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Biochemical and Growth Inhibitory Effects of the erythro and threo Isomers of -Fluoromethotrexate, a Methotrexate Analogue Defective in Polyglutamylation¹

Grace Cancer Drug Center, Roswell Park Memorial Institute, Buffalo, New York 14263 [J. J. M.]; Department of Chemistry, Rensselaer Polytechnic Institute, Troy, New York 12180 [M. G., N. L., C. V., J. K. C.]; and Wadsworth Center for Laboratories and Research, New York State Department of Health, Empire State Plaza, Albany, New York 12201 [M. G., Z. N., J. G.]

We previously reported (J. Galivan et al., Proc. Natl. Acad. Sci. USA, 82: 2598–2602, 1985) the synthesis and characterization of DL-erythro,threo--fluoromethotrexate (FMTX). The individual diastereomers, DL-erythro-FMTX (eFMTX) and DL-threo-FMTX (tFMTX), and their radiolabeled counterparts have now been prepared and characterized. Transport of eFMTX (K_m = 9.3 µM; V_max = 7.5 pmol/min/10⁷ cells) was similar to that of methotrexate (MTX: K_m = 6.6-9.9 µM; V_max = 11.4-14.2 pmol/min/10⁷ cells), while tFMTX (K_m = 65.1 µM; V_max = 8.4 pmol/min/10⁷ cells) was transported less efficiently. Both isomers were able to saturate intracellular dihydrofolate reductase and accumulate further as unbound intracellular drug. Based on competition experiments and studies with MTX transport-defective cell lines, both isomers utilized the reduced folate/MTX transport system. Efflux half-times for the isomers were similar to those of MTX. Each isomer was equivalent to MTX in its ability to inhibit dihydrofolate reductase activity and bind to intracellular dihydrofolate reductase when the intracellular drug concentration was limiting. Both isomers had drastically diminished capacity to be metabolized to poly(-glutamyl) metabolites by isolated folylpolyglutamate synthetase and in whole cells; tFMTX was metabolized to a slightly lesser extent than eFMTX. Using the CCRF-CEM human leukemia and H35 rat hepatoma cell lines, the growth-inhibitory effects of eFMTX were almost the same as those of MTX during continuous exposure, while tFMTX was slightly less potent. This difference in growth-inhibitory potency of the two isomers correlated with their ability to inhibit de novo thymidylate synthesis in the H35 cell line. These results indicate that both diastereomers of FMTX are similar in their properties to MTX, except that both are incapable of being readily converted to polyglutamate derivatives. As a result of these properties, both isomers could be used under appropriate conditions in comparative studies with MTX to define the roles of MTX polyglutamates.

¹ This work was supported in part by Grants CA13038 and CA24538 (J. J. M.), CA28097 (J. K. C.), and CA25933 (J. G.) from the National Cancer Institute, Public Health Service, Department of Health and Human Services, and Institutional Research Grant IN-54Z from the American Cancer Society (J. J. M.). J. J. M. is a Leukemia Society Scholar. A preliminary account of this work was presented at the Eighth International Symposium on Pteridines and Folic Acid Derivatives, Montreal, Canada, June 1986 (1).

² To whom requests for reprints should be addressed, at Grace Cancer Drug Center, Roswell Park Memorial Institute, 666 Elm St., Buffalo, NY 14263.

³ Present address: Departments of Chemistry and Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109-1055.

Received 12/19/88. Revised 4/20/89. Accepted 5/10/89.

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