Structure determination of metabolites isolated from urine and bile after administration of AY4166, a novel d-phenylalanine-derivative hypoglycemic agent

Abstract Molecular structures of 10 metabolites, which were isolated from urine (M1-M8) or bile (M9 and M10) after administration of AY4166 ( N-(trans -4-isopropylcyclohexanecarbonyl)- d -phenylalanine), a novel amino acid derivative with hypoglycemic activity, have been elucidated by mass spectrometry and nuclear magnetic resonance. Four of these (M1, M2, M3 and M8) were determined to be hydroxyl derivatives of AY4166, two (M9 and M10) were carboxylate derivatives via oxidization of M2 and M3, three (M4, M5 and M6) were glucronic acid conjugates and the other (M7) was a dehydro derivative. The estimated structures for M1, M2, M3, M7, M8, M9 and M10 were confirmed by the coincidence of the retention time of HPLC, MS and 1 H NMR spectra between the isolated metabolites and authentic synthesized substances. For three glucronic acid conjugates, M4, M5 and M6, structural confirmation was performed by a selective enzymatic digestion with β-glucronidase. M1 and M2/3 were about 5–6 and 3 times less potent than AY4166, respectively, and M7 was almost as potent as AY4166.