In vitro cannabinoid receptor activity, metabolism, and detection in seized samples of CH-PIATA, a new indole-3-acetamide synthetic cannabinoid.

The rapidly evolving synthetic cannabinoid receptor agonist (SCRA) market poses significant challenges for forensic scientists. Since the enactment of a generic ban in China, a variety of new compounds have emerged capable of evading the legislation by carrying new structural features. One recent example of a SCRA with new linker and head moieties is CH-PIATA (CH-PIACA, CHX-PIATA, CHX-PIACA). CH-PIATA bears an additional methylene spacer in the linker moiety between the indole core and the traditional carbonyl component of the linker. This study describes detections in 2022 of this new SCRA in the United States, Belgium, and Scottish prisons. CH-PIATA was detected once in a seized powder by Belgian customs and 12 times in Scottish prisons in infused papers or resin. The metabolites of CH-PIATA were investigated via in vitro human liver microsome (HLM) incubations and eight metabolites were identified, dominated by oxidative biotransformations. A blood sample from the United States was confirmed to contain a mixture of SCRAs including CH-PIATA via presence of the parent and at least five of the metabolites identified from HLM incubations. Furthermore, this paper evaluates the intrinsic in vitro cannabinoid 1 and 2 (CB1 and CB2) receptor activation potential of CH-PIATA reference material and the powder seized by Belgian customs by means of ß-arrestin 2 recruitment assays. Both the reference and the seized powder showed a weak activity at both CB receptors with signs of antagonism found. Based on these results, the expected harm potential of this newly emerging substance remains limited.

Detection in seized samples, analytical characterization, and in vitro metabolism of the newly emerged 5-bromo-indazole-3-carboxamide synthetic cannabinoid receptor agonists.

Synthetic cannabinoid receptor agonists (SCRAs) are a diverse class of new psychoactive substances (NPS) and new structural scaffolds have emerged on the recreational drug market since the enactment of Chinese SCRA analog controls in 2021. This study reports the first SCRAs to be detected with a bromide at the 5 position (5'Br) on the phenyl ring of the indazole core and without a tail moiety. ADB-5'Br-INACA (ADMB-5'Br-INACA) and MDMB-5'Br-INACA were detected in seized samples from Scottish prisons, Belgian customs, and US forensic casework. The brominated analog with a tail moiety, ADB-5'Br-BUTINACA (ADMB-5'Br-BUTINACA), was also detected in Scottish prisons and US forensic casework. The metabolites of these compounds and the predicted compound MDMB-5'Br-BUTINACA were identified through incubation with primary human hepatocytes to aid in their toxicological identification. The bromide on the indazole remains intact on metabolites, allowing these compounds to be easily distinguished in toxicological samples from their non-brominated analogs. Glucuronidation was more common for tail-less analogs than their butyl tail-containing counterparts. Forensic toxicologists are advised to update their analytical methods with the characteristic ions for these compounds, as well as their anticipated urinary markers: amide hydrolysis and monoOH at tert-butyl metabolites (after ß-glucuronidase treatment) for ADB-5'Br-INACA; monoOH at tert-butyl and amide hydrolysis metabolites for ADB-5'Br-BUTINACA; and ester hydrolysis metabolites with additional metabolites for MDMB-5'Br-INACA and MDMB-5'Br-BUTINACA. Toxicologists should remain vigilant to the emergence of new SCRAs with halogenation of the indazole core and tail-less analogs, which have already started to emerge.

In vitro cannabinoid activity profiling of generic ban-evading brominated synthetic cannabinoid receptor agonists and their analogs.

Following the enactment of a generic ban in China in 2021, the synthetic cannabinoid market has been evolving, now encompassing even wider structural diversity. Compounds carrying a brominated core such as ADB-5'Br-BUTINACA (ADMB-B-5Br-INACA) and tail-less analogs, such as ADB-5'Br-INACA (ADMB-5Br-INACA), MDMB-5'Br-INACA, and ADB-INACA (ADMB-INACA), have been detected since late 2021. This study investigated the cannabinoid receptor (CB) activation potential of synthesized (S)-enantiomers of these substances, as well as of two predicted analogs MDMB-5'Br-BUTINACA (MDMB-B-5Br-INACA) and ADB-5'F-BUTINACA (ADMB-B-5F-INACA), using CB1 and CB2 ß-arrestin 2 recruitment assays and a CB1 intracellular calcium release assay. Surprisingly, the tail-less (S)-ADB-5'Br-INACA and (S)-MDMB-5'Br-INACA retained CB activity, albeit with a decreased potency compared to their tailed counterparts (S)-ADB-5'Br-BUTINACA and (S)-MDMB-5'Br-BUTINACA, respectively, which were potent and efficacious CB1 agonists. Also, at CB2 , tail-less analogs showed a lower potency but increased efficacy. Removing the bromine substitution ((S)-ADB-INACA) resulted in a reduced activity at CB1 ; however, this effect was less prominent at CB2 . Looking at tailed analogs, replacing the bromine with a fluorine substitution ((S)-ADB-5'F-BUTINACA) resulted in an increased potency and efficacy at both receptors. Furthermore, as ADB-5'Br-INACA and MDMB-5'Br-INACA have been frequently detected together in Scottish prisons, this study also evaluated the CB1 receptor activation potential of different mixtures of their respective reference standards, showing no unexpected cannabimimetic effect of combining both substances. Lastly, two powders seized by Belgian Customs and confirmed to contain ADB-5'Br-INACA and MDMB-5'Br-INACA, respectively, were assessed for CB activity. Based on the comparison with their reference standards, varying degrees of purity were suspected.

Visible-Light Photoswitchable Benzimidazole Azo-Arenes as ß-Arrestin2-Biased Selective Cannabinoid 2 Receptor Agonists.

The cannabinoid 2 receptor (CB2 R) has high therapeutic potential for multiple pathogenic processes, such as neuroinflammation. Pathway-selective ligands are needed to overcome the lack of clinical success and to elucidate correlations between pathways and their respective therapeutic effects. Herein, we report the design and synthesis of a photoswitchable scaffold based on the privileged structure of benzimidazole and its application as a functionally selective CB2 R "efficacy-switch". Benzimidazole azo-arenes offer huge potential for the broad extension of photopharmacology to a wide range of optically addressable biological targets. We used this scaffold to develop compound 10 d, a "trans-on" agonist, which serves as a molecular probe to study the ß-arrestin2 (ßarr2) pathway at CB2 R. ßΑrr2 bias was observed in CB2 R internalization and ßarr2 recruitment, while no activation occurred when looking at Gα16 or mini-Gαi . Overall, compound 10 d is the first light-dependent functionally selective agonist to investigate the complex mechanisms of CB2 R-ßarr2 dependent endocytosis.

Dual-Acting Small Molecules: Subtype-Selective Cannabinoid Receptor 2 Agonist/Butyrylcholinesterase Inhibitor Hybrids Show Neuroprotection in an Alzheimer's Disease Mouse Model.

We present the synthesis and characterization of merged human butyrylcholinesterase (hBChE) inhibitor/cannabinoid receptor 2 (hCB2R) ligands for the treatment of neurodegeneration. In total, 15 benzimidazole carbamates were synthesized and tested for their inhibition of human cholinesterases, also with regard to their pseudoirreversible binding mode and affinity toward both cannabinoid receptors in radioligand binding studies. After evaluation in a calcium mobilization assay as well as a ß-arrestin 2 (ßarr2) recruitment assay, two compounds with balanced activities on both targets were tested for their immunomodulatory effect on microglia activation and regarding their pharmacokinetic properties and blood-brain barrier penetration. Compound 15d, containing a dimethyl carbamate motif, was further evaluated in vivo, showing prevention of Aß25-35-induced learning impairments in a pharmacological mouse model of Alzheimer's disease for both short- and long-term memory responses. Additional combination studies proved a synergic effect of BChE inhibition and CB2R activation in vivo.

Off-target activity of NBOMes and NBOMe analogs at the µ opioid receptor.

New psychoactive substances (NPS) are introduced on the illicit drug market at a rapid pace. Their molecular targets are often inadequately elucidated, which contributes to the delayed characterization of their pharmacological effects. Inspired by earlier findings, this study set out to investigate the µ opioid receptor (MOR) activation potential of a large set of psychedelics, substances which typically activate the serotonin (5-HT2A) receptor as their target receptor. We observed that some substances carrying the N-benzyl phenethylamine (NBOMe) structure activated MOR, as confirmed by both the NanoBiT® ßarr2 recruitment assay and the G protein-based AequoScreen® Ca2+ release assay. The use of two orthogonal systems proved beneficial as some aspecific, receptor independent effects were found for various analogs when using the Ca2+ release assay. The specific 'off-target' effects at MOR could be blocked by the opioid antagonist naloxone, suggesting that these NBOMes occupy the same common opioid binding pocket as conventional opioids. This was corroborated by molecular docking, which revealed the plausibility of multiple interactions of 25I-NBOMe with MOR, similar to those observed for opioids. Additionally, structure-activity relationship findings seen in vitro were rationalized in silico for two 25I-NBOMe isomers. Overall, as MOR activity of these psychedelics was only noticed at high concentrations, we consider it unlikely that for the tested compounds there will be a relevant opioid toxicity in vivo at physiologically relevant concentrations. However, small modifications to the original NBOMe structure may result in a panel of more efficacious and potent MOR agonists, potentially exhibiting a dual MOR/5-HT2A activation potential.

In vitro characterization of the pyrazole-carrying synthetic cannabinoid receptor agonist 5F-3,5-AB-PFUPPYCA and its structural analogs.

The synthetic cannabinoid receptor agonist (SCRA) market is undergoing important changes since the enactment of the 2021 class-wide generic SCRA ban in China, one of the most important source countries for new psychoactive substances (NPS). Recently, various compounds with new structural features, synthesized to bypass this legislation, have entered the recreational drug market. Certain monocyclic pyrazole-carrying "FUPPYCA" SCRAs have been sporadically detected since 2015 without gaining further popularity. However, as evidenced by their recent detection in Scottish prisons, 5F-3,5-AB-PFUPPYCA and 3,5-ADB-4en-PFUPPYCA have re-emerged, potentially triggered by the new legislative ban. The aim of this study was to characterize the in vitro intrinsic CB1 and CB2 receptor activation potential of 5F-3,5-AB-PFUPPYCA and 3,5-ADB-4en-PFUPPYCA, as well as 4 analogs (5F-3,5-ADB-PFUPPYCA, 3,5-AB-CHMFUPPYCA, 5,3-AB-CHMFUPPYCA and 5,3-ADB-4en-PFUPPYCA) using live cell ß-arrestin 2 recruitment assays. Most analogs were essentially inactive at either CB1 or CB2, with only 3,5-AB-CHMFUPPYCA, 5,3-AB-CHMFUPPYCA and 5,3-ADB-4en-PFUPPYCA showing a limited activation potential at CB1. Furthermore, the importance of the position of the tail structure was demonstrated, with 5,3 regioisomers being more active than their 3,5 analogs. Moreover, all compounds exhibited antagonistic behavior at both receptors, which may be associated with their structural resemblance to cannabinoid antagonists and inverse agonists. Although the 3,5 regioisomers of these "FUPPYCA" SCRAs circumvent the Chinese ban, it is unlikely that these SCRAs will pose a major threat to public health, given the lack of pronounced CB receptor activity.

Development of an Indole-Amide-Based Photoswitchable Cannabinoid Receptor Subtype 1 (CB1R) "Cis-On" Agonist.

Activation of the human cannabinoid receptor type 1 (hCB1R) with high spatiotemporal control is useful to study processes involved in different pathologies related to nociception, metabolic alterations, and neurological disorders. To synthesize new agonist ligands for hCB1R, we have designed different classes of photoswitchable molecules based on an indole core. The modifications made to the central core have allowed us to understand the molecular characteristics necessary to design an agonist with optimal pharmacological properties. Compound 27a shows high affinity for CB1R (Ki (cis-form) = 0.18 µM), with a marked difference in affinity with respect to its inactive "trans-off" form (CB1R Ki trans/cis ratio = 5.4). The novel compounds were evaluated by radioligand binding studies, receptor internalization, sensor receptor activation (GRABeCB2.0), Western blots for analysis of ERK1/2 activation, NanoBiT ßarr2 recruitment, and calcium mobilization assays, respectively. The data show that the novel agonist 27a is a candidate for studying the optical modulation of cannabinoid receptors (CBRs), serving as a new molecular tool for investigating the involvement of hCB1R in disorders associated with the endocannabinoid system.

Cannabinoid receptor activation potential of the next generation, generic ban evading OXIZID synthetic cannabinoid receptor agonists.

In recent years, several nations have implemented various measures to control the surge of new synthetic cannabinoid receptor agonists (SCRAs) entering the recreational drug market. In July 2021, China put into effect a new generic legislation, banning SCRAs containing one of seven general core scaffolds. However, this has driven manufacturers towards the synthesis of SCRAs with alternative core structures, exemplified by the recent emergence of "OXIZID SCRAs." Here, using in vitro ß-arrestin2 recruitment assays, we report on the CB1 and CB2 potency and efficacy of five members of this new class of SCRAs: BZO-HEXOXIZID, BZO-POXIZID, 5-fluoro BZO-POXIZID, BZO-4en-POXIZID, and BZO-CHMOXIZID. All compounds behaved as full agonists at CB1 and partial agonists at CB2 . Potencies ranged from 84.6 to 721 nM at CB1 and 2.21 to 25.9 nM at CB2 . Shortening the n-hexyl tail to a pentyl tail enhanced activity at both receptors. Fluorination of this pentyl analog did not yield a higher receptor activation potential, whereas an unsaturated tail resulted in decreased potency and efficacy at CB1 . The cyclohexyl methyl analog BZO-CHMOXIZID was the most potent compound at both receptors, with EC50 values of 84.6 and 2.21 nM at CB1 and CB2 , respectively. Evaluation of the activity of a seized powder containing BZO-4en-POXIZID suggested a high purity, in line with high-performance liquid chromatography coupled to diode-array detection (HPLC-DAD), gas chromatography coupled to mass spectrometry (GC-MS), liquid chromatography coupled to time-of-flight mass spectrometry (LC-QTOF-MS), and Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) analysis. Furthermore, all tested compounds showed a preference for CB2 , except for BZO-POXIZID. Overall, these findings inform public health officials, law enforcement agencies, and clinicians on these newly emerging SCRAs.

A new cannabinoid receptor 1 selective agonist evading the 2021 "China ban": ADB-FUBIATA.

Following the class-wide ban of synthetic cannabinoid receptor agonists (SCRAs) in China, SCRAs carrying new core and linker structures, aimed at circumventing the recent Chinese generic legislation, have appeared on the recreational drug market. A very recent example is (S)-2-(2-(1-(4-fluorobenzyl)-1H-indol-3-yl)acetamido)-3,3-dimethylbutanamide (ADB-FUBIATA), which is structurally closely related to the potent SCRA ADB-FUBICA, but carries an additional methylene in the linker region of the molecule. ADB-FUBIATA has recently been identified in seized materials in China, Russia, the United States, and also Belgium; however, its pharmacological characteristics were unknown. The aim of this study was to evaluate the intrinsic cannabinoid receptor (hCB1 and hCB2 ) activation potential of this previously unknown substance via two distinct yet similar in vitro ß-arrestin2 recruitment assays, based on the NanoLuc Binary Technology®. At CB1 , a potency of 635 nM (EC50 ) was found, with an efficacy (Emax ) of 141% relative to the reference compound CP55,940. On the other hand, ADB-FUBIATA had almost no activity at CB2 , indicative of a clear CB1 selectivity. Interestingly, this activation pattern differs markedly from that observed for ADB-FUBICA, which was previously found to be potent and efficacious at both cannabinoid receptors. Additionally, the bioassays were applied to a seized powder containing ADB-FUBIATA, as analytically confirmed by high-performance liquid chromatography coupled to diode-array detection (HLPC-DAD), gas chromatography coupled to mass spectrometry (GC-MS), liquid chromatography couple to time-of-flight mass spectrometry (LC-QTOF-MS), Fourier transform infrared spectroscopy (FTIR), and nuclear magnetic resonance (NMR). The EC50 and Emax values obtained for this powder were very similar to those of the ADB-FUBIATA analytical standard, suggesting a high purity of the powder, although analytical techniques did reveal that the sample was not entirely pure.

Structure-activity relationships of valine, tert-leucine, and phenylalanine amino acid-derived synthetic cannabinoid receptor agonists related to ADB-BUTINACA, APP-BUTINACA, and ADB-P7AICA.

Synthetic cannabinoid receptor agonists (SCRAs) remain one the most prevalent classes of new psychoactive substances (NPS) worldwide, and examples are generally poorly characterised at the time of first detection. We have synthesised a systematic library of amino acid-derived indole-, indazole-, and 7-azaindole-3-carboxamides related to recently detected drugs ADB-BUTINACA, APP-BUTINACA and ADB-P7AICA, and characterised these ligands for in vitro binding and agonist activity at cannabinoid receptor subtypes 1 and 2 (CB1 and CB2), and in vivo cannabimimetic activity. All compounds showed high affinity for CB1 (K i 0.299-538 nM) and most at CB2 (K i = 0.912-2190 nM), and most functioned as high efficacy agonists of CB1 and CB2 in a fluorescence-based membrane potential assay and a ßarr2 recruitment assay (NanoBiT®), with some compounds being partial agonists in the NanoBiT® assay. Key structure-activity relationships (SARs) were identified for CB1/CB2 binding and CB1/CB2 functional activities; (1) for a given core, affinities and potencies for tert-leucinamides (ADB-) > valinamides (AB-) ≫ phenylalaninamides (APP-); (2) for a given amino acid side-chain, affinities and potencies for indazoles > indoles ≫ 7-azaindoles. Radiobiotelemetric evaluation of ADB-BUTINACA, APP-BUTINACA and ADB-P7AICA in mice demonstrated that ADB-BUTINACA and ADB-P7AICA were cannabimimetic at 0.1 mg kg-1 and 10 mg kg-1 doses, respectively, as measured by pronounced decreases in core body temperature. APP-BUTINACA failed to elicit any hypothermic response up to the maximally tested 10 mg kg-1 dose, yielding an in vivo potency ranking of ADB-BUTINACA > ADB-P7AICA > APP-BUTINACA.

NNL-3: A Synthetic Intermediate or a New Class of Hydroxybenzotriazole Esters with Cannabinoid Receptor Activity?

Synthetic cannabinoid receptor agonists (SCRAs) remain a prolific class of new psychoactive substances (NPS) and continue to expand rapidly. Despite the recent identification of hydroxybenzotriazole (HOBt) containing SCRAs in synthetic cannabis samples, there is currently no information regarding the pharmacological profile of these NPS with respect to human CB1 and CB2 receptors. In the current study, a series consisting of seven HOBt indole-, indazole-, and 7-azaindole-carboxylates bearing a range of N-alkyl substituents were synthesized and pharmacologically evaluated. Competitive binding assays at CB1 and CB2 demonstrated that all analogues except a 2-methyl-substituted derivative had low affinity for CB1 (Ki = 3.80-43.7 µM) and CB2 (Ki = 2.75-18.2 µM). A fluorometric functional assay revealed that 2-methylindole- and indole-derived HOBt carboxylates were potent and efficacious agonists of CB1 (EC50 = 12.0 and 63.7 nM; Emax = 118 and 120%) and CB2 (EC50 = 10.9 and 321 nM; Emax = 91 and 126%). All other analogues incorporating indazole and 7-azaindole cores and bearing a range of N1-substituents showed relatively low potency for CB1 and CB2. Additionally, a reporter assay monitoring ß-arrestin 2 (ßarr2) recruitment to the receptor revealed that the 2-methylindole example was the most potent and efficacious at CB1 (EC50 = 131 nM; Emax = 724%) and the most potent at CB2 (EC50 = 38.2 nM; Emax = 51%). As with the membrane potential assay, the indazole and other indole HOBt carboxylates were considerably less potent at both receptors, and analogues comprising a 7-azaindole core showed little activity. Taken together, these data suggest that NNL-3 demonstrates little CB1 receptor activity and is unlikely to be psychoactive in humans. NNL-3 is likely an unintended SCRA manufacturing byproduct. However, the synthesis of NNL-3 analogues proved simple and general, and some of these showed potent cannabimetic profiles in vitro, indicating that HOBt esters of this type may represent an emerging class of SCRA NPS.