Identification of 2,3-diaminophenazine and of o-benzoquinone dioxime as the major in vitro metabolites of benzofuroxan

Abstract

1. The results of an in vitro study of the metabolism of benzofuroxan using either cytosolic or microsomal fractions obtained from rat liver are reported.

2. Benzofuroxan was incubated with an appropriate volume of cytosol or microsomal suspension; control incubations were performed without the β-nicotinamide adenine dinucleotide phosphate-generating system or, alternatively, by using the subcellular fractions inactivated by heating. Incubation mixtures were analysed by high-performance liquid chromatography. Two principal metabolites (M1, M2) were identified in the cytosolic fraction only. The dependence of M2 formation on thiol cofactors, incubation time and protein concentration was examined.

3. The two metabolites were isolated and characterized by their ¹H-, ¹³C-nuclear magnetic resonance, infrared and mass spectra. The structures of o-benzoquinonedioxime (2) and 2,3-diaminopleuozuc (3), were arranged to M1 and M2 respectively. The proposed structures were confirmed by the identity of the metabolites with authentic samples obtained by synthesis. X-ray analysis showed that the dioxime metabolite had an amphy configuration.

4. A metabolic scheme for the formation of the two products is proposed.