9 Inhibitors with

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Design of Novel 3-Pyrimidinylazaindole based CDK2/9 Inhibitors with Potent In-vitro and In-vivo Antitumor Efficacy in a Triple-Negative Breast Cancer Model Umed Singh, Gousia Chashoo, Sameer U. Khan, Priya Mahajan, Amit Nargotra, Girish Mahajan, Amarinder Singh, Anjna Sharma, Mubashir Javeed 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 J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00663 • Publication Date (Web): 16 Nov 2017 Downloaded from http://pubs.acs.org on November 19, 2017

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Journal of Medicinal Chemistry

Madishetti, Sreedhar; CSIR-Indian Institute of Integrative Medicine, Pharmacokinetic & Pharmacodynamic Division; Academy of Scientific and Innovative Research Nandi, Utpal; CSIR-Indian Institute of Integrative Medicine, Pharmacokinetic & Pharmacodynamic Division; Academy of Scientific and Innovative Research Singh, Gurdarshan ; CSIR-Indian Institute of Integrative Medicine, Pharmacokinetic & Pharmacodynamic Division; Academy of Scientific and Innovative Research Mondhe, Dilip ; Indian Institute of Integrative Medicine CSIR, Cancer Pharmacology Division; Academy of Scientific and Innovative Research Bhushan, Shashi; Indian Pharmacopoeia Commission; CSIR-Indian Institute of Integrative Medicine, Cancer Pharmacology Division Malik, Fayaz; CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-180001, India, Cancer Pharmacology Division; Academy of Scientific and Innovative Research Mignani, Serge; Université Paris Descartes, PRES Sorbonne Paris Cité, CNRS UMR 860, Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologique Vishwakarma, Ram; Indian Institute of Integrative Medicine, Medicinal Chemistry Division; Academy of Scientific and Innovative Research Singh, Parvinder; Indian Institute of Integrative Medicine, Medicinal Chemistry Division; Academy of Scientific and Innovative Research

ACS Paragon Plus Environment


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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,¶∆ Sameer U. Khan,¶∆ Priya Mahajan,┴ Amit Nargotra,┴ Girish Mahajan,¶ Amarinder Singh,‡ Anjna Sharma,‡ Mubashir J. Mintoo,¶ Santosh Kumar Guru,¶ Hari Prasad 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, ¶,ɸ 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;

ɸ

Indian Pharmacopoeia Commission, Sector-23, Raj Nagar,

Ghaziabad-201002; ±Université Paris Descartes, PRES Sorbonne Paris Cité, CNRS UMR 860, Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologique, 45, rue des Saints Pères, 75006 Paris, France.


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



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Introduction Fundamentally, our understanding towards biological processes has opened up new opportunities to tackle diseases, and the recent advancement highlighting the kinases as 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 are found deregulated in number of pathological conditions such as wide variety of human cancers, neurodegenerative disorders, inflammation, renal and infectious diseases including malaria.1,2,3 These CDKs form a CDK/cyclin complex for their functioning and human genome encodes 21 CDKs (1−11a, 11b−20) and over 15 cyclins (A−L, O, T, and Y), 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, CDKs involved in transcriptional regulation are 7, 8, 9, and 11 and their cyclins are C, H, L, and T.1,2 Deregulation in the CDKs is found in number of malignancies and therefore, number of small molecule CDK inhibitors were designed and reported. From >20 years, CDK inhibitors have been investigated for anti-cancer potential. In the initial phase, most of the candidates are pan-CDK inhibitor 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 that too has 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, and several selective inhibitors have been reported in the recent past and most of them have been found to be an inhibitor of CDK4 and 6 and the successful candidate are Pfizer’s Palbociclib,7,8 Lilly’s Abemaciclib9 and Novartis’s Ribociclib10.


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The reason for their success is based on the fact that CDK4 and 6 regulate tumorsuppressor 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 US-FDA,12 while abemaciclib9 got fast tract approval and ribociclib got approval for priority review after Phase-III trial.13 Recently, the FDA approved ribociclib in combination with an aromatase inhibitors for the first-line treatment of advanced breast cancer.36 However, recent studies reported that cancers and embryonic cell lines lacking CDK2, CDK4 and CDK6 kinases proliferate normally, suggested that they are functionally redundant and their function can be controlled by other cell-cycle kinases.14,15 On the other hand, 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 focused by many groups.16,17,21,23 Moreover, efforts towards understanding the mechanism of first CDKbased clinical candidate flavopiridol revealed that CDK9-mediated pathway is the primary mechanism responsible for its anti-cancer activity such as in chronic lymphocytic leukemia.19-21 In addition to this, first orally-active CDK inhibitor R-roscovitine was also reported to perform its function through transcriptional CDK.4,22 During the past decade, attempts have been made towards the exploitation of transcriptional CDK for anti-cancer 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 anti-cancer efficacy.24 In this direction, 3-pyrimidinylazaindole derivatives namely meriolins represent the hybrid structure of two natural products based CDK inhibitors namely variolins and meridianins (both coming from marine natural sources), known for



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inhibiting both cell cycle (CDK2) and transcriptional (CDK9) CDKs (Fig 1). In addition, meriolins also possessed potent activity in cell-based assay 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 of nanomolar potency in-vitro and good in-vivo efficacy of meriolins, they have poor physico-chemical and PK properties and, consequently, limits further clinical development. Based on these data, we designed and synthesized novel 3-pyrimidinylazaindole CDK2/9 inhibitors with potent in-vitro and in-vivo antitumor efficacy such as in triple negative breast cancer model (TNBC) which is responsible for a large proportion of breast cancer deaths, duo to its generally aggressive clinical course.28,29 Therefore, we have started medicinal chemistry of 3-pyrimidinylazaindole skeleton (4, Figure 1) to discover new generation analogs having potency against both cell-cycle and transcriptional CDKs along with better physico-chemical and PK properties.

Fig. 1: Literature precedent and present strategy


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Results and discussion Design and Chemistry: A medicinal chemistry programme was initiated with an aim to identify the sites for modification which should not hampered CDKs potency and used for improving physicochemical and pharmacokinetic properties. The inhibition of CDKs requires 7-azaindole as key pharmacophore, which involved N1 and N7 in interaction with the hinge residues i.e. Leu83, Glu81 in CDK2 and Cys106, Asp104 in CDK9 and best analogs among them are 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 transcription respectively. The targeted kinase domain of CDK2 and CDK9 shares similar secondary structure, the active site is present in between the two lobes, an upper small lobe constitutes mainly helices and lower larger lobe comprises of β-sheets. These two proteins shares 40% sequence identity calculated via Omega 3 online database.30 All the insilico studies of 3-pyrimidinylazaindole derivative (meriolin 3, most potent derivative) with CDK2/cyclin A and CDK9/cyclin T were carried out using Schrodinger suite 2015. Binding pocket analysis of the standard molecule (merolin 3) with respect to CDK2 (PDB Id: 3BHT)26 and CDK9 (PDB Id : 4IMY)31 protein infers that there is a scope of modification such as introduction of hydrogen bond acceptor, hydrogen bond donor and hydrophobic regions, which are depicted in Figure 2. We decided to prepare several original 3pyrimidinylazaindole derivatives 4a, 4b, and 4c corresponding to substitution(s) in position(s) 5, 2’ and 1, respectively (Figure 1). All of these compounds were tested in biochemical assays of cyclin-dependent kinase inhibition (CDK2/9).



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H2N COOH

Lys33

O Hydrophobic region

H3N

COOH

HO NH2

N OMe

N O

NH2 COOH Leu83

OH

N H

N

HO

Glu51

NH2

NH2 C

COOH COOH Asp145

O HO

NH2 Glu81

Hinge region

A

B

H2N COOH

COOH

Lys48

H2N

O

Phe103 H3N N OMe

COOH

HO

NH2 Glu66 NH2

N

O

OH NH2

N HOOC

NH2

SH Cys106

COOH Asp167

N H O

OH

H2N COOH Asp104

Fig. 2: Binding pocket analysis of CDK2 and CDK9 active and potential sites of modifications of 3pyrimidinylazaindole

Approach 1: Design and Synthesis of 4a It is worth mentioning that in case of meriolins, most potent analogs described in the literature are meriolin 3 and 5, where hydrophobic substitutions were present at 4th- position of 7-azaindole ring. The substitution at 4th-position of 7-azaindole improves the activity significantly in contrast to the parent classes namely meriolin1 and meridianins. However, interestingly, our analysis suggested that substitution at 5th positions of ring A is also acceptable and could be utilized for the improvement of physico-chemical 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 in to discriminate, as far as possible, between enthalpic efficiency and entropy profile (H driven > S driven) but to have both, and, consequently,


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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 transformations. Thus, as shown in Scheme 1, the synthesis of ring A analogs began with a simple Suzuki cross coupling reaction between 5-bromo-7-azaindole 5 and arylhetero-aryl boronic acids (6a-s)34,35 which gave 5-aryl/hetro-aryl substituted 7-azaindoles (7a-s) in good yields (60-95 %). Iodination of 7a-s in the presence of molecular iodine gave 8a-s which further treated with Boc anhydride to synthesized 9a-s in good yield (90-97 %). Next, Masuda borylation reaction on 9a-s generated key intermediate 10a-s (80-94 %), which on coupling with 4-chloro-2(amino)pyrimidine 11 under Suzuki conditions gave final targeted compounds 4aa-4as (3965 %).

4ah

4ao

4ab

4ai

4ap

4ac

4aj

4aq

4ad

4ak

4ar

4aa: Ar' =



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4ae

4al

4af

4am

4ag

F

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4as

4an

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

Approach 2: Design and Synthesis of 4b The molecular docking studies suggested that modification at amino group (2’position) of ring C is feasible. Taking this information into account, synthesis of ring C analogs was made by incorporating 2-aryl/hetroarylethane amines in place of amino group of ring C (Scheme 2). The 7-azaindole 12 was used as starting material and treated with iodine under basic condition to generate 3-iodo-7-azaindole 13 (95 %) followed by the protection of NH of azoles with Boc anhydride to generate N-Boc-3-iodo-7-azaindole 14 (98 %). Then the regioselective Masuda borylation of the 14 gave boronate ester 15 with 80 % yield. Next, Suzuki coupling reaction was performed between boronate ester 15 and 4-chloro-2(methylthio)pyrimidine 16 affording 3-pyrimidinyl-7-azaindole 17 with thiomethyl group at 2’-position in 66 % yield. The oxidation of thiomethyl group of 17 was performed with metachloroperbenzoic acid (m-CPBA) to generate sulfone 18 with 90 % yield. The generated sulfones 18 were then treated with different 2-aryl/heteroarylethane amines 19 to furnish the target compounds 4ba-4bd with 50-68 % yield.36 In addition to this, several seconadary amines (20) were also introduced and generated corresponding analogs 4be-4bh with 52-60 % yield in order to know the importance of H-bond donor group on the biochemical assays.


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4ba: R

4bd

4bg

4bb

4be

4bh

4bc

4bf

Scheme 2: 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 oC, 3 h; d). Pd(PPh3)4, Cs2CO3, anhydrous MeOH, 100 oC, 35 h; e). m-CPBA, CH2Cl2, rt, 1 h.; f). THF, 85 oC, overnight.

Approach 3: Design and Synthesis of 4c As shown in Scheme 3, in order to know the effect of the disruption of hinge region bonding, a series of analogs were prepared where sulfonamide moieties were introduced on ring B. Synthesis of ring B modified analogs were prepared by coupling intermediate 15 with 4-chloro-2-(amino)pyrimidine 11 under Suzuki conditions which gave compound 21 with 60 % yield. The compound 21 on treatment with different aryl/heteroaryl sulfonylchlorides 22 furnish required compounds 4ca-4cg with 65-80 % yield (Scheme 3).



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4ca: Ar =

4ce

4cb

4cf

4cc

4cg

4cd

Scheme 3: Reagents and conditions: a). 11, Pd(PPh3)4,Cs2CO3, anhydrous MeOH, 100 oC, 49 h, 60%; b) (i). 21, NaH, anhydrous DMF, rt, 2 h.; (ii). 22, 0-rt, overnight, 65-80 %.

Biological Evaluation: Cell-free enzyme activity and drug-like properties: Cyclin-dependent kinase inhibition. The versatile biological activities exhibited by 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 the synthesized compounds were screened against CDK2/cyclin A and CDK9/cyclin T at 500 nM concentration. The compounds showed > 60 % inhibition were further studies for IC50 determination. The activity profile is shown in Table 1. As the design was not only to improve the affinity but also drug-like


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parameters, therefore, an important metrics of ligand efficiency (LE, binding energy/nonhydrogen atoms, K.cal//mol per non-hydrogen atom) which represents a simple an useful guide to optimize fragment and lead selection in the discover process, and to avoid, as far as possible, the inflation in physicochemical properties.37,38 The suggested LE value was brought above 0.3 also maintained throughout the optimization process.

Table 1: Biological activity and drug-like profile of synthesized analogs 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

% inhibition @ 500 nM (CDK2/cyclinA)

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

IC50 (nM) (CDK2/cyclinA)

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

% inhibition @ 500 nM (CDK9/cyclinT)

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


IC50 (nM) (CDK9/cyclinT)

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

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


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4ca 4cb 4cc 4cd 4ce 4cf 4cg Meriolin 1 Meriolin 3* Flavopiridol

22.6 28.1 28.4 31.3 36.3 48.7 55.2 -

90 11 106

40.0 31.2 50.9 54.4 44.2 52.2 50.3 -

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30 6 3.7

0.60/0.64 0.64/0.60

*Reported in literature

In approach 1, ring A modified analogs (4aa-4as), substituted aryl- and heteroarylmoieties to the 5-position of ring A were introduced and all the analogs have shown excellent nanomolar activity against CDK2/cyclin A and CDK9/cyclin T except 4am along with acceptable LE value (0.64-0.27 for CDK2 and 0.53-0.28 for CDK9). The molecular docking studies revealed that these analogs retain bioactive conformation and showed similar interactions (with Asp145, Leu83, Glu81, Glu51, Lys33 in case of CDK2; Asp167, Cys106, Asp104, Phe103, Glu66, Lys48 in case of CDK9) in comparison to meriolin 3. The substituted groups at 5th-position are lies in a cavity where they surrounded (within the 4Å 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 are responsible for high potency (nM range). Moreover from MD simulation studies it has been observed Leu134 and Ile10 in CDK2; Leu156 and Ile25 in CDK9 form hydrophobic contacts (exist for more than 40 % during the 10 ns simulation run, detail provided in supporting information as Figure S1). In case of aryl rings, monocyclic analogs (4aa-4ak) have shown better activity than bicyclic analogs (4al4am). However, in case of heteroaryl rings, both mono- and bicyclic- analogs (4an-4as) have shown good activity with respect to CDK2 and CDK9. The residues interaction of one of the best analog 4ab with CDK2/cyclin A and CDK9/cyclin T are shown in Figure 3.


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A

B

C

D

Fig 3: Main interaction of 4ab with CDKs: A* and B* represents the interaction of 4ab with CDK2/CyclinA; C* and D* represents the interaction of 4ab with CDK9/Cyclin T. Where A* and C* showing 3D model with full protein core, B* and D* showing 3D model with amino acid residues only

After this, using the approach 2, several structural alterations in relation to the substitution at the amino group of the ring C were made to examine whether the introduction of secondary amines and tertiary amines would affect the CDK inhibition activity. Interestingly, substitution of 3-amino group of ring C with 2-aryl/hetroarylethane amines (4ba-4bd) gave encouraging result and all analogs have shown nano-molar 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). On the other hand, the substitution of 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 linker (4ba-4bd) are



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found active, which orient the analogs in such a way that aryl/heteroaryl rings lies in the solvent accessible area.This conformation further increase the chances of interaction with the residues resides in this region of binding site. In addition, this substitution orients the pyrimidine-2-amine, resulted in the disruption of its NH2 interaction with Glu51 in CDK2 and Glu66 in CDK9. However, the secondary amines substituted analogs 4ba-4bd engaged their interaction as hydrogen bond donor with other amino acid residues viz., Asp145 in case CDK2 and Asp167 in case of CDK9, whereas in 4be-4bh, the compounds with tert-amines, free-NH is not available to form interaction with the acidic residues (Asp145, Glu51 in CDK2 and Asp167, Glu66 in CDK9) thus reduces its binding affinity and could be the reason for decline in the activity. The residues interaction of 4bb (A-D) and 4be (E-F) with CDK2/cyclin A and CDK9/cyclin T are showed in Figure 4.


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A

B

C

D

E

F

Fig 4: Main interaction of ring C modified analogs with CDKs: A* and B* represents the interaction of 4bb with CDK2/Cyclin A; C* and D* represents the interaction of 4bb with CDK9/Cyclin T; E represents the interaction of 4be with CDK2/Cyclin A; F represents the interaction of 4be with CDK9/Cyclin T. Where A*, C*, E* and F* showing 3D model with full protein core, B* and D* showing 3D model with amino acid residues only

In approach 3, ring B modified analogs disrupts the H-bonding interactions between N7/N1 and Leu-83/Glu-81 was made by incorporation of bulkier group (4ca-4cg), leads to



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abolition of activity against CDK2/cyclin A and CDK9/cyclin T. As seen in Figure 5, substitution of bulkier group at N1 position orients the molecules by at 180° and disrupts the bioactive conformation and reason for the abolition of activity. A

B

Fig 5: Interaction of ring B modified analogs with CDKs: A represents the interaction of 4ca with CDK2/Cyclin A; B and) represents the interaction of 4ca with CDK9/Cyclin T. Based on the in-vitro screening results of 3-pyrimidinylazaindole analogs 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 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 (Figure 3-5).

Fig 6: Structure activity relationship in 3-pyrimidinylazaindole analog series


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Cell-based in vitro activity: The eight most potent biochemical (CDK2/9) analogs (4ab-4ad, 4an-4ap, 4ba-4bb and meriolins) were further evaluated against two different human cancer cell lines videlicet HCT-116 (colorectal) and SHSY-5Y (neuroblastoma) for their anti-proliferative potential using an MTT cytotoxicity assay. The results are summarized in Table 2. All the tested analogs have shown interesting antiproliferative activities, however, the analogs 4ab has shown the most promising activity against both the tested cell-lines videlicet HCT-116 (IC50 of 0.2 µM) and SH-SY5Y (IC50 of 0.8 µM) with compare to meriolin1 whereas 4ab showed comparable cytotoxicity profile with meriolin348 against tested cell line.

Table 2: In-vitro cell proliferation activity of 4a and 4b against HCT-116 and SHSY-5Y Compound ID 4ab 4ac 4ad 4an 4ao 4ap 4ba 4bb Meriolin1 Meriolin3 a

HCT-116

SH-SY5Y

(IC50 µM)

(IC50 µM)

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

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

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 the compounds have shown



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high solubility in SGF. Interestingly, three analogs 4ab, 4ac and 4ap have also shown comparatively better profile in other tested media also.

Table 3: Solubility profile of active compounds Compound ID 4ab 4ac 4an 4ap 4ba Meriolin1 a

Solubility (in µg/ml)a Water 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-2-amine (4ao) TLC (MeOH: DCM 1:9) Rf = 0.5; Yield: 44 %; Color/nature: yellow/solid; m.p. = 237-239 o

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 (d, J = 5.2 Hz, 1H), 6.75 (s, 2H);

13

C NMR (126 MHz, DMSO-d6) δ 162.8, 162.4, 156.2,

148.4, 142.0, 139.5, 129.6, 127.6, 126.9, 126.5, 124.7, 120.3, 117.8, 112.4, 104.8; HRMS (ESI-TOF) calc’d for C15H12N5S [M + H+] 294.0813; found 294.0821; HPLC-purity 96 % (tR = 30.8, Method A). 4-(5-(Pyridin-3-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine (4ap) TLC (MeOH: DCM 1:9) Rf = 0.4; Yield: 45 %; Color/nature: yellow/solid; m.p. = 238-240 o

C; 1H NMR (400 MHz, DMSO-d6) δ 12.40 (s, 1H), 9.21 (s, 1H), 9.04 (s, 1H), 8.73 – 8.54

(m, 2H), 8.46 (s, 1H), 8.19 (dd, J = 25.9, 6.3 Hz, 2H), 7.60 – 7.47 (m, 1H), 7.13 (d, J = 5.1 Hz, 1H), 6.70 (s, 2H); 13C NMR (126 MHz, DMSO-d6) δ 163.1, 162.1, 156.8, 148.9, 148.0, 147.8, 142.4, 134.6, 134.2, 129.7, 128.7, 126.4, 123.9, 117.8, 112.7, 105.0; HRMS (ESITOF) calc’d for C16H13N6 [M + H+] 289.1202; found 289.1195; HPLC-purity 97 % (tR = 26.38, Method A). 4-(5-(Benzo[b]thiophen-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine (4aq)



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TLC (MeOH: DCM 1:9) Rf = 0.4; Yield: 40 %; Color/nature: yellow/solid; m.p. = 265-267 o

C; 1H NMR (400 MHz, DMSO-d6) δ 12.44 (s, 1H), 9.27 (s, 1H), 8.71 (s, 1H), 8.46 (d, J =

1.9 Hz, 1H), 8.18 (d, J = 5.2 Hz, 1H), 8.08 – 7.96 (m, 2H), 7.88 (d, J = 7.6 Hz, 1H), 7.45– 7.36 (m, 2H), 7.12 (d, J = 5.3 Hz, 1H), 6.67 (s, 2H);

13

C NMR (126 MHz, DMSO-d6) δ

163.2, 162.0, 157.0, 149.1, 141.6, 140.5, 138.5, 130.06, 130.02, 127.7, 124.8, 124.4, 123.4, 123.3, 122.3, 119.8, 117.8, 112.7, 105.05; HRMS (ESI-TOF) calc’d for C19H14N5S [M + H+] 344.0970; found 344.0945; HPLC-purity 98 % (tR = 29.68, Method A). 4-(5-(Benzofuran-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine (4ar) TLC (MeOH: DCM 1:9) Rf = 0.4; Yield: 39 %; Color/nature: yellow/solid; m.p. = 264-266 o

C, 1H NMR (400 MHz, DMSO-d6) δ 12.45 (s, 1H), 9.37 (d, J = 2.1 Hz, 1H), 8.90 (d, J = 2.1

Hz, 1H), 8.46 (d, J = 2.7 Hz, 1H), 8.19 (d, J = 5.3 Hz, 1H), 7.69 (d, J = 7.8 Hz, 2H), 7.63 (s, 1H), 7.41 – 7.30 (m, 2H), 7.12 (d, J = 5.3 Hz, 1H), 6.69 (s, 2H);

13

C NMR (126 MHz,

DMSO-d6) δ 163.4, 161.8, 157.3, 154.6, 154.1, 149.1, 140.5, 129.8, 128.9, 126.9, 124.2, 123.2, 120.7, 119.5, 117.6, 112.8, 111.1, 105.0, 101.2; HRMS (ESI-TOF) calc’d for C19H14N5O [M + H+] 328.1198; found 328.1200; HPLC-purity 99 % (tR = 29.71, Method A). 4-(5-(5-Methoxy-1H-indol-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine (4as) TLC (MeOH: DCM 1:9) Rf = 0.3; Yield: 45 %; Color/nature: yellow/solid; m.p. = 210-212 o

C; 1H NMR (400 MHz, DMSO-d6) δ 12.38 (s, 1H), 11.45 (s, 1H), 9.32 (d, J = 1.9 Hz, 1H),

8.89 (d, J = 2.1 Hz, 1H), 8.46 (d, J = 2.7 Hz, 1H), 8.25 (d, J = 5.3 Hz, 1H), 7.40 (d, J = 8.7 Hz, 1H), 7.19 (d, J = 5.3 Hz, 1H), 7.09 (dd, J = 14.0, 1.7 Hz, 2H), 6.82 (dd, J = 8.7, 2.4 Hz, 1H), 6.72 (s, 2H), 3.84 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 163.5, 161.8, 157.4, 153.6, 148.5, 141.3, 137.0, 132.2, 129.2, 129.1, 126.3, 121.9, 117.7, 112.7, 111.8, 111.3, 105.0, 101.5, 98.2, 55.2; HRMS (ESI-TOF) calc’d for C20H17N6O [M + H+] 357.1464; found 357.1467; HPLC-purity 97 % (tR = 30.65, Method A). 1b. Procedure for the synthesis of ring C (4ba-4bh) modified analogs


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1ba. Synthesis of final compounds (4ba-4bh) meta-Chloroperbenzoic acid (m-CPBA) (1.5 mmol) was added portion wise to an ice cooled solution of 17 (1.0 mmol) in dichloromethane and resulting reaction mixture was stirred at 25 0

C for 2 h. After completion of reaction, monitored by thin layer chromatography and

reaction mixture was washed with saturated solution of sodium bicarbonate, extracted with dichloromethane and concentrated invaccuo to obtain beige solid 18, which was used without further purification for the next step. To the solution of 18 in tetrahydrofuran (THF), amines (primary (19), and secondary (20) (2.0 equiv) was added and refluxed at 85 oC for overnight and cooled to room temperature solvents were removed in vaccuo and the residue was absorbed onto celite and purified chromatographically on silica gel with dichloromethane-methanol-aqueous ammonia (isocratic or stepwise gradient) to obtained pure compounds (4ba-4bh). N-Phenethyl-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine (4ba) TLC (MeOH: DCM 1:9) Rf = 0.6; Yield: 65 %; Color/nature: white/solid; m.p. = 220-222 oC; 1

H NMR (400 MHz, CDCl3) δ 11.62 (s, 1H), 8.82 (d, J = 7.8 Hz, 1H), 8.40 (dd, J = 4.7, 1.4

Hz, 1H), 8.25 (d, J = 5.3 Hz, 1H), 8.04 (s, 1H), 7.39 – 7.18 (m, 7H), 6.90 (d, J = 5.3 Hz, 1H), 3.83 (dd, J = 13.3, 6.8 Hz, 2H), 3.02 (t, J = 7.1 Hz, 2H);

13

C 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) calc’d 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; Yield: 60 %; Color/nature: yellow/solid; m.p. = 224-226 o

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



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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) calc’d 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; Yield: 68 %; Color/nature: light yellow/solid; m.p. = 226228 oC; 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);

13

C 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) calc’d 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; Yield: 50 %; Color/nature: light yellow/solid; m.p. = 233235 oC; 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);

13

C 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) calc’d 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; Yield: 52 %; Color/nature: light yellow/solid; m.p. = 265267 oC, 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 =


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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) calc’d 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; Yield: 52 %; Color/nature: light yellow/solid; m.p. = 270272 oC; 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) calc’d 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; Yield: 60 %; Color/nature: light yellow/solid; m.p. 264-266 o

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); 13C 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) calc’d 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; Yield: 54 %; Color/nature: light yellow/solid; m.p. 258260 oC ; 1H NMR (400 MHz, CDCl3) δ 11.19 (s, 1H), 8.70 – 8.62 (m, 1H), 8.31 (dd, J = 4.8,



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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) calc’d for C16H19N6 [M + H+] 295.1671; found 295.1670; HPLCpurity 99 % (tR = 4.98, Method C). 1c. Procedure for the synthesis of ring B (4ca-4cg) modified analogs 1ca. Synthesis of final compounds 4ca-4cg 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 an hour. The resulting mixture was cooled to 0 oC and corresponding sulfonyl chloride (22) (1.5 mmol) was added dropwise and resulting reaction mixture was stirred at room temperature for overnight. It was suspended in ice-cooled water and extracted with ethylacetate (3-4 times), the solvents removed in-vaccuo 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; Yield: 70 %; Color/nature: off white/solid; m.p. = 240242 oC 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);

13

C 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 (ESI-TOF) calc’d 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)


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TLC (MeOH: DCM 0.5:9.5) Rf = 0.6; Yield: 70 %; Color/nature: off white/solid; m.p. = 257259 oC; 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) calc’d 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-2amine (4cc) TLC (MeOH: DCM 0.5:9.5) Rf = 0.6; Yield: 77 %; Color/nature: off white/solid; m.p. = 251253 oC; 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);

13

C 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) calc’d 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-2amine (4cd) TLC (MeOH: DCM 0.5:9.5) Rf = 0.6; Yield: 80 %; Color/nature: off white/solid; m.p. = 245247 oC; 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) calc’d for C18H13F3N5O3S [M + H+] 436.0691; found 436.0698; HPLC-purity 99 % (tR = 8.54, Method D).



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N-(4-((3-(2-Aminopyrimidin-4-yl)-1H-pyrrolo[2,3-b]pyridin-1yl)sulfonyl)phenyl)acetamide (4ce) TLC (MeOH: DCM 0.5:9.5) Rf = 0.5; Yield: 71 %; Color/nature: off white/solid; m.p. = 234236 oC; 1H NMR (400 MHz, 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);

13

C 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) calc’d 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-3yl)pyrimidin-2-amine (4cf) TLC (MeOH: DCM 0.5:9.5) Rf = 0.6; Yield: 79 %; Color/nature: off white/solid; m.p. = 229231 oC, 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) calc’d 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-2amine (4cg). TLC (MeOH: DCM 0.5:9.5) Rf = 0.4; Yield: 65 %; Color/nature: yellow/solid; m.p. = 228230 oC; 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,


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130.3, 127.2, 126.5, 120.9, 119.9, 117.4, 114.0, 107.6, 36.6; HRMS (ESI-TOF) calc’d for C15H14N7O2S [M + H+] 356.0930; found 356.0922; HPLC-purity 98 % (tR= 8.8, Method D). 1d. General procedure for the synthesis of intermediates (9a-s) {1,1-Bis(diphenylphosphino)ferrocene dichloride palladium(II)} (0.05 eq.) 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) ratio. The resulting mixture was stirred at 80 oC for 6-12 h. After cooling to at 25 oC, all solvents were evaporated invaccuo and the residue was filtered onto 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 DMF was dropped to the solution of substituted 7-azaindole 7a-s (1 mmol) and potassium hydroxide (2.5 mmol) in 20 mL DMF at 0 oC - rt and the mixture was stirred for 0.5-1 h. The reaction mixture was then poured on 50 ml ice water containing 1 % ammonia and 0.2 % sodium disulfite. The precipitate was filtered, washed with ice water and dried in vaccuo 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 dichloromethane, 4-dimethylaminopyridine (0.1 mmol) was added and di-tert-butyl dicarbonate (1.2 mmol), dissolved in 20 mL dichloromethane, was added drop wise for 30 min. The mixture was stirred for 0.5-1 h. at 25 oC, washed with 20 mL 0.1 N HCl, and the aqueous phase was extracted with dichloromethane (2 x 50 mL). The combined organic layers were dried with sodium sulphate, 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 of 9a-s, which solidifies upon storage in refrigerator. tert-Butyl 3-Iodo-5-(4-(trifluoromethyl)phenyl)-1H-pyrrolo[2,3-b]pyridine-1-carboxylate (9a).



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Overall yield 72 % (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-1-carboxylate (9b) Overall yield 70 % (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]pyridine-1-carboxylate (9c) Overall yield 63 % (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[2,3-b]pyridine-1carboxylate (9d) Overall yield 68 % (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) Overall yield 63 % (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) Overall yield 66 % (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-1-carboxylate (9g)


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Overall yield 73 % (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) Overall yield 61 % (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) Overall yield 69 % (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) Overall yield 61 % (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-1-carboxylate (9k) Overall yield 67 % (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]pyridine-1-carboxylate (9l) Overall yield 64 % (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]+.



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tert-Butyl 3-Iodo-5-(2-methoxynaphthalen-1-yl)-1H-pyrrolo[2,3-b]pyridine-1carboxylate (9m) Overall yield 59 % (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) Overall yield 68 % (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-1-carboxylate (9o) Overall yield 68 % (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]+. tert-Butyl 3-Iodo-5-(pyridin-3-yl)-1H-pyrrolo[2,3-b]pyridine-1-carboxylate (9p) Overall yield 58 % (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) Overall yield 60 % (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]pyridine-1-carboxylate (9r)


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Overall yield 68 % (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[2,3b]pyridine-1-carboxylate (9s) Overall yield 56 % (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); m/z 589. 1e. Synthesis of key intermediate (14) A solution of iodine (1 mmol) in 20 mL DMF was dropped to the solution of 7-azaindole 12 (1 mmol) and potassium hydroxide (2.5 mmol) in 20 mL DMF at 0 oC-rt and the mixture was stirred for 30 min. The reaction mixture was then poured on 50 ml ice water containing 1 % ammonia and 0.2 % sodium disulfite. The precipitate was filtered, washed with ice water and dried in vaccuo to obtain a yellow solid 13. The obtained solid was used without further purification for the next step. It was suspended in 10 mL dichloromethane, 4dimethylaminopyridine (0.1 mmol) was added and di-tert-butyl dicarbonate (1.2 mmol), dissolved in 20 mL dichloromethane, was added drop wise for 30 min. The mixture was stirred for 30 min. at 25 oC, washed with 20 mL 0.1 N HCl, and the aqueous phase was extracted with dichloromethane (2 x 50 mL). The combined organic layers were dried with sodium sulphate, 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 refrigerator. tert-Butyl 3-Iodo-1H-pyrrolo[2,3-b]pyridine-1-carboxylate (14)



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TLC (EtOAC: Hexane 1:9) Rf = 0.6; Color/nature: 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); m/z 344. 1f. 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 argon atmosphere in a dry screw-cap vessel with septum. Then, 5 mL of dry dioxane were 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 to at 25 oC (water bath), 5 mL of dry methanol, 1.00 mmol of 2-thiomethyl-4chloropyrimidine (16) 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 to at 25 oC (water bath) the solvents were removed in vaccuo 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 17 can be further purified by suspending in dichloromethane, sonication in ultrasound bath for 0.5-1.0 h, filtration and drying in vaccuo overnight 12 h to obtained the compounds 17. 3-(2-(Methylthio)pyrimidin-4-yl)-1H-pyrrolo[2,3-b]pyridine (17) TLC (MeOH: DCM 1:9) Rf = 0.6; Yield: 58 %; Color/nature: 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) calc’d C12H11N4S for [M + H+] 243.0704; found 243.0702. 1g. Synthesis of compound 21


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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 argon atmosphere in a dry screw-cap vessel with septum. Then, 5 mL of dry dioxane were 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 to at 25 oC (water bath), 5 mL of dry methanol, 1.00 mmol of 2-amino-4chloropyrimidine (11) 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 to at 25 oC (water bath) the solvents were removed in vaccuo 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 21 can be further purified by suspending in dichloromethane, sonication in ultrasound bath for 0.5-1.0 h, filtration and drying in vaccuo overnight 12 h to obtained the compounds 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; Yield 60 %; mp 222 °C; 1H NMR δ (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 δ (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. 2. Experimental section:Biology 2a. In-vitro biochemical assay 2aa. Radioactive assay: General protocol for kinase assay. All assays except asterisk marked (*) were carried out using a radioactive (33P-ATP) filter-binding assay.41



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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 pH7.5, 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 incubated for 30 min at room temperature. Assays were stopped by 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 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml 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, 0.1mM EDTA, 10mM DTT, 1mg/ml BSA, 0.3 mM YSPTSPSYSPTSPSYSPTSPKKK, 10 mM magnesium acetate and 0.05 mM [33P-γ-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays were stopped by 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 2ab. Luminescence assay for CDK: Compounds 4ah and 4ak were assayed by following 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 co-relates with 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 well ideal for both primary screening as well as kinase selectivity profiling. Briefly, the assay is performed in a white 96 well plates taking both reaction mixture (kinase reaction in the presence of substrate) and blank control (kinase reaction in the absence of


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substrate) into consideration. The respective CDK/Cyclin reaction is initiated by the addition of 5 µl of 250 µM ATP Assay Solution (1ml of ATP Assay Solution is prepared by adding 25 µl ATP Solution (10 mM) to 500 µl of 2X buffer and 475 µl of dH20) bringing the final volume up to 25 µl and the reaction mixture is incubated at 30 oC for 15 min. After the incubation period, the reaction termination and the remaining ATP depletion is done by adding 25 µl of ADP-Glo Reagent to each well and the reaction mixture is further incubated at ambient temperature for another 40 min. After this, a 50 µl of Kinase Detection Reagent (Prepared by mixing Kinase Detection Buffer with Lyophillized Kinase Detection Substrate) is added to each well and the plate is incubated again for 30 min. Finally, the 96-well reaction plate is read on a Luminescence plate reader and the ADP produced (nmol) in the presence and absence of substrate is determined. Percent Kinase Inhibition is calculated as: % Kinase Activity = Luminescence of Test- Luminescence of Blank X 100 Luminescence of Control-Luminescence of Blank

% Kinase Inhibition= 100- % Kinase Activity 2b. In-vitro antiproliferative activity/cell line assay 2ba. Cell Culture and Growth Conditions A panel of Human cancer cell lines were procured from 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 streptomycin and 100 units/ml penicillin in 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. Cells were cultured in RPMI-1640 (Sigma) supplemented with 10 % fetal bovine serum, sodium pyruvate, nonessential amino acids, and 1X Anti biotic anti mycotic (Gibco,) at 37 °C in a humidified atmosphere with 5% CO2. Tumor cell suspension



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with more than 90% viability was prepared from subconfluent cultures by the treatment of trypsin-EDTA solution (Invitrogen).43 2bb. Method for Sulforhodamine B Assay (SRB Assay) The assay was carried out in which cell suspension of optimum cell density was seeded in 96 well flat bottom plates (NUNC). Inoculation densities per well used in the screen for T47D (12000), HCT-116 (7000). 100 µL of cell suspension was plated. The cells were then exposed to 100 µM concentration of test materials containing complete growth medium along with AKBA as positive control for 24 hours. The plates were again incubated under the same conditions for another 48 hours at 37 °C. Further, cells were fixed with ice cold TCA (trichloroacetic acid) for 1 hour at 4 °C. After 1 hour, the plates were rinsed three times with water and allowed to air dry. After drying, 100µl of 0.4% SRB dye was added for half an hour at room temperature. Plates were then washed 3 times with 1% vol/vol acetic acid to remove the unbound SRB. After drying at room temperature, the bound dye was solublized 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 mins to solublize 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 % Cell Viability= 100 × (T – T0) / (C – T0) % Growth inhibition = 100 - % Cell viability. T: Absorbance of Test sample, T0 : Absorbance of Blank, C: Absorbance of Control

2bc. Methods for 4T1 mouse breast cancer cell line MTT Cell proliferation assay Cell viability was measured by MTT assay. Briefly cells were trypsinised and seeded in 96 well plate. After 24 hours of incubation, cells were treated with different concentrations of


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4ab and meriolins for 48 hours. Cells were incubated with MTT reagent at final concentration of 0.25 mg/ml for the last four hours before termination and absorbance was measured at 570 nm. 2c. Determination of thermodynamic equilibrium solubility by 96-well plate-based assay The compounds 4ab, 4ac, 4an, 4ao, 4ba and meriolin1 were dissolved in methanol to get 2000 µg/mL 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) is in solid form in the beginning of the experiment. Thereafter, 200 µl of the dissolution medium (water) was added to the wells and 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 aluminium foil and were kept overnight for equilibration. Later, the plates were centrifuged at 3000 rpm for 15 min (Jouan centrifuge BR4i). Samples of 50 µl was withdrawn into UV 96-well plates (Corning® 96 Well Clear Flat Bottom UV-Transparent Microplate) for analyses with plate reader at 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 saturation point and the corresponding concentration was noted.44,45 2d. In-vivo pharmacokinetic: Pharmacokinetic studies were performed following single dose administration of Compound 4ab (2.5 mg/kg, IV; 5 mg/kg, IP), meriolin1 (2.5 mg/kg, IV) and meriolin3 (2.5 mg/kg, IV) in male BALB/c mice. Each study done by using total ten animals, divided into two groups (n=5) for sparse sampling. Formulation was prepared using 5% DMSO, 2.5% absolute alcohol, 2.5% solutol, and normal saline (q.s) for dose administration. Blood samples were collected at 0.083h (IV only), 0.25h, 0.5h 1h, 2h, and 4h after i.v./i.p. dosing. Plasma was separated and processed for estimation of Compound 4ab and meriolin1 by LC-



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MS/MS. Plasma concentration vs. time profile data was analyzed using PK solution software (Summit Research Services, Colorado, USA) by non-compartmental method for the pharmacokinetic parameters.46

2e. Maximum tolerance dose study of 4ab Ehrlich ascites carcinoma (EAC) cells were collected from the peritoneal cavity of the swiss mice harbouring 8-10 days old ascitic tumor. 1x107 EAC cells were injected intramuscularly in right thigh of 24 swiss male mice selected for the experiment on day 0. The next day, animals were randomized and divided into three groups. Two treatment groups contained 7 animals each and one control group contained 10 animals. Treatment was given as follows: Group I: 4ab (15, 30, 45 mg/kg iv and 15 mg/kg ip) from day 1-9 The second treatment group was treated with 5-fluorouracil (22 mg/kg, i.p) from day 1-9 and it served as positive control. The control group was similarly administered normal saline (0.2 ml, ip) from day 1-9. On day 9 & 13, tumor bearing thigh of each animal was shaved and longest and shortest diameters of the tumor were measured with the help of vernier caliper. Tumor weight of each animal was calculated using the following formula. Length (mm) x [width(mm)]2 Tumor weight (mg) = ------------------------------------------2 The percent tumor growth inhibition was calculated on day 13 by comparing the average values of treated groups with that of control group. Tumor growth in saline treated control animals was taken to be 100 %. 2f. Anti tumor 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


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were injected with 1×106 4T1 cells per mice by way of a second mammary fat pad. When tumor volumes approached almost 100 mm3, mice were divided into four experimental groups of with five mice in each group. Injections of PBS (Diluent), 4ab (15 mg/kg), Meriolin 3 (15 mg/kg), Doxorubicin (5 mg/kg) as positive control were given in 200 µL total volume at three days interval for 21 days through tail vein (iv). Animal weight and tumor measurements were done two times per week. The effect of treatment on tumor regression was determined by measuring tumor volume. Tumor volume was calculated using the formula (L×B2/2) mm3, (L indicates length; B width) and Verniear Caliper. The experiments were repeated twice. 2g. Molecular modeling studies All 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 firstly the crystal structure information of cocrystallized ligands of CDK2 and CDK9 were collected from the Protein Data Bank (PDB) and the coordinates of those PDB Ids were selected which shares similar co-crystallized 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 molecular modeling studies. Before initiating the docking studies the selected crystal structure were prepared using the Protein Preparation wizard.49,50 Grids were generated at active site, identified on the bases of already co-crystallized ligand to the receptor using receptor grid generation module of Glide. For standardizing the docking protocol the stereoisomers and conformers of co-crystallized ligand were generated and minimized using the OPLS-2005 force field and docked on to the active site of protein through docking module Glide.51 To validate the docking protocol, the conformation of co-crystallized ligand



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was compared with the top pose obtained through Extra precision (XP) and Standard precision (SP), and on comparing it was identified at XP similar orientation was attained to the co-crystallized ligand with an RMSD distance of >1A⁰. Thus all the molecular docking studies were performed at XP. Molecular dynamic simulation The docked complex of 4ab with CDK2 and CDK9 were subjected to MD simulation studies to understand important interactions involved in providing stability of the protein-ligand complexes. To perform MD simulations, 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- counter ion 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. MD simulation was carried out at NPT ensemble with 1 bar pressure and 300 K temperature using Nose-Hover 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 1e-09. 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 MaestroDesmond interoperability tool52 (version 4.1, Schrodinger, LLC, 2015). 2h. Western blotting: Materials and methods Antibodies: Monoclonal antibody Mcl-1(#94296), p-Rb (#8516), polyclonal antibody pRpb1-CTD (Ser2/5)(#4735) were purchased from Cell signaling technology, monoclonal beta actin (#A3854) and protease inhibitors were obtained from sigma. Non listed chemicals used from Sigma.


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ASSOCIATED CONTENT *S Supporting Information The Supporting Information is available free of charge. Spectras of all compounds Molecular formula string (CSV)

AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected] Author contributions ∆

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

ORCID Umed Singh: 0000-0003-0683-7533 Ram A. Vishwakarma: 0000-0002-0752-6238 Parvinder Pal Singh: 0000-0001-8824-7945 Notes The authors declare no competing financial interest. IIIM Communication Number. IIIM/2034/2017

ACKNOWLEDGMENTS The authors acknowledge the financial support of CSIR through Research Grants BSC0205, MLP5005. 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., P.K. thanks CSIR; S.U.K. thanks DST; and P.M. thanks ICMR for their fellowships.



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ABBREVIATIONS USED 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; Fig, figure; HER2, human epidermal growth factor receptor 2; iv, intravenous; ip, intraperitoneal; MD, molecular dynamics; MTD, maximum tolerated dose; PR, progesterone receptor; RB, retinoblastoma proteins; t1/2, elimination half-life; TNBC, triple negative breast cancer; Vd, volume of distribution.

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A mouse model for triple-negative breast cancer tumor-initiating cells



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Graphical Abstract:

N C N

F

A N

NH2

B N H 4ab In-vivo profile Effect of 4ab on tumor volume

In-vitro biochemical assay In-vitro cell line assay

HCT-116, IC 50 = 0.2 SH SY5Y, IC50 = 0.8 ~ 33 (IC 50 HEK/IC 50 MCF-7)

Safety ratio Solubility profile

Pharmacokinetic

CDK2/cyclinA, IC 50 = 0.0055 CDK9/cyclinT, IC50 = 0.024

Water = 72.467 g/ml PBS = 98.250 g/ml SGF = 53.733 g/ml SIF = 138.233 g/ml

t1/2 = 2.5 h, AUC(0-t) = 437.1 ng*hr/ml, AUC(0-inf.) = 794.6 ng*hr/ml


9 Inhibitors with

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