Enzyme-assisted synthesis and structural characterization of pure benzodiazepine glucuronide epimers

Eur J Pharm Sci. 2010 Feb 19;39(4):233-40. doi: 10.1016/j.ejps.2009.12.005. Epub 2009 Dec 29.

Abstract

The three hydroxybenzodiazepines oxazepam, temazepam, and lorazepam used for their anxiolytic, sedative, and anticonvulsant properties are metabolized by glucuronidation, which is the predominant pathway in the clearance mechanism of exogenous and endogenous substances during phase II metabolism. The aim of this study was the synthesis of benzodiazepine-O-glucuronides as analytical reference substances. All benzodiazepines are prescribed clinically as racemic formulations. The resulting conjugates from the coupling reactions with glucuronic acid are epimeric pairs of glucuronides. Due to the importance of stereochemical factors in drug disposition it is necessary to separate the diastereomeric forms after synthesis. An enzyme-assisted synthesis was developed and optimized by using microsomal UGT from fresh swine liver to receive multimilligram amounts of the benzodiazepine glucuronides, which were not accessible by standard synthetic procedures, like the Koenigs-Knorr- and Williamson-ether-synthesis. Swine liver microsomes were prepared by homogenization and differential centrifugation of liver tissue. In the presence of liver microsomes the benzodiazepines and cofactor UDPGA were incubated for 24h. After incubation the microsomes were removed by protein precipitation and the residual benzodiazepines by liquid-liquid extraction (dichloromethane). The epimeric pairs of benzodiazepine glucuronides were separated by preparative high performance liquid chromatography (HPLC) followed by solid phase extraction (SPE) to obtain the pure benzodiazepine glucuronide epimers. The synthesis products were characterized by mass spectroscopy and nuclear magnetic resonance (NMR) spectroscopy.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Benzodiazepines / chemical synthesis*
  • Benzodiazepines / chemistry
  • Benzodiazepines / metabolism
  • Chromatography, High Pressure Liquid
  • Glucuronides / chemical synthesis*
  • Glucuronides / chemistry
  • Glucuronides / metabolism
  • Glucuronosyltransferase / chemical synthesis
  • Glucuronosyltransferase / chemistry
  • Glucuronosyltransferase / metabolism
  • Magnetic Resonance Spectroscopy
  • Microsomes, Liver / drug effects
  • Microsomes, Liver / enzymology
  • Rats
  • Stereoisomerism
  • Swine
  • Uridine Diphosphate Glucuronic Acid / chemistry

Substances

  • Glucuronides
  • Benzodiazepines
  • Uridine Diphosphate Glucuronic Acid
  • Glucuronosyltransferase