SYNTHESIS OF 5-METHYL-5-DEAZA NONCLASSICAL ANTIFOLATES AS INHIBITORS OF DIHYDROFOLATE REDUCTASES AND AS POTENTIAL ANTIPNEUMOCYSTIS, ANTITOXOPLASMA, AND ANTITUMOR AGENTS

A series of 5-methyl-6-(anilinomethyl)pyrido[2,3-d]pyrimidines 4-9 wer e synthesized as 5-deaza nonclassical antifolates containing trimethox y, dichloro-, or trichlorophenyl substitutions and a N-H, N-CH3, or N- CHO at the 10-position. The compounds were evaluated as inhibitors of dihydrofolate reductases (DHFR) from Pneumocystis carinii (P. carinii) , Toxoplasma gondii (T. gondii), rat liver (RL), and Lactobacillus cas ei (L. casei); as inhibitors of T. gondii and P. carinii cell growth i n culture; and as antitumor agents. The compounds were prepared by mod ifications of procedures for classical 5-deaza folates. oxy-N-methylan ilino)methyl]pyrido[2,3-d]pyrimidine (5a) exhibited high potency as we ll as selectivity (compared to RL DHFR) for P. carinii and T. gondii D HFR. Compound 5a is one of the most potent and selective nonclassical folate inhibitors of T. gondii DHFR known. The N-10 formyl analogue -t rimethoxyanilino)methyl]pyrido-[2,3-d]pyrimidine (6a) had decreased po tency, but it maintained high selectivity for T. gondii DHFR. The corr esponding chloro-substituted analogues maintained potency or had decre ased potency; N-10 substitution did not increase potency or selectivit y to the extent observed in the 3',4',5'-trimethoxy series. Partial re duction of the B ring to afford the dihydro analogue anilino)methyl]-5 ,8-dihydropyrido[2,3-d]pyrimidine (7), its 5,6,7,8-tetrahydropyrido[2, 3-d]pyrimidine analogue 8, and )methyl]-5,6,7,8-tetrahydropyrido[2,3-d ]pyrimidine (9) resulted in a significant decrease in potency. In T. g ondii cell culture inhibitory studies, '-trimethoxyanilino)methyl]pyri do[2,3-d]pyrimidine (4a), 5a, and 6a were less potent compared to thei r DHFR inhibitory potencies. Against P. carinii cells in culture, 4a a nd 5a at 10 mug/mL were as effective as the clinically used combinatio n of trimethoprim/sulfamethoxazole (50/250 mug/mL). With the exception of the B ring reduced analogues 7-9, all of the compounds were signif icantly cytotoxic to leukemia CCRF-CEM cells in culture. The chloro-su bstituted analogues, in general, were more potent against a variety of other tumor cells in culture than the trimethoxy analogues. These res ults were corroborated by the preclinical tumor screening program at t he National Cancer Institute where the most potent compound ,4'-dichlo roanilino)methyl]pyrido[2,3-d]pyrimidine (4b) was found to inhibit the growth of 26 tumor cell lines at an IG50 < 1.00 X 10(-8) M.