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.

Evaluating signaling bias for synthetic cannabinoid receptor agonists at the cannabinoid CB2 receptor.

The rapid structural evolution and emergence of novel synthetic cannabinoid receptor agonists (SCRAs) in the recreational market remains a key public health concern. Despite representing one of the largest classes of new psychoactive substances, pharmacological data on new SCRAs is limited, particularly at the cannabinoid CB2 receptor (CB2 ). Hence, the current study aimed to characterize the molecular pharmacology of a structurally diverse panel of SCRAs at CB2 , including 4-cyano MPP-BUT7AICA, 4F-MDMB-BUTINACA, AMB-FUBINACA, JWH-018, MDMB-4en-PINACA, and XLR-11. The activity of SCRAs was assessed in a battery of in vitro assays in CB2 -expressing HEK 293 cells: G protein activation (Gαi3 and GαoB ), phosphorylation of ERK1/2, and ß-arrestin 1/2 translocation. The activity profiles of the ligands were further evaluated using the operational analysis to identify ligand bias. All SCRAs activated the CB2 signaling pathways in a concentration-dependent manner, although with varying potencies and efficacies. Despite the detection of numerous instances of statistically significant bias, compound activities generally appeared only subtly distinct in comparison with the reference ligand, CP55940. In contrast, the phytocannabinoid THC exhibited an activity profile distinct from the SCRAs; most notably in the translocation of ß-arrestins. These findings demonstrate that CB2 is able to accommodate a structurally diverse array of SCRAs to generate canonical agonist activity. Further research is required to elucidate whether the activation of CB2 contributes to the toxicity of these compounds.

Subtle Structural Modification of a Synthetic Cannabinoid Receptor Agonist Drastically Increases its Efficacy at the CB1 Receptor.

The emergence of synthetic cannabinoid receptor agonists (SCRAs) as illicit psychoactive substances has posed considerable public health risks, including fatalities. Many SCRAs exhibit much higher efficacy and potency compared with the phytocannabinoid Δ9-tetrahydrocannabinol (THC) at the cannabinoid receptor 1 (CB1R), leading to dramatic differences in signaling levels that can be toxic. In this study, we investigated the structure-activity relationships of aminoalkylindole SCRAs at CB1Rs, focusing on 5F-pentylindoles containing an amide linker attached to different head moieties. Using in vitro bioluminescence resonance energy transfer assays, we identified a few SCRAs exhibiting significantly higher efficacy in engaging the Gi protein and recruiting ß-arrestin than the reference CB1R full agonist CP55940. Importantly, the extra methyl group on the head moiety of 5F-MDMB-PICA, as compared to that of 5F-MMB-PICA, led to a large increase in efficacy and potency at the CB1R. This pharmacological observation was supported by the functional effects of these SCRAs on glutamate field potentials recorded in hippocampal slices. Molecular modeling and simulations of the CB1R models bound with both of the SCRAs revealed critical structural determinants contributing to the higher efficacy of 5F-MDMB-PICA and how these subtle differences propagated to the receptor-G protein interface. Thus, we find that apparently minor structural changes in the head moiety of SCRAs can cause major changes in efficacy. Our results highlight the need for close monitoring of the structural modifications of newly emerging SCRAs and their potential for toxic drug responses in humans.

Interaction of Synthetic Cannabinoid Receptor Agonists with Cannabinoid Receptor I: Insights into Activation Molecular Mechanism.

Synthetic cannabinoid receptor agonists (SCRAs) have become a wide group of new psychoactive substances since the 2010s. For the last few years, the X-ray structures of the complexes of cannabinoid receptor I (CB1) with SCRAs as well as the complexes of CB1 with its antagonist have been published. Based on those data, SCRA-CB1 interactions are analyzed in detail, using molecular modeling and molecular dynamics simulations. The molecular mechanism of the conformational transformation of the transmembrane domain of CB1 caused by its interaction with SCRA is studied. These conformational changes allosterically modulate the CB1-Gi complex, providing activation of the Gi protein. Based on the X-ray-determined structures of the CB1-ligand complexes, a stable apo conformation of inactive CB1 with a relatively low potential barrier of receptor activation was modeled. For that model, molecular dynamic simulations of SCRA binding to CB1 led to the active state of CB1, which allowed us to explore the key features of this activation and the molecular mechanism of the receptor's structural transformation. The simulated CB1 activation is in accordance with the previously published experimental data for the activation at protein mutations or structural changes of ligands. The key feature of the suggested activation mechanism is the determination of the stiff core of the CB1 transmembrane domain and the statement that the entire conformational transformation of the receptor to the active state is caused by a shift of alpha helix TM7 relative to this core. The shift itself is caused by protein-ligand interactions. It was verified via steered molecular dynamics simulations of the X-ray-determined structures of the inactive receptor, which resulted in the active conformation of CB1 irrespective of the placement of agonist ligand in the receptor's active site.

"Spice"-related deaths in and around Munich, Germany: A retrospective look at the role of synthetic cannabinoid receptor agonists in our post-mortem cases over a seven-year period (2014-2020).

Synthetic cannabinoid receptor agonists (SCRAs, "Spice") are a diverse group of recreational drugs, with their structural and pharmacological variability still evolving. Forensic toxicologists often rely on previous reports to assess their role in intoxication cases. This work provides detailed information on the "Spice"-related fatalities around Munich, Germany, from 2014 to 2020. All cases underwent an autopsy. Pharmaceutical and illicit drugs were detected and quantified in post-mortem peripheral blood or liver by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Based on circumstantial evidence, only those cases for which a prior consumption was suspected underwent additional analyses for SCRAs and other new psychoactive substances in post-mortem blood, liver or antemortem specimens. Drug concentrations, pathological findings at autopsy and case histories were considered to assess and rank the SCRAs' involvement in each death. Concentration ranges for the individual substances in blood were defined and their distribution patterns over the investigated period were determined and correlated with their legal status and local police seizures. We identified 41 different SCRAs among 98 fatalities. 91.8% were male, at a median age of 36 years. SCRAs played a causative role in 51%, contributory role in 26%, and an insignificant role in 23% of cases. In correlation with local police seizures and legal status, 5F-ADB was the most prevalent in our cases, followed by 5F-MDMB-PICA and AB-CHMINACA. Cumyl-CBMICA and 5F-MDMB-P7AICA were among the least frequently detected SCRAs. "Spice"-related fatalities and SCRAs' causative role have significantly decreased among our cases since the German New Psychoactive Substances Act.

Novel Cannabinoid Receptor 2 (CB2) Low Lipophilicity Agonists Produce Distinct cAMP and Arrestin Signalling Kinetics without Bias.

Cannabinoid Receptor 2 (CB2) is a promising target for treating inflammatory diseases. We designed derivatives of 3-carbamoyl-2-pyridone and 1,8-naphthyridin-2(1H)-one-3-carboxamide CB2-selective agonists with reduced lipophilicity. The new compounds were measured for their affinity (radioligand binding) and ability to elicit cyclic adenosine monophosphate (cAMP) signalling and ß-arrestin-2 translocation with temporal resolution (BRET-based biosensors). For the 3-carbamoyl-2-pyridone derivatives, we found that modifying the previously reported compound UOSS77 (also known as S-777469) by appending a PEG2-alcohol via a 3-carbomylcyclohexyl carboxamide (UOSS75) lowered lipophilicity, and preserved binding affinity and signalling profile. The 1,8-naphthyridin-2(1H)-one-3-carboxamide UOMM18, containing a cis configuration at the 3-carboxamide cyclohexyl and with an alcohol on the 4-position of the cyclohexyl, had lower lipophilicity but similar CB2 affinity and biological activity to previously reported compounds of this class. Relative to CP55,940, the new compounds acted as partial agonists and did not exhibit signalling bias. Interestingly, while all compounds shared similar temporal trajectories for maximal efficacy, differing temporal trajectories for potency were observed. Consequently, when applied at sub-maximal concentrations, CP55,940 tended to elicit sustained (cAMP) or increasing (arrestin) responses, whereas responses to the new compounds tended to be transient (cAMP) or sustained (arrestin). In future studies, the compounds characterised here may be useful in elucidating the consequences of differential temporal signalling profiles on CB2-mediated physiological responses.

Structure-Based Virtual Screening and Molecular Dynamics Simulation Assessments of Depsidones as Possible Selective Cannabinoid Receptor Type 2 Agonists.

The discovery of natural drug metabolites is a leading contributor to fulfilling the sustainable development goal of finding solutions to global health challenges. Depsidones are a class of polyketides that have been separated from lichens, fungi, sponges, and plants and possess various bioactivities, including cytotoxic, antimicrobial, antimalarial, antituberculosis, acetylcholinesterase and α-glucosidase inhibition, and anti-inflammatory effects. Endocannabinoid receptors (CB1 and CB2) are G-protein-coupled receptors (GPCRs), and their activation mediates many physiological processes. CB1 is the dominant subtype in the central nervous system, while CB2 is mainly expressed in the immune system. The two receptors exhibit high heterogeneity, making developing selective ligands a great challenge. Attempts to develop CB2 selective agonists for treating inflammatory diseases and neuropathic pain have not been successful due to the high homology of the binding sites of the CB receptors. In this work, 235 depsidones from various sources were investigated for the possibility of identifying CB2-selective agonists by performing multiple docking studies, including induced fit docking and Prime/molecular mechanics-generalized Born surface area (MM-GBSA) calculations to predict the binding mode and free energy. Simplicildone J (10), lobaric acid (110), mollicellin Q (101), garcinisidone E (215), mollicellin P (100), paucinervin Q (149), and boremexin C (161) had the highest binding scores (-12.134 kcal/mol, -11.944 kcal/mol, -11.479 kcal/mol, -11.394 kcal/mol, -11.322 kcal/mol, -11.305 kcal/mol, and -11.254 kcal/mol, respectively) when screened against the CB2 receptor (PDB ID: 6KPF). The molecular dynamic simulation was performed on the compounds with the highest binding scores. The computational outcomes show that garcinisidone E (215) and paucinervin Q (149) could be substantial candidates for CB2 receptor activation and warrant further in vivo and in vitro investigations.

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.

Photochemical Fingerprinting Is a Sensitive Probe for the Detection of Synthetic Cannabinoid Receptor Agonists; toward Robust Point-of-Care Detection.

With synthetic cannabinoid receptor agonist (SCRA) use still prevalent across Europe and structurally advanced generations emerging, it is imperative that drug detection methods advance in parallel. SCRAs are a chemically diverse and evolving group, which makes rapid detection challenging. We have previously shown that fluorescence spectral fingerprinting (FSF) has the potential to provide rapid assessment of SCRA presence directly from street material with minimal processing and in saliva. Enhancing the sensitivity and discriminatory ability of this approach has high potential to accelerate the delivery of a point-of-care technology that can be used confidently by a range of stakeholders, from medical to prison staff. We demonstrate that a range of structurally distinct SCRAs are photochemically active and give rise to distinct FSFs after irradiation. To explore this in detail, we have synthesized a model series of compounds which mimic specific structural features of AM-694. Our data show that FSFs are sensitive to chemically conservative changes, with evidence that this relates to shifts in the electronic structure and cross-conjugation. Crucially, we find that the photochemical degradation rate is sensitive to individual structures and gives rise to a specific major product, the mechanism and identification of which we elucidate through density-functional theory (DFT) and time-dependent DFT. We test the potential of our hybrid "photochemical fingerprinting" approach to discriminate SCRAs by demonstrating SCRA detection from a simulated smoking apparatus in saliva. Our study shows the potential of tracking photochemical reactivity via FSFs for enhanced discrimination of SCRAs, with successful integration into a portable device.

Synthetic cannabinoid receptor agonists are monoamine oxidase-A selective inhibitors.

Synthetic cannabinoid receptor agonists (SCRAs) are one of the fastest growing classes of recreational drugs. Despite their growth in use, their vast chemical diversity and rapidly changing landscape of structures make understanding their effects challenging. In particular, the side effects for SCRA use are extremely diverse, but notably include severe outcomes such as cardiac arrest. These side effects appear at odds with the main putative mode of action, as full agonists of cannabinoid receptors. We have hypothesized that SCRAs may act as MAO inhibitors, owing to their structural similarity to known monoamine oxidase inhibitors (MAOI's) as well as matching clinical outcomes (hypertensive crisis) of 'monoaminergic toxicity' for users of MAOIs and some SCRA use. We have studied the potential for SCRA-mediated inhibition of MAO-A and MAO-B via a range of SCRAs used commonly in the UK, as well as structural analogues to prove the atomistic determinants of inhibition. By combining in silico and experimental kinetic studies we demonstrate that SCRAs are MAO-A-specific inhibitors and their affinity can vary significantly between SCRAs, most notably affected by the nature of the SCRA 'head' group. Our data allow us to posit a putative mechanism of inhibition. Crucially our data demonstrate that SCRA activity is not limited to just cannabinoid receptor agonism and that alternative interactions might account for some of the diversity of the observed side effects and that these effects can be SCRA-specific.

Pharmacology, prevalence in Germany, and analytical data of cyclobutylmethyl- and norbornylmethyl-type synthetic cannabinoid receptor agonists.

Synthetic cannabinoid receptor agonists (SCRAs) are distributed on the drug market to produce THC-like effects while evading routine drug testing and legislation. The cyclobutylmethyl (CBM) and norbornylmethyl (NBM) side chain specifically circumvented the German legislation and led to the emergence of exploratory SCRAs in 2019-2021. The NBM SCRAs were detected post-amendment of the new psychoactive substances act in 2020, which scheduled all CBM SCRAs. All six SCRAs are full agonists at the cannabinoid receptor 1 compared with Δ9 -tetrahydrocannabinol and CP-55,940. The CBM SCRAs showed binding affinities of Ki : 29.4-0.65 nm and potencies of EC50 : 483-40.1 nm (CBMICA << CBMINACA < CBMeGaClone). The norbornyl derivatives exhibited high affinities (Ki : 1.87-0.25 nm), with indazole being the most affine. Functional activity data confirmed that the indazole derivative tends to be the most potent of all three NBM SCRAs (EC50 : 169-1.78 nm). The sterically demanding NBM side chain increased the affinity and activity of almost all core structures. Future studies should be conducted on similarly voluminous side chain moieties. The 'life cycle' of all SCRAs on the drug market was less than a year. Notably, Cumyl-CBMICA was the most prevalent while also having the weakest cannabimimetic properties. Quantification of Cumyl-CBMICA during peak consumption in late 2019 and early 2020 revealed an increase in the concentration on the herbal material, which, together with forum entries and blog posts, corroborates the low in vitro cannabimimetic properties. Seizure prevalence data indicate that almost all SCRAs continue to be identified in 2022, potentially due to remaining stocks.

Comprehensive Characterization of a Systematic Library of Alkyl and Alicyclic Synthetic Cannabinoids Related to CUMYL-PICA, CUMYL-BUTICA, CUMYL-CBMICA, and CUMYL-PINACA.

Over 200 synthetic cannabinoid receptor agonists (SCRAs) have been identified as new psychoactive substances. Effective monitoring and characterization of SCRAs are hindered by the rapid pace of structural evolution. Ahead of possible appearance on the illicit drug market, new SCRAs were synthesized to complete a systematic library of cumyl-indole- (e.g., CUMYL-CPrMICA, CUMYL-CPMICA) and cumyl-indazole-carboxamides (e.g., CUMYL-CPrMINACA, CUMYL-CPMINACA), encompassing butyl, pentyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, and cyclohexylmethyl tails. Comprehensive pharmacological characterization was performed with three assay formats, monitoring the recruitment of either wild-type or C-terminally truncated (ßarr2d366) ß-arrestin2 to the activated cannabinoid 1 receptor (CB1) or monitoring Gßγ-mediated membrane hyperpolarization. Altered compound characterization was observed when comparing derived potency (EC50) and efficacy (Emax) values from both assays monitoring the same or a different signaling event, whereas ranges and ranking orders were similar. Structure-activity relationships (SAR) were assessed in threefold, resulting in the identification of the pendant tail as a critical pharmacophore, with the optimal chain length for CB1 activation approximating an n-pentyl (e.g., cyclopentylmethyl or cyclohexylmethyl tail). The activity of the SCRAs encompassing cyclic tails decreased with decreasing number of carbons forming the cyclic moiety, with CUMYL-CPrMICA showing the least CB1 activity in all assay formats. The SARs were rationalized via molecular docking, demonstrating the importance of the optimal steric contribution of the hydrophobic tail. While SAR conclusions remained largely unchanged, the differential compound characterization by both similar and different assay designs emphasizes the importance of detailing specific assay characteristics to allow adequate interpretation of potencies and efficacies.

N-[1,3-Dialkyl(aryl)-2-oxoimidazolidin-4-ylidene]-aryl(alkyl)sulphonamides as Novel Selective Human Cannabinoid Type 2 Receptor (hCB2R) Ligands; Insights into the Mechanism of Receptor Activation/Deactivation.

Cannabinoid type 1 (hCB1) and type 2 (hCB2) receptors are pleiotropic and crucial targets whose signaling contributes to physiological homeostasis and its restoration after injury. Being predominantly expressed in peripheral tissues, hCB2R represents a safer therapeutic target than hCB1R, which is highly expressed in the brain, where it regulates processes related to cognition, memory, and motor control. The development of hCB2R ligands represents a therapeutic opportunity for treating diseases such as pain, inflammation and cancer. Identifying new selective scaffolds for cannabinoids and determining the structural determinants responsible for agonism and antagonism are priorities in drug design. In this work, a series of N-[1,3-dialkyl(aryl)-2-oxoimidazolidin-4-ylidene]-aryl(alkyl)sulfonamides is designed and synthesized and their affinity for human hCB1R and hCB2R is determined. Starting with a scaffold selected from the NIH Psychoactive Drug Screening Program Repository, through a combination of molecular modeling and structure-activity relationship studies, we were able to identify the chemical features leading to finely tuned hCB2R selectivity. In addition, an in silico model capable of predicting the functional activity of hCB2R ligands was proposed and validated. The proposed receptor activation/deactivation model enabled the identification of four pure hCB2R-selective agonists that can be used as a starting point for the development of more potent ligands.

Elucidation of partial activation of cannabinoid receptor type 2 and identification of potential partial agonists: Molecular dynamics simulation and structure-based virtual screening.

Cannabinoid receptor type 2 (CB2R) is a member of the class A G protein-coupled receptor (GPCRs) family and a component of the endocannabinoid system that is modulated by the psychoactive chemical from Cannabis sativa, partial agonist Δ9-tetrahydrocannabinol (Δ9-THC). Selective activation of CB2R allows for the treatment of inflammatory and immune-related conditions without the psychotropic effects of CB1R. While CB2R-selective agonists are available, CB2R partial agonists are scarce. Hence, the pharmacological difference between CB2R full agonists and partial agonists remains to be deciphered, prompting the search for novel partial agonists. Here, using an induced-fit docking approach, we built a partial agonist Δ9-THC bound CB2R system from the inactive CB2R structure (PDB ID: 5ZTY) and performed microsecond molecular dynamics (MD) simulations. The simulations reveal an upward shift of the "toggle switch" W6.48(258) and minor outward movement of the transmembrane helix 6 (TM6). Dynamic network model identifies a possible communication path between the ligand and the toggle switch" W6.48(258). Furthermore, to identify potential CB2R partial agonists, we conducted structure-based virtual screening of ZINC15 "Druglike" library containing 17,900742 compounds against 3 conformations derived from MD simulation of CB2R complexed with partial agonist Δ9-THC using Glide virtual screening protocol comprising various filters with increasing accuracy. Nine diverse compounds predicted to have high MM-GBSA binding energy scores and good ADMET properties (including high gastrointestinal absorption and low toxicity) are proposed as potential CB2R partial agonists.

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.

Synthesis and In Vitro Characterization of Selective Cannabinoid CB2 Receptor Agonists: Biological Evaluation against Neuroblastoma Cancer Cells.

1,8-naphthyridine-3-carboxamide structures were previously identified as a promising scaffold from which to obtain CB2R agonists with anticancer and anti-inflammatory activity. This work describes the synthesis and functional characterization of new 1,8-naphthyridin-2(1H)-one-3-carboxamides with high affinity and selectivity for CB2R. The new compounds were able to pharmacologically modulate the cAMP response without modulating CB2R-dependent ß-arrestin2 recruitment. These structures were also evaluated for their anti-cancer activity against SH-SY5Y and SK-N-BE cells. They were able to reduce the cell viability of both neuroblastoma cancer cell lines with micromolar potency (IC50 of FG158a = 11.8 µM and FG160a = 13.2 µM in SH-SY5Y cells) by a CB2R-mediated mechanism. Finally, in SH-SY5Y cells one of the newly synthesized compounds, FG158a, was able to modulate ERK1/2 expression by a CB2R-mediated effect, thus suggesting that this signaling pathway might be involved in its potential anti-cancer effect.

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.

Defining Steric Requirements at CB1 and CB2 Cannabinoid Receptors Using Synthetic Cannabinoid Receptor Agonists 5F-AB-PINACA, 5F-ADB-PINACA, PX-1, PX-2, NNL-1, and Their Analogues.

Synthetic cannabinoid receptor agonists (SCRAs) are a diverse class of new psychoactive substances (NPS). They commonly comprise N-alkylated indole, indazole, or 7-azaindole scaffolds with amide-linked pendant amino acid groups. To explore the contribution of the amino acid side chain to the cannabinoid pharmacology of SCRA NPS, a systematic library of side chain-modified SCRAs was prepared based on the recent detections of amino acid derivatives 17 (5F-AB-PINACA), 18 (5F-ADB-PINACA), 15 (PX-1), 19 (PX-2), and 20 (NNL-1). In vitro binding affinities and functional activities at cannabinoid type 1 and 2 receptors (CB1 and CB2, respectively) were determined for all the library members using radioligand competition experiments and a fluorescence-based membrane potential assay. Binding affinities and functional activities varied widely across compounds (Ki = 0.32 to >10 000 nM, EC50 = 0.24-1259 nM), with several clear structure-activity relationships (SARs) emerging. Affinity and potency at CB1 changed as a function of the heterocyclic core (indazole > indole > 7-azaindole) and the pendant amino acid side chain (tert-butyl > iso-propyl > iso-butyl > benzyl > ethyl > methyl > hydrogen). Ensemble docking at CB1 revealed a clear steric basis for observed SAR trends. Interestingly, although 15 (PX-1) and 19 (PX-2) have been detected in recreational drug markets, they failed to induce centrally CB1-mediated effects (e.g., hypothermia) in mice using radiobiotelemetry. Together, these data provide insights regarding structural contributions to the cannabimimetic profiles of 17 (5F-AB-PINACA), 18 (5F-ADB-PINACA), 15 (PX-1), 19 (PX-2), 20 (NNL-1), and other SCRA NPS.

Mechanistic origin of partial agonism of tetrahydrocannabinol for cannabinoid receptors.

Cannabinoid receptor 1 (CB1) is a therapeutically relevant drug target for controlling pain, obesity, and other central nervous system disorders. However, full agonists and antagonists of CB1 have been reported to cause serious side effects in patients. Therefore, partial agonists have emerged as a viable alternative as they can mitigate overstimulation and side effects. One of the key bottlenecks in the design of partial agonists, however, is the lack of understanding of the molecular mechanism of partial agonism itself. In this study, we examine two mechanistic hypotheses for the origin of partial agonism in cannabinoid receptors and predict the mechanistic basis of partial agonism exhibited by Δ9-Tetrahydrocannabinol (THC) against CB1. In particular, we inspect whether partial agonism emerges from the ability of THC to bind in both agonist and antagonist-binding poses or from its ability to only partially activate the receptor. We used extensive molecular dynamics simulations and Markov state modeling to capture the THC binding in both antagonist and agonist-binding poses in the CB1 receptor. Furthermore, we predict that binding of THC in the agonist-binding pose leads to rotation of toggle switch residues and causes partial outward movement of intracellular transmembrane helix 6 (TM6). Our simulations also suggest that the alkyl side chain of THC plays a crucial role in determining partial agonism by stabilizing the ligand in the agonist and antagonist-like poses within the pocket. Taken together, this study provides important insights into the mechanistic origin of the partial agonism of THC.

Fenchone Derivatives as a Novel Class of CB2 Selective Ligands: Design, Synthesis, X-ray Structure and Therapeutic Potential.

A series of novel cannabinoid-type derivatives were synthesized by the coupling of (1S,4R)-(+) and (1R,4S)-(-)-fenchones with various resorcinols/phenols. The fenchone-resorcinol derivatives were fluorinated using Selectfluor and demethylated using sodium ethanethiolate in dimethylformamide (DMF). The absolute configurations of four compounds were determined by X-ray single crystal diffraction. The fenchone-resorcinol analogs possessed high affinity and selectivity for the CB2 cannabinoid receptor. One of the analogues synthesized, 2-(2',6'-dimethoxy-4'-(2″-methyloctan-2″-yl)phenyl)-1,3,3-trimethylbicyclo[2.2.1]heptan-2-ol (1d), had a high affinity (Ki = 3.51 nM) and selectivity for the human CB2 receptor (hCB2). In the [35S]GTPγS binding assay, our lead compound was found to be a highly potent and efficacious hCB2 receptor agonist (EC50 = 2.59 nM, E(max) = 89.6%). Two of the fenchone derivatives were found to possess anti-inflammatory and analgesic properties. Molecular-modeling studies elucidated the binding interactions of 1d within the CB2 binding site.