9 Inhibitors with Potent

Article Cite This: J. Med. Chem. 2017, 60, 9470−9489

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Design of Novel 3‑Pyrimidinylazaindole CDK2/9 Inhibitors with Potent In Vitro and In Vivo Antitumor Efficacy in a Triple-Negative Breast Cancer Model Umed Singh,† Gousia Chashoo,¶,Δ Sameer U. Khan,¶,Δ Priya Mahajan,⊥ Amit Nargotra,⊥ Girish Mahajan,¶ Amarinder Singh,‡ Anjna Sharma,‡ Mubashir J. Mintoo,¶ Santosh Kumar Guru,¶ Hariprasad Aruri,† Thanusha Thatikonda,† Promod Sahu,‡ Pankaj Chibber,‡ Vikas Kumar,§ Sameer A. Mir,¶ Sonali S. Bharate,§ Sreedhar Madishetti,‡ Utpal Nandi,‡ Gurdarshan Singh,‡ Dilip Manikrao Mondhe,¶ Shashi Bhushan,¶,∥ Fayaz Malik,*,¶ Serge Mignani,#,† Ram A. Vishwakarma,† and Parvinder Pal Singh*,† †

Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Academy of Scientific and Innovative Research, Canal Road, Jammu, Jammu & Kashmir-180001, India ¶ Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Academy of Scientific and Innovative Research, Canal Road, Jammu, Jammu & Kashmir-180001, India ⊥ Discovery Informatics, CSIR-Indian Institute of Integrative Medicine, Academy of Scientific and Innovative Research, Canal Road, Jammu, Jammu & Kashmir-180001, India ‡ Pharmacokinetic & Pharmacodynamic Division, CSIR-Indian Institute of Integrative Medicine, Academy of Scientific and Innovative Research, Canal Road, Jammu, Jammu & Kashmir-180001, India § Preformulation Division, CSIR-Indian Institute of Integrative Medicine, Academy of Scientific and Innovative Research, Canal Road, Jammu, Jammu & Kashmir-180001, India ∥ Indian Pharmacopoeia Commission, Sector-23, Raj Nagar, Ghaziabad-201002, India # PRES Sorbonne Paris Cité, CNRS UMR 860, Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologique, Université Paris Descartes, 45, rue des Saints Péres, 75006 Paris, France S Supporting Information *

ABSTRACT: In the present study, a novel series of 3pyrimidinylazaindoles were designed and synthesized using a bioinformatics strategy as cyclin-dependent kinases CDK2 and CDK9 inhibitors, which play critical roles in the cell cycle control and regulation of cell transcription. The present approach gives new dimensions to the existing SAR and opens a new opportunity for the lead optimizations from comparatively inexpensive starting materials. The study led to the identification of the alternative lead candidate 4ab with a nanomolar potency against CDK2 and CDK9 and potent antiproliferative activities against a panel of tested tumor cell lines along with a better safety ratio of ∼33 in comparison to reported leads. In addition, the identified lead 4ab demonstrated a good solubility and an acceptable in vivo PK profile. The identified lead 4ab showed an in vivo efficacy in mouse triple-negative breast cancer (TNBC) syngeneic models with a TGI (tumor growth inhibition) of 90% without any mortality growth inhibition in comparison to reported leads.

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INTRODUCTION Fundamentally, our understanding of biological processes has opened up new opportunities to tackle diseases and the recent advancement highlighting the kinases as the most promising targets for therapeutic intervention in various therapeutic domains. Out of the 518 kinases, cyclin-dependent kinases (CDKs) have attracted major interest, despite the complexity of their roles, as they regulate the cell division, apoptosis, transcription, and differentiation and as they are found to © 2017 American Chemical Society

deregulate in a number of pathological conditions such as a wide variety of human cancer, neurodegenerative disorders, inflammation, and renal and infectious diseases including malaria.1−3 These CDKs form a CDK/cyclin complex for their function, and the human genome encodes 21 CDKs (1− 11a and 11b−20) and over 15 cyclins (A−L, O, T, and Y), Received: May 11, 2017 Published: November 16, 2017 9470

DOI: 10.1021/acs.jmedchem.7b00663 J. Med. Chem. 2017, 60, 9470−9489

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Figure 1. Literature precedent and present strategy.

first orally active CDK inhibitor R-roscovitine was also reported to perform its function through a transcriptional CDK.4,22 During the past decade, attempts have been made toward the exploitation of transcriptional CDKs for anticancer potential. Intensive attempts are going on to explore diverse scaffolds (pyrimidines, pyrazole, pyridines, phenyl triazines, and fused pyrazolopyrimidine) for the search of CDK9-based inhibitors.23 Therefore, molecules capable of inhibiting cell cycle and transcriptional CDKs may provide superior anticancer efficacy.24 In this direction, 3-pyrimidinylazaindole derivatives, namely, meriolins, represent the hybrid structure of two natural product-based CDK inhibitors, namely, variolins and meridianins (both coming from marine natural sources), known for inhibiting both cell cycle (CDK2) and transcriptional (CDK9) CDKs (Figure 1). In addition, meriolins also possess a potent activity in cell-based assays against several cancer cell lines. The in vivo efficacy of meriolins was also established, where meriolin 3 significantly inhibited the tumor growth in two mice xenograft cancer models such as Ewings sarcoma and LS174T colorectal carcinoma.25−27 Despite the nanomolar potency in vitro and good in vivo efficacy of meriolins, they have poor physicochemical and PK properties, which consequently limit further clinical development. On the basis of these data, we designed and synthesized novel 3-pyrimidinylazaindole CDK2/9 inhibitors with potent in vitro and in vivo antitumor efficacy such as in a triple-negative breast cancer (TNBC) model, which is responsible for a large proportion of breast cancer deaths due to its generally aggressive clinical course.28,29 Therefore, we have started a medicinal chemistry study on the 3-pyrimidinylazaindole skeleton (4, Figure 1) to discover new generation analogues that have potency against both cell cycle and transcriptional CDKs along with better physicochemical and PK properties.

which according to their function are further differentiated into cell cycle regulation CDKs and transcription CDKs (also known as RNA processing).3 The CDKs involved in the cell cycle regulation are 1, 2, 3, 4, and 6, and their cyclins are A, B, D, and E, respectively.3 However, the CDKs involved in transcriptional regulation are 7, 8, 9, and 11, and their cyclins are C, H, L, and T, respectively.1,2 Deregulation in the CDKs is found in a number of malignancies, and therefore, a number of small molecule CDK inhibitors were designed and reported. In the last 20 years, CDK inhibitors have been investigated for their anticancer potential. In the initial phase, most of the candidates are pan-CDK inhibitors, and those are roscovitine,4 olomucine,5 flavopiridol (Alvocidib),2 and the flavone derivative P276-00.6 Among these, only one candidate, flavopiridol, has been approved, and it too has an orphan status. The poor selectivity, lack of understanding of their mechanism of action, and narrow therapeutic window restrict their entry into market.2 The boom of designing selective inhibitors also prevailed in this domain. Several selective inhibitors have been reported in the recent past, and most of them have been found to be an inhibitor of CDK4 and CDK6. The successful candidates are Pfizer’s palbociclib,7,8 Lilly’s abemaciclib,9 and Novartis’ ribociclib.10 The reason for their success is based on the fact that CDK4 and CDK6 regulate tumor-suppressor retinoblastoma proteins (Rb), which is found to be critical for cell duplication and deregulation in most of the cancer types.10 Palbociclib11 represents the first CDK inhibitor approved by the U.S. FDA,12 while abemaciclib9 got a fast tract approval and ribociclib got approval for priority review after the phase III trial.13 Recently, the FDA approved ribociclib in combination with aromatase inhibitors for the first-line treatment of advanced breast cancer.28 However, recent studies reported that cancer and embryonic cell lines lacking CDK2, CDK4, and CDK6 proliferate normally, suggesting that they are functionally redundant and that their function can be controlled by other cell cycle kinases.14,15 However, inhibiting transcriptional CDKs is also gaining significant interest for effective anticancer therapy because of their role in controlling short-lived mitotic regulatory kinases and apoptosis regulators.16−18 Among the transcriptional CDKs, CDK9 has also been the focus of many groups.16,17,21,23 Moreover, efforts toward understanding the mechanism of the first CDK-based clinical candidate flavopiridol revealed that the CDK9-mediated pathway is the primary mechanism responsible for its anticancer activity such as in chronic lymphocytic leukemia.19−21 In addition to this, the

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RESULTS AND DISCUSSION Design and Chemistry. A medicinal chemistry program was initiated with an aim to identify the sites for modification, which should not hamper the potency of CDKs, used for improving physicochemical and pharmacokinetic properties. The inhibition of CDKs requires 7-azaindole as the key pharmacophore, which involves N1 and N7 in the interaction with the hinge residues, i.e., Leu83, Glu81 in CDK2 and Cys106, Asp104 in CDK9, and the best analogue among them is substituted 3-pyrimidinylazaindole. As per our strategy, an in silico approach was followed to design inhibitors of CDK2 and CDK9, which are majorly involved in cell cycle regulation and

9471

DOI: 10.1021/acs.jmedchem.7b00663 J. Med. Chem. 2017, 60, 9470−9489

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Figure 2. Binding pocket analysis of CDK2 and CDK9 active and potential sites of modifications of 3-pyrimidinylazaindole.

transformations. Thus, as shown in Scheme 1, the synthesis of ring A analogues began with a simple Suzuki cross-coupling reaction between 5-bromo-7-azaindole 5 and heteroaryl/aryl boronic acids (6a−s),34,35 which gave 5-aryl/hetroaryl substituted 7-azaindoles (7a−s) in good yields (60−95%). Iodination of 7a−s in the presence of molecular iodine gave 8a−s, which were further treated with Boc anhydride to synthesize 9a−s in good yields (90−97%). Next, the Masuda borylation reaction on 9a−s generated key intermediates 10a−s (80−94%), which were coupled with 4-chloro-2-(amino)pyrimidine 11 under Suzuki conditions to give the final targeted compounds 4aa−4as (39−65%). Approach 2: Design and Synthesis of 4b. The molecular docking studies suggested that modification at the amino group (2′-position) of ring C is feasible. Taking this information into account, the synthesis of ring C analogues was made by incorporating 2-aryl/hetroarylethane amines in place of the amino group of ring C (Scheme 2). 7-Azaindole 12 was used as a starting material and was treated with iodine under basic conditions to generate 3-iodo-7-azaindole 13 (95%) followed by the protection of the NH of azoles with Boc anhydride to generate N-Boc-3-iodo-7-azaindole 14 (98%). Then the regioselective Masuda borylation of 14 gave boronate ester 15 in 80% yield. Next, the Suzuki coupling reaction was performed between boronate ester 15 and 4-chloro-2(methylthio)pyrimidine 16 affording 3-pyrimidinyl-7-azaindole 17 with a thiomethyl group at the 2′-position in 66% yield. The oxidation of the thiomethyl group of 17 was performed with meta-chloroperbenzoic acid (m-CPBA) to generate sulfone 18 in 90% yield. The generated sulfones 18 were then treated with different 2-aryl/heteroarylethane amines 19 to furnish the target compounds 4ba−4bd in 50−68% yields.36 In addition to this, several secondary amines (20) were also introduced, and the corresponding analogues 4be−4bh were generated in 52− 60% yields, which showed the importance of the H-bond-donor group on the biochemical assays. Approach 3: Design and Synthesis of 4c. As shown in Scheme 3, in order to know the effect of the disruption of hinge

transcription, respectively. The targeted kinase domain of CDK2 and CDK9 shares a similar secondary structure; the active site is present in between the two lobes, the upper small lobe constitutes mainly helices and the lower larger lobe comprises β-sheets. These two proteins share 40% sequence identity calculated via the Omega 3 online database.30 All of the in silico studies of the 3-pyrimidinylazaindole derivative (meriolin 3, most potent derivative) with CDK2/cyclin A and CDK9/cyclin T were carried out using the 2015 Schrodinger suite. Binding pocket analysis of the standard molecule (meriolin 3) with respect to CDK2 (PDB Id: 3BHT)26 and CDK9 (PDB Id: 4IMY)31 infers that there is a scope of modification such as the introduction of a hydrogen bond acceptor, hydrogen bond donor, and hydrophobic regions, which are depicted in Figure 2. We decided to prepare several original 3-pyrimidinylazaindole derivatives 4a, 4b, and 4c corresponding to the substitutions in positions 5, 2′, and 1, respectively (Figure 1). All of these compounds were tested in biochemical assays of cyclin-dependent kinase inhibition (CDK2/9). Approach 1: Design and Synthesis of 4a. It is worth mentioning that in the case of meriolins, the most potent analogues described in the literature are meriolin 3 and 5, where hydrophobic substitutions were present at the 4-position of the 7-azaindole ring. The substitution at the 4-position of 7azaindole improves the activity significantly in contrast to the parent classes, namely, meriolin 1 and meridianins. However, interestingly, our analysis suggested that substitution at the 5positions of ring A is also acceptable and could be utilized for the improvement of physicochemical and pharmacokinetic properties by the introduction, for instance, of substituted aryl and heteroaryls to increase specific polar interactions (e.g., hydrogen bonds) and also by hydrophobic groups. The strategy is to discriminate, as far as possible, between the enthalpic efficiency and entropy profile (ΔH driven > ΔS driven) but to have both and consequently to have a good balanced affinity between these two thermodynamic functions.32,33 In addition, we chose this site for substitution due to the easy chemical 9472

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Scheme 1. Synthesis of Ring A Analoguesa

Reagents and conditions: (a) PdCl2(dppf), K2CO3, dioxane/H2O, 80 °C, 6−12 h; (b) I2, KOH, DMF, rt, 0.5−1 h; (c) Boc anhydride, DMAP, DCM, rt, 0.5−1 h; (d) Pd(PPh3)4, anhydrous Et3N, anhydrous dioxane, HBPin, 80 °C, 3−4 h; (e) Pd(PPh3)4, Cs2CO3, anhydrous MeOH, 100 °C, 35−49 h. a

cyclin T at a concentration of 500 nM. The compounds that showed >60% inhibition were further studied for the IC50 determination. The activity profiles are shown in Table 1. As the design was not only to improve the affinity but also to improve the drug-like parameters, an important metric of ligand efficiency (LE, binding energy/non-hydrogen atoms, Kcal/mol per non-hydrogen atom) was used, which represents a simple and useful guide to optimize fragments and to avoid, as far as possible, the inflation in physicochemical properties and represents a lead selection in the discovery process.37,38 The suggested LE value that was brought above 0.3 was also maintained throughout the optimization process. In approach 1, ring A modified analogues (4aa−4as), substituted aryl and heteroaryl moieties to the 5-position of ring A, were introduced, and all of the analogues have shown excellent nanomolar activity against CDK2/cyclin A and

region bonding, a series of analogues were prepared where sulfonamide moieties were introduced on ring B. The synthesis of ring B modified analogues was prepared by coupling intermediate 15 with 4-chloro-2-(amino)pyrimidine 11 under Suzuki conditions, which gave compound 21 in 60% yield. Treatment of compound 21 with different aryl/heteroaryl sulfonyl chlorides 22 furnished the required compounds 4ca− 4cg in 65−80% yields (Scheme 3). Biological Evaluation. Cell-Free Enzyme Activity and Drug-Like Properties. Cyclin-Dependent Kinase Inhibition. The versatile biological activities exhibited by the meriolin class indicate that such molecules hold the key to new drug discovery. Therefore, the preparation of new analogues with improved physiochemical properties could yield new leads in drug development. As per our plan, all of the synthesized compounds were screened against CDK2/cyclin A and CDK9/ 9473

DOI: 10.1021/acs.jmedchem.7b00663 J. Med. Chem. 2017, 60, 9470−9489

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Scheme 2. Synthesis of Ring C Analoguesa

a

Reagents and conditions: (a) I2, KOH, DMF, rt, 30 min; (b) Boc anhydride, DMAP, DCM, rt, 30 min; (c) Pd(PPh3)4, anhydrous Et3N, HBPin, anhydrous dioxane, 80 °C, 3 h; (d) Pd(PPh3)4, Cs2CO3, anhydrous MeOH, 100 °C, 35 h; (e) m-CPBA, CH2Cl2, rt, 1 h; (f) THF, 85 °C, overnight.

CDK9/cyclin T, except 4am, along with acceptable LE values (0.64−0.27 for CDK2 and 0.53−0.28 for CDK9). The molecular docking studies revealed that these analogues retain bioactive conformations and showed similar interactions (with Asp145, Leu83, Glu81, Glu51, and Lys33 in the case of CDK2; Asp167, Cys106, Asp104, Phe103, Glu66, and Lys48 in the case of CDK9) in comparison to meriolin 3. The substituted groups at the 5-position lay in a cavity where they are surrounded (within the 4 Å vicinity) by several residues, viz., Leu134, Phe82, Lys89, Val18, and Ile10 in CDK2; Leu156, Glu107, Asp109, Phe105, Val33, and Ile25 in CDK9, just before the solvent accessible region, and they are responsible for the high potency (nM range). Moreover, from the MD simulation studies, it was observed that Leu134 and Ile10 in CDK2 and Leu156 and Ile25 in CDK9 form hydrophobic contacts (exists for more than 40% during the 10 ns simulation run, details provided in Supporting Information as Figure S1). In the case of aryl rings, monocyclic analogues (4aa−4ak) showed better activity than bicyclic analogues (4al−4am). However, in the case of heteroaryl rings, both mono and bicyclic analogues (4an−4as) showed good activity with respect to CDK2 and CDK9. The residue interactions of one of the best analogues, 4ab, with CDK2/cyclin A and CDK9/cyclin T are shown in Figure 3. After this, using approach 2, several structural alterations in relation to the substitution at the amino group of ring C were

made to examine whether the introduction of secondary amines and tertiary amines would affect the CDK inhibition activity. Interestingly, substitution of the 3-amino group of ring C with 2-aryl/hetroarylethane amines (4ba−4bd) gave encouraging results, and all analogues showed a nanomolar potency against both CDK2/cyclin A and CDK9/cyclin T along with good LE values (0.37−0.45 for CDK2 and 0.35−0.44 for CDK9). However, the substitution of the 3-amino group of ring C with tertiary amines (4be−4bh) resulted in the reduction of activity against both CDK2/cyclin A and CDK9/cyclin T. The binding pocket analysis of CDK2 and CDK9 with respect to meriolins suggested that there is less space around the pyrimidine ring (ring C) for substitution. However, the substitution with aryl/ heteroaryl rings having linkers (4ba−4bd) was found to be active, which orients the analogues in such a way that the aryl/ heteroaryl rings lay in the solvent accessible area. This conformation further increases the chances of interaction with the residues residing in this region of the binding site. In addition, this substitution orients the pyrimidine-2-amine, resulting in the disruption of its NH2 interaction with Glu51 in CDK2 and Glu66 in CDK9. However, the secondary amine substituted analogues 4ba−4bd engaged in their interaction as hydrogen bond donors with other amino acid residues, viz., Asp145 in the case of CDK2 and Asp167 in the case of CDK9, whereas in 4be−4bh, the compounds with tert-amines and freeNH are not available to form interactions with the acidic 9474

DOI: 10.1021/acs.jmedchem.7b00663 J. Med. Chem. 2017, 60, 9470−9489

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Scheme 3. Synthesis of Ring B Modified Analoguesa

Reagents and conditions: (a) 11, Pd(PPh3)4, Cs2CO3, anhydrous MeOH, 100 °C, 49 h, 60%; (b) (i) 21, NaH, anhydrous DMF, rt, 2 h, (ii) 22, 0 °C to rt, overnight, 65−80%.

a

viz. HCT-116 (IC50 of 0.2 μM) and SH-SY5Y (IC50 of 0.8 μM), compared to meriolin 1, whereas 4ab showed a comparable cytotoxicity profile with meriolin 348 against the tested cell line. Solubility Study (Thermodynamic Equilibrium Solubility). Based on the in vitro cell-free and cell-based activity, five compounds (4ab−4ac, 4an, 4ap, and 4ba) were further studied for their solubility profile in aqueous and biological fluids, namely, phosphate buffer saline (PBS), stimulated gastric fluids (SGF), and stimulated intestinal fluids (SIF). The results are summarized in Table 3. All of the compounds showed a high solubility in SGF. Interestingly, three analogues 4ab, 4ac, and 4ap also showed comparatively better profiles in other tested media. In Vivo PK Profile of 4ab. On the basis of biochemical and cellular activities and a drug-like profile (LE, solubility), compound 4ab was further selected for the PK studies (via iv and ip routes). In the PK study, compound 4ab showed a moderate PK profile but was comparatively better than the parent compounds (meriolins). In the iv route (2.5 mg/kg), compound 4ab showed a half-life of 2.5 h and an AUC of 437.1 ng/mL × h. However, via the ip route (5.0 mg/kg), the test compound showed a half-life (t1/2) of 4.4 h and a Cmax of 162.2 ng/mL. The plasma concentration of meriolin 1 dropped to the lower level of quantitation within 1 h after iv administration, and it was maintained up to 10 h. The high volume of distribution (Vd) suggested that the test material resides more in tissues than in blood. However, the clearance (CL) was found to be quite high but was comparatively lower than that of meriolin 1 (Table 4). Meriolin 3 showed a short exposure time in the experimental time frame, so the PK parameters could not

residues (Asp145, Glu51 in CDK2 and Asp167, Glu66 in CDK9), thus reducing its binding affinity. This could be the reason for the decline in activity. The residue interactions of 4bb (A−D) and 4be (E−F) with CDK2/cyclin A and CDK9/ cyclin T are shown in Figure 4. In approach 3, ring B modified analogues disrupting the Hbonding interactions between N7/N1 and Leu-83/Glu-81 were made by the incorporation of a bulkier group (4ca−4cg), leading to the abolition of activity against CDK2/cyclin A and CDK9/cyclin T. As seen in Figure 5, substituting the bulkier group at the N1 position orients the molecules by 180° and disrupts the bioactive conformation and reason for the abolition of activity. Based on the in vitro screening results of the 3pyrimidinylazaindole analogues against CDK2/cyclin A and CDK9/cyclin T, key structural features essential for inhibition have been identified and summarized in Figure 6, showing the crucial role of positions 1, 2′, and 5 with a specific chemical nature of substituents R1, R2, and R3. The position and the nature of the groups are related to their bindings with CDK2/9 kinases (Figures 3−5). Cell-Based In Vitro Activity. The eight most potent biochemical (CDK2/9) analogues (4ab−4ad, 4an−4ap, 4ba− 4bb, and meriolins) were further evaluated against two different human cancer cell lines, viz. HCT-116 (colorectal) and SHSY5Y (neuroblastoma), for their antiproliferative potential using an MTT cytotoxicity assay. The results are summarized in Table 2. All of the tested analogues showed interesting antiproliferative activities; however, the analogue 4ab showed the most promising activity against both of the tested cell lines, 9475

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group had 7 animals; the results are summarized in Table 6. The test analogue 4ab at a dose of 15 mg/kg was well-tolerated and also showed a tumor growth inhibition (TGI) of 33.60%. However, a dose of 30 mg/kg showed a better efficacy of the TGI of 45.96% with some mortality. Moreover, a dose of 45 mg/kg was found highly toxic, and all of the animals died. After these results, the tolerability of 4ab was also evaluated via the ip route at an acceptable dose of 15 mg/kg. Interestingly, the dose was found acceptable, and 45.57% of the TGI was observed. These in vivo experiments suggested that a dose of 15 mg/kg was safe and could be considered for further relevant in vivo efficacy studies. In Vitro Cell Proliferation Assay Activity of 4ab against a Panel of Breast Cancer Cell Lines. In recent years, CDK inhibitors were explored for the much needed treatment of triple-negative breast cancer,28,29 which represents the most aggressive subtype of breast cancer with a poor prognosis and short survival time. Currently, there is no target-based therapy against this cancer, and radiotherapy and chemotherapy are the only available treatment options for patients. Therefore, the identified potent lead was further tested against breast cancer cell lines, five human such as MDAMB-231, MDAMB-468, T47D, BT549, and MCF-7 and one mouse such as 4T1 (Table 7). Compound 4ab showed a potent activity against all of the tested cell lines, with the most potent activity against the MCF7 cell line. The test analogue 4ab also showed less activity against the normal cell line HEK. The safety ratio (IC50 HEK/ IC50 MCF-7) of 4ab is ∼33 versus 6 for meriolin 1 and 0.94 for meriolin 3. In Vivo Evaluation of 4ab against Mouse Mammary Triple-Negative Breast Cancer. In the present study, we used the mouse cell line 4T1 for establishing the in vivo efficacy against breast cancer. The 4T1 cell line represents the highly proliferative and metastatic tumors in immune competent BALB/c mice and also mimics human triple-negative breast cancer.39 It was observed that 4ab induced significant dosedependent tumor regression in the 4T1 mouse syngeneic model (Figure 7A). Compound 4ab at a dose of 15 mg/kg was able to cause a tumor growth inhibition (TGI) of 90.58 with no mortality, comparable to that of meriolin 3 (TGI = 94.96 with mortality of 2/5 mice) and anthracycline doxorubicin (TGI = 97.35 without mortality at 5 mg/kg). Doxorubicin is used for the treatment in metastatic triple-negative breast cancer.40 During the study, it was observed that the mice treated with 4ab looked healthy and that the treatment did not affect the weight of animals; however, the mice treated with doxorubicin showed a loss of weight (Figure 7B). On the basis of these results, compound 4ab appeared very interesting and represented another alternative lead with a better safety profile in comparison to meriolin 3. Effect of 4ab on Cellular Protein Expression. After the cytotoxicity study of 4ab on the 4T1 cell line, we assayed the effect of 4ab on the expression of proteins important for cell cycle regulation.25,26 In this direction, we tested the effect of 4ab on phosphorylation of CDK targets in the mice mammary breast cancer cell line 4T1 in a concentration-dependent manner for 24 h. (The densitometry analysis is provided in the Supporting Information as Figure S2.) It is clear from the results (Figure 8) that 4ab inhibits the phosphorylation of RNA polymerase II (p-Rpb1-CTD) (CDK9 site, 8A) and the retinoblastoma protein (CDK2/CDK4 sites, 8B). Compound 4ab was initially seen to be very active in the cell-free kinase assay inhibiting CDK2 and CDK9 in a low nanomolar

Table 1. Biological Activity and Drug-Like Profile of Synthesized Analogues of 3-Pyrimidinylazaindoles

compounds 4aa 4ab 4ac 4ad 4ae 4af 4ag 4ah 4ai 4aj 4ak 4al 4am 4an 4ao 4ap 4aq 4ar 4as 4ba 4bb 4bc 4bd 4be 4bf 4bg 4bh 4ca 4cb 4cc 4cd 4ce 4cf 4cg meriolin 1 meriolin 3a flavopiridol a

% inhibition at 500 nM (CDK2/ cyclin A)

IC50 (nM) (CDK2/ cyclin A)

% inhibition at 500 nM (CDK9/ cyclin T)

91.3 95.8 97.2 96.2 95.4 90.3 89.8 84.8 93.4 78.7 79.3 79.2 24.2 97.8 95.1 98.2 92.0 87.9 90.2 92.5 97.7 89.2 89.7 78.3 74.0 73.3 71.6 22.6 28.1 28.4 31.3 36.3 48.7 55.2

9.0 5.5 4.0 5.0 6.0 10 15 92 10 24 75 182 1089 3.0 1.0 5.0 12 23 16 10 4.0 22 10 160 300 231 350

86.9 90.0 92.3 88.6 90.1 87.9 87.3 82.6 86.5 93.7 70.4 84.0 47.4 87.6 93.5 89.9 86.4 85.0 82.4 92.4 91.0 85.0 88.0 77.2 71.9 72.9 70.5 40.0 31.2 50.9 54.4 44.2 52.2 50.3

90 11 106

IC50 (nM) (CDK9/ cyclin T) 61 24 22 27 28 52 40 146 47 45 213 159 625 42 35 42 82 93 342 20 38 59 52 450 800 642 700

30 6 3.7

LE CDK2/9 0.42/0.38 0.49/0.45 0.50/0.45 0.42/0.38 0.39/0.32 0.45/0.41 0.46/0.44 0.41/0.40 0.42/0.38 0.42/0.41 0.40/0.38 0.35/0.36 0.27/0.28 0.55/0.48 0.64/0.53 0.51/0.45 0.45/0.40 0.41/0.38 0.39/0.32 0.45/0.44 0.44/0.39 0.37/0.35 0.40/0.37 0.46/0.43 0.42/0.39 0.43/0.40 0.40/0.38

0.60/0.64 0.64/0.60

Reported in literature.

be calculated. On the basis of these results, compound 4ab was selected for further evaluation of the in vivo antitumor activity. Selectivity Study of 4ab. Compounds 4ab and meriolin 3 were investigated against a panel of 19 protein kinases at concentrations of 100 and 500 nM in in vitro activity assays. 4ab showed poor inhibition at these two concentrations against most of the kinases except VEGFR1, DYRK1a, and CDK4. However, meriolin 3 also showed a poor inhibition at these concentrations against most of the kinases except S6K1 and DYRK1a (Table 5).26 Maximum Tolerance Dose Study of 4ab. In the preliminary study, the maximum tolerated dose (MTD) was evaluated for the identified analogue 4ab in the solid Ehrlich (male swiss mice) model. In addition to this, the in vivo efficacy was evaluated. Initially, three doses (15, 30, and 45 mg/kg via iv route) were evaluated where the dosing was done for 2 weeks. 5-Fluorouracil (5-FU) was used as a positive control, and each 9476

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Figure 3. Main interaction of 4ab with CDKs. Panels A* and B* represent the interaction of 4ab with CDK2/cyclin A. Panels C* and D* represent the interaction of 4ab with CDK9/cyclin T. Panels A* and C* show 3D models with full protein cores. Panels B* and D* show 3D models with amino acid residues only.

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concentration (Table 1) also shown to be effective on inhibiting site specific phosphorylation of the CDK substrate in a cell culture. In agreement with the extreme sensitivity of CDK2 to 4ab in in vitro, the in vivo phosphorylation of p-Rb, its CDK2 specific site, was most sensitive to 4ab (Figure 8B). We next also analyzed the level of Mcl-1; 4ab also effects the expression of Mcl-1 (antiapoptotic protein usually overexpressed in cancer cells). When the 4T1 cell line was exposed to 4ab in a concentration-dependent manner for 24 h, it showed a complete disappearance of Mcl-1 at 3.125 μM (8C).

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EXPERIMENTAL SECTION

All chemicals for this study were purchased from Sigma-Aldrich, India. 1 H NMR data were recorded on a 400 or 500 MHz Bruker NMR instrument, and 13C NMR data were recorded on a 101 or 126 MHz Bruker NMR instrument. Chemical data for protons were reported in parts per million (ppm, scale) downfield from tetramethylsilane and were referenced to the residual proton in the NMR solvent (CDCl3, δ 7.26; DMSO-d6, δ 2.5 and 3.5; or other solvents as mentioned). All of the NMR spectra were processed by either MestReNova or Bruker software. Mass spectra were recorded with HRMS or LC−MS instrument. Melting points were recorded on a digital melting point apparatus and were uncorrected. The purity of all of the final compounds (used for biological screening) was determined by using the HPLC-Agilent Technologies 1260 infinity series or Shimadzu lab solutions system using the following methods: Method A used the Enable C18 G column (Spinco, 5 μm, 4.6 × 250 mm), and the gradient mixture of water (A)/acetonitrile (B) was used as a mobile phase over 55 min with a flow rate of 0.8 mL/min. Method B used the RP-18 column (Merck, 5 μm, 4 × 250 mm), and the gradient mixture of water (A)/acetonitrile (B) was used as a mobile phase over 60 min with a flow rate of 0.9 mL/min. Method C used the RP-18 column (Merck, 5 μm, 4 × 250 mm), and the gradient mixture of water (A)/ acetonitrile (B) was used as a mobile phase over 50 min with a flow rate of 0.5 mL/min. Method D used the RP-18 column (Merck, 5 μm, 4 × 250 mm), and the gradient mixture of water (A)/acetonitrile (B) was used as a mobile phase over 50 min with a flow rate of 0.3 mL/ min. The HPLC purity of all of the final compounds were >95%. Experimental Procedures for the Synthesis of Ring A (4aa− 4as) Modified Analogues. Synthesis of Final Compounds 4aa− 4as. Tetrakis(triphenylphosphane)palladium(0) (3 mol %) and intermediates 5-substituted tert-butyl 3-iodo-1H-pyrrolo[2,3-b]pyridine-1-carboxylate (9a−s) (1.00 mmol) were placed under an argon atmosphere in a dry screw-cap vessel with a septum. Then, 5 mL

CONCLUSION

In this study, we describe the design and the medicinal chemistry/synthesis of original 3-pyrimidinylazaindole derivatives. Several compounds inhibit CDK2 and CDK9 activity at low nanomolar concentrations and exhibit a very potent antiproliferative activity in a panel of tumor cells. Optimization led to the discovery of 4ab, the most potent analogue with nanomolar potency against CDK2 and CDK9. 4ab showed a good drug-like profile (e.g., aqueous solubility and ligandefficiency metric), along with an in vivo pharmacokinetic profile, and also demonstrated a good in vivo efficacy in a mouse mammary triple-negative breast cancer model. The present study provided the opportunity for the optimization of this class in terms of a drug-like profile and also gave an interesting and promising new development candidate against TNBC. 9477

DOI: 10.1021/acs.jmedchem.7b00663 J. Med. Chem. 2017, 60, 9470−9489

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Figure 4. Main interaction of ring C modified analogues with CDKs. Panels A* and B* represent the interaction of 4bb with CDK2/cyclin A. Panels C* and D* represent the interaction of 4bb with CDK9/cyclin T. Panel E represents the interaction of 4be with CDK2/cyclin A. Panel F represents the interaction of 4be with CDK9/cyclin T. Panels A*, C*, E*, and F* show 3D models with full protein cores. Panels B* and D* show 3D models with amino acid residues only. ultrasound bath for 0.5−1.0 h, filtrating, and drying in vacuo overnight for 12 h to obtain the compounds 4aa−4as. The purity of all of the compounds was determined by using method A. 4-(5-(4-(Trifluoromethyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine (4aa): TLC (MeOH/DCM 1:9) Rf = 0.5; 63% yield; off-white solid; mp 267−269 °C; 1H NMR (400 MHz, DMSOd6) δ 12.38 (s, 1H), 9.22 (d, J = 2.1 Hz, 1H), 8.67 (d, J = 2.2 Hz, 1H), 8.44 (s, 1H), 8.16 (d, J = 5.3 Hz, 1H), 8.05 (d, J = 8.1 Hz, 2H), 7.85 (d, J = 8.2 Hz, 2H), 7.11 (d, J = 5.3 Hz, 1H), 6.61 (s, 2H); 13C NMR (126 MHz, DMSO-d6) δ 163.4, 161.9, 157.3, 149.1, 142.8, 142.5, 129.5, 129.0, 127.9, 127.8, 127.3 (q, J = 31.5 Hz), 125.6 (q, J = 3.78 Hz), 124.4 (q, J = 272.1 Hz), 117.8, 112.8, 104.9; 19F NMR (376.5 MHz, DMSO-d6) δ −60.85 (s, 3F); HRMS (ESI-TOF) calcd for C18H13F3N5 for [M + H]+ 356.1123, found 356.1121; HPLC purity 98% (tR = 31.8, method A).

of dry dioxane was added, and the mixture was degassed with argon. Dry triethylamine (10.0 mmol, 10.0 equiv) and 4,4,5,5-tetramethyl1,3,2-dioxaborolane (1.50 mmol, 1.50 equiv) were successively added to the mixture, which was stirred at 80 °C (preheated oil bath) for 3−4 h to obtain 10a−s (monitored by TLC). Then, after cooling the mixture to 25 °C (water bath), tetrakis(triphenylphosphane)palladium(0) (3 mol %), dry methanol (5 mL), 2-amino-4-chloro pyrimidine (11, 1.00 mmol), and cesium carbonate (2.50 mmol, 2.50 equiv) were successively added, and the mixture was stirred at 100 °C for 35−49 h. Then, after cooling the mixture to 25 °C (water bath), the solvents were removed in vacuo, and the residue was absorbed onto Celite and purified chromatographically on silica gel with dichloromethane/methanol/aqueous ammonia (isocratic or stepwise gradient). The obtained bis(hetero)aryls 4aa−4as can be further purified by suspending in dichloromethane, sonicating in an 9478

DOI: 10.1021/acs.jmedchem.7b00663 J. Med. Chem. 2017, 60, 9470−9489

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Figure 5. Interaction of ring B modified analogues with CDKs. Panel A represents the interaction of 4ca with CDK2/cyclin A. Panel B represents the interaction of 4ca with CDK9/cyclin T.

Figure 6. Structure−activity relationship in the 3-pyrimidinylazaindole analogue series. 12.31 (s, 1H), 9.10 (d, J = 2.0 Hz, 1H), 8.58 (d, J = 2.1 Hz, 1H), 8.40 (d, J = 2.7 Hz, 1H), 8.15 (d, J = 5.3 Hz, 1H), 7.89−7.75 (m, 2H), 7.33 (t, J = 8.8 Hz, 2H), 7.10 (d, J = 5.3 Hz, 1H), 6.60 (s, 2H); 13C NMR (126 MHz, DMSO-d6) δ 163.5, 161.9, 161.6 (d, J = 240.6 Hz), 157.3, 148.7, 142.3, 135.2 (d, J = 2.5 Hz), 129.3, 129.1 (d, J = 7.5 Hz), 128.5, 128.3, 117.7, 115.6 (d, J = 21.4 Hz), 112.7, 105.0; 19F NMR (376.5 MHz, DMSO-d6) δ −119 (septet, 1F); HRMS (ESI-TOF) calcd for C17H13FN5 [M + H]+ 306.1155, found 306.1153; HPLC purity 99% (tR = 17.89, method A). 4-(5-(4-Chlorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2amine (4ac): TLC (MeOH/DCM 1:9) Rf = 0.5; 62% yield; yellow solid; mp 306−308 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.32 (s, 1H), 9.13 (d, J = 1.9 Hz, 1H), 8.60 (d, J = 1.9 Hz, 1H), 8.40 (d, J = 2.4 Hz, 1H), 8.16 (d, J = 5.3 Hz, 1H), 7.84 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 8.3 Hz, 2H), 7.09 (d, J = 15.8 Hz, 1H), 6.59 (s, 2H); 13C NMR (126 MHz, DMSO-d6) δ 163.4, 161.9, 157.2, 148.8, 142.2, 137.6, 131.9, 129.4, 128.9, 128.8, 128.4, 128.2, 117.7, 112.7, 105.0; HRMS (ESITOF) calcd for C17H18ClN5[M + H]+ 322.0859, found 322.0860; HPLC purity 98% (tR = 30.43, method A). 4-(5-(4-(Trifluoromethoxy)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine (4ad): TLC (MeOH/DCM 1:9) Rf = 0.5; 60% yield; light-yellow solid; mp 245−247 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.36 (s, 1H), 9.15 (d, J = 1.8 Hz, 1H), 8.63 (d, J = 1.9 Hz, 1H), 8.42 (d, J = 2.6 Hz, 1H), 8.16 (d, J = 5.3 Hz, 1H), 7.86 (d, J = 7.8 Hz, 1H), 7.77 (s, 1H), 7.65 (t, J = 8.0 Hz, 1H), 7.40 (d, J = 8.0 Hz, 1H), 7.11 (d, J = 5.3 Hz, 1H), 6.61 (s, 2H); 13C NMR (126 MHz, DMSO-d6) δ 163.4, 161.8, 157.2, 148.9, 142.5, 141.2, 130.8, 129.6, 128.6, 127.9, 126.4, 119.7, 119.2, 117.6, 112.9, 105.0; 19F NMR (376.5 MHz, DMSO-d6) δ −56.53 (s, 3F); HRMS (ESI-TOF) calcd for C18H13F3N5O [M + H]+ 372.1072, found 372.1059; HPLC purity 98% (tR = 31.05, method A). 4-(5-(4-Methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin2-amine (4ae): TLC (MeOH/DCM 1:9) Rf = 0.5; 59% yield; yellow solid; mp 235−237 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.24 (s, 1H), 9.05 (s, 1H), 8.55 (d, J = 2.2 Hz, 1H), 8.37 (d, J = 2.8 Hz, 1H), 8.15 (d, J = 5.3 Hz, 1H), 7.74 (d, J = 8.7 Hz, 2H), 7.08 (t, J = 7.5 Hz,

Table 2. In Vitro Cell Proliferation Activity of 4a and 4b against HCT-116 and SHSY-5Y compound

HCT-116 (IC50 μM)

SHSY-5Y (IC50 μM)

4ab 4ac 4ad 4an 4ao 4ap 4ba 4bb meriolin 1 meriolin 3

0.2 0.5 1.5 0.7 0.6 0.7 0.56 9.5 1.8 0.1

0.8 0.9 0.4 0.9 3.3 1.0 ND ND ND 0.12

a

Dose−response curves were generated from duplicate 10-point serial dilutions of inhibitory compounds. IC50 values were derived by a nonlinear regression analysis.

Table 3. Solubility Profile of Active Compounds solubility (in μg/mL)a

a

compound

water

PBS

SGF

SIF

4ab 4ac 4an 4ap 4ba meriolin 1

72.4 57.4 20 80 96% (tR = 28.27, method A). 4-(5-(Thiophen-3-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2amine (4ao): TLC (MeOH/DCM 1:9) Rf = 0.5; 44% yield; yellow solid; mp 237−239 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.31 (s, 1H), 9.17 (s, 1H), 8.73 (s, 1H), 8.41 (s, 1H), 8.15 (d, J = 5.2 Hz, 1H), 8.05 (s, 1H), 7.78 (d, J = 4.4 Hz, 1H), 7.71 (d, J = 2.3 Hz, 1H), 7.12 9482

DOI: 10.1021/acs.jmedchem.7b00663 J. Med. Chem. 2017, 60, 9470−9489

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= 13.3, 6.8 Hz, 2H), 3.02 (t, J = 7.1 Hz, 2H); 13C NMR (126 MHz, CDCl3) δ 162.3, 162.2, 157.2, 149.1, 143.3, 139.4, 131.2, 128.9, 128.6, 127.0, 126.4, 118.7, 117.2, 113.9, 105.6, 42.8, 35.9; HRMS (ESI-TOF) calcd for C19H18N5 [M + H]+ 316.1562, found 316.1568; HPLC purity 99% (tR = 23.97, method B). N-(4-Methoxyphenethyl)-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine (4bb): TLC (MeOH/DCM 1:9) Rf = 0.6; 60% yield; yellow solid; mp 224−226 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.15 (s, 1H), 8.81 (s, 1H), 8.31 (d, J = 2.1 Hz, 1H), 8.23 (d, J = 3.5 Hz, 1H), 8.11 (d, J = 4.7 Hz, 1H), 7.19−7.06 (m, 3H), 7.00 (d, J = 5.3 Hz, 2H), 6.82 (d, J = 8.3 Hz, 2H), 3.66 (s, 3H), 2.78 (t, J = 7.0 Hz, 2H), 2.44−2.35 (m, 2H); 13C NMR (126 MHz, DMSO-d6) δ 165.0, 162.7, 158.0, 155.0, 151.4, 149.7, 143.9, 138.0, 132.1, 130.0, 129.0, 118.3, 117.2, 114.2, 105.1, 55.4, 43.2, 34.9; HRMS (ESI-TOF) calcd for C20H20N5O [M + H]+ 346.1668, found 346.1687; HPLC purity 99% (tR = 23.26, method B). N-(3,4-Dimethoxyphenethyl)-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine (4bc): TLC (MeOH/DCM 1:9) Rf = 0.6; 68% yield; light-yellow solid; mp 226−228 °C; 1H NMR (400 MHz, CDCl3) δ 10.74 (s, 1H), 8.81 (d, J = 7.7 Hz, 1H), 8.40 (d, J = 4.6 Hz, 1H), 8.26 (d, J = 5.0 Hz, 1H), 8.02 (s, 1H), 7.23 (dd, J = 7.9, 4.8 Hz, 3H), 6.90 (d, J = 5.3 Hz, 1H), 6.82 (d, J = 14.8 Hz, 2H), 3.86 (d, J = 7.9 Hz, 6H), 3.83−3.77 (m, 2H), 2.96 (t, J = 7.0 Hz, 2H); 13C NMR (126 MHz, DMSO-d6) δ 165.3, 161.9, 158.3, 154.9, 151.5, 150.0, 144.1, 138.2, 137.3, 132.2, 130.1, 128.9, 127.3, 118.6, 117.4, 114.4, 105.3, 56.4, 43.4, 34.9; HRMS (ESI-TOF) calcd for C21H22N5O2 [M + H]+ 376.1773, found 376.1754; HPLC purity 99% (tR = 23.32, method B). N-(2-(1H-Indol-3-yl)ethyl)-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine (4bd): TLC (MeOH/DCM 1:9) Rf = 0.5; 50% yield; light-yellow solid; mp 233−235 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H), 10.85 (s, 1H), 8.88 (s, 1H), 8.39 (d, J = 2.6 Hz, 1H), 8.28 (d, J = 3.4 Hz, 1H), 8.20 (s, 1H), 7.60 (d, J = 7.4 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.23 (s, 1H), 7.08 (t, J = 6.4 Hz, 4H), 7.02−6.92 (m, 2H), 4.07−3.90 (m, 2H), 3.03 (t, 2H); 13C NMR (101 MHz, DMSO-d6) δ 162.3, 161.8, 157.1, 149.2, 143.3, 136.2, 128.5, 127.3, 122.5, 120.8, 118.3, 118.1, 117.8, 116.6, 112.2, 111.3, 104.6, 41.6, 25.2; HRMS (ESI-TOF) calcd for C21H19N6 [M + H]+ 355.1671, found 355.1697; HPLC purity 96% (tR = 21.93, method B). 3-(2-(Pyrrolidin-1-yl)pyrimidin-4-yl)-1H-pyrrolo[2,3-b]pyridine (4be): TLC (MeOH/DCM 1:9) Rf = 0.6; 52% yield; light-yellow solid; mp 265−267 °C; 1H NMR (400 MHz, CDCl3) δ 10.16 (s, 1H), 8.90 (dd, J = 7.9, 1.5 Hz, 1H), 8.39 (dd, J = 4.7, 1.5 Hz, 1H), 8.30 (d, J = 5.3 Hz, 1H), 7.99 (s, 1H), 7.26−7.20 (m, 1H), 6.81 (d, J = 5.3 Hz, 1H), 3.87−3.54 (m, 4H), 2.06 (t, J = 6.7 Hz, 4H); 13C NMR (126 MHz, DMSO-d6) δ 162.1, 160.7, 157.4, 149.7, 143.9, 130.8, 129.1, 118.3, 117.4, 113.1, 104.3, 46.8, 25.5; DEPT NMR (126 MHz, DMSO-d6) δ 156.9, 143.4, 130.3, 128.6, 116.9, 103.8, 46.3, 25.0; HRMS (ESI-TOF) calcd for C15H16N5 [M + H]+ 266.1406, found 266.1402; HPLC purity 95% (tR = 24.09, method B). 3-(2-(Piperidin-1-yl)pyrimidin-4-yl)-1H-pyrrolo[2,3-b]pyridine (4bf): TLC (MeOH/DCM 1:9) Rf = 0.6; 52% yield; light-yellow solid; mp 270−272 °C; 1H NMR (400 MHz, CDCl3) δ 10.15 (s, 1H), 8.75 (d, J = 8.1 Hz, 1H), 8.39 (d, J = 3.7 Hz, 1H), 8.35−8.24 (m, 1H), 8.00 (s, 1H), 7.35−7.27 (m, 1H), 6.81 (dd, J = 8.5, 5.0 Hz, 1H), 3.92 (d, J = 4.6 Hz, 4H), 3.51 (dd, J = 8.6, 3.1 Hz, 1H), 1.70−1.58 (m, 5H); 13C NMR (126 MHz, DMSO-d6) δ 162.4, 160.5, 158.1, 149.9, 144.2, 131.3, 129.5, 118.3, 117.5, 113.3, 105.0, 49.8, 24.5, 23.5; DEPT NMR (126 MHz, DMSO-d6) δ 158.1, 144.2, 131.3, 129.5, 117.5, 105.0, 49.8, 24.5, 23.5; HRMS (ESI-TOF) calcd for C16H18N5 [M + H]+ 280.1562, found 280.1560; HPLC purity 96% (tR = 26.24, method B). 4-(4-(1H-Pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-yl)morpholine (4bg): TLC (MeOH/DCM 1:9) Rf = 0.6; 60% yield; light-yellow solid; mp 264−266 °C; 1H NMR (400 MHz, CDCl3) δ 11.01 (s, 1H), 8.66 (d, J = 7.9 Hz, 1H), 8.40−8.30 (m, 2H), 8.25 (d, J = 5.2 Hz, 1H), 7.97 (s, 1H), 6.83 (d, J = 5.2 Hz, 1H), 3.82 (dt, J = 29.9, 4.2 Hz, 8H); 13 C NMR (126 MHz, DMSO-d6) δ 171.6, 170.8, 162.2, 157.5, 149.7, 143.9, 130.4, 129.6, 117.5, 111.6, 105.8, 66.5, 44.5; DEPT NMR (126 MHz, DMSO-d6) δ 157.5, 143.9, 130.4, 117.5, 111.5, 105.9, 66.4, 44.5;

HRMS (ESI-TOF) calcd for C15H16N5O [M + H]+ 282.1355, found 282.1354; HPLC purity 95% (tR = 6.42, method C). 3-(2-(4-Methylpiperazin-1-yl)pyrimidin-4-yl)-1H-pyrrolo[2,3-b]pyridine (4bh): TLC (MeOH/DCM 1:9) Rf = 0.6; 54% yield; lightyellow solid; mp 258−260 °C; 1H NMR (400 MHz, CDCl3) δ 11.19 (s, 1H), 8.70−8.62 (m, 1H), 8.31 (dd, J = 4.8, 1.5 Hz, 1H), 8.23 (d, J = 5.2 Hz, 1H), 7.95 (s, 1H), 7.19−7.14 (m, 1H), 6.79 (d, J = 5.2 Hz, 1H), 3.99−3.86 (m, 4H), 2.58−2.49 (m, 4H), 2.34 (s, 3H); 13C NMR (126 MHz, DMSO-d6) δ 161.7, 161.4, 157.1, 149.1, 143.4, 129.8, 129.0, 117.5, 117.0, 112.5, 105.1, 54.2, 45.4, 43.1; DEPT NMR (126 MHz, DMSO-d6) δ 157.1, 143.4, 129.8, 129.0, 117.0, 105.1, 54.2, 45.4, 43.1; HRMS (ESI-TOF) calcd for C16H19N6 [M + H]+ 295.1671, found 295.1670; HPLC purity 99% (tR = 4.98, method C). Procedure for the Synthesis of Ring B (4ca−4cg) Modified Analogues. Synthesis of Final Compounds 4ca−4cg. A solution of compound 21 (1 mmol in DMF) was dropwise added to a suspension of sodium hydride (1 mmol) in anhydrous DMF, and the resulting reaction mixture was stirred at room temperature for 1 h. The resulting mixture was cooled to 0 °C; the corresponding sulfonyl chloride (22) (1.5 mmol) was added dropwise, and the resulting reaction mixture was stirred at room temperature overnight. It was suspended in icecooled water and extracted with ethyl acetate (3−4 times). The solvents were removed in vacuo and purified chromatographically on silica gel with hexane/ethyl acetate to obtain title compounds 4ca− 4cg. 4-(1-((4-Fluorophenyl)sulfonyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine (4ca): TLC (MeOH/DCM 0.5:9.5) Rf = 0.6; 70% yield; off-white solid; mp 240−242 °C; 1H NMR (400 MHz, CDCl3) δ 8.70 (dd, J = 8.0, 1.6 Hz, 1H), 8.48 (dd, J = 4.8, 1.5 Hz, 1H), 8.37− 8.26 (m, 3H), 7.30 (dd, J = 8.0, 4.8 Hz, 2H), 7.18 (t, J = 8.6 Hz, 2H), 6.99 (d, J = 5.2 Hz, 1H), 5.10 (s, 2H); 13C NMR (126 MHz, CDCl3) δ 167.2, 165.1, 162.8, 161.0, 157.8, 147.4, 145.6, 133.7, 131.6, 131.5, 131.4, 126.7, 120.7, 119.8, 117.3, 116.6, 116.4, 107.5; HRMS (ESITOF) calcd for C17H13FN5O2S [M + H]+ 370.0774, found 370.0775; HPLC purity 99% (tR = 9.06, method D). 4-(1-((4-Bromophenyl)sulfonyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine (4cb): TLC (MeOH/DCM 0.5:9.5) Rf = 0.6; 70% yield; off-white solid; mp 257−259 °C; 1H NMR (400 MHz, CDCl3) δ 8.70 (dd, J = 8.0, 1.6 Hz, 1H), 8.48 (dd, J = 4.8, 1.5 Hz, 1H), 8.39− 8.26 (m, 2H), 8.13 (d, J = 8.7 Hz, 2H), 7.65 (d, J = 7.0 Hz, 2H), 7.31 (dd, J = 8.0, 4.8 Hz, 1H), 6.98 (d, J = 5.2 Hz, 1H), 5.08 (s, 2H); 13C NMR (126 MHz, DMSO-d6) δ 163.4, 159.6, 158.4, 146.8, 145.3, 136.1, 132.8, 132.5, 129.7, 129.3, 127.3, 120.4, 120.0, 117.2, 105.9; HRMS (ESI-TOF) calcd for C17H13BrN5O2S [M + H]+ 429.9973, found 429.9979; HPLC purity 99% (tR = 9.16, method D). 4-(1-((4-(Trifluoromethyl)phenyl)sulfonyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine (4cc): TLC (MeOH/DCM 0.5:9.5) Rf = 0.6; 77% yield; off-white solid; mp 251−253 °C; 1H NMR (400 MHz, CDCl3) δ 8.70 (dd, J = 8.0, 1.6 Hz, 1H), 8.49 (dd, J = 4.8, 1.5 Hz, 1H), 8.41 (d, J = 8.3 Hz, 2H), 8.34 (d, J = 5.2 Hz, 1H), 8.32 (s, 1H), 7.78 (d, J = 8.4 Hz, 2H), 7.32 (dd, J = 8.0, 4.8 Hz, 1H), 6.99 (d, J = 5.2 Hz, 1H), 5.10 (s, 2H); 13C NMR (101 MHz, CDCl3) δ 163.0, 160.7, 158.3, 147.5, 145.8, 141.2, 136.1, 135.8, 131.7, 129.0, 126.5, 126.3, 124.3, 120.8, 120.1, 118.0, 107.6; HRMS (ESI-TOF) calcd for C18H13F3N5O2S [M + H]+ 420.0742, found 420.0744; HPLC purity 99% (tR = 9.11, method D). 4-(1-((4-(Trifluoromethoxy)phenyl)sulfonyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine (4cd): TLC (MeOH/DCM 0.5:9.5) Rf = 0.6; 80% yield; off-white solid; mp 245−247 °C; 1H NMR (400 MHz, CDCl3) δ 8.70 (d, J = 7.9 Hz, 1H), 8.49 (d, J = 4.5 Hz, 1H), 8.34 (dd, J = 11.1, 4.8 Hz, 4H), 7.38−7.28 (m, 3H), 6.99 (d, J = 5.2 Hz, 1H), 5.07 (s, 2H); 13C NMR (126 MHz, CDCl3) δ 163.1, 160.6, 158.5, 153.4, 147.4, 145.7, 135.8, 131.6, 130.7, 126.3, 120.8, 120.6, 119.8, 117.6, 107.7; DEPT NMR (126 MHz, CDCl3) δ 158.6, 145.7, 131.6, 130.7, 126.4, 120.6, 119.8, 107.7; HRMS (ESI-TOF) calcd for C18H13F3N5O3S [M + H]+ 436.0691, found 436.0698; HPLC purity 99% (tR = 8.54, method D). N-(4-((3-(2-Aminopyrimidin-4-yl)-1H-pyrrolo[2,3-b]pyridin-1-yl)sulfonyl)phenyl)acetamide (4ce): TLC (MeOH/DCM 0.5:9.5) Rf = 0.5; 71% yield; off-white solid; mp 234−236 °C; 1H NMR (400 MHz, 9483

DOI: 10.1021/acs.jmedchem.7b00663 J. Med. Chem. 2017, 60, 9470−9489

Journal of Medicinal Chemistry

Article

DMSO-d6) δ 10.46 (s, 1H), 9.00 (d, J = 9.5 Hz, 1H), 8.73 (s, 1H), 8.43 (d, J = 4.7 Hz, 1H), 8.27 (d, J = 5.3 Hz, 1H), 8.11 (d, J = 9.0 Hz, 2H), 7.79 (d, J = 9.0 Hz, 2H), 7.40 (dd, J = 8.0, 4.8 Hz, 1H), 7.29 (d, J = 5.3 Hz, 1H), 6.77 (s, 2H), 2.06 (s, 3H); 13C NMR (126 MHz, DMSO-d6) δ 169.2, 163.4, 159.8, 158.2, 146.8, 145.2, 144.9, 132.3, 129.9, 129.3, 127.4, 120.2, 119.7, 118.5, 116.7, 105.9, 24.12; HRMS (ESI-TOF) calcd for C19H17N6O3S [M + H]+ 409.1083, found 409.1088; HPLC purity 97% (tR = 8.68, method D). 4-(1-((2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)sulfonyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine (4cf): TLC (MeOH/DCM 0.5:9.5) Rf = 0.6; 79% yield; off-white solid; mp 229−231 °C; 1H NMR (400 MHz, CDCl3) δ 8.68 (d, J = 8.0 Hz, 1H), 8.49 (d, J = 3.7 Hz, 1H), 8.31 (d, J = 5.0 Hz, 2H), 7.79−7.72 (m, 2H), 7.30 (d, J = 4.8 Hz, 1H), 6.98 (d, J = 5.3 Hz, 1H), 6.92 (d, J = 8.2 Hz, 1H), 5.18 (s, 2H), 4.34−4.17 (m, 4H); 13C NMR (126 MHz, CDCl3) δ 162.8, 161.1, 157.7, 149.0, 147.3, 145.5, 143.5, 131.4, 129.5, 127.0, 122.1, 120.6, 119.6, 117.8, 117.7, 116.9, 107.3, 64.5, 63.9; HRMS (ESI-TOF) calcd for C19H16N5O4S [M + H]+ 410.0923, found 410.0929; HPLC purity 96% (tR = 9.2, method D). 4-(1-((1-Methyl-1H-imidazol-5-yl)sulfonyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine (4cg): TLC (MeOH/DCM 0.5:9.5) Rf = 0.4; 65% yield; yellow solid; mp 228−230 °C; 1H NMR (400 MHz, CDCl3) δ 8.76 (dd, J = 8.0, 1.6 Hz, 1H), 8.47−8.25 (m, 3H), 7.33−7.24 (m, 1H), 7.11 (d, J = 0.9 Hz, 1H), 7.01 (dd, J = 7.9, 2.9 Hz, 2H), 5.10 (s, 2H), 4.35 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 163.0, 160.8, 158.3, 148.0, 145.3, 139.4, 132.1, 130.3, 127.2, 126.5, 120.9, 119.9, 117.4, 114.0, 107.6, 36.6; HRMS (ESI-TOF) calcd for C15H14N7O2S [M + H]+ 356.0930, found 356.0922; HPLC purity 98% (tR = 8.8, method D). General Procedure for the Synthesis of Intermediates (9a− s). {1,1-Bis(diphenylphosphino)ferrocene dichloride palladium(II)} (0.05 equiv) was added to the suspension of 5-bromo-7-azaindole 5 (1 mmol), substituted boronic acid 6a−s (1.5 mmol), and potassium carbonate (2.5 mmol) in dioxane and water (2.5:1 mL). The resulting mixture was stirred at 80 °C for 6−12 h. After cooling the mixture to 25 °C, all solvents were evaporated in vacuo, and the residue was filtered onto a thin pad of Celite and purified chromatographically on silica gel with hexane/ethyl acetate to obtain 7a−s. A solution of iodine (1 mmol) in 20 mL of DMF was dropped into the solution of substituted 7-azaindoles 7a−s (1 mmol) and potassium hydroxide (2.5 mmol) in 20 mL of DMF at 0 °C to rt, and the mixture was stirred for 0.5−1 h. The reaction mixture was then poured into 50 mL of ice water containing 1% ammonia and 0.2% sodium disulfite. The precipitate was filtered, washed with ice water, and dried in vacuo to obtain a yellow solid (8a−s). The obtained solid was used without further purification for the next step. It was suspended in 10 mL of dichloromethane, and 4-dimethylaminopyridine (0.1 mmol) was added; di-tert-butyl dicarbonate (1.2 mmol), dissolved in 20 mL of dichloromethane, was added dropwise for 30 min. The mixture was stirred for 0.5−1 h at 25 °C and washed with 20 mL of 0.1 N HCl, and the aqueous phase was extracted with dichloromethane (2 × 50 mL). The combined organic layers were dried with sodium sulfate; the solvents were removed under reduced pressure, and the residue was adsorbed onto Celite and purified chromatographically on silica gel with hexane/ethyl acetate to obtain 9a−s, which solidify upon storage in a refrigerator. tert-Butyl 3-Iodo-5-(4-(trifluoromethyl)phenyl)-1H-pyrrolo[2,3-b]pyridine-1-carboxylate (9a): 72% overall yield (three steps); TLC (EtOAC/hexane 1:9) Rf = 0.5; 1H NMR (400 MHz, CDCl3) δ 8.74 (d, J = 2.1 Hz, 1H), 7.88 (d, J = 2.2 Hz, 1H), 7.86 (s, 1H), 7.76 (s, 4H), 1.69 (s, 9H); LC−MS (ESI) m/z 489 [M + H]+. tert-Butyl 5-(4-Fluorophenyl)-3-iodo-1H-pyrrolo[2,3-b]pyridine-1carboxylate (9b): 70% overall yield (three steps); TLC (EtOAC/ hexane 1:9) Rf = 0.5; 1H NMR (400 MHz, CDCl3) δ 8.69 (d, J = 1.8 Hz, 1H), 7.86−7.78 (m, 2H), 7.60 (dd, J = 8.4, 5.3 Hz, 2H), 7.19 (t, J = 8.6 Hz, 2H), 1.69 (s, 9H); LC−MS (ESI) m/z 439 [M + H]+. tert-Butyl 5-(4-Chlorophenyl)-3-iodo-1H-pyrrolo[2,3-b]pyridine1-carboxylate (9c): 63% overall yield (three steps); TLC (EtOAC/ hexane 1:9) Rf = 0.5; 1H NMR (400 MHz, CDCl3) δ 8.69 (d, J = 2.0

Hz, 1H), 7.88−7.77 (m, 2H), 7.57 (d, J = 8.5 Hz, 2H), 7.47 (d, J = 8.4 Hz, 2H), 1.69 (s, 9H); LC−MS (ESI) m/z 455 [M + H]+. tert-Butyl 3-Iodo-5-(4-(trifluoromethoxy)phenyl)-1H-pyrrolo[2,3b]pyridine-1-carboxylate (9d): 68% overall yield (three steps); TLC (EtOAC/hexane 1:9) Rf = 0.5; 1H NMR (400 MHz, CDCl3) δ 8.71 (d, J = 2.1 Hz, 1H), 7.87−7.81 (m, 2H), 7.59−7.45 (m, 3H), 7.28 (s, 1H), 1.69 (s, 9H); LC−MS (ESI) m/z 505 [M + H]+. tert-Butyl 3-Iodo-5-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine-1-carboxylate (9e): 63% overall yield (three steps); TLC (EtOAC/hexane 1:9) Rf = 0.5; 1H NMR (400 MHz, CDCl3) δ 8.69 (d, J = 2.1 Hz, 1H), 7.85−7.75 (m, 2H), 7.57 (d, J = 8.8 Hz, 2H), 7.03 (d, J = 8.8 Hz, 2H), 3.87 (s, 3H), 1.68 (s, 9H); LC−MS (ESI) m/z 451 [M + H]+. tert-Butyl 3-Iodo-5-(4-(methylthio)phenyl)-1H-pyrrolo[2,3-b]pyridine-1-carboxylate (9f): 66% overall yield (three steps); TLC (EtOAC/hexane 1:9) Rf = 0.5; 1H NMR (400 MHz, CDCl3) δ 8.71 (d, J = 2.1 Hz, 1H), 7.82 (d, J = 2.3 Hz, 2H), 7.57 (d, J = 8.5 Hz, 2H), 7.38 (d, J = 8.5 Hz, 2H), 2.54 (s, 3H), 1.68 (s, 9H); LC−MS (ESI) m/ z 467 [M + H]+. tert-Butyl 5-(3-Fluorophenyl)-3-iodo-1H-pyrrolo[2,3-b]pyridine-1carboxylate (9g): 73% overall yield (three steps); TLC (EtOAC/ hexane 1:9) Rf = 0.5; 1H NMR (400 MHz, CDCl3) δ 8.72 (d, J = 1.9 Hz, 1H), 7.84 (d, J = 3.0 Hz, 2H), 7.49−7.39 (m, 2H), 7.34 (d, J = 9.8 Hz, 1H), 7.10 (t, J = 8.4 Hz, 1H), 1.69 (s, 9H); LC−MS (ESI) m/z 439 [M + H]+. tert-Butyl 3-Iodo-5-(m-tolyl)-1H-pyrrolo[2,3-b]pyridine-1-carboxylate (9h): 61% overall yield (three steps); TLC (EtOAC/hexane 1:9) Rf = 0.5; 1H NMR (400 MHz, CDCl3) δ 8.73 (d, J = 2.1 Hz, 1H), 7.88−7.79 (m, 2H), 7.47−7.33 (m, 3H), 7.22 (d, J = 7.4 Hz, 1H), 2.46 (s, 3H), 1.69 (s, 9H); LC−MS (ESI) m/z 435 [M + H]+. tert-Butyl 3-Iodo-5-(3-(trifluoromethyl)phenyl)-1H-pyrrolo[2,3-b]pyridine-1-carboxylate (9i): 69% overall yield (three steps); TLC (EtOAC/hexane 1:9) Rf = 0.5; 1H NMR (400 MHz, CDCl3) δ 8.73 (d, J = 2.1 Hz, 1H), 7.87 (s, 3H), 7.82 (d, J = 7.5 Hz, 1H), 7.65 (dt, J = 15.3, 7.7 Hz, 2H), 1.69 (s, 9H); LC−MS (ESI) m/z 489 [M + H]+. tert-Butyl 3-Iodo-5-(2-(methylthio)phenyl)-1H-pyrrolo[2,3-b]pyridine-1-carboxylate (9j): 61% overall yield (three steps); TLC (EtOAC/hexane 1:9) Rf = 0.5; 1H NMR (400 MHz, CDCl3) δ 8.56 (d, J = 2.0 Hz, 1H), 7.83 (s, 1H), 7.77 (d, J = 2.1 Hz, 1H), 7.41−7.30 (m, 2H), 7.26−7.22 (m, 2H), 2.36 (s, 3H), 1.68 (s, 9H); LC−MS (ESI) m/z 467 [M + H]+. tert-Butyl 5-(2-Ethylphenyl)-3-iodo-1H-pyrrolo[2,3-b]pyridine-1carboxylate (9k): 67% overall yield (three steps); TLC (EtOAC/ hexane 1:9) Rf = 0.5; 1H NMR (400 MHz, CDCl3) δ 8.61 (d, J = 2.1 Hz, 1H), 7.88−7.79 (m, 2H), 7.41−7.35 (m, 2H), 7.31−7.23 (m, 2H), 2.52 (q, J = 7.5 Hz, 2H), 1.07−1.01 (m, 3H), 1.69 (s, 9H); LC−MS (ESI) m/z 449 [M + H]+. tert-Butyl 3-Iodo-5-(naphthalen-1-yl)-1H-pyrrolo[2,3-b]pyridine1-carboxylate (9l): 64% overall yield (three steps); TLC (EtOAC/ hexane 1:9) Rf = 0.5; 1H NMR (400 MHz, CDCl3) δ 8.65 (d, J = 1.9 Hz, 1H), 7.98−7.89 (m, 2H), 7.88 (s, 1H), 7.82 (dd, J = 8.4, 5.4 Hz, 2H), 7.61−7.39 (m, 4H), 1.71 (s, 9H); LC−MS (ESI) m/z 471 [M + H]+. tert-Butyl 3-Iodo-5-(2-methoxynaphthalen-1-yl)-1H-pyrrolo[2,3b]pyridine-1-carboxylate (9m): 59% overall yield (three steps); TLC (EtOAC/hexane 1:9) Rf = 0.5; 1H NMR (400 MHz, CDCl3) δ 8.51 (d, J = 1.9 Hz, 1H), 7.94 (d, J = 9.1 Hz, 1H), 7.85 (t, J = 4.5 Hz, 2H), 7.73 (d, J = 1.9 Hz, 1H), 7.51−7.30 (m, 4H), 3.83 (s, 3H), 1.70 (s, 9H); LC−MS (ESI) m/z 501 [M + H]+. tert-Butyl 5-(Furan-3-yl)-3-iodo-1H-pyrrolo[2,3-b]pyridine-1-carboxylate (9n): 68% overall yield (three steps); TLC (EtOAC/hexane 1:9) Rf = 0.5; 1H NMR (400 MHz, CDCl3) δ 8.66 (d, J = 1.9 Hz, 1H), 7.81 (s, 2H), 7.73 (d, J = 1.9 Hz, 1H), 7.54 (s, 1H), 6.79 (s, 1H), 1.64 (d, J = 31.1 Hz, 9H); LC−MS (ESI) m/z 411 [M + H]+. tert-Butyl 3-Iodo-5-(thiophen-3-yl)-1H-pyrrolo[2,3-b]pyridine-1carboxylate (9o): 68% overall yield (three steps); TLC (EtOAC/ hexane 1:9) Rf = 0.5; 1H NMR (400 MHz, CDCl3) δ 8.76 (d, J = 2.1 Hz, 1H), 7.84 (d, J = 2.1 Hz, 1H), 7.81 (s, 1H), 7.55 (dd, J = 2.7, 1.6 Hz, 1H), 7.46 (dd, J = 2.1, 1.4 Hz, 2H), 1.68 (s, 9H); LC−MS (ESI) m/z 427 [M + H]+. 9484

DOI: 10.1021/acs.jmedchem.7b00663 J. Med. Chem. 2017, 60, 9470−9489

Journal of Medicinal Chemistry

Article

tert-Butyl 3-Iodo-5-(pyridin-3-yl)-1H-pyrrolo[2,3-b]pyridine-1-carboxylate (9p): 58% overall yield (three steps); TLC (EtOAC/hexane 3:7) Rf = 0.5; 1H NMR (400 MHz, DMSO) δ 9.02 (d, J = 1.9 Hz, 1H), 8.78 (d, J = 2.1 Hz, 1H), 8.64 (dd, J = 4.7, 1.4 Hz, 1H), 8.27−8.21 (m, 1H), 8.09 (s, 1H), 8.03 (d, J = 2.1 Hz, 1H), 7.54 (dd, J = 7.8, 4.8 Hz, 1H), 1.63 (s, 9H); LC−MS (ESI) m/z 422 [M + H]+. tert-Butyl 5-(Benzo[b]thiophen-2-yl)-3-iodo-1H-pyrrolo[2,3-b]pyridine-1-carboxylate (9q): 60% overall yield (three steps); TLC (EtOAC/hexane 1:9) Rf = 0.5; 1H NMR (400 MHz, CDCl3) δ 8.88 (d, J = 2.1 Hz, 1H), 7.94 (d, J = 1.2 Hz, 1H), 7.84 (dd, J = 14.1, 9.3 Hz, 3H), 7.63 (s, 1H), 7.44−7.30 (m, 2H), 1.69 (s, 9H); LC−MS (ESI) m/z 477 [M + H]+. tert-Butyl 5-(Benzofuran-2-yl)-3-iodo-1H-pyrrolo[2,3-b]pyridine1-carboxylate (9r): 68% overall yield (three steps); TLC (EtOAC/ hexane 1:9) Rf = 0.5; 1H NMR (400 MHz, CDCl3) δ 8.92 (d, J = 2.3 Hz, 1H), 7.98 (d, J = 1.5 Hz, 1H), 7.84−7.63 (m, 4H), 7.44−7.30 (m, 2H), 1.69 (s, 9H); LC−MS (ESI) m/z 461 [M + H]+. tert-Butyl 5-(1-(tert-Butoxycarbonyl)-5-methoxy-1H-indol-2-yl)3-iodo-1H-pyrrolo[2,3-b]pyridine-1-carboxylate (9s): 56% overall yield (three steps); TLC (EtOAC/hexane 1:9) Rf = 0.5; 1H NMR (400 MHz, CDCl3) δ 8.62 (d, J = 1.9 Hz, 1H), 8.16 (d, J = 9.1 Hz, 1H), 7.86 (s, 1H), 7.77 (d, J = 2.0 Hz, 1H), 7.07 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 9.1, 2.5 Hz, 1H), 6.60 (s, 1H), 3.90 (s, 3H), 1.71 (s, 9H), 1.35 (s, 9H); LC−MS (ESI) m/z 589 [M + H]+. Synthesis of Key Intermediate (14). A solution of iodine (1 mmol) in 20 mL of DMF was dropped into the solution of 7-azaindole 12 (1 mmol) and potassium hydroxide (2.5 mmol) in 20 mL of DMF at 0 °C to rt, and the mixture was stirred for 30 min. The reaction mixture was then poured into 50 mL of ice water containing 1% ammonia and 0.2% sodium disulfite. The precipitate was filtered, washed with ice water, and dried in vacuo to obtain 13 as a yellow solid. The obtained solid was used without further purification for the next step. It was suspended in 10 mL of dichloromethane; 4dimethylaminopyridine (0.1 mmol) was added, and di-tert-butyl dicarbonate (1.2 mmol), dissolved in 20 mL of dichloromethane, was added dropwise for 30 min. The mixture was stirred for 30 min at 25 °C and washed with 20 mL of 0.1 N HCl, and the aqueous phase was extracted with dichloromethane (2 × 50 mL). The combined organic layers were dried with sodium sulfate; the solvents were removed under reduced pressure, and the residue was adsorbed onto Celite and purified chromatographically on silica gel with hexane/ethyl acetate to obtain 14, which solidifies upon storage in a refrigerator. tert-Butyl 3-Iodo-1H-pyrrolo[2,3-b]pyridine-1-carboxylate (14): TLC (EtOAC/hexane 1:9) Rf = 0.6; oily liquid; 1H NMR (400 MHz, CDCl3) δ 8.12 (d, J = 7.6 Hz, 1H), 7.72 (s, 1H), 7.44−7.24 (m, 2H), 1.66 (s, 9H); LC−MS (ESI) m/z 344 [M + H]+. Synthesis of Compound 17. Tetrakis(triphenylphosphane)palladium(0) (3 mol %) and tert-butyl 3-iodo-1H-pyrrolo[2,3b]pyridine-1-carboxylate (14) (1.00 mmol) were placed under an argon atmosphere in a dry screw-cap vessel with a septum. Then, 5 mL of dry dioxane was added, and the mixture was degassed with argon. Dry triethylamine (10.0 mmol, 10.0 equiv) and 4,4,5,5-tetramethyl1,3,2-dioxaborolane (1.50 mmol, 1.50 equiv) were successively added to the mixture, which was stirred at 80 °C (preheated oil bath) for 3 h to obtain 15 (monitored by TLC). Then, after cooling the mixture to 25 °C (water bath), dry methanol (5 mL), 2-thiomethyl-4chloropyrimidine (16) (1.00 mmol), and cesium carbonate (2.50 mmol, 2.50 equiv) were successively added, and the mixture was stirred at 100 °C for 35 h. Then, after cooling the mixture to 25 °C (water bath), the solvents were removed in vacuo, and the residue was absorbed onto Celite and purified chromatographically on silica gel with dichloromethane/methanol/aqueous ammonia (isocratic or stepwise gradient). The obtained bis(hetero)aryl 17 could be further purified by suspending in dichloromethane, sonicating in an ultrasound bath for 0.5−1.0 h, filtrating, and drying in vacuo overnight for 12 h to obtain the compound 17. 3-(2-(Methylthio)pyrimidin-4-yl)-1H-pyrrolo[2,3-b]pyridine (17): TLC (MeOH/DCM 1:9) Rf = 0.6; 58% yield; yellow solid; 1H NMR (400 MHz, DMSO-d6) δ 12.47 (s, 1H), 8.77 (dd, J = 7.9, 1.6 Hz, 1H), 8.58 (d, J = 2.8 Hz, 1H), 8.48 (d, J = 5.4 Hz, 1H), 8.33 (dd, J

= 4.7, 1.6 Hz, 1H), 7.65 (d, J = 5.4 Hz, 1H), 7.27 (dd, J = 7.9, 4.7 Hz, 1H), 2.62 (s, 3H); 13C NMR (126 MHz, DMSO-d6) δ 171.1, 161.6, 156.4, 149.3, 143.7, 130.1, 117.5, 117.2, 111.5, 111.1, 13.7; HRMS (ESI-TOF) m/z calcd for C12H11N4S [M + H]+ 243.0704, found 243.0702. Synthesis of Compound 21. Tetrakis(triphenylphosphane)palladium(0) (3 mol %) and tert-butyl 3-iodo-1H-pyrrolo[2,3b]pyridine-1-carboxylate (14) (1.00 mmol) were placed under an argon atmosphere in a dry screw-cap vessel with a septum. Then, 5 mL of dry dioxane was added, and the mixture was degassed with argon. Dry triethylamine (10.0 mmol, 10.0 equiv) and 4,4,5,5-tetramethyl1,3,2-dioxaborolane (1.50 mmol, 1.50 equiv) were successively added to the mixture, which was stirred at 80 °C (preheated oil bath) for 3 h to obtain 15 (monitored by TLC). Then, after cooling the mixture to 25 °C (water bath), dry methanol (5 mL), 2-amino-4-chloropyrimidine (11) (1.00 mmol), and cesium carbonate (2.50 mmol, 2.50 equiv) were successively added, and the mixture was stirred at 100 °C for 35 h. Then, after cooling the mixture to 25 °C (water bath), the solvents were removed in vacuo, and the residue was absorbed onto Celite and purified chromatographically on silica gel with dichloromethane/methanol/aqueous ammonia (isocratic or stepwise gradient). The obtained bis(hetero)aryl 21 could be further purified by suspending in dichloromethane, sonicating in an ultrasound bath for 0.5−1.0 h, filtrating, and drying in vacuo for 12 h to obtain the compound 21. 4-(1H-Pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine (21) (meriolin 1): TLC Rf = 0.5 (10% MeOH/DCM); yellow solid; 60% yield; mp 222 °C; 1H NMR (500 MHz, DMSO) δ 12.20 (s, 1H), 8.93 (dd, J = 7.9, 1.5 Hz, 1H), 8.35 (d, J = 2.1 Hz, 1H), 8.29 (dd, J = 4.6, 1.6 Hz, 1H), 8.14 (d, J = 5.3 Hz, 1H), 7.19 (dd, J = 7.9, 4.7 Hz, 1H), 7.07 (d, J = 5.3 Hz, 1H), 6.52 (s, 2H); 13C NMR (126 MHz, DMSO) δ 163.40, 162.04, 157.09, 149.19, 143.42, 130.72, 128.44, 117.78, 116.70, 112.42, 104.97; HRMS (ESI+) m/z calcd for C11H10N5 [M + H]+ 212.0936, found 212.0918. In Vitro Biochemical Assay/Radioactive Assay. General Protocol for Kinase Assay. All assays, except asterisk marked (*), were carried out using a radioactive (33P-ATP) filter-binding assay.41 CDK-2/Cyclin A (5−20 mU diluted in 50 mM Hepes pH 7.5, 1 mM DTT, 0.02% Brij35, 100 mM NaCl) was assayed against histone H1 in a final volume of 25.5 μL containing 50 mM Hepes pH 7.5, 1 mM DTT, 0.02% Brij35, 100 mM NaCl, histone H1 (1 mg/mL), 10 mM magnesium acetate, and 0.02 mM 33P-g-ATP (500−1000 cpm/ pmole), and the mixture was incubated for 30 min at room temperature. Assays were stopped by the addition of 5 μL of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.41 CDK-9/Cyclin T1 (5−20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/mL of BSA, 0.1% mercaptoethanol) was assayed against a substrate peptide (YSPTSPSYSPTSPSYSPTSPKKK) in a final volume of 25.5 μL containing 50 mM Tris pH 7.5, 0.1 mM EDTA, 10 mM DTT, 1 mg/mL of BSA, 0.3 mM YSPTSPSYSPTSPSYSPTSPKKK, 10 mM magnesium acetate, and 0.05 mM 33P-γATP (50−1000 cpm/pmole), and the mixture was incubated for 30 min at room temperature. Assays were stopped by the addition of 5 μL of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.41 Luminescence Assay for CDK. Compounds 4ah and 4ak were assayed by following the luminescence assay42 against CDK2 and CDK9. ADP-Glo Kinase Assay is a luminescent kinase assay that measures ADP formed from a kinase reaction; ADP is converted into ATP, which is converted into light by Ultra-Glo Luciferase. The luminescent signal positively correlates with the ADP amount and kinase activity. The assay is well-suited for measuring the effects chemical compounds have on the activity of a broad range of purified kinases, making it ideal for both primary screening and kinase selectivity profiling. Briefly, the assay was performed in white 96-well plates taking both reaction mixture (kinase reaction in the presence of a substrate) and blank control (kinase reaction in the absence of a substrate) into consideration. The respective CDK/cyclin reaction was initiated by 9485

DOI: 10.1021/acs.jmedchem.7b00663 J. Med. Chem. 2017, 60, 9470−9489

Journal of Medicinal Chemistry

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the addition of 5 μL of 250 μM ATP assay solution (ATP assay solution (1 mL) prepared by adding 25 μL of ATP solution (10 mM) to 500 μL of 2× buffer and 475 μL of H2O-d), bringing the final volume up to 25 μL, and the reaction mixture was incubated at 30 °C for 15 min. After the incubation period, the reaction was terminated, and the remaining ATP depletion was done by adding 25 μL of the ADP-Glo reagent to each well. The reaction mixture was further incubated at ambient temperature for another 40 min. After this, 50 μL of the kinase detection reagent (prepared by mixing the kinase detection buffer with the lyophilized kinase detection substrate) was added to each well, and the plate was incubated again for 30 min. Finally, the 96-well reaction plate was read on a luminescence plate reader, and the ADP produced (nmol) in the presence and in the absence of a substrate was determined. Percent kinase inhibition was calculated as % kinase activity =

Determination of Thermodynamic Equilibrium Solubility by a 96-Well Plate-Based Assay. The compounds 4ab, 4ac, 4an, 4ao, 4ba, and meriolin 1 were dissolved in methanol to get 2000 μg/mL of the stock solution. The stock solution was introduced into 96-well plates and allowed to evaporate at room temperature to ensure that the compound (1, 2, 4, 8, 16, 25, 40, 80, 160, and 300 μg) was in solid form in the beginning of the experiment. Thereafter, 200 μL of the dissolution medium (water) was added to the wells, and the plates were shaken horizontally at 300 rpm (Eppendorf Thermoblock Adapter, North America) for 4 h at room temperature (25 ± 1 °C). The plates were covered with aluminum foil and were kept overnight for equilibration. Later, the plates were centrifuged at 3000 rpm for 15 min (Jouan centrifuge BR4i). The samples (50 μL) were withdrawn into UV 96-well plates (Corning 96-Well Clear-Flat Bottom UVTransparent Microplate) for analyses with the plate reader at the corresponding λmax of the sample (SpectraMax Plus384). The analysis was performed in triplicate for each compound. The solubility curve of concentration (μg/mL) vs absorbance was plotted to find out the saturation point, and the corresponding concentration was noted.44,45 In Vivo Pharmacokinetic. Pharmacokinetic studies were performed following the single-dose administration of compound 4ab (2.5 mg/ kg, iv; 5 mg/kg, ip), meriolin 1 (2.5 mg/kg, iv), and meriolin 3 (2.5 mg/kg, iv) in male BALB/c mice. Each study was done by using total ten animals, divided into two groups (n = 5) for sparse sampling. The formulation was prepared using 5% DMSO, 2.5% absolute alcohol, 2.5% Solutol, and normal saline (quantum satis (q.s.)) for dose administration. The blood samples were collected at 0.083 h (iv only), 0.25 h, 0.5 h, 1 h, 2 h, and 4 h after iv/ip dosing. The plasma was separated and processed for the estimation of compound 4ab and meriolin 1 by LC−MS/MS. The plasma concentration vs time profile data was analyzed using PK Solutions software (Summit Research Services, Colorado, USA) by the noncompartmental method for the pharmacokinetic parameters.46 Maximum Tolerance Dose Study of 4ab. Ehrlich ascites carcinoma (EAC) cells were collected from the peritoneal cavity of the swiss mice harboring 8−10 day old ascitic tumors. EAC cells (1 × 107) were injected intramuscularly in the right thigh of 24 swiss male mice selected for the experiment on day 0. On the next day, the animals were randomized and divided into 3 groups; 2 treatment groups contained 7 animals each, and 1 control group contained 10 animals. Treatment was given as follows: The first treatment group was treated with 4ab (15, 30, 45 mg/kg, iv; 15 mg/kg, ip) on day 1−9. The second treatment group was treated with 5-fluorouracil (22 mg/kg, i.p) on day 1−9, and it served as a positive control. The control group was similarly administered normal saline (0.2 mL, ip) on day 1−9. On days 9 and 13, the tumor-bearing thigh of each animal was shaved, and the longest and shortest diameters of the tumor were measured with the help of a vernier caliper. The tumor weight of each animal was calculated using the following formula:

luminescence of test − luminescence of blank × 100 luminescence of control − luminescence of blank

% kinase inhibition = 100 − % kinase activity In Vitro Antiproliferative Activity/Cell Line Assay. Cell Culture and Growth Conditions. A panel of human cancer cell lines were procured from the U.S. National Cancer Institute (NCI). The human cancer cell lines were grown in tissue culture flasks in complete growth medium (RPMI-1640) supplemented with 10% fetal bovine serum, 100 μg/mL of streptomycin, and 100 units/mL of penicillin in a carbon dioxide incubator (New Brunswick, Galaxy 170R, Eppendorf) at 37 °C, 5% CO2, and 98% RH. The 4T1 mouse breast cancer cell line was obtained from ATCC. The cells were cultured in RPMI-1640 (Sigma) supplemented with 10% fetal bovine serum, sodium pyruvate, nonessential amino acids, and 1× Antibiotic-Antimycotic (Gibco) at 37 °C in a humidified atmosphere with 5% CO2. A tumor cell suspension with more than 90% viability was prepared from subconfluent cultures by the treatment of a trypsin/EDTA solution (Invitrogen).43 Method for Sulforhodamine B Assay (SRB Assay). The assay was carried out in the cell suspension of optimum cell density and was seeded in 96-well flat-bottom plates (NUNC). Inoculation densities per well were used to screen for T47D (12 000) and HCT-116 (7000), and 100 μL of the cell suspension was plated. The cells were then exposed to a concentration of 100 μM of test materials containing complete growth medium along with AKBA as a positive control for 24 h. The plates were again incubated under the same conditions for another 48 h at 37 °C. Further, the cells were fixed with ice-cold TCA (trichloroacetic acid) for 1 h at 4 °C. After 1 h, the plates were rinsed three times with water and allowed to air dry. After drying the plates, 100 μL of 0.4% SRB dye was added for 0.5 h at room temperature. The plates were then washed 3 times with 1% v/v acetic acid to remove the unbound SRB. After drying the plates at room temperature, the bound dye was solubilized by adding 100 μL of 10 mM Tris (tris(hydroxymethyl)aminomethane) buffer (pH 10.4) to each well. The plates were kept on the shaker for 5 min to solubilize the protein bound dye. Finally, OD was taken at 540 nm in a microplate reader (Thermo Scientific). IC50 was determined by plotting OD against concentration.43

tumor weight (mg) =

length (mm) × [width (mm)]2 2

The percent tumor growth inhibition was calculated on day 13 by comparing the average values of the treated groups with that of the control group. The tumor growth in saline-treated control animals was taken to be 100%. Antitumor Activity of 4ab in Animal Model Balb/c Mice. Female Balb/C mice (Institutional Animal Ethical Committee approval (IAEC) no. 68/93/8/16) were maintained in core animal facilities under an institute-approved animal protocol. Mice were injected with 1 × 106 4T1 cells per mice by way of a secondary mammary fat pad. When the tumor volumes approached almost 100 mm3, the mice were divided into 4 experimental groups with 5 mice in each group. Injections of PBS (diluent), 4ab (15 mg/kg), meriolin 3 (15 mg/kg), and doxorubicin (5 mg/kg) as a positive control were given in a total volume of 200 μL at 3 day intervals for 21 days through the tail vein (iv). Animal weight and tumor measurements were done two times per week. The effect of treatment on tumor regression was determined

% cell viability = 100 × (T − T0)/(C − T0)

% growth inhibition = 100 − % cell viability where T is the absorbance of the test sample, T0 is the absorbance of the blank, and C is the absorbance of the control. Methods for 4T1 Mouse Breast Cancer Cell Line/MTT Cell Proliferation Assay. The cell viability was measured by the MTT assay. Briefly, the cells were trypsinized and seeded in a 96-well plate. After 24 h of incubation, the cells were treated with different concentrations of 4ab and meriolins for 48 h. The cells were incubated with the MTT reagent at a final concentration of 0.25 mg/mL for the last 4 h before termination, and the absorbance was measured at 570 nm. 9486

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ORCID

by measuring the tumor volume. The tumor volume was calculated using the formula (L × B2/2) mm3 (L indicates length; B indicates width) and a vernier caliper. The experiments were repeated twice. Molecular Modeling Studies. All of the molecular docking studies of 3-pyrimidinylazaindole and its derivatives against CDK2 and CDK9 were carried out using the Schrodinger suite 2015 molecular modeling software. To conduct molecular docking studies, first the crystal structure information on the cocrystallized ligands of CDK2 and CDK9 were collected from the Protein Data Bank (PDB), and the coordinates of those PDB Ids were selected, which share a similar cocrystallized ligand structure to meriolin (standard molecule). 3BHT26 PDB Id (CDK2/cyclin A in complex with the inhibitor meriolin 3) for CDK2 and 4IMY31 PDB Id (CDK9/cyclin T1 in complex with the adenosine monophosphate) for CDK9 were selected for carrying out the molecular modeling studies. Before initiating the docking studies, the selected crystal structures were prepared using the Protein Preparation Wizard.49,50 Grids were generated at the active site, identified on the bases of the already cocrystallized ligand to the receptor using the receptor grid generation module of Glide. For standardizing the docking protocol, the stereoisomers and conformers of the cocrystallized ligand were generated and minimized using the OPLS-2005 force field and docked on to the active site of the protein through the docking module, Glide.51 To validate the docking protocol, the conformation of the cocrystallized ligand was compared with the top pose obtained through extra precision (XP) and standard precision (SP), and by comparison, it was identified at XP that a similar orientation was attained to the cocrystallized ligand with an RMSD distance of >1Å. Thus, all of the molecular docking studies were performed at XP. Molecular Dynamic Simulation. The docked complex of 4ab with CDK2 and CDK9 was subjected to MD simulation studies to understand important interactions involved in providing stability of the protein−ligand complexes. To perform the MD simulations, a SPC (simple point charge) solvent model with cubic boundary conditions within a radius of 12 Å was used to define the core, and the whole complex was neutralized by adding Na+ and Cl− counterions to stabilize the complex to perform simulation studies. These complexes were further minimized using a hybrid method of the steepest descent (SD) and Broyden−Fletcher−Goldfarb−Shanno algorithms (LBFGS) with a convergence threshold of 1 kcal/mol/Å and 2000 iterations. The MD simulation was carried out at NPT ensemble with a pressure of 1 bar and a temperature of 300 K using the Nosé−Hoover chain thermostat and Martyna−Tobias−Klein barostat methods. Coulombic interactions were defined by a short-range cut off radius of 9.0 Å and by a long-range smooth particle mesh Ewald tolerance to 1 × 10−9. The whole model system was relaxed before a simulation run of 10 ns with a recording interval of 1.2 ps (for energy) and 4.8 ps (for trajectory) using the Maestro−Desmond interoperability tool52 (version 4.1, Schrodinger, LLC, 2015). Western Blotting: Materials and Methods. Monoclonal antibody Mcl-1 (no. 94296), p-Rb (no. 8516), and polyclonal antibody p-Rpb1CTD (Ser2/5) (no. 4735) were purchased from Cell Signaling Technology, and monoclonal beta actin (no. A3854) and protease inhibitors were obtained from Sigma. The nonlisted chemicals were used from Sigma.

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Amit Nargotra: 0000-0003-3561-219X Ram A. Vishwakarma: 0000-0002-0752-6238 Parvinder Pal Singh: 0000-0001-8824-7945 Author Contributions Δ

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the financial support of CSIR and DST/SJF through Research Grants BSC0205/MLP5005 and GAP2130. U.S., V.K. thanks UGC. G.C., G.M., A.S., A.S., M.J.M., S.K.G., H.A., T.T., P.K.S., and P.K. thanks CSIR. S.U.K. thanks DST, and P.M. thanks ICMR for their fellowships. IIIM Communication no. IIIM/2034/2017.

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ABBREVIATIONS USED 5-FU, 5-fluorouracil; AUC0−t, area under the plasma concentration−time curve from 0 to the last measurable time point; AUC0−∞, area under the plasma concentration−time curve from time zero to infinity; Cmax, maximum observed plasma concentration; C0, extrapolated concentration at zero time point; CL, clearance; CDK, cyclin-dependent kinase; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; LE, ligand efficiency; iv, intravenous; ip, intraperitoneal; MD, molecular dynamics; MTD, maximum tolerated dose; PR, progesterone receptor; q.s., quantum satis; Rb, retinoblastoma proteins; SGF, stimulated gastric fluids; SIF, stimulated intestinal fluids; t1/2, elimination half-life; TGI, tumor growth inhibition; TNBC, triple-negative breast cancer; Vd, volume of distribution

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00663. Spectra of all compounds (PDF) Molecular formula strings (CSV)

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G.C. and S.U.K. contributed equally.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. 9487

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