M + H]<sup>+</sup> 411.9365, found 411.9365.

3.2.2. Synthesis of 3,6-Dibromocarbazole-4-butyramide Derivatives (**4**–**27**)

#### 3.2.3. General Procedure for the Synthesis of

4-(3,6-Dibromocarbazol-9-yl)-N-(2-Morpholin-4-Ylethyl)Butyramide (**4**), and for Compounds **5**–**27**

A 50 mL three-neck round-bottom flask was charged with 4-(3,6-Dibromo-carbazol-9-yl)-butyric acid **2** (0.4111 g, 1.0 mmol), HOBT (0.2027 g, 1.5 mmol), and EDAC (0.2876 g, 1.5 mmol). The mixture was dissolved in CH2Cl2 (10 mL), stirred for 30 min, and 2-(4-morpholinyl)ethanamine **2** (0.133 g, 1.0 mmol) was added. After 15 min, Et3N (0.43 mL, 3.0 mmol) was added and the mixture was stirred at room temperature for 16 h. After completion of the reaction (analyzed by TLC), water was added (30 mL) and the product was extracted using dichloromethane (3 × 10 mL). The organic layer was washed with brine and dried with Na2SO4, and filtered and concentrated under reduced pressure. The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.382 g, 0.73 mmol, 73%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.26. 1H NMR (400 MHz, CDCl3) δ 2.0 (2H, m), 2.22 (2H, t, *J* = 6.8 Hz), 2.35 (2H, t, *J* = 4 Hz), 2.50 (4H, t, *J* = 1.6 Hz), 3.19 (2H, m), 3.56 (4H, t, *J* = 4.4 Hz), 4.39 (2H, t, *J* = 6.8 Hz), 7.60 (2H, d, *J* = 1.6 Hz), 8.0 (2H, bs), 8.46 (2H, bs); 13C NMR (100 MHz, CDCl3) 24.0, 31.2, 34.3, 41.8, 53.15, 53.2, 54.9, 57.2, 66.1, 111.3, 111.5, 122.9, 123.4, 128.8, 139.0, 161.0. HR-FTMS (ESI) *m*/*z* calcd. for C22H25Br2N3O2, [M + H]<sup>+</sup> 524.0366, found 524.0367.

#### Synthesis of 4-(3,6-Dibromocarbazol-9-yl)-*N*-(2-methoxyethyl)butyramide (**5**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.096 g, 0.21 mmol, 63%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.56. 1H NMR (400 MHz, CDCl3) δ 1.63 (2H, bs), 2.15 (2H, t, *J* = 6.0 Hz), 2.21 (2H, m), 3.63 (3H, s), 3.46 (2H, d, *J* = 2.0 Hz), 4.39 (2H, t, *J* = 6.4 Hz), 7.35 (2H, d, *J* = 8.8 Hz), 7.57 (2H, dd, *J* = 1.6, 8.8 Hz), 8.16 (2H, *d*, *J* = 1.6 Hz); 13C NMR (100 MHz, CDCl3) 24.1, 32.4, 39.2, 42.3, 58.7, 71.0, 110.5, 112.1, 123.2, 123.4, 129.1, 139.2, 171.6. HR-FTMS (ESI) *m*/*z* calcd. for C19H20Br2N2O2, [M + H]<sup>+</sup> 468.9944, found 468.9947.

#### Synthesis of 4-(3,6-Dibromocarbazol-9-yl)-*N*-(3-imidazol-1-yl-propyl)butyramide (**6**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.078 g, 0.15 mmol, 49%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.22. 1H NMR (400 MHz, CDCl3) δ 1.96 (2H, t, *J* = 6.8 Hz), 2.07 (2H, t, *J* = 6.4 Hz), 2.20 (2H, m), 3.23 (2H, q, *J* = 6.4 Hz), 3.98 (2H, t, *J* = 6.8 Hz), 4.38 (2H, t, *J* = 6.8 Hz), 6.93 (1H, bs), 7.05 (1H, Bs), 7.33 (2H, d, *J* = 8.4 Hz), 7.51 (1H, bs), 7.56 (2H, dd, *J* = 2.0, 8.8 Hz), 8.15 (2H, d, *J* = 1.6 Hz); 13C NMR (100 MHz, CDCl3) 24.0, 31.0, 32.3, 36.9, 42.2, 44.8, 110.5, 112.2, 112.3, 123.3, 123.4, 129.1, 129.3, 129.4, 139.3, 171.9. HR-FTMS (ESI) *m*/*z* calcd. for C22H22Br2N4O, [M + H]<sup>+</sup> 519.0213, found 519.0211.

#### Synthesis of 4-(3,6-Dibromocarbazol-9-yl)-*N*-(2-piperidin-1-ylethyl)butyramide (**7**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.1033 g, 0.2 mmol, 59%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.47. 1H NMR (400 MHz, CDCl3) δ 1.43 (2H, q, *J* = 4. 9 Hz), 1.52 (4H, m), 2.11 (4H, dt*, J* = 3.3, 11.7 Hz), 2.34 (3H, bs), 2.38 (3H, t, *J* = 6.0 Hz), 3.31 (2H, q, *J* = 5.5 Hz), 4.29 (2H, t, *J* = 6.4 Hz), 6.05 (1H, bs), 7.28 (2H, d, *J* = 8.7 Hz), 7.5 (2H, dd, *J* = 1.9, 8.7 Hz), 8.06 (2H, d, *J* = 1.9 Hz); 13C NMR (100 MHz, CDCl3) 24.0, 24.2, 32.2, 35.9, 42.2, 54.2, 57.0, 110.5, 112.0, 123.1, 123.2, 128.9, 139.2, 171.4. HR-FTMS (ESI) *m*/*z* calcd. for C23H27Br2N3O, [M + H]<sup>+</sup> 522.0573, found 522.0572.

#### Synthesis of 4-(3,6-Dibromocarbazol-9-yl)-*N*-(2-piperazin-1-ylethyl)butyramide (**8**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.1425 g, 0.27 mmol, 71%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.23. 1H NMR (400 MHz, CDCl3) δ 1.9 (1H, bs), 2.11 (2H, t, *J* = 6.4 Hz), 2.20 (2H, m), 2.38 (3H, bs), 2.42 (4H, t, *J* = 5.9 Hz), 2.84 (4H, t, *J* = 4.7 Hz), 3.32 (2H, q, *J* = 5.4 Hz), 4.38 (2H, t, *J* = 6.7 Hz), 7.33 (2H, d, *J* = 8.7 Hz), 7.54 (2H, dd, *J* = 1.9, 10.6 Hz), 8.14 (2H, s); 13C NMR (100 MHz, CDCl3) 24.0, 32.2, 35.6, 42.4, 45.9, 54.1, 57.0, 110.6, 112.1, 123.2, 123.5, 139.4, 171.4. HR-FTMS (ESI) *m*/*z* calcd. for C23H27Br2N3O, [M + H]<sup>+</sup> 523.0526, found 523.0526.

#### Synthesis of 4-(3,6-Dibromocarbazol-9-yl)-*N*-(3-morpholin-4-ylpropyl)butyramide (**9**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.1496 g, 0.28 mmol, 69%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.59. 1H NMR (400 MHz, CDCl3) δ 1.56 (2H, m, *J* = 6.0 Hz), 1.95 (2H, t, *J* = 6.8 Hz), 2.14 (4H, m), 2.23 (2H, bs), 2.36 (2H, t, *J* = 6.4 Hz), 3.25 (2H, q, *J* = 5.6 Hz), 3.42 (1H, bs), 4.31 (2H, t, *J* = 6.8 Hz), 7.31 (2H, d, *J* = 8.8 Hz), 7.54 (2H, dd, *J* = 2.0, 8.8 Hz), 8.12 (2H, d, *J* = 1.6 Hz); 13C NMR (100 MHz, CDCl3) 24.0, 24.4, 32.3, 39.6, 42.1, 53.5, 58.0, 66.8, 110.6, 112.1, 123.2, 123.4, 129.1, 139.3, 171.1. HR-FTMS (ESI) *m*/*z* calcd. for C23H27Br2N3O2, [M + H]<sup>+</sup> 538.0522, found 538.0521.

### Synthesis of 4-(3,6-Dibromocarbazol-9-yl)-*N*-(4-diethylamino-1-methylbutyl)butyramide (**10**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.0063 g, 0.040 mmol, 9.1%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.21. 1H NMR (400 MHz, CDCl3) δ 0.89 (2H, m), 1.19 (6H, t, *J* = 7.6 Hz), 1.64 (2H, m), 2.20 (3H, bs), 2.25 (2H, t, *J* = 6.8 Hz), 2.62 (2H, t, *J* = 6.4 Hz), 3.29 (4H, m), 3.60 (2H, t, *J* = 7.20 Hz), 4.37 (2H, t, *J* = 7.20 Hz), 7.33 (2H, d, *J* = 8.8 Hz), 7.55 (2H, dd, *J* = 2.0, 8.8 Hz), 8.13 (2H, d, *J* = 2.0 Hz), 9.28 (1H, bs); 13C NMR (100 MHz, CDCl3) 8.5, 21.2, 21.3, 24.4, 29.7, 32.7, 33.6, 42.6, 44.6, 47.0, 52.3, 110.7, 112.0, 123.2, 123.5, 129.1, 139.4, 172.0. HR-FTMS (ESI) *m*/*z* calcd. for C25H33Br2N3O2, [M + H]<sup>+</sup> 552.1043, found 552.1044.

Synthesis of 4-[4-(3,6-Dibromocarbazol-9-yl)-butyrylamino]piperidine-1-carboxylic acid ethyl ester (**11**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.1254 g, 0.22 mmol, 85.3%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.63. 1H NMR (400 MHz, CDCl3) δ 1.25 (3H, t, *J* = 7.2 Hz), 1.88 (2H, dd, *J* = 2.8, 12.4 Hz), 2.08 (2H, t, *J* = 6.4 Hz), 2.19 (2H, m, *J* = 6.8 Hz), 2.88 (2H, t, *J* = 12.0 Hz), 3.92 (1H, m, *J* = 3.6 Hz), 4.12 (4H, q, *J* = 6.8 Hz), 4.37 (2H, t, *J* = 6.8 Hz), 5.16 (1H, d, *J* = 7.6 Hz), 7.32 (2H, d, *J* = 8.8 Hz), 7.55 (2H, dd, *J* = 2.0, 8.8 Hz), 8.14 (2H, d, *J* = 1.6 Hz); 13C NMR (100 MHz, CDCl3) 14.7, 24.0, 32.0, 32.4, 42.2, 42.7, 46.8, 61.4, 110.5, 112.2, 123.3, 123.5, 129.2, 139.3, 155.4, 170.1. HR-FTMS (ESI) *m*/*z* calcd. for C24H27Br2N3O3, [M + H]<sup>+</sup> 566.0471, found 566.0470.

Synthesis of 4-(3,6-Dibromocarbazol-9-yl)-1-morpholin-4-ylbutan-1-one (**12**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.0768 g, 0.16 mmol, 52%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.94. 1H NMR (400 MHz, CDCl3) δ 2.15 (4H, t, *J* = 6.0 Hz), 2.21 (2H, m, *J* = 6.8 Hz), 3.16 (2H, t, *J* = 4.8 Hz), 3.50 (2H, t, *J* = 4.4 Hz), 3.64 (4H, dt, *J* = 2.8, 6.4 Hz), 7.31 (2H, d, *J* = 8.8 Hz), 7.55 (2H, dd, *J* = 1.6, 8.4 Hz), 8.14 (2H, d, *J* = 1.6 Hz); 13C NMR (100 MHz, CDCl3) 23.6, 28.6, 41.5, 41.9, 45.1, 65.9, 66.0, 111.3, 111.5, 122.9, 123.41, 128.8, 139.0, 170.0. HR-FTMS (ESI) *m*/*z* calcd. for C20H20Br2N2O2, [M + H]<sup>+</sup> 480.9944, found 480.9945.

Synthesis of 4-(3,6-Dibromocarbazol-9-yl)-1-thiomorpholin-4-ylbutan-1-one (**13**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.0712 g, 0.41 mmol, 35%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.60. 1H NMR (400 MHz, CDCl3) δ 2.11 (2H, t, *J* = 6.3 Hz), 2.21 (2H, m), 2.34 (2H, t, *J* = 5.0 Hz), 2.62 (2H, t, *J* = 5.2 Hz), 3.46 (2H, t, *J* = 5.0 Hz), 3.89 (2H, t, *J* = 5.0 Hz), 4.41 (2H, t, *J* = 6.7 Hz), 7.32 (2H, d, *J* = 8.6 Hz), 7.56 (2H, dd, *J* = 1.9, 8.7 Hz), 8.15 (2H, d, *J* = 1.7 Hz); 13C NMR (100 MHz, CDCl3) 23.5, 27.3, 27.4, 29.0, 42.1, 44.3, 47.8, 110.6, 112.2, 123.3, 123.5, 129.1, 139.4, 169.9. HR-FTMS (ESI) *m*/*z* calcd. for C20H20Br2N3OS, [M + H]<sup>+</sup> 496.9715, found 496.9713.

Synthesis of 1-[4-(3,6-Dibromocarbazol-9-yl)butyryl]piperidine-4-carbonitrile (**14**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.0775 g, 0.16 mmol, 44%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.97. 1H NMR (400 MHz, CDCl3) δ 1.63 (2H, t, *J* = 5.6 Hz), 1.85 (1H, m), 2.15 (2H, m), 2.84 (2H, m), 3.14 (2H, dt, *J* = 5.2, 13.6 Hz), 3.35 (2H, dt, *J* = 6.0, 14.4 Hz), 3.63 (2H, dq, *J* = 4.4, 13.6 Hz), 3.76 (2H, dq, *J* = 3.2, 13.6 Hz), 4.41 (2H, m), 7.31 (2H, d, *J* = 8.4 Hz), 7.54 (2H, dd, *J* = 1.6, 8.4 Hz), 8.14 (2H, d, *J* = 1.6 Hz); 13C NMR (100 MHz, CDCl3) 23.4, 26.2, 28.1, 28.6, 28.7, 39.5, 42.1, 43.0, 110.6, 112.2, 120.5, 123.3, 123.5, 129.1, 139.4, 169.8. HR-FTMS (ESI) *m*/*z* calcd. for C22H21Br2N3O, [M + H]<sup>+</sup> 504.0104, found 504.0104.

### Synthesis of 4-(3,6-Dibromocarbazol-9-yl)-1-(4-phenylpiperazin-1-yl)butan-1-one (**15**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.2658 g, 0.27 mmol, 69%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.53. 1H NMR (400 MHz, CDCl3) δ 1.55 (2H, bs), 2.22 (2H, m), 2.97 (2H, t, *J* = 5.3 Hz), 3.15 (2H, t, *J* = 4.9 Hz), 3.34 (2H, t, *J* = 4.5 Hz), 3.30 (2H, t, *J* = 4.7 Hz), 4.43 (2H, t, *J* = 3.4 Hz), 6.92 (2H, t, *J* = 7.9 Hz), 7.29 (3H, t, *J* = 7.9 Hz), 7.34 (2H, d, *J* = 8.7 Hz), 7.54 (2H, dd, *J* = 1.8, 8.6 Hz), 8.15 (2H, d, *J* = 1.7 Hz); 13C NMR (100 MHz, CDCl3) 23.6, 28.9, 41.6, 42.2, 45.1, 49.4, 49.5, 110.6, 112.1, 116.8, 120.7, 123.3, 123.5, 129.2, 129.3, 139.4, 150.9, 170.0. HR-FTMS (ESI) *m*/*z* calcd. for C26H25Br2N3O, [M + H]<sup>+</sup> 556.0417, found 556.0417.

#### Synthesis of 1-(4-Acetylpiperazin-1-yl)-4-(3,6-dibromocarbazol-9-yl)butan-1-one (**16**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.0775 g, 0.15 mmol, 62%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.67. 1H NMR (400 MHz, CDCl3) δ 1.57 (3H, s), 2.08 (2H, bs), 2.12 (2H, bs), 2.21 (2H, bs), 3.19 (2H, t, *J* = 5.3 Hz), 3.45 (2H, t, *J* = 6.2), 3.64 (2H, t, *J* = 5.0 Hz), 4.42 (2H, t, *J* = 6.6 Hz), 7.32 (2H, dd, *J* = 4.2, 8.7 Hz), 7.54 (2H, dd, *J* = 1.9, 8.7 Hz), 8.14 (2H, d, *J* = 1.8 Hz); 13C NMR (100 MHz, CDCl3) 21.4, 28.8, 31.0, 41.5, 42.0, 42.2, 44.8, 45.9, 110.5, 112.2, 123.4, 129.1, 139.3, 169.3, 170.4. HR-FTMS (ESI) *m*/*z* calcd. for C22H23Br2N3O2, [M + H]<sup>+</sup> 522.0209, found 522.0209.

Synthesis of 4-[4-(3,6-Dibromocarbazol-9-yl)butyryl]piperazine-1-carboxylic acid tert-butyl ester (**17**)

The crude oil was purified via column chromatography over silica gel and hexane-ethyl acetate (1:1) as the mobile phase, and the product obtained as a white solid (0.1977 g, 0.4 mmol, 80%). TLC analysis in hexane-ethyl acetate (1:1), *Rf* = 0.35. 1H NMR (400 MHz, CDCl3) δ 1.47 (9H, s), 2.20 (4H, m), 3.16 (2H, bs), 3.26 (2H, bs), 3.40 (2H, t, *J* = 5.2 Hz), 3.60 (2H, t, *J* = 4.4 Hz), 4.41 (2H, t, *J* = 6.4 Hz), 7.32 (2H, d, *J* = 8.4 Hz), 7.54 (2H, dd, *J* = 1.6, 8.4 Hz), 8.14 (2H, d, *J* = 2.0 Hz); 13C NMR (100 MHz, CDCl3) 23.5, 28.4, 29.0, 41.4, 42.2, 45.0, 80.4, 110.5, 112.2, 123.3, 123.5, 129.1, 139.4, 154.5, 170.2. HR-FTMS (ESI) *m*/*z* calcd. for C25H29Br2N3O3, [M + Na]<sup>+</sup> 602.0447, found 602.0446.

Synthesis of 4-(3,6-Dibromocarbazol-9-yl)-1-piperazin-1-ylbutan-1-one (**18**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.0383 g, 0.080 mmol, 80%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.38. 1H NMR (400 MHz, CDCl3) δ 1.25 (1H, s), 2.18 (4H, m), 2.68 (2H, t, *J* = 4.8 Hz), 2.84 (2H, t, *J* = 5.2 Hz), 3.16 (2H, t, *J* = 4.8 Hz), 3.61 (2H, t, *J* = 5.2 Hz), 4.41 (2H, t, *J* = 6.4 Hz), 7.32 (2H, d, *J* = 8.4 Hz), 7.54 (2H, dd*, J* = 2.0, 8.8 Hz), 8.14 (2H, d, *J* = 1.6 Hz); 13C NMR (100 MHz, CDCl3) 22.6, 23.6, 42.2, 42.7, 45.8, 46.0, 46.3, 110.6, 112.1, 123.3, 123.5, 129.1, 139.4, 170.0. HR-FTMS (ESI) *m*/*z* calcd. for C20H21Br2N3O, [M + H]<sup>+</sup> 480.0104, found 480.0101.

Synthesis of 4-(3,6-Dibromocarbazol-9-yl)-1-[4-(2-morpholin-4-ylethyl)piperazin-1-yl]butan-1-one (**19**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.2036 g, 0.34 mmol, 76.4%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.52. 1H NMR (400 MHz, CDCl3) δ 2.12 (2H, t, *J* = 6.0 Hz), 2.18 (2H, t, *J* = 7.2 Hz), 2.24 (2H, m), 2.44 (2H, t, *J* = 5.2 Hz), 2.48 (4H, t, *J* = 4.4 Hz), 3.17 (2H, t, *J* = 4.8 Hz), 3.63 (2H, t, *J* = 5.2 Hz), 3.70 (4H, t, *J* = 4.8 Hz), 4.41 (2H, t, *J* = 6.4 Hz), 7.28 (2H, d, *J* = 6.0 Hz), 7.37 (2H, dd, *J* = 6.4 Hz, 8.8 Hz), 8.15 (2H, d, *J* = 2.1 Hz); 13C NMR (100 MHz, CDCl3) 23.5, 28.7, 41.6, 42.1, 45.0, 53.2, 53.3, 54.1, 55.4, 56.3, 66.9, 110.7, 112.1, 123.3, 123.4, 129.2, 139.4, 169.8. HR-FTMS (ESI) *m*/*z* calcd. for C26H32Br2N4O2, [M + H]<sup>+</sup> 593.0944, found 593.0945.

### Synthesis of 2-{4-[4-(3,6-Dibromocarbazol-9-yl)-butyryl]piperazin-1-yl}-*N*-isopropylacetamide (**20**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.2273 g, 0.39 mmol, 87.3%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.68. 1H NMR (400 MHz, CDCl3) δ 1.17 (6H, d, *J* = 6.6 Hz), 2.10 (2H, t, *J* = 6.3 Hz), 2.21 (2H, m), 2.26 (2H, t, *J* = 4.7 Hz), 2.47 (2H, t, *J* = 4.5 Hz), 3.15 (2H, t, *J* = 4.5 Hz), 3.64 (2H, t, *J* = 4.5 Hz), 4.10 (1H, m), 4.43 (2H, t, *J* = 6.6 Hz), 6.73 (1H, bs), 7.31 (2H, d, *J* = 8.7 Hz), 7.54 (2H, dd, *J* = 1.9, 8.7 Hz), 8.14 (2H, d, *J* = 1.9 Hz); 13C NMR (100 MHz, CDCl3) 22.8, 23.4, 28.7, 40.8, 41.6, 42.0, 45.0, 53.0, 53.1, 61.5, 110.64, 112.1, 123.3, 123.5, 129.2, 139.4, 168.4, 170.0. HR-FTMS (ESI) *m*/*z* calcd. for C25H30Br2N4O2, [M + H]<sup>+</sup> 579.0788, found 579.0790.

Synthesis of 4-(3,6-Dibromocarbazol-9-yl)-1-[4-(2-dimethylaminoethyl)piperazin-1-yl]butan-1-one (**21**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.0228 g, 0.04 mmol, 7%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.50. 1H NMR (400 MHz, CDCl3) δ 1.25 (6H, s), 2.12 (2H, t, *J* = 6.0 Hz), 2.19 (2H, m), 2.24 (4H, t, *J* = 5.6 Hz), 2.43 (4H, t, *J* = 4.8 Hz), 3.19 (2H, t, *J* = 4.4 Hz), 3.64 (2H, t, *J* = 4.4 Hz), 4.40 (2H, t, *J* = 6.4 Hz), 7.31 (2H, d, *J* = 8.8 Hz), 7.53 (2H, dd, *J* = 1.6, 8.4 Hz,), 8.21 (2H, d, *J* = 2.0 Hz); 13C NMR (100 MHz, CDCl3) 23.5, 28.7, 41.5, 42.1, 45.0, 45.6, 53.1, 53.3, 56.1, 56.5, 110.6, 112.1, 123.2, 123.4, 129.2, 139.4, 170.0. HR-FTMS (ESI) *m*/*z* calcd. for C24H30Br2N4O, [M + H]<sup>+</sup> 551.0839, found 551.0842.

Synthesis of 4-(3,6-Dibromocarbazol-9-yl)-*N*-(4-morpholin-4-ylphenyl)butyramide (**22**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.0689 g, 0.12 mmol, 40%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.80. 1H NMR (400 MHz, DMSO*-d6*) δ 2.05 (2H, t, *J* = 6.8 Hz), 2.30 (2H, m), 3.02 (4H, t, *J* = 4.8 Hz), 3.71 (4H, t, *J* = 4.4 Hz), 4.44 (2H, t, *J* = 6.8 Hz), 6.86 (2H, d, *J* = 8.8 Hz), 7.41 (2H, d, *J* = 8.8 Hz), 7.61 (2H, dd, *J* = 0.8, 8.8 Hz), 7.64 (2H, d, *J* = 8.4 Hz), 8.48 (2H, s), 9.62 (1H, s); 13C NMR (100 MHz, DMSO-*d6*) 24.1, 32.7, 42.0, 48.9, 66.1, 111.3, 111.6, 115.4, 120.3, 122.9, 123.4,128.8, 131.5, 139.0, 147.0, 169.7. HR-FTMS (ESI) *m*/*z* calcd. for C26H25Br2N3O2, [M + H]<sup>+</sup> 572.0366, found 572.0365.

### Synthesis of 4-(3,6-Dibromocarbazol-9-yl)-*N*-(3,4,5-trimethoxyphenyl)butyramide (**23**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.051 g, 0.10 mmol, 30%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.17. 1H NMR (400 MHz, DMSO*-d6*) δ 2.08 (2H, m), 2.28 (2H, t, *J* = 7.2 Hz), 3.60 (3H, s), 3.71 (3H, s), 3.72 (3H, s), 4.46 (2H, t, *J* = 6.8 Hz), 6.90 (2H, s), 7.60 (2H, dd, *J* = 1.6, 8.4 Hz), 7.64 (2H, d, *J* = 8.8 Hz), 8.47 (2H, d, *J* = 1.6 Hz), 9.71 (1H, s); 13C NMR (100 MHz, DMSO-*d6*) 6.96, 24.0, 32.0, 42.0, 55.6, 60.0, 96.9, 111.3, 111.6, 122.9, 123.4, 128.8, 133.3, 135.2, 139.1, 152.6, 170.1. HR-FTMS (ESI) *m*/*z* calcd. for C25H24Br2N2O4, [M + H]<sup>+</sup> 577.0155, found 577.0142.

#### Synthesis of 4-(3,6-Dibromocarbazol-9-yl)-*N*-quinolin-3-ylbutyramide (**24**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.0275 g, 0.052 mmol, 15%). TLC analysis in hexane-ethyl acetate (1:1), *Rf* = 0.20. 1H NMR (400 MHz, DMSO*-d6*) δ 2.13 (2H, m), 2.44 (2H, t, *J* = 6.8 Hz), 4.50 (2H, t, *J* = 6.5 Hz), 7.41 (1H, t, *J* = 7.4 Hz), 7.54 (1H, t, *J* = 8.0 Hz), 7.62 (1H, t, *J* = 7.9 Hz), 7.67 (1H, d, *J* = 8.6 Hz), 7.71 (1H, d, *J* = 8.3 Hz), 7.92 (2H, t, *J* = 8.4 Hz), 7.98 (2H, d, *J* = 8.4 Hz), 8.47 (2H, s), 8.64 (1H, s), 8.82 (1H, s), 10.3 (1H, s); 13C NMR (100 MHz, DMSO-*d6*) 23.9. 32.9, 42.0, 109.6, 111.4, 111.7, 119.2, 122.0, 123.0, 123.5, 124.5, 127.0, 127.4, 127.6, 127.7, 127.8, 128.5, 128.9, 132.8, 139.1, 144.1, 144.5, 171.3. HR-FTMS (ESI) *m*/*z* calcd. for C25H19Br2N3O, [M + H]<sup>+</sup> 537.9947, found 537.9946.

#### Synthesis of 4-(3,6-Dibromocarbazol-9-yl)-*N*-(1H-indol-6-yl)butyramide (**25**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.1152 g, 0.22 mmol, 38%). TLC analysis in hexane-ethyl acetate (1:1), *Rf* = 0.50. 1H NMR (400 MHz, DMSO*-d6*) δ 2.08 (2H, m), 2.35 (2H, t, *J* = 7.1 Hz), 4.47 (2H, t, *J* = 7.0 Hz), 6.33 (1H, s), 6.97, (1H, d, *J* = 9.1 Hz), 7.24 (1H, s), 7.40 (1H, d, *J* = 8.4 Hz), 7.61 (2H, d, *J* = 7.2 Hz), 7.66 (2H, d, *J* = 8.7 Hz), 7.95 (1H, s), 8.49 (2H, s), 9.75 (1H, s); 13C NMR (100 MHz, DMSO-*d6*) 24.2, 32.9, 42.1, 100.9, 102.2, 111.3, 111.6, 112.3, 119.7, 123.0, 123.5, 123.7, 124.8, 128.9, 133.3, 135.9, 139.1. HR-FTMS (ESI) *m*/*z* calcd. for C24H19Br2N3O, [M + H]<sup>+</sup> 525.9947, found 9946.

#### Synthesis of *N*-(3H-Benzoimidazol-5-yl)-4-(3,6-dibromocarbazol-9-yl)butyramide (**26**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.100 g, 0.20 mmol, 44%). TLC analysis in hexane-ethyl acetate (1:1), *Rf* = 0.70. 1H NMR (400 MHz, DMSO*-d6*) δ 2.16 (2H, m, *J* = 7.2 Hz), 3.18 (2H, t, *J* = 7.6 Hz), 4.52 (2H, t, *J* = 7.2 Hz), 6.66 (1H, dd, *J* = 2.0, 8.8 Hz), 7.60 (1H, dd, *J* = 2.0, 8.8 Hz), 7.68 (1H, d, *J* = 8.8 Hz), 8.05 (1H, s), 8.47 (1H, dd, *J* = 1.6, 8.0 Hz); 13C NMR (100 MHz, DMSO-*d6*) 23.6, 32.0, 42.3, 97.2, 111.8, 112.0, 114.3, 117.2, 122.3, 123.5, 124.0, 129.4, 139.5, 141.0, 141.2, 151.7, 173.0. HR-FTMS (ESI) *m*/*z* calcd. for C23H18Br2N4O, [M + H]<sup>+</sup> 526.9900, found 526.9904.

#### Synthesis of 4-(3,6-Dibromocarbazol-9-yl)-*N*-(4-methoxyphenyl)butyramide (**27**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.1830 g, 0.35 mmol, 94%). TLC analysis in hexane-ethyl acetate (1:1), *Rf* = 0.64. 1H NMR (400 MHz, DMSO*-d6*) δ 2.05 (2H, m), 2.30 (2H, t, *J* = 7.2 Hz), 3.70 (3H, s), 4.45 (2H, t, *J* = 7.2 Hz), 6.84 (2H, d, *J* = 9.2 Hz), 7.44 (2H, d, *J* = 9.2 Hz), 7.60 (2H, dd, *J* = 2.0, 8.8 Hz), 7.65 (2H, d, *J* = 8.8 Hz), 8.48 (2H, d, *J* = 2.0 Hz), 9.68 (1H, s); 13C NMR (100 MHz, DMSO-*d6*) 24.1, 32.7, 42.0, 55.1, 111.3, 111.6, 113.8, 120.7, 123.0, 123.5, 128.7, 132.3, 139.1, 155.1, 169.9. HR-FTMS (ESI) *m*/*z* calcd. for C23H20Br2N2O2, [M + H]<sup>+</sup> 516.9944, found 516.9941.

#### 3.2.4. Synthesis of N-Alkyl-5-Bromoindole Derivatives (**31**–**34**)

#### 3.2.5. General Procedure for the Synthesis of 4-(5-Bromoindol-1-yl)Butyric Acid (**29**)

A 50 mL three-neck round-bottom flask, equipped with a reflux condenser, was charged with 5-bromoindole **28** (0.1960 g, 1.0 mmol), Cs2CO3 (0.9770 g, 3 mmol) and ethyl 4-bromobutyrate (0.43 mL, 3.0 mmol), dissolved in DMF (3 mL). After 15 min of stirring at room temperature, the reaction mixture was refluxed at 100 ◦C for 16 h. After completion of the reaction (analyzed by TLC), water (1 mL) and KOH (1.0 mmol) were added and the reaction mixture refluxed at 80 ◦C for 2 h. After the reaction was completed (analyzed by TLC), the mixture was allowed to reach room temperature and 1N HCl solution was added until reach neutral pH. The mixture was washed with water (20 mL) and the product was extracted using dichloromethane (3 × 10 mL). The organic layer was washed with brine and dried with Na2SO4, and filtered and concentrated under reduced pressure. The crude oil was purified via column chromatography over silica gel and 10% CH2Cl2 in MeOH, and the product obtained as a white solid for the precursor 4-(5-Bromoindol-1-yl)Butyric acid **29** (0.2260 g, 0.801 mmol, 80%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.25. 1H NMR (400 MHz, CDCl3) δ 2.16 (2H, m), 2.34 (2H, t, *J* = 7.2 Hz), 4.20 (2H, t, *J* = 6.80 Hz), 6.44 (1H, d, *J* = 2.4 Hz), 7.08 (1H, 2, *J* = 3.2 Hz), 7.22 (1H, d, *J* = 6.4 Hz), 7.29 (1H, dd, *J* = 2.0, 8.8 Hz), 7.74 (1H, d, *J* = 1.6 Hz); 13C NMR (100 MHz, CDCl3) 25.1, 30.5, 45.3, 101.1, 110.7, 112.8, 123.5, 124.5, 128.9, 130.3, 134.6, 177.4. HR-FTMS (ESI) *m*/*z* calcd. for C12H12BrNO2, [M + H]<sup>+</sup> 284.0104, found 284.0102.

#### 3.2.6. General Procedure for the Synthesis of

4-(5-Bromoindol-1-yl)-N-(3-Morpholin-4-ylpropyl)Butyramide (**31**), and for Compounds **32**–**34**

A 50 mL three-neck round-bottom flask was charged with 4-(5-Bromoindol-1-yl)Butyric acid **29** (0.098 g, 0.35 mmol), HOBT (0.2027 g, 1.5 mmol), and EDAC (0.2876 g, 1.5 mmol). The mixture was dissolved in DMF (5 mL), stirred for 30 min, and 3-morpholinopropylamine (0.0512 mL, 0.35 mmol) was added. After 15 min, Et3N (0.43 mL, 3.0 mmol) was added and the mixture stirred at room temperature for 16 h. After the reaction was completed (analyzed by TLC), water was added (30 mL) and the product was extracted using dichloromethane (3 × 10 mL). The organic layer was washed with brine and dried with Na2SO4, and filtered and concentrated under reduced pressure. The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.0748 g, 0.18 mmol, 53%). TLC analysis in hexane-ethyl acetate (1:1), *Rf* = 0.36. 1H NMR (400 MHz, CDCl3) δ 1.62 (2H, m), 2.01 (2H, t, *J* = 6.8 Hz), 2.21 (2H, m), 2.35 (4H, bs), 2.42 (2H, t, *J* = 6.4 Hz), 3.32 (2H, q, *J* = 5.6 Hz), 3.47 (4H, bs), 4.39 (2H, t, *J* = 6.8 Hz), 7.0 (1H, bs), 7.26 (1H, d, *J* = 1.2 Hz), 7.32 (1H, d, *J* = 8.8 Hz), 7.55 (1H, dd, *J* = 2.0, 8.8 Hz), 8.14 (1H, d, *J* = 1.6 Hz); 13C NMR (100 MHz, CDCl3) 24.5, 25.8, 32.6, 39.6, 45.2, 53.6, 58.0, 67.0, 101.1, 110.9, 112.6, 123.4, 124.3, 128.8, 130.2, 134.9, 171.2. HR-FTMS (ESI) *m*/*z* calcd. for C19H26BrN3O2, [M + H]<sup>+</sup> 410.1261, found 410.1257.

#### Synthesis of 1-[4-(5-Bromoindol-1-yl)butyryl]piperidine-4-carbonitrile (**32**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.0356 g, 0.10 mmol, 24.4%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.76. 1H NMR (400 MHz, DMSO-*d6*) δ 1.58 (4H, m), 1.81 (2H, bs), 1.94 (2H, t, *J* = 7.2 Hz), 2.26 (2H, t, *J* = 7.2 Hz), 3.07 (1H, m), 3.50 (2H, dt, *J* = 4.8, 15.2 Hz), 3.78 (2H, dt, *J* = 4.8, 13.6Hz), 4.18 (2H, t, *J* = 6.8Hz), 6.42 (1H, d, *J* = 3.2 Hz), 7.24 (1H, dd, *J* = 2.0, 8.4 Hz), 7.41 (1H, d, *J* = 3.2 Hz), 7.47 (1H, d, *J* = 8.8 Hz), 7.73 (1H, d, *J* = 2.0 Hz); 13C NMR (100 MHz, DMSO-*d6*) 22.7, 25.3, 28.2, 29.4, 29.7, 39.5, 43.1, 45.3, 101.0, 111.0, 112.7, 120.6, 123.5, 124.4, 128.9, 130.2, 134.9, 169.9. HR-FTMS (ESI) *m*/*z* calcd. for C18H20BrN3O, [M + H]<sup>+</sup> 376.0842, found 376.0837.

#### Synthesis of 4-(5-Bromoindol-1-yl)-N-(3-imidazol-1-yl-propyl)butyramide (**33**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.1255 g, 0.32 mmol, 57%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.44. 1H NMR (400 MHz, CDCl3) δ 1.81 (2H, m, *J* = 6.8 Hz), 1.98 (2H, m, *J* = 6.8 Hz), 2.06 (2H, t, *J* = 6.4 Hz), 3.01 (2H, q, *J* = 5.6 Hz), 3.95 (2H, t, *J* = 6.8 Hz), 4.17 (2H, t, *J* = 7.2 Hz), 6.42 (1H, d, *J* = 2.8 Hz), 6.95 (1H, bs), 7.23 (2H, dd, *J* = 1.6, 8.4 Hz), 7.40 (1H, d, *J* = 2.8 Hz), 7.45 (1H, d, *J* = 8.8 Hz), 7.72 (1H, d, *J* = 2.0 Hz), 7.90 (1H, t, *J* = 5.2 Hz); 13C NMR (100 MHz, CDCl3) 25.7, 31.0, 32.2, 36.5, 44.2, 45.6, 101.0, 111.0, 112.5, 119.1, 123.3, 124.4, 128.7, 128.8, 130.2, 134.7, 136.9, 172.4. HR-FTMS (ESI) *m*/*z* calcd. for C18H21BrN4O, [M + H]<sup>+</sup> 391.0951, found 391.0956.

#### Synthesis of 4-(5-Bromoindol-1-yl)-N-(2-piperidin-1-ylethyl)butyramide (**34**)

The crude oil was purified via column chromatography over silica gel and 10% methanol in dichloromethane, and the product obtained as a white solid (0.1967 g, 0.50 mmol, 50%). TLC analysis in CH2Cl2-MeOH (9:1), *Rf* = 0.47. 1H NMR (400 MHz, DMSO-*d6*) δ 1.35 (2H, m, *J* = 5.2 Hz), 1.47 (4H, m, *J* = 5.6 Hz), 1.96 (2H, m, *J* = 6.4 Hz), 2.01 (2H, t, *J* = 6.0 Hz), 2.30 (2H, t, *J* = 6.8 Hz), 2.35 (2H, bs), 3.13 (2H, q, *J* = 6.4 Hz), 4.18 (2H, t, *J* = 6.4 Hz) 7.35 (1H, dd, *J* = 2.0, 8.8 Hz), 7.54 (2H, d, *J* = 2.4 Hz), 7.56 (1H, s), 7.68 (1H, s), 7.71 (1H, t, *J* = 5.6 Hz); 13C NMR (100 MHz, DMSO-*d6*) 24.1, 25.5, 25.7, 32.4, 35.7, 45.8, 54.3, 57.2, 100.8, 109.4, 111.0, 118.5, 120.3, 128.9, 129.0, 134.7, 171.7. HR-FTMS (ESI) *m*/*z* calcd. for C25H30Br2N4O2, [M + H]<sup>+</sup> 394.1312, found 394.1313.

#### *3.3. Biological Evaluation*

#### 3.3.1. Cell Culture

MCF-7 and MDA-MB-231 cells were cultured in Minimum Essential Medium Eagle (MEME) 10% FBS supplemented with Earle's Balanced Salt Solution (EBSS), Non-essential Amino Acids (NEAA), Sodium Pyruvate, Pen/Strep, and L-glutamine 37 ◦C in 5% CO2.

#### 3.3.2. Sulforhodamine B (SRB) Assay

A stock solution of compounds was prepared at 50 mM in 100% DMSO. For cell preparation, a flask of 75 cm<sup>2</sup> or 25 cm<sup>2</sup> was used for 2.6 <sup>×</sup> 105 cells/mL or 1.44 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL, respectively, with an 80–90% confluence. Cells were washed with PBS and trypsinized. The concentration of cells was determined using a 1:2 dilution with Trypan Blue and a hemocytometer. After cell count, the concentration was adjusted to have a 7.0–10.0 <sup>×</sup> 104 cells/mL. Approximately 100 <sup>μ</sup>L of cells suspension, compounds, control positive and control negative were added in triplicates to a 96 well plate. The positive control used was doxorubicin, and the negative control was DMSO 0.1%. All compounds at 50, 25, 12.5, 6.3, and 1.6 μM were incubated with cells at 37 ◦C for 48 hrs. For fixation, cold TCA 50% was used and incubated at 4 ◦C for 1 hr. Wells were washed and dried prior to tincture with 100 μL of SRB 0.4%. To remove excess SRB, acetic acid was used. For analysis, TRIS-Base Solution (pH = 10.5) was used and shaken prior to reading using an ELISA reader at 540 nm and the software SoftMax Pro 4.8. For each compound, 50% growth inhibition (GI50) was calculated from sigmoidal dose-response curves (variable-slope) that were generated with data obtained from experiments carried out in triplicates (GI50 values were generated with GraphPad Prism V. 6.02, GraphPad Software, Inc.).

#### 3.3.3. Wound Healing Assay (Scratch Method) Using MDA-MB-231 Cancer Cells

Prior to assays, cells were grown until 80–90% confluence was observed. We used a 75 cm2 flask for 2.6 <sup>×</sup> 10<sup>5</sup> cells/mL in 10 mL, and for a 25 cm<sup>2</sup> flask 1.44 <sup>×</sup> 105 cells/mL in 5 mL. The cells were washed with PBS to remove all traces of FBS. We added trypsin at 2 mL for a 25 cm<sup>2</sup> flask or 4 mL for a 75 cm2 flask, and incubated 5–10 min at 37 ◦C. At the end of the incubation time, cells were re-suspended and counted with hemocytometer using 1:2 dilutions with Trypan Blue. Subsequently, cell viability was calculated. In a 12 multiwell plate. Cells were seeded at 1.5–2.2 <sup>×</sup> 105 cells/mL in 1 mL and incubated for 24 h. Cells were then rinsed with PBS and incubated in starving media (0.5% FBS) overnight. All controls and drugs were tested in triplicate. The vehicle control for each drug was prepared according to the drug's DMSO concentration. Drugs were diluted and the final concentration at each well was 10 μM (or GI50/5 on MDA-MB-231 cells). The wound was made using a sterile pipette tip of 200 μL. Cells were then rinsed very gently with media without FBS and media with vehicle controls or drugs were added. After a 24 h incubation, the gap distance was evaluated using the software Lumera Infinity Analyze 6.4.0. Pictures were taken at 0, 8, 12, and 24 h using a 10X objective in an Inverted Laboratory Microscope Leica DM IL LED, and an Infinity1-3 3.1 Megapixel USB 2.0 camera CMOS. The percentage of migration was calculated using the following formula: ..

100 − [ (X0⁄ X0)]\*100 for time 0 h measurements ..

100 − [ (X24⁄⁄ X0)]\*100 for time 24 h measurements

#### 3.3.4. Actin Polymerization Assay

MDA-MB-231 cells were treated with vehicle or compound derivatives at 10 μM (or at concentrations that do not affect cell viability) for 24 h at 37 ◦C in 5% CO2. Cells were fixed in 3.7% of formaldehyde, permeabilized using 0.2% Triton, and stained with Rhodamine Phalloidin to visualize F-actin. Fluorescence micrographs were acquired at 60x in an Eclipse E400 fluorescence microscope using a DS-Qi2 monochrome digital camera from Nikon for fluorescence imaging.

#### **4. Conclusions**

In this study, a new series of *N*-alkyl-3,6-dibromocarbazole and *N*-alkyl-5-bromoindole derivatives were designed, synthesized, and biologically evaluated for their anti-cancer and anti-migration effect on MCF-7 and MDA-MB-231 cancer cells. In addition, the effect of compounds on the actin cytoskeleton of MDA-MB-231 cancer cells was explored. From the results, we have established the Structure-Activity Relationships of novel derivatives of the lead compound Wiskostatin. We found that the carbazole moiety is needed for in vitro anti-cancer and anti-migratory potency. The replacement of the carbazole by an indole ring resulted in loss of activity. Moreover, the aliphatic or aromatic amide group was extended 3-carbon atoms away from the carbazole moiety. The most potent anti-proliferative compounds were those bearing a 3,6-dibromocarbazole group with GI50 values between 7–32 μM and 4.7–23 μM on MCF-7 and MDA-MB-231 cancer cells, respectively. The most promising results in terms of anti-migratory effect was exhibited by carbazole derivatives **10**, **23**, **14–16**, and **24** with migration inhibition in the range of 10–20% on the highly metastatic breast cancer cells MDA-MB-231. Future studies will improve these derivatives and also test their effects at higher concentrations and shorter times of migration to reduce potential cytotoxic effects, which may confound inhibition of cell migration. The carbazole derivatives **10**, **14**, and **15** were further examined as actin polymerization inhibitors and results demonstrated reduction in actin polymerization and extension of F-actin based structures. Further studies of mechanism of actions focused on the effect of these novel compounds in the Cdc42/N-WASP/Arp2/3 pathway is needed to fully characterize and analyze the potential of this new series of compounds as in vitro and in vivo anti-metastatic drugs.

**Supplementary Materials:** The following are available online. 1H and 13C NMR spectral data for representative compounds. Figure S1: 1H NMR Spectral Data of 2; Figure S2: 13C NMR Spectral Data of 2; Figure S3: 1H NMR Spectral Data of 7; Figure S4: 13C NMR Spectral Data of 7; Figure S5: 1H NMR Spectral Data of 12; Figure S6: 13C NMR Spectral Data of 12; Figure S7: 1H NMR Spectral Data of 14; Figure S8: 13C NMR Spectral Data of 14; Figure S9: 1H NMR Spectral Data of 16; Figure S10: 1H NMR Spectral Data of 22; Figure S11: 13C NMR Spectral Data of 22; Figure S12: 1H NMR Spectral Data of 24; Figure S13: 13C NMR Spectral Data of 24; Figure S14: 1H NMR Spectral Data of 29; Figure S15: 13C NMR Spectral Data of 29; Figure S16: 1H NMR Spectral Data of 33; Figure S17: 13C NMR Spectral Data of 33.

**Author Contributions:** K.B., Z.R., and A.F. performed the experiments and data collection; J.B. performed data analysis of GCMS; S.D. developed a methodology for actin assay experiments; and E.H. supervised, administrated, and was in charge of funding acquisition of the project. All authors read and approved the final manuscript.

**Funding:** This research was funded by the National Institutes of Health (MBRS-SCORE NIH/NIGMS SC2GM116712).

**Acknowledgments:** We thank Fernando González-Illán from the US FDA for assisting with the high-resolution electrospray ionization mass spectrometry data and analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Sample Availability:** Not available.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Novel Homo-Bivalent and Polyvalent Compounds Based on Ligustrazine and Heterocyclic Ring as Anticancer Agents**

### **Jiawen Wang 1,**†**, Ge Hong 2,3,**†**, Guoliang Li 2, Wenzhi Wang <sup>2</sup> and Tianjun Liu 1,2,3,\***


#### Academic Editor: Qiao-Hong Chen

Received: 7 November 2019; Accepted: 6 December 2019; Published: 9 December 2019

**Abstract:** Bivalent and polyvalent inhibitors can be used as antitumor agents. In this experiment, eight ligustrazine dimers and seven ligustrazine tetramers linked by alkane diamine with different lengths of carbon chain lengths were synthesized. After screening their antiproliferation activities against five cancer cell lines, most ligustrazine derivatives showed better cytotoxicity than the ligustrazine monomer. In particular, ligustrazine dimer **8e** linked with decane-1,10-diamine exhibited the highest cytotoxicity in FaDu cells with an IC50 (50% inhibiting concentration) value of 1.36 nM. Further mechanism studies suggested that **8e** could induce apoptosis of FaDu cells through the depolarization of mitochondrial membrane potential and S-phase cell cycle arrest. Inspired by these results, twenty-seven additional small molecule heterocyclic dimers linked with decane-1,10-diamine and nine cinnamic acid dimers bearing ether chain were synthesized and screened. Most monocyclic and bicyclic aromatic systems showed highly selective anti-proliferation activity to FaDu cells and low toxicity to normal MCF 10A cells. The structure-activity relationship revealed that the two terminal amide bonds and the alkyl linker with a chain length of 8–12 carbon were two important factors to maintain its antitumor activity. In addition, the ADMET calculation predicted that most of the potent compounds had good oral bioavailability.

**Keywords:** bivalency; polyvalency; antitumor; apoptosis; cell cycle

#### **1. Introduction**

Natural products play a vital role in the development of the drug, especially anticancer drugs [1]. Ligustrazine (2,3,5,6-tetramethylpyrazine, TMP), an important component of the Chinese traditional medicinal herb Chuanxiong (*Ligusticum chuanxiong Hort)*, has been of wide clinical use for cardiovascular and cerebrovascular diseases [2]. Recently, TMP has been reported to possess anticancer activity in vitro and in vivo, inducing cancer cell apoptosis and cell cycle modulation [3,4]. More importantly, the introduction of different substituted groups into ligustrazine has helped to improve the antitumor activity. Many ligustrazine derivatives with potent antitumor activity have been developed in the past few years, like monocarbonyl ligustrazine-curcumin hybrids [5], ligustrazine-betulinic acid hybrids [6], and ligustrazine-rhein derivatives [7]. This advancement stimulated our interest in using TMP as the scaffold to synthesize new antitumor agents.

In the interaction between host and guest molecules, both bivalency and polyvalency could enhance the affinity and activity of monovalent ligand [8,9]. This phenomenon has attracted attention in drug design. Many effective bivalent anti-cancer drugs have been found, such as artemisinin-derivative dimers [10], jesterone dimer [11], indole-3-carbinol dimer [12], bis-daunorubicin [13]. Previous work in this field has designed and synthesized several ligustrazine dimers linked by cyclohexanone and oxime [14], curcumin [15], or triterpenes [16], which had a good cytotoxic effect on human cancer cells. The result showed that the dimerization of ligustrazine could obviously improve the antitumor potency of its monomer.

With an effort to developing new antitumor agents, we designed and synthesized a series of novel TMP dimers and tetramers linked with different alkyl diamines and screened their anti-proliferation potential on a panel of human cancer cell lines, including HeLa, Hep G2, MCF-7, FaDu, and A549. Based on these experimental results, according to the importance of small molecular heterocyclic rings in drug discovery [17], additional dimers series of aromatic rings instead of ligustrazine, a total of 51 new compounds were synthesized, screened, and their structure-activity relationship was briefly discussed. Meanwhile, through morphological observation and flow cytometric analysis, the antitumor mechanisms of the most potent one were preliminarily discussed in this study. ADMET properties of all compounds were also evaluated to explore the drug-likeness.

#### **2. Results and Discussion**

#### *2.1. Chemistry*

In this study, 51 novel designed compounds were synthesized. Series **6a**–**g** and **8a**–**f** were obtained according to the synthetic method described in Schemes 1 and 2. The important intermediate 2-chloromethyl-3,5,6-trimethyl-pyrazine **5** was prepared from ligustrazine by the tandem reaction: Boekelheide reaction [18] and deprotection and the chlorination reaction [19]. The key intermediate 2-carboxylic acid-3,5,6-trimethyl-pyrazine **7** was synthesized by the one-pot reaction, as previously reported [20]. To explore the length effect, the TMP moieties, with 2–12 methylene alkyl chain as a linker, were synthesized.

**Scheme 1.** Synthesis of the TMP complexes **6a**–**6i**. *Reagents and conditions***:** (a) 30% H2O2, CH3COOH, 95 ◦C, 2 h; (b) (CH3CO)2O, 140 ◦C, 2.5 h; (c) CH3ONa, CH3OH, r.t., 0.5 h; (d) SOCl2, CH2Cl2, r.t., 2.5 h; (e) n-diaminoalkane (piperazine and monoethanolamine), K2CO3, CH3CN, 95 ◦C, 2 h.

**Scheme 2.** Synthesis of the Bis-TMP hybrids **8a**–**8f**. *Reagents and conditions***:** (f) KMnO4, H2O, 37 ◦C, 24 h; (g) n-diamino-alkane, EDCI/DMAP, r.t., 12 h.

Using heteroaromatic acids with decane-1,10-diamine as raw materials, the target compounds **9e**–**35e** (outlined in Scheme 3) linked by amide chain were synthesized with EDCI (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and DMAP (4-Dimethylaminopyridine) in anhydrous CH2Cl2 at room temperature. Series **9d**, **9f**–**l** (shown in Scheme 4) linked by ether chain or amide chain were synthesized by the above method. All the newly synthesized compounds were characterized by 1H-NMR, 13C-NMR, and high-resolution mass spectra (HRMS). Details were provided in the experimental part. The purity of the compounds was over 95% measured by HPLC (Figure 1).

**Scheme 3.** Synthesis of compounds **9e–35e**. *Reagents and conditions***:** (h) decane-1,10-diamine, EDCI/DMAP, r.t., 12 h.

**Scheme 4.** Synthesis of compounds **9d**, **9f**–**9m**. *Reagents and conditions***:** (i) cinnamic acid, EDCI/DMAP, r.t., 12 h.

**Figure 1.** The purity of compound **8e** as a representative analyzed by HPLC (Kromasil C18 column, eluted by methanol/water (80/20) containing 0.1% trifluoroacetic acid at a flow rate of 1 mL/min).

#### *2.2. Anti-Proliferative Activity In Vitro*

For the newly synthesized compounds, their anti-proliferative activity on human cancer cell lines, HeLa (cervical carcinoma), Hep G2 (hepatoma carcinoma), MCF-7 (breast carcinoma), FaDu (head and neck carcinoma), A549 (lung carcinoma), and normal mammary epithelial cell line MCF 10A, was screened by MTT assay [21] with doxorubicin (DOX) as the positive control. The IC50 values of these compounds are summarized in Table 1. Among the ligustrazine derivatives, **6a**–**g** and **8a**–**f**, the tetramers **6f** and **6g** had broad-spectrum cytotoxic activities in all cell lines (IC50 6.57–20.83 μM), and the cytotoxicity activities increased with the increase of carbon chain length. The dimeric ligustrazine **8d**, **8e**, **8f** showed the most promising anticancer activity in most tested tumor cell lines with IC50 values between 0.00136 and 6.35 μM, a much better result than DOX and TMP. In particular, compound **8e**, two ligustrazine rings linked with decane-1,10-diamine, exhibited the best anti-proliferative ability in cells (except Hep G2), and compared with other linkers, its IC50 values were 1.42 ± 0.71 μM, 0.037 ± 0.001 μM, 0.00136 ± 0.00035 μM, 1.05 ± 0.05 μM, and 0.047 ± 0.008 μM against HeLa, MCF-7, FaDu, A549, and MCF 10A, respectively. In light of these results, we replaced TMP with aromatic heterocycles similar to ligustrazine in electronic space. The novel small aromatic molecule and heterocyclic dimers **9e**–**35e** were synthesized, and their anti-proliferative ability was evaluated. Compounds **9e**, **10e**, **11e**, **17e**, **18e**, **21e**, **22e, 23e,** and **25e,** aromatic acid dimers (cinnamoyl, nicotinoyl, benzoyl, quinoline-3-carboxyl, quinoline-6-carboxyl, benzofuran-2-carboxyl, benzothiophene-2-carboxyl, indol-3-carboxyl, chlorinated cinnamoyl), were linked by decane-1,10-diamine, which had potent anti-proliferative ability in FaDu or A549 cell lines. Their IC50 in FaDu cell lines were less than 1 μM, in the range of 20 nM (benzothiophene-2-carboxyl) and 697 nM (benzofuran-2-carboxyl). Also, they had nearly no toxicity in normal cells like MCF 10A. In series **9d**–**n**, compounds **9d**–**f** (IC50 < 1 μM), two cinnamic acids linked by C8, C10, C12 chain with two terminal amide bonds, were more effective than compounds **9g**–**i** (IC50 > 20 μM), two cinnamic acids linked by the same alkyl chain with two terminal ester bonds instead of amide bonds. This demonstrated the vital role of two-terminal amide bonds in antitumor activity. Compared with compounds **9d**–**f**, the inhibition of compounds **9j**–**m**, two cinnamic acids linked by the similar length ether chain instead of alky chain, was weaker in tumor cells (IC50 > 20 μM), which confirmed that the alkyl chain played an important role in the structure. Then, we evaluated the position effect of the substitution at the ortho, meta, or para positions of the same aromatic ring. Compounds **25**–**35e** and the unsubstituted compounds **9e** and **11e** were all derivatives of cinnamic acid or benzoic acid with different substituents and electronic effects. The results of the compounds **26**–**27e** or **28**–**29e** confirmed that the substituents had a general effect on the activity, -OH, -OCH3, -NH2, and other electron donor groups could significantly reduce the activity, with the influence of -OH greater than that of -OCH3. However, the electron attraction group like -Cl (**25e**) had no obvious effect. The compounds with para-substituted analogs (**31e**, para position, IC50 = 5.594 μM) exhibited better cell growth inhibitory activities in FaDu cells than ortho- and meta- substituted analogs (**30e**, **32e**, ortho and meta position, IC50 > 20 μM). It is worth noting that para -OCH3 substitution could enhance anticancer activity. The IC50 value of compound **34e** was 5.853 ± 0.408 μM, the IC50 value of para- OCH3 was half that of the parent compound **33e**, and the IC50 value of compound **33e** was 10.393 ± 0.949 μM. Generally, there were three crucial elements in the structure-activity relationship of these systems: alkyl chain linker, two-terminal amide bonds, and heteroaromatic substituents. This SAR (structure-activity relationship) study would help to discover more effective compounds in the future.

To study the selective antiproliferative activities of bivalent and polyvalent inhibitors in normal cells and cancer cell lines, the cytotoxicity of compounds in FaDu cells and normal mammary epithelial MCF 10A cells was measured. The results listed in Table 1 showed that although the ligustrazine dimers **8d** and **8e** had high cytotoxicity in MCF 10A, their IC50 values were 2.69 ± 0.46 and 0.047 ± 0.008 μM, while the IC50 values in FaDu cells were 110 ± 30 and 1.36 ± 0.035 nM, respectively, which were far lower than that in MCF 10A. The selective index (SI) between MCF10A and Fadu (IC50MCF 10A/IC50FaDu) was 24.45 and 34.56, respectively. In addition, the heteroaromatic acid dimers **9e**–**12e**, **21e**–**23e,** and **25e** had the similar activity, which was stronger than doxorubicin in FaDu cells, and had high selectivity in FaDu cells and MCF 10A cells with SI (IC50MCF 10A/IC50FaDu) values over 28.69. In contrast, the SI (IC50MCF 10A/IC50FaDu) value of doxorubicin was 0.43. These results encouraged us to further investigate the possible cellular mechanisms.


**Table 1.** Inhibitory effects of synthetic compounds on the growth of different types of human cancer cells and normal mammary epithelial cells.

[a] HeLa (cervical cancer cell line); [b] Hep G2 (hepatocarcinoma cell line); [c] MCF-7 (breast cancer cell line); [d] FaDu (head and neck squamous carcinoma cell line); [e] A549 (epithelial cancer cell line); [f] MCF 10A (normal mammary epithelial cell); [g] SI: selectivity index. It was calculated as: SI = IC50 (MCF 10A)/IC50 (FaDu); [h] NC: not calculated.

#### *2.3. Colony Formation Assay*

As ligustrazine dimer **8e** had the most potent antiproliferative activity in all synthesized compounds, the colony formation assay in FaDu cells was conducted. As shown in Figure 2, FaDu cells treated with compound **8e** at concentrations 0.75 nM, 1.50 nM, 3 nM, 6 nM for 10 days formed smaller and fewer colonies compared to untreated control group, indicating that compound **8e** had a dose-dependent inhibition on the proliferation of FaDu cells, and the colony inhibition rate reached around 85.4% at 6 nM.

**Figure 2.** Effect of **8e** on the colony formation in FaDu cells. (**A**) Representative images of FaDu colonies after treatment at the indicated concentrations for 10 days; (**B**) The percentage of colonies compared with control group, *n* = 3. \*\*: *p* < 0.01; \*\*\*: *p* < 0.001 versus control.

#### *2.4. Live*/*Dead Staining*

Live-dead double staining experiment was conducted to visually evaluate the cell toxicity of compound **8e**, the most potent one obtained by MTT assay. Propidium iodide (PI)-stained dead cells in red and calcein AM-stained living cells in green. The number of dead cells increased with the dose of **8e** while living cells decreased. Simultaneously, the densities of **8e**-treated cells were much lower than that of the control group. The concentration-dependent loss of FaDu cell viability was consistent with the MTT results (Figure 3).

**Figure 3.** The confocal images of FaDu cells after incubation with compound **8e** at concentrations of 0, 2, 5, and 10 nM for 24 h, respectively. Viable cells were stained in green with Calcein-AM, and dead cells were stained in red with PI. The scale bar was 100 μm.

#### *2.5. Morphological Observation by Hoechst 33,342 Staining*

To investigate whether the anti-proliferation behavior of **8e** was related to the apoptosis pathway, the interaction between **8e**- and FaDu cells was analyzed through Hoechst 33,342 staining with a laser scanning confocal microscope. As shown in Figure 4, cells without treatment as the control group exhibited almost negligible apoptotic characteristics with homogenous and round blue-stained nuclei. On the contrary, the typical apoptotic nuclei with irregular nuclear morphology and condensation of chromatin (much brighter stained cells) were observed, and most FaDu cells were shrunken and rounded up from the culture dish after exposure to **8e**. The result demonstrated that inducing apoptosis in FaDu cells might be one cause of cell growth inhibition by compound **8e**.

**Figure 4.** Hoechst staining of FaDu cells treated with compound **8e,** (**A**) Control group; (**B**) 20 nM group.

#### *2.6. Apoptosis Analysis by Annexin V-FITC*/*PI Staining*

To substantiate whether the cell death induced by the ligustrazine dimer **8e** was associated with apoptosis, the interaction of FaDu cells with **8e** was further analyzed by an annexin V-FITC/PI staining, and the apoptosis ratios were quantitated by flow cytometry. The cells were divided into four quadrants: Q1 represented necrotic cells (annexin V−/PI+), Q2 represented late apoptotic cells (annexin V+/PI+), Q3 represented early apoptotic cells (annexin V+/PI−), Q4 represented living cells (annexin V−/PI−). As shown in Figure 5, after being exposed to different concentrations of **8e** (2, 5, 10 nM) for 24 h in FaDu cells, the apoptotic cells (including the early and late apoptosis ratios) increased gradually from 8.23% of the control to 34.2%, 57.7%, 79.5%, respectively. The results revealed that **8e** could induce FaDu cells' apoptosis in a dose-dependent manner. This was consistent with other reports showing TMP and its derivatives could induce apoptosis of cancer cells [22–24].

**Figure 5.** Flow cytometry analysis of apoptosis using annexin V-FITC/PI double staining in FaDu cells treated with compound **8e** at the indicated concentrations for 24 h. The percentage of cells was given in the respective quadrants.

#### *2.7. Mitochondrial Membrane Potential (*ΔΨ*m) Analysis*

Mitochondria play an essential role in cell apoptotic progression by regulating mitochondrial membrane potential (ΔΨm). Depolarization of ΔΨm is considered an indicator of cell apoptosis [25]. To better understand the mechanism of **8e**-induced FaDu cells apoptosis, the depolarization of ΔΨm was quantitated by JC-1 (tetraethylbenzimidazolylcarbocyanine iodide) dye. As a mitochondrial-specific dual-fluorescence probe, JC-1 selectively entered the mitochondria. In healthy cells with high ΔΨm, it accumulated in the mitochondrial matrix as J-aggregates with red fluorescence, while in the reduced ΔΨm cells, it remained in the cytoplasm as a monomer with green fluorescence. The change of ΔΨm could be easily detected by the change of fluorescence color. Flow cytometric analysis of FaDu cells treated with different concentrations of **8e** (2, 5, 10 nM) for 24 h revealed that the ratio of green to red fluorescence was significantly higher than that of the control group in a concentration-dependent manner, indicating that the number of lower ΔΨm cells increased (Figure 6). These results suggested that **8e** could induce apoptosis of FaDu cells by decreasing ΔΨm.

**Figure 6.** Flow cytometry analysis of the mitochondrial membrane potential using the JC-1 (tetraethylbenzimidazolylcarbocyanine iodide) dye in FaDu cells treated with compound **8e** at the indicated concentrations for 24 h. The percentage of cells was given in the respective quadrants.

#### *2.8. Cell Cycle Analysis*

In order to investigate whether **8e** induced cell cycle disturbances in FaDu cells, the flow cytometric analysis was performed with PI staining. The fluorescence intensity was proportional to the content of double-stranded DNA. The separation of cells in G0/G1, S, and G2/M was based upon the distribution of DNA content. Cytometric profiles of the PI-stained DNA showed that after being treated with **8e** at different concentrations (2, 5, 10 nM) for 24 h, the cell cycle of FaDu cells was arrested. As shown in Figure 7, the percentage of cells in the S phase (33.7%, 44.3%, and 54.7%) was markedly improved compared with the control group (28.1%), and this effect was concentration-dependent in FaDu cells. The population of the G0/G1 phase decreased from 51.9% to 41.8%, 44.2%, and 32.4%, respectively. These data indicated that **8e** significantly arrested cell cycle at S phase. This was different from the previously reported cell cycle arrest in the G0/G1 phase induced by TMP [4], which revealed that the dimerization of TMP could change the modulation of the cell cycle of its monomer.

**Figure 7.** Flow cytometry analysis for cell cycle distribution of FaDu cells. (**A**) Representative cell cycle distribution of FaDu cells with compound 8e treatment (0, 2, 5, and 10 nM) for 24 h. The tall peak (**left**) represents cells in the G0/G1 phase, the small peak (**right**) represents cells in the G2/M phase, and the middle area represents cells in the S phase. (**B**) Quantification of the percentage of cell cycle distribution.

#### *2.9. In Silico ADMET Prediction*

ADMET prediction is becoming increasingly crucial as most drugs fail in clinical trials owing to poor pharmacokinetic parameters [26,27]. Table 2 shows the ADMET calculated descriptors for our synthesized compounds and two biplots shown in Figure 8. All compounds had 0–4 HBD (hydrogen bond donor) and 4–10 HBA (hydrogen bond acceptor), which were under Lipinski's rule of five (HBD < 5, HBA < 10). PPB (plasma protein binding) was one of the key properties related to drug efficacy. Except for compounds **10**–**15e**, **17**–**24e,** and **9d,** most of the compounds had weak (<90%) binding with plasma proteins in the blood, which were predicted to be non-inhibitors of cytochrome P450 2D6, and participated in the good- metabolism of phase I metabolism. T1/<sup>2</sup> of the target molecules was in the range of 1.63–2.29 h, showing the characteristic of rapid metabolism. The HT (hepatotoxicity) values estimated the hepatoxicity of the chemical compounds. HT values for series **6a**–**i**, **13**–**14e**, **20e**, **29e**, **31**–**35e**, **9f**, **9i**, **9k,** and **9m** were zero, and there was no hepatoxicity, while other compounds might be hepatotoxic. Moreover, the LD50 (median lethal dose) predicted that series **8a**–**f**, **9**–**35e,** and **9d**–**m** were over 500 mg/kg, implying they were less toxic. In addition, an ADMET model formed with descriptors 2D polar surface area (PSA) and AlogP98 could predict human intestinal absorption and the blood-brain barrier penetration (BBB) at 95% and 99% confidence level. In the absorption plot, compounds **6a**–**f**, **6h**–**i**, **8a**–**f**, **9**–**11e**, **13**–**19e**, **23e**, **30**–**33e**, **9d**, **9g,** and **9j**–**m** were predicted to be easily absorbed by the intestine at 99% confidence level. While in the BBB plot, the most potent compounds **8e** and **8f** were fallen out of the 99% ellipse (undefined), suggesting its poor blood-brain barrier penetration ability. Therefore, these molecules might possess low or no side effects of the central nervous system (CNS). In conclusion, ADMET prediction of the compounds might be helpful for further designing new drugs with favorable oral bioavailability.


**Table 2.** The ADMET properties of all synthetic compounds.

<sup>a</sup> HBD (hydrogen bond donor), <sup>b</sup> HBA (hydrogen bond acceptor), <sup>c</sup> RBN (number of rotatable bonds), <sup>d</sup> logP (log of the octanol/water partition coefficient), <sup>e</sup> log S (log of the aqueous solubility), and <sup>f</sup> PSA (polar surface area) were predicted using Discovery Studio 2.0 software. <sup>g</sup> HIA (human intestinal absorption), <sup>h</sup> PPB (plasma protein binding), <sup>I</sup> CYP3D4 (CYP3D4 inhibition), <sup>j</sup> T1/2 (half lifetime), <sup>k</sup> HT (human hepatoxicity), and <sup>l</sup> LD50 (median lethal dose) were calculated via http://admet.scbdd.com/home/index/

**Figure 8.** The plot of PSA (polar surface area) versus AlogP98 for the newly synthesized compounds showing the 95% and 99% confidence limit ellipses corresponding to the intestinal absorption and blood-brain barrier models.

#### **3. Conclusions**

Using ligustrazine, aromatic, or heteroaromatic acids as the main raw materials and linked by alkyl or ether chain, bivalent antitumor agents were synthesized and screened from five different human cancer cell lines and one normal mammary epithelial cell line. SAR revealed that the two terminal amide bonds and the alky chain were the dominant factors with 10 C atom chain showing the most potent antitumor effect. Among different aromatic moieties, the ligustrazine dimer **8e** was the best candidate; its IC50 was 1.36 nM in FaDu cell, it showed a broad-spectrum antitumor activity with IC50 in the range of 0.00136–1.42 μM, and the selective ratio MCF 10A/FaDu was 34.56. The subsequent fluorescence staining and flow cytometry analysis indicated that **8e** could induce apoptosis through depolarization of the mitochondrial membrane potential in FaDu cells. Further mechanism investigation showed that **8e** could arrest cell cycle at the S phase in FaDu cells. Furthermore, ADMET predicted that most of the potent compounds followed Lipinski's law and became 'drug-like' molecules, further confirming that the dimerization of ligustrazine could improve the antitumor activity of its monomer. The highly selective inhibition of these dimers on the proliferation of FaDu cells might be related to the effect on a specific target in FaDu cells. Based on the characteristics of FaDu cells, further targeted screening would give insight into the selectivity of such potent compounds.

#### **4. Materials and Methods**

#### *4.1. Chemistry*

1H-NMR and 13C-NMR spectra were recorded on a Mercury Vx-300 (300 MHz) or AVANCE III (400 MHz). Chemical shifts were reported in ppm (δ) using the residue solvent line as the internal standard (for TMS: 0 ppm, 1H and 13C; for CHCl3-*d*: 7.26 ppm, 1H and 77.16 ppm, 13C). Coupling constants were given in Hertz (*J*). High-resolution MS (HRMS) were recorded on an Agilent 6520 Q-TOF LC/MS. Melting points were uncorrected and were determined on a digital melting point apparatus (Shenguang WRS-1B, shanghai, China). Flash column chromatography was carried out by using silica gel (200–300 mesh). Reactions were monitored by thin-layer chromatography (TLC) on silica gel GF254 plates (Qingdao Haiyang Chemical Plant, Qingdao, China). Reagents and solvents were used as purchased from commercial sources without further purification. The purity of the synthesized compounds was over 95% analyzed by HPLC (Waters 2695 Alliance system, Waters Corp., Milford, MA, USA), with the Kromasil C18 column eluted by methanol/water (80/20) containing 0.1% trifluoroacetic acid at a flow rate of 1 mL/min. Compounds **4** and **5** were prepared, as reported previously [19]. 1H-NMR, 13C-NMR and HR-MS spectra of compounds **6a**–**9m** can be seen in supplementary materials.

#### *4.2. General Synthetic Procedure for* **6a**–**g**

A mixture of alkane-diamine (10 mmol), TMP-Cl (50 mmol), KOH (60 mmol), and dimethylformamide (35 mL) was heated to reflux. The reaction progress was monitored via TLC. After 2 h, the reaction mixture was cooled to room temperature. Subsequently, the solid was filtered off, and the filtrate was added with H2O (100 mL) and extracted with ethyl acetic acid three times. The organic layers were combined and washed with H2O and brine, and then separated and dried over anhydrous Na2SO4 for 8 h. The solvent was removed under reduced pressure; the resulting residue was separated on flash column chromatography eluted by a mixture of petroleum ether/acetone in a volume ratio of 7:1–3:1, and further recrystallized from acetone to give pale white solid.

#### 4.2.1. N1,N1,N2,N2-Tetrakis((3,5,6-Trimethylpyrazin-2-yl)Methyl)Ethane-1,2-Diamine (**6a**)

Pale white solid, yield 28%, m.p. 207.6–208.3 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 3.60 (s, 8H), 2.67 (s, 4H), 2.43 (s, 12H), 2.41 (s, 12H), 2.23 (s, 12H); 13C-NMR (101 MHz, DMSO) δ: 149.91, 149.63, 148.37, 147.85, 58.75, 52.14, 50.83, 21.61, 21.42, 20.65; HRMS (ESI) *m*/*z*: 597.4134 [M + H]+, calcd. for [C34H49N10] <sup>+</sup> 597.4142.

#### 4.2.2. N1,N1,N3,N3-Tetrakis((3,5,6-Trimethylpyrazin-2-yl)Methyl)Propane-1,3-Diamine (**6b**)

Pale white solid, yield 27%, m.p. 113–114.3 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 3.65 (s, 8H), 2.46 (s, 12H), 2.43 (s, 12H), 2.41–2.38 (m, 4H), 2.32 (s, 12H), 1.81–1.71 (m, 2H). 13C-NMR (101 MHz, DMSO) δ: 149.93, 149.53, 148.51, 147.82, 58.63, 53.36, 21.61, 21.44, 20.70; HRMS (ESI) *m*/*z*: 611.4286 [M + H]+, calcd. for [C35H51N10] <sup>+</sup> 611.4298.

#### 4.2.3. N1,N1,N4,N4-Tetrakis((3,5,6-Trimethylpyrazin-2-yl)Methyl)Butane-1,4-Diamine (**6c**)

Pale white solid, yield 37%, m.p. 129.2–129.5 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 3.62 (s, 8H), 2.44 (s, 24H), 2.40–2.37 (m, 4H), 2.30 (s, 12H), 1.37 (s, 4H); 13C-NMR (101 MHz, DMSO) δ: 150, 149.53, 148.60, 147.79, 58.54, 54.85, 24.39, 21.64, 21.47, 20.71; HRMS (ESI) *m*/*z*: 625.4451 [M + H]+, calcd. for [C36H53N10] <sup>+</sup> 625.4455.

#### 4.2.4. N1,N1,N6,N6-Tetrakis((3,5,6-Trimethylpyrazin-2-yl)Methyl)Hexane-1,6-Diamine (**6d**)

Pale white solid, yield 43%, m.p. 146.7–147.9 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 3.67 (s, 8H), 2.46–2.45 (m, 24H), 2.43–2.40 (m, 4H), 2.34 (s, 12H), 1.43 (br, 4H), 1.04 (br, 4H); 13C-NMR (101 MHz, DMSO) δ: 150.05, 149.48, 148.71, 147.77, 58.64, 54.93, 27.34, 26.56, 21.65, 21.48, 20.74; HRMS (ESI) *m*/*z*: 653.4765 [M + H]<sup>+</sup>, calcd. for [C38H57N10] <sup>+</sup> 653.4768.

#### 4.2.5. N1,N1,N8,N8-Tetrakis((3,5,6-Trimethylpyrazin-2-yl)Methyl)Octane-1,8-Diamine (**6e**)

Pale white solid, yield 48%, m.p. 107.8–108.6 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 3.67 (s, 8H), 2.45 (br, 24H), 2.42–2.38 (m, 4H), 2.34 (s, 12H), 1.48–1.40 (m, 4H), 1.05 (br, 8H); 13C-NMR (101 MHz, CDCl3) δ: 150.09, 149.47, 148.75, 147.76, 58.69, 54.90, 29.52, 27.46, 26.55, 21.65, 21.47, 20.73; HRMS (ESI) *m*/*z*: 681.5076 [M + H]<sup>+</sup>, calcd. for [C40H61N10] <sup>+</sup> 681.5081.

4.2.6. N1,N1,N10,N10-Tetrakis((3,5,6-Trimethylpyrazin-2-yl)Methyl)Decane-1,10-Diamine (**6f**)

Pale white solid, yield 76%, m.p. 67.4–68 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 3.69 (s, 8H), 2.46 (br, 24H), 2.41–2.39 (m, 4H), 2.35 (s, 12H), 1.47 (br, 4H), 1.11 (br, 12H); 13C-NMR (101 MHz, DMSO) δ: 150.10, 149.45, 148.78, 147.76, 58.71, 54.92, 29.73, 29.52, 27.48, 26.57, 21.64, 21.47, 20.74; HRMS (ESI) *m*/*z*: 709.5378 [M + H]<sup>+</sup>, calcd. for [C42H65N10] <sup>+</sup> 709.5394.

4.2.7. N1,N1,N12,N12-Tetrakis((3,5,6-Trimethylpyrazin-2-yl)Methyl)Dodecane-1,12-Diamine (**6g**)

Pale white solid, yield 76%, m.p. 84.7–85.3 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 3.69 (s, 8H), 2.46 (br, 24H), 2.44 (br, 4H), 2.35 (s, 12H), 1.47 (br, 4H), 1.15–1.12 (br, 16H); 13C-NMR (101 MHz, DMSO) δ: 150.10, 149.44, 148.78, 147.74, 58.72, 54.89, 29.75, 29.53, 27.46, 26.56, 21.64, 21.47, 20.73; HRMS (ESI) *m*/*z*: 737.5689 [M + H]<sup>+</sup>, calcd. for [C44H69N10] <sup>+</sup> 737.5707.

#### *4.3. 1,4-Bis((3,5,6-Trimethylpyrazin-2-yl)Methyl)Piperazine (***6h***)*

A mixture of piperazine (0.800 g, 9.29 mmol), TMP-Cl (3.80 g, 22.29 mmol), KOH (1.56 g, 27.66 mmol), and dimethylformamide (20 mL) was heated under reflux for 2 h. The reaction was monitored by TLC. The reaction was quenched, filtered, and the filtrate was added with 100 mL H2O and extracted with ethyl acetate three times. The organic layers were combined, washed with H2O and brine, and then dried over anhydrous Na2SO4 for 8 h. The solvent was evaporated in vacuo. The resulting residue was separated on flash column chromatography with a mixture of petroleum ether/acetone 7:1–3:1(volume ratio) as eluent, and then further recrystallized from acetone to afford compound **6h**.

Pale white solid, yield 62%, m.p. 155.8–156.3 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 3.58 (s, 4H), 2.55 (s, 8H), 2.47 (br, 18H); 13C-NMR (101 MHz, DMSO) δ: 150.01, 149.57, 148.10, 147.98, 61.88, 53.26, 50.70, 21.61, 21.51, 21.04; HRMS (ESI) *m*/*z*: 355.2610 [M + H]<sup>+</sup>, calcd. for [C20H31N6] <sup>+</sup> 355.2610.

#### *4.4. 2-(Bis((3,5,6-Trimethylpyrazin-2-yl)Methyl)Amino)Ethanol (***6i***)*

A mixture of ethanolamine (0.500 g, 8.19 mmol), TMP-Cl (3.35 g, 19.65 mmol), K2CO3 (3.39 g, 24.56 mmol), and acetonitrile (20 mL) was heated under reflux for 3 h. The product formation was monitored via TLC. After cooling, the mixture was filtered, and the filtrate was evaporated in vacuo. The residue was purified by flash column chromatography with an eluent of petroleum ether/acetone = 10:1–3:1 to afford compound **6i**.

Light yellow oil, yield 72%; 1H-NMR (300 MHz, CDCl3) δ: 3.82 (13s, 4H), 3.66 (t, *J* = 5 Hz, 2H), 2.87 (t, *J* = 5.1 Hz, 2H), 2.41 (s, 6H), 2.37 (s, 6H), 2.35 (s, 6H); 13C-NMR (101 MHz, DMSO) δ: 149.54, 148.94, 148.27, 147.87, 59.67, 58.15, 57.36, 21.41, 21.21, 20.52; HRMS (ESI) *m*/*z*: 330.2290 [M + H]+, calcd. for [C18H28N5O]<sup>+</sup> 330.2294.

#### *4.5. General Synthetic Procedure for* **8a***–***f**

Compound **7** was gained according to the method described by Wu [20]. A mixture of tetramethylpyrazine acid (4.40 mmol), EDCI (4.72 mmol), triethylamine (12 mmol), DMAP (2.40 mmol), and anhydrous dichloromethane (10 mL) was stirred to dissolve, then diamino-alkane (2 mmol) was added and stirred at room temperature until the reaction was finished (monitored by TLC). The reaction mixture was washed with brine and water, extracted with dichloromethane (20 mL), dried over anhydrous sodium sulfate for 8 h, filtered, and the solvent was evaporated. The resulting residue was purified by flash column chromatography with an eluent of petroleum ether/acetone = 15:1–3:1(volume ratio) to give pale white solid.

#### 4.5.1. N,N -(Propane-1,3-Diyl)Bis(3,5,6-Trimethylpyrazine-2-Carboxamide) (**8a**)

Pale white solid, yield 16%, m.p. 125.4–126.4 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 8.30 (t, *J* = 5.9 Hz, 2H), 3.58–3.52 (m, 4H), 2.92 (s, 6H), 2.56 (s, 6H), 2.50 (s, 6H), 1.99–1.90 (m, 2H); 13C-NMR (101 MHz, CDCl3) δ: 165.57, 154.09, 151.29, 147.80, 139.09, 36.89, 29.87, 22.94, 22.07, 21.47; HRMS (ESI) *m*/*z*: 371.2189 [M + H]<sup>+</sup>, calcd. for [C19H27N6O2] <sup>+</sup> 371.2195.

#### 4.5.2. N,N -(Butane-1,4-Diyl)Bis(3,5,6-Trimethylpyrazine-2-Carboxamide) (**8b**)

Pale white solid, yield 30%, m.p. 168.4–168.6 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 8.07 (t, *J* = 5.5 Hz, 2H), 3.50–3.48 (m, 4H), 2.91 (s, 6H), 2.56 (s, 6H), 2.52 (s, 6H), 1.75–1.73 (m, 4H); 13C-NMR (101 MHz, CDCl3) δ: 165.25, 154.15, 151.38, 147.75, 139.07, 50.90, 39.13, 27.49, 22.96, 22.06, 21.48; HRMS (ESI) *m*/*z*: 385.2352 [M + H]<sup>+</sup>, calcd. for [C20H29N6O2] <sup>+</sup> 385.2352.

#### 4.5.3. N,N -(Hexane-1,6-Diyl)Bis(3,5,6-Trimethylpyrazine-2-Carboxamide) (**8c**)

Pale white solid, yield 47%, m.p. 114.2–114.7 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 8.02 (t, *J* = 5.4 Hz, 2H), 3.45–3.38 (m, 4H), 2.90 (s, 6H), 2.55 (s, 6H), 2.51 (s, 6H), 1.67–1.63 (m, 4H), 1.47–1.43 (m, 4H); 13C-NMR (101 MHz, CDCl3) δ: 165.12, 154.03, 151.35, 147.63, 139.11, 39.33, 29.74, 26.82, 22.98, 22.06, 21.48; HRMS (ESI) *m*/*z*: 413.2662 [M + H]<sup>+</sup>, calcd. for [C22H33N6O2] <sup>+</sup> 413.2665.

#### 4.5.4. N,N -(Octane-1,8-Diyl)Bis(3,5,6-Trimethylpyrazine-2-Carboxamide) (**8d**)

Pale white solid, yield 68%, m.p. 113.6–113.8 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 8 (t, *J* = 5.3 Hz, 2H), 3.45–3.38 (m, 4H), 2.91 (s, 6H), 2.56 (s, 6H), 2.52 (s, 6H), 1.65–1.60 (m, 4H), 1.37–1.37 (m, 8H); 13C-NMR (101 MHz, CDCl3) δ: 165.10, 154.02, 151.40, 147.65, 139.20, 39.45, 29.81, 29.34, 27.11, 23.01, 22.08, 21.53; HRMS (ESI) *m*/*z*: 441.2975 [M + H]<sup>+</sup>, calcd. for [C24H37N6O2] <sup>+</sup> 441.2978.

#### 4.5.5. N,N -(Decane-1,10-Diyl)Bis(3,5,6-Trimethylpyrazine-2-Carboxamide) (**8e**)

Pale white solid, yield 66%, m.p. 110.3–110.6 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 8 (t, *J* = 5.2 Hz, 2H), 3.44–3.37 (m, 4H), 2.91 (s, 6H), 2.55 (s, 6H), 2.51 (s, 6H), 1.64–1.57 (m, 4H), 1.34–1.30 (m, 12H); 13C-NMR (101 MHz, CDCl3) δ: 165.07, 153.97, 151.33, 147.66, 139.21, 39.47, 29.80, 29.56, 29.39, 27.14, 22.94, 22.02, 21.49; HRMS (ESI) *m*/*z*: 469.3287 [M + H]<sup>+</sup>, calcd. for [C26H41N6O2] <sup>+</sup> 469.3291.

#### 4.5.6. N,N -(Dodecane-1,12-Diyl)Bis(3,5,6-Trimethylpyrazine-2-Carboxamide) (**8f**)

Pale white solid, yield 72%, m.p. 98.3–99.1 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 8 (t, *J* = 4.9 Hz, 2H), 3.45–3.38 (m, 4H), 2.91 (s, 6H), 2.56 (s, 6H), 2.52 (s, 6H), 1.67–1.57 (m, 4H), 1.34–1.27 (m, 16H); 13C-NMR (101 MHz, CDCl3) δ: 165.07, 153.98, 151.37, 147.62, 139.22, 39.49, 29.83, 29.66, 29.44, 27.18, 22.99, 22.07, 21.51; HRMS (ESI) *m*/*z*: 497.3602 [M + H]<sup>+</sup>, calcd. for [C28H45N6O2] <sup>+</sup> 497.3604.

#### *4.6. General Synthetic Procedure for* **9e***–***35e**

Compounds **9e**–**35e** were obtained by using one-pot reaction. A mixture of aromatic acid (6.30 mmol), EDCI (7.50 mmol), DMAP (0.60 mmol), and anhydrous dichloromethane (20 mL) was stirred to dissolve, then decane-diamine (3 mmol) was added and stirred at room temperature for 12 h. The mixture solution was filtered under reduced pressure. After that, the residue was washed with little amount of CH2Cl2 and water successively, and dried to give the solid. Then, the residue was purified on preparative TLC eluted with chloroform/methanol = 40:1–7:1 to yield compounds **26e**, **28e, 30e,** and **31e**.

#### 4.6.1. (2E,2 E)-N,N -(Decane-1,10-Diyl)Bis(3-Phenylacrylamide) (**9e**)

Pale white solid, yield 62%, m.p. 169.8–170.5 ◦C; 1H-NMR (300 MHz, CDCl3+CD3OD) δ: 7.54 (d, *J* = 15.7 Hz, 2H), 7.49–7.46 (m, 4H), 7.36–7.31 (m, 6H), 6.47 (d, *J* = 15.6 Hz, 2H), 3.33–3.28 (m, 4H), 1.57–1.54 (m, 4H), 1.52–1.28 (m, 12H); 13C-NMR (101 MHz, CDCl3+CD3OD) δ: 150.10, 149.45, 148.78, 147.76, 58.71, 54.92, 29.73, 29.52, 27.48, 26.57, 21.64, 21.47, 20.74; HRMS (ESI) *m*/*z*: 433.2851 [M + H]+, calcd. for [C28H45N6O2] <sup>+</sup> 433.2855

#### 4.6.2. N,N -(Decane-1,10-Diyl)Dinicotinamide (**10e**)

Pale white solid, yield 67%, m.p. 167.4–167.8 ◦C; 1H-NMR (300 MHz, CDCl3+CD3OD) δ: 8.84 (s, 2H), 8.55 (d, *J* = 3.9 Hz, 2H), 8.11 (dt, *J* = 8, 1.7 Hz, 2H), 7.35 (dd, *J* = 7.9, 4.9 Hz, 2H), 3.34 (t, *J* = 7.2 Hz, 4H), 1.58–1.51 (m, 4H), 1.27–1.24 (m, 12H); 13C-NMR (101 MHz, CDCl3+CD3OD) δ: 165.85, 151.29, 147.53, 135.93, 130.87, 123.79, 40.16, 29.28, 29.12, 26.86; HRMS (ESI) *m*/*z*: 383.2441 [M + H]+, calcd. for [C28H45N6O2] <sup>+</sup> 383.2447.

#### 4.6.3. N,N -(Decane-1,10-Diyl)Dibenzamide (**11e**)

Pale white solid, yield 70%, m.p. 154.3–154.6 ◦C; 1H-NMR (300 MHz, CDCl3+CD3OD) δ: 7.77–7.73 (m, 4H), 7.51–7.33 (m, 6H), 3.42–3.35 (m, 4H), 1.63–1.56 (m, 4H), 1.34–1.30 (m, 12H); 13C-NMR (101 MHz, CDCl3+CD3OD) δ: 168.39, 134.55, 131.36, 128.45, 126.86, 40.02, 29.36, 29.27, 29.13, 26.84. HRMS (ESI) *m*/*z*: 381.2702 [M + H]<sup>+</sup>, calcd. for [C24H33N2O2] <sup>+</sup> 381.2542.

#### 4.6.4. N,N -(Decane-1,10-Diyl)Bis(2-Naphthamide) (**12e**)

Pale white solid, yield 64%, m.p. 163.9–164 ◦C; 1H-NMR (300 MHz, CDCl3+CD3OD) δ: 8.22–8.17 (m, 2H), 7.89–7.82 (m, 4H), 7.54–7.39 (m, 8H), 3.49–3.44 (m, 4H), 1.67–1.60 (m, 4H), 1.38–1.34 (m, 12H); 13C-NMR (101 MHz, CDCl3 + CD3OD) δ: 170.38, 134.50, 133.56, 130.33, 129.96, 128.22, 126.89, 126.29, 125.10, 124.77, 124.65, 39.94, 29.36, 29.30, 29.11, 26.85. HRMS (ESI) *m*/*z*: 481.2853 [M + H]+, calcd. for [C32H37N2O2] <sup>+</sup> 481.2855.

#### 4.6.5. N,N -(Decane-1,10-Diyl)Diisonicotinamide (**13e**)

Pale white solid, yield 60%, m.p. 175.6–176.3 ◦C; 1H-NMR (300 MHz, CDCl3+CD3OD) δ: 8.62 (d, *J* = 5.6 Hz, 4H), 7.71 (dd, *J* = 6.1, 2.8 Hz, 4H), 3.41–3.33 (m, 4H), 1.64–1.57 (m, 4H), 1.33–1.31 (m, 12H); 13C-NMR (101 MHz, CDCl3+CD3OD) δ: 165.88, 149.48, 142.45, 121.46, 40.04, 29.16, 28.99, 26.71. HRMS (ESI) *m*/*z*: 383.2445 [M + H]<sup>+</sup>, calcd. for [C22H31N4O2] <sup>+</sup> 383.2447.

#### 4.6.6. N,N -(Decane-1,10-Diyl)Bis(Furan-2-Carboxamide) (**14e**)

Pale white solid, yield 62%, m.p. 132.6–133.3 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 7.41 (dd, *J* = 1.7, 0.8 Hz, 2H), 7.09 (dd, *J* = 3.5, 0.7 Hz, 2H), 6.48 (dd, *J* = 3.5, 1.7 Hz, 2H), 6.37 (br, 2H), 3.45–3.38 (m, 4H), 1.65–1.55 (m, 4H), 1.36–1.30 (m, 12H); 13C-NMR (101 MHz, CDCl3) δ: 158.53, 148.38, 143.77, 114.02, 112.22, 39.29, 29.80, 29.48, 29.33, 26.98. HRMS (ESI) *m*/*z*: 383.1944 [M + Na]+, calcd. for [C20H28N2NaO4] <sup>+</sup> 383.1947.

#### 4.6.7. N,N -(Decane-1,10-Diyl)Dipicolinamide (**15e**)

Pale white solid, yield 68%, m.p. 156.6–156.9 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 9.35 (d, *J* = 1.4 Hz, 2H), 8.69 (d, *J* = 2.5 Hz, 2H),.8.47–8.46 (m, 2H), 7.77 (br, 2H), 3.47–3.40 (m, 4H), 1.65–1.55 (m, 4H), 1.34–1.26 (m, 12H); 13C-NMR (101 MHz, CDCl3) δ: 163.01, 147.26, 144.76, 144.55, 142.58, 39.61, 29.68, 29.51, 29.34, 27.03. HRMS (ESI) *m*/*z*: 383.2441 [M + H]<sup>+</sup>, calcd. for [C22H31N4O2] <sup>+</sup> 383.2447

#### 4.6.8. N,N -(Decane-1,10-Diyl)Bis(Pyrazine-2-Carboxamide) (**16e**)

Pale white solid, yield 70%, m.p. 77.6–80.1 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 8.54–8.51 (m, 2H), 8.20–8.17 (m, 2H), 8.05 (br, 2H), 7.85–7.80 (m, 2H), 7.42–7.38 (m, 2H), 3.49–3.43 (m, 4H), 1.68–1.58 (m, 4H), 1.38–1.26 (m, 12H); 13C-NMR (101 MHz, CDCl3) δ: 164.31, 150.25, 148.09, 137.49, 126.13, 122.34, 39.61, 29.77, 29.56, 29.41, 27.11. HRMS (ESI) *m*/*z*: 385.2350 [M + H]<sup>+</sup>, calcd. for [C20H29N6O2] <sup>+</sup> 385.2352.

#### 4.6.9. N,N -(Decane-1,10-Diyl)Bis(Quinoline-3-Carboxamide) (**17e**)

Pale white solid, yield 59%, m.p. 206.7–207.1 ◦C; 1H-NMR (300 MHz, DMSO-*d*<sup>6</sup> + CD3OD) δ: 8.57 (d, *J* = 1.8 Hz, 2H), 8.09 (s, 2H), 7.39 (dd, *J* = 8.3, 3.1 Hz, 4H), 7.17 (t, *J* = 7.6 Hz, 2H), 7 (t, *J* = 7.4 Hz, 2H), 2.68 (t, *J* = 7 Hz, 4H), 0.99–0.88 (m, 4H), 0.76–0.62 (m, 12H). 13C-NMR (101 MHz, CDCl3) δ: 165.92, 148.01, 136.52, 131.47, 128.81, 127.93, 127.56, 127.35, 127.06, 40.11, 29.20, 29.15, 29.04, 26.78. HRMS (ESI) *m*/*z*: 483.2756 [M + H]<sup>+</sup>, calcd. for [C30H35N4O2] <sup>+</sup> 483.2760.

#### 4.6.10. N,N -(Decane-1,10-Diyl)Bis(Quinoline-6-Carboxamide) (**18e**)

Pale yellow solid, yield 73%, m.p. 197.7–198.3 ◦C; 1H-NMR (300 MHz, CDCl3 + CD3OD) δ: 8.71 (dd, *J* = 4.3, 1.6 Hz, 2H), 8.19 (d, *J* = 1.6 Hz, 2H), 8.16 (dd, *J* = 8.3, 1.3 Hz, 2H), 7.95 (dd, *J* = 8.8, 1.9 Hz, 2H), 7.91 (s, 2H), 7.35 (dd, *J* = 8.3, 4.3 Hz, 2H), 3.28 (t, *J* = 7.2 Hz, 4H), 1.53–1.45 (m, 5H), 1.22–1.13

(m, 12H). 13C-NMR (101 MHz, CDCl3 + CD3OD) δ: 167.43, 151.15, 148.21, 137.99, 132.82, 128.38, 127.79, 127.71, 127.64, 121.81, 40.14, 29.19, 29.04, 26.78. HRMS (ESI) *m*/*z*: 483.2758 [M + H]+, calcd. for [C30H35N4O2] <sup>+</sup> 483.2760.

#### 4.6.11. N,N -(Decane-1,10-Diyl)Bis(1H-Benzo[d]Imidazole-6-Carboxamide) (**19e**)

Brick red solid, yield 75%, m.p. 229.7–230.3 ◦C; 1H-NMR (300 MHz, CDCl3+ CD3OD) δ: 8.11 (s, 2H), 8.07 (s, 2H), 7.73 (dd, *J* = 8.5, 1.6 Hz, 2H), 7.61 (d, *J* = 8.5 Hz, 2H), 3.42 (t, *J* = 7.1 Hz, 4H), 1.70–1.56 (m, 4H), 1.42–1.25 (m, 12H). 13C-NMR (101 MHz, CDCl3+ CD3OD) δ: 168.61, 142.46, 128.70, 121.15, 39.32, 28.66, 28.62, 28.50, 26.19. HRMS (ESI) *m*/*z*: 461.2662 [M + H]<sup>+</sup>, calcd. for [C26H33N6O2] + 461.2665.

4.6.12. N,N -(Decane-1,10-Diyl)Bis(2-Oxo-2H-Chromene-3-Carboxamide) (**20e**)

Pale white solid, yield 60%, m.p. 208.9–209.3 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 8.90 (s, 2H), 8.80 (s, 2H), 7.71–7.61 (m, 4H), 7.43–7.32 (m, 4H), 3.45 (dd, *J* = 13, 7 Hz, 4H), 1.63 (dt, *J* = 14.4, 7.2 Hz, 4H), 1.44–1.26 (m, 12H). 13C-NMR (101 MHz, CDCl3 + CD3OD) δ: 161.74, 161.44, 154.23, 148.38, 134.16, 129.80, 125.30, 118.46, 117.97, 116.38, 39.69, 29.20, 29.02, 29.01, 26.77. HRMS (ESI) *m*/*z*: 517.2338 [M + H]<sup>+</sup>, calcd. for [C30H33N2O6] <sup>+</sup> 517.2339.

4.6.13. N,N -(Decane-1,10-Diyl)Bis(Benzofuran-2-Carboxamide) (**21e**)

Pale white solid, yield 43%, m.p. 135.8–136.3 ◦C; 1H-NMR (300 MHz, DMSO-*d*6) δ: 8.64 (s, 2H), 7.74 (d, *J* = 7.7 Hz, 2H), 7.62 (d, *J* = 8.2 Hz, 2H), 7.49 (s, 2H), 7.43 (t, *J* = 7.5 Hz, 2H), 7.30 (t, *J* = 7.4 Hz, 2H), 3.25 (dd, *J* = 13.1, 6.6 Hz, 4H), 1.58–1.43 (m, 4H), 1.37–1.14 (m, 12H). 13C-NMR (101 MHz, DMSO-*d*6) δ: 157.94, 154.13, 149.37, 127.17, 126.61, 123.59, 122.63, 111.70, 109.02, 38.68, 29.01, 28.91, 28.71, 26.39. HRMS (ESI) *m*/*z*: 461.2438 [M + H]<sup>+</sup>, calcd. for [C28H33N2O4] <sup>+</sup> 461.2430.

4.6.14. N,N -(Decane-1,10-Diyl)Bis(Benzo[b]Thiophene-2-Carboxamide) (**22e**)

Pale yellow solid, yield 46%, m.p. 175–175.7 ◦C; 1H-NMR (300 MHz, DMSO-*d*6) δ: 8.68 (s, 2H), 8.04 (s, 2H), 8.01–7.95 (m, 2H), 7.93–7.87 (m, 2H), 7.41 (p, *J* = 7 Hz, 4H), 3.25 (dd, *J* = 12.8, 6.6 Hz, 4H), 1.58–1.44 (m, 4H), 1.37–1.21 (m, 12H). 13C-NMR (101 MHz, DMSO-*d*6) δ: 161.28, 140.28, 140.07, 139.16, 126, 125.02, 124.80, 124.33, 122.72, 28.99, 28.92, 28.72, 26.43. HRMS (ESI) *m*/*z*: 493.1976 [M + H]+, calcd. for [C28H33N2O2S2] <sup>+</sup> 493.1983.

4.6.15. N,N -(Decane-1,10-Diyl)Bis(1-Methyl-1H-Indole-3-Carboxamide) (**23e**)

Pale white solid, yield 43%, m.p. 192.8–193.2 ◦C; 1H-NMR (300 MHz, DMSO-*d*<sup>6</sup> + CD3OD) δ: 8.08 (d, *J* = 7.7 Hz, 2H), 7.86 (s, 2H), 7.41 (d, *J* = 8 Hz, 2H), 7.20–7.13 (m, 2H), 7.13–7.06 (m, 2H), 3.78 (s, 6H), 3.22 (t, *J* = 7.1 Hz, 4H), 1.55–1.43 (m, 4H), 1.32–1.22 (m, 12H). 13C-NMR (101 MHz, CDCl3 + CD3OD) δ: 166.26, 137.01, 131.75, 125.56, 122.27, 121.03, 120.13, 109.63, 39.26, 32.62, 29.33, 29.07, 28.94, 26.66. HRMS (ESI) *m*/*z:* 487.3064 [M + H]<sup>+</sup>, calcd. for [C30H39N4O2] <sup>+</sup> 487.3073.

4.6.16. N,N -(Decane-1,10-Diyl)Bis(1H-Indole-6-Carboxamide) (**24e**)

Pale white solid, yield 47%, m.p. 168.4–169.2 ◦C; 1H-NMR (300 MHz, DMSO-*d*6) δ: 11.32 (s, 2H), 8.30 (s, 2H), 7.92 (s, 2H), 7.59–7.41 (m, 6H), 6.46 (s, 2H), 3.25 (dd, *J* = 12.7, 6.4 Hz, 4H), 1.61–1.43 (m, 4H), 1.38–1.21 (m, 12H). 13C-NMR (101 MHz, DMSO) δ: 167.04, 135.24, 129.61, 127.74, 127.66, 119.27, 117.90, 111.07, 101.14, 39.21, 29.27, 29, 28.83, 26.55. HRMS (ESI) *m*/*z*: 459.2760 [M + H]+, calcd. for [C28H35N4O2] <sup>+</sup> 459.2760.

4.6.17. (2E,2 E)-N,N -(Decane-1,10-Diyl)Bis(3-(4-Chlorophenyl)Acrylamide) (**25e**)

Pale white solid, yield 64%, m.p. 230.3–230.9 ◦C; 1H-NMR (300 MHz, CDCl3 +CF3COOD) δ: 7.64 (d, *J* = 13 Hz, 2H), 7.54–7.38 (m, 8H), 6.51 (d, *J* = 11.9 Hz, 2H), 3.62–3.37 (m, 4H), 1.73–1.54 (m, 4H), 1.46–1.18 (m, 12H). 13C-NMR (101 MHz, CDCl3 + CF3COOD) δ: 129.71, 118.78, 115.96, 113.13, 110.30, 29.22, 28.99, 26.64. HRMS (ESI) *m*/*z*: 501.2075 [M + H]<sup>+</sup>, calcd. for [C28H35Cl2N2O2] <sup>+</sup> 501.2076.

4.6.18. (2E,2 E)-N,N -(Decane-1,10-Diyl)Bis(3-(3-Hydroxy-4-Methoxyphenyl)Acrylamide) (**26e**)

Pale yellow solid, yield 35%, m.p. 192.9–195.3 ◦C; 1H-NMR (300 MHz, CDCl3 + CD3OD) δ: 7.42 (d, *J* = 15.3 Hz, 2H), 7.05 (s, 2H), 6.98 (d, *J* = 8.1 Hz, 2H), 6.83 (d, *J* = 8.3 Hz, 2H), 6.33 (d, *J* = 15.6 Hz, 2H), 3.89 (s, 6H), 3.29 (t, *J* = 7 Hz, 4H), 1.55–1.50 (m, 4H), 1.31–1.27 (m, 12H). 13C-NMR (101 MHz, CDCl3 + CD3OD) δ: 167.37, 148.94, 146.05, 140.42, 128.11, 121.06, 118.25, 113.07, 111, 55.57, 39.50, 29.13, 28.98, 26.69. HRMS (ESI) *m*/*z*: 525.2951 [M + H]<sup>+</sup>, calcd. for [C30H41N2O6] <sup>+</sup> 525.2965.

4.6.19. (2E,2 E)-N,N -(Decane-1,10-Diyl)Bis(3-(3,4-Dimethoxyphenyl)Acrylamide) (**27e**)

Pale white solid, yield 59%, m.p. 168.9–169.9 ◦C; 1H-NMR (300 MHz, DMSO-*d*6) δ: 7.95 (br, 2H), 7.30 (br, 2H), 7.11 (br, 4H), 6.97 (br, 2H), 6.50 (br, 2H), 3.78 (br, 12H), 3.14 (br, 4H), 1.42 (br, 4H), 1.27 (br, 12H). 13C-NMR (101 MHz, CDCl3+ CD3OD) δ: 167.15, 150.31, 148.90, 140.25, 127.89, 121.77, 118.56, 111.04, 109.74, 55.66, 55.58, 39.47, 29.16, 29.14, 28.99, 26.69. HRMS (ESI) *m*/*z:* 553.3272 [M + H]+, calcd. for [C32H45N2O6] <sup>+</sup> 553.3278.

4.6.20. (2E,2 E)-N,N -(Decane-1,10-Diyl)Bis(3-(4-Hydroxy-3,5-Dimethoxyphenyl)Acrylamide) (**28e**)

Pale yellow solid, yield 33%, m.p. 179.8–180.5 ◦C; 1H-NMR (300 MHz, CDCl3 + CD3OD) δ: 7.44 (d, *J* = 15.5 Hz, 3H), 6.76 (s, 4H), 6.37 (d, *J* = 15.6 Hz, 2H), 3.88 (s, 12H), 3.30 (t, *J* = 7.1 Hz, 4H), 1.61–1.49 (m, 4H), 1.38–1.24 (m, 12H). 13C-NMR (101 MHz, CDCl3 + CD3OD) δ: 166.94, 147.48, 141.03, 136.87, 126.16, 118.46, 104.99, 56.23, 39.63, 29.39, 29.21, 29.08, 26.79. HRMS (ESI) *m*/*z*: 585.3164 [M+H]+, calcd. for [C32H45N2O8] <sup>+</sup> 585.3176

4.6.21. (2E,2 E)-N,N -(Decane-1,10-Diyl)Bis(3-(3,4,5-Trimethoxyphenyl)Acrylamide) (**29e**)

Pale white solid, yield 67%, m.p. 193.4–194.3 ◦C; 1H-NMR (300 MHz, CDCl3 + CD3OD) δ: 7.45 (d, *J* = 15.4 Hz, 2H), 6.76 (s, 4H), 6.44 (d, *J* = 15.6 Hz, 2H), 3.88 (s, 12H), 3.85 (s, 6H), 3.31 (t, *J* = 7.1 Hz, 4H), 1.63–1.47 (m, 4H), 1.40–1.23 (m, 12H). 13C-NMR (101 MHz, CDCl3 + CD3OD) δ: 166.73, 153.26, 140.53, 139.33, 130.65, 120.18, 104.99, 60.81, 56, 39.63, 29.28, 29.21, 29.05, 26.76. HRMS (ESI) *m*/*z*:613.3472 [M + H]<sup>+</sup>, calcd. for [C34H49N2O8] <sup>+</sup> 613.3489.

4.6.22. N,N -(Decane-1,10-Diyl)Bis(2-Aminobenzamide) (**30e**)

Pale white solid, yield 67%, m.p. 90.2–91 ◦C; 1H-NMR (300 MHz, CDCl3 + CD3OD) δ: 7.36 (dd, *J* = 7.8, 1.2 Hz, 2H), 7.22–7.15 (m, 2H), 6.79–6.60 (m, 4H), 3.35 (dt, *J* = 9.7, 5.5 Hz, 4H), 1.66–1.49 (m, 4H), 1.41–1.24 (m, 12H). 13C-NMR (101 MHz, CDCl3 + CD3OD) δ: 173.92, 151.77, 135.96, 131.40, 121.48, 121.28, 120.96, 43.54, 33.31, 33.26, 33.11, 30.83. HRMS (ESI) *m*/*z*:411.2759 [M + H]+, calcd. for [C24H35N4O2] <sup>+</sup> 411.2760.

4.6.23. N,N -(Decane-1,10-Diyl)Bis(4-Aminobenzamide) (**31e**)

Pale yellow solid, yield 31%, m.p. 186.7–187.7 ◦C; 1H-NMR (300 MHz, CDCl3 + CD3OD) δ: 7.57 (d, *J* = 8.6 Hz, 4H), 6.66 (d, *J* = 8.6 Hz, 4H), 3.34 (t, *J* = 7.2 Hz, 4H), 1.63–1.53 (m, 4H), 1.31–1.26 (m, 12H). 13C-NMR (101 MHz, CDCl3 + CD3OD) δ: 168.44, 150.01, 128.49, 123.39, 114.04, 39.77, 29.33, 29.18, 29.06, 26.76. HRMS (ESI) *m*/*z*: 411.2755 [M + H]<sup>+</sup>, calcd. for [C24H35N4O2] <sup>+</sup> 411.2760.

4.6.24. N,N -(Decane-1,10-Diyl)bis(3-Aminobenzamide) (**32e**)

Pale white solid, yield 57%, m.p. 135.7–136.3 ◦C; 1H-NMR (300 MHz, CDCl3 + CD3OD) δ: 7.30–6.97 (m, 6H), 6.83 (dd, *J* = 20.8, 7.2 Hz, 2H), 3.36 (t, *J* = 6.8 Hz, 4H), 1.67–1.47 (m, 4H), 1.46–1.06 (m, 12H). 13C-NMR (101 MHz, CDCl3 + CD3OD) δ: 168.75, 146.84, 135.56, 129.27, 118.09, 116.55, 113.73, 77.48, 77.16, 76.84, 49.31, 49.09, 48.88, 48.67, 48.45, 39.93, 29.29, 29.22, 29.09, 26.79. HRMS (ESI) *m*/*z*: 411.2758 [M + H]<sup>+</sup>, calcd. for [C24H35N4O2] <sup>+</sup> 411.2760

#### 4.6.25. N,N -(Decane-1,10-Diyl)Bis(3,5-Dimethoxybenzamide) (**33e**)

Pale white solid, yield 56%, m.p. 143.4–144.2 ◦C; 1H-NMR (300 MHz, CDCl3 + CD3OD) δ: 6.79 (d, *J* = 2.1 Hz, 4H), 6.43 (t, *J* = 1.9 Hz, 2H), 3.69 (s, 12H), 3.25 (t, *J* = 7.2 Hz, 4H), 1.50–1.45 (m, 4H), 1.25–1.15 (m, 12H). 13C-NMR (101 MHz, CDCl3 + CD3OD) δ: 168.01, 160.73, 136.74, 104.94, 103.34, 55.40, 40.03, 29.28, 29.26, 29.11, 26.82. HRMS (ESI) *m*/*z:* 501.2959 [M + H]<sup>+</sup>, calcd. for [C28H41N2O6] <sup>+</sup> 501.2965.

#### 4.6.26. N,N -(Decane-1,10-Diyl)Bis(3,4,5-Trimethoxybenzamide) (**34e**)

Pale white solid, yield 63%, m.p. 194–194.9 ◦C; 1H-NMR (300 MHz, CDCl3 + CD3OD) δ: 6.91 (s, 4H), 3.72 (s, 12H), 3.67 (s, 6H), 3.22–3.16 (m, 4H), 1.50–1.36 (m, 4H), 1.22–1.08 (m, 12H). 1H-NMR (300 MHz, CDCl3 + CD3OD). δ: 6.91 (s, 4H), 3.72 (s, 12H), 3.67 (s, 6H), 3.22–3.16 (m, 4H), 1.50–1.36 (m, 4H), 1.22–1.08 (m, 12H). 13C-NMR (101 MHz, CDCl3 + CD3OD) δ: 167.81, 152.77, 129.84, 104.42, 60.53, 55.85, 40.01, 29.17, 29.03, 26.73. HRMS (ESI) *m*/*z:* 561.3169 [M + H]<sup>+</sup>, calcd. for [C30H45N2O8] <sup>+</sup> 561.3176.

#### 4.6.27. N,N -(Decane-1,10-Diyl)Bis(2,3,4-Trimethoxybenzamide) (**35e**)

Pale white solid, yield 26%, m.p. 112.3–112.5 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 7.94 (s, 2H), 7.89 (d, *J* = 8.9 Hz, 2H), 6.76 (d, *J* = 9 Hz, 2H), 3.96 (s, 6H), 3.90 (s, 6H), 3.87 (s, 6H), 3.44 (dd, *J* = 12.7, 6.9 Hz, 4H), 1.62 (dt, *J* = 14.3, 7.1 Hz, 4H), 1.41–1.29 (m, 12H). 13C-NMR (101 MHz, CDCl3) δ: 164.90, 156.35, 152.42, 141.83, 126.76, 119.31, 107.67, 61.71, 61.08, 56.15, 39.71, 29.78, 29.65, 29.43, 27.24. HRMS (ESI) *m*/*z:* 561.3172 [M + H]<sup>+</sup>, calcd. for [C30H45N2O8] <sup>+</sup> 561.3176.

#### *4.7. General Synthetic Procedure for* **9d***,* **9f**

A mixture of cinnamic acid (6.30 mmol), EDCI (7.50 mmol), DMAP (0.60 mmol), and anhydrous dichloromethane (20 mL) was stirred to dissolve, then diamino-alkane (3 mmol) was added and stirred at room temperature for 12 h. The mixture solution was filtered under reduced pressure. After that, the residue was washed with dichloromethane and water successively, and then dried to give a pale white solid. TLC indicated that it was a single point.

#### 4.7.1. (2E,2 E)-N,N -(Octane-1,8-Diyl)Bis(3-Phenylacrylamide) (**9d**)

Pale white solid, yield 63%, m.p. 186–186.4 ◦C; 1H-NMR (300 MHz, CDCl3 + CD3OD) δ: 7.58–7.29 (m, 12H), 6.53 (d, *J* = 15.7 Hz, 2H), 3.31 (t, *J* = 7.1 Hz, 4H), 1.59–1.55 (m, 4H), 1.35 (s, 8H). 13C-NMR (101 MHz, CDCl3 + CD3OD) δ: 166.96, 140.39, 134.78, 129.42, 128.59, 127.54, 120.60, 39.43, 29.04, 28.85, 26.57. HRMS (ESI) *m*/*z:* 405.2537 [M + H]<sup>+</sup>, calcd. for [C26H33N2O2] <sup>+</sup> 405.2542.

#### 4.7.2. (2E,2 E)-N,N -(Dodecane-1,12-Diyl)Bis(3-Phenylacrylamide) (**9f**)

Pale white solid, yield 49%, m.p. 164.7–165 ◦C; 1H-NMR (300 MHz, CDCl3 + CD3OD) δ: 7.56–7.49 (m, 6H), 7.40–7.28 (m, 6H), 6.53 (d, *J* = 15.7 Hz, 2H), 3.30 (t, *J* = 7.1 Hz, 4H), 1.62–1.51 (m, 4H), 1.38–1.23 (m, 16H). 13C-NMR (101 MHz, CDCl3 + CD3OD) δ: 166.94, 140.22, 134.73, 129.29, 128.47, 127.42, 120.54, 39.42, 29.19, 29.03, 28.99, 26.66. HRMS (ESI) *m*/*z:* 461.3166 [M + H]<sup>+</sup>, calcd. for [C30H41N2O2] <sup>+</sup> 461.3168.

#### *4.8. General Synthetic Procedure for* **9g***–***9i**

A mixture of cinnamic acid (6.30 mmol), EDCI (7.50 mmol), DMAP (0.60 mmol), and anhydrous dichloromethane (20 mL) was stirred to dissolve, then alkane-diol (3 mmol) was added and stirred at room temperature for 12 h. The mixture solution was filtered under reduced pressure. After that, the residue was washed with dichloromethane and water successively, subsequently, purified by preparative TLC eluted with petroleum ether/ethyl acetate = 5:1 to give pale white solid.

#### 4.8.1. Octane-1,8-Diyl (2E,2 E)-Bis(3-Phenylacrylate) (**9g**)

Pale white solid, yield 35%, m.p. 61.1–62.5 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 7.68 (d, *J* = 16 Hz, 2H), 7.58–7.46 (m, 4H), 7.43–7.30 (m, 6H), 6.45 (d, *J* = 16 Hz, 2H), 4.21 (t, *J* = 6.7 Hz, 4H), 1.77–1.68 (m, 4H), 1.46–1.40 (m, 8H). 13C-NMR (101 MHz, CDCl3) δ: 167.22, 144.72, 134.62, 130.35, 129.01, 128.19, 118.43, 64.79, 29.31, 28.85, 26.05. HRMS (ESI) *m*/*z:* 407.2223 [M + H]<sup>+</sup>, calcd. for [C26H31O4] <sup>+</sup> 407.2222.

#### 4.8.2. Decane-1,10-Diyl (2E,2 E)-Bis(3-Phenylacrylate) (**9h**)

Pale white solid, yield 33%, m.p. 118.6–119.2 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 7.68 (d, *J* = 16 Hz, 2H), 7.57–7.48 (m, 4H), 7.38 (dd, *J* = 6.6, 3.6 Hz, 6H), 6.45 (d, *J* = 16 Hz, 2H), 4.21 (t, *J* = 6.7 Hz, 4H), 1.77–1.62 (m, 4H), 1.48–1.25 (m, 12H). 13C-NMR (101 MHz, CDCl3) δ: 167.23, 144.69, 134.64, 130.35, 129.01, 128.19, 118.46, 64.85, 29.58, 29.40, 28.88, 26.12. HRMS (ESI) *m*/*z:* 435.2534 [M + H]+, calcd. for [C28H35O4] <sup>+</sup> 435.2535.

#### 4.8.3. Dodecane-1,12-Diyl (2E,2 E)-Bis(3-Phenylacrylate) (**9i**)

Pale white solid, yield 28%, m.p. 63.1–64 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 7.68 (d, *J* = 16 Hz, 2H), 7.52 (dt, *J* = 4.6, 3.2 Hz, 4H), 7.42–7.33 (m, 6H), 6.45 (d, *J* = 16 Hz, 2H), 4.21 (t, *J* = 6.7 Hz, 4H), 1.77–1.65 (m, 4H), 1.43–1.25 (m, 16H). 13C-NMR (101 MHz, CDCl3) δ: 167.22, 144.68, 134.64, 130.34, 129.01, 128.18, 118.47, 64.87, 29.68, 29.66, 29.42, 28.87, 26.12. HRMS (ESI) *m*/*z:* 463.2851 [M + H]+, calcd. for [C30H39O4] <sup>+</sup> 463.2828.

#### *4.9. General Synthetic Procedure for* **9j***–***9m**

A mixture of cinnamic acid (6.30 mmol), EDCI (7.50 mmol), DMAP (0.60 mmol), and anhydrous dichloromethane (20 mL) was stirred to dissolve, then diamino-ether (3 mmol) was added and stirred at room temperature for 12 h. Then, the solid was filtered off, the filtrate was added with H2O (50 mL) and extracted by dichloromethane three times. The combined organic layers were washed with H2O and brine, dried with anhydrous sodium sulfate for 8 h, filtered, and evaporated. The resulting residue was purified by preparative TLC with petroleum ether/ethyl acetate = 2:1–1:1 to give pale white solid or light yellow oil.

#### 4.9.1. (2E,2 E)-N,N -((Ethane-1,2-Diylbis(Oxy))Bis(Ethane-2,1-Diyl))Bis(3-Phenylacrylamide) (**9j**)

Pale white solid, yield 78%, m.p. 117.9–118.5 ◦C; 1H-NMR (300 MHz, CDCl3) δ: 7.62 (d, *J* = 15.6 Hz, 2H), 7.51–7.41 (m, 4H), 7.37–7.27 (m, 6H), 6.48 (d, *J* = 15.6 Hz, 2H), 3.65–3.60 (m, 12H). 13C-NMR (101 MHz, CDCl3) δ: 166.26, 141.19, 134.95, 129.77, 128.92, 127.92, 120.82, 70.50, 70.05, 39.63. HRMS (ESI) *m*/*z*: 409.2121 [M + H]<sup>+</sup>, calcd. for [C24H29N2O4] <sup>+</sup> 409.2127.

#### 4.9.2.

(2E,2 E)-N,N -(((Oxybis(Ethane-2,1-Diyl))Bis(Oxy))Bis(Ethane-2,1-Diyl))Bis(3-Phenylacrylamide) (**9k**)

Light yellow oil, yield 28%; 1H-NMR (300 MHz, CDCl3) δ: 7.60 (d, *J* = 15.6 Hz, 2H), 7.45 (dd, *J* = 6.5, 3.1 Hz, 4H), 7.32–7.30 (m, 4H), 6.71 (t, *J* = 5.7 Hz, 2H), 6.47 (d, *J* = 15.6 Hz, 2H), 3.65 (s, 8H), 3.63–3.54 (m, 8H). 13C-NMR (101 MHz, CDCl3) δ: 166.17, 140.96, 134.98, 129.70, 128.89, 127.86, 120.93, 70.49, 70.27, 70, 39.59. HRMS (ESI) *m*/*z:* 453.2387 [M + H]<sup>+</sup>, calcd. for [C26H33N2O5] <sup>+</sup> 453.2389.

#### 4.9.3. (Ethane-1,2-Diylbis(Oxy))Bis(Ethane-2,1-Diyl) (2E,2 E)-Bis(3-Phenylacrylate) (**9l**)

Light yellow oil, yield 65%; 1H-NMR (300 MHz, CDCl3) δ: 7.70 (d, *J* = 16 Hz, 2H), 7.54–7.48 (m, 4H), 7.40–7.34 (m, 6H), 6.48 (d, *J* = 16 Hz, 2H), 4.42–4.36 (m, 4H), 3.84–3.77 (m, 4H), 3.73 (s, 4H). 13C-NMR (101 MHz, CDCl3) δ: 167.03, 145.21, 134.50, 130.44, 129.01, 128.23, 118, 70.79, 69.48, 63.78. HRMS (ESI) *m*/*z:* 411.1805 [M + H]<sup>+</sup>, calcd. for [C24H27O6] <sup>+</sup> 411.1808.

#### 4.9.4. ((Oxybis(Ethane-2,1-Diyl))Bis(Oxy))Bis(Ethane-2,1-Diyl) (2E,2 E)-bis(3-Phenylacrylate) (**9m**)

Light yellow oil, yield 87%; 1H-NMR (300 MHz, CDCl3) δ: 7.70 (d, *J* = 16 Hz, 2H), 7.55–7.48 (m, 4H), 7.38 (dd, *J* = 3.8, 2.7 Hz, 6H), 6.48 (d, *J* = 16 Hz, 2H), 4.37 (dd, *J* = 5.5, 4.1 Hz, 4H), 3.81–3.74 (m, 4H), 3.70 (s, 8H). 13C-NMR (101 MHz, CDCl3) δ: 167.03, 145.19, 134.52, 130.45, 129.02, 128.23, 118.04, 77.48, 77.36, 77.16, 76.84, 70.82, 70.79, 69.42, 63.80. HRMS (ESI) *m*/*z*: 472.2337 [M + NH4] <sup>+</sup>, calcd. for [C26H34NO7] <sup>+</sup> 472.2335.

#### *4.10. Biological Assays*

#### 4.10.1. Cell Culture

The tested cells were obtained from Shanghai Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). HeLa and Hep G2 cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries), 100 U/mL penicillin-streptomycin (PS; gibco, Life Technologies, Carlsbad, CA, USA). MCF-7 and A549 cell lines were maintained in RPMI-1640 media (gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS and 100 U/mL PS. FaDu cell lines were cultured in minimum essential medium (MEM; Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS and 100 U/mL PS. MCF 10A cell lines were maintained in Mammary MEGM kit (Lonza/Clonetics) supplemented with 100 ng/mL cholera toxin (Sigma, Shanghai, China). Cell lines were grown at 37 ◦C in a humidified atmosphere of 5% CO2.

#### 4.10.2. Cell Viability Assay

The growth-inhibitory effects against HeLa, Hep G2, MCF-7, FaDu, and A549 cancer cells and normal cell lines MCF 10A were determined by MTT assay [21]. All of the synthesized compounds and the positive control drug doxorubicin were dissolved in 0.1% DMSO. Cells were seeded in 96 well plates at a density of 1 <sup>×</sup> 104 cells per well and incubated for 24 h at 37 ◦C in a humidified atmosphere of 5% CO2. The cells were then incubated with various concentrations of drugs for 48 h in a 5% CO2 incubator at 37 ◦C. After treatment with 1 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution for 4 h, the formazan crystals were dissolved in 100 μL DMSO in each well. Plates were read on a microplate reader (Thermo Varioskan Flash 3001) at 490 nm. The experiment was performed in triplicate. The inhibitory concentration at 50% (IC50) was calculated based on concentration-inhibition relationships via regression using SPSS software (Version 17.0).

#### 4.10.3. Colony Formation Assay

FaDu cells were seeded in a 6-well plate with 500 cells per well and incubated for 24 h. Then, the cells were treated with compound **8e** at various final concentrations of 0, 0.75, 1.50, 3 nM. Following treatment for 10 days, the cells were washed with PBS twice and fixed with 4% paraformaldehyde for 10 min at room temperature. Then, the cell colonies were visualized by staining with 0.1% crystal violet for 10 min. The image was photographed with Handy camera, and the numbers of cell colonies were analyzed using the open Image J software (Developed by National Institutes of Health). A group of >50 cells was defined as one colony. Triplicate wells were set up for each concentration.

#### 4.10.4. Live/Dead Staining

The Calcein AM/propidium iodide (PI) double staining was performed following the manufacturer's instructions (Dojindo Laboratory, Kumamoto, Japan). Briefly, FaDu cells were seeded in 6-well plates overnight. Then, the **8e** solutions at final concentrations of 2.5 nM, 5 nM, and 10 nM were added. After incubation for 24 h, the cells were collected, washed with PBS, and stained by a mixture of 2 μM Calcein-AM (live cells, green) and 4.5 μM PI (dead cells, red) solution in the dark for 30 min at 37 ◦C (excitation 490 nm). Then, the cells were rinsed with PBS twice and re-suspended in the cell medium. Confocal fluorescence images were acquired using a laser scanning confocal microscope (LSCM, Carl-Zeiss LSM 710).

#### 4.10.5. Hoechst 33,342 Staining

For Hoechst staining, FaDu cells were seeded onto a glass-bottom of cell culture dish at a density of 1 <sup>×</sup> <sup>10</sup><sup>5</sup> cells. After incubation for 24 h, the media containing 20 nM of the compound **8e** was used to replace the culture medium. Then, the cells were stained with Hoechst 33,342 solution (10 μg/mL in the culture medium, Beyotime Institute of Biotechnology, Shanghai, China) at 37 ◦C in the dark for 20 min after treatment with **8e**. Then, the cells were washed with the serum-free medium to remove excess dye. LSCM (Carl-Zeiss LSM 710) was used to capture the images of nuclear morphological changes to find apoptotic cells.

#### 4.10.6. Flow Cytometric Analysis of Apoptosis by Annexin V-FITC/PI Staining

Apoptosis in FaDu cells was evaluated by an annexin V-FITC/PI apoptosis detection kit (Beyotime Institute of Biotechnology, Shanghai, China). Briefly, FaDu cells were treated with various concentrations of compound **8e** (2, 5, 10 nM) or DMSO as vehicle control for 24 h at 37 ◦C; then, the cells were washed twice with cold 1× PBS and centrifuged at 1000 rpm for 5 min. The harvested cells were resuspended gently in 400 μL binding buffer, containing 5 μL annexin V-FITC and 10 μL PI. After incubating for 15 min in the dark at room temperature, the cells were analyzed with flow cytometry (CyFlow® Cube 6, Sysmex). Data were shown as pseudo color graphs and analyzed using FlowJo Software (Tree Star Inc, Ashland, OR).

#### 4.10.7. Mitochondrial Membrane Potential (ΔΨm) Analysis

The mitochondrial membrane potential (ΔΨm) was detected by a mitochondrial membrane potential assay kit with JC-1 (Beyotime Institute of Biotechnology, Shanghai, China). Briefly, after treatment with various concentrations of compound **8e** (2, 5, 10 nM) for 24 h, FaDu cells were harvested and stained with 500 μL 1× JC-1 dye solution at 37 ◦C for 20 min in the dark. Then, the treated cells were washed twice and resuspended by 1× JC-1 staining buffer. The fluorescence of approximately <sup>1</sup> <sup>×</sup> 104 cells was analyzed using flow cytometry (CyFlow® Cube 6, Sysmex).

#### 4.10.8. Cell Cycle Analysis

The cell cycle analysis was carried out by a cell cycle and apoptosis analysis kit with a propidium iodide (PI) staining method (Beyotime Institute of Biotechnology, Shanghai, China). Briefly, FaDu cells were incubated with the above-mentioned doses of compound **8e** for 24 h; all samples were collected, washed once with cold 1× PBS, and fixed by 70% ice-cold ethanol at 4 ◦C for 12 h. Then, the fixed cells were washed once with cold 1× PBS and stained with 500 μL 1× PI dye solution (containing 10 μL RNase A) at 37 ◦C for 30 min in the dark. The red fluorescence was detected using a flow cytometer (CyFlow® Cube 6, Sysmex). The data were analyzed using FlowJo Software (Tree Star Inc, Ashland, OR, USA).

#### 4.10.9. In Silico ADMET Prediction

A computational study of all synthesized compounds was carried out for the prediction of ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties. In this module, thirteen mathematical models, such as HBD (hydrogen bond donor), HBA (hydrogen bond acceptor), RBN (number of rotatable bonds), logP (log of the octanol/water partition coefficient), log S (log of the aqueous solubility), and PSA (polar surface area) were predicted via Discovery Studio 2.0 Software (Studio 2.5, Accelrys, Co. Ltd., San Diego, CA, USA) and HIA (human intestinal absorption), PPB (plasma protein binding), CYP3D4 (CYP3D4 inhibition), T1/<sup>2</sup> (half lifetime), HT (human hepatotoxicity), and LD50 (median lethal dose) were calculated using web-based applications and then analyzed to

predict the drug likeliness profile (Xiangya School of Pharmaceutical Sciences and Central South University, http://admet.scbdd.com/home/index/) [28]. We also screened our novel derivatives through the prediction model by Egan et al. [29,30]. Two molecular descriptors, AlogP98 and PSA, were computed and plotted in a 2D plane to comprehensively evaluate the absorption and blood-brain penetration of the compounds.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1420-3049/24/24/4505/s1, 1H-NMR, 13C-NMR and HR-MS spectra of compounds.

**Author Contributions:** Ideas and experiment design: J.W., G.H., and T.L.; Chemistry and Biology: J.W., G.H., G.L., and W.W.; Analysis and interpretation of data: J.W., G.H., G.L., and W.W.; Writing and review of the manuscript: J.W., G.H., G.L., W.W., and T.L.; Study supervision: T.L. and G.H.

**Funding:** This research was funded by the national major science and technology special project for "significant new drugs development" (2018ZX09711001-005-018), "the medical and health science and technology innovation project of Chinese Academy of Medical Science" (2017-I2M-3-021/2019-I2M-1-005), "the Tianjin special support program for talent development" (TJTZJH-GCCCXCYTD-1-30).

**Acknowledgments:** The authors gratefully acknowledge the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College and information technology center for affording the Discovery Studio 2.0 Software.

**Conflicts of Interest:** The authors declare that they have no competing interests.

#### **References**


**Sample Availability:** Samples of all compounds are available from the authors.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **Novel Chrysin-De-Allyl PAC-1 Hybrid Analogues as Anticancer Compounds: Design, Synthesis, and Biological Evaluation**

**Buthina A. Al-Oudat 1,**†**, Hariteja Ramapuram 2,**†**, Saloni Malla 2, Suaad A. Audat 3, Noor Hussein 2, Jenna M. Len 2, Shikha Kumari 4, Mel F. Bedi 5, Charles R. Ashby, Jr. <sup>6</sup> and Amit K. Tiwari 2,\***


#### Academic Editor: Qiao-Hong Chen

Received: 2 June 2020; Accepted: 30 June 2020; Published: 4 July 2020

**Abstract:** New chrysin-De-allyl-Pac-1 hybrid analogues, tethered with variable heterocyclic systems (**4a**–**4o**), were rationally designed and synthesized. The target compounds were screened for in vitro antiproliferative efficacy in the triple-negative breast cancer (TNBC) cell line, MDA-MB-231, and normal human mammary epithelial cells (HMECs). Two compounds, **4g** and **4i**, had the highest efficacy and selectivity towards MDA-MB-231 cells, and thus, were further evaluated by mechanistic experiments. The results indicated that both compounds **4g** and **4i** induced apoptosis by (1) inducing cell cycle arrest at the G2 phase in MDA-MB-231 cells, and (2) activating the intrinsic apoptotic pathways in a concentration-dependent manner. Physicochemical characterizations of these compounds suggested that they can be further optimized as potential anticancer compounds for TNBC cells. Overall, our results suggest that **4g** and **4i** could be suitable leads for developing novel compounds to treat TNBC.

**Keywords:** triple-negative breast cancer; cytotoxicity; chrysin analogues; flavonoid; anticancer compounds

#### **1. Introduction**

Breast cancer is the second leading cause of death among all cancers affecting women [1]. Triple-negative breast cancer (TNBC) lacks the expression of hormone receptors (estrogen (ER) or progesterone (PR)) and/or human epidermal growth factor receptor 2 (HER2), which are more amenable to targeted therapy [2]. TNBC often presents as a high-grade invasive ductal carcinoma (IDC) in

patients, accounting for one-fourth of all breast cancer deaths. Consequently, there is an urgent need for the development of compounds that are efficacious and safe for the treatment of breast cancer. Flavonoids are ubiquitous naturally occurring polyphenolic compounds that are commonly found in fruits and vegetables [3]. Flavonoids comprise several classes of low molecular weight compounds, including flavanones, anthocyanidins, flavonols, flavanols, isoflavones, dihydroflavonols, and flavones [4–6]. Chrysin (5, 7-dihydroxyflavone, Figure 1) is a natural flavone found in many plant extracts, such as the blue passionflower, as well as in honey and propolis [7,8]. Chrysin has been reported to have properties, such as antioxidant [9], antihypertensive [10], antibacterial [11], anti-inflammatory [12], antiviral [13], antiallergic [14], antidiabetic [15], anxiolytic [16], and anticancer efficacy [17,18]. The activation of apoptosis plays a major role in producing the anticancer efficacy of chrysin [19–21]. Mechanistically, apoptosis involves a cascade of initiator and effector caspases [22]. Among these caspases, caspase-3 and caspase-7 are downstream executioner caspases that play an essential role in inducing apoptosis by cleaving a variety of cellular substrates [23]. Therefore, caspase-dependent apoptosis pathways represent targets for the development of efficacious anticancer drugs. Recently, a number of studies have been done to augment the pharmacological activity of chrysin by producing synthetic analogues [24–29]. Moreover, there are studies indicating that chrysin-based compounds have in vitro efficacy in breast cancer cells [30–32]. The compound, de-allyl procaspase-activating compound 1 (PAC-1), induces apoptosis in different types of cancer cells by activating caspase-3 and/or caspase-7 [33–36]. Previously, we reported that molecular hybridization between chrysin and de-allyl PAC-1 can be used to produce novel hybrid molecules with cytotoxic efficacy [29]. Molecular hybridization is a process that comprises the amalgamation of two or more pharmacophoric moieties of different bioactive molecules into a single molecular framework [37,38].

**Figure 1.** Chemical structures of chrysin, de-allyl PAC-1, and a representative designed hybrid molecule **4a**.

In this study, new chrysin-de-allyl PAC-1 hybrid analogues, substituted with variable aromatic heterocyclic cores, were synthesized and evaluated to identify a more potent bioactive hybrid against breast cancer cells (Figure 1). The in silico parameters of the synthesized compounds were calculated to predict their pharmacokinetic profile and drug-likeness using SwissSimilarity ADME, a web tool [39]. Subsequently, the target compounds were screened for antiproliferative efficacy in the human breast cancer cell line MDA-MB-231, using the MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium

Bromide) colorimetric assay. Finally, we determined the effects of the most potent compounds on the cell cycle.

#### **2. Results and Discussions**

#### *2.1. Chemistry*

The target compounds (**4a**–**4o**) were prepared according to Scheme 1, starting from the commercially available chrysin, using previously published synthetic procedures [29]. Briefly, chrysin was added to methyl 2-bromoacetate at a low temperature in the presence of K2CO3, yielding the desired alkylation product **2**. The conversion of ester **2** into hydrazide **3** was accomplished using 80% hydrazine hydrate and a few drops of hydrochloric acid at low temperature. 1H and 13C NMR data confirmed the formation of hydrazide **3** as a pure single product. Target compounds (**4a**–**4o**) were obtained by adding hydrazide **3** to an appropriate aldehyde in the presence of a catalytic amount of hydrochloric acid at room temperature. All analogues gave adequate analytical and spectroscopic data, which were in full accordance with their structures. 1H and 13C NMR spectra showed that compounds **4a**–**4o** existed as geometrical isomers (E/Z isomers). The E:Z ratio for each compound was determined from 1H NMR spectra utilizing the integration of the neat methylene group peaks that appeared as two separated singlets for each isomer. Target compounds were analyzed using SwissSimilarity (Swiss Institute of Bioinformatics, Lausanne, Switzerland). Compounds **4a**–**4j** and **4n**–**4o** were the most structurally similar to apigenin, a flavonoid similar to chrysin. The physicochemical properties of the compounds (**4a**–**4o**) are shown in Table 1.

**Scheme 1.** Reagents and conditions: (**a**) methyl 2-bromoacetate, K2CO3, DMF, 0 ◦C, 3 h; (**b**) hydrazine hydrate 80%, EtOH, 0 ◦C, HCl (cat.), 1 h; (**c**) different substituted aldehydes, CH3OH, HCl (cat), rt, 1 h.

**Table 1.** Physiochemical properties of the compounds **4a**–**4o** collected from Swiss ADME.



**Table 1.** *Cont.*

ESOL Class: Estimated Solubility Class [40]. GI Absorption: Gastrointestinal Absorption. BBB Permeant: Blood Brain Barrier Permeant. Lipinski Violations examines orally active compounds to determine ranges for high probability to be an oral drug. Lead-likeness Violations examines compounds' likeliness to become a lead based on a rule-based method [41]. PAINS Alerts: compounds that give false positives where the compound nonspecifically binds to numerous biological targets, instead of one desired target [42]. Log P: partition coefficient measuring lipophilicity [41].

#### *2.2. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide-Based Cytotoxicity Assay*

The synthesized chrysin derivatives were evaluated for cytotoxic efficacy in the human breast cancer cell line, MDA-MB-231, using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and doxorubicin was used as a reference anticancer drug. The IC50 values (concentration of compound in μM required to reduce 50% of cell viability) are shown in Table 2.


**Table 2.** The effects of chrysin derivatives (**4a**–**4o**) on the survival of the MDA-MB-231 cancer cell line.


Cell survival was determined by MTT assay as described in materials and methods. The IC50 values are represented as mean ± SD of two independent experiments performed in triplicate. <sup>a</sup> The data was taken from [29].

As shown in Table 2, the structure–activity relationship (SAR) results indicated that the cytotoxic efficacy of the synthesized compounds was affected by the modifications on the benzene ring (ring D) of the parent compound **4a**. Compound **4a**, possessing a hydroxyl group at C-2 of ring D, had cytotoxic efficacy, with an IC50 value of 6.8 μM. To determine the importance of the hydroxyl group at the C-2 position, compound **4b** was synthesized, and its efficacy was similar to compound **4a**. The methylation of the hydroxyl group in **4a** yielded compound **4c**, which had a lower efficacy than **4a** and **4b**, whereas ethylation of the hydroxyl group, which yielded compound **4d,** produced a significant decrease in cytotoxic efficacy, compared to compound **4a**. Previously, we reported that the shifting of the hydroxyl group from C-2 to C-4 (**4e**) produced a significant reduction in the cytotoxic efficacy (IC50 > 50 μM). However, methylation of the hydroxyl group in **4e** yielded compound **4f,** which was ~5 times more efficacious than **4e [29]**. The addition of a second hydroxyl group at the C-4 position in **4a** resulted in compound **4g** (IC50 = 5.98 μM), which had cytotoxic efficacy similar to compound **4a**. Interestingly, the methylation of the two hydroxyl groups in **4g**, yielding compound **4h,** significantly decreased the cytotoxic efficacy. The addition of a third hydroxyl group to **4g** at position 3 of ring D, yielding compound **4i**, did not significantly alter the antiproliferative efficacy of the compound, whereas adding a third hydroxyl group at position 6 of ring D yielded a totally inactive compound, **4j**. Next, we determined the effect of ring D on the cytotoxic efficacy of the synthesized compounds. Therefore, compounds **4k**–**4m** were synthesized, where ring D was replaced with aromatic heterocyclic moieties. Compounds **4k**, **4l**, and **4m**, containing furan, 3-pyridine, and 2-pyridine moieties, respectively, did not have significant cytotoxic efficacy. Furthermore, adding another fused benzene ring to ring D, while keeping the C-2 hydroxyl group (2-hydroxynaphthalene), yielded the inactive compound, **4n**. The addition of a nitro group at the C5-position of ring D in **4a** yielded compound **4o**, which had an efficacy similar to compound **4a** (IC50 = 7.9 μM).

Next, the cytotoxicity and selectivity of the most active compounds, **4g** and **4i**, were determined in a panel of cell lines, including the normal cell lines, HMECs, and cancer cell lines, BT-20, U-251, and HCT116, as shown in Figure 2 and Table 3. Compound **4g** had antiproliferative efficacy in the BT-20, U-251, and HCT116, with IC50 values of 5.32, 7.64, and 2.68, respectively. In contrast, compound **4i** had IC50 values ranging from 10–25 μM. The results indicated that although compound **4g** is more potent than compound **4i** in the three cancer cell lines, compound **4i** is more selective for the cancer cells than the normal cells.

**Figure 2.** *Cont*.

**B** 

**Figure 2.** The efficacy and selectivity of **4g** and **4i** in MDA-MB-231, BT-20, U-251, and HCT116, and compared to the normal cell line, HMEC (**A**) the viability curves for cancer cells (MDA-MB-231, BT-20, U-251, HCT116), and the normal cell line, HMEC; (**B**) the IC50 values of **4g** and **4i** for these cell lines are also compared to the normal cell line. Cell survival was determined using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC50 values are represented as the means ± SD of three independent experiments performed in triplicate with \* *p* < 0.05, \*\*\*\* *p* < 0.0001 vs. control group.



Cell survival was determined by MTT assay as described in the materials and methods. The IC50 values (μM) are represented as the mean ± SEM of three independent experiments performed in triplicate. The compounds were screened on breast (MDA-MB-231, BT-20), brain (U-251), and colon (HCT116) cancer cell lines and normal human mammary epithelial cells (HMECs).

#### *2.3. 4g and 4i Induce Apoptosis and G2 Cell Cycle Arrest in a Triple-Negative Breast Cancer Cell Line*

Apoptosis, a type of programmed cell death, is one of the major mechanisms by which chemotherapeutic drugs produce their therapeutic efficacy [43]. Morphologically, apoptosis is characterized by cellular shrinkage, which is accompanied with nuclear chromatin condensation and fragmentation followed by blebbing of the plasma membrane. This leads to the formation of small apoptotic bodies that have an intact cellular membrane and unaltered organelle integrity. These bodies are then released in the extracellular environment and removed by the process of phagocytosis [44,45]. Apoptosis can occur by two pathways: The extrinsic pathway and intrinsic pathway. In either pathway, when the cell is exposed to certain extrinsic or intrinsic stimuli, the integrity of the inner mitochondrial membrane of the cell is compromised, resulting in the loss of the mitochondrial membrane potential, and causing the release of several apoptotic factors, including cytochrome c [46,47]. Numerous studies indicate that during apoptosis, phosphatidylserine (PS), in the cytoplasmic side of the plasma membrane, is translocated to the extracellular cell surface [48]. The flipped anionic PS binds to the Ca2<sup>+</sup>-dependent phospholipid-binding protein, annexin V [49]. We discovered and reported several potent apoptosis-inducing compounds [50–57]. In this study, the result of our morphological studies in MDA-MB-231 cells after incubation with our lead compounds, **4g** and **4i,** indicated that apoptosis was occurring (Figure 3A). At a concentration of 20 μM, both compounds decreased the number of adherent

MDA-MB-231 cells and induced cellular shrinkage. The cells were rounded and loosely attached, and apoptotic bodies were present. Similarly, the incubation of MDA-MB-231 cells with compounds **4g** or **4i** for 24 h produced a significant loss of the mitochondrial membrane potential. For compound **4g**, the population of cells undergoing apoptosis increased from 14.30% at 0 μM to 26.56% and 58.05% at 5 and 10 μM, respectively (*p* value < 0.0001; Figure 3B,C). Compound **4i** also produced a significant shift in the apoptotic cell population in quadrant II, from 14.78% at 0 μM to 32.25% and 42.56% at 5 and 10 μM, respectively (*p* value < 0.0001, Figure 3B,C). Cell cycle analysis indicated that compounds **4g** and **4i** produced a significant disruption in the cell cycle of MDA-MB-231 cells (Figure 3D). Data obtained using flow cytometry indicated that, when incubated with vehicle alone, MDA-MB-231 cells had a normal cell cycle (5.43%, 83.4%, 4.63%, and 4.46% in the subG1, G1, S, and G2 phases, respectively). However, incubation of MDA-MB-231 cells with compound **4g** resulted in a significant shift towards the G2 phase (59.12% and 54.13% for 5 and 10 μM, respectively (*p* value < 0.0001, Figure 3E).

**Figure 3.** *Cont*.

**Figure 3.** *Cont*.

**Figure 3.** Effects of **4g** and **4i** on cellular morphology, mitochondrial membrane potential, and cell cycle (**A**) Morphological observations of MDA-MB-231 cells incubated with 0, 5, and 20 μM concentrations of **4g** and **4i** at different time intervals of 0, 24, 48, and 72 h, respectively; (**B**) The MDA-MB-231 cells in complete medium were incubated with **4g** and **4i** at 0, 5, or 10 μM for 24 h. Cells were then incubated with the reagents of the MitoTracker Red and Alexa Fluor 488 annexin V kits for flow cytometry. Representative results of MDA-MB-231 cells from two independent experiments, each performed in triplicate, are shown; (**C**) Histograms quantitatively summarize the results following incubation with **4g** and **4i**, respectively; (**D**) The induction of cell cycle arrest in MDA-MB-231 cells by **4g** and **4i** is shown. The MDA-MB-231 cells were incubated with different concentrations (0, 5, and 10 μM) of **4g** and **4i** for 24 h and were subjected to cell cycle analysis by flow cytometry of PI (X axis)/cell counts (Y axis); (**E**) A histogram quantitatively summarizing the change in % of cells in each phase of the cell cycle due to incubation with **4g** and **4i**. The data represents means ± SEM of three independent experiments performed in triplicate with \*\*\*\* *p* < 0.0001 vs. control group.

**A** 

#### *2.4. Compounds 4g and 4i Activate Apoptosis by Activating the Intrinsic Apoptotic Pathway*

Apoptosis can be induced by the activation of two major pathways: The intrinsic and extrinsic apoptotic pathways [22]. The activation of the intrinsic apoptotic pathway induces the activation of proapoptotic proteins, such as apoptosis regulator Bak (Bcl-2 homologous antagonist/killer) and Bax (Bcl-2-associated X protein) [58]. The activated the Bax and Bak proteins subsequently permeabilize the mitochondrial outer membrane by forming pores on its outer surface [58–60]. Consequently, cytochrome c (Cyt C) is released into the cytosol, where it combines with the adaptor protein (Apaf-1) to form an apoptosome [22]. The initiator caspases (i.e., caspase-2, caspase-8, caspase-9, or caspase-10) are activated and recruited to large protein complexes, resulting in the cleavage of the executioner caspases, caspase-3 or caspase-7 [61].

Since MDA-MB-231 cells incubated with compounds **4g** and **4i** had a decrease in the mitochondrial membrane potential, which is an early event of intrinsic apoptosis, i.e., altered permeability of the inner mitochondrial membrane, we conducted experiments to determine if these compounds altered the expression of key apoptotic proteins, including cytochrome c, in MDA-MB-231 cells using Western blotting analysis. Our results indicated that compounds **4g** at 5 μM, and compound **4i** at 10 μM produced a significant increase in the expression of cytochrome c, compared to cells incubated with vehicle (Figure 4A,B). This may be due to an increase in the expression of Bak following incubation with **4g** and **4i** (Figure 4A,B), compared to cells incubated in the absence of lead compounds. In addition, both compounds (**4g** at 5 μM and **4i** at 10 μM) produced significant cleavage of the initiator caspase, caspase 9, in MDA-MB-231 cells, compared to cells incubated with vehicle (Figure 4A,B). These events activated caspase 7 in breast cancer cells incubated with both concentrations of **4g** and 10 μM of **4i**, compared to cells incubated with vehicle (Figure 4A,B). In contrast, there was no significant change in the mammalian target of rapamycin (mTOR) expression, indicating that cell death induced by **4g** and **4i** in MDA-MB-231 cells is not due to autophagy (Figure 4A,B). Thus, our results suggest that cytochrome c release induces intracellular initiator caspase activation, followed by the activation of executioner caspases, thus activating apoptotic cell death machinery through intrinsic the pathway.

**Figure 4.** *Cont*.

**Figure 4.** The effect on apoptosis induction at different concentrations of **4g** and **4i** in MDA-MB-231 cells. (**A**) Western blots for the proteins caspase 3, caspase 7, cleaved caspase 7, caspase 9, cleaved caspase 9, Bak, cyt C and mTOR following incubation with **4g** and **4i** at 0, 5, or 10 μM. The values of the proteins were normalized to β-actin levels. (**B**) Histograms summarizing the levels of each protein are also shown. All the data are presented as the means ± SEM of three independent studies with \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. control group. Cleaved is abbreviated as Clvd.

#### **3. Materials and Method**

#### *3.1. Chemistry*

All chemicals and solvents were procured from commercial sources (reagent grade) and were used without further purification. The reaction progress was monitored by thin layer chromatography (TLC) using precoated TLC plates of silica gel 60 F254. 1H-NMR and 13C-NMR spectra were recorded using a 400 MHz Bruker Avance Ultrashield spectrometer. The spectra were obtained in ppm using automatic calibration to the residual proton peak of the solvent, dimethyl-sulphoxide (DMSO-d6). The 1H NMR data are presented as follows: Chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constants (Hz), and integration. The 13C NMR analyses were reported in terms of the chemical shift. The 1H and 13C NMR spectra for all compounds are included in the supporting information (Figure S1). HRMS data were acquired using a Thermo QExactive Plus mass spectrometer equipped with an electrospray ionization source (Thermo Fisher Scientific, Greensboro, NC, USA).

3.1.1. Synthesis of methyl 2-((5-Hydroxy-4-oxo-2-phenyl-4*H*-chromen-7-yl)oxy)acetate (**2**)

The title compound was synthesized as previously described [62].

3.1.2. Synthesis of 2-((5-Hydroxy-4-oxo-2-phenyl-4*H*-chromen-7-yl)oxy)acetohydrazide (**3**)

The title compound was synthesized according to our previously published procedure [29].

3.1.3. General Procedure for the Synthesis of *N*'-arylidene-2-((5-Hydroxy-4-oxo-2-phenyl-4*H*-chromen-7-yl)oxy)acetohydrazide (**4a**–**4o**)

To a stirred suspension of hydrazide **3** (1.0 g, 3.0 mmol) in anhydrous methanol (60 mL), the appropriate aldehyde (3.0 mmol) was added, along with a few drops of concentrated hydrochloric acid. After one hour, the reaction was completed and the formed precipitate was separated by filtration, washed with methanol, and dried in air to give pure compounds in good yields.

#### (*E,Z*)-2-((5-Hydroxy-4-oxo-2-phenyl-4*H*-chromen-7-yl)oxy)-*N*'-(2-hydroxybenzylidene)acetohydrazide (**4a**)

The product was obtained as a pale-yellow powder. Yield (64%). 1H NMR (DMSO-*d*6) (*E:Z* = 1:1): δ 12.84–12.81 (m, 2H), 11.84 (s, 1H), 11.62 (s, 1H), 10.99 (s, 1H), 10.05 (s, 1H), 8.57 (s, 1H), 8.33 (s, 1H), 8.12–8.10 (d, *J* = 8 Hz, 4H), 7.77–7.55 (m, 8H), 7.31–7.24 (m, 2H), 7.07–6.85 (m, 8H), 6.51–6.42 (m, 2H), 5.31 (s, 2H), 4.87 (s, 2H). 13C NMR (DMSO-*d*6): δ 182.11, 182.06, 167.94, 164.42, 163.65, 163.61, 163.48, 163.37, 161.17, 161.07, 157.36, 157.22, 156.45, 148.27, 141.63, 132.18, 132.12, 131.59, 131.29, 130.57, 130.54, 129.18, 129.15, 129.11, 126.56, 126.47, 120.01, 119.40, 118.65, 116.39, 116.14, 105.44, 105.39, 105.31, 105.09, 98.81, 98.72, 93.62, 93.46, 66.57, 65.36. HRMS (ESI, *m*/*z*): calculated for C24H19N2O6 [M + H]<sup>+</sup> 431.1237; found 431.1238.

### (*E,Z*)-*N'*-Benzylidene-2-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetohydrazide (**4b**)

The synthesis and full characterization of the title compound have been previously reported [29].

(*E,Z*)-2-((5-Hydroxy-4-oxo-2-phenyl-4*H*-chromen-7-yl)oxy)-*N*'-(2-methoxybenzylidene)acetohydrazide (**4c**)

The product was obtained as a yellow powder. Yield (55%). 1H NMR (DMSO-*d*6) (*E:Z* = 1:0.5): δ 12.84–12.80 (m, 1.5H), 11.64 (s, 1.5H), 8.68 (s, 0.5H), 8.37 (s, 1H), 8.11–8.09 (d, *J* = 8 Hz, 3H), 7.91–7.89 (d, *J* = 8 Hz, 1H), 7.82–7.80 (d, *J* = 8 Hz, 0.5H), 7.63–7.55 (m, 4.5H), 7.44–7.4 (m, 1.5H), 7.12–7.0 (m, 4.5H), 6.88–6.84 (m, 1.5H), 6.5–6.42 (m, 1.5H), 5.32 (s, 2H), 4.82 (s, 1H), 3.86 (s, 4.5H). 13C NMR (DMSO-*d*6): δ182.09, 182.03, 168.09, 164.43, 163.71, 163.60, 163.48, 163.22, 161.13, 161.05, 157.80, 157.64, 157.21, 143.56, 139.73, 132.15, 132.10, 131.72, 131.48, 130.55, 129.09, 126.45, 125.67, 125.53, 121.94, 120.72, 120.66, 111.84, 111.77, 105.42, 105.31, 105.06, 98.77, 98.70, 93.62, 93.45, 66.65, 65.36, 55.68, 54.87. HRMS (ESI, *m*/*z*): calculated for C25H21N2O6 [M + H]<sup>+</sup> 445.1394; found 445.1392.

(*E,Z*)-*N'*-(2-Ethoxybenzylidene)-2-((5-hydroxy-4-oxo-2-phenyl-4*H*-chromen-7-yl)oxy)acetohydrazide (**4d**)

The product was obtained as a light-brown powder. Yield (80%). 1H NMR (DMSO-d6) (*E:Z* = 1:0.5): δ 12.84–12.80 (m, 1.5H), 11.68–11.62 (m, 1.5H), 8.66 (s, 0.5H), 8.40 (s, 1H), 8.11–8.10 (d, *J* = 4 Hz, 3H), 7.90–7.80 (m, 1.5H), 7.60–7.57 (m, 4.5H), 7.41–7.37 (m, 1.5H), 7.10–6.84 (m, 6H), 6.5–6.42 (m, 1.5H), 5.31 (s, 2H), 4.82 (s, 1H), 4.14–4.09 (q, *J* = 8 Hz, 3H), 1.39–1.35 (t, *J* = 8 Hz, 4.5H).13C NMR (DMSO-*d*6): δ 182.08, 182.03, 168.03, 164.42, 163.77, 163.57, 163.46, 163.23, 161.14, 161.04, 157.20, 157.13, 156.98, 143.46, 140.01, 132.10, 131.67, 131.42, 130.54, 129.09, 126.45, 125.76, 125.58, 122.14, 122.09, 120.61, 112.75, 105.41, 105.29, 105.05, 98.71, 93.63, 93.45, 66.62, 65.36, 63.80,14.63. HRMS (ESI, *m*/*z*): calculated for C26H23N2O6 [M + H]<sup>+</sup> 459.1550; found 459.1548.

(*E,Z*)-2-((5-Hydroxy-4-oxo-2-phenyl-4*H*-chromen-7-yl)oxy)-*N*'-(4-hydroxybenzylidene)acetohydrazide (**3e**)

The synthesis and full characterization of the title compound were previously published [29].

(*E,Z*)-2-((5-Hydroxy-4-oxo-2-phenyl-4*H*-chromen-7-yl)oxy)-*N*'-(4-methoxybenzylidene)acetohydrazide (**3f**)

The synthesis and full characterization of the title compound were previously published [29].

(*E,Z*)-*N'*-(2,4-Dihydroxybenzylidene)-2-((5-hydroxy-4-oxo-2-phenyl-4*H*-chromen-7 yl)oxy)acetohydrazide (**4g**)

The product was obtained as an off-white powder. Yield (85%). 1H NMR (DMSO-d6) (*E:Z* = 1:0.8): δ 12.84–12.80 (m, 1.8H), 11.67 (s, 1H), 11.43 (s, 0.8H), 11.15 (s, 1H), 9.98 (s, 1.8H), 9.83 (s, 0.8H), 8.42 (s, 1H), 8.20 (s, 0.8H), 8.12–8.10 (d, *J* = 8 Hz, 3.6H), 7.63–7.53 (m, 6.2H), 7.33–7.31 (d, *J* = 8 Hz, 1H), 7.08–7.06 (d, *J* = 8 Hz, 1.8H), 6.89 (d, *J* = 2 Hz, 1H), 6.8 (d, *J* = 2.4 Hz, 0.8H), 6.5 (d, *J* = 2 Hz, 1H), 6.4 (d, *J* = 2 Hz, 0.8H), 6.36–6.30 (m, 3.6H), 5.26 (s, 1.6H), 4.83 (s, 2H). 13C NMR (DMSO-*d*6): δ182.10, 182.05, 167.42, 164.44, 163.68, 163.64, 163.50, 162.86, 161.14, 161.04, 160.86, 160.53, 159.35, 158.07, 157.23, 149.28, 142.68, 132.18, 132.12, 131.10, 130.55, 129.15, 129.12, 128.27, 126.47, 111.49, 110.37, 107.84, 107.77, 105.45, 105.37, 105.32, 105.07, 102.60, 102.37, 98.81, 98.72, 93.63, 93.45, 66.59, 65.33. HRMS (ESI, *m*/*z*): calculated for C24H19N2O7 [M + H]<sup>+</sup> 447.1186; found 447.1187.

(*E,Z*)-*N'*-(2,4-Dimethoxybenzylidene)-2-((5-hydroxy-4-oxo-2-phenyl-4*H*-chromen-7 yl)oxy)acetohydrazide (**4h**)

The product was obtained as a yellow powder. Yield (96%). 1H NMR (DMSO-*d*6) (*E:Z* = 1:0.5): δ 12.81 (s, 1.5H), 11.49 (s, 1.5H), 8.58 (s, 0.5H), 8.27 (s, 1H), 8.11–8.10 (m, 3H), 7.84–7.57 (m, 6H), 7.05 (s, 1.5H), 6.88–6.84 (m, 1.5H), 6.63–6.41 (m, 4.5H), 5.29 (s, 2H), 4.79 (s, 1H), 3.86–3.82 (m, 9H). 13C NMR (DMSO-*d*6): δ182.04, 167.79, 164.43, 163.69, 163.55, 163.44, 162.91, 162.55, 162.33, 161.09, 161.02, 159.19, 159.02, 157.18, 143.62, 139.82, 132.10, 130.53, 129.09, 126.78, 126.43, 114.76, 106.41, 105.38, 105.28, 105.02, 98.68, 98.24, 98.07, 93.59, 93.42, 66.66, 65.35, 55.74, 55.40.HRMS (ESI, *m*/*z*): calculated for C26H23N2O7 [M + H]<sup>+</sup> 475.1499; found 475.1499.

(*E,Z*)-2-((5-Hydroxy-4-oxo-2-phenyl-4*H*-chromen-7-yl)oxy)-*N*'-(2,3,4 trihydroxybenzylidene)acetohydrazide (**4i**)

The product was obtained as a yellow powder. Yield (85%). 1H NMR (DMSO-*d*6) (*E:Z* = 1:0.5): δ 12.84–12.80 (m, 1.5H), 11.71 (s, 1H), 11.47 (s, 0.5H), 11.18 (s, 1H), 9.57–9.51 (m, 1.5H), 9.37 (s, 0.5H), 8.54–8.50 (m, 1.5H), 8.38 (s, 1H), 8.18–8.10 (m, 3.5H), 7.61–7.59 (m, 4.5H), 7.07–7.0 (m, 2H), 6.90–6.78 (m, 2.5H), 6.51–6.38 (m, 3H), 5.26 (s, 1H), 4.85 (s, 2H). 13C NMR (DMSO-*d*6): δ 182.12, 182.07, 167.34, 164.42, 163.68, 163.54, 162.93, 161.16, 161.07, 157.25, 150.45, 148.91, 148.42, 147.47, 146.65, 144.18, 132.76, 132.71, 132.21, 132.16, 130.56, 129.18, 126.49, 121.11, 118.36, 112.11, 110.70, 107.84, 107.73, 105.46, 105.40, 105.34, 105.10, 98.83, 98.75, 93.65, 93.48, 66.59, 65.32. HRMS (ESI, *m*/*z*): calculated for C24H19N2O8 [M + H]<sup>+</sup> 463.1134; found 463.1133.

(*E,Z*)-2-((5-Hydroxy-4-oxo-2-phenyl-4*H*-chromen-7-yl)oxy)-*N*'-(2,4,6 trihydroxybenzylidene)acetohydrazide (**4j**)

The product was obtained as a brown powder. Yield (76%). 1H NMR (DMSO-*d*6) (*E:Z* = 1:0.2): δ 12.83–12.79 (m, 1.2H), 11.65 (s, 1H), 11.48 (s, 0.2H), 10.99 (s, 2H), 10.32 (s, 0.4H), 9.85 (s, 1.2H), 8.72 (s, 1H), 8.41 (s, 0.2H), 8.09–8.08 (m, 2.4H), 7.59–7.58 (m, 3.6H), 7.04 (m, 1.2H), 6.88–6.84 (m, 1.2H), 6.5–6.42 (m, 1.2H), 5.86–5.83 (m, 2.4H), 5.2 (s, 0.4H), 4.82 (s, 2H). 13C NMR (DMSO-d6): δ 182.08, 166.56, 164.28, 163.61, 163.51, 162.55, 161.74, 161.46, 161.12, 161.04, 159.73, 159.24, 157.20, 147.32, 144.33, 132.15, 130.54, 129.13, 126.45, 105.43, 105.37, 105.11, 98.85, 98.79, 94.37, 93.61, 93.50, 66.58, 65.26. HRMS (ESI, *m*/*z*): calculated for C24H19N2O8 [M + H]<sup>+</sup> 463.1134; found 463.1134.

(*E,Z*)-*N'*-(Furan-2-ylmethylene)-2-((5-hydroxy-4-oxo-2-phenyl-4*H*-chromen-7-yl)oxy)acetohydrazide(**4k**)

The product was obtained as a yellow powder. Yield (87%). 1H NMR (DMSO-*d*6) (*E:Z* = 1:0.5): δ 12.83–12.79 (m, 1.5H), 11.63–11.57 (m, 1.5H), 8.23 (s, 0.5H), 8.11–8.09 (d, *J* = 8 Hz, 3H), 7.92 (s, 1H), 7.85 (s, 1.5H), 7.63–7.55 (m, 4.5H), 7.06–7.05 (m, 1.5H), 6.95–6.83 (m, 3H), 6.64 (m, 1.5H), 6.49–6.39 (m, 1.5H), 5.24 (s, 2H), 4.83 (s, 1H). 13C NMR (DMSO-d6): δ182.03, 167.97, 164.34, 163.66, 163.59, 163.46, 163.35, 161.13, 161.02, 157.21, 149.12, 148.94, 145.34, 145.10, 137.86, 134.20, 132.11, 130.54, 129.09, 126.45, 113.95, 113.78, 112.16, 105.42, 105.29, 105.08, 98.70, 93.60, 93.38, 66.65, 65.14.HRMS (ESI, *m*/*z*): calculated for C22H17N2O6 [M + H]<sup>+</sup> 405.1081; found 405.1077.

(*E,Z*)-2-((5-Hydroxy-4-oxo-2-phenyl-4*H*-chromen-7-yl)oxy)-*N*'-(pyridin-3-ylmethylene)acetohydrazide (**4l**)

The product was obtained as a yellow powder. Yield (86%). 1H NMR (DMSO-d6) (*E:Z* = 1:0.5): δ 12.82(bs, 1.5H), 11.98 (bs, 1.5H), 8.43 (s, 0.5H), 8.30–8.28 (d, *J* = 8 Hz, 3H), 8.13–7.96 (m, 7H), 7.62–7.55 (m, 4.5H), 7.07–7.06 (m, 1.5H), 6.88 (s, 1.5H), 6.5–6.45 (m, 1.5H), 5.39 (s, 2H), 4.9 (s, 1H). 13C NMR (DMSO-d6): δ 182.12, 168.50, 164.42, 163.67, 163.55, 161.08, 157.27, 150.51, 148.52, 145.40, 141.22, 133.75, 132.20, 130.59, 129.96, 129.16, 126.51, 123.94, 105.36, 105.12, 98.76, 93.55, 66.61, 65.40. HRMS (ESI, *m*/*z*): calculated for C23H18N3O5 [M + H]<sup>+</sup> 416.1241; found 416.1240.

(*E,Z*)-2-((5-Hydroxy-4-oxo-2-phenyl-4*H*-chromen-7-yl)oxy)-*N*'-(pyridin-2 ylmethylene)acetohydrazide (**4m**)

The product was obtained as a brown powder. Yield (52%). 1H NMR (DMSO-*d*6) (*E:Z* = 1:0.5): δ 12.84–12.81 (m, 1.5H), 11.89–11.87 (m, 1.5H), 8.61 (m, 1.5H), 8.35 (s, 0.5H), 8.11–8.03 (m, 5H), 7.94–7.87 (m, 2H), 7.62–7.55 (m, 4.5H), 7.44–7.41 (m, 1.5H), 7.07–7.06 (m, 1.5H), 6.88 (m, 1.5H), 6.51–6.45 (m, 1.5H), 5.37 (s, 2H), 4.88 (s, 1H). 13C NMR (DMSO-*d*6): δ 182.02, 168.45, 164.34, 163.75, 163.65, 163.59, 163.48, 161.13, 161.05, 157.21, 152.93, 152.78, 149.51, 149.44, 148.26, 144.45, 136.86, 136.75, 132.09, 130.53, 129.07, 126.44, 124.52, 124.34, 119.98, 119.90, 105.42, 105.30, 105.09, 98.70, 93.63, 93.48, 66.61, 65.30. HRMS (ESI, *m*/*z*): calculated for C23H18N3O5 [M + H]<sup>+</sup> 416.1241; found 416.1238.

(*E,Z*)-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)-*N*'-((2-hydroxynaphthalen-1 yl)methylene)acetohydrazide (**4n**)

The product was obtained as a yellow powder. Yield (54%). 1H NMR (DMSO-*d*6) (*E:Z* = 1:0.5): δ 12.86–12.81 (m, 1.5H), 12.46 (s, 1H), 11.91 (s, 1H), 11.68 (s, 0.5H), 10.77 (s, 0.5H), 9.40 (s, 1H), 8.89 (s, 0.5H), 8.77–8.75 (d, *J* = 8 Hz, 0.5H), 8.30–8.28 (d, *J* = 8 Hz, 1H), 8.12–8.09 (m, 3H), 7.94–7.84 (m, 3H), 7.63–7.58 (m, 6H), 7.42–7.36 (m, 1.5H), 7.24–7.21 (m, 1.5H), 7.08–7.06 (m, 1.5H), 6.94–6.88 (m, 1.5H), 6.55–6.46 (m, 1.5H), 5.38 (s, 1H), 4.94 (s, 2H). 13C NMR (DMSO-*d*6): δ182.09, 182.03, 167.52, 164.38, 163.64, 163.54, 163.49, 163.17, 161.17, 161.06, 157.95, 157.23, 156.88, 147.37, 143.29, 132.91, 132.52, 132.16, 132.10, 131.59, 131.25, 130.53, 129.13, 128.89, 128.68, 128.13, 127.80, 127.75, 126.46, 123.54, 123.40, 120.97, 118.75, 118.13, 110.08, 108.45, 105.45, 105.33, 105.10, 98.87, 98.73, 93.68, 93.46, 66.72, 65.59.HRMS (ESI, *m*/*z*): calculated for C28H21N2O6 [M + H]<sup>+</sup> 481.1394; found 481.1394.

(*E,Z*)-2-((5-Hydroxy-4-oxo-2-phenyl-4*H*-chromen-7-yl)oxy)-*N*'-((2-hydroxy-5 nitrobenzylidene)acetohydrazide (**4o**)

The product was obtained as a yellow powder. Yield (91%). 1H NMR (DMSO-*d*6) (*E:Z* = 1:0.75): δ 12.85–12.80 (m, 1.75H), 11.97 (bs, 2H), 11.78 (s, 1.5H), 8.64–8.54 (m, 2.75H), 8.33 (s, 1H), 8.15–8.10 (m, 5.25H), 7.64–7.55 (m, 5.25H), 7.08–7.04 (m, 3.25H), 6.90–6.86 (m, 1.75H), 6.52–6.44 (m, 1.75H), 5.39 (s, 2H), 4.89 (s, 1.5H). 13C NMR (DMSO-*d*6): δ 182.08, 182.03, 168.28, 164.42, 163.71, 163.61, 163.46, 162.04, 161.14, 161.04, 157.20, 144.30, 139.91, 138.65, 132.16, 132.10, 130.55, 129.12, 129.08, 126.73, 126.45, 123.37, 121.68, 120.98, 119.95, 117.10, 116.72, 105.43, 105.37, 105.30, 105.08, 98.78, 98.73, 93.62, 93.46, 66.53, 65.42. HRMS (ESI, *m*/*z*): calculated for C24H18N3O8 [M + H]<sup>+</sup> 476.1088; found 476.1089.

#### *3.2. Biological Studies*

#### 3.2.1. Cell Lines and Cell Culture

A panel of cancer cell lines, including breast (MDA-MB-231, BT20), brain (U251), and colon (HCT116), as well as a normal cell line (human mammary epithelial cells: HMECs), were grown as adherent monolayers in flasks with Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin in a humidified incubator with 5% CO2 at 37 ◦C.

#### 3.2.2. MTT Assay

The (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay was used to determine the cytotoxicity of the 15 chrysin derivatives in the above-mentioned cell lines. Briefly, cells were harvested with 0.05% trypsin, 2.21 mM ethylenediaminetetraacetic acid (EDTA), 1× from Corning (Corning, NY, USA), and suspended at a final density of 5 <sup>×</sup> 103 cells/well. Cells were seeded (180 μL/well) into 96-well plates. Initially, four different concentrations of each compound were added to find the compounds with the greatest antiproliferative efficacy in the MDA-MB-231 cell line (0, 1, 10, and 100 μM). Subsequently, eight different concentrations (0.1, 0.3, 1, 3, 10, 30, and 100 μM) of each compound were added to the remaining cell lines mentioned above. After 68 h of incubation, 20 μL of the MTT solution (4 mg/mL) were added to each well, and the plates were incubated for 4 h. This allowed viable cells to biotransform the yellow-colored MTT into dark-blue formazan crystals. Subsequently, the medium was discarded, and 150 μL of DMSO were added to each well to dissolve the formazan crystals. The absorbance was determined at 590 nm using a DTX 880 multimode detector (Beckman Coulter life sciences, IN, USA). The IC50 ± SD concentrations were calculated from three experiments performed in triplicate/duplicate. The IC50 values were calculated from the cell survival percentages obtained for each compound tested at different concentrations. Similarly, the cytotoxicity of the test compounds was compared to the normal cell line (HMEC).

#### 3.2.3. Cell Cycle, Apoptosis, and Mitochondrial Membrane Potential Analysis

MDA-MB-231 cells were plated into 6-well plates at 2.5 <sup>×</sup> 105 cells/well. The cells were incubated with 0, 5, or 10 μM of compounds **4g** or **4i** and incubated for 12 h. Next, the cells were trypsinized with 0.05% trypsin, 2.21 mM EDTA, 1×, washed, counted, and resuspended in 1 mL of ice-cold PBS. The cells then were stained with propidium iodide (PI) dye and incubated for at least 15 min. The distribution of the cells in each cell cycle phase for the different concentrations was measured using a BD Accuri™ C6 flow cytometer from BD Biosciences (Becton-Dickinson, San Jose, CA, USA) and analyzed using FCS Express 5 plus De Novo software (Glendale, CA, USA).

MitoTracker Red and Alexa Fluor 488 annexin V kits for flow cytometry (Molecular Probes Inc., Invitrogen, Eugene, OR) were used to measure the mitochondrial membrane potential and apoptosis in MDA-MB-231 cells. Briefly, cells were seeded into 6-well plates and incubated with 0, 5, or 10 μM of compounds **4g** and **4i** for 12 h. The cells were then lysed using 0.05% trypsin, 2.21 mM EDTA, 1×, counted, and 4 μL of 10 μM of the MitoTracker Red working solution were added to 1 mL of the harvested cells. The cells were incubated at 37 ◦C with 5% CO2 for 30 min. The cells were washed once with PBS and resuspended in 100 μL of the annexin binding buffer. The cell suspensions were incubated with 5 μL of Alexa Fluor 488 annexin V for 15 min. This was followed by the addition of 400 μL of the annexin-binding buffer. Finally, flow cytometry was used to detect the fluorescence of stained cells at the following excitation/emission maxima: Alexa Fluor® 488 annexin V: 499/521 nm; MitoTracker® Red: 579/599 nm with the BD Accuri™ C6 flow cytometer from BD Biosciences (Becton-Dickinson, San Jose, CA, USA) and analyzed using FCS express 5 plus De Novo software (Glendale, CA, USA).

#### 3.2.4. Protein Expression Analysis Using Western Blot

To measure the expression of Bak, cytochrome c, caspase-7, caspase-9, and mTOR, Western blotting was performed by lysing MDA-MB-231 cells using a lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1% Triton, 0.1% SDS) containing a protease inhibitor cocktail that consisted of Aprotinin, Bestatin, E-64, Leupeptin, and Pepstatin A (Sigma-Aldrich Life Science, St. Louis, MO, USA). The bicinchoninic acid (BCA) quantification assay was used to determine the protein levels in the cell extracts (G-BIOSCIENCES, St. Louis, MO, USA). The extracted proteins were loaded onto a 10–20% tris-glycine gel. After separation, the proteins were transferred from the gel onto a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked using 5% milk in Tris-buffered saline Tween 20 for 30 min and incubated overnight with primary antibodies against Bak (1:1000), cytochrome C (1:1000), caspase-3 (1:1000), caspase 7 (1:1000), caspase 9 (1:1000), mTOR (1:1000), or B-actin (1:2000) in 5% BSA (bovine serum albumin) at 4 ◦C. The next day, membranes were washed and incubated with horseradish peroxidase-labelled (HRP) anti-rabbit secondary antibody (1:5000 dilutions). The membrane was incubated with the antibody for an additional 1 h. Subsequently, the membranes were washed and developed by Clarity Western ECL substrate (Bio-Rad; Hercules, CA, USA). Protein was detected using a ChemiDoc Imaging System (Bio-Rad). Densitometry analyses of the blots for the detected protein were quantified using the ImageJ software. Data was calculated as ratios of protein/β-actin.

#### **4. Conclusions**

In conclusion, a series of novel chrysin derivatives were designed, synthesized, and characterized. Upon screening these compounds for their antiproliferative efficacy in the TNBC cell line, MDA-MB-231, and normal breast HMEC cells, two compounds, **4g** and **4i**, had the highest efficacy and selectivity towards MDA-MB-231 cells. Upon investigating the mechanism by which these compounds produce cytotoxicity, it was determined that **4g** and **4i** cause the death of MDA-MB-231 cells by inducing apoptosis, producing cell cycle arrest at the G2 phase, and activating the intrinsic apoptotic pathway. Physicochemical characterizations of these compounds suggested that they can be further optimized as potential anticancer compounds for TNBC cells. The compounds were determined to have some solubility issues that need to be overcome in the future design of additional compounds. Overall, our results suggest that **4g** and **4i** could be suitable leads for developing novel compounds to treat TNBC.

**Supplementary Materials:** The following are available online, Figure S1: Chemical characterization of compounds **4a**–**4o**.

**Author Contributions:** Conceptualization, B.A.A.-O., S.A.A. and A.K.T.; methodology, B.A.A.-O., S.A.A., H.R., S.M., N.H. and J.M.L.; software, S.M. and N.H.; validation, S.M. and N.H.; formal analysis, B.A.A.-O.; investigation, H.R., S.M., N.H., J.M.L.; resources, B.A.A.-O. and A.K.T.; data curation, B.A.A.-O., H.R., S.M., N.H. and J.M.L.; writing—original draft preparation, B.A.A. and S.M.; writing—review and editing, B.A.A.-O., S.A.A., S.M., S.K., M.F.B., C.R.A.J. and A.K.T.; visualization, A.K.T.; supervision, A.K.T.; project administration, A.K.T.; funding acquisition, B.A.A.-O., and A.K.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the Deanship of Scientific Research at Jordan University of Science and Technology (grant # 20190360 to BAA). This manuscript has been supported, in part, by University of Toledo startup grants (F110760 to AKT) and Susan G. Komen Breast Cancer Foundation (CCR18548498 to AKT).

**Acknowledgments:** The authors acknowledge the financial support given by the Deanship of Research at the Jordan University of Science and Technology (grant # 20190360 to BAA). The high-resolution mass spectrometry data were acquired from the Triad Mass Spectrometry Laboratory at the University of North Carolina at Greensboro, Greensboro, NC, USA. This manuscript has been supported, in part, by the University of Toledo startup grants (F110760 to AKT) and Susan G. Komen Breast Cancer Foundation (CCR18548498 to AKT).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Sample Availability:** Samples of the compounds **4a**, **4g**, **4i** and **4o** are available from the authors on request.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Design and Synthesis of Flavonoidal Ethers and Their Anti-Cancer Activity In Vitro**

**Lu Jin 1,2, Meng-Ling Wang 1, Yao Lv 3, Xue-Yi Zeng 1,2, Chao Chen 1,2, Hai Ren 1,2, Heng Luo 1,2,\* and Wei-Dong Pan 1,2,\***


Academic Editor: Qiao-Hong Chen Received: 30 March 2019; Accepted: 3 May 2019; Published: 6 May 2019

**Abstract:** Flavonoids are well-characterized polyphenolic compounds with pharmacological and therapeutic activities. However, most flavonoids have not been developed into clinical drugs, due to poor bioavailability. Herein, we report a strategy to increase the drugability of flavonoids by constructing C(sp2)-O bonds and stereo- as well as regioselective alkenylation of hydroxyl groups of flavonoids with ethyl-2,3-butadienoate allenes. Twenty-three modified flavonoid derivatives were designed, synthesized, and evaluated for their anti-cancer activities. The results showed that compounds **4b**, **4c**, **4e**, **5e**, and **6b** exhibited better in vitro inhibitory activity against several cancer cell lines than their precursors. Preliminary structure–activity relationship studies indicated that, in most of the cancer cell lines evaluated, the substitution on position 7 was essential for increasing cytotoxicity. The results of this study might facilitate the preparation or late-stage modification of complex flavonoids as anti-cancer drug candidates.

**Keywords:** flavonoids; allene; E-stereoselective; regioselective; anti-cancer activity

#### **1. Introduction**

Consisting of more than 9000 compounds, flavonoids represent the most widely distributed polyphenols in nature [1,2]. In addition, flavonoids are ubiquitous in some medicinal plants and herbal remedies used in traditional medicine around the world, especially in China [2–6]. The characteristic skeleton of flavonoids is a phenylbenzopyrone moiety (C6-C3-C6), which could be further categorized according to the saturation level and the presence or absence of the central pyran ring. Many studies have confirmed that flavonoids are naturally occurring, pharmacologically active molecules. However, most flavonoids have not been developed as clinical drugs because of poor bioavailability (less than 5%), toxicity, and induction or inhibition of some metabolic enzymes [7]. To overcome these problems, structural modifications of the flavonoid skeletons have attracted great interest [8].

Allenes are highly reactive and have been used extensively in organic chemistry [9–15], due to the electronic or steric effects and their 1,2-diene functionality. The transition-metal catalyzed chemistry of allenes has become a recent focus of research [16,17]. We proposed that carbopalladation of allenes would provide a highly convenient method for the formation of a conjugated aryl ether, following β-H elimination by the introduction of a π-allyl palladium species.

In this research, we have developed a friendly and straightforward protocol to access various novel flavonoidal ethers by employing allenes as important multifunctional modules. Notably, both electrophilic groups and aliphatic chains can be embedded in the flavonoid scaffold smoothly with the above mentioned protocol. Therefore, more than 20 flavonoidal ethers were obtained successfully and evaluated for anti-cancer activities.

#### **2. Materials and Methods**

#### *2.1. Instruments and Materials*

1H- and 13C-NMR spectra were obtained on a 400 MHz (Varian, Inc) or 500 MHz (WIPM, China) spectrometer in CDCl3 or DMSO-*d6* (tetramethylsilane (TMS) as internal standard). Chemical shifts (δ) were expressed in parts per million (ppm), relative to TMS (0 ppm). High resolution mass spectra (HRMS) were recorded on a ThermoFisher QE Focus apparatus. All the solvents were dried using standard methods and distilled before being used. Reagents and solvents were purchased from commercial sources. Solvents were purified according to the guidelines in the Purification of Laboratory Chemicals.

#### *2.2. General Procedure for the Synthesis of Flavonoidal Ether Derivatives*

#### 2.2.1. Synthesis of Allenes

Allenes were synthesized according to previously described protocols [16–18].

#### 2.2.2. Optimization of Reaction Conditions

We selected chrysin **2a** and ethyl ester allene **1a** as the model substrates in this study and carried out extensive screening of the reaction conditions. In an initial attempt, a variety of factors including types of Pd catalysts, bases, solvents, and ligands were examined. After extensive optimization, the product (**3a**) was obtained with an isolated yield of 41.7% in the presence of 10 mol% of PPh3/Pd(dba)2 as a catalyst, K2CO3 (2.0 equiv.) as a base, in a solution of MeCN under argon at 80 ◦C (Table 1, entry 1). Further investigations of the solvent indicated that the yield could be slightly improved to 43.0% by replacing MeCN with DMF (Table 1, entry 6). Furthermore, absence of Pd catalysts and ligands resulted in lower yield (Table 1, entry 14). None of the desired product was obtained when the base was abstracted away (Table 1, entries 15 and 16). Interestingly, only 11.2% yield of the expected product was obtained at lower temperatures (Table 1, entry 17). It was worth noting that **3a** was the only isomer isolated, indicating a highly regio- and stereoselective process, in which the steric effect of the carbonyl or the hydrogen bond effect between the carbonyl and oxygen atom at position 7 may be critical for the highly regio- and stereoselective process.

#### **Table 1.** Optimization of reaction conditions.


Conditions: **1a** (0.4 mmol), **2a** (0.2 mmol), isolated yield. [a] *<sup>N</sup>*,*N*-Dimethylaniline; [b] *<sup>N</sup>*,*N*-Dimethylacetamide; [c] Reaction occurred at rt.

#### 2.2.3. General Procedure for the Synthesis of Target Derivatives **3a**–**3i**, **4a**–**4g**, **5a**–**5e**

Chrysin **2a** (127.12 mg, 0.5 mmol), Pd(dba)2 (5.75 mg, 0.01 mmol), K2CO3 (138.21 mg, 1.0 mmol), PPh3 (13.11 mg, 0.05 mmol), allene (112.13 mg, 1.0 mmol), and anhydrous DMF (5 mL) were added to a 30 mL tube under argon, which was then sealed. The resulting mixture was stirred at 80 ◦C for 12 h. After completion, the reaction solution was cooled down to room temperature before 80 mL water was added, followed by the addition of 1 N HCl aqueous solution at 0 ◦C, until the pH value reached 7. Ethyl acetate (100 mL) was added to the reaction solution and the organic layer was washed sequentially with a large amount of water (4 × 200 mL), and then brine, before being dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether–ethylacetate, 5:1) to give **3a** (78.8 mg, 43% yield). Compounds **3b**–**3i, 4a**–**4g,** and **5a**–**5e** were prepared according to similar procedures in which the double-substitution is a byproduct. The products **4a**–**4g** were generated in a shorter time compared with the other products (within 10 h).

#### 2.2.4. General Synthetic Procedure for **6a**–**6b** Target Derivatives

The reaction with 1.0 mmol scale was carried out to assess the scalability of flavonoidal ether formation (Scheme 1, **4a**). Under standard conditions, the yield was lower than that of the small-scale reaction. The reaction solution contained the following components: **4a** (62.8 mg, 0.02 mmol), 1 mL methanol, and 1 N sodium hydroxide solution (2.0 equiv). The solution was then stirred at 60 ◦C for 5 min and 1 mL water was added before heating at 60 ◦C for 12 h. After completion, the reaction solution was cooled down to room temperature and 40 mL water as well as 30 mL ethyl acetate was added sequentially. Aqueous HCl (1 N) was added to the water layer at 0 ◦C until the pH value reached 4. Other post-processing procedures were similar to those performed to synthesize **4a**. The desired product **6a** was finally obtained (41.2 mg, 61% yield). The product **6b** was prepared according to the same general procedure.

**Scheme 1.** Synthesis of flavonoidal ether and derivatization.

*Ethyl (E)-3-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl) oxy) but-2-enoate* (**3a**); yellow powder; yield 43.0%; HRMS(ESI): calcd. C21H17O6, *m*/*z* 365.1031 [M <sup>−</sup> H]−, found 365.1025; 1H-NMR (CDCl3, 500 MHz) δ(ppm): 7.88 (d, *J* = 7.0 Hz, 2H), 7.59–7.51 (m, 3H), 6.73 (s, 1H), 6.68 (d, *J* = 2.0 Hz, 1H), 6.49 (d, *J* = 2.0 Hz, 1H), 5.16 (s, 1H), 4.12 (q, *J* = 7.0 Hz, 2H), 2.49 (s, 3H), 1.23 (t, *J* = 7.0 Hz, 3H); 13C-NMR (CDCl3, 125 MHz) δ(ppm): 182.9, 170.5, 167.1, 164.7, 162.6, 159.4, 157.4, 132.3, 131.0, 129.3, 126.5, 108.6, 106.3, 104.8, 100.0, 99.8, 60.0, 18.2, 14.4.

*Ethyl (E)-3-((5,6-dihydroxy-4-oxo-2-phenyl-4H-chromen-7-yl) oxy) but-2-enoate* (**3b**); yellow powder; yield 43.9 %; HRMS(ESI): calcd. C21H17O7, *m*/*z* 381.0980 [M <sup>−</sup> H]−, found 381.0970; 1H-NMR (DMSO-*d*6, 400 MHz) δ(ppm): 13.02 (s, 1H), 8.04 (d, *J* = 7.2 Hz, 2H), 7.59–7.52 (m, 3H), 6.97 (s, 1H), 6.69 (s, 1H), 4.80 (s, 1H), 3.97 (q, *J* = 7.2 Hz, 2H), 2.40 (s, 3H), 1.09 (t, *J* = 7.2 Hz, 3H); 13C-NMR (DMSO-*d*6, 100 MHz) δ(ppm): 182.3, 170.7, 166.6, 163.7, 156.7, 154.2, 152.4, 132.2, 130.7, 129.2, 126.5, 123.8, 105.0, 104.4, 94.8, 93.3, 59.2, 17.6, 14.2.

*Diethyl 3,3 -((5-hydroxy-4-oxo-2-phenyl-4H-chromene-6,7-diyl) bis (oxy)) (2E,2 E)-bis (but-2-enoate)* (**3c**); yellow powder; yield 29.3%; HRMS(ESI): calcd. C27H27O9, *m*/*z* 495.1650 [M + H]+, found 495.1647; 1H-NMR (DMSO-*d*6, 500 MHz) δ(ppm): 11.76 (s, 1H), 8.04 (d, *J* = 6.0 Hz, 2H), 7.60–7.57 (m, 3H), 7.16 (s, 1H), 6.78 (s, 1 H), 4.77 (s, 1H), 4.63 (s, 1H), 4.01–3.94 (m, 4H), 2.44 (s, 3H), 2.39 (s, 3H), 1.16–1.03 (m, 6H); 13C-NMR (DMSO-*d*6, 125 MHz) δ(ppm): 174.7, 171.6, 170.4, 166.3, 166.2, 161.0, 155.4, 155.3, 143.0, 131.7, 131.3, 130.8, 129.1, 126.3, 109.8, 107.5, 102.7, 94.5, 93.9, 59.3, 59.1, 17.6, 17.3, 14.1.

*Ethyl (E)-3-(5-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2-methoxyphenoxy)-2-methylacrylate* (**3d**); yellow powder; yield 25.9%; HRMS(ESI): calcd. C22H19O8, *<sup>m</sup>*/*<sup>z</sup>* 411.1085 [M <sup>−</sup> H]−, found 411.1078; 1H-NMR (DMSO-*d6*, 500 MHz) δ(ppm): 12.87 (s, 1H), 10.88 (s, 1H), 8.03 (dd, *J* = 9.0, 2.5 Hz, 1H), 7.87 (d, *J* = 2.5 Hz, 1H), 7.33 (d, *J* = 9 Hz, 1H), 6.95 (s, 1H), 6.52 (d, *J* = 2.0 Hz, 1H), 6.20 (d, *J* = 2.0 Hz, 1H), 4.66 (s, 1H), 3.99 (q, *J* = 7.0 Hz, 2H), 3.88 (s, 3H), 2.43 (s, 3H), 1.10 (t, *J* = 7.0 Hz, 3H); 13C-NMR (DMSO-*d6*, 125 MHz) δ(ppm): 181.8, 171.6, 166.3, 164.3, 162.1, 161.4, 157.4, 153.9, 141.1, 126.0, 123.6, 121.1, 113.8, 104.3, 103.8, 99.0, 94.6, 94.2, 59.2, 56.3, 17.7, 14.1.

*Ethyl (E)-3-((2-(3-(((E)-4-ethoxy-4-oxobut-2-en-2-yl) oxy)-4-methoxyphenyl)-5-hydroxy-4-oxo-4H-chromen-7-yl) oxy) but-2-enoate* (**3e**); yellow powder; yield 21.2%; HRMS(ESI): calcd. C28H29O10, *m*/*z* 525.1755 [M + H]+, found 525.1759; 1H-NMR (DMSO-*d*6, 500 MHz) δ(ppm): 12.94 (s, 1H), 8.05 (dd, *J* = 8.5, 2.0 Hz, 1H), 7.86 (d, *J* = 2.0 Hz, 1H), 7.32 (d, *J* = 8.5 Hz, 1H), 7.05 (s, 1H), 6.95 (d, *J* = 2.0 Hz, 1H), 6.51 (d, *J* = 2.0 Hz, 1H), 5.05 (s, 1H), 4.66 (s, 1H), 4.05–3.95 (m, 4H), 3.90 (s, 3H), 2.42 (d, *J* = 3.5 Hz, 6H), 1.21–1.10 (m, 6H). 13C-NMR (DMSO-*d*6, 125 MHz) δ(ppm): 181.8, 171.6, 166.3, 164.3, 162.1, 161.4, 157.4, 153.9, 141.1, 126.0, 123.6, 121.1, 113.8, 104.3, 103.8, 99.0, 94.6, 94.2, 59.2, 56.3, 39.7, 39.5, 39.4, 17.7, 14.1.

*Ethyl (E)-3-((5-hydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-7-yl) oxy) but-2-enoate* (**3f**); yellow powder; yield 30.3%; HRMS(ESI): calcd. C21H17O7, *<sup>m</sup>*/*<sup>z</sup>* 381.0980 [M <sup>−</sup> H]−, found 381.0986; 1H-NMR (DMSO-*d*6, 400 MHz) δ(ppm): 13.10 (s, 1H), 10.45 (s, 1H), 7.97 (d, *J* = 8.0 Hz, 2H), 6.98–6.91 (m, 4H), 6.56 (s, 1H), 5.05 (s, 1H), 4.03 (q, *J* = 7.2 Hz, 2H), 2.42 (s, 3H), 1.14 (t, *J* = 7.2 Hz, 3H); 13C-NMR (DMSO-*d*6, 100 MHz) δ(ppm): 182.3, 170.5, 166.1, 164.7, 161.6, 161.5, 158.4, 156.8, 128.8, 120.8, 116.0, 107.9, 104.0, 103.4, 100.2, 98.7, 59.4, 17.7, 14.1.

*Ethyl (E)-3-(4-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl) phenoxy) but-2-enoate* (**3g**); yellow powder; yield 28.3%; HRMS(ESI): calcd. C21H17O7, *m*/*z* 381.0980 [M <sup>−</sup> H]−, found 381.0976; 1H-NMR (DMSO-*d6*, 400 MHz) δ(ppm): 12.82 (s, 1H), 10.96 (s, 1H), 8.17 (d, *J* = 8.8 Hz, 2H), 7.34 (d, *J* = 8.4 Hz, 2H), 6.99 (s, 1H), 6.52 (d, *J* = 2.0 Hz, 1H), 6.22 (d, *J* = 2.0 Hz, 1H), 4.79 (s, 1H), 4.01 (q, *J* = 7.2 Hz, 2H), 2.45 (s, 3H), 1.12 (t, *J* = 7.2 Hz, 3H); 13C-NMR (DMSO-*d6*, 100 MHz) δ(ppm): 181.9, 171.7, 166.2, 164.5, 162.4, 161.5, 157.5, 155.6, 128.8, 128.3, 122.2, 105.3, 103.9, 99.1, 96.9, 94.2, 59.3, 17.8, 14.2.

*Ethyl 2-(5-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2-methylbenzo [d] [1,3] dioxol-2-yl) acetate* (**3h**); yellow powder; yield 30.1%; HRMS(ESI): calcd. C21H17O8, *<sup>m</sup>*/*<sup>z</sup>* 397.0929 [M <sup>−</sup> H]−, found 397.0923; 1H-NMR (CDCl3, 400 MHz) δ(ppm): 12.62 (s, 1H), 7.43 (dd, *J* = 8.4, 1.6 Hz, 1H), 7.26 (d, *J* = 1.2 Hz, 1H), 6.88 (d, *J* = 8.4 Hz, 1H), 6.52 (s, 1H), 6.49 (d, *J* = 1.2 Hz, 1H), 6.38 (d, *J* = 1.6 Hz, 1H), 4.20 (q, *J* = 7.2 Hz, 2H), 3.06 (s, 2H), δ 1.88 (s, 3H), 1.25 (t, *J* = 7.2 Hz, 3H); 13C-NMR (CDCl3, 100 MHz) δ(ppm): 182.5, 168.3, 164.1, 164.0, 162.1, 157.9, 150.5, 148.1, 124.8, 121.6, 117.6, 108.9, 106.4, 104.9, 104.0, 100.0, 94.6, 61.4, 44.0, 25.0, 14.1.

*Ethyl 2-(2-methyl-5-(3,5,7-trihydroxy-4-oxo-4H-chromen-2-yl) benzo [d] [1,3] dioxol-2-yl) acetate* (**3i**); yellow powder; yield 30.1%; HRMS(ESI): calcd. C21H19O9, *m*/*z* 415.1024 [M + H]+, found 415.1018; 1H-NMR (DMSO-*d6*, 500 MHz) δ(ppm): 12.40 (s, 1H), 10.83 (s, 1H), 9.57 (s, 1H), 7.74 (dd, *J* = 8.0, 1.5 Hz, 1H), 7.62 (d, *J* = 1.5 Hz, 1H), 7.03 (d, *J* = 8.0 Hz, 1H), 6.47 (d, *J* = 2.0 Hz, 1H), 6.19 (d, *J* = 2.0 Hz, 1H), 4.02 (q, *J* = 7.5 Hz, 2H), 3.13 (s, 2H), 1.78 (s, 3H), 1.10 (t, *J* = 7.5 Hz, 3H); 13C-NMR (DMSO-*d6*, 125 MHz) δ(ppm): 176.0, 167.6, 164.1, 160.7, 156.2, 148.2, 147.1, 145.9, 136.3, 124.5, 122.6, 117.3, 108.3, 107.3, 103.1, 98.3, 93.6, 60.3, 43.1, 24.7, 13.9.

*Benzyl (E)-3-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl) oxy) but-2-enoate* (**4a**); yellow powder; yield 33.0%; HRMS(ESI): calcd. C26H21O6, *m*/*z* 429.1333 [M + H]+, found 429.1324; 1H-NMR (DMSO-*d6*, 400 MHz) δ(ppm): 12.93 (s, 1 H), 8.12 (d, *J* = 6.8 Hz, 2H), 7.74–7.56 (m, 3H), 7.32 (m, 5H), 7.07 (d, *J* = 2.0 Hz, 1H), 6.63 (d, *J* = 2.0 Hz, 1H), 5.10 (s, 1H), 5.07 (s, 2H), 2.46 (s, 3H); 13C-NMR (DMSO-*d6*, 100 MHz) δ(ppm): 182.7, 171.3, 166.1, 164.2, 161.6, 158.7, 157.1, 136.3, 132.5, 130.4, 129.3, 128.5, 128.3, 128.1, 126.8, 108.3, 105.8, 104.4, 100.7, 98.3, 65.3, 17.9.

*Dibenzyl 3,3 -((4-oxo-2-phenyl-4H-chromene-5,7-diyl) bis (oxy)) (2E,2 E)-bis (but-2-enoate)* (**4b**); white powder; yield 29.8%; HRMS(ESI): calcd. C37H31O8, *m*/*z* 603.2013 [M + H]+, found 603.2010; 1H-NMR (CDCl3, 400 MHz) δ(ppm): 7.85 (d, *J* = 6.8 Hz, 2H), 7.52 (d, *J* = 7.6 Hz, 3H), 7.33 (m, *J* = 10H), 7.12 (d, *J* = 2.0 Hz, 1H), 6.71 (d, *J* = 2.0 Hz, 1H), 6.65 (s, 1H), 5.24 (s, 1H), 5.12 (s, 2H), 5.03 (s, 2H), 4.73 (s, 1H), 2.66 (s, 3H), 2.52 (s, 3H); 13C-NMR (CDCl3, 100 MHz) δ(ppm): 175.6, 174.0, 170.6, 167.0, 166.6, 162.3, 158.9, 157.5, 153.0, 136.3, 136.1 133.1, 131.9, 129.2, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 126.3, 114.7, 113.3, 108.9, 108.0, 100.2, 95.2, 66.1, 65.8, 18.6, 18.3.

*Dibenzyl 3,3 -((5-hydroxy-4-oxo-2-phenyl-4H-chromene-6,7-diyl) bis (oxy)) (2E,2 E)-bis (but-2-enoate)* (**4c**); yellow powder; yield 24.2%; HRMS(ESI): calcd. C37H30O9Na, *m*/*z* 641.1782 [M + Na]+, found 641.1779; 1H-NMR (DMSO-*d6*, 400 MHz) δ(ppm): 13.17 (s, 1H), 8.16-8.11 (m, 2H), 7.76–7.53 (m, 3H), 7.39-7.23 (m, 10H), 7.18 (s, 1H), 5.14 (s, 1H), 5.04 (s, 1H), 5.01 (s, 2H), 4.96 (s, 1H), 2.43 (s, 3H), 2.42 (s, 3H); 13C-NMR (DMSO-*d6*, 100 MHz) δ(ppm): 182.9, 170.4, 170.3, 166.1, 165.8, 164.5, 153.7, 152.9, 150.5, 136.1, 130.3, 129.2, 128.4, 128.2, 128.1, 128.0, 126.7, 109.4, 105.4, 101.8, 98.0, 94.3, 65.2, 65.1, 17.5, 17.4.

*Benzyl (E)-3-((5,6-dihydroxy-4-oxo-2-phenyl-4H-chromen-7-yl) oxy) but-2-enoate* (**4d**); yellow powder; yield 16.0%; HRMS(ESI): calcd. C26H20O7Na, *m*/*z* 467.1101 [M + Na]+, found 467.1092; 1H-NMR (DMSO-*d6*, 500 MHz) δ(ppm): 13.06 (s, 1H), 11.37 (s, 1H), 8.08 (d, *J* = 6.0 Hz, 2H), 7.62–7.56 (m, 3H), 7.34-7.28 (m, 5H), 7.02 (s, 1H), 6.72 (s, 1H), 5.03 (s, 2H), 4.87 (s, 1H), 2.44 (s, 3H); 13C-NMR (DMSO-*d6*, 125 MHz) δ(ppm): 182.3, 171.2, 166.4, 163.7, 156.6, 154.2, 152.4, 136.3, 132.2, 130.7, 129.2, 128.4, 128.3, 128.0, 126.6, 123.7, 105.0, 104.4, 94.8, 92.9, 65.1, 17.7.

*Benzyl (E)-3-((5-hydroxy-2-(3-hydroxy-4methoxyphenyl)-4-oxo-4H-chromen-7yl) oxy) but-2-enoate* (**4e**); yellow powder; yield 32.5%; HRMS(ESI): calcd. C27H23O8, *m*/*z* 475.1387 [M + H]+, found 475.1375; 1H-NMR (DMSO-*d6*, 400 MHz) δ(ppm): 13.07 (s, 1H), 9.48 (s, 1H), 7.58 (d, *J* = 8.4 Hz, 1H), 7.46 (d, *J* = 1.6 Hz, 1H), 7.32 (s, 5H), 7.08 (d, *J* = 8.4 Hz, 1H), 6.98 (s, 1H), 6.90 (s, 1H), 6.58 (d, *J* = 1.6 Hz, 1H), 5.10 (s, 1H), 5.06 (s, 2H), 3.87 (s, 3H), 2.45 (s, 3H); 13C-NMR (DMSO-*d6*, 100 MHz) δ(ppm): 182.3, 171.3, 166.0, 164.5, 161.6, 158.4, 156.9, 151.5, 146.8, 132.2, 128.4, 128.2, 128.0, 122.6, 119.1, 113.3, 112.1, 108.0, 104.2, 104.1, 100.4, 98.1, 65.2, 55.8, 17.9.

*Benzyl (E)-3-(4-(3,5,7-trihydroxy-4-oxo-4H-chromen-2-yl) phenoxy) but-2-enoate* (**4f**); yellow powder; yield 23.6%; HRMS(ESI): calcd. C26H20O8Na, *m*/*z* 483.1050 [M + Na]+, found 483.1047; 1H-NMR (DMSO-*d6*, 400 MHz) δ(ppm): 12.24 (s, 1H), 10.96 (s, 1H), 10.40 (s, 1H), 7.78–7.76 (m, 2H), 7.32–7.30 (m, 5H), 6.95–6.93 (m, 2H), 6.48 (d, *J* = 2.0 Hz, 1H), 6.23 (d, *J* = 2.0 Hz, 1H), 5.28 (s, 1H), 5.01 (s, 2H), 2.44 (s, 3H); 13C-NMR (DMSO-*d6*, 100 MHz) δ(ppm): 175.0, 170.0, 166.3, 164.5, 161.1, 160.8, 156.8, 156.5, 136.2, 131.0, 130.1, 128.4, 128.3, 128.0, 119.5, 115.9, 104.1, 98.9, 94.8, 94.2, 65.1, 17.6.

*Benzyl (E)-3-(4-(7-(((E)-3-(benzyloxy)-2-methyl-3-oxoprop-1-en-1-yl) oxy)-5-hydroxy-4-oxo-4H-chromen-2-yl) phenoxy)-2-methylacrylate* (**4g**); yellow powder; yield: 64.9%; HRMS(ESI) calcd. C37H31O9, *m*/*z* 619.1963 [M + H]+, found 619.1954; 1H-NMR (CDCl3, 500 MHz) δ (ppm): 12.75 (s, 1H), 7.91 (d, *J* = 7.5 Hz, 2H), 7.33 (s, 10H), 7.18 (d, *J* = 8.0 Hz, 2H), 6.68 (d, *J* = 14.5 Hz, 2H), 6.50 (s, 1H), 5.20 (s, 1H), 5.11 (d, *J* = 7.5 Hz, 4H), 5.01 (s, 1H), 2.53 (d, *J* = 11.5 Hz, 6H); 13C-NMR (CDCl3, 125MHz) δ (ppm):182.7, 172.3, 171.2, 167.0, 166.9, 163.7, 162.6, 159.3, 157.3, 156.5, 136.19, 136.1, 128.6, 128.5, 128.4, 128.3, 122.4, 108.6, 106.2, 105.0, 100.1, 99.1, 97.5, 66.0, 65.9, 18.5, 18.3.

*Ethyl (E)-3-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl) oxy) hex-2-enoate* (**5a**); yellow powder; yield 32.0%; HRMS(ESI): calcd. C23H22O6Na, *m*/*z* 417.1309 [M + Na]+, found 417.1305; 1H-NMR (CDCl3, 400 MHz) δ(ppm): 12.79 (s, 1H), 7.89 (dd, *J* = 8.0, 2.0 Hz, 2H), 7.57–7.50 (m, 3H), 6.74 (s, 1H), 6.67 (d, *J* = 2.0 Hz, 1H), 6.49 (d, *J* = 2.0 Hz, 1H), 5.08 (s, 1H), 4.11 (dd, *J* = 2H), 2.94–2.90 (m, 2H), 1.74 (m, *J* = 15.2, 7.6 Hz, 2H), 1.23 (t, *J* = 7.2 Hz, 3H), 1.05 (t, *J* = 7.2 Hz, 3H); 13C-NMR (CDCl3, 100 MHz) δ(ppm): 182.9, 174.4, 166.9, 164.7, 162.6, 159.7, 157.5, 132.3, 131.0, 129.3, 126.5, 108.6, 106.3, 105.0, 100.1, 99.2, 60.0, 32.9, 20.9, 14.4, 13.9.

*Ethyl (E)-3-((7-hydroxy-4-oxo-2-phenyl-4H-chromen-5-yl) oxy) hex-2-enoate* (**5b**); yellow powder; yield 40.0%; HRMS(ESI): calcd. C23H23O6, *m*/*z* 395.1489 [M + H]+, found 395.1482; 1H-NMR (DMSO-*d6*, 400 MHz) δ(ppm): 8.26–8.01 (m, 2H), 7.56 (d, *J* = 7.2 Hz, 3H), 6.98 (d, *J* = 2.2 Hz, 1H), 6.74 (s, 1H), 6.49 (d, *J* = 2.2 Hz, 1H), 4.49 (s, 1H), 3.94 (dd, *J* = 14.4, 7.2 Hz, 2H), 2.94–2.90 (m, 2H), 1.76 (m, *J* = 2H), 1.09–0.99 (m, 6H), 1.00 (t, *J* = 7.2 Hz, 3H); 13C-NMR (DMSO-*d6*, 100 MHz) δ(ppm): 176.1, 174.6, 166.2, 162.6, 160.7, 158.8, 151.8, 131.6, 130.9, 129.1, 126.2, 109.5, 108.6, 107.7, 101.5, 93.7, 32.6, 59.1, 19.9, 14.1, 13.8.

*Ethyl (E)-3-((7-(((Z)-1-ethoxy-1-oxohex-2-en-3-yl) oxy)-6-hydroxy-4-oxo-2-phenyl-4H-chromen-5-yl) oxy) hex-2-enoate* (**5c**); yellow powder; yield 7.0%; HRMS(ESI): calcd. C31H35O9, *m*/*z* 551.2276 [M + H]+, found 551.2260; 1H-NMR (CDCl3, 400 MHz) δ(ppm): 8.40 (s, 1H), 7.83 (d, *J* = 6.4 Hz, 2H), 7.55–7.48 (m, 3H), 7.11 (s, 1H), 6.60 (s, 1H), 4.90 (s, 1H), 4.70 (s, 1H), 4.10–4.03 (m, 4H), 3.05–3.02 (m, 2H), 2.93–2.90 (m, 2H), 1.87–1.72 (m, 4H), 1.21–1.16 (m, *J* = 6H), 1.07–1.02 (m, 6H); 13C-NMR (CDCl3, 125 MHz) δ(ppm): 176.5, 175.5, 173.6, 167.3, 167.1, 162.5, 156.0, 154.6, 144.1, 132.0, 131.6, 131.0, 129.2, 126.3, 111.4, 107.8, 102.7, 95.6, 94.5, 60.1, 59.9, 33.6, 33.1, 21.2, 20.7, 14.4, 14.2.

*Ethyl (E)-3-((5,6-dihydroxy-4-oxo-2-phenyl-4H-chromen-7-yl) oxy) hex-2-enoate* (**5d**); yellow powder; yield 11.2%; HRMS(ESI): calcd. C23H21O7, *m*/*z* 409.1293 [M <sup>−</sup> H]−, found 409.1289; 1H-NMR (DMSO-*d6*, 500 MHz) δ(ppm): 13.03 (s, 1H), 11.36 (s, 1H), 8.07 (d, *J* = 7.5 Hz, 2H), 7.61–7.56 (m, 3H), 7.01 (s, 1H), 6.70 (s, 1H), 4.74 (s, 1H), 3.98 (dd, *J* = 14.5, 6.0 Hz, 2H), 2.86 (t, *J* = 7.0 Hz, 2H), 1.71 (dd, *J* = 15.0, 7.5 Hz, 2H), 1.10 (t, *J* = 7.0 Hz, 3H), 1.00 (t, *J* = 7.5 Hz, 3H); 13C-NMR (DMSO-*d6*, 100 MHz) δ(ppm): 182.3, 173.8, 166.3, 163.6, 156.7, 154.1, 152.4, 132.1, 130.7, 129.2, 126.5, 123.7, 104.9, 104.4, 94.7, 92.8, 59.1, 32.3, 20.4, 14.1, 13.5.

*Diethyl 3,3 -((5-hydroxy-4-oxo-2-phenyl-4H-chromene-6,7-diyl) bis (oxy)) (2E,2 E)-bis (hex-2-enoate)* (**5e**); yellow powder; yield 14.8%; HRMS(ESI): calcd. C31H34O9Na, *m*/*z* 573.2095 [M + Na]+, found 573.2106; 1H-NMR (CDCl3, 500 MHz) δ(ppm): 12.96 (s, 1H), 7.89 (d, *J* = 7.5 Hz, 2H), 7.60–7.53 (m, 3H), 6.76 (d, *J* = 9.0 Hz, 1H), 5.03 (s, 1H), 4.85 (s, 1H), 4.13–4.06 (m, 4H), 2.93–2.87 (m, 4H), 1.77–1.69 (m, 4H), 1.21 (dd, *J* = 15.2, 7.6 Hz, 6H), 1.06–1.00 (m, 6H); 13C-NMR(CDCl3, 125 MHz) δ(ppm): 182.9, 174.0, 173.8, 167.1, 166.7, 165.2, 154.1, 153.8, 151.8, 132.5, 130.9, 129.4, 128.4, 126.6, 109.5, 105.9, 100.7, 98.7, 94.5, 60.0, 59.7, 33.1, 32.8, 21.0, 20.9, 14.4, 14.0, 13.9.

*(E)-3-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl) oxy) but-2-enoic acid* (**6a**); yellow powder; yield 69.4%; HRMS(ESI): calcd. C19H13O6, *m*/*z* 337.0718 [M + H]+, found 337.0711; 1H-NMR (DMSO-*d*6, 500 MHz) δ(ppm): 12.93 (s, 1H), 8.14 (d, *J* = 6.4 Hz, 2H), 7.76–7.58 (m, 3H), 7.15 (s, 1H), 7.05 (d, *J* = 2.0 Hz, 1H), 6.61 (d, *J* = 2.0 Hz, 1H), 5.09 (s, 1H), 2.41 (s, 3H); 13C-NMR (DMSO-*d*6, 125 MHz) δ(ppm): 182.8, 169.7, 167.6, 164.3, 161.7, 159.3, 157.2, 132.6, 130.6, 129.4, 126.9, 108.1, 105.9, 104.2, 100.7, 100.3, 17.7.

*2-(2-methyl-5-(3,5,7-trihydroxy-4-oxo-4H-chromen-2-yl) benzo[d] [1,3] dioxol-2-yl) acetic acid* (**6b**); yellow powder; yield 43.7%; HRMS(ESI): calcd. C19H15O9, *m*/*z* 387.0711 [M + H]+, found 387.0707; 1H-NMR (DMSO-*d6*, 500 MHz) δ(ppm): 12.40 (s, 1 H), 7.73 (dd, *J* = 10.5, 1.5 Hz, 1H), 7.61 (d, *J* = 1.5 Hz, 1H), 7.03 (d, *J* = 10.5 Hz, 1H), 6.46 (d, *J* = 2.5 Hz, 1H), 6.19 (d, *J* = 2.5 Hz, 1H), 3.02 (s, 2H), 1.78 (s, 3H); 13C-NMR (DMSO-*d*6, 125 MHz) δ(ppm): 217.5, 176.0, 172.1, 169.2, 164.1, 160.7, 156.2, 148.2, 147.1, 146.0, 136.2, 124.8, 122.9, 117.6, 108.4, 107.3, 103.1, 98.3, 93.7, 24.5, 21.1.

#### *2.3. Anti-Cancer Activity Assay*

#### 2.3.1. Cell Lines and Cell Culture

The human leukemia cell lines K562 and HEL, the non-small cell lung cancer A549, and the prostate cancer cell line PC3 were obtained from the Key Laboratory of Chemistry for Natural Product of Guizhou Province and Chinese Academy of Science (Guiyang, China). Cells were cultured as a monolayer in DMEM (Hyclone, Germany), supplemented with 10% heat-inactivated research-grade fetal bovine serum (Hyclone, Germany) and penicillin/streptomycin (Sigma, St. Louis, MO, USA) at 37 ◦C in a humidified atmosphere, containing 5% CO2.

#### 2.3.2. Cytotoxic Activity Assay

The cytotoxic activity of compounds was measured by MTT assay, using adriamycin as the positive control. Cells were seeded in 96-well microculture plates at a density of 1 <sup>×</sup> 104 cells/well and left to adhere for 24 h. The cells were then exposed to different concentrations of the compounds for 48 h. MTT was added to each well at a final concentration of 0.5 mg/mL and cells were incubated at 37 ◦C for an additional 4 h. The medium was then discarded and 200 μL Tris-DMSO solution was added to each well. The dark blue formazan crystals formed were dissolved by slight shaking, and the absorbance was measured at 490 nm, using an ELISA plate reader.

#### 2.3.3. Statistical Analysis

The data were analyzed using SPSS 18.0 and reported as mean ± standard deviation (SD) of the number of experiments indicated. For all the measurements, one-way ANOVA followed by a Student's *t*-test was used to assess the statistical significance of the differences between each group. The statistical significance of the difference between two groups was assessed using the LSD method. *p* < 0.05 was considered to indicate statistical significance. The data are presented as the mean ± standard error of the mean (SEM) of three assays.

#### **3. Results and Discussion**

#### *3.1. Synthesis*

The optimal reaction conditions were established (Table 1, entry 6**)**. The substrate scope was further extended to other types of allenes (Figure 1). As expected, various substituent groups led to a variety of products. When using either the monoallene (ethyl ester/benzyl ester) or diallene, the Pd catalyst efficiently promoted this C-O functionalization. Intriguingly, the application of triethylamine as a base instead of potassium carbonate was demonstrated to be more efficient for target product purification (**4e**, **4f**). However, completion of the reaction is hard to achieve. Starting flavonoids with OH groups at positions 3 and 4 easily generated the 1,3-dioxolane byproducts, instead of the desired ones. The key intermediates **1a**–**1c** were prepared according to reported methods [18–20].

**Figure 1.** Types of allenes.

Under optimal conditions, as illustrated in Figure 2, a variety of flavonoid substrates was investigated. For instance, flavones and flavonols were well tolerated, providing ample opportunities for further anti-cancer activity evaluation of the derivatives. We found that the property of flavonoid and the substituted phenolic hydroxyl groups had a great impact on the formation of the desired products. Notably, it was more challenging to separate the analog products when more than two hydroxyl groups were presented. Finally, the target products (**3a**–**3i**, **4a**–**4g**, **5a**–**5e**, **6a**, **6b**) were successfully purified by silica gel column chromatography. The configuration of the C=C bond in **3a** and **4a** was determined by the NOE (Nuclear Overhauser Effect) study. The structures of final compounds were characterized by 1H-, 13C-NMR, and MS spectrometry.

**Figure 2.** Substrate scope of flavonoids and allenes.

#### *3.2. Anti-Cancer Activity*

The anti-tumor activity of all derivatives in vitro was summarized in Table 2. Results showed that the inhibitory effect of compound **4e** on the growth of K562 was six times stronger (*p* < 0.01) than that of the substrate, diosmetin, which indicated that the anti-tumor activity of the diosmetin derivative was associated with the more lipophilic benzyl ester than -OCH3 moiety at R6 position of the substrate. Compounds **5e**, **3h**, and **6b** showed better activity (*p* < 0.01) on the proliferation of HEL than that of the substrates, baicalein, kaempferol, and quercetin, respectively. These results suggested that the anti-leukemia activity may be improved by increasing steric hindrance, such as by introducing propyl and 1,3-dioxolane groups onto baicalein. Compound **6b** exhibited an enhanced inhibitory activity on the growth of leukemia cells (K562 and HEL) and non-small cell lung cancer A549 compared with those of **3i**, which can be speculated as the enhanced water solubility associated with the carboxyl group. We then synthesized compounds **3e**, **4b**, and **4c** to improve the anti-cancer activity (*p* < 0.01) on the proliferation of PC3 compared with the substrates, diosmetin, chrysin, and baicalein, respectively. The results suggested that introducing an ethyl and benzyl esters could increase their anti-cancer activity.


**Table 2.** The anti-cancer activities of the synthetic derivatives.

**3h** 16.3 ± 3.4 −16.7 ± 2.3 58.6 ± 3.9 \*\* −15.9 ± 2.6 **3i** 26.2 ± 3.5 −60.4 ± 2.7 24.9 ± 3.8 42.2 ± 2.1


**Table 2.** *Cont.*

Note: \*\* represents *p* < 0.01, vs. the inhibition of the substrate to the cancer cell lines. The data represented the average of three independent experiments.

#### **4. Conclusions**

Twenty-three flavonoidal ether derivatives were synthesized without the steps of introducing protective groups followed by deprotection. The in vitro tumor growth inhibitory activities of all the derivatives were assayed using four human cancer cell lines, K562, A549, HEL, and PC3. In general, compounds containing 1,3-dioxolane, such as **6b**, possessed broad-spectrum inhibitory activity against the above four cancer cells. Preliminary structure–activity relationship studies indicated that the position-7 substituents were essential for the cytotoxicities of the derivatives. These results provided new insight into developing flavonoid-derived anti-cancer agents.

**Author Contributions:** Conceptualization, L.J. and M.-L.W.; Methodology, L.J. and X.-Y.Z.; Software, L.J., X.-Y.Z.; Validation, X.-Y.Z. and C.C.; Formal analysis, L.J.; Resources, W.-D.P. and H.L.; Data curation, L.J., Y.L., and M.-L.W.; Writing—original draft preparation, L.J.; Writing—review and editing, L.J., H.R., W.-D.P., and H.L.; Project administration, W.-D.P. and H.L.; Funding acquisition, W.-D.P. and H.L.

**Funding:** Supported by the National Natural Science Foundation of China (No. 81660580, 81702914) and the Science and Technology Department of Guizhou Province, China (QKHRC[2016]4037, QKHJC[2017]1412).

**Acknowledgments:** We would like to acknowledge the help of my colleagues Ni Zhang, Xiao-Pan Ma, Chang-Fen Chen, and Sai Jiang.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


**Sample Availability:** Samples of the compounds are available from the authors.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Design, Synthesis, and Mechanism of Dihydroartemisinin–Coumarin Hybrids as Potential Anti-Neuroinflammatory Agents**

#### **Haonan Yu 1,**†**, Zhuang Hou 1,**†**, Xiaoguang Yang 1, Yanhua Mou 2,\* and Chun Guo 1,\***


Received: 6 April 2019; Accepted: 24 April 2019; Published: 28 April 2019

**Abstract:** Cancer patients frequently suffer from cancer-related fatigue (CRF), which is a complex syndrome associated with weakness and depressed mood. Neuroinflammation is one of the major inducers of CRF. The aim of this study is to find a potential agent not only on the treatment of cancer, but also for reducing CRF level of cancer patients. In this study, total-thirty new Dihydroartemisinin–Coumarin hybrids (DCH) were designed and synthesized. The in vitro cytotoxicity against cancer cell lines (HT-29, MDA-MB-231, HCT-116, and A549) was evaluated. Simultaneously, we also tested the anti-neuroinflammatory activity of DCH. DCH could inhibit the activated microglia N9 release of NO, TNF-α, and IL-6. The docking analysis was shown that MD-2, the coreceptor of TLR4, might be one of the targets of DCH.

**Keywords:** anticancer; anti-neuroinflammation; coumarin; dihydroartemisinin

#### **1. Introduction**

Chemotherapy is an important method in the therapy of cancer, but the adverse side effects of anticancer agents should also be focused on. Gastrointestinal injury induced by chemotherapeutic agents could result in bacterial/endotoxin translocation from the gut to the systemic blood circulation [1,2]. The released endotoxin would cause a systemic inflammatory response including neuroinflammation [3]. Cancer patients frequently suffer from cancer-related fatigue (CRF), which is a complex syndrome associated with weakness and depressed mood. Diana M., Norden et al. demonstrated that the neuroinflammation is one of the major reasons which induce CRF [4]. Currently, there were no effective method to reduce CRF [5]. The aim of this study is to find a potential anti-neuroinflammatory agents which might reduce CRF of cancer patients.

Central nervous system (CNS) consists of two major types of cells, nerve cells and glial cells. Microglia is a major glial cell and plays an important role as the resident macrophage population in the CNS [6,7]. There has been wide recognition that the fully activated microglial cells are neurotoxic. They release reactive oxygen species, nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), and other potentially cytotoxic molecules [8,9]. It means that activation of microglia could induce neuroinflammation.

Lipopolysaccharide (LPS), also known as endotoxin, is a natural Toll-like receptor 4 (TLR4) ligand and it is also a potent microglial activator that can trigger the production of inflammatory mediators. TLR-4, a transmembrane receptor, plays a vital role in the initiation and acceleration of the inflammatory response induced by LPS [10,11]. TLR4 alone does not directly bind LPS and needs

the coreceptor MD-2 [12]. MD-2 is linked with the extracellular domain of TLR4 and is indispensable for LPS recognition [13]. MD-2 is a member of the MD-2-related lipid-recognition protein family [14]. MD-2 directly binds to LPS in its hydrophobic cavity with high affinity [15].

Artemisinin and its derivatives have been the first-line antimalarial drug since the late 1990s. Dihydroartemisinin (DHA) is the primary bioactive metabolite of artemisinin and its derivatives, but not artemisinin per se [16]. Additionally, DHA has higher bioavailability and less side effects than artemisinin [17]. Recent research showed that besides antimalarial activity, DHA also exhibits anticancer and anti-inflammatory activity [18–21]. As the anti-inflammatory agent, DHA could decrease LPS-induced protein expression of TLR4 [22].

Coumarins are widely distributed in different parts of plants. There are various pharmacological activities of coumarins, which includes anti-inflammatory [23,24] and anticancer activity [25,26]. As the anticancer agents, coumarins were reported to exhibit negligible or mild side effects [27,28]. Substitution at the C-3 or C-4 position of coumarins exhibit good cytotoxic activity in various cancer cell lines [26,29–31]. Additionally, as anti-inflammatory agents, some kinds of coumarins could also target TLR4/MD-2 complex [32,33].

In our previous study, Dihydroartemisinin–Coumarin hybrids (DCH) were designed and synthesized referring to the principle of hybridization [34,35]. Each of them contains coumarin moiety, DHA moiety and a linker. DCH could inhibit HT-29 cells proliferation and arrest the G0/G1 phase of HT-29 cells [35]. They could also suppress cancer cell migration, and induce apoptosis and ferroptosis of HT-29 cancer cells [35]. It also has been confirmed that the DCH having 1,2,3-triazole linker generally exhibit better cytotoxicity exhibition than the others [35].

With this background, total-thirty new DCH were designed and synthesized, and the synthetic approaches were shown in Figure 1. All of them contained 1,2,3-triazole linker. We focused on the modifications of positions 3 and 4 of the coumarin moiety. Due to the new synthesized DCH were for the cancer patients, we evaluated the in vitro cytotoxicity against cancer cell lines (HT-29, MDA-MB-231, HCT-116, and A549). In addition, DCH could inhibit the activated N9 cell release of NO, TNF-α and IL-6. Furthermore, to investigate the mechanism of the anti-neuroinflammatory activity, docking analysis was applied. The results might support that the anti-neuroinflammatory activity was related to inhibiting TLR4/MD-2 complex.

**Figure 1.** Structures of new designed Dihydroartemisinin–Coumarin hybrids.

#### **2. Results and Discussion**

#### *2.1. Chemistry*

The synthesis of the intermediates **a**–**p** and **I**–**IV** is presented in Scheme 1. The intermediates **I** and **II** were obtained by 2-bromoethanol or 3-bromo-1-propanol, with dihydroartemisinin (DHA), respectively, followed by treating the product with sodium azide. The intermediates **III** and **IV** were synthesized by propargyl alcohol or 3-butyn-1-ol with DHA, respectively. The required 3-methylcoumarin derivatives were obtained by substitution of salicylaldehyde with propionic anhydride in the presence of sodium propionate and triethylamine. The substituted-3-bromomethylcoumarins were prepared by the radical bromination of 3-methylcoumarin derivatives through the reaction with

N-Bromosuccinimide (NBS) in the presence of dibenzoylperoxide [36]. The intermediates **a**–**e** were synthesized by substituted-3-bromomethylcoumarins via azidation reaction. The required substituted-4-chloromethylcoumarins were prepared by the Pechmann cyclisation [37] of substituted phenols with 4-chloroethylacetoacetate using sulfuric acid as the condensing agent. The intermediates **f**–**i** were synthesized by substituted-4-chloromethylcoumarin via azidation reaction. To synthesize the intermediates **j**–**p**, several substituted-4-hydroxycoumarin were prepared by condensing the substituted phenol with malonic acid [38]. Then the products were treated with 3-bromo-1-propyne in the presence of potassium carbonate.

**Scheme 1.** Synthetic routes of intermediates. Reagents and conditions: (1) BF3 . Et2O, CH2Cl2, 0 ◦C; (2) NaN3, DMF, 60 ◦C; (3) CH3CH2COONa, Et3N, reflux; (4) NBS, BPO, CCl4, reflux; (5) 70% H2SO4, r.t.; (6) ZnCl2, POCl3, 65 ◦C; and (7) K2CO3, DMF, 60 ◦C.

The synthesis of the desired new compounds was presented in Scheme 2. The target compounds **1a**–**1e** and **2a**–**2e** were obtained by the reaction of the intermediates **III** or**IV** with **a**–**e** via click chemistry, respectively. The target compounds **3f**–**3i** and **4f**–**4i** were synthesized by intermediates **III** or **IV** with **f**–**i** via click chemistry, respectively. The target compounds **5j**, **5l**–**5o**, and **6j**–**6p** were prepared by the reaction of the intermediates **I** or **II** with **j**–**p** via click chemistry, respectively.

**Scheme 2.** Synthetic routes of target compounds. Reagents and conditions: (1) CuI, triethylamine, dichloromethane, r.t.

#### *2.2. Biological Assay*

#### 2.2.1. In Vitro Cytotoxic Activity of the Compounds

As the anti-neuroinflammatory agents for cancer patients, all newly synthesized compounds were evaluated in vitro for antitumor activity against human breast cancer cell line MDA-MB-231, colorectal cancer cells HT-29 and HCT-116, and lung cancer cell line A549 by using the standard MTT method, respectively. Doxorubicin (DOX) and DHA were used as the reference standard. Additionally, owing to the rapid proliferation, the tumor cells actually was always in hypoxia condition. Therefore, the in vitro cytotoxicity was evaluated in two different conditions (hypoxia and normoxia). All data are summarized in Table 1. Most of the target compounds exhibited moderate activities against the tested cell lines and they showed a general promotion under hypoxia conditions.

The structure–activity relationship was evaluated: (i) the series of compounds **5** and **6** generally exhibited better cytotoxicity activity than that of the series of compounds **3** and **4**, which indicated that the 4-oxygen group in the coumarin moiety as a part of linker could improve the antitumor activity; (ii) although compounds **3** and **4** did not exhibit good activity against three cell lines (MDA-MB-231, HCT-116, and A549), they showed good activity against HT-29 like the other derivatives. Therefore, 4-methyl group in the coumarin moiety could improve the selectivity of DCH against HT-29.

In addition, almost all of the target compounds exhibited good cytotoxicity activity against HT-29. Therefore, HT-29 was selected for the next explorations.


**Table 1.** IC50 (μM) of tested samples in MDA-MB-231, HT-29, HCT-116, and A549 cell lines.

#### 2.2.2. Morphological Changes and Effect of Compound 5n on Cell Viability

As shown in A–F of Figure 2, there showed a concentration-dependent cell death after treatment with compound **5n** for 48 h. As the concentration up to 100 μM, the compound **5n** could obviously inhibit the proliferation of HT-29. As shown in the (G) of Figure 2, after HT-29 cells were treated with different concentrations of **5n** for 48 h, the cell viability decreased in a dose-dependent manner.

**Figure 2.** Inhibition of compound **5n** on cell viability in HT-29 cells by MTT staining. Cells were treated with (**A**) 0 μM, (**B**) 6.25 μM, (**C**) 12.5 μM, (**D**) 25.0 μM, (**E**) 50.0 μM, and (**F**) 100.0 μM compound **5n** for 48 h. Representative images were shown; 200×. (**G**) Cells were treated with 6.25, 12.5, 25, 50, and 100 μM compound **5n** for 48 h. Data were shown as mean ± SD. \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 compared with group 0 μM.

#### 2.2.3. Anti-Neuroinflammatory Activity

The anti-neuroinflammatory activity of DCH was evaluated and then the representative compounds were selected for the further study. As shown in Table 2, murine microglial N9 cells were treated with 1μg/mL LPS for 48 h and the NO level in the medium was measured as the preliminary screening. Most of the tested compounds exhibited good anti-inflammatory activity, and especially the IC50 of some compounds were even less than 1 μM. Among them, compound **5n** had the best activity and the IC50 reached 0.22 μM. In addition, in order to exclude the death influence, MTT-IC20 of all the compounds were measured. The results showed that DCH did not exhibit cytotoxic activity in N9 cells. The exact reason was still unclear, but we supposed that it might be related to the homeostasis of cells. The tumor cells actually were always in hypoxia condition, in which DCH could promote the cytotoxic activity against tumor cells. In brief, DCH could reduce the release of NO caused by LPS-induced microglial cells.


**Table 2.** IC50 of tested samples in LPS-induced N9 cells for the inhibition of NO; IC20 of tested samples in N9 cells for cytotoxic activity.

#### NO Capture Capability

Sodium nitroprusside (SNP) is a NO donor. In this experiment, the reason of the DCH reducing NO level was preliminary explored. As shown in Figure 3, the results showed that the compounds only partially reduced NO levels by direct NO capture. That means the main reason of the NO level reduction is due to species-specific effect.

**Figure 3.** Compound 6l and 5n had NO radical scavenging activity. 6l (0.01 μM, 0.1 μM, 1 μM, and 10 μM) or **5n** (0.01 μM, 0.1 μM, 1 μM, and 10 μM) was added to a concentration of 0.5 mM SNP solution. After reacted for 1 h under normal sun light at room temperature, Griess reagent was added and the optical density was observed at 540 nm. Data were shown as mean ± SD. \*\*\* *p* < 0.001 compared with control group.

Effect of Representative Compounds on TNF-α and IL-6 Release

As shown in Table 3, four kinds of DCH were involved to measure the effect on the release of TNF-α and IL-6 from the microglia cells which induced by LPS for 48 h. The results showed that compounds **2b**, **2c**, **5n**, and **6p** could inhibit the release of TNF-α with the inhibition rate of 22% to 32% and the release of IL-6 with the inhibition rate of 26% to 43%. The results indicated that the inhibition effect of DCH on NO release was not only by nonspecific effects, but also by slightly influencing the synthesis and release of upstream inflammatory pathway.

**Table 3.** The inhibition of compounds (1 μM) on cytokines release (48 h) of the murine microglial cell line N9 induced by lipopolysaccharide (LPS) (1 μg/mL).

