In vitro metabolism of seven arolyl-derived fentanyl-type new psychoactive substances

Over the past decade, fentanyl-type new psychoactive substances (F-NPS) have emerged as the most representative synthetic opioids in third-generation drugs. These substances are characterized by their "low" fatal dose and parent drug levels in biological matrices, "fast" rates of derivatization and metabolism, and "many" derivatization sites and analogs. The low levels of parent fentanyl NPS in biological matrices complicate their detection, necessitating the use of characteristic metabolites as biomarkers for forensic analysis. Moreover, the ongoing emergence of arolyl-derived F-NPS further challenges forensic laboratories in accurately identifying the parent drug from its metabolites. To address this issue, in this study, the in vitro phase I metabolism of seven arolyl-derived F-NPS was studied using a human liver microsome model. Metabolites were analyzed by liquid chromatography-ion trap tandem time-of-flight mass spectrometry. Using density functional theory, the structural characteristics and their effects on amide hydrolysis, N-dealkylation, and oxidation metabolism were clarified. Amide hydrolysis was influenced by the positive charge of the carbonyl carbon and the 2-substituent effect on the aryl groups. N-dealkylation, β-monohydroxylation, N-oxidation, and phenyl group monohydroxylation in the tail were less affected by structural changes in the head. The former two were the major metabolites and exhibited competition. The relative contents of N-oxidation and phenyl group monohydroxylation in the tail were relatively stable at 4% and 13%, respectively. Furthermore, the β-C adjacent to the nitrogen on the piperidine ring was susceptible to oxidation, leading to the formation of the monohydroxylation metabolite. The results of this study may enhance our understanding of the in vitro metabolism of arolyl-derived F-NPS, and potentially all F-NPS, providing important data and theoretical support for predicting their in vivo metabolism in the future.