Biochemical and growth inhibitory effects of the erythro and threo isomers of gamma-fluoromethotrexate, a methotrexate analogue defective in polyglutamylation.

We previously reported (J. Galivan et al., Proc. Natl. Acad. Sci. USA, 82: 2598-2602, 1985) the synthesis and characterization of DL-erythro,threo-gamma-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 (Km = 9.3 microM; Vmax = 7.5 pmol/min/10(7) cells) was similar to that of methotrexate (MTX: Km = 6.6-9.9 microM; Vmax = 11.4-14.2 pmol/min/10(7) cells), while tFMTX (Km = 65.1 microM; Vmax = 8.4 pmol/min/10(7) 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(gamma-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.