In vitro formation and properties of beta- and gamma-hydroxy-N-nitrosohexamethyleneimine

Stable hydroxylated metabolites of N-nitrosohexamethyleneimine (NO-HEX) account for about one-third of the metabolites produced in in vitro reactions using uninduced rat liver microsomes post-microsomal supernatant. The ratio of gamma- to beta-hydroxyNO-HEX thus produced is 3:1. Each of these isomers exists in two conformeric forms that can be separated easily at room temperature by h.p.l.c. on C18 columns. The ratio of isomers does not vary with substrate concentration or time of reaction, which suggests that one liver enzyme is responsible for the formation of both isomers. Different ratios of conformeric forms of gamma-hydroxyNO-HEX produced by liver and lung microsomes indicate that these organs have different enzymes that perform the same functions. Anti-syn (E-Z) configurational assignments for the conformers were based on data obtained from 13C-n.m.r. studies. Although isolated conformers were stable at room temperature, when heated above 70 degrees C or dissolved in aprotic solvents, after several hours an equilibrium mixture was formed. The conformers of beta-hydroxyNO-HEX reached equilibrium in less than half the time required by gamma-hydroxy conformers. The results of i.r. spectroscopic studies suggest that hydrogen bonding may be involved in the stabilization of the conformers. Upon modification of the hydroxy group separable conformers are not detected; this is consistent with the suggestion that the hydroxy group may be involved in the stabilization of separable conformeric forms. We speculate that the occurrence of relatively stable and separable conformers may be due to intramolecular hydrogen bonding between the hydroxy and nitroso groups.