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 (ß-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.

In vitro µ-opioid receptor activation potential of U10 and ß-U10, positional isomers of the synthetic opioid naphthyl U-47700.

New synthetic opioids (NSOs) with diverse chemical structures continue to appear on recreational drug markets worldwide. U-type opioids have become one of the largest groups of non-fentanyl-related NSOs. Starting in 2020, a previously unreported U-compound coined "ß-U10" (2-naphthyl U-47700; N-[2-(dimethylamino)cyclohexyl]-N-methylnaphthalene-2-carboxamide) was identified in Australia and the United States. ß-U10 is a positional isomer of α-U10 (1-naphthyl U-47700), more commonly known as "U10." Here, the first comparative in vitro pharmacological characterization of naphthyl U-47700 (U10 and ß-U10), together with the structural analogue U-47700 and fentanyl, is reported. Application of a cell-based µ-opioid receptor (MOR) activation (ß-arrestin 2 recruitment) assay demonstrated ß-U10 (EC50 = 348 nM; Emax = 150% vs. hydromorphone) to be less potent than U-47700 (EC50 = 116 nM; Emax = 154%) and fentanyl (EC50 = 9.35 nM; Emax = 146%) but considerably more active than the α-isomer (EC50 value in the µM range). For the latter, maximum receptor activation could not be reached at 100 µM. The difference in MOR activation potential for U10 and ß-U10 stresses the importance of (analytical) differentiation between closely related analytes. The emergence of ß-U10 on the recreational drug market is an example of the continuing emergence of non-fentanyl-related NSOs and further emphasizes the need to closely monitor fluctuations in the drug supply.

Alkoxy chain length governs the potency of 2-benzylbenzimidazole 'nitazene' opioids associated with human overdose.

RATIONALE: Novel synthetic opioids (NSOs) are emerging in recreational drug markets worldwide. In particular, 2-benzylbenzimidazole 'nitazene' compounds are problematic NSOs associated with serious clinical consequences, including fatal respiratory depression. Evidence from in vitro studies shows that alkoxy chain length can influence the potency of nitazenes at the mu-opioid receptor (MOR). However, structure-activity relationships (SARs) of nitazenes for inducing opioid-like effects in animal models are not well understood compared to relevant opioids contributing to the ongoing opioid crisis (e.g., fentanyl). OBJECTIVES: Here, we examined the in vitro and in vivo effects of nitazene analogues with varying alkoxy chain lengths (i.e., metonitazene, etonitazene, isotonitazene, protonitazene, and butonitazene) as compared to reference opioids (i.e., morphine and fentanyl). METHODS AND RESULTS: Nitazene analogues displayed nanomolar affinities for MOR in rat brain membranes and picomolar potencies to activate MOR in transfected cells. All compounds induced opioid-like effects on locomotor activity, hot plate latency, and body temperature in male mice, and alkoxy chain length markedly influenced potency. Etonitazene, with an ethoxy chain, was the most potent analogue in MOR functional assays (EC50 = 30 pM, Emax = 103%) and across all in vivo endpoints (ED50 = 3-12 µg/kg). In vivo SARs revealed that ethoxy, isopropoxy, and propoxy chains engendered higher potencies than fentanyl, whereas methoxy and butoxy analogues were less potent. MOR functional potencies, but not MOR affinities, were positively correlated with in vivo potencies to induce opioid effects. CONCLUSIONS: Overall, our data show that certain nitazene NSOs are more potent than fentanyl as MOR agonists in mice, highlighting concerns regarding the high potential for overdose in humans who are exposed to these compounds.

Comparative neuropharmacology of structurally distinct non-fentanyl opioids that are appearing on recreational drug markets worldwide.

BACKGROUND: The emergence of novel synthetic opioids (NSOs) is contributing to the opioid overdose crisis. While fentanyl analogs have historically dominated the NSO market, a shift towards non-fentanyl compounds is now occurring. METHODS: Here, we examined the neuropharmacology of structurally distinct non-fentanyl NSOs, including U-47700, isotonitazene, brorphine, and N-desethyl isotonitazene, as compared to morphine and fentanyl. Compounds were tested in vitro using opioid receptor binding assays in rat brain tissue and by monitoring forskolin-stimulated cAMP accumulation in cells expressing the human mu-opioid receptor (MOR). Compounds were administered subcutaneously to male Sprague-Dawley rats, and hot plate antinociception, catalepsy score, and body temperature changes were measured. RESULTS: Receptor binding results revealed high MOR selectivity for all compounds, with MOR affinities comparable to those of morphine and fentanyl (i.e., nM). All drugs acted as full-efficacy MOR agonists in the cyclic AMP assay, but nitazene analogs had greater functional potencies (i.e., pM) compared to the other drugs (i.e., nM). When administered to rats, all compounds induced opioid-like antinociception, catalepsy, and body temperature changes, but nitazenes were the most potent. Similar to fentanyl, the nitazenes had faster onset and decline of in vivo effects when compared to morphine. In vivo potencies to induce antinociception and catalepsy (i.e., ED50s) correlated with in vitro functional potencies (i.e., EC50s) but not binding affinities (i.e., Kis) at MOR. CONCLUSIONS: Collectively, our findings indicate that non-fentanyl NSOs pose grave danger to those individuals who use opioids. Continued vigilance is needed to identify and characterize synthetic opioids as they emerge in clandestine drug markets.

Detection, chemical analysis, and pharmacological characterization of dipyanone and other new synthetic opioids related to prescription drugs.

The emergence of structurally diverse new synthetic opioids (NSOs) has caused the opioid crisis to spiral to new depths. Little information is available about the pharmacology of most novel opioids when they first emerge. Here, using a ß-arrestin 2 recruitment assay, we investigated the in vitro µ-opioid receptor (MOR) activation potential of dipyanone, desmethylmoramide, and acetoxymethylketobemidone (O-AMKD) - recent NSOs that are structurally related to the prescription opioids methadone and ketobemidone. Our findings indicate that dipyanone (EC50=39.9 nM; Emax=155% vs. hydromorphone) is about equally active as methadone (EC50=50.3 nM; Emax=152%), whereas desmethylmoramide (EC50=1335 nM; Emax=126%) is considerably less active. A close structural analogue of ketobemidone (EC50=134 nM; Emax=156%) and methylketobemidone (EC50=335 nM; Emax=117%), O-AMKD showed a lower potency (EC50=1262 nM) and efficacy (Emax=109%). Evaluation of the opioid substitution product buprenorphine and its metabolite norbuprenorphine confirmed the increased in vitro efficacy of the latter. In addition to in vitro characterization, this report details the first identification and full chemical analysis of dipyanone in a seized powder, as well as a postmortem toxicology case from the USA involving the drug. Dipyanone was quantified in blood (370 ng/mL), in which it was detected alongside other NSOs (e.g., 2-methyl AP-237) and novel benzodiazepines (e.g., flualprazolam). While dipyanone is currently not commonly encountered in forensic samples worldwide, its emergence is worrisome and representative of the dynamic NSO market. Graphical Abstract.

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

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-ß-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.

First identification, chemical analysis and pharmacological characterization of N-piperidinyl etonitazene (etonitazepipne), a recent addition to the 2-benzylbenzimidazole opioid subclass.

N-Piperidinyl etonitazene ('etonitazepipne') represents a recent addition to the rapidly expanding class of 2-benzylbenzimidazole 'nitazene' opioids. Following its first identification in an online-sourced powder and in biological samples from a patient seeking help for detoxification, this report details its in-depth chemical analysis and pharmacological characterization. Analysis of the powder via different techniques (LC-HRMS, GC-MS, UHPLC-DAD, FT-IR) led to the unequivocal identification of N-piperidinyl etonitazene. Furthermore, we report the first activity-based detection and analytical identification of N-piperidinyl etonitazene in authentic samples. LC-HRMS analysis revealed concentrations of 1.21 ng/mL in serum and 0.51 ng/mL in urine, whereas molecular networking enabled the tentative identification of various (potentially active) urinary metabolites. In addition, we determined that the extent of opioid activity present in the patient's serum was equivalent to the in vitro opioid activity exerted by 2.5-10 ng/mL fentanyl or 10-25 ng/mL hydromorphone in serum. Radioligand binding assays in rat brain tissue revealed that the drug binds with high affinity (Ki = 14.3 nM) to the µ-opioid receptor (MOR). Using a MOR-ß-arrestin2 activation assay, we found that N-piperidinyl etonitazene is highly potent (EC50 = 2.49 nM) and efficacious (Emax = 183% versus hydromorphone) in vitro. Pharmacodynamic evaluation in male Sprague Dawley rats showed that N-piperidinyl etonitazene induces opioid-like antinociceptive, cataleptic, and thermic effects, its potency in the hot plate assay (ED50 = 0.0205 mg/kg) being comparable to that of fentanyl (ED50 = 0.0209 mg/kg), and > 190 times higher than that of morphine (ED50 = 3.940 mg/kg). Taken together, our findings indicate that N-piperidinyl etonitazene is a potent opioid with the potential to cause harm in users.

Toxicological and pharmacological characterization of novel cinnamylpiperazine synthetic opioids in humans and in vitro including 2-methyl AP-237 and AP-238.

The recent scheduling actions of fentanyl-related substances in both the United States and China have sparked the emergence and proliferation of other generations of "legal" opioids that are structurally distinct from fentanyl, including the recently emerged class of cinnamylpiperazines. In contrast to fentanyl, which contains a piperidine core and a phenethyl moiety, the primary structural components of cinnamylpiperazines are the piperazine core and a cinnamyl moiety. This manuscript reports on the toxicological profile for antemortem and postmortem cases where a cinnamylpiperazine was detected. Samples were quantitatively confirmed using liquid chromatography tandem mass spectrometry. The cases were received between February 2020 and April 2021. Concentrations of 2-methyl AP-237 from four postmortem cases ranged from 820 to 5800 ng/mL, and concentrations of AP-238 from two postmortem cases were 87 and 120 ng/mL. µ-Opioid receptor (MOR) activation potential for 2-methyl AP-237, AP-237, para-methyl AP-237, and AP-238 were studied using a ßarr2 recruitment assay. Efficacies (Emax, relative to hydromorphone) and potencies (EC50) were derived and of the compounds tested AP-238 was the most potent compound in the panel with an EC50 of 248 nM. 2-Methyl AP-237 was found to be the most efficacious drug (Emax = 125%) of the tested cinnamylpiperazines; however, it had substantially less efficacy than fentanyl. The in vitro MOR activation potential of the studied cinnamylpiperazines was lower than that of fentanyl and other novel synthetic opioids (NSOs), in line with the relatively higher concentrations observed in postmortem toxicology samples-an important observational link between in vitro pharmacology and in vivo toxicology.

In vitro functional assays as a tool to study new synthetic opioids at the µ-opioid receptor: Potential, pitfalls and progress.

New psychoactive substances (NPS), formerly also referred to as "designer drugs", are often synthetic derivatives of existing psychoactive drugs, their diverse structures aiming at circumventing legislation and detection while their effects mimic those of traditional drugs of abuse. Of these, the group of new synthetic opioids (NSOs) has been one of the fastest growing NPS subclasses in the last couple of years, with over 70 new compounds detected in Europe since 2009. Apart from effects such as euphoria and analgesia, opioid use is associated with severe side effects such as constipation and respiratory depression. The µ-opioid receptor (MOR), a class A G protein-coupled receptor, is responsible for most of the therapeutic and adverse opioid effects. Insight into the pharmacology of opioids can aid the implementation of proactive harm reduction strategies, as well as the development of safer opioid analgesics. This review aims at assembling the available information on in vitro MOR agonism of the emerging class of new synthetic opioids, with a special focus on functional assays monitoring G protein and ß-arrestin pathways.

Characterization of recent non-fentanyl synthetic opioids via three different in vitro µ-opioid receptor activation assays.

New synthetic opioids (NSOs) are one of the fastest growing groups of new psychoactive substances. Amid this dynamic landscape, insight into the pharmacology of NSOs is important to estimate the harm potential of newly emerging drugs. In this work, we determined the µ-opioid receptor (MOR) affinity and activation potential of seven poorly characterized non-fentanyl NSOs (N-ethyl-U-47700, 3,4-difluoro-U-47700, U-47931E/bromadoline, 2,4-difluoro-U-48800, U-62066/spiradoline, 2F-viminol, ketobemidone) and a panel of nine reference opioids. MOR affinity was determined via [3H]-DAMGO binding in rat brain tissue homogenates, and was found to correlate well with different functional parameters. MOR activation potential was studied at different levels of receptor signaling using three distinct assays (NanoBiT® MOR-ß-arrestin2/mini-Gαi and AequoScreen®). The most active compounds were ketobemidone (EC50 32.8-528 nM; Emax 105-271%, relative to hydromorphone) and N-ethyl-U-47700 (EC50 241-767 nM; Emax 139-247%). The same opioids showed the strongest MOR affinity. As most of the other NSOs only weakly activated MOR in the three assays (EC50 values in the high nM-µM range), they likely do not pose a high overdose risk. 2F-viminol (EC50 2.2-4.5 µM; Emax 21.2-61.5%) and U-47931E/bromadoline (EC50 0.55-2.9 µM; Emax 52.8-85.9%) were partial agonists compared to hydromorphone, and maximum receptor activation was not reached for 2,4-difluoro-U-48800 (EC50 > 22 µM). We further highlight the importance of considering specific assay characteristics upon interpretation of potencies, efficacies and biased agonism. As absolute values may greatly differ between assays with varying experimental set-ups, a comparison of functional parameters to those of well-characterized reference agonists is considered the most informative.

Phenethyl-4-ANPP: A Marginally Active Byproduct Suggesting a Switch in Illicit Fentanyl Synthesis Routes.

Profiling of the illicit fentanyl supply is invaluable from surveillance and intelligence perspectives. An important strategy includes the study of chemical attribution signatures (e.g., trace amounts of synthesis precursors, impurities/byproducts in seized material and metabolites in biological samples). This information provides valuable insight into the employed synthesis routes at the heart of illicit fentanyl manufacture (previously mainly the so-called Janssen or Siegfried methods), allowing to track and ultimately regulate crucial precursors. This report focuses on phenethyl-4-anilino-N-phenethylpiperidine (phenethyl-4-ANPP), a formerly unknown compound that was identified for the first time in a fentanyl powder sample seized in April 2019, followed by its identification in a biological sample in December 2019. Between 2019-Q4 and 2020-Q3, phenethyl-4-ANPP was detected in 25/1,054 fentanyl cases in the USA. There are currently no reports on how this compound may have ended up in illicit drug preparations and whether its presence may have potential in vivo relevance. We propose three possible fentanyl synthesis routes that, when badly executed in a single reaction vessel, may involve the formation of phenethyl-4-ANPP. We hypothesize that the presence of the latter is the result of a shift in fentanyl synthesis routes in an attempt to circumvent restrictions on previously used precursors. Using a cell-based µ-opioid receptor recruitment assay, we show that the extent of MOR activation caused by 100 µM phenethyl-4-ANPP is comparable to that exerted by a roughly 100,000-fold lower concentration of fentanyl (0.001 µM or 0.336 ng/mL). Negligible in vitro opioid activity, combined with its low abundance in fentanyl preparations, most likely renders phenethyl-4-ANPP biologically irrelevant in vivo. However, as clandestine operations are constantly changing shape, monitoring of fentanyl attributions remains pivotal in our understanding and control of illicit fentanyl manufacture and supply.

The Rise and Fall of Isotonitazene and Brorphine: Two Recent Stars in the Synthetic Opioid Firmament.

Synthetic opioids constitute one of the fastest growing groups of new psychoactive substances (NPS) worldwide. With fentanyl analogues being increasingly controlled via class-wide scheduling, many non-fentanyl related opioids are now emerging on the recreational opioid market, rendering the landscape highly complex and dynamic. While new compounds are entering the supply in rapid and unpredictable manners, some recent patterns have become apparent. Many of these newly emerging opioids are being pirated from early patent literature and/or research papers, synthesized and sold online through various channels. Burdened by the identification of every newly emerging drug, many toxicology labs struggle to keep up. Moreover, by the time a "new" drug is controlled via legislative measures, illicit drug markets will have already adapted and diversified as manufacturers work to avoid the restricted product(s). Hence, the typical life-cycle of an NPS opioid is generally short (less than 6 months to one year), with only a few drugs escalating to significant numbers of detections. In this review, we summarize the key events in the emergence, rise, and subsequent decline of two non-fentanyl opioids - isotonitazene and brorphine. These two opioids sequentially dominated the NPS opioid market in 2019 and 2020. Both isotonitazene and brorphine remained in circulation for over a year, each contributing to hundreds of deaths and adverse events. By detailing the life-cycles of these opioids from their earliest synthesis as described in scientific literature to their subsequent rise and fall on recreational markets, this review illustrates the new characteristic life-cycle of synthetic opioids in the 'post-fentanyl-analogue' era.

Systematic evaluation of a panel of 30 synthetic cannabinoid receptor agonists structurally related to MMB-4en-PICA, MDMB-4en-PINACA, ADB-4en-PINACA, and MMB-4CN-BUTINACA using a combination of binding and different CB1 receptor activation assays. Part III: The G protein pathway and critical comparison of different assays.

The present work is the last of a three-part study investigating a panel of 30 systematically designed synthetic cannabinoid receptor agonists (SCRAs) including features such as the 4-pentenyl tail and varying head groups including amides and esters of l-valine (MMB, AB), l-tert-leucine (ADB), and l-phenylalanine (APP), as well as adamantyl (A) and cumyl moieties (CUMYL). Here, we evaluated these SCRAs for their capacity to activate the human cannabinoid receptor 1 (CB1 ) via indirect measurement of G protein recruitment. Furthermore, we comparatively evaluated the results obtained from three in vitro assays, based on the recruitment of ß-arrestin 2 (ßarr2 assay) or Gαi protein (mini-Gαi assay), or binding of [35 S]-GTPγS. The observed efficacies (Emax ) varied depending on the conducted assay. Statistical analysis suggests that the population means of the relative intrinsic activity (RAi ) significantly differ for the [35 S]-GTPγS assay and the other two assays, but the population means of the ßarr2 and mini-Gαi assays were not statistically different. Our data suggest that differences observed between the ßarr2 and mini-Gαi assays are the best predictor for 'biased agonism' towards ßarr or G protein recruitment in our study. SCRAs carrying an ADB or MPP moiety as a head group tended to produce elevated Emax values in the ßarr2 assay, which might result in a tendency of these compounds to cause pronounced tolerance in users-a hypothesis that should be evaluated further by future studies. In general, a comparison of efficacies derived from different assays is difficult and should only be conducted very cautiously.

Synthesis, Chemical Characterization, and µ-Opioid Receptor Activity Assessment of the Emerging Group of "Nitazene" 2-Benzylbenzimidazole Synthetic Opioids.

Several 2-benzylbenzimidazole opioids (also referred to as "nitazenes") recently emerged on the illicit market. The most frequently encountered member, 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. In this study, ten nitazenes and four metabolites were synthesized, analytically characterized via four different techniques, and pharmacologically evaluated using two cell-based ß-arrestin2/mini-Gi recruitment assays monitoring µ-opioid receptor (MOR) activation. On the basis of absorption spectra and retention times, high-performance liquid chromatography coupled to diode-array detection (HPLC-DAD) allowed differentiation between most analogues. Time-of-flight mass spectrometry (LC-QTOF-MS) identified a fragment with m/z 100.11 for 12/14 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 very high potency of the N-desethylisotonitazene metabolite, rivaling the potency of etonitazene and exceeding that of isotonitazene itself. Supported by its identification in fatalities, this likely has in vivo consequences. These results improve our understanding of this emerging group of opioids by laying out an analytical framework for their detection, as well as providing important new insights into their MOR activation potential.

First Report on Brorphine: The Next Opioid on the Deadly New Psychoactive Substance Horizon?

New psychoactive substances continue to appear on the drug market. Until recently, new synthetic 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 help for 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 characterization 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 brorphine was determined by a cell-based µ-opioid receptor activation assay, resulting in an EC50 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 and a hydroxy-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.

In vitro functional characterization of a panel of non-fentanyl opioid new psychoactive substances.

The landscape of new psychoactive substances (NPS) is constantly evolving, with new compounds entering the illicit drug market at a continuous pace. Of these, opioid NPS form a threat given their high potency and prevalence. Whereas previously, the use of fentanyl and fentanyl derivatives was the main point of attention, legislations have reacted accordingly, which may have been a driving force towards the (ab)use of alternative µ-opioid receptor (MOR) agonists. In contrast to fentanyl (analogues), details on these novel non-fentanyl opioid NPS are scarce. We investigated the biological activity of a panel of 11 'alternative', newly emerging MOR agonists (2-methyl-AP-237, AP-237, bromadol, brorphine, butorphanol, isotonitazene, mitragynine, 7-OH-mitragynine, MT-45, piperidylthiambutene, and tianeptine) using two closely related in vitro MOR activation bio-assays, monitoring either G protein (mini-Gi), or ß-arrestin2 (ßarr2) recruitment. Activity profiles were obtained for all tested compounds, with values for potency (EC50) ranging from 1.89 nM (bromadol) to > 3 µM (AP-237 and tianeptine). Bromadol, brorphine, isotonitazene, piperidylthiambutene, and tianeptine had the highest efficacy (Emax) values, exceeding that of the reference compound hydromorphone ≥ 1.3-fold (ßarr2 assay) and > 2.6-fold (mini-Gi assay). Information on the recruitment of two distinct signaling molecules additionally enabled evaluation of biased agonism; none of the evaluated opioids being significantly biased. Taken together, this study is the first to systematically investigate the in vitro biological activity of a diverse panel of emerging non-fentanyl opioid NPS at MOR. Given the known danger of (fatal) intoxications with many opioid NPS, it is important to continuously monitor and characterize newly emerging compounds.