6-SUBSTITUTED 2,4-DIAMINOPYRIDO[3,2-D]PYRIMIDINE ANALOGS OF PIRITREXIM AS INHIBITORS OF DIHYDROFOLATE-REDUCTASE FROM RAT-LIVER, PNEUMOCYSTIS-CARINII, AND TOXOPLASMA-GONDII AND AS ANTITUMOR AGENTS

The synthesis and biological activity are reported for 21 6-substitute d 2,40diaminopyrido[3,2-d]pyrimidine analogues (4-24) of piritrexim (P TX) as inhibitors of dihydrofolate reductase (DHFR) and as antitumor a gents. Recombinant DHFR from Pneumocystis carinii (pc) and native DHFR from Toxoplasma gondii (tg) were the target enzymes tested; these org anisms are responsible for fatal opportunistic infections in AIDS pati ents. Rat liver (rl) DHFR served as the mammalian reference enzyme to determine selectivity for the pathogenic DHFR. The synthesis of S9-bri dged compounds 4-6 was achieved by aryl displacement of 2,4-diamino-6- chloropyrido[3,2-d]pyrimidine (27) with thiol nucleophiles. Oxidation of 4-6 with hydrogen peroxide in glacial acetic acid afforded the corr esponding sulfone, analogues 7-9. The N9-bridged compounds 10-24 were synthesized from their precursor 3-amino-6-(arylamino)-2-pyridinecarbo nitriles via a thermal cyclization with chloroformamidine hydrochlorid e. Unlike the S9-bridged compounds, the arylamino side chains of the N 9-bridged analogues were introduced prior to the formation of the 2,4- diaminopyrido[3,2-d]pyrimidine nucleus. A reversed two-atom-bridged an alogue (25) was also synthesized using a synthetic strategy similar to that utilized for compounds 10-24. The IC50 values of these compounds against pcDHFR ranged from 0.0023 x 10(-6) M for hyl-3',4'-dimethoxya nilino)pyrido[3,2-d]pyrimidine (21), which was the most potent, to 90. 4 x 10(-6) M for 2,4- amino-6-(4'-methoxyanilina)pyrido[3,2-d]pyrimidi ne (12), which was the least potent. The three S9-bridged compounds te sted were more potent than the corresponding sulfone-bridged compounds for all three DHFRs. N9-Methylation increased the potency by as much as 17 000-fold (compounds 15 and 21). None of the analogues were selec tive for pcDHFR. Against tgDHFR the most potent analogue was again 21 with an IC50 value of 0.00088 x 10(-6) M and the least potent was 12 w ith an IC50 Of 2.8 x 10(-6) M. N9-Methylation afforded an increase in potency of up to 770-fold (compound 15 NH vs 21 N-CH3) compared to the corresponding N9-H analogue. In contrast to pcDHFR, several analogues had a greater selectivity ratio for tgDHFR compared to trimetrexate ( TMQ) or PTX, most notably 3',4'-dimethoxyphenyl)thio]pyrido[3,2-d]pyri midine (4), no-6-[(2'-methoxyphenyl)sulfonyl][3,2-d]pyrimidine (7), an d 2,4-diamino-6-(2', 5'-dimethoxyanilino)pyrido[3,2-d]pyrimidine (14) which combined relatively high potency at 10(-7)-10(-8) M along with s electivity ratios of 3.97, 6.67, and 4.93, respectively. Several analo gues synthesized had better selectivity ratios than TMQ or PTX for bot h pcDHFR and tgDHFR, and the potencies of the N9-methylated compounds were comparable to or greater than that of TMQ or PTX. Selected compou nds were evaluated as inhibitors of the growth of a variety of tumor c ells in culture. The N9-CH3 analogues were, in general, highly potent with GI(50) values in the nanomolar range. The N9-H and S9 analogues w ere less potent with GI(50) values in the millimolar to micromolar ran ge.