Nineteen previously undescribed 2,4-diamino-6-(arylmethyl)-5,6,7, 8-tetrahydroquinazolines (5a-m, 10-12) were synthesized as part of a larger effort to assess the therapeutic potential of lipophilic dihydrofolate reductase (DHFR) inhibitors against opportunistic infections of AIDS. Condensation of appropriately substituted (arylmethyl)triphenylphosphoranes with 4, 4-ethylenedioxycyclohexanone, followed by hydrogenation (H2/Pd-C) and acidolysis, yielded the corresponding 4-(arylmethyl)cyclohexanones, which were then condensed with cyanoguanidine to form the tetrahydroquinazolines. Three simple 2, 4-diamino-6-alkyl-5,6,7,8-tetrahydroquinazoline model compounds (9a-c) were also prepared in one step from commercially available 4-alkylcyclohexanones by this method. Enzyme inhibition assays against rat liver DHFR, Pneumocystis carinii DHFR, and the bifunctional DHFR-TS enzyme from Toxoplasma gondii were carried out, and the selectivity ratios IC50(rat)/IC50(P. carinii) and IC50(rat)/IC50(T. gondii) were compared. The three most potent inhibitors of P. carinii DHFR were the 2,5-dimethoxybenzyl (5j), 3, 4-dimethoxybenzyl (5k), and 3,4,5-trimethoxybenzyl (5l) analogues, with IC50 values of 0.057, 0.10, and 0.091 microM, respectively. The remaining compounds generally had IC50 values in the 0.1-1.0 microM range. However all the compounds were more potent against the rat liver enzyme than the P. carinii enzyme and thus were nonselective. The T. gondii enzyme was always more sensitive than the P. carinii enzyme, with most of the analogues giving IC50 values of 0.01-0.1 microM. Moderate 5-10-fold selectivity for T. gondii versus rat liver DHFR was observed with five compounds, the best combination of potency and selectivity being achieved with the 2-methoxybenzyl analogue 5d, which had an IC50 of 0.014 microM and a selectivity ratio of 8.6. One compound (5l) was tested for antiproliferative activity against P. carinii trophozoites in culture at a concentration of 10 microgram/mL and was found to completely suppress growth over 7 days. The suppressive effect of 5l was the same as that of trimethoprim (10 microgram/mL) + sulfamethoxazole (250 microgram/mL), a standard clinical combination for the treatment of P. carinii pneumonia in AIDS patients. Four compounds (5a,h,k,l) were tested against T. gondii tachyzoites in culture and were found to have a potency (IC50 = 0.1-0.5 microM) similar to that of pyrimethamine (IC50 = 0.69 microM), a standard clinical agent for the treatment of cerebral toxoplasmosis in AIDS patients. Compound 5h was also active against T. gondii infection in mice when given qdx8 by peritoneal injection at doses ranging from 62.5 (initial dose) to 25 mg/kg. Survival was prolonged to the same degree as with 25 mg/kg clindamycin, another widely used drug against toxoplasmosis. Three compounds (5j-l) were tested for antiproliferative activity against human tumor cells in culture. Among the 25 cell lines in the National Cancer Institute panel for which data were confirmed in two independent experiments, the IC50 for at least two of these compounds was <10 microM against 17 cell lines (68%) and in the 0. 1-1 microM range against 13 cell lines (52%). One compound (5j) had an IC50 of <0.01 microM against four of the cell lines. The activity profiles of 5k,l were generally similar to that of 5j except that there were no cells against which the IC50 was <0.01 microM.