2,4-DIAMINO-5-SUBSTITUTED-QUINAZOLINES AS INHIBITORS OF A HUMAN DIHYDROFOLATE-REDUCTASE WITH A SITE-DIRECTED MUTATION AT POSITION-22 AND OFTHE DIHYDROFOLATE REDUCTASES FROM PNEUMOCYSTIS-CARINII AND TOXOPLASMA-GONDII

2,4-Diaminoquinazoline antifolates with a lipophilic side chain at the 5-position, and in one case with a das sic al (p-aminobenzoyl)-L-glut am ate side chain, were synthesized as potentially selective inhibitor s of a site-directed mutant of human dihydrofolate reductase (DHFR) co ntaining phenylalanine instead of leucine at position 22. This mutant enzyme is approximately 100-fold more resistant than native enzyme to the classical antifolate methotrexate (MTX), yet shows minimal cross r esistance to the nonclassical antifolates piritrexim (PTX) and trimetr exate (TMQ). Although they were much less potent than trimetrexate and piritrexim, the lipophilic 5-substituted analogues were all found to bind approximately 10 times better to the mutant DHFR than to the wild -type enzyme. The potency of the analogue with a classical (p-aminoben zoyl)-L-glutamate side chain was similarly diminished in comparison wi th MTX, but the difference in its binding affinity to the two DHFR spe cies was only 5-fold. Thus, by making subtle structural changes in the antifolate molecule, it may be possible to attack resistance due to m utational alterations in the active site of the target enzyme. Also, t o test the hypothesis that DHFR from Pneumocystis carinii and Toxoplas ma gondii may have a less sterically restrictive active site than the enzyme from mammalian cells, inhibition assays using several of the li pophilic analogues in the series were carried out against the P. carin ii and T. gondii reductases in comparison with the enzyme from rat liv er. In contrast to their preferential binding to mutant versus wild-ty pe human DHFR, binding of these analogues to the P. carinii and T. gon dii enzymes was weaker than binding to rat enzyme. It thus appears tha t, if the active site of the DHFR from these parasites is less sterica lly restrictive than the active site of the mammalian enzyme, this dif ference cannot be successfully exploited by moving the side chain from the 6-position to the 5-position.