Analogs of tetrahydrofolic acid. XII. On the relative contribution by the 2‐amino and 4‐hydroxy groups of 2‐amino‐5‐(3‐anilinopropyl)‐6‐methyl‐4‐pyrimidinol to inhibition of folic reductase and thymidylate synthetase

The structure of 2‐amino‐5‐(3‐anilinopropyl)‐6‐methyl‐4‐pyrimidinol (II), an inhibitor of both dihydrofolic reductase and thymidylate synthetase, has been modified by replacement of the 2‐amino group by hydrogen or the 4‐hydroxyl group by hydrogen, mercapto or amino. Enzymic evaluation showed that 5‐(3‐anilinopropyl)‐2,4‐diamino‐6‐methyl‐pyrimidine was a 170‐fold more effective inhibitor of dihydrofolic reductase than thymidylate synthetase. This specificity is due to the fact that replacement of the 4‐hydroxyl group of II with 4‐amino enhances binding to dihydrofolic reductase by 350‐fold, but binding to thymidylate synthetase is unchanged. No significant cross‐over specificity was noted with the six compounds studied. Replacement of the 4‐hydroxyl group of II by hydrogen gave a compound (XVII) that was a slightly more effective inhibitor of dihydrofolic reductase than II, but was slightly less effective on thymidylate synthetase. Binding to dihydrofolic reductase and to thymidylate synthetase could be increased by 17‐fold and 5‐fold, respectively, by replacement of the 4‐hydroxyl group of II by 4‐mercapto (XVI). It was concluded that the mode of binding in the N‐3 and 4‐hydroxyl region of II was not the same for the two enzymes. Synthesis of the 2‐amino‐, the 4‐hydroxy‐ and the 4‐mercaptopyrimidyl analogs of II are described. Also described are the partial purification of and assays for dihydrofolic reductase and thymidylate synthetase by procedures more familiar to the organic chemist.