title: Reactive metabolite trapping screens and potential pitfalls: bioactivation of a homomorpholine and formation of an unstable thiazolidine adduct.
abstract: Successful early attrition of potential problematic compounds is of great importance in the pharmaceutical industry. The lead compound in a recent project targeting neuropathic pain was susceptible to metabolic bioactivation, which produced reactive metabolites and showed covalent binding to protein. Therefore, as a part of the backup series for this compound several structural modifications were explored to mediate the reactive metabolite and covalent binding risk. A homomorpholine containing series of compounds was identified without compromising potency. However, when these compounds were incubated with human liver microsomes in the presence of GSH, Cys-Gly adducts were identified, instead of intact GSH conjugates. This article examines the formation of the Cys-Gly adduct with AZX ([M+H]+ 486) as a representative compound for this series. The AZX-Cys-Gly-adduct ([M+H]+ 662) showed evidence of ring contraction by formation of a thiazolidine-glycine and was additionally shown to be unstable. During its isolation for structural characterization by 1H NMR spectroscopy, it was found to have decomposed to a product with [M+H]+ 446. The characterization and identification of this labile GSH-derived adduct using LC-MS/MS and 1H NMR are described, along with observations around stability. In addition, various structurally related trapping reagents were employed in an attempt to further investigate the reaction mechanism along with a methoxylamine trapping experiment to confirm the structure of the postulated reactive intermediate.
authors.affiliation: DMPK Department, Alderley Park, AstraZeneca UK Ltd. , Macclesfield, Cheshire SK10 4TG, United Kingdom.
authors.forename: Eva M
authors.initials: EM
authors.lastname: Lenz
authors.forename: Scott
authors.initials: S
authors.lastname: Martin
authors.forename: Ralf
authors.initials: R
authors.lastname: Schmidt
authors.forename: Pierre-Emmanuel
authors.initials: PE
authors.lastname: Morin
authors.forename: Robin
authors.initials: R
authors.lastname: Smith
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Weston
authors.forename: Malken
authors.initials: M
authors.lastname: Bayrakdarian
mesh_terms: D001410:Bacillus megaterium / Q000201:enzymology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D006801:Humans; D008862:Microsomes, Liver / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D009025:Morpholines / Q000737:chemistry / Q000378:metabolism*; D053778:Thiazolidines / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D009025:Morpholines; D053778:Thiazolidines; D003577:Cytochrome P-450 Enzyme System