In vitro metabolism of Benzyl-4CN-BUTINACA and MDMB-4CN-BUTINACA using human hepatocytes and LC-QToF-MS analysis

Synthetic cannabinoid receptor agonists (SCRAs) are a large and continuously evolving group of new psychoactive substances (NPS). Recently, many different nitrile-containing SCRAs have emerged on the illicit market, two of which have been found to release cyanide during metabolism. This can produce symptoms similar to those of cyanide poisoning, contributing to the toxicity of these SCRAs. Notified by the EU Early Warning System in 2020, Benzyl-4CN-BUTINACA (Benzyl-4CN-BINACA, BZ-4CN-BUTINACA) is the most recent nitrile-containing SCRA to emerge. This study characterized the metabolism of Benzyl-4CN-BUTINACA and the prophetic compound MDMB-4CN-BUTINACA for the first time using ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QToF-MS) following incubation with primary human hepatocytes (HHeps; 5 µmol/L, up to 5 h). For Benzyl-4CN-BUTINACA, nine metabolites (no phase II metabolites) were identified and 12 for MDMB-4CN-BUTINACA, including only two minor phase II metabolites. By far the most abundant metabolites for Benzyl-4CN-BUTINACA were metabolites with a dihydrodiol on the indazole core (B1) and decyanation to a carboxylic acid (B2). The metabolites with ester hydrolysis (M1) and ester hydrolysis with dehydrogenation (M2) were the most abundant for MDMB-4CN-BUTINACA. Decyanation was less prevalent for these compounds than for other nitrile-containing SCRAs, such as Cumyl-4CN-BUTINACA, with only 29.0% and 1.78% of metabolites of Benzyl-4CN-BUTINACA and MDMB-4CN-BUTINACA, respectively, having a loss of cyanide. However, the second major metabolite of Benzyl-4CN-BUTINACA was a decyanation metabolite, making the potential CN formation not negligible.