Functional evaluation of carboxy metabolites of synthetic cannabinoid receptor agonists featuring scaffolds based on L-valine or L-tert-leucine.

Indole- and indazole-based synthetic cannabinoid receptor agonists (SCRAs), featuring valine or tert-leucine substituents, are commonly abused new psychoactive substances (NPS). A major metabolic pathway for these SCRAs is hydrolysis of the terminal amide or methylester functionalities. Although these hydrolysis products were already detected as main ingredients in some "legal highs," these metabolites are often poorly characterized. Here, we report a systematic investigation of the activity of 7 common hydrolysis metabolites of 15 SCRAs featuring scaffolds based on L-valine or L-tert-leucine in direct comparison to their parent compounds. An activity-based cannabinoid receptor 1 (CB₁ ) bio-assay was used for activity profiling of SCRAs and their metabolites in a stable HEK293T cell system. The recruitment of β-arrestin2 to the activated CB₁ (each fused to one part of a split Nanoluciferase) was provoked by adding the (putative) SCRAs. Luminescence of the functionally complemented luciferase was monitored by a 96-well plate-reader. The major hydrolysis metabolites of 5F-AB-PINACA, ADB-CHMICA, ADB-CHMINACA, ADB-FUBICA, and their methyl- and ethylester derivatives showed no detectable CB₁ activation at concentrations up to 1 μM. On the other hand, metabolites of 5F-ADB-PINACA, AB-CHMINACA, and ADB-FUBINACA did retain activity, although significantly reduced as compared to the parent compounds (EC₅₀ values >100 nM). Activity-based characterization of SCRAs and their metabolites at CB₁ may not only allow a better insight into the complex interplay between SCRAs and their metabolites in intoxications, but may also allow application of the concept of "activity equivalents" present in biological fluids or, alternatively, in confiscated materials.