Identification and characterisation of 2-aminopyridine inhibitors of checkpoint kinase 2 Stephen Hilton a,  , Sebastien Naud a , John J. Caldwell a , Kathy Boxall a , Samantha Burns a , Victoria E. Anderson a , Laurent Antoni a , Charlotte E. Allen a , Laurence H. Pearl b , Antony W. Oliver b , G. Wynne Aherne a , Michelle D. Garrett a , Ian Collins a, * a Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK b Section of Structural Biology, Chester Beatty Laboratories, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK article info Article history: Received 14 September 2009 Revised 16 November 2009 Accepted 27 November 2009 Available online 6 December 2009 Keywords: CHK2 Kinase inhibitor High-throughput screening Crystallography abstract 5-(Hetero)aryl-3-(4-carboxamidophenyl)-2-aminopyridine inhibitors of CHK2 were identified from high throughput screening of a kinase-focussed compound library. Rapid exploration of the hits through straightforward chemistry established structure–activity relationships and a proposed ATP-competitive binding mode which was verified by X-ray crystallography of several analogues bound to CHK2. Variation of the 5-(hetero)aryl substituent identified bicyclic dioxolane and dioxane groups which improved the affinity and the selectivity of the compounds for CHK2 versus CHK1. The 3-(4-carboxamidophenyl) sub- stituent could be successfully replaced by acyclic x-aminoalkylamides, which made additional polar interactions within the binding site and led to more potent inhibitors of CHK2. Compounds from this ser- ies showed activity in cell-based mechanistic assays for inhibition of CHK2. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Checkpoint kinase 2 (CHK2) is a serine/threonine kinase which plays an important part in the complex signalling networks respon- sible for the maintenance of mammalian genomic integrity and re- pair of damaged DNA. 1–4 Signalling through CHK2 is activated in cells in response to DNA damage caused by external agents such as ionising radiation or genotoxic chemotherapy. Double strand breaks in DNA lead to activation of the DNA-damage transducer ataxia and telangiectasia mutated (ATM). ATM phosphorylates CHK2 on Thr68, 5,6 leading to homodimerisation of the protein and activation through trans-autophosphorylation 7,8 on Thr383 and Thr387 and cis-autophosphorylation 9,10 on Ser516. In turn, activated CHK2 phosphorylates multiple substrates that control cell cycle progres- sion and DNA repair. In cells with a functional p53 tumour suppres- sor pathway, phosphorylation of HDMX by CHK2 stabilises p53, leading to a G1 cell cycle arrest and apoptotic cell death if the dam- aged DNA is not repaired. 11,12 Similarly, phosphorylation by CHK2 stabilizes the transcription factor E2F-1, promoting apoptosis. 13 CHK2 also signals to the Cdc25 family of phosphatases that control cell cycle progression in S, G2 and M phases through regulation of cy- clin dependent kinases. 3,4 At the same time, CHK2 activates several DNA damage repair pathways. In particular, phosphorylation of BRCA1 promotes double strand break repair, 14 while phosphoryla- tion of the transcription factor FOXM1 leads to increased expression of proteins involved in the homologous recombination and base excision repair mechanisms. 15 There is current interest in the therapeutic potential of inhibi- tors of CHK2 in several distinct contexts, but especially in cancer where DNA-damaging agents remain a central component of treat- ment regimes. 1,2,16 The p53-mediated apoptotic response contrib- utes to cell death in normal tissue in response to ionising radiation and chemotherapies that cause double strand DNA breaks. In contrast, many tumours lack a functional p53 pathway. 17 There is therefore the potential for CHK2 inhibition to selectively reduce p53-mediated cell death in normal tissue and suppress the side-effects of these therapies. In support of this, chk2 À/À trans- genic mice show resistance to apoptosis after exposure to ionising radiation. 18,19 Importantly, no increased tumourigenesis is seen in these CHK2-deficient animals, in contrast to p53-deficient mice. A radioprotective effect on isolated mouse thymocytes and human T- cells has been observed with selective small molecule inhibitors of CHK2. 20,21 It is also plausible that inhibition of CHK2 alone could exert an antitumour effect. In some cancer cell lines, CHK2 is highly acti- vated. 3,22 There is the possibility that inhibition of CHK2 activity would lead to cell death through inactivation of DNA repair path- ways that are critical for the survival of the aberrant cells. Related to the inhibition of DNA repair pathways is the recent finding that replication of the human hepatitis C virus (HCV) is suppressed by 0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2009.11.058 * Corresponding author. Tel.: +44 208 722 4317; fax: +44 208 722 4216. E-mail address: ian.collins@icr.ac.uk (I. Collins).   Present address: The School of Pharmacy, University of London, UK. Bioorganic & Medicinal Chemistry 18 (2010) 707–718 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc