10-Propargylaminopterin and alkyl homologues of methotrexate as inhibitors of folate metabolism.

Reported antifolate activity against leukemia L1210 by N-[14-[[(2-amino-4-hydroxy-6-quinazolinyl)methyl]-propargylamino]benzoyl]]-L-glu tamic acid through potent inhibition of thymidylate synthase (EC 2.1.1.45) prompted us to include the propargyl group in a study of the effect on folate metabolism and membrane transport of replacing the 10-methyl group of methotrexate with other groups. Along with the propyl (8a) and octyl (8b) homologues of methotrexate, the propargyl compound 8c was prepared for evaluation. Syntheses of 8a,b were achieved by a standard multistep sequence involving preparation of the side-chain precursors via tosylated intermediates and then their alkylation with 6-(bromomethyl)-2,4-pteridinediamine hydrobromide. The side-chain precursor to 8c was prepared by direct alkylation of diethyl N-(4-aminobenzoyl)-L-glutamate with propargyl bromide and was separated from unchanged amine and dipropargyl coproduct by a combination of methods, including dry-column chromatography and recrystallization. Subsequent steps leading to 8c were like those used to prepare 8a,b. Biological evaluations of the three compounds consisted of studies of their effects on enzyme inhibition [(dihydrofolate reductase (EC 1.5.1.3) and thymidylate synthase)], L1210 cell growth inhibition, cellular membrane transport with various murine cell types (L210, S180, Ehrlich, and epithelial), in vivo (mice) activity vs. L1210 leukemia and S180 ascites, and plasma clearance in mice. The in vivo results vs. S180 ascites offered evidence that 8c might have a better therapeutic index against this tumor than methotrexate, but no other result from either of these compounds suggested significant superiority over methotrexate.