Nine novel 2,4-diamino-5-methyl-6-substituted-pyrido[2,3-d]pyrimidines, 2-10, were synthesized as potential inhibitors of Pneumocystis carinii dihydrofolate reductase (pcDHFR) and Toxoplasma gondii dihydrofolate reductase (tgDHFR). Compounds 2-5 were designed as conformationally restricted analogues of trimetrexate (TMQ), in which rotation around tau 3 was constrained by incorporation of the side chain nitrogen as part of an indoline or an indole ring. Analogue 6, which has an extra atom between the side chain nitrogen and the phenyl ring, has its nitrogen as part of a tetrahydroisoquinoline ring. Analogues 7-9 are epiroprim (Ro 11-8958) analogues and contain a pyrrole ring as part of the side chain substitution on the phenyl ring similar to epiroprim. These analogues were designed to investigate the role of the pyrrole substitution on the phenyl ring of 2,4-diamino-5-methyl-6-(anilinomethyl)pyrido[2,3-d]pyrimidines. Molecular modeling indicated that a pyrrole substituent in the ortho position of the side chain phenyl ring was most likely to interact with pcDHFR in a manner similar to the pyrrole moiety of epiroprim. Analogue 10, in which a phenyl ring replaced a methoxy group, was synthesized to determine the contribution of a phenyl ring on selectivity, lipophilicity, and cell penetration. The synthesis of analogues 2-4 was achieved via reductive amination of 2,4-diamino-5-methyl 6-carboxaldehyde with the appropriately substituted indolines. The indolines were obtained from the corresponding indoles via NaCNBH3 reductions. Analogues 5-10 were synthesized by nucleophilic displacement of 2,4-diamino-5-methyl-6-(bromomethyl)-pyrido[2,3-d]pyrimidine with the 5-methoxyindolyl anion, 6,7-dimethoxytetrahydroisoquinoline, the appropriately substituted pyrroloaniline or 2-methoxy-5-phenylaniline. The pyrroloanilines were synthesized in two steps by treating the substituted nitroanilines with 2,5-dimethoxy-tetrahydrofuran to afford the nitropyrrole intermediates, followed by reduction of the nitro group with Raney Ni. The analogues were more potent than trimethoprim and epiroprim and more selective than TMQ and piritrexim against pcDHFR and tgDHFR. Compounds 5 and 10 had IC50 values of 1 and 0.64 microM, respectively, for the inhibition of the growth of T. gondii cells in culture, and showed excellent culture IC50/enzyme IC50 ratios, which were correlated with their calculated log P values, indicating a direct relationship between calculated lipophilicity and cell penetration.
Twenty-two 2,6-diamino-8-substituted purines (2-23) were synthesized, in which rotation around the two flexible bonds of trimethoprim (TMP), linking the pyrimidine ring to the side chain phenyl ring, was restricted by incorporation into a purine ring, in an attempt to increase the potency and selectivity of TMP against dihydrofolate reductase (DHFR) from the organisms that often cause fatal opportunistic infections in patients with AIDS, i.e., Pneumocystis carinii (pc) and Toxoplasma gondii (tg). The syntheses of analogues 2-20 were achieved via a one-pot reaction of 2,4,5,6-tetraaminopyrimidine and the appropriately substituted benzaldehyde or phenyl acetaldehyde, in acidic methoxyethanol. Analogues 21-23 were synthesized via nucleophilic displacement of 2,6-diamino-8-(chloromethyl)purine with the appropriate anilines or 2-naphthalenethiol. The compounds were evaluated as inhibitors of pcDHFR and tgDHFR with rat liver (rl) DHFR as the mammalian reference enzyme. Compound 11, the 3',4'-dichlorophenyl analogue, was as potent as TMP and had a selectivity ratio of 13 for pcDHFR, which ranked it as one of the three most selective inhibitors of pcDHFR (compared to rlDHFR) known to date. It also displayed a selectivity ratio of 38 for tgDHFR. None of the other analogues showed any improvement compared to TMP in potency or selectivity. In the preclinical in vitro screening program of the National Cancer Institute, compound 11 showed a GI50 of 10(-6) M for the inhibition of the growth of 17 tumor cell lines.
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