In vitro toxicokinetics and metabolic profiling of methoxycathinones and methylthiocathinones using human liver systems and hyphenated mass spectrometry

Ring-substituted synthetic cathinones represent a major subgroup within new psychoactive substances. This study investigated the in vitro toxicokinetics of the three 4-methoxy-substituted representatives 4MeO-NE-BP (4'-methoxy-N-ethylbutyrophenone), 4MeO-αP-BP (4'-methoxy-α-pyrrolidinobutyrophenone), and 4MeO-αP-VP (4'-methoxy-α-pyrrolidinovalerophenone) and the three related novel 4-methylthio analogs 4MeS-NE-BP (4'-methylthio-N-ethylbutyrophenone), 4MeS-αP-BP (4'-methylthio-α-pyrrolidinobutyrophenone), and 4MeS-αMor-PrP (4'-methylthio-2-morpholinopropiophenone). This included plasma protein binding (PPB), phase I and phase II metabolism in pooled human liver S9 fraction (pHLS9) and HepaRG cells, and monooxygenases activity. Methoxycathinones exhibited lower PPB (~ 40-60%) compared to methylthiocathinones (~ 85%). Predominant phase I metabolic reactions included O-/S-demethylation and hydroxylation, with additional transformations such as N-dealkylation, N-oxidation, and oxo reduction. Phase II conjugation reactions, such as glucuronidation and sulfation, were observed post-demethylation. Overall, 42 and 45 metabolites were identified in pHLS9 and HepaRG systems, respectively, with metabolite number increasing alongside alkyl chain length and heterocyclic substitution. All compounds were substrates for multiple monooxygenases, suggesting a low risk for drug-drug interactions. Based on metabolic stability and abundance, parent compounds and O-/S-desmethyl and hydroxylated metabolites might be proposed as urinary screening targets in clinical and forensic toxicology, as well as doping control settings.