Oxidative metabolism of N-isopropyl-alpha-(2-methylhydrazino)-p-toluamide hydrochloride (procarbazine) by rat liver microsomes.

Abstract

The oxidative metabolism of procarbazine ( N -isopropyl-α-(2-methylhydrazino)- p -toluamide hydrochloride) by rat liver microsomes has been studied utilizing three different biochemical assays. Using high-pressure liquid chromatography, it was observed that the major, stable microsomal metabolite produced was the azo derivative, N -isopropyl-α-(2-methylazo)- p -toluamide, which can undergo a rapid chemical conversion to an aldehyde, p -formyl- N -isopropylbenzamide, and methylhydrazine upon the addition of acid. The rate of metabolism could most conveniently be determined spectrophotometrically by reacting the resultant methylhydrazine with p -dimethylaminobenzaldehyde. Alternatively, p -formyl- N -isopropyl benzamide could be extracted directly from the acid-treated mixture with an organic solvent, and its concentration could be determined spectrophotometrically. Rates of metabolism using all three assays ranged from 13.4 to 14.1 nmol/min/mg of microsomal protein. Inhibitor and induction studies indicated that the cytochrome P-450-dependent monooxygenase system is responsible for this oxidation reaction. Several possible mechanisms were considered: either a direct dehydrogenation or a preliminary N -oxidation by cytochrome P-450 followed by a rapid dehydration step. The azo derivative of procarbazine was further oxidized to two isomeric azoxy derivatives, N -isopropyl-α-(2-methyl-NNO-azoxy)- p -toluamide and N -isopropyl-α-(2-methyl-ONN-azoxy)- p -toluamide, at a metabolic rate which is 10 to 15% as large as the rate of procarbazine oxidation. In addition, the affinity of the azo derivative for the monooxygenase is high relative to that of the parent hydrazine. The reaction was inhibited by cytochrome P-450 inhibitors and was sensitive to the in vitro addition of several thiono-sulfur-containing compounds. The azoxy compounds were also metabolized by microsomal fractions from liver and yielded p -formyl- N -isopropylbenzamide as a product. This complex in vitro metabolic scheme is analogous to the in vivo metabolism of 1,2-dimethylhydrazine and suggests that procarbazine may be metabolically activated to yield alkylating agents capable of expressing carcinogenic and/or toxic effects by a mechanism common for at least these two N,N′ -disubstituted hydrazines.