Effect of N-9-methylation and bridge atom variation on the activity of 5-substituted 2,4-diaminopyrrolo[2,3-d]pyrimidines against dihydrofolate reductases from Pneumocystis carinii and Toxoplasma gondii

The effect of N-9-methylation and bridge atom variation on inhibitory potency and selectivity of 2,4-diaminopyrrolo[2,3-d]pyrimidines against dihydrofolate reductases (DHFR) was studied. Specifically three nonclassical 2,4-diamino-5-((N-methylanilino)methyl)pyrrolo[2,3-d]pyrimidines with 2',5'-dimethoxyphenyl (2), 3',4'-dichlorophenyl (3), 1'-naphthyl (4), one classical analogue with a 4'-L-glutamate substituent (10), and four nonclassical 2,4-diamino-5-((phenylthio)methyl)pyrrolo[2,3-d]pyrimidines with 3',4'-dimethoxyphenyl (5), 3',4'-dichlorophenyl (6), 1'-naphthyl (7), and 2'-naphthyl (8) substituents were synthesized. The classical and nonclassical analogues were obtained by displacement of the intermediate 2,4-diamino-5-bromomethylpyrrolo[2,3-d]pyrimidine, 14, with appropriately substituted N-methylaniline, thiophenols, or 4-(N-methylamino)benzoyl-L-glutamate. Compounds 2-8 and 10 were evaluated against Pneumocystis carinii (pc), Toxoplasma gondii (tg), and rat liver (rl) DHFRs. The N-methyl and thiomethyl analogues were more inhibitory than their corresponding anilino-methyl analogues (previously reported) against all three DHFRs. The inhibitory potency of these analogues was greater against rlDHFR than against tgDHFR which resulted in a loss of selectivity for tgBHFR compared to the N-9-H analogues. The classical N-9-methyl analogue 10 was more potent and about a-fold more selective against tgDHFR than its corresponding desmethyl analogue. All of the analogues, 2-8 and 10, were more selective than trimetrexate (TMQ) against pcDHFR (except 4) and significantly more selective than TMQ against tgDHFR.