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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 613863¹

Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones Cientificas, Velazquez 144, 28006 Madrid, Spain [C. I., L B.]; Groupe de Recherche sur les Interactions des Proteines en Pharmacologie, Faculté de Pharmacie, 27Bd. Jean Moulin, 13385 Marseille Cedex 5, France [D. L., V. P., P. G., C. B.]; and Organic Chemistry Research Department, Southern Research Institute, Birmingham, Alabama [G. A. R., C. T.]

² To whom requests for reprints should be addressed.

CI 980 (NSC 613862; [S-(–)]) and NSC 613863 [R-(+)] are the two chiral isomers of ethyl 5-amino l,2-dihydro-2-methyl-3-phenylpyrido[3,4-b]pyrazin-7-ylcarbamate (NSC 370147), which is a mitotic inhibitor with in vivo and in vitro activity against murine multidrug-resistant sublines. We have characterized the inhibition of in vitro microtubule assembly by the S (CI 980) and R (NSC 613863) enantiomers, their actions on the cytoplasmic 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 compounds, which also induced a slow depolymerization of preassembled microtubules. Half inhibitory concentrations were 0.4–0.7 µM and 1.6–2.1 µM for the S and R isomers, respectively. Excess of both drugs induced polymerization of liganded tubulin into abnormal polymers similar to colchicine. The cytoplasmic microtubules of PtK2 cells were disrupted by both compounds in a concentration- and time-dependent manner, which was observed by indirect immunofluorescence microscopy and quantified by an enzyme-linked immunoassay of cytoskeletal tubulin. Half inhibitory concentrations were 6 nM S isomer, 100 nM R isomer, and 1 µM colchicine. Twenty nM S isomer or 500–700 nM R isomer gave nearly maximal effect. At these concentrations, half maximal microtubule depolymerization took place after 2 h of treatment. After drug removal, slow microtubule assembly and nearly complete reorganization of the cytoplasmic microtubules 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 EL4 leukemias. PtK2 cells also stopped in mitosis after a 24-h incubation with 50 nM R isomer or S nM S isomer. The inhibition of cell division was irreversible in the leukemic cells, while PtK2 cells partially resumed growth. Although the interactions of CI 980 with microtubules in vitro are not very different from other drugs, it is a most potent cellular microtubule and mitotic inhibitor.

¹ This work was supported by Grant PB 87-0230 from the Direccion General de Investigacion Cientifica y Tecnica, French-Spanish grants, and grants from Association pour la Recherche sur le Cancer Federation de Centres de lutte Contre Cancer.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7/16/93. Accepted 10/29/93.

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