In vitro structure-activity relationships and forensic case series of emerging 2-benzylbenzimidazole 'nitazene' opioids.

2-Benzylbenzimidazole 'nitazene' opioids are presenting a growing threat to public health. Although various nitazenes were previously studied, systematic comparisons of the effects of different structural modifications to the 2-benzylbenzimidazole core structure on μ-opioid receptor (MOR) activity are limited. Here, we assessed in vitro structure-activity relationships of 9 previously uncharacterized nitazenes alongside known structural analogues. Specifically, we focused on MOR activation by 'ring' substituted analogues (i.e., N-pyrrolidino and N-piperidinyl modifications), 'desnitazene' analogues (lacking the 5-nitro group), and N-desethyl analogues. The results from two in vitro MOR activation assays (β-arrestin 2 recruitment and inhibition of cAMP accumulation) showed that 'ring' modifications overall yield highly active drugs. With the exception of 4'-OH analogues (which are metabolites), N-pyrrolidino substitutions were generally more favorable for MOR activation than N-piperidine substitutions. Furthermore, removal of the 5-nitro group on the benzimidazole ring consistently caused a pronounced decrease in potency. The N-desethyl modifications showed important MOR activity, and generally resulted in a slightly lowered potency than comparator nitazenes. Intriguingly, N-desethyl isotonitazene was the exception and was consistently more potent than isotonitazene. Complementing the in vitro findings and demonstrating the high harm potential associated with many of these compounds, we describe 85 forensic cases from North America and the United Kingdom involving etodesnitazene, N-desethyl etonitazene, N-desethyl isotonitazene, N-pyrrolidino metonitazene, and N-pyrrolidino protonitazene. The low-to-sub ng/mL blood concentrations observed in most cases underscore the drugs' high potencies. Taken together, by bridging pharmacology and case data, this study may aid to increase awareness and guide legislative and public health efforts.