title: 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)-1H-pyrazol-5-yl)-N-ethyl-5-methyl-3-oxo-4-(3-(trifluoromethyl)phenyl)-3,4-dihydropyrazine-2-carboxamide (AZD9819). The rearranged oxidation product 2-(1-(4-cyanophenyl)-1H-pyrazol-5-yl)-5-(N-ethylacetamido)-N-(3-(trifluoromethyl)phenyl)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)O2 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.
authors.affiliation: AstraZeneca, R&D Boston, Waltham, Massachusetts (C.G., V.P.H.); AstraZeneca, R&D Mölndal, Mölndal, Sweden (R.J.L., E.J.); AstraZeneca, R&D Charnwood, Loughborough, United Kingdom (R.J.L., A.S.W.); AstraZeneca, R&D Södertälje, Södertälje, Sweden (P.H.S); AstraZeneca, R&D Wilmington, Wilmington, Delaware (C.G.); AstraZeneca, R&D Lund, Lund, Sweden (E.J., G.H.); SP Process Development, Södertälje, Sweden (P.H.S.); and Applied Physical Chemistry, Royal Institute of Technology, Stockholm, Sweden (P.H.S.) Chungang.gu@astrazeneca.com.
authors.forename: Chungang
authors.initials: C
authors.lastname: Gu
authors.affiliation: AstraZeneca, R&D Boston, Waltham, Massachusetts (C.G., V.P.H.); AstraZeneca, R&D Mölndal, Mölndal, Sweden (R.J.L., E.J.); AstraZeneca, R&D Charnwood, Loughborough, United Kingdom (R.J.L., A.S.W.); AstraZeneca, R&D Södertälje, Södertälje, Sweden (P.H.S); AstraZeneca, R&D Wilmington, Wilmington, Delaware (C.G.); AstraZeneca, R&D Lund, Lund, Sweden (E.J., G.H.); SP Process Development, Södertälje, Sweden (P.H.S.); and Applied Physical Chemistry, Royal Institute of Technology, Stockholm, Sweden (P.H.S.).
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Lewis
authors.affiliation: AstraZeneca, R&D Boston, Waltham, Massachusetts (C.G., V.P.H.); AstraZeneca, R&D Mölndal, Mölndal, Sweden (R.J.L., E.J.); AstraZeneca, R&D Charnwood, Loughborough, United Kingdom (R.J.L., A.S.W.); AstraZeneca, R&D Södertälje, Södertälje, Sweden (P.H.S); AstraZeneca, R&D Wilmington, Wilmington, Delaware (C.G.); AstraZeneca, R&D Lund, Lund, Sweden (E.J., G.H.); SP Process Development, Södertälje, Sweden (P.H.S.); and Applied Physical Chemistry, Royal Institute of Technology, Stockholm, Sweden (P.H.S.).
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Wells
authors.affiliation: AstraZeneca, R&D Boston, Waltham, Massachusetts (C.G., V.P.H.); AstraZeneca, R&D Mölndal, Mölndal, Sweden (R.J.L., E.J.); AstraZeneca, R&D Charnwood, Loughborough, United Kingdom (R.J.L., A.S.W.); AstraZeneca, R&D Södertälje, Södertälje, Sweden (P.H.S); AstraZeneca, R&D Wilmington, Wilmington, Delaware (C.G.); AstraZeneca, R&D Lund, Lund, Sweden (E.J., G.H.); SP Process Development, Södertälje, Sweden (P.H.S.); and Applied Physical Chemistry, Royal Institute of Technology, Stockholm, Sweden (P.H.S.).
authors.forename: Per H
authors.initials: PH
authors.lastname: Svensson
authors.affiliation: AstraZeneca, R&D Boston, Waltham, Massachusetts (C.G., V.P.H.); AstraZeneca, R&D Mölndal, Mölndal, Sweden (R.J.L., E.J.); AstraZeneca, R&D Charnwood, Loughborough, United Kingdom (R.J.L., A.S.W.); AstraZeneca, R&D Södertälje, Södertälje, Sweden (P.H.S); AstraZeneca, R&D Wilmington, Wilmington, Delaware (C.G.); AstraZeneca, R&D Lund, Lund, Sweden (E.J., G.H.); SP Process Development, Södertälje, Sweden (P.H.S.); and Applied Physical Chemistry, Royal Institute of Technology, Stockholm, Sweden (P.H.S.).
authors.forename: Vinayak P
authors.initials: VP
authors.lastname: Hosagrahara
authors.affiliation: AstraZeneca, R&D Boston, Waltham, Massachusetts (C.G., V.P.H.); AstraZeneca, R&D Mölndal, Mölndal, Sweden (R.J.L., E.J.); AstraZeneca, R&D Charnwood, Loughborough, United Kingdom (R.J.L., A.S.W.); AstraZeneca, R&D Södertälje, Södertälje, Sweden (P.H.S); AstraZeneca, R&D Wilmington, Wilmington, Delaware (C.G.); AstraZeneca, R&D Lund, Lund, Sweden (E.J., G.H.); SP Process Development, Södertälje, Sweden (P.H.S.); and Applied Physical Chemistry, Royal Institute of Technology, Stockholm, Sweden (P.H.S.).
authors.forename: Eskil
authors.initials: E
authors.lastname: Johnsson
authors.affiliation: AstraZeneca, R&D Boston, Waltham, Massachusetts (C.G., V.P.H.); AstraZeneca, R&D Mölndal, Mölndal, Sweden (R.J.L., E.J.); AstraZeneca, R&D Charnwood, Loughborough, United Kingdom (R.J.L., A.S.W.); AstraZeneca, R&D Södertälje, Södertälje, Sweden (P.H.S); AstraZeneca, R&D Wilmington, Wilmington, Delaware (C.G.); AstraZeneca, R&D Lund, Lund, Sweden (E.J., G.H.); SP Process Development, Södertälje, Sweden (P.H.S.); and Applied Physical Chemistry, Royal Institute of Technology, Stockholm, Sweden (P.H.S.).
authors.forename: Gösta
authors.initials: G
authors.lastname: Hallström
mesh_terms: D000577:Amides / Q000097:blood / Q000737:chemistry; D000818:Animals; D005260:Female; D006801:Humans; D008054:Lipid Peroxides / Q000097:blood* / Q000737:chemistry; D008297:Male; D010084:Oxidation-Reduction; D053491:Proteinase Inhibitory Proteins, Secretory / Q000097:blood* / Q000737:chemistry*; D011719:Pyrazines / Q000097:blood / Q000737:chemistry; D011720:Pyrazoles / Q000097:blood / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar
chemical_list: C000622435:AZD9819; D000577:Amides; D008054:Lipid Peroxides; D053491:Proteinase Inhibitory Proteins, Secretory; D011719:Pyrazines; D011720:Pyrazoles