Mechanism-based competitive inhibitors of glyoxalase I: Intracellular delivery, in vitro antitumor activities, and stabilities in human serum and mouse serum

S-(N-Aryl-N-hydroxycarbamoyl)glutathione derivatives (GSC(O)N(OH)C6H4X, where GS = glutathionyl and X = H (1), Cl (2), Br (3)) have been proposed as possible anticancer agents, because of their ability to strongly inhibit the methylglyoxal-detoxifying enzyme glyoxalase I. In order to test this hypothesis, the in vitro antitumor activities of these compounds and their [glycyl, glutamyl] diethyl ester prodrug forms (1(Et)(2)-3(Et)(2)) have been examined. All three diethyl esters inhibit the growth of L1210 murine leukemia and B16 melanotic melanoma in culture, with GI(50) values in the micromolar concentration range. Cell permeability studies with L1210 cells indicate that growth inhibition is associated with rapid diffusion of the diethyl esters into the cells, followed by enzymatic hydrolysis of the ethyl ester functions to give the inhibitory diacids. In contrast, the corresponding diacids neither readily diffuse into nor significantly inhibit the growth of these cells. Consistent with the hypothesis that cell growth inhibition is due to competitive inhibition of glyoxalase I, preincubation of L1210 cells with 2(Et)(2) increases the sensitivity of these cells to the inhibitory effects of exogenous methylglyoxal. Compound 2(Et)(2) is much less toxic to nonproliferating murine splenic lymphocytes, possibly reflecting reduced sensitivity to methylglyoxal and/or reduced chemical stability of the diacid inside these cells. Finally, a plasma esterase-deficient murine model has been identified that should allow in vivo testing of the diethyl esters.