Characterization of novel nitazene recreational drugs: Insights into their risk potential from in vitro µ-opioid receptor assays and in vivo behavioral studies in mice.

2-Benzylbenzimidazole derivatives or 'nitazenes' are increasingly present on the recreational drug market. Here, we report the synthesis and pharmacological characterization of 15 structurally diverse nitazenes that might be predicted to emerge or grow in popularity. This work expands the existing knowledge about 2-benzylbenzimidazole structure-activity relationships (SARs), while also helping stakeholders (e.g., forensic toxicologists, clinicians, policymakers) in their risk assessment and preparedness for the potential next generation of nitazenes. In vitro µ-opioid receptor (MOR) affinity was determined via competition radioligand ([H]DAMGO) binding assays in rat brain tissue. MOR activation (potency and efficacy) was studied by means of a cell-based β-arrestin 2 recruitment assay. For seven nitazenes, including etonitazene, opioid-like pharmacodynamic effects (antinociception, locomotor activity, body temperature changes) were evaluated after subcutaneous administration in male C57BL/6 J mice. The results showed that all nitazenes bound to MOR with nanomolar affinities, and the functional potency of several of them was comparable to or exceeded that of fentanyl. In vivo, dose-dependent effects were observed for antinociception, locomotor activity, and body temperature changes in mice. SAR insights included the high opioid-like activity of methionitazene, iso-butonitazene, sec-butonitazene, and the etonitazene analogues 1-ethyl-pyrrolidinylmethyl N-desalkyl etonitazene and ethylene etonitazene. The most potent analogue of the panel across all functional assays was α'-methyl etonitazene. Taken together, through critical pharmacological evaluation, this work provides a framework for strengthened preparedness and risk assessments of current and future nitazenes that have the potential to cause harm to users.