~) Pergamon Bioorganic & Medicinal ChemistryLetters, Vol.7. No. 21. pp. 2697-2702,1997 © 1997 Elsevier Science Ltd All rightsreserved.Printed in GreatBritain PII: S0960-894X(97) 10076-2 0960-894X/97$17.00+ 0~00 SYNTHESIS AND ACTIVITY OF 2,6,9-TRISUBSTITUTED PURINES Steven R. Schow, Richard L. Mackman,t Cheri L. Blum, Eric Brooks, Amy G. Horsma, Alison Joly, Suresh S. Kerwar, Gavin Lee,* Dov Shiffman, Marek G. Nelson, Xingbo Wang, Michael M. Wick, Xiaoming Zhang,* and Robert T. Lum* CV Therapeutics Inc., 3172 Porter Drive, Palo Alto, CA 94304 Abstract: The preparation of a series of 2,6,9-trisubstituted purines and the structure-activity data for the inhibition of cyclin dependent kinase, CDK2 are presented. © 1997ElsevierScienceLtd. Recent advances in molecular and cellular biology have greatly contributed to our understanding of the mechanisms of cell proliferation and of specific steps of the cell cycle as cells progress through mitosis. 1These studies have demonstrated that the cell cycle is tightly regulated by time dependent activation a family of serine/threonine kinases. These kinases are multiprotein complexes consisting of (a) a phosphorylated kinase called cyclin dependent kinase (CDK) and (b) a regulatory protein called cyclin. Different cyclin-CDK combinations control cell cycle steps such as growth, DNA replication, and cell division. 2 One key member of the CDK family of enzymes is CDK2. CDK2/cyclin A activity has been shown to be essential for mammalian cell cycle progression at the G1/S boundary. Olomoucine is a specific inhibitor of CDK2 with an IC50 of approximately 7 gM, 3 and in vivo studies using mammalian cells in culture demonstrate that olomoucine inhibits cell proliferation at an approximate concentration of 50 gg/mL. We report here the results of program to identify potent inhibitors of CDK2 to evaluate as potential therapeutic agents for controlling aberrant cell proliferation in a variety of diseases. 4 Chemistry and Library Generation There have been several recent reports on the preparation of combinatorial libraries of purine derivatives using solid-phase methodology,s Rather than trying to generate the most diverse set of structures available, we prepared a modest sized (ca 3000 compounds) biased library that appears to be a general resource for lead generation in kinase inhibition. 6 Solution-phase methodology was used for the preparation of the target compounds. The general reaction pathway is outlined in Scheme 1, and is based upon the differential reactivity of 2,6-dichloropurine. 7 The 6-chloro group was displaced by refluxing with a primary amine or aniline in butanol for several hours. This reaction gave quantitative yields of analog 1. Alkylation of the 9-position was achieved by treating 1 with Nail in DMF followed by addition of an alkyl halide to afford compound 2. No N-7 alkylation was observed. As noted by Nugiel,5bthe displacement of the 2-chloro proved to be the most difficult. The optimal conditions used a 1:1 mixture of N-methylpyrrolidinone and amine at 135 °C for 24 to 40 h. This allowed for the introduction of a variety of amines into 2-position. Even with these forcing conditions, anilines and certain sterically hindered amines would not displace the 2 chlorine. 2697