Research paper
Discovery of 4-(((4-(5-chloro-2-(((1s,4s)-4-((2-methoxyethyl)amino)cyclohexyl)amino)pyridin-4-yl)thiazol-2-yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile (JSH-150) as a novel highly selective and potent CDK9 kinase inhibitor

https://doi.org/10.1016/j.ejmech.2018.09.025Get rights and content

Highlights

  • Highly potent CDK9 inhibitor 40 (IC50 = 1 nM).

  • Achieved 300–10000 fold selectivity over other CDK kinase family members.

  • High selectivity over other 468 kinases/mutants (KINOMEscan S score(1) = 0.01).

  • Potently inhibited the phosphorylation of RNA Pol II Ser2 (EC50: <100 nM).

Abstract

Through a structure-guided rational drug design approach, we have discovered a highly selective inhibitor compound 40 (JSH-150), which exhibited an IC50 of 1 nM against CDK9 kinase in the biochemical assay and achieved around 300–10000-fold selectivity over other CDK kinase family members. In addition, it also displayed high selectivity over other 468 kinases/mutants (KINOMEscan S score(1) = 0.01). Compound 40 displayed potent antiproliferative effects against melanoma, neuroblastoma, hepatoma, colon cancer, lung cancer as well as leukemia cell lines. It could dose-dependently inhibit the phosphorylation of RNA Pol II, suppress the expression of MCL-1 and c-Myc, arrest the cell cycle and induce the apoptosis in the leukemia cells. In the MV4-11 cell-inoculated xenograft mouse model, 10 mg/kg dosage of 40 could almost completely suppress the tumor progression. The high selectivity and good in vivo PK/PD profile suggested that 40 would be a good pharmacological tool to study CDK9-mediated physiology and pathology as well as a potential drug candidate for leukemia and other cancers.

Introduction

Cyclin-dependent kinases (CDKs) are a class of serine/threonine protein kinase which play critical roles in the regulation of cell cycle or/and transcription [1,2]. To date, 21 different CDKs (1−11a, 11b−20) have been identified in the human genome and they can be divided into two main groups based on their primary roles [3]. CDK1, CDK2, CDK4, and CDK6 have been found to regulate the cell cycle progression upon binding to cyclin proteins. CDK7, CDK8, CDK9 and CDK11 are key players in transcription regulation [4,5]. These CDKs promote RNA synthesis for cell growth, differentiation and viral pathogenesis [6,7] etc. Among them, CDK9 is a key regulator of transcription elongation in eukaryotic cells and has been considered as a potential drug target for several diseases including cardiac hypertrophy and certain cancers such as a variety of blood cancers as well as solid tumors [8]. CDK9 phosphorylates Ser2 residues in the carboxy-terminal domain (CTD) of RNA Pol II to initiate the transcription elongation [9]. Activation of CDK9 kinase will promote the expression of antiapoptotic factor myeloid cell leukemia 1 (MCL-1) [10], which leads to malignant cell transformation. Inhibition of CDK9 activity will result in the down-regulation of this short-lived antiapoptotic protein MCL-1, and consequently the induction of caspase-dependent apoptosis which is important for tumor control [[11], [12], [13], [14]].

Flavopiridol (alvocidib, 1), the first CDK inhibitor entered into clinical trials, exhibited antileukemic activity in CLL patients via potent inhibition of the CDK9-meditated down-regulation of antiapoptotic proteins transcription [15,16]. This has stimulated broad research interests for CDK9 and a number of small molecule inhibitors have been discovered and moved into clinical investigation (Fig. 1). However, the high structural homology of ATP binding pocket among CDKs made it a great challenge for developing highly selective CDK9 kinase inhibitors. Most of the currently known CDK9 inhibitors are non-selective inhibitors. For example, 1 exhibited high potency against CDK1, 2, 4, 5, 6 and 9; 2 (SNS-032) was potent to CDK 2, 7 and 9 [17,18]; compound 3 (RGB286638) targeted CDK1, 2, 3, 4, 5 and 9. Compound 4 (LY2857785), which was evaluated in phase I clinical trials for the treatment of solid tumors, was relatively more selective but also simultaneously inhibited CDK7, 8, 9 [19,20]. Other multiple CDK inhibitors including 5 [21] (AZD5438, CDK1/2/9), 6 [22] (AT7519, CDK1/2/4/6/9) and 7 [[23], [24], [25]] (SCH727965, CDK1/2/5/9) have also been tested in clinical trials. The pyrimidine core scaffold-based CDK9 inhibitors including 8 (CDKI-73) [13] and 9 also potently inhibited both CDK7 and CDK9. However, given the fact that most of the CDK family members play critical roles both in physiology and pathology conditions, e.g., CDK7 is directly upstream of CDK9 and both of them control transcription, non-specific inhibition of these kinases would increase the potential of the adverse events in the clinic and also made it hard to dissect the specific roles in the cellular context. Therefore, a highly selective CDK9 inhibitor is urgently desired for both of the physiological and pathological study. Here we reported our medicinal chemistry efforts starting from 9's core scaffold which led to the discovery of a highly selective and potent CDK9 inhibitor 40 (JSH-150). It is worth mentioning that during preparation of this manuscript, a structurally similar inhibitor 10 (NVP-2), which also exhibited high selectivity and potency against CDK9 kinase, was reported as a chemical biology research tool for studying protein degradation [26].

Section snippets

Design rationale

Compound 9 is a potent inhibitor of CDK9 with an IC50 of 14 nM, but also displays an IC50 value of 92 nM for CDK7 [27]. In order to compare the binding modes of 9 in CDK7 and CDK9, we docked 9 into the CDK7 kinase (PDB ID: 1UA2) [7] (Fig. 2A–B). The results showed that the aminopyrimidine formed two hydrogen bonds in the hinge binding area with Met94 and Cys106 of CDK7 and CDK9 respectively. The diazepane moiety of 9 formed a hydrogen bond with Leu143 and Thr29 of CDK7 and CDK9 respectively. In

Conclusions

Starting from the core scaffold of CDK7/9 inhibitor 9, a structure-guided drug design approach led to the discovery of a novel highly potent and selective CDK9 kinase inhibitor 40. It exhibited single digit nM potency against CDK9 kinase in the biochemical assay. Compared to most of the currently known CDK kinase inhibitors, 40 achieved superior selectivity among CDK subfamily, especially the high selectivity between CDK9 and CDK7 kinase (over 1700-fold), both of which are critical

Chemistry

All reagents and solvents were purchased from commercial sources and used as obtained. 1H NMR spectra were recorded with a Bruker 400 MHz NMR spectrometer and referenced to deuterated methanol (CD3OD), deuterium dimethyl sulfoxide (DMSO‑d6) or deuterium chloroform (CDCl3). Chemical shifts are expressed in ppm. In the NMR tabulation, s indicates singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and br, broad peak. LC/MS experiments were performed on an Agilent 6224 TOF using an ESI

Author contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grants U1432250, 81471773, 81473088, U1532154, 81703559, 81703007, 21708044), the National Key Research and Development Program of China (Grant 2016YFA0400900), the Major Science and Technology Program of Anhui Province (Grant 16030801114), the China Postdoctoral Science Foundation (Grants 2018M630721, 2018T110633, 2018T110634), the Natural Science Foundation of Anhui Province (Grants 1808085MH268, 1708085MH208,

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