Structural determination of glucuronide conjugates and a carbamoyl glucuronide conjugate of carvedilol: Use of acetylation reactions as an aid to determine positions of glucuronidation

Carvedilol is metabolized via both oxidation and conjugation pathways in dog and rat to more than 12 different products. Several glucuronide conjugates of the parent drug were identified. The drug contains an aliphatic hydroxyl, an aliphatic amine and a carbazole amine, all of which are potential sites for conjugation with glucuronic acid. In order to determine the positions of glucuronidation, a strategy involving acetylation of the metabolites was devised. The metabolites were acetylated using acetic anhydride in either pyridine or aqueous solution, and the products were analyzed by fast atom bombardment mass spectrometry. Carvedilol was acetylated at both the hydroxyl and the aliphatic amine in pyridine and at only the aliphatic amine in aqueous solution. The carbazole nitrogen was unreactive under both conditions. Based on the acetylation patterns observed for the metabolites in pyridine or aqueous solution, the positions of conjugation were determined. Each of the five glucuronide metabolites of carvedilol formed in dog and rat was analyzed and the structures included two diastereomeric carbamoyl glucuronide metabolites formed from addition of CO2 and glcuronic acid to carvedilol, a carbazole-N-linked glucuronide, and two diastereomeric O-linked glucuronides. This approach should be generally applicable in many cases to determine the structures of glucuronide conjugates for compounds which contain more than one potential glucuronidation site.