Characterization of the metabolites of TUG-891 in rat, dog, and human hepatocytes using ultra-high-performance liquid chromatography tandem mass spectrometry and nuclear magnetic resonance spectroscopy

Rationale: TUG-891 is a potent and selective agonist of the long chain Free Fatty Acid Receptor 4. However, its metabolic profiles have not been revealed. The aim of this study was to investigate the in vitro metabolism of TUG-891 in hepatocytes.

Methods: TUG-891 at a concentration of 20 μM was incubated with rat, dog, and human hepatocytes at 37°C for 120 min. The samples were analyzed using ultra-high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry. The structures of the metabolites were proposed according to their MS/MS product ions. Furthermore, M4 and M5 were biosynthesized using human liver microsomes, and their structures were characterized using ¹³ C-NMR spectroscopy.

Results: Under the current conditions, eight metabolites were detected and structurally identified using high-resolution LC/MS and MS/MS spectra. The metabolites M4 and M5 were unambiguously confirmed to be TUG-891 alcohol and TUG-891 acid, respectively, using ¹³ C-NMR spectroscopy. Our results revealed that hydroxylation of methyl group at C-21 position to form TUG-891 alcohol (M5) followed by oxidation to yield TUG-891 aldehyde (M7) and carboxylic acid (M4) were the major metabolism processes. Phase II metabolism processes included glucuronidation and sulphation.

Conclusions: Hydroxylation at the C-21 position was the primary metabolic site of TUG-891. This study provided an overview of the metabolic profiles of TUG-891 in hepatocytes.