Structure–metabolism relationships of 4-pentenyl synthetic cannabinoid receptor agonists using in vitro human hepatocyte incubations and high-resolution mass spectrometry

The rapid structural evolution and extensive metabolism of synthetic cannabinoid receptor agonists (SCRAs) following consumption makes their detection in biological samples challenging, especially in urine. In vitro metabolite identification studies are an essential tool for identifying analytical targets to confirm SCRA consumption in clinical and forensic toxicology casework. Systematic studies on structurally related SCRAs allow structure–metabolism relationships (SMRs) to be determined, helping to predict the metabolites of emerging and future compounds. In this study, a series of amino acid-derived 4-pentenyl SCRAs and the OXIZID 4-pentenyl SCRA BZO-4en-POXIZID were incubated at 5 µM with pooled human hepatocytes for up to 3 h. Metabolites were identified using liquid chromatography–quadrupole time-of-flight mass spectrometry (LC–QTOF-MS) and SMRs investigated using the proportion that each type of biotransformation contributed to the total abundance of metabolites for each parent compound. Metabolites were mainly produced via terminal amide/ester hydrolysis, dihydrodiol formation on the tail, hydroxylation and N-dealkylation, but also by ketone formation, dehydrogenation, glucuronidation and combinations thereof. Methyl valinate (MMB) and ethyl valinate (EMB) SCRAs underwent extensive ester hydrolysis (90.9–96.0%), which was less dominant for methyl tert-leucinate (MDMB) SCRAs (47.6–60.7%), with dihydrodiol formation (24.3–27.7%) and hydroxylation (6.2–17.3%) becoming more prominent. For AB-4en-PICA and tert-leucinamide (ADB) SCRAs, hydroxylation was the major metabolic pathway (47.9–58.4%), while tail dihydrodiol formation was most prominent for BZO-4en-POXIZID (61.5%). Clear SMRs were identified for 4-pentenyl SCRAs and can be used to recommend biomarkers of consumption and aid prediction of metabolites of emerging and future SCRAs.