Comparative in vitro metabolic profiling of six novel methylthiophene cathinones using different human liver systems and LC-HRMS/MS

Abstract Synthetic cathinones represent a major class of new psychoactive substances associated with severe toxicological risks. Therapeutic cathinone derivatives such as amfepramone (appetite suppressant) were partially removed from the market due to severe side effects. p -Tolyl-substituted synthetic cathinones are primarily known as new psychoactive substances and are associated with serious adverse events and abuse liability. Bioisosteric replacement of the p -tolyl ring with a 5′-methylthiophene moiety has recently emerged as a structural optimization strategy in this class and has led to the evaluation of methylthiophenes, a novel class of cathinone analogues. Knowledge of their hepatic biotransformation is essential for reliable toxicological detection and risk assessment. This study investigated the in vitro hepatic metabolism of six novel methylthiophene cathinones, 5Me-NM-BT (5-methyl- N -methyl-butanothiophenone), 5Me-NM-VT (5-methyl- N -methyl-valerothiophenone), 5Me-NE-BT (5-methyl- N -ethyl-butanothiophenone), 5Me-NE-VT (5-methyl- N -ethyl-valerothiophenone), 5Me- α P-BT (5-methyl- α -pyrrolidino-butanothiophenone), and 5Me- α P-VT (5-methyl- α -pyrrolidino-valerothiophenone) using pooled human liver microsomes (pHLM), pooled human liver S9 fraction (pHLS9), and HepG2 cells. Metabolites were characterized using reversed-phase and hydrophilic interaction liquid chromatography coupled to high-resolution tandem mass spectrometry. Additionally, recombinant human monooxygenases were screened to identify isoenzymes mediating initial phase I steps. In total, 48 phase I and two phase II metabolites could be tentatively identified. Within each subgroup, the compounds showed similar metabolic profiles. Isozyme mapping revealed consistent contributions of various enzymes, including CYP1A2, CYP2B6, CYP2C19, CYP2D6, and CYP3A4 suggesting a low risk of metabolism-based drug–drug interactions or single-enzyme polymorphism impact. Hydroxylated metabolites at the 5′-methyl group were consistently abundant across all models and, in combination with the respective parent compounds, represent promising analytical targets for toxicological screening.