2,4-DIAMINO-5-DEAZA-6-SUBSTITUTED PYRIDO[2,3-D]PYRIMIDINE ANTIFOLATESAS POTENT AND SELECTIVE NONCLASSICAL INHIBITORS OF DIHYDROFOLATE REDUCTASES

Fifteen novel nonclassical and two classical 2,4-diamino-6-(benzylamin o)pyrido[2,3-d]pyrimidine antifolates were synthesized as potential in hibitors of Pneumocystis carinii, (pc) Toxoplasma gondii, (tg) rat liv er (rl), and human (h) recombinant dihydrofolate reductases (DHFR). Th ese analogues lack a 5-methyl substitution which has been shown to be important for increased hDHFR inhibitory activity. In addition, they c ontain a reversal of the C9-N10 bridge present in folates and most ant ifolates. The synthesis of the compounds involved the reaction of 2,4, 6-triaminopyrimidine with the sodium salt of nitromalonaldehyde to aff ord the key intermediate 2,4-diamino-6-nitropyrido[2,3-d]pyrimidine (7 ), in a single step. Reduction of 7 to the 2,4,6-triaminopyrido[2,3-d] pyrimidine (8), followed by reductive amination with the appropriate b enzaldehydes or phenylacetaldehydes afforded the target compounds. N9 methylation of these analogues was carried out using formaldehyde and sodium cyanoborohydride. The analogues demonstrated significant inhibi tion of pcDHFR and tgDHFR. N9 methylation significantly increased DHFR inhibitory potency. Compound 11, the 3',4',5'-trimethoxy-substituted analogue with a selectivity ratio of 9.4 for tgDHFR (compared to rlDHF R) was the most selective analogue of the nonclassical series. Compoun d 22, the N9 methyl 2',5'-dimethoxy-substituted analogue was the most potent analogue against tgDHFR (IC50 = 6.3 nM) and was the second most selective analogue for tgDHFR (compared to rlDHFR) in the nonclassica l series. The naphthyl-substituted analogues 23-25 were generally more potent against rlDHFR than against pcDHFR and tgDHFR. Selected analog ues were also evaluated against Streptococcus faecium (sf) DHFR, Esche richia coli (ec) DHFR, Lactobacillus casei (Ic) DHFR and tgDHFR with h DHFR as the mammalian reference, under slightly different assay condit ions than those employed for rlDHFR. Analogues 11 and 22 had selectivi ty ratios of greater than 100 for tgDHFR (compared to hDHFR). Analogue 22 in particular, was the most selective analogue of the nonclassical series against tgDHFR (selectivity ratio = 303.5) with excellent pote ncy (28 nM). Analogue 11, also displayed significant selectivity for s fDHFR (selectivity ratio = 4902). Compound 22 was evaluated in vivo fo r the inhibition of the growth of T.gondii trophozoites in mice, where at 50 mg/kg orally, it demonstrated distinct prolongation of survival without toxicity. Compounds 11, 12, and 21-23 were evaluated as antit umor agents in the National Cancer Institutes preclinical in vitro scr eening program. Compounds 12, 22, and 23 showed GI(50)s for tumor grow th inhibition in the 10(-6)-10(-7) M range.