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Oxidative Metabolism of N-Isopropyl--(2-methylazo)-p-toluamide (Azoprocarbazine) by Rodent Liver Microsomes¹

Department of Biochemistry, The University of Texas Health Science Center at Dallas, Dallas, Texas 75235

N-Isopropyl--(2-methylazo)-p-toluamide (azoprocarbazine) is oxidized by rat liver microsomes in the presence of reduced nicotinamide adenine dinucleotide phosphate and oxygen to yield two major metabolites as determined by high-pressure liquid chromatography. The isolated products are the two isomeric azoxy derivatives, N-isopropyl--(2-methyl-NNO-azoxy)-p-toluamide (benzylazoxy) and N-isopropyl--(2-methyl-ONN-azoxy)-p-toluamide (methylazoxy), since the mass spectra of the metabolites are distinctly different from each other but are identical to those of the respective chemically synthesized azoxy standards. In addition, p-formyl-N-isopropylbenzamide is also formed in measurable quantities. Liver microsomes from untreated rabbits or rats form only the methylazoxy derivative; the benzylazoxy isomer is nearly undetectable. Animal pretreatment with phenobarbital or 5,6-benzoflavone results in a marked increase in the rate of methylazoxy formation catalyzed by rat or rabbit liver microsomes. The rate of benzylazoxy formation is also stimulated by phenobarbital pretreatment but is unaffected by 5,6-benzoflavone pretreatment. The rate of formation of p-formyl-N-isopropylbenzamide as well is increased by animal pretreatment with either 5,6-benzoflavone or phenobarbital. Kinetic evaluation of these data suggests the possible involvement of more than one species of cytochrome P-450 in these reactions. Production of both benzylazoxy- and methylazoxyprocarbazine by rat liver microsomes is inhibited by a number of specific cytochrome P-450 inhibitors. Azoprocarbazine elicits a very weak spectral complex (type II) with rat liver microsomal cytochrome P-450. These results strongly suggest that cytochrome P-450-dependent monooxygenase(s) are involved in the N-oxidation of azoprocarbazine to yield two azoxy isomers of procarbazine. Incubation of liver microsomal protein with [¹⁴C]azoprocarbazine, oxygen, and reduced nicotinamide adenine dinucleotide phosphate results in a time-dependent increase in covalent binding of labeled material to microsomal protein. More protein-bound label is obtained with [methyl-¹⁴C]azoprocarbazine than with [ring-¹⁴C]azoprocarbazine, suggesting that the molecule can be metabolically activated to a moiety which preferentially binds the methyl portion of its structure to microsomal protein in vitro.

¹ This work was supported in part by Grant I-616 from the Robert A. Welch Foundation and by Grant B-336 from the American Cancer Society.

² Robert A. Welch Postdoctoral Fellow.

³ Research Career Development Awardee HL 00255. To whom requests for where reprints should be addressed.

Received 2/20/80. Accepted 6/25/80.