Class Of New Psychoactive Substances Research Articles

Structure-Activity Relationships, Deuteration, and Fluorination of Synthetic Cannabinoid Receptor Agonists Related to AKB48, 5F-AKB-48, and AFUBIATA.

Synthetic cannabinoid receptor agonists (SCRAs) are a growing class of new psychoactive substances (NPS) commonly derived from an N-alkylated indole, indazole, or 7-azaindole scaffold. Diversification of this core (at the 3-position) with amide-linked pendant amino acid groups and modular N-alkylation (of the indole/indazole/7-azaindole core) ensures that novel SCRAs continue to enter the illicit drug market rapidly. In response to the large number of SCRAs that have been detected, pharmacological evaluation of this NPS class has become increasingly common. Adamantane-derived SCRAs have consistently appeared throughout the market since 2011, and as such, a systematic set of these derivatives was synthesized and pharmacologically evaluated. Deuterated and fluorinated adamantane derivatives were prepared to evaluate typical hydrogen bioisosteres, as well as evaluation of the newly detected AFUBIATA.

Synthesis and Functional Evaluation of Synthetic Cannabinoid Receptor Agonists Related to ADB-HEXINACA.

ADB-HEXINACA has been recently reported as a synthetic cannabinoid receptor agonist (SCRA), one of the largest classes of new psychoactive substances (NPSs). This compound marks the entry of the n-hexyl tail group into the SCRA landscape, which has continued in the market with recent, newly detected SCRAs. As such, a proactive characterization campaign was undertaken, including the synthesis, characterization, and pharmacological evaluation of ADB-HEXINACA and a library of 41 closely related analogues. Two in vitro functional assays were employed to assess activity at CB1 and CB2 cannabinoid receptors, measuring Gβγ-coupled agonism through a fluorescence-based membrane potential assay (MPA) and β-arrestin 2 (βarr2) recruitment via a live cell-based nanoluciferase complementation reporter assay. ADB-HEXINACA was a potent and efficacious CB1 agonist (CB1 MPA pEC50 = 7.87 ± 0.12 M; Emax = 124 ± 5%; βarr2 pEC50 = 8.27 ± 0.14 M; Emax = 793 ± 42.5), as were most compounds assessed. Isolation of the heterocyclic core and alkyl tails allowed for the comprehensive characterization of structure-activity relationships in this compound class, which were rationalized in silico via induced fit docking experiments. Overall, most compounds assessed are possibly emerging NPSs.

Pharmacological evaluation of new generation OXIZID synthetic cannabinoid receptor agonists

Synthetic cannabinoid receptor agonists (SCRAs) remain one the largest classes of new psychoactive substances, and are increasingly associated with severe adverse effects and death compared to the phytocannabinoid Δ9-tetrahydrocannabinol (THC). In the attempt to circumvent the rapid emergence of novel SCRAs, several nations have implemented ‘generic’ legislations, or ‘class-wide’ bans based on common structural scaffolds. However, this has only encouraged the incorporation of new chemical entities, including distinct core and linker structures, for which there is a dearth of pharmacological data. The current study evaluated five emergent OXIZID SCRAs for affinity and functional activity at the cannabinoid CB1 receptor (CB1) in HEK 293 cells, as well as pharmacological equivalence with THC in drug discrimination in mice. All OXIZID compounds behaved as agonists in Gαi protein activation and β-arrestin 2 translocation assays, possessing low micromolar affinity at CB1. All ligands also substituted for THC in drug discrimination, where potencies broadly correlated with in vitro activity, with the methylcyclohexane analogue BZO-CHMOXIZID being the most potent. Notably, MDA-19 (BZO-HEXOXIZID) exhibited partial efficacy in vitro, generating an activity profile most similar to that of THC, and partial substitution in vivo. Overall, the examined OXIZIDs were comparatively less potent and efficacious than previous generations of SCRAs. Further toxicological data will elucidate whether the moderate cannabimimetic activity for this series of SCRAs will translate to severe adverse health effects as seen with previous generations of SCRAs.

A comprehensive LC-MS/MS method for simultaneous analysis of 65 synthetic cannabinoids in human hair samples and application to forensic investigations

Synthetic cannabinoids (SCs) represent a diverse class of new psychoactive substances characterized by extensive substance variety and severe abuse implications. The current situation of synthetic cannabinoid abuse in China is getting worse, with an increasing number of SC variants emerging. Therefore, it is imperative to improve synthetic cannabinoid detecting methods to align with the prevalent abuse situation in the region. In this study, a reliable and validated liquid chromatography-tandem mass spectrometry method was developed for the qualitative and quantitative analysis of 65 SC analogues in human hair samples. The validation results demonstrated satisfactory linearity (r ≥ 0.99) within the range of 25–2500 pg/mg for each SC analogue. The method exhibited limits of detection ranging from 10 to 15 pg/mg and limits of quantification ranging from 25 to 40 pg/mg. The relative standard deviations of intra-day precision and inter-day precision were below 15 %. Furthermore, negligible matrix effects were observed, with recovery rates ranging from 85.70 % to 119.43 %. Analysis of abuser demographics revealed that the primary group engaged in SC analogue abuse consisted of adolescents, predominantly males, accounting for 79.5 % of cases. Among the suspected individuals, ADB-BUTINACA and MDMB-4en-PINACA were the most frequently detected substances. The present study develops a highly sensitive analytical method and provides a comprehensive overview of the prevalence of SC abuse in the eastern region of China.

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 β-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.

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 invitro assays in CB2 -expressing HEK 293 cells: G protein activation (Gαi3 and GαoB ), phosphorylation of ERK1/2, and β-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 β-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.

Development of a Dispersive Liquid-Liquid Microextraction for Synthetic Cathinones in Biological Fluids Based on Principles of Green Analytical Toxicology.

The present work describes a practical application of Green Analytical Toxicology (GAT) during the development of an eco-friendly dispersive liquid-liquid microextraction (DLLME) avoiding the use of highly toxic chlorinated solvents that are commonly used in this type of the technique. The purpose was to further consolidate GAT guidelines during method development. Thus, a full method optimization using a multivariate statistical approach and validation were performed. To that end, synthetic cathinones (SCs), one of the major classes of new psychoactive substances, were the target analytes due to their relevance and chemical diversity. Furthermore, whole blood and urine samples were the matrices of choice due to their clinical relevance. The sample preparation step prior to DLLME consisted of protein precipitation of whole blood samples, while urine specimens were centrifuged and diluted with ultrapure water. Then, borate buffer, sodium chloride and ethyl acetate:acetonitrile were added and vortexed. Finally, vials were centrifuged and the organic layer was transferred to autosampler vials, evaporated to dryness and resuspended with mobile phase prior to injection into the ultra-high performance liquid chromatography-tandem mass spectrometry system. Once optimized, the proposed DLLME was fully validated: 0.2 and 1 ng/mL as the limit of detection and 1 and 10 ng/mL as the limit of quantitation for urine and blood samples, respectively. The linear range was established as 1-100 and 10-1,000 ng/mL for urine and blood samples, respectively (r2 > 0.99), while the bias and precision were within acceptable limits (≥80%). The matrix effect was of 1.9-260.2% and -12.3-139.6%; while the recovery was of 27.4-60.0% and 13.0-55.2%; the process efficiency ranged from 45.0% to 192.0% and 17.9% to 58.4% for whole blood and urine, respectively. Finally, the method was applied to real case samples as proof of applicability. Thus, a simple, cheap and fast eco-friendly technique to analyze SCs in two biological specimens was described.

Neurochemical and Cardiovascular Effects of 4-Chloro Ring-Substituted Synthetic Cathinones in Rats.

Synthetic cathinones are a class of new psychoactive substances that display psychomotor stimulant properties, and novel cathinone analogs continue to emerge in illicit drug markets worldwide. The aim of the present study was to characterize the pharmacology of 4-chloro ring-substituted cathinones that are appearing in illicit drug markets compared with the effects of 4-methylmethcathinone (mephedrone). Synaptosomes were prepared from rat caudate for dopamine transporter (DAT) assays or from whole brain minus caudate and cerebellum for norepinephrine transporter (NET) and serotonin transporter (SERT) assays. Findings from transporter uptake inhibition and release assays showed that mephedrone and 4-chloromethcathinone (4-CMC) function as substrates at DAT, NET, and SERT, with similar potency at all three transporters. In contrast, 4-chloro-α-pyrrolidinopropiophenone (4-CαPPP) was an uptake inhibitor at DAT and NET, with similar potency at each site, but had little activity at SERT. 4-Chloroethcathinone (4-CEC) was a low-potency uptake inhibitor at DAT and NET but a substrate at SERT. In rats implanted with telemetry transmitters, mephedrone and 4-CMC increased blood pressure, heart rate, and locomotor activity to a similar extent. 4-CEC and 4-CαPPP were less potent at increasing blood pressure and had modest stimulatory effects on heart rate and activity. 4-CMC also transiently decreased temperature at the highest dose tested. All three 4-chloro ring-substituted cathinones are biologically active, but only 4-CMC has potency comparable to mephedrone. Collectively, our findings suggest that 4-CMC and other 4-chloro cathinones may have abuse potential and adverse effects in humans that are analogous to those associated with mephedrone. SIGNIFICANCE STATEMENT: The 4-chloro ring-substituted cathinones all produced significant cardiovascular stimulation, with 4-chloromethcathinone (4-CMC) showing potency similar to mephedrone. All of the drugs are likely to be abused given their effects at the dopamine transporter, particularly 4-CMC.

Acute, chronic, and post-mortem toxicity: a review focused on three different classes of new psychoactive substances.

New psychoactive substances (NPS) are not controlled under the Single Convention on Narcotic Drugs of 1961 or the 1971 Convention, but they may pose a public health threat. Knowledge of the main properties and toxicological effects of these substances is lacking. According to the current Drugs Law (Law n. 11.343/2006), the Brazilian Surveillance Agency issues directives for forbidden substances in Brazil, and structural classes of synthetic cannabinoids, cathinones, and phenylethylamines are considered illicit drugs. Considering that data on these controlled substances are scattered, the main objective of this work was to collect and organize data to generate relevant information on the toxicological properties of NPS. We carried out a literature review collecting information on the acute, chronic, and post-mortem toxicity of these classes of NSP. We searched info in five scientific databases considering works from 2017 to 2021 and performed a statistical evaluation of the data. Results have shown a general lack of studies in this field given that many NPS have not had their toxicity evaluated. We observed a significant difference in the volume of data concerning acute and chronic/post-mortem toxicity. Moreover, studies on the adverse effects of polydrug use are scarce. More in-depth information about the main threats involving NPS use are needed.

Rapid Detection of Synthetic Cannabinoid Receptor Agonists Impregnated into Paper by Raman Spectroscopy

Abstract English The last decade has witnessed the emergence of new psychoactive substances that are analogues of classical drugs of abuse in order to escape the regulations surrounding the latter drugs. These drugs were of both herbal and synthetic origin and were advertised initially as ‘legal highs’; thus, they were perceived as safe by users. Hence, upon their emergence, they were not controlled by the Misuse of Drugs Act 1971, which contributed to their popularity and increased sales online and within street markets. In 2016, the Psychoactive Substance Act introduced a blanket ban on all new psychoactive substances except for caffeine, alcohol, and nicotine. This in turn, contributed to the change in the sale of new psychoactive substances products that have been sold as concealed in different matrices, including herbal products, papers, fabrics, and textiles. Concealing drugs in paper has been very popular, especially since the drug product is of lightweight and can be sent via postal services. However, new psychoactive concealed in papers are toxic not only to the users; but also, to the person handling them (i.e. mail employees). One of the classes of new psychoactive substances that have been commonly concealed in papers and that have been linked to toxicity and hospitalization cases is synthetic cannabinoids. Therefore, there is a need to identify new psychoactive substances concealed in papers non-destructively and rapidly to prevent toxicity linked to them. Handheld Raman spectroscopy offers this advantage as it is of lightweight and carries the sample to the matrix. Therefore, this work used handheld Raman spectroscopy for identifying synthetic cannabinoids concealed in papers using Raman spectroscopy combined with machine learning analytics. Synthetic cannabinoid and paper samples were measured non-destructively using a handheld Raman spectrometer equipped with a 1064 nm laser wavelength. Spectral data was exported into Matlab 2020b where machine learning analytics including identification and prediction was. The results showed that Raman spectroscopy could identify specific synthetic cannabinoids in papers that were either deposited on the surface of the paper or diffused inside the paper substrate. When machine learning analytics were applied to the Raman spectra of the papers, quantitative information was obtained regarding the amount of synthetic cannabinoid deposited on the paper surface. In summary, handheld Raman spectroscopy could identify and quantify synthetic cannabinoids on paper rapidly and non-destructively. Future work involves testing other classes of new psychoactive substance and applying deep learning analytics

Off-target pharmacological profiling of synthetic cannabinoid receptor agonists including AMB-FUBINACA, CUMYL-PINACA, PB-22, and XLR-11.

Synthetic cannabinoid receptor agonists (SCRAs) are a diverse class of new psychoactive substances that have been associated with multiple instances and types of toxicity. Some SCRAs appear to carry a greater toxicological burden than others, or compared to the prototypical cannabis-derived agonist Δ9-tetrahydrocannabinol (Δ9-THC), despite a common primary mechanism of action via cannabinoid 1 (CB1) receptors. "Off-target" (i.e., non-CB1 receptor) effects could underpin this differential toxicity, although there are limited data around the activity of SCRAs at such targets. A selection of 7 SCRAs (AMB-FUBINACA, XLR11, PB-22, AKB-48, AB-CHMINICA, CUMYL-PINACA, and 4F-MDMB-BUTINACA), representing several distinct chemotypes and toxicological profiles, underwent a 30 μM single-point screen against 241 G protein-coupled receptor (GPCR) targets in antagonist and agonist mode using a cellular β-arrestin recruitment assay. Strong screening "hits" at specific GPCRs were followed up in detail using concentration-response assays with AMB-FUBINACA, a SCRA with a particularly notable history of toxicological liability. The single-point screen yielded few hits in agonist mode for any compound aside from CB1 and CB2 receptors, but many hits in antagonist mode, including a range of chemokine receptors, the oxytocin receptor, and histamine receptors. Concentration-response experiments showed that AMB-FUBINACA inhibited most off-targets only at the highest 30 μM concentration, with inhibition of only a small subset of targets, including H1 histamine and α2B adrenergic receptors, at lower concentrations (≥1 μM). AMB-FUBINACA also produced concentration-dependent CB1 receptor signaling disruption at concentrations higher than 1 μM, but did not produce overt cytotoxicity beyond CP55,940 or Δ9-THC in CB1 expressing cells. These results suggest that while some "off-targets" could possibly contribute to the SCRA toxidrome, particularly at high concentrations, CB1-mediated cellular dysfunction provides support for hypotheses concerning on-target, rather than off-target, toxicity. Further investigation of non-GPCR off-targets is warranted.

Synthesis of emerging cathinones and validation of a SPE GC-MS method for their simultaneous quantification in blood.

Over the past 15years, synthetic cathinones have emerged as an important class of new psychoactive substances (NPS) worldwide. The proliferation of these psychostimulants and their sought-after effects among recreational drug users pose a serious threat to public health and enormous challenges to forensic laboratories. For forensic institutions, it is essential to be one step ahead of covert laboratories, foreseeing the structural changes possible to introduce in the core skeleton of cathinones while maintaining their stimulating activity. In this manner, it is feasible to equip themselves with standards of possible new cathinones and validated analytical methods for their qualitative and quantitative detection. Therefore, the aim of the work herein described was to synthesize emerging cathinones based on the evolving patterns in the illicit drug market, and to develop an analytical method for their accurate determination in forensic situations. Five so far unreported cathinones [4'-methyl-N-dimethylbuphedrone (4-MDMB), 4'-methyl-N-ethylbuphedrone (4-MNEB), 4'-methyl-N-dimethylpentedrone (4-MDMP), 4'-methyl-N-dimethylhexedrone (4-MDMH), and 4'-methyl-N-diethylbuphedrone (4-MDEB)] and a sixth one, 4'-methyl-N-ethylpentedrone, already reported to EMCDDA and also known as 4-MEAP, were synthesized and fully characterized by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). An analytical method for the simultaneous quantification of these cathinones in blood, using solid phase extraction (SPE) combined with gas chromatography-mass spectrometry (GC-MS) was developed and validated. The results prove that this methodology is selective, linear, precise, and accurate. For all target cathinones, the extraction efficiency was higher than 73%, linearity was observed in the range of 10 (lower limit of quantification, LLOQ) to 800ng/mL, with coefficients of determination higher than 0.99, and the limits of detection (LODs) were 5ng/mL for all target cathinones. The stability of these cathinones in blood matrices is dependent on the storage conditions; 4-MNEBis the most stable compound and 4-MDMH isthe least stable compound. The low limits obtained allow the detection of the compounds in situations where they are involved, even if present at low concentrations.

Metabolic study of methoxpropamine (MXPr) in mice and determination of its metabolites in biological samples by UHPLC-QTOF-HRMS

The main metabolites of methoxpropamine (MXPr) and the evaluation of their formation and excretion over time, using biological samples from mice, was investigated. MXPr is an arylcyclohexylamine dissociative drug structurally similar to 3-MeO-PCE, ketamine and deschloroketamine, which was recently identified on the European illegal market and added to the class of New Psychoactive Substances (NPS). Forensic laboratories are often required to identify new drugs and their metabolites, albeit the latter are yet not known or their reference standards are lacking. Therefore, we performed the investigation of methoxpropamine (MXPr) metabolism, in order to elucidate the distribution of the parent drug and its metabolites in urine and plasma over time. Furthermore, body hair was collected and analysed. Initially, urine samples from 16 mice were collected every hour for 6 consecutive hours. Furthermore, one sample was collected after 12 and 24 hours from the administration of increasing doses of MXPr (1-3-10 mg/kg). After 16 days of wash out, two doses of 1 and 3 mg/kg were administered and plasma samples were collected. After adding ketamine-d4, urine and plasma were diluted 1:3 with methanol/acetonitrile (95:5) and 5 μL were injected into the UHPLC-QTOF-HRMS. Based on the typical phase I and II metabolic reaction, the metabolic pattern was hypothesized. The tentative metabolites were identified by means of the fragmentation patterns, the exact masses of both their precursor and fragment ions. Finally, body hair collected one month before and after the injection of MXPr was analyzed following a specific procedure for keratin matrix. A wide array of metabolites was identified in urine, including normethoxpropamine, desmethylmethoxpropamine, dihydromethoxpropamine, desmethyl-normethoxpropamine, dihydro-desmethylmethoxpropamine and phase II glucuronide conjugates. Furthermore, the metabolic pathway that produces desmethylmethoxpropamine-glucoronide was identified as the preferential pathway for the elimination of MXPr from the body. In fact, 1 h after the administration, an abundant peak of excretion was observed with an average intensity 16 times higher than the free phase desmetyl-MXPr. The presence of normethoxpropamine (NorMXPr) in urine and plasma is also relevant, following the typical metabolic pathways based on dealkylation. Since norketamine is known to be pharmacologically active, so it may be for NorMXPr, thus probably contributing to the toxicity of this substance. In addition, desmethylmethoxpropamine-glucoronide, together with other minor products of metabolism, were detectable in urine up to 24 h after injection. Finally, MXPr and the major phase I metabolites were observed also in plasma (average concentration for the parent drug: 1.0 ng/mL) and hair (average concentration for the parent drug: 0.18 ng/mg). Few phases I and II metabolites were confirmed to be present in body hair, approximately one month after body administration. Some interesting differences were observed in the metabolites generated over time between female and male mice, a phenomenon that could be related to sexually dimorphic metabolism, both involving phase I and phase II enzymes. A comprehensive workflow for the detection of MXPr and its I and II phase metabolites are presented. Data acquisition included a TOF-MS high-resolution scan combined with TOF-MS/MS acquisition and demonstrated a considerable capability to detect and identify target compounds with high resolution levels. Thanks to this study, the identification of MXPr metabolites and their excretion times from the body was possible. The introduction of new target compounds will improve the efficiency of toxicological screening analysis on real samples and will extend the window of detection of the dissociative drug MXPr in biological matrices. In the future, human urine samples will be analyzed to confirm the detectability of the tentative metabolites.

Development of a new green microextraction technique, switchable hydrophilicity solvent-based homogenous liquid–liquid microextraction, to analyze synthetic cannabinoids in plasma

Synthetic cannabinoid receptor agonists (SCRA) are currently the most popular class of new psychoactive substances (NPS) in several countries, including Brazil. Notwithstanding that, they pose a threat to public health, as little of their toxicological properties is known. Moreover, these SCRAs have different presentations compared to classic cannabinoids and are not usually detected by conventional analytical methods due to their chemical variety. Thus, the development of new methodologies fit to analyze SCRAs is extremely warranted. In that regard, the use of environmentally benign or green approaches has been a trend in the last decades and has gained attention in Forensic Toxicology – so much so that guidelines have been established for that purpose, e.g. green analytical toxicology (GAT). With that in mind, the aim of this work was to develop and validate a quantitative method to analyze SCRAs in plasma by a novel green technique that agrees with the principles of GAT. A switchable hydrophilicity solvent-based homogenous liquid–liquid microextraction (SHS-HLLME) was developed and statistical tools were used to optimize it. Analysis of Variance (ANOVA) was chosen to find the best switchable hydrophilicity solvent (SHS) and Full Factorial and Central Composite design to study the following variables: volume of SHS:HCl 6 M mixture (200–500 μL) and NaOH 10 M (200–500 μL), salting-out effect, and time of extraction (1–5 min). The ANSI/ASB Standard 036, 1st edition 2019 guidelines were used for method validation. All analyses were performed using a UPLC-MS/MS with the multiple reaction monitoring approach. Ethical Committee approval No. 46404121.8.3001.0067. Funded by CNPq 142056/2020-0, CAPES/INSPEQT 16/2020, and FAPESP 21/09857-8. ANOVA showed that between two SHS tested, N,N-dimethylcyclohexylamine and dipropylamine, the latter yielded higher analyte recovery ( P < 0.05). Moreover, the Design of Experiment tools herein used showed that the salting-out effect and time of extraction had no major impact on analyte recovery; in contrast, 500 μL of both SHS:HCl mixture and NaOH yielded higher analyte recovery ( P < 0.05). Once optimized, the proposed technique was successfully validated according to international guidelines with the following parameters: limits of detection: 0.01–0.08 ng/mL; limit of quantitation: 0.1 ng/mL; linearity: 0.1–10 ng/mL; bias and precision < 15%; no carryover was observed and the method proved to be selective for the aimed SCRAs. The matrix effect, recovery efficiency, and process efficiency studies are being currently carried out. The microextraction technique herein developed is based on an acid-base neutralization between HCl and NaOH, making it an approach significantly more sustainable when compared to conventional choices that rely entirely on organic solvents. In that regard, only 250 μL of dipropylamine was required–an organic solvent considerably less toxic than those used in common extractions, organochlorides for example. In addition, a small volume was used compared to more conventional techniques in forensic analyses, such as solid-phase extraction (> 500 μL). Hence, we were able to successfully detect and quantitate 31 SCRAs at extremely low concentrations (0.01–0.1 ng/mL, respectively) using only 300 μL of plasma, showing that the SHS-HLLME can be used for the analysis of multiple SCRAs in a single run, thus could be a valuable tool in forensic cases. We demonstrated for the first time the application of the novel SHS-HLLME for synthetic cannabinoids. The microextraction technique herein developed has promising applications in Forensic Toxicology, as it consists of a simple and fast procedure that yields high sensitivity with only a few microliters of a biological sample. Also, it avoids the use of highly toxic chemicals and generates less residue, thus it fits the growing trend of using sustainable analytical methodologies, established by GAT and other similar guidelines.

Identification of the novel synthetic cannabinoid-type new psychoactive substance, CH-PIACA, in seized material

Synthetic cannabinoids (SCs) remain the largest class of new psychoactive substances (NPS) according to the European Monitoring Centre for Drugs and Drug Addiction (EMDCDDA) and while the number of new analogues that are reported for the first time each year declines, SCs are reported the most compared to other NPS classes. This decline can be seen as a result of legislative changes by different jurisdictions which have sometimes transitioned to a more generalized approach when controlling substances by defining common structural scaffolds rather than explicit structures. While the consequences of such legislative changes have been expected over the years, the introduction of so called “class-wide” bans has put further pressure on clandestine laboratories to synthesize compounds which are out of the scope of the legislation and thus these compounds are initially harder to detect and/or identify in forensic chemistry or medico-legal contexts in the absence of analytical data. Recently, a synthetic cannabinoid with an indole-3-acetamide core-linker scaffold, AD-18 (i.e. ADB-FUBIATA or ADB-FUBIACA), was reported for the first time in China in 2021. Here, an additional cannabinoid with the indole-3-acetamide scaffold, N-cyclohexyl-2-(1-pentyl-1H-indol-3-yl)acetamide (tentatively CH-PIACA) was structurally characterized using gas chromatography – mass spectrometry (GC-MS), liquid chromatography – high-resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR) spectroscopy. A beige powder in a zip-lock bag was seized by The Danish Customs Agency and subsampled by combining three aliquots of approximately 1 g after homogenization with a mortar and pestle. Sample solutions for GC-MS and UHPLC-QTOF-MS were prepared by dissolving the material in methanol and 25% methanol + 1% formic acid, respectively. For NMR, 3 mg of the powder was dissolved in DMSO-d6 and filtered into an NMR tube. Routine GC-MS analysis of the seized material yielded a single major peak at 22.156 min with a molecular ion at m/z 326.3 and fragment ions at m/z 200, 144 and 130 in the electron ionization mass spectrum (EI-MS). In addition, a major component was observed at 11.91 min in the UHPLC-QTOF-MS screening which had a precursor ion at m/z 327.2450 corresponding to a compound with a molecular formula of C21H30N2O. Similar product ions were observed in the electrospray ionization tandem MS (ESI-MS/MS) spectrum with m/z 245.1644, 200.1441, 144.0808, 130.0651, 100.1120 and 83.0853. NMR spectroscopy with 1H, 13 C, COSY, HMBC and HSQC indicated that the structure was likely to be a substituted indole with a pentyl chain and N-cyclohexylacetamide at the 1- and 3 positions. This structure was corroborated by the EI-MS and ESI-MS/MS data with the neutral loss of N-cyclohexyl-carboxamide (C7H13NO, 127.0997 Da) via cleavage of the bond between the α and β carbons forming m/z 200.1441, while the product ion at m/z 245.1644 corresponded to the loss of cyclohexene from the amide nitrogen. The loss of the N-cyclohexyl-carboxamide was also followed by the loss of the pentyl chain from the nitrogen of the indole ring to yield m/z 130.0651, while m/z 100.1120 corresponded to the cyclohexylammonium cation. The product ion at m/z 144.0808 was proposed to be due to the loss of both the N-cyclohexyl-carboxamide and cleavage of the pentyl chain to liberate 1-butene. A new SC with the indole-3-acetamide scaffold was identified in a seized material in Denmark and characterized using GC-MS, UHPLC-QTOF-MS and NMR spectroscopy. This marks the second SC with this scaffold that has been reported with further analogues anticipated to emerge in the near future.

Human methylone, MDMA and metabolites’ plasma concentrations by a new LC-MS/MS method after controlled administration of methylone and MDMA

Synthetic cathinones are a class of new psychoactive substances (NPS) with structural and pharmacological similarity to amphetamines. Methylone, a synthetic cathinone NPS, is the β-keto analog of MDMA. There are no controlled administration data for methylone in humans. The aim of this research was development of an LC-MS/MS method to simultaneously quantify methylone, MDMA and their metabolites 4-hydroxy-3-methoxy-N-methylcathinone (HMMC), 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxymeth-amphetamine (HMMA) and 4-hydroxy-3-methoxyamphetamine (HMA) in human plasma and application of the method to the first controlled oral methylone administration study. Briefly, 10 μL 100 ng/mL internal standard mixture (methylone-d3, MDA-d5 and MDMA-d5) and 2 μL NH2 2% in H2O (pH 9) were added to 100 μL plasma, tubes were stirred in a roller mixer for 10 min and centrifuged at 3500 rpm for 5 min. Supernatants were collected into clean tubes, 100 μL acidic methanol (1% HCl) was added to prevent evaporative losses, and samples dried under nitrogen for approximately 30 min. Samples were reconstituted in 100 μL mobile phase A:B (95:5) (0.1% formic acid in water:acetonitrile) and transferred into autosampler vials for LC–MS/MS analysis. Analytes separation was obtained on a UHPLC 1290 Infinity II (Agilent Technologies Italia S.p.a.) coupled to a mass spectrometer (6470A Triple Quadrupole LC-MS) equipped with an electrospray ionization source operating in positive-ion mode (ESI+). Limits of quantification (LLOQ) were 5 (methylone) to 500 (MDMA) μg/L and 0.5 to 50 μg/L for their metabolites. A randomized double blinded study was conducted in 12 male volunteers (aged 18–45 years old; weight ranging from 50 to 90 kg) and divided into 3 sessions, with single oral doses of 50, 100, 150 or 200 mg methylone, 100 mg MDMA or placebo in each session. Plasma samples were collected before and 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10 and 24 h after dosing. The analytical method was validated over five successive days in plasma following the most recent criteria for bioanalytical method development and validation. The method was linear for all analytes over the calibration range. Intra- and inter-assay accuracies were ±15%, while intra- and inter-assay precision were ±10%. Methylone plasma concentrations increased in a dose-related manner. Methylone Cmax were 138, 243, 320 and 530 μg/L following the 50, 100, 150 and 200 mg doses, respectively, while corresponding area under the curve (AUC) were 1065, 2486, 3511 and 5489 min ×μg/L. Methylone Tmax were 1 h for the lowest 50 mg dose and 2 h for the remaining doses. The last detected methylone concentrations (Clast) at 24 h were 10.5, 18.8, 36.4 and 51.8 μg/L (for 50, 100, 150 and 200 mg respectively). For methylone's HMMC metabolite, Cmax were 6.6, 16.9, 28.3, and 32.7 μg/L, while AUC were 36.9, 90.1, 129 and 173 min ×μg/L. HMMC exhibited rapid kinetics with Tmax of 1–1.25 h for all doses. As for Clast, HMMC concentrations were 0.5, 0.4, 1.3 and 0.8 μg/L (for 50, 100, 150 and 200 mg, respectively) 24 h after drug administration. Methylone is a popular drug of abuse, yet no studies addressed its metabolism and pharmacokinetics in humans. A validated LC-MS/MS method for simultaneous quantification of methylone, MDMA and their metabolites (HMMC, MDA, HMMA and HMA) in human plasma is presented. Considering the pharmacokinetic study for methylone and HMMC, concentrations following the 100 mg dose were approximately doubled compared to the 50 mg dose, and after the 200 mg dose were about four times higher than the lowest dose (within ±20%), suggesting linear pharmacokinetics for both analytes.

N-Ethylnorketamine has anesthetic and analgesic effects with abuse liability

Arylcyclohexylamines is an ever-growing class of new psychoactive substances, including an increasing number of ketamine analogs. N-Ethylnorketamine (NENK) is a new synthetic ketamine analog that has emerged as an abused drug, but little is known about the pharmacological profile of NENK. In this study, we investigated the anesthetic and analgesic activity, abuse liability of NENK compared with ketamine. The ED50 values of anesthetic activity for NENK and ketamine were 96.9, 69.4 mg/kg, respectively. The ED50 values of analgesic activity for NENK and ketamine were 45.9 and 23.6 mg/kg, respectively. NENK induced significant conditioned place preference at a minimum dose of 10.0 mg/kg in mice, an effect comparable to that of ketamine (3.0 mg/kg). Acute injections of NENK or ketamine at 30.0 mg/kg enhanced locomotor activity, and repeated treatments with this dose induced locomotor sensitization after withdrawal. Taken together, these results clearly demonstrated that NENK has lower anesthetic and analgesic activity compared to ketamine, but has significant abuse liability.

Identification of the synthetic cannabinoid-type new psychoactive substance, CH-PIACA, in seized material.

Synthetic cannabinoids (SCs) remain the largest class of new psychoactive substances (NPS), and while the number of NPS that are reported to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) for the first time each year declines, the number of newly reported SCs still exceeds other NPS classes. This decline can be seen as a result of legislative changes by different jurisdictions which have sometimes transitioned to a more generalized approach when controlling substances by defining common structural scaffolds rather than explicit structures. While the consequences of such legislative changes have been expected over the years, the introduction of so‐called “class‐wide” bans puts further pressure on clandestine laboratories to synthesize compounds which are out of the scope of the legislation, and thus, these compounds are initially harder to detect and/or identify in the absence of analytical data. Recently, a SC with an indole‐3‐acetamide core‐linker scaffold, AD‐18 (i.e., ADB‐FUBIATA or ADB‐FUBIACA), was reported for the first time in China in 2021. Here, an additional cannabinoid with the indole‐3‐acetamide scaffold, N‐cyclohexyl‐2‐(1‐pentyl‐1H‐indol‐3‐yl)acetamide (CH‐PIACA), is reported which was identified for the first time in a seized material in Denmark. Structural characterization was performed using gas chromatography–mass spectrometry (GC–MS), liquid chromatography‐high‐resolution mass spectrometry (LC‐HRMS), and nuclear magnetic resonance (NMR) spectroscopy.

Putative Synthetic Cannabinoids MEPIRAPIM, 5F-BEPIRAPIM (NNL-2), and Their Analogues Are T-Type Calcium Channel (CaV3) Inhibitors.

Synthetic cannabinoid receptor agonists (SCRAs) are a large and growing class of new psychoactive substances (NPSs). Two recently identified compounds, MEPIRAPIM and 5F-BEPIRAPIM (NNL-2), have not been confirmed as agonists of either cannabinoid receptor subtype but share structural similarities with both SCRAs and a class of T-type calcium channel (CaV3) inhibitors under development as new treatments for epilepsy and pain. In this study, MEPIRAPIM and 5F-BEPIRAPIM and 10 systematic analogues were synthesized, analytically characterized, and pharmacologically evaluated using in vitro cannabinoid receptor and CaV3 assays. Several compounds showed micromolar affinities for CB1 and/or CB2, with several functioning as low potency agonists of CB1 and CB2 in a membrane potential assay. 5F-BEPIRAPIM and four other derivatives were identified as potential CaV3 inhibitors through a functional calcium flux assay (>70% inhibition), which was further confirmed using whole-cell patch-clamp electrophysiology. Additionally, MEPIRAPIM and 5F-BEPIRAPIM were evaluated in vivo using a cannabimimetic mouse model. Despite detections of MEPIRAPIM and 5F-BEPIRAPIM in the NPS market, only the highest MEPIRAPIM dose (30 mg/kg) elicited a mild hypothermic response in mice, with no hypothermia observed for 5F-BEPIRAPIM, suggesting minimal central CB1 receptor activity.

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.