Abstract

A series of lipid-soluble dialkyl esters of methotrexate and 3',5'-dichloromethotrexate have been prepared and their properties as substrates for rabbit liver aldehyde oxidase and inhibitors for mouse leukemia L1210 dihydrofolate reductase examined. The short-chain esters are highly active as substrates for aldehyde oxidase, being converted to the corresponding 7-hydroxy derivatives; the Michaelis constants for the hydroxylation reaction are in the range of 0.002-0.005 mM, 10- to 100-fold lower than for previously described substrates for this enzyme. Increases in the ester chain length are accompanied by increased lipid solubility and decreased substrate activity. With the nonesterified parent compound, methotrexate, the Michaelis constant decreases and oxidation rate increases with decreasing pH throughout the physiological pH range; with the esters, on the other hand, a well-defined maximum in apparent affinity and reaction rate is seen at pH 8. This disparity in behavior for the two types of substrates supports the hypothesis that the substrate activity of the nonesterified parent compound is a property of the undissociated carboxylic acid, rather than the anionic form, and that the apparent enhancement in substrate activity seen with the esters is due to their inability to form the latter. As inhibitors of dihydrofolate reductase, the dialkyl esters are 2- to 10-fold less effective than methotrexate; inhibitory activity increases with increasing chain length. with the maximum inhibition being observed with the di-n-amyl and di-n-octyl esters of methotrexate.