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Synthesis and Identification of Products Derived from the Metabolism of the Carcinostatic 1-(2-Chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea by Rat Liver Microsomes¹

Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331

Liver microsomal metabolism of 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea in the presence of reduced nicotinamide adenine dinucleotide phosphate and O₂ was shown to produce seven metabolites that included the parent urea. A cytochrome P-450-dependent monohydroxylation of the cyclohexyl ring occurred in 3 positions, cis-3, trans-3, and cis-4, and on the methyl group to form a trans-4-hydroxymethyl derivative. In addition, monohydroxylation of the 2-chloroethyl carbon attached to the N-1 urea nitrogen yielded an -hydroxy metabolite. A ring-hydroxylated derivative remained unidentified while the structures of all other such derivatives were established by comparison with compound synthesized, purified by high-pressure liquid chromatography, and characterized by mass spectral and nuclear magnetic resonance analyses. It was tentatively concluded that some parent urea is formed by a cytochrome P-450-dependent reaction because of a requirement for reduced nicotinamide adenine dinucleotide phosphate and inhibition by CO.

Microsomes from rats pretreated with phenobarbital showed about a 3-fold increase in hydroxylation rate while phenobarbital-treated mice microsomes were induced 8-fold. However, in both species, the induced hydroxylation rate was about 4 nmol/min/mg protein. When microsomes from phenobarbital-induced rats were used, a mixture of 80% CO:20% O₂ decreased the rate of formation of all metabolites to 14% of that in 80% N₂:20% O₂.

¹ This investigation was supported by Contract N01-23201, awarded by the National Cancer Institute, Department of Health, Education, and Welfare.

² On leave from Oral Roberts University. Current address: Department of Biochemistry, Oral Roberts University, Tulsa, Okla. 74171.

³ To whom requests for reprints should be addressed.

Received 8/23/78. Accepted 11/21/78.