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

T. Pallmann, U. Jonas, M. Wagner, M. Thevis, H. Kaeferstein, M. A. Rothschild, K. Bender

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

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 24 h. 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.

Original languageEnglish
Pages (from-to)233-240
Number of pages8
JournalEuropean Journal of Pharmaceutical Sciences
Volume39
Issue number4
DOIs
StatePublished - 19 Feb 2010
Externally publishedYes

Keywords

  • Benzodiazepine glucuronides
  • Benzodiazepines
  • Enzyme-assisted synthesis
  • Liver microsomes
  • Lorazepam
  • Oxazepam
  • Temazepam
  • UGT

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