*Review* **TRK Inhibitors: Tissue-Agnostic Anti-Cancer Drugs**

**Sun-Young Han**

Research Institute of Pharmaceutical Sciences and College of Pharmacy, Gyeongsang National University, Jinju-si 52828, Korea; syhan@gnu.ac.kr

**Abstract:** Recently, two tropomycin receptor kinase (Trk) inhibitors, larotrectinib and entrectinib, have been approved for Trk fusion-positive cancer patients. Clinical trials for larotrectinib and entrectinib were performed with patients selected based on the presence of Trk fusion, regardless of cancer type. This unique approach, called tissue-agnostic development, expedited the process of Trk inhibitor development. In the present review, the development processes of larotrectinib and entrectinib have been described, along with discussion on other Trk inhibitors currently in clinical trials. The on-target effects of Trk inhibitors in Trk signaling exhibit adverse effects on the central nervous system, such as withdrawal pain, weight gain, and dizziness. A next generation sequencing-based method has been approved for companion diagnostics of larotrectinib, which can detect various types of Trk fusions in tumor samples. With the adoption of the tissue-agnostic approach, the development of Trk inhibitors has been accelerated.

**Keywords:** Trk; NTRK; tissue-agnostic; larotrectinib; entrectinib; Trk fusion

### **1. Introduction**

Tropomyosin receptor kinases (Trk) are tyrosine kinases encoded by neurotrophic tyrosine/tropomyosin receptor kinase (NTRK) genes [1]. Chromosomal rearrangement of NTRK genes is found in cancer tissues [2]. The resulting fusion proteins containing part of the Trk protein have a constitutively active form of kinase that transduces deregulating signals. There is active progress in the development of small molecule inhibitors against Trk kinases in the field of cancer therapeutics [1]. Currently, larotrectinib and entrectinib are two approved drugs for Trk fusion-positive cancers in the market [3,4]. The timeline for the clinical development of the two Trk inhibitors is shown in Figure 1. *Pharmaceuticals* **2021**, *14*, 632 2 of 11

**Figure 1.** Timeline for the clinical development of larotrectinib and entrectinib. TPM3, tropomyosin 3; NTRK1, neurotrophic tyrosine receptor kinase 1; TrkA, tropomyosin receptor kinase A; NGF, nerve growth factor; FDA, US Food and Drug Administration. **Figure 1.** Timeline for the clinical development of larotrectinib and entrectinib. TPM3, tropomyosin 3; NTRK1, neurotrophic tyrosine receptor kinase 1; TrkA, tropomyosin receptor kinase A; NGF, nerve growth factor; FDA, US Food and Drug Administration.

Targeted cancer therapies that act on specific molecules have become mainstream

The concept of targeted therapy expanded to the term precision medicine, personalized medicine, or stratified medicine, meaning "targeting drugs for each genetic profile" [7]. In contrast to the traditional "one-size-fits-all" approach, individualizing pharmacotherapy was emphasized upon due to the factors of disease heterogeneity and genetic variability [10]. Biomarkers that can predict therapeutic responses are important elements in precision medicine. Therefore, the diagnosis of biomarkers has become an important step in precision medicine, generating new terms such as companion diagnostics (CDx) or drug-diagnostic co-development [7]. With the adoption of CDx, it was possible to enroll only selected patients who were likely to respond to drug therapy. Clinical research involving a relatively small number of patients, enabled by screening out of non-responders, is called enrichment trial [11]. Trastuzumab was the first drug developed using a CDx approach. A diagnostic assay (HercepTest), which tests the expression of HER2 in breast tumors was developed and approved together with the drug [12]. Another representative example of CDx and enrichment trials is crizotinib, an anaplastic lymphoma kinase (ALK) inhibitor for non-small cell lung cancer (NSCLC) patients [13]. ALK fusion proteins caused by chromosomal rearrangement are found in approximately 4% of NSCLC patients, and these ALK fusion proteins have been reported to induce tumorigenesis. Enrichment clinical trials were conducted for crizotinib development in ALK fusion-positive NSCLC patients, and the number of patients in phase I trials was only 143. Diagnostic tests for ALK gene rearrangement were developed in conjunction with crizotinib development, and the approval of drugs and diagnostic tests were linked and included in the drug labeling.

Before the concept of tissue-agnostic drug was introduced, the development process of precision medicine included only one type of tumor. In the case of crizotinib, only NSCLC patients were included in the clinical trials, even though ALK gene fusion was originally found in anaplastic large cell lymphoma (ALCL) as well [14]. ALK translocation has also been discovered in rare tumors called inflammatory myofibroblastic tumors

specific to the human epidermal growth factor receptor 2 (HER2) protein [7]. The US Food and Drug Administration (FDA) approval for trastuzumab was obtained in 1998 for metastatic breast cancer overexpressing HER2 protein [8]. Imatinib is a small-molecule drug targeting the fusion protein BCR-ABL. The fusion protein is generated by chromosomal rearrangement in chronic myeloid leukemia cells. The remarkable efficacy of these two drugs paved the way for the era of targeted cancer therapy, and this made kinase family

**Citation:** Han, S.-Y. TRK Inhibitors: Tissue-Agnostic Anti-Cancer Drugs. *Pharmaceuticals* **2021**, *14*, 632. https:// doi.org/10.3390/ph14070632

Academic Editors: Mary J. Meegan, Niamh M. O'Boyle and Jean Jacques Vanden Eynde

Received: 18 May 2021 Accepted: 25 June 2021 Published: 29 June 2021

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

**Copyright:** © 2021 by the author. 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/).

**2. Tissue-Agnostic Drug Development** 

proteins major targets for cancer therapy [9].

A unique process of drug development, known as tissue-agnostic development, was employed for larotrectinib and entrectinib approval. Patients for tissue-agnostic clinical trials were selected based on the presence of NTRK gene rearrangement, independent of tumor type [5,6]. Trk fusion-positive tumors of several cancer types were tested for Trk inhibitors, and excellent efficacy of these drugs was shown in tissue-agnostic trials.

In this review, the development process and pharmacological efficacy of current Trk inhibitors in the market will be described, along with some discussion on the Trk inhibitors currently in clinical development. In addition, we will review the development process of tissue-agnostic drugs. Finally, we aim to provide perspectives learned from the pioneering approach of tissue-agnostic therapy for Trk inhibitors.

### **2. Tissue-Agnostic Drug Development**

Targeted cancer therapies that act on specific molecules have become mainstream strategies for anti-cancer drug development. There are two milestones for targeted cancer drug development: trastuzumab and imatinib. Trastuzumab is a monoclonal antibody specific to the human epidermal growth factor receptor 2 (HER2) protein [7]. The US Food and Drug Administration (FDA) approval for trastuzumab was obtained in 1998 for metastatic breast cancer overexpressing HER2 protein [8]. Imatinib is a small-molecule drug targeting the fusion protein BCR-ABL. The fusion protein is generated by chromosomal rearrangement in chronic myeloid leukemia cells. The remarkable efficacy of these two drugs paved the way for the era of targeted cancer therapy, and this made kinase family proteins major targets for cancer therapy [9].

The concept of targeted therapy expanded to the term precision medicine, personalized medicine, or stratified medicine, meaning "targeting drugs for each genetic profile" [7]. In contrast to the traditional "one-size-fits-all" approach, individualizing pharmacotherapy was emphasized upon due to the factors of disease heterogeneity and genetic variability [10]. Biomarkers that can predict therapeutic responses are important elements in precision medicine. Therefore, the diagnosis of biomarkers has become an important step in precision medicine, generating new terms such as companion diagnostics (CDx) or drug-diagnostic co-development [7]. With the adoption of CDx, it was possible to enroll only selected patients who were likely to respond to drug therapy. Clinical research involving a relatively small number of patients, enabled by screening out of non-responders, is called enrichment trial [11]. Trastuzumab was the first drug developed using a CDx approach. A diagnostic assay (HercepTest), which tests the expression of HER2 in breast tumors was developed and approved together with the drug [12]. Another representative example of CDx and enrichment trials is crizotinib, an anaplastic lymphoma kinase (ALK) inhibitor for non-small cell lung cancer (NSCLC) patients [13]. ALK fusion proteins caused by chromosomal rearrangement are found in approximately 4% of NSCLC patients, and these ALK fusion proteins have been reported to induce tumorigenesis. Enrichment clinical trials were conducted for crizotinib development in ALK fusion-positive NSCLC patients, and the number of patients in phase I trials was only 143. Diagnostic tests for ALK gene rearrangement were developed in conjunction with crizotinib development, and the approval of drugs and diagnostic tests were linked and included in the drug labeling.

Before the concept of tissue-agnostic drug was introduced, the development process of precision medicine included only one type of tumor. In the case of crizotinib, only NSCLC patients were included in the clinical trials, even though ALK gene fusion was originally found in anaplastic large cell lymphoma (ALCL) as well [14]. ALK translocation has also been discovered in rare tumors called inflammatory myofibroblastic tumors (IMTs) [15]. Clinical trials of crizotinib for patients with ALCL are ongoing. If clinical research was implemented regardless of tumor type, crizotinib could be used in ALCL and IMT patients as well as in subsets of NSCLC patients. In this way, clinical research with patient selection based on molecular features would have benefitted more cancer patients.

Therefore, a biomarker-guided drug development process has been proposed and successfully applied to three FDA-approved drugs. The immune checkpoint inhibitor, pembrolizumab, and two Trk inhibitors, larotrectinib and entrectinib, underwent tissueagnostic development. Tissue-agnostic drugs target specific genetic molecular features regardless of tumor sites [16]. Terms such as histology-agnostic, tumor-agnostic, siteagnostic, pan-tumor therapies are used, depending on the literature [17]. If specific genetic aberrations are found across several tumor types, tissue-agnostic drug development can be utilized. Select ongoing tumor-agnostic developments with several cancer targets are listed in Table 1.


**Table 1.** Select tissue-agnostic developments in clinical trials.

ALK, anaplastic lymphoma kinase; ROS1, c-ros proto-oncogene 1; RET, ret proto-oncogene; SRC, src protooncogene; FGFR, fibroblast growth factor receptor; BRAF, B rapidly accelerated fibrosarcoma; PD-1, programmed cell death protein 1.

As a type of clinical research, encompassing different tumor types with the same molecular features is called a basket trial. Unlike enrichment trial, which generally consists of patients with a single tumor type, in basket trials, patients are selected based on their molecular characteristics, regardless of tumor histology. Sometimes, basket trials are viewed as a set of sub-trials [27]. Hypothetically, if crizotinib is developed using a basket trial, the basket trial would be composed of sub-trial 1 with NSCLC, sub-trial 2 with ALCL, and sub-trial 3 with IMT, all with ALK fusion-positive tumors. The results will be analyzed either by tumor type within the sub-trials or altogether.

Traditional clinical trials are based on randomization in new treatment vs. standard of care to avoid selection bias. With the introduction of targeted therapy, molecular segmentation of cancer resulted in a small patient population. And this became a challenge for conducting clinical trials [28]. In crizotinib phase 3 clinical trials in Europe, for example, there was a patient selection process from NSCLC patients. A total of 4967 NSCLC patients were screened, and 347 ALK fusion-positive patients were selected and randomized. The clinical benefit of crizotinib over chemotherapy was shown with overall response rates (65% vs. 20%) and a median PFS (7.7 months vs. 3 months) [29]. Given the large number of patients to be screened and the high overall response rate, the requirement of randomization was called into question. With the introduction of drug development in a tissue-agnostic way, FDA approval could be granted based on the nonrandomized trials. Pembrolizumab obtained FDA approval based on the clinical trials with 149 patients [30], larotrectinib with 55 patients [3], and entrectinib with 54 patients [4]. Given the extremely low prevalence of NTRK fusion (0.31%) [31], it would take a much more extended period to recruit patients for randomized clinical trials.

Tumor-agnostic approach cannot be adopted for all oncogenic alterations [6,16]. The B rapidly accelerated fibrosarcoma (BRAF) inhibitor vemurafenib is very effective in melanoma and NSCLC patients with the BRAF V600 mutation, an activating mutation of BRAF. However, only 5% of colorectal cancers harboring the BRAF V600 mutation respond to vemurafenib therapy [32]. Several clinical studies have been conducted on trastuzumab for tumors with HER2 mutations or amplification; the clinical benefits differed depending on the tumor type. A subset of colorectal cancer with HER2 amplification (5%) showed an overall response rate (ORR) of 30% only for lapatinib plus trastuzumab therapy [33]. Despite the 20 years of clinical research on various tumor types with HER2 aberration, the indications for trastuzumab are only breast cancer and gastroesophageal cancer. These

studies clearly show that not all biomarkers can be developed in a tissue-agnostic manner. Besides Trk and PD-1 inhibitors already approved by the FDA, the targets for potential tissue-agnostic drugs in clinical development are ret proto-oncogene (RET), ALK, fibroblast growth factor receptor (FGFR), Axl, ros proto-oncogene 1 (ROS1), and BRAF (Table 1) [17]. It is interesting to note that oncogenic alterations caused by chromosomal rearrangements, RET, ALK, FGFR, and ROS1 fusion proteins account for the majority of cancer targets in tissue-agnostic therapy.

Besides regulatory reasons, there are several factors why drug development processes have been restricted to one cancer type before pembrolizumab. There are different available therapies and unmet medical needs for each tumor type, factors that are considered substantially for development decisions and drug approval. In addition, the endpoints of drug efficacy for each tumor type are different. As some drug discovery experts term as 'low hanging fruit', generally a tumor type with no known therapy and urgent unmet medical need is first taken up for clinical research, subsequently followed by development in another tumor type.

With the introduction of tissue-agnostic drug development approaches, several tumor types can be subjected to clinical trials at the same time. Tissue-agnostic drug development is also good news for rare cancer patients. Due to the small number of patients, it is not easy to conduct clinical trials for cancers with low incidence. Tissue-agnostic drug development enables the participation of rare cancer patients in clinical trials; therefore, rare cancer patients can benefit from this new paradigm of drug approval process [28].

### **3. Trk Inhibitors**

### *3.1. Trk and Cancer*

The Trk family is comprised of three isoforms, TrkA, TrkB, and TrkC, encoded by NTRK1, NTRK2, and NTRK3, respectively. The Trk family is abundantly expressed in the nervous system. Ligands for Trk cell surface receptor tyrosine kinase are nerve growth factor (NGF) for TrkA, brain-derived neurotropic factor or neurotropin 4 for TrkB, and neurotropin 3 for TrkC [1]. Downstream signaling for Trk receptor kinases is primarily mediated by the phospholipase Cγ, mitogen-activated protein kinase, and phosphoinositol-3 kinase pathways.

As implicated by the expression pattern and cognate ligands, neuronal development and differentiation have been reported as major functions of Trk pathways. The importance of TrkA in neuronal development is shown in case of genetic diseases with loss-of-function NTRK genes. Hereditary disorder called congenital insensitivity to pain (CIPA) is reported to have NTRK1 gene mutations [34]. The absence of TrkA during fetal development results in the loss of pain sensing in TrkA-deficient mice [35,36], suggesting the crucial role of TrkA signaling in nociceptive reception [37]. In case of TrkB, impairment of TrkB signaling causes hyperphagia and consequent obesity [38].

Various mechanisms of Trk activation exist in cancer, including somatic mutations, activating splice variants, Trk overexpression, and NTRK fusion [1]. The most common mechanism of Trk activation in cancer is fusion involving NTRK1, NTRK2, and NTRK3. Trk fusion proteins are generated by chromosomal rearrangements between NTRK genes, including the kinase domain, with different partner genes. The resulting fusion proteins are chimeras with a constitutively activated Trk kinase, independent of ligand binding [39].

The first identified NTRK fusion was tropomyosin 3 (TPM3)-NTRK1, which was found in patients with colorectal cancer [40]. Subsequently, Trk fusion proteins with different partners have been identified in a variety of cancer types. The NTRK fusions include translocated promoter region (TPR)-NTRK1 in thyroid cancer [41], tripartite motif containing 24 (TRIM24)-NTRK2 [42] and ETS variant transcription factor 6 (ETV6)-NTRK3 in fibrosarcoma [43].

### *3.2. Larotrectinib*

Larotrectinib, also known as ARRY-470, LOXO-101, and Vitrakvi®, is the first FDAapproved Trk inhibitor with high potency and selectivity. Larotrectinib inhibits the in vitro kinase activity of TrkA by blocking ATP-binding sites with an half maximal inhibitory concentration (IC50) of 10 nM [44]. Kinase selectivity analyses with 226 kinases indicated that larotrectinib is highly selective for TrkA, TrkB, and TrkC. Except for one kinase, TNK2, inhibition of no other notable kinases was observed. Larotrectinib potently suppressed the growth of cancer cells harboring TrkA and TrkB fusion proteins in vitro and in vivo [45].

Based on the impressive preclinical efficacy, clinical trials of larotrectinib started in 2014. Approval of larotrectinib is based on three clinical studies: an adult phase 1 trial (NCT02122913; LOXO-TRK-14001), a pediatric phase 1/2 trial called SCOUT (NCT02637687; LOXO-TRK-15003), and an adult/adolescent phase 2 basket trial called NAVIGATE (NCT02576431; LOXO-TRK-15002). Five journal articles have been published to date on these clinical trials. Deobele et al. described a case of a patient with soft-tissue sarcoma treated with larotrectinib in the LOXO-TRK-14001 trial [45]. Cases of five patients in the SCOUT clinical trial were discussed in the paper by Dubois et al. [46], and the overall phase 1 study results of the SCOUT trial with 24 pediatric solid tumor patients were published by Laetsch et al. [47]. The combined analyses of the three clinical trials stated above (LOXO-TRK-14001, SCOUT, and NAVIGATE) were published for 55 patients from 2015 to 2017 (data cut-off), and larotrectinib was approved on the basis of these results [48]. Clinical research continued, and data from 2014 to 2019 with 159 patients were analyzed and reported in 2020 by Hong et al. [49].

According to a recent report by Hong et al. [49], 159 patients with Trk fusion-positive cancers were treated with larotrectinib, with ages ranging from less than 1 month to 84 years. There were 153 evaluable patients, and the ORR was 79% (121 patients), consisting of complete response in 16% (24 patients) and partial response in 63% (97 patients). More than 16 tumor types were included in the clinical research, and clinical benefits were observed in a wide range of tumor types indicating tumor-agnostic activity. Trk fusions for NTRK1, NTRK2, and NTRK3 were included with 29 distinct fusion partners. The response rate was independent of the Trk subtype and upstream fusion partners. The adverse events of larotrectinib treatment were predominantly grade 1 and 2, indicating that long-term administration is feasible.

### *3.3. Entrectinib*

Entrectinib, also called RXDX-101, NMS-E628, and Rozlyreck®, is an orally available inhibitor of TrkA/B/C, ROS1, and ALK [4]. Potent in vitro kinase activity for TrkA/B/C, ROS1, and ALK exhibited IC<sup>50</sup> values between 1 nM and 12 nM [50]. The growth of cancer cell lines addicted to these kinases was suppressed upon entrectinib treatment in vitro and in vivo. Entrectinib was designed to have intracranial activity; thus, penetration into the central nervous system (CNS) has been demonstrated in preclinical models. The brain/plasma ratio of entrectinib in mice was 0.43 [50].

Three representative clinical trials of entrectinib are ALKA-372-001 (EudraCT 2012- 000148-88), STARTRK-1 (NCT02097810), and STARTRK-2 (NCT02568267). ALKA-372-001 and STARTRK-1 are phase 1 dose-escalation studies, while STARTRK-2 is a phase 2 basket trial. The interim results of ALKA-372-001 and STARTRK-1 in 119 patients were published in 2017 [51]. Integrated analyses of the three clinical trials were reported in 2020 [52]. Another key trial is STARTRK-NG (NCT02650401), which is a phase 1/1b multicenter, dose-escalation study in patients aged 2–21 years with recurrent or refractory solid tumors and primary CNS tumors [4]. The STARTRK-1, -2, and -NG trials are ongoing.

Pooled analyses of ALKA-372-001, STARTRK-1, and STARTRK-2 with a data cut-off date in May 2018 were performed [52]. Efficacy-evaluable patients included 54 adults with NTRK fusion-positive solid tumors. Ten different types of tumor types were included, with the predominant types being sarcoma (13 (24%) patients) and NSCLC (10 (19%)). Among the 54 patients, 12 (22%) had baseline CNS disease and 31 (57%) had an objective response, comprising of 4 (7%) complete responses and 27 (50%) partial responses. 9 patients (17%) showed stable disease. The median response duration was 10 months. Among the 11 patients with brain metastases at baseline, six patients had measurable disease for intracranial response: four with complete response or partial response, one with stable disease, and one with progressive disease. The overall safety-evaluable population was 355 patients, and the most common grade 3 or 4 adverse events were weight gain and anemia. The most serious adverse events were nervous system disorders, reported in 10 (3%) of the 355 patients. Overall, entrectinib achieved anti-tumor activity against tumors harboring NTRK1, NTRK2, and NTRK3 fusions, including CNS activity.

### *3.4. Trk Inhibitors in Clinical Development*

Other Trk inhibitors have been developed to overcome resistance mutations. Selitrectinib (LOXO-195, BAY 2731954) and repotrectinib (TPX-0005) are next-generation Trk inhibitors with efficacy against Trk with acquired resistance. Resistance mutations in the amino acid substitution of the Trk kinase domain have been reported in clinical cases [53]. The most common mutations are "solvent-front" mutations, termed after the hydrophilic solvent-exposed portion of the kinase domain ATP-binding site. TrkA G595R and TrkC G623R are solvent-front mutations, and TrkB mutations have not been reported to date [1]. In addition, gatekeeper mutations (TrkA F589L) and xDFG (Aspartate-Phenylalanine-Glycine) site mutations of TrkA (G667S) and TrkC (G696A) were identified in patients.

Selitrectinib has been developed in parallel with clinical trials to prepare for the emergence of resistance to larotrectinib [54]. Selitrectinib showed potent activity against TrkA/C solvent-front- and xDFG site-mutated forms as well as TrkA/C wild-type in in vitro and in vivo xenograft experiments. Kinase profiling of selitrectinib showed that it is highly selective for Trk kinase. A Phase1/2 clinical trial for selitrectinib is ongoing (NCT03215511), and interim results have been reported [20]. Patient selection was based on presence of tumor with TRK fusion and tumor progression or intolerance to prior Trk inhibitors. A total of 31 patients were analyzed and an ORR of 34% was reported. Trk mutations were identified in 20 patients, including 14 solvent-front, 4 gatekeeper, and 2 xDFG mutations, and a complete or partial response was observed in 9 patients.

Repotrectinib is a next-generation TKI inhibitor designed to inhibit the solvent-front mutations of Trk, ROS1, and ALK. In addition to TrkA G595R and TrkC G623R, repotrectinib is also active against solvent-front mutations of ROS1 and ALK in vitro and in vivo [18]. A phase 1/2 clinical trial of repotrectinib (TRIDENT-1, NCT03093116) is ongoing for TKIrefractory patients.

### *3.5. Adverse Effects of Trk Inhibitors on the Central Nervous System*

Given the physiological roles of Trk signaling in the neuronal system, the effects of Trk inhibitors in the CNS are expected. It is reasonable to expect and prepare for the adverse effects of Trk inhibitors on the CNS. As described above, congenital insensitivity to pain is caused by NTRK1 mutation [34]. Based on the role of NTRK1 in pain sensing, NGF/TrkA is a target for analgesics, and several small molecules and antibodies modulating the NGF/TrkA pathway are under development [55].

Trk signaling is known to play a role in nerve growth during the fetal period, while NGF function is reported to induce pain in adulthood [55]. From the beginning of Trk inhibitor development, there was a concern for adverse CNS effects, and thus, adverse events were closely observed and characterized [56]. Representative adverse events related to neurological systems include withdrawal pain, weight gain, and dizziness [48,52].

Patients who discontinued Trk inhibitor therapy experienced symptoms of pain. Full-body ache, muscle pain, allodynia, and concurrent flares of pre-existing pain are described as withdrawal symptoms [56]. The mechanisms of withdrawal pain are not clear, but it is presumed to be caused by increased expression of transient receptor potential vanilloid I, a nociceptive mediator [57]. Weight gain was observed in more than 50% of patients, as expected from the role of the brain-derived neurotropic factor (BDNF)-

TrkB pathway in appetite centers [38,58,59]. Hyperphagia and consequent obesity were observed upon BDNF-TrkB axis impairment in both mice and humans. Dizziness was caused upon TrkB and TrkC inhibition. Mutant mice with low levels of BDNF in the cerebellum developed ataxia [60]. Mice with NTRK3 gene knockout exhibited abnormal movement and posture [61]. These adverse events in the CNS caused by on-target inhibition of Trk inhibitors should be monitored carefully.

### *3.6. Identification of NTRK Fusions*

Obviously, the diagnostic identification of NTRK fusion genes is an important process in tissue-agnostic Trk inhibitor development. Patient selection with NTRK fusion-positive cancers is a key point in screening responders and non-responders. NTRK fusions can be evaluated using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), reverse transcriptase polymerase chain reaction (RT-PCR), and next-generation sequencing (NGS) [2].

IHC using antibodies against Trk proteins can be utilized for identification of NTRK fusion, as Trk proteins are poorly expressed in normal adult tissues [62]. Positive staining in the IHC test can be interpreted as the presence of NTRK fusions. Information about cellular localization of fused proteins can also be obtained from IHC results. The localization of fusion proteins depends on the normal localization of the fusion partner [62]. For example, LMNA-NTRK1 fusion result in nuclear membrane staining due to the nuclear membrane protein lamin A/C encoded by the LMNA gene, while ETV6-NTRK3 fusions exhibit nuclear staining due to the ETV6-encoding protein located in the nucleus. IHC can be used for diagnosis in conjunction with other diagnostic methods.

FISH is a highly sensitive and specific tool for the detection of fused genes generated from chromosomal rearrangements, such as ALK, ROS1, and RET. In general, FISH has many advantages, such as high sensitivity and quick turn-around time. In case of NTRK1, 2, and 3 fusions, three separate assays are required for each gene. When chromosomal rearrangements involve non-canonical sites or intra-chromosomal rearrangements, FISH can lead to false negative results [63].

RT-PCR uses primers recognizing the 50 -fusion partner and NTRK kinase domain. Since there are numerous fusion partners, RT-PCR has limitations in clinical applications. Furthermore, fused genes with novel fusion partners cannot be detected using RT-PCR [64].

NGS offers the advantage of simultaneous assessment of multiple oncogenes. NGS is a highly sensitive and specific assay via which unknown NTRK fusions can be identified. DNA- and RNA-based NGS assays are currently available. Sometimes DNA-based NGS can fail to detect NTRK fusions because of the large intronic regions. RNA-based NGS assays can overcome this disadvantage of DNA-based NGS, as the results are not affected by intron size. However, the unstable nature of RNA is a major limitation of this assay. Currently, NGS assay sequencing of mature mRNA is considered the gold standard for NTRK fusion detection [64].

In 1998, the FDA approved the first CDx for drug-diagnostic co-development, HercepTest for trastuzumab therapy [65]. According to the regulatory guidance issued by the FDA in 2014, CDx testing is mandatory and must be performed before the use of the corresponding therapeutic product [66]. The FDA has approved an NGS-based CDx test for larotrectinib (Foundation Medicine Inc., F1CDx) [65]. It is a DNA-based NGS assay and was approved based on the retrospective testing of available tumor samples from patients in the three clinical trials of larotrectinib described above. Currently, there is no FDA-approved CDx for entrectinib.

### **4. Conclusions**

Novel Trk inhibitors, larotrectinib and entrectinib, exhibit impressive clinical activity in cancer patients with Trk fusions. The tissue-agnostic drug development approach made it possible for a relatively efficient clinical development process. Although the tissue-agnostic approach cannot be applied to all cancer targets, these processes can expedite some of the drug development projects with unique biomarkers that enable patient selection. The adoption of the tissue-agnostic approach is expected to increase, resulting in an accelerated development process and the possibility of developing therapy for rare cancers.

**Funding:** This research was funded by the National Research Foundation, Government of Korea (grant number 2021R1A2C1007790).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data sharing not applicable.

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

### **Abbreviations**


### **References**


## *Article* **Discovery of a Novel Template, 7-Substituted 7-Deaza-4**0 **-Thioadenosine Derivatives as Multi-Kinase Inhibitors**

**Karishma K. Mashelkar <sup>1</sup> , Woong Sub Byun <sup>1</sup> , Hyejin Ko <sup>1</sup> , Kisu Sung <sup>1</sup> , Sushil K. Tripathi <sup>1</sup> , Seungchan An <sup>1</sup> , Yun A Yum <sup>1</sup> , Jee Youn Kwon <sup>1</sup> , Minjae Kim <sup>1</sup> , Gibae Kim <sup>1</sup> , Eun-Ji Kwon <sup>1</sup> , Hyuk Woo Lee <sup>2</sup> , Minsoo Noh <sup>1</sup> , Sang Kook Lee <sup>1</sup> and Lak Shin Jeong 1,\***


**Abstract:** The development of anticancer drugs remains challenging owing to the potential for drug resistance. The simultaneous inhibition of multiple targets involved in cancer could overcome resistance, and these agents would exhibit higher potency than single-target inhibitors. Protein kinases represent a promising target for the development of anticancer agents. As most multi-kinase inhibitors are heterocycles occupying only the hinge and hydrophobic region in the ATP binding site, we aimed to design multi-kinase inhibitors that would occupy the ribose pocket, along with the hinge and hydrophobic region, based on ATP-kinase interactions. Herein, we report the discovery of a novel 40 -thionucleoside template as a multi-kinase inhibitor with potent anticancer activity. The in vitro evaluation revealed a lead **1g** (7-acetylene-7-deaza-40 -thioadenosine) with potent anticancer activity, and marked inhibition of TRKA, CK1δ, and DYRK1A/1B kinases in the kinome scan assay. We believe that these findings will pave the way for developing anticancer drugs.

**Keywords:** 7-deaza-40 -thioadenosine derivatives; multi-kinase inhibitor; anticancer; nucleoside

### **1. Introduction**

Although cancer has been extensively investigated, drug resistance remains a major challenge in the clinical progress of anticancer drugs [1]. It is frequently responsible for treatment failure in patients with cancer undergoing monotherapy. Under these circumstances, a polypharmacological strategy may overcome the drug resistance crisis. The question then arises: How would it work? Cancer cells are dependent not only on a single oncogene but also on cells enclosing it. Therefore, inhibition of a single target produces mutations that promote cancer cell survival, in advanced cancers [2]. Rationally designed multi-target inhibitors that could hit more than one oncogenic target may surpass the effect mediated by single-target inhibitors, as they would obstruct cancer cell proliferation and, secondly, block the microenvironment that facilitates oncogenesis [3]. This would more comprehensively inhibit the pathway involved, simultaneously reducing the negative impact on tumor cells to acquire a resistance mutation. Accordingly, the synergistic effect of inhibiting multiple targets would induce less resistance and greater efficacy [3]. As cancer is a polygenic disease, it is worth noting that a single drug acting synchronously on multiple targets is advantageous over the combined use of individual single-target drugs [3]. Accordingly, smaller doses are required for simultaneous targets to produce desired effects as the molecule will be concurrently present in tissues (at the site of action).

**Citation:** Mashelkar, K.K.; Byun, W.S.; Ko, H.; Sung, K.; Tripathi, S.K.; An, S.; Yum, Y.A.; Kwon, J.Y.; Kim, M.; Kim, G.; et al. Discovery of a Novel Template, 7-Substituted 7-Deaza-40 - Thioadenosine Derivatives as Multi-Kinase Inhibitors. *Pharmaceuticals* **2021**, *14*, 1290. https://doi.org/10.3390/ ph14121290

Academic Editors: Mary J. Meegan and Niamh M O'Boyle

Received: 12 October 2021 Accepted: 8 December 2021 Published: 10 December 2021

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On the other hand, combination therapy complicates the dosing schedule, increases the risk of toxicity arising from drug-drug interactions, and negatively impacts patient adherence, contrary to multi-target drugs [4,5]. These well-established facts motivate our interest in the current study. Polypharmacology has become more appealing in recent years and is currently a hot topic in this field [6–8].

One main factor underlying cancer growth is the presence of kinase mutations. Protein kinases play a crucial role in cellular functions by mediating protein phosphorylation. These enzymes transfer the terminal phosphoryl group of adenosine triphosphate (ATP) to a protein substrate, ultimately resulting in processes, such as signal transduction, gene regulation, and metabolism. Therefore, dysregulation of kinases is often associated with several diseases, including cancer [9]. The protein kinase domain is the most common domain encoded by cancer genes [10] and is linked to cancer onset and progression [10,11]. Several multi-kinase inhibitors are currently in clinical use, indicating growing attention for multi-kinase inhibitors [12]; for example, multi-targeted receptor tyrosine kinase inhibitors, such as sorafenib—approved for the treatment of renal cell carcinoma (RCC) and hepatocellular carcinoma, and sunitinib—approved for the treatment of RCC and imatinib-resistant gastrointestinal stromal tumor have been developed [3,13,14]. Recently, the multi-target kinase inhibitor, entrectinib was approved by the Food and Drug Administration for the treatment of ROS1 (c-ros oncogene 1)-positive, metastatic non-small cell lung cancer and solid tumors with neurotrophic receptor tyrosine kinase (NTRK) fusions [15,16].

The majority of the kinase inhibitors are heterocycles, which are ATP-competitive [17–19], i.e., they act by competing with ATP to bind to the ATP-binding site of kinases, and therefore, block the phosphorylation process. The ATP binding site of protein kinases is illustrated in Figure 1A. The catalytic domain of all protein kinases encompasses two lobes, linked by a flexible hinge region. ATP binds to the cleft, between the two lobes, a highly conserved catalytic structure in protein kinases [20], where the transfer of γ-phosphate of ATP to protein substrate is catalyzed by kinases in their active DFG-in conformation. The adenine ring from ATP forms two hydrogen bonds with the amino acids in the hinge region [21]. The ATP pocket also contains the unoccupied hydrophobic pockets and a hydrophilic ribose region. Most of the ATP-competitive inhibitors known, commonly occupy the hinge and hydrophobic regions I, II [22,23] but rarely the ribose pocket. Nevertheless, it is worthy to note that, occupying the ribose region results in improved binding towards kinases as demonstrated by Gandin et al. [23]. Since ATP binding site is conserved in protein kinases, it could be a challenging task to design selective multi-kinase inhibitors [24] as it can often lead to off-target interactions [22,25]. It may be advantageous in treating polygenic diseases like cancer, where polypharmacological agents are more effective [3]. However, to maintain the safety profile of a multi-kinase inhibitor, only specific kinases should be targeted [22]. It would be of great importance to find out the combination of kinases whose inhibition would result in therapeutic benefits without unwanted side effects [26,27]. The state-of-the-art of kinase inhibitors in human trials have been provided by Klaeger et al. [28].

In the present study, we aimed to design multi-kinase inhibitors that would interact with the hinge region, hydrophobic pocket I, also known as buried region adjacent to the hinge region and ribose pocket at the same time. The interactions with the hydrophobic pocket have frequently been utilized to achieve inhibitor selectivity over kinases [23]. On this basis, we attempted to design novel kinase inhibitors with a nucleoside skeleton as it's an ATP-mimic by modifying the hydrophobic residue (R), based on ATP-kinase interactions, as illustrated in Figure 1B. These compounds are expected to simultaneously inhibit several kinases, given the sequence and structural homology among the ATP binding sites of kinases [20]. Nevertheless, we wanted to determine the most suitable substituent for the hydrophobic pocket which is not occupied by ATP, whether acting as a pharmacophore for selective kinase inhibition. To achieve this goal, we selected a 7-deazaadenine scaffold, as it serves as a good template for functionalization at the 7 position to occupy the adjacent hydrophobic pocket, thereby enhancing the interactions with the kinase. It is interesting to note how a subtle structural variation in the nucleobase

of adenosine exerts cytotoxic biological properties, as demonstrated by a natural product tubercidin (7-deazaadenosine) [29,30]. The 7-substituted-tubercidin analogs also showed very interesting anti-cancer activity [31]. The sugar pocket is predominantly hydrophilic and conserved in most protein kinases. It is well known that the bioisosteric replacement of oxygen with sulfur on furanose imparts chemotherapeutic properties to its respective sulfur analog [32–35] with metabolic stability [36]. Like the ribose ring in ATP, the hydrophilic polar hydroxyl group of the 4-thiosugar moiety will form a hydrogen bond with the sugar region enabling the molecule to fit in it, resulting in enhanced binding [23]. *Pharmaceuticals* **2021**, *14*, x FOR PEER REVIEW 3 of 25

**Figure 1.** The rationale for the design of multi-kinase inhibitors with a nucleoside skeleton. (**A**) Schematic representation displaying catalytic domain of typical protein kinases binding ATP. Important interactions are highlighted with either colored or dashed lines, indicating hydrophobic interaction/hydrogen bonding. (**B**) Rational design of kinase inhibitors based on ATP-kinase interactions. **Figure 1.** The rationale for the design of multi-kinase inhibitors with a nucleoside skeleton. (**A**) Schematic representation displaying catalytic domain of typical protein kinases binding ATP. Important interactions are highlighted with either colored or dashed lines, indicating hydrophobic interaction/hydrogen bonding. (**B**) Rational design of kinase inhibitors based on ATP-kinase interactions.

In the present study, we aimed to design multi-kinase inhibitors that would interact with the hinge region, hydrophobic pocket I, also known as buried region adjacent to the hinge region and ribose pocket at the same time. The interactions with the hydrophobic pocket have frequently been utilized to achieve inhibitor selectivity over kinases [23]. On this basis, we attempted to design novel kinase inhibitors with a nucleoside skeleton as it's an ATP-mimic by modifying the hydrophobic residue (R), based on ATP-kinase interactions, as illustrated in Figure 1B. These compounds are expected to simultaneously inhibit several kinases, given the sequence and structural homology among the ATP binding sites of kinases [20]. Nevertheless, we wanted to determine the most suitable substituent for the hydrophobic pocket which is not occupied by ATP, whether acting as a pharmacophore for selective kinase inhibition. To achieve this goal, we selected a 7-deazaadenine All the synthesized compounds were evaluated for their anticancer activity by employing a sulforhodamine B (SRB) colorimetric assay and the most potent compound **1g** (7-acetylene-7-deaza-40 -thioadenosine) was screened for kinase panel assay. Accordingly, compound **1g** was found to inhibit TRKA (neurotrophic tyrosine receptor kinase 1, NTRK1), DYRK1A/1B (dual specificity tyrosine-phosphorylation-regulated kinase 1A and 1B), and CK1δ (casein kinase 1 delta, CSNK1D) kinases, reportedly associated with overexpression in cancer cells [11,37–39]. To the best of our knowledge, we report for the first time the design and synthesis of 7-substituted 7-deaza-40 -thionucleoside analogs that are supposed to simultaneously occupy hinge, hydrophobic, and ribose regions and their structure-activity relationship as a multi-kinase inhibitor against TRKA, DYRK1A, DYRK1B, and CK1δ with potent anticancer activity. *Pharmaceuticals* **2021**, *14*, x FOR PEER REVIEW 4 of 25

#### scaffold, as it serves as a good template for functionalization at the 7-position to occupy **2. Results and Discussion 2. Results and Discussion**

#### the adjacent hydrophobic pocket, thereby enhancing the interactions with the kinase. It is *2.1. Chemistry 2.1. Chemistry*

interesting to note how a subtle structural variation in the nucleobase of adenosine exerts cytotoxic biological properties, as demonstrated by a natural product tubercidin (7- The structures of synthesized compounds are represented in Figure 2. The structures of synthesized compounds are represented in Figure 2.

ploying a sulforhodamine B (SRB) colorimetric assay and the most potent compound **1g Figure 2.** The structures of target nucleoside analogs were modified at the C7 position. **Figure 2.** The structures of target nucleoside analogs were modified at the C7 position.

(7-acetylene-7-deaza-4'-thioadenosine) was screened for kinase panel assay. Accordingly, compound **1g** was found to inhibit TRKA (neurotrophic tyrosine receptor kinase 1, NTRK1), DYRK1A/1B (dual specificity tyrosine-phosphorylation-regulated kinase 1A and As shown in Scheme 1, to synthesize the final nucleoside **1**, we first synthesized the glycosyl donor **9** from commercially available D-ribose. As shown in Scheme 1, to synthesize the final nucleoside **1**, we first synthesized the glycosyl donor **9** from commercially available D-ribose.

1B), and CK1δ (casein kinase 1 delta, CSNK1D) kinases, reportedly associated with overexpression in cancer cells [11,37–39]. To the best of our knowledge, we report for the first

structure-activity relationship as a multi-kinase inhibitor against TRKA, DYRK1A,

**Scheme 1.** Synthesis of glycosyl donor **9** from D-ribose. Reagents and conditions: (a) (i) MsCl, pyridine, 0 °C, 4 h; (ii) KOH, H2O, rt, 12 h; (b) TBDPSCl, imidazole, DMAP, CH2Cl2, 0 °C to rt, 12 h; (c)

D-Ribose was converted to 2,3-*O*-isopropylidene-D-ribonic γ-lactone (**2**) using the two steps protocol as described previously [40]. First, D-ribose was converted to D-ribonolactone using bromine/water in the presence of potassium carbonate and later treated with acetone in the presence of a catalytic amount of concentrated sulfuric acid at room temperature to afford **2**. Following the reported general protocol [41] for thiosugar **7**, the inversion of configuration at the C4 chiral center of D-ribonolactone was achieved by treat-

9H2O, DMF, 90

NaBH4, THF/MeOH, 0 °C to rt, 2 h; (d) MsCl, Et3N, CH2Cl2, 0 °C to rt, 2 h; (e) Na2S.

°C, 15 h; (f) *m*CPBA, CH2Cl2, −78 °C, 45 min; (g) acetic anhydride, 110 °C, 4 h.

DYRK1B, and CK1δ with potent anticancer activity.

**2. Results and Discussion** 

*2.1. Chemistry* 

The structures of synthesized compounds are represented in Figure 2.

**Figure 2.** The structures of target nucleoside analogs were modified at the C7 position.

glycosyl donor **9** from commercially available D-ribose.

**Scheme 1.** Synthesis of glycosyl donor **9** from D-ribose. Reagents and conditions: (a) (i) MsCl, pyridine, 0 °C, 4 h; (ii) KOH, H2O, rt, 12 h; (b) TBDPSCl, imidazole, DMAP, CH2Cl2, 0 °C to rt, 12 h; (c) NaBH4, THF/MeOH, 0 °C to rt, 2 h; (d) MsCl, Et3N, CH2Cl2, 0 °C to rt, 2 h; (e) Na2S. 9H2O, DMF, 90 °C, 15 h; (f) *m*CPBA, CH2Cl2, −78 °C, 45 min; (g) acetic anhydride, 110 °C, 4 h. **Scheme 1.** Synthesis of glycosyl donor **9** from D-ribose. Reagents and conditions: (a) (i) MsCl, pyridine, 0 ◦C, 4 h; (ii) KOH, H2O, rt, 12 h; (b) TBDPSCl, imidazole, DMAP, CH2Cl<sup>2</sup> , 0 ◦C to rt, 12 h; (c) NaBH<sup>4</sup> , THF/MeOH, 0 ◦C to rt, 2 h; (d) MsCl, Et3N, CH2Cl<sup>2</sup> , 0 ◦C to rt, 2 h; (e) Na2S .9H2O, DMF, 90 ◦C, 15 h; (f) *m*CPBA, CH2Cl<sup>2</sup> , −78 ◦C, 45 min; (g) acetic anhydride, 110 ◦C, 4 h.

D-Ribose was converted to 2,3-*O*-isopropylidene-D-ribonic γ-lactone (**2**) using the two steps protocol as described previously [40]. First, D-ribose was converted to D-ribonolactone using bromine/water in the presence of potassium carbonate and later treated with acetone in the presence of a catalytic amount of concentrated sulfuric acid at room temperature to afford **2**. Following the reported general protocol [41] for thiosugar **7**, the inversion of configuration at the C4 chiral center of D-ribonolactone was achieved by treat-D-Ribose was converted to 2,3-*O*-isopropylidene-D-ribonic γ-lactone (**2**) using the two steps protocol as described previously [40]. First, D-ribose was converted to Dribonolactone using bromine/water in the presence of potassium carbonate and later treated with acetone in the presence of a catalytic amount of concentrated sulfuric acid at room temperature to afford **2**. Following the reported general protocol [41] for thiosugar **7**, the inversion of configuration at the C4 chiral center of D-ribonolactone was achieved by treating **2** initially with mesyl chloride and subjecting it to base hydrolysis using aqueous potassium hydroxide solution to give **3** with inverted stereochemistry. Protection of the C5 hydroxyl of **3** with *tert*-butyldiphenylsilyl (TBDPS), followed by reduction of the resulting compound **4** with NaBH4, afforded diol **5**. Sulfur heterocyclization was performed by converting **5** to dimesylate **6**, immediately reacted with sodium sulfide nonahydrate at 90 ◦C to give **7** at a 28% overall yield from **2**. Compound **7** was subjected to *m*CPBA oxidation at −78 ◦C to give sulfoxide **8** (82% yield). Pummerer rearrangement of **8** occurred upon heating with acetic anhydride to afford glycosyl donor **9** as a 1:1.6 α/β anomeric mixture.

> The glycosyl donor **9** was then condensed with silylated 7-deaza-7-iodo-6-chloropurine under heating at 80 ◦C for 1 h in the presence of a Lewis acid, TMSOTf, to afford the desired β-stereoisomer **10** as a single stereoisomer (40% yield; Scheme 2); however, the same reaction at room temperature failed to afford the desired product. The β configuration of condensed nucleoside **10** was easily determined by 2D NOESY experiments. The NOESY spectrum revealed a correlation between 10 -H and 40 -H, as well as between 10 -H and one of the two methyl groups of the acetonide group. A correlation between 50 -H and H-8 was also observed, confirming the presence of the β-D-anomer (see the Supporting Information). Ammonolysis of **10** in *tert*-butanolic ammonia at 90 ◦C produced key intermediate

**11**, which was ready for functionalization with hydrophobic groups at the C7 position via palladium-catalyzed cross-coupling reactions. Pd-catalyzed Stille coupling of 7-iodo derivative **11** with 2-tributylstannylfuran and 2-tributylstannylthiophene in the presence of PdCl2(PPh3)<sup>2</sup> yielded 7-furanyl and 7-thiofuranyl derivatives **12a** and **12b**, respectively. Removal of acetonides of **12a** and **12b** with 50% aqueous trifluoroacetic acid (TFA) afforded the final nucleosides, **1a**, and **1b**, respectively. mediate **11**, which was ready for functionalization with hydrophobic groups at the C7 position via palladium-catalyzed cross-coupling reactions. Pd-catalyzed Stille coupling of 7-iodo derivative **11** with 2-tributylstannylfuran and 2-tributylstannylthiophene in the presence of PdCl2(PPh3)2 yielded 7-furanyl and 7-thiofuranyl derivatives **12a** and **12b**, respectively. Removal of acetonides of **12a** and **12b** with 50% aqueous trifluoroacetic acid (TFA) afforded the final nucleosides, **1a**, and **1b**, respectively.

ing **2** initially with mesyl chloride and subjecting it to base hydrolysis using aqueous potassium hydroxide solution to give **3** with inverted stereochemistry. Protection of the C5 hydroxyl of **3** with *tert*-butyldiphenylsilyl (TBDPS), followed by reduction of the resulting compound **4** with NaBH4, afforded diol **5**. Sulfur heterocyclization was performed by converting **5** to dimesylate **6**, immediately reacted with sodium sulfide nonahydrate at 90 °C to give **7** at a 28% overall yield from **2**. Compound **7** was subjected to *m*CPBA oxidation at −78 °C to give sulfoxide **8** (82% yield). Pummerer rearrangement of **8** occurred upon heating with acetic anhydride to afford glycosyl donor **9** as a 1:1.6 α/β anomeric mixture. The glycosyl donor **9** was then condensed with silylated 7-deaza-7-iodo-6-chloropurine under heating at 80 °C for 1 h in the presence of a Lewis acid, TMSOTf, to afford the desired β-stereoisomer **10** as a single stereoisomer (40% yield; Scheme 2); however, the same reaction at room temperature failed to afford the desired product. The β configuration of condensed nucleoside **10** was easily determined by 2D NOESY experiments. The NOESY spectrum revealed a correlation between 1'-H and 4'-H, as well as between 1'-H and one of the two methyl groups of the acetonide group. A correlation between 5'-H and H-8 was also observed, confirming the presence of the β-D-anomer (see the Supporting Information). Ammonolysis of **10** in *tert*-butanolic ammonia at 90 °C produced key inter-

*Pharmaceuticals* **2021**, *14*, x FOR PEER REVIEW 5 of 25

**Scheme 2.** Synthesis of 7-substituted 7-deaza-4'-thioadenosine derivatives **1a–f**. Reagents and conditions: (a) 7-Deaza-7 iodo-6-chloropurine, BSA, TMSOTf, CH3CN, rt to 80 °C, 1 h; (b) NH3/*tert*-BuOH, 90 °C, 12 h; (c) corresponding 2-tributylstannylheteroaryl, PdCl2(PPh3)2, THF, MW, 70 °C, 1 h; (d) 50% TFA/H2O, THF, rt, 12 h; (e) corresponding boronic ester, PdCl2(PPh3)2, Na2CO3, DMF/H2O, MW, 70 °C, 1 h. **Scheme 2.** Synthesis of 7-substituted 7-deaza-40 -thioadenosine derivatives **1a–f**. Reagents and conditions: (a) 7-Deaza-7-iodo-6-chloropurine, BSA, TMSOTf, CH3CN, rt to 80 ◦C, 1 h; (b) NH3/*tert*-BuOH, 90 ◦C, 12 h; (c) corresponding 2-tributylstannylheteroaryl, PdCl<sup>2</sup> (PPh<sup>3</sup> )2 , THF, MW, 70 ◦C, 1 h; (d) 50% TFA/H2O, THF, rt, 12 h; (e) corresponding boronic ester, PdCl<sup>2</sup> (PPh<sup>3</sup> )2 , Na2CO<sup>3</sup> , DMF/H2O, MW, 70 ◦C, 1 h.

Further Suzuki coupling reactions were performed to introduce other hydrophobic groups, such as vinyl, phenyl, and 4-substituted-phenyl to **11**. Coupling of **11** with vinyl boronic ester in the presence of PdCl2(PPh3)2 in DMF/H2O gave **12c** in 85% yield. In this Suzuki coupling reaction, water was used as a co-solvent to avoid side reactions, resulting Further Suzuki coupling reactions were performed to introduce other hydrophobic groups, such as vinyl, phenyl, and 4-substituted-phenyl to **11**. Coupling of **11** with vinyl boronic ester in the presence of PdCl2(PPh3)<sup>2</sup> in DMF/H2O gave **12c** in 85% yield. In this Suzuki coupling reaction, water was used as a co-solvent to avoid side reactions, resulting from Heck coupling [42]. The desired Suzuki coupled product was obtained as a single product. Similar Suzuki reactions of **11** with phenyl and 4-substituted phenyl boronic esters afforded 7-phenyl and 7-(4-substituted)phenyl derivatives, **12d**–**f**. Treatment of **12c**–**f** with 50% aqueous TFA yielded the final nucleosides **1c**–**f**, respectively.

Next, to introduce other linear hydrophobic groups, such as acetylene, Sonogashira coupling was employed, as shown in Scheme 3. Treatment of **11** with trimethylsilyl acetylene in the presence of a palladium catalyst and copper iodide afforded **13** (93% yield). Removal of the silyl groups of **13** with 1 M TBAF solution in THF afforded **14**, which was treated with 2 N HCl to yield the 7-acetylene derivative **1g**. The molecular structure of **1g** was confirmed by a single X-ray crystal analysis (CCDC 1575257); further evidence supporting the β-configuration is provided in the Supporting Information [43]. Several 2-substituted acetylene analogs **15a**–**d** were also synthesized from **11** by employing the same Sonogashira coupling conditions. The final propylene, butylene, *tert*-butyl acetylene, and cyclopropyl acetylene analogs **1h**–**k** were obtained by treating **15a**–**d** with 50% aqueous TFA at room temperature.

from Heck coupling [42]. The desired Suzuki coupled product was obtained as a single product. Similar Suzuki reactions of **11** with phenyl and 4-substituted phenyl boronic esters afforded 7-phenyl and 7-(4-substituted)phenyl derivatives, **12d**–**f**. Treatment of **12c**–**f**

Next, to introduce other linear hydrophobic groups, such as acetylene, Sonogashira coupling was employed, as shown in Scheme 3. Treatment of **11** with trimethylsilyl acetylene in the presence of a palladium catalyst and copper iodide afforded **13** (93% yield). Removal of the silyl groups of **13** with 1 M TBAF solution in THF afforded **14**, which was treated with 2 N HCl to yield the 7-acetylene derivative **1g**. The molecular structure of **1g** was confirmed by a single X-ray crystal analysis (CCDC 1575257); further evidence supporting the β-configuration is provided in the Supporting Information [43]. Several 2-substituted acetylene analogs **15a**–**d** were also synthesized from **11** by employing the same Sonogashira coupling conditions. The final propylene, butylene, *tert*-butyl acetylene, and cyclopropyl acetylene analogs **1h**–**k** were obtained by treating **15a**-**d** with 50% aqueous

with 50% aqueous TFA yielded the final nucleosides **1c**–**f**, respectively.

**Scheme 3.** Synthesis of 7-substituted 7-deaza-4'-thioadenosine derivatives **1g–k**. Reagents and conditions: (a) corresponding alkyne, PdCl2(PPh3)2, CuI, Et3N, DMF, MW, 50 °C, 1 h; (b) 1 M TBAF, THF, rt, 40 min; (c) 2 N HCl, THF, rt, 15 h; (d) 50% TFA/H2O, THF, rt, 12 h. **Scheme 3.** Synthesis of 7-substituted 7-deaza-40 -thioadenosine derivatives **1g**–**k**. Reagents and conditions: (a) corresponding alkyne, PdCl<sup>2</sup> (PPh<sup>3</sup> )2 , CuI, Et3N, DMF, MW, 50 ◦C, 1 h; (b) 1 M TBAF, THF, rt, 40 min; (c) 2 N HCl, THF, rt, 15 h; (d) 50% TFA/H2O, THF, rt, 12 h.

#### *2.2. Biological Evaluation 2.2. Biological Evaluation*

TFA at room temperature.

#### 2.2.1. Antiproliferative Activity 2.2.1. Antiproliferative Activity

All synthesized compounds **1a**–**k** were evaluated for their antiproliferative activity against six different cancer cell lines, including human lung (A549), colon (HCT116), breast (MDA-MB-231), liver (SK-HEP-1), stomach (SNU638), and prostate (PC-3) cancer cells, using SRB colorimetric assay[44,45]. As demonstrated in Table 1, compounds **1a** and **1b** possessing furanyl and thiofuranyl moieties at the 7-position, respectively, exhibited All synthesized compounds **1a**–**k** were evaluated for their antiproliferative activity against six different cancer cell lines, including human lung (A549), colon (HCT116), breast (MDA-MB-231), liver (SK-HEP-1), stomach (SNU638), and prostate (PC-3) cancer cells, using SRB colorimetric assay [44,45]. As demonstrated in Table 1, compounds **1a** and **1b** possessing furanyl and thiofuranyl moieties at the 7-position, respectively, exhibited moderate antiproliferative activity. Compound **1c**, with a vinyl substituent at the 7-position, displayed potent anticancer activity, whereas bulky groups, such as phenyl (**1d**) and substituted phenyls (**1e** and **1f**) showed low to no anticancer activity. In contrast, the linear acetylene moiety (**1g**) at the 7-position exhibited excellent anticancer activity in the nanomolar range. Surprisingly, 2-substituted acetylene derivatives **1h**–**k** abolished antiproliferative activity. This result demonstrates that a small and linear hydrophobic group, such as acetylene is necessary for potent anticancer activity. Since compound **1g** exhibited the most potent antiproliferative activity against cancer cells (IC<sup>50</sup> = 0.004–0.06 µM), the antiproliferative activity of **1g** against normal cells was additionally evaluated in primary dermal fibroblast cells. Compound **1g** also showed considerable growth inhibition in cultured human normal dermal fibroblast cells (IC<sup>50</sup> = 0.15 µM). Although rather toxic, compound **1g** seems to be more potent in the antiproliferative activity of cancer cells than normal cells. **Table 1.** Anticancer activity of the final 7-substituted 7-deaza-40 -thionucleosides **1a**–**k** against several human cancer cell lines. eral human cancer cell lines.

moderate antiproliferative activity. Compound **1c**, with a vinyl substituent at the 7-position, displayed potent anticancer activity, whereas bulky groups, such as phenyl (**1d**) and substituted phenyls (**1e** and **1f**) showed low to no anticancer activity. In contrast, the linear acetylene moiety (**1g**) at the 7-position exhibited excellent anticancer activity in the nanomolar range. Surprisingly, 2-substituted acetylene derivatives **1h**–**k** abolished antiproliferative activity. This result demonstrates that a small and linear hydrophobic group, such as acetylene is necessary for potent anticancer activity. Since compound **1g** exhibited the most potent antiproliferative activity against cancer cells (IC50 = 0.004–0.06 μM), the antiproliferative activity of **1g** against normal cells was additionally evaluated in primary dermal fibroblast cells. Compound **1g** also showed considerable growth inhibition in cultured human normal dermal fibroblast cells (IC50 = 0.15 μM). Although rather toxic, compound **1g** seems to be more potent in the antiproliferative activity of cancer cells than

**Table 1.** Anticancer activity of the final 7-substituted 7-deaza-4'-thionucleosides **1a-k** against sev-

*Pharmaceuticals* **2021**, *14*, x FOR PEER REVIEW 7 of 25



**1k** >50 >50 30.6 8.31 >50 >50 Etoposide *h* 0.36 1.11 4.9 0.91 0.41 23.4 Gemcitabine *<sup>i</sup>* 0.3 0.2 1.1 0.2 0.1 3.6 *<sup>a</sup>* Measured using SRB assay. *<sup>b</sup>* Human lung cancer cells. *<sup>c</sup>* Human colon cancer cells. *<sup>d</sup>* Human breast cancer cells. *<sup>e</sup>* Human liver cancer cells. *<sup>f</sup>* Human stomach cancer cell. *<sup>g</sup>* Human prostate cancer cells. *<sup>h</sup>* Etoposide was used as positive control. *<sup>i</sup>* Gemcitabine was used as positive control [44].

#### *<sup>a</sup>*Measured using SRB assay. *<sup>b</sup>*Human lung cancer cells. *<sup>c</sup>*Human colon cancer cells. *<sup>d</sup>* Human 2.2.2. Kinome Scan Profile

breast cancer cells. *e* Human liver cancer cells. *f* Human stomach cancer cell. *g* Human prostate cancer cells. *<sup>h</sup>*Etoposide was used as positive control. *<sup>I</sup>*Gemcitabine was used as positive control [44]. 2.2.2. Kinome Scan Profile To characterize the kinase inhibition profile of the most potent compound **1g**, it was profiled against a panel of 96 kinases at a concentration of 1 μM (Figure 3). The results revealed that compound **1g** exhibited strong inhibitory activities against four kinases at 1 μM (<20% activity remaining), i.e., TRKA (NTRK1), DYRK1B, and CK1δ (CSNK1D) To characterize the kinase inhibition profile of the most potent compound **1g**, it was profiled against a panel of 96 kinases at a concentration of 1 µM (Figure 3). The results revealed that compound **1g** exhibited strong inhibitory activities against four kinases at 1 µM (<20% activity remaining), i.e., TRKA (NTRK1), DYRK1B, and CK1δ (CSNK1D) among the panel (see the Supporting Information for tabular representation of kinome scan data, Table S1). Since compound **1g** showed strong inhibition of DYRK1B, it was evaluated for its isoform, DYRK1A inhibition. Compound **1g** displayed potent inhibition of DYRK1A (IC<sup>50</sup> = 43 nM, see the Supporting information, Table S2). These kinases are reportedly involved in cancer initiation and progression [11,37–39] and represent promising targets for cancer therapy. Based on this result, it could be inferred that the hydrophobic pocket in the ATP binding site of these four kinases could accommodate only small and linear hydrophobic groups such as acetylene for kinase inhibition. Next, the concentrationdependent inhibitory activities of compounds **1a**–**k** were investigated and the half-maximal inhibitory concentration (IC50) was determined against four kinases, TRKA, DYRK1A, DYRK1B, and CK1δ (see the Supporting information, Table S2). The kinase inhibition trend observed for **1a**–**k** was almost similar to that of their antiproliferative activity. In general, compounds **1a**, **1b**, and **1g** exhibited excellent kinase inhibition activities, whereas compounds **1c** and **1d** exhibited moderate kinase inhibition. Among compounds tested, compounds **1a** and **1b** with 7-furanyl and 7-thiofuranyl substituents, respectively showed excellent kinase inhibition activity against NTRK1, whereas compounds **1a** and **1g** with 7-furanyl and 7-acetylene substituents, respectively showed excellent kinase inhibition activity against DYRK1A and DYRK1B. Compounds **1b** and **1d** with 7-thiofuranyl and 7-phenyl substituents, respectively showed excellent inhibition against CSNK1D. However,

sterically demanding compounds **1e** and **1f** and the 2-substituted acetylene derivatives **1h**–**k** demonstrated weak to no inhibitory activity. Compound **1g** showed the best antiproliferative activity, but compound **1a** was discovered as the best inhibitory compound against the above-mentioned four kinases, indicating that the anti-cancer effect of **1g** might occur by unexpected mode of action. However, sterically demanding compounds **1e** and **1f** and the 2-substituted acetylene derivatives **1h**–**k** demonstrated weak to no inhibitory activity. Compound **1g** showed the best antiproliferative activity, but compound **1a** was discovered as the best inhibitory compound against the above-mentioned four kinases, indicating that the anti-cancer effect of **1g** might occur by unexpected mode of action.

among the panel (see the Supporting Information for tabular representation of kinome scan data, Table S1). Since compound **1g** showed strong inhibition of DYRK1B, it was evaluated for its isoform, DYRK1A inhibition. Compound **1g** displayed potent inhibition of DYRK1A (IC50 = 43 nM, see the Supporting information, Table S2). These kinases are reportedly involved in cancer initiation and progression [11,37–39] and represent promising targets for cancer therapy.Based on this result, it could be inferred that the hydrophobic pocket in the ATP binding site of these four kinases could accommodate only small and linear hydrophobic groups such as acetylene for kinase inhibition. Next, the concentration-dependent inhibitory activities of compounds **1a–k** were investigated and the halfmaximal inhibitory concentration (IC50) was determined against four kinases, TRKA, DYRK1A, DYRK1B, and CK1δ (see the Supporting information, Table S2). The kinase inhibition trend observed for **1a**–**k** was almost similar to that of their antiproliferative activity. In general, compounds **1a**, **1b**, and **1g** exhibited excellent kinase inhibition activities, whereas compounds **1c** and **1d** exhibited moderate kinase inhibition. Among compounds tested, compounds **1a** and **1b** with 7-furanyl and 7-thiofuranyl substituents, respectively showed excellent kinase inhibition activity against NTRK1, whereas compounds **1a** and **1g** with 7-furanyl and 7-acetylene substituents, respectively showed excellent kinase inhibition activity against DYRK1A and DYRK1B. Compounds **1b** and **1d** with 7-thiofuranyl and 7-phenyl substituents, respectively showed excellent inhibition against CSNK1D.

*Pharmaceuticals* **2021**, *14*, x FOR PEER REVIEW 8 of 25

**Figure 3.** Kinase inhibition profile of compound **1g**. Kinome scan assays were performed for compound **1g** (1 μM) against 96 kinases. \*\*—strong inhibition of target kinases (<20% activity remaining). **Figure 3.** Kinase inhibition profile of compound **1g**. Kinome scan assays were performed for compound **1g** (1 µM) against 96 kinases. \*\*—strong inhibition of target kinases (<20% activity remaining).

#### 2.2.3. Antiproliferative Activity against KM12 and ACHN Cell Lines 2.2.3. Antiproliferative Activity against KM12 and ACHN Cell Lines

The effect of **1g** on cell growth was also determined in KM12, a colon cancer cell line that highly expresses NTRK1/2/3 and DYRK3. Compared to doxorubicin (IC50 = 0.12 μM), compound **1g** (IC50 = 0.07 μM) showed marked activity against the cell growth of KM12. Likewise, IC50 of **1g** on cell growth of ACHN, a renal cancer cell line, expressing CK1δ and DYRK2 was approximately 0.04 μM, when compared with 0.04 μM of doxorubicin, suggesting the highly effective and selective anti-proliferative activity of **1g** in cancer cell lines expressing NTRK, DYRK2 or CK1δ; However, we can't exclude the possibility that anticancer phenotype might be driven through some other mechanism. The effect of **1g** on cell growth was also determined in KM12, a colon cancer cell line that highly expresses NTRK1/2/3 and DYRK3. Compared to doxorubicin (IC<sup>50</sup> = 0.12 µM), compound **1g** (IC<sup>50</sup> = 0.07 µM) showed marked activity against the cell growth of KM12. Likewise, IC<sup>50</sup> of **1g** on cell growth of ACHN, a renal cancer cell line, expressing CK1δ and DYRK2 was approximately 0.04 µM, when compared with 0.04 µM of doxorubicin, suggesting the highly effective and selective anti-proliferative activity of **1g** in cancer cell lines expressing NTRK, DYRK2 or CK1δ; However, we can't exclude the possibility that anti-cancer phenotype might be driven through some other mechanism.

### 2.2.4. Metabolic Stability and CYP Inhibition

2.2.4. Metabolic Stability and CYP Inhibition Based on anticancer and kinase inhibition data, compound **1g** was examined for its microsomal stability and CYP isozyme inhibition in vitro (Table 2). Compound **1g** was metabolically stable in human liver microsomes. Moreover, it showed no CYP isozyme inhibition against the five major drug-metabolizing cytochrome P450 isozymes. Based on anticancer and kinase inhibition data, compound **1g** was examined for its microsomal stability and CYP isozyme inhibition in vitro (Table 2). Compound **1g** was metabolically stable in human liver microsomes. Moreover, it showed no CYP isozyme inhibition against the five major drug-metabolizing cytochrome P450 isozymes.


**Table 2.** Human liver microsomal stability and CYP isozyme inhibition activity of **1g**.

*<sup>a</sup>* % Remaining during 30 min. *<sup>b</sup>* IC<sup>50</sup> < 1 µM—potent inhibition; 1 µM < IC<sup>50</sup> < 10 µM—moderate inhibition; IC<sup>50</sup> > 10 µM—no or weak inhibition. *<sup>c</sup>* Percentage of enzyme remaining after inhibition. *<sup>d</sup>* CYP3A4 inhibitor (0.1 µM).

### *2.3. Docking Analysis*

Next, to justify the rationale of this design strategy, the ligand-bound DYRK1A (PDB ID: 7A51) [46] and TRKA (PDB ID: 5JFV) [47] crystal structures were used for the molecular docking study (Figure 4). The docked pose of compound **1g** into the ATP binding site of DYRK1A and TRKA, as depicted in Figure 4A and 4B respectively, revealed that the purine ring formed two key hydrogen bonds with hinge residue Leu 241, and Glu 239 of DYRK1A, whereas in the case of TRKA the purine ring of **1g** formed hydrogen bonding with Met 592 and Glu 590, in a manner comparable with that of ATP. Furthermore, hydrogen bonding interaction was seen between 50 -OH of **1g** and Glu 291 of DYRK1A (Figure 4C). Similarly, hydrogen bonding interaction between 50 -OH of **1g** and Glu 518 of TRKA was

observed. In addition, the acetylene moiety occupied the hydrophobic pocket as supposed and displayed hydrophobic interactions with gatekeeper residue, Phe 238 and Val 306 of DYRK1A (Figure 4C). While, in the TRKA: **1g** docked pose acetylene was found to form hydrophobic interactions with the gatekeeper residue, Phe 589 and Phe 669 (Figure 4D). Additionally, a hydrophobic interaction of 4-thio of the sugar moiety with Val 173 and Val 524 of DYRK1A and TRKA, respectively was observed. Thus, it can be concluded that compound **1g** fits markedly well in the ATP binding sites of DYRK1A and TRKA, thus providing a new scaffold that inhibits the activity of these enzymes. *Pharmaceuticals* **2021**, *14*, x FOR PEER REVIEW 11 of 25

**Figure 4.** (**A**) Proposed binding mode of **1g** (represented in green) in the ATP binding site of (**A**) DYRK1A (represented in pale cyan; PDB ID: 7A51). (**B**) TRKA (represented in gray; PDB ID: 5JFV). Hydrogen bonding is represented as yellow dashed lines. Non-polar hydrogen atoms are omitted. (**B**) 2D interaction diagram of **1g** (represented in green) with (**C**) DYRK1A amino acid residues. (**D**) TRKA amino acid residues. **Figure 4.** (**A**) Proposed binding mode of **1g** (represented in green) in the ATP binding site of (**A**) DYRK1A (represented in pale cyan; PDB ID: 7A51). (**B**) TRKA (represented in gray; PDB ID: 5JFV). Hydrogen bonding is represented as yellow dashed lines. Non-polar hydrogen atoms are omitted. (**B**) 2D interaction diagram of **1g** (represented in green) with (**C**) DYRK1A amino acid residues. (**D**) TRKA amino acid residues.

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

#### *3.1. General Methods 3.1. General Methods*

Proton (1H) and carbon (13C) NMR spectra were recorded on a JEOL JNM-GCX (400/100 MHz), Bruker AMX-500 (500/125 MHz), or JEOL JNM-ECA 600 (600/150 MHz) spectrometer. Chemical shifts are given in parts per million (*δ*), calibrated to the solvent peak, and coupling constants (*J*) in hertz (Hz). High-resolution mass (HRMS) measure-Proton (1H) and carbon (13C) NMR spectra were recorded on a JEOL JNM-GCX (400/100 MHz), Bruker AMX-500 (500/125 MHz), or JEOL JNM-ECA 600 (600/150 MHz) spectrometer. Chemical shifts are given in parts per million (*δ*), calibrated to the solvent peak, and coupling constants (*J*) in hertz (Hz). High-resolution mass (HRMS) measure-

ments were recorded on a Thermo LCQ XP instrument. UV spectra were recorded in

determined on a Barnstead electrothermal 9100 instrument and are uncorrected. Microwave-assisted reactions were conducted in Biotage Initiator+ US/JPN (part no. 356007) microwave reactor. The TLC spots were examined under ultraviolet light at 254 nm and further visualized by *p*-anisaldehyde or phosphomolybdic acid stain solution. Column chromatography was performed using silica gel (Kieselgel 60, 70–230 mesh, Merck). The

ments were recorded on a Thermo LCQ XP instrument. UV spectra were recorded in methanol on a U-3000 made by Hitachi. Optical rotations were measured on Jasco III in an appropriate solvent and [α] 25 <sup>D</sup> values are given in 10−<sup>1</sup> deg cm<sup>2</sup> g −1 . Melting points were determined on a Barnstead electrothermal 9100 instrument and are uncorrected. Microwave-assisted reactions were conducted in Biotage Initiator+ US/JPN (part no. 356007) microwave reactor. The TLC spots were examined under ultraviolet light at 254 nm and further visualized by *p*-anisaldehyde or phosphomolybdic acid stain solution. Column chromatography was performed using silica gel (Kieselgel 60, 70–230 mesh, Merck). The purity of all tested compounds was determined by high-performance liquid chromatography (HPLC) analysis, confirming ≥95% purity.

### *3.2. Chemical Synthesis*

3.2.1. (3aR,6S,6aR)-6-(Hydroxymethyl)-2,2-dimethyldihydrofuro[3,4-d][1,3] dioxol-4(3aH)-one (**3**)

To an ice-cooled solution of **2** (50 g, 0.265 mol) in pyridine (330 mL), methanesulfonyl chloride (52.34 g, 0.45 mol) was dropwise added under a nitrogen atmosphere and the solution was stirred at room temperature for 4 h. The reaction was quenched with slow addition of saturated aqueous NaHCO<sup>3</sup> (520 mL) with stirring until no effervescence and extracted with dichloromethane (2 L). The combined organic layer was washed successively with water and brine, dried (MgSO4), and concentrated in vacuo below 25 ◦C to give the crude product. To this crude mesylate, a solution of potassium hydroxide (36.74 g, 0.65 mol) in water (250 mL) was added using a dropping funnel, maintaining the temperature below 30 ◦C. This reaction mixture was stirred at room temperature for 12 h and then adjusted to pH 3.0 to 4.0 by adding 3 M hydrochloric acid (260 mL). The acidic solution was concentrated under reduced pressure to afford a solid mass. The solid mass was triturated with acetone (2 L) and heated to 50 ◦C for 30 min. The acetone was decanted, dried over anhydrous MgSO4, and filtered. The filtrate was concentrated to obtain crude **3** (*R*<sup>f</sup> = 0.45, TLC eluent = CH2Cl2/MeOH, 19:1).

3.2.2. (3aR,6S,6aR)-6-(((*tert*-butyldiphenylsilyl)oxy)methyl)-2,2-dimethyldihydrofuro[3,4 d][1,3]dioxol-4(3aH)-one (**4**)

To a solution of **3** in methylene chloride (240 mL), imidazole (8.78 g, 129 mmol) was added, followed by dropwise addition of *tert*-butyldiphenylsilyl chloride (26 g, 94.26 mmol) at 0 ◦C. After being stirred at room temperature for 12 h, the reaction mixture was partitioned between methylene chloride (3 × 600 mL) and water (760 mL). The layers were separated and the combined organic layer was dried (MgSO4), filtered, and evaporated to give crude **4** (*R*<sup>f</sup> = 0.50, TLC eluent = hexane/ethyl acetate, 4:1).

3.2.3. (S)-2-((*tert*-butyldiphenylsilyl)oxy)-1-((4S,5R)-5-(hydroxymethyl)-2,2-dimethyl-1,3 dioxolan-4-yl)ethan-1-ol (**5**)

The crude **4** was dissolved in THF-MeOH (285 mL-54 mL) and to this, sodium borohydride (14.64 g, 387.07 mmol) was added portion wise at 0 ◦C. After stirring for 2 h at room temperature, the reaction mixture was quenched with glacial acetic acid (26 mL) and evaporated. The residue was diluted with 20% aqueous potassium sodium tartrate (500 mL) and the aqueous layer was extracted with ethyl acetate (3 × 600 mL). The organic layer was washed with brine, dried over MgSO4, and evaporated to obtain crude **5** (*R*<sup>f</sup> = 0.50, TLC eluent = hexane/ethyl acetate, 3:2).

3.2.4. (S)-2-((*tert*-butyldiphenylsilyl)oxy)-1-((4R,5R)-2,2-dimethyl-5- (((methylsulfonyl)oxy)methyl)-1,3-dioxolan-4-yl)ethyl Methanesulfonate (**6**)

To a solution of **5** in methylene chloride (277 mL), 4-dimethylaminopyridine (0.36 g, 2.96 mmol) and triethylamine (106.40 g, 1051.53 mmol) were added and the solution was cooled to 0 ◦C. To this methanesulfonyl chloride (59.11 g, 516.09 mmol) was added dropwise and the reaction mixture was stirred at room temperature for 2 h. The reac-

tion was quenched with saturated aqueous NaHCO<sup>3</sup> (300 mL) until no effervescence and extracted with methylene chloride (3 × 520 mL). The combined organic layer was washed with brine (150 mL), dried (MgSO4), and passed through silica to remove any inorganic impurities and evaporated below 25 ◦C to give crude di-*O*-mesylate **6** (*R*<sup>f</sup> = 0.55, TLC eluent = hexane/ethyl acetate, 3:2).

### 3.2.5. *tert*-Butyl(((3aS,4R,6aR)-2,2-dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4 yl)methoxy)diphenylsilane (**7**)

To a stirred solution of **6** in DMF (1.5 L) crushed Na2S·9H2O (43.37g, 180.60 mmol) was added and the reaction mixture was transferred to a preheated bath at 90 ◦C. After being stirred for 15 h, the reaction mixture was cooled to room temperature and quenched with water (1 L). The aqueous layer was extracted with n-hexane (3 × 1 L). The organic layer was combined, washed with brine, dried over anhydrous MgSO4, and concentrated. The residue was purified by column chromatography (silica gel, hexane/ethyl acetate, 19:1) to afford **7** (31.6 g, 28% in 5 steps) as pale yellow syrup; [α] 25 <sup>D</sup> 45.17 (*c* 14.5, CH3OH); UV (CH3OH) *λ*max 264.94 nm; <sup>1</sup>H NMR (CDCl3, 500 MHz): *δ* 7.67–7.63 (m, 4H), 7.42–7.35 (m, 6H), 4.79 (d, *J* = 1.7 Hz, 2H), 3.76 (dd, *J* = 10.6, 4.9 Hz, 1H), 3.60 (dd, *J* = 10.6, 6.8 Hz, 1H), 3.38–3.35 (m, 1H), 3.13–3.10 (m, 1H), 2.82 (merged dd, *J*<sup>1</sup> = *J*<sup>2</sup> = 12.6 Hz, 1H), 1.50 (s, 3H), 1.30 (s, 3H), 1.04 (s, 9H); <sup>13</sup>C NMR (CDCl3, 125 MHz): *δ* 135.56, 135.55, 133.0, 132.9, 129.8, 129.7, 127.7, 110.6, 85.9, 83.7, 65.9, 55.4, 38.3, 26.8, 26.4, 24.5, 19.1; HRMS (ESI-Q-TOF) *m*/*z* [M + NH4] + for C24H36NO3SSi calculated 446.218, found 446.2175.

3.2.6. (3aS,4R,5S,6aR)-4-(((*tert*-butyldiphenylsilyl)oxy)methyl)-2,2 dimethyltetrahydrothieno[3,4-d][1,3]dioxole 5-oxide (**8**)

A solution of 3-chloroperbenzoic acid (5.56 g, 32.25 mmol) in methylene chloride (110 mL) was added dropwise to a stirred solution of **7** (27.65 g, 64.50 mmol) in methylene chloride (110 mL) at −78 ◦C under a nitrogen atmosphere and stirred at the same temperature for 45 min. The reaction mixture was quenched with saturated NaHCO<sup>3</sup> solution and extracted with methylene chloride (3 × 650 mL). The combined organic layer was washed with brine, dried over MgSO4, evaporated, and the residue was purified by silica gel column chromatography (hexane/ethyl acetate, 2:3) to afford **8** (23 g, 82%) as a colorless syrup; [α] 25 <sup>D</sup> 17.87 (*c* 3.75, CH3OH); UV (CH3OH) *λ*max 264.94 nm; <sup>1</sup>H NMR (CDCl3, 500 MHz): *δ* 7.59–7.54 (m, 4H), 7.46–7.43 (m, 2H), 7.40–7.37 (m, 4H), 5.21 (t, *J* = 5.5 Hz, 1H), 4.97 (d, *J* = 5.3 Hz, 1H), 4.09 (dd, *J* = 11.2, 2.5 Hz, 1H), 3.82 (dd, *J* = 11.2, 3.3 Hz, 1H), 3.58 (s, 1H), 3.39 (merged dd, *J*<sup>1</sup> = *J*<sup>2</sup> = 14.8 Hz, 1H), 3.25 (dd, *J* = 14.8, 6.0 Hz, 1H), 1.56 (s, 3H), 1.33 (s, 3H), 1.01 (s, 9H); <sup>13</sup>C NMR (CDCl3, 125 MHz): *δ* 135.4, 135.3, 131.7, 131.6, 130.1, 130.0, 127.9, 127.8, 112.2, 86.3, 84.7, 74.7, 61.7, 58.3, 26.7, 26.6, 24.3, 18.9; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C24H33O4SSi calculated 445.1863, found 445.1862.

3.2.7. (3aR,6R,6aS)-6-(((*tert*-butyldiphenylsilyl)oxy)methyl)-2,2 dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl acetate (**9**)

A solution of **8** (16 g, 35.98 mmol) in acetic anhydride (142 mL) was transferred to a preheated bath at 110 ◦C and stirred at the same temperature for 4 h. After concentration under reduced pressure, the residue was neutralized with aqueous sat. NaHCO<sup>3</sup> until pH 7.0 and stirred for 15 min. To the solution brine was added and extracted with ethyl acetate (3 × 600 mL). The organic layer was combined and washed with brine, dried (MgSO4), filtered, and evaporated under reduced pressure. The crude residue obtained was purified by silica gel column chromatography (hexane/ethyl acetate, 19:1) to give **9** (12.6 g, 72%) as a colorless syrup: 1:1.6 α/β mixture of anomers; UV (CH3OH) *λ*max 259.85 nm; <sup>1</sup>H NMR (CDCl3, 500 MHz): *δ* 7.70–7.62 (m, 6H), 7.43–7.40 (m, 3H), 7.39–7.36 (m, 7H), 6.09 (d, *J* = 5.3 Hz, 0.4H), 5.90 (s, 1H), 4.99 (d, *J* = 5.4 Hz, 1H), 4.82–4.80 (m, 0.6H), 4.64–4.61 (m, 1.5H), 3.82–3.80 (m, 1H), 3.77–3.72 (m, 1.6H), 3.60–3.52 (m, 2H), 2.13 (s, 1.4H), 1.81 (s, 3H), 1.53 (s, 1.9H), 1.48 (s, 3H), 1.30 (s, 1.6H), 1.28 (s, 3H), 1.06 (s, 9H), 1.04 (s, 5H); HRMS (ESI-Q-TOF) *m*/*z* [M + Na]<sup>+</sup> for C26H34NaO5SSi calculated 509.1788, found 509.1791.

3.2.8. 7-((3aR,4R,6R,6aS)-6-(((*tert*-butyldiphenylsilyl)oxy)methyl)-2,2 dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-4-chloro-5-iodo-7H-pyrrolo[2,3 d]pyrimidine (**10**)

*N*,*O*-Bis(trimethylsilyl)acetamide (BSA, 2.5 mL, 10.27 mmol) was added to a stirred suspension of 4-chloro-5-iodo-7*H*-pyrrolo[2,3-d]pyrimidine (2.6 g, 9.33 mmol) in anhydrous acetonitrile (67 mL) under nitrogen atmosphere. The resulting suspension was stirred at room temperature for 10 min until a homogeneous solution was obtained. To this clear solution a solution of **9** (5 g, 10.27 mmol) in anhydrous acetonitrile (50 mL) were added followed by dropwise addition of trimethylsilyl trifluoromethanesulfonate (1.5 mL, 8.40 mmol). The reaction mixture was stirred at room temperature for 15 min before transferring it to a preheated bath at 80 ◦C. After stirring at the same temperature for 1 h, the reaction mixture was cooled to room temperature and diluted with ethyl acetate (700 mL). The organic layer was washed with aqueous sat. NaHCO<sup>3</sup> (3 × 250 mL) and brine (100 mL), dried over MgSO4, filtered, and concentrated. The residue was purified by column chromatography (silica gel, hexane/ethyl acetate, 50:3) to give **10** (2.9 g, 40%) as a pale yellow sticky mass; [α] 25 <sup>D</sup> −7.03 (*c* 0.6, CH3OH); UV (CH3OH) *λ*max 310.78 nm; <sup>1</sup>H NMR (CD3OD, 500 MHz): *δ* 8.46 (s, 1H), 7.87 (s, 1H), 7.64–7.58 (m, 4H), 7.40–7.33 (m, 4H), 7.31–7.28 (m, 2H), 6.24 (d, *J* = 2.2 Hz, 1H), 5.10 (dd, *J* = 5.5, 2.2 Hz, 1H), 4.96 (dd, *J* = 5.5, 2.1 Hz, 1H), 3.88 (dd, *J* = 10.4, 7.2 Hz, 1H), 3.81 (dd, *J* = 10.4, 7.3 Hz, 1H), 3.75 (td, *J* = 7.2, 2.1 Hz, 1H), 1.55 (s, 3H), 1.28 (s, 3H), 1.04 (S, 9H); <sup>13</sup>C NMR (CD3OD, 100 MHz): *δ* 154.2, 152.5, 152.3, 137.5, 137.4, 136.2, 135.0, 134.7, 131.9, 131.8, 129.7, 129.6, 119.9, 114.0, 90.9, 87.0, 70.1, 67.7, 58.9, 53.5, 28.3, 28.1, 26.1, 20.8; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C30H34ClIN3O3SSi calculated 706.0818, found 706.0798.

3.2.9. 7-((3aR,4R,6R,6aS)-6-(((*tert*-butyldiphenylsilyl)oxy)methyl)-2,2 dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-4 amine (**11**)

A solution of **10** (2.9 g, 4.11 mmol) in saturated solution of NH3/*t*-BuOH (30 mL) contained in a stainless steel bomb reactor was transferred to a preheated bath at 90 ◦C and stirred at the same temperature for 24 h. The steel bomb containing reaction mixture was cooled to room temperature and solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate, 13:7) to obtain **11** (2.42 g, 86%) as a sticky mass; [α] 25 <sup>D</sup> −45.49 (*c* 2.4, CH3OH); UV (CH3OH) *λ*max 286.04 nm; <sup>1</sup>H NMR (CDCl3, 500 MHz): *δ* 8.22 (s, 1H), 7.64–7.62 (m, 4H), 7.43–7.40 (m, 2H), 7.37–7.33 (m, 5H), 6.21 (d, *J* = 2.7 Hz, 1H), 5.88 (br s, 2H), 4.88 (dd, *J* = 5.6, 2.8 Hz, 1H), 4.78 (dd, *J* = 5.6, 2.8 Hz, 1H), 3.86–3.79 (m, 2H), 3.77–3.75 (m, 1H), 1.58 (s, 3H), 1.27 (s, 3H), 1.07 (s, 9H); <sup>13</sup>C NMR (CDCl3, 125 MHz): *δ* 156.7, 151.9, 150.1, 135.5, 135.5, 132.9, 132.8, 129.9, 129.8, 127.8, 127.0, 112.4, 104.5, 89.0, 84.0, 66.3, 65.1, 55.7, 50.5, 27.3, 26.8, 25.1, 19.2; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C30H36IN4O3SSi calculated 687.1317, found 687.1301.

General Procedure of Stille Coupling for the Synthesis of **12a** and **12b**. To the compound **11** (1 equiv) in a microwave vial equipped with a septum, catalyst PdCl2(PPh3)<sup>2</sup> (15 mol %) was added and degassed THF (2.8 mL/mmol) under nitrogen atmosphere. The resulting solution was degassed for 5 min and a corresponding 2-(tributylstannyl)hetaryl (2.5 equiv) was added. After stirring the reaction mixture in a microwave for 1 h at 70 ◦C, it was quenched by adding water and brine. The aqueous layer was extracted with ethyl acetate thrice and organics were concentrated. The residue was purified by column chromatography.

3.2.10. 7-((3aR,4R,6R,6aS)-6-(((*tert*-butyldiphenylsilyl)oxy)methyl)-2,2 dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-5-(furan-2-yl)-7H-pyrrolo[2,3 d]pyrimidin-4-amine (**12a**)

Compound **12a** (0.16 g, 88%) was obtained from **11** (0.2 g, 0.29 mmol) as a pale yellow sticky mass; silica gel column chromatography (hexane/ethyl acetate, 3:2); [α] 25 <sup>D</sup> −31.0 (*c* 0.2, CH3OH); UV (CH3OH) *λ*max 291.76 nm; <sup>1</sup>H NMR (CDCl3, 400 MHz): *δ* 8.27 (s, 1H),

7.64–7.61 (m, 4H), 7.47–7.46 (m, 1H), 7.39–7.36 (m, 3H), 7.33–7.29 (m, 4H), 6.45–6.44 (m, 1H), 6.28–6.27 (m, 2H), 5.98 (br s, 2H), 4.95 (dd, *J* = 5.9, 3.2 Hz, 1H), 4.82 (dd, *J* = 5.9, 3.2 Hz, 1H), 3.92–3.82 (m, 2H), 3.79–3.75 (m, 1H), 1.59 (s, 3H), 1.28 (s, 3H), 1.05 (s, 9H); <sup>13</sup>C NMR (CDCl3, 100 MHz): *δ* 157.2, 152.9, 151.2, 149.0, 141.3, 135.77, 135.74, 133.17, 133.11, 130.1, 130.0, 127.9, 120.0, 112.7, 112.1, 107.3, 105.6, 100.9, 89.2, 84.2, 66.3, 65.3, 55.8, 27.6, 27.0, 25.4, 19.5; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C34H39N4O4SSi calculated 627.2456, found 627.2463.

3.2.11. 7-((3aR,4R,6R,6aS)-6-(((*tert*-butyldiphenylsilyl)oxy)methyl)-2,2 dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-5-(thiophen-2-yl)-7H-pyrrolo[2,3 d]pyrimidin-4-amine (**12b**)

Compound **12b** (0.21 g) was afforded from **11** (0.25 g, 0.36 mmol) in 92% yield as colorless sticky mass; silica gel column chromatography (hexane/ethyl acetate, 3:2); [α] 25 D <sup>−</sup>51.36 (*<sup>c</sup>* 0.31, CH3OH); UV (CH3OH) *<sup>λ</sup>*max 287.13 nm; <sup>1</sup>H NMR (CDCl3, 400 MHz): *<sup>δ</sup>* 8.28 (s, 1H), 7.62–7.59 (m, 4H), 7.37–7.34 (m, 3H), 7.32–7.27 (m, 4H), 7.23 (s, 1H), 7.10–7.07 (m, 1H), 7.00–6.99 (m, 1H), 6.28 (d, *J* = 2.7 Hz, 1H), 5.41 (br s, 2H), 4.94 (dd, *J* = 5.5, 3.2 Hz, 1H), 4.81 (dd, *J* = 5.9, 3.2 Hz, 1H), 3.91–3.82 (m, 2H), 3.79–3.74 (m, 1H), 1.59 (s, 3H), 1.28 (s, 3H), 1.03 (s, 9H); <sup>13</sup>C NMR (CDCl3, 100 MHz): *δ* 156.7, 152.1, 150.7, 135.68, 135.63, 129.97, 129.94, 128.0, 127.8, 126.5, 125.8, 121.9, 112.6, 109.4, 102.1, 89.0, 84.1, 66.2, 65.3, 55.6, 27.5, 26.9, 25.3, 19.3; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C34H39N4O3S2Si calculated 643.2227, found 643.2218.

General Procedure for the Synthesis of **1a** and **1b**. To an ice-cooled solution of **12a** and **12b** (1 equiv) in THF (3 mL/mmol) 50% aqueous trifluoroacetic acid (19.5 mL/mmol) was dropwise added and the resulting mixture was stirred at room temperature for 12 h. Acidic solution was basified using a weakly basic anion-exchange resin (Dowex® 66 free base) and stirred for an additional 3 h, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography (CH2Cl2/MeOH, 47:3) to give **1a** and **1b** respectively.

3.2.12. (2R,3R,4S,5R)-2-(4-Amino-5-(furan-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5- (hydroxymethyl)tetrahydrothiophene-3,4-diol (**1a**)

It was obtained in 75% yield as white solid; mp 197–199 ◦C; [α] 25 <sup>D</sup> −21.14 (*c* 0.05, CH3OH); UV (CH3OH) *λ*max 290.77 nm; <sup>1</sup>H NMR (DMSO-*d*6, 500 MHz): *δ* 8.13 (s, 1H), 7.95 (s, 1H), 7.78 (s, 1H), 6.90 (br s, 2H, D2O exchange), 6.72 (d, *J* = 3.0 Hz, 1H), 6.62–6.61 (m, 1H), 6.17 (d, *J* = 7.0 Hz, 1H), 5.43 (d, *J* = 6.4 Hz, 1H, D2O exchange), 5.27 (d, *J* = 4.4 Hz, 1H, D2O exchange), 5.18 (t, *J* = 5.5 Hz, 1H, D2O exchange), 4.48–4.45 (m, 1H), 4.19–4.18 (m, 1H), 3.78–3.75 (m, 1H), 3.63–3.58 (m, 1H), 3.29–3.28 (m, 1H); <sup>13</sup>C NMR (DMSO-*d*6, 100 MHz): *δ* 157.6, 152.5, 151.6, 149.1, 142.3, 120.7, 112.3, 106.8, 105.6, 99.4, 77.7, 73.6, 63.9, 61.3, 53.4; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C15H17N4O4S calculated 349.0965, found 349.0974; purity ≥95%.

3.2.13. (2R,3R,4S,5R)-2-(4-Amino-5-(thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5- (hydroxymethyl)tetrahydrothiophene-3,4-diol (**1b**)

It was afforded in 81% yield as white solid; mp 166–172 ◦C; [α] 25 <sup>D</sup> −23.93 (*c* 0.09, CH3OH); UV (CH3OH) *λ*max 286.41 nm; <sup>1</sup>H NMR (CD3OD, 500 MHz): *δ* 8.13 (s, 1H), 7.66 (s, 1H), 7.42 (d, *J* = 5.0 Hz, 1H), 7.14–7.11 (m, 2H), 6.22 (d, *J* = 5.9 Hz, 1H), 4.52 (dd, *J* = 5.7, 3.7 Hz, 1H), 4.29 (merged dd, *J*<sup>1</sup> = *J*<sup>2</sup> = 3.8 Hz, 1H), 3.88–3.80 (m, 2H), 3.48 (dd, *J* = 9.4, 4.8 Hz, 1H); <sup>13</sup>C NMR (CD3OD, 125 MHz): *δ* 159.5, 153.5, 152.6, 137.5, 129.8, 128.5, 127.6, 124.5, 111.6, 103.5, 80.8, 76.3, 65.1, 64.5, 54.8; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C15H17N4O3S<sup>2</sup> calculated 365.0737, found 365.0749; purity ≥95%.

General Procedure of Suzuki Coupling for the Synthesis of **12c**–**f**. A degassed mixture of DMF/H2O (6.9 mL/2.7 mL/mmol) was added to a microwave vial containing compound **11** (1 equiv), corresponding boronic ester (1.2 equiv), PdCl2(PPh3)<sup>2</sup> (6 mol %), and sodium carbonate (2 equiv). The resulting reaction mixture was heated in a microwave at 70 ◦C for 1 h. After quenching with water, the reaction mixture was extracted with ethyl

acetate thrice. The organic layers were combined, washed with brine, dried over MgSO4, filtered, and evaporated. The residue obtained was purified by column chromatography.

3.2.14. 7-((3aR,4R,6R,6aS)-6-(((*tert*-butyldiphenylsilyl)oxy)methyl)-2,2 dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-5-vinyl-7H-pyrrolo[2,3-d]pyrimidin-4 amine (**12c**)

Compound **12c** was obtained in 85% yield as yellow sticky mass; silica gel column chromatography (hexane/ethyl acetate, 13:7); [α] 25 <sup>D</sup> −38.33 (*c* 1.1, CH3OH); UV (CH3OH) *λ*max 288.95 nm; <sup>1</sup>H NMR (CD3OD, 400 MHz): *δ* 8.03 (s, 1H), 7.64 (t, *J* = 9.2 Hz, 4H), 7.42–7.39 (m, 3H), 7.37–7.31 (m, 4H), 6.91 (dd, *J* = 17.2, 10.8 Hz, 1H), 6.21 (s, 1H), 5.44 (d, *J* = 17.6 Hz, 1H), 5.20 (d, *J* = 10.8 Hz, 1H), 5.07–5.05 (m, 1H), 4.96–4.95 (m, 1H), 3.93–3.89 (m, 1H), 3.86–3.81 (m, 1H), 3.73–3.69 (m, 1H), 1.55 (s, 3H), 1.29 (s, 3H), 1.04 (s, 9H); <sup>13</sup>C NMR (CD3OD, 100 MHz): *δ* 157.8, 151.1, 150.1, 135.37, 135.35, 133.0, 132.7, 129.74, 129.71, 128.1, 127.58, 127.54, 119.4, 115.8. 114.4, 112.0, 101.4, 88.4, 84.5, 66.0, 65.5, 55.7, 26.3, 26.0, 24.0, 18.7; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C32H39N4O3SSi calculated 587.2507, found 587.2506.

3.2.15. 7-((3aR,4R,6R,6aS)-6-(((*tert*-butyldiphenylsilyl)oxy)methyl)-2,2 dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-5-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (**12d**)

It was obtained in 91% yield as colorless sticky mass; silica gel column chromatography (hexane/ethyl acetate, 1:1); [α] 25 <sup>D</sup> −46.89 (*c* 0.14, CH3OH); UV (CH3OH) *λ*max 283.50 nm; <sup>1</sup>H NMR (CD3OD, 400 MHz): *δ* 8.11 (s, 1H), 7.62–7.59 (m, 4H), 7.43–7.41 (m, 3H), 7.39–7.33 (m, 4H), 7.29–7.26 (m, 4H), 7.23 (s, 1H), 6.27 (d, *J* = 2.7 Hz, 1H), 5.09 (dd, *J* = 5.4, 2.7 Hz, 1H), 4.96 (dd, *J* = 5.5, 2.3 Hz, 1H), 3.92–3.82 (m, 2H), 3.75–3.71 (m, 1H), 1.57 (s, 3H), 1.30 (s, 3H), 1.01 (s, 9H); <sup>13</sup>C NMR (CD3OD, 100 MHz): *δ* 157.4, 151.2, 150.1, 135.38, 135.34, 134.1, 132.9, 132.7, 129.7, 128.7, 128.6, 127.55, 127.50, 127.1, 120.7, 117.9, 111.9, 101.3, 88.6, 84.7, 66.5, 65.6, 56.0, 26.2, 25.9, 24.0, 18.6; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C36H41N4O3SSi calculated 637.2663, found 637.2646.

3.2.16. 4-(4-(4-Amino-7-((3aR,4R,6R,6aS)-6-(((*tert*-butyldiphenylsilyl)oxy)methyl)-2,2 dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5 yl)phenyl)thiomorpholine 1,1-dioxide (**12e**)

It was afforded in 89% yield as colorless sticky mass; silica gel column chromatography (hexane/ethyl acetate, 11:9); [α] 25 <sup>D</sup> −28.78 (*c* 0.38, CH3OH); UV (CH3OH) *λ*max 270.04 nm; <sup>1</sup>H NMR (CDCl3, 400 MHz): *δ* 8.27 (s, 1H), 7.61–7.59 (m, 4H), 7.40–7.34 (m, 4H), 7.32–7.27 (m, 4H), 7.14 (s, 1H), 6.94–6.92 (m, 2H), 6.31 (d, *J* = 3.6 Hz, 1H), 5.28 (br s, 2H), 4.99 (dd, *J* = 5.9, 3.2 Hz, 1H), 4.82 (dd, *J* = 5.9, 2.7 Hz, 1H), 3.93–3.86 (m, 4H), 3.84–3.82 (m, 2H), 3.79–3.75 (m, 1H), 3.13–3.11 (m, 4H), 1.60 (s, 3H), 1.29 (s, 3H), 1.03 (s, 9H); <sup>13</sup>C NMR (CDCl3, 100 MHz): *δ* 156.6, 151.5, 150.7, 146.9, 135.66, 135.62, 133.0, 130.3, 129.96, 129.92, 127.8, 126.5, 120.6, 116.8, 116.6, 112.7, 101.9, 88.9, 84.0, 66.0, 65.2, 55.5, 50.5, 47.6, 27.5, 26.9, 25.3, 19.3; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C40H48N5O5S2Si calculated 770.2861, found 770.2865.

3.2.17. N-(4-(4-Amino-7-((3aR,4R,6R,6aS)-6-(((*tert*-butyldiphenylsilyl)oxy)methyl)-2,2 dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5 yl)phenyl)ethanesulfonamide (**12f**)

Compound **12f** was obtained in 87% yield as colorless sticky mass; silica gel column chromatography (hexane/ethyl acetate, 2:3); [α] 25 <sup>D</sup> −48.16 (*c* 0.09, CH3OH); UV (CH3OH) *λ*max 286.04 nm; <sup>1</sup>H NMR (CDCl3, 500 MHz): *δ* 8.29 (s, 1H), 7.60–7.59 (m, 4H), 7.39–7.34 (m, 2H), 7.32–7.30 (m, 4H), 7.28–7.27 (m, 2H), 7.25–7.24 (m, 2H), 7.20 (s, 1H), 6.66 (br s, 1H), 6.30 (d, *J* = 2.9 Hz, 1H), 5.37 (br s, 2H), 4.97 (dd, *J* = 5.6, 3.0 Hz, 1H), 4.82 (dd, *J* = 5.6, 2.7 Hz, 1H), 3.90–3.82 (m, 2H), 3.80–3.78 (m, 1H), 3.15 (q, *J* = 7.3 Hz, 2H), 1.60 (s, 3H), 1.39 (t, *J* = 7.3 Hz, 3H), 1.29 (s, 3H), 1.02 (s, 9H); <sup>13</sup>C NMR (CDCl3, 100 MHz): *δ* 156.3, 151.1, 150.7, 136.4, 135.63, 135.60, 132.8, 130.9, 130.0, 127.8, 121.3, 120.9, 116.4, 112.6, 101.7, 89.1, 84.1, 66.4, 65.2, 55.6, 46.3, 27.5, 26.9, 25.3, 19.3, 8.4; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C38H46N5O5S2Si calculated 744.2704, found 744.2698.

3.2.18. (2R,3R,4S,5R)-2-(4-Amino-5-vinyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5- (hydroxymethyl)tetrahydrothiophene-3,4-diol (**1c**)

Compound **12c** (0.12 g, 0.20 mmol) was converted to **1c** as described for **1a**, affording white solid (47.5 mg, 78%); silica gel column chromatography (CH2Cl2/MeOH, 19:1); mp 110–112 ◦C; [α] 25 <sup>D</sup> <sup>−</sup>41.86 (*<sup>c</sup>* 0.20, CH3OH); UV (CH3OH) *<sup>λ</sup>*max 288.50 nm; <sup>1</sup>H NMR (DMSO-*d*6, 500 MHz): *δ* 8.04 (s, 1H), 7.77 (s, 1H), 7.10 (dd, *J* = 17.2, 10.9 Hz, 1H), 6.70 (br s, 2H, D2O exchange), 6.12 (d, *J* = 7.0 Hz, 1H), 5.59 (d, *J* = 17.1 Hz, 1H), 5.38 (d, *J* = 6.4 Hz, 1H, D2O exchange), 5.25 (d, *J* = 4.4 Hz, 1H, D2O exchange), 5.17 (t, *J* = 5.5 Hz, 1H, D2O exchange), 5.11 (d, *J* = 11.0 Hz, 1H), 4.46–4.41 (m, 1H), 4.18–4.14 (m, 1H), 3.77–3.72 (m, 1H), 3.61–3.55 (m, 1H), 3.27–3.24 (m, 1H); <sup>13</sup>C NMR (CD3OD, 100 MHz): *δ* 157.8, 150.9, 150.6, 128.3, 119.9, 115.3, 113.9, 78.6, 74.1, 63.0, 62.2, 52.6; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C13H17N4O3S calculated 309.1016, found 309.1018; purity ≥95%.

3.2.19. (2R,3R,4S,5R)-2-(4-Amino-5-phenyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5- (hydroxymethyl)tetrahydrothiophene-3,4-diol (**1d**)

Compound **1d** was prepared from **12d** (0.16 g, 0.25 mmol) as described for **1a**, affording white solid (72.5 mg, 81%); silica gel column chromatography (CH2Cl2/MeOH, 19:1); mp 102–104 ◦C; [α] 25 <sup>D</sup> <sup>−</sup>46.30 (*<sup>c</sup>* 0.06, CH3OH); UV (CH3OH) *<sup>λ</sup>*max 280.95 nm; <sup>1</sup>H NMR (DMSO-*d*6, 500 MHz): *δ* 8.14 (s, 1H), 7.66 (s, 1H), 7.50–7.46 (m, 4H), 7.38–7.34 (m, 1H), 6.18 (d, *J* = 6.9 Hz, 1H), 5.42 (d, *J* = 6.4 Hz, 1H), 5.28 (d, *J* = 4.5 Hz, 1H), 5.16 (t, *J* = 5.5 Hz, 1H), 4.51–4.47 (m, 1H), 4.19–4.16 (m, 1H), 3.77–3.71 (m, 1H), 3.61–3.56 (m, 1H), 3.29–3.25 (m, 1H); <sup>13</sup>C NMR (CD3OD, 125 MHz): *δ* 159.5, 153.1, 152.8, 136.6, 130.9, 130.7, 129.2, 123.4, 119.7, 103.2, 80.9, 76.4, 65.2, 64.5, 54.8; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C17H19N4O3S calculated 359.1172, found 359.1177; purity ≥95%.

3.2.20. 4-(4-(4-Amino-7-(3,4-dihydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-7Hpyrrolo[2,3-d]pyrimidin-5-yl)phenyl)thiomorpholine 1,1-dioxide (**1e**)

It was obtained from **12e** (0.1 g, 0.12 mmol) as described for **1a**, as white solid (47.1 mg, 80%); silica gel column chromatography (CH2Cl2/MeOH, 93:7); mp 188–192 ◦C; [α] 25 D <sup>−</sup>24.29 (*<sup>c</sup>* 0.05, CH3OH); UV (CH3OH) *<sup>λ</sup>*max 270.04 nm; <sup>1</sup>H NMR (DMSO-*d*6, 500 MHz): *δ* 8.13 (s, 1H), 7.54 (s, 1H), 7.35 (merged dd, *J*<sup>1</sup> = *J*<sup>2</sup> = 8.4 Hz, 2H), 7.13 (merged dd, *J*<sup>1</sup> = *J*<sup>2</sup> = 8.5 Hz, 2H), 6.18 (d, *J* = 6.9 Hz, 1H), 5.40 (d, *J* = 6.4 Hz, 1H), 5.26 (d, *J* = 4.3 Hz, 1H), 5.16 (t, *J* = 5.4 Hz, 1H), 4.49–4.47 (m, 1H), 4.18–4.17 (m, 1H), 3.87–3.82 (m, 4H), 3.75–3.72 (m, 1H), 3.60–3.57 (m, 1H), 3.29–3.27 (m, 1H), 3.17–3.12 (m, 4H); <sup>13</sup>C NMR (DMSO-*d*6, 125 MHz): *δ* 157.2, 151.6, 151.0, 146.2, 129.4, 125.1, 120.1, 116.2, 116.0, 100.3, 77.3, 73.3, 63.3, 60.9, 52.8, 49.7, 46.5; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C21H26N5O5S<sup>2</sup> calculated 492.137, found 492.1388; purity ≥95%.

3.2.21. N-(4-(4-Amino-7-(3,4-dihydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-7Hpyrrolo[2,3-d]pyrimidin-5-yl)phenyl)ethanesulfonamide (**1f**)

It was afforded from **12f** (90 mg, 0.12 mmol) as described for **1a**, as white solid (44.6 mg, 80%); silica gel column chromatography (CH2Cl2/MeOH, 91:9); mp 127–132 ◦C; [α] 25 D <sup>−</sup>35.60 (*<sup>c</sup>* 0.05, CH3OH); UV (CH3OH) *<sup>λ</sup>*max 283.86 nm; <sup>1</sup>H NMR (DMSO-*d*6, 500 MHz): *<sup>δ</sup>* 9.87 (s, 1H), 8.14 (s, 1H), 7.60 (s, 1H), 7.42 (merged dd, *J*<sup>1</sup> = *J*<sup>2</sup> = 8.2 Hz, 2H), 7.31 (merged dd, *J*<sup>1</sup> = *J*<sup>2</sup> = 8.1 Hz, 2H), 6.18 (d, *J* = 6.9 Hz, 1H), 5.40 (d, *J* = 6.4 Hz, 1H), 5.26 (d, *J* = 4.3 Hz, 1H), 5.15 (t, *J* = 5.4 Hz, 1H), 4.49–4.46 (m, 1H), 4.19–4.17 (m, 1H), 3.76–3.72 (m, 1H), 3.61–3.57 (m, 1H), 3.29–3.27 (m, 1H), 3.13 (q, *J* = 7.2 Hz, 2H), 1.22 (t, *J* = 7.2 Hz, 3H); <sup>13</sup>C NMR (DMSO-*d*6, 125 MHz): *δ* 157.2, 151.6, 151.1, 137.1, 129.7, 129.2, 120.7, 119.8, 115.9, 100.1, 77.3, 73.3, 63.3, 60.9, 52.8, 45.0, 8.02; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C19H24N5O5S<sup>2</sup> calculated 466.1213, found 466.1225; purity ≥95%.

General Procedure of Sonogashira Coupling for the Synthesis of **13**. To a microwave vial equipped with a septum, containing **11** (1 equiv), CuI (25 mol %), and PdCl2(PPh3)<sup>2</sup> (10 mol %) a degassed mixture of DMF-Et3N (6.1 mL/mmol, 4:1) was added. The resulting reaction mixture was degassed with nitrogen for 5 min before adding corresponding alkyne (1.1 equiv) and heated in a microwave at 50 ◦C for 1 h. The reaction mixture was partitioned between ethyl acetate and water. The combined organic layer was dried over MgSO4, filtered, and evaporated. The resulting residue was purified by silica gel chromatography to afford the respective compounds.

3.2.22. 7-((3aR,4R,6R,6aS)-6-(((*tert*-butyldiphenylsilyl)oxy)methyl)-2,2 dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-5-((trimethylsilyl)ethynyl)-7Hpyrrolo[2,3-d]pyrimidin-4-amine (**13**)

The desired compound **13** was obtained from **11** (1.67 g, 2.43 mmol) in 93% yield as sticky mass; silica gel column chromatography (hexane/ethyl acetate, 4:1); [α] 25 <sup>D</sup> −74.11 (*c* 0.65, CH3OH); UV (CH3OH) *λ*max 284.59 nm; <sup>1</sup>H NMR (CDCl3, 500 MHz): *δ* 8.24 (s, 1H), 7.65–7.62 (m, 4H), 7.42–7.39 (m, 3H), 7.37–7.34 (m, 4H), 6.19 (d, *J* = 2.6 Hz, 1H), 5.73 (br s, 2H), 4.89 (dd, *J* = 5.6, 2.6 Hz, 1H), 4.78 (dd, *J* = 5.6, 2.3 Hz, 1H), 3.87–3.84 (m, 1H), 3.79–3.73 (m, 2H), 1.57 (s, 3H), 1.27 (s, 3H), 1.07 (s, 9H), 0.24 (s, 9H); <sup>13</sup>C NMR (CDCl3, 100 MHz): *δ* 157.3, 153.0, 149.7, 135.7, 135.6, 133.0, 132.9, 130.0, 127.9, 126.9, 112.5, 104.0, 98.4, 97.7, 96.2, 89.3, 84.2, 66.5, 65.2, 56.0, 27.4, 26.9, 25.2, 19.3, 0.01; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C35H45N4O3SSi<sup>2</sup> calculated 657.2745, found 657.2739.

3.2.23. ((3aS,4R,6R,6aR)-6-(4-Amino-5-ethynyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2 dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)methanol (**14**)

To the stirred solution of **13** (0.58 g, 0.88 mmol) in anhydrous THF (5.8 mL) a solution of 1 M TBAF in THF (2.64 mL, 2.64 mmol) was added under nitrogen atmosphere and the resulting reaction mixture was stirred at room temperature for 40 min. The reaction was quenched with saturated aqueous NH4Cl solution (5 mL) and extracted with ethyl acetate (3 × 150 mL). The combined organic layer was washed with brine (100 mL), dried over MgSO4, filtered, and concentrated to give the residue. Upon silica gel column chromatography (methylene chloride/methanol, 97:3), compound **14** (0.27 g, 90%) was obtained as pale yellow syrup; [α] 25 <sup>D</sup> <sup>−</sup>51.62 (*<sup>c</sup>* 1.15, CH3OH); UV (CH3OH) *<sup>λ</sup>*max 280.95 nm; <sup>1</sup>H NMR (CD3OD, 500 MHz): *δ* 8.12 (s, 1H), 7.78 (s, 1H), 6.27 (d, *J* = 3.0 Hz, 1H), 5.14 (dd, *J* = 5.3, 3.1 Hz, 1H), 4.97 (dd, *J* = 5.4, 2.2 Hz, 1H), 3.79–3.74 (m, 2H), 3.73 (s, 1H), 3.71–3.68 (m, 1H), 1.58 (s, 3H), 1.32 (s, 3H); <sup>13</sup>C NMR (CD3OD, 125 MHz): *δ* 159.9, 154.4, 151.2, 129.7, 114.1, 105.2, 97.7, 91.1, 87.4, 77.9, 68.7, 65.8, 57.7, 28.6, 26.2; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C16H19N4O3S calculated 347.1172, found 347.1168.

3.2.24. (2R,3R,4S,5R)-2-(4-Amino-5-ethynyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5- (hydroxymethyl)tetrahydrothiophene-3,4-diol (**1g**)

To a solution of **14** (0.21 g, 0.60 mmol) in THF (6 mL) 2 N HCl solution (6 mL) was added dropwise at 0 ◦C and the reaction mixture was stirred at room temperature for 15 h. A weakly basic anion-exchange resin (Dowex® 66 free base) was added to the resulting solution to neutralize HCl and stirred for additional 3 h. The solution was filtered, evaporated, and the residue was purified by silica gel column chromatography (methylene chloride/methanol, 47:3) to afford **1g** (0.13 g, 72%) as white solid; mp 235–237 ◦C; [α] 25 D <sup>−</sup>47.30 (*<sup>c</sup>* 0.05, CH3OH); UV (CH3OH) *<sup>λ</sup>*max 280.59 nm; <sup>1</sup>H NMR (DMSO-*d*6, 500 MHz): *<sup>δ</sup>* 8.12 (s, 1H), 7.93 (s, 1H), 6.06 (d, *J* = 6.9 Hz, 1H), 5.43 (d, *J* = 6.3 Hz, 1H), 5.27 (d, *J* = 4.3 Hz, 1H), 5.17 (t, *J* = 5.3 Hz, 1H), 4.45–4.42 (m, 1H), 4.28 (s, 1H), 4.16–4.14 (m, 1H), 3.76–3.71 (m, 1H), 3.61–3.57 (m, 1H), 3.28–3.25 (m, 1H); <sup>13</sup>C NMR (DMSO-*d*6, 150 MHz): *δ* 157.3, 152.6, 149.8, 127.6, 102.2, 94.0, 83.0, 77.34, 77.30, 73.2, 63.1, 61.1, 53.0; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C13H15N4O3S calculated 307.0859, found 307.0863; purity ≥95%.

3.2.25. 7-((3aR,4R,6R,6aS)-6-(((*tert*-butyldiphenylsilyl)oxy)methyl)-2,2 dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-5-(prop-1-yn-1-yl)-7H-pyrrolo[2,3 d]pyrimidin-4-amine (**15a**)

Following the procedure described to synthesize **13**, compound **11** (200 mg, 0.29 mmol) afforded **15a** (164 mg, 96%) as sticky mass; silica gel column chromatography (hexane/ethyl acetate, 3:1); [α] 25 <sup>D</sup> <sup>−</sup>63.53 (*<sup>c</sup>* 0.195, CH3OH); UV (CH3OH) *<sup>λ</sup>*max 282.77 nm; <sup>1</sup>H NMR (CDCl3, 400 MHz): *δ* 8.22 (s, 1H), 7.66–7.63 (m, 4H), 7.41–7.40 (m, 2H), 7.38–7.34 (m, 4H), 7.30 (s, 1H), 6.20 (d, *J* = 2.8 Hz, 1H), 5.74 (br s, 2H), 4.90 (dd, *J* = 5.6, 2.8 Hz, 1H), 4.80 (dd, *J* = 6.0, 5.8 Hz, 1H), 3.88 (dd, *J* = 10, 6.8 Hz, 1H), 3.82–3.75 (m, 2H), 2.07 (s, 3H), 1.58 (s, 3H), 1.28 (s, 3H), 1.08 (s, 9H); <sup>13</sup>C NMR (CDCl3, 100 MHz): *δ* 157.0, 152.3, 149.4, 135.7, 135.6, 133.0, 132.9, 129.9, 127.8, 125.8, 112.6, 103.8, 97.0, 89.1, 88.4, 84.1, 72.5, 66.3, 65.2, 55.9, 27.4, 26.9, 25.2, 19.3, 4.6; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C33H39N4O3SSi calculated 599.2507, found 599.2512.

3.2.26. 5-(But-1-yn-1-yl)-7-((3aR,4R,6R,6aS)-6-(((*tert*-butyldiphenylsilyl)oxy)methyl)-2,2 dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-4 amine (**15b**)

It was obtained from **11** (234 mg, 0.34 mmol) as described for **13**, as sticky mass (189 mg, 91%); silica gel column chromatography (hexane/ethyl acetate, 7:3); [α] 25 <sup>D</sup> −71.76 (*c* 0.06, CH3OH); UV (CH3OH) *λ*max 283.50 nm; <sup>1</sup>H NMR (CDCl3, 500 MHz): *δ* 8.22 (s, 1H), 7.65–7.62 (m, 4H), 7.42–7.39 (m, 2H), 7.37–7.34 (m, 4H), 7.30 (s, 1H), 6.19 (d, *J* = 2.7 Hz, 1H), 5.68 (br s, 2H), 4.89 (dd, *J* = 5.7, 2.8 Hz, 1H), 4.79 (dd, *J* = 5.6, 2.8 Hz, 1H), 3.87–3.85 (m, 1H), 3.80–3.77 (m, 1H), 3.76–3.74 (m, 1H), 2.43 (q, *J* = 14.9, 7.5 Hz, 2H), 1.57 (s, 3H), 1.27 (s, 3H), 1.23 (t, *J* = 7.5 Hz, 3H), 1.06 (s, 9H); <sup>13</sup>C NMR (CDCl3, 100 MHz): *δ* 157.0, 152.4, 149.4, 135.6, 135.5, 132.99, 132.9, 129.8, 127.8, 127.7, 125.6, 112.5, 103.8, 96.8, 94.1, 89.0, 84.0, 72.7, 66.1, 65.1, 55.8, 27.3, 26.8, 25.1, 19.2, 13.9, 13.2; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C34H41N4O3SSi calculated 613.2663, found 613.2669.

3.2.27. 7-((3aR,4R,6R,6aS)-6-(((*tert*-butyldiphenylsilyl)oxy)methyl)-2,2 dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-5-(3,3-dimethylbut-1-yn-1-yl)-7Hpyrrolo[2,3-d]pyrimidin-4-amine (**15c**)

For the synthesis of **15c**, compound **11** (300 mg, 0.43 mmol) was treated as described for **13**, yielding sticky mass (224 mg, 80%); silica gel column chromatography, hexane/ethyl acetate, 13:7; [α] 25 <sup>D</sup> <sup>−</sup>67.09 (*<sup>c</sup>* 0.265, CH3OH); UV (CH3OH) *<sup>λ</sup>*max 283.86 nm; <sup>1</sup>H NMR (CDCl3, 500 MHz): *δ* 8.21 (s, 1H), 7.64 (t, *J* = 6.4 Hz, 4H), 7.42–7.40 (m, 2H), 7.38–7.34 (m, 4H), 7.32 (s, 1H), 6.20 (d, *J* = 2.6 Hz, 1H), 5.83 (br s, 2H), 4.88 (dd, *J* = 5.6, 2.7 Hz, 1H), 4.77 (dd, *J* = 5.5, 2.5 Hz, 1H), 3.87 (dd, *J* = 9.7, 6.2 Hz, 1H), 3.79–3.73 (m, 2H), 1.57 (s, 3H), 1.31 (s, 9H), 1.26 (s, 3H), 1.07 (s, 9H); <sup>13</sup>C NMR (CDCl3, 100 MHz): *δ* 157.2, 152.6, 149.6, 135.7, 135.6, 133.0, 132.9, 129.9, 127.9, 125.3, 112.5, 103.9, 101.1, 96.9, 89.2, 84.1, 72.1, 66.2, 65.2, 55.9, 31.0, 28.3, 27.4, 26.9, 25.2, 19.3; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C36H45N4O3SSi calculated 641.2976, found 641.2991.

3.2.28. 7-((3aR,4R,6R,6aS)-6-(((*tert*-butyldiphenylsilyl)oxy)methyl)-2,2 dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-5-(cyclopropylethynyl)-7H-pyrrolo[2,3 d]pyrimidin-4-amine (**15d**)

It was obtained from **11** (214 mg, 0.31 mmol) as described for **13**, as sticky mass (180 mg, 92%); silica gel column chromatography, hexane/ethyl acetate, 13:7; [α] 25 <sup>D</sup> −70.38 (*c* 0.10, CH3OH); UV (CH3OH) *λ*max 284.50 nm; <sup>1</sup>H NMR (CDCl3, 400 MHz): *δ* 8.21 (s, 1H), 7.66–7.63 (m, 4H), 7.44–7.40 (m, 2H), 7.39–7.34 (m, 4H), 7.31 (s, 1H), 6.19 (d, *J* = 3.2 Hz, 1H), 5.93 (br s, 2H), 4.88 (dd, *J* = 5.9, 3.2 Hz, 1H), 4.79 (dd, *J* = 5.9, 2.7 Hz, 1H), 3.89–3.85 (m, 1H), 3.82–3.73 (m, 2H), 1.58 (s, 3H), 1.49–1.44 (m, 1H), 1.27 (s, 3H), 1.07 (s, 9H), 0.92–0.85 (m, 2H), 0.79–0.75 (m, 2H); <sup>13</sup>C NMR (CDCl3, 100 MHz): *δ* 156.4, 149.0, 135.6, 135.5, 132.9, 132.8, 129.9, 127.8, 126.2, 112.5, 103.8, 97.1, 89.1, 84.1, 68.0, 66.3, 65.1, 55.8, 27.3, 26.8, 25.1, 19.2, 8.7, 0.2; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C35H41N4O3SSi calculated 625.2663, found 625.2667.

3.2.29. (2R,3R,4S,5R)-2-(4-Amino-5-(prop-1-yn-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5- (hydroxymethyl)tetrahydrothiophene-3,4-diol (**1h**)

Compound **15a** (150 mg, 0.25 mmol) was converted to **1h** (66.4 mg, 83%) yielding white solid, using the procedure described for **1a**; silica gel column chromatography (CH2Cl2/MeOH, 47:3); mp 190–192 ◦C; [α] 25 <sup>D</sup> −51.14 (*c* 0.05, CH3OH); UV (CH3OH) *λ*max 282.77 nm; <sup>1</sup>H NMR (DMSO-*d*6, 500 MHz): *δ* 8.10 (s, 1H), 7.75 (s, 1H), 6.05 (d, *J* = 6.7 Hz, 1H), 5.40 (d, *J* = 6.2 Hz, 1H), 5.25 (d, *J* = 4.1 Hz, 1H), 5.16 (t, *J* = 5.3 Hz, 1H), 4.42–4.39 (m, 1H), 4.16–4.13 (m, 1H), 3.75–3.70 (m, 1H), 3.61–3.56 (m, 1H), 3.27–3.24 (m, 1H), 2.08 (s, 3H); <sup>13</sup>C NMR (DMSO-*d*6, 125 MHz): *δ* 157.4, 152.5, 149.7, 125.8, 102.1, 95.7, 88.4, 77.3, 73.2, 72.6, 63.1, 61.1, 52.9, 4.2; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C14H17N4O3S calculated 321.1016, found 321.1013; purity ≥95%.

3.2.30. (2R,3R,4S,5R)-2-(4-Amino-5-(but-1-yn-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5- (hydroxymethyl)tetrahydrothiophene-3,4-diol (**1i**)

Following the procedure described for **1a**; compound **15b** (176 mg, 0.28 mmol) yielded **1i** (80.4 mg, 86%) as white solid; silica gel column chromatography (CH2Cl2/MeOH, 47:3); mp 161–162 ◦C; [α] 25 <sup>D</sup> −32.48 (*c* 0.05, CH3OH); UV (CH3OH) *λ*max 283.86 nm; <sup>1</sup>H NMR (DMSO-*d*6, 500 MHz): *δ* 8.10 (s, 1H), 7.76 (s, 1H), 6.06 (d, *J* = 6.8 Hz, 1H), 5.40 (*J* = 6.3 Hz, 1H), 5.26 (*J* = 4.4 Hz, 1H), 5.16 (t, *J* = 5.4 Hz, 1H), 4.43–4.40 (m, 1H), 4.16–4.13 (m, 1H), 3.75–3.70 (m, 1H), 3.60–3.56 (m, 1H), 3.27–3.24 (m, 1H), 2.47 (q, *J* = 14.9, 7.4 Hz, 2H), 1.17 (t, *J* = 7.4 Hz, 3H); <sup>13</sup>C NMR (CD3OD, 100 MHz): *δ* 157.6, 151.6, 149.2, 126.0, 103.1, 97.0, 93.8, 78.8, 74.2, 72.0, 63.0, 62.5, 52.7, 12.9, 12.5; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C15H19N4O3S calculated 335.1172, found 335.1157; purity ≥95%.

3.2.31. (2R,3R,4S,5R)-2-(4-Amino-5-(3,3-dimethylbut-1-yn-1-yl)-7H-pyrrolo[2, 3-d]pyrimidin-7-yl)-5-(hydroxymethyl)tetrahydrothiophene-3,4-diol (**1j**)

Compound **15c** (200 mg, 0.31 mmol) was converted to **1j** (91.8 mg, 82%) as white solid, by following the procedure described for **1a**; silica gel column chromatography (CH2Cl2/MeOH, 47:3); mp 144–146 ◦C; [α] 25 <sup>D</sup> −61.72 (*c* 0.06, CH3OH); UV (CH3OH) *λ*max 287.50 nm; <sup>1</sup>H NMR (DMSO-*d*6, 500 MHz): *δ* 8.11 (s, 1H), 7.76 (s, 1H), 6.06 (d, *J* = 7.0 Hz, 1H), 5.39 (d, *J* = 6.4 Hz, 1H), 5.27 (d, *J* = 4.4 Hz, 1H), 5.16 (t, *J* = 5.5 Hz, 1H), 4.45–4.41 (m, 1H), 4.16–4.13 (m, 1H), 3.76–3.71 (m, 1H), 3.61–3.55 (m, 1H), 3.27–3.24 (m, 1H), 1.31 (s, 9H); <sup>13</sup>C NMR (DMSO-*d*6, 100 MHz): *δ* 157.5, 152.6, 149.8, 125.7, 102.2, 100.3, 95.3, 77.3, 73.2, 72.5, 63.2, 60.9, 52.9, 30.6, 27.8; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C17H23N4O3S calculated 363.1485, found 363.1492; purity ≥95%.

3.2.32. (2R,3R,4S,5R)-2-(4-Amino-5-(cyclopropylethynyl)-7H-pyrrolo[2,3-d]pyrimidin-7 yl)-5-(hydroxymethyl)tetrahydrothiophene-3,4-diol (**1k**)

Compound **15d** (156 mg, 0.24 mmol) was converted to **1k** (71.4 mg, 86%) as white solid, by following the procedure described for **1a**; silica gel column chromatography (CH2Cl2/MeOH, 47:3); mp 166–168 ◦C; [α] 25 <sup>D</sup> −50.34 (*c* 0.05, CH3OH); UV (CH3OH) *λ*max 284.22 nm; <sup>1</sup>H NMR (CD3OD, 400 MHz): *δ* 8.09 (s, 1H), 7.67 (s, 1H), 6.11 (d, *J* = 5.6 Hz, 1H), 4.44 (dd, *J* = 5.6, 3.6 Hz, 1H), 4.24 (merged dd, *J*<sup>1</sup> = *J*<sup>2</sup> = 4.0 Hz, 1H), 3.89–3.79 (m, 2H), 3.49–3.44 (m, 1H), 1.57–1.50 (m, 1H), 0.94–0.88 (m, 2H), 0.78–0.74 (m, 2H); <sup>13</sup>C NMR (CD3OD, 100 MHz): *δ* 159.2, 153.2, 150.7, 127.8, 104.7, 98.3, 97.1, 80.2, 75.6, 69.1, 64.5, 64.0, 54.2, 9.1, 0.9; HRMS (ESI-Q-TOF) *m*/*z* [M + H]<sup>+</sup> for C16H19N4O3S calculated 347.1172, found 347.1159; purity ≥95%.

### *3.3. Cell Proliferation Inhibition Assay (SRB Assay)*

Human lung cancer cells (A549), colorectal cancer (HCT116) cells, breast cancer cells (MDA-MB-231), liver cancer cells (SK-HEP-1), and prostate cancer cells (PC-3) were pur-

chased from the American Type Culture Collection (Manassas, VA, USA). Human gastric cancer cells (SNU-638) were purchased from the Korean Cell Line Bank (Seoul, Korea). Cells were cultured in medium (Dulbecco's modified Eagle's medium for MDA-MB-231 and SK-HEP-1 cells; Roswell Park Memorial Institute 1640 for A549, HCT116, SNU-638, PC-3 cells) supplemented with penicillin-streptomycin and 10% fetal bovine serum at 37 ◦C in a humidified incubator with 5% carbon dioxide. Cells were seeded at a density of 4–7 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/mL in 96-well culture plates, and then treated with indicated compounds for 72 h. At the end of the experiment, cells were fixed with 10% trichloroacetic acid (TCA) solution and subjected to sulforhodamine B (SRB) assay to determine cell proliferation [42]. The percentage of cell proliferation was calculated with the following formula:

Cell proliferation (%) = 100 × [(A treated − A zero day)/(A control − A zero day)],

where A is the average absorbance. The IC<sup>50</sup> values were calculated through non-linear regression analysis using TableCurve 2D v5.01 (Systat Software Inc., San Jose, CA, USA). All experiments were performed in triplicate and data shown are representative of two or three independent experiments.

### Cell Culture

The human colon cancer (KM12) and renal cancer (ACHN) cell lines were obtained from the Korean Cell Line Bank (Seoul, Korea). Cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics-antimycotics (PSF: 100 units/mL penicillin G sodium, 100 µg/mL streptomycin and 250 ng/mL amphotericin B). All cells were incubated at 37 ◦C in a humidified atmosphere containing 5% CO<sup>2</sup> and subcultured twice a week.

### *3.4. Kinome Scan Assays*

The kinome scan assays were carried out at Eurofins DiscoverX Corporation. For kinome scan profiling of compound **1g**, it was screened at 1µM against 96 kinases (*N* = 2 independent experiments) [26]. The results for binding interactions are reported as % inhibition, where higher values indicate strong affinity; see the Supporting Information, Table S1 for full kinome profile. For kinase inhibition profile of compounds **1a**–**k** [see the Supporting information, Table S2, whose IC<sup>50</sup> values were determined using an 11-point 3-fold serial dilution of each test compound using their KINOMEscan assay and *K*<sup>i</sup> was determined by the Cheng-Prusoff equation..

### *3.5. Metabolic Stability*

Phosphate buffer (0.1 M, pH 7.4) containing human liver microsomes (0.5 mg/mL) and test compound (a final concentration of 1 µM) were pre-incubated for 5 min at 37 ◦C. NADPH regeneration system solution was added to it and incubated for 30 min at 37 ◦C. Acetonitrile solution containing chlorpropamide was added at the end of the reaction. The sample was centrifuged for 5 min (14,000× *g* rpm, 4 ◦C) and the supernatant was injected into the LC-MS/MS system for the analysis. The amount of substrate that remained after the reaction was analyzed using the Shimadzu Nexera XR system and TSQ vantage (Thermo). Kinetex C18 column (2.1 × 100 mm, 2.6 µm particle size; Phenomenex) was used for HPLC. The mobile phase used contained 0.1% formic acid in distilled water (A) and 0.1% formic acid containing acetonitrile (B). Xcalibur (version 1.6.1) was used for data analysis. Verapamil was used as a positive control [48,49].

### *3.6. CYP Inhibition Assay*

Human liver microsomes (0.25 mg/mL), 0.1 M phosphate buffer (pH 7.4), a cocktail of five coenzyme substrates (Phenacetin 50 µM, Diclofenac 10 µM, S-mephenytoin 100 µM, Dextromethorphan 5 µM, Midazolam 2.5 µM), and test compound (10 µM concentration) was pre-incubated for 5 min at 37 ◦C. NADPH generation system solution was added and incubated for 15 min at 37 ◦C. In order to terminate the reaction, acetonitrile solu-

tion containing an internal standard (Terfenadine) was added and centrifuged for 5 min (14,000× *g* rpm, 4 ◦C). The supernatant was injected into the LC-MS/MS system to analyze the metabolites of the substrates simultaneously. Metabolites of each substrate produced during the reaction were analyzed using the Shimadzu Nexera XR system and TSQ vantage (Thermo). Kinetex C18 column (2.1 × 100 mm, 2.6 µm particle size; Phenomenex, USA) was used for HPLC. The mobile phase used contained 0.1% formic acid in distilled water (A) and 0.1% formic acid containing acetonitrile (B). The generated metabolites were quantified using MRM (Multiple Reaction Monitoring) and Xcalibur (version 1.6.1) was used for data analysis [50,51].

### *3.7. Computational Docking Simulation*

Ligand binding site for docking was defined as a 30 Å<sup>3</sup> grid box for DYRK1A and <sup>20</sup> <sup>×</sup> <sup>24</sup> <sup>×</sup> 20 Å<sup>3</sup> grid box for TRKA centered on the centroid of co-crystallized native ligands. The crystal structures of DYRK1A (PDB ID: 7A51) [46] and TRKA (PDB ID: 5JFV) [47] were downloaded from RCSB PDB and computational docking was performed using AutoDock Vina version 1.5.6 (The Scripps Research Institute, La Jolla, CA, USA) [52]. For the macromolecule-ligand pair, the binding model of the ligand with the lowest binding free energy (kcal/mol) was used for further analysis. Figure to show the molecular modeling results were visualized using PyMOL (Schrödinger, LLC, New York, NY, USA) [53]. LIGPLOT<sup>+</sup> (version 2.2.4) was used to view the interactions between amino acid residues of enzyme and compound [54].

### **4. Conclusions**

Protein kinases represent a promising target for the development of anticancer agents due to their association with cancer growth and progression [10–12]. In the present study, we designed molecules using the nucleoside skeleton with the intention to simultaneously occupy the hinge and the hydrophobic region I (buried region), along with the ribose region of the ATP-binding site. We sought to identify whether the hydrophobic pocket I acts as a pharmacophore in kinase inhibition. Thus, we designed and synthesized 7 substituted 7-deaza-40 -thioadenosine derivatives **1** with a nucleoside skeleton by modifying the hydrophobic residue (R), based on ATP-kinase interactions. Among all the synthesized compounds, compound **1g** with acetylene at the 7-position of 7-deaza-40 -thioadenosine (R = acetylene) exhibited markedly potent anticancer activity in vitro against six different cancer cell lines and potent kinase inhibition of TRKA, DYRK1A/1B, and CK1δ at a concentration of 1 µM among the panel of 96 kinases. The results showed that the C-7 substituent of 7-deazaadenine was optimal for substituting extremely small and linear acetylene, indicating that a very small linear hydrophobic group is required to inhibit TRKA, DYRK1A/1B, and CK1δ. These results will contribute greatly to the further development of new anticancer agents with multi-kinase inhibition.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/ph14121290/s1, <sup>1</sup>H and <sup>13</sup>C NMR spectra, HRMS (ESI-Q-TOF) data for **1g**, Figure S1: ORTEP diagram of compound **1g** showing thermal ellipsoid at 50% probability, X-ray crystallographic data for **1g**, HPLC chromatograms, Table S1: Kinome scan data of compound **1g**, Table S2: Kinase inhibition profile of **1a**–**k** against TRKA, CK1δ, and DYRK1A/1B.

**Author Contributions:** Conceptualization, L.S.J.; methodology, K.K.M., K.S., S.K.T., Y.A.Y., H.W.L., J.Y.K. and M.K.; software, K.K.M. and G.K.; validation, L.S.J.; formal analysis, K.K.M., W.S.B., H.K. and S.A.; investigation, K.K.M., W.S.B., H.K., S.A., E.-J.K., M.N. and S.K.L.; data curation, K.K.M., W.S.B., H.K. and S.A.; writing—original draft preparation, K.K.M. and L.S.J.; writing—review and editing, L.S.J.; supervision, L.S.J.; project administration, L.S.J.; funding acquisition, L.S.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Research Foundation (NRF) grants (NRF-2021R1A2B5B02001544) of Korea.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article and supplementary files.

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

### **References**


### *Article* **Exploring the Anti-Cancer Mechanism of Novel 3,4**0 **-Substituted Diaryl Guanidinium Derivatives**

**Viola Previtali <sup>1</sup> , Helene B. Mihigo <sup>1</sup> , Rebecca Amet <sup>2</sup> , Anthony M. McElligott <sup>3</sup> , Daniela M. Zisterer <sup>2</sup> and Isabel Rozas 1,\***


Received: 16 November 2020; Accepted: 16 December 2020; Published: 21 December 2020

**Abstract:** We previously identified a guanidinium-based lead compound that inhibited BRAF through a hypothetic type-III allosteric mechanism. Considering the pharmacophore identified in this lead compound (i.e., "lipophilic group", "di-substituted guanidine", "phenylguanidine polar end"), several modifications were investigated to improve its cytotoxicity in different cancer cell lines. Thus, several *lipophilic groups* were explored, the *di-substituted guanidine* was replaced by a secondary amine and the phenyl ring in the *polar end* was substituted by a pyridine. In a structure-based design approach, four representative derivatives were docked into an in-house model of an active triphosphate-containing BRAF protein, and the interactions established were analysed. Based on these computational studies, a variety of derivatives was synthesized, and their predicted drug-like properties calculated. Next, the effect on cell viability of these compounds was assessed in cell line models of promyelocytic leukaemia and breast, cervical and colorectal carcinomas. The potential of a selection of these compounds as apoptotic agents was assessed by screening in the promyelocytic leukaemia cell line HL-60. The toxicity against non-tumorigenic epithelial MCF10A cells was also investigated. These studies allowed for several structure-activity relationships to be derived. Investigations on the mechanism of action of representative compounds suggest a divergent effect on inhibition of the MAPK/ERK signalling pathway.

**Keywords:** 3,40 -bis-guanidino; 3-amino-40 -guanidino; diphenyl ether; phenyl pyridyl ether; intramolecular hydrogen bond; cancer cell viability; HL-60; BRAF; apoptosis

### **1. Introduction**

Various interlinked signalling pathways are involved in cell proliferation, apoptosis or survival, and these processes are even more relevant in the case of tumour formation [**?** ]. Oncogenic mutations in one of these signalling pathways, the Ras/RAF/MEK/ERK (MAPK/ERK) pathway, are observed frequently in many cancers [**? ?** ]. Additionally, since mutations in RAF kinases are common events, these kinases (i.e., ARAF, BRAF and CRAF) have become very interesting therapeutic targets [**? ?** ].

Most of the protein kinase inhibitors developed so far are ATP competitive and, based on the conformation of the protein kinase they bind to, have been broadly classified as type I (bind to the αC-helix-IN/DFG-IN conformation), type II (bind to the αC-helix-IN/DFG-OUT conformation), or type I/II (bind to the αC-helix-OUT/DFG-IN conformation) [**?** ]. In addition, there are allosteric inhibitors, known as type III protein kinase inhibitors, which do not compete with ATP. These inhibitors tend

to exhibit the highest degree of kinase selectivity because they exploit binding sites and regulatory mechanisms unique to particular kinases [**?** ]. an ATP analogue (AMP-PCP) and MEK has been resolved (PDB 6U2G) [11], we had previously constructed a model that reproduced an active form of this kinase including ATP to model potential type III inhibitors [12].This in-house model allowed us to explore the potential allosteric inhibition

*Pharmaceuticals* **2020**, *13*, x FOR PEER REVIEW 2 of 26

inhibitors, known as type III protein kinase inhibitors, which do not compete with ATP. These inhibitors tend to exhibit the highest degree of kinase selectivity because they exploit binding sites

Previously, we had identified a 3,4′-bis-guanidinium diphenyl derivative (**1**, Figure 1) that demonstrated strong cytotoxicity, mediated through induction of apoptosis, in colorectal cancer cells containing wild type(wt)-BRAF and mutated V600EBRAF [8,9]. Compound **1** also inhibited ERK1/2 signalling, EGFR activation, as well as Src, STAT3 and Akt phosphorylation. We also showed that **1** did not inhibit ATP binding to BRAF, but a radiometric assay of BRAF activity indicated that this was inhibited in vitro. From these studies, we hypothesised that **1** could inhibit BRAF as a type III inhibitor. We propose that the positively charged guanidines present in compound **1** could interact

and regulatory mechanisms unique to particular kinases [7].

Previously, we had identified a 3,40 -bis-guanidinium diphenyl derivative (**1**, Figure **??**) that demonstrated strong cytotoxicity, mediated through induction of apoptosis, in colorectal cancer cells containing wild type(wt)-BRAF and mutated V600EBRAF [**? ?** ]. Compound **1** also inhibited ERK1/2 signalling, EGFR activation, as well as Src, STAT3 and Akt phosphorylation. We also showed that **1** did not inhibit ATP binding to BRAF, but a radiometric assay of BRAF activity indicated that this was inhibited in vitro. From these studies, we hypothesised that **1** could inhibit BRAF as a type III inhibitor. We propose that the positively charged guanidines present in compound **1** could interact with the negatively charged phosphates of the ATP present in the active state of all kinases [**?** ]. through ATP binding of compound **1** and it was considered a good target model for structure-based design when no crystallographic data was available. Accordingly, in this article, we first present a computational study of a series of guanidine-based di-aromatic systems representative of all the compounds proposed with a simplified version of our in-house active BRAF model to explore their potential as type-III allosteric inhibitors. Additionally, we describe the preparation of the compounds proposed and the study of the anti-proliferative and pro-apoptotic activity on cancerous cell lines as well as their effect on the MAPK/ERK signalling pathway.

**2. Results**  Considering the structure of compound **1** ("link" -blue box-, "polar moiety" -red box- and "hydrophobic moiety" -green box-; see Figure 1), we explored a number of modifications in the different moieties, while maintaining the diaryl ether core due to its versatility and suitability for synthesis. First, several "hydrophobic" substituents have been considered (green box in Figure 1), Considering that only very recently a crystal structure of a BRAF protein kinase in complex with an ATP analogue (AMP-PCP) and MEK has been resolved (PDB 6U2G) [**?** ], we had previously constructed a model that reproduced an active form of this kinase including ATP to model potential type III inhibitors [**?** ].This in-house model allowed us to explore the potential allosteric inhibition through ATP binding of compound **1** and it was considered a good target model for structure-based design when no crystallographic data was available.

since in our previously reported computational model [12], this section of the molecule seems to interact with a hydrophobic pocket. Next, based on previous computational studies [9], the effect of the length of the molecule on its activity has been studied by replacing the disubstituted guanidinium in the "link" by a secondary amine (blue box in Figure 1). Additionally, we explored the effect of substituting one of the phenyl rings by a pyridine to lock the orientation of the guanidinium by means of intramolecular hydrogen bonds (IMHBs) [13]. Finally, in the "polar" region, different guanidine Accordingly, in this article, we first present a computational study of a series of guanidine-based di-aromatic systems representative of all the compounds proposed with a simplified version of our in-house active BRAF model to explore their potential as type-III allosteric inhibitors. Additionally, we describe the preparation of the compounds proposed and the study of the anti-proliferative and pro-apoptotic activity on cancerous cell lines as well as their effect on the MAPK/ERK signalling pathway.

#### surrogates (i.e., isourea and sulfamide) have been tested (red box in Figure 1). **2. Results**

Considering the structure of compound **1** ("link" -blue box-, "polar moiety" -red box- and "hydrophobic moiety" -green box-; see Figure **??**), we explored a number of modifications in the different moieties, while maintaining the diaryl ether core due to its versatility and suitability for synthesis. First, several "hydrophobic" substituents have been considered (green box in Figure **??**), since in our previously reported computational model [**?** ], this section of the molecule seems to interact with a hydrophobic pocket. Next, based on previous computational studies [**?** ], the effect of the length of the molecule on its activity has been studied by replacing the disubstituted guanidinium in the "link" by a secondary amine (blue box in Figure **??**). Additionally, we explored the effect of substituting one of the phenyl rings by a pyridine to lock the orientation of the guanidinium by means of intramolecular hydrogen bonds (IMHBs) [**?** ]. Finally, in the "polar" region, different guanidine surrogates (i.e., isourea and sulfamide) have been tested (red box in Figure **??**).

#### *2.1. Molecular Modelling Studies 2.1. Molecular Modelling Studies*

We had previously reported the docking of compound **1** into our in-house ATP-containing BRAF model [**?** ]; here, we are using a simplified model containing triphosphate (TP) instead of ATP since the interactions of interest only occur with the phosphates and adjacent hydrophobic pocket. Thus, in order to validate our TP-containing BRAF model, we first docked compound **1** (Figure **??**) and considering that similar outcomes were observed, we used the TP-containing structure and similar conditions for the rest of the computational studies. Accordingly, a set of representative structures of all the compounds proposed were selected; compound **2** contains a pyridine instead of a phenyl ring as it was in compound **1**, in compound **3** the di-substituted guanidine of **1** has been changed to a secondary amine and compound **4** shows both modifications (Figure **??**). We had previously reported the docking of compound **1** into our in-house ATP-containing BRAF model [12]; here, we are using a simplified model containing triphosphate (TP) instead of ATP since the interactions of interest only occur with the phosphates and adjacent hydrophobic pocket. Thus, in order to validate our TP-containing BRAF model, we first docked compound **1** (Figure 1) and considering that similar outcomes were observed, we used the TP-containing structure and similar conditions for the rest of the computational studies. Accordingly, a set of representative structures of all the compounds proposed were selected; compound **2** contains a pyridine instead of a phenyl ring as it was in compound **1**, in compound **3** the di-substituted guanidine of **1** has been changed to a secondary amine and compound **4** shows both modifications (Figure 2).

*Pharmaceuticals* **2020**, *13*, x FOR PEER REVIEW 3 of 26

**Figure 2.** Structures of the proposed new derivatives of **1** (compounds **2**, **3** and **4**), which docking to the in-house triphosphate (TP)-containing BRAF simplified model was studied. **Figure 2.** Structures of the proposed new derivatives of **1** (compounds **2**, **3** and **4**), which docking to the in-house triphosphate (TP)-containing BRAF simplified model was studied.

As in our previous modelling studies [12], we found that compound **1** interacts with BRAF through an allosteric region found near the ATP binding site and with the TP system. The main interactions established involved bifurcated hydrogen bonds (HBs) between the mono-substituted guanidinium and two negatively charged O atoms of a phosphate (NH…O distances: 1.6, 2.9 and 2.7 Å, Figure S1) and a parallel HB interaction between the di-substituted guanidinium and the carboxylate of Glu648 (NH…O distances: of 2.0 and 2.6 Å, Figure S1). Both sets of HBs are reinforced by ionic interactions due to the charged nature of guanidinium, phosphate and carboxylate groups. Additionally, interactions are observed in the lipophilic pocket with the (4-Cl,3-CF3)Ph moiety; thus, the CF3 group interacts with Met620 and the Cl with Trp619 (Figure S1). As in our previous modelling studies [**?** ], we found that compound **1** interacts with BRAF through an allosteric region found near the ATP binding site and with the TP system. The main interactions established involved bifurcated hydrogen bonds (HBs) between the mono-substituted guanidinium and two negatively charged O atoms of a phosphate (NH . . . O distances: 1.6, 2.9 and 2.7 Å, Figure S1) and a parallel HB interaction between the di-substituted guanidinium and the carboxylate of Glu648 (NH . . . O distances: of 2.0 and 2.6 Å, Figure S1). Both sets of HBs are reinforced by ionic interactions due to the charged nature of guanidinium, phosphate and carboxylate groups. Additionally, interactions are observed in the lipophilic pocket with the (4-Cl,3-CF3)Ph moiety; thus, the CF<sup>3</sup> group interacts with Met620 and the Cl with Trp619 (Figure S1).

Likewise, when derivative **2** is docked into the TP-BRAF simplified model, it forms ionically reinforced bifurcated HBs between the mono-substituted guanidinium and two negatively charged O atoms of TP (Figure 3). Likewise, a bifurcated HB interaction between the di-substituted guanidinium and Glu648 and van der Waals contacts at the lipophilic pocket with the (4-Cl,3-CF3)Ph moiety were also found for compound **2** (Figure 3). The newly introduced pyridine seems to form a HB with Asn137 as well as an IMHB that locks the mono-substituted guanidinium (Figure 3). This conformational restriction could result in increased affinity. Likewise, when derivative **2** is docked into the TP-BRAF simplified model, it forms ionically reinforced bifurcated HBs between the mono-substituted guanidinium and two negatively charged O atoms of TP (Figure **??**). Likewise, a bifurcated HB interaction between the di-substituted guanidinium and Glu648 and van der Waals contacts at the lipophilic pocket with the (4-Cl,3-CF3)Ph moiety were also found for compound **2** (Figure **??**). The newly introduced pyridine seems to form a HB with Asn137 as well as an IMHB that locks the mono-substituted guanidinium (Figure **??**). This conformational restriction could result in increased affinity.

Replacement of the di-substituted guanidine by an -NH- leads to a significantly shorter molecule as in system **3** (Figure **??**). Upon docking to the TP-containing simplified BRAF, we observed that the mono-substituted guanidinium still forms the expected bifurcated HBs. Compound **3** also fits within the hydrophobic pocket of the target establishing weaker contacts (longer interaction distances) with Met620 and Trp619. Additionally, the newly introduced -NH- group forms a HB with one of the O atoms of Glu648 (Figure S2).

Finally, the docking study of compound **4** (Figure **??**) into the aforementioned target reproduce the results observed for the mono-substituted guanidinium pyridine system in analogue **2** by establishing bifurcated HBs with the TP, IMHB between guanidinium and pyridine as well as contacts between the

pyridine N and Asn137. Additionally, as the -NH- shortens the structure, similar interactions to those seen for compound **3** are observed, i.e., the -NH- group forms a HB with Glu648 and contacts within the hydrophobic pocket are found (Figure S3). *Pharmaceuticals* **2020**, *13*, x FOR PEER REVIEW 4 of 26

**Figure 3.** Docking of derivative **2** in the TP-containing BRAF simplified model indicating the bifurcated (up left, bottom left and right) and single (up right) hydrogen bond (HB) interactions **Figure 3.** Docking of derivative **2** in the TP-containing BRAF simplified model indicating the bifurcated (up left, bottom left and right) and single (up right) hydrogen bond (HB) interactions observed. Distances are expressed in Å.

observed. Distances are expressed in Å. Replacement of the di-substituted guanidine by an -NH- leads to a significantly shorter molecule as in system **3** (Figure 2). Upon docking to the TP-containing simplified BRAF, we observed that the mono-substituted guanidinium still forms the expected bifurcated HBs. Compound **3** also fits within the hydrophobic pocket of the target establishing weaker contacts (longer interaction distances) with All this work was carried out prior to the recent publication of the crystal structure of BRAF containing an analogue of ATP (AMP-PCP) [**?** ], and in order to validate our in-house BRAF-ATP model [**?** ] we now superimposed both the reported crystal structure with our model finding an RMSD of 2.53 Å between them and with both ATP-like systems occupying the same pocket and with very similar orientations (Figures S4 and S5). This level of similarity gives us confidence on the docking studies performed with our BRAF-TP model.

Met620 and Trp619. Additionally, the newly introduced -NH- group forms a HB with one of the O atoms of Glu648 (Figure S2). Finally, the docking study of compound **4** (Figure 2) into the aforementioned target reproduce the results observed for the mono-substituted guanidinium pyridine system in analogue **2** by establishing bifurcated HBs with the TP, IMHB between guanidinium and pyridine as well as contacts between the pyridine N and Asn137. Additionally, as the -NH- shortens the structure, The G-scores obtained for the best-poses obtained were very similar for the four compounds studied (around −7.7 kcal/mol); hence, the interaction with the target was favoured in all the cases but did not help to discriminate among the four compounds. In summary, according to the docking studies all the compounds proposed seem to establish favourable interactions with BRAF, suggesting a possible type III allosteric binding.

#### similar interactions to those seen for compound **3** are observed, i.e., the -NH- group forms a HB with *2.2. Synthesis*

Glu648 and contacts within the hydrophobic pocket are found (Figure S3). All this work was carried out prior to the recent publication of the crystal structure of BRAF containing an analogue of ATP (AMP-PCP) [11], and in order to validate our in-house BRAF-ATP model [12] we now superimposed both the reported crystal structure with our model finding an RMSD of 2.53 Å between them and with both ATP-like systems occupying the same pocket and with very similar orientations (Figures S4 and S5). This level of similarity gives us confidence on the docking studies performed with our BRAF-TP model. The G-scores obtained for the best-poses obtained were very similar for the four compounds studied (around −7.7 kcal/mol); hence, the interaction with the target was favoured in all the cases The synthesis of the analogues of compound **1**, 3,40 -bis-guanidino diphenyl ethers **5**–**11**, required the preparation of the corresponding *N*-aryl-*N'*-Boc-protected thioureas (**12**–**16**) following a procedure previously developed in the group starting from *N*,*N'*-bis*-*(*tert*-butoxycarbonyl)thiourea **17** (Scheme **??**) [**?** ]. Given the diverse electron-withdrawing effect of differently substituted commercially available anilines, different yields of the corresponding thioureas **12**–**16** were obtained (37–57%, see details in ESI). The corresponding Boc-protected mono-guanidines **18** and **19** were prepared by reaction of commercially available 3,40 -dianiline ether **20** and *N*,*N'*-bis*-*(*tert*-butoxycarbonyl)thiourea **17** in the presence of HgCl<sup>2</sup> and NEt<sup>3</sup> yielding **18** and **19** as a mixture that was separated by column chromatography in good and moderate yields (Scheme **??**) [**?** ].

but did not help to discriminate among the four compounds. In summary, according to the docking studies all the compounds proposed seem to establish favourable interactions with BRAF, suggesting a possible type III allosteric binding. Next, the *N*-aryl-*N'*-Boc-protected thioureas **12**–**16** were reacted with **18** or **19** under our standard conditions (HgCl<sup>2</sup> and NEt3) to yield the corresponding Boc-protected 3-arylguanidino-4<sup>0</sup> -guanidino (**21**–**25**) and 3-guanidino-40 -arylguanidino (**26**–**27**) diphenyl ether derivatives (Scheme **??**).

*2.2. Synthesis*  The synthesis of the analogues of compound **1**, 3,4′-bis-guanidino diphenyl ethers **5–11**, required In order to prepare compound **3** and its analogues **28**–**30**, the nitrophenyl precursor **31** was synthesised by a SNAr reaction between commercially available 3-aminophenol and 1-fluoro-4 nitrobenzene [**?** ]. Compounds **32**–**35** were synthesized using a Buchwald–Hartwig cross-coupling;

the preparation of the corresponding *N*-aryl-*N'*-Boc-protected thioureas (**12–16**) following a procedure previously developed in the group starting from *N*,*N'*-bis*-*(*tert*-butoxycarbonyl)thiourea

(37–57%, see details in ESI). The corresponding Boc-protected mono-guanidines **18** and **19** were

the success of this reaction depends on variables such as ligand, Pd source, base and solvent [**?** ]. The conditions chosen (Pd2(dba)<sup>3</sup> 3 mol%, BINAP 3 mol%, NaOtBu 1.4 eq. in dry toluene (2 mL mmol−<sup>1</sup> ) at 90 ◦C) afforded the proposed compounds **32**–**35** in high yields (62–87%, Scheme **??**). prepared by reaction of commercially available 3,4′-dianiline ether **20** and *N*,*N'*-bis*-*(*tert*butoxycarbonyl)thiourea **17** in the presence of HgCl2 and NEt3 yielding **18** and **19** as a mixture that was separated by column chromatography in good and moderate yields (Scheme 1) [14].

*Pharmaceuticals* **2020**, *13*, x FOR PEER REVIEW 5 of 26

**Scheme 1.** Preparation of mono-substituted 3,4'-bis-guanidinium diphenyl ether derivatives. **Scheme 1.** Preparation of mono-substituted 3,4'-bis-guanidinium diphenyl ether derivatives.

Next, the *N*-aryl-*N'*-Boc-protected thioureas **12–16** were reacted with **18** or **19** under our standard conditions (HgCl2 and NEt3) to yield the corresponding Boc-protected 3-arylguanidino-4′ guanidino (**21–25**) and 3-guanidino-4′-arylguanidino (**26–27**) diphenyl ether derivatives (Scheme 1). In order to prepare compound **3** and its analogues **28–30**, the nitrophenyl precursor **31** was synthesised by a SNAr reaction between commercially available 3-aminophenol and 1-fluoro-4- With the intention of probing the effect of branching in the diphenyl ether core, we prepared the fluoro derivative **36** for which we synthesised precursor **37** by using commercially available 3-bromo -5-fluorophenol that in the presence of K2CO<sup>3</sup> and DMF quantitatively reacted with 4-fluoronitrobenzene. Then, **37** was used for the Buchwald–Hartwig coupling with 4-chloro-3-(trifluoromethyl)aniline to afford compound **38** in good yield (Scheme **??**).

nitrobenzene [15]. Compounds **32–35** were synthesized using a Buchwald–Hartwig cross-coupling; the success of this reaction depends on variables such as ligand, Pd source, base and solvent [16]. The conditions chosen (Pd2(dba)3 3 mol%, BINAP 3 mol%, NaOtBu 1.4 eq. in dry toluene (2 mL mmol−1) at 90 °C) afforded the proposed compounds **32–35** in high yields (62–87%, Scheme 2). With the intention of probing the effect of branching in the diphenyl ether core, we prepared the fluoro derivative **36** for which we synthesised precursor **37** by using commercially available 3-bromo-5-fluorophenol that in the presence of K2CO3 and DMF quantitatively reacted with 4- Precursors **32**–**35** and **38** were then subjected to selective reduction of the nitro group to the amine that will serve as handle for the introduction of the guanidine moiety. Nitro reduction of compounds **34** and **35** was achieved using catalytic hydrogenation (H2, Pd/C 10 mol%) yielding aniline derivatives **41** and **42**; however, in the case of chloro-derivatives **32**, **33,** and **38**, selective reduction was achieved with the use of tin(II) chloride dihydrate (SnCl2·2H2O) to produce anilines **39**, **40,** and **43** [**?** ]. Utilising our standard conditions, a guanidine moiety was introduced affording Boc-protected mono-guanidines **44**–**48** (Scheme **??**).

fluoronitrobenzene. Then, **37** was used for the Buchwald–Hartwig coupling with 4-chloro-3- (trifluoromethyl)aniline to afford compound **38** in good yield (Scheme 2). In order to prepare the 3,40 -bis-guanidino phenyloxypyridines **2**, **4,** and **49**–**52**, the starting 5-(3 aminophenoxy)pyridin-2-amine (**53**) was synthesized. Thus, SNAr between 5-bromo-2-nitropyridine and 3-nitrophenol yielded the previously reported mixture of isomers (**54** and **55**) [**?** ], which were

separated by chromatography. Further hydrogenation of **54** gave the desired product **53** in good yield (Scheme *Pharmaceuticals*  **??**) [**? ? 2020** ]. , *13*, x FOR PEER REVIEW 6 of 26

**Conditions:** (i) 3-Aminophenol or, for 37, 3-bromo-5-fluorophenol, K2CO3, DMF, 80 °C, 12 h; (ii) R-Br, Pd2(dba)3, BINAP, NaOt Bu, toluene, 90 °C, 24 h; (iii) for 38, 4-chloro-3-(trifluoromethyl)aniline, Pd2(dba)3, BINAP, NaO<sup>t</sup> Bu, toluene, 90 °C 24 h; (iv) H2, Pd/C, EtOH, rt, 24 h; (v) SnCl2.2H2O, EtOAc, 70 °C, 3 h; (vi) *N,N'*-bis-(*tert*-butoxycarbonyl)-*S*-methylisothiourea, HgCl2, NEt3, CH2Cl2, 0 °C to rt; (vii) 0.2M HCl/dioxane, 55 °C, 8 h.

**Scheme 2.** Preparation of 3-amino,4'-guanidine diphenyl ether derivatives. **Scheme 2.** Preparation of 3-amino,4'-guanidine diphenyl ether derivatives. *Pharmaceuticals* **2020**, *13*, x FOR PEER REVIEW 7 of 26

**Scheme 3.** Preparation of 3,4'-bis-guanidine pehylpyridyl ether derivatives. **Scheme 3.** Preparation of 3,4'-bis-guanidine pehylpyridyl ether derivatives.

Preparation of the corresponding precursors for compound **4** required the synthesis of 3-((6 nitropyridin-3-yl)oxy)aniline **66** through a SNAr between 5-bromo-2-nitropyridine and 3-

Preparation of the arylanilino nitropyridine **67** was next attempted following the previous

(*tert*-butoxycarbonyl)-*S*-methylisothiourea to obtain Boc-protected compound **69** (Scheme 4).

aminophenol (Scheme 4).

Compound **53** was then reacted with Boc-protected thioureas **12**–**16**, under our standard conditions to yield Boc-protected mono-guanidines **56**–**60** [**?** ]. Subsequent guanidylation at position 2 of the pyridine ring was carried out with commercial *N,N'*-bis-(*tert*-butoxycarbonyl)-*S*-methylisothiourea to obtain Boc-protected compounds **61**–**65** (Scheme **??**).

Preparation of the corresponding precursors for compound **4** required the synthesis of 3-((6 nitropyridin-3-yl)oxy)aniline **66** through a SNAr between 5-bromo-2-nitropyridine and 3-aminophenol (Scheme **??**). *Pharmaceuticals* **2020**, *13*, x FOR PEER REVIEW 8 of 26

**Scheme 4.** Preparation of 3-amino-4'-guanidine phenylpiridyl ether derivatives. **Scheme 4.** Preparation of 3-amino-4'-guanidine phenylpiridyl ether derivatives.

With the aim of introducing diversity to the "polar" moiety of compound **3**, we also explored the substitution of the guanidinium system by an isouronium cation or a sulfamide (Scheme 5). Thus, preparation of the 3-anilino-4'-*O*-isourea phenylozybenzene derivative **70** involved the use of the previously synthesised compound **71** [20] as starting material for the synthesis of the intermediate **72** through the mentioned conditions for a Buchwald–Hartwig coupling (Pd2(dba)3, Xantphos, Cs2CO3, Preparation of the arylanilino nitropyridine **67** was next attempted following the previous conditions described for the synthesis of **32**–**35** and **38** with poor results. However, Pd-catalysed reaction using of Xantphos and Cs2CO<sup>3</sup> yielded compound **67** in good yield. Next, SnCl2·2H2O reduction conditions afforded compound **68** that was then subjected to guanidylation using *N,N'*-bis-(*tert*-butoxycarbonyl)-*S*-methylisothiourea to obtain Boc-protected compound **69** (Scheme **??**).

toluene, 90 °C, 24 h). With the aim of introducing diversity to the "polar" moiety of compound **3**, we also explored the substitution of the guanidinium system by an isouronium cation or a sulfamide (Scheme **??**). Thus, preparation of the 3-anilino-4'-*O*-isourea phenylozybenzene derivative **70** involved the use of the previously synthesised compound **71** [**?** ] as starting material for the synthesis of the intermediate **72** through the mentioned conditions for a Buchwald–Hartwig coupling (Pd2(dba)3, Xantphos, Cs2CO3, toluene, 90 ◦C, 24 h).

Compound **72** was then deprotected with montmorillonite KSF to obtain the corresponding phenol **73**, which OH was then amidylated using standard conditions previously described by us [**?** ] to yield the corresponding Boc-protected isouronium **74** (Scheme **??**). Sulfamide **75** was synthesised by treating amine **40** with sulfamoyl chloride (previously prepared from chlorosulfonyl isocyanate and formic acid [**? ?** ]) to afford compound **75** in good yield (Scheme **??**).

All Boc-protected precursors were deprotected using HCl 4M/dioxane to yield compounds **2**–**11**, **28**–**30**, **36**, **49**–**52** (**????????**) and SnCl<sup>4</sup> to obtain compound **70**. The purity of all the final hydrochloride salts was determined by HPLC, where a minimum purity of 95% was required before proceeding to biological testing (ESI).

**Scheme 5.** Preparation of 3-amino-4'-isourea and 3-amino-4'-sulfamido diphenyl ether derivatives.

toluene, 90 °C, 24 h).

**Scheme 4.** Preparation of 3-amino-4'-guanidine phenylpiridyl ether derivatives.

With the aim of introducing diversity to the "polar" moiety of compound **3**, we also explored the substitution of the guanidinium system by an isouronium cation or a sulfamide (Scheme 5). Thus, preparation of the 3-anilino-4'-*O*-isourea phenylozybenzene derivative **70** involved the use of the

**Scheme 5. Scheme 5.**  Preparation of 3-amino-4'-isourea and 3-amino-4'-sulfamido diphenyl ether derivatives. Preparation of 3-amino-4'-isourea and 3-amino-4'-sulfamido diphenyl ether derivatives.

### *2.3. Predicted Physicochemical and In Vitro ADME Properties*

Attention to physicochemical and pharmacokinetic properties of active molecules should be given at the early stages of their design in order to shorten their development to a drug. However, these properties are not always easy to be experimentally evaluated and, hence, computational approaches represent a good solution to get a general idea of the potential of compounds as drugs. Thus, to assess the drug-likeness of the compounds studied, we utilized SwissADME [**?** ] and ChemAxon's Marvin [**?** ] to computationally evaluate the mentioned properties and the calculated values are reported in Tables S1–S3 (ESI). All proposed compounds have a molecular weight (MW) <500 Da, except for 3,40 -bis-guanidine phenyloxybenzenes **7**, **9**, **10** and **11** or phenyloxypyridines **50** and **52**, in which the MW exceeds by 7–10 units this threshold; such small exceptions are considered acceptable [**?** ]. The consensus logPo/<sup>w</sup> [**?** ] for all synthesised compounds is reported in Table S2 (ESI) indicating that, overall, all the compounds have a logP < 5. Topological Polar Surface Area (TPSA), which is an indicator of HB formation and a commonly used metric for a drug's ability to permeate cells, was calculated for all the synthesised molecules and all of them have values <140 Å<sup>2</sup> , which is the limit suggested in the literature for poor cell membrane permeation [**? ? ?** ]. Specifically, shorter 3-amino-40 -guanidines have much lower TPSA (80–100 Å<sup>2</sup> ) than 3,40 -bis-guanidinium analogues (120–135 Å<sup>2</sup> ).

The BOILED egg graph presents a correlation between calculated logP and calculated TPSA and is an intuitive simultaneous prediction of two key ADME parameters, i.e., the passive gastrointestinal (GI) absorption and brain access (blood brain barrier, BBB) [**? ?** ]. Compounds that fall into the white part of the graph are likely to undergo GI absorption and those that fall into the yellow part of the graph are likely to be brain permeant. Accordingly, all of our derivatives could undertake passive GI absorption (except pentafluorosulfanyl derivative **30** and sulfamide **75**), but none of them can cross the BBB (Figure S6). In addition, SwissADME enables the estimation for a chemical to be a substrate of the permeability glycoprotein (P-gp), which is the most important ATP-binding cassette transporter responsible for an active efflux through biological membranes, e.g., from the GI wall to the lumen or exiting the brain) [**? ?** ]. Thus, as reported in the legend of Figure S6, red dots indicate that all of our compounds, except compounds **49**–**51** (blue dots), are non-substrates of P-gp (Table S3).

The theoretical pKaH values of all the synthesised compounds calculated with Marvin are reported in Table S1. The results indicate that exchanging the *para* mono-substituted guanidinium (compound **3**) by an isouronium moiety (compound **70**) results in a pKaH decrease (less basic molecule). Interestingly, the pyridine derivatives (**49**–**52**, **2** and **4**) show lower pKaH values than their diphenyl counterparts (**6**–**9**, **1** and **3**, respectively), probably due to the IMHB formed between the *para* guanidinium and the pyridine ring.

Solubility is not an easy parameter to model in silico, but SwissADME gives an estimation of the solubility class based on three predictors, two topological and one fragmental method [**?** ]. According to this program, all our molecules are poorly soluble in aqueous environments (Table S2). For the preclinical evaluation of our molecules, their solubility in EtOH/DMSO is still acceptable, but future work will have to be carried out to achieve full water solubility.

Other numerical descriptors are used to assess drug-likeness including the number of HB acceptors and donors (HBAs and HBDs) or the number of rotatable bonds (RotB) and the results obtained for this set of compounds are shown in Table S1 [**?** ]. According to these descriptors, in general, all studied compounds fulfil drug-like conditions.

### *2.4. Biochemical Studies*

### 2.4.1. Cell Viability in Cancerous and Non-Cancerous Cell Lines

Cell viability and proliferation assays were used to evaluate the in vitro cytotoxicity of all synthesised compounds in a variety of different cancer cell lines using the alamarBlue® viability assay and the results are presented in **????**. The sensitivity of cancer cells to drugs can often be compromised by PIK3CA, Ras and BRAF mutations. To determine whether such mutations are critical to the efficacy of our new compounds we tested them in a range of cancerous cell lines expressing different mutations. Firstly, we used the HL-60 (human promyelocytic leukaemia, NRas mutated) cell line for general cytotoxicity screening of all compounds. Next, the most active derivatives were studied in MCF-7 (breast adenocarcinoma, Ras/RAF wild type, and PIK3CA mutant), HeLa (cervical carcinoma, Ras/RAF and PIK3CA wild type), as well as HCT-116 and HKH-2 (colorectal carcinoma, KRas mutant and mutated KRas disrupted, respectively) cell lines [**?** ]. Lastly, toxicity against MCF-10A, which is a non-tumorigenic epithelial cell line, was also assessed for one of the most promising compounds (**4**). The graphs representing the viability results with the HL-60, MCF-7, HeLa, HCT-116, and HKH-2 cancer cell lines for compound **1** and derivatives **2**, **3,** and **4** are shown in Figures S7–S10 (ESI). Sorafenib (a known inhibitor of protein kinases including VEGFR, PDGFR and RAF [**? ?** ]) was used as a positive control in all viability assays.


**Table 1.** Effect on the viability of HL-60 cells (IC50, µM) of compounds **1**–**11**, **28**–**30**, **36**, **49**–**52**, **70**, **75** and sorafenib with alamarBlue® assays.

(a) Cells were seeded at a density of 2 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL (HL-60) in a 96-well plate and treated with different concentrations of the compounds dissolved in EtOH or DMSO (1% *v*/*v* and 0.1% *v*/*v*, respectively). Sorafenib was used as a reference and tested in the same manner. Once treated, cells were incubated for 72 h at 37 ◦C after which they were treated with alamarBlue® and left in darkness in an incubator for 5 h. The resulting fluorescence (λexcitation = 544 nm, λemission = 590 nm) was read using a plate reader from which percentage viability was calculated. IC<sup>50</sup> values were calculated using Prism GraphPad Prism software from at least three independent experiments performed in triplicate. Highlighted in grey are those IC<sup>50</sup> values better than or similar to the control used, Sorafenib.


**Table 2.** Effect in viability of MCF-7, HeLa, HCT116, and HKH-2 cancer cells (IC50, µM) of compounds **1**–**4**, **70** and sorafenib on the alamarBlue® assays.

(a) Cells were seeded at a density of 25 <sup>×</sup> <sup>10</sup><sup>3</sup> cells/mL (MCF-7 and HeLa) or 1 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL (HCT-116 and HKH-2) in a 96-well plate and treated with different concentrations of the compounds dissolved in EtOH or DMSO (1% *v*/*v* and 0.1% *v*/*v*, respectively). Sorafenib was used as a reference and tested in the same manner. Once treated, cells were incubated for 72 h at 37 ◦C after which they were treated with alamarBlue® and left in darkness in an incubator for 5 h. The resulting fluorescence (λexcitation = 544 nm, λemission = 590 nm) was read using a plate reader from which percentage viability was calculated. IC<sup>50</sup> values were calculated using Prism GraphPad Prism software from at least three independent experiments performed in triplicate. Highlighted in grey are those IC<sup>50</sup> values better than or similar to the control used, Sorafenib, in each particular cell line.

The results obtained with the HL-60 cell line (Table **??**) for the 3,40 -bis-guanidine derivatives **5**–**11** show, in general, more cytotoxicity than the previously tested compound **1** [**?** ]. However, compounds **5** and **6**, which carry 3-F and 3,4-diF phenyl groups, respectively, give IC<sup>50</sup> values above 100 µM; this drop in activity is a clear indication of the importance of the size and nature of the substituents on the phenyl ring [**?** ]. The presence of 2-F and 4-I substituents in the hydrophobic moiety of derivative **7** resulted in an IC<sup>50</sup> value (8.63 µM) similar to **1**. Substitution of the 4-Cl in compound **1** by a 4-Br (i.e., compound **9**), gave a four-fold increase in activity, indicating that the halogen in this hydrophobic moiety could establish a halogen-bond with a Lewis base in the protein binding site. Additionally, compound **8** had decreased activity because of the absence of the 3-CF<sup>3</sup> substituent; this is an indication that such a big and polarized halogen atom can only result in a beneficial increment in activity when a bulky lipophilic substituent as CF<sup>3</sup> is present at position 3.

Cytotoxicity results of compounds **10** and **11** (lipophilic moiety in 40 -position of the phenyloxyphenyl core instead of the 3-position) with HL-60 cells show that compound **10** maintains a similar IC<sup>50</sup> value as **7**; however, **11** has a reduction of activity compared to **9**.

Compound **3**, which is a shorter version of **1** (-NH- link instead of a di-substituted guanidinium) shows increased cytotoxicity in HL-60 cells (IC<sup>50</sup> = 3.08 µM). Similar to what was observed for the 3,40 -bis-guanidine derivatives, removal of the 3-CF<sup>3</sup> group in the lipophilic section caused decreased activity in the shorter analogue **28** (7.50 µM). Interestingly, removal of the 4-halogen in this lipophilic section did not affect the IC<sup>50</sup> value of compounds **29** and **30** compared to their analogue **3**. This could be explained by the compensation of bulky and lipophilic effects when going from trifluoromethyl (-CF3) to bulkier pentafluorosulfanyl (-SF5) substitution.

The 3,40 -bis-guanidines phenyloxypyridines **49**–**52**, **2** and **4** show, overall, increased HL-60 cytotoxicity than the previously discussed derivatives. Compound **2**, the pyridine analogue of **1**, has an IC<sup>50</sup> value of 2.36 µM, a four-fold increased activity compared to **1**. Likewise, compound **52** with a 3-CF3-4-Br phenyl system, shows a low IC<sup>50</sup> of 1.53 µM. Surprisingly, the introduction of a pyridinoguanidinium system as in compound **49** (11.61 µM) instead of a phenylguanidinium moiety as in **3** (>100 µM) results in increased HL-60 cytotoxicity. Even though this is not the most active compound of the series, it is a clear indication of the importance of the pyridinoguanidinium system in improving cytotoxicity in HL-60 cells.

Interestingly, compound **4**, which includes both a shorter -NH- link and the pyridinoguanidinium moiety has a relatively low IC<sup>50</sup> value of 3.48 µM. Compound **70**, with a -NH- link and an isouronium instead of the para guanidinium, shows similar cell viability as the guanidinium analogue **3**. From these results we can deduce that the isouronium cation has a similar behaviour to the guanidinium cation, as we had previously observed in the 3,40 -bis-guanidinium series [**?** ]. Finally, compound **75**, where the

*para* guanidinium is replaced by a sulfamide, shows a decreased cytotoxicity in HL-60 cells to 9.14 µM, indicating the importance of the guanidinium or isouronium cations.

The IC<sup>50</sup> values obtained for selected compounds in the MCF-7 cell line (Table **??**, Figure S8) were similar to the results obtained for the HL-60 cell line with most values in the low µM range. Compounds **3** and **4** (IC<sup>50</sup> = 2.02 and 3.73 µM, respectively) are still the most cytotoxic agents compared to compound **1** (IC<sup>50</sup> of 9.30 µM). The pyridine ring present in compounds **4** and **2** still appears responsible for the improved activity, even though less accentuated than in the HL-60 cell line. Instead, the isouronium version of compound **3**, compound **70**, has an increased IC<sup>50</sup> in MCF-7 cells compared with HL-60 cells indicating a certain degree of cell selectivity.

We have also evaluated the effect of compounds **2**, **3**, **4,** and **1** on the viability of the HeLa cell line and the results are reported in Table **??** (Figure S9). In this cell line, compound **4** showed again the lowest IC<sup>50</sup> value (1.33 µM). The rest of the compounds maintained similar cytotoxic activity in HeLa cells compared to HL-60 and MCF-7 cells.

It is known that sorafenib was originally developed as an inhibitor of the Ras effector RAF, and there are studies showing that sorafenib enhances the therapeutic efficacy of rapamycin in certain colorectal cancers [**?** ]. The IC<sup>50</sup> results in Table **??** (Figure S10) show that compound **1** has more potency in the HCT116 *KRAS* mutated cancer cell line (9.96 µM) than in the HKH-2 *KRAS* wt isogenic form (19.18 µM). Remarkably, its cytotoxic effect is like that of sorafenib in HCT116 (6.79 µM), but not in HKH-2, where sorafenib has a much lower IC50. Similar activity in both cell lines is reached with compound **2**, while **4** is revealed to be the most active of the series with an IC<sup>50</sup> of 4.59 µM in HCT116 and 2.88 µM in HKH-2. Interestingly, compound **3**, which has a phenyl group instead of the 2-pyridinyl of compounds **2** and **4**, shows poor cytotoxic activity in both cell lines.

Searching for a relationship between physicochemical properties and cytotoxicity, we observed a trend between the calculated logP and the HL-60 IC<sup>50</sup> values obtained for all synthesised compounds (except the inactive **5** and **6**, IC<sup>50</sup> > 100 µM) (Figure S11). This supports our hypothesis that the hydrophobic moiety of our molecules interacts with a specific allosteric hydrophobic pocket in protein kinases, justifying the use of bulkier and more lipophilic substituents (larger logP) in order to obtain improved anti-cancer activity (lower IC<sup>50</sup> values).

Therefore, some structure–activity relationships (SARs) can be drawn from the cell viability assays (Figure **??**): (i) the hydrophobic moiety is necessary for the activity; particularly, bulky and lipophilic substituents improve the cytotoxicity of these compounds (I- or Br- substitution at the 4-position better than Cl-), and a substituent at the 3-position is required to maintain efficacy (CF<sup>3</sup> or SF5); (ii) replacement of the di-substituted guanidinium in compound **1** (position 3 of the phenyloxyaryl core) by a shorter -NH- link results in compounds with better cytotoxicity; (iii) a mono-substituted guanidinium group as in compound **1** (position 40 of the phenyloxyaryl core) gives the greatest cytotoxic activity; additionally, incorporating a 2-pyridinyl instead of a phenyl group, attached to this guanidinium facilitates forming *Pharmaceuticals*  an IMHB that seems to a **2020** ffect positively the cytotoxic activity. , *13*, x FOR PEER REVIEW 13 of 26

**Figure 4.** Structure Activity Relationship (SAR) deduced from the analysis of the HL-60 cytotoxicity **Figure 4.** Structure Activity Relationship (SAR) deduced from the analysis of the HL-60 cytotoxicity results.

results. Considering the promising cytotoxicity results obtained with the cancer cell lines, we also Considering the promising cytotoxicity results obtained with the cancer cell lines, we also evaluated the potential toxicity of the most efficient derivative (compound **4**) in the non-cancerous cell

evaluated the potential toxicity of the most efficient derivative (compound **4**) in the non-cancerous

We have previously reported that compound **1** induces 68.9 ± 0.1% apoptosis in HL-60 cells at a 10 μM concentration after 48 h [8]. Accordingly, in order to evaluate the apoptotic effect of the most relevant derivatives (**2**, **3**, and **4**), HL-60 cells were treated with these compounds (at 5 μM, 5 μM, and 4 μM concentration, respectively) for 48 h, stained with annexin V-FITC/PI and analysed using flow

The results presented in Figures 5 and S13 show that, under these conditions, a more potent induction of apoptosis was observed with **3** (82.5 ± 11.3%) and **4** (92.7 ± 5.5%) compared to compound **2** (30.7 ± 11.6%). As the alamarBlue assay indirectly assesses cytotoxicity by quantifying cell viability and proliferation, these data suggest that compound **2** may have a predominantly cytostatic

**3 2 4**

Annexin V - FITC

10 and 1 μM the compound does not appear to affect in any way cell viability (Figure S12).

2.4.2. Apoptosis Assay in HL-60

cytometry (Figure 5).

mechanism of action.

Propidium Iodide – PerCP-cy5.5

results.

line MCF10A (human mammary epithelial cell line). We observed that this compound **4** only shows toxicity towards MCF10A at a high concentration of 100 µM while at lower concentrations of 10 and 1 µM the compound does not appear to affect in any way cell viability (Figure S12). We have previously reported that compound **1** induces 68.9 ± 0.1% apoptosis in HL-60 cells at a 10 μM concentration after 48 h [8]. Accordingly, in order to evaluate the apoptotic effect of the most relevant derivatives (**2**, **3**, and **4**), HL-60 cells were treated with these compounds (at 5 μM, 5 μM, and 4 μM concentration, respectively) for 48 h, stained with annexin V-FITC/PI and analysed using flow

10 and 1 μM the compound does not appear to affect in any way cell viability (Figure S12).

*Pharmaceuticals* **2020**, *13*, x FOR PEER REVIEW 13 of 26

**Figure 4.** Structure Activity Relationship (SAR) deduced from the analysis of the HL-60 cytotoxicity

Considering the promising cytotoxicity results obtained with the cancer cell lines, we also evaluated the potential toxicity of the most efficient derivative (compound **4**) in the non-cancerous cell line MCF10A (human mammary epithelial cell line). We observed that this compound **4** only shows toxicity towards MCF10A at a high concentration of 100 μM while at lower concentrations of

N N H NH2

**guanidinium ~ isouronium > sulfamide**

NH2

**pyridine > benzene**

X O

Cl

**bulky and lipophilic substituents**

F3C

**-NH > gua**

#### 2.4.2. Apoptosis Assay in HL-60 cytometry (Figure 5).

**- I > -Br > -Cl**

**-SF5 > -CF3**

We have previously reported that compound **1** induces 68.9 ± 0.1% apoptosis in HL-60 cells at a 10 µM concentration after 48 h [**?** ]. Accordingly, in order to evaluate the apoptotic effect of the most relevant derivatives (**2**, **3**, and **4**), HL-60 cells were treated with these compounds (at 5 µM, 5 µM, and 4 µM concentration, respectively) for 48 h, stained with annexin V-FITC/PI and analysed using flow cytometry (Figure **??**). The results presented in Figures 5 and S13 show that, under these conditions, a more potent induction of apoptosis was observed with **3** (82.5 ± 11.3%) and **4** (92.7 ± 5.5%) compared to compound **2** (30.7 ± 11.6%). As the alamarBlue assay indirectly assesses cytotoxicity by quantifying cell viability and proliferation, these data suggest that compound **2** may have a predominantly cytostatic mechanism of action.

**Figure 5.** Annexin V-FITC vs. PI flow cytometry analysis of HL-60 cancer cells treated with compounds **3** (5 µM), **2** (5 µM) and **4** (4 µM) for 48 h. These figures are representative of three independent experiments. The viable cells, early apoptotic, necrotic and late apoptotic cells are represented by the lower left, lower right, upper left and upper right quadrants, respectively.

The results presented in Figure **??** and Figure S13 show that, under these conditions, a more potent induction of apoptosis was observed with **3** (82.5 ± 11.3%) and **4** (92.7 ± 5.5%) compared to compound **2** (30.7 ± 11.6%). As the alamarBlue assay indirectly assesses cytotoxicity by quantifying cell viability and proliferation, these data suggest that compound **2** may have a predominantly cytostatic mechanism of action.

### 2.4.3. Effect on the MAPK/ERK Pathway

Taking into account the positive binding results obtained in the docking studies to the BRAF-ATP model and the promising cytotoxicity shown in several cancer cells, we next explored the effect of compounds with IC<sup>50</sup> near to that of sorafenib (**1**, **2**, **3**, **4**, **9,** and **52**) on the MAPK/ERK pathway. Thus, using Western immunoblot analysis of HL-60 extracts, we investigated the expression and phosphorylation levels of ERK (as an indication of ERK activation), which is the downstream effector of the Ras/BRAF signalling pathway (Figure **??**).

2.4.3. Effect on the MAPK/ERK Pathway

of the Ras/BRAF signalling pathway (Figure 6).

**Figure 5.** Annexin V-FITC vs. PI flow cytometry analysis of HL-60 cancer cells treated with compounds **3** (5 μM), **2** (5 μM) and **4** (4 μM) for 48 h. These figures are representative of three independent experiments. The viable cells, early apoptotic, necrotic and late apoptotic cells are represented by the lower left, lower right, upper left and upper right quadrants, respectively.

Taking into account the positive binding results obtained in the docking studies to the BRAF-ATP model and the promising cytotoxicity shown in several cancer cells, we next explored the effect of compounds with IC50 near to that of sorafenib (**1**, **2**, **3**, **4**, **9,** and **52**) on the MAPK/ERK pathway. Thus, using Western immunoblot analysis of HL-60 extracts, we investigated the expression and

**Figure 6.** Western immunoblot of HL-60 cell extracts following incubation with compounds **2**, **3**, **4**, **9**, **52** and **1** (as a control). HL60 cells were seeded at 2 × 105 cells/mL and were treated with either vehicle [0.5% EtOH (v/v)], compounds **52**, **2**, **9**, **3** and **4** (5 μM) or compound **1** (5 and (\*) 10 μM, as in reference [9]) for 16 h. Cells were lysed and equal amounts of protein were loaded and separated on 15% SDS-PAGE gels, transferred to PVDF membrane and probed with antibodies against total and phosphorylated ERK. Anti-GAPDH was used as a loading control. Results are representative of 2 independent experiments. **Figure 6.** Western immunoblot of HL-60 cell extracts following incubation with compounds **2**, **3**, **4**, **<sup>9</sup>**, **<sup>52</sup>** and **<sup>1</sup>** (as a control). HL60 cells were seeded at 2 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL and were treated with either vehicle [0.5% EtOH (*v*/*v*)], compounds **52**, **2**, **9**, **3** and **4** (5 µM) or compound **1** (5 and (\*) 10 µM, as in reference [**?** ]) for 16 h. Cells were lysed and equal amounts of protein were loaded and separated on 15% SDS-PAGE gels, transferred to PVDF membrane and probed with antibodies against total and phosphorylated ERK. Anti-GAPDH was used as a loading control. Results are representative of 2 independent experiments.

We observed that compounds **3** and **4** do not appear to interfere with the Ras/BRAF signalling pathway; however, compounds **2**, **9,** and **52** have a similar effect as lead compound **1** (both at 5 and 10 μM) in inhibiting ERK phosphorylation and therefore inhibiting the Ras/BRAF pathway. These results may suggest that the potent cytotoxicity observed with these derivatives may be due to different mechanisms of action. Therefore, compounds **2**, **9,** and **52**, which are all 3,4′-bis-guanidino phenyloxy(phenyl or pyridyl) analogues of **1** with similar lipophilic moieties (3-CF3,4-(Br/Cl)-Ph), may exert their cytotoxicity by interfering with the Ras/BRAF pathway. On the contrary, compounds **3** and **4** (shorter 3-amino-4′-guanidino phenyloxy(phenyl or pyridyl) derivatives) may act in a different signalling pathway or in the same pathway but through a different mechanism from compound **1** [6]. Pulikakos and co-workers have reported that the biochemical effect of a RAF inhibitor on ERK signalling would be the combined outcome of different mechanisms [34], clearly indicating the complexity of the biological target and opening the door to future studies to understand the mechanism of action of these compounds. We observed that compounds **3** and **4** do not appear to interfere with the Ras/BRAF signalling pathway; however, compounds **2**, **9,** and **52** have a similar effect as lead compound **1** (both at 5 and 10 µM) in inhibiting ERK phosphorylation and therefore inhibiting the Ras/BRAF pathway. These results may suggest that the potent cytotoxicity observed with these derivatives may be due to different mechanisms of action. Therefore, compounds **2**, **9,** and **52**, which are all 3,40 -bis-guanidino phenyloxy(phenyl or pyridyl) analogues of **1** with similar lipophilic moieties (3-CF3,4-(Br/Cl)-Ph), may exert their cytotoxicity by interfering with the Ras/BRAF pathway. On the contrary, compounds **3** and **4** (shorter 3-amino-40 -guanidino phenyloxy(phenyl or pyridyl) derivatives) may act in a different signalling pathway or in the same pathway but through a different mechanism from compound **1** [**?** ]. Pulikakos and co-workers have reported that the biochemical effect of a RAF inhibitor on ERK signalling would be the combined outcome of different mechanisms [**?** ], clearly indicating the complexity of the biological target and opening the door to future studies to understand the mechanism of action of these compounds.

#### **3. Discussion 3. Discussion**

Considering the promising results previously obtained for lead compound **1** we explored several modifications to improve its cytotoxicity in different cancer cell lines. Accordingly, we designed a variety of compounds where different changes have been introduced: several lipophilic groups were considered; the di-substituted guanidine was replaced by a secondary amine; the phenyl ring was exchanged with a pyridine; and the mono-substituted guanidine in the polar side was switched to an isourea or a sulfanylamide group. Considering the promising results previously obtained for lead compound **1** we explored several modifications to improve its cytotoxicity in different cancer cell lines. Accordingly, we designed a variety of compounds where different changes have been introduced: several lipophilic groups were considered; the di-substituted guanidine was replaced by a secondary amine; the phenyl ring was exchanged with a pyridine; and the mono-substituted guanidine in the polar side was switched to an isourea or a sulfanylamide group.

Molecular docking was utilised to understand the interactions between the proposed biological target and model compounds **1–4**. Thus, considering the putative activity of **1** as a type-III kinase inhibitor, compounds **1–4** were docked into an in-house model of an active TP-containing BRAF Molecular docking was utilised to understand the interactions between the proposed biological target and model compounds **1**–**4**. Thus, considering the putative activity of **1** as a type-III kinase inhibitor, compounds **1**–**4** were docked into an in-house model of an active TP-containing BRAF kinase. All final poses exhibit similar interactions between the lipophilic moiety and the lipophilic pocket, as well as between the mono-substituted guanidinium and one of the phosphate groups of TP.

Based on this computational study, 3,40 -bis-guanidino diphenyl ether derivatives **5**–**11** were prepared by reacting the corresponding 3,40 -diamino diphenyl ether with conveniently substituted Boc-protected thioureas. Additionally, synthesis of the 3-amino-40 -guanidino diphenyl ethers **3**, **28**–**30** and **36** required the preparation of the corresponding starting diamines. Furthermore, preparation of 3,40 -bis-guanidino and 3-amino-40 -guanidino phenylpyridyl ether derivatives **2**, **49**–**52,** and **4**, required different synthetic approaches involving Buchwald–Hartwig coupling. Finally, the 3-amino-40 isouronium **70** and 3-amino-40 -sulfonamido **75** derivatives were prepared following specific synthetic routes. All compounds were obtained as hydrochloride salts and their purity was determined to be >95% by HPLC before proceeding to biological testing.

A screening of the cytotoxicity of all compounds was performed in the HL-60 cell line and the more potent compounds were selected for further biological evaluation in MCF-7, HeLa, HCT116, and HKH-2 cell lines. These cell viability studies revealed that these compounds can inhibit cell proliferation in the low µM range, showing up to a nine-fold increase in cytotoxicity compared to lead compound **1**. All modifications explored generated SAR information which helped to understand the structural requirements for a more potent cytotoxicity. Cytotoxicity of compound **4** was also evaluated against non-tumorigenic MCF10A and the results show that, at active concentration in cancer cell lines, compound **4** has no toxic effects to non-tumorigenic cells.

Flow cytometry in HL-60 cell lines was also performed to assess the apoptotic effect of compounds **2**, **3**, and **4**. The results are in agreement with the corresponding structures; thus, mono-guanidinium compounds (**3** and **4**) induce a stronger apoptotic effect in HL-60 cells (82% and 92%, respectively) compared to the bis-guanidinium compounds (**1** and **2**, around 60%<sup>7</sup> and 30%, respectively).

With the aim of determining whether compounds **1**–**4**, **9,** and **52** exert their anticancer activity by interfering with the Ras/BRAF pathway (as previously suggested by us for lead compound **1**), Western immunoblot analysis was performed with HL-60 cell extracts measuring the activation of the downstream effector ERK. Thus, we observed that compounds **1**, **2**, **9,** and **52** (3,40 -bis-guanidine phenyloxy(phenyl or pyridyl) derivatives) abrogates ERK activation, suggesting potential inhibition of the Ras/BRAF pathway; however, compounds **3** and **4** (shorter derivatives) do not act in the same way.

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

### *4.1. Computational Details*

### 4.1.1. Ligand Optimization

All ligands were fully optimized at DFT level using the M06-2X functional with the 6-311+G\* basis set as implemented in the Gaussian16 package [**?** ]. Frequency calculations were performed at the same computational level to confirm that the resulting optimized structures were energetic minima. The effect of water solvation was accounted for by using the SCRF-PCM approach implemented in the Gaussian16 package including dispersion, repulsion and cavitation energy terms of the solvent in the optimization.

### 4.1.2. Docking Experiments

The program AutoDock Vina 4.2 was used to carry out docking studies [**?** ]. The ligands were flexibly docked into the rigid in-house TP-containing BRAF model (see details in ESI). The corresponding docking scores (G-scores) were measured in kcal/mol and are only indicative of the quality of the interaction with the target; they do not provide a quantitative measure of binding. Poses obtained from the docking were visualised with VMD [**?** ].

### *4.2. Chemistry*

### 4.2.1. 1-(4-Fluorophenyl)-2-(3-(4-guanidinophenoxy)phenyl)guanidine dihydrochloride (5)

Following Method A (see ESI), **21** (80 mg, 0.12 mmol) was dissolved in 4 M HCl in dioxane (0.54 mL, 2.16 mmol) and in additional dioxane (0.07 mL) until a final concentration of 0.2 M was reached. After 8 h stirring at 55 ◦C, the reaction was adjudged complete (TLC), solvents were evaporated and the residue was purified by silica gel (CH3Cl:MeOH) chromatography to afford the pure hydrochloride salt as a white-yellow solid (35 mg, 76%). Mp: decomp. > 180 ◦C. δ<sup>H</sup> (400 MHz, CD3OD): 7.00 (dd, *J* = 8.3, 2.4, 1H, H-4), 7.04 (t, *J* = 2.1 Hz, 1H, H-2), 7.13–7.17 (m, 3H, H-8 and H-80 and H-6), 7.19–7.24 (m, 2H, H-13 and H-130 ), 7.31 (d, *J* = 8.9 Hz, 2H, H-9 and H-90 ), 7.36–7.39 (m, 2H, H-12 and H-120 ), 7.47 (t, *J* = 8.1 Hz, 1H, H-5). δ<sup>C</sup> (100 MHz, CD3OD): 116.5 (CH Ar, C-2), 117.8 (d, *J* = 23.3 Hz, 2 CH Ar, C-13 and C-130 ), 118.6 (CH Ar, C-4), 121.1 (CH Ar, C-6), 121.5 (2 CH Ar, C-8 and C-80 ), 129.95 (d, *J* = 8.8 Hz, 2 CH Ar, C-12 and C-120 ), 128.96 (2 CH Ar, C-9 and C-90 ), 131.6 (qC), 132.3 (d, *J* = 3.1 Hz, qC, C-11), 132.4 (CH Ar, C-5), 138.0 (qC), 156.6 (qC), 157.4 (qC), 158.4 (qC), 159.5 (qC), 163.2 (d, *<sup>J</sup>* <sup>=</sup> 246.1 Hz, qC, C-14). <sup>δ</sup><sup>F</sup> (376 MHz, CD3OD): <sup>−</sup>115.93 (m). <sup>ν</sup>max(ATR)/cm−<sup>1</sup> : 3110 (NH), 3052 (NH), 2922, 2330, 2134, 1655 (C=N), 1582 (C=N), 1505 (C-N), 1486, 1404, 1212 (C-O), 1066 (C-F), 834, 792, 552. HRMS (*m*/*z* ESI+): found: 379.1687 (M<sup>+</sup> + H), C20H20N6OF requires: 379.1683. HPLC: 99.7% (*t*R: 22.9 min).

### 4.2.2. 1-(3:4-Di-fluorophenyl)-2-(3-(4-guanidinophenoxy)phenyl)guanidine dihydrochloride (6)

Following Method A (see ESI), **22** (53 mg, 0.08 mmol) was dissolved in 4 M HCl in dioxane (0.36 mL, 1.37 mmol) and in additional dioxane (0.06 mL) until a final concentration of 0.2 M was reached. After 8 h stirring at 55 ◦C, the reaction was adjudged complete (TLC), solvents were evaporated and the residue was purified by silica gel chromatography (CHCl3:MeOH) to afford the pure hydrochloride salt as a white solid (34 mg, 90%). Mp: 158–160 ◦C. δ<sup>H</sup> (400 MHz, CD3OD): 7.00 (dd, *J* = 8.3, 2.4 Hz, 1H, H-4), 7.05 (t, *J* = 2.2 Hz, 1H, H-2), 7.13–7.20 (m, 4H, H-8 and H-80 , H-6 and H-16), 7.32 (d, *J* = 8.9 Hz, 2H, H-9 and H-90 ), 7.34–7.41 (m, 2H, H-12 and H-15), 7.47 (t, *J* = 8.1 Hz, 1H, H-5). δ<sup>C</sup> (100 MHz, CD3OD): 116.3 (d, *J* = 19.7 Hz, C-12 or C-15), 116.5 (CH Ar, C-2), 118.6 (CH Ar, C-4), 119.5 (d, *J* = 18.8 Hz, C-12 or C-15), 121.1 (CH Ar, C-6), 121.5 (2 CH Ar, C-8 and C-80 ), 123.4 (dd, *J* = 6.7, 3.7 Hz, C-16), 129.0 (2 CH Ar, C-9 and C-90 ), 131.6 (qC), 132.4 (CH Ar, C-5), 133.0 (dd, *J* = 8.3, 3.6 Hz, qC, C-11), 137.9 (qC), 150.8 (dd, *J* = 247.8, 12.6 Hz, qC, C-13 or C-14), 151.8 (dd, *J* = 248.7, 13.7 Hz, qC, C-13 or C-14), 156.5 (qC), 157.4 (qC), 158.3 (qC), 159.5 (qC). δ<sup>F</sup> (376 MHz, CD3OD): −137.23 (m), −141.12 (m). νmax(ATR)/cm−<sup>1</sup> : 3228 (NH), 3040 (NH), 2923, 2853, 1655 (C=N), 1579 (C=N), 1505, 1485, 1401, 1259, 1211 (C-F), 1149, 972, 825, 771, 694, 649, 609, 587. HRMS (*m*/*z* ESI+): found: 397.1598 (M<sup>+</sup> + H), C20H19N6OF<sup>2</sup> requires: 397.1588. HPLC: 96.8% (*t*R: 23.2 min).

### 4.2.3. 1-(2-Fluoro-4-iodophenyl)-2-(3-(4-guanidinophenoxy)phenyl)guanidine dihydrochloride (7)

Following Method A (see ESI), **23** (357 mg, 0.44 mmol) was dissolved in 4 M HCl in dioxane (2 mL, 7.92 mmol) and in additional dioxane (0.2 mL) until a final concentration of 0.2 M was reached. After 8 h stirring at 55 ◦C, the reaction was adjudged complete (TLC), solvents were evaporated and the residue was purified by silica gel chromatography (CH3Cl:MeOH) to afford the pure hydrochloride salt as a white solid (210 mg, 83%). Mp: decomp. > 180 ◦C. δ<sup>H</sup> (400 MHz, CD3OD): 6.99–7.01 (m, 2H, H-2 and H-4), 7.11–7.14 (m, 1H, H-6), 7.15 (d, *J* = 8.9 Hz, 2H, H-8 and H-80 ), 7.20 (t, *J* = 8.2 Hz, 1H, H-5), 7.33 (d, *J* = 8.9 Hz, 2H, H-9 and H-90 ), 7.44–7.49 (m, 1H, H-15), 7.64–7.66 (m, 1H, H-16), 7.69 (dd, *J* = 9.6, 1.8 Hz, 1H, H-13). δ<sup>C</sup> (100 MHz, CD3OD): 93.4 (d, *J* = 7.5 Hz, qC, C-14), 116.4 (CH Ar, C-2), 118.6 (CH Ar, C-4), 121.0 (CH Ar, C-6), 121.5 (2 CH Ar, C-8 and C-80 ), 124.1 (d, *J* = 12.4 Hz, qC, C-11), 127.3 (d, *J* = 22.2 Hz, CH Ar, C-13), 128.9 (2 CH Ar, C-9 and C-90 ), 130.9 (CH Ar, C-5), 131.5 (qC), 132.4 (CH Ar, C-15), 136.0 (d, *J* = 3.9 Hz, CH Ar, C-16), 137.9 (qC), 156.4 (qC), 158.0 (d, *J* = 254.4 Hz, qC, C-12), 157.3 (qC), 158.3 (qC), 159.5 (qC). δ<sup>F</sup> (376, CD3OD):—121.10 (t, *J* = 8.9 Hz). νmax(ATR)/cm−<sup>1</sup> : 3318 (NH), 3098 (NH), 2958, 1661 (C=N), 1620, 1579, 1485, 1214 (C-F), 1149, 625, 609, 576 (C-I), 566. HRMS (*m*/*z* ESI+): found 505.0646 (M<sup>+</sup> + H), C20H19N6OFI requires: 505.0649. HPLC: 99.2% (*t*R: 25.3 min).

### 4.2.4. 1-(4-Bromophenyl)-2-(3-(4-guanidinophenoxy)phenyl)guanidine dihydrochloride (8)

Following Method A (see ESI), **24** (212 mg, 0.29 mmol) was dissolved in 4 M HCl in dioxane (1.30 mL, 5.22 mmol) and in additional dioxane (0.12 mL) until a final concentration of 0.2 M was reached. After 8 h stirring at 55 ◦C, the reaction was adjudged complete (TLC), solvents were evaporated and the residue was purified by flash chromatography to afford the pure hydrochloride salt as a light-yellow solid (128 mg, 87%). Mp: decomp. > 110 ◦C. δ<sup>H</sup> (400 MHz, CD3OD): 6.99 (dd, *J* = 8.2, 2.3 Hz, 1H, H-4), 7.03 (t, *J* = 2.2 Hz, 1H, H-2), 7.12–7.16 (m, 3H, H-6 and H-8 and H-80 ), 7.27 (d, *J* = 8.7 Hz, 2H, H-12 and H-120 or H-13 and H-130 ), 7.31 (d, *J* = 8.8 Hz, 2H, H-9 and H-90 ), 7.46 (t, *J* = 8.1 Hz, 1H, H-5), 7.61 (d, *J* = 8.7 Hz, 2H, H-12 and H-120 or H-13 and H-130 ). δ<sup>C</sup> (100 MHz, CD3OD): 116.3 (CH Ar, C-2), 118.5 (CH Ar, C-4), 120.9 (CH Ar, C-6), 121.5 (2 CH Ar, C-8 and C-80 ), 121.7 (qC, C-14), 127.9 (2 CH Ar, C-12 and C-120 or C-13 and C-130 ), 128.9 (2 CH Ar, C-9 and C-90 ), 131.5 (qC), 132.4 (CH Ar, C-5), 134.1 (CH Ar, C-12 and C-120 or C-13 and C-130 ), 135.8 (qC), 138.0 (qC), 156.2 (qC), 157.4 (qC), 158.3 (qC), 159.5 (qC). νmax(ATR)/cm−<sup>1</sup> : 3124 (NH), 3044 (NH), 1655, 1571 (C=N), 1504 (C=N), 1484, 1405, 1213 (C-O), 1070 (C-Br), 1010, 617-567. HRMS (*m*/*z* APCI+): found: 439.0857 (M<sup>+</sup> + H), C20H20BrN6O requires: 439.0876. HPLC: 99.8% (*t*R: 24.8 min).

### 4.2.5. 1-(4-Bromo-3-(trifluoromethyl)phenyl)-2-(3-(4-guanidinophenoxy)phenyl)guanidine dihydrochloride (9)

Following Method A (see ESI), **25** (566 mg, 0.70 mmol) was dissolved in 4 M HCl in dioxane (3.15 mL, 12.6 mmol) and in additional dioxane (0.35 mL) until a final concentration of 0.2 M was reached. After 8 h stirring at 55 ◦C, the reaction was adjudged complete (TLC), solvents were evaporated and the residue was purified by silica gel chromatography (CHCl3:MeOH) to afford the pure hydrochloride salt as a white solid (361 mg, 89%). Mp: decomp. >136 ◦C. δ<sup>H</sup> (400 MHz, CD3OD): 6.99 (dd, *J* = 8.0, 1.9 Hz, 1H, H-4), 7.06 (t, *J* = 2.1 Hz, 1H, H-2), 7.14–7.16 (m, 3H, H-8 and H-80 and H-6), 7.32 (d, *J* = 8.9 Hz, 2H, H-9 and H-90 ), 7.46 (t, *J* = 8.1 Hz, 1H, H-5), 7.51 (dd, *J* = 8.6, 2.4 Hz, 1H, H-16), 7.74 (d, *J* = 2.4 Hz, 1H, H-12), 7.88 (d, *J* = 8.6 Hz, 1H, H-15). δ<sup>C</sup> (100 MHz, CD3OD): 116.2 (CH Ar, C-2), 118.3 (qC, C-14), 118.5 (CH Ar, C-4), 120.8 (CH Ar, C-6), 121.5 (2 CH Ar, C-8 and C-80 ), 123.9 (d, *J* = 260.3 Hz, qCF3), 125.3 (m, CH Ar, C-12), 128.9 (2 CH Ar, C-9 and C-9<sup>0</sup> ), 130.5 (CH Ar, C-16), 131.6 (qC), 132.3 (d, *J* = 31.7 Hz, qC, C-13), 132.4 (CH Ar, C-5), 136.9 (qC), 137.8 (CH Ar, C-15), 138.0 (qC), 156.1 (qC), 157.3 (qC), 158.3 (qC), 159.6 (qC). δ<sup>F</sup> (376 MHz, CD3OD):—64.73 (s). νmax(ATR)/cm−<sup>1</sup> : 3119 (NH), 3053 (NH), 1663 (C=O), 1584 (C=N), 1478, 1412, 1320 (C-F), 1238, 1214, 1174, 1129 (CF3), 1099 (C-Br), 1023, 828, 581—558. HRMS (*m*/*z* ESI+): found 507.0766 (M<sup>+</sup> + H), C21H19N6OF3Br requires: 507.0756. HPLC: 99.9% (*t*R: 26.3 min).

### 4.2.6. 1-(2-Fluoro-4-iodophenyl)-2-(4-(3-guanidinophenoxy)phenyl)guanidine dihydrochloride (10)

Following Method A (see ESI), **26** (310 mg, 0.39 mmol) was dissolved in 4 M HCl in dioxane (1.73 mL, 6.93 mmol) and in additional dioxane (0.17 mL) until a final concentration of 0.2 M was reached. After 8 h stirring at 55 ◦C, the reaction was adjudged complete (TLC), solvents were evaporated and the residue was purified by flash chromatography to afford the pure hydrochloride salt as a white solid (198 mg, 88%). Mp: decomp. >150 ◦C. δ<sup>H</sup> (400 MHz, CD3OD): 6.97 (t, *J* = 2.1 Hz, 1H, H-2), 7.00 (dd, *J* = 8.2, 2.3 Hz, 1H, H-4), 7.08 (dd, *J* = 7.6, 1.5 Hz, 1H, H-6), 7.16 (d, *J* = 8.8 Hz, 2H, H-8 and H-80 ), 7.22 (t, *J* = 8.2 Hz, 1H, H-5 or H-15), 7.36 (d, *J* = 8.8 Hz, 2H, H-9 and H-90 ), 7.47 (t, *J* = 8.1 Hz, 1H, H-5 or H-15), 7.66 (d, *J* = 9.1 Hz, 1H, H-16), 7.70 (dd, *J* = 9.6, 1.7 Hz, 1H, H-13). δ<sup>C</sup> (100 MHz, CD3OD): 93.5 (d, *J* = 7.5 Hz, qC, C-14), 116.6 (CH Ar, C-2), 118.6 (CH Ar, C-4), 121.3 (CH Ar, C-6), 121.6 (2 CH Ar, C-8 and C-80 ), 124.0 (d, *J* = 12.5 Hz, qC, C-11), 127.3 (d, *J* = 22.2 Hz, C-13), 128.7 (2 CH Ar, C-9 and C-90 ), 131.1 (CH Ar, C-5 or C-15), 131.6 (qC), 132.4 (CH Ar, C-5 or C-15), 136.0 (d, *J* = 3.9 Hz, C-16), 137.7 (qC), 156.8 (qC), 157.3 (qC), 157.9 (qC), 158.2 (d, *J* = 254.5 Hz, qC, C-12), 159.5 (qC). δ<sup>F</sup> (376 MHz, CD3OD): <sup>−</sup>121.04 (t, *<sup>J</sup>* <sup>=</sup> 8.8 Hz). <sup>ν</sup>max(ATR)/cm−<sup>1</sup> : 3335 (NH), 3265 (NH), 3180 (NH), 3052, 2868, 2325, 1616 (C=N), 1560 (C=N), 1504, 1400, 1226 (C-F), 1162, 1109, 971, 875, 789, 684—573 (C-I). HRMS (*m*/*z* ESI+): found: 505.0645 (M<sup>+</sup> + H), C20H19N6OFI requires: 505.0649. HPLC: 99.9% (*t*R: 25.9 min).

4.2.7. 1-(4-Bromo-3-(trifluoromethyl)phenyl)-2-(4-(3-guanidinophenoxy)phenyl)guanidine dihydrochloride (11)

Following Method A (see ESI), 27 (148 mg, 0.18 mmol) was dissolved in 4 M HCl in dioxane (0.81 mL, 3.24 mmol) and in additional dioxane (0.10 mL) until a final concentration of 0.2 M was reached. After 8 h stirring at 55 ◦C, the reaction was adjudged complete (TLC), solvents were evaporated and the residue was purified by flash chromatography to afford the pure hydrochloride salt as a white solid (100 mg, 94%). Mp: decomp. > 95 ◦C. δ<sup>H</sup> (400 MHz, CD3OD): 6.98–7.02 (m, 2H, H-2 and H-4), 7.09 (ddd, 1H, *J* = 8.0, 1.9, 0.9 Hz, H-6), 7.17 (d, *J* = 8.9 Hz, 2H, H-8 and H-80 ), 7.41 (d, *J* = 9.0 Hz, 2H, H-9 and H-90 ), 7.49 (t, *J* = 8.0 Hz, 1H, H-5), 7.55 (dd, *J* = 8.5, 2.6 Hz, 1H, H-16), 7.77 (d, *J* = 2.6 Hz, 1H,

H-12), 7.91 (d, *J* = 8.6 Hz, 1H, H-15). δ<sup>C</sup> (100 MHz, CD3OD): 116.6 (CH Ar, C-2 or C-4), 118.5 (qC, C-14), 118.6 (CH Ar, C-2 or C-4), 121.3 (CH Ar, C-6), 121.6 (2 CH Ar, C-8 and C-80 ), 123.9 (d, *J* = 272.8 Hz, qCF3), 125.5 (q, *J* = 5.6 Hz, CH Ar, C-12), 128.5 (2 CH Ar, C-9 and C-9<sup>0</sup> ), 130.8 (CH Ar, C-16), 131.7 (qC), 132.3 (q, *J* = 31.6 Hz, qC, C-13), 132.4 (CH Ar, C-5), 136.7 (qC), 137.7 (qC), 137.8 (CH Ar, C-15), 156.5 (qC), 157.3 (qC), 157.9 (qC), 159.6 (qC). δ<sup>F</sup> (376 MHz, CD3OD):—64.30 (s). νmax(ATR)/cm−<sup>1</sup> : 3309 (NH), 3116 (NH), 3053 (NH), 2837, 2280, 1663 (C=N), 1577 (C=N), 1505, 1486, 1405, 1320, 1258 (C-O), 1214 (CF3), 1129, 1023 (C-Br), 829, 595—575. HRMS (*m*/*z* ESI+): found: 507.0750 (M<sup>+</sup> + H), C21H19N6OBrF<sup>3</sup> requires: 507.0756. HPLC: 99.9% (*t*R: 26.6 min).

### 4.2.8. 1-(4-(3-((4-Chlorophenyl)amino)phenoxy)phenyl)guanidine hydrochloride (28)

Following Method A (see ESI), **44** (97 mg, 0.18 mmol) was dissolved in 4 M HCl in dioxane (0.53 mL, 2.10 mmol) and in additional dioxane (0.9 mL) until a final concentration of 0.2 M was reached. After 8 h stirring at 55 ◦C, the reaction was adjudged complete (TLC), solvents were evaporated and the residue was purified by silica gel chromatography (CHCl3:MeOH) to afford the pure hydrochloride salt as a purple solid (69 mg, 99%). Mp: 50–52 ◦C. δ<sup>H</sup> (400 MHz, CD3OD): 6.51 (dd, *J* = 8.3, 1.9 Hz, 1H, H-4), 6.73 (t, *J* = 2.2 Hz, 1H, H-2), 6.85 (dd, *J* = 7.8, 1.8 Hz, 1H, H-6), 7.05 (d, *J* = 8.9 Hz, 2H, H-12 and H-120 ), 7.08 (d, *J* = 8.9 Hz, 2H, H-8 and H-80 ), 7.18 (d, *J* = 8.9 Hz, 2H, H-13 and H-130 ), 7.22 (t, *J* = 8.2 Hz, 1H, H-5), 7.27 (d, *J* = 8.9 Hz, 2H, H-9 and H-90 ). δ<sup>C</sup> (100 MHz, CD3OD): 108.6 (CH Ar, C-2), 111.8 (CH Ar, C-4), 113.7 (CH Ar, C-6), 120.0 (2 CH Ar, C-12 and C-120 ), 120.7 (2 CH Ar, C-8 and C-80 ), 126.1 (qC, C-14), 128.8 (2 CH Ar, C-9 and C-90 ), 130.1 (2 CH Ar, C-13 and C-130 ), 130.6 (qC), 131.5 (CH Ar, C-5), 143.4 (qC), 146.7 (qC), 158.4 (qC), 158.5 (qC), 158.9 (qC). νmax(ATR)/cm−<sup>1</sup> : 3297 (N-H), 3126 (N-H), 1688, 1586 (C=N), 1502, 1485 (C-N), 1325, 1216 (C-O), 1142 (C-Cl), 997, 972, 823, 770, 689, 604, 588, 570. HRMS (*m*/*z* ESI+): found 353.1177 (M<sup>+</sup> + H. C19H18N4OCl requires: 353.1169). HPLC: 98.0% (*t*R: 32.3 min).

### 4.2.9. 1-(4-(3-((4-Chloro-3-(trifluoromethyl)phenyl)amino)phenoxy)phenyl)guanidine hydrochloride (3)

Following Method A (see ESI), **45** (112 mg, 0.18 mmol) was dissolved in 4 M HCl in dioxane (0.54 mL, 2.16 mmol) and in additional dioxane (0.36 mL) until a final concentration of 0.2 M was reached. After 8 h stirring at 55 ◦C, the reaction was adjudged complete (TLC), solvents were evaporated and the residue was purified by flash chromatography to afford the pure hydrochloride salt as a light brown solid (59 mg, 80%). Mp: 58–60 ◦C. δ<sup>H</sup> (400 MHz, CD3OD): 6.62 (dd, *J* = 8.7, 2.3 Hz, 1H, H-4), 6.78 (t, *J* = 2.2 Hz, 1H, H-2), 6.90 (dd, *J* = 8.1, 2.1 Hz, 1H, H-6), 7.11 (d, *J* = 8.9 Hz, 2H, H-8 and H-8'), 7.24 (dd, *J* = 8.8, 2.8 Hz, 1H, H-16), 7.27–7.31 (m, 3H, H-9, H-9' and H-5) 7.36–7.39 (m, 2H, H-12, H-15). δ<sup>C</sup> (100 MHz, CD3OD): 109.8 (CH Ar, C-2), 113.1 (CH Ar, C-4), 114.8 (CH Ar, C-6), 116.1 (q, *J* = 5.6 Hz, CH Ar, C-12), 121.0 (2 CH Ar, C-8 and C-8'), 121.5 (CH Ar, C-16), 122.1 (qC, C-14), 124.4 (d, *J* = 272.5 Hz, qCF3), 129.0 (2 CH Ar, C-9 and C-9'), 129.6 (q, *J* = 31.0, qC, C-13), 131.0 (qC), 131.8 (CH Ar, C-5), 133.4 (CH Ar, C-15), 144.4 (qC), 145.3 (qC), 158.2 (qC), 158.4 (qC), 159.2 (qC). δ<sup>F</sup> (376 MHz, CD3OD):—64.18 (s). νmax(ATR)/cm−<sup>1</sup> : 3295 (NH), 3163, 2923, 2853, 2400, 1664 (C=O), 1595 (C=N), 1504, 1482, 1441, 1333, 1258, 1217 (CF3), 1127, 1112 (C-Cl), 1027, 999, 977, 825. HRMS (*m*/*z* ESI+): found 421.1044 (M<sup>+</sup> + H. C20H17ClF3N4O requires: 421.1043). HPLC: 97.8% (*t*R: 32.9 min).

### 4.2.10. 1-(4-(3-((3-(Trifluoromethyl)phenyl)amino)phenoxy)phenyl)guanidine hydrochloride (29)

Following Method A (see ESI), **46** (371 mg, 0.63 mmol) was dissolved in 4 M HCl in dioxane (1.90 mL, 7.56 mmol) and in additional dioxane (1.25 mL) until a final concentration of 0.2 M was reached. After 6 h stirring at 55 ◦C, the reaction was adjudged complete (TLC), solvents were evaporated and the residue was purified by flash chromatography to afford the pure hydrochloride salt as a white solid (242 mg, 90%). Mp: 93–95 ◦C. δ<sup>H</sup> (400 MHz, CD3OD): 6.59 (dd, *J* = 8.1, 2.3 Hz, 1H, H-4), 6.78 (t, *J* = 2.2 Hz, 1H, H-2), 6.90 (dd, *J* = 7.9, 1.9 Hz, 1H, H-6), 7.07–7.13 (m, 3H, H-8 and 80 and H-12 or H-14), 7.26–7.30 (m, 5H, H-9 and 90 , H-12 or H-14, H-16 and H-5 or H-15), 7.37 (t, *J* = 8.3 Hz, 1H, H-5 or H-15). δ<sup>C</sup> (100 MHz, CD3OD): 109.4 (CH Ar, C-2), 112.6 (CH Ar, C-4), 113.9 (q, *J* = 4.0 Hz, CH Ar, C-12 or C-14), 114.6 (CH Ar, C-6), 117.3 (q, *J* = 4.0 Hz, CH Ar, C-12 or C-14), 119.9 (d, *J* = 280.7 Hz, qCF3), 120.9 (2 CH Ar, C-8 and C-8<sup>0</sup> ), 121.2 (CH Ar, C-16), 128.9 (2 CH Ar, C-9 and C-90 ), 130.8 (qC), 131.1 (CH Ar, C-5 or C-15), 131.7 (CH Ar, C-5 or C-15), 132.6 (d, *J* = 31.9 Hz, qC, C-13), 145.7 (qC), 145.9 (qC), 158.3 (qC), 158.4 (qC), 159.1 (qC). <sup>δ</sup><sup>F</sup> (376 MHz, CD3OD): <sup>−</sup>64.42 (s). <sup>ν</sup>max(ATR)/cm−<sup>1</sup> : 3301 (NH), 3135 (NH), 1665, 1587 (C=N), 1490, 1486, 1335 (C-N), 1216 (C-O), 1161, 1116 (CF3), 1067 (C-Cl), 976, 836, 785, 689. HRMS (*m*/*z* ESI+): found 387.1438 (M<sup>+</sup> + H. C20H18N4OF<sup>3</sup> requires: 387.1433). HPLC: 97.5% (*t*R: 31.7 min).

4.2.11. 1-(4-(3-((3-(Pentafluorosulfanyl)phenyl)amino)phenoxy)phenyl)guanidine hydrochloride (30)

Following Method A (see ESI), **47** (272 mg, 0.42 mmol) was dissolved in 4 M HCl in dioxane (1.27 mL, 5.06 mmol) and in additional dioxane (0.83 mL) until a final concentration of 0.2 M was reached. After 6 h stirring at 55 ◦C, the reaction was adjudged complete (TLC), solvents were evaporated and the residue was purified by silica gel chromatography (CHCl3:MeOH) to afford the pure hydrochloride salt as an orange solid (127 mg, 62%). Mp: 104–106 ◦C. δ<sup>H</sup> (400 MHz, CD3OD): 6.61 (dd, *J* = 8.2, 2.3 Hz, 1H, H-4), 6.78 (t, *J* = 2.2. Hz, 1H, H-2), 6.90 (dd, *J* = 8.1, 2.1 Hz, 1H, H-6), 7.11 (d, *J* = 8.9 Hz, H-8 and H-80 ), 7.22–7.31 (m, 5H, H-9 and H-90 , H-5 or H-15, H-14 and H-16), 7.34–7.38 (m, 1H, H-5 or H-15), 7.44 (t, *J* = 2.2 Hz, 1H, H-12). δ<sup>C</sup> (100 MHz, CD3OD): 109.6 (CH Ar, C-2), 112.9 (CH Ar, C-4), 114.6 (CH Ar, C-6), 114.9 (p, *J* = 4.6 Hz, CH Ar, C-12), 118.0 (p, *J* = 4.7 Hz, CH Ar, C-14), 120.8 (CH Ar, C-16), 120.9 (2 CH Ar, C-8 and C-80 ), 128.8 (2 CH Ar, C-9 and C-90 ), 130.6 (CH Ar, C-5 or C-15), 130.8 (qC), 131.8 (CH Ar, C-5 or C-15), 145.6 (qC), 145.7 (qC), 155.9 (p, *J* = 16.4 Hz, qC, C-13), 158.2 (qC), 158.3 (qC), 159.2 (qC). <sup>δ</sup><sup>F</sup> (376 MHz, CD3OD): <sup>−</sup>64.34 (s). <sup>ν</sup>max(ATR)/cm−<sup>1</sup> : 3273 (NH), 3150 (NH), 1669, 1593 (C=N), 1487 (C-N), 1218 (C-O), 834 (SF5), 567. HRMS (*m*/*z* ESI+): found 445.1124 (M<sup>+</sup> + H. C19H18N4OSF<sup>5</sup> requires: 445.1121). HPLC: 95.4% (*t*R: 32.3 min).

4.2.12. 1-(4-(3-((4-Chloro-3-(trifluormethyl)phenyl)amino)-5-fluorophenoxy)phenyl)guanidine hydrochloride (36)

Following Method A (see ESI), **48** (166 mg, 0.26 mmol) was dissolved in 4 M HCl in dioxane (0.78 mL, 3.12 mmol) and in additional dioxane (0.51 mL) until a final concentration of 0.2 M was reached. After 8 h stirring at 55 ◦C, the reaction was adjudged complete (TLC), solvents were evaporated and the residue was purified by silica gel chromatography (CHCl3:MeOH) to afford the pure hydrochloride salt as a light brown solid (114 mg, 92%). Mp: 92–94 ◦C. δ<sup>H</sup> (600 MHz, CD3OD): 6.31 (dt, *J* = 9.9, 2.1 Hz, 1H, H-4), 6.55 (s, 1H, H-2), 6.58 (dt, *J* = 10.7, 2.0 Hz, 1H, H-6), 7.16 (d, *J* = 8.8 Hz, 2H, H-8 and H-80 ), 7.29 (dd, *J* = 8.8, 2.7 Hz, 1H, H-16), 7.32 (d, *J* = 8.8 Hz, 2H, H-9 and H-90 ), 7.40 (d, *J* = 2.6 Hz, 1H, H-12), 7.43 (d, *J* = 8.7 Hz, 1H, H-15). δ<sup>C</sup> (150 MHz, CD3OD): 99.4 (d, *J* = 25.7 Hz, CH Ar, C-4), 100.3 (d, *J* = 25.5 Hz, CH Ar, C-6), 103.8 (d, *J* = 2.6 Hz, CH Ar, C-2), 117.4 (q, *J* = 5.5 Hz, CH Ar, C-12), 121.8 (2 CH Ar, C-8 and C-80 ), 122.7 (CH Ar, C-16), 123.4 (qC, C-14), 124.3 (d, *J* = 272.2 Hz, qCF3), 128.9 (2 CH Ar, C-9 and C-9<sup>0</sup> ), 129.8 (q, *J* = 31.0, qC, C-13), 131.7 (qC), 133.5 (CH Ar, C-15), 143.4 (qC), 146.7 (d, *J* = 13.2 Hz, qC, C-1 or C-3), 157.1 (qC), 158.4 (qC), 160.7 (d, *J* = 13.7 Hz, qC, C-1 or C-3), 165.8 (d, *<sup>J</sup>* <sup>=</sup> 243.4 Hz, qC, C-5). <sup>δ</sup><sup>F</sup> (376 MHz, CD3OD): <sup>−</sup>64.67 (s), <sup>−</sup>112.53 (s). <sup>ν</sup>max(ATR)/cm−<sup>1</sup> : 3285 (NH), 3139 (NH), 1666, 1601 (C=N), 1504, 1476, 1323 (CF3), 1216 (C-O), 1112 (C-F), 1020 (C-Cl), 994, 823, 660. HRMS (*m*/*z* ESI+): found 439.0945 (M<sup>+</sup> + H. C20H16N4OF4Cl requires: 439.0943). HPLC: 95.7% (*t*R: 33.1 min).

4.2.13. 1-(3,4-Di-fluorophenyl)-3-(3-((6-guanidinopyridin-3-yl)oxy)phenyl)guanidine dihydrochloride (49)

Following Method A (see ESI), **61** (176 mg, 0.25 mmol) was dissolved in 4 M HCl in dioxane (1.14 mL, 4.54 mmol) and in additional dioxane (0.11 mL) until a final concentration of 0.2 M was reached. After 8 h stirring at 55 ◦C, the reaction was adjudged complete (TLC), solvents were evaporated and the residue was purified by silica gel chromatography (CHCl3:MeOH) to afford the pure hydrochloride salt as a white solid (101 mg, 84%). Mp: decomp. above 110 ◦C. δ<sup>H</sup> (400 MHz, CD3OD): 6.62 (dd, *J* = 7.5,

2.4 Hz, 1H, H-4), 6.77–6.81 (m, 1H, H-17), 6.93 (dd, *J* = 8.0, 1.8 Hz, 1H, H-6), 6.99 (d, *J* = 8.9 Hz, 1H, H-9), 7.00–7.13 (m, 3H, H-2, H-13 and H-16), 7.25 (t, *J* = 8.1 Hz, 1H, H-5), 7.49 (dd, *J* = 8.9, 2.9 Hz, 1H, H-8), 8.05 (d, *J* = 2.9 Hz, 1H, H-11). δ<sup>C</sup> (100 MHz, CD3OD): 112.0 (d, *J* = 18.8 Hz, CH Ar, C-13 or C-16), 112.4 (CH Ar, C-2), 113.0 (CH Ar, C-4), 116.4 (CH Ar, C-9), 118.0 (CH Ar, C-6), 118.1 (d, *J* = 17.9 Hz, CH Ar, C-13 or C-16), 119.1 (dd, *J* = 5.6, 3.1 Hz, CH Ar, C-17), 131.3 (CH Ar, C-5), 131.4 (CH Ar, C-8), 138.5 (CH Ar, C-11), 143.9 (dd, *J* = 7.4, 2.2 Hz, qC, C-12), 146.6 (qC), 147.2 (dd, *J* = 240.4, 12.9 Hz, qC, C-14 or C-15), 150.1 (qC), 151.1 (qC), 151.4 (dd, *J* = 245.1, 13.4 Hz, qC, C-14 or C-15), 152.5 (qC), 157.4 (qC), 159.0 (qC). <sup>δ</sup><sup>F</sup> (376 MHz, CD3OD): <sup>−</sup>139.72 (m), <sup>−</sup>149.19 (m). <sup>ν</sup>max(ATR)/cm−<sup>1</sup> : 3313 (NH), 3154 (NH), 2922, 2861, 1682, 1625 (C=N), 1507, 1473 (C-F), 1375, 1228 (C-F), 1166, 1146 (C-O), 866, 830, 770, 570, 557. HRMS (*m*/*z* ESI+): found 398.1548 (M<sup>+</sup> + H. C19H18N7OF<sup>2</sup> requires: 398.1541). HPLC: 99.8% (*t*R: 23.6 min).

4.2.14. 1-(2-Fluoro-4-iodophenyl)-3-(3-((6-guanidinopyridin-3-yl)oxy)phenyl)guanidine dihydrochloride (50)

Following Method A (see ESI), **62** (104 mg, 0.13 mmol) was dissolved in 4 M HCl in dioxane (0.58 mL, 2.32 mmol) and in additional dioxane (0.10 mL) until a final concentration of 0.2 M was reached. After 8 h stirring at 55 ◦C, the reaction was adjudged complete (TLC), solvents were evaporated and the residue was purified by silica gel chromatography (CHCl3:MeOH) to afford the pure hydrochloride salt as a white solid (67 mg, 89%). Mp: decomp. above 120 ◦C. δ<sup>H</sup> (400 MHz, CD3OD): 6.98 (ddd, *J* = 8.3, 2.4, 0.7 Hz, 1H, H-4), 7.02 (t, *J* = 2.2 Hz, 1H, H-2), 7.11–7.18 (m, 3H, H-5 or H-16, H-6 and H-9), 7.46 (t, *J* = 8.1 Hz, 1H, H-5 or H-16), 7.61–7.64 (m, 2H, H-17 and H-8), 7.67 (dd, *J* = 9.7, 1.8 Hz, 1H, H-14), 8.16 (d, *J* = 2.9 Hz, 1H, H-11). δ<sup>C</sup> (100 MHz, CD3OD): 92.7 (qC, C-15), 115.5 (CH Ar, C-2), 115.6 (CH Ar, C-9), 117.6 (CH Ar, C-4), 120.9 (CH Ar, C-6), 127.2 (d, *J* = 22.2 Hz, CH Ar, C-14), 127.3 (d, *J* = 14.1 Hz, qC, C-12), 130.6 (CH Ar, C-5 or C-16), 132.1 (CH Ar, C-8), 132.5 (CH Ar, C-5 or C-16), 136.0 (CH Ar, *J* = 3.9 Hz, C-17), 138.7 (qC), 139.1 (CH Ar, C-11), 149.2 (qC), 151.2 (qC), 156.2 (qC), 156.9 (qC), 157.9 (qAr, *J* = 253.9 Hz, C-13), 159.4 (qC). δ<sup>F</sup> (376 MHz CD3OD):—121.55 (t, *J* = 8.3 Hz). νmax(ATR)/cm−<sup>1</sup> : 3277 (NH), 3122 (NH), 2923, 2849, 1680, 1660, 1623–1570 (C=N), 1474 (C-F), 1227 (C-O), 1160, 1026, 945, 600 (C-I). HRMS (*m*/*z* ESI+): found 506.0609 (M<sup>+</sup> + H. C19H18N7OFI requires: 506.0602). HPLC: 98.1% (*t*R: 25.7 min).

### 4.2.15. 1-(4-Bromophenyl)-3-(3-((6-guanidinopyridin-3-yl)oxy)phenyl)guanidine dihydrochloride (51)

Following Method A (see ESI), **63** (184 mg, 0.24 mmol) was dissolved in 4 M HCl in dioxane (1.08 mL, 4.4 mmol) and in additional dioxane (0.12 mL) until a final concentration of 0.2 M was reached. After 8 h stirring at 55 ◦C, the reaction was adjudged complete (TLC), solvents were evaporated and the residue was purified by silica gel chromatography (CHCl3:MeOH) to afford the pure hydrochloride salt as a white solid (113 mg, 92%). Mp: decomp. above 124 ◦C. δ<sup>H</sup> (400 MHz, CD3OD): 6.99–7.01 (m, 1H, H-4), 7.04 (bs, 1H, H-2), 7.14–7.16 (m, 2H, H-6 and H-9), 7.28 (d, *J* = 8.5 Hz, 2H, H-13 and H-130 or H-14 and H-140 ), 7.48 (t, *J* = 8.1 Hz, 1H, H-5), 7.60–7.63 (m, 3H, H-13 and H-130 or H-14 and H-140and H-8), 8.16 (d, *J* = 2.6 Hz, 1H, H-11). δ<sup>C</sup> (100 MHz, CD3OD): 115.6 (CH Ar, C-6 or C-9), 115.8 (CH Ar, C-2), 118.0 (CH Ar, C-4), 121.1 (CH Ar, C-6 or C-9), 121.7 (qC, C-15), 127.9 (CH Ar, C-13 and C-130 or C-14 and C-140 ), 132.1 (CH Ar, C-8), 132.5 (CH Ar, C-5), 134.1 (CH Ar, C-13 and C-130 or C-14 and C-140 ), 135.7 (qC), 138.2 (qC), 139.0 (CH Ar, C-11), 149.2 (qC), 151.2 (qC), 156.1 (qC), 156.8 (qC), 159.4 (qC). νmax(ATR)/cm−<sup>1</sup> : 3256 (NH), 3114 (NH), 2971, 1680, 1660, 1619 (C=N), 1566 (C=N), 1474, 1376 (C-N), 1226 (C-O), 1069 (C-Br), 1010, 832, 637—584. HRMS (*m*/*z* ESI+): found 440.0837 (M<sup>+</sup> + H. C19H19N7OBr requires: 440.0834). HPLC: 97.5% (*t*R: 25.2 min).

4.2.16. 1-(4-Bromo-3-(trifluoromethyl)phenyl)-3-(3-((6-guanidinopyridin-3-yl)oxy)phenyl) guanidine dihydrochloride (52)

Following Method A (see ESI), **64** (358 mg, 0.44 mmol) was dissolved in 4 M HCl in dioxane (2 mL, 7.97 mmol) and in additional dioxane (0.2 mL) until a final concentration of 0.2 M was reached. After 8 h stirring at 55 ◦C, the reaction was adjudged complete (TLC), solvents were evaporated and the residue was purified by silica gel chromatography (CHCl3:MeOH) to afford the pure hydrochloride salt as a white solid (225 mg, 88%). Mp: decomp. above 170 ◦C. δ<sup>H</sup> (400 MHz, CD3OD): 6.66 (dd, *J* = 8.1, 2.4 Hz, 1H, H-4), 6.96 (dd, *J* = 8.0, 1.9 Hz, 1H, H-6), 7.04 (d, *J* = 8.9 Hz, 1H, H-9), 7.08 (t, *J* = 2.1 Hz, 1H, H-2), 7.20 (dd, *J* = 8.6, 2.5 Hz, 1H, H-17), 7.28 (t, *J* = 8.1 Hz, 1H, H-5), 7.48 (d, *J* = 2.5, 1H, H-13), 7.56 (dd, *J* = 8.9, 2.9 Hz, 1H, H-8), 7.60 (d, *J* = 8.6 Hz, 1H, H-16), 8.10 (d, *J* = 2.7 Hz, 1H, H-11). δ<sup>C</sup> (100 MHz, CD3OD): 111.8 (d, *J* = 1.8 Hz, qC, C-15), 112.4 (CH Ar, C-2), 113.3 (CH Ar, C-4), 115.5 (CH Ar, C-9), 117.8 (CH Ar, C-6), 122.4 (q, *J* = 5.5 Hz, CH Ar, C-13), 124.4 (d, *J* = 272.6 Hz, qCF3), 127.7 (CH Ar, C-17), 130.9 (d, *J* = 30.8 Hz, qC, C-14), 131.4 (CH Ar, C-5), 131.6 (CH Ar, C-8), 136.6 (CH Ar, C-16), 138.6 (CH Ar, C-11), 146.1 (qC), 147.2 (qC), 148.9 (qC), 151.8 (qC), 152.4 (qC), 156.9 (qC), 159.0 (qC). δ<sup>F</sup> (376 MHz, CD3OD):—63.96 (s). νmax(ATR)/cm−<sup>1</sup> : 3281 (NH), 3142 (NH), 2922, 2849, 1680, 1625 (C=N), 1566 (C=N), 1473, 1319, 1227 (CF3), 1129 (C-Br), 1023, 832, 592-583. HRMS (*m*/*z* ESI+): found 508.0710 (M<sup>+</sup> + H. C20H18N7OF3Br requires: 508.0708). HPLC: 99.8% (*t*R: 27.5 min).

4.2.17. 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(3-((6-guanidinopyridin-3-yl)oxy)phenyl) guanidine dihydrochloride (2)

Following Method A (see ESI), **65** (113 mg, 0.15 mmol) was dissolved in 4 M HCl in dioxane (0.67 mL, 2.70 mmol) and in additional dioxane (0.10 mL) until a final concentration of 0.2 M was reached. After 6 h stirring at 55 ◦C, the reaction was adjudged complete (TLC), solvents were evaporated and the residue was purified by flash chromatography to afford the pure hydrochloride salt as a white solid (63 mg, 79%). Mp: 169–171 ◦C. δ<sup>H</sup> (400 MHz, CD3OD): 6.93 (dd, *J* = 8.3, 2.3 Hz, 1H, H-4), 7.06 (t, *J* = 2.1 Hz, 1H, H-2), 7.11–7.13 (m, 2H, H-6 and H-9), 7.44 (t, *J* = 8.2 Hz, 1H, H-5), 7.53 (dd, *J* = 8.6, 2.5 Hz, 1H, H-17), 7.60–7.65 (m, 2H, H-16 and H-8), 7.69 (d, *J* = 2.4 Hz, 1H, H-13), 8.15 (d, *J* = 2.9 Hz, 1H, H-11). δ<sup>C</sup> (100 MHz, CD3OD): 115.1 (CH Ar, C-2), 115.6 (CH Ar, C-9), 117.1 (CH Ar, C-4), 120.5 (CH Ar, C-6), 123.9 (d, *J* = 272.5 Hz, qCF3) 124.5 (q, *J* = 5.5 Hz, CH Ar, C-13), 129.9 (qC, C-15), 130.1 (CH Ar, C-17), 130.2 (d, *J* = 34.7 Hz, qC, C-14), 132.0 (CH Ar, C-8 or C-16), 132.3 (CH Ar, C-5), 134.0 (CH Ar, C-8 or C-16), 138.3 (qC), 139.0 (CH Ar, C-11), 139.7 (qC), 149.1 (qC), 151.3 (qC), 155.5 (qC), 156.9 (qC), 159.3 (qC). δ<sup>F</sup> (376 MHz, CD3OD):—64.24 (s). νmax(ATR)/cm−<sup>1</sup> : 3297 (NH), 3121 (NH), 2923, 2854, 1625 (C=N), 1581 (C=N), 1474 (CF3), 1320, 1227 (C-O), 1130 (C-Cl), 1032, 832, 589, 557. HRMS (*m*/*z* ESI+): found: 464.1222 (M<sup>+</sup> + H. C20H18N7OF3Cl requires: 464.1213). HPLC: 96.9% (*t*R: 26.8 min).

4.2.18. 1-(5-(3-((4-Chloro-3-(trifluoromethyl)phenyl)amino)phenoxy)pyridin-2-yl)guanidine hydrochloride (4)

Following Method A (see ESI), **69** (200 mg, 0.32 mmol) was dissolved in 4 M HCl in dioxane (0.96 mL, 3.86 mmol) and in additional dioxane (0.65 mL) until a final concentration of 0.2 M was reached. After 8 h stirring at 55 ◦C, the reaction was adjudged complete (TLC), solvents were evaporated and the residue was purified by silica gel chromatography (CHCl3:MeOH) to afford the pure hydrochloride salt as a white solid (136 mg, 93%). Mp: 89–91 ◦C. δ<sup>H</sup> (400 MHz, CD3OD): 6.60 (dd, *J* = 8.2, 2.3 Hz, 1H, H-4), 6.75 (t, *J* = 2.2 Hz,1H, H-2), 6.90 (dd, *J* = 8.1, 2.0 Hz, 1H, H-6), 7.08 (d, *J* = 8.9 Hz, 1H, H-9), 7.24 (dd, *J* = 8.7, 2.7 Hz, 1H, H-17), 7.29 (t, *J* = 8.1 Hz, 1H, H-5), 7.37–7.39 (m, 2H, H-13 and H-16), 7.59 (dd, *J* = 8.9, 2.9 Hz, 1H, H-8), 8.13 (d, *J* = 2.9 Hz, 1H, H-11). δ<sup>C</sup> (100 MHz, CD3OD): 108.7 (CH Ar, C-2), 112.2 (CH Ar, C-4), 114.7 (CH Ar, C-6), 115.5 (CH Ar, C-9), 116.2 (q, *J* = 5.5 Hz, CH Ar, C-13), 121.8 (CH Ar, C-17), 122.4 (qC, C-15), 124.3 (d, *J* = 272.4 Hz, qCF3), 129.6 (d, *J* = 31.0 Hz, qC, C-14), 131.8 (CH Ar, C-8), 132.0 (CH Ar, C-5), 133.4 (CH Ar, C-16), 138.7 (CH Ar, C-11), 144.2 (qC), 145.6 (qC), 148.8 (qC), 151.8 (qC), 156.9 (qC), 159.4 (qC). δ<sup>F</sup> (376 MHz, CD3OD):—64.15 (s). νmax(ATR)/cm−<sup>1</sup> : 3264 (NH), 2923, 2863, 1684, 1629, 1595, 1474 (C=N), 1400, 1332, 1229 (C-O), 1129 (CF3), 1115 (C-Cl), 977, 998. HRMS (*m*/*z* ESI+): found 422.0987 (M<sup>+</sup> + H. C19H16N5OClF<sup>3</sup> requires: 422.0995). HPLC: 98.6% (*t*R: 33.3 min).

### 4.2.19. 4-(3-((4-Chloro-3-(trifluoromethyl)phenyl)amino)phenoxy)phenyl carbamimidate hydrochloride (70)

To a stirred solution of **74** (205 mg, 0.33 mmol, 1eq) in EtOAc was added SnCl<sup>4</sup> (0.15 mL, 1.32 mmol, 4 eq). After 2 h of stirring at room temperature, the solvent and the excess of SnCl<sup>4</sup> were evaporated in vacuo. The remaining liquid was purified by silica gel chromatography(CHCl3:Acetone) to afford the pure hydrochloride salt (130 mg, 86%) as a colourless gum. δ<sup>H</sup> (400 MHz, DMSO-*d6*): 6.61 (dd, *J* = 7.6, 2.3 Hz, 1H, H-4), 6.76 (t, *J* = 2.2 Hz, 1H, H-2), 6.92 (dd, *J* = 7.8, 1.7 Hz, 1H, H-6), 7.16 (d, *J* = 9.1 Hz, 2H, H-8 and H-80 or H-9 and H-90 ), 7.30–7.34 (m, 2H, H-5 and H-16), 7.37 (d, *J* = 9.0 Hz, 2H, H-8 and H-80 or H-9 and H-90 ), 7.40 (d, *J* = 2.7 Hz, 1H, H-12), 7.50 (d, *J* = 8.8 Hz, 1H, H-15), 8.61 (bs, 4H, NH), 8.88 (bs, NH). δ<sup>C</sup> (100 MHz, DMSO-*d6*): 107.8 (CH Ar, C-2), 111.2 (CH Ar, C-4), 113.1 (CH Ar, C-6), 114.7 (q, *J* = 5.5 Hz, CH Ar, C-12), 119.4 (qC, C-14), 120.4 (CH Ar, C-5 or C-16), 120.5 (2 CH Ar, C-8 and C-80 or C-9 and C-90 ), 122.0 (d, *J* = 227.5 Hz, qCF3), 123.1 (2 CH Ar, C-8 and C-80 or C-9 and C-90 ), 127.2 (d, *J* = 30.7 Hz, qC, C-13), 130.9 (CH Ar, C-5 or C-16), 132.7 (CH Ar, C-15), 142.8 (qC), 143.5 (qC), 145.2 (qC), 155.2 (qC), 157.6 (qC), 161.1 (qC). δ<sup>F</sup> (376 MHz, CD3OD):—61.59 (s). νmax(ATR)/cm−<sup>1</sup> : 3285 (NH), 2924, 2854, 1693, 1655, 1593 (C=N), 1481 (C-N), 1400, 1258, 1231, 1195, 1175 (C-O), 1128 (CF3), 1111 (C-Cl), 1027, 824, 681, 665. HRMS (*m*/*z* ESI+): found: 422.0883 (M<sup>+</sup> + H. C20H16N3O2ClF<sup>3</sup> requires: 422.0883). HPLC: 96.2% (*t*R: 32.2 min).

### 4.2.20. 4'-Sulfonamide-3-[4-chloro-3 trifluoromethylphenylamino]diphenylether (75)

Compound **40** (100 mg, 0.26 mmol, 1 eq.), sulfamoyl chloride (30 mg, 0.26 mmol, 1 eq.) and NEt<sup>3</sup> (0.05 mL, 0.29 mmol, 1.1 eq.) were dissolved in CH2Cl<sup>2</sup> (2 mL) and stirred at overnight at room temperature. The mixture was then washed with water and the organic layer extracted with EtOAc, washed with brine, dried over MgSO4, concentrated under vacuum and purified by silica gel chromatography (hexanes:EtOAc) to get **75** as a light brown solid (95 mg, 80%). Mp: 124–126 ◦C. δ<sup>H</sup> (400 MHz, CD3OD): 6.54 (dd, *J* = 8.2, 2.3 Hz, 1H, H-4), 6.69 (t, *J* = 2.2 Hz, 1H, H-2), 6.84 (dd, *J* = 8.1, 2.1 Hz, 1H, H-6), 6.99 (d, *J* = 9.0 Hz, 2H, H-8 and H-80 ), 7.21–7.26 (m, 4H, H-9 and H-90 , H-16, H-5), 7.34–7.38 (m, 2H, H-12, H-15). δ<sup>C</sup> (100 MHz, CD3OD): 109.1 (CH Ar, C-2), 112.2 (CH Ar, C-4), 113.8 (CH Ar, C-6), 116.1 (q, *J* = 5.5 Hz, C-12, CH Ar), 121.1 (2 CH Ar, C-8 and C-80 ), 121.2 (CH Ar, C-16), 121.9 (qC, C-14), 123.2 (2 CH Ar, C-9 and C-90 ), 124.4 (d, *J* = 272.5 Hz, qCF3), 129.7 (d, *J* = 30.9 Hz, qC, C-13), 131.5 (CH Ar, C-5), 133.3 (CH Ar, C-15), 136.1 (qC), 144.6 (qC), 145.1 (qC), 154.4 (qC), 160.5 (qC). δ<sup>F</sup> (376 MHz, CD3OD):—64.19 (s). νmax (ATR)/cm−<sup>1</sup> : 3404 (NH), 3279 (NH), 1596 (S=O), 1489 (S=O), 1143 (C-O), 1153 (CF3), 1125 (C-N), 830 (C-Cl), 821. HRMS (*m*/*z* ESI−): found: 456.0397 (M−—H. C19H14N3O3SClF<sup>3</sup> requires: 456.0397). HPLC: 99.3% (*t*R: 35.4 min).

### *4.3. Biochemistry*

### 4.3.1. Cell Viability Studies (alamarBlue)

Cells were counted and seeded in 96-well plates at a density of 2 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL for HL-60, 2.5 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/mL for MCF-7, MCF10A and HeLa, 1 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL for HCT116 and HKH-2, all of them in their respective media. The 96-well plates were then treated with a 1:100 dilution of stock concentrations of drugs or EtOH (1% *v*/*v*)/DMSO (0.1% *v*/*v*) as vehicle control in triplicate. Three blank wells containing 200 µL RPMI with no cells were also set-up as blanks. After a 72 h incubation, 20 µL of alamarBlue was added to each well. The plates were incubated in darkness at 37 ◦C for 4–5 h using a Molecular Devices microplate reader, the fluorescence (F) was then read at an excitation wavelength of 544 nm and an emission wavelength of 590 nm. Cell viability was then determined by subtracting the mean blank fluorescence (Fb) from the treated sample fluorescence (Fs) and expressing this as a percentage of the fluorescence of the blanked vehicle control (Fc). This is demonstrated in the equation below. The results were then plotted as a nonlinear regression, sigmoidal dose-response curves on Prism, from which the IC<sup>50</sup> value for each drug was determined.

### 4.3.2. Flow Cytometry

Apoptosis was analysed using annexin V fluorescein isothiocyanate (FITC) and propidium iodide (PI). HL-60 cells were seeded at a density of 2 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL in 12 well plates. Cells were then treated with either vehicle (0.5% ethanol), **2** (5 µM), **3** (5 µM), or **4** (4 µM) for 48 h. Following treatment, HL60 cells were collected and washed with annexin V binding buffer (5 mM HEPES, 70 mM NaCl, 1.25 mM CaCl2 pH 7.4) and stained with annexin V-FITC (iQ Corporation, Groningen, The Netherlands) for 20 min. Following washing with annexin V binding buffer, cells were resuspended in PI (0.5 µg/mL) in binding buffer and analysed on BD FACS Canto II flow cytometer (BD Sciences) using FloJo software (Ashland, OR, USA).

### 4.3.3. Western Blotting

HL60s were seeded in T25 flasks at 50 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/mL and cells were treated with either vehicle [0.5% EtOH (*v*/*v*)], **1**–**4**, **9** or **52** (5 µM), as well as **1** (10 µM as reported [**?** ]). After 16 h, cells were collected and washed with PBS. Cell pellets were re-suspended in cold cell lysis buffer (radio-immunoprecipitation assay buffer) supplemented with 1% phosphatase inhibitor cocktail 2 and 3 (Sigma) and 10% protease inhibitor (Roche). Cells were lysed for 30 min on ice. Protein concentration was then determined by BCA assay. Lysates were boiled with Laemmli sample buffer [Tris-HCL 50 mM (pH 6.7), glycerol 10% (*w*/*v*), sodium dodecyl sulphate 2% (*w*/*v*), bromophenol blue 0.02% (*w*/*v*)] containing DTT 50 µM for 10 min at 90 ◦C. Moreover, 20 µg of lysates were resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF transfer membrane (EMD Millipore). Membranes were blocked with 5% non-fat milk and probed with primary antibodies for ERK and phospho-ERK (Cell Signalling). Anti-GAPDH was used as a loading control (Calbiochem).

### **5. Conclusions**

Taking all of these results into account, the following SARs were drawn in terms of HL-60 cytotoxicity; for example, for a good cytotoxicity bulky substituents (4-Br/Cl and 3-CF3) in the phenyl ring of the hydrophobic moiety are needed; replacement of the di-substituted guanidinium as in compound **1** by a shorter -NH- link is also beneficial; a mono-substituted guanidinium group at the position 40 of the phenyloxyaryl core gives good cytotoxic activity; additionally, substituting one of the phenyl rings by a 2-pyridinyl to facilitate IMHB seems also to increase the cytotoxic activity in the 3,40 -bis-guanidinium series. On the negative side, in the bis-guanidinium diphenyl ether series, when only one substituent is kept in the phenyl ring of the hydrophobic moiety (4-Br or 4-F as in **8** or **5**) or both are very small (3,4-diF as in **6**), cytotoxicity decreases or is completely abolished. This is not the case either in the amino-guanidinium diphenyl ether or in the bis-guanidinium phenyloxypyridine series where mono-substituted or di-fluoro phenyl rings in the hydrophobic moiety still exhibit good HL-60 cytotoxicity (i.e., compounds **28**–**30** or **49** and **51**).

Future work will be required to investigate the molecular target(s) of our guanidinium derivatives, but nonetheless, while compound **2**, **9,** and **52** seem to be improved derivatives of previous *lead* molecule **1**, compounds **3** and **4** can be considered excellent *hit* molecules in the search for new anticancer therapies.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1424-8247/13/12/485/s1, synthetic details (general information, general procedures, characterisation of intermediates); computational details (Figure S1, Figure S2, Figure S3, Figure S4 and Figure S5); theoretical physicochemical and pharmacokinetic parameters (Table S1, Table S2, and Table S3, Figure S6); biochemical protocols (Figure S7, Figure S8, Figure S9, Figure S10, Figure S11, Figure S12, Figure S13); NMR spectra of final salts; and HPLC chromatograms of final salts.

**Author Contributions:** Conceptualization, V.P., A.M.M., D.M.Z., and I.R.; Formal analysis, V.P., H.B.M., R.A., and I.R.; Funding acquisition, A.M.M., D.M.Z., and I.R.; Investigation, V.P., H.B.M., and R.A.; Methodology, V.P.; Supervision, A.M.M., D.M.Z., and I.R.; Writing—original draft, V.P., and I.R.; Writing—review and editing, V.P., H.B.M., R.A., A.M.M., D.M.Z., and I.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the School of Chemistry at Trinity College Dublin (V.P.), the Irish Research Council (GOIPG/2017/834, H.B.M.), and the John Scott PhD fellowship from the School of Biochemistry & Immunology and the School of Medicine at Trinity College Dublin (R.A.).

**Acknowledgments:** Thanks are given to the Irish Centre for High-End Computing (ICHEC) for the provision of computational facilities.

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

### **References**


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