Reactive Metabolite TrappingScreens and PotentialPitfalls: Bioactivation of a Homomorpholine and Formation of an UnstableThiazolidine Adduct.

Successful early attrition of potentialproblematic compounds isof great importance in the pharmaceutical industry. The lead compoundin a recent project targeting neuropathic pain was susceptible tometabolic bioactivation, which produced reactive metabolites and showedcovalent binding to protein. Therefore, as a part of the backup seriesfor this compound several structural modifications were explored tomediate the reactive metabolite and covalent binding risk. A homomorpholinecontaining series of compounds was identified without compromisingpotency. However, when these compounds were incubated with human livermicrosomes in the presence of GSH, Cys-Gly adducts were identified,instead of intact GSH conjugates. This article examines the formationof the Cys-Gly adduct with AZX ([M+H]+486) as a representativecompound for this series. The AZX-Cys-Gly-adduct ([M+H]+662) showed evidence of ring contraction by formation of a thiazolidine-glycineand was additionally shown to be unstable. During its isolation forstructural characterization by 1H NMR spectroscopy, itwas found to have decomposed to a product with [M+H]+446.The characterization and identification of this labile GSH-derivedadduct using LC-MS/MS and 1H NMR are described, along withobservations around stability. In addition, various structurally relatedtrapping reagents were employed in an attempt to further investigatethe reaction mechanism along with a methoxylamine trapping experimentto confirm the structure of the postulated reactive intermediate. [ABSTRACT FROM AUTHOR]