Characterizing the in vitro metabolic features of nine alicyclic fentanyl-type new psychoactive substances

New psychoactive substances (NPS) represent a third-generation class of drugs characterized by high addictive potential, enhanced potency, low lethal doses, rapid in vivo metabolic rates, and fast structural diversification. Among these, fentanyl-type NPS (F-NPS), which are derived from the opioid analgesic "fentanyl", present a category of NPS that simultaneously exhibit all five characteristics (in addition to nitazenes). Consequently, global F-NPS use poses a critical challenge, as metabolic data from F-NPS have often proven insufficient to support law enforcement agencies in combating emerging analogs. In this study, an in vitro human liver microsome model was used to incubate nine alicyclic F-NPS derivatives and the metabolites was analyzed by using liquid chromatography-ion trap tandem time-of-flight mass spectrometry. We systematically characterized the metabolic profiles of the alicyclic F-NPS and elucidated the structural determinants that influence metabolism. Our findings revealed that sp³-carbon monohydroxylation occurring at the alicyclic ring, a distinctive metabolic pathway in this subclass, is governed by the C-H Laplacian bond order, intermediate radical stability, alicyclic ring rigidity, and steric hindrance, while also competing with analogous metabolic pathways at other sites. Notably, alicyclic dehydrogenation, a previously unreported metabolic pathway, correlated with the stability of alicyclic alkenes and conjugative stabilization through adjacent carbonyl groups. N-oxidation metabolism was significantly modulated by N-dealkylation metabolism at adjacent positions and substituents on the phenyl group of the core. Furthermore, >N-CH₂-CH₂- serves as the primary metabolic hotspot within the F-NPS skeleton structure, with the total relative contents of corresponding metabolites accounting for > 80 % (and up to 98 %). The monohydroxylation of the phenyl group adhered to an electrophilic substitution mechanism governed by the electron density and both the theoretical calculation results and mass spectrometric data consistently indicated that sp²-carbon monohydroxylation occurred exclusively at the phenethyl group in the tail. The findings provide data and analytical strategies for the development of a universal F-NPS metabolic prediction model.