Analysis of lorazepam and its 30-glucuronide in human urine by capillary electrophoresis: evidence for the formation of two distinct diastereoisomeric glucuronides

Lorazepam (LOR) is a 3-hydroxy-1,4-benzodiazepine that is chiral and undergoes enantiomerization at room temperature. In humans, about 75% of the administered dose of LOR is excreted in the urine as its 30-glucuronide. CE-MS with negative ESI was used to confirm the presence of LOR-30-glucuronide in urines that stemmed from a healthy individual who ingested 1 or 2 mg LOR, whereas free LOR could be detected in extracts prepared from enzymatically hydrolyzed urines. As the 30-glucuronidation reaction occurs at the chiral center of the molecule, two diastereoisomers can theoretically be formed, molecules that can no longer interconvert. The stereoselective formation of LOR glucuronides in humans and in vitro was investigated. MEKC analysis of extracts of the nonhydrolyzed urines suggested the presence of the two different LOR glucuronides in the urine. The formation of the same two diastereoisomers was also observed in vitro employing incubations of LOR with human liver microsomes in the presence of uridine 5'-diphospho-glucuronic acid as coenzyme. The absence of other coenzymes excluded the formation of phase I or other phase II metabolites of LOR. Both results revealed a stereoselectivity, one diastereoisomer being formed in a higher amount than the other. After enzymatic hydrolysis using beta-glucuronidase, these peaks could not be detected any more. Instead, LOR was monitored. Analysis of the extracts prepared from enzymatically hydrolyzed urines by MEKC in the presence of 2-hydroxypropyl-beta-CD revealed the enantiomerization process of LOR (observation of two peaks of equal magnitude connected with a plateau zone). The data presented provide for the first time the evidence of the stereoselectivity of the LOR glucuronidation in humans.

Synthesis of (5' S )-5'- C -Alkyl-2'-deoxynucleosides

We describe the synthesis of (5 S )-5- C -butylthymidine ( 5a ), of the (5 S )-5- C -butyl- and the (5 S )-5- C -isopentyl derivatives 16a and 16b of 2-deoxy-5-methylcytidine, as well as of the corresponding cyanoethyl phosphoramidites 9a , b and 14a , b , respectively. Starting from thymidin-5-al 1 , the alkyl chain at C(5) is introduced via Wittig chemistry to selectively yield the ( Z )-olefin derivatives 3a and 3b ( Scheme 2 ). The secondary OH function at C(5) is then introduced by epoxidation followed by regioselective reduction of the epoxy derivatives 4a and 4b with diisobutylaluminium hydride. In the latter step, a kinetic resolution of the diastereoisomer mixture 4a and 4b occurs, yielding the alkylated nucleoside 2a and 2b , respectively, with (5 S )-configuration in high diastereoisomer purity (de=94%). The corresponding 2-deoxy-5-methylcytidine derivatives are obtained from the protected 5-alkylated thymidine derivatives 7a and 7b via known base interconversion processes in excellent yields ( Scheme 3 ). Application of the same strategy to the purine nucleoside 2-deoxyadenine to obtain 5- C -butyl-2-deoxyadenosine 25 proved to be difficult due to the sensitivity of the purine base to hydride-based reducing agents ( Scheme 4 ).