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/roscovitine lead structure were synthesized and evaluated for their ability to inhibit starfish oocyte CDK1/cyclin B, neuronal CDK5/p35 and erk1 kinases in purified 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 different 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 0.65 microM, 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 (LD50-6.7 microM versus 42.7 microM for olomoucine, 24-h contact). Furthermore, unlike olomoucine, compound 21 was effective upon short exposure (LD50= 25.3 microM, 2-h contact). The available data suggest that the affinity for CDKs and the cytotoxic potential of the drugs are inter-related. However, no straightforward cell cycle phase specificity 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. olomoucilnc and compound 21 were unable to reversibly arrest cell cycle progression in asynchronous growing HeLa cells. Some irreversible hlock in GI and G2 phase occurred at high olomoucine concentration, correlated with induced cell death. Moreover, chmronic exposure to lethal doses of compound 21 resulted in massive nuclear fragmentation, evocative of mitotic catastrophe with minour amounts of apoptosis only. It was also found that olomoucine and compound 21 reversibly block the intracellular uptake of nuicleosides with high efficiency.
Legraverend
M
M
UMR 176 CNRS-IC, Institut Curie-Recherche, Centre Universitaire, Orsay, France.
Ludwig
O
O
Bisagni
E
E
Leclerc
S
S
Meijer
L
L
Giocanti
N
N
Sadri
R
R
Favaudon
V
V
D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D051357:Cyclin-Dependent Kinase 2; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology*; D006367:HeLa Cells / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D020128:Inhibitory Concentration 50; D007701:Kinetin; D007928:Lethal Dose 50; D009705:Nucleosides / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011687:Purines / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D016196:S Phase / Q000187:drug effects; D013329:Structure-Activity Relationship; D013936:Thymidine / Q000493:pharmacokinetics
C121642:2-(2-(hydroxymethyl)pyrrolidinyl)-6-((3-iodophenyl)methylamino)-9-isopropylpurine; D004791:Enzyme Inhibitors; D009705:Nucleosides; D011687:Purines; C090046:olomoucine; D051360:Cyclin-Dependent Kinase 5; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495905:CDK5 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D007701:Kinetin; D013936:Thymidine