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Discovery of SY-5609: A Selective, Noncovalent Inhibitor of CDK7
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  • Jason J. Marineau*
    Jason J. Marineau
    Syros Pharmaceuticals Inc., 35 Cambridge Park Drive, Fourth Floor, Cambridge, Massachusetts 02140, United States
    *Email: [email protected]. Phone: (617)-674-9075.
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    Orcidhttps://orcid.org/0000-0002-4321-0038
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    Syros Pharmaceuticals Inc., 35 Cambridge Park Drive, Fourth Floor, Cambridge, Massachusetts 02140, United States
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    Shanhu Hu
    Syros Pharmaceuticals Inc., 35 Cambridge Park Drive, Fourth Floor, Cambridge, Massachusetts 02140, United States
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    Syros Pharmaceuticals Inc., 35 Cambridge Park Drive, Fourth Floor, Cambridge, Massachusetts 02140, United States
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    Paraza Pharma Inc., 2525 Avenue Marie-Curie, Montreal, Quebec H4S 2E1, Canada
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    Syros Pharmaceuticals Inc., 35 Cambridge Park Drive, Fourth Floor, Cambridge, Massachusetts 02140, United States
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    Syros Pharmaceuticals Inc., 35 Cambridge Park Drive, Fourth Floor, Cambridge, Massachusetts 02140, United States
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Journal of Medicinal Chemistry

Cite this: J. Med. Chem. 2022, 65, 2, 1458–1480
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https://doi.org/10.1021/acs.jmedchem.1c01171
Published November 2, 2021

Copyright © 2021 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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CDK7 has emerged as an exciting target in oncology due to its roles in two important processes that are misregulated in cancer cells: cell cycle and transcription. This report describes the discovery of SY-5609, a highly potent (sub-nM CDK7 Kd) and selective, orally available inhibitor of CDK7 that entered the clinic in 2020 (ClinicalTrials.gov Identifier: NCT04247126). Structure-based design was leveraged to obtain high selectivity (>4000-times the closest off target) and slow off-rate binding kinetics desirable for potent cellular activity. Finally, incorporation of a phosphine oxide as an atypical hydrogen bond acceptor helped provide the required potency and metabolic stability. The development candidate SY-5609 displays potent inhibition of CDK7 in cells and demonstrates strong efficacy in mouse xenograft models when dosed as low as 2 mg/kg.

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SPECIAL ISSUE

This article is part of the New Horizons in Drug Discovery - Understanding and Advancing Kinase Inhibitors special issue.

Introduction

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Cyclin-dependent kinase 7 (CDK7) is a serine-threonine kinase that plays key roles in both transcription and regulation of the cell cycle. This duality of function makes it an important signaling node controlling many of the hallmarks of cancer (1) and an attractive oncology target. (2,3) Selective drug targeting within the cyclin-dependent kinase (CDK) family has historically proven challenging, but the recent clinical success of CDK4/6 inhibitors provides an example of the potential benefits of selective CDK inhibition. (4) Herein, we describe the discovery of SY-5609, a potent, highly selective, noncovalent inhibitor of CDK7. Optimization began from a series of potent aminopyrimidine CDK inhibitors. Key substitutions, including a critical dimethyl phosphine oxide, were identified to attain the desired selectivity and ADME profile. SY-5609 inhibits CDK7 in both the CAK and TFIIH complexes, resulting in reduced transcription, cell cycle arrest, and induction of apoptosis in cancer cell lines. Daily oral dosing of SY-5609 is well tolerated and induces regression of the HCC70 cell line derived xenograft mouse model. SY-5609 is currently being evaluated in a phase 1 clinical trial (NCT04247126) in patients with select solid tumors.
CDK7, in its trimeric complex with Cyclin H and MAT1, serves as the mammalian cyclin activating kinase (CAK) and is responsible for phosphorylation of the T-loops of other cyclin-dependent kinases (CDKs) including the cell cycle CDKs: CDK1, 2, 4, and 6. Appropriate temporal activation of these CDKs is responsible for coordinating the progression of cells through the cell cycle. (4) Additionally, CDK7 has been shown to activate transcriptional CDKs. (5,6) The CDK7 trimer is also found in the multisubunit general transcription factor TFIIH complex. As part of this complex, CDK7 phosphorylates the C-terminal domain (CTD) of the largest subunit RBP1 of RNA polymerase II (PolII) at Ser5 and Ser7 of its YSPTSPS heptad repeat. This phosphorylation facilitates promoter escape and the initiation of transcription. Beyond this function on PolII, CDK7 is also known to directly phosphorylate or to modulate the expression of many important oncogenic transcription factors such as c-Myc. (7) These features are critical to the amplified transcription and aberrant growth characteristic of cancer and make CDK7 an attractive target in oncology indications. Indeed, five CDK7 inhibitors have entered clinical trials (SY-1365, (8) CT7001, (9) LY3405105, SY-5609, and XL102 (10)). (2,3)
Previous experience with the covalent inhibitor SY-1365, (8) the first selective CDK7 inhibitor in clinical trials, demonstrated the potential of CDK7 inhibition in a variety of cancer types and inspired development of a highly selective and orally available CDK7 inhibitor. This was accomplished via a medicinal chemistry program which optimized a series of highly potent, slow off-rate CDK7 binders. The desired selectivity was obtained without the need for a covalent warhead, and SY-5609 was identified with the required oral PK profile. SY-5609 induces G2/M cell cycle arrest in treated cells and triggers apoptosis in cancer cell lines. Notably, SY-5609 is capable of inhibiting CDK7 in vivo and demonstrates regression in murine xenograft models.

Results and Discussion

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Initial Structure Activity Relationship (SAR) Exploration

During the course of the medicinal chemistry program leading to SY-1365, (8) compound 1 (Figure 1) was identified. This compound exhibited highly potent inhibition of CDK7 and 39% bioavailability after oral dosing in mice. Its selectivity, as assessed in a small panel of cell cycle and transcriptional CDKs (CDK2, CDK9, and CDK12), however, was limited. Docking studies using the publicly available CDK7 X-ray crystal structure (PDB: 1UA2) suggested that in addition to the aminopyrimidine hinge binding, the basic nitrogen of the (S)-3-aminopiperidine ring could make a salt bridge with Asp97 of CDK7, which is conserved across the CDK family and potentially explains the potent binding. To accurately characterize the affinity of these molecules, a surface plasmon resonance (SPR) assay using immobilized CDK7/CyclinH dimer was developed, enabling measurement of compound binding kinetics, including off-rates (koff) reported here and determining subnanomolar Kd values for this series. Optimization of these scaffolds began with the goal of identifying an orally available compound capable of highly selective CDK7 inhibition.

Figure 1

Figure 1. Structure of compound 1 and predicted interactions with CDK7.

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Compound 1, a 2-amino-4-indolylpyrimidine kinase inhibitor (Table 1) was identified as a potent CDK7 inhibitor with limited selectivity. Replacement of the 5-Cl substituent with trifluoromethyl afforded compound 2, which was more potent but slightly less selective. Improved selectivity could be obtained by substitution at the 5-position of the pyrimidine ring with an ethyl group (3), but at the cost of reduced cellular potency and increased koff from 0.0038 s–1 for compound 1 to 0.015 s–1 for compound 3. Slow off-rate binding kinetics became an important dimension in compound optimization, with faster off-rate compounds such as 3 displaying significantly weaker antiproliferation effects.
Table 1. Potency, Selectivity, and Cellular Activity of Inhibitorsa
a

IC50 values with CDK7/CycH/MAT1 are at or below the limit of detection in the enzymatic activity assay, so SPR Kd values were employed for SAR.

Substitution at the 7-position of the indole ring in compound 2 with a methyl sulfone to generate compound 4 provided an increase in potency and slight increase in selectivity, particularly over CDK2. Given limitations in the availability of CDK7 crystal structures (11) and to help confirm the putative CDK binding mode and inspire analogues, which could provide increased selectivity over the off targets, the X-ray cocrystal structure of 4 with CDK2 at 1.7 Å resolution was determined (Figure 2, PDB: 7RA5). The increased potency of 4 likely derives from multiple polar contacts in the sugar binding pocket. One sulfonyl oxygen coordinates with the catalytic lysine (Lys33) while the other makes a bidentate water-mediated interaction to both Gln131 and the backbone carbonyl of Glu12 in the P-loop. The indole NH hydrogen bonds with Asp145. These interactions with the catalytic lysine and aspartate are likely conserved between kinases and explain the increased potency but lack of selectivity. Also conserved in addition to the contacts with the hinge residue (Leu83) are the interaction of the 5-CF3 moiety with the phenylalanine gatekeeper residue (Phe80) and a salt bridge between the basic piperidine nitrogen and the conserved aspartic acid (Asp86). Installation of a nitrile at the 6-position of the indole ring to project toward the P-loop afforded compound 5. Compound 5 exhibited increased selectivity over CDK2, 9 and 12, likely through differential interactions with the P-loop. A similar effect was observed for compound 7, in which a methyl sulfone was attached to the 6-position of the indole. Both compounds resulted in a decrease in cellular potency.

Figure 2

Figure 2. X-ray crystal structure of compound 4 with CDK2 (PDB: 7RA5).

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An alternate path to increased selectivity lay with the piperidine ring. Substitution of this ring with gem-dimethyl groups increased the selectivity relative to compound 2 at the 6-position (6) but not at the 5-position (8). While more selective, compound 6 exhibited a slight decrease in CDK7 affinity relative to compound 2 from 0.15 nM to 0.34 nM (6). Compound 9 was designed to incorporate the uncommon sulfone isostere dimethyl phosphine oxide at the 7-position of the indole. (12) This modification increased the selectivity over CDK2, CDK9, and CDK12 relative to the 7-methylsulfonyl compound 4. Compound 9 also exhibited more potent antiproliferative activity in HCC70 cells. Lastly, it was observed that substitution of the 6-position of the indole with small heterocycles such as 3,5-dimethylisoxazole (10) also resulted in an increase in selectivity over the off-target CDKs compared to compound 2.

Development of ADME and PK SAR

Initial compounds in the series (1 and 2) were profiled to assess their ADME properties and found to have acceptable metabolic stability and permeability (Table 2). Upon dosing in mice (1 mg/kg intravenous (IV) and 5 mg/kg oral (po)), these compounds were found to have moderate unbound clearance and significant oral bioavailability (39% and 100%, respectively). The 5-ethyl substitution on the pyrimidine ring (3), which had increased the CDK selectivity, resulted in decreased permeability and almost no oral bioavailability. The 7-methylsulfonyl substitution rendered compound 4 stable upon incubation in mouse liver microsomes and it exhibited moderate clearance. Compounds 5 and 7, with their desirable selectivity profile, unfortunately exhibited increased unbound clearance. This was particularly surprising for 6-methylsulfonyl compound 7, which was stable in liver microsomes. This disconnect and its low LogD (1.33) suggested a contribution from extrahepatic clearance mechanisms. This polarity also likely contributed to the low permeability of 7, which had no oral bioavailability. Selective compounds 9 and 10 also provided limited oral bioavailability, likely due to the polarity of the phosphine oxide 9 (LogD = 0.78), which resulted in low permeability and due to the increased metabolic clearance of 10 in line with its higher lipophilicity.
Table 2. ADME and PK Properties

Identification of SY-5102 and SY-5609

The properties described above provided selective CDK7 inhibitors and those with the desired PK profile and oral availability but not both. On the basis of the existing SAR, combination analogues were designed (Chart 1). These sought to leverage both the substituents that provided the desired potency and selectivity with those that afforded the required metabolic stability and oral bioavailability.

Chart 1

Chart 1. Advanced Analogues Leading to SY-5609
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Compounds 11 and 12 were designed to determine whether the metabolic stability of the 7-position substituents (4) could be combined with the improved selectivity available via substitution at the 6-position (5). Gratifyingly, compound 11 retained the desired potency and metabolic stability of 4 and afforded a similar selectivity profile to 5 (Table 3). This selectivity could be further improved by ethyl substitution at the 5-position of the pyrimidine (12), but with a corresponding decrease in metabolic stability. Both compounds displayed low permeability and were not orally bioavailable, likely in part due to the increasing polarity of these combination analogues (11 LogD = 1.05, 12 LogD = 0.49). Instead, utilizing the more lipophilic 6-position substituent of 10 (3,5-dimethylisoxazolyl) in place of the nitrile to drive selectivity, we found that azaindole substitution at the 7-position also afforded an increase in potency. The resulting compound SY-5102, despite increased unbound clearance, afforded our initial in vivo tool to explore the potential of a selective oral CDK7 inhibitor. When dosed twice daily (BID) in an HCC70 cell line derived xenograft mouse model, SY-5102 showed dose-dependent tumor growth inhibition including tumor regression at 4 mg/kg po (Figure 3).
Table 3. Profile of Advanced Analoguesa
 CDK7 SPR Kd (nM)Enzymatic Activity IC50 (nM)Anti-Proliferation EC50 (nM)LogDMouse Microsomal Stability, Clint (mL/min/kg)MDCK Papp A-B (10–6cm/s)Mouse Unbound IV Cl (mL/min/kg)Mouse po %F
  CDK2CDK9CDK12HCC70     
110.082846033151.05<381.863685
120.1516389072152870.49891.123940
SY-51020.03189907592.6<384.6271236
130.06a77157215612.71643.3259728
SY-56090.07a55241919170212<383.9123947
a

Long dissociation SPR.

Figure 3

Figure 3. Efficacy of SY-5102 in HCC70 Xenograft. SY-5102 was orally dosed twice daily in HCC70 cell line derived xenograft models at 2.5 and 4 mg/kg for 21 days. Ten mice per arm. Tumor volumes and bodyweight were measured twice weekly. One-tail t test was performed to assess significance of antitumor effect of SY-5102 (**** p < 0.0001).

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With SY-5102 demonstrating that an oral, noncovalent, selective CDK7 inhibitor could achieve strong tumor growth inhibition, we sought a compound with improved PK to support once daily dosing and even higher selectivity. Returning to the original SAR, the promise of the phosphine oxide moiety in compound 9 was apparent. Its strong hydrogen bonding and high stability have led to increased adoption of this functionality in preclinical discovery programs and even marketed drugs. (13) Combination of 9 with the nitrile at the indole 6-position could provide the desired selectivity profile, but it was likely that permeability and oral bioavailability would suffer. Adding some increased lipophilicity using the gem-dimethyl piperidine rings in compound 13 showed that this type of substitution could bring the physiochemical properties back into a desirable range to rescue permeability and, to some extent, oral bioavailability. Metabolic stability, however, suffered somewhat and the selectivity was not as high as hoped. Leveraging previous SAR from compound 6 and since molecular modeling suggested that the piperidine 6-position would provide a better vector toward the nonconserved Val100, lipophilic substitutions at that position were explored. Moving the gem-dimethyl group to the 6-position led to the discovery of SY-5609. As before (6, Table 1), this resulted in an improvement in selectivity against CDK2, CDK9, and CDK12. This compound exhibits potent (Kd = 0.07 nM), slow-off (koff = 0.0032 s–1) binding to CDK7/Cyclin H. This likely contributes to more potent HCC70 antiproliferative activity than, for instance, compound 12 (287 nM HCC70 EC50 value) with a CDK7/Cyclin H off-rate of 0.0057 s–1. Compound 12 also exhibits approximately 2-fold weaker CDK7 binding and is less selective. SY-5609 not only potently inhibits HCC70 cell proliferation (1 nM EC50 value) but also exhibits increased lipophilicity and permeability (LogD = 2, MDCK Papp 3.91 cm/s) compared to compounds like 9 and 11. It is stable in mouse liver microsomes and exhibits significant oral bioavailability (47%F).

Chemical Synthesis

The compounds described in this paper can be obtained following the synthetic sequences outlined in Schemes 1–8. (14−16) The synthesis of compounds 1 and 3 is depicted in Scheme 1. Intermediate 16 was obtained from commercially available 14 and 15 through a Friedel–Crafts reaction. After indole protection, compound 17 was subjected to a SNAr reaction with chiral amine 18 to afford common intermediate 19. Sequential deprotection of the phenylsulfonyl and Boc groups from 19 provided the final compound 1. To obtain 3, intermediate 19 was first reacted with potassium vinyltrifluoroborate 20 via Suzuki-Miyaura cross-coupling reaction to produce 21, followed by hydrogenation. Two deprotections then furnished 3.

Scheme 1

Scheme 1. Synthesis of Compounds 1 and 3a
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aReagents and conditions: (a) 15, AlCl3, DCE, 80 °C, 16 h, 98%; (b) PhSO2Cl, NaOtBu, THF, 0 °C, 1 h, 69%; (c) 18, DIPEA, NMP, 135 °C, 45 min, μw, 64%; (d) (i) 5N aq. NaOH, dioxane, 70 °C, 3–6 h, (ii) HCl/EtOAc, rt, 50–85%; (e) 20, Pd(OAc)2, cataCXium, Cs2CO3, toluene/water, 120 °C, 4 h, 61%; (f) Pd/C, H2, EtOH, 1 atm, rt, 16 h, quant. yield.

Further analogs (2, 6, and 8) were synthesized (Scheme 2) by reacting commercially available 4-chloro-2-(methylthio)-5-(trifluoromethyl)pyrimidine 23 and the boronic acid 24 to provide intermediate 25, followed by oxidation with m-CPBA to common intermediate 26. Subsequent SNAr reactions between 26 and chiral amines 18, 27, or 28a provided the corresponding Boc, Bn, or Cbz-protected piperidine intermediate 29 (PG, protecting group). Compound 6 was obtained after sequential deprotection of the benzyl group under hydrogenation conditions using both Pd/C and Pd(OH)2/C, and basic hydrolysis of the phenylsulfonamide. Basic hydrolysis of the phenylsulfonamide of 29 followed by acid deprotection of the tert-butylcarbamate (Boc) or carboxybenzyl (Cbz) groups afforded compounds 2 and 8, respectively.

Scheme 2

Scheme 2. Synthesis of Compounds 2, 6, and 8a
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aReagents and conditions: (a) 24, Pd(PPh3)4, Cs2CO3, dioxane/water, 100 °C, 1.5 h, 54%; (b) m-CPBA, DCM/THF, rt, 16 h, 95%; (c) chiral amine 18, 27, or 28a, DIPEA, THF, rt, 16 h, 27–50%; (d) (i) 5N aq. NaOH, dioxane, 70 °C, 3 h, (ii) TFA/DCM, rt, 42%; (e) (i) Pd/C, Pd(OH)2/C, H2, EtOH, 1 atm, rt, 16 h, 70%; (ii) 5N aq. NaOH, dioxane, 70 °C, 16 h, 25%; (f) (i) 5N aq. NaOH, dioxane, 70 °C, 3 h; (ii) HBr/AcOH, DCM, rt, 1 h, 66%; (g) NaH, BnBr, 33%; (h) ZrCl4, MeMgBr, 83%; (i) TFA, DCM, quant. yield; (j) Pd/C, H2, 30 psi, quant. yield; (k) CbzCl, NaHCO3, 93%; (l) HCl, EtOAc, quant. yield.

Scheme 3 presents the synthetic strategy for compounds 5, 7, and 10 with varying substituents at the C-6 position of the indole. Common intermediate 32 was prepared in 3 steps: Friedel–Crafts reaction, N-protection, and SNAr with amine 18. Further indole deprotection provided bromide 33, which was subjected to a Pd-catalyzed cyanation to afford nitrile (5) after Boc deprotection. Alternatively, 33 was converted to the methyl sulfone via an Ullmann reaction with CuI to afford 7. Compound 10 was obtained through Suzuki coupling between 32 and 3,5-dimethylisoxazol-4-yl-4-boronic acid, followed by two deprotections.

Scheme 3

Scheme 3. Synthesis of Compounds 5, 7, and 10a
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aReagents and conditions: (a) (i) 6-bromo-1H-indole, AlCl3, DCE, 80 °C, 2 h, 26%; (ii) NaOtBu, PhSO2Cl, THF, 0 °C to rt, 16 h, 86%; (b) 18, DIPEA, NMP, 140 °C, 1 h, 86%; (c) 5N aq. NaOH, dioxane, 75 °C, 1 h, quant. yield; (d) (i) Zn, Zn(CN)2, Pd2dba3, XPhos, DMAc, 90 °C, 30 min, 95%; (ii) TFA/DCM, 0 °C, 30 min, 62%; (e) (i) CH3SO2Na, CuI, NMP, 140 °C, 2 h, 9%; (ii) 4 M HCl/EtOAc, rt, 1 h, 29%; (f) (i) 3,5-dimethylisoxazol-4-yl-4-boronic acid, Pd(PPh3)4, Cs2CO3, dioxane/water, 100 °C, 1 h, 90%; (ii) 5N aq. NaOH, dioxane, 70 °C, 3 h; (iii) TFA/DCM, 0 °C, 30 min, 60%.

The synthesis of compounds 4 and 9 to vary substituents at the C-7 position of indole is shown in Scheme 4. First, Boc-protected pinacol boronic ester 36 was prepared from 7-bromoindole 34 in two steps: Boc-protection and Ir-catalyzed C–H borylation. Suzuki coupling between 36 and 37 provided the common intermediate 38, where the reaction was heated until full Boc-deprotection of indole occurred. The subsequent Ullmann reaction with sodium methanesulfinate or Pd-catalyzed coupling with dimethylphosphine oxide afforded the corresponding C7-substituted compounds 4 and 9, respectively, after final Boc deprotection.

Scheme 4

Scheme 4. Synthesis of Compounds 4 and 9a
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aReagents and conditions: (a) Boc2O, DMAP, MeCN, rt, 16 h, quant. yield; (b) B2Pin2, [Ir(OMe)(COD)]2, 4,4′-ditert-butyl-2,2′-bipyridine, MTBE, 100 °C, 7 h; (c) 18, ZnCl2, TEA, DCE/tBuOH, rt, 16 h, 29%; (d) Pd(PPh3)4, Cs2CO3, dioxane/water, 95 °C, 8 h, 21%; (e) (i) CH3SO2Na, CuI, NMP, 140 °C, 2 h, 70%; (ii) TFA/DCM, rt, 1 h, 74%; (f) (i) P(O)Me2, Pd(OAc)2, XantPhos, K3PO4, DMF, 150 °C, 45 min, μw; (ii) TFA/DCM, rt, 1 h, 69%.

For compound SY-5102 (Scheme 5), the synthesis of intermediate 43 was straightforward starting from commercially available 6-bromo-7-azaindole 39. The boronic ester 42 was synthesized using a 4-step reaction sequence beginning with Suzuki coupling to 3,5-dimethylisoxazol-4-yl-4-boronic acid followed by N-protection, bromination, and Pd-catalyzed Miyaura borylation. The final two deprotections of the phenylsulfonyl and Boc groups provided SY-5102 as an HCl salt.

Scheme 5

Scheme 5. Synthesis of SY-5102a
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aReagents and conditions: (a) 3,5-dimethylisoxazol-4-yl-4-boronic acid, Pd(dppf)Cl2·DCM, NaHCO3, dioxane/water, 100 °C, 2 h, quant. yield; (b) (i) NBS, DCM, 0 °C, 1 h, 49%; (ii) NaH, PhSO2Cl, DMF/THF, 0 °C, 1 h, 68%; (c) B2Pin2, Pd(dppf)Cl2·DCM, KOAc, dioxane, 100 °C, 1 h, quant. yield; (d) 37, Pd(dppf)Cl2, Na2CO3, dioxane/water, 100 °C, 12 h, 23%; (e) (i) 2N aq. NaOH, MeOH, 60 °C, 1 h; (ii) 4 M HCl/EtOAc, rt, 1 h, 22%.

Synthesis of SY-5609 requires two main fragments, the chiral amine 45 and the functionalized 4-indolyl-5-trifluoromethyl-2-chloropyrimidine 49 (Scheme 6). The chiral amine can be generated by zirconium chloride mediated addition of methylmagnesium bromide to piperidone 44, (17) followed by deprotection. Synthesis of intermediate 49 begins from 46 via Bartoli indole synthesis of 47. Primary amide formation, followed by dehydration to the nitrile, affords 48, which is coupled with dichloropyrimidine 30 through a Friedel–Crafts reaction to provide 49 as the major regioisomer. The chiral amine 45 is then installed through SNAr reaction with 49, followed by the final step, the dimethylphosphine oxide installation via palladium catalyzed coupling, to provide SY-5609. Intermediate 49 was also used for the synthesis of compound 13 (Scheme 6), using the chiral amine 28b.

Scheme 6

Scheme 6. Synthesis of SY-5609 and Compound 13a
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aReagents and conditions: (a) (i) MeMgBr, ZrCl4, THF, −10 °C to rt, 16 h; (ii) TFA, DCM, rt, 16 h; (b) VinylMgBr, THF, −78 °C to rt, 16 h, 79%; (c) (i) CDI, NH4OH, DMF, 0 °C, 10 min; (ii) MsCl, TEA, DCM, 0 °C, 5 min, 83%; (d) 30, AlCl3, DCE, 80 °C, 4 h, 42%; (e) (i) 45, DIPEA, NMP, 130 °C, 3 h; (ii) P(O)Me2, Pd(OAc)2, XantPhos, K3PO4, DMF, 150 °C, 45 min, μw, 42%; (f) 28b, DIPEA, NMP, 130 °C, 4 h, quant. yield; (g) (i) P(O)Me2, Pd(OAc)2, XantPhos, K3PO4, DMF, 145 °C, 45 min, μw, 24%; (ii) Pd/C, H2, Boc2O, EtOH, 1 atm, rt, 48 h; (iii) TFA/DCM, rt, 16 h, 23%; (h) (i) SOCl2, MeOH; (ii) Boc2O, 74%; (i) LiHMDS, CH3I, 38%; (j) (i) NaBH4, EtOH; (ii) MsCl, TEA; (iii) BnNH2, DME, 14%; (k) HCl, quant. yield.

The synthesis of compounds 11 and 12 that contain different C5 substituents on pyrimidine was carried out as shown in Schemes 7 and 8. In case of compound 11, the methylsulfonyl group at indole C7 was installed at the beginning of the synthesis. Thus, 1-bromo-2-fluoro-3-nitrobenzene 51 reacted with sodium thiomethoxide to provide 52, followed by oxidation with m-CPBA and a Bartoli reaction for the indole formation. Next, the bromide in indole 53 was converted into nitrile group through the cyanation reaction using CuCN. Bromination, Boc protection, and Pd-catalyzed borylation reactions provided intermediate 56, which was used for the Suzuki coupling with 37 as described in Scheme 4.

Scheme 7

Scheme 7. Synthesis of Compound 11a
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aReagents and conditions: (a) NaSMe, DMF, 0 to 15 °C, 2 h, 80%; (b) (i) m-CPBA, DCM, rt, 2 h, 65%; (ii) VinylMgBr, THF, −78 °C, 2 h, 42%; (c) CuCN, DMF, 140 °C, 1 h, 80%; (d) (i) NBS, DMF, rt, 2 h, 66%; (ii) Boc2O, DMAP, DIPEA, THF, 80 °C, 4 h, 75%; (e) B2Pin2, Pd(dppf)Cl2, KOAc, dioxane, 80 °C, 4 h, 21%; (f) (i) 37, Pd(PPh3)4, Na2CO3, dioxane/water, 100 °C, 4 h; (ii) TFA/DCM, rt, 30 min, 26%.

Scheme 8

Scheme 8. Synthesis of Compound 12a
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aReagents and conditions: (a) VinylMgBr, THF, −78 to 0 °C, 2 h, 61%; (b) (i) 14, AlCl3, DCE, 80 °C, 12 h, 41%; (ii) SEM-Cl, NaH, DMF, 0 °C to rt, 1.5 h, 52%; (c) (i) m-CPBA, DCM, rt, 2 h, 79%; (ii) CuCN, DMF, 80 °C, 3 h, 63%; (d) 18, DIPEA, NMP, 90 °C, 1 h, 53%; (e) (i) 20, XPhos Pd G1, K3PO4, THF/water, 80 °C, 12 h, 43%; (ii) Pd/C, H2, TEA, MeOH, 15 psi, 15 °C, 1 h, 60%; (f) H2SO4, dioxane, 40 °C, 3 h; then K2CO3, MeCN, 15 °C, 1 h, 10%.

Alternatively, for the synthesis of compound 12 (Scheme 8), intermediate 52 was converted to indole 57 first. Next, Friedel–Crafts reaction followed by SEM protection provided corresponding intermediate 58, which was oxidized to the sulfone 59 with m-CPBA. Then the synthesis was continued following the same strategy as described above in Scheme 1.

Computational Modeling

The publicly available X-ray crystal structure of CDK7 (PDB: 1UA2) (11) proved sufficient to rationalize the potent binding of this series as described above for compound 1. However, this structure lacks the binding partners cyclin H and MAT1, which are important for the catalytic activity of CDK7. (11) Furthermore, the C-helix and the activation loop are both in conformations characteristic of an inactive kinase. (11) This makes detailed analysis of the interactions that lead to the high selectivity of SY-5609 difficult. During preparation of this manuscript, atomic resolution structures of the human CAK complex (CDK7/cyclin H/MAT1) were reported as determined by cryo-electron microscopy. (18,19) This afforded the opportunity to examine the likely binding mode of SY-5609 with active CDK7. Three structures were published with ATP-γ-S as the ligand (PDB: 6XBZ), covalently bound inhibitor THZ1 (20) as the ligand (PDB: 6XD3) and ICE0942/CT7001 as the ligand (PDB: 7B5Q).
The dimethylphosphine oxide moiety on SY-5609 adopts a rigid conformation with the oxygen from the dimethylphosphine oxide coplanar to the indole NH to which it hydrogen bonds in a pseudotricycle. This low energy conformer is also observed in a small molecule X-ray crystal structure of SY-5609 (Supplementary Figure S4) and likely also contributes to the improved permeability of SY-5609. This ring system is proximal to the flexible kinase P-loop, particularly residues from Gly19 to Ala 24. Given the similar hinge binding contacts, lack of covalent attachment, and high resolution, the PDB structure 7B5Q was chosen for modeling the interaction of SY-5609 with CDK7. Docking using the default parameters in Glide (21) was found to rotate the phosphine oxide away from the indole NH rather than preserving the H-bond. To preserve the internal H-bond, the geometry of SY-5609 observed by small molecule X-ray crystallography was rigidly docked to the kinase domain of CDK7 in its trimeric complex with Cyclin H and MAT1. In this docked structure, the expected hydrogen bond interactions between the hinge residue Met94 and the aminopyrimidine are observed. The 6,6-dimethylpiperidine nitrogen forms a salt bridge with Asp97 and the 5-trifluoromethyl group of the pyrimidine packs against the Phe91 gatekeeper residue, consistent with previous observations in this series. To investigate the contribution of water mediated interactions and protein flexibility, the rigidly docked complex of SY-5609, CDK7, cyclin H, and MAT1 was utilized as the starting point for a 50 ns molecular dynamics simulation. Protein and ligand RMSD indicated that the system was well equilibrated. Analysis of this simulation (Figure 4) indicated that the intramolecular hydrogen bond between the phosphine oxide and the indole is stable during the simulation and this carbonyl also forms water mediated hydrogen bonds to the catalytic lysine, Lys41 and Asp155. This aspartic acid is also hydrogen bonded to the indole NH. The piperidine nitrogen forms a stable water network in addition to its salt bridge to Asp97. These interactions likely explain the potent binding of SY-5609, but they involve conserved residues throughout the CDK family and do not provide a rationale for the selectivity. Some of the selectivity of SY-5609 may be accounted for by the 6,6-dimethyl substitution on the piperidine ring, which projects toward hydrophobic Val100 at the beginning of the K-helix, a residue that is unique to CDK7. This bulky substitution likely clashes with the polar Lys89 in CDK2. In the case of the other off targets CDK9 and CDK12, the corresponding residue is a much smaller, less hydrophobic glycine. (22,23) In this case, it is likely that the gem-dimethyl groups make Van Der Waals contacts with the valine. This contact may be locked in place by the neighboring salt bridge and is lost in CDK9 and 12, providing increased selectivity over these targets as also shown for compound 6 (Table 1). The selectivity of SY-5609, however, is much higher than that of compound 6 and not entirely explained by these interactions. Rigid docking of SY-5609 to the off-targets, CDK2, CDK9, and CDK12, failed to elucidate any additional differential interactions. The key indole substitutions of the nitrile at the 6-position and the rigid dimethylphosphine oxide at the 7-position likely drive selectivity by exploiting the conformational preferences of the P-loop. There is even evidence of a transient water-mediated interaction between the C-6 nitrile and the backbone carbonyl of Glu20. The aligned sequence in Figure 4, highlights differences between residues 22 and 25. Ala24, in particular, is unique to CDK7 and likely alters the flexibility of the loop and provides more hydrophobic contact surface on the lower side proximal to the methyl group of the phosphine oxide. The different P-loop conformations are evident in an overlay of the docked structure of SY-5609/CDK7 and the kinase domains of the other CDKs. The CDK7 P-loop diverges outward from the binding site around Gly19, and the end of the loop around Phe23 is tipped up relative to the other CDKs. This likely allows CDK7 to accommodate the substituted indole ring of SY-5609 better than the other CDKs while preserving the key polar interactions. CDK7 P-loop flexibility was previously also suggested to be important for the selectivity of ICE0942/CT7001. (9,19) Molecular dynamics simulations (50 ns) were conducted for the SY-5609 docked complexes of CDK2/cyclin A (PDB: 1PKD), CDK9/cyclin T1 (PDB: 3BLQ), and CDK12/cyclin K (PDB: 6B3E) as well as for the apo complexes of all 4 CDKs. To explore the hypothesis, loop conformation was monitored by the measurement of the mean phi and psi dihedral angles of the residues corresponding Gly19 and Ala24 for each simulation (Supplementary Figure S1). Only for CDK7 were all four measurements comparable between the apo and SY-5609-bound state suggesting that little rearrangement of the P-loop is required to accommodate the ligand, which likely contributes to the observed selectivity.

Figure 4

Figure 4. Computational analysis of the putative binding mode of SY-5609 to CDK7.

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CDK Family and Kinome Selectivity

Initial data clearly show that SY-5609 exhibits potent binding to CDK7 and high selectivity over the primary off-targets of CDK2, CDK9, and CDK12. To confirm the breadth of this selectivity, both SY-5102 and SY-5609 were profiled in a panel of 28 CDK family assays including CDK1, 2, 4–6, 7, 8, 9, 12,13, 16, 17, 19, and 20. Improved selectivity was observed with SY-5609 over SY-5102 across all family kinases tested. Only with CDK7/CycH/MAT1 does SY-5609 have a sub-500 nM IC50. Since the IC50 values with CDK7/CycH/MAT1 are at or below the assay limit (due to enzyme concentration), selectivity could be defined by comparing off target potencies with SPR Kd values for CDK7/cyclin H. This resulted in a selectivity window that increased from 306 fold for SY-5102 to 4340 fold for SY-5609, over CDK16/cyclin Y, which was the closest off-target for both compounds (Supplementary Table S1). The broader kinome selectivity was also measured in a panel of 485 kinases, and SY-5609 was again more selective, with only 10 kinases exhibiting greater than 70% inhibition at 1 μM SY-5609 (Figure 5). Combined, these data suggest that significant engagement of other kinases is unlikely at therapeutically relevant concentrations.

Figure 5

Figure 5. Kinome selectivity of SY-5102 and SY-5609. Kinases that were inhibited at least 70% with 1 μM SY-5102 or SY-5609 in a panel of 485 kinases (SelectScreen Biochemical Kinase Profiling) are shown. The size of the circle depicting each kinase indicates the percentage inhibition, as shown. Kinome trees were generated using KinMapbeta. (24) Illustration reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com) (Supplementary Table S2).

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Cellular Activity of SY-5609

Since inhibition of CDK7 has shown promising antitumor effects in triple negative breast cancer (TNBC) (25) and ovarian (OVA) cancer cells, (26) a small cell panel composed of TNBC, OVA cancer cell lines, and two normal fibroblast cell lines were tested against SY-5609. SY-5609 demonstrated strong antiproliferative effects in cancer cell lines, while it showed over 50% antiproliferative effect in two normal fibroblast cell lines only at the highest dose tested (Figure S2).
In HCC70 cell line, SY-5609 exhibits the expected effects on both the cell cycle and transcriptional targets of CDK7. (2) Treatment with SY-5609 for 24 and 48 h results in inhibition of the phosphorylation of CDK2 at Thr160 (Figure 6A) via loss of CAK function. Inhibition of CDK7 in TFIIH results in reduced phosphorylation of Ser5 in the heptad repeats of the C-terminal domain of RNA polymerase II and a reduction of the levels of c-Myc (Figure 6A). Interestingly, treatment of SY-5609 only leads to mild MCL1 protein reduction at 48 h but not earlier time points (Figure 6A). Treatment with SY-5609 induces apoptosis at 48 and 72 h treatment as measured by Annexin V/PI staining in multiple cancer cell lines but not a primary fibroblast line (Figure 6B). A G2/M cell cycle arrest is also observed (Figure 6C), a logical consequence of the loss of CAK function, which is consistent with Cyclin B1 accumulation observed at 24 and 48 h SY-5609 treatment (Figure 6A).

Figure 6

Figure 6. Cellular effects of treatment with SY-5609. Immunoblotting quantification is presented in Table S3. (A) HCC70 cells were treated with indicated dosage of SY-5609, protein samples were collected at 6, 24, and 48 h post treatment. Phospho-CDK2 T160, total CDK2, c-MYC, MCL1, cyclinB1, RNAPII Ser5 phosphorylation, and total RNAPII protein level were measured by immunoblotting. Vinculin was used as loading control for RNA pol II and Ser5 phosphorylation, while tubulin was used as loading control for the rest of the targets. All immunoblot data are quantified by LICOR. Experiments were performed independently at least three times. (B) HCC70, MDA-MB-468, CAOV3 and OVCAR3, and HDF (adult) cells were treated with SY-5609 for 48 and 72 h and percentage of cells undergoing apoptosis determined by flow cytometry following annexin V-FITC and propidium iodide (PI) staining. Data show mean population from two independent experiments with standard deviation as error bars. (C) HCC70 cells were treated with indicated doses of SY-5609 and fixed after 48 h. DNA was stained with Fxcycle violet stain. Single cell population was determined by FSC-W/FSC-H and SSC-W/SSC-H gating, and G1 and G2/M population was gated as shown on each figure. Experiments were performed independently at least three times.

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In Vivo Efficacy and Pharmacodynamic Effects of SY-5609

Given its promising biochemical, cellular, and pharmacokinetic profile, SY-5609 was tested in an HCC70 cell line derived xenograft mouse model. Daily oral dosing of 2 mg/kg SY-5609 in mice provided a plasma exposure of 261.28 ng h/mL with a Cmax of 50.67 ng/mL (103 nM) and an elimination half-life of 3.33 h. This induced tumor regression over the 21-day dosing period and was well tolerated. No regrowth of tumor was observed out to day 28 (Figure 7).

Figure 7

Figure 7. In vivo efficacy and tolerability of SY-5609. SY-5609 was orally dosed once daily in HCC70 cell line derived xenograft models at 2 mg/kg for 21 days. Ten mice per arm. Tumor volumes and bodyweight were measured twice weekly. One-tail t test was performed to assess significance of antitumor effect of SY-5609 (**** p < 0.0001).

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In a satellite arm designed to measure the pharmacodynamic response, markers involving cell cycle and transcriptional responses to CDK7 inhibition were monitored (Figure 8). At 4 h following a single 2 mg/kg dose, RNA polymerase II Ser5 phosphorylation and CDK2 Thr160 phosphorylation were both decreased, while c-Myc and MCL1 protein levels remained essentially unchanged. Expression of c-Myc mRNA was also decreased, providing evidence of rapid onset of inhibition of the transcriptional functions of CDK7. The reductions in CDK2 p-T160 and p-Ser5 were preserved after 7 daily doses, and the levels of c-Myc and MCL1 proteins were also reduced at steady state. These results are consistent with the observed effects of CDK7 in cells (Figure 6) and exemplify the effects on cell cycle (pCDK2), transcription (pSer5 and c-Myc), and apoptosis (MCL1). It is clear from the efficacy study that this dose level causes tumor growth inhibition even by day 7 (Figure 7).

Figure 8

Figure 8. Pharmacodynamic effects of SY-5609 in HCC70 tumor 4 h after a single dose or after 7 days of QD dosing. Four hours post SY-5609 single dose or after 7 day of QD dosing, tumor and plasma samples were collected from mice, 3 mice per arm per time point. Protein and RNA were harvested from tumor samples. Phospho-CDK2 T160, total CDK2, c-MYC, MCL1, cyclinB1, RNAPII 5 phosphorylation, and total RNAPII protein level were measured by immunoblotting. Vinculin was used as loading control for RNA pol II and Ser5 phosphorylation, while tubulin was used as loading control for the rest of the targets. c-MYC mRNA level was measured by NanoString.

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Conclusions

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Inspired by the promise of CDK7 inhibition in cancer therapy, we report the discovery of SY-5609. Utilizing a key salt bridge to Asp97 and a critical phosphine oxide substitution, this compound exhibits potent and highly selective CDK7 inhibition with a PK profile supportive of daily oral dosing. Treatment of HCC70 cells with SY-5609 results in the decrease of cell cycle CAK targets (pCDK2 Thr160) and TFIIH targets (pSer5 of the RNA polymerase II CTD) in a dose-dependent fashion. This results in a G2/M cell cycle arrest and downregulation of important oncogenes like c-Myc, and ultimately, leads to apoptosis in cancer cells. Daily dosing of SY-5609 at 2 mg/kg induces regression in the HCC70 cell line derived xenograft mouse model of triple negative breast cancer. Observed decreases in pCDK2 Thr160, RNA polymerase II pSer5, c-Myc, and MCL1 describe a pharmacodynamic response consistent with CDK7 inhibition in vivo. SY-5609 was selected for clinical development and is currently being studied in a Phase 1 trial in patients with select solid tumors (NCT04247126).

Experimental Section

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Chemistry

General Methods and Compound Characterization

Unless otherwise noted, reagents and solvents were obtained from commercial suppliers and were used without further purification. The reactions were run under an atmosphere of nitrogen or argon unless otherwise noted. Reaction progress was monitored using a variety of LC instruments equipped with electrospray positive ionization detectors. 1H NMR spectra were recorded at 400 or 500 MHz, and chemical shifts are reported in parts per million (ppm, δ) downfield from tetramethylsilane (TMS). Coupling constants (J) are reported in Hz. Spin multiplicities are described as s (singlet), br (broad singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). Chromatography was performed on CombiFlash Rf+ Teledyne ISCO systems. Normal phase purification used silica gel columns obtained from Silicycle (FLH-R10030B-ISO40) and reverse phase used Biotage SNAP Cartridge KP C18-HS. All compounds were analyzed by LC–MS (Waters 2695. MS: Waters Micromass ZQ. Waters 996 PDA detector) or UPLC-UV-MS (Waters Acquity UPLC. MS: Waters SQD. PDA: Waters Acquity PDA eλ detector), where the final compounds reported had purities greater than 95% based on 1H NMR and an estimated UV purity by (UP)LC–MS. High resolution chromatography was performed using the Accela 1250 UPLC and a 2.1 mm × 30 mm C18 RP column (2.5 μm particles). The mobile phases consisted of 0.1% formic acid in water for mobile phase A and 0.1% formic acid in isopropanol/acetonitrile (1:4) for mobile phase B. High resolution mass spectrometer was a Q-Exactive operated at a resolution of 70 000, calibrated the same day and using scan range of 150 to 850 m/z.

Synthesis of 3-(2,5-Dichloropyrimidin-4-yl)-1H-indole (16)

To a solution of 2,4,5-trichloropyrimidine 14 (520 mg, 2.83 mmol) in dichloroethane (30 mL) was added aluminum chloride (415 mg, 3.1 mmol). The resulting suspension was stirred at 80 °C for 30 min. Reaction mixture was then cooled down to rt and indole 15 (300 mg, 2.57 mmol) was added. The resulting solution was stirred at 80 °C for 16 h. The reaction mixture was cooled down to rt, and ice cold water (15 mL) was then added followed by vigorous stirring for 30 min. The resulting slurry was further diluted with MeTHF (200 mL) and water (60 mL). Aqueous layer was extracted with MeTHF (twice), the organics were combined, dried over Na2SO4, and concentrated under vacuum to afford 16 (665 mg, 2.52 mmol, 98% yield) as a yellow solid.

Synthesis of 3-(2,5-Dichloropyrimidin-4-yl)-1-(phenylsulfonyl)-1H-indole (17)

To a solution of 16 (665 mg, 2.52 mmol) in dry THF (10 mL) was added sodium tert-butoxide, 2 M in THF (1.5 mL, 3 mmol), and the resulting mixture was stirred for 30 min at 0 °C. Benzenesulfonyl chloride (0.38 mL, 3 mmol) was then added, and the reaction mixture was stirred for 30 min at 0 °C. The mixture was diluted with EtOAc and washed with water. The organic phase was separated, dried over Na2SO4, and concentrated under vacuum to provide 17 (700 mg, 1.73 mmol, 69% yield) as a beige solid.

Synthesis of (S)-tert-Butyl 3-((5-chloro-4-(1-(phenylsulfonyl)-1H-indol-3-yl)pyrimidin-2-yl)amino)piperidine-1-carboxylate (19)

A solution of 17 (700 mg, 1.73 mmol), (S)-tert-butyl 3-aminopiperidine-1-carboxylate 18 (382 mg, 1.91 mmol), and DIPEA (0.6 mL, 3.46 mmol) in NMP (9 mL) was heated for 45 min at 135 °C in a microwave reactor. The mixture was diluted with EtOAc (200 mL), washed with water (50 mL) and brine (50 mL), dried over Na2SO4, filtered, and concentrated under vacuum. The obtained residue was purified by silica gel chromatography (EtOAc in DCM, 0 to 100% gradient) to provide 19 (694 mg, 1.22 mmol, 64% yield) as a light pink solid.

Synthesis of 5-Chloro-4-(1H-indol-3-yl)-N-[(3S)-3-piperidyl]pyrimidin-2-amine (1)

To a solution of 19 (260 mg, 0.46 mmol) in dioxane (5 mL) was added 5N aq. NaOH (1.0 mL, 5.0 mmol). The reaction mixture was stirred at 70 °C until complete conversion (3 h). The mixture was concentrated, then diluted with MeTHF and water. The crude product was extracted from water with MeTHF (twice), combined organic phase was dried over Na2SO4, filtered, and concentrated under vacuum. Obtained crude (S)-tert-butyl 3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)piperidine-1-carboxylate (200 mg) was redissolved in EtOAc (3 mL) and HCl/EtOAc (4 M, 2.3 mL) was added. The mixture was stirred at rt until complete conversion (1 h), where solid was precipitated out and then filtered. The obtained solid was redissolved in water (10 mL) and washed with EtOAc (2 × 10 mL), this organic phase was discarded. The aqueous phase was then basified to pH = 9 with NaOH providing a white solid, which was filtered, washed with water (3 × 10 mL), and dried under reduced pressure to afford compound 1 (128 mg, 0.39 mmol, 85% yield) as a white solid. LC–MS (ESI+) m/z 328.2 (MH+). 1H NMR (MeOD-d4, 400 MHz) δ 8.61–8.59 (m, 1H), 8.43 (s, 1H), 8.16 (s, 1H), 7.46–7.44 (m, 1H), 7.24–7.18 (m, 2H), 4.01 (br s, 1H), 3.26–3.23 (m, 1H), 2.94–2.91 (m, 1H), 2.63–2.55 (m, 2H), 2.14 (br s, 1H), 1.78–1.77 (m, 1H), 1.65–1.53 (m, 2H).

Synthesis of (S)-tert-Butyl 3-((4-(1-(phenylsulfonyl)-1H-indol-3-yl)-5-vinylpyrimidin-2-yl)amino)piperidine-1-carboxylate (21)

To a degassed solution of 19 (300 mg, 0.528 mmol), Cs2CO3 (527 mg, 1.58 mmol) and potassium vinyltrifluoroborate 20 (217 mg, 1.58 mmol) in (2:1) toluene/water (6 mL) were added Pd(OAc)2 (6 mg, 0.0264 mmol, 5 mol %) and di(1-adamantyl)-n-butylphosphine (cataCXium; 20 mg, 0.053 mmol, 10 mol %). The reaction mixture was heated at 120 °C for 4 h. The cooled mixture was diluted with EtOAc (50 mL) and water (50 mL), and the aq. layer was extracted twice with EtOAc. The combine organic phase was dried over Na2SO4, filtered, and concentrated under vacuum. The residue was purified by normal phase chromatography on silica gel (EtOAc in DCM, 0 to 60% gradient) to provide 21 (179 mg, 0.32 mmol, 61% yield) as a yellow foam.

Synthesis of (S)-tert-Butyl 3-((5-ethyl-4-(1-(phenylsulfonyl)-1H-indol-3-yl)pyrimidin-2-yl)amino)piperidine-1-carboxylate (22)

To a degassed (with nitrogen) solution of 21 (179 mg, 0.32 mmol) in ethanol (10 mL) was added Pd/C, 10% w/w (34 mg, 0.032 mmol), and the mixture was filled with hydrogen through 3 vacuum/hydrogen cycles. The reaction mixture was stirred under hydrogen atmosphere (1 atm) at rt for 16 h, then filtered on Celite pad and concentrated under reduced pressure. The obtained compound 22 (180 mg, 0.32 mmol) was used in the next step without further purification.

Synthesis of (S)-5-Ethyl-4-(1H-indol-3-yl)-N-(piperidin-3-yl)pyrimidin-2-amine hydrochloride (3)

See the preparation of compound 1 described above. The solution of (S)-tert-butyl 3-((5-ethyl-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)piperidine-1-carboxylate (100 mg, 0.23 mmol) in HCl/EtOAc (10 mL) was stirred at rt for 1 h. After completion, the mixture was concentrated. The residue was triturated with EtOAc (10 mL) and hexane (10 mL), then dried under vacuum to afford compound 3 (44 mg, 120.49 umol, 0.12 mmol, 50% yield; HCl salt) as a yellow solid. LC–MS (ESI+) m/z 322.2 (MH+). 1H NMR (MeOD-d4, 400 MHz) δ 8.45–8.44 (m, 1H), 8.30 (s, 1H), 8.03 (s, 1H), 7.53–7.34 (m, 1H), 7.33–7.30 (m, 2H), 4.56 (br s, 1H), 3.59–3.58 (m, 1H), 3.33–3.25 (m, 2H), 3.11–3.10 (m, 1H), 2.96–2.90 (m, 2H), 2.32–2.31 (m, 1H), 2.12–2.11 (m, 1H), 1.99–1.97 (m, 1H), 1.83–1.81 (m, 1H), 1.35–1.31 (m, 3H).

Synthesis of 3-(2-(Methylthio)-5-(trifluoromethyl)pyrimidin-4-yl)-1-(phenylsulfonyl)-1H-indole (25)

4-Chloro-2-(methylthio)-5-(trifluoromethyl)pyrimidine 23 (27) (2.0 g, 8.75 mmol) and (1-(phenylsulfonyl)-1H-indol-3-yl)boronic acid 24 (2.77 g, 9.19 mmol) were dissolved in degassed 2:1 mixture of dioxane/water (120/60 mL) at rt and degassed. Then Cs2CO3 (5.7 g, 17.5 mmol) was added. The mixture was degassed again and then Pd(PPh3)4 (1 g, 0.87 mmol) was added followed by degassing. The reaction mixture was stirred at 100 °C for 1.5 h. Reaction mixture (brown–green) was cooled and diluted with aq. NaHCO3 solution, and the crude product was extracted 3 times with EtOAc. Combined organic phase was dried over Na2SO4, filtered, and concentrated. The residue was purified by normal phase chromatography on silica gel (EtOAc in hexanes, 0 to 100% gradient) to provide 25 (2.13 g, 4.73 mmol, 54% yield) as a greenish foam. LC–MS (ESI+) m/z 450.09 (MH+). 1H NMR (500 MHz, CDCl3) δ 8.83 (s, 1H), 8.10 (s, 1H), 8.04 (d, J = 8.4 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.94 (dd, J = 8.5, 1.1 Hz, 2H), 7.60–7.56 (m, 1H), 7.48 (t, J = 7.9 Hz, 2H), 7.43–7.38 (m, 1H), 7.35–7.31 (m, 1H), 2.62 (s, 3H).

Synthesis of 3-(2-(Methylsulfonyl)-5-(trifluoromethyl)pyrimidin-4-yl)-1-(phenylsulfonyl)-1H-indole (26)

To a solution of 25 (2.13 g, 4.73 mmol) in DCM/THF (25/25 mL) was added m-CPBA (≥77%) (3.18 g, 14.18 mmol). The reaction mixture was stirred at rt for 16 h. The mixture was then concentrated and purified by normal phase chromatography on silica gel (EtOAc in hexanes, 0 to 100% gradient) to provide 26 (2.15 g, 4.47 mmol, 95% yield) as a yellowish semisolid. LC–MS (ESI+) m/z 482.09 (MH+).

Synthesis of (S)-1-Benzyl-6,6-dimethylpiperidin-3-amine 2,2,2-trifluoroacetate (27)

To a solution of (S)-tert-butyl (6-oxopiperidin-3-yl)carbamate 44 (28) (250 mg, 1.17 mmol) in THF (12 mL) at 0 °C was added NaH, 60% oil dispersion (94 mg, 2.92 mmol), and the resulting suspension was stirred for 30 min, at which point (bromomethyl)benzene (0.35 mL, 2.92 mmol) was added over 5 min. The reaction was allowed to warm to rt for overnight. Reaction was cooled to 0 °C, quenched with water, and then concentrated. The residue was redissolved in EtOAc (15 mL) and water (15 mL), and the aqueous layer was extracted with EtOAc (twice). Combined organic phase was then dried over Na2SO4, filtered, and concentrated. The crude product was purified by normal phase chromatography on silica gel (MeOH in DCM, 0 to 5% gradient) to provide (S)-tert-butyl (1-benzyl-6-oxopiperidin-3-yl)carbamate (116 mg, 0.38 mmol, 33% yield) as a colorless oil. LC–MS (ESI+) m/z 305.96 (MH+). (S)-tert-Butyl (1-benzyl-6-oxopiperidin-3-yl)carbamate (43.0 mg, 0.14 mmol) was dissolved in THF (1.4 mL) and cooled to −10 °C. ZrCl4 (39 mg, 0.17 mmol) was added, and the reaction mixture was stirred for 30 min at this temperature. Then 3 M MeMgBr (0.30 mL, 0.91 mmol) in ether was added to the reaction mixture and the solution allowed to slowly warm to rt and stir until complete conversion (16 h). The solution was quenched with 30% aq. NaOH and extracted with DCM (3 × 50 mL). The organics were combined, dried over Na2SO4, filtered, and concentrated to afford (S)-tert-butyl (1-benzyl-6,6-dimethylpiperidin-3-yl)carbamate (37 mg, 0.12 mmol, 83%) as an orange oil, which was used as is in the next step. LC–MS (ESI+) m/z 320.00 (MH+). To a stirring solution of (S)-tert-butyl (1-benzyl-6,6-dimethylpiperidin-3-yl)carbamate (87 mg, 0.27 mmol) in DCM (5 mL) was added TFA (2.1 mL, 27.3 mmol). The reaction mixture was stirred at rt until complete conversion (2 h). The resulting solution was concentrated under reduced pressure and azeotroped 3 times with DCM to provide compound 27 (60 mg, quant. yield) as an orange oil, which was used in the next step without further purification. LC–MS (ESI+) m/z 219.86 (MH+).

Synthesis of (S)-1-Benzyl-5,5-dimethylpiperidin-3-amine hydrochloride (28b)

To a solution of (S)-5-oxopyrrolidine-2-carboxylic acid (10.0 g, 77.45 mmol) in MeOH (100 mL) was added SOCl2 (11.24 mL, 154.9 mmol). The reaction mixture was stirred at rt for 1 h, then concentrated. The residue was diluted with EtOAc (250 mL) and TEA (20 mL). The precipitate was filtered out. The filtrate was concentrated to afford (S)-methyl 5-oxopyrrolidine-2-carboxylate (13.0 g, crude) as a yellow oil, which was used in the next step without further purification. To a solution of (S)-methyl 5-oxopyrrolidine-2-carboxylate (13.0 g, 90.82 mmol) and DMAP (1.33 g, 10.9 mmol) in EtOAc (150 mL) was added di-tert-butyl dicarbonate (25.77 g, 118.07 mmol). The mixture was stirred at rt for 16 h. The reaction mixture was washed with 0.5 M aq. HC1 (50 mL), sat. aq. NaHCO3 (150 mL), and brine (500 mL). The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was recrystallized from MTBE (250 mL) to afford (S)-1-tert-butyl 2-methyl 5-oxopyrrolidine-1,2-dicarboxylate (14.0 g, 57.55 mmol, 74.3% yield over 2 steps) as a red solid. To a solution of (S)-1-tert-butyl 2-methyl 5-oxopyrrolidine-1,2-dicarboxylate (14.0 g, 57.55 mmol, 1.00 equiv) in THF (400 mL) was added dropwise LiHMDS (1 M in THF, 120.9 mL, 120.9 mmol) at −78 °C. After addition, the mixture was stirred at this temperature for 0.5 h, and then iodomethane (10.75 mL, 172.65 mmol) was added dropwise at −78 °C. The resulting mixture was stirred at rt for 1 h. The reaction mixture was diluted with sat. aq. NH4Cl (400 mL) and extracted with EtOAc (3 × 200 mL). The combined organic phase was washed with brine (2 × 600 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by normal phase chromatography on silica gel (EtOAc in hexanes, 0 to 50% gradient) to afford (S)-1-tert-butyl 2-methyl 4,4-dimethyl-5-oxopyrrolidine-1,2-dicarboxylate (6.0 g, 22.11 mmol, 38.4% yield) as a yellow solid. To a solution of (S)-1-tert-butyl 2-methyl 4,4-dimethyl-5-oxopyrrolidine-1,2-dicarboxylate (5.24 g, 19.31 mmol) in THF (10 mL) was added portion wise NaBH4 (2.19 g, 57.94 mmol) at 0 °C. After addition, EtOH (12.42 mL, 213.03 mmol) was added dropwise at 0 °C. The resulting mixture was stirred at rt for 16 h. The reaction mixture was diluted with sat. aq. NH4Cl (150 mL) and extracted with EtOAc (3 × 80 mL). The combined organic phase was washed with brine (2 × 250 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to afford (S)-tert-butyl (1,5-dihydroxy-4,4-dimethylpentan-2-yl)carbamate (6.46 g) as a yellow oil, which was used without further purification. To a solution of (S)-tert-butyl (1,5-dihydroxy-4,4-dimethylpentan-2-yl)carbamate (6.46 g, 26.12 mmol) and TEA (14.5 mL, 104.5 mmol) in EtOAc (60 mL) was added dropwise MsCl (6.07 mL, 78.36 mmol) at 0 °C. The reaction mixture was stirred at rt for 1 h. The mixture was poured into water (150 mL) and then extracted with EtOAc (3 × 20 mL). The combined organic phase was washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to afford (S)-4-((tert-butoxycarbonyl)amino)-2,2-dimethylpentane-1,5-diyl dimethanesulfonate (10.4 g) as yellow oil, which was used as is in the next step. A mixture of (S)-4-((tert-butoxycarbonyl)amino)-2,2-dimethylpentane-1,5-diyl dimethanesulfonate (10.4 g, 25.8 mmol) and phenylmethanamine (9.03 mL, 82.56 mmol) in DME (100 mL) was stirred at 70 °C for 16 h. The reaction mixture was diluted with water (500 mL) and extracted with EtOAc (3 × 200 mL). The combined organic phase was washed with brine (2 × 500 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by normal phase chromatography on silica gel (EtOAc in hexanes, 0 to 100% gradient) to afford (S)-tert-butyl (1-benzyl-5,5-dimethylpiperidin-3-yl)carbamate (1.0 g, 3.1 mmol, 14% yield over 3 steps) as a yellow oil. The obtained product (1.0 g) was then treated with 4 M HCl in EtOAc (7 mL) and the reaction mixture was stirred at rt for 1 h. The mixture was concentrated to provide (S)-1-benzyl-5,5-dimethylpiperidin-3-amine hydrochloride 28b (790 mg, quant. yield) as an off-white solid that was used in the next step without further purification. LC–MS (ESI+) m/z 219.4 (MH+).

Synthesis of (S)-Benzyl 5-amino-3,3-dimethylpiperidine-1-carboxylate hydrochloride (28a)

To a solution of (S)-tert-butyl (1-benzyl-5,5-dimethylpiperidin-3-yl)carbamate [an intermediate in the synthesis of 28b] (1.0 g, 3.14 mmol) in EtOH (10 mL) was added Pd/C, 10 wt % on activated carbon (1 g) under nitrogen. The suspension was degassed under vacuum and purged with hydrogen 3 times. The mixture was stirred under hydrogen pressure (30 psi) at rt for 16 h. The reaction mixture was filtered on Celite pad, and the filtrate was concentrated to afford (S)-tert-butyl (5,5-dimethylpiperidin-3-yl)carbamate (500 mg, crude) as a yellow oil. To a solution of (S)-tert-butyl (5,5-dimethylpiperidin-3-yl)carbamate (500 mg, 2.19 mmol) and NaHCO3 (1.29 g, 15.33 mmol) in THF (5 mL) and water (5 mL) was added dropwise benzyl carbonochloridate (0.47 mL, 3.29 mmol). The mixture was stirred at rt for 30 min. The reaction mixture was diluted with water (100 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phase was washed with brine (2 × 200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by normal phase chromatography on silica gel (EtOAc in hexanes, 0 to 50% gradient) to afford (S)-benzyl 5-((tert-butoxycarbonyl)amino)-3,3-dimethylpiperidine-1-carboxylate (740 mg, 2.04 mmol, 93% yield) as a yellow oil. The obtained product was then treated with 4 M HCl in EtOAc (5 mL), and the reaction mixture was stirred at rt for 1 h. The mixture was concentrated to provide (S)-benzyl 5-amino-3,3-dimethylpiperidine-1-carboxylate hydrochloride 28a (525 mg, quant. yield) as a yellow solid, which was used as such in the next step.

General Method for Synthesis of 29

Compound 26 (1 equiv), chiral amine 18, 27, or 28a (1 equiv), and anhydrous DIPEA (3 equiv) were dissolved in anhydrous THF (0.06 M). The reaction mixture was stirred at rt until complete conversion (typically, 16 h). The mixture was then concentrated under reduced pressure, and the residue was purified by normal phase chromatography on silica gel (EtOAc in DCM, 0 to 100% gradient) to provide 29 (27–50% yield).

Synthesis of (S)-4-(1H-Indol-3-yl)-N-(piperidin-3-yl)-5-(trifluoromethyl)pyrimidin-2-amine hydrochloride (2)

See the preparation of compound 1. To a solution of (S)-tert-butyl 3-((4-(1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl)amino)piperidine-1-carboxylate (340 mg, 0.73 mmol) in DCM (20 mL) was added TFA (4 mL), and the reaction mixture was stirred at rt until complete conversion (3 h). The mixture was then concentrated under reduced pressure, and the residue was purified by prep-HPLC (MeCN in aq. HCl buffer) to afford compound 2 (126 mg, 0.31 mmol, 42% yield; HCl salt) as a yellow solid after lyophilization. LC–MS (ESI+) m/z 362.2 (MH+). 1H NMR (MeOD-d4, 400 MHz) δ 8.57 (s, 1H), 8.35 (s, 1H), 8.08 (br s, 1H), 7.50 (s, 1H), 7.28 (br s, 2H), 4.53 (br s, 1H), 3.60–3.56 (m, 1H), 3.34–3.31 (m, 1H), 3.15 (br s, 1H), 3.09–3.07 (m, 1H), 2.13–2.09 (m, 1H), 1.93–1.89 (m, 1H), 1.86–1.80 (m, 2H).

Synthesis of (S)-N-(6,6-Dimethylpiperidin-3-yl)-4-(1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-amine (6)

To a solution of (S)-N-(1-benzyl-6,6-dimethylpiperidin-3-yl)-4-(1-(phenylsulfonyl)-1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-amine (46 mg, 0.07 mmol) in EtOH (7 mL) was added a mixture of Pd/C (10 wt % on activated carbon) and Pd(OH)2/C (20% dispersion) (5 mg of each). The reaction mixture was stirred under a positive pressure of hydrogen (1 atm) at rt for 16 h. The reaction mixture was filtered through Celite pad and concentrated under reduced pressure to provide (S)-N-(5,5-dimethylpiperidin-3-yl)-4-(1-(phenylsulfonyl)-1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-amine (27 mg, 0.05 mmol, 70% yield) as a light yellow solid. LC–MS (ESI+) m/z 530.30 (MH+). To this obtained crude product in dioxane (5 mL), 5 N aq. NaOH (0.83 mL, 4.1 mmol) was added. The resulting solution was heated at 75 °C until complete conversion (16 h). The solution was concentrated under reduced pressure and directly purified by reverse phase chromatography (MeCN in aq. 10 mM ammonium formate, 0 to 100% gradient) to afford compound 6 (5 mg, 0.013 mmol, 25% yield) as a light yellow solid after lyophilization. LC–MS (ESI+) m/z 390.2 (MH+). 1H NMR (500 MHz, DMSO) δ 11.82 (br s, 1H), 8.54 (d, J = 19.2 Hz, 1H), 8.39 (br s, 1H), 8.24 (d, J = 7.9 Hz, 1H), 7.83 (s, 1H), 7.72 (d, J = 8.1 Hz, 1H), 7.49 (t, J = 9.0 Hz, 1H), 7.20 (t, J = 7.5 Hz, 1H), 7.14 (t, J = 7.4 Hz, 1H), 4.00–3.87 (m, 1H), 2.96 (dd, J = 12.6, 4.1 Hz, 1H), 2.74 (dd, J = 22.9, 13.0 Hz, 1H), 1.93–1.77 (m, 1H), 1.75–1.62 (m, 1H), 1.60–1.51 (m, 1H), 1.45–1.32 (m, 1H), 1.21–0.99 (m, 6H).

Synthesis of (S)-N-(5,5-Dimethylpiperidin-3-yl)-4-(1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-amine (8)

See the preparation of compound 1. To a solution of (S)-benzyl 5-((4-(1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-3,3-dimethylpiperidine-1-carboxylate (200 mg, 0.38 mmol) in DCM (3 mL) was added HBr, 33 wt % in AcOH (0.6 mL, 3.82 mmol). The reaction mixture was stirred at rt for 1 h. The mixture was then poured into MTBE (10 mL), and the formed solid was filtered to collect cake as a yellow solid, which was purified by prep-HPLC (formic acid buffer) to obtain compound 8 (110 mg, 0.25 mmol, 66% yield; FA) as a white solid after lyophilization. LC–MS (ESI+) m/z 390.2 (MH+). 1H NMR (400 MHz, MeOD-d4) δ 8.53 (s, 2H), 8.40–8.17 (m, 1H), 7.81 (s, 1H), 7.45 (d, J = 7.89 Hz, 1H), 7.26–7.13 (m, 2H), 4.68–4.37 (m, 1H), 3.50 (br s, 1H), 2.99–2.95 (m, 1H), 2.71–2.68 (m, 2H), 1.97 (br s, 1H), 1.51 (br s, 1H), 1.20 (s, 3H), 1.08 (s, 3H).

Synthesis of 6-Bromo-3-(2-chloro-5-(trifluoromethyl)pyrimidin-4-yl)-1-(phenylsulfonyl)-1H-indole (31)

To a solution of 2,4-dichloro-5-(trifluoromethyl)pyrimidine 30 (4.87 g, 22.44 mmol) in dichloroethane (29 mL) was added aluminum chloride (3.26 g, 24.49 mmol). The resulting suspension was stirred at 70 °C for 15 min to provide a clear yellow solution. Reaction mixture was then cooled to 60 °C followed by addition of 6-bromo-1H-indole (4.0 g, 20.40 mmol). The resulting orange solution was stirred at 80 °C for 2 h. The reaction mixture was cooled to rt and quenched with ice-cold water followed by stirring for 30 min. The resulting slurry was further diluted with EtOAc and water and then filtered. Aqueous layer was extracted with EtOAc (twice), and the organics were combined, dried over Na2SO4, and concentrated under vacuum. The crude red solid was dissolved in minimal amount of MeOH and, after stirring for 30 min at rt, was then filtered to provide the desired regioisomer 6-bromo-3-(2-chloro-5-(trifluoromethyl)pyrimidin-4-yl)-1H-indole (2.20 g, 5.85 mmol, 26% yield) as a beige solid. To a solution of 6-bromo-3-(2-chloro-5-(trifluoromethyl)pyrimidin-4-yl)-1H-indole (1.19 g, 3.16 mmol) in THF (15.8 mL) was added sodium tert-butoxide, 2 M in THF (1.90 mL, 3.79 mmol), and the resulting mixture was stirred for 30 min at 0 °C. Benzenesulfonyl chloride (0.48 mL, 3.79 mmol) was then added. Reaction mixture was stirred for 15 min at 0 °C and then at rt for overnight. The mixture was diluted with EtOAc and washed with water. The organic phase was separated, dried over Na2SO4, and concentrated under vacuum to provide compound 31 (1.40 g, 2.71 mmol, 86% yield) as a beige solid, which was used in the next step without further purification.

Synthesis of (S)-tert-Butyl 3-((4-(6-bromo-1-(phenylsulfonyl)-1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl)amino)piperidine-1-carboxylate (32)

A mixture of 31 (1.0 g, 1.94 mmol), (S)-tert-butyl 3-aminopiperidine-1-carboxylate 18 (0.39 g, 1.94 mmol) and DIPEA (1.02 mL, 5.82 mmol) in NMP (10 mL) was stirred at 140 °C for 1 h. The mixture was diluted with water (100 mL) and extracted with EtOAc (2 × 50 mL). Combined organic phase was washed with brine (2 × 100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by normal phase chromatography on silica gel (EtOAc in hexanes, 0 to 100% gradient) to afford compound 32 (1.13 g, 1.66 mmol, 86% yield) as a yellow solid.

Synthesis of (S)-tert-Butyl 3-((4-(6-bromo-1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl)amino)piperidine-1-carboxylate (33)

To a stirring solution of 32 (1.02 g, 1.49 mmol) in dioxane (50 mL) was added 5N aq. NaOH (24 mL, 119.3 mmol). The reaction mixture was heated at 75 °C until complete conversion (1 h). The solution was concentrated under reduced pressure to remove dioxane and extracted with MeTHF (3 × 100 mL). The organics were combined, dried over Na2SO4, filtered, and concentrated to provide 33 (810 mg, quant. yield) as a yellow foam. LC–MS (ESI+) m/z 540.2 (MH+).

Synthesis of (S)-3-(2-(Piperidin-3-ylamino)-5-(trifluoromethyl)pyrimidin-4-yl)-1H-indole-6-carbonitrile (5)

A solution of 33 (806 mg, 1.49 mmol) in degassed DMAc (10 mL) was placed in a vial under argon atmosphere and degassed for 30 min. In another 20 mL vial, Zn (20 mg, 0.3 mmol), Pd2(dba)3 (137 mg, 0.15 mmol), XPhos (142 mg, 0.30 mmol), and ZnCN (210 mg, 1.79 mmol) were premixed in degassed DMAc (10 mL) and stirred at 95 °C for 10 min. Degassed solution of 33 was added to the vial with all other reagents, and the reaction mixture was stirred at 95 °C for 30 min. The mixture was cooled to rt, passed through Celite pad, diluted with MeTHF, and washed twice with brine. Organic phase was dried over Na2SO4, filtered, and concentrated. The residue was purified by normal phase chromatography on silica gel (EtOAc in DCM/hexanes (1:1), 0 to 100% gradient) to provide (S)-tert-butyl 3-((4-(6-cyano-1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl)amino)piperidine-1-carboxylate (685 mg, 1.4 mmol, 95% yield) as a yellowish foam. LC–MS (ESI+) m/z 487.3 (MH+). To a cooled, 0 °C solution of (S)-tert-butyl 3-((4-(6-cyano-1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl)amino)piperidine-1-carboxylate (1.23 g, 2.53 mmol) in DCM (25 mL) was added TFA (5.8 mL, 75.9 mmol). The reaction mixture was stirred at 0 °C for 30 min and then concentrated under reduced pressure. The residue was then redissolved in MeTHF and rinsed with sat. aq. NaHCO3 (twice). The organic phase was dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by reverse phase chromatography (0–100% MeCN in water, 0.1% formic acid buffers) to provide compound 5 (610 mg, 1.58 mmol, 62% yield) as a white solid after lyophilization. LC–MS (ESI+) m/z 387.2 (MH+). 1H NMR (500 MHz, CD3OD) δ 8.63 (s, 1H), 8.54 (s, 1H), 8.06 (br s, 1H), 7.91 (s, 1H), 7.46 (d, J = 8.1 Hz, 1H), 4.34 (br s, 1H), 3.61–3.51 (m, 1H), 3.31–3.25 (m, 1H), 3.07–2.95 (m, 2H), 2.26–2.17 (m, 1H), 2.14–2.04 (m, 1H), 1.92–1.72 (m, 2H).

Synthesis of (S)-4-(6-(Methylsulfonyl)-1H-indol-3-yl)-N-(piperidin-3-yl)-5-(trifluoromethyl)pyrimidin-2-amine (7)

To a solution of 33 (2.2 g, 4.1 mmol) in NMP (20 mL), CuI (3 g, 16 mmol) and sodium methanesulfinate (1.67 g, 16.4 mmol) were added. Nitrogen was bubbled through the reaction mixture for few minutes and then the mixture, in a sealed vial, was heated at 140 °C for 2 h. The reaction mixture was directly purified by reverse phase chromatography (MeCN in aq. 10 mM ammonium formate, pH 3.8, 0 to 100% gradient) to afford (S)-tert-butyl 3-((4-(6-(methylsulfonyl)-1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl)amino)piperidine-1-carboxylate (200 mg, 0.37 mmol, 9% yield) as a light brown solid. Then obtained compound was treated with 4 M HCl in EtOAc (2 mL), and the reaction mixture was stirred at rt for 1 h. The mixture was concentrated under reduced pressure. The residue was adjusted to pH = 8 with sat. aq. NaHCO3, and crude product was extracted with ethyl acetate (3 × 20 mL). The combined organic phase was washed with brine (2 × 100 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by prep-HPLC (MeCN in water, formic acid buffers) to obtain compound 7 (48 mg, 0.11 mmol, 29% yield) as a white solid after lyophilization. LC–MS (ESI+) m/z 440.2 (MH+). 1H NMR (400 MHz, MeOD-d4) δ 8.59 (s, 1H), 8.49 (s, 1H), 8.12–8.02 (m, 2H), 7.71–7.69 (m, 1H), 4.34 (br s, 1H), 3.56–3.53 (m, 1H), 3.31–3.28 (m, 1H), 3.14 (s, 3H), 3.05- 2.93 (m, 2H), 2.22–2.01 (m, 2H), 1.91–1.69 (m, 2H).

Synthesis of (S)-4-(6-(3,5-Dimethylisoxazol-4-yl)-1H-indol-3-yl)-N-(piperidin-3-yl)-5-(trifluoromethyl)pyrimidin-2-amine (10)

Compound 32 (3.0 g, 4.4 mmol) and 3,5-dimethylisoxazol-4-yl-4-boronic acid (1.24 g, 8.8 mmol) were dissolved in degassed 2:1 mixture of dioxane and water (90 mL) at rt. Cs2CO3 (2.87 g, 8.8 mmol) was added and the mixture was degassed. Then Pd(PPh3)4 (508 mg, 0.44 mmol) was added, and the reaction mixture was stirred at 100 °C for 1 h. Reaction mixture was cooled to rt and diluted with water, and the crude product was extracted twice with EtOAc. The combined organics were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by normal phase chromatography on silica gel (EtOAc in DCM/hexanes (1:1), 0 to 100% gradient) to provide (S)-tert-butyl 3-((4-(6-(3,5-dimethylisoxazol-4-yl)-1-(phenylsulfonyl)-1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl)amino)piperidine-1-carboxylate (2.78 g, 3.98 mmol, 90% yield) as a pale yellow solid. LC–MS (ESI+) m/z 697.3 (MH+). Then obtained (S)-tert-butyl 3-((4-(6-(3,5-dimethylisoxazol-4-yl)-1-(phenylsulfonyl)-1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl)amino)piperidine-1-carboxylate (2.78 g, 3.98 mmol) was dissolved in dioxane (24 mL) followed by addition of 5N aq. NaOH (24 mL, 120.0 mmol). The reaction mixture was heated at 75 °C for 3 h. After being concentrated under vacuum, the reaction mixture was diluted with water and crude product was extracted with MeTHF (3×). The combined organic phase was then dried over Na2SO4, filtered, and concentrated to afford a white solid (2.21 g, quant.). LC–MS (ESI+) m/z 558.3 (MH+). Next, the obtained (S)-tert-butyl 3-((4-(6-(3,5-dimethylisoxazol-4-yl)-1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl)amino)piperidine-1-carboxylate (2.21 g, 3.98 mmol) was dissolved in DCM (20 mL) and TFA (8.14 mL, 120 mmol) was added. The reaction mixture was stirred at rt for 1 h, then concentrated and coevaporated several times with DCM. The residue was dissolved in MeTHF and neutralized with sat. aq. NaHCO3. The organic phase was rinsed with sat. aq. NaHCO3, then dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified twice by reverse phase chromatography (0–100% MeCN in water, 0.1% formic acid buffers and then 0–100% MeCN in 10 mM aq. ammonium formate buffer, pH 3.8) to afford compound 10 (1.10 g, 2.4 mmol, 60% yield over 2 steps) as a white solid after lyophilization. LC–MS (ESI+) m/z 457.3 (MH+). 1H NMR (500 MHz, DMSO) δ 11.90 (br s, 1H), 8.60 (s, 0.5H; rotamer), 8.56 (s, 0.5H; rotamer), 8.49 (d, J = 8.1 Hz, 0.5H; rotamer), 8.31 (s, 1H), 8.29 (d, J = 8.4 Hz, 0.5H; rotamer), 7.91–7.83 (m, 2H), 7.49–7.44 (m, 1H), 7.18–7.10 (m, 1H), 4.06 (br s, 1H), 3.23–3.16 (m, 1H), 3.01–2.90 (m, 1H), 2.69–2.57 (m, 2H), 2.43 (s, 3H), 2.25 (s, 3H), 2.06.1.93 (m, 1H), 1.76 (br s, 1H), 1.62–1.45 (m, 2H).

Synthesis of tert-Butyl 7-bromo-1H-indole-1-carboxylate (35)

To a solution of 7-bromoindole 34 (10.0 g, 51 mmol) in MeCN (100 mL) was added Boc2O (12.25 g, 56.11 mmol) and DMAP (621 mg, 5.1 mmol), and the mixture was stirred at rt for 16 h. The mixture was concentrated, redissolved in EtOAc, and rinsed the organics with water and brine (2×), and then organic phase was dried over Na2SO4, filtered, and concentrated to afford 35 (15.1 g, quant.) as a brown oil, which was used in the next step without further purification. LC–MS (ESI+) m/z 240.1/242.0 (M-tBu+). 1H NMR (500 MHz, CDCl3) δ 7.54–7.49 (m, 3H), 7.09 (t, J = 7.8 Hz, 1H), 6.56 (d, J = 3.6 Hz, 1H), 1.66 (s, 9H).

Synthesis of tert-Butyl 7-bromo-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-1-carboxylate (36)

A tube was charged with [Ir(OMe)(COD)]2 (57 mg, 0.086 mmol; 0.73 mol %), 4,4′-ditert-butyl-2,2′-bipyridine (dtbpy; 47 mg, 0.174 mmol; 1.46 mol %), and bis(pinacolato)diboron (B2Pin2; 1.5 g, 5.9 mmol) and then cycled a few times with vacuum/nitrogen. Compound 35 (3.5 g, 11.82 mmol) in degassed MTBE (12 mL) was then added. The tube was sealed, and the reaction mixture was stirred at 100 °C for 7 h. The mixture was diluted with EtOAc and washed twice with brine. The organic phase was dried over Na2SO4 and concentrated under reduced pressure to a brown semisolid of crude 36 (4.8 g, 68% purity), which was used as is in the next step. 1H NMR (500 MHz, CDCl3) δ 7.97 (dd, J = 7.8, 0.8 Hz, 1H), 7.92 (s, 1H), 7.51 (d, J = 7.9 Hz, 1H), 7.12 (t, J = 7.8 Hz, 1H), 1.66 (s, 9H), 1.37 (s, 12H).

Synthesis of (S)-tert-Butyl 3-((4-chloro-5-(trifluoromethyl)pyrimidin-2-yl)amino)piperidine-1-carboxylate (37)

This synthesis was performed following the procedure from WO 2014/124230. (30) To a solution of 2,4-dichloro-5-(trifluoromethyl)pyrimidine 30 (1 g, 4.61 mmol) in DCE/t-BuOH (1/1, 10 mL) at rt was added dry ZnCl2 (735 mg, 5.4 mmol) and triethylamine (693 μL, 4.968 mmol). The reaction mixture was stirred at rt for 1 h (pH should not be >7). To this mixture, (S)-tert-butyl 3-aminopiperidine-1-carboxylate 18 (1 g, 5 mmol) was added, and stirring continued at rt for 16 h. TLC (hex/EtOAc = 5/1) showed formation of the major compound (0.2 Rf) and the minor regioisomer (0.25 Rf). Reaction mixture was concentrated, and the obtained residue was purified by normal phase chromatography on silica gel (EtOAc in DCM, 0 to 100% gradient) to provide the desired regioisomer 37 (510 mg, 1.34 mmol, 29% yield) as a white solid. LC–MS (ESI+) m/z 381.21 (MH+). 1H NMR (500 MHz, CDCl3) δ 8.47 (br s, 1H), 5.65 (br s, 1H), 4.05 (br s, 1H), 3.67 (d, J = 11.8 Hz, 1H), 3.40 (br s, 3H), 1.97–1.88 (m, 1H), 1.76–1.66 (m, 2H), 1.61 (br s, 1H), 1.43 (s, 9H).

Synthesis of (S)-tert-Butyl 3-((4-(7-bromo-1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl)amino)piperidine-1-carboxylate (38)

Tetrakis(triphenylphosphine)palladium (433 mg, 0.375 mmol) and Cs2CO3 (7.33 g, 22.5 mmol) were added to a stirring solution of 37 (3.0 g, 7.88 mmol) and crude 36 (3.2 g, 7.5 mmol) in a previously degassed 2:1 mixture of dioxane/H2O (120 mL). The resulting mixture was heated at 95 °C until complete conversion to mono-Boc protected product 38 (8 h, monitored by LC–MS). The reaction mixture was allowed to cool to rt; then water and EtOAc were added and the phases were separated. The aqueous layer was extracted with EtOAc (2×), and the combined organic phase was dried over Na2SO4, filtered, and evaporated to dryness. The residue was purified first by reverse phase chromatography (0–100% MeCN in 10 mM aq. ammonium formate, pH 3.8) and then by normal phase chromatography on silica gel (EtOAc in DCM/hexanes (1:1), 0 to 100% gradient) to afford 38 (1 g, 1.85 mmol, 21% yield) as an off-white solid. LC–MS (ESI+) m/z 543.2 (MH+).

Synthesis of (S)-4-(7-(Methylsulfonyl)-1H-indol-3-yl)-N-(piperidin-3-yl)-5-(trifluoromethyl)pyrimidin-2-amine (4)

To a solution of 38 (1.0 g, 1.85 mmol) in NMP (18.5 mL) was added Cul (1.37 g, 7.22 mmol) and sodium methanesulfinate (755 mg, 7.40 mmol). N2 was bubbled through the reaction mixture for few minutes, and then the mixture was heated at 140 °C for 2 h. The mixture was then directly purified by reverse phase chromatography (0–100% MeCN in aq. 10 mM ammonium formate buffer, pH 3.8) to provide (S)-tert-butyl 3-((4-(7-(methylsulfonyl)-1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl)amino)piperidine-1-carboxylate (698 mg, 1.3 mmol, 70% yield) as an off-white foam. LC–MS (ESI+) m/z 540.4 (MH+). Next, a solution of obtained compound (698 mg, 1.3 mmol) in DCM (13 mL) was treated with TFA (2.51 mL, 30.7 mmol). The mixture was stirred at rt for 1 h, then concentrated under reduced pressure. The residue was redissolved in MeTHF and washed 3 times with sat. aq. NaHCO3. The organic phase was dried over Na2SO4, concentrated, and residue was purified by reverse phase chromatography (0 to 100% MeCN in water, 0.1% formic acid buffers) to afford compound 4 (422 mg, 0.96 mmol, 74% yield) as a white solid. LC–MS (ESI+) m/z 440.3 (MH+). 1H NMR (500 MHz, MeOD) δ 8.80–8.67 (m, 1H), 8.62 (s, 1H), 8.54 (s, 1H), 7.96 (br s, 1H), 7.79 (d, J = 7.4 Hz, 1H), 7.40 (t, J = 7.8 Hz,1H), 4.33–4.28 (m, 1H), 3.60–3.47 (m, 1H), 3.29–3.17 (m, 1H), 3.22 (s, 3H), 3.01–2.90 (m, 2H), 2.19 (d, J = 7.5 Hz, 1H), 2.10–1.99 (m, 1H), 1.89–1.68 (m, 2H).

Synthesis of (S)-Dimethyl(3-(2-(piperidin-3-ylamino)-5-(trifluoromethyl)pyrimidin-4-yl)-1H-indol-7-yl)phosphine oxide (9)

Compound 38 (50 mg, 0.093 mmol), XantPhos (5.4 mg, 0.0093 mmol; 10 mol %), palladium(II) acetate (1.0 mg, 0.0047 mmol; 5 mol %), and K3PO4 (22 mg, 0.102 mmol) were combined in a microwave vial under N2. Dimethylphosphine oxide (9 mg, 0.11 mmol) was dissolved in anhydrous DMF (0.3 mL), and the mixture was degassed before being combined with the other reactants. The reaction was heated at 150 °C in a microwave for 45 min. The reaction was cooled to rt, diluted with MeTHF, washed with sat. aq. NaHCO3 and brine, and then organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The obtained residue was then redissolved in DCM (1 mL) and treated with TFA (0.22 mL, 2.79 mmol). The mixture was stirred for at rt for 1 h, then concentrated under reduced pressure, and the residue was purified by reverse phase chromatography (0–100% MeCN in aq. 10 mM ammonium formate buffer, pH 3.8) to afford compound 9 (28 mg, 0.064 mmol, 69% yield) as a white solid after lyophilization. LC–MS (ESI+) m/z 438.2 (MH+). 1H NMR (500 MHz, DMSO) δ 8.63 (d, J = 7.5 Hz, 0.5H; rotamer), 8.59 (d, J = 16.5 Hz, 1H), 8.41 (d, J = 7.5 Hz, 0.5H; rotamer), 8.35 (br s, 1H), 7.99–7.89 (m, 2H), 7.53–7.47 (m, 1H), 7.33–7.23 (m, 1H), 4.12 (br s, 1H), 3.28–3.18 (m, 1H), 3.05–2.94 (m, 1H), 2.75–2.63 (m, 2H), 2.06–1.94 (m, 1H), 1.82 (s, 3H), 1.80 (s, 3H), 1.80–1.74 (br s, 1H), 1.65–1.45 (m, 2H). 31P NMR (203 MHz, DMSO) δ 37.59 (s).

Synthesis of 3,5-Dimethyl-4-(1H-pyrrolo[2,3-b]pyridin-6-yl)isoxazole (40)

To a solution of 6-bromo-1H-pyrrolo[2,3-b]pyridine 39 (6.0 g, 30.5 mmol) in degassed 2:1 mixture of dioxane/water (150 mL) were added (3,5-dimethylisoxazol-4-yl)boronic acid (4.72 g, 33.5 mmol) and NaHCO3 (5.12 g, 60.9 mmol), and the resulted suspension was degassed for 30 min. Pd(dppf)Cl2 DCM complex (620 mg, 0.76 mmol; 2.5 mol %) was then added, and the reaction mixture was stirred at 100 °C for 2 h. After cooling down to rt, the mixture was extracted with EtOAc (3 × 30 mL), the combined organic phase was then washed with sat. aq. NaHCO3 (2 × 20 mL) and brine (2 × 20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain compound 40 (6.5 g, 30.5 mmol, quant. yield) as a tan solid, which was used in the next step without further purification. LC–MS (ESI+) m/z 213.9 (MH+).

Synthesis of 4-(3-Bromo-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-6-yl)-3,5-dimethylisoxazole (41)

To a suspension of 40 (0.9 g, 4.22 mmol) in DCM (10 mL) was added N-bromosuccinimide (NBS; 0.67 g, 3.80 mmol), and the reaction mixture was stirred at 0 °C for 1 h. The mixture was then poured into sat. aq. Na2S2O3 and stirred vigorously for 30 min; the crude product was then extracted 3 times with EtOAc. The combined organic phase was washed with brine (twice), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by normal phase chromatography on silica gel (10–50% EtOAc in hexanes) to provide a pink solid of 4-(3-bromo-1H-pyrrolo[2,3-b]pyridin-6-yl)-3,5-dimethylisoxazole (600 mg, 2.05 mmol, 49%). To a solution of obtained compound (600 mg, 2.05 mmol) in DMF (9 mL) and THF (1 mL) at 0 °C was added 60% NaH dispersion in mineral oil (98.6 mg, 2.46 mmol) followed by benzenesulfonyl chloride (0.34 mL, 2.67 mmol), and reaction mixture was stirred at this temperature for 1 h. The mixture was poured into water (20 mL), and the formed solid was filtered and dried under vacuum to provide compound 41 (600 mg, 1.39 mmol, 68%) as a white solid, which was used in the next step without further purification.

Synthesis of 3,5-Dimethyl-4-(1-(phenylsulfonyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)isoxazole (42)

A mixture of 41 (540 mg, 1.24 mmol), bis-(pinacolato)diboron (475.8 mg, 1.88 mmol), Pd(dppf)Cl2 DCM complex (91.4 mg, 0.124 mmol), and KOAc (245.2 mg, 2.5 mmol) in dioxane (6 mL) was degassed and then stirred at 100 °C for 1 h. Cooled to rt, the mixture was then poured into water (20 mL), and the crude product was extracted with EtOAc (3 times). The combined organic phase was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to provide compound 42 (600 mg, quant. yield) as a brown oil, which was used without purification. LC–MS (ESI+) m/z 480.1 (MH+).

Synthesis of (S)-tert-Butyl 3-((4-(6-(3,5-dimethylisoxazol-4-yl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl)amino)piperidine-1-carboxylate (43)

A mixture of 42 (600 mg, 1.24 mmol), (S)-tert-butyl 3-((4-chloro-5-(trifluoromethyl)pyrimidin-2-yl)amino)piperidine-1-carboxylate 37 (476.6 mg, 1.25 mmol), Pd(dppf)Cl2 (91.6 mg, 0.125 mmol; 10 mol %), and Na2CO3 (265.3 mg, 2.50 mmol) in dioxane (10 mL) and water (2 mL) was degassed and then was stirred at 100 °C for 12 h. The mixture was cooled to rt and poured into water (30 mL), then extracted with EtOAc (3 times). The combined organic phase was washed with brine, then dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by normal phase chromatography on silica gel (10–30% EtOAc in hexanes) to obtain compound 43 (200 mg, 0.28 mmol, 23% yield) as a yellow solid. UPLC–MS (ESI+) m/z 698.6 (MH+).

Synthesis of (S)-4-(6-(3,5-Dimethylisoxazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-N-(piperidin-3-yl)-5-(trifluoromethyl)pyrimidin-2-amine hydrochloride (SY-5102)

To a solution of 43 (200 mg, 0.287 mmol) in MeOH (1 mL) was added 2N aq. NaOH (0.8 mL, 5.58 mmol), and the mixture was stirred at 60 °C for 1 h. The mixture was concentrated under reduced pressure to a yellow solid. UPLC–MS (ESI+) m/z 558.5 (MH+). The obtained residue was redissolved in EtOAc (1 mL) and then treated with 4 M HCl in EtOAc (2 mL, 8.0 mmol). After stirring at rt for 1 h, the mixture was concentrated under reduced pressure, and the crude product was purified by prep-HPLC (MeCN in water HCl buffer) to afford SY-5102 (31.6 mg, 0.064 mmol, 22% yield, HCl salt) as a yellow solid after lyophilization. LC–MS (ESI+) m/z 458.0 (MH+). 1H NMR (MeOD-d4, 400 MHz) δ 9.45–8.82 (m, 1H), 8.68 (br s, 1H), 8.41–8.18 (m, 1H), 7.80–7.48 (m, 1H), 4.79–4.29 (m, 1H), 3.66 (d, J = 10.4 Hz, 1H), 3.44–3.33 (m, 1H), 3.28–3.03 (m, 2H), 2.62 (s, 3H), 2.45 (s, 3H), 2.35–2.20 (m, 1H), 2.19–1.93 (m, 2H), 1.87–1.85 (m, 1H).

Synthesis of (S)-6,6-Dimethylpiperidin-3-amine (45)

(S)-tert-Butyl (6-oxopiperidin-3-yl)carbamate 44 (1.0 g, 4.67 mmol) (prepared in house following published procedure (28)) was dissolved in THF (47 mL), and the solution was cooled to −10 °C. Zirconium(IV) chloride (2.61 g, 11.22 mmol) was added, and the mixture was stirred for 30 min at this temperature. Methylmagnesium bromide solution, 3 M in ether (20.25 mL, 60.75 mmol) was added, and then reaction mixture was allowed to slowly warm up to rt and then stirred for 16 h. The solution was quenched with 30% aq. NaOH, diluted with EtOAc, filtered, and then extracted 3 times with EtOAc. The organics were combined, dried over sodium sulfate, filtered, and concentrated in vacuo to provide the crude product as a yellow oil that was used without purification. The obtained oil was dissolved in DCM (47 mL), and TFA (3.58 mL, 46.73 mmol) was added. The reaction mixture was stirred at rt for 16 h. The mixture was concentrated in vacuo and coevaporated three times with DCM to provide compound 45 as a brown oil, which was used in the next step without further purification. 1H NMR (500 MHz, MeOD) δ 3.08 (ddd, J = 12.5, 4.3, 1.7 Hz, 1H), 2.99 (td, J = 9.9, 5.0 Hz, 1H), 2.80 (dd, J = 12.5, 10.0 Hz, 1H), 1.96–1.88 (m, 1H), 1.75 (ddd, J = 8.2, 4.6, 2.7 Hz, 1H), 1.68–1.51 (m, 2H), 1.40–1.34 (m, 1H), 1.33–1.30 (m, 1H), 1.28 (d, J = 5.6 Hz, 6H).

Synthesis of 7-Bromo-1H-indole-6-carboxylic acid (47)

To a stirred vinylmagnesium bromide solution, 1.0 M in THF (159 mL, 159 mmol) cooled to −78 °C, a solution of 2-bromo-3-nitrobenzoic acid 46 (10.0 g, 39.8 mmol) in THF (159 mL) was added dropwise over a period of 1 h. The reaction mixture was allowed to reach rt and was stirred at this temperature for 16 h. The reaction mixture was then poured into sat. aq. NH4Cl (150 mL) and acidified to a pH 2, using aq. 1 M HCl. The crude product was extracted with ethyl acetate (3 × 200 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was then triturated with DCM (100 mL) and dried for overnight with a flow of air to provide compound 47 (7.58 g, 31.58 mmol, 79% yield) as a light brown solid. LC–MS (ES-) m/z 238/240.

Synthesis of 7-Bromo-1H-indole-6-carbonitrile (48)

To a stirred solution of acid 47 (6.58 g, 27.4 mmol) in DMF (54.8 mL) at 0 °C, CDI (8.89 g, 54.8 mmol) was added portion wise. The mixture was stirred for 5 min, and the intermediate was observed by LC–MS. Then NH4OH (39.5 mL, 274 mmol) was added at 0 °C, and the solution was stirred for 5 min. The reaction was then quenched with sat. aq. NH4Cl (25 mL) and brine (25 mL), then diluted with 2-MeTHF (50 mL). The phases were separated, and the organic layer was washed again with sat. aq. NH4Cl (25 mL) and brine (25 mL). The organic layer was then dried over Na2SO4, filtered, and concentrated in vacuo to provide crude 7-bromo-1H-indole-6-carboxamide, which was carried over to the next step assuming the quantitative yield. LC–MS (ESI+) m/z 239/241 (MH+). To a suspension of obtained 7-bromo-1H-indole-6-carboxamide in DCM (315 mL) at 0 °C was added Et3N (44.1 mL, 315 mmol), and the resulting orange solution was stirred at this temperature until a homogeneous solution was obtained. MsCl (12.2 mL, 157 mmol) was then added dropwise, and the solution was stirred at 0 °C for 5 min. The mixture was diluted with EtOAc and washed with sat. aq. NaHCO3. The aqueous layer was extracted twice more with EtOAc, and the organic layers were combined, washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by filtering through a pad of silica gel (eluting with EtOAc) to provide compound 48 (5.80 g, 26.24 mmol, 83% yield) as a brown solid. LC–MS (ES-) m/z 219/221. NMR (400 MHz, DMSO): δ 7.73 (dd, J = 7.2, 4.7 Hz, 2H), 7.43 (d, J = 8.2 Hz, 1H), 6.72 (dd, J = 3.0, 1.9 Hz, 1H).

Synthesis of 7-Bromo-3-(2-chloro-5-(trifluoromethyl)pyrimidin-4-yl)-1H-indole-6-carbonitrile (49)

To a solution of 2,4-dichloro-5-trifluoromethylpyrimidine 30 (3.66 mL, 27.2 mmol) in DCE (36.2 mL) was added AlCl3 (1.83 g, 13.6 mmol), and the resulting suspension was stirred at 80 °C for 30 min. 7-Bromo-1H-indole-6-carbonitrile 48 (2.00 g, 9.05 mmol) was added to the mixture, and the red solution was stirred at 80 °C until full conversion (4 h). The reaction mixture was then diluted with 2-MeTHF (100 mL) and washed with water (100 mL). The aqueous layer was extracted with 2-MeTHF (100 mL), and the organic extracts were combined, dried over Na2SO4, filtered, and concentrated in vacuo. Formation of two possible regioisomers was observed with the ratio 3:1 (desired/undesired). The residue was purified by reverse phase chromatography (MeCN in water, 15 to 80% gradient) to provide the desired regioisomer 49 (1.51 g, 3.76 mmol, 42% yield) as a beige solid. LC–MS (ES-) m/z 400.7. 1H NMR (500 MHz, DMSO) δ 13.00 (br s, 1H), 9.17 (s, 1H), 8.35 (d, J = 8.4 Hz, 1H), 8.16 (d, J = 2.6 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H).

Synthesis of (S)-3-(2-((1-Benzyl-5,5-dimethylpiperidin-3-yl)amino)-5-(trifluoromethyl)pyrimidin-4-yl)-7-bromo-1H-indole-6-carbonitrile (50)

7-Bromo-3-(2-chloro-5-(trifluoromethyl)pyrimidin-4-yl)-1H-indole-6-carbonitrile 49 (168 mg, 0.418 mmol), (S)-1-benzyl-5,5-dimethylpiperidin-3-amine 28b (128 mg, 0.585 mmol), and DIPEA (0.221 mL, 1.26 mmol) were dissolved in NMP (2 mL). The reaction mixture was stirred at 130 °C in an oil bath until full conversion (4 h). The mixture was cooled down to rt, diluted with EtOAc, and washed with sat. aq. LiCl. The organic layer was separated, dried over sodium sulfate, filtered, and concentrated in vacuo to provide compound 50 (240 mg, 0.411 mmol, quant. yield), which was used in the next step without further purification. LC–MS (ESI+) m/z 581.3/583.4 (MH+).

Synthesis of (S)-7-(Dimethylphosphoryl)-3-(2-((5,5-dimethylpiperidin-3-yl)amino)-5-(trifluoromethyl)pyrimidin-4-yl)-1H-indole-6-carbonitrile (13)

Compound 50 (240 mg, 0.411 mmol), Xantphos (24.3 mg, 41.1 umol), palladium(II) acetate (4.66 mg, 20.6 umol), and K3PO4 (96.0 mg, 0.452 mmol) were combined in a 2.5 mL microwave vial under nitrogen. Dimethylphosphine oxide (39.2 mg, 0.494 mmol) was dissolved in anhydrous DMF (1 mL), and the solution was degassed before being combined with the other reactants in a microwave vial. The sealed vial with the reaction mixture was then submitted to heat in a microwave reactor at 145 °C for 45 min. The reaction mixture was cooled down to rt, diluted with 2-MeTHF, and washed with sat. aq. NaHCO3 and brine. The organic layer was separated, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by reverse phase chromatography (MeCN in aq. 10 mM ammonium formate, pH 3.8, 0 to 100% gradient) to provide (S)-3-(2-((1-benzyl-5,5-dimethylpiperidin-3-yl)amino)-5-(trifluoromethyl)pyrimidin-4-yl)-7-(dimethylphosphoryl)-1H-indole-6-carbonitrile (58.0 mg, 0.10 mmol, 24% yield) as a pale brown oil. LC–MS (ESI+) m/z 581.4 (MH+). To a stirring solution of obtained compound (58.0 mg, 0.10 mmol) in EtOH (12.5 mL) were added Pd/C 10% w/w (1.1 mg, 0.01 mmol) and Boc2O (65.5 mg, 0.30 mmol). The reaction mixture was evacuated and backfilled with nitrogen for 3 times before being filled with hydrogen. The reaction mixture was then stirred at rt for overnight under hydrogen atm (1 atm.). After 16 h, an incomplete conversion was observed; thus, the reaction mixture was filtered through a pad of Celite and concentrated under reduced pressure. The reaction was then repeated with the residue as described above. After almost complete consumption of starting material (48 h), the reaction mixture was filtered through a pad of Celite and concentrated in vacuo to provide the crude product (S)-tert-butyl 5-((4-(6-cyano-7-(dimethylphosphoryl)-1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-3,3-dimethylpiperidine-1-carboxylate, which was engaged in the next step. Thus, the obtained oil was redissolved in DCM (5 mL), and TFA (0.23 mL, 3.0 mmol) was added. The reaction mixture was stirred at rt for 16 h. The mixture was then concentrated in vacuo, and the residue was purified by reverse phase chromatography (MeCN in aq. 10 mM ammonium formate, pH 3.8, 0 to 100% gradient) to provide compound 13 (11.11 mg, 0.023 mmol, 23% yield over two steps) as a white solid. LC–MS (ESI+) m/z 491.3 (MH+). 1H NMR (500 MHz, DMSO) δ 12.12 (br s, 1H), 8.74 (d, J = 7.5 Hz, 0.5H), 8.65–8.54 (m, 1.5H), 8.25 (s, 1H), 8.21–8.18 (m, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.67 (td, J = 8.4, 3.0 Hz, 1H), 4.18–4.01 (m, 1H), 3.17–3.04 (m, 2H), 2.30–2.21 (m, 2H), 2.07–2.01 (m, 6H), 1.76–1.64 (m, 1H), 1.35–1.20 (m, 1H), 1.03 (s, 3H), 0.89–0.85 (m, 3H). Rotamers.

Synthesis of (S)-7-(Dimethylphosphoryl)-3-(2-((6,6-dimethylpiperidin-3-yl)amino)-5-(trifluoromethyl)pyrimidin-4-yl)-1H-indole-6-carbonitrile (SY-5609)

7-Bromo-3-(2-chloro-5-(trifluoromethyl)pyrimidin-4-yl)-1H-indole-6-carbonitrile 49 (200 mg, 0.498 mmol), (S)-6,6-dimethylpiperidin-3-amine 45 (95.8 mg, 0.747 mmol), and DIPEA (0.174 mL, 0.996 mmol) were dissolved in NMP (4 mL). The reaction mixture was stirred at 130 °C in an oil bath until full conversion (3 h). The mixture was cooled down to rt, loaded directly to C18 column, and purified by reverse phase chromatography (MeCN (0.1% FA) in water (0.1% FA), 0 to 100% gradient) to provide (S)-7-bromo-3-(2-((6,6-dimethylpiperidin-3-yl)amino)-5-(trifluoromethyl)pyrimidin-4-yl)-1H-indole-6-carbonitrile (245 mg, 0.497 mmol, quant. yield) as a beige solid. LC–MS (ESI+) m/z 493.2/495.2 (MH+). Then the obtained compound (180 mg, 0.365 mmol), Xantphos (21.5 mg, 36.5 umol), palladium(II) acetate (4.14 mg, 18.2 umol), and K3PO4 (85.2 mg, 0.401 mmol) were combined in a 2.5 mL microwave vial under nitrogen. Dimethylphosphine oxide (73 mg, 0.912 mmol) was dissolved in anhydrous DMF (1 mL), and the solution was degassed before being combined with the other reactants in a microwave vial. The sealed vial with the reaction mixture was then submitted to heat in a microwave reactor at 150 °C for 45 min. The reaction mixture was cooled down to rt, loaded directly to C18 column, and purified by reverse phase chromatography (MeCN in aq. 10 mM ammonium formate, pH 3.8, 15 to 35% gradient) to provide compound SY-5609 (76 mg, 0.155 mmol, 42% yield) as an off-white solid. HR-MS (C23H26F3N6OP): [MH]+ theor.: 491.1931, [MH]+ exp: 491.1933. 1H NMR (500 MHz, DMSO) δ 12.12 (br s, 1H), 8.74 (d, J = 8.8 Hz, 0.5H), 8.63 (s, 0.5H), 8.60 (s, 0.5H), 8.49 (d, J = 8.5 Hz, 0.5H), 8.34 (brs, 1H), 8.21 (s, 0.5H), 8.16 (s, 0.5H), 7.95–7.87 (m, 1H), 7.74–7.68 (m, 1H), 3.93 (br s, 1H), 3.02–2.94 (m, 1H), 2.75 (t, J = 9.8 Hz, 1H), 2.05 (s, 3H), 2.02 (s, 3H), 1.90–1.80 (m, 1H), 1.77–1.64 (m, 1H), 1.62–1.51 (m, 1H), 1.47–1.35 (m, 1H), 1.16–1.08 (m, 6H). Rotamers. 31P NMR (203 MHz, DMSO) δ 40.20 (s).

Synthesis of (2-Bromo-6-nitrophenyl)(methyl)sulfane (52)

A 20% aq. solution of NaSMe (10.35 g, 29.55 mmol) was added to a solution of 1-bromo-2-fluoro-3-nitrobenzene 51 (5.0 g, 22.73 mmol) in DMF (50 mL) at 0 °C within 10 min. The reaction mixture was stirred for 1 h 50 min at 15 °C. The mixture was added dropwise into water (200 mL) and stirred for 30 min, then filtered, and the obtained solid was dried under reduced pressure to afford compound 52 (4.5 g, 18.2 mmol, 80% yield) as a white solid, which was used in the next step without further purification.

Synthesis of 6-Bromo-7-(methylsulfonyl)-1H-indole (53)

To a mixture of 52 (6.0 g, 24.2 mmol) in DCM (100 mL) was added m-CPBA (≤77%) (14.9 g, 60.5 mmol) in one portion at rt. The reaction mixture was stirred at rt for 2 h. The mixture was poured into water (300 mL) and extracted with EtOAc (2 × 150 mL). The combined organic phase was washed with sat. aq. Na2SO3 (3 × 200 mL), brine (2 × 200 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by normal phase chromatography on silica gel (DCM in hexanes, 10 to 100% gradient) to afford a yellow solid of 1-bromo-2-(methylsulfonyl)-3-nitrobenzene (4.4 g, 15.7 mmol, 65% yield). To the obtained compound (4.4 g, 15.7 mmol) in THF (100 mL) at −78 °C was added dropwise 1 M bromo(vinyl)magnesium in THF (78.5 mL, 78.5 mmol). The mixture was stirred at −78 °C for 2 h. The mixture was then poured into water (300 mL) and extracted with EtOAc (2 × 150 mL). The combined organic phase was washed with brine (2 × 200 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by normal phase chromatography on silica gel (EtOAc in hexanes, 10 to 100% gradient) to afford compound 53 (1.8 g, 6.57 mmol, 42% yield) as a yellow solid.

Synthesis of 7-(Methylsulfonyl)-1H-indole-6-carbonitrile (54)

To a solution of 53 (700 mg, 2.55 mmol) in DMF (20 mL) was added CuCN (685 mg, 7.65 mmol) in one portion. The reaction mixture was stirred at 140 °C for 1 h. The mixture was poured into water (100 mL) and extracted with EtOAc (2 × 50 mL). The combined organic phase was washed with brine (2 × 100 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by normal phase chromatography on silica gel (EtOAc in hexanes 10 to 60% gradient) to afford 54 (450 mg, 2.04 mmol, 80% yield) as a yellow solid.

Synthesis of tert-Butyl 3-bromo-6-cyano-7-(methylsulfonyl)-1H-indole-1-carboxylate (55)

To a mixture of 54 (450 mg, 2.04 mmol) in DMF (20 mL) was added NBS (340 mg, 2.24 mmol) in one portion at rt. The reaction mixture was stirred at rt for 2 h. The mixture was poured into water (100 mL) and extracted with EtOAc (2 × 50 mL). The combined organic phase was washed with brine (2 × 50 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by normal phase chromatography on silica gel (EtOAc in hexanes, 10 to 60% gradient) to afford 3-bromo-7-(methylsulfonyl)-1H-indole-6-carbonitrile (400 mg, 1.34 mmol, 66% yield) as a white solid. To a mixture of obtained compound (400 mg, 1.34 mmol) and Boc2O (438 mg, 2.01 mmol) in THF (20 mL) were added DIPEA (0.47 mL, 2.67 mmol) and DMAP (33 mg, 0.27 mmol, 20 mol %). The mixture was stirred at 80 °C for 4 h. The residue was poured into water (50 mL) and extracted with EtOAc (2 × 30 mL). The combined organic phase was washed with brine (2 × 50 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by normal phase chromatography on silica gel (EtOAc in hexanes, 0 to 100% gradient) to provide 55 (400 mg, 1 mmol, 75% yield) as a white solid.

Synthesis of tert-Butyl 6-cyano-7-(methylsulfonyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-1-carboxylate (56)

To a degassed mixture of 55 (400 mg, 1 mmol), B2Pin2 (305 mg, 1.2 mmol), and KOAc (196.3 mg, 2 mmol) in dioxane (10 mL) was added Pd(dppf)Cl2 (73.2 mg, 0.1 mmol, 10 mol %) at rt. The reaction mixture was then stirred at 80 °C for 4 h. The residue was poured into water (20 mL) and extracted with EtOAc (2 × 10 mL). The combined organic phase was washed with brine (2 × 10 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by prep-HPLC (MeCN in water) to afford 56 (95 mg, 0.21 mmol, 21% yield) as a yellow oil.

Synthesis of (S)-7-(Methylsulfonyl)-3-(2-(piperidin-3-ylamino)-5-(trifluoromethyl)pyrimidin-4-yl)-1H-indole-6-carbonitrile (11)

To a degassed mixture of 56 (95 mg. 0.21 mmol) and (S)-tert-butyl 3-((4-chloro-5-(trifluoromethyl)pyrimidin-2-yl)amino)piperidine-1-carboxylate 37 (120 mg, 0.315 mmol) in dioxane (10 mL) and water (2 mL) were added Pd(PPh3)4 (25.3 mg, 0.021 mmol, 10 mol %) and Na2CO3 (44.5 mg, 0.42 mmol). The reaction mixture was stirred at 100 °C for 4 h. The residue was poured into water (30 mL) and extracted with EtOAc (2 × 20 mL). The combined organic phase was washed with brine (2 × 20 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by normal phase chromatography on silica gel (EtOAc in hexanes, 0 to 100% gradient) to afford compound (S)-tert-butyl 3-((4-(6-cyano-7-(methylsulfonyl)-1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl)amino)piperidine-1-carboxylate (80 mg, 0.14 mmol) as a yellow solid. To a solution of obtained compound in DCM (2 mL) was added TFA (0.4 mL). The reaction mixture was stirred at rt for 30 min and then concentrated under reduced pressure to dryness. The residue was purified by reverse phase chromatography (MeCN in water, 0.1% formic acid buffers, 0 to 100% gradient) to provide compound 11 (28 mg, 0.055 mmol, 26% yield) as a white solid. LC–MS (ESI+) m/z 465.2 (MH+). 1H NMR (MeOD, 400 MHz) δ 8.84 (br s, 1H), 8.65 (s, 1H), 8.52 (br s, 1H), 8.16 (br s, 1H), 7.73 (d, J = 8.60 Hz, 1H), 4.36–4.25 (m, 1H), 3.62–3.47 (m, 1H), 3.43 (s, 3H), 3.28–3.20 (m, 1H), 3.02–2.91 (m, 2H), 2.19–2.16 (m, 1H), 2.10–2.04 (m, 1H), 1.90–1.68 (m, 2H).

Synthesis of 6-Bromo-7-(methylthio)-1H-indole (57)

To a mixture of (2-bromo-6-nitrophenyl)(methyl)sulfane 52 (10 g, 40.3 mmol) in THF (200 mL) at −78 °C was added dropwise 1 M in THF bromo(vinyl)magnesium (201.6 mL, 201.6 mmol). The reaction mixture was then stirred at 0 °C for 2 h. The mixture was poured into cold water (500 mL) and extracted with EtOAc (2 × 300 mL). The combined organic phase was washed with brine (2 × 300 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by normal phase chromatography on silica gel (EtOAc in hexanes, 0 to 100% gradient) to afford 57 (6 g, 24.8 mmol, 61% yield) as a yellow oil.

Synthesis of 6-Bromo-3-(2,5-dichloropyrimidin-4-yl)-7-(methylthio)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indole (58)

To a solution of 2,4,5-trichloropyrimidine 14 (2.73 g, 14.8 mmol) in DCE (30 mL) was added AlCl3 (2.48 g, 18.6 mmol) in one portion, and the obtained suspension was stirred at 80 °C for 30 min. Then indole 57 (3 g, 12.4 mmol) was added, and the reaction mixture was stirred at 80 °C for 12 h. The mixture was poured into cold water (200 mL) and extracted with EtOAc (2 × 200 mL). The combined organic phase was washed with brine (2 × 200 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by normal phase chromatography on silica gel (EtOAc in hexanes, 0 to 100% gradient) to afford 6-bromo-3-(2,5-dichloropyrimidin-4-yl)-7-(methylthio)-1H-indole (2 g, 5.14 mmol, 41% yield) as a brown solid. Next, to the obtained compound (2 g) in DMF (50 mL) at 0° was added NaH, 60% oil dispersion (411 mg, 10.28 mmol) in few portions. The mixture was stirred at 0 °C for 30 min followed by addition of SEM-Cl (1.37 mL, 7.71 mmol). The resulting mixture was then stirred at rt for 1 h. The mixture was poured into cold water (100 mL) and extracted with EtOAc (2 × 50 mL). The combined organic phase was washed with brine (2 × 50 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by normal phase chromatography on silica gel (EtOAc in hexanes, 0 to 100% gradient) to provide compound 58 (1.4 g, 2.7 mmol, 52% yield) as a yellow solid.

Synthesis of 3-(2,5-Dichloropyrimidin-4-yl)-7-(methylsulfonyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indole-6-carbonitrile (59)

To a solution of 58 (1.2 g, 2.31 mmol) in DCM (50 mL) at 0 °C was added m-CPBA (≤77%) (1.3 g, 5.78 mmol) in one portion. The reaction mixture was then stirred at rt for 2 h. The mixture was poured into cold water (200 mL) and extracted with EtOAc (2 × 100 mL). The combined organic phase was washed with sat. aq. K2CO3 (2 × 50 mL), brine (2 × 100 mL), dried over Na2SO4, filtered, and concentrated in vacuo to afford 6-bromo-3-(2,5-dichloropyrimidin-4-yl)-7-(methylsulfonyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indole (1 g, 1.81 mmol, 79% yield) as a white solid, which was used without purification. Then to a solution of obtained compound (700 mg, 1.27 mmol) in DMF (20 mL) was added CuCN (170 mg, 1.91 mmol) in one portion, and the reaction mixture was stirred at 80 °C for 3 h. The mixture was poured into cold water (50 mL) and extracted with EtOAc (2 × 50 mL). The combined organic phase was washed with brine (2 × 100 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by normal phase chromatography on silica gel (EtOAc in hexanes, 10 to 100% gradient) to afford compound 59 (400 mg, 0.8 mmol, 63% yield) as a yellow solid.

Synthesis of (S)-tert-Butyl 3-((5-chloro-4-(6-cyano-7-(methylsulfonyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indol-3-yl)pyrimidin-2-yl)amino)piperidine-1-carboxylate (60)

To a solution of 59 (200 mg, 0.4 mmol) in NMP (2 mL) were added (S)-tert-butyl 3-aminopiperidine-1-carboxylate 18 (320 mg, 1.6 mmol) and DIPEA (0.21 mL, 1.2 mmol). The reaction mixture was stirred at 90 °C for 1 h, then poured into water (10 mL) under stirring, while yellow solid was formed. The solid was filtered, washed with water (2 × 2 mL), and dried under vacuum to provide compound 60 (140 mg, 0.21 mmol, 53% yield), which was used in the next step without further purification.

Synthesis of (S)-tert-Butyl 3-((4-(6-cyano-7-(methylsulfonyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indol-3-yl)-5-ethylpyrimidin-2-yl)amino)piperidine-1-carboxylate (61)

To a degassed solution of 60 (140 mg, 0.21 mmol) in THF (1 mL) and water (0.2 mL) were added potassium vinyltrifluoroborate 20 (140 mg, 1.05 mmol), K3PO4 (90 mg, 0.42 mmol), and XPhos Pd G1 (23.3 mg, 0.0315 mmol, 15 mol %). The reaction mixture was degassed and then stirred at 80 °C for 12 h. The mixture was concentrated under reduced pressure, and the residue was then diluted with water (10 mL) and extracted with EtOAc (2 × 10 mL). The combined organic phase was washed with brine (2 × 5 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by normal phase chromatography on silica gel (EtOAc in hexanes, 0 to 100% gradient) to afford (S)-tert-butyl 3-((4-(6-cyano-7-(methylsulfonyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indol-3-yl)-5-vinylpyrimidin-2-yl)amino)piperidine-1-carboxylate (59 mg, 0.09 mmol, 43% yield) as a yellow solid. Next, the obtained compound was dissolved in MeOH (5 mL), and Pd/C wt. 10% (50 mg) and triethylamine (25 μL, 0.18 mmol) were added. The resulting suspension was degassed under vacuum and purged with hydrogen several times. The reaction mixture was then stirred under hydrogen atm. (15 psi) at 15 °C for 1 h. The mixture was filtered through Celite pad, and the filtrate was concentrated to afford compound 61 (36 mg, 0.05 mmol, 60% yield), which was used in the next step without further purification.

Synthesis of (S)-3-(5-Ethyl-2-(piperidin-3-ylamino)pyrimidin-4-yl)-7-(methylsulfonyl)-1H-indole-6-carbonitrile (12)

To a solution of 61 (36 mg, 0.05 mmol) in 1,4-dioxane (0.5 mL) was added H2SO4 (27 μL, 0.5 mmol), and the reaction mixture was stirred at 40 °C for 3 h. The reaction mixture was concentrated under reduced pressure, then redissolved in MeCN (1 mL) followed by addition of K2CO3 (0.2 g, powder). The mixture was then stirred at 15 °C for another 1 h. The mixture was poured into water (10 mL) and extracted with EtOAc (2 × 10 mL). The combined organic phase was washed with brine (2 × 10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The obtained residue was purified by reverse phase chromatography (MeCN in water, 0.1% formic acid buffers) to afford compound 12 (2.3 mg, 0.005 mmol, 10% yield, FA) as a white solid. LC–MS (ESI+) m/z 425.3 (MH+). 1H NMR (400 MHz, MeOH-d4) δ 8.76–8.68 (m, 1H), 8.57–8.47 (m, 1H), 8.27 (s, 1H), 8.10 (s, 1H), 7.70 (d, J = 8.3 Hz, 1H), 4.26–4.15 (m, 1H), 3.52–3.50 (m, 1H), 3.42 (s, 3H), 3.27–3.21 (m, 1H), 2.99 (d, J = 10.1 Hz, 2H), 2.75 (d, J = 7.5 Hz, 2H), 2.21–2.12 (m, 1H), 2.10–1.99 (m, 1H), 1.90–1.67 (m, 2H), 1.21 (t, J = 7.5 Hz, 3H).

Computational Chemistry

All computational modeling utilized the Schrodinger Suite, Version 12.4, Release 2020–2 (Schrodinger, LLC, New York, NY). (21) Crystal Structures of CDK7/Cyclin H/Mat 1 (PDB: 1UA2: A, 7B5Q: H,I,J), CDK2/cyclin A (PDB: 1pkd: A,B), CDK9/Cyclin T1 (PDB 3BLQ: A,B) and CDK12 (PDB: 6B3E: A,B) were obtained from the Protein Data Bank. All proteins were prepared using the default settings in the Protein Preparation Wizard. Ionization states for side chain heteroatoms were applied using Epik at pH 7.0 ± 2. Ligands were built in Maestro and prepared for docking using LigPrep with protonation states assigned using Epik. Conformational search and minimization utilized Macromodel the OPLS3e force field. Molecular dynamics simulations were conducted using the Desmond Molecular Dynamics System from Schrodinger and the default settings. The prepared protein complexes were placed in a minimum volume orthorhombic box using System Builder and used the SPC water model and the OPLS3e force field. The system was neutralized and then relaxed before being subjected to a production simulation of 50 ns at 300k and 1.01 bar. The system was analyzed using the Simulation interaction Diagram tool in the Schrodinger Suite and dihedral angles were calculated using the trajectory_dihedral.py script.

SPR

For SPR experiments, CDK7/cyclin H dimer was expressed using baculovirus pFastBac expression vectors in SF9 insect cells. Individual virus stocks were produced for full length CDK7 (1–346, accession P50613) containing an N-terminal TEV-cleavable 6His tag and full-length Cyclin H (1–323, accession P51946). For expression of the CDK7/Cyclin H complex, SF9 cells were coinfected with separate viruses expressing CDK7 and Cyclin H. The CDK7/Cyclin H complex was purified with a Nickel affinity column, followed by TEV protease cleavage. The complex was further purified by size exclusion gel-filtration chromatography to yield CDK7/cyclin H dimer complex.
Binding kinetics and affinities to CDK7/Cyclin H dimer were measured using a Biacore T200 surface plasmon resonance (SPR) instrument (GE Healthcare). The CDK7/cyclin H dimer was amine-coupled to a CM5 sensor chip at pH 6.5 in 10 mM MES buffer at a concentration of 12.5 μg/mL with a flow rate of 10 μL/min. Target protein was immobilized on two flow cells for 12–16 s to achieve immobilized protein levels of 200–400 response units. The remaining two flow cells were used as reference, with no immobilized protein.
Each compound concentration cycle was run at 100 μL/min with 70 s contact time, 300 s dissociation time, 60 s regeneration with 10 mM glycine pH 9.5, and 400 s stabilization time. Compound titration followed a 9-step, 2-fold serial dilution with top concentration of 20 nM in 10 mM HEPES buffer at pH 7.5 with 150 mM NaCl, 0.05% Surfactant P20, and 0.0002% DMSO. Compounds that were found to have a slow off-rate were followed up with a 4-step, 3-fold serial dilution with a top concentration of 10 nM and a 1800 s dissociation time, all other conditions were consistent.
Data analyses are carried out using Biacore T200 evaluation software v2.0 (GE Healthcare). For each compound, 0 nM blank buffer controls and reference flow-cell data were subtracted to remove background and systematic noise. Compound titrations were globally fit by a 1:1 langmuir binding model. Best-fit values for compound binding on-rate (kon, M–1 s–1), dissociation off-rate (koff, s–1), and compound affinities (Kd) for CDK7/Cyclin H compound interactions were obtained using the following equation:
(1)
where A refers to the compound concentration (M), B refers to the ligand density (RU), and Kd is calculated using the following equation:
(2)

Kinase Enzymatic Activity Assay

Compound potencies were determined for each CDK by measuring the loss of activity of the kinase to phosphorylate a 5-FAM labeled peptide substrate by mobility shift with a LabChip EZ Reader (PerkinElmer, Waltham MA), as previously described. (8) IC50 values for each CDK/cyclin dimer were determined in the presence of ATP at the Km concentration: 100 μM ATP with 0.5 nM CDK2/Cyclin E1, 30 μM ATP with 8 nM CDK9/Cyclin T1, and 30 μM ATP with 50 nM CDK12/Cyclin K.

Kinase Selectivity

CDK selectivity was measured in the 33PanQinase Activity Assay (ProQinase GmbH, Freiburg Germany). Each compound was tested at 10 concentrations spanning the range of 1 × 10–05 M to 3 × 10–10 M against the panel of 28 CDK protein kinases. Assay conditions were specific to the activity of the kinase, as predetermined for the 33PanQinase Activity Assay. For the CDK7 assay, conditions were, 6.6 nM CDK7/CycH/MAT1 with 2 μg peptide substrate and 3 μM ATP (near ATP-Km apparent as measured in the PanQinase Activity Assay with equimolar Mn2+ and Mg2+, wherein tighter binding of Mn2+-ATP over Mg2+-ATP is expected to result in higher affinity for ATP). (29)
Broad kinase selectivity was determined by SelectScreen Biochemical Kinase Profiling (ThermoFisher Scientific, Madison WI). Compounds were screened at 1.0 μM to determine % inhibition against a panel of 485 kinases.

Cell Culture

HCC70, MDA-MB-468, CAOV3, and OVCAR3 cell lines were obtained from ATCC. HDF (adult) fibroblast cells were obtained from Sigma. GM07492 WT fibroblast cells were obtained from Coriell Institute for Medical Research. All cell lines were cultured at 5% CO2, 37 °C following vendor instructions.

Antiproliferation Assay

Logarithmically growing cells were plated the day before treatment then incubated with compound for 72 h. Compound was dosed at 4,1.26, 0.4, 0.126, 0.04, 0.0126, 0.004, 0.00126, 0.0004, and 0.000126 μM (duplicate measurements per dose and time point). For regular compound screening, CellTiter-Glo 2.0 cell viability assay (Promega #G9243) was used for readout. For the experiment in Figure S2, CyQUANT Direct Cell Proliferation Assay (C35012, ThermoFisher Scientific) was used for readout. Antiproliferative effect of compounds was calculated against averaged DMSO group signal on the same plate.

Animal Study

Six-to-eight-week-old Balb/c nude female mice (Shanghai Lingchang BioTech Co. Ltd.) were implanted subcutaneously with HCC70 cells (Shanghai Chempartner). After tumor cell inoculation, tumor volumes were measured twice weekly using a caliper. For efficacy arms, once tumors had reached 150–200 mm3, mice were randomized into groups for dosing, 10 mice per arm. Animal body weight and tumor volume were measured twice weekly. Tumor growth inhibition (TGI) was calculated as follows:
(3)
For PK/PD arms, once tumor reached 500 mm3, mice were randomized into groups for dosing. After single dose or 7 day of dosing, plasma samples were collected at 1, 2, 4, 8 h post dosing and tumor samples were collected at 2, 4, 8 h post dosing.
In the SY-5102 study, mice were dosed via oral gavage at 4 or 2.5 mg/kg BID for 21 days followed by 7 day post treatment observation period. In the SY-5609 study, mice were dosed via oral gavage at 2 mg/kg QD for 21 days followed by 7 day post treatment observation period.
Animal experiments were conducted in AALAC-accredited facilities at ChemPartner (Shanghai, China). They were carried out in compliance with humane treatment of research animals under an experimental protocol approved by the Institutional Animal Care and Use Committee (IACUC) of ChemPartner and review team of Syros Pharmaceuticals.

ADME Profiling

Mouse microsomal stability, mouse plasma protein binding, MDCK permeability, and LogD assays were performed by WuXi Apptec Incorporated.

Pharmacokinetics

Pharmacokinetic studies were performed by WuXi Apptec Incorporated. With the exception of SY-5609 and Compounds 13, which substituted 15% SBE-β-CD, compounds were formulated in 15% Captisol in water at a concentration of 0.2 to 0.5 mg/mL for intravenous injection and 0.5 to 1 mg/mL for oral gavage. Male CD1 mice (three per dose group) were dosed via tail vein injection or oral gavage according to their bodyweight. Blood was collected from the saphenous vein and centrifuged to obtain plasma. Blood was collected at 0.25, 0.5, 1, 2, 4, 6, 10, and 24 h post administration to obtain a full PK time-course. Plasma was processed for bioanalysis, which was performed using LC–MS/MS analysis (Waters Aquity UPLC and API 5500 or API 4000 triple quad mass spectrometer). All studies were conducted following an approved IACUC protocol.

Immunoblotting

Cells were lysed with RIPA lysis and extraction buffer (ThermoFisher: 89901), with 1× Halt Protease and Phosphatase Inhibitor Cocktail (100×) Life technologies: 78440 and 1× Benzonase Nuclease (1000×), Sigma-Aldrich: E1014–25KU) for 20 min on a rotator at 4 °C. After clarification of lysates, protein concentration was quantified with Pierce BCA protein assay kit (ThermoFisher Scientific: 23225).
The 4× LDS buffer (NuPAGE LDS lysis buffer NP0008, final concentration 2×) and DTT (final concentration 50 mM) were added to protein lysates, and lysates were incubated at 95 °C for 5 min before loading on to NuPAGE Novex 4–12% Bis-Tris Protein Gels (Life Technologies: WG1402Bx10) or 3–8% Tris-Acetate protein gels (Life Technologies: WG1602BOX).
Gels were run in NuPAGE MES SDS Running Buffer (Life Technologies: NP0002) or NuPAGETris-Acetate SDS Running Buffer (Life Technologies: LA0041) at constant voltage 160 V for about 1.5 h or until loading dyes ran out, then transferred at constant Amp 360 mA in 1× NuPage transfer buffer with 20% methanol for 1 h. Membranes were blocked in blocking buffer (Odyssey Blocking Buffer (PBS): 927–40000) for 1 h, and incubated with primary antibodies overnight at 4 °C on shaker.
The next day, membranes were probed with IRDye 800CW Secondary Antibodies (1:10000) in 1× Odyssey Blocking Buffer with 0.2% Tween-20 for 1 h, analyzed, and quantified on LI-COR Odyssey imaging system. Antibodies against MCL1 (4572), p-CDK2 (Thr160) (2561), CDK2 (2546), RNA pol II (14958), and Cyclin B1 (12231) were purchased from Cell Signaling Technology. Antibody against α-tubulin (T6199) was purchased from Sigma-Aldrich. Antibody against c-MYC (ab32072) was purchased from Abcam. Antibody against RNA pol II phospho-Ser5 (04–1572) was purchased from Millipore Sigma.

Apoptosis Assay

Logarithmically growing cells were seeded the day before treatment and grown to 60 to 80% confluence on the day of treatment. Cells were dosed with either DMSO or the indicated dosage of SY-5609 for 48 or 72 h in a humidified 37 °C incubator with 5% CO2 before staining with Annexin V and propidium iodide (10010–02, Southernbiotech, ApoScreen Annexin V Apoptosis Kit) following vendor’s recommendations. Samples were analyzed by flow cytometry, and results were analyzed using FlowJo V10 (Becton, Dickinson and Company).

Cell Cycle Analysis

Cells were plated at 1 million per 10 cm dish the day before treatment. The next day, cells were treated with the indicated dosages of SY-5609. Then 48 h after treatment, cells were lifted with Accumax cell detachment solution (Millipore Sigma: SCR006) and fixed with histochoice (Sigma: 2904) at rt for 20 min. Then cells were stained with FxCycle violet stain (ThermoFisher Scientific: F10347) for 30 min at rt before FACS analysis.
Cell cycle analyses were done using FlowJo software where a single cell population was selected and G1 and G2/M populations were gated in each sample.

PD Sample Analysis

For immunoblotting analysis, tumor samples were lysed with RIPA lysis and extraction buffer (ThermoFisher: 89901) with 1× Halt Protease and Phosphatase Inhibitor Cocktail (100×) Life technologies: 78440 and 1× Benzonase Nuclease (1000×), Sigma-Aldrich: E1014–25KU) and homogenized using Precellys Evolution homogenizer in Tissue homogenizing CKMix-2 mL (P000918-LYSK0-A) tubes. After homogenization, lysates were rotated for 20 min on a rotator at 4 °C. Protein concentration was quantified with Pierce BCA protein assay kit (ThermoFisher Scientific: 23225) after lysate clarification. Tumor samples were analyzed using immunoblotting method described above.
Precellys (KT03961–1–009.2) kits were used to homogenize tumor tissues. Total RNA were extracted following mini prep kit from Qiagen (Qiagen, RNeazy kit: 74134). Then 200 ng of total RNA was used for NanoString XT element nCounter assay via custom code sets. NanoString results were processed according to NanoString analysis guidelines (Gene Expression Data Analysis Guidelines MAN-C0011). Results passing QC were first normalized using the average arithmetic mean of positive control and then normalized using average geometric mean of reference control genes.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c01171.

  • Additional figures illustrating dihedral angles from molecular dynamics simulations, CDK selectivity of SY-5102 and SY-5609, kinase selectivity of SY-5102 and SY-5609, cellular antiproliferation panel for SY-5609, quantification of immunoblotting from Figure 6, immunoblot images from Figure 8, small molecule X-ray structure of SY-5609, protein cocrystal structure of Compound 4 with CDK2, NMR spectra and LC–MS chromatograms for all compounds (PDF)

  • Molecular formula strings (CSV)

  • PDB ID Codes: Compound 4 with CDK2 (PDB: 7RA5)

Accession Codes

CCDC 2093192 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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Author Information

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  • Corresponding Author
  • Authors
    • Kristin B. Hamman - Syros Pharmaceuticals Inc., 35 Cambridge Park Drive, Fourth Floor, Cambridge, Massachusetts 02140, United States;  Present Address: Merck and Co Inc., 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States
    • Shanhu Hu - Syros Pharmaceuticals Inc., 35 Cambridge Park Drive, Fourth Floor, Cambridge, Massachusetts 02140, United States
    • Sydney Alnemy - Syros Pharmaceuticals Inc., 35 Cambridge Park Drive, Fourth Floor, Cambridge, Massachusetts 02140, United States
    • Janessa Mihalich - Syros Pharmaceuticals Inc., 35 Cambridge Park Drive, Fourth Floor, Cambridge, Massachusetts 02140, United States
    • Anzhelika Kabro - Paraza Pharma Inc., 2525 Avenue Marie-Curie, Montreal, Quebec H4S 2E1, Canada
    • Kenneth Matthew Whitmore - Paraza Pharma Inc., 2525 Avenue Marie-Curie, Montreal, Quebec H4S 2E1, Canada
    • Dana K. Winter - Paraza Pharma Inc., 2525 Avenue Marie-Curie, Montreal, Quebec H4S 2E1, Canada
    • Stephanie Roy - Paraza Pharma Inc., 2525 Avenue Marie-Curie, Montreal, Quebec H4S 2E1, Canada
    • Stephane Ciblat - Paraza Pharma Inc., 2525 Avenue Marie-Curie, Montreal, Quebec H4S 2E1, Canada
    • Nan Ke - Syros Pharmaceuticals Inc., 35 Cambridge Park Drive, Fourth Floor, Cambridge, Massachusetts 02140, United States
    • Anneli Savinainen - Syros Pharmaceuticals Inc., 35 Cambridge Park Drive, Fourth Floor, Cambridge, Massachusetts 02140, United States;  Present Address: Aura Biosciences Inc., 85 Bolton St, Cambridge, Massachusetts 02140, United States
    • Ashraf Wilsily - Syros Pharmaceuticals Inc., 35 Cambridge Park Drive, Fourth Floor, Cambridge, Massachusetts 02140, United States
    • Goran Malojcic - Syros Pharmaceuticals Inc., 35 Cambridge Park Drive, Fourth Floor, Cambridge, Massachusetts 02140, United States;  Present Address: Disease Area Oncology, Novartis Institutes for Biomedical Research, Basel, Switzerland
    • Robert Zahler - Syros Pharmaceuticals Inc., 35 Cambridge Park Drive, Fourth Floor, Cambridge, Massachusetts 02140, United States;  Present Address: PharmD Consulting LLC, Pennington, New Jersey 08534, United States
    • Darby Schmidt - Syros Pharmaceuticals Inc., 35 Cambridge Park Drive, Fourth Floor, Cambridge, Massachusetts 02140, United States;  Present Address: Inzen Therapeutics, Cambridge, Massachusetts 02139, United States
    • Michael J. Bradley - Syros Pharmaceuticals Inc., 35 Cambridge Park Drive, Fourth Floor, Cambridge, Massachusetts 02140, United States;  Present Address: RADD Pharmaceuticals Inc., 285 Riverside Avenue, Suite 250, Westport, Connecticut 06880, United States
    • Nigel J. Waters - Syros Pharmaceuticals Inc., 35 Cambridge Park Drive, Fourth Floor, Cambridge, Massachusetts 02140, United States;  Present Address: Black Diamond Therapeutics, 1 Main Street, 10th Floor, Cambridge, Massachusetts 02142, United States
    • Claudio Chuaqui - Syros Pharmaceuticals Inc., 35 Cambridge Park Drive, Fourth Floor, Cambridge, Massachusetts 02140, United States
  • Notes
    The authors declare the following competing financial interest(s): Jason Marineau, Claudio Chuaqui, Kristin Hamman, Shanhu Hu, Sydney Alnemy, Janessa Mihalich, Nan Ke, Anneli Savinainen, Ashraf Wilsily, Goran Malojcic, Darby Schmidt, Michael Bradley, and Nigel Waters are (or were) employees and equity holders of Syros Pharmaceuticals Inc. at the time the study was conducted.

Acknowledgments

Click to copy section linkSection link copied!

We thank Wojciech Dworakowski, John Carulli, and Eric Olson for the helpful discussions and critical reading of the manuscript.

Abbreviations Used

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MeCN

acetonitrile

CAK

cyclin activating kinase

CDK

cyclin-dependent kinase

CTD

C-terminal domain

EtOAc

ethyl acetate

MCL

myeloid cell leukemia

MDCK

Madin-Darby canine kidney

MeTHF

2-methyl tetrahydrofuran

OVA

ovarian cancer

RBP

retinol binding protein

SNAr

nucleophilic aromatic substitution

SPR

surface plasmon resonance

ns

nanoseconds

TFIIH

transcription factor II H

TNBC

triple negative breast cancer

References

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This article references 30 other publications.

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    Proceedings of the National Academy of Sciences of the United States of America (2020), 117 (37), 22849-22857CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The human CDK-activating kinase (CAK), a complex composed of cyclin-dependent kinase (CDK) 7, cyclin H, and MAT1, is a crit. regulator of transcription initiation and the cell cycle. It acts by phosphorylating the C-terminal heptapeptide repeat domain of the RNA polymerase II (Pol II) subunit RPB1, which is an important regulatory event in transcription initiation by Pol II, and it phosphorylates the regulatory T-loop of CDKs that control cell cycle progression. Here, the authors detd. the three-dimensional (3D) structure of the catalytic module of human CAK, revealing the structural basis of its assembly and providing insight into CDK7 activation in this context. The unique third component of the complex, MAT1, substantially extends the interaction interface between CDK7 and cyclin H, explaining its role as a CAK assembly factor, and it forms interactions with the CDK7 T-loop, which may contribute to enhancing CAK activity. The authors also detd. the structure of the CAK in complex with the covalently bound inhibitor THZ1 to provide insight into the binding of inhibitors at the CDK7 active site and to aid in the rational design of therapeutic compds.
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    Resolution structure 2.5 Å-of human CDK-activating kinase bound to the clinical inhibitor ICEC0942
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    Biophysical Journal (2021), 120 (4), 677-686CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    The human CDK-activating kinase (CAK), composed of CDK7, cyclin H, and MAT1, is involved in the control of transcription initiation and the cell cycle. Because of these activities, it has been identified as a promising target for cancer chemotherapy. A no. of CDK7 inhibitors have entered clin. trials, among them ICEC0942 (also known as CT7001). Structural information can aid in improving the affinity and specificity of such drugs or drug candidates, reducing side effects in patients. Here, we have detd. the structure of the human CAK in complex with ICEC0942 at 2.5 Å-resoln. using cryogenic electron microscopy. Our structure reveals conformational differences of ICEC0942 compared with previous X-ray crystal structures of the CDK2-bound complex, and highlights the crit. ability of cryogenic electron microscopy to resolve structures of drug-bound protein complexes without the need to crystalize the protein target.
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    Background: Annotations of the phylogenetic tree of the human kinome is an intuitive way to visualize compd. profiling data, structural features of kinases or functional relationships within this important class of proteins. The increasing vol. and complexity of kinase-related data underlines the need for a tool that enables complex queries pertaining to kinase disease involvement and potential therapeutic uses of kinase inhibitors. Results: Here, we present KinMap, a user-friendly online tool that facilitates the interactive navigation through kinase knowledge by linking biochem., structural, and disease assocn. data to the human kinome tree. To this end, preprocessed data from freely-available sources, such as ChEMBL, the Protein Data Bank, and the Center for Therapeutic Target Validation platform are integrated into KinMap and can easily be complemented by proprietary data. The value of KinMap will be exemplarily demonstrated for uncovering new therapeutic indications of known kinase inhibitors and for prioritizing kinases for drug development efforts. Conclusions: KinMap represents a new generation of kinome tree viewers which facilitates interactive exploration of the human kinome. KinMap enables generation of high-quality annotated images of the human kinome tree as well as exchange of kinome-related data in scientific communications. Furthermore, KinMap supports multiple input and output formats and recognizes alternative kinase names and links them to a unified naming scheme, which makes it a useful tool across different disciplines and applications. A web-service of KinMap is freely available at http://www.kinhub.org/kinmap/.
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    Triple-neg. breast cancer (TNBC) is a highly aggressive form of breast cancer that exhibits extremely high levels of genetic complexity and yet a relatively uniform transcriptional program. We postulate that TNBC might be highly dependent on uninterrupted transcription of a key set of genes within this gene expression program and might therefore be exceptionally sensitive to inhibitors of transcription. Utilizing kinase inhibitors and CRISPR/Cas9-mediated gene editing, we show here that triple-neg. but not hormone receptor-pos. breast cancer cells are exceptionally dependent on CDK7, a transcriptional cyclin-dependent kinase. TNBC cells are unique in their dependence on this transcriptional CDK and suffer apoptotic cell death upon CDK7 inhibition. An "Achilles cluster" of TNBC-specific genes is esp. sensitive to CDK7 inhibition and frequently assocd. with super-enhancers. We conclude that CDK7 mediates transcriptional addiction to a vital cluster of genes in TNBC and CDK7 inhibition may be a useful therapy for this challenging cancer.
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    Zhang, Z.; Peng, H.; Wang, X.; Yin, X.; Ma, P.; Jing, Y.; Cai, M. C.; Liu, J.; Zhang, M.; Zhang, S.; Shi, K.; Gao, W. Q.; Di, W.; Zhuang, G. Preclinical efficacy and molecular mechanism of targeting CDK7-dependent transcriptional addiction in ovarian cancer. Mol. Cancer Ther. 2017, 16, 1739– 1750,  DOI: 10.1158/1535-7163.MCT-17-0078
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    Preclinical Efficacy and Molecular Mechanism of Targeting CDK7-Dependent Transcriptional Addiction in Ovarian Cancer
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    Molecular Cancer Therapeutics (2017), 16 (9), 1739-1750CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)
    Ovarian cancer remains a significant cause of gynecol. cancer mortality, and novel therapeutic strategies are urgently needed in clinic as new treatment options. We previously showed that BET bromodomain inhibitors displayed promising efficacy for the treatment of epithelial ovarian cancer by downregulating pivot transcription factors. However, the potential antitumor activities and mol. mechanisms of other epigenetic or transcriptional therapies have not been systematically detd. Here, by performing an unbiased high-throughput drug screen to identify candidate compds. with antineoplastic effects, we identified THZ1, a recently developed covalent CDK7 inhibitor, as a new transcription-targeting compd. that exerted broad cytotoxicity against ovarian tumors. Mechanistically, CDK7 represented a previously unappreciated actionable vulnerability in ovarian cancer, and CDK7 inhibition led to a pronounced dysregulation of gene transcription, with a preferential repression of E2F-regulated genes and transcripts assocd. with super-enhancers. Our findings revealed the mol. underpinnings of THZ1 potency and established pharmaceutically targeting transcriptional addiction as a promising therapeutic strategy in aggressive ovarian cancer. Mol Cancer Ther; 16(9); 1739-50. ©2017 AACR.
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    Biochemistry (2010), 49 (14), 3092-3100CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Various mutations in leucine-rich repeat kinase 2 (LRRK2) have been linked to susceptibility for both familial and idiopathic late-onset Parkinson's disease (PD). In this study, it was demonstrated that phosphorylation of MBP and LRRKtide by the LRRK2 G2019S mutant was activated by Mn2+ in vitro. This enhanced G2019S kinase activity was due to the combination of an increase in kinase and a decrease in ATPase activity by Mn2+. Compared to 10 mM Mg2+, 1 mM Mn2+ reduced ATP Km for G2019S from 103 to 1.8 μM and only modestly reduced kcat (2.5-fold); as a result, the Mn2+ increased its kcat/Km by 22-fold. This change in ATP Km was due in large part to an increase in nucleotide affinity. While Mn2+ also increased ATP affinity and had similar effects on kcat/Km for LRRK2 WT and R1441C enzymes, it reduced their kcat values significantly by 13-17-fold. Consequently, the difference in the kinase activity between G2019S and other LRRK2 variants was enhanced from about 2-fold in Mg2+ to 10-fold in Mn2+ at satg. ATP concns. relative to its Km. Furthermore, while Mg2+ yielded optimal Vmax values at Mg2+ concn. greater than 5 mM, the optimal Mn2+ concn. for activating LRRK2 catalysis was in the micromolar range with increasing Mn2+ above 1 mM causing a decrease in enzyme activity. Finally, despite the large but expected differences in IC50 tested at 100 μM ATP, the apparent Ki values of a small set of LRRK2 ATP-competitive inhibitors were within 5-fold between Mg2+- and Mn2+-mediated reactions except AMP-CPP, an ATP analog.
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Cite this: J. Med. Chem. 2022, 65, 2, 1458–1480
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Published November 2, 2021

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  • Abstract

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    Figure 1

    Figure 1. Structure of compound 1 and predicted interactions with CDK7.

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    Figure 2

    Figure 2. X-ray crystal structure of compound 4 with CDK2 (PDB: 7RA5).

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    Chart 1

    Chart 1. Advanced Analogues Leading to SY-5609
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    Figure 3

    Figure 3. Efficacy of SY-5102 in HCC70 Xenograft. SY-5102 was orally dosed twice daily in HCC70 cell line derived xenograft models at 2.5 and 4 mg/kg for 21 days. Ten mice per arm. Tumor volumes and bodyweight were measured twice weekly. One-tail t test was performed to assess significance of antitumor effect of SY-5102 (**** p < 0.0001).

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    Scheme 1

    Scheme 1. Synthesis of Compounds 1 and 3a
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    aReagents and conditions: (a) 15, AlCl3, DCE, 80 °C, 16 h, 98%; (b) PhSO2Cl, NaOtBu, THF, 0 °C, 1 h, 69%; (c) 18, DIPEA, NMP, 135 °C, 45 min, μw, 64%; (d) (i) 5N aq. NaOH, dioxane, 70 °C, 3–6 h, (ii) HCl/EtOAc, rt, 50–85%; (e) 20, Pd(OAc)2, cataCXium, Cs2CO3, toluene/water, 120 °C, 4 h, 61%; (f) Pd/C, H2, EtOH, 1 atm, rt, 16 h, quant. yield.

    Scheme 2

    Scheme 2. Synthesis of Compounds 2, 6, and 8a
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    aReagents and conditions: (a) 24, Pd(PPh3)4, Cs2CO3, dioxane/water, 100 °C, 1.5 h, 54%; (b) m-CPBA, DCM/THF, rt, 16 h, 95%; (c) chiral amine 18, 27, or 28a, DIPEA, THF, rt, 16 h, 27–50%; (d) (i) 5N aq. NaOH, dioxane, 70 °C, 3 h, (ii) TFA/DCM, rt, 42%; (e) (i) Pd/C, Pd(OH)2/C, H2, EtOH, 1 atm, rt, 16 h, 70%; (ii) 5N aq. NaOH, dioxane, 70 °C, 16 h, 25%; (f) (i) 5N aq. NaOH, dioxane, 70 °C, 3 h; (ii) HBr/AcOH, DCM, rt, 1 h, 66%; (g) NaH, BnBr, 33%; (h) ZrCl4, MeMgBr, 83%; (i) TFA, DCM, quant. yield; (j) Pd/C, H2, 30 psi, quant. yield; (k) CbzCl, NaHCO3, 93%; (l) HCl, EtOAc, quant. yield.

    Scheme 3

    Scheme 3. Synthesis of Compounds 5, 7, and 10a
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    aReagents and conditions: (a) (i) 6-bromo-1H-indole, AlCl3, DCE, 80 °C, 2 h, 26%; (ii) NaOtBu, PhSO2Cl, THF, 0 °C to rt, 16 h, 86%; (b) 18, DIPEA, NMP, 140 °C, 1 h, 86%; (c) 5N aq. NaOH, dioxane, 75 °C, 1 h, quant. yield; (d) (i) Zn, Zn(CN)2, Pd2dba3, XPhos, DMAc, 90 °C, 30 min, 95%; (ii) TFA/DCM, 0 °C, 30 min, 62%; (e) (i) CH3SO2Na, CuI, NMP, 140 °C, 2 h, 9%; (ii) 4 M HCl/EtOAc, rt, 1 h, 29%; (f) (i) 3,5-dimethylisoxazol-4-yl-4-boronic acid, Pd(PPh3)4, Cs2CO3, dioxane/water, 100 °C, 1 h, 90%; (ii) 5N aq. NaOH, dioxane, 70 °C, 3 h; (iii) TFA/DCM, 0 °C, 30 min, 60%.

    Scheme 4

    Scheme 4. Synthesis of Compounds 4 and 9a
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    aReagents and conditions: (a) Boc2O, DMAP, MeCN, rt, 16 h, quant. yield; (b) B2Pin2, [Ir(OMe)(COD)]2, 4,4′-ditert-butyl-2,2′-bipyridine, MTBE, 100 °C, 7 h; (c) 18, ZnCl2, TEA, DCE/tBuOH, rt, 16 h, 29%; (d) Pd(PPh3)4, Cs2CO3, dioxane/water, 95 °C, 8 h, 21%; (e) (i) CH3SO2Na, CuI, NMP, 140 °C, 2 h, 70%; (ii) TFA/DCM, rt, 1 h, 74%; (f) (i) P(O)Me2, Pd(OAc)2, XantPhos, K3PO4, DMF, 150 °C, 45 min, μw; (ii) TFA/DCM, rt, 1 h, 69%.

    Scheme 5

    Scheme 5. Synthesis of SY-5102a
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    aReagents and conditions: (a) 3,5-dimethylisoxazol-4-yl-4-boronic acid, Pd(dppf)Cl2·DCM, NaHCO3, dioxane/water, 100 °C, 2 h, quant. yield; (b) (i) NBS, DCM, 0 °C, 1 h, 49%; (ii) NaH, PhSO2Cl, DMF/THF, 0 °C, 1 h, 68%; (c) B2Pin2, Pd(dppf)Cl2·DCM, KOAc, dioxane, 100 °C, 1 h, quant. yield; (d) 37, Pd(dppf)Cl2, Na2CO3, dioxane/water, 100 °C, 12 h, 23%; (e) (i) 2N aq. NaOH, MeOH, 60 °C, 1 h; (ii) 4 M HCl/EtOAc, rt, 1 h, 22%.

    Scheme 6

    Scheme 6. Synthesis of SY-5609 and Compound 13a
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    aReagents and conditions: (a) (i) MeMgBr, ZrCl4, THF, −10 °C to rt, 16 h; (ii) TFA, DCM, rt, 16 h; (b) VinylMgBr, THF, −78 °C to rt, 16 h, 79%; (c) (i) CDI, NH4OH, DMF, 0 °C, 10 min; (ii) MsCl, TEA, DCM, 0 °C, 5 min, 83%; (d) 30, AlCl3, DCE, 80 °C, 4 h, 42%; (e) (i) 45, DIPEA, NMP, 130 °C, 3 h; (ii) P(O)Me2, Pd(OAc)2, XantPhos, K3PO4, DMF, 150 °C, 45 min, μw, 42%; (f) 28b, DIPEA, NMP, 130 °C, 4 h, quant. yield; (g) (i) P(O)Me2, Pd(OAc)2, XantPhos, K3PO4, DMF, 145 °C, 45 min, μw, 24%; (ii) Pd/C, H2, Boc2O, EtOH, 1 atm, rt, 48 h; (iii) TFA/DCM, rt, 16 h, 23%; (h) (i) SOCl2, MeOH; (ii) Boc2O, 74%; (i) LiHMDS, CH3I, 38%; (j) (i) NaBH4, EtOH; (ii) MsCl, TEA; (iii) BnNH2, DME, 14%; (k) HCl, quant. yield.

    Scheme 7

    Scheme 7. Synthesis of Compound 11a
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    aReagents and conditions: (a) NaSMe, DMF, 0 to 15 °C, 2 h, 80%; (b) (i) m-CPBA, DCM, rt, 2 h, 65%; (ii) VinylMgBr, THF, −78 °C, 2 h, 42%; (c) CuCN, DMF, 140 °C, 1 h, 80%; (d) (i) NBS, DMF, rt, 2 h, 66%; (ii) Boc2O, DMAP, DIPEA, THF, 80 °C, 4 h, 75%; (e) B2Pin2, Pd(dppf)Cl2, KOAc, dioxane, 80 °C, 4 h, 21%; (f) (i) 37, Pd(PPh3)4, Na2CO3, dioxane/water, 100 °C, 4 h; (ii) TFA/DCM, rt, 30 min, 26%.

    Scheme 8

    Scheme 8. Synthesis of Compound 12a
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    aReagents and conditions: (a) VinylMgBr, THF, −78 to 0 °C, 2 h, 61%; (b) (i) 14, AlCl3, DCE, 80 °C, 12 h, 41%; (ii) SEM-Cl, NaH, DMF, 0 °C to rt, 1.5 h, 52%; (c) (i) m-CPBA, DCM, rt, 2 h, 79%; (ii) CuCN, DMF, 80 °C, 3 h, 63%; (d) 18, DIPEA, NMP, 90 °C, 1 h, 53%; (e) (i) 20, XPhos Pd G1, K3PO4, THF/water, 80 °C, 12 h, 43%; (ii) Pd/C, H2, TEA, MeOH, 15 psi, 15 °C, 1 h, 60%; (f) H2SO4, dioxane, 40 °C, 3 h; then K2CO3, MeCN, 15 °C, 1 h, 10%.

    Figure 4

    Figure 4. Computational analysis of the putative binding mode of SY-5609 to CDK7.

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    Figure 5

    Figure 5. Kinome selectivity of SY-5102 and SY-5609. Kinases that were inhibited at least 70% with 1 μM SY-5102 or SY-5609 in a panel of 485 kinases (SelectScreen Biochemical Kinase Profiling) are shown. The size of the circle depicting each kinase indicates the percentage inhibition, as shown. Kinome trees were generated using KinMapbeta. (24) Illustration reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com) (Supplementary Table S2).

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    Figure 6

    Figure 6. Cellular effects of treatment with SY-5609. Immunoblotting quantification is presented in Table S3. (A) HCC70 cells were treated with indicated dosage of SY-5609, protein samples were collected at 6, 24, and 48 h post treatment. Phospho-CDK2 T160, total CDK2, c-MYC, MCL1, cyclinB1, RNAPII Ser5 phosphorylation, and total RNAPII protein level were measured by immunoblotting. Vinculin was used as loading control for RNA pol II and Ser5 phosphorylation, while tubulin was used as loading control for the rest of the targets. All immunoblot data are quantified by LICOR. Experiments were performed independently at least three times. (B) HCC70, MDA-MB-468, CAOV3 and OVCAR3, and HDF (adult) cells were treated with SY-5609 for 48 and 72 h and percentage of cells undergoing apoptosis determined by flow cytometry following annexin V-FITC and propidium iodide (PI) staining. Data show mean population from two independent experiments with standard deviation as error bars. (C) HCC70 cells were treated with indicated doses of SY-5609 and fixed after 48 h. DNA was stained with Fxcycle violet stain. Single cell population was determined by FSC-W/FSC-H and SSC-W/SSC-H gating, and G1 and G2/M population was gated as shown on each figure. Experiments were performed independently at least three times.

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    Figure 7

    Figure 7. In vivo efficacy and tolerability of SY-5609. SY-5609 was orally dosed once daily in HCC70 cell line derived xenograft models at 2 mg/kg for 21 days. Ten mice per arm. Tumor volumes and bodyweight were measured twice weekly. One-tail t test was performed to assess significance of antitumor effect of SY-5609 (**** p < 0.0001).

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    Figure 8

    Figure 8. Pharmacodynamic effects of SY-5609 in HCC70 tumor 4 h after a single dose or after 7 days of QD dosing. Four hours post SY-5609 single dose or after 7 day of QD dosing, tumor and plasma samples were collected from mice, 3 mice per arm per time point. Protein and RNA were harvested from tumor samples. Phospho-CDK2 T160, total CDK2, c-MYC, MCL1, cyclinB1, RNAPII 5 phosphorylation, and total RNAPII protein level were measured by immunoblotting. Vinculin was used as loading control for RNA pol II and Ser5 phosphorylation, while tubulin was used as loading control for the rest of the targets. c-MYC mRNA level was measured by NanoString.

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      Recent studies suggest that targeting transcriptional machinery can lead to potent and selective anticancer effects in cancers dependent on high and constant expression of certain transcription factors for growth and survival. Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of the CDK-activating kinase complex. Its function is required for both cell-cycle regulation and transcriptional control of gene expression. CDK7 has recently emerged as an attractive cancer target because its inhibition leads to decreased transcript levels of oncogenic transcription factors, especially those associated with super-enhancers. Here, we describe a selective CDK7 inhibitor SY-1365, which is currently in clinical trials in populations of patients with ovarian and breast cancer (NCT03134638). In vitro, SY-1365 inhibited cell growth of many different cancer types at nanomolar concentrations. SY-1365 treatment decreased MCL1 protein levels, and cancer cells with low BCL2L1 (BCL-XL) expression were found to be more sensitive to SY-1365. Transcriptional changes in acute myeloid leukemia (AML) cell lines were distinct from those following treatment with other transcriptional inhibitors. SY-1365 demonstrated substantial antitumor effects in multiple AML xenograft models as a single agent; SY-1365-induced growth inhibition was enhanced in combination with the BCL2 inhibitor venetoclax. Antitumor activity was also observed in xenograft models of ovarian cancer, suggesting the potential for exploring SY-1365 in the clinic in both hematologic and solid tumors. Our findings support targeting CDK7 as a new approach for treating transcriptionally addicted cancers. SIGNIFICANCE: These findings demonstrate the molecular mechanism of action and potent antitumor activity of SY-1365, the first selective CDK7 inhibitor to enter clinical investigation.
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      9
      Inhibitor selectivity for cyclin-dependent kinase 7: A structural, thermodynamic, and modeling study
      Hazel, Pascale; Kroll, Sebastian H. B.; Bondke, Alexander; Barbazanges, Marion; Patel, Hetal; Fuchter, Matthew J.; Coombes, R. Charles; Ali, Simak; Barrett, Anthony G. M.; Freemont, Paul S.
      ChemMedChem (2017), 12 (5), 372-380CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Deregulation of the cell cycle by mechanisms that lead to elevated activities of cyclin-dependent kinases (CDKs) is a feature of many human diseases, cancer in particular. Here, we identified small-mol.-wt. inhibitors that selectively inhibit CDK7, the kinase that phosphorylates cell-cycle CDKs to promote their activities. To investigate the selectivity of these inhibitors, we used a combination of structural, biophys., and modeling approaches. We detd. the crystal structures of CDK7-selective compds. ICEC 0942 and ICEC 0943 bound to CDK2, and used these to build models of inhibitor binding to CDK7. Mol. dynamics (MD) simulations of inhibitors bound to CDK2 and CDK7 generated possible models of inhibitor binding. To exptl. validate these models, we gathered isothermal titrn. calorimetry (ITC) binding data for recombinant wild-type and binding site mutants of CDK7 and CDK2. We identified specific residues of CDK7, notably Asp-155, that were involved in detg. inhibitor selectivity. The MD simulations also showed that the flexibility of the G-rich and activation loops of CDK7 is likely an important determinant of inhibitor specificity similar to CDK2.
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    10. 10
      Study of XL102 as single-agent and combination therapy in subjects with solid tumors. ClinicalTrials.gov; U.S. National Library of Medicine, 2021. https://ClinicalTrials.gov/show/NCT04726332.
      There is no corresponding record for this reference.
    11. 11
      Lolli, G.; Lowe, E. D.; Brown, N. R.; Johnson, L. N. The crystal structure of human CDK7 and its protein recognition properties. Structure 2004, 12, 2067– 2079,  DOI: 10.1016/j.str.2004.08.013
      11
      The Crystal Structure of Human CDK7 and Its Protein Recognition Properties
      Lolli, Graziano; Lowe, Edward D.; Brown, Nick R.; Johnson, Louise N.
      Structure (Cambridge, MA, United States) (2004), 12 (11), 2067-2079CODEN: STRUE6; ISSN:0969-2126. (Cell Press)
      CDK7, a member of the cyclin-dependent protein kinase family, regulates the activities of other CDKs through phosphorylation on their activation segment and hence contributes to control of the eukaryotic cell cycle. CDK7 also assists in the regulation of transcription as part of the transcription factor TFIIH complex. For max. activity and stability, CDK7 requires phosphorylation, assocn. with cyclin H, and assocn. with a third protein, MAT1. We have detd. the crystal structure of human CDK7 in complex with ATP at 3Å resoln. The kinase is in the inactive conformation, similar to that obsd. for inactive CDK2. The activation segment is phosphorylated at Thr170 and is in a defined conformation that differs from that in phospho-CDK2 and phospho-CDK2/cyclin A. The functional properties of the enzyme against CDK2 and CTD as substrates are characterized through kinase assays. Expts. confirm that CDK7 is not a substrate for kinase-assocd. phosphatase.
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    12. 12
      Finkbeiner, P.; Hehn, J. P.; Gnamm, C. Phosphine oxides from a medicinal chemist’s perspective: physicochemical and in vitro parameters relevant for drug discovery. J. Med. Chem. 2020, 63, 7081– 7107,  DOI: 10.1021/acs.jmedchem.0c00407
      12
      Phosphine Oxides from a Medicinal Chemist's Perspective: Physicochemical and in Vitro Parameters Relevant for Drug Discovery
      Finkbeiner, Peter; Hehn, Joerg P.; Gnamm, Christian
      Journal of Medicinal Chemistry (2020), 63 (13), 7081-7107CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Phosphine oxides and related phosphorus-contg. functional groups such as phosphonates and phosphinates are established structural motifs that are still underrepresented in today's drug discovery projects, and only few examples can be found among approved drugs. In this account, the physicochem. and in vitro properties of phosphine oxides and related phosphorus-contg. functional groups are reported and compared to more commonly used structural motifs in drug discovery. Furthermore, the impact on the physicochem. properties of a real drug scaffold is exemplified by a series of phosphorus-contg. analogs of imatinib. We demonstrate that phosphine oxides are highly polar functional groups leading to high soly. and metabolic stability but occasionally at the cost of reduced permeability. We conclude that phosphine oxides and related phosphorus-contg. functional groups are valuable polar structural elements and that they deserve to be considered as a routine part of every medicinal chemist's toolbox.
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    13. 13
      Huang, W. S.; Liu, S.; Zou, D.; Thomas, M.; Wang, Y.; Zhou, T.; Romero, J.; Kohlmann, A.; Li, F.; Qi, J.; Cai, L.; Dwight, T. A.; Xu, Y.; Xu, R.; Dodd, R.; Toms, A.; Parillon, L.; Lu, X.; Anjum, R.; Zhang, S.; Wang, F.; Keats, J.; Wardwell, S. D.; Ning, Y.; Xu, Q.; Moran, L. E.; Mohemmad, Q. K.; Jang, H. G.; Clackson, T.; Narasimhan, N. I.; Rivera, V. M.; Zhu, X.; Dalgarno, D.; Shakespeare, W. C. Discovery of brigatinib (AP26113), a phosphine oxide-containing, potent, orally active inhibitor of anaplastic lymphoma kinase. J. Med. Chem. 2016, 59, 4948– 4964,  DOI: 10.1021/acs.jmedchem.6b00306
      13
      Discovery of Brigatinib (AP26113), a Phosphine Oxide-Containing, Potent, Orally Active Inhibitor of Anaplastic Lymphoma Kinase
      Huang, Wei-Sheng; Liu, Shuangying; Zou, Dong; Thomas, Mathew; Wang, Yihan; Zhou, Tianjun; Romero, Jan; Kohlmann, Anna; Li, Feng; Qi, Jiwei; Cai, Lisi; Dwight, Timothy A.; Xu, Yongjin; Xu, Rongsong; Dodd, Rory; Toms, Angela; Parillon, Lois; Lu, Xiaohui; Anjum, Rana; Zhang, Sen; Wang, Frank; Keats, Jeffrey; Wardwell, Scott D.; Ning, Yaoyu; Xu, Qihong; Moran, Lauren E.; Mohemmad, Qurish K.; Jang, Hyun Gyung; Clackson, Tim; Narasimhan, Narayana I.; Rivera, Victor M.; Zhu, Xiaotian; Dalgarno, David; Shakespeare, William C.
      Journal of Medicinal Chemistry (2016), 59 (10), 4948-4964CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      In the treatment of echinoderm microtubule-assocd. protein-like 4 (EML4)-anaplastic lymphoma kinase pos. (ALK+) non-small-cell lung cancer (NSCLC), secondary mutations within the ALK kinase domain have emerged as a major resistance mechanism to both first- and second-generation ALK inhibitors. This report describes the design and synthesis of a series of 2,4-diarylaminopyrimidine-based potent and selective ALK inhibitors culminating in identification of the investigational clin. candidate brigatinib. A unique structural feature of brigatinib is a phosphine oxide, an overlooked but novel hydrogen-bond acceptor that drives potency and selectivity in addn. to favorable ADME properties. Brigatinib displayed low nanomolar IC50s against native ALK and all tested clin. relevant ALK mutants in both enzyme-based biochem. and cell-based viability assays and demonstrated efficacy in multiple ALK+ xenografts in mice, including Karpas-299 (anaplastic large-cell lymphomas [ALCL]) and H3122 (NSCLC). Brigatinib represents the most clin. advanced phosphine oxide-contg. drug candidate to date and is currently being evaluated in a global phase 2 registration trial.
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    14. 14
      Bradley, M.; Ciblat, S.; Kabro, A.; Marineau, J. J.; Chuaqui, C. Inhibitors of cyclin-dependent kinase 7 (CDK7). Patent WO2020093011A1, 2020.
      There is no corresponding record for this reference.
    15. 15
      Marineau, J. J.; Chuaqui, C.; Ciblat, S.; Kabro, A.; Piras, H.; Whitmore, K. M.; Lund, K.-L. Preparation of substituted aminopyrimidines as inhibitors of cyclin-dependent kinase 7 (CDK7). Patent WO2019143719A1, 2019.
      There is no corresponding record for this reference.
    16. 16
      Marineau, J. J.; Zahler, R.; Ciblat, S.; Winter, D. K.; Kabro, A.; Roy, S.; Schmidt, D.; Chuaqui, C.; Malojcic, G.; Piras, H.; Whitmore, K. M.; Lund, K.-I.; Sinko, B.; Sprott, K. Preparation of inhibitors of cyclin dependent kinase 7 (CDK7). Patent WO2018013867A1, 2018.
      There is no corresponding record for this reference.
    17. 17
      Panday, S. K.; Langlois, N. Enantioselective synthesis of (S)-5-aminopiperidin-2-one from (S)-pyroglutaminol. Tetrahedron Lett. 1995, 36, 8205– 8208,  DOI: 10.1016/00404-0399(50)17557-
      17
      Enantioselective synthesis of (S)-5-aminopiperidin-2-one from (S)-pyroglutaminol
      Panday, Sharad Kumar; Langlois, Nicole
      Tetrahedron Letters (1995), 36 (45), 8205-8CODEN: TELEAY; ISSN:0040-4039. (Elsevier)
      (5S)-5-aminopiperidin-2-one and several derivs. were synthesized from (S)-pyroglutaminol through ring opening and Mitsunobu reaction as the key steps.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXptFeitb0%253D&md5=000b9e9296f97404f8f6745698c351e4
    18. 18
      Greber, B. J.; Perez-Bertoldi, J. M.; Lim, K.; Iavarone, A. T.; Toso, D. B.; Nogales, E. The cryoelectron microscopy structure of the human CDK-activating kinase. Proc. Natl. Acad. Sci. U. S. A. 2020, 117, 22849– 22857,  DOI: 10.1073/pnas.2009627117
      18
      The cryoelectron microscopy structure of the human CDK-activating kinase
      Greber, Basil J.; Perez-Bertoldi, Juan M.; Lim, Kif; Iavarone, Anthony T.; Toso, Daniel B.; Nogales, Eva
      Proceedings of the National Academy of Sciences of the United States of America (2020), 117 (37), 22849-22857CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The human CDK-activating kinase (CAK), a complex composed of cyclin-dependent kinase (CDK) 7, cyclin H, and MAT1, is a crit. regulator of transcription initiation and the cell cycle. It acts by phosphorylating the C-terminal heptapeptide repeat domain of the RNA polymerase II (Pol II) subunit RPB1, which is an important regulatory event in transcription initiation by Pol II, and it phosphorylates the regulatory T-loop of CDKs that control cell cycle progression. Here, the authors detd. the three-dimensional (3D) structure of the catalytic module of human CAK, revealing the structural basis of its assembly and providing insight into CDK7 activation in this context. The unique third component of the complex, MAT1, substantially extends the interaction interface between CDK7 and cyclin H, explaining its role as a CAK assembly factor, and it forms interactions with the CDK7 T-loop, which may contribute to enhancing CAK activity. The authors also detd. the structure of the CAK in complex with the covalently bound inhibitor THZ1 to provide insight into the binding of inhibitors at the CDK7 active site and to aid in the rational design of therapeutic compds.
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    19. 19
      Greber, B. J.; Remis, J.; Ali, S.; Nogales, E. 2.5 A-resolution structure of human CDK-activating kinase bound to the clinical inhibitor ICEC0942. Biophys. J. 2021, 120, 677– 686,  DOI: 10.1016/j.bpj.2020.12.030
      19
      Resolution structure 2.5 Å-of human CDK-activating kinase bound to the clinical inhibitor ICEC0942
      Greber, Basil J.; Remis, Jonathan; Ali, Simak; Nogales, Eva
      Biophysical Journal (2021), 120 (4), 677-686CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      The human CDK-activating kinase (CAK), composed of CDK7, cyclin H, and MAT1, is involved in the control of transcription initiation and the cell cycle. Because of these activities, it has been identified as a promising target for cancer chemotherapy. A no. of CDK7 inhibitors have entered clin. trials, among them ICEC0942 (also known as CT7001). Structural information can aid in improving the affinity and specificity of such drugs or drug candidates, reducing side effects in patients. Here, we have detd. the structure of the human CAK in complex with ICEC0942 at 2.5 Å-resoln. using cryogenic electron microscopy. Our structure reveals conformational differences of ICEC0942 compared with previous X-ray crystal structures of the CDK2-bound complex, and highlights the crit. ability of cryogenic electron microscopy to resolve structures of drug-bound protein complexes without the need to crystalize the protein target.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXjtVSrs7s%253D&md5=16cffe07b3fe538abaa36eb66202406e
    20. 20
      Kwiatkowski, N.; Zhang, T.; Rahl, P. B.; Abraham, B. J.; Reddy, J.; Ficarro, S. B.; Dastur, A.; Amzallag, A.; Ramaswamy, S.; Tesar, B.; Jenkins, C. E.; Hannett, N. M.; McMillin, D.; Sanda, T.; Sim, T.; Kim, N. D.; Look, T.; Mitsiades, C. S.; Weng, A. P.; Brown, J. R.; Benes, C. H.; Marto, J. A.; Young, R. A.; Gray, N. S. Targeting transcription regulation in cancer with a covalent CDK7 inhibitor. Nature 2014, 511, 616– 620,  DOI: 10.1038/nature13393
      20
      Targeting transcription regulation in cancer with a covalent CDK7 inhibitor
      Kwiatkowski, Nicholas; Zhang, Tinghu; Rahl, Peter B.; Abraham, Brian J.; Reddy, Jessica; Ficarro, Scott B.; Dastur, Anahita; Amzallag, Arnaud; Ramaswamy, Sridhar; Tesar, Bethany; Jenkins, Catherine E.; Hannett, Nancy M.; McMillin, Douglas; Sanda, Takaomi; Sim, Taebo; Kim, Nam Doo; Look, Thomas; Mitsiades, Constantine S.; Weng, Andrew P.; Brown, Jennifer R.; Benes, Cyril H.; Marto, Jarrod A.; Young, Richard A.; Gray, Nathanael S.
      Nature (London, United Kingdom) (2014), 511 (7511), 616-620CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Tumor oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state, but direct pharmacol. inhibition of transcription factors has so far proven difficult. However, the transcriptional machinery contains various enzymic cofactors that can be targeted for the development of new therapeutic candidates, including cyclin-dependent kinases (CDKs). Here the authors present the discovery and characterization of a covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell-line profiling indicates that a subset of cancer cell lines, including human T-cell acute lymphoblastic leukemia (T-ALL), have exceptional sensitivity to THZ1. Genome-wide anal. in Jurkat T-ALL cells shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and the key role of RUNX1 in the core transcriptional regulatory circuitry of these tumor cells. Pharmacol. modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumor types that are dependent on transcription for maintenance of the oncogenic state.
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    21. 21
      Schrödinger Release 2020–2; Schrödinger, L.: New York, NY, 2020.
      There is no corresponding record for this reference.
    22. 22
      Kyte, J.; Doolittle, R. F. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 1982, 157, 105– 132,  DOI: 10.1016/0022-2836(82)90515-0
      22
      A simple method for displaying the hydropathic character of a protein
      Kyte, Jack; Doolittle, Russell F.
      Journal of Molecular Biology (1982), 157 (1), 105-32CODEN: JMOBAK; ISSN:0022-2836.
      A computer program that progressively evaluates the hydrophilicity and hydrophobicity of a protein along its amino acid sequence was devised. A hydropathy scale takes into consideration the hydrophilic and hydrophobic properties of each of the 20 amino acid side chains. Correlation was demonstrated between the plotted values and known structures detd. by crystallog.
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    23. 23
      Zamyatnin, A. A. Protein volume in solution. Prog. Biophys. Mol. Biol. 1972, 24, 107– 123,  DOI: 10.1016/0079-6107(72)90005-3
      23
      Protein volume in solution
      Zamyatnin A A
      Progress in biophysics and molecular biology (1972), 24 (), 107-23 ISSN:0079-6107.
      There is no expanded citation for this reference.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaE3s%252Fnt1Gluw%253D%253D&md5=1eb0435b9ebb74a00ff9517a292cd5c5
    24. 24
      Eid, S.; Turk, S.; Volkamer, A.; Rippmann, F.; Fulle, S. KinMap: a web-based tool for interactive navigation through human kinome data. BMC Bioinf. 2017, 18, 16– 21,  DOI: 10.1186/s12859-016-1433-7
      24
      KinMap: a web-based tool for interactive navigation through human kinome data
      Eid, Sameh; Turk, Samo; Volkamer, Andrea; Rippmann, Friedrich; Fulle, Simone
      BMC Bioinformatics (2017), 18 (), 16/1-16/6CODEN: BBMIC4; ISSN:1471-2105. (BioMed Central Ltd.)
      Background: Annotations of the phylogenetic tree of the human kinome is an intuitive way to visualize compd. profiling data, structural features of kinases or functional relationships within this important class of proteins. The increasing vol. and complexity of kinase-related data underlines the need for a tool that enables complex queries pertaining to kinase disease involvement and potential therapeutic uses of kinase inhibitors. Results: Here, we present KinMap, a user-friendly online tool that facilitates the interactive navigation through kinase knowledge by linking biochem., structural, and disease assocn. data to the human kinome tree. To this end, preprocessed data from freely-available sources, such as ChEMBL, the Protein Data Bank, and the Center for Therapeutic Target Validation platform are integrated into KinMap and can easily be complemented by proprietary data. The value of KinMap will be exemplarily demonstrated for uncovering new therapeutic indications of known kinase inhibitors and for prioritizing kinases for drug development efforts. Conclusions: KinMap represents a new generation of kinome tree viewers which facilitates interactive exploration of the human kinome. KinMap enables generation of high-quality annotated images of the human kinome tree as well as exchange of kinome-related data in scientific communications. Furthermore, KinMap supports multiple input and output formats and recognizes alternative kinase names and links them to a unified naming scheme, which makes it a useful tool across different disciplines and applications. A web-service of KinMap is freely available at http://www.kinhub.org/kinmap/.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjsFCjtLc%253D&md5=29737c7cda8d9caf75018e0d29e9a459
    25. 25
      Wang, Y.; Zhang, T.; Kwiatkowski, N.; Abraham, B. J.; Lee, T. I.; Xie, S.; Yuzugullu, H.; Von, T.; Li, H.; Lin, Z.; Stover, D. G.; Lim, E.; Wang, Z. C.; Iglehart, J. D.; Young, R. A.; Gray, N. S.; Zhao, J. J. CDK7-dependent transcriptional addiction in triple-negative breast cancer. Cell 2015, 163, 174– 86,  DOI: 10.1016/j.cell.2015.08.063
      25
      CDK7-dependent transcriptional addiction in triple-negative breast cancer
      Wang, Yubao; Zhang, Tinghu; Kwiatkowski, Nicholas; Abraham, Brian J.; Lee, Tong Ihn; Xie, Shaozhen; Yuzugullu, Haluk; Von, Thanh; Li, Heyuan; Lin, Ziao; Stover, Daniel G.; Lim, Elgene; Wang, Zhigang C.; Iglehart, J. Dirk; Young, Richard A.; Gray, Nathanael S.; Zhao, Jean J.
      Cell (Cambridge, MA, United States) (2015), 163 (1), 174-186CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Triple-neg. breast cancer (TNBC) is a highly aggressive form of breast cancer that exhibits extremely high levels of genetic complexity and yet a relatively uniform transcriptional program. We postulate that TNBC might be highly dependent on uninterrupted transcription of a key set of genes within this gene expression program and might therefore be exceptionally sensitive to inhibitors of transcription. Utilizing kinase inhibitors and CRISPR/Cas9-mediated gene editing, we show here that triple-neg. but not hormone receptor-pos. breast cancer cells are exceptionally dependent on CDK7, a transcriptional cyclin-dependent kinase. TNBC cells are unique in their dependence on this transcriptional CDK and suffer apoptotic cell death upon CDK7 inhibition. An "Achilles cluster" of TNBC-specific genes is esp. sensitive to CDK7 inhibition and frequently assocd. with super-enhancers. We conclude that CDK7 mediates transcriptional addiction to a vital cluster of genes in TNBC and CDK7 inhibition may be a useful therapy for this challenging cancer.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFKqtrjP&md5=34441125e931816d29e419f3f7dc0346
    26. 26
      Zhang, Z.; Peng, H.; Wang, X.; Yin, X.; Ma, P.; Jing, Y.; Cai, M. C.; Liu, J.; Zhang, M.; Zhang, S.; Shi, K.; Gao, W. Q.; Di, W.; Zhuang, G. Preclinical efficacy and molecular mechanism of targeting CDK7-dependent transcriptional addiction in ovarian cancer. Mol. Cancer Ther. 2017, 16, 1739– 1750,  DOI: 10.1158/1535-7163.MCT-17-0078
      26
      Preclinical Efficacy and Molecular Mechanism of Targeting CDK7-Dependent Transcriptional Addiction in Ovarian Cancer
      Zhang, Zhenfeng; Peng, Huixin; Wang, Xiaojie; Yin, Xia; Ma, Pengfei; Jing, Ying; Cai, Mei-Chun; Liu, Jin; Zhang, Meiying; Zhang, Shengzhe; Shi, Kaixuan; Gao, Wei-Qiang; Di, Wen; Zhuang, Guanglei
      Molecular Cancer Therapeutics (2017), 16 (9), 1739-1750CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)
      Ovarian cancer remains a significant cause of gynecol. cancer mortality, and novel therapeutic strategies are urgently needed in clinic as new treatment options. We previously showed that BET bromodomain inhibitors displayed promising efficacy for the treatment of epithelial ovarian cancer by downregulating pivot transcription factors. However, the potential antitumor activities and mol. mechanisms of other epigenetic or transcriptional therapies have not been systematically detd. Here, by performing an unbiased high-throughput drug screen to identify candidate compds. with antineoplastic effects, we identified THZ1, a recently developed covalent CDK7 inhibitor, as a new transcription-targeting compd. that exerted broad cytotoxicity against ovarian tumors. Mechanistically, CDK7 represented a previously unappreciated actionable vulnerability in ovarian cancer, and CDK7 inhibition led to a pronounced dysregulation of gene transcription, with a preferential repression of E2F-regulated genes and transcripts assocd. with super-enhancers. Our findings revealed the mol. underpinnings of THZ1 potency and established pharmaceutically targeting transcriptional addiction as a promising therapeutic strategy in aggressive ovarian cancer. Mol Cancer Ther; 16(9); 1739-50. ©2017 AACR.
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    27. 27
      Holmes, I. P.; Bergman, Y.; Lunniss, G. E.; Nikac, M.; Choi, N.; Hemley, C. F.; Walker, S. R.; Foitzik, R. C.; Ganame, D.; Lessene, R. Preparation of pyrimidine derivatives useful as FAK inhibitors. U.S. Patent US20130017194A1, 2013.
      There is no corresponding record for this reference.
    28. 28
      Albers, R.; Ayala, L.; Clareen, S. S.; Delgado Mederos, M. M.; Hilgraf, R.; Hedge, S.; Hughes, K.; Kois, A.; Plantevin-Krenitsky, V.; McCarrick, M.; Nadolny, L.; Palanki, M.; Sahasrabudhe, K.; Sapienza, J.; Satoh, Y.; Sloss, M.; Sudbeck, E.; Wright, J. Preparation of haloaryl substituted aminopurines for use as a prodrug in the treatment of cancers, cardiovascular or renal diseases. Patent WO2006076595A1, 2006.
      There is no corresponding record for this reference.
    29. 29
      Lovitt, B.; Vanderporten, E. C.; Sheng, Z.; Zhu, H.; Drummond, J.; Liu, Y. Differential effects of divalent manganese and magnesium on the kinase activity of leucine-rich repeat kinase 2 (LRRK2). Biochemistry 2010, 49, 3092– 3100,  DOI: 10.1021/bi901726c
      29
      Differential Effects of Divalent Manganese and Magnesium on the Kinase Activity of Leucine-Rich Repeat Kinase 2 (LRRK2)
      Lovitt, Brian; Vander Porten, Erica C.; Sheng, Zejuan; Zhu, Haitao; Drummond, Jake; Liu, Yichin
      Biochemistry (2010), 49 (14), 3092-3100CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Various mutations in leucine-rich repeat kinase 2 (LRRK2) have been linked to susceptibility for both familial and idiopathic late-onset Parkinson's disease (PD). In this study, it was demonstrated that phosphorylation of MBP and LRRKtide by the LRRK2 G2019S mutant was activated by Mn2+ in vitro. This enhanced G2019S kinase activity was due to the combination of an increase in kinase and a decrease in ATPase activity by Mn2+. Compared to 10 mM Mg2+, 1 mM Mn2+ reduced ATP Km for G2019S from 103 to 1.8 μM and only modestly reduced kcat (2.5-fold); as a result, the Mn2+ increased its kcat/Km by 22-fold. This change in ATP Km was due in large part to an increase in nucleotide affinity. While Mn2+ also increased ATP affinity and had similar effects on kcat/Km for LRRK2 WT and R1441C enzymes, it reduced their kcat values significantly by 13-17-fold. Consequently, the difference in the kinase activity between G2019S and other LRRK2 variants was enhanced from about 2-fold in Mg2+ to 10-fold in Mn2+ at satg. ATP concns. relative to its Km. Furthermore, while Mg2+ yielded optimal Vmax values at Mg2+ concn. greater than 5 mM, the optimal Mn2+ concn. for activating LRRK2 catalysis was in the micromolar range with increasing Mn2+ above 1 mM causing a decrease in enzyme activity. Finally, despite the large but expected differences in IC50 tested at 100 μM ATP, the apparent Ki values of a small set of LRRK2 ATP-competitive inhibitors were within 5-fold between Mg2+- and Mn2+-mediated reactions except AMP-CPP, an ATP analog.
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    30. 30
      Haq, N.; Niu, D.; Petter, R. C.; Qiao, L.; Singh, J.; Zhu, Z. Pyrimidine derivatives as ERK inhibitors and their preparation. Patent WO2014124230A2, 2014.
      There is no corresponding record for this reference.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c01171.

    • Additional figures illustrating dihedral angles from molecular dynamics simulations, CDK selectivity of SY-5102 and SY-5609, kinase selectivity of SY-5102 and SY-5609, cellular antiproliferation panel for SY-5609, quantification of immunoblotting from Figure 6, immunoblot images from Figure 8, small molecule X-ray structure of SY-5609, protein cocrystal structure of Compound 4 with CDK2, NMR spectra and LC–MS chromatograms for all compounds (PDF)

    • Molecular formula strings (CSV)

    • PDB ID Codes: Compound 4 with CDK2 (PDB: 7RA5)

    Accession Codes

    CCDC 2093192 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.


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