In Vitro Evaluation of a Novel 2,6,9‐Trisubstituted Purine Acting As a Cyclin‐Dependent Kinase Inhibitor

Abstract

NICOLE GIOCANTI,a RAMIN SADRI,a MICHEL LEGRAVEREND,b ODILE LUDWIG,b EMILE BISAGNI,bSOPHIE LECLERC,c LAURENT MEIJER,c AND VINCENT FAVAUDONa.d aU 350 INSERM and bUMR 176 CNRS, Institut Curie, Centre Universitaire, 91405 Orsay, France cUPR 9042 CNRS, Station Biologique, 29682 Roscoff, France The frequent deregulation of cell cycle progression in cancer1 has prompted an active search for kinase inhibitors with high affinity and specificity for cyclin-dependent kinases (Cdks). Three major classes of Cdk-targeting drugs have been identified to date, including butyrolactone I,2 polyhydroxylated flavones such as flavopiridol,3 and substituted purines.4 The first substituted purine derivative acting as a selective Cdk inhibitor, olomoucine, has been identified from screening against Cdk1/cyclin B complex.5 Olomoucine competitively inhibits Cdk1, Cdk2, Cdk5, and, to a lesser extent, Erk1.5 Recent results have pointed to unexpected pharmacologic properties of 2,6,9trisubstituted purines derived from the olomoucine lead structure.6,7 To investigate the question in more detail, we developed a program for synthesis and evaluation of new compounds in this series. Twenty-seven derivatives were synthesized and assayed for specific inhibition of Cdk1/cyclin B from starfish oocytes and human recombinant Cdk5/p35 complex. In agreement with earlier results,5 data showed that a strong correlation exists between inhibitory efficiencies against Cdk1 and Cdk5. In contrast, all compounds were only marginally active against Erk1 and Erk2 kinases. One compound in the series, ML-1437, proved much more active than olomoucine against purified Cdk1/cyclin B, Cdk5/p35, and Cdk2/cyclin E. It also showed pronounced cytotoxicity against human cervix carcinoma HeLa cells in vitro, even on short exposure. Growing IMR-90 (human normal fibroblasts), LoVo (human colon adenocarcinoma), and SQ-20B (human head and neck squamous carcinoma) cells gave similar results, but drug resistance increased rapidly as cells (SQ-20B and IMR-90) reached confluence. These results suggest that the affinity for Cdks and the cytotoxic potential of the drugs are interrelated (FIG. 1, TABLE 1). With the exception of pronounced lengthening of S phase transit during early-S in synchronized HeLa cells, ML-1437 at subtoxic concentration proved unable to produce reversible arrest of the cell cycle progression. When observed, arrest in the G1 and G2 phases of the cell cycle correlated with induced cell death, and chronic exposure to lethal doses of the drug resulted in massive micronucleation in relation to mitotic cell death, with no evidence of endoreduplication (polyploidization) or ap-