Received for publication April 10, 2008.
Revised July 2, 2008.
Accepted for publication July 2, 2008.

Extractive Biotransformation for Production of Metabolites of Poorly Soluble Compounds. Synthesis of 32-Hydroxy-Rifalazil

Abstract

A novel reaction system was developed for the production of metabolites of poorly water-soluble parent compounds using mammalian liver microsomes. The system includes the selection and use of an appropriate hydrophobic polymeric resin as a reservoir for the hydrophobic parent compounds and its metabolites. The utility of the extractive biotransformation approach was demonstrated for the production of a low yielding, synthetically-challenging 32-hydroxylated metabolite of the antibiotic rifalazil, using mouse liver microsomes. In order to address the low solubility and reactivity of rifalazil in the predominantly aqueous microsomal catalytic system, a variety of strategies were tested for the enhanced delivery of hydrophobic substrates, including the addition of mild detergents, polyvinylpyrrolidone, glycerol, bovine serum albumin, and hydrophobic polymeric resins. The latter strategy was identified as the most suitable for the production of 32-hydroxy-rifalazil, resulting in up to 13-fold enhancement of the volumetric productivity compared to the standard aqueous system operating at the solubility limit of rifalazil. The production process was optimized for a wide range of reaction parameters; the most important for improving volumetric productivity included the type and amount of the polymeric resin, cofactor recycling system, concentrations of the biocatalyst and rifalazil, reaction temperature, and agitation rate. The optimized extractive biotransformation system was used to synthesize 32-hydroxy-rifalazil on a multimilligram scale.

Key words: antibiotics, cytochrome P450 catalyzed oxidations, drug development, mass spectrometry, metabolite identification, microsomes