Received: 29 August 2019 Revised: 12 November 2019 Accepted: 13 November 2019 DOI: 10.1002/dta.2738 RESEARCH ARTICLE Report on a novel emerging class of highly potent benzimidazole NPS opioids: Chemical and in vitro functional characterization of isotonitazene Peter Blanckaert1† | Annelies Cannaert2† | Katleen Van Uytfanghe2 | Fabian Hulpia3 | Eric Deconinck4 | Serge Van Calenbergh3 | Christophe Stove2 1Scientific Direction Epidemiology and Public Health, Section Lifestyle and Chronic Diseases, Abstract Belgian Early Warning System Drugs This paper reports on the identification and full chemical characterization of (BEWSD), Sciensano, Brussels, Belgium isotonitazene (N,N-diethyl-2-[5-nitro-2-({4-[(propan-2-yl)oxy]phenyl}methyl)-1H-ben- 2Laboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical zimidazol-1-yl]ethan-1-amine), a potent NPS opioid and the first member of the Sciences, Ghent University, Ghent, Belgium benzimidazole class of compounds to be available on online markets. Interestingly, 3Laboratory for Medicinal Chemistry, Department of Pharmaceutics, Faculty of this compound was sold under the name etonitazene, a structural analog. Identifica- Pharmaceutical Sciences, Ghent University, tion of isotonitazene was performed by gas chromatography mass spectrometry Ghent, Belgium (GC–MS) and liquid chromatography time-of-flight mass spectrometry (LC-QTOF- 4Scientific Direction Chemical and Physical Health Risks, Service of Medicines and Health MS), the latter identifying an exact-mass m/z value of 411.2398. All chromatographic Products, Sciensano, Brussels, Belgium data indicated the presence of a single, highly pure compound. Confirmation of the 1 13 Correspondence specific benzimidazole regio-isomer was performed using H and C NMR spectros- copy, after which the chemical characterization was finalized by recording Fourier- Christophe Stove, Laboratory of Toxicology, Department of Bioanalysis, Faculty of transform (FT-IR) spectra. A live cell-based reporter assay to assess the in vitro bio- Pharmaceutical Sciences, Ghent University, logical activity at the μ-opioid receptor (MOR) revealed that isotonitazene has a high Ottergemsesteenweg 460, 9000, Ghent, Belgium. potency (EC50 of 11.1 nM) and efficacy (Emax 180% of that of hydromorphone), thus Email: [email protected] confirming that this substance is a strong opioid. Isotonitazene has not been previ- Funding information ously detected, either in powder form, or in biological fluids. The high potency and Bijzonder Onderzoeksfonds, Grant/Award efficacy of isotonitazene, combined with the fact that this compound was being sold Numbers: 01J15517, 01N00814, PDO026-18; Research Foundation-Flanders, Grant/Award undiluted, represents an imminent danger to anyone aiming to use this powder. Number: 12Y9520N; Hercules Foundation, Grant/Award Number: AUGE/17/22 KEYWORDS new psychoactive substances, synthetic opioids, isotonitazene, NPS, NPS opioids 1 | INTRODUCTION market for a relatively long time. The first synthetic opioids appearing for sale online included AH-7921 and MT-45 in 2012 and 2013, For about a decade large and increasing numbers of new psychoactive respectively.1-3 From around 2014 increasing numbers of novel fenta- substances (NPS) with similar effects to classic illicit drugs have nyl analogs started to appear for sale, both in clear web online mar- started to appear in Europe, escaping legislation by using chemical kets as well as on darknet markets. Also fentanyl derivatives already structures that are similar to, yet differ from, those of known illicit used in human or veterinary medicine were increasingly being sold drugs. Well-known examples include synthetic cannabinoids and illegally, including alfentanil, sufentanil, remifentanil, and carfentanil.4,5 cathinones. Substances with opioid activity remained absent from the The arrival of these fentanyl analogs was accompanied by an increas- ing and alarming number of deaths, with the USA especially being hit † 6-9 Contributed equally. hard by this opioid epidemic. While the death toll in Europe Drug Test Anal. 2020;1–9. wileyonlinelibrary.com/journal/dta © 2019 John Wiley & Sons, Ltd. 1 2 BLANCKAERT ET AL. remained relatively limited, compared with the USA, also here, an 2 | MATERIALS AND METHODS increasing number of fatalities linked to the use of synthetic opioids has been observed.4,10,11 The sample was obtained during routine online monitoring of drug Because of the relatively simple synthesis, in addition to excellent markets, performed continuously in the framework of the functioning online precursor availability, the large majority of NPS opioids appe- of the Belgian Early Warning System Drugs (BEWSD), located at aring until the end of 2018 were derivatives of fentanyl (marked in Sciensano, previously known as the Scientific Institute of Public blue and with horizontal stripes in Figure 1).12 From 2018 onwards, Health. these numbers dropped and only one novel fentanyl analog was iden- tified in 2019. This strong reduction was most probably a result of the introduction of new legislation in China in 2018.13 Similarly, the num- 2.1 | Materials ber of newly detected U-compounds (U-47700 is the best known example10,14) increased until 2018, and started to decrease from then All reagents used during the analyses were at least of HPLC grade. For on. In contrast, from the end of 2018 onwards, there has been a NMR analysis, deuterated dimethyl sulfoxide (DMSO-d6, 99.8%) was prominent increase in the general “others” category (examples include purchased from Eurisotop (Saint-Aubin, FR). Hydromorphone was 2F-viminol, 2-Me-AP-237, and the molecule discussed in this paper, purchased as hydromorphone HCl from Fagron (Nazareth, Belgium). isotonitazene) (Figure 1), suggesting the resilience of Asian suppliers Fentanyl and isotonitazene (1 mg) were obtained as a free base from after the recent tightening of domestic and international regulations LGC Chemicals (Wesel, Germany) and Cayman Chemicals (Ann Arbor, of fentanyl-type opioids. Michigan, US), respectively. Dulbecco's modified Eagle's medium In this paper we report on the identification and full characteri- (DMEM; GlutaMAX™), Opti-MEM® I reduced serum medium, zation of a novel NPS opioid sourced online: isotonitazene (N,N- penicillin–streptomycin (5.000 U/mL), and amphotericin B (250 μg/mL) diethyl-2-[5-nitro-2-({4-[(propan-2-yl)oxy]phenyl}methyl)-1H-ben- were purchased from Thermo Fisher Scientific (Pittsburg, PA, USA). zimidazol-1-yl]ethan-1-amine; chemical structure can be found in Fetal bovine serum (FBS) and poly-D-lysine were supplied by Sigma Figure 2), which is the first detected member of a new benzimid- Aldrich (Overijse, Belgium). The Nano-Glo® Live Cell reagent, which azole class of NPS opioids. This type of compound is not new – was used for the readout of the bioassay, was procured from Promega the synthesis of benzimidazole derivatives with analgesic activity (Madison, WI, USA). was first reported in 1957.15,16 Since then, other, simplified synthe- sis pathways have been described, including a relatively simple one-pot, three-component synthesis at high yield.17 Although sev- 2.2 | Sample preparation eral derivatives were patented,18-21 no clinically approved therapeu- tics have made it to the market. This benzimidazole class of The sample was obtained from an online supplier in June 2019 as a compounds (Figure 2) differs radically in structure from other white homogenous powder, sold as etonitazene. It was provided in a potent analgesics.22 The most potent compound in this class, with small sealed plastic bag (Supplementary Data, Figure S1), which itself an estimated reported potency of a hundred to a thousand times was again sealed inside an aluminum pouch. It was used as provided, that of morphine, is etonitazene.23,24 Interestingly, the compound after short-term storage in the freezer (range −20Cto−30C) for that was obtained online was wrongly sold under this name. In analysis. For all the chromatographic analyses and the determination addition to the full chemical characterization and identification, we of the biological activity, 5.44 mg of the powder was dissolved in also report here the in vitro functional characterization of this com- 0.544 mL of methanol as a stock solution. For NMR analysis, a sample pound, using a μ-opioid receptor activation assay. (5.1 mg) was dissolved in DMSO-d6 (~ 0.75 mL). FIGURE 1 NPS opioids formally notified to the European monitoring Centre for Drugs and Drug Addiction (EMCDDA) 2009 - June 2019 BLANCKAERT ET AL. 3 FIGURE 2 Chemical structures of analgesic nitrobenzimidazole derivatives (etonitazene, isotonitazene, and clonitazene), hydromorphone and fentanyl 2.3 | Instrumentation started at 80C for 1 min, was ramped at 20C/min to 200C, then raised by 4C/min to 260C and by 30C/min to 300C, which 2.3.1 | Liquid chromatography time-of-flight mass was held for 8 min more. The transfer line temperature and ion spectrometry (LC-QTOF-MS) source temperature were set at 300 and 230C, respectively. The MS quadrupole temperature was set at 150C and an ionization Chromatographic separation was accomplished with an Agilent energy of 70 eV was used. The mass spectrometer operated in 1290 Infinity LC system and a Phenomenex Kinetex C18-column SCAN-mode, scanning the range of 50–700 Da. (2.6 μm, 3 × 50 mm), maintained at 30C. The high resolution mass spectrometry (HRMS) system was a 5600+ QTOF from Sciex, with an electrospray ionization (ESI) source and Analyst TF 1.7.1 2.3.3 | High-performance liquid chromatography software, from the same provider. The LC-HRMS analysis was diode array detector (HPLC-DAD) performed using the same settings to those described,25 obtaining a TOF-MS full scan combined with a data dependent acquisition of Reversed-phase separation of the extract was performed on a product ion spectra. Ten μL of a 1/10 000 dilution of the stock LaChrom HPLC system from Merck-Hitachi (Tokyo, Japan), using a solution in 0.05/50/25/25 formic acid/water/methanol/acetonitrile Merck Purospher® Star RP-8 endcapped column (5 μm, 125 mm × containing 2.5 mM ammonium formate was injected (resulting in 4.6 mm) fitted with a Merck Purospher® Star RP-8 endcapped an absolute amount of 10 ng).