NNL-3: A Synthetic Intermediate or a New Class of Hydroxybenzotriazole Esters with Cannabinoid Receptor Activity?

Synthetic cannabinoid receptor agonists (SCRAs) remain a prolific class of new psychoactive substances (NPS) and continue to expand rapidly. Despite the recent identification of hydroxybenzotriazole (HOBt) containing SCRAs in synthetic cannabis samples, there is currently no information regarding the pharmacological profile of these NPS with respect to human CB₁ and CB₂ receptors. In the current study, a series consisting of seven HOBt indole-, indazole-, and 7-azaindole-carboxylates bearing a range of N-alkyl substituents were synthesized and pharmacologically evaluated. Competitive binding assays at CB₁ and CB₂ demonstrated that all analogues except a 2-methyl-substituted derivative had low affinity for CB₁ (K_i = 3.80-43.7 μM) and CB₂ (K_i = 2.75-18.2 μM). A fluorometric functional assay revealed that 2-methylindole- and indole-derived HOBt carboxylates were potent and efficacious agonists of CB₁ (EC₅₀ = 12.0 and 63.7 nM; E_max = 118 and 120%) and CB₂ (EC₅₀ = 10.9 and 321 nM; E_max = 91 and 126%). All other analogues incorporating indazole and 7-azaindole cores and bearing a range of N1-substituents showed relatively low potency for CB₁ and CB₂. Additionally, a reporter assay monitoring β-arrestin 2 (βarr2) recruitment to the receptor revealed that the 2-methylindole example was the most potent and efficacious at CB₁ (EC₅₀ = 131 nM; E_max = 724%) and the most potent at CB₂ (EC₅₀ = 38.2 nM; E_max = 51%). As with the membrane potential assay, the indazole and other indole HOBt carboxylates were considerably less potent at both receptors, and analogues comprising a 7-azaindole core showed little activity. Taken together, these data suggest that NNL-3 demonstrates little CB1 receptor activity and is unlikely to be psychoactive in humans. NNL-3 is likely an unintended SCRA manufacturing byproduct. However, the synthesis of NNL-3 analogues proved simple and general, and some of these showed potent cannabimetic profiles in vitro, indicating that HOBt esters of this type may represent an emerging class of SCRA NPS.