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.

Photochemotherapy using pyridopsoralens.

Aiming to decrease the acute side effects and genotoxic hazards of PUVA, pyrido (3,4-C) psoralen (PP) and 7-methyl pyrido (3,4-C) psoralen (MPP) were synthesized and studied. Their UVA maximum absorption lies at 325 and 330 nm, respectively. Their photostability is comparable to that of 8-MOP. They complex to DNA in the dark, and, in the presence of UVA, produce only monoadditions to DNA, as shown by fluorescence and DNA denaturation-renaturation studies. In diploid eukaryotic yeast they are more effective than 8-MOP for the induction of lethal effects and mitochondrial damage. Their mutagenic activity per unit dose of UVA is in the same range as that of 8-MOP. However, per viable cell they are clearly less mutagenic than 8-MOP. This difference is also observed for recombinogenic activity. No oxygen effect is observed. In mammalian cells the following ranges of effectiveness are found: inhibition of DNA synthesis in human fibroblasts: MPP greater than PP greater than 8-MOP; mutagenic activity in V79 Chinese hamster cells: MPP greater than PP greater than 8-MOP; cell transforming ability in C3H embryonic mouse cells: MPP greater than 8-MOP greater than PP as a function of UVA dose, and: 8-MOP greater than MPP greater than PP as a function of survival; induction of sister chromatic exchanges (SCE) per unit dose: MPP greater than PP greater than 8-MOP in the linear part of the induction curve, and : 8-MOP greater than PP greater than MPP at the maximum level of SCE obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Antitumor amino-substituted pyrido[3',4':4,5]pyrrolo[2,3-g]isoquinolines and pyrido[4,3-b]carbazole derivatives: synthesis and evaluation of compounds resulting from new side chain and heterocycle modifications.

New modifications of 10-[[3-(diethylamino)propyl]amino]-6-methyl-5H-pyrido[3',4':4,5]pyrrolo[2,3-g]i sisoquinoline (1b) and 1-[[3-(diethylamino)propyl]amino]-9-methoxy-5,11-dimethyl-6H-pyrido[4,3-b]carba zole (4b), which display important antitumor properties, were performed either on the side chain or on the intercalating heterocycle. Side chains were introduced by direct substitution of the corresponding chloro derivatives and 6-N-methyl-9-hydroxypyrido[4,3-b]carbazoles analogues were prepared via 9-O-benzoyl-1-chloroellipticines. Evaluation of all new compounds shows no significant increase of in vitro cytotoxicity and percent ILS on the L1210 leukemia system by comparison with the model compounds 1b and 4b.

Photochemotherapy (PUVA) of psoriasis using 3-carbethoxypsoralen, a non-carcinogenic compound in mice.

The carcinogenic risk of photochemotherapy (PUVA) with bi-functional furocoumarins such as 8-methoxypsoralen (8-MOP) which form cross-links in cellular DNA has initiated a search for active but less hazardous psoralens. A new compound, 3-carbethoxypsoralen (3-CPs), studied in the yeast Saccharomyces cerevisiae (eukaryote), has been shown to be very photoactive on DNA and to form only mono-additions to DNA. These lesions appear to be more easily repaired than the cross-links induced by 8-MOP. 3-CPs produces less nuclear genetic events such as nuclear mutations and mitotic crossovers, but more cytoplasmic 'petite' mutations (damage to mitochondrial DNA) than 8-MOP. In mice it was demonstrated that after local or intra-peritoneal administration, in contrast to 8-MOP, 3-CPs is non-toxic, non-erythematogenic, and non-carcinogenic. A study of ten psoriatic patients had shown that local applications of 3-CPs plus UV-A exhibit about the same therapeutic activity for the clearing of psoriatic lesions as local treatment with 8-MOP plus UV-A, but without any localized hyperpigmentation.