A versatile method for the unequivocal synthesis of 6-substituted pteridines wa.z used for the preparation of several methotrexate analogs in which the glutamate moiety was modified in order to enhance lipophilic character. Tu-o 8-oxido derivatives were also prepared. These compounds were tested for in c'itro inhibitory activity against Strepiococcus faecium and as enzyme inhibitors against dihydrofolate reductase derived from Lactobacilluo caaei, chicken liver, and L1210-FR8 cells. Several compounds showed significant bacterial inhibition. but all were found to be less inhibitory than methotrexate against these enzymes. Only the diethyl ester of methotiexate showed significant in civo biological activity against L1210 leukemic mice, comparable to that of methotrexate. The pteridine 8-oxide function was found to have a detrimental biological effect in the two compounds of this type tested in these assays. An efficient synthesis of 4-amino-4-deoxy-N1°-methylpteroic acid and its ethyl ester is also described. The ester was found to have prophylactic antimalarial activity in the sporozoite-induced Plasmodium gallinaceurn chick assay. For more than 20 years, methotrexate (4-amino-4deoxy-N1*-methylpteroylglutamic acid, amethopterin, MTX) has been widely used as one of the most effective clinical antitumor agents, especially in the treatment of acute childhood le~kemia.~ Its mode of action as a folic acid antagonist4 is recognized to involve strong inhibition of dihydrofolate reductase and interference with the synthesis of metabolites that play a role in important biochemical one-carbon transfer reactions, including the biosynthesis of nucleotides. Despite some remarkable successes in induction of remission in acute leukemia and in certain solid tumors, the clinical usefulness of MTX is limited by the development of drug resistance, due in part to incieased levels of enzymes in the folic acid cycle and restricted cellular uptake of the dr~g.~,~ Except when given intrathecally,i MTX is ineffective against tumors of the central nervous system, Tt is also ineffective against malarial parasites, which lack an active transport mechanism for folates.x MTX is transported across tumor cell membranes by an active carrier-mediated mechanism, possibly sharing the same mechanism used for natural f~lates.~-'l Since this mechanism probably involves the carboxyl groups of the glutamate moiety, any significant chemical modification at this sire should change the overall transport properties of the molecule without greatly affecting the enzyme-inhibit ory properties of the 2,4diaminopyrimi do system. common to many types of folate antagonists. Replacement of the carboxyl groups in MTX by less polar groups such dS CHzOH or CH3 might result in a sacrifice of the active transport mechanism. However, this might be offset by the likelihood that the less ionic nature of the reduced species would enhance the lipophilic nature of the molecule, as would other structural modifications such as the complete replacement of the glutamate moiety by large alkylamine resiciues. Such molecules could still enter a cell by passive diffusion, as is the case with other small