Enzymatic assays for 2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline, a promising new "nonclassical" antifolate.

2,4-Diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)-methyl]quinazoline (I) is a promising new "nonclassical" antifolate. Inhibition of dihydrofolate reductase from bacterial (Lactobacillus casei)and mammalian (beef liver) sources was employed to develop useful enzymatic assays for this compound. A linear relationship was obtained by plotting the I concentration versus 1/V. The resultant standard curves maintained linearity particularly well between the 30 and 70% control range, with a correlation coefficient of 0.99 for both enzyme systems. The two enzyme systems are characterized by differences in sensitivity, stability, and day-to-day variation. The ID50 for the beef liver reductase system was 1.6 X 10(-9) M (+/-0.03); for the L. casei system, it was 1.35 X 10(-8) M (+/- 0.2). The apparent advantage for the beef liver enzyme was offset somewhat by its relative instability and its higher day-to-day variability. Studies in mice demonstrated that these assays are suitable for pharmacokinetic studies in vivo. Such studies indicated that I has a serum t1/2 of 45 min in mice; a similar serum t1/2 (50 min) was estimated in studies with 14C-labeled I in position 6.