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Pharmacological evaluation and forensic case series of N-pyrrolidino etonitazene (etonitazepyne), a newly emerging 2-benzylbenzimidazole ‘nitazene’ synthetic opioid

An Organ Toxicity and Mechanisms to this article was published on 21 April 2022


Novel synthetic opioids continue to emerge on recreational drug markets worldwide. In response to legislative bans on fentanyl analogues, non-fentanyl structural templates, such as 2-benzylbenzimidazoles (‘nitazenes’), are being exploited to create new μ-opioid receptor (MOR) agonists. Here, we pharmacologically characterize an emerging cyclic analogue of etonitazene, called N-pyrrolidino etonitazene (etonitazepyne), using in vitro and in vivo methods. A series of analytically confirmed fatalities is described to complement preclinical findings. Radioligand binding assays in rat brain tissue revealed that N-pyrrolidino etonitazene has high affinity for MOR (Ki = 4.09 nM) over δ-opioid (Ki = 959 nM) and κ-opioid (Ki = 980 nM) receptors. In a MOR-β-arrestin2 activation assay, N-pyrrolidino etonitazene displayed high potency (EC50 = 0.348 nM), similar to etonitazene (EC50 = 0.360 nM), and largely exceeding the potencies of fentanyl (EC50 = 14.9 nM) and morphine (EC50 = 290 nM). When administered s.c. to male Sprague Dawley rats, N-pyrrolidino etonitazene induced opioid-like antinociceptive, cataleptic, and thermic effects. Its potency in the hot plate test (ED50 = 0.0017 mg/kg) was tenfold and 2,000-fold greater than fentanyl (ED50 = 0.0209 mg/kg) and morphine (ED50 = 3.940 mg/kg), respectively. Twenty-one overdose fatalities associated with N-pyrrolidino etonitazene were found to contain low blood concentrations of the drug (median = 2.2 ng/mL), commonly in the context of polysubstance use. N-Pyrrolidino etonitazene was reported as a cause of death in at least two cases, demonstrating toxicity in humans. We demonstrate that N-pyrrolidino etonitazene is an extremely potent MOR agonist that is likely to present high risk to users. Continued vigilance is required to identify and characterize emergent 2-benzylbenzimidazoles, and other non-fentanyl opioids, as they appear in the marketplace.

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Dr. Donna Iula (Cayman Chemical) is kindly acknowledged for supplying the reference standard for N-pyrrolidino etonitazene to the Stove lab. The authors further acknowledge Dr. P. Blanckaert and Dr. E. Deconinck for gifting of one of the sourced powders. The authors would like to acknowledge medical examiner and forensic toxicology partners for their involvements with sample submission and inclusion of case histories: Dr. Tonya Mitchell of the Office of the Chief Medical Examiner, WV; Dr. Aaron Shapiro of the British Columbia Provincial Toxicology Centre, Canada; Dr. Robert Buchsbaum of the Florida 4th District Chief Medical Examiner, FL; Dr. Rebecca Asch-Kendrick of the Midwest Medical Examiner’s Office, MN; and Claire Mincher at the Knox County Medical Examiner’s Office, TN. The authors would like to acknowledge CFSRE scientists for their involvement with toxicology cases including Kelly Cunha and Lindsey Domonoski.


This work was supported by the Research Foundation-Flanders (FWO) [1S81522N to M.V., and G069419N to C.S.] and the Ghent University Special Research Fund (BOF) [01J15517 to C.S.]. The research program of Dr. Baumann is generously funded by the Intramural Research Program of the National Institute on Drug Abuse, NIH, grant number ZIA 000523–13. Funding for the case study portion of this work was received from the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice, award number 2020-DQ-BX-0007, “Real-Time Sample-Mining and Data-Mining Approaches for the Discovery of Novel Psychoactive Substances (NPS).” The opinions, findings, conclusions and/or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect those of the Department of Justice.

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Correspondence to Christophe P. Stove.

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Vandeputte, M.M., Krotulski, A.J., Walther, D. et al. Pharmacological evaluation and forensic case series of N-pyrrolidino etonitazene (etonitazepyne), a newly emerging 2-benzylbenzimidazole ‘nitazene’ synthetic opioid. Arch Toxicol 96, 1845–1863 (2022).

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  • New synthetic opioids (NSOs)
  • 2-benzylbenzimidazole ‘nitazene’ opioids
  • Forensic toxicology
  • μ- opioid receptor
  • In vitro and in vivo characterization
  • New psychoactive substances (NPS)