Synthesis, chemical characterization, and µ-opioid receptor activity assessment of the emerging group of nitazene new synthetic opioids

Several benzimidazole opioids (commonly known as nitazenes) have recently started to emerge on the illicit market. The most frequently encountered member, the highly potent isotonitazene, has been identified in multiple fatalities since its appearance in 2019. Although recent scheduling efforts targeted isotonitazene, many other analogues remain unregulated. Being structurally unrelated to fentanyl, little is known about the harm potential of these compounds. This study describes the synthesis, analytical characterization, and µ-opioid receptor (MOR) activity of nine nitazenes and three metabolites. Chemical characterization was performed by liquid chromatography coupled to diode array detection (LC-DAD), gas chromatography-mass spectrometry (GC-MS), and time-of-flight mass spectrometry (LC-QTOF-MS). The MOR potency and efficacy of the nitazenes was determined by means of two cell-based β-arrestin2/mini-Gi recruitment assays. Based on absorption spectra and retention times, HPLC-DAD allowed differentiation between most analogues. LC-QTOF-MS identified a fragment with m/z 100.11 for 11/12 compounds, which could serve as a basis for MS-based nitazene screening. MOR activity determination confirmed that nitazenes are generally highly active, with potencies and efficacies of several analogues exceeding that of fentanyl. Particularly relevant is the unexpected (extremely) high potency of the N-desethyl-isotonitazene metabolite, rivalling the potency of etonitazene and exceeding that of isotonitazene itself. Supported by its identification in fatalities, this likely has in vivo consequences. The increasing emergence of nitazenes illustrates the continuously evolving nature of the illicit drug market. Nitazenes are potent MOR agonists. As their use may pose an imminent threat to any user, intensive monitoring and increased awareness are of critical importance.