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Mechanism of Action of 5,8-Dideazaisofolic Acid and Other Quinazoline Antifols in Human Colon Carcinoma Cells¹

Departments of Pharmacology and Medicine, Yale University School of Medicine, New Haven, Connecticut 06510 [J. J. M., A. F. S., J. R. B.], and Department of Pharmaceutical Sciences, College of Pharmacy, Medical University of South Carolina, Charleston, South Carolina 29425 [J. B. H.]

The clonal cytotoxic effects and mechanism of action of a new series of 2-amino-4-hydroxyquinazoline folate analogues (5,8-dideazafolates) have been assessed using the human colon tumor cell line HCT-8. Of these compounds only 5-methyl-5,8-dideazafolate was potentially more effective than a compound previously identified, 5,8-dideazaisofolate (H-338, NSC 289517). HCT-8 sublines resistant to methotrexate, 5-fluorodeoxyuridine, and H-338 were either minimally or not cross-resistant to the other agents. The cytotoxicity of H-338 was strongly dependent on the time of exposure; at exposure times shorter than 8 h it was essentially nontoxic. Thymidine alone, as well as leucovorin or folic acid, protected against the cytotoxic effects of H-338. This is consistent with thymidylate synthase (TS) as its only locus of action. Studies with dihydrofolate reductase and TS isolated from HCT-8 cells indicated that these quinazolines were weaker inhibitors of dihydrofolate reductase than was methotrexate, but they were not particularly potent TS inhibitors. However, synthetic poly--glutamate derivatives of quinazolines showed dramatically increased TS, but not dihydrofolate reductase, inhibition. TS inhibition increased as the polyglutamate chain length increased. Using isolated HCT-8 folylpolyglutamate synthetase, all the parent quinazolines containing L-glutamate were found to be substrates. With H-338, the results indicated that tetraglutamate or longer derivatives could be synthesized intracellularly. These results are consistent with our hypothesis that cytotoxicity by such quinazolines necessarily involves "lethal synthesis" from a prodrug; i.e., the nontoxic parent drug must be converted to polyglutamates before TS inhibition and subsequent cytotoxicity can occur.

¹ This work was supported in part by USPHS Grants CA-24887 and CA-25014 awarded by the National Cancer Institute as part of the National Large Bowel Cancer Project.

² Scholar of the Leukemia Society of America. To whom requests for reprints should be addressed, at Grace Cancer Drug Center, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, NY 14263.

³ American Cancer Society Professor of Medicine and Pharmacology.

Received 3/16/87. Revised 7/29/87. Accepted 8/20/87.