Metabolic Stability and Biotransformation of Muscarinic Receptor Antagonists: An Integrated In Vitro Study

Background/Objectives: Metabolic stability is an important determinant of the pharmacokinetic properties of drug candidates, as metabolic instability may limit systemic exposure and lead to the formation of metabolites with distinct pharmacological properties. Here, we characterized the phase I metabolic stability and biotransformation pathways of five phenyl-substituted benzimidazole-based muscarinic acetylcholine receptor (mAChR) antagonists using an integrated in vitro and in silico approach. Methods: Metabolic stability, microsomal binding, and metabolite formation were investigated in human liver microsomes using liquid chromatography coupled to high-resolution and tandem mass spectrometry. GLORY, GLORYx, and FAME3R were used to rank possible sites of metabolism, while SwissADME was applied to estimate physicochemical and pharmacokinetic properties of the parent compounds and putative metabolites. Molecular docking was further used to explore potential effects of metabolic transformations on target interactions. Results: Compound 4 showed the most rapid metabolic turnover, whereas 1 displayed the highest intrinsic metabolic stability after correction for microsomal binding. Hydroxylation was the most frequently observed phase I transformation, with N-demethylation consistently detected across the series. Several putative metabolites were predicted to have properties consistent with P-glycoprotein (P-gp) substrate recognition and reduced brain penetration, whereas the parent compounds were not predicted to be P-gp substrates except for 4. Conclusions: The study defines major metabolic transformation pathways within this benzimidazole-based mAChR antagonist series and identifies metabolic liabilities that may be relevant to further compound optimization. The combined experimental and computational approach provides a framework for interpreting metabolic profiles and generating hypotheses for subsequent pharmacokinetic and structure activity investigations.