title: Phosphocholine conjugation: an unexpected in vivo conjugation pathway associated with hepatitis c ns5b inhibitors featuring a bicyclo[1.1.1]pentane.
abstract: During a medicinal chemistry campaign to identify inhibitors of the hepatitis C virus nonstructural protein 5B (RNA-dependent RNA polymerase), a bicyclo[1.1.1]pentane was introduced into the chemical scaffold to improve metabolic stability. The inhibitors bearing this feature, 5-(3-(bicyclo[1.1.1]pentan-1-ylcarbamoyl)-4-fluorophenyl)-2-(4-fluorophenyl)-N-methyl-6-(3,3,3-trifluoropropyl)furo[2,3-b]pyridine-3-carboxamide (1) and 5-(3-(bicyclo[1.1.1]pentan-1-ylcarbamoyl)phenyl)-2-(4-fluorophenyl)-N-methyl-6-(3,3,3-trifluoropropyl)furo[2,3-b]pyridine-3-carboxamide (2), exhibited low turnover in incubations with liver S9 or hepatocytes (rat, human), with hydroxylation of the bicyclic moiety being the only metabolic pathway observed. In subsequent disposition studies using bile-duct-cannulated rats, the metabolite profiles of bile samples revealed, in addition to multiple products of bicyclopentane-oxidation, unexpected metabolites characterized by molecular masses that were 181 Da greater than those of 1 or 2. Further LC/MSn and NMR analysis of the isolated metabolite of 1 demonstrated the presence of a phosphocholine (POPC) moiety bound to the methine carbon of the bicyclic moiety through an ester bond. The POPC conjugate of the NS5B inhibitors was assumed to result from two sequential reactions: hydroxylation of the bicyclic methine to a tertiary alcohol and addition of POPC by CDP-choline: 1,2-diacylglycerol cholinephosphotransferase, an enzyme responsible for the final step in the biosynthesis of phosphatidylcholine. However, this pathway could not be recapitulated using CDP-choline-supplemented liver S9 or hepatocytes due to inadequate formation of the hydroxylation product in vitro. The observation of this unexpected pathway prompted concerns about the possibility that 1 and 2 might interfere with routine phospholipid synthesis. These results demonstrate the participation in xenobiotic metabolism of a process whose function is ordinarily limited to the synthesis of endogenous compounds.
authors.affiliation: Bristol-Myers Squibb; xiaoliang.zhuo@bms.com.
authors.forename: Xiaoliang
authors.initials: X
authors.lastname: Zhuo
authors.affiliation: Bristol-Myers Squibb;
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Cantone
authors.affiliation: Bristol-Myers Squibb;
authors.forename: Yingzi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Bristol-Myers Squibb;
authors.forename: John E
authors.initials: JE
authors.lastname: Leet
authors.affiliation: Bristol-Myers Squibb;
authors.forename: Dieter M
authors.initials: DM
authors.lastname: Drexler
authors.affiliation: Bristol-Myers Squibb;
authors.forename: Kap-Sun
authors.initials: KS
authors.lastname: Yeung
authors.affiliation: Bristol-Myers Squibb;
authors.forename: Xiaohua Stella
authors.initials: XS
authors.lastname: Huang
authors.affiliation: Bristol-Myers Squibb;
authors.forename: Kyle J
authors.initials: KJ
authors.lastname: Eastman
authors.affiliation: Bristol-Myers Squibb;
authors.forename: Kyle E
authors.initials: KE
authors.lastname: Parcella
authors.affiliation: Bristol-Myers Squibb;
authors.forename: Kathleen W
authors.initials: KW
authors.lastname: Mosure
authors.affiliation: Bristol-Myers Squibb;
authors.forename: Matthew G
authors.initials: MG
authors.lastname: Soars
authors.affiliation: Bristol-Myers Squibb;
authors.forename: John F
authors.initials: JF
authors.lastname: Kadow
authors.affiliation: Bristol-Myers Squibb Company.
authors.forename: Benjamin M
authors.initials: BM
authors.lastname: Johnson
keywords: NMR; antivirals; drug design; mass spectrometry/MS; metabolite disposition; metabolite identification