Abstract

Four lipophilic 2,4-diaminopyrimidine antifolates, 2,4-diamino-5-(1-adamantyl)-6-ethylpyrimidine (DAEP), 2,4-diamino-5-(1-adamantyl)-6-methylpyrimidine (DAMP), 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (DDMP), and 2,4-diamino-5-(p-chlorophenyl)-6-ethylpyrimidine (PRM), were examined in cultures of several mouse and human cell lines and compared with methotrexate (MTX). Unlike MTX, the diaminopyrimidines not only inhibited dihydrofolate reductase, but were found to have a second, folate-independent site of action as judged by growth inhibition in a medium supplemented with the products of folate-dependent reactions (hypoxanthine, thymidine, and glycine). With respect to this site of inhibition, DAEP, DAMP, and DDMP were equipotent, with an ID₅₀ for all cells in the range of 5-50 µM. The second site did not concern DNA polymerases α,β, or γ (EC 2.7.7.7), and excess methionine did not alleviate the inhibition. When folates in the medium were present at concentrations adequate for optimal growth (folic acid and N⁵-methy-H₄ folic acid at 1 µM, folinic acid at 0.01 µM), the growth inhibitory potency of DAMP was the same. Increased concentrations of folinic acid protected the cells against DAMP to a certain degree, but not as well as hypoxanthine and thymidine. This is unlike MTX, which is competitively antagonized by folinic acid. Folinic acid and the products could also, to a limited degree, rescue cells preexposed to DAMP. Under folate-dependent conditions the growth of human cells was, on the average, four to eight times less sensitive to inhibition by DAEP, DAMP, and DDMP than that of mouse cells. This is unlike MTX. Neither the velocity or extent of cellular uptake of diaminopyrimidines nor the level of dihydrofolate reductase (EC 1.5.1.3) or the activity of TMP synthetase (EC 2.1.1.6) correlated with these sensitivities. No cellular metabolism of DAMP or DDMP was detected. The apparent affinities of the diaminopyrimidines to human and mouse dihydrofolate reductases may help explain the different sensitivities of cells to growth inhibition. While DAEP was the most potent growth inhibitor, with an ID₅₀ in the 1 to 40 nM range for all cells examined, the average ID₅₀ values for DAEP, DAMP, DDMP, and PRM related as 1:4:13:300. The true K_i values for DAEP, DAMP, and DDMP were 0.14, 0.68, and 2.1 nM, respectively, for the dihydrofolate reductase purified from MTX-resistant S-180 cells and 0.14, 0.43, and 1.0 nM, respectively, for the enzyme of MTX-resistrant KB cells. There was a positive correlation between the ID₅₀ values for growth inhibition by the various compounds and the K_i values for dihydrofolate reductase.