**Anticancer Inhibitors**

Editors

**Marialuigia Fantacuzzi Alessandra Ammazzalorso**

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

*Editors* Marialuigia Fantacuzzi Pharmacy "G. d'Annunzio" University of Chieti-Pescara Chieti Italy Alessandra Ammazzalorso Pharmacy "G. d'Annunzio" University of Chieti-Pescara Chieti Italy

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Molecules* (ISSN 1420-3049) (available at: www.mdpi.com/journal/molecules/special issues/anticancer inhibitor).

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### **Contents**


Reprinted from: *Molecules* **2021**, *26*, 3528, doi:10.3390/molecules26123528 . . . . . . . . . . . . . . **131**

### **Yaomin Wang, Chen Xia, Lianfu Chen, Yi Charlie Chen and Youying Tu** Saponins Extracted from Tea (*Camellia Sinensis*) Flowers Induces Autophagy in Ovarian Cancer Cells Reprinted from: *Molecules* **2020**, *25*, 5254, doi:10.3390/molecules25225254 . . . . . . . . . . . . . . **153 Youying Tu, Lianfu Chen, Ning Ren, Bo Li, Yuanyuan Wu and Gary O. Rankin et al.** Standardized Saponin Extract from Baiye No.1 Tea (*Camellia sinensis*) Flowers Induced S Phase Cell Cycle Arrest and Apoptosis via AKT-MDM2-p53 Signaling Pathway in Ovarian Cancer Cells Reprinted from: *Molecules* **2020**, *25*, 3515, doi:10.3390/molecules25153515 . . . . . . . . . . . . . . **165 Henrik Franzyk and Søren Brøgger Christensen** Targeting Toxins toward Tumors Reprinted from: *Molecules* **2021**, *26*, 1292, doi:10.3390/molecules26051292 . . . . . . . . . . . . . . **183**

### **About the Editors**

### **Marialuigia Fantacuzzi**

Marialuigia Fantacuzzi is an Assistant Professor in Medicinal Chemistry at the Department of Pharmacy, "G. d'Annunzio"University of Chieti-Pescara (Italy). She received a degree in Pharmaceutical Chemistry and Technology and a Ph.D. in Pharmaceutical Sciences from the same university. Her research interests are mainly focused on design, synthesis, biological evaluation, and docking study of compounds of pharmaceutical interest useful for the treatment of inflammatory pathology, metabolic syndrome, neurodegenerative diseases, and cancer.

Her scientific activity is certified by 61 papers in international peer reviewed journals and different communications in scientific meetings. Her commitment to pharmaceutical chemistry is rounded out by her role as a reviewer for major international medicinal chemistry journals, as well as her participation as an editorial board member and as a Special Issue guest editor.

### **Alessandra Ammazzalorso**

Alessandra Ammazzalorso is an Assistant Professor in Medicinal Chemistry at the Department of Pharmacy, "G. d'Annunzio"University of Chieti-Pescara (Italy). She obtained her degree in Pharmaceutical Chemistry and Technology and Ph.D. in Pharmaceutical Sciences. Her research activity is devoted to the synthesis of small molecules endowed with biological activity. In detail, her studies are focused on compounds targeting Peroxisome Proliferator-Activated Receptors, with special attention to the antitumor potential of their agonists and antagonists. She is also involved in the identification of novel inhibitors of nitric oxide synthase and aromatase as anticancer agents. Her research activity is documented by 74 peer reviewed papers, several contributions to scientific meetings, and participation in international and national research projects. She is also a reviewer for several high-ranked medicinal chemistry journals, and she serves as an editorial board member for different international journals.

### *Editorial* **Anticancer Inhibitors**

**Alessandra Ammazzalorso \* and Marialuigia Fantacuzzi \***

Department of Pharmacy, "G. d'Annunzio" University of Chieti-Pescara, Via dei Vestini 31, 66100 Chieti, Italy

**\*** Correspondence: alessandra.ammazzalorso@unich.it (A.A.); marialuigia.fantacuzzi@unich.it (M.F.); Tel.: +39-08713554682 (A.A.); +39-08713554684 (M.F.)

### **1. Introduction**

Cancer is a multifactorial disorder caused by several aberrations in gene expression that generate a homeostatic imbalance between cell division and death. Because of the worldwide increasing burden and the complexity of the mechanisms involved, considerable efforts have been devoted to cancer management. Many chemotherapeutics have been developed, but most of them have failed in cancer treatment. Antitumor drugs can be divided in non-specific (cytotoxic) drugs and specific drugs (targeted). Due to the inability of non-specific drugs to selectively target tumor cells, targeted therapy has grown more in recent years, allowing researchers to identify drugs characterized by a high specificity towards receptors and enzymes involved in cancer proliferation [1–7]. Since multiple pathogenetic mechanisms are involved in the development of cancer, the characterization of different types of cancers, which distinguishes them from healthy cells and other cancers, allows for the identification of specific targets for each individual tumor.

The Special Issue "Anticancer Inhibitors" covers twelve contributes (nine original research papers and three reviews). As guest editors, we briefly report an overview of these contributions.

### **2. Results**

The roles of cyclin-dependent kinases (CDKs) in different cancers allows for targeting specific kinases to obtain a selective action in the cell cycle and gene transcription. In the last years, CDK4/6 inhibitors revealed a therapeutic role for the treatment of breast cancer. Structural modifications of the three FDA-approved CDK4/6 inhibitors furnished novel molecules currently under clinical investigation as antitumor drugs. The novel generation of PROTACs (proteolysis targeting chimeras) has also been reviewed, allowing for a selective degradation of CDK4 or CDK6 by varying the chemical structures of inhibitors and linkers [8].

Al-Salem et al. synthesized a series of isatin-hydrazones with cytotoxic effects against MCF-7 and A2780 cell lines. Structure–activity relationship studies highlighted the structural modifications mainly responsible for the CDK2 IC50s nanomolar range activity. In silico ADME demonstrated the recommended drug likeness properties, while computational predictions of the binding mode confirmed type II ATP competitive inhibition [9].

Phosphatidylinositide-3-kinase (PI3K)/Akt signaling pathway inhibitors have undergone pre-clinical evaluation as a promising therapy for cancer treatment; the combination use of LY294002 and tamoxifen in breast cancer MCF-7 cells was indagated by Abdallah and coworkers. A synergistic cytotoxic effect of the combination, achieved by the induction of apoptosis and cell cycle arrest through cyclin D1, pAKT, caspases, and Bcl-2 signaling pathways, was found helpful to develop novel and effective therapeutic combination against breast cancer and reduce the toxicity and resistance of LY294002 and tamoxifen [10].

Konkol'ová et al. synthesized novel tacrine–coumarin hybrids as inhibitors of topoisomerase, and enzyme involved in DNA metabolism. Novel compounds inhibit the metabolic

**Citation:** Ammazzalorso, A.; Fantacuzzi, M. Anticancer Inhibitors. *Molecules* **2022**, *27*, 4650. https:// doi.org/10.3390/molecules27144650

Received: 1 July 2022 Accepted: 16 July 2022 Published: 21 July 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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 (https:// creativecommons.org/licenses/by/ 4.0/).

activity of A549 cell line in a time- and dose-dependent manner, increase the accumulation of cells in the G0/G1 phase, and topoisomerase I inhibition was confirmed as the mechanism of action of this class of hybrids [11].

Tanuma et al. synthesized novel azaindole–piperidine or azaindole–piperazine to develop effective and safer (no gastrointestinal symptoms and thrombocytopenia) anticancer nicotinamide phosphoribosyltransferase (NAMPT) inhibitors [12].

Tilayov et al. combined rational and combinatorial engineering approaches for transforming dimeric stem cell factor (SCF) into ligands with different agonistic potencies by engineering variants with a reduced dimerization potential and an increased affinity for c-Kit. The combinatorial site-directed engineering of both ligand–ligand and ligand–receptor interactions provides the means to generate improved therapeutic mediators and to gain insights into the dynamics of receptor tyrosine kinases (RTK)–ligand interactions [13].

The involvement of Cathepsin K in non-small-cell lung cancer has been investigated by Yang et al. through in vitro experiments of cell proliferation, migration, and invasion in human cell line A549. The results showed that Cathepsin K was overexpressed, promoting the proliferation, migration, invasion, and activation of the mammalian target of the rapamycin (mTOR) signaling pathway [14].

The review by Chen collects the literature evidence about both pro-oncogenic and tumor-suppressive effects of ZMYND8 (zinc finger myeloid, nervy, and deformed epidermal autoregulatory factor 1-type containing 8) in various types of cancer [15].

Rimpelová et al. studied the effect of statins on the expression of genes, whose products are implicated in cancer inhibition. The study on MiaPaCa-2 pancreatic cancer cells analyzes the genes involved in the metabolism of lipids and steroids that were affected by statin treatment [16].

The pivotal role played by natural products in the discovery and development of novel anticancer agents is well known [17]. The anticancer effect of saponins from tea (*Camellia sinensis*) was investigated by Wang et al. The extracted saponins decreased cell viability and induced morphological changes in OVCAR-3 cells. The autophagic effect occurred independently from Akt/mTOR/p70S6K pathway signaling, but it is linked to ERK activation and ROS generation [18]. A further evaluation on a high purity standardized saponin extract, namely, Baiye No.1 tea flower saponin, demonstrated its potential to be used as a nutraceutical for the prevention and treatment of ovarian cancer [19].

Franzyk and Christensen reviewed the recent literature reports on advanced prodrug concepts targeting toxins to cancer tissues. These strategies include antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT), lectin-directed enzyme-activated prodrug therapy (LEAPT), and antibody-drug conjugated therapy (ADC). In addition, recent examples of protease-targeting chimeras (PROTACs) were also analyzed and discussed; these methods involve ubiquitination enzyme complexes that undergo proteolytic degradation to release the drug. Overall, these innovative strategies of tumor targeting may lead to future new anticancer drugs that are urgently needed for. However, many of these recently developed targeting principles remain to result in approved drugs, which emphasizes the need for further research [20].

### **3. Conclusions**

This collection contributes to improving the knowledge on anticancer inhibitors, focusing on the synthesis and evaluation of novel compounds able to inhibit enzymes involved in tumorigenesis and proliferation, the role of transcription factors, the use of natural molecules as lead compounds for anti-cancer drug development, and, finally, the search for innovative strategies to overcome pharmacokinetic limitations.

**Funding:** This research received no external funding.

**Acknowledgments:** The Guest Editors wishes to thank all the authors for their contributions to this Special Issue, all the reviewers for their work in evaluating the submitted articles, and the editorial staff of *Molecules* for their kind assistance.

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

### **References**


### *Review* **Development of CDK4/6 Inhibitors: A Five Years Update**

**Alessandra Ammazzalorso , Mariangela Agamennone , Barbara De Filippis and Marialuigia Fantacuzzi \***

Unit of Medicinal Chemistry, Department of Pharmacy, "G. d'Annunzio" University, 66100 Chieti, Italy; alessandra.ammazzalorso@unich.it (A.A.); mariangela.agamennone@unich.it (M.A.); barbara.defilippis@unich.it (B.D.F.)

**\*** Correspondence: marialuigia.fantacuzzi@unich.it; Tel.: +39-0871-3554684

**Abstract:** The inhibition of cyclin dependent kinases 4 and 6 plays a role in aromatase inhibitor resistant metastatic breast cancer. Three dual CDK4/6 inhibitors have been approved for the breast cancer treatment that, in combination with the endocrine therapy, dramatically improved the survival outcomes both in first and later line settings. The developments of the last five years in the search for new selective CDK4/6 inhibitors with increased selectivity, treatment efficacy, and reduced adverse effects are reviewed, considering the small-molecule inhibitors and proteolysis-targeting chimeras (PROTACs) approaches, mainly pointing at structure-activity relationships, selectivity against different kinases and antiproliferative activity.

**Keywords:** cyclin-dependent kinase; cancer; resistance; small molecule inhibitors; PROTACs

### **1. Introduction**

Breast cancer (BC) is the most recurrent cancer in women worldwide, impacting 2.1 million women each year according to World Health Organization [1]. BC is a heterogeneous disease due to genetic factors that are reflected in different phenotypes. BC can be divided into different subtypes: luminal, in which estrogen receptors (ER) and/or progesterone receptors (PR) are expressed, further divided into luminal A and luminal B subtypes depending on the expression of Ki67 (low levels in A and high in B); HER2+, in which the human epidermal growth factor receptor 2 (HER2) is overexpressed and ER and PR are lacking; triple negative BC (TN), in which the previous targets are not expressed. The estrogen-receptor positive (ER+) BC is the most common type, with the prevalence of about 60% of cases in pre-menopausal women and 75% in post-menopausal women [2–5].

The anti-hormonal treatment involves the suppression or reduction of the estrogen effects and can be carried out using drugs that limit the production of these hormones, such as aromatase inhibitors (AIs), or act on the ER receptor, such as selective ER modulators (SERM) or down-regulators (SERD) [6–8]. The adjuvant therapy consists of 5–10 years of ER-directed endocrine therapy that result in a reduction of mortality in ER+ BC of more than 40%. Resistance to endocrine therapy leading to early-stage ER+ BC is common and decisive in the setting of advanced disease [9,10].

The aromatization reaction in the final step of estrogen biosynthesis is unique, therefore this reaction becomes an excellent target for inhibiting the synthesis of estrogens without affecting the production of other steroids. In recent decades, several aromatase inhibitors have been developed to adequately suppress estrogen production and have been used in the treatment of estrogen-dependent BC [11–16].

Given the high percentage of resistance to aromatase inhibitor treatment, new therapeutic strategies have been identified to make the treatment of ER+ BC more effective. One of the mechanisms involved in the resistance concerns the activation of cyclin-dependent kinases (CDKs) as an ER-independent growth signal, that involves the important protein kinase signaling pathway (PI3K/AKT/mTOR) (Figure 1) [17,18].

**Citation:** Ammazzalorso, A.; Agamennone, M.; De Filippis, B.; Fantacuzzi, M. Development of CDK4/6 Inhibitors: A Five Years Update. *Molecules* **2021**, *26*, 1488. https://doi.org/10.3390/ molecules26051488

Academic Editor: Margherita Brindisi

Received: 13 February 2021 Accepted: 6 March 2021 Published: 9 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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 (https:// creativecommons.org/licenses/by/ 4.0/).

**Figure 1.** Schematic representation of the cyclinD/CDK4/6 involvement in overpassing resistance to aromatase inhibitors. Aromatase converts androgens (A) in estrogens (E) that bind to ER receptor. The recruitment of co-activators (co-A) allows the binding to ERE element on the target genes and the activation of the transcription. AIs block the production of estrogen inhibiting the ER-driven activation of cell cycle progression. The activation of cyclinD/CDK4/6 complex, mediated by the protein kinase signaling pathway (PI3K/AKT/mTOR), stimulates cell proliferation independently from aromatase. The use of CDK4/6 inhibitor blocks this alternative activation pathway.

> CDKs, a family of serine/threonine kinases, regulate cell cycle progression into the four distinct phases G1, S (DNA synthesis), G2 and M, and are crucially involved in the regulation of cell division and proliferation. CDK stability, activation and downstream phosphorylation is controlled by cyclin counterpart and endogenous inhibitors. To date, 21 CDKs are known and their role in different types of cancer has been reported by many research groups [19–21]. In particular, CDK1, 2 and 4 regulate the transition of the cell cycle steps, CDK 7, 8, 9 and 11 regulate the gene transcription, while CDK 6 regulates both [22–24].

> Mitogenic, hormonal, and growth factors allow the cyclin D to bind CDK4/6, forming the complex that regulates the phosphorylation status of retinoblastoma protein (Rb). The phosphorylated-Rb determines the dissociation of the transcription factor E2F that binds to DNA and promotes the expression of different genes, regulates DNA replication and cell division, with the transition from G1 to S phase (Figure 1). Several endogenous factors control cell proliferation, including INK4 family proteins (p16, p15, p18, p19), cyclin inhibitory proteins and kinase inhibitory proteins (KIPs, p21, p27) [25,26].

> The dysregulation of cell cycle caused by the overexpression or gain-of-function mutations in the CDKs and cyclins or the loss of endogenous inhibitors expression and function, is recurrent in many cancer diseases. In BC, but also in other type of cancer, a dysregulation of this process leads to a proliferative stimulus, and a significant role in this mechanism is played by the overexpression of cyclin D. Inhibition of cell cycle using CDK4/6 inhibitor has emerged as antitumor treatment in BC in association to the hormonal therapy to overpass resistance to AIs and avoiding relapses [27–32].

Over the years several CDK inhibitors have been developed and tested in different types of cancer [33–37]. First generation inhibitors (flavopiridol and roscovitine) demonstrated an inadequate balance between efficacy and toxicity, due to their action on several kinases (pan-inhibitors) [38–40]. Second generation inhibitors (dinaciclib) were developed with the aim to increase selectivity and potency, but demonstrated limited efficacy and considerable toxicity in clinical studies. The toxicity of these compounds is associated with the multi-target activity against isoforms fundamental for the proliferation (CDK1) and survival (CDK9) of normal cells [41–44]. However, in recent years, the interest in identifying inhibitors of specific kinases with targeted action on tumor cells and with less toxic effects, has led to the discovery of selective CDK4/6 inhibitors [30,45]. Third generation CDK inhibitors selectively inhibit CDK4/6 with potent efficacy and reduced toxicity, such as the FDA approved palbociclib (**1**), ribociclib (**2**), and abemaciclib (**3**) (Figure 2) [46–49].

**Figure 2.** FDA approved CDK4/6 inhibitors palbociclib, ribociclib, and abemaciclib.

The search for even more selective CDK4/6 inhibitors is still a challenge in the development of novel anticancer therapies. To date, three ATP-competitive CDK 4/6 selective inhibitors (trilaciclib, lerociclib and SHR-6390) are under advanced clinical trials. Trilaciclib (G1T28, **4**, Figure 3) is currently ongoing phase II trials in patients with small cell lung cancer (NCT02514447, NTC02499770) and with hormone receptor negative BC (NCT02978716), while entered in phase III for metastatic colorectal cancer (NCT04607668) [50–53]. Phase II (NCT02983071) trial of lerocliclib (G1T38, **5**, Figure 3) examined the effect in combination with fulvestrant in hormone receptor-positive, HER2-negative locally advanced metastatic BC while another phase II study combined lerociclib with osimertinib in EGFRmutant non-small cell lung cancer (NCT03455829) [54,55]. SHR-6390 (**6**, Figure 3) is currently ongoing phase II trial in patients with hormone receptor positive, ErbB2 negative BC (NCT03966898) [56,57]. Moreover, an abemaciclib related compound, BPI-16350 (**7**, Figure 3), recently entered phase I clinical trial (NCT03791112) in patients with advanced solid tumor and the estimated study completion date is in December 2021 [58].

Based on their mode of action, kinase inhibitors can be divided into two types: ATPcompetitive and non-competitive inhibitors. The reported CDK4/6 small molecule inhibitors abemaciclib, palbociclib and ribociclib are ATP-competitive inhibitors, forming hydrogen bonds in the ATP binding site with the kinase "hinge" residues (Val101 in CDK6) and hydrophobic interactions in the region normally occupied by the ATP adenine ring (Figure 4A,C,D) [59–61].

**Figure 3.** Chemical structure of ATP-competitive CDK 4/6 selective inhibitors in clinical trials.

**Figure 4.** (**A**,**C**,**D**) 2D interaction diagram of the three approved CDK4/6 selective inhibitors as observed in their X-ray complexes into CDK6: (**A**) palbociclib (PDB ID: 5L2I); (**C**) ribociclib (PDB ID: 5L2T); (**D**) abemaciclib (PDB ID: 5L2S). (**B**) 3D representation of the X-ray binding geometry of palbociclib (stick, yellow C atoms) into the CDK6 binding site (dark green solid surface). Protein residues involved in key H-bond interactions are represented as stick; H-bonds are depicted as magenta dashed lines.

According to the study of Chen et al., the interactions enhancing the CDK inhibition and selectivity over other kinases are the H-bonds with the sidechains of His100 and

Lys43, as shown for abemaciclib (Figure 4B) and a solvent-mediated interaction of a positively charged atom of the ligand and a solvent-exposed ridge consisting of Asp104 and Thr107 [62].

Given the great similarity in the structure of the approved compounds, several research groups have tried to identify different compounds with increased CDK4/6 selectivity, reduced adverse effects while maintaining or improving treatment efficacy. In this review we focused the attention on the last five years progress in the optimization of clinically approved CDK4/6 inhibitors, primarily tested for their BC anti-tumor activity. The main works, considering both the small-molecule inhibitors and the PROteolysis-TArgeting Chimeras (PROTACs) are summarized.

### **2. Small-Molecule Inhibitors**

Small molecule inhibitors (SMIs) are compounds of ≤500 Da size, often administered orally, useful in cancer diseases. The traditional chemotherapy includes single or combination-therapy of drug targeting dividing tumor cells. The principal drawback of this non-targeted approach is the non-selective action on both normal and cancer cells. SMIs bind to specific molecular targets (targeted therapy) and selectively eliminate malignant cells [63,64]. The small size allows to use these molecules towards extracellular, surface, or intracellular proteins, including anti-apoptotic proteins that play a key role in cell growth and promotion of metastases. The most interesting targets in the antitumor field are mainly kinases such as serine/threonine/tyrosine kinases, matrix metalloproteinases (MMP), and CDKs [63–68].

The following SMIs are classified based on their chemical scaffold.

### *2.1. Thiazolyl-Pyrimidine Derivatives*

Starting from the structure of abemaciclib and considering the fundamental interactions with the hinge region of CDK4/6, Tadesse et al. synthesized three series of compounds keeping constant pyrimidine, pyridine, and the amino linker to maintain the coplanarity of the two rings for ATP-mimetic kinase inhibitors (Figure 5), that represents the characteristic moiety of the three approved inhibitors [69].

The thiazole C2 amino moiety, introduced in previously synthesized CDK9 inhibitors, established a H-bond with the highly conserved Asp (Asp163 in the CDK6) residue among CDKs family of the Asp-Phe-Gly motif in the ATP-binding pocket, improving strong hydrophobic interaction of the methyl thiazole with the gatekeeper residues [70]. For this reason, the 2-amine-thiazole was introduced in C4 of the pyrimidine scaffold. The pharmacophore of the Tadesse's lead compound is the *N*-(5-(piperidin-1-yl)pyridin-2-yl)- 4-(thiazol-5-yl)pyrimidine (**8**–**10**, Figure 5). The main modifications of the first series (**8**, Figure 5) concerned the mono or di-substitution of the amine group (R1, R2) with methyl, ciclopentyl, phenyl or *iso*-propyl; the introduction of a fluorine atom in C5 of pyrimidine (R4) to optimize the pharmacokinetic properties; the N4 (R5) of the piperazine was substituted with a variety of ionizable groups. The most active compound (**8a**, Figure 5) contains a morpholine ring and a cyclopentyl substitution of the amine (K<sup>i</sup> CDK4 = 0.004 µM, K<sup>i</sup> CDK6 = 0.030 µM). Its antiproliferative activity was assessed by MTT assay in human leukemia Rb positive MV4-11 (GI<sup>50</sup> = 0.209 µM), and in human breast cancer Rb negative MDA-MB-453 (GI<sup>50</sup> = 3.683 µM) cell lines. The SAR analysis shows that the amino group in the thiazole ring could accept only a mono-substitution and the cyclopentyl is better than alkyl chain or aromatic ring; the introduction of the electron withdrawing trifluoromethyl increases the toxicity; the substitution of the second nitrogen atom of the piperazine with carbon and the exocyclic primary amino decreases activity and selectivity, while the introduction of oxygen increases the selectivity toward CDK4/6.

**Figure 5.** CDK4/6 inhibitors based on thiazolyl-pyrimidine scaffold.

μ μ μ μ μ μ μ μ μ μ μ μ In the second series of derivatives (**9**, Figure 5), the amino group on thiazole ring was replaced with alkyl, ether or thioether substituents, a cyano group or chlorine atom on C5 of pyrimidine was introduced, and the C4 of piperidine was replaced by an oxygen atom or secondary or tertiary amine substituted with alkyl or acetyl group [71]. Among the synthesized compounds, **9a** and **9b** (Figure 5) emerged with good CDK inhibition values (K<sup>i</sup> CDK4 = 0.010 and 0.007 µM, K<sup>i</sup> CDK6 = 1.67 and 0.042 µM, respectively) and antiproliferative activity in MV4–11 (GI<sup>50</sup> = 0.591 µM and 0.456 µM, respectively). The antiproliferative activity of compounds **9a**–**b** was also evaluated in a panel of human cancer cell lines including breast, colon, ovary, pancreas, prostate, leukemia and melanoma. Noteworthy, the different antiproliferative activity in the MDA-MB-453 (GI<sup>50</sup> = 3.32 µM and 4.17 µM, respectively) and the corrensponding Rb-deficient MDA-468 (GI<sup>50</sup> = 8.03 µM and 7.16 µM, respectively) confirms the mechanism of action of these compounds which act in the presence of the intact Rb function. The potent effect on the growth of melanoma M249 (GI<sup>50</sup> = 0.47 µM and 0.91 µM, respectively) and of resistant to dabrafenib M249R (GI<sup>50</sup> = 0.27 µM and 0.91 µM, respectively) cell lines paves the way for a possible therapeutic use in melanoma. The SAR analysis highlighted that the presence of the nitrogen atom of the pyridine is fundamental for CDK4/6 selectivity, and the substitution of the amine group in C2 of the thiazole with alkyl, thioether or ether prevents the H-bond with the conserved Asp163, increasing the selectivity.

In another series of derivatives (**10**, Figure 5) the substitution of the pyridine with a benzene ring, the mono-substitution of the amino group of the thiazole, the introduction of fluorine atom or cyano group in C5 of pyrimidine, and the alkylation of the piperidine nitrogen or the introduction of morpholine or piperidine were studied [72]. Compound **10a** (Figure 5) was the most active in biological assays, with good CDK 4/6 inhibition (K<sup>i</sup> CDK4 = 0.002 µM, K<sup>i</sup> CDK6 = 0.279 µM), selectivity over other kinases and good pharmacokinetic profile. The antiproliferative activity was tested in different cancer cell lines such as leukemia or solid cancers (breast, colorectal, melanoma, ovarian, prostate).

### *2.2. Benzimidazolyl-Pyrimidine Derivatives*

μ μ

Zha et al. focused their attention to the substitution of the abemaciclib pyridine with a benzoimidazolyl in C4 and the introduction of tetrahydro-naphthyridine as substituent of the amino linker, with the aim to discover conformationally restricted analogs sharing improved activity and selectivity [73]. Compound **11** (Figure 6), discovered through the combination of structure-based drug design and traditional medicinal chemistry approaches, retains all key contacts between abemaciclib and CDK6, such as the edge-to-face interaction of the benzimidazole ring and Phe98 of the gatekeeper and the H-bonds of the amino pyrimidine with residues in the hinge loop. The tetrahydro-naphthyridine forms an additional water-mediated H-bond between the aromatic nitrogen with His100 and a salt-bridge interaction of the physiologically protonated nitrogen and Asp104 (Figure 4D) [19,74].

**Figure 6.** CDK4/6 inhibitors based on benzimidazolyl-pyrimidine scaffold.

The authors explored the removal of the nitrogen in pyridine, in 2,4-pyrimidine or the substitution with a 4,6-pyrimidine, confirming that the nitrogen atoms are fundamental for CDK6 selectivity over CDK1. Although compound **11** exhibited good activity (IC<sup>50</sup> CDK4 = 1.5 nM) and a selectivity index of 311, the very poor pharmacokinetic properties required further optimization. In compounds of the series **12** (Figure 6) a cyclic or acyclic alkyl substitution of the *iso*-propyl of the imidazole of compound **11** was introduced without a substantial improvement of activity, suggesting that the hydrophobic cleft of the protein could not host rigid or bulky groups. Compounds of the series **13** (Figure 6) contain a variety of substituents on the protonable nitrogen of tetrahydro-naphthyridine. The hydrophilic substitutions maintain the inhibition potency, with amide analogues suffering from poor exposure; the introduction of a *N*-alkyl piperidine improves the inhibitory activity and selectivity. In fact, compound **13a** (Figure 6) emerged as the best compound, with enzymatic CDK4 IC<sup>50</sup> of 1.4 nM and selectivity CDK1/CDK4 around 850 (IC<sup>50</sup> CDK1 = 1180 nM), good antiproliferative activity in Colo-205 cell line (IC<sup>50</sup> = 0.057 µM),

favourable *in vitro* metabolic properties (microsomal stability and CYP isoforms inhibition) and robust pharmacokinetic properties in mice and rats.

μ

Wang et al. synthesized and tested a library of abemaciclib analogs [75]. The tetracycle scaffold (piperazine, pyridine, pyrimidine and benzimidazole) was kept constant, while small substituents were introduced on piperazine nitrogen (ethyl, 2-fluorethyl, cyclopropyl, *iso*-propyl), in C6 of pyridine (methyl), in C5 of pyrimidine (fluorine), and in C4 of benzimidazole (fluorine). The benzimidazole ring was transformed in a tricycle by connecting N1 and C2, inserting a cyclopentyl, cyclohexyl or cycloheptene [75].

A large library of 23 compounds (**14**, Figure 7) was tested for CDK1 and CDK4/6 activity. Compounds demonstrated null activity versus CDK1, but a remarkable inhibition of CDK4/6, with IC<sup>50</sup> ranging from 0.6 to 340 nM. Compound **14a** (IC<sup>50</sup> CDK4 = 7.4 nM and IC<sup>50</sup> CDK6 = 0.9 nM) was further tested for hERG channel inhibition, showing low heart toxicity. The pharmacokinetic parameters of **14a** (Cmax, AUC, T1/2, MRT, CL/f, V2/F) demonstrated drug-like properties for following development. A docking study revealed that the di-methyl cyclopentyl group contributed to favourable H-bond between the nitrogen atom of the imidazole-condensed cycle and the amine group of Lys43.

**Figure 7.** CDK4/6 inhibitors based on benzimidazolyl-cycloalkyl-pyrimidine scaffold.

The studies on Colo-205 subcutaneous xenografts tumor model in BALB/c nude mice revealed that compound **14a** was not well tolerated and had a narrow therapeutic window [73]. For this reason, Shi's research group synthesized two novel series of benzimidazolyl-pyrimidine containing the tetrahydro-naphthyridines with a dimethylaminoethyl group as substituent on protonable nitrogen of the bicycle: in the series **15** (Figure 8) the nitrogen atom of imidazole was substituted with alkyl or cycloalkyl, while in the series **16** (Figure 8) the protonable nitrogen of the ethylamine chain was substituted [76]. Compound **16a** (Figure 8) demonstrated nanomolar *in vitro* activity (IC<sup>50</sup> CDK4 = 0.71 nM, IC<sup>50</sup> CDK6 = 1.10 nM) with high kinase selectivity, excellent metabolic properties, good pharmacokinetic properties, low toxicity, and desirable antitumor efficacy in MCF-7, Colo-205, and A549 xenograft murine models. Even though compounds of series **16** possessed fair CDK4/6 activity in the range of nanomolar (IC<sup>50</sup> = 0.999–6.14 nM), many of them failed in antiproliferative activity in MCF-7, T-47D, ZR-75-1, and Colo-205 cell lines.

The SAR analysis demonstrated that, with respect to *N*-*iso*-propyl (**16a**), the *N*-methyl or *N*-ethyl substitution of the imidazole decreased the activity more than others alkyl or cycloalkyl groups in terms of CDK 4/6 activity. The cycloalkyl, probably for its steric hindrance, also decreases the antiproliferative activities. Considering the substitution of *N*-methyl on the nitrogen of the ethylamine chain on tetrahydro-naphthyridine, the introduction of a bulkier group was unproductive in terms of CDK4/6 activity and selectivity over CDK2.

**Figure 8.** Structure of Shi's research group of benzimidazolyl-pyrimidine derivatives.

### *2.3. Pyrido-Pyrimidine Derivatives*

μ μ μ Considering the pyrido [2,3-*d*]pyrimidine scaffold of palbociclib, Abbas et al. synthesized two series of 7-thienylpyrido[2,3-*d*]pyrimidines (**17** and **18**, Figure 9) and tested them for CDK6 inhibition and cytotoxicity against breast, lung, and prostate cancer cell lines [77]. In the series **17**, the 2-aryldiene hydrazinyl moiety was introduced in the scaffold and the aryl group was substituted in *para*-position. The most active compound of this series resulted **17a**, containing a *para*-methoxy group on the benzene ring. In fact, it demonstrated a CDK6 IC<sup>50</sup> value of 115.38 nM and a good cytotoxicity against breast MCF-7 (IC<sup>50</sup> = 1.59 µM), prostate PC-3 (IC<sup>50</sup> = 0.01 µM), lung A-549 (IC<sup>50</sup> = 2.48 µM) cancer cells. μ μ μ μ μ μ

**Figure 9.** CDK6 inhibitors based on the pyrido-pyrimidine scaffold.

The series **18** is constituted by a fused ring to pyrimidine forming a tricyclic pyridothyazolopyrimidine, substituted in C2 with a *para*-substituted benzylidene. Compound **18a** emerged as the most potent CDK6 inhibitor (IC<sup>50</sup> = 726.25 nM) and cytotoxic against

MCF-7 (IC<sup>50</sup> = 0.01 µM), prostate PC-3 (IC<sup>50</sup> = 1.37 µM), lung A-549 (IC<sup>50</sup> = 1.69 µM) cancer cell lines.

### *2.4. Imidazo-Pyrido-Pyrimidine Derivatives*

The pyrido-pyrimidine scaffold of palbociclib was fused with imidazole in two novel series of CDK4/6 inhibitors containing the fused tricyclic ring of imidazo[10,2′ :1,6]pyrido[2,3 d]pyrimidine (**19** and **20**, Figure 10) [78]. ′

**Figure 10.** Structure of imidazo[10,2 ′ ′ :1,6]pyrido[2,3-d]pyrimidine derivatives.

In the series **19**, the fused tri-heteroaryl structure was substituted with a methyl in C5 and C8, cyano in C6, while the amino group in C2 was substituted by phenyl or parasubstituted phenyl groups. This type of modification did not sufficiently improve the activity, and compound **19a** with the piperazine in *para* position showed modest inhibition values (IC<sup>50</sup> CDK4 = 26.50 nM, IC<sup>50</sup> CDK6 = 33.60 nM). Keeping constant the piperazine moiety, in the series **20** the C6 and C8 positions were changed by the introduction of methyl, *iso*-propyl, *terz*-butyl, cyclopentyl, cyclohexyl, phenyl, ethylester, or pyrrolidine-1-carbonyl in C8, while the cyano group in C6 was replaced with the acetyl one. Compound **20a** was the best one of the series in terms of inhibition (CDK4 IC<sup>50</sup> = 0.8 nM, CDK6 IC<sup>50</sup> = 2.0 nM).

The piperazine ring of compound **20a** was also replaced by saturated heterocycle, distanced by a methyl and a carbonyl linker; alternatively the piperazinyl-pyridine portion was replaced with a fused bicycle. None of these changes improved the inhibition of kinases [79]. Compound **20a** demonstrated good activities on Colo-205 (IC<sup>50</sup> = 56.4 nM), and glioma U87MG (IC<sup>50</sup> = 84.6 nM) cell lines, favourable *in vitro* metabolic properties (microsomal stability, CYP isoforms inhibition), acceptable pharmacokinetic profiles in mice and rats, antitumor efficacy with controllable observed side effects in xenograft in vivo studies.

### *2.5. Pyrazolo-Quinazoline Derivatives*

Considering the inhibition activity of different kinases (Aurora-A, CDK2, Polo-like Kinase 1) of the 4,4-dimethyl-4,5-dihydro-1*H*-pyrazolo[4,3-h]quinazoline [80], Zhao et al. synthesized a series of 4,5-dihydro-1*H*-pyrazolo[4,3-h]quinazolines (**21**, Figure 11) and tested their inhibition of CDK4/6 [79]. The amine group in C2 position of the quinazoline was substituted with pyridine or benzene ring. Compounds containing the pyridine confirmed that the nitrogen atom in this position affects not only the inhibitory activity, but also the cellular activity against MCF-7 cell line. In fact, the pyridine derivatives were more active as CDK4/6 inhibitors and displayed improved cellular activity.

**Figure 11.** Structure of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives.

μ μ μ μ Compound **21a** was the best one of this series, showing good activity on CDK4/6 (IC<sup>50</sup> CDK4 = 0.01 µM, IC<sup>50</sup> CDK6 = 0.026 µM) and high selectivity against CDK2 (IC<sup>50</sup> CDK2 = 0.70 µM), anti-proliferative activity in MCF-7 cell line (IC<sup>50</sup> = 0.19 µM) and other solid tumors (colorectal, liver, pancreatic), favorable pharmacokinetic parameters (T1/2, CL, AUC, V, Cmax).

### **3. PROTACS**

The therapeutic use of small-molecule inhibitors to target proteins such as transcription factors, non-enzymatic, and scaffolding proteins, has several limitations because these targets lack appropriate active site to be occupied that directly modulate protein functions [81]. Moreover, high systemic drug exposures in the use of small molecules that bind to the active site of a protein are required to achieve site occupation, which may lead to an increase in adverse effects caused by binding to off-target sites [82]. Other complications in the prolonged use of small molecule inhibitors are the possible mutation of the target protein and the establishment of resistance to the therapy, the overexpression of such protein to balance the inhibition drug-mediated, and the accumulation. These mechanisms are associated with the partial or overall suppression of the downstream signaling pathways [83].

A strategy to circumvent the problem of binding site occupancy to regulate the inhibition of a protein and the possibility of significantly expand the number of proteins that can be inhibited, is the use of small-molecule-induced protein degradation. In this way, the pharmaceutical advantages deriving from the use of small molecules are preserved and the proteins generally considered "undraggable" are removed [84]. These hybrid molecules, generally called PROteolysis-TArgeting Chimeras (PROTACs), are constituted by two small binding molecules connected by a linker (Figure 12): one domain is directed to the targeted protein, while the other domain binds E3 ubiquitin ligase. The complex allows the binding of the proteolytic ubiquitin on the target protein, and its consequent degradation of the targeted protein in proteasome. PROTACs act catalytically and are not destroyed as small molecule suicide inhibitors that permanently bind target macromolecules [85,86].

**Figure 12.** A schematic representation of proteolysis targeting chimera. The PROteolysis-TArgeting Chimera (PROTAC) is composed by a portion that binds to the ubiquitin ligase and a small molecule that binds to target protein, joined by a linker. When the targeted protein binds the small molecule, and the other part binds to E3 ligase, a ternary complex is formed. The following poliubiquitination of the target allows the proteasome to degrade the target protein and regenerate the PROTAC.

PROTAC strategy is widely applied to degrade proteins related to immune disorders, neurodegenerative diseases, viral infections, and cancer diseases [87–89]. In this paragraph the application of PROTAC strategy to CDK4/6 inhibitors is summarized.

The use of this approach could be exploited to selectively inhibit CDK6 with respect to CDK4, which have specific functions, could derive from the use of PROTACs. In fact, the binding site of ATP in kinases 4 and 6 possesses a high structural similarity, that could hardly be circumvented with the use of small molecules. All the reviewed studies have in common the binding of the E3-binding portion (E3 ligase ligand) to the nitrogen atom of the piperazine of the three approved CDK4/6 inhibitors. In fact, the crystallographic studies of palbociclib, ribociclib and abemaciclib show that the piperazine ring is projected towards the solvent (Figure 4B), in an optimal position to act as an anchor point.

Among the first studies reporting a PROTAC active towards CDK4/6, emerges the work of Zhao and Burgess [90], who combined palbocilib and ribocilib with pomalidomide (cereblon (CRBN), E3 ligase ligand) by means of a linker containing a triazole ring (**22a**–**b**, Figure 13). Studies on MDA-MB-231, a triple negative breast cancer cell line, showed that CDK4 is degraded more efficiently and PROTAC containing palbociclib (**22a**) is more potent (DC<sup>50</sup> CDK4 = 12.9 nM, DC<sup>50</sup> CDK6 = 34.1 nM) than **22b** (DC<sup>50</sup> CDK4 = 97 nM, DC<sup>50</sup> CDK6 = 300 nM). The same CDK degradation and cytotoxicity studies conducted on MCF-7 showed that **22a**–**b** are less efficient towards this cell line with respect to the triple negative cell line.

In the same period, Rana et al. synthesized a chimera series of palbociclib and pomalidomide by changing the length and the composition of the flexible linker (**23**, Figure 14) [91].

**Figure 13.** Chemical structures of palbociclib or ribociclib/pomalidomide PROTACs.

**Figure 14.** Chemical structures of palbociclib/pomalidomide PROTACs.

All compounds with shorter linker degrade CDK6 partly, while the PROTAC containing the longest linker (**23a**, Figure 14) selectively degraded CDK6 at the single dose of 500 nM in pancreatic cancer MiaPaCa2 cells with respect to other cyclin-dependent kinases including CDK4. Two hypotheses on the selective behavior of this PROTAC could be found in the less stable ternary complex palbociclib-E3 ligase-CDK4, that avoids the degradation, or in the fast deubiquitination of CDK4. The quantitative degradation of only CDK6 (CDK4 was not affected) was observed for compound **23a** in a dose-response study at 4 and 24 h in Human Pancreatic Nestin-Expressing ductal (HPNE) and MiaPaCa2 cells at 100 nM.

Another library of PROTACs containing CDK4/6 inhibitor and pomalidomide was synthesized by Su et al. (**24**, Figure 15), in which the influence of the length and rigidity of the linker, the spatial orientation of the target protein and the E3 ligase, and the binding affinity of PROTAC to CDK4 and 6 were studied [92].

PROTACs containing ribociclib did not degrade CDK6, while for the others best results in selectively degradation CDK6 was obtained with shorter linkers. In particular, the linker anchoring group to CDK inhibitors (amide, triazole, or methylene) did not influence the activity while to the other side, the best anchoring group to E3 ligase was the amino group, demonstrating that the flexibility of this portion is fundamental to correctly interact. The most potent PROTAC **24a** possesses a DC<sup>50</sup> value of 2.1 nM in glioblastoma U251 cells and demonstrated good potency also in hematopoietic cancer cells, including multiple myeloma MM.1S (IC<sup>50</sup> 10 nM).

**Figure 15.** PROTACs containing CDK4/6 inhibitors and pomalidomide synthesized by Su et al.

Jiang and co-workers prepared a library of palbociclib, ribociclib, and abemaciclib PROTACs (**25**–**27**, Figure 16) connected to pomalidomide through an alkyl or polyethylene glycol (PEG) linker [93].

**Figure 16.** PROTACs containing CDK4/6 inhibitors and pomalidomide synthesized by Jiang et al.

PROTACs of each CDK4/6 inhibitor demonstrated degrading activity of both CDK4 and 6, but abemaciclib-PROTACs also induced the degradation of the off-target CDK9,

that should be avoided [60]. The type of the linker (length and structure) and the CDK4/6 inhibitor of the PROTAC influenced the selectivity of degradation at 100 nM: compound **25a** (alkyl linker conjugated to palbociclib) indifferently degraded both CDK4 and CDK6, **25b** (extended PEG-3 linker conjugated to palbociclib) selectively hit CDK6, while **26a** (4-carbon alkyl linker conjugated to ribociclib) was selectively toward CDK4.

Compounds containing the imide group were tested for their capability to inhibit Ikaros (IKZF1) and Aiolos (IKZF3), well-established targets of imide-based degraders [94–96]. Compounds **25a**–**b** and **26a** degraded also IKZF1/3, resulting in an enhnanced antiproliferative effect on mantle cell lymphoma lines.

Compound **25c** was previously synthesized by Brand and co-workers and studied for its ability to selectively degrade CDK6 over CDK4, in particular the correlation between the use of the CDK6 degrader in acute myeloid leukemia cells was investigated [97].

In a recent study, Anderson et al. evaluated the effect of other E3 ligase, such as von Hippel-Lindau (VHL) and Inhibitor of Apoptosis (IAP) instead of CRBN, on the selective degradation of CDK4/6, maintaining the anchoring on the nitrogen of piperazine of palbociclib and using different types of linkers (**28**, Figure 17) [98].

**Figure 17.** PROTACs palbociclib and E3 ligase ligands, such as von Hippel-Lindau (VHL) and Inhibitor of Apoptosis (IAP) ligands and pomalidomide.

The dose-response study in Jurkat cells after 24 h revealed that the degradation of CDK4 and CDK6 occurred independently of the type of E3 ligases (VHL, CRBN, and IAP binder), with a CDK4 pDC<sup>50</sup> in the range of 6.2–8.0 and CDK6 pDC<sup>50</sup> in the range of 7.7–9.1. It is important to note that all of them show a greater degradation power towards CDK6, probably due to a better stability of the formed ternary complex.

Compounds **28a**–**b**, containing VHL and IAP, are the less potent degraders (**28a**: pDC<sup>50</sup> CDK4 = 5.6; pDC<sup>50</sup> CDK6 = 5.3; **28b**: pDC<sup>50</sup> CDK4 = 6.7; pDC<sup>50</sup> CDK6 = 5.8), probably due to the linker nature. The most potent CDK4/6 degrader is **25a**, previously reported by Jiang (pDC<sup>50</sup> CDK4 = 8.0, pDC<sup>50</sup> CDK6 = 9.1) [93].

Compounds **25a** was taken into account by Steinbach et al. to synthesize novel palbociclib based PROTACs by changing the E3 ligase portion and inserting various linkers (**29**–**31**, Figure 18) [99]. In the series **29**, pomalidomide was linked by an amide linker to the palbociclib piperidine, avoiding the protonation of the previously synthesized tertiary amine that could affect activity and selectivity. The linkers were polyethylene or alkyl chain of different size. These compounds were tested in multiple myeloma (MM.1S) cell lines at 0.1 µM and the activity of PROTACs was shown as the percentage of remaining CDK levels (D). The degradation percentage (D) of CDK6 for compounds **29a**–**c** was in the range of 7.7–8.4 and the selectivity over CDK4 in the range of 1.9–3.3. In the series **30** and **31**, palbociclib was linked to VHL ligand functionalized in two different positions to create an amide or a phenoxy group in the E3 ligase ligand side, while in the other side of the linker there was an amine group. Compound **30a** showed a degradation percentage 1.7 and a selectivity CDK4 ratio of 19, while compound **31a** showed a comparable degradation activity (D CDK6 = 1.4) but an improved selectivity (DCDK4/DCDK6 = 31). PROTACs **30a** and **31a** were also tested in different cancer cell lines (multiple mieloma, acute myeloid leukemia, acute lymphoid blastic leukemia) inhibiting cell proliferation. μ

**Figure 18.** PROTACs containing palbociclib and E3 ligase ligand, such as pomalidomide and VHL ligand.

### **4. Conclusions**

Since 2015, the arsenal of drug against breast cancer is enriched with third-generation CDK4/6 inhibitors. Three compounds (palbociclib, ribociclib, abemaciclib) have been approved by the FDA for the treatment of breast cancer in association with endocrine therapy. These ATP-competitive compounds share a common portion interacting with the ATPbinding site; in fact, they contain the pyridine-amine-pyrimidine scaffold, that determines the formation of more than one H-bond with the hinge residue of the target kinases.

In the last five years, a number of small molecule inhibitors have been synthesized and tested in order to identify compounds more potent, selective, and with improved pharmacokinetic parameters. The main heteroaromatic scaffold, that represents the central part of the molecule, was kept constant, while different groups or additional cycles were introduced on the terminal portions.

The use of PROTACs (proteolysis targeting chimeras), composed combining the CDK4/6 inhibitor small molecule and an E3 ligase ligand, is a novel approach to selectively degrade the targeted kinases. The anchoring point in CDK inhibitor is the nitrogen of the piperazine, which is extended towards the outside of the binding site, without interfering with the ATP-binding site. The majority of studies have been done on palbociclib and pomalidomide, by varying the type (nature and length) of the linker, although studies on the other two approved CDK inhibitors and different E3 ligases are reported. These studies have shown that it is possible to selectively degrade CDK4 or CDK6, depending on the type of inhibitor and linker, although the single inhibitor acts to a comparable extent on the two kinases.

Moreover, in addition to the study on breast cancer, the actions on other cancer cell lines have been explored. The development of new CDK inhibitors or degraders will certainly continue over the next years and possibly will allow to treat other forms of cancer with improved potency and less side effects.

**Author Contributions:** Conceptualization and supervision: M.F.; literature review: M.F., A.A., M.A., B.D.F. figures: M.F., A.A., M.A., B.D.F. writing and review: M.F., A.A., M.A., B.D.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding. The APC was funded by MDPI.

**Data Availability Statement:** The data presented in this study are available in this article.

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

### **Abbreviations**


### **References**


*Article*

## **A Series of Isatin-Hydrazones with Cytotoxic Activity and CDK2 Kinase Inhibitory Activity: A Potential Type II ATP Competitive Inhibitor**

**Huda S. Al-Salem 1, \*, Md Arifuzzaman 2 , Hamad M. Alkahtani 1 , Ashraf N. Abdalla 3 , Iman S. Issa 1 , Aljawharah Alqathama 4 , Fatemah S. Albalawi <sup>1</sup> and A. F. M. Motiur Rahman 1, \***


Academic Editor: Marialuigia Fantacuzzi Received: 2 September 2020; Accepted: 18 September 2020; Published: 25 September 2020

**Abstract:** Isatin derivatives potentially act on various biological targets. In this article, a series of novel isatin-hydrazones were synthesized in excellent yields. Their cytotoxicity was tested against human breast adenocarcinoma (MCF7) and human ovary adenocarcinoma (A2780) cell lines using MTT assay. Compounds **4j** (IC<sup>50</sup> = 1.51 ± 0.09 µM) and **4k** (IC<sup>50</sup> = 3.56 ± 0.31) showed excellent activity against MCF7, whereas compound **4e** showed considerable cytotoxicity against both tested cell lines, MCF7 (IC<sup>50</sup> = 5.46 ± 0.71 µM) and A2780 (IC<sup>50</sup> = 18.96± 2.52 µM), respectively. Structure-activity relationships (SARs) revealed that, halogen substituents at 2,6-position of the C-ring of isatin-hydrazones are the most potent derivatives. In-silico absorption, distribution, metabolism and excretion (ADME) results demonstrated recommended drug likeness properties. Compounds **4j** (IC<sup>50</sup> = 0.245 µM) and **4k** (IC<sup>50</sup> = 0.300 µM) exhibited good inhibitory activity against the cell cycle regulator CDK2 protein kinase compared to imatinib (IC<sup>50</sup> = 0.131 µM). A molecular docking study of **4j** and **4k** confirmed both compounds as type II ATP competitive inhibitors that made interactions with ATP binding pocket residues, as well as lacking interactions with active state DFG motif residues.

**Keywords:** isatin-hydrazones; cytotoxicity; CDK2 inhibitor; ATP competitive inhibitor; ADME analysis

### **1. Introduction**

Development of anticancer drugs is essential due to the increasing number of morbidity and mortality by cancer day-by-day all over the world. According to the International Agency for Research on Cancer, in 2018, around 18 million people were infected; 9.6 million people among them had died due to life threatening cancer [1,2]. It is rather alarming that cancer morbidity cases may increase to 29.5 million by 2040 [3]. Having said that, it is very much challenging to develop an anticancer drug due to the long and expensive synthesis/isolation process and the huge lack of opportunities to conduct clinical trials. Moreover, most of the anticancer drugs currently available are lacking specificity and have adverse effects. In this context, developing novel anticancer agents with great efficacy and

high specificity becomes imperative. To overcome these challenges, researchers should develop a drug molecule with potent biological activity and low/no toxicity, study its mode of action, in silico properties and in vitro/vivo metabolism, conduct a toxicity evaluation [4,5], study its topoisomerase inhibitory activity [6–8] and enzyme inhibitory activity [9], etc., all of which are some of the key evaluation practices for the development of potential anticancer therapeutics. Regarding enzyme inhibitory activities, cyclin-dependent kinases (CDKs) are considered as a vital feature, inciting various key transitions in the cell cycle for cancer cells, in addition to instructing apoptosis, transcription and exocytosis. CDKs are active only when bound to their regulator proteins, cyclins. CDK activity is tightly controlled for successful cell division. Since abnormal cell division represents cancer pathology, controlling CDK activity has been shown as a promising therapeutic strategy. In particular, CDK2 plays an important role in DNA replication. Therefore, therapeutic strategies based on the inhibition of CDKs work as an encouraging viewpoint for anticancer drug discovery. With that being said, to consider a compound, such as a drug molecule, as a treatment, it is still necessary to first test their drug likeness properties as well as analyze their physiological descriptors, such as absorption, distribution, metabolism and excretion (ADME). ADME is an important physiological descriptor of chemical compounds used for selecting potential drug targets. However, testing a wide range of compounds directly in the clinical or pre-clinical phase is extensively time consuming and costly. Moreover, ADME is considered as the last step of drug development, where many drugs (approximately 60%) fail after all the procedures. To tackle these problems, recent experiments have utilized in silico ADME tools as the first step to shortlist the amount of target compounds by calculating predicted ADME properties and discarding the compounds with unsatisfactory ADME values from the drug designing pipeline [10].

Isatin (**1**) is an organic compound first discovered in 1840 by Erdmann and Laurent from the oxidation of indigo dye [11,12]. It was considered as a synthetic product until isolated from natural sources, such as *Couroupita guianensis* [13], *Isatis tinctoria* [14] and *Calanthe discolor* [15], and from many other sources [16–18]. It has been reported that tryptophan obtained from food sources is usually converted to indole by gastrointestinal bacteria, which is further oxidized in the liver by CYP450 to isatin, therefore, isatin is present as an endogenous molecule in humans [19,20]. Various substituents on the isatin nucleus displayed numerous biological activities [21–36], including antimicrobial activity[31,37], topoisomerase inhibitory activity [7,38], epidermal growth factor receptor (EGFR) inhibitory activity [39], inhibitory activities on histone deacetylase (HDAC) [40,41], carbonic anhydrase [42–44], tyrosine kinase [45–47], cyclin-dependent kinases (CDKs) [9,48,49], adenylate cyclase inhibition [50] and protein tyrosine phosphatase (Shp2) [51]. A number of isatin-based marketed drugs and potential anticancer agents [41] are illustrated in Figure 1. Considering the importance of the development of anticancer therapeutics and the various biological properties of isatin and isatin nucleus-containing derivatives, a series of isatin-hydrazones were designed and synthesized, their cytotoxicities against two different cancer cell lines, namely MCF7 (human breast adenocarcinoma) and A2780 (human ovary adenocarcinoma), were evaluated, their structure–activity relationships (SARs) were studied, their ADME properties were studied using in silico ADME tools and cyclin-dependent kinases 2 inhibitory activities were performed using an enzyme inhibition assay. Additionally, docking simulations were conducted in order to explore the behavior of the synthesized compounds within the active site of CDK2 to justify its binding mechanism.

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

### *2.1. Synthesis of Isatin-Hydrazones (***4***)*

Synthesis of 3-((substituted)benzylidene)hydrazono)indolin-2-one (**4**) was straightforward, as illustrated in Scheme 1 [36,52]. In the first step, a mixture of isatin (**1**) and hydrazine hydrate was refluxed in ethanol and isatin monohydrazone (**2**) was obtained in quantitative yields (~99%). Subsequently, the isatin monohydrazone (**2**) was refluxed with substituted aryl aldehydes (**3**) in the presence of a catalytic amount of glacial acetic acid in absolute ethanol to

obtain 3-((substituted)benzylidene)hydrazono)indolin-2-one (**4**) in good to excellent yields (75–98%). The structures of the synthesized compounds were confirmed using IR, NMR (1H and <sup>13</sup>C) and mass spectral data, as well as reported values that are known.

**Figure 1.** Isatin moiety containing active and potential drugs. 

‐ ‐ ‐ **Scheme 1.** Synthesis of 3-((substituted)benzylidene)hydrazono)indolin-2-one (4).

‐ ‐ ‐

### *2.2. Biological Evaluation*

‐ ‐ ‐

### 2.2.1. Cytotoxicity

The cytotoxicity of the synthesized compounds **4a**–**k** was evaluated against two different cancer cell lines, namely MCF7 and A2780, and the results are summarized in Table 1. Among the tested compounds, the isatin-hydrazone **4j** exhibited the highest inhibitory activity against MCF7 cell lines (1.51 ± 0.09 µM). It should be noted that **4k** (3.56 ± 0.31), **4e** (5.46 ± 0.71), **4i** (7.77 ± 0.008) and **4f** (9.07 ± 0.59) showed moderate inhibitory activity against MCF7 cell lines. In the case of A2780 cell lines, however, only the halogen-substituted compounds **4e, 4j, 4k** and **4f** showed a little inhibitory activity. Nevertheless, all of the tested compounds were more sensitive towards MCF7 compared to A2780 cell lines.

μ

‐

‐


˃ ˃ ˃ ˃

˃ ˃ ˃

**Table 1.** Cytotoxicity of **4a**–**k** against MCF7 and A2780 cell lines.

Figure 2 shows the dose–response curves for compounds **4j** and **4k,** which were the most cytotoxic compounds against the breast cancer cells lines (MCF7) at a concentration of 1.51 and 3.56 µM, respectively. The IC<sup>50</sup> values interpolated from dose–response data with five different concentrations were 0.1, 1, 10, 25 and 50 µM. μ μ

**Figure 2.** Dose–response curve of the most cytotoxic compounds against MCF7 cell lines.

### 2.2.2. Structure–Activity Relationships (SARs) Study of **4a**–**k**

‐ ‐ ‐ μ ‐ The SARs study revealed that the cytotoxicity of **4a**–**k** increased or decreased in the same fashion as increases or decreases in halogen substitution in the aromatic C-ring. It is also related to the position of the substituents. As depicted in Figure 3, the bromo substituent at 4-position of the C-ring gave IC<sup>50</sup> = 15.7 µM against MCF7 cell lines (Table 1, entry **4g**), while at 3-position, it increased to IC<sup>50</sup> = 9.07 µM (Table 1, entry **4f**). Interestingly, the bromo substituent's cytotoxicity at 2-position, increased dramatically to IC<sup>50</sup> = 5.46 µM (Table 1, entry **4e**). Surprisingly, while 2- and 6-positions of the C-ring having respective chloro- and fluoro- substituents, the IC<sup>50</sup> of compound **4k** was 3.56 µM (Table 1, entry **4k**). More surprisingly, with both 2- and 6-positions of the C-ring with chloro- substituents, compound **4j** exhibited the highest cytotoxicity of IC<sup>50</sup> = 1.51 µM (Table 1, entry **4j**) which is two-fold more than the control anticancer drug doxorubicin (IC<sup>50</sup> = 3.1 µM) (Table 1, entry doxorubicin). On the other hand, the methyl substituent at the C-ring also affects cytotoxicity against MCF7 cell lines. The methyl substituent at 4-position gave IC<sup>50</sup> = 32.48 µM (Table 1, entry **4c**) and it increased at 3-position to IC<sup>50</sup> = 14.65 µM (Table 1, entry **4b**), whereas at 2-position, the IC<sup>50</sup> value was 10.82 µM

(Table 1, entry **4a**). On the other hand, A2780 cell lines were inhibited by the halogenated derivatives **4e**, **4j**, **4k** and **4f**. In this case, 2-bromo substituted derivatives showed higher activity than the other three. μ ‐ 

‐ μ

‐ μ ‐

μ ‐

‐ ‐ ‐ μ

μ ‐ ‐

‐

‐ μ

‐ ‐ ‐ ‐ μ

**Figure 3.** Structure–activity relationship (SAR) analysis of compounds **4a**–**k**.

### 2.2.3. CDK2 Protein Kinase Inhibitory Activity of **4a**–**k**

‐ The promising cytotoxicity of **4**, especially **4j** and **4k,** motivated us to study further inhibitory activities against CDK2 protein kinase. As summarized in Table 2, **4j** and **4k** exhibited good inhibitory activity against cyclin-dependent kinase 2 (CDK2), which is half of that of the known kinase inhibitor imatinib.

**Table 2.** Inhibitory activities of compounds **4j** and **4k** against CDK2 protein kinase.


\* IC<sup>50</sup> values are the mean ± SD of triplicate measurements.

### *2.3. In Silico Drug Likeness Property Analysis*

‐ Rational drug designing is the most significant part in modern drug discovery approaches. In this regard, computational ADME (absorption, distribution, metabolism and excretion) analysis can help us select the best drug in terms of cost, time and efficiency. Applying computational chemistry tools, in vitro and in vivo ADME prediction is now much more convenient and it can aid pharmaceutical industries to screen thousands of compounds within a short time [53]. Here, synthesized compounds (**4a**–**k**) were screened for predicted ADME values and the results are summarized in Table 3. Since high molecular weight compounds are always less effective in terms of intestinal absorption [54,55], our designed and synthesized isatin-hydrazones' (**4a**–**k**) molecular weights were kept low, in between 263–328 Da. Compounds **4a**–**k** showed hydrogen bond donor (HBD) values of 1, except **4i** which had a HBD value of 2 (recommended value = <5), and hydrogen bond acceptor (HBA) values of 5, except **4i** which had HBA value = 6.5, **4h** with a HBA value = 5.75 and **4d** with **a** HBA value = 5.5 (recommended value = <10). On the other hand, doxorubicin (Doxo) showed a HBD value of 5 and HBA value of 15, which indicates that synthesized isatin-hydrazones are superior to Doxo in respect to HBD and HBA values. A parameter was established in 2002 to check the bioavailability of a drug using octanol/water partition coefficient and solubility scoring (recommended values for octanol/water partition coefficient are −2 – 6.5 and solubility scoring are −6.5 – 0.5 mol/dm−3) [56]. The octanol/water partition coefficient for hydrazones **4a**–**k** is in between 1.79–3.12 and solubility score is −3.39 – −4.35, respectively. Doxo

showed a score within the reference values of −0.49 and −2.37, respectively. The hERG K<sup>+</sup> channel blockers are potentially toxic for the heart, thus the recommended range for predicted logIC<sup>50</sup> values for blockage of hERG K<sup>+</sup> channels (loghERG) is > −5 [57]. Intriguingly, **4a**–**c** and **4e**–**k** showed higher values for loghERG score (> −5.58–−5.91) than Doxo (−6.02), except **4d,** which was similar to Doxo, which proved their (**4a**–**k)** toxicity to be lower than Doxo. The Caco-2 cell, considered as the reliable in vitro model to estimate oral drug absorption and transdermal delivery [58], was high (>1310) for all compounds except **4i** (487). Interestingly, Doxo had a much lower value (2.29) than **4a**–**k**, which signifies the improved oral drug absorption and transdermal delivery efficiency of the studied compounds compared to Doxo.


**Table 3.** Analysis of drug likeness and pharmacokinetic properties by QikProp for compounds **4a–k**.

<sup>a</sup> Molecular weight in Daltons (acceptable range: <500); <sup>b</sup> hydrogen bond donor (acceptable range: ≤5); <sup>c</sup> hydrogen bond acceptor (acceptable range: ≤10); <sup>d</sup> predicted octanol/water partition coefficient (acceptable range: −2–6.5); <sup>e</sup> predicted aqueous solubility, S in mol/dm−3 (acceptable range: −6.5–0.5); <sup>f</sup> predicted IC<sup>50</sup> value for blockage of hERG K+ channels (concern: below −5); <sup>g</sup> Caco−2 value, permeability to Caco−2 (human colorectal carcinoma) cells in vitro; <sup>h</sup> blood−brain barrier permeability (acceptable range: ~−0.4); <sup>i</sup> predicted apparent Madin–Darby canine kidney (MDCK) cell permeability in nm/sec, QPPMDCK= >500 is great, <25 is poor; <sup>j</sup> predicted human oral absorption on 0% to 100% scale (<25% is poor and >80% is high); <sup>k</sup> Doxo = Doxorubicin.

The blood–brain barrier separates the CNS from blood, and a successful compound must pass into the blood stream, which depends on several factors, such as molecular weight, which must be below 480 [59]. Since our synthesized compounds have low molecular weights and fall within the recommended values, this, therefore, showed significant results. Madin–Darby canine kidney (MDCK) cell permeability is considered as the measurement of blood–brain barrier permeability, where greater than 500 is of great value and less than 25 indicates a very poor result according to Jorgensen's rule of 3 [60]. Except compound **4i** (227), all the other compounds gave much higher MDCK values (> 666 to 3162) than Doxo (0.766 only). The synthesized compounds also gave a predicted human oral absorption rate of 100%, except compound **4i** which gave 86%. On the other hand, Doxo showed a predicted human oral absorption rate of 0%. Taken together, all the designed compounds, **4a**–**k**, of this study showed higher predicted ADME values than Doxo.

### *2.4. Architecture of the CDK2 Active Site*

Developing new inhibitors against CDK2 mainly involves designing compounds that can act as ATP competitive inhibitors by binding to the ATP binding cleft of CDK2. According to the active and inactive state of the protein kinase, two different types of inhibitors can be designed: type I and type II inhibitors. Type I inhibitors mainly bind to the ATP binding pocket of an active kinase, whereas type II inhibitors bind to the inactive kinase [61]. From the recently published crystal structure of CDK2 in a complex with the inhibitor CVT-313, it was found that active kinase inhibition depends solely on the interaction with the DFG motif, which comprises Asp145-Phe146- Gly147. Leu83, Asp86 and Asp145 form the ATP binding site of CDK2 through hydrogen bonds, where Asp145 belongs to the DFG motif. Outside of the active site, the residues Glu81–Leu83 hinge linker sequence is responsible for flexibility of the kinase. The phosphorylation of the C-terminal domain contains the catalytic residue (Glu51) required for the phosphorylation of Thr160 in the T-loop for its activation. The activation segment is composed of the conserved DFG motif (Asp145-Phe146- Gly147) and the APE motif (Ala170-Pro171-Glu172). The unique PSTAIRE motif (Pro45–Glu51) in CDK2 that has a key role in its interaction with the cyclin subunit is found in the N-terminal domain [61]. To investigate whether the synthesized compounds (**4j** and **4k**, based on best cytotoxicity assay and enzyme inhibition assay against CDK2 protein kinase) are type I or type II inhibitors, and also to check their binding mechanism with CDK2, we performed a molecular docking analysis.

### *2.5. In Silico Binding Mechanism Analysis*

From the docking analysis of compounds **4j** and **4k** with CDK2, it was clearly observed that both compounds interacted with the ATP binding pocket residues Leu83 and Asp86 but not with Asp145 of the DFG motif (Figure 4A,D). **4j** and **4k** thus can act as ATP competitive type II inhibitor by binding to inactive kinase [61]. Moreover, both the compounds showed a similar fashion of interactions, which involved several hydrogen bonds and ionic interactions with the binding site cavity surrounding residues, such as Ile10, Val18, Ala31, Val64, Glu81, Phe82, Leu134 and Ala144, which is consistent with the molecular docking analysis of 3,6-disubstituted pyridazines; 6-N,6-N-dimethyl-9-(2-phenylethyl)purine-2,6-diamine as CDK2 inhibitors (2, 3) [62,63]. However, compound **4j** formed one additional interaction with Lys89, which was absent in case of compound **4k**. It may bind to inactive kinase, which can be analyzed by finding no interaction between the compounds with catalytic residue Glu51 that is responsible for the phosphorylation of Thr160 for activation of kinase function. The interacting residues, although they do not belongs to the ATP binding pocket, formed a pathway for the compounds to bind properly to the ATP binding pocket. Glu81 to Leu83, on the other hand, forms the hinge region responsible for the flexibility of the protein kinase. From Figure 4C,F, it is visible how these residues form the binding cleft and pathway for the compounds to occupy the ATP binding site of CDK2 protein kinase. Figure 4B,E show the 3D interaction pattern and formation of binding cleft. From the docking analysis, it can be concluded that both **4j** and **4k** served as ATP competitive type II inhibitors by interacting with ATP binding pocket residues and with the residues that paved the way for the compounds to bind to the CDK2 ATP binding pocket. Table 4 summarizes the compounds and names the interacting residues, along with the types of interactions and the docking score of each compound.


**Table 4.** Docking score, interacting residues and types of interaction mediated by **4j** and **4k** with the ATP binding pocket of CDK2 protein kinase.

**Figure 4.** (**A**) 2D docking pose of **4j** within the active site of CDK2; (**B**) 3D docking pose of **4j** within **Figure 4.** (**A**) 2D docking pose of **4j** within the active site of CDK2; (**B**) 3D docking pose of **4j** within the active site of CDK2; (**C**) binding pocket formed by interacting residues of active site of CDK2 surrounding **4j**; (**D**) 2D docking pose of **4k** within the active site of CDK2; (**E**) 3D docking pose of **4k** within the active site of CDK2; (**F**) binding pocket formed by interacting residues of active site of CDK2 surrounding **4k**. Compounds are shown in red color, protein in cyan and interacting residues in blue color.

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

### *3.1. General*

− π‐ Chemicals and solvents were of commercial reagent grade (Sigma-Aldrich, St. Louis, MO, USA) and were used without further purification. The progress of reactions and purity of reactants and products were checked using pre-coated silica gel 60 aluminum TLC sheets with fluorescent indicator

π‐σ

UV254 of Macherey-Nagel, and detection was carried out with an ultraviolet light (254 nm) (Merck, Darmstadt, Germany). Melting points were measured using an Electrothermal IA9100 melting point apparatus (Stone, Stafforshire, ST15 OSA, UK). Infrared (IR) spectra (as KBr pellet) were recorded on a FT-IR Spectrum BX device from Perkin Elmer (Ayer Rajah Crescent, Singapore). <sup>1</sup>H NMR spectra were recorded using a Bruker 600 MHz spectrometer (Reinstetten, Germany) and DMSO-d<sup>6</sup> was used as a solvent. Chemical shifts were expressed in parts per million (ppm) relative to TMS as an internal standard. Mass spectra were taken with an Agilent 6410 Triple Quad mass spectrometer fitted with an electrospray ionization (ESI) ion source (Agilent Technologies, Palo Alto, CA, USA).

### *3.2. (Z)-3-Hydrazonoindolin-2-one (***2***)*

A mixture of isatin (0.1 mole) and hydrazine hydrate (1.2 equiv.) in methanol was refluxed for 1h and cooled to room temperature. The precipitate was filtered, washed with cold methanol and dried at room temperature in open air to give isatin monohydrazone in quantitative yield (~99%). A yellow powder was obtained (~99%). Mp. = 230–231 ◦C (Lit. [64] Mp. = 231–232 ◦C).

### *3.3. General Procedure for the Synthesis of 3-[benzylidene(substituted)hydrazono]indolin-2-ones 4a–k*

A mixture of isatin monohydrazone (**2**, 5 mmol) and 4-methylbenzaldehyde (**3a**, 5 mmol) in absolute ethanol (15 mL) was added to a few drops of glacial acetic acid. The reaction mixture was refluxed for 4 h. The completion of the reaction was monitored by TLC. The precipitate solid was filtered, washed with cold ethanol and air dried, and was then further purified by recrystallization using ethanol, obtained **4a** as yellow powder. Please see in Supplementary Materials for NMR (1H & <sup>13</sup>C) and MS spectra of compound **4** (in Supplementary Materials).

### 3.3.1. 3-((2-Methylbenzylidene)hydrazono)indolin-2-one (**4a**)

Yellow powder (80%). Mp. = 198–199 ◦C. IR (KBr) νmax(cm−<sup>1</sup> ): 3238 (N-H), 2910 (C-H), 1728 (C=O), 1612 (C=N). <sup>1</sup>H NMR (DMSO-d6, 600 MHz) (ppm), δ 2.52 (s, 3H, -CH3), 6.89 (t, 1H, ArH), 7.01 (t, 1H, ArH), 7.32–7.44 (m, 4H, ArH), 7.89 (t, 1H, ArH), 8.06 (t, 1H, ArH), 8.80 (s, 1H), 10.88 (s, 1H, -NH). <sup>13</sup>C NMR (DMSO-d6, 150 MHz) (ppm), δ 165.02, 159.58, 150.71, 145.45, 139.72, 134.20, 132.31, 131.78, 129.00, 127.96, 127.06, 122.83, 116.81, 111.35 and 19.58. ESI mass *m*/*z* = 264 [M + H]+; 286 [M + Na]+.

### 3.3.2. 3-((3-Methylbenzylidene)hydrazono)indolin-2-one (**4b**)

Yellow powder (82%). Mp. = 183–184 ◦C. <sup>1</sup>H NMR (DMSO-d6, 600 MHz) (ppm), δ 2.39 (s, 3H, -CH3), 6.89 (t, 1H, ArH), 7.02 (t, 1H, ArH), 7.39 (m, 2H, ArH). 7.44 (t, 1H, ArH), 7.56 (m, 2H, ArH), 7.88 (t, 1H, ArH), 8.53 (s, 1H), 10.86 (s, 1H, -NH). <sup>13</sup>C NMR (DMSO-d6, 150 MHz) (ppm), δ 164.93, 160.61, 150.64, 145.46, 139.02, 134.19, 133.83, 133.28, 129.87, 129.58, 129.20, 126.34, 122.86, 116.82, 111.32 and 21.36. ESI mass *m*/*z* = 264 [M + H]+; 286 [M + Na]+.

### 3.3.3. 3-((4-Methylbenzylidene)hydrazono)indolin-2-one (**4c**)

Orange powder (75%). Mp. = 230–231 ◦C. (Lit. [65] mp. = 231 ◦C) IR (KBr) νmax(cm−<sup>1</sup> ): 3182 (N-H), 2839 (C-H), 1716 (C=O), 1612 (C=N). <sup>1</sup>H NMR (DMSO-d6, 600 MHz) (ppm), δ 2.38 (s, 3H, -CH3), 6.88 (t, 1H, ArH), 7.02 (t, 1H, ArH), 7.37 (m, 3H, ArH), 7.86 (m, 2H, ArH), 7.93 (t, 1H, ArH), 8.58 (s, 1H), 10.86 (s, 1H, -NH). <sup>13</sup>C NMR (DMSO-d6, 150 MHz) (ppm), δ 165.02, 161.34, 150.91, 145.4, 142.96, 134.11, 131.29, 130.3, 129.39, 129.26, 122.83, 116.91, 111.28 and 21.73. ESI mass *m*/*z* = 264 [M + H]+; 286 [M + Na]+.

### 3.3.4. 3-((4-(Methylthio)benzylidene)hydrazono)indolin-2-one (**4d**)

Red crystals (79%). Mp. = 204–205 ◦C. IR (KBr) νmax(cm−<sup>1</sup> ): 3278 (N-H), 2920 (C-H), 1732 (C=O), 1612 (C=N). <sup>1</sup>H NMR (DMSO-d6, 600 MHz) (ppm), δ 2.53 (s, 3H, S-CH3), 6.88 (t, 1H, ArH), 7.02 (t, 1H, ArH), 7.33–7.50 (m, 3H, ArH). 7.77–7.95 (m, 3H, ArH). 8.59 (s, 1H), 10.84 (s, 1H, -NH). <sup>13</sup>C NMR

(DMSO-d6, 150 MHz) (ppm), δ 165.07, 161.47, 151.01, 145.40, 144.69, 134.09, 130.09, 129.75, 129.28, 129.13, 126.05, 125.93, 122.79, 116.97, 111.27 and 14.50. ESI mass *m*/*z* = 296 [M + H]+; 318 [M + Na]+.

### 3.3.5. 3-((2-Bromobenzylidene)hydrazono)indolin-2-one (**4e**)

Yellow powder (93%). Mp. = 233–234 ◦C. IR (KBr) νmax(cm−<sup>1</sup> ): 3194 (N-H), 2818 (C-H), 1730 (C=O), 1535 (C=N). <sup>1</sup>H NMR (DMSO-d6, 600 MHz) (ppm), δ 6.89 (d, *J*=1.2 Hz, 1H, ArH), 7.02 (t, 1H, ArH), 7.40 (t, 1H, ArH), 7.51 (t, 1H, ArH), 7.59 (t, 1H, ArH), 7.80–7.58 (m, 2H, ArH), 7.22 (t, 1H, ArH), 8.72 (s, 1H), 10.90 (s, 1H, -NH). <sup>13</sup>C NMR (DMSO-d6, 150 MHz) (ppm), δ 164.75, 158.16, 150.99, 145.73, 134.56, 134.18, 134.13, 132.23, 129.25, 129.09, 125.60, 122.93, 116.62 and 111.45. ESI mass *m*/*z* = 328 [M(79Br) + H]+, 330 [M(81Br) + H]+; 350 [M(79Br) + Na]+, 352 [M(81Br) + Na]+.

### 3.3.6. 3-((3-Bromobenzylidene)hydrazono)indolin-2-one (**4f**)

Yellowish brown powder (92%). Mp. = 182–183 ◦C. IR (KBr) νmax(cm−<sup>1</sup> ): 3412 (N-H), 2920 (C-H), 1714 (C=O), 1676 (C=N). <sup>1</sup>H NMR (DMSO-d6, 600 MHz) (ppm), δ 6.89 (t, 1H, ArH), 7.01 (t, 1H, ArH), 7.39–7.53 (m, 2H, ArH), 7.71–7.87 (m, 2H, ArH), 7.99–8.10 (m, 1H, ArH), 8.54 (s, 1H), 10.91 (s, 1H, -NH). <sup>13</sup>C NMR (DMSO-d6, 150 MHz) (ppm), δ 164.77, 161.07, 158.28, 150.52, 145.60, 136.16, 134.98, 134.42, 131.97, 131.84, 131.60, 131324, 129.16, 127.72, 127.56, 122.92, 116.64 and 111.41. ESI mass *m*/*z* = 328 [M(79Br) + H]+, 330 [M(81Br) + H]+; 350 [M(79Br) + Na]+, 352 [M(81Br) + Na]+.

### 3.3.7. 3-((4-Bromobenzylidene)hydrazono)indolin-2-one (**4g**)

Orange powder (90%). Mp. = 267–268 ◦C. IR (KBr) νmax(cm−<sup>1</sup> ): 3169 (N-H), 2879 (C-H), 1735 (C=O), 1616 (C=N). <sup>1</sup>H NMR (DMSO-d6, 600 MHz) (ppm), δ 6.89 (t, 1H, ArH), 7.01 (t, 1H, ArH), 7.39 (t, 1H, ArH), 7.77 (t, 2H, ArH), 7.83 (t, 1H, ArH), 7.90 (t, 2H, ArH), 8.58 (s, 1H), 10.89 (s, 1H, -NH). <sup>13</sup>C NMR (DMSO-d6, 150 MHz) (ppm), δ 164.87, 159.42, 150.73, 145.52, 134.35, 133.01, 132.75, 131.05, 129.25, 126.18, 122.90, 116.72 and 111.38. ESI mass *m*/*z* = 328 [M(79Br) + H]+, 330 [M(81Br) + H]+; 350 [M(79Br) + Na]+, 352 [M(81Br) + Na]+.

### 3.3.8. 3-((4-Methoxy-2,6-dimethylbenzylidene)hydrazono)indolin-2-one (**4h**)

Orange powder (90%). Mp. = 251–252 ◦C. IR (KBr) νmax(cm-1): 3182 (N-H), 2839 (C-H), 1716 (C=O), 1612 (C=N). <sup>1</sup>H NMR (DMSO-d6, 600 MHz) (ppm), δ 2.18 (s, 3H, -CH3) 2.52 (s, 3H, -CH3) 3.85 (t, 3H, OCH3), 6.88 (d, *J* = 9, 2H, ArH), 7.03 (t, 1H, ArH), 7.36 (t, 1H, ArH), 7.85 (t, 1H, ArH), 8.02 (t, 1H, ArH), 8.78 (s, 1H), 10.82 (s, 1H, -NH). <sup>13</sup>C NMR (DMSO-d6, 150 MHz) (ppm), δ 165.32, 161.75, 160.97, 150.83, 145.19, 140.40, 133.81, 130.36, 129.01, 124.59, 123.77, 122.79, 117.04, 113.28, 111.17, 56.06, 19.70 and 16.19. ESI mass *m*/*z* = 308 [M + H]+; 330 [M + Na]+.

### 3.3.9. 3-((2-Hydroxy-4-methoxybenzylidene)hydrazono)indolin-2-one (**4i**)

Reddish brown (98%). Mp. = 242–243 ◦C. IR (KBr) νmax(cm−<sup>1</sup> ): 3188 (O-H), 2910 (C-H), 1724 (C=O), 1620 (C=N). <sup>1</sup>H NMR (DMSO-d6, 600 MHz) (ppm), δ 3.81 (s, 3H, OCH3), 6.52 (t, 1H, ArH), 6.59 (t, 1H, ArH), 6.87 (t, 1H, ArH), 7.04 (t, 1H, ArH), 7.40 (t, 1H, ArH), 7.55 (t, 1H, ArH), 7.60 (t, 1H, ArH), 8.97 (s, 1H), 10.9 (s, 1H, -NH), 12.31 (s, 1H, -OH). <sup>13</sup>C NMR (DMSO-d6, 150 MHz) (ppm), δ 167.89, 165.07, 163.03, 159.79, 150.21, 144.64, 135.36, 133.90, 122.69, 120.40, 111.69, 111.20, 108.06, 101.54 and 56.08. ESI mass *m*/*z* = 296 [M + H]+; 318 [M + Na]+.

### 3.3.10. 3-((2,6-Dichlorobenzylidene)hydrazono)indolin-2-one (**4j**)

Orange powder (98%). Mp. = 286–287 ◦C. IR (KBr) νmax(cm−<sup>1</sup> ): 3165 (N-H), 2812 (C-H), 1730 (C=O), 1618 (C=N). <sup>1</sup>H NMR (DMSO-d6, 600 MHz) (ppm), δ 6.89 (t, 1H, ArH), 6.97 (t, 1H, ArH), 7.39–7.55 (m, 2H, ArH), 7.65 (t, 2H, ArH), 7.83 (t, 1H, ArH), 8.71 (s, 1H), 10.91 (s, 1H, -NH). <sup>13</sup>C NMR (DMSO-d6, 150 MHz) (ppm), δ 164.67, 155.50, 150.49, 145.80, 134.76, 134.72, 133.03, 129.98, 128.85, 122.77, 116.48 and 111.46. ESI mass *m*/*z* = 318 [M(35Cl) + H]+, 320 [M(37Cl) + H]+; 340 [M(35Cl) + Na]+, 342 [M(37Cl) + Na]+.

### 3.3.11. 3-((2-Chloro-6-fluorobenzylidene)hydrazono)indolin-2-one (**4k**)

Reddish brown (75%). Mp. = 277–778 ◦C. IR (KBr) νmax(cm−<sup>1</sup> ): 3165 (N-H), 2852 (C-H), 1732 (C=O), 1620 (C=N). <sup>1</sup>H NMR (DMSO-d6, 600 MHz) (ppm), δ 6.89 (t, 1H, ArH), 6.99 (t, 1H, ArH), 7.39–7.45 (m, 2H, ArH), 7.52 (t, 1H, ArH), 7.62 (t, 1H, ArH), 7.94 (t, 1H, ArH), 8.73 (s, 1H), 10.90 (s, 1H, -NH). <sup>13</sup>C NMR (DMSO-d6, 150 MHz) (ppm), δ 164.80, 162.18, 160.46, 154.25, 150.99, 145.77, 135.57, 134.74, 134.35, 134.29, 128.86, 127.07, 122.78, 119.84, 119.76, 116.67, 116.58, 116.44 and 111.43. ESI mass *m*/*z* = 302 [M(35Cl) + H]+, 304 [M(37Cl) + H]<sup>+</sup> 324 [M(35Cl) + H]+, 326 [M(37Cl) + H]+.

### *3.4. Cytotoxicity*

The cytotoxicity of the synthesized compounds was evaluated by MTT assay, as previously described [66]. Two cancer cell lines, MCF7 (human breast adenocarcinoma) and A2780 (human ovary adenocarcinoma), were used in this study, which were obtained from the ATCC (Rockville, MD, USA). They were sub-cultured in RPMI-1640 media (supplemented with 10% FBS and 1% antibiotics) at 37 ◦C and 5% CO2. Additionally, compounds were prepared at the same medium to obtain serial dilutions (50, 25, 19,1 and 0.1 µM). The two cell lines were separately cultured in 96-well plates (3 × 10<sup>3</sup> /well) and incubated at 37 ◦C overnight. The following day, before treating the cells with the compounds, each well of the T0 plate was treated with 50 µL MTT solution (2 mg/mL in phosphate buffered saline) and then incubated for 2–4 h. The media were aspirated, and the formazan crystals were solubilized by adding 150 µL DMSO. Absorbance was read on a multi-plate reader (BioRad) at 550 mm. Optical density of the purple formazan A550 was proportional to the number of viable cells. Compound concentration causing 50% inhibition (IC50) compared to control cell growth (100%) was determined. The data were obtained from triplicates and analyzed using statistical software.

### *3.5. In Vitro Cyclin Dependent Kinase2 (CDK2) Inhibitory Activity*

The CDK2 Assay Kit is designed to measure CDK2/CyclinA2 activity for screening and profiling applications, using Kinase-Glo® MAX as a detection reagent. The CDK2 Assay Kit comes in a convenient 96-well format, with enough purified recombinant CDK2/CyclinA2 enzyme, CDK substrate peptide, ATP and kinase assay buffer for 100 enzyme reactions [67]. The assay was performed according to the protocol supplied from the CDK2 Assay kit #79599. The CDK2/CyclinA2 activity at a single dose concentration of 10µM was performed, where the Kinase-Glo MAX luminescence kinase assay kit (Promega#V6071) was used. The compounds were diluted in 10% DMSO and 5 µL of the dilution was added to a 50 µL reaction so that the final concentration of DMSO was 1% in all of the reactions. All of the enzymatic reactions were conducted at 30 ◦C for 40 min. The 50 µL reaction mixture contained 40 mM Tris, pH 7.4, 10 mM MgCl2, 0.1 mg/mL BSA, 1 mM DTT, 10 mM ATP, Kinase substrate and the enzyme (CDK2/CyclinA2). After the enzymatic reaction, 50 µL of Kinase-Glo® MAX Luminescence kinase assay solution was added to each reaction and the plates were incubated for 5 min at room temperature. Luminescence signal was measured using a Bio Tek Synergy 2 microplate reader.

### *3.6. Molecular Docking and In-Silico ADME Analysis*

For molecular docking purposes, the Protein Data Bank (PDB) structure corresponding to the CDK2 protein kinase was downloaded from the Research Collaboratory for Structural Bioinformatics (RCSB) PDB database (https://www.rcsb.org/) in PDB format. The PDB ID used for CDK2 protein kinase was 2BHY. Proteins and compounds were prepared for docking by using an established procedure [68]. Discovery Studio was used for making 2D interaction figures. Pymol was used to generate the 3D and surface representation figures. For the in silico ADME analysis, all the compounds' structures were prepared with the LigPrep module of Schrodinger Maestro and ADME was calculated by the Qikprop module of the same software package [69].

### **4. Conclusions**

A series of novel isatin-hydrazones (**4a**–**b and 4d**–**k**), with a known compound **4c,** were designed and synthesized with good to moderate yields for cytotoxicity evaluation for the development of potent anticancer therapeutics. Among the compounds, **4j** showed a two-fold increase in cytotoxicity compared to the known cancer drug doxorubicin, and **4k** showed a similar cytotoxicity. The IC<sup>50</sup> value of compound **4j** was 1.51 and for **4k** it was 3.56 µM, whereas doxorubicin had a 3.1 µM concentration against human breast adenocarcinoma (MCF7) cell lines. The most active compounds, **4j** and **4k,** were further evaluated for their inhibitory activities against CDK2 protein kinase. As expected, **4j** and **4k** exhibited good inhibitory activity against cyclin-dependent kinase 2 (CDK2) 0.2456 and 0.3006 µM, respectively, which is comparable to kinase imatinib 0.1512 µM. Highly recommended predicted ADME values were obtained than the known doxorubicin. The molecular docking study of **4j** and **4k** with CDK2 protein kinase revealed that they interacted with ATP binding pocket residues and lacked interactions with the active state DFG motif residues; therefore, **4j** and **4k** can be considered as ATP competitive type II inhibitors against CDK2 protein kinase. In conclusion, these simple molecules, isatin-hydrazones **4j** and **4k,** can be used as potential agents for anticancer therapeutics in further mechanism and toxicity studies.

**Supplementary Materials:** The following are available online, Figure S1.: Proton (1H) Spectra of **4a**, Figure S2.: Carbon (13C) Spectra of **4a,** Figure S3.: Proton (1H) Spectra of **4g**, Figure S4.: Carbon (13C) Spectra of **4g,** Figure S5.: Proton (1H) Spectra of **4h**, Figure S6.: Carbon (13C) Spectra of **4h,** Figure S7.: Proton (1H) Spectra of **4i**, Figure S8.: Carbon (13C) Spectra of **4i,** Figure S9.: Proton (1H) Spectra of **4j**, Figure S10.: Carbon (13C) Spectra of **4j,** Figure S11.: Proton (1H) Spectra of **4k**, Figure S12.: Carbon (13C) Spectra of **4k,** Figure S13.: Mass Spectra of **4a**, Figure S14.: Mass Spectra of **4b**, Figure S15.: Mass Spectra of **4c**, Figure S16.: Mass Spectra of **4d**, Figure S17.: Mass Spectra of **4e**, Figure S18.: Mass Spectra of **4f**, Figure S19.: Mass Spectra of **4g**, Figure S20.: Mass Spectra of **4g**, Figure S21.: Mass Spectra of **4i**, Figure S22.: Mass Spectra of **4j**, Fifure S23.: Mass Spectra of **4k**.

**Author Contributions:** Conceptualization, H.S.A.-S. and A.F.M.M.R.; methodology, I.S.I., F.S.A., H.M.A., M.A., A.N.A. and A.A.; software, M.A.; validation, A.F.M.M.R. and H.S.A.-S.; formal analysis, I.S.I., F.S.A., H.M.A., A.N.A. and A.A.; investigation, H.S.A.-S. and A.F.M.M.R.; resources, H.S.A.-S.; data curation, M.A., I.S.I., F.S.A., H.M.A., M.A. and A.N.A.; writing—original draft preparation, A.F.M.M.R.; writing—review and editing, A.F.M.M.R. and H.S.A.-S.; visualization, I.S.I., F.S.A., H.M.A., M.A., A.N.A. and M.A.; supervision, A.F.M.M.R.; project administration, H.S.A.-S.; funding acquisition, H.S.A.-S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the King Saud University, Research Center of the Female Campus for Scientific and Medical Studies.

**Acknowledgments:** This research project was supported by a grant from the research center of the Female Campus for Scientific and Medical Studies, King Saud University, Riyadh, Saudi Arabia. The authors are thankful to Mrs. Yin Wencui for English editing.

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

### **References**


**Sample Availability:** Samples of the compounds **4a**–**k** 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*

## **Tamoxifen and the PI3K Inhibitor: LY294002 Synergistically Induce Apoptosis and Cell Cycle Arrest in Breast Cancer MCF-7 Cells**

**Mohamed E. Abdallah 1 , Mahmoud Zaki El-Readi 1,2, \* , Mohammad Ahmad Althubiti 1 , Riyad Adnan Almaimani 1 , Amar Mohamed Ismail 3 , Shakir Idris 4 , Bassem Refaat 4 , Waleed Hassan Almalki 5 , Abdullatif Taha Babakr 1 , Mohammed H. Mukhtar 1 , Ashraf N. Abdalla 5,6, \* and Omer Fadul Idris 3**


Academic Editors: Marialuigia Fantacuzzi and Alessandra Ammazzalorso Received: 27 June 2020; Accepted: 21 July 2020; Published: 24 July 2020

**Abstract:** Breast cancer is considered as one of the most aggressive types of cancer. Acquired therapeutic resistance is the major cause of chemotherapy failure in breast cancer patients. To overcome this resistance and to improve the efficacy of treatment, drug combination is employed as a promising approach for this purpose. The synergistic cytotoxic, apoptosis inducing, and cell cycle effects of the combination of LY294002 (LY), a phosphatidylinositide-3-kinase (PI3K) inhibitor, with the traditional cytotoxic anti-estrogen drug tamoxifen (TAM) in breast cancer cells (MCF-7) were investigated. LY and TAM exhibited potent cytotoxic effect on MCF-7 cells with IC<sup>50</sup> values 0.87 µM and 1.02 µM. The combination of non-toxic concentration of LY and TAM showed highly significant synergistic interaction as observed from isobologram (IC50: 0.17 µM, combination index: 0.18, colony formation: 9.01%) compared to untreated control. The percentage of early/late apoptosis significantly increased after treatment of MCF-7 cells with LY and TAM combination: 40.3%/28.3% (*p* < 0.001), compared to LY single treatment (19.8%/11.4%) and TAM single treatment (32.4%/5.9%). In addition, LY and TAM combination induced the apoptotic genes Caspase-3, Caspase-7, and p53, as well as p21 as cell cycle promotor, and significantly downregulated the anti-apoptotic genes Bcl-2 and survivin. The cell cycle assay revealed that the combination induced apoptosis by increasing the pre-G1: 28.3% compared to 1.6% of control. pAKT and Cyclin D1 protein expressions were significantly more downregulated by the combination treatment compared to the single drug treatment. The results suggested that the synergistic cytotoxic effect of LY and TAM is achieved by the induction of apoptosis and cell cycle arrest through cyclin D1, pAKT, caspases, and Bcl-2 signaling pathways.

**Keywords:** breast cancer; tamoxifen; LY294002; synergism; apoptosis; cell cycle

### **1. Introduction**

Worldwide, breast cancer (BC) has the highest incidence rate (24.2%) of all cancers in women with more than two million newly diagnosed cases and almost 627,000 deaths (15%) occurred in 2018 [1]. In Saudi Arabia, BC has the largest number of incidences between females (3629 cases: 29.7% of total malignancies) [2]. Nevertheless, frequent tumor recurrence results in poor prognosis of BC patient of which less than 5% survive for more than ten years [3]. Discovering new therapies with improved pharmacokinetics is therefore required for improving the outcome of BC treatment [4].

BC is classified according to the gene expression of estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) into five major molecular subtypes, which are different in growth and prognosis. Theses subtypes include luminal A (ER+/HER2−/low levels of Ki-67 protein), luminal B (ER+/HER2−/+/high levels of Ki-67 protein), triple-negative/basal-like (ER−/HER2−), HER2 enriched (HR−/HER2+), and normal-like BC, which is similar to luminal A, but with poor prognosis [5]. Luminal A and luminal B breast cancer are the most dominant subtype, affecting more than 73% of total BC patients [6].

Tamoxifen (TAM) is the oldest and most-prescribed selective estrogen receptor modulator, that has been approved to treat women and men diagnosed with hormonal receptor (HR+), early-stage BC after surgery to reduce the risk of the cancer recurrence as well as treatment of advanced-stage or metastatic HR<sup>+</sup> BC patients [7–9]. The combination of high efficacy in both pre- and postmenopausal women and a good tolerability profile of TAM lead to maintain its position as drug of choice for most patients with HR<sup>+</sup> breast cancer [7]. In addition, TAM has been used as chemopreventive agent to reduce breast cancer risk in women, who haven't been diagnosed, but are at higher-than-average risk for incidence of BC [10]. These high therapeutic benefits of TAM by binding with the ER causing apoptotic effect on the mammary cells [7,11].

The development of both de novo and acquired resistance to TAM is a significant problem. Recent advances in our understanding of the molecular mechanisms that contribute to resistance have provided means to predict patient responses to TAM and develop rational approaches for combining therapeutic agents with TAM to avoid or desensitize the resistant phenotype [12]. Overcoming the anti ER drug resistance can be achieved by the introduction of new drug classes and combinations that can synergistically improve the efficacy and decrease the effective dose, hence decrease the side effects. Long term estrogen deprivation (LTED) treatment among ER+ BC cells results in adaptive increase in ER expression, which is followed by activation of multiple tyrosine kinases. Combination therapy with the ER down-regulator fulvestrant and the broad kinase inhibitor dasatinib exhibited synergistic activity against LTED cells, by a reduction of cell proliferation, survival, and invasion [13].

In addition, the phosphatidylinositide-3-kinase (PI3K)/Akt signaling pathway is considered as the ideal pathway to explain the transmission of anti-apoptotic signals for cancer cell survival and regulate cell growth, proliferation, transcription, and metabolic processes [14]. The activation of PI3K/Akt signaling pathway is associated with poor prognosis in BC [14]. Inhibitors of PI3K/Akt have undergone pre-clinical evaluation with encouraging results and considered to be one of the most promising targeted therapies for cancer treatment [15]. LY294002 (LY) is a morpholine containing compound with potent inhibitory action for numerous proteins, and a strong inhibition of PI3Ks, which causes induction of apoptosis in tumor cells, but the precise mechanism of its antitumor activity is not completely well understood, as it was also shown to inhibit the invasiveness of cancer cells by downregulating the expression of MMP-2, MMP-9, and VEGF, and reducing MVD [16]. LY, among other PI3K inhibitors, did not reach the clinical trials phase because of its weak drug ability and toxicity [17].

This study was conducted to show the effects of LY or TAM alone or in combination against MCF-7 (ER+) cells. The underlying mechanisms of the possible synergistic effects of this combination are further explored in order to develop a novel and effective therapeutic combination against BC and to reduce the toxicity and resistance of LY and TAM.

### **2. Results**

### *2.1. LY294002 and Tamoxifen Synergistically Inhibited Breast Cancer Cells Proliferation*

The ability of LY to improve the cytotoxicity of tamoxifen on MCF-7 breast cancer was evaluated using an MTT assay. Figure 1A shows dose-response curve of MTT assay. The down deviation of curve was observed in MCF-7 cell treated with LY + TAM combination comparing to each of LY or TAM alone. The combination showed significant synergistic interaction with decrease in IC<sup>50</sup> in MCF-7 cells (0.17 µM) comparing to LY (0.87 µM) and TAM (1.02 µM) treated cells (Figure 1B). Non-toxic concentration 100 nM (85% live cells) from LY and TAM was selected to use in all experimental sets. The synergistic effect of the combination was elucidated from isobologram and combination index value (0.18) Figure 1C. To confirm the synergistic interaction of LY and TAM, the plate colony formation assay was performed. As shown in Figure 2, the combination of LY and TAM exhibited a significant lower percentage of colony formation (9.1%) compared to the control, where LY treated cells showed (27.3%) and TAM showed (36.4%) compared to the control MCF-7 cells. A non-significant difference between LY and TAM treated cells was observed.

**Figure 1.** (**A**): Dose response curve, (**B**): IC<sup>50</sup> values, (**C**): isobolgram and combination index of MCF-7 cells treated with different concentrations of LY, TAM, and LY + TAM combination for 72 h. The IC<sup>50</sup> was calculated using GraphPad Prism V6 by fitting of sigmodal four parameter curve. The data expressed as mean ± SD (*n* = 3, of three experiments). Statistical differences, compared with the control cells, were assessed by a one-way ANOVA with the Tukey's post-hoc multiple comparison test (GraphPad Prism). *p* < 0.001 (\*\*\*) was taken as significant.

**Control LY TAM LY + TAM**

Tukey's post **Figure 2.** Colony formation assay of MCF-7 cells treated with LY, TAM, and LY + TAM combination. MCF-7 cells were treated for 24 h with the experimental set and cells were seeded in 6-well plates (200 cells/well) and incubated for 14 days. The colonies were counted after staining with methylene blue. The colony formation of the treatment set was quantified as a percentage related to untreated control. Statistical differences, compared with the control cells, were assessed by a one-way ANOVA with the Tukey's post-hoc multiple comparison test (GraphPad Prism).). *p* < 0.05 (\*), *p* < 0.001 (\*\*\*) was taken as significant.

### *2.2. LY294002 and Tamoxifen Induced Apoptosis in Breast Cancer Cells*

In order to elucidate the underlying mechanism of the synergistic inhibition of BC cell growth by LY and TAM combination, apoptosis analysis was performed through annexin V FITC/PI double staining. The data revealed that each of LY and TAM were able to induce early/late apoptosis 19.8%/11.4% and 32.4/5.9%, respectively (Figure 3). However, the combination of LY with TAM significantly increased the early/late apoptosis to 40.3/28.3% (*p* < 0.001). To explore the molecular mechanism of increasing in the apoptotic MCF-7 cells, anti-apoptotic and apoptotic genes were measured by immunofluorescence in MCF-7 cells. As shown in Figure 4, the treatment of MCF-7 cells by LY + TAM increased the expression of Caspase-3 and decreased the expression of Bcl-2 compared to the cells treated with either LY or TAM alone. In addition, Figure 5A shows that LY +TAM significantly increased the expression of Caspase-3 3.2 and 9.2-times more compared to TAM and LY alone, respectively. Moreover, caspase-7 was overexpressed in MCF-7 cells 3.4 and 12.6 times higher in treated cells with LY +TAM compared to cells treated with TAM and LY single treatment, respectively. The combination also significantly induced the expression of both p53 and p21: 4 and 2 times more compared to LY, and 6.3 and 3.6 times more compared to TAM, respectively. Additionally, the combination decreased the Bcl-2, BAX, and survivin 2.8 times, 2.5 times, and 3 times more than single treatment with TAM, and 3.1 times, 2.8 times, and 4.46 times more than single treatment LY, respectively. Finally, LY and TAM did not exhibit any change in HER-2 gene, while the combination decreased the expression of HER-2 to 0.45 folds compared to untreated control (Figure 5B)

**Figure 3.** The induction of apoptosis in MCF-7 cells treated with (**A**): control, (**B**): LY, (**C**): TAM, and (**D**): LY + TAM combination for 24 h. Followed by Annexin V FITC/PI staining. The scattered plot *X* axis: FL1 for Annexin V, *Y* axis: FL3 for PI. (**E**): Columns represent the flow cytometry data analysis as means of the percentages of vital, early apoptotic, late apoptotic, and narcotic cells (*n* = 3 of three independent experiments).

**Figure 4.** The induction of apoptosis in MCF-7 cells treated with LY, TAM, and LY + TAM combination 24 h. Images taken with confocal microscope (EVOS FL, scale bar 20 nM) to evaluate the expression of apoptotic (Caspase-3) and antiapoptotic (Bcl-2) markers. The images show green and red color staining for Caspase-3 and Bcl-2, respectively. Overlay images represent the fluorescence intensity of both apoptotic markers.

**Figure 5.** The expression of apoptosis genes in MCF-7 cell after treatment with LY, TAM, and LY+ TAM combination for 24 h. The total RNA was extracted and the mRNA levels of upregulated genes (**A**) and downregulated genes (**B**) was quantified using RT-PCR. The data represented the mean of the fold change related to untreated control (fold change = 1 dashed line).

### *2.3. LY294002 and Tamoxifen Induced Cell Cycle Arrest in Breast Cancer Cells*

The effect of LY, TAM, and LY + TAM combination on the DNA content of MCF-7 cells was assessed using PI staining. The treatments lead to a significant increase in the apoptotic pre-G1cell population phase from 1.6% (untreated control) to 8.1%, 9.8%, and 28.3% in the treated cells with LY, TAM, and LY + TAM, respectively as shown in Figure 6. The cell population of G0/G<sup>1</sup> phase decreased from 69.6% to 53.2%, 55.4%, and 50.6% after treatment with LY, TAM, and their combination, respectively. A non-significant decrease in S phase cell population was observed in all experimental set compared to untreated cells (6.8%). The G2/M cell population was reduced in cells treated with LY (23.7%), TAM (13.7%, *p* < 0.05), and combination (12%, *p* < 0.05) compared to untreated cells (19.1%) (Figure 6).

(**E**)

**Figure 6.** Histograms represent DNA cell cycle distribution in MCF-7 cells treated with (**A**): control, (**B**): LY, (**C**): TAM, and (**D**): LY + TAM combination for 24 h. (**E**): columns represent the flow cytometry data analysis as means of the percentages of pre-G<sup>1</sup> , G<sup>0</sup> /G<sup>1</sup> , S, and G<sup>2</sup> /M (*n* = 3, three independent experiments).

### *2.4. pAKT and Cyclin D1 Decreased in MCF-7 Cells Treated with LY294002 and Tamoxifen*

To explore the molecular targeting of PI3K signaling, AKT, pAKT, and cyclin D1 were assessed after 24 h of the treatment with combination. A Significant decrease of pAKT and cyclin D1 was seen after treatment with LY, ATM, and LY + TAM combination compared to untreated control (Figure 7).

ANOVA with the Tukey's post **Figure 7.** MCF-7 cells were incubated with control, LY, TAM, and LY + TAM for 24 h. Proteins from total cell lysate were separated by SDS-PAGE gel electrophoresis, and immunoblotted with antibodies against AKT, phosphorylated AKT, and cyclin D1. The color density was quantified using densitometry). Statistical differences, compared with the control cells, were assessed by a one-way ANOVA with the Tukey's post-hoc multiple comparison test (GraphPad Prism). *p* < 0.05 (\*), *p* < 0.01 (\*\*) and *p* < 0.001 (\*\*\*) were taken as significant.

### **3. Discussion**

The phosphatidylinositide-3-kinase (PI3K)/Akt signaling pathway regulates many biological processes including cancer cell growth and metastasis [18,19]. Consequently, aberrant activation of the PI3K/Akt signaling pathway is frequently associated with progressive BC, which could be resistant to anticancer therapies [20]. It has been estimated that upregulation of PI3K signalling is involved in around 70% of BC cases [21]. Several PI3K/Akt signaling inhibitors have been effective in inhibiting progression of tumors during pre-clinical and clinical trials and approved by United States Food and Drug Administration (FDA) [22,23]. However, most of these inhibitors have demonstrated only modest clinical efficacy as monotherapies in BC because of drawbacks in their pharmacokinetics and tolerability [23]. Therefore, the combination of PI3K/Akt signaling inhibitors with radiation or chemotherapy is considered as a dynamic research area, approached to overcome therapeutic resistance and enhance treatment efficacy [24]. In a previous study, the tumor associated macrophages were shown to accelerate the endocrine resistance of MCF-7 cells treated with TAM, due to activation of the PI3K/Akt/mTOR signaling pathway [25]. Thus, the co-targeting of ER and PI3K/Akt pathway may stand as a new therapeutic target.

This study was conducted to evaluate the possible synergistic cytotoxic combination effect of LY: as a specific phosphatidylinositide-3-kinase (PI3K) inhibitor, and TAM: as an established BC/ER + drug. MCF-7 cells were used as a model of ER <sup>+</sup> BC. Our results uncovered that the non-toxic dose

of LY and TAM synergistically enhanced their cytotoxicity and clonogenecity against MCF-7 with a significant decrease in IC<sup>50</sup> and combination index (Figures 1 and 2). The cytotoxicity of LY and TAM as single treatments and in combination were previously evaluated on A2780 (ovarian cancer) and MRC-5 (normal fibroblast). The IC<sup>50</sup> of LY were 21.2 µM and 35.7 µM, and for TAM were 10.4 µM and 11.4 µM, and for the combination of LY and TAM were 4.7 and 24.2 µM, respectively [26–28]. When that result was compared with the result of this study, LY and TAM were found to be more effective in MCF-7 cells compared to A2780 cells. In other previous studies, the co-treatment of LY and TAM significantly enhanced the cytotoxicity against lung and brain cancer cells compared to treatment with TAM alone [27,29].

In the second part of this study, the underlying mechanism of the synergistic apoptosis-inducing effect resulting from the treatment of MCF-7 cells with LY and TAM combination, could be explained by the increase of released apoptotic molecules or the decrease of released anti-apoptotic ones. Some of these key apoptotic molecules, which are used as indicators of apoptosis in BC are caspase-3, -7, and p53 [30–33] and p21 as cell cycle promotors [34]. In addition to the Bcl-2 family, which are also considered as important anti-apoptotic genes, their overexpression is frequently related to cancer development [35]. The LY and TAM combination decreased the expression of Bcl-2 and increased the expression of caspase-3 in MCF-7 cells as observed from immunofluorescence experiment (Figure 4). These data were confirmed by determination of mRNA levels of apoptosis genes Bcl-2, BAX, surviving, HER2, p53, p21, caspase-3, and caspse-7 after treatment of MCF-7 cells with LY, TAM, and their combination (Figure 5).

The downregulation of Bcl-2 and survivin was more significant by treatment with the combination more compared to single treatments, which may partly explain the weaker effect of LY or TAM on the induction of apoptosis. While, the non-significant downregulation of BAX might be explained by indirect interaction of LY, TAM or combination with BID/BIM, which required to initiate membrane permeabilization and apoptosis [36]. Survivin is a pro-survival gene and its overexpression is observed in most cancers. It is associated with resistance to chemotherapy and radiation, thus possibly leading to the failure of therapy and poor prognosis [37,38]. Therefore, through the downregulation of anti-apoptotic genes (Bcl-2 and surviving) and overexpression of apoptotic genes (p53 and caspases) the resistance against TAM in breast cancer cells can be reversed.

In this study, the treatment with LY, TAM, and their combination increased the expression of caspase-3 in MCF-7 cells. There are debate in the expression of caspase-3 in MCF-7. Several studies measured levels of caspase-3 indirectly via fluorometric assay systems and by western blotting analyses and reported that directly the presence of this protease in MCF-7 cells [39,40]. However, other reports stated that MCF-7 cells do not express caspase-3 [41]. Our results supposed that the expression of caspase-3 is deficient in untreated MCF-7 cells, but by treatment with LY, TAM, and their combination, the expression of genes was increased as an indication of apoptosis induction by their treatment.

In this study, the induction of apoptosis by LY, TAM, and their combination was confirmed by annexin V/PI double staining using flow cytometry (Figure 3). The pattern of cell death in LY treated cells was suggest necrosis rather than apoptosis Figure 3B. The LY dose (100 nM) that was used in this experiment might be too high to induce necrosis. Previously, it has been reported that LY induced apoptosis and necrosis in mice depending on the treated dose [42].

The combination of LY and TAM increased the percentage of cells in pre-G<sup>1</sup> cell cycle phase and decreased the percentage of cells in G0/G1, S and G2/M phases compared to untreated cells (Figure 6), thus indicating the down regulation of the cell cycle regulation genes. It has been previously reported that one of major problems in BC is the occurrence of cross-resistance that develops due to the change in the expression of DNA damage repair or cell cycle genes [43]. The phosphorylation of AKT can mediate BC resistance to therapy [44,45]. We have shown in this study a significant decrease in the levels of pAKT and cyclin D1 after treatment with LY and TAM combination better than the decreasing effect of LY or TAM alone (Figure 7). These results indicated that the downregulation of the pAKT signaling

pathway could also be responsible for the synergistic cytotoxic effect of LY and TAM combination in MCF-7.

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

### *4.1. Compounds and Reagents*

LY294002 and tamoxifen were purchased from Selleckchem, Houston, TX, USA. All reagents and kits used in this study were purchased from Sigma-USA, unless other manufacturer is mentioned.

### *4.2. Cell Culture*

MCF-7 cells (breast adenocarcinoma) was obtained from the ATCC. For sub-culture, RPMI-1640 media (10% FBS; 1% Antibiotic-Antimycotic, Gibco) was used. Cells were kept at 95% humidity, 37 ◦C, and 5% CO<sup>2</sup> for up to 10 passages. Mycoplasma was tested monthly using the bio-luminescence kit (Lonza, Visp, witzerland) and read by a multi-plate reader.

### *4.3. Cytotoxicity and Combination Studies*

MTT assay was used for evaluation the cytotoxic effects of LY and TAM and their combination according to previous reports [46,47]. MCF-7 cells were cultured in 96-well (1 × 10<sup>3</sup> /well). Cells were treated with several concentrations (0.001–50 µM) of LY and TAM. Cells were incubated for 72 h, followed by addition of MTT for 3 h (Life technologies). The formazan crystals were dissolved in DMSO (100 µL) and the light absorbance was measured used BIORAD PR 4100 microplate reader at λmax 570 nm. IC<sup>50</sup> were calculated using GraphPad Prism. Nontoxic concentrations (~80% of viable cells) from LY (100 nM) and TAM (100 nM) were used for all combination experiments of this study.

### *4.4. Clonogenic Survival Assay*

The cytotoxicity of LY, TAM and the synergistic cytotoxic effect of their combination was confirmed by clonogenic survival assay according to previous report [48]. Briefly, low density MCF-7 cells (2 × 10<sup>2</sup> cells/well) were cultivated in 2 mL media in 6-well plates in duplicates. Plates were incubated at 37 ◦C overnight to allow attachment. Cells were treated with either LY (100 nM), TAM (100 nM) or their combinations. Plates were incubated at 37 ◦C for 24 h, then medium containing compounds were aspirated, and replaced with 2 mL fresh media. Plates were checked under the microscope every 2 days, and cells forming a colony were counted. After 14 days, colonies which containing at least 50 cells were counted. Following the aspiration of media, cells were washed with cold PBS, then fixed with cold methanol for 5 min at room temperature. Cells were stained with 0.5% *v*/*v* methylene blue in methanol: H2O (1:1) for 15 min. Colonies were washed with PBS and H2O. Plates were left to dry, before counting colonies.

### *4.5. Apoptosis Assay Using Flow Cytometric Analysis*

The ability of LY, TAM and their combinations to induce apoptosis in BC cells was quantified by flow cytometry using annexin V FITC/PI (propidium iodide) double staining assay following previous report [49,50]. MCF-7 cells (5 × 10<sup>5</sup> cells/well) were cultivated in 6 well plates for 24 h. LY (100 nM), TAM (100 nM), and their combinations were incubated with cells for further 24 h, before harvested cells were labeled by annexin V FITC/PI apoptosis detection kit (Invitrogen) according to the manufacturer's instruction. Apoptotic cells (early and late) were quantified as % by flow cytometer (FC500, Beckman Coulter, Miami, FL, USA).

### *4.6. Immunofluorescence Staining*

The induction of apoptosis in MCF-7 cells by LY, TAM, and their combinations were confirmed by immunofluorescence staining assay to determine the co-localization of the antiapoptotic marker (Bcl-2) and the apoptotic marker (Caspase-3). MCF-7 cells were treated with LY, TAM, and their combinations

for 24 h. MCF-7 cells (5 × 10<sup>3</sup> /chamber) were seeded and incubated for 24 h. Then cells were blocked with normal donkey serum (30 min), followed by incubation with the primary mouse monoclonal and rabbit polyclonal IgG antibodies (1:200, 3 h) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for the detection of Bcl-2 and Caspase-3, respectively. Then, slides were incubated with a mixture of tagged cross-adsorbed donkey anti-mouse (Alexa Fluor 488) and anti-rabbit (Alexa Fluor 555) IgG secondary antibodies (Thermo Fisher Scientific) for 60 min. Slide sections were counterstained with ProLong Diamond Anti-fade Mountant including 4′ ,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, Waltham, MA, USA). EVOS FL microscopy (Thermo Fisher Scientific, Waltham, MA, USA) was used for slide examination. Digital images were taken with 40× objective.

### *4.7. Quantitative Real-Time PCR*

For more elucidation of the apoptotic effects of LY, TAM, and their combinations in BC, the RT-PCR platform (Applied Biosystems 7500 Fast Real Time PCR System, Waltham, MA, USA) was applied. RT-PCR was used to quantify the expression of the apoptosis genes: caspase-3, caspse-7, p53, p21, Bcl-2, BAX, Survivin, and Her2 in MCF-7 cells [51]. Briefly, MCF-7 cells (1 × 10<sup>6</sup> cells/well) were cultivated in 6 well plates for 24 h, then cells were treated with LY, TAM, and their combinations for 24 h. Total RNA was isolated according to manufactory instruction. The RT-PCR experiment was conducted with a mixture of cDNA, 2X SYBR Green I Master mix, PCR-grade water, forward and reversed human primers of selective genes, and GAPDH as housekeeping gene (Applied-Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) (Table 1).


**Table 1.** Sequence of primers used in RT-PCR.

The RT- PCR program was run in 41 cycles of denaturation at 95 ◦C for 15 s followed by annealing/extension at 60 ◦C for 60 s (*n* = 3). Standard comparative method was used to evaluate the genes expression, where the raw Ct values were converted into relative expression levels (fold change: 2−∆∆*C*<sup>t</sup> ).

### *4.8. Cell Cycle Analysis*

Cell cycle analysis was applied to explore the underlying mechanisms of cytotoxic effects of LY, TAM, and their combinations in BC. MCF-7 cells (5 × 10<sup>5</sup> cells/well) were treated with LY, TAM, and their combinations for 24 h. Cells were then fixed in 70% ethanol and processed for cell cycle analysis, after staining with propidium iodide (PI, Santa Cruz). FC500, Beckman Coulter, Miami, FL,

USA flow cytometer was used for analyzing a total of 20,000 single-cells, with the aid of Expo 32 software, Miami, FL, USA [52].

### *4.9. Western Immunoblotting*

Identification of the expression change of cell cycle proteins (AKT, pAKT, CyclinD1, and GAPDH) was confirmed by immunoblotting assay. MCF7 cells (1 × 10<sup>6</sup> cells/well of 6 well plate) were treated with LY, TAM, and their combinations for 24 h. Lysis buffer was used to isolate total proteins. The Bradford Method was used to determine the concentration of total proteins, which were electrophoresed using a polyacrylamide gel and transferred to membrane. The membrane was incubated with AKT, pAKT cyclin D1 antibodies (Cell signalling) for 2 h at room temperature and secondary antibody GAPDH for 1 h. Horseradish peroxidase (HRP)-conjugated secondary antibodies were used to visualize the immunoreactivity by chemiluminescence, and images were captured by a scanner (GeneGenome, Syngene Bioimaging, Cambridge, CB4 1TF, United Kingdom) [51].

### *4.10. Statistics*

Statistical differences were assessed by one-way ANOVA with the Tukey's post-hoc multiple comparison test. *p* < 0.05 (\*), *p* < 0.01 (\*\*), *p* < 0.001 (\*\*\*), and *p* < 0.0001 (\*\*\*\*) were taken as significant.

### **5. Conclusions**

Our results demonstrated that the synergistic cytotoxic effect of LY and TAM is achieved by the induction of apoptosis and cell cycle distribution through cyclin D1, pAKT, caspases, and Bcl-2 signaling pathways, all which might help in reversing the resistance of MCF-7 cells to TAM and decrease the toxicity of LY. Further in vivo and genetic studies are needed to explore more information about the efficacy and molecular targeting of this combination.

**Author Contributions:** Conceptualization, M.E.A. and O.F.I.; Experimental part M.A.A., B.R., R.A.A., A.T.B., A.M.I., M.H.M., and S.I.; Statistical analysis and proof reading, W.H.A.; design and writing, M.E.A., M.Z.E.-R., and A.N.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

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

### **References**


**Sample Availability:** Samples of the compounds are 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* **Tacrine-Coumarin Derivatives as Topoisomerase Inhibitors with Antitumor Effects on A549 Human Lung Carcinoma Cancer Cell Lines**

**Eva Konkol'ová 1,2 , Monika Hudáˇcová 1 , Slávka Hamul'aková 3 , Rastislav Jendželovský 4 , Jana Vargová 4 , Juraj Ševc <sup>4</sup> , Peter Fedoroˇcko <sup>4</sup> and Mária Kožurková 1,5, \***


**Abstract:** A549 human lung carcinoma cell lines were treated with a series of new drugs with both tacrine and coumarin pharmacophores (derivatives **1a**–**2c**) in order to test the compounds' ability to inhibit both cancer cell growth and topoisomerase I and II activity. The ability of human topoisomerase I (*h*TOPI) and II to relax supercoiled plasmid DNA in the presence of various concentrations of the tacrine-coumarin hybrid molecules was studied with agarose gel electrophoresis. The biological activities of the derivatives were studied using MTT assays, clonogenic assays, cell cycle analysis and quantification of cell number and viability. The content and localization of the derivatives in the cells were analysed using flow cytometry and confocal microscopy. All of the studied compounds were found to have inhibited topoisomerase I activity completely. The effect of the tacrine-coumarin hybrid compounds on cancer cells is likely to be dependent on the length of the chain between the tacrine and coumarin moieties (**1c**, **1d** = tacrine-(CH<sup>2</sup> )8–9 -coumarin). The most active of the tested compounds, derivatives **1c** and **1d**, both display longer chains.

**Keywords:** tacrine-coumarin derivatives; DNA; topoisomerases I, II; cytotoxicity; lung carcinoma cells; A549

### **1. Introduction**

Coumarins have attracted a great deal of attention due to the wide range of their biological properties [1–4]. Recent research has focused attention on the anticancer activity of coumarin and coumarin-derived compounds due to their high level of biological activity and low toxicity [5–7]. Coumarins are commonly used in the treatment of prostate cancer, colon, renal cell carcinoma and leukemia in particular [8–10]. Further research has also led to irusostat (a potent coumarin-based irreversible inhibitor) compounds entering clinical trials for possible future use in the treatment of breast cancer [11–13]. Lung cancer is one of the most commonly diagnosed malignant tumors and is the leading cause of cancer death throughout the world. The currently available therapies in the treatment of advanced lung cancer, primarily radiotherapy and chemotherapy, are still inadequate. While highly effective FDA-approved drugs such as, e.g., efitinib, erlotinib, and bevacizumab are now available for targeted therapy/chemotherapy, these drugs can cause side effects [14]. Therefore, there is an urgent need for the development of novel drugs for treating this

**Citation:** Konkol'ová, E.; Hudáˇcová, M.; Hamul'aková, S.; Jendželovský, R.; Vargová, J.; Ševc, J.; Fedoroˇcko, P.; Kožurková, M. Tacrine-Coumarin Derivatives as Topoisomerase Inhibitors with Antitumor Effects on A549 Human Lung Carcinoma Cancer Cell Lines. *Molecules* **2021**, *26*, 1133. https://doi.org/10.3390/ molecules26041133

Academic Editors: Marialuigia Fantacuzzi and Alessandra Ammazzalorso

Received: 27 January 2021 Accepted: 18 February 2021 Published: 20 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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 (https:// creativecommons.org/licenses/by/ 4.0/).

disease. A549 human lung carcinoma cells are a well characterized cellular model for this purpose [15,16].

Different mechanisms are thought to be responsible for the anticancer activity of coumarins, including the blocking of the cell cycle, the induction of cell apoptosis, the modulation of the estrogen receptor, or the inhibition of DNA-associated enzymes such as telomerase and topoisomerase (TOP). Topoisomerase enzymes play an important role in DNA metabolism, and the search for novel enzyme inhibitors is an important target in the development of new anticancer drugs [17,18].

The relevance and significance of these compounds is obvious, and the agents have attracted considerable attention through the development of novel biologically active molecules. The approach is based on the highly effective combination principle of drug design and involves the coupling of coumarins with other bioactive molecules [19–22]. The activity of tacrine (9-amino-1,2,3,4-tetrahydroacridine) in neurological disorders such as Alzheimer's disease is now well established. Numerous studies have confirmed that the drug is an effective inhibitor of acetylcholinesterase [22–25] and it has also been reported that it is not clastogenic in mammalian cells [26]. Tacrine is a relatively weak catalytic inhibitor of TOPII (in comparison with 9-aminoacridine), which has been found to inhibit topoisomerase and DNA synthesis, thereby resulting in mitochondrial DNA depletion and apoptosis [27–29]. Hybrid molecules, formed by the combination of two or more pharmacophores, is an emerging concept in the field of medicinal chemistry and drug discovery that has attracted substantial attraction in the past few years [30]. – ' – –

The aim of this study is to show that these structurally novel tacrine-coumarin compounds, derivatives **1a**–**1d** and **2a**–**2c**, may exhibit anticancer properties and also to examine the antiproliferative and topoisomerase activities of the derivatives in more detail. – –

### **2. Results**

### *2.1. Topoisomerase Relaxation Assay*

The ability of human topoisomerase I (*h*TOPI) to relax supercoiled plasmid DNA in the presence of various concentrations of tacrine-coumarin hybrid molecules was studied with agarose gel electrophoresis and the results are shown in Figure 1. The results clearly show that supercoiled plasmid DNA (line *p*BR322) was fully relaxed under normal conditions with *h*TOPI (*h*TOPI line + *p*BR322). However, relaxation induced by *h*TOPI was inhibited when the concentration of the studied compounds (lines **1a**–**2c**) was gradually increased. All of the studied compounds were found to have caused partial inhibition of topoisomerase activity at a concentration of 30 × 10 <sup>−</sup><sup>6</sup> M and complete inhibition was detected at a concentration of 60 × 10 <sup>−</sup><sup>6</sup> M. – − −

– — — – **Figure 1.** Electrophoresis agarose gel showing inhibitory effects of tacrine-coumarin compounds **1a**–**2c** on human topoisomerase I (*h*TOPI) activity. Supercoiled plasmid DNA (*p*BR322—negative control) was incubated for 30 min with 2 U of *h*TOPI in the absence (lines *h*TOPI + *p*BR322—positive control) and presence of varying concentrations of compounds **1a-2c** (lines **1a**–**2c**).

> In order to evaluate whether the compounds can also inhibit topoisomerase IIα ac-In order to evaluate whether the compounds can also inhibit topoisomerase IIα activity, the decatenation of catenated plasmid DNA was performed in the presence of the

compounds. However, no significant inhibitory effect was detected, even at the highest concentration of 100 × 10 <sup>−</sup><sup>6</sup> M concentration (data not shown) and therefore we suggest that compounds **1a**–**2c** are unable to inhibit the topoisomerase IIα enzyme. – topoisomerase IIα enzyme.

–

### *2.2. Intracellular Localization and Cytotoxicity Assays*

−

Flow cytometric analysis of the content of the derivatives present in A549 cells revealed the cumulative fluorescence of derivatives **1b**–**1d** and **2b** from the green (FL-1) to the red (FL-3) channel (Figure 2). Compound **1c** was found to display the highest level of fluorescence. The presence of compounds **1a**–**2c** in A549 human adherent lung carcinoma cells were analysed by observing the fluorescence of the compounds in the green channel (*Ex* = 488 nm, *Em* = 510–560 nm). This analysis was performed in order to detect the accumulation of the compounds within the cells by exploiting their natural fluorescence. This allowed us to correlate the accumulation of the derivatives with their observed effects on the cellular parameters. The accumulation of the compounds was then investigated in more detail with respect to their specific intracellular distribution using confocal microscopy. – –

– **Figure 2.** Flow cytometric analysis of intracellular level of derivatives **1a**–**2c**. Intrinsic fluorescence of compounds was detected after excitation at 488 nm and the emission was measured using a 530/30 nm band-pass filter (FL-1), 585/42 band-pass filter (FL-2) and 670 nm long-pass filter (FL-3). The results are presented as the mean values ± SD of three independent experiments; statistical significance \* *p* < 0.05 for each experimental group compared to the untreated control.

According to our results (Figure 3), compound **1d** displayed the highest rate of detection in cells, with compounds **1c** and **1b** also showing weaker levels of detection. In other samples, the fluorescence of the derivatives could not be distinguished from the autofluorescence of the cancer cells. At the cellular level, the analyzed compounds were distributed in the cytoplasm with no interference with the cell nucleus. Based on mitochondrial staining and the overall distribution of the signal, we could not confirm the accumulation of the derivatives in the mitochondria or in the other organelles or membranes (data not shown).

### *2.3. MTT Assay*

nuclear labelling with Draq5. Scale bar = 25 μm.

The ability of the studied compounds to inhibit the metabolic activity of A549 cancer cell lines was determined using an MTT assay. Results were obtained from three independent experiments and each experiment was carried out in triplicate. As is evident from Figure 4, the compounds were found to have inhibited metabolic activity in a timeand dose-dependent manner, and the highest efficiency was recorded in the case of the experimental group treated with compounds **1c** and **1d**.

–

–

–

−

–

– topoisomerase IIα enzyme.

–

–

–

– – – nuclear labelling with Draq5. Scale bar = 25 μm. **Figure 3.** Confocal microscopy images of A549 cancer cell lines after 24 h incubation with compounds **1a**–**2c**. The microphotographs show the representative images of the samples with merged channels. Compounds **1a**–**2c** were visualized in cells with a 488 nm laser and the fluorescence was captured at the range of 510–560 nm (green insets). Red insets show nuclear labelling with Draq5. Scale bar = 25 µm.

– **Figure 4.** Effect of tacrine-coumarin hybrid compounds **1a**–**2c** on metabolic activity evaluated by MTT assay in A549 cancer cell lines. MTT assays are expressed as percentages of the untreated control. The results are presented as the mean values ± SD of three independent experiments; statistical significance (\*): *p* < 0.05 for each experimental group compared to the untreated control.

− − The results obtained from the MTT assay were also used to determine IC<sup>50</sup> values for each compound which are listed in Table 1. The IC<sup>50</sup> values show that A549 cancer cells are more sensitive to the action of compounds **1c** and **1d** (IC<sup>50</sup> = 27.04 and 21.22 × 10 <sup>−</sup><sup>6</sup> M, respectively after 48 h) than to the other compounds from this series (IC<sup>50</sup> > 50 × 10 <sup>−</sup><sup>6</sup> M). Furthermore, these data corroborate the results obtained from the viability assay and the quantification of total cell number.

**–**

**−**


**Table 1.** IC<sup>50</sup> values of tacrine-coumarin hybrid molecules **1a–2c** in A549 cancer cell lines.

n.d.—not detected, a IC50—the concentration of the compound at which 50% of metabolic activity is inhibited. — —

### *2.4. Quantification of Cell Number and Viability*

The influence of the tacrine-coumarin compounds on total cell numbers was investigated after 24 h of treatment with the derivatives. As is shown in Figure 5, the total cell number decreased sharply (by more than 50%) in the case of cells treated with compounds **1c** and **1d**.

– **Figure 5.** Effect of tacrine-coumarin hybrid compounds **1a**–**2c** on viability and total cell numbers in A549 cancer cell lines. The viability and total cell number were evaluated 24 h after the addition of the derivatives and are expressed as a percentage of the viable, eosin negative cells or as a percentage of the untreated control of the total cell number, respectively. The results are presented as the mean values ± SD of three independent experiments; statistical significance \* *p* < 0.05 for each experimental group is compared to the untreated control.

A simultaneous analysis of viability (Figure 5) showed that higher concentrations of compounds **1c** and **1d** had a weaker but nonetheless significant effect on cell survival. These results indicate that compounds **1c** and **1d** can influence total cell numbers and viability in a concentration-dependent manner.

### *2.5. Cell Cycle Distribution*

The influence of the tacrine-coumarin hybrid molecules on the cell cycle distribution of cancer cells was investigated using flow cytometry. Data were collected from three independent experiments. As is shown in Table 2, the percentage of the cells at G0/G<sup>1</sup> in the control group is 53.77 ± 1.43. The A549 cells were incubated with different concentrations of the studied compounds, and after 24 h incubation, the cells treated with compounds **1b** (at a higher concentration), **1c** and **1d** displayed an increased percentage of cells at the G0/G<sup>1</sup> phase.

–

**−**

**n** 


**Table 2.** Effect of tacrine-coumarin hybrid compounds **1a**–**2c** on cell cycle distribution.

\* Statistical significance: *p* < 0.05 for each experimental group compared to untreated control.

### *2.6. Clonogenic Assay*

A549 cell lines were treated with two different concentrations of these derivatives. As is shown in Figure 6, no significant decrease in colony formation was observed, while a limited reduction was observed in the presence of a higher concentration of compound **1d**.

(**b**)

– **Figure 6.** Clonogenic assay of A549 cancer cell lines. Cells were untreated (control) or treated with different concentrations of tacrine-coumarin hybrid derivatives **1a**–**2c** for 24 h. (**a**) The experimental and (**b**) graphical presentation of the results. The results of the subsequent 7-day cultivation are presented as the mean values ± SD of three independent experiments.

### **3. Discussion**

DNA topoisomerases are crucial nuclear enzymes which control the topology of DNA by cleaving and re-joining the phosphodiester backbone of the DNA strand during various genetic processes. Clinical topoisomerase inhibitors act by generating topoisomeraselinked DNA breaks, blocking the religation of the cleavage complexes when a single drug molecule binds tightly at the interface of the topoisomerase-DNA cleavage complex [31]. As is well known, relaxed forms of supercoiled DNA migrate into a gel more slowly than non-relaxed DNA; this means that only the supercoiled band should be visible when topoisomerase activity is inhibited [32,33]. However, relaxation induced by *h*TOPI was inhibited when the concentration of the studied compounds was gradually increased, suggesting that the tacrine-coumarin compounds may cause a concentration-dependent inhibition of *h*TOPI. All of the studied compounds were found to have caused a complete inhibition at a concentration of 60 × 10−<sup>6</sup> M. In order to evaluate whether the compounds can also inhibit topoisomerase IIα activity, the decatenation of catenated plasmid DNA was performed in the presence of the compounds. However, no significant inhibitory effect was detected, even at the highest concentration, and therefore we suggest that compounds **1a**–**2c** are unable to inhibit the topoisomerase IIα enzyme. It is important to understand that the cytotoxicity of topoisomerase inhibitors is due to the trapping of topoisomerase cleavage complexes, a process which should be distinguished from the associated topoisomerase catalytic inhibition. With the exception of molecularly defined settings, it is the topoisomerase cleavage complexes that kill the cancer cell [31]. TOPI plays an important role during the cell division process and we hypothesize that the inhibition of TOPI by tacrine-coumarin compounds can also influence cell division in A549 cell lines.

The presence of compounds **1a**–**2c** in A549 human adherent lung carcinoma cells was analysed by observing the fluorescence of the compounds in the green channel. As our results show, compound **1d** displayed the highest rate of detection in cells. In other samples, the fluorescence of derivatives was not distinguishable from the autofluorescence of the cancer cells. In cells, the compounds were distributed in the cytoplasm. Based on mitochondrial staining and the overall distribution of the signal, we were unable to confirm the accumulation of the compounds in the mitochondria or in other organelles or membranes. These observations suggest that no specific interaction through DNA binding is responsible for the observed cytotoxicity of these compounds. Flow cytometric analysis of the content of the derivatives present in the A549 cells revealed that compound **1c** was found to display the highest level of fluorescence.

The influence of the tacrine-coumarin compounds on total cell numbers was investigated after 24 h of treatment with the derivatives. The total cell number decreased sharply in the case of cells treated with compounds **1c** and **1d**. No significant changes were observed for cells treated with the other compounds from the series, but a simultaneous analysis of viability showed that higher concentrations of compounds **1c** and **1d** had a weaker but nonetheless significant effect on cell survival. These results indicate that compounds **1c** and **1d** can influence total cell numbers and viability in a concentration-dependent manner.

The ability of the studied compounds to inhibit the metabolic activity of A549 cancer cell lines was determined using an MTT assay. The compounds were found to have inhibited metabolic activity in a time- and dose-dependent manner, with the highest efficacy being recorded in the case of the experimental groups treated with compounds **1c** and **1d**. The IC<sup>50</sup> values show that A549 cancer cells are more sensitive to the action of compounds **1c** and **1d** after 48 h than to the other compounds from this series. Furthermore, these data corroborate the results obtained from the viability assay and the quantification of total cell number. Tacrine was found to be a weak antiproliferative agent but we determined that the combination of tacrine and coumarin in a single molecule is more efficient against the cancer cell line.

Solarova et al. [34] have tested the cytotoxic and/or anti-cancer activities of tacrinecoumarin heterodimers **1a**–**2c** on 4T1 (mouse mammary carcinoma), MCF-7 (human breast adenocarcinoma), HCT116 (human colorectal carcinoma), A549 (human lung carcinoma),

NMuMG (normal mouse mammary gland cells) and HUVEC (human endothelial cells isolated from umbilical vein) cell lines. Based on the obtained IC<sup>50</sup> values, compounds **1a**–**2c** showed moderate to significant activity in the µM range. The A549 tumor cells proved to be the most resistant, with a proliferation not significantly different from the other cell lines after the administration of tacrine-coumarin derivatives **1b**–**1d**. Among the synthesized compounds, the tacrine-coumarin heterodimer with nine methylene groups between the two amino groups in the side chain exhibited the greatest efficacy. The authors proposed that tacrine-coumarin heterodimers **1a**–**2c** with longer side chains (replacing some methylene groups with amine moiety) had decreased the anticancer activity. The effect of the tacrine-coumarin hybrid compounds on the cancer cells is likely dependent on the length of the chain between the tacrine and coumarin moiety (compounds **1c**, **1d** = tacrine-(CH2)8-9-coumarin). However, when the -CH<sup>2</sup> chain is interrupted by -NH groups, only a moderate inhibition effect on proliferation is recorded. Our attention was focused only on one A549 cancer cell line with the purpose of studying these compounds in more detail. According to our results, derivatives **1c** and **1d** displayed the best antiproliferative effect, a result which is similar to those reported by Solarova et al. The compounds with a greater length of chain between the tacrine and coumarin molecules showed an insignificant effect (**2a**–**2c**). As further evidence of the significance of hydrocarbon length, the antiproliferative activity increased in the order **1b** < **2b** < **1c** < **1d**.

A novel *bis*-tacrine and its congeners was tested for its potential as an anticancer agent by Hu et al. [35] An in-vitro cytotoxic evaluation of the compounds was carried out against a panel of 60 human cancer cell lines. Of the novel compounds, the butyl-linked *bis*-tacrine exhibited the strongest cytotoxic profile against non-small lung cancer cells. Congeners bearing a longer alkyl chain were on average 30- to 100-times less cytotoxic against these cancer cells.

We also investigated the influence of tacrine-coumarin hybrid molecules **1a**–**2c** on the cell cycle distribution of cancer cells. The A549 cells, which were treated with compounds **1b**–**1d**, displayed an increased percentage of cells at the G0/G<sup>1</sup> phase. The data demonstrate that these compounds were also capable of inhibiting cells in the G0/G<sup>1</sup> phase. These results are in agreement with those of Roldán-Pena et al. [36] who designed a series of tacrine-based homo- and heterodimer compounds incorporating an antioxidant tether which displayed antiproliferative activity. The compounds exhibited excellent in vitro antiproliferative activities against a panel of 6 human tumor cell lines, while cell cycle experiments indicated the accumulation of cells in the G<sup>1</sup> phase of the cycle. A study by Janoˇcková et al. [37] examined the effect of 7-MEOTA tacrine urea heterodimers on HL-60 cell lines and their results clearly demonstrated a significant accumulation of cells in the G<sup>1</sup> phase.

In our study, the effects of derivatives **1c** and **1d** were found to be more prominent on the proliferation of cancer cells (demonstrated as a decline in total cell number) than on the viability of the cells. This agrees with the increased accumulation of cells in the G0/G<sup>1</sup> phase. Finally, the inhibition of Topo I observed for the tacrine-coumarin compounds may also influence cell division in the A549 cell line. While all of these observed effects have a strong impact on the proliferation of cells (and consequently on the total cell number), this does not necessarily mean that the compounds also exert a cytotoxic effect (i.e., the compounds had not impaired cell viability to such a significant degree). Thus, the decreased cell number is primarily the result of inhibited proliferation rather than any cytotoxic effect of tacrine-coumarin hybrid compounds **1c** and **1d**.

In order to test the effect of the studied compounds on colony formation or clonogenic ability, we performed experiments with clonogenic assays. This is a simple technique which can identify biological alterations leading to irreversible losses of proliferative capacity and thus the loss of cells' ability to form new colonies [38]. The changes were accompanied by a corresponding reduction in the percentage of cells in the S and G2/M phases. No significant decrease in colony formation was observed; a limited reduction was observed in the presence of a higher concentration of compound **1d**.

When we compare the results of all of the biological techniques used in this study, it is possible to suggest that the effect of the tacrine-coumarin hybrid compounds on cancer cells likely depends on the length of the chain between the tacrine and coumarin moiety. However, when the -CH<sup>2</sup> chain is interrupted by -NH groups, only a moderate inhibition effect on proliferation is recorded.

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

### *4.1. Compounds*

All chemicals and reagents were purchased from Sigma-Aldrich Chemie (Hamburg, Germany) and used without further purification. Human topoisomerase I- hTOPI, TOPOII (Inspiralis, Ltd., Norwich, UK), Ham Nutrient Mixture (Sigma-Aldrich, St. Louis, MO, USA) foetal bovine serum (Biosera, Boussens, France) and antibiotics (Antibiotic-Antimycotic 100 × and 50 × 10 <sup>−</sup><sup>3</sup> g L <sup>−</sup><sup>1</sup> gentamicin; Biosera), MitoTrackerTM Red, DRAQ5TM, ProLongTM Gold Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA). MTT (3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, St. Louis, MO, USA) were used in the study. − − – –

The studied tacrine-coumarin hybrids (derivatives **1a**–**1d** and **2a**–**2c**) **1a**: *N*1-{6-[(1,2,3,4 tetra-hydroacridin-9-yl)amino]hexyl}-2-(7-hydroxy-2-oxo-2*H*-chromen-4-yl)acetamide, **1b**: *N*1-{7-[(1,2,3,4-tetrahydroacridin-9-yl)amino]heptyl}-2-(7-hydroxy-2-oxo-2*H*-chromen-4-yl) acetamide, 1c: *N*1-{8-[(1,2,3,4-tetrahydroacridin-9-yl)amino]octyl}-2-(7-hydroxy-2-oxo-2*H*chromen-4-yl)acetamide, 1d: *N*1-{9-[(1,2,3,4-tetrahydroacridin-9-yl) amino]nonyl}-2-(7 hydroxy-2-oxo-2*H*-chromen-4-yl)acetamide, **2a**: 2-(7-hydroxy-2-oxo-2*H*-chromen-4-yl)-*N*- [6-(1,2,3,4-tetrahydroacridin-9-ylamino)hexyl]acetamide, **2b:** *N*1-[3-({3-[(1,2,3,4-tetrahydroacridin -9-yl)amino]propyl}amino)propyl]-2-(7-hydroxy-2-oxo-2*H*-chromen-4-yl)acetamide, **2c:** 2- (7-hydroxy-2-oxo-2*H*-chromen-4-yl)-*N*-[3-[2-[3-(1,2,3,4-tetrahydroacridin-9-ylamino) propylamino]ethylamino]propyl]acetamide (Figure 7) [1] were dissolved in dimethyl sulfoxide (DMSO, Fluka) to a final concentration of 5 × 10 <sup>−</sup><sup>2</sup> M. −

– – **Figure 7.** Structure of tacrine-coumarin hybrid molecules (derivatives **1a**–**1d** and **2a**–**2c**) [1].

### *4.2. Topoisomerase Relaxation Assay*

– − − The effects of compounds **1a**–**2c** on the relaxation of plasmid DNA with human topoisomerase I (*h*TOPI) were investigated using negatively supercoiled plasmid *p*BR322 (0.5 × 10 <sup>−</sup><sup>6</sup> g) incubated for 30 min at 37 ◦C with 2 units of *h*TOPI (Inspiralis, Ltd., Norwich, UK) in both the presence and absence of the studied tacrine-coumarin hybrid molecules at concentrations of 5, 30 and 60 × 10 <sup>−</sup><sup>6</sup> M, respectively. The method used to perform the experiment of TOPOII has been published previously [37].

### *4.3. Cell Culture*

ture Collection (ATCC, Rockville, MD, USA). The cells were incubated in Kaighn's modi- − − <sup>−</sup> well μ Human lung carcinoma cell lines A549 were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were incubated in Kaighn's modification of F-12 Ham Nutrient Mixture supplemented with 10% fetal bovine serum (FBS) and antibiotics (1% Antibiotic-Antimycotic 100 × and 50 × 10 <sup>−</sup><sup>3</sup> g L <sup>−</sup><sup>1</sup> gentamicin; Biosera) at 37 ◦C, 95% humidity and 5% CO2. The cells (10,000/cm−<sup>2</sup> ) were seeded on

12-well µ-Chamber slides (ibidi GmbH, Martinsried, Germany) on 6, 12 and 96-well plates (TPP, Trasadingen, Switzerland) and left to settle for 24 h. This incubation method has been published previously [39].

### *4.4. Intracellular Localization and Cytotoxicity Assays*

The derivatives were visualized in cells with an Argon Laser at 488 nm and fluorescence was captured at a range of 510–560 nm with identical exposure parameters used for all samples. Microphotographs were taken with a 100 × oil lens and were then captured and analysed using LAS AF software (Leica Microsystems, Mannheim, Germany).

Floating and adherent cells were harvested both 6 and 24 h after treatment with the derivatives, washed in PBS and resuspended in Hank's balanced salt solution (HBSS). Intracellular levels of derivatives were detected using a BD FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) and determined based on fluorescence excitation at 488 nm. Fluorescence was detected via a 530/30 nm band-pass filter (FL-1), 585/42 band-pass filter (FL-2) and 670 nm long-pass filter (FL-3). The results were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA).

MTT assays were added to the cells in a 96-well plate (at a final concentration of 0.5 g L−<sup>1</sup> ) 24 and 48 h after treatment with the derivatives [39]. The absorbance (λ = 584 nm) was measured using a BMG FLUOstar Optima (BMG Labtechnologies GmbH, Offenburg, Germany). The results were evaluated as percentages of the absorbance of the untreated control. IC<sup>50</sup> values for each derivative were extrapolated from a sigmoidal fit to the metabolic activity data using OriginPro 8.5.0 SR1 (OriginLab Corp., Northampton, MA, USA).

For an assessment of total cell numbers and viability within individual experimental groups, floating and adherent cells were harvested 24 h after treatment with the studied derivatives and evaluated using a Bürker chamber (Paul Marienfeld GmbH&Co.KG, Lauda-Königshofen) with eosin staining. The total cell number was expressed as a percentage of the untreated control of the total cell number. Viability was expressed as a percentage of viable, eosin negative cells.

Details of the experiment with flow cytometric analysis have been published previously [38]. The DNA content was analysed using a BD FACSCalibur flow cytometer (Becton Dickinson) with a 488 nm argon-ion excitation laser, and fluorescence was detected via a 585/42 nm band-pass filter (FL-2). ModFit 3.0 software (Verity Software House, Topsham, ME, USA) was used to generate DNA content frequency histograms and to quantify the percentage of cells in the individual cell cycle phases.

### *4.5. Clonogenic Assay*

The cells were counted using a Bürker chamber with eosin staining and 800 viable cells per well were seeded in 6-well plates. After 7 days of incubation under standard conditions, the cells in the plates were fixed and stained with 1% methylene blue dye in methanol. Visualized colonies were scanned, counted and the results were evaluated as percentages of the untreated control.

### *4.6. Statistical Analysis*

Data were analyzed using a one-way ANOVA with Tukey´s post-test and are expressed as the mean ± standard deviation (SD) of at least three independent experiments. The experimental groups treated with the derivatives were compared with the control group: \* *p* < 0.05.

### **5. Conclusions**

This study has investigated a series of novel derivatives with both tacrine and coumarin pharmacophores, compounds **1a**–**2c**. Our results suggest that the novel derivatives had completely inhibited topoisomerase activity at a concentration of 60 × 10−<sup>6</sup> M. The presence and content of the novel tacrine-coumarin hybrid molecules after intro-

duction to A549 human adherent lung carcinoma cell lines were also investigated using confocal microscopy. Only compound **1d** was found to be present in the cell lines to a substantial degree. The IC<sup>50</sup> values which were determined in this assay show that A549 cancer cell lines are more sensitive to the effect of compounds **1c** and **1d** (IC<sup>50</sup> = 27.04 and 21.22 × 10−<sup>6</sup> M, respectively after 48 h) than to the other compounds in the series. A simultaneous analysis of viability showed that higher concentrations of compounds **1c** and **1d** had a weaker but nonetheless significant effect on cell survival. These results indicate that compounds **1c** and **1d** are capable of influencing total cell numbers and viability in a concentration-dependent manner. The findings presented in this paper suggest that these tacrine-coumarin molecules exhibit promising potential as topoisomerase I inhibitors with anticancer activity against A549 human adherent lung carcinoma cells in addition to their well-known anticholinesterase effects [1] and may also serve as BSA-interacting agents [40]. These features would be of considerable use in the development of drugs with enhanced or more selective effects and greater clinical efficacy.

**Author Contributions:** Material preparation, data collection and analysis were performed by E.K., M.H., S.H. and M.K.; R.J., J.V., J.Š., P.F. performed and analyzed the cancer cell line experiments. The first draft of the manuscript was written by M.K. and all authors commented on earlier versions of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Grant Project of the Ministry of Education, Science, Research and Sport of the Slovak Republic VEGA 1/0016/18, MH CZ-DRO (UHHK, 00179906) and Operational Programs Research and Innovations for the Medical University Scientific Park in Košice project (MediPark, Košice-Phase II.), ITMS2014+313011D103; Operational Program Integrated Infrastructure, project "NANOVIR", ITMS: 313011AUW7, cofinanced by the European Fund of Regional Progress.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors are grateful to Gavin Cowper for assistance with the manuscript.

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

**Sample Availability:** Samples of the compounds **1c** and **1d** are available from the authors.

### **References**

