Structure Elucidation and Initial Characterization of a N-glucuronide Metabolite (M1) of Tuvusertib

The investigational DNA-damage response inhibitor tuvusertib showed the formation of a direct glucuronide in hepatocyte incubations. Early metabolite identification workflows were unable to assign the site of modification. Thus, a synthesis approach was established to facilitate structure elucidation as well as an initial characterization regarding drug–drug interaction and safety. Different methods for generating sufficient amounts of the glucuronide were assessed and after purification structure elucidation of the glucuronide was performed via nuclear magnetic resonance spectroscopy (NMR). A series of in vitro experiments were conducted to determine its potential risk for drug–drug interaction. Confirmation of its identity was achieved by co-chromatography with hepatic incubations, which initially showed the presence of the metabolite. Chemical synthesis showed the most promising route for the formation of the proposed glucuronide and NMR determined the modification to be at the primary amino group attached to the pyrazolo-pyrimidine moiety. The retention time as well as the mass spectra in the co-chromatography aligned with the glucuronide of interest in the hepatocyte incubation, confirming the synthesized metabolite to be M1. Subsequent in vitro experiments showed that M1 did not inhibit any major CYP enzyme. The tuvusertib metabolite M1 was identified as an N-glucuronide demonstrating no direct liabilities associated with drug–drug interactions (DDI) or safety implications. Furthermore, M1 can be used in bioanalyses as retention time marker or reference material in further clinical evaluations.