Pharmacological inhibition of cyclin-dependent kinases has emerged as a possible treatment option for various cancer types. We recently identified substituted imidazo[1,2-c]pyrimidin-5(6H)-ones as inhibitors of cyclin-dependent kinase 2 (CDK2). Here, we report the synthesis of derivatives modified at positions 2, 3, 6 or 8 prepared using Suzuki-Miyaura cross-coupling, halogenation, Dimroth-type rearrangement and alkylation as the main synthetic methods. The compounds displayed micro- to submicromolar inhibition of CDK2/cyclin E activity. Binding of the most potent compound 3b to CDK2 was determined using isothermal titration calorimetry. The co-crystal structure of 3b in complex with fully active CDK2 was solved, revealing the binding mode of 3b in the ATP pocket and a hydrogen bonding interaction with hinge region residue Leu83. Evaluation against leukaemia cell lines revealed low cytotoxicity, which is in line with the high selectivity towards CDK2. This study demonstrates that substituted imidazo[1,2-c]pyrimidines can be exploited for future kinase inhibitor development.

Department of Experimental Biology Faculty of Science Palacký University Šlechtitelů 27 78371 Olomouc Czech Republic

Faculty of Science University of Hradec Rokitanského 62 50003 Hradec Králové Czech Republic

Institute of Molecular Genetics The Czech Academy of Sciences Vídeňská 1083 14220 Prague Czech Republic

Institute of Organic Chemistry and Biochemistry The Czech Academy of Sciences Flemingovo nám 2 16610 Prague 6 Czech Republic

Research Institute for Organic Syntheses Rybitví 296 53354 Pardubice Rybitví Czech Republic

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