Synthesis and in vitro evaluation of novel 2,6,9-trisubstituted purines acting as cyclin-dependent kinase inhibitors

Novel C-2, C-6, N-9 trisubstituted purines derived from the olomoucine/rosc ovitine lead structure were synthesized and evaluated for their ability to inhibit starfish oocyte CDK1/cyclin B, neuronal CDK5/p35 and erk1 kinases i n purified extracts. Structure-activity relationship studies showed that in creased steric bulk at N-9 reduces the inhibitory potential whereas substit ution of the aminoethanol C-2 side chain by various groups of different siz e (methyl, propyl, butyl, phenyl, benzyl) only slightly decreases the activ ity when compared to (R)-roscovitine. Optimal inhibitory activity against C DK5, CDK1 and CDK2, with IC50 values of 0.16, 0.45 and 0.65 mu M, respectiv ely, was obtained with compound 21 containing a (2R)-pyrrolidin-2-yl-methan ol substituent at the C-2 and a 3-iodobenzylamino group at the C-6 of the p urine. Compound 21 proved cytotoxic against human tumor HeLa cells (LD50 = 6.7 mu M versus 42.7 mu M for olomoucine, 24-h contact). Furthermore, unlik e olomoucine, compound 21 was effective upon short exposure (LD50 = 25.3 mu M, 2-h contact). The available data suggest that the affinity for CDKs and the cytotoxic potential of the drugs are inter-related. However, no straig htforward cell cycle phase specificity of the cytotoxic response to 21 was observed in synchronized HeLa cells. With the noticeable exception of prono unced lengthening of the S-phase transit by 21 applied during early-S in sy nchronized HeLa cells, and in striking contrast with earlier reports on stu dies using plant or echinoderm cells, olomoucine and compound 21 were unabl e to reversibly arrest cell cycle progression in asynchronous growing HeLa cells. Some irreversible block in G1 and G2 phase occurred at high olomouci ne concentration, correlated with induced cell death. Moreover, chronic exp osure to lethal doses of compound 21 resulted in massive nuclear fragmentat ion, evocative of mitotic catastrophe with minor amounts of apoptosis only. It was also found that olomoucine and compound 21 reversibly block the int racellular uptake of nucleosides with high efficiency. (C) 1999 Elsevier Sc ience Ltd. All rights reserved.