INHIBITION OF MICROTUBULES AND CELL-CYCLE ARREST BY A NEW 1-DEAZA-7,8-DIHYDROPTERIDINE ANTITUMOR DRUG, CI-980, AND BY ITS CHIRAL ISOMER, NSC-613863

CI 980 (NSC 613862; [S-(-)]) and NSC 613863 [R-(+)] are the two chiral isomers of ethyl 5-amino -methyl-3-phenylpyrido[3,4-b]pyrazin-7-ylcar bamate (NSC 370147), which is a mitotic inhibitor with in vivo and in vitro activity against murine multidrug-resistant sublines. We have ch aracterized the inhibition of in vitro microtubule assembly by the S ( CI 980) and R (NSC 613863) enantiomers, their actions on the cytoplasm ic microtubule network of epithelial-like PtK2 cells, and on the cell cycle of different human and murine leukemias and PtK2 cells. Assembly of purified tubulin, or tubulin plus microtubule-associated proteins, into microtubules was substoichiometrically inhibited by both compoun ds, which also induced a slow depolymerization of preassembled microtu bules. Half inhibitory concentrations were 0.4-0.7 muM and 1.6-2.1 muM for the S and R isomers, respectively. Excess of both drugs induced p olymerization of liganded tubulin into abnormal polymers similar to co lchicine. The cytoplasmic microtubules of PtK2 cells were disrupted by both compounds in a concentration- and time-dependent manner, which w as observed by indirect immunofluorescence microscopy and quantified b y an enzyme-linked immunoassay of cytoskeletal tubulin. Half inhibitor y concentrations were 6 nM S isomer, 100 nM R isomer, and 1 muM colchi cine. Twenty nM S isomer or 500-700 nM R isomer gave nearly maximal ef fect. At these concentrations, half maximal microtubule depolymerizati on took place after 2 h of treatment. After drug removal, slow microtu bule assembly and nearly complete reorganization of the cytoplasmic mi crotubules of PtK2 cells were observed (24 h). One nM S enantiomer or 25 nM R enantiomer induced mitotic arrest in 8 h in U937, HL60, and EL 4 leukemias. PtK2 cells also stopped in mitosis after a 24-h incubatio n with 50 nM R isomer or 5 nM S isomer. The inhibition of cell divisio n was irreversible in the leukemic cells, while PtK2 cells partially r esumed growth. Although the interactions of CI 980 with microtubules i n vitro are not very different from other drugs, it is a most potent c ellular microtubule and mitotic inhibitor.