Institute of Legal Medicine, University Hospital of Cologne, Melatengürtel 60-62, D-50823 Cologne, Germany.
Eur J Pharm Sci. 2010 Feb 19;39(4):233-40. doi: 10.1016/j.ejps.2009.12.005. Epub 2009 Dec 29.
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.
用于抗焦虑、镇静和抗惊厥特性的三种羟基苯并二氮䓬类药物奥沙西泮、替马西泮和劳拉西泮通过葡萄糖醛酸化代谢，这是外源性和内源性物质在 II 期代谢中清除机制中的主要途径。本研究的目的是合成苯并二氮䓬-O-葡糖苷作为分析参考物质。所有苯并二氮䓬类药物在临床上均作为外消旋制剂开处方。与葡萄糖醛酸偶联反应产生的缀合物是葡糖苷的差向异构体对。由于立体化学因素在药物处置中的重要性，有必要在合成后分离非对映异构体形式。通过使用新鲜猪肝中的微粒体 UGT 开发并优化了酶辅助合成，以接收毫克量的苯并二氮䓬葡糖苷，这些葡糖苷无法通过标准合成方法获得，如 Koenigs-Knorr 和 Williamson-醚合成。猪肝微粒体通过肝组织的匀浆和差速离心制备。在肝微粒体存在下，将苯并二氮䓬类药物和辅因子 UDPGA 孵育 24 小时。孵育后，通过蛋白质沉淀去除微粒体，通过液液萃取（二氯甲烷）去除残留的苯并二氮䓬类药物。通过制备高效液相色谱（HPLC）和固相萃取（SPE）分离苯并二氮䓬葡糖苷的差向异构体对，以获得纯的苯并二氮䓬葡糖苷差向异构体。通过质谱和核磁共振（NMR）光谱对合成产物进行了表征。
