Discovery and SAR of novel pyrazolo[1,5-a]pyrimidines as inhibitors of CDK9

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Abstract

The serine–threonine kinase CDK9 is a target of emerging interest for the development of anti-cancer drugs. There are multiple lines of evidence linking CDK9 activity to cancer, including the essential role this kinase plays in transcriptional regulation through phosphorylation of the C-terminal domain (CTD) of RNA polymerase II. Indeed, inhibition of CDK9 has been shown to result in a reduction of short-lived proteins such as the pro-survival protein Mcl-1 in malignant cells leading to the induction of apoptosis. In this work we report our initial studies towards the discovery of selective CDK9 inhibitors, starting from the known multi-kinase inhibitor PIK-75 which possesses potent CDK9 activity. Our series is based on a pyrazolo[1,5-a]pyrimidine nucleus and, importantly, the resultant lead compound 18b is devoid of the structural liabilities present in PIK-75 and possesses greater selectivity.

Introduction

The Cyclin-Dependant Kinases (CDKs) are a family of serine–threonine kinases comprising more than 13 members. A number of CDKs (subtypes 1–4, 6 and 11) play a direct role in the regulation of the cell cycle,1, 2, 3, 4 while CDK subtypes 7–9 have been shown to control RNA polymerase II mediated transcription.1 CDK9 is expressed in a range of human tissues and is highly expressed in terminally differentiated cells.4 CDK9 is stabilised by the formation of heterodimers; these are either (i) transient complexes with chaperones such as HSP70, HSP90 and Cdc37 or (ii) active heterodimers with the T and K cyclins. The active heterodimers form the complex p-TEFb (positive transcription elongation factor b) where the cyclin forms the regulatory subunit and CDK9 the active enzyme component.4 The p-TEFb complex plays two roles in the successful initiation and elongation of RNA transcription: phosphorylation of the RNA polymerase II C-terminus domain and usurping N-TEF (negative transcription elongation factor) leading to stable elongation of the RNA transcript.5

Inhibition of specific CDKs or CDK subsets has been the focus of considerable research and drug development activity over the last two decades with a number of inhibitors of varying selectivity profiles progressing to clinical trials, predominantly for the treatment of cancer.3, 6 While a number of pan-specific CDK inhibitors (e.g., flavopiridol (1), SNS-032 (2), dinaciclib (3) and AT7519 (4)) have been examined in clinical trials, dose-limiting toxicities have been reported most likely due to their broad target inhibition.7, 8, 9 More recently selective targeting of CDK9 for the treatment of various cancers has gained interest, since blocking RNA synthesis by the inhibition of CDK9 leads to a reduction in the levels of short-lived pro-survival proteins such as Mcl-1 and the induction of apoptosis.3, 4 Of the many CDK inhibitors reported to possess potent activity against the CDK9 subtype2, 3, 6, 10 few have been reported as CDK9 selective,11, 12 one of which is the recently disclosed LY2857785 (5).13 The structures for some of the inhibitors with potent CDK9 activity are shown in Figure 1.

As part of a phenotypic screening study looking for drugs targeting Mcl-1 in acute myeloid leukaemia (AML), we identified the literature standard PI3K p110α (PI3Kα) inhibitor PIK-75 (6)14, 15 as being particularly active against a panel of AML cell lines and primary samples.16 Further studies demonstrated that this compound also possessed significant CDK7 and CDK9 inhibitory activity and that this combination of activities was driving much of the PIK-75 mediated cytotoxicity in AML. Studies from other laboratories have also demonstrated that PIK-75 possesses significant CDK inhibitory activity resulting in the induction of apoptosis in diverse cancer cell lines.17, 18, 19 In addition, our group showed in AML that the direct inhibition of CDK9 by PIK-75 causes blockade of the transcription of the MCL-1 gene thus reducing levels of the short-lived pro-survival protein, Mcl-1, resulting in rapid cell death, given the critical survival role of Mcl-1 in AML.20, 21 Similarly, Lemke et al. showed that PIK-75 leads to a reduction of the short-lived proteins Mcl-1 and c-FLIP, and subsequently a re-sensitisation of TRAIL resistant cancer cells to TRAIL.18

These data encouraged us to initiate a drug discovery programme focused on the development of selective CDK9 inhibitors using PIK-75 as a chemical starting point. This compound, originally described by Hayakawa et al. as a selective inhibitor of the kinase PI3Kα, shows significant activity against a number of kinases including CDK7, CDK9 and FLT3.14, 15, 16 Furthermore, the compound possesses two non-drug-like structural motifs—a hydrazone linkage and aryl nitro moiety—that are perceived as developmental liabilities due to potential toxicity concerns.22, 23 Presumably as a consequence of these issues there has been little work published exploring variations on the PIK-75 structure24, 25, 26, 27, 28 which offered the possibility to rapidly develop novel chemical matter. Below, we describe our preliminary work on a series of novel pyrazolo[1,5-a]pyrimidines that show an improved selectivity profile compared to PIK-75 and that are devoid of the structural liabilities of the original lead compound.

Section snippets

Synthesis

A series of sulfonylhydrazones were synthesised (Scheme 1) starting from commercially available 5-chloropyrazolo[1,5-a]pyrimidine (7) which was converted to the carbaldehyde (8) under Vilsmeier–Haack conditions.25 A one pot, two-step procedure was then used to convert 8 to the desired arylsulfonylhydrazones 9au via condensation of 8 with methyl hydrazine, followed by sulfonylation with various sulfonyl chlorides. Use of 2,6-lutidine as base allowed isolation of the desired product in high

Conclusions

The development of selective inhibitors of CDK9 represents a novel approach for the treatment of cancer by targeting the transcriptional machinery. In this work we have demonstrated that the prototypical PI3Kα inhibitor PIK-75 (6), which is known to also inhibit CDK9, can be used for the design of novel CDK9 inhibitors with improved selectivity over PI3Kα. Importantly we have shown that the two major structural liabilities with PIK-75—the hydrazone and aryl nitro moieties—can be substituted

Chemistry

NMR spectra were recorded on a Bruker AV-II DRX 300 NMR spectrometer at 300 K; chemical shifts are reported in δ ppm using residual solvent as the internal standard. Liquid chromatography mass spectroscopy (LCMS) was carried out on a Waters ZQ 3100 using reverse phase HPLC (column: XBridge™ C18 5 μm 4.6 × 100 mm), Solvent A: Water 0.1% Formic Acid, Solvent B: Acetonitrile 0.1% Formic Acid, Gradient: 10–100% B over 10 min, flow rate: 1.5 mL/min, detection: 100–600 nm and ESI in positive mode with source

Acknowledgements

This work is supported by scholarships, fellowships and grants from the Australian National Health and Medical Research Council (Research Fellowship to M.W.P.; Independent Research Institutes Infrastructure Support Scheme Grants 9000220 and 9000225), Victorian State Government Operational Infrastructure Support (OIS) Grants, Australian Cancer Research Foundation, and Dyson Bequest funding (Dunn Fellowship to C.J.B.). We thank Prof. D.C.S. Huang for helpful advice and discussion. Ms. Dana Buczek

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