Phosphocholine Conjugation: An Unexpected In Vivo Conjugation Pathway Associated with Hepatitis C NS5B Inhibitors Featuring A Bicyclo[1.1.1]Pentane

• DOI: 10.1124/dmd.115.069062
• Publication Date: 2016-03-10T03:11:44Z
• Authors: Xiaoliang Zhuo; Joseph L. Cantone; Yingzi Wang; John E. Leet; Dieter M. Drexler; Kap-Sun Yeung; Xiaohua Stella Huang; Kyle J. Eastman; Kyle E. Parcella; Kathleen W. Mosure; Matthew G. Soars; John F. Kadow; Benjamin M. Johnson
• Keywords: Moiety; Phosphocholine; Hydroxylation

During a medicinal chemistry campaign to identify inhibitors of the hepatitis C virus nonstructural protein 5B (RNA-dependent RNA polymerase), bicyclo[1.1.1]pentane was introduced into chemical scaffold improve metabolic stability. The 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), hydroxylation bicyclic moiety being only pathway observed. In subsequent disposition studies using bile-duct-cannulated rats, metabolite profiles bile samples revealed, addition multiple products bicyclopentane-oxidation, unexpected metabolites characterized by molecular masses that were 181 Da greater than those 1 2. Further LC/MSn NMR analysis isolated demonstrated presence phosphocholine (POPC) bound methine carbon through an ester bond. POPC conjugate NS5B assumed result from two sequential reactions: tertiary alcohol CDP-choline: 1,2-diacylglycerol cholinephosphotransferase, enzyme responsible for final step biosynthesis phosphatidylcholine. However, could not be recapitulated CDP-choline-supplemented due inadequate formation product vitro. observation prompted concerns about possibility 2 might interfere routine phospholipid synthesis. These results demonstrate participation xenobiotic metabolism process whose function is ordinarily limited synthesis endogenous compounds.