Lipid Peroxide–Mediated Oxidative Rearrangement of the Pyrazinone Carboxamide Core of Neutrophil Elastase Inhibitor AZD9819 in Blood Plasma Samples

Abstract

This study focused on the mechanistic interpretation of ex vivo oxidation of a candidate drug in blood plasma samples. An unexpected lipid peroxide-mediated epoxidation followed by a dramatic rearrangement led to production of a five-membered oxazole derivative from the original six-membered pyrazinone-carboxamide core of a human neutrophil elastase inhibitor, 6-(1-(4-cyanophenyl)-oxazole-4-carboxamide was characterized by accurate-mass tandem mass spectrometry fragmentations, by two-dimensional NMR and X-ray crystallography of an authentic standard, and by incorporation of an 18 O atom from molecular 18 O 2 to the location predicted by our proposed mechanism. The lipid peroxide-mediated oxidation was demonstrated by using human low-density lipoprotein (LDL) in pH 7.4 phosphate buffer and by inhibiting the oxidation with ascorbic acid or L-glutathione, two antioxidants effective in both plasma and the LDL incubation. A nucleophilic mechanism for the epoxidation of AZD9819 by lipid hydroperoxides explains the prevention of its ex vivo oxidation by acidification of the plasma samples. The discovery of the lipid peroxide-dependent oxidation of an analyte and the means of prevention could provide valuable information for biotransformation and bioanalysis.

Late-occurring and Long-circulating Metabolites of GABAAα2,3 Receptor Modulator AZD7325 Involving Metabolic Cyclization and Aromatization: Relevance to MIST Analysis and Application for Patient Compliance

“…In a previous experience, generating putative structures with envisaged rearrangement mechanisms was an effective way of assisting structural identification on a dramatically rearranged oxidation product in plasma for a candidate drug acting as a human neutrophil elastase inhibitor (Gu et al, 2015). Two intuitive thoughts have guided the proposal for possible mechanisms in the present study.…”

The Importance of Tracking “Missing” Metabolites: How and Why?

“…This is due to the synthetic complexity of the proposed structures or because the proposed structures are simply unable to be provided. When 1 H NMR information is very limited at core structures before and after biotransformation/rearrangement, 2D NMR data and educated guesses on possible formation mechanisms to draft putative structures for verification can constitute an effective approach to eventually confirm metabolite structure. , As illustrated previously, the biosynthesis and NMR characterizations and chemical synthesis of the proposed AZD7325 M9 metabolite standard, pimasertib M554 standard, and navoximod 306-P1 metabolite standard were essential for definitive metabolite structure elucidation.…”

Radiolabel Uncovers Nonintuitive Metabolites of BIIB104: Novel Release of [14C]Cyanide from 2-Cyanothiophene and Subsequent Formation of [14C]Thiocyanate

“…It has been previously reported that comparing observed carbon chemical shifts with predicted carbon chemical shifts of putative structures can be an effective approach for structural elucidation when 1 H NMR information is limited at the chemical structure of biotransformation (Gu et al, 2015).…”