Pii: S0968-0896(99)00064-4

ÐNovel C-2, C-6, N-9 trisubstituted purines derived from the olomoucine/roscovitine lead structure were synthesized and evaluated for their ability to inhibit star®sh oocyte CDK1/cyclin B, neuronal CDK5/p35 and erk1 kinases in puri®ed extracts. Structure±activity relationship studies showed that increased steric bulk at N-9 reduces the inhibitory potential whereas substitution of the aminoethanol C-2 side chain by various groups of di€erent size (methyl, propyl, butyl, phenyl, benzyl) only slightly decreases the activity when compared to (R)-roscovitine. Optimal inhibitory activity against CDK5, CDK1 and CDK2, with IC50 values of 0.16, 0.45 and 0.65mM, respectively, was obtained with compound 21 containing a (2R)-pyrrolidin-2-yl-methanol substituent at the C-2 and a 3-iodobenzylamino group at the C-6 of the purine. Compound 21 proved cytotoxic against human tumor HeLa cells (LD50=6.7mM versus 42.7mM for olomoucine, 24-h contact). Furthermore, unlike olomoucine, compound 21 was e€ective upon short exposure (LD50=25.3 mM, 2-h contact). The available data suggest that the anity for CDKs and the cytotoxic potential of the drugs are inter-related. However, no straightforward cell cycle phase speci®city of the cytotoxic response to 21 was observed in synchronized HeLa cells. With the noticeable exception of pronounced lengthening of the S-phase transit by 21 applied during early-S in synchronized HeLa cells, and in striking contrast with earlier reports on studies using plant or echinoderm cells, olomoucine and compound 21 were unable to reversibly arrest cell cycle progression in asynchronous growing HeLa cells. Some irreversible block in G1 and G2 phase occurred at high olomoucine concentration, correlated with induced cell death. Moreover, chronic exposure to lethal doses of compound 21 resulted in massive nuclear fragmentation, evocative of mitotic catastrophe with minor amounts of apoptosis only. It was also found that olomoucine and compound 21 reversibly block the intracellular uptake of nucleosides with high eciency. # 1999 Elsevier Science Ltd. All rights reserved.