Journal of Analytical Toxicology, 2020;44:937–946 doi:10.1093/jat/bkaa094 Advance Access Publication Date: 3 August 2020 Article Article First Report on Brorphine: The Next Opioid on the Deadly New Psychoactive Substance Downloaded from https://academic.oup.com/jat/article/44/9/937/5879253 by Ghent University user on 07 May 2021 Horizon? Nick Verougstraete1,2,#, Marthe M. Vandeputte1,#, Cathelijne Lyphout3, Annelies Cannaert1, Fabian Hulpia4, Serge Van Calenbergh4, Alain G. Verstraete2,5 and Christophe Stove1,* 1Laboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium; 2Department of Laboratory Medicine, Ghent University Hospital, Ghent, Belgium; 3Emergency Department, Ghent University Hospital, Ghent, Belgium; 4Laboratory for Medicinal Chemistry, Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium and 5Department of Diagnostic Sciences, Ghent University, Ghent, Belgium #These authors contributed equally to this work. *Author to whom correspondence should be addressed. Email: [email protected] Abstract New psychoactive substances continue to appear on the drug market. Until recently, new syn- thetic opioids, which are among the most dangerous new psychoactive substances, primarily encompassed analogs of the potent analgesic fentanyl. Lately, also other new synthetic opioids have increasingly started to surface. This is the first report on the identification and full chemical characterization of brorphine, a novel potent synthetic opioid with a piperidine benzimidazolone structure. A powder, identified as brorphine, was obtained from a patient seeking medical helpfor detoxification. Brorphine was also found in a serum sample of the patient. Liquid chromatography– high-resolution mass spectrometry (LC–HRMS) identified an exact mass of m/z 400.1020 and 402.1005 for the compound, corresponding to both bromine isotopes. Further chemical character- ization was performed by gas chromatography–mass spectrometry, liquid chromatography–diode array detection and Fourier-transform infrared spectroscopy analyses. Finally, the structure was confirmed by performing 1H-NMR and 13C-NMR spectroscopy. In vitro biological activity of bror- phine was determined by a cell-based µ-opioid receptor activation assay, resulting in an EC 50 of 30.9 nM (13.5 ng/mL) and an Emax of 209% relative to hydromorphone, confirming the high potency and efficacy of this compound. In a serum sample of the patient, brorphine andahydroxy- metabolite were found using the LC–HRMS screening method. The presence of opioid activity in the serum was also confirmed via the activity-based opioid screening assay. The occurrence of brorphine is yet another example of how the illicit drug market is continuously evolving in an attempt to escape international legislation. Its high potency poses a serious and imminent health threat for any user. Introduction illicit drugs and are sold as (allegedly) legal alternatives. By the end New psychoactive substances (NPS) intend to mimic the (pharma- of 2019, 731 NPS had been notified to the European Early Warning cological) effects of known prescription/controlled medications or System (1, 2) and 950 to the United Nations Office on Drugs © The Author(s) 2020. Published by Oxford University Press on behalf of Society of Forensic Toxicologists, Inc. All rights reserved. For permissions, please e-mail: [email protected] 937 938 Verougstraete et al. Downloaded from https://academic.oup.com/jat/article/44/9/937/5879253 by Ghent University user on 07 May 2021 Figure 1. Structural similarities between (A) fentanyl, (B) benzylfentanyl and (C) the molecule discussed in this report, brorphine (1-(1-(1-(4- bromophenyl)ethyl)piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazol-2-one). and Crime (UNODC) (3). Although the rate of appearance of para position were important for the high potency. Using two NPS detections in Europe seems to slow down, approximately 50 distinct assays, monitoring either G protein signaling or β-arrestin new substances are still reported annually. As NPS are typically coupling, brorphine was found to be a full agonist, showing relatively cheap, easily available over the internet and often very some bias toward G protein signaling. Although no in vivo data potent, many health problems are attributable to their use (1, 2). NPS are available for brorphine itself, intraperitoneal injection of sev- were involved in about 9.1% of the drug-related hospital emergency eral piperidine benzimidazolone derivatives demonstrated that this presentations in Europe during the period 2014–2017. Although class of compounds is capable of crossing the blood–brain barrier cathinones and synthetic cannabinoids were the most prevalent, new (17, 21). synthetic opioids have also made their entry in Europe (4). Brorphine has been mentioned on drug fora for a while and has A few years ago, new synthetic opioids primarily encompassed been reported to circulate in the American Midwest since the second analogs of the potent analgesic fentanyl, the latter having been asso- half of 2019 (22), although no formal reports on its identification ciated with tens of thousands of deaths in the United States during (either as a powder or in authentic cases) are available. At the time of the past few years (5–7). Whilst fentanyl, as well as its analogs, writing, we are not aware of any brorphine cases that were reported remains present on the illicit market, the introduction of legislation to the UNODC Early Warning Advisory or the European Monitor- scheduling these substances has resulted in a marked decline in the ing Centre for Drugs and Drug Addiction. Therefore, in this article, number of new “fentalogs” (8). Coinciding with the decrease in the we report on the identification and full chemical characterization of number of new fentanyl-related substances, other new synthetic opi- brorphine, sourced online and used by a patient querying admission oids have started to surface (9), including 2F-viminol, 2-Me-AP-237 for detoxification. In addition, besides the unequivocal identification and isotonitazene (10), the latter having been associated with several of brorphine in serum samples from this patient, its MOR agonistic hundreds of deaths in just a few months in the United States (11, 12). activity was demonstrated, both as a powder and in the authentic Brorphine [1-(1-(1-(4-bromophenyl)ethyl)piperidin-4-yl)-1, patient sample. 3-dihydro-2H-benzo[d]imidazol-2-one] is a piperidine benzimida- zolone derivative and could be considered a fentanyl analog Case History (Figure 1). However, it is not internationally scheduled and is even outside the scope of generic legislations aiming at covering The patient, who provided written informed consent, was a 24- fentanyl analogs (13, 14). Relatively little is known about this year-old male who presented with withdrawal symptoms to the novel NPS opioid. Already back in 1967, the opioid activity of emergency department, querying an admission for detoxification. piperidine benzimidazolones was reported by Janssen (15). The syn- His complaints consisted of generalized pain, situated mainly in thesis of a structural analog of brorphine, where p-bromophenyl the chest, abdomen and muscles. He had a normal blood pres- is substituted for an o-chlorophenyl group, was reported in the sure with a tachycardia of 114/minute, oxygen saturation of 98% ◦ 1990s, in an attempt to develop an opioid-receptor-like 1 antagonist. and a temperature of 37.3 C. He presented with confusion and Despite its high binding affinity, this compound lacked selectivity bradyphrenia, generalized weakness and cramping. On physical and antagonistic activity (16). Recently, the synthesis and in vitro examination, he was clammy and sweaty. A CT scan of the brain, pharmacological characterization of brorphine were reported by chest X-ray and ECG were all normal. Apart from a slightly ele- Kennedy et al. in the search for µ-opioid receptor (MOR) lig- vated gamma-glutamyltransferase of 111 U/L, there were no remark- ands biased toward G protein signaling (17). Biased agonism at able abnormalities in the blood lab results. Anamnesis revealed MOR has received quite some interest, given the reported—but cur- that he had a medical history of opioid misuse with the use of, rently heavily questioned—association of β-arrestin signaling with among others, the synthetic opioids 2-Me-AP-237 and intravenous adverse events like respiratory depression and G protein signaling O-acetylmethylketobemidone (a derivative of the synthetic opioid with the desired (analgesic) effects of opioids (18–20). Interestingly, ketobemidone). He had an episode of abstinence of one year, up in the report of Kennedy et al., brorphine was among the most to six months before presentation. One to two months before pre- potent compounds, with a potency exceeding that of morphine (17). sentation, he started using brorphine orally four times a day, as Both the N-1-phenylethyl substituent and the bromo group in well as etizolam orally (last use of etizolam was a few days before First Report on Brorphine 939 presentation). He reported that the use of brorphine resulted in a Patient samples pretty strong effect, similar to oxycodone or fentanyl, with a result- Two serum samples, taken approximately 60 hours apart, were ing “high” that lasted quite long, and the development of tolerance. available for (toxicological) analysis. The first sample was collected He also reported to suffer