DESIGN AND MECHANISM OF ACTION OF A NOVEL CYTOTOXIC 1,2,3-TRIAZENE-CONTAINING HETEROCYCLE, 3,5-DIMETHYL-PYRIDO-1,2,3,5-TETRAZEPIN-4-ONE (PYRZ), IN THE HUMAN EPITHELIAL OVARIAN-CANCER CELL-LINE NIH-OVCAR-3 IN-VITRO

The mechanism of action of the novel heterocycle 3,5-dimethyl-pyrido-1 ,2,3,5-tetrazepin-4-one (PYRZ), structurally related to temozolomide, was studied in the human ovarian tumour cell line OVCAR-3. Our results showed that, despite its marked structural similarities to temozolomi de, PYRZ presents properties that are atypical of 1,2,3-triazene-conta ining alkylating agents. In a Maxam-Gilbert DNA sequencing assay, PYRZ showed background levels of DNA alkylation, in contrast to temozolomi de which strongly alkylated DNA preferentially at guanine residues. At high concentrations, PYRZ inhibited the synthesis of DNA, RNA and pro tein 3 h after treatment, in contrast to temozolomide which, in previo us work, was found to preferentially inhibit DNA synthesis in OVCAR-3 cells. In cells exposed to PYRZ, alkaline sucrose density-gradient cen trifugation showed a dose-dependent increase in DNA fragmentation only 12 and 24 h after treatment. PYRZ induced increasing accumulation of cells in late S and G(2)+M6-24 h after treatment. This also contrasts with previous work that showed delayed cell cycle arrest induced by te mozolomide in OVCAR-3 cells and in the murine leukaemia L1210 cells. C ell-killing kinetics by PYRZ showed a series of sigmoidal dose-respons e curves with 50-90% cell killing attained as early as 24 h after trea tment in the 25-100 mu M dose range. (IC50 clonogenic assay 18 mu M). The results suggest that the mechanism of cell killing by PYRZ may be different from that of its parent drug temozolomide, and other alkyl-t riazene-containing molecules of the same class.