text: In a continuing effort to design small-molecule inhibitors of dihydrofolate reductase (DHFR) that combine the enzyme-binding selectivity of 2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine (trimethoprim, TMP) with the potency of 2,4-diamino-5-methyl-6-(2',5'-dimethoxybenzyl)pyrido[2,3-d]pyrimidine (piritrexim, PTX), seven previously undescribed 2,4-diamino-5-[2'-methoxy-5'-(substituted benzyl)]pyrimidines were synthesized in which the substituent at the 5'-position was a carboxyphenyl group linked to the benzyl moiety by a bridge of two or four atoms in length. The new analogues were all obtained from 2,4-diamino-5-(5'-iodo-2'-methoxybenzyl)pyrimidine via a Sonogashira reaction, followed, where appropriate, by catalytic hydrogenation. The new analogues were tested as inhibitors of DHFR from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), and Mycobacterium avium (Ma), three life-threatening pathogens often found in AIDS patients and individuals whose immune system is impaired as a result of treatment with immunosuppressive chemotherapy or radiation. The selectivity index (SI) of each compound was obtained by dividing its     inhibitory concentration (IC(  )) against Pc, Tg, or Ma DHFR by its IC(  ) against rat DHFR. 2,4-Diamino-[2'-methoxy-5'-(3-carboxyphenyl)ethynylbenzyl]pyrimidine (28), with an IC(  ) of 23 nM and an SI of 28 in the Pc DHFR assay, had about the same potency as PTX and was     times more potent than TMP. As an inhibitor of Tg DHFR, 28 had an IC(  ) of 5.5 nM (   -fold lower than that of TMP and similar to that of PTX) and an SI value of     (2-fold better than TMP and vastly superior to PTX). Against Ma DHFR, 28 had IC(  ) and SI values of 1.5 nM and    , respectively, compared with     nM and     for TMP. Although it had 2.5-fold lower potency than 28 against Ma DHFR (IC(  ) = 3.7 nM) and was substantially weaker against Pc and Tg DHFR, 2,4-diamino-[2'-methoxy-5'-(4-carboxyphenyl)ethynylbenzyl]pyrimidine (29), with the carboxy group at the para rather than the meta position, displayed     -fold selectivity against the Ma enzyme and was the most selective 2,4-diamino-5-(5'-substituted benzyl)pyrimidineinhibitor  of this enzyme we have encountered to date. Additional bioassay data for these compounds are also reported.