Liquid chromatography-mass spectrometry and liquid chromatography-NMR characterization of in vitro metabolites of a potent and irreversible peptidomimetic inhibitor of rhinovirus 3C protease

In vitro metabolism of AG7088 [trans-(4S,2'R,5'S,3'''S)-4-{2'-4-(4-fluorobe nzyl)-6'-methyl-5'-[(5"-methylisoxazole-3"-carbonylamino]-4-oxoheptanoylami no}-5-(2'''-oxopyrrolidin-3-'''-yl)pent-2-enoic acid ethyl ester] was studi ed in liver microsomes isolated from mice, rats, rabbits, dogs, monkeys, an d humans. The structures of the metabolites were characterized by liquid ch romatography (LC)-tandem mass spectrometry and LC-NMR methods. Hydrolysis o f the ethyl ester to produce metabolite M4 (AG7185) is the predominant path way in all species, with the greatest activity observed in rodents and rabb its, followed by monkeys, dogs, and humans. Several hydroxylation products were identified as minor metabolites, including diastereomers M1 and M2, wi th a hydroxy group at the P1-lactam moiety, and M3, with a hydroxy group at the methyl position of the methylisoxazole ring. Rodent and rabbit liver m icrosomes formed almost exclusively the acid metabolite M4 (AG7185), with v ery little hydroxylated metabolites, whereas monkey liver microsomes formed more secondary metabolites (i.e., acid analogs of the hydroxylated metabol ites). The overall metabolic profile of AG7088 formed in dog liver microsom es closely resembled that of human liver microsomes; therefore, this specie s may be the most appropriate animal model relative to humans for exposure to AG7088 and its metabolites.