Metabolism and cytotoxicity studies of the two hallucinogens 1cP-LSD and 4-AcO-DET in human liver and zebrafish larvae models using LC-HRMS/MS and a high-content screening assay.

The continuous emergence of new psychoactive substances (NPS) attracted a great deal of attention within recent years. Lately, the two hallucinogenic NPS 1cP-LSD and 4-AcO-DET have appeared on the global market. Knowledge about their metabolism to identify potential metabolic targets for analysis and their cytotoxic properties is lacking. The aim of this work was thus to study their in vitro and in vivo metabolism in pooled human liver S9 fraction (pHLS9) and in zebrafish larvae (ZL) by means of liquid chromatography-high-resolution tandem mass spectrometry. Monooxygenases involved in the initial metabolic steps were elucidated using recombinant human isozymes. Investigations on their cytotoxicity were performed on the human hepatoma cell line HepG2 using a multiparametric, fluorescence-based high-content screening assay. This included measurement of CYP-enzyme mediated effects by means of the unspecific CYP inhibitor 1-aminbenzotriazole (ABT). Several phase I metabolites of both compounds and two phase II metabolites of 4-AcO-DET were produced in vitro and in vivo. After microinjection of 1cP-LSD into the caudal vein of ZL, three out of seven metabolites formed in pHLS9 were also detected in ZL. Twelve 4-AcO-DET metabolites were identified in ZL after exposure via immersion bath and five of them were found in pHLS9 incubations. Notably, unique metabolites of 4-AcO-DET were only produced by ZL, whereas 1cP-LSD specific metabolites were found both in ZL and in pHLS9. No toxic effects were observed for 1cP-LSD and 4-AcO-DET in HepG2 cells, however, two parameters were altered in incubations containing 4-AcO-DET together with ABT compared with incubations without ABT but in concentrations far above expected in vivo concentration. Further investigations should be done with other hepatic cell lines expressing higher levels of CYP enzymes.

Analytical and behavioral characterization of 1-dodecanoyl-LSD (1DD-LSD).

1-Acetyl-N,N-diethyllysergamide (1A-LSD, ALD-52) was first synthesized in the 1950s and found to produce psychedelic effects similar to those of LSD. Evidence suggests that ALD-52 serves as a prodrug in vivo and hydrolysis to LSD is likely responsible for its activity. Extension of the N1-alkylcarbonyl chain gives rise to novel lysergamides, which spurred further investigations into their structure-activity relationships. At the same time, ALD-52 and numerous homologues have emerged as recreational drugs ("research chemicals") that are available from online vendors. In the present study, 1-dodecanoyl-LSD (1DD-LSD), a novel N1-acylated LSD derivative, was subjected to analytical characterization and was also tested in the mouse head-twitch response (HTR) assay to assess whether it produces LSD-like effects in vivo. When tested in C57BL/6J mice, 1DD-LSD induced the HTR with a median effective dose (ED50) of 2.17 mg/kg, which was equivalent to 3.60 µmol/kg. Under similar experimental conditions, LSD has 27-fold higher potency than 1DD-LSD in the HTR assay. Previous work has shown that other homologues such as ALD-52 and 1-propanoyl-LSD also have considerably higher potency than 1DD-LSD in mice, which suggests that hydrolysis of the 1-dodecanoyl moiety may be comparatively less efficient in vivo. Further investigations are warranted to determine whether the increased lipophilicity of 1DD-LSD causes it to be sequestered in fat, thereby reducing its exposure to enzymatic hydrolysis in plasma and tissues. Further clinical studies are also required to assess its activity in humans and to test the prediction that it could potentially serve as a long-acting prodrug for LSD.

Insights into the metabolism of CH-PIATA - a novel synthetic cannabinoid featuring an acetamide linker.

The recent change from the popular carboxamide to an acetamide (ATA) linker scaffold in synthetic cannabinoid receptor agonists (SCRAs) can be interpreted as an attempt to circumvent legal regulations, setting new analytical challenges. Metabolites of N-cyclohexyl-2-(1-pentyl-1H-indol-3-yl)acetamide: CH-PIATA, the second ATA type SCRA detected in the EU, were investigated in urine and serum samples by LC-HRMS-MS and LC-MS-MS. Two different in vitro models: a pHLM assay and HepG2-cells as well as an in silico prediction by GLORYx freeware assisted in metabolite formation/identification. CH-PIATA was extensively metabolized, leading to metabolites formed primarily by mono- and dihydroxylation. For urine and serum specimens, monohydroxylation at the indole core or the methylene spacer of the acetamide linker (M1.8), carboxylic acid formation at the N-pentyl side chain (M3.1), and degradation of the latter leading to a tentatively identified N-propionic acid metabolite (M5.1) are suggested as reliable markers for substance intake. The N-propionic acid metabolite could not be confirmed in the in vitro assays as it includes multiple consecutive metabolic reactions. Furthermore, CH-PIATA could be detected as parent substance in blood samples, but not in urine. Both in vitro assays and the in silico tool proved suitable for predicting metabolites of CH-PIATA. Considering effort and costs, pHLM incubations seem to be more effective for metabolite prediction in forensic toxicology. The highlighted phase I metabolites serve as reliable urinary targets for confirming CH-PIATA use. The in silico approach is advantageous when reference material is unavailable.

EMCDDA framework and practical guidance for naming cathinones.

Cathinones are often sold as "legal" alternatives to controlled stimulants such as amphetamine, MDMA and cocaine. Cathinones are the second largest group of new psychoactive substances (NPS), with close to 170 monitored by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). Although all cathinones are related to the parent compound cathinone, one of the psychoactive principles in khat, assigning consistent, informative and user-friendly common names to these substances is challenging. Over time different naming approaches have been applied, leading to cathinones being known by several names. This work provides a framework and practical examples for the consistent naming of cathinones which is easy to understand and can be applied by the forensic community, researchers, clinical practitioners, and policy makers. The scope of the issue and rationale for earlier naming approaches are also discussed. The new naming framework has been developed based on established naming approaches and centered around the common "cathinone," and "phenone" motifs/scaffolds. The proposed framework establishes clear rules to derive the EMCDDA framework names for cathinones. Each name is, in turn, composed by a principal name containing a parent letter, derived after the "cathinone" or the "phenone" scaffold. Additional substitutions are prepended to the principal name. The framework also provides for exceptions for several cathinones and structural analogs scheduled under UN and EU legislation.

Analytical and behavioral characterization of N-ethyl-N-isopropyllysergamide (EIPLA), an isomer of N6 -ethylnorlysergic acid N,N-diethylamide (ETH-LAD).

Preclinical investigations have shown that N-ethyl-N-isopropyllysergamide (EIPLA) exhibits lysergic acid diethylamide (LSD)-like properties, which suggests that it might show psychoactive effects in humans. EIPLA is also an isomer of N6 -ethylnorlysergic acid N,N-diethylamide (ETH-LAD), a lysergamide known to produce psychedelic effects in humans that emerged as a research chemical. EIPLA was subjected to analysis by various forms of mass spectrometry, chromatography (GC, LC), nuclear magnetic resonance (NMR) spectroscopy, and GC condensed-phase infrared spectroscopy. The most straightforward differentiation between EIPLA and ETH-LAD included the evaluation of mass spectral features that reflected the structural differences (EIPLA: N6 -methyl and N-ethyl-N-isopropylamide group; ETH-LAD: N6 -ethyl and N,N-diethylamide group). Proton NMR analysis of blotter extracts suggested that EIPLA was detected as the base instead of a salt, and two blotter extracts suspected to contain EIPLA revealed the detection of 96.9 ± 0.5 µg (RSD: 0.6%) and 85.8 ± 2.8 µg base equivalents based on LC-MS analysis. The in vivo activity of EIPLA was evaluated using the mouse head-twitch response (HTR) assay. Similar to LSD and other serotonergic psychedelics, EIPLA induced the HTR (ED50 = 234.6 nmol/kg), which was about half the potency of LSD (ED50 = 132.8 nmol/kg). These findings are consistent with the results of previous studies demonstrating that EIPLA can mimic the effects of known psychedelic drugs in rodent behavioral models. The dissemination of analytical data for EIPLA was deemed justifiable to aid future forensic and clinical investigations.

Pharmacological profile, phase I metabolism, and excretion time profile of the new synthetic cathinone 3,4-Pr-PipVP.

1-(2,3-Dihydro-1H-inden-5-yl)-2-(piperidin-1-yl)pentan-1-one (3,4-Pr-PipVP), a novel synthetic cathinone (SCat), was first identified in 2022 in Germany. The product was marketed as 1-(bicyclo[4.2.0]octa-1,3,5-trien-3-yl)-2-(pyrrolidin-1-yl)pentan-1-one (3,4-EtPV), a substance not covered by the German New Psychoactive Substances Act (NpSG). Although originally intended to be an exploratory new synthetic cathinone containing the novel bicyclo[4.2.0]octatrienyl function, the compound was subsequently confirmed to contain an indanyl ring system scheduled under generic legislation like the NpSG. However, it is one of only a few marketed SCats carrying a piperidine ring. Inhibition experiments involving norepinephrine, dopamine, and serotonin transporters showed that 3,4-Pr-PipVP was a low potency blocker at all three monoamine transporters compared to related substances such as MDPV. Additionally, pharmacokinetic data were collected from pooled human liver microsomes incubations and from the analysis of authentic urine samples received after oral administration of 5 mg 3,4-Pr-PipVP hydrochloride. Phase I metabolites were tentatively identified in vitro and in vivo using liquid chromatography-time-of-flight mass spectrometry. Main metabolites were formed by metabolic reduction of the carbonyl function with and without additional hydroxylations at the propylene bridge of the molecule. Keto-reduced H2 -3,4-Pr-PipVP and H2 -piperidine-OH-3,4-Pr-PipVP as well as aryl-OH-3,4-Pr-PipVP, and indanyl-OH-piperidine-OH-3,4-Pr-PipVP are suggested as most suitable biomarkers for the detection of 3,4-Pr-PipVP since they were detected for much longer than the parent compound. 3,4-Pr-PipVP could be detected for up to 21 h whereas its metabolites were detectable for up to about 4 days.

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.

Analytical profile of the lysergamide 1cP-AL-LAD and detection of impurities.

The development of novel lysergamides continues to occur, based on both the needs of psychedelic medicine and commercial interest in new recreational substances. The present study continues the authors' research on novel lysergamides and describes the analytical profile of 1-cyclopropanoyl-AL-LAD (IUPAC name: 1-(cyclopropanecarbonyl)-N,N-diethyl-6-(prop-2-en-1-yl)-9,10-didehydroergoline-8ß-carboxamide; 1cP-AL-LAD), using various chromatographic, mass spectrometric, and spectroscopic methods. Analysis of a powdered sample of 1cP-AL-LAD, obtained from an online vendor, by high performance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry in full scan/AutoMS/MS mode revealed the detection of 17 impurities based on high-resolution tandem mass spectral data; tentative determination of their identity was based on mass spectral grounds alone, though detection of AL-LAD and 1P-AL-LAD was confirmed using available reference standards. Other tentative compound identifications included 1-acetyl-AL-LAD and several other substances potentially reflecting oxidation of the N6 -allyl group as well as other positions on the ergoline ring system. These data may assist those interested in the chemistry of lysergamides. Finally, 1cP-AL-LAD was also detected in samples of "blotters" sold online for recreational use.

EMCDDA framework and practical guidance for naming synthetic cannabinoids.

Synthetic cannabinoids (SCs), often sold as "legal" replacements for cannabis, are the largest group of new psychoactive substances monitored by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). Currently, close to 240 structurally heterogeneous SCs are monitored through the European Union (EU) Early Warning System, and attributing consistent, informative, and user-friendly names to SCs has been a challenge in the past. Over time, several naming conventions have been employed with the aim of making SCs more easily recognizable by non-chemists, including regulators. To achieve this, the names assigned need to contain detailed information on the structural features present in the substance. This work provides a theoretical framework and a practical hands-on guideline for consistent naming of SCs, which is easy to understand and can be applied by the forensic community, researchers, clinical practitioners, and policy-makers. The proposed framework builds on the established letter code system for molecular building blocks (core, linker, linked group, and tail) implemented by the EMCDDA in 2013 and has been expanded to incorporate additional structural features through substitution. The scope of the issue of attributing semi-systematic code names is illustrated, and earlier approaches used for naming SCs are discussed. The concepts and rules of the EMCDDA framework are described through a flowchart that provides a basis for naming new SCs, a graphical overview of the chemical diversity of SCs, and a detailed list of the SCs identified in the EU by the Early Warning System of the EMCDDA for reference.

A new synthetic cathinone: 3,4-EtPV or 3,4-Pr-PipVP? An unsuccessful attempt to circumvent the German legislation on new psychoactive substances.

Synthetic cathinones comprise psychostimulants with desired effects like euphoria, increased vigilance, appetite suppression, and-mainly depending on certain structural features-entactogenic properties. 3,4-EtPV (1-(bicyclo[4.2.0]octa-1,3,5-trien-3-yl)-2-(pyrrolidin-1-yl)pentan-1-one) was first mentioned in an online drug forum in September 2021, where its imminent synthesis was announced. The goal was to produce a legal alternative to the phenylethylamines already banned by the German NpSG. In February and June 2022, two samples labeled with the name and molecular structure of 3,4-EtPV were analyzed. The molecular structure of the obviously mislabeled compound was elucidated and comprehensively characterized within the ADEBAR project. The synthetic cathinone identified differed from the declared 3,4-EtPV by a 3,4-propylene bridge instead of a 3,4-ethylene bridge and a piperidine ring instead of a pyrrolidine ring. The short name 3,4-Pr-PipVP (3,4-propylene-2-(1-piperidinyl)valerophenone) was suggested as a semisystematic name in collaboration with the European Monitoring Centre for Drugs and Drug Addiction. Herein, the results of the analyses are discussed and will enable forensic laboratories to update their databases quickly and identify 3,4-Pr-PipVP confidently. 3,4-Pr-PipVP is already scheduled under the German NpSG. This study highlights that there are ongoing efforts to deliberately circumvent generic definitions given, for example, in the German NpSG and that (unintentional?) mislabeling can be an issue. The end user purchasing substances online can never be sure that the material actually supplied will be the one ordered, and he might receive an illicit drug instead of an uncontrolled one. Furthermore, the purity is always unknown, creating health risks due to unexpected effects and potencies.

In Vitro and In Vivo Toxicometabolomics of the Synthetic Cathinone PCYP Studied by Means of LC-HRMS/MS.

Synthetic cathinones are one important group amongst new psychoactive substances (NPS) and limited information is available regarding their toxicokinetics and -dynamics. Over the past few years, nontargeted toxicometabolomics has been increasingly used to study compound-related effects of NPS to identify important exogenous and endogenous biomarkers. In this study, the effects of the synthetic cathinone PCYP (2-cyclohexyl-1-phenyl-2-(1-pyrrolidinyl)-ethanone) on in vitro and in vivo metabolomes were investigated. Pooled human-liver microsomes and blood and urine of male Wistar rats were used to generate in vitro and in vivo data, respectively. Samples were analyzed by liquid chromatography and high-resolution mass spectrometry using an untargeted metabolomics workflow. Statistical evaluation was performed using univariate and multivariate statistics. In total, sixteen phase I and one phase II metabolite of PCYP could be identified as exogenous biomarkers. Five endogenous biomarkers (e.g., adenosine and metabolites of tryptophan metabolism) related to PCYP intake could be identified in rat samples. The present data on the exogenous biomarker of PCYP are crucial for setting up analytical screening procedures. The data on the endogenous biomarker are important for further studies to better understand the physiological changes associated with cathinone abuse but may also serve in the future as additional markers for an intake.

A simplified strategy to assess the cytotoxicity of new psychoactive substances in HepG2 cells using a high content screening assay - Exemplified for nine compounds.

New psychoactive substances (NPS) are an issue of global concern posing a serious threat to the healthcare systems. Consumption of some NPS has been associated with toxic effects on the liver amongst others. However, data concerning their (cyto-)toxicity are usually not available. For a straightforward assessment of their cytotoxic potential, a simplified strategy measuring six different cytotoxicity indicating parameters simultaneously by a high content screening assay (HCSA) was developed. Its applicability was further investigated using nine NPS from heterogeneous chemical classes. HepG2 cells were incubated with NPS for 48 h at a low and high concentration (7.81 and 125 µM), respectively. To study metabolism-mediated effects on their cytotoxicity, cells were additionally incubated with the unspecific cytochrome (CYP) P450 inhibitor 1-aminobenzotriazole. Four fluorescence dyes were used to monitor cell count, nuclear size, and nuclear intensity (all Hoechst33342), mitochondrial membrane potential (TMRM), cytoplasmic calcium levels (CAL-520), and plasma membrane integrity (TOTO-3). Amongst the investigated NPS, ephylone, CUMYL-CBMICA, and dibutylone showed a strong cytotoxic potential, affecting two parameters at 7.81 µM. 5-MeO-MiPT showed moderate effects by impairing one parameter at 7.81 and one at 125 µM. Furthermore, at the high concentration of 5-MeO-MiPT, an effect of metabolism on cytotoxicity was observed. The HCSA confirmed the cytotoxic potential of ephylone and 5-MeO-MiPT, as the investigated concentrations were in the range of their published blood concentrations which induced liver damages after intake. The mitochondrial membrane potential was the parameter with the highest sensitivity and thus considered as suitable "cytobiomarker". In turn, parameters showing a high variability or unexpected effects such as cytosolic calcium levels and plasma membrane integrity might be omitted in the future. Even though 5-MeO-MiPT showed metabolism-based effects, HepG2 are known to have limited metabolic activity compared to cell lines such as HepaRG. Therefore, in further experiments cell lines with higher CYP-expression needs to be included and findings compared. Nevertheless, the simplified HCSA-based strategy allowed to screen NPS from diverse chemical groups for a first assessment of the cytotoxic properties of the parent compound. This information is crucial for a thorough risk assessment of NPS not only for public health authorities.

The ADEBAR project: European and international provision of analytical data from structure elucidation and analytical characterization of NPS.

Novel substances for which none or limited analytical data are available constitute a challenge for police and customs forensic laboratories. The time-consuming process of structural elucidation and acquisition of analytical data has been centralized in the ADEBAR project in Germany, co-funded since 2017 by the EU's Internal Security Fund. The project aims to comprehensively characterize substances relevant for forensic-toxicological casework within the analytical competence network. The analytical datasets are distributed digitally through European and (inter)national channels. Additionally, pharmacological evaluation allows for estimating in vivo potency and potential harm required as scientific evidence for legislative amendments. The ADEBAR project contributes to the availability of analytical data on new substances relevant to the daily work of police and customs laboratories. Since the inception of the ADEBAR project, 549 samples have been registered, and 302 substance reports notified to the EMCDDA, including numerous spectrometric and spectroscopic data. In addition, 3,619 mass spectra have been accumulated in ADEBAR mass spectra databases. A central institution for the structure elucidation and acquisition of valid, high-quality analytical data for police and customs forensic laboratories and forensic medicine institutes is important in the future because there does not seem to be an end to the dynamic of novel NPS appearing on the drug market.

Analytical profile, in vitro metabolism and behavioral properties of the lysergamide 1P-AL-LAD.

Lysergic acid diethylamide (LSD) is known to induce powerful psychoactive effects in humans, which cemented its status as an important tool for clinical research. A range of analogues and derivatives has been investigated over the years, including those classified as new psychoactive substances. This study presents the characterization of the novel lysergamide N,N-diethyl-1-propanoyl-6-(prop-2-en-1-yl)-9,10-didehydroergoline-8ß-carboxamide (1P-AL-LAD) using various mass spectrometric, gas- and liquid chromatographic and spectroscopic methods. In vitro metabolism studies using pooled human liver microsomes (pHLM) confirmed that 1P-AL-LAD converted to AL-LAD as the most abundant metabolite consistent with the hypothesis that 1P-AL-LAD may act as a prodrug. Fourteen metabolites were detected in total; metabolic reactions included hydroxylation of the core lysergamide ring structure or the N6 -allyl group, formation of dihydrodiol metabolites, N-dealkylation, N1 -deacylation, dehydrogenation, and combinations thereof. The in vivo behavioral activity of 1P-AL-LAD was evaluated using the mouse head twitch response (HTR), a 5-HT2A -mediated head movement that serves as a behavioral proxy in rodents for human hallucinogenic effects. 1P-AL-LAD induced a dose-dependent increase in HTR counts with an inverted U-shaped dose-response function, similar to lysergic acid diethylamide (LSD), psilocybin, and other psychedelics. Following intraperitoneal injection, the median effective dose (ED50 ) for 1P-AL-LAD was 491 nmol/kg, making it almost three times less potent than AL-LAD (174.9 nmol/kg). Previous studies have shown that N1 -substitution disrupts the ability of lysergamides to activate the 5-HT2A receptor; based on the in vitro metabolism data, 1P-AL-LAD may induce the HTR because it acts as a prodrug and is metabolized to AL-LAD after administration to mice.

Structure elucidation of the novel synthetic cannabinoid Cumyl-Tosyl-Indazole-3-Carboxamide (Cumyl-TsINACA) found in illicit products in Germany.

New chemical moieties continue to appear in synthetic cannabimimetics (SC), the largest group of new psychoactive substances in the EU. We describe the first comprehensive characterisation of the novel SC Cumyl-Tosyl-Indazole-3-Carboxamide (Cumyl-TsINACA) (N-[2-phenylpropan-2-yl]-1-tosyl-1H-indazole-3-carboxamide) from seized case samples. Structure elucidation was performed within the EU-project ADEBAR plus to facilitate confident identification by other researchers and practitioners worldwide. Characteristic MS fragmentations include the cleavage of the sulfonamide bond (S-N), the aryl sulfone bond (C-S) and the elimination rearrangement of SO2 in the side chain. Cumyl-TsINACA is a full receptor agonist at hCB1 (Emax  = 228%) with very weak binding affinity (Ki  = 292 nm) and low functional activity (EC50  = 31 µm). Thermal degradation of Cumyl-TsINACA was observed under GC conditions. The degree to which the tosyl side chain is cleaved due to pyrolysis primarily depends on solvent, the use of glass wool in the liner and injector temperature. The determination of the constitution by NMR spectroscopy was ambiguous due to the high number of neighbouring, non-proton-bearing atoms. Therefore, other possible structures compatible with the NMR correlations were generated using the WebCocon software. The unambiguous structural evidence was finally obtained by spectra comparison after the synthesis of Cumyl-TsINACA. The low thermal stability, as well as the low affinity and potency, renders this compound unfavourable for the use as a psychoactive substance. Thus, we do not expect widespread adoption of this SC.

Return of the lysergamides. Part VII: Analytical and behavioural characterization of 1-valeroyl-d-lysergic acid diethylamide (1V-LSD).

The psychopharmacological properties of the psychedelic drug lysergic acid diethylamide (LSD) have attracted the interest of several generations of scientists. While further explorations involving novel LSD-type compounds are needed to assess their potential as medicinal drugs, the emergence of novel derivatives as recreational drugs has also been observed. 1-Valeroyl-LSD (also known as 1-valeryl-LSD, 1-pentanoyl-LSD, 1V-LSD, or "Valerie") is a new N1 -acylated LSD derivative that recently appeared on the online market, and it could be viewed as a higher homolog of ALD-52, 1P-LSD, and 1B-LSD. The present study included the analytical characterization and involved various methods of mass spectrometry (MS), gas and liquid chromatography (GC and LC), nuclear magnetic resonance (NMR) spectroscopy, GC-solid-state infrared (GC-sIR) analysis, and Raman spectroscopy. The in vivo activity of 1V-LSD was assessed using the mouse head-twitch response (HTR), a 5-HT2A -mediated head movement that serves as a behavioral proxy in rodents for human hallucinogenic effects. Similar to LSD and other psychedelic drugs, the HTR induced by 1V-LSD was dose dependent, and the median effective dose for 1V-LSD was 373 nmol/kg, which was about a third of the potency of LSD (ED50  = 132.8 nmol/kg). Lysergamides containing the N1 -substituent typically act as weak partial agonists at the 5-HT2A receptor and are believed to serve as prodrugs for LSD. 1V-LSD is also likely to be hydrolyzed to LSD and serve as a prodrug, but studies to assess the biotransformation and receptor pharmacology of 1V-LSD should be performed to fully elucidate its mechanism of action.

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

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

New synthetic cannabinoids carrying a cyclobutyl methyl side chain: Human Phase I metabolism and data on human cannabinoid receptor 1 binding and activation of Cumyl-CBMICA and Cumyl-CBMINACA.

Synthetic cannabinoids (SCs) represent a large group of new psychoactive substances (NPS), sustaining a high prevalence on the drug market since their first detection in 2008. Cumyl-CBMICA and Cumyl-CBMINACA, the first representatives of a new subclass of SCs characterized by a cyclobutyl methyl (CBM) moiety, were identified in July 2019 and February 2020. This work aimed at evaluating basic pharmacological characteristics and human Phase I metabolism of these compounds. Human Phase I metabolites were tentatively identified by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QToF-MS) of urine samples and confirmed by a pooled human liver microsome (pHLM) assay. The basic pharmacological evaluation was performed by applying a competitive ligand binding assay and a functional activation assay (GTPγS) using cell membranes carrying the human cannabinoid receptor 1 (hCB1 ). Investigation of the human Phase I metabolism resulted in the identification of specific urinary markers built by monohydroxylation or dihydroxylation. Although Cumyl-CBMICA was primarily hydroxylated at the indole ring, hydroxylation of Cumyl-CBMINACA mainly occurred at the CBM moiety. Both substances acted as agonists at the hCB1 receptor, although substantial differences could be observed. Cumyl-CBMINACA showed higher binding affinity (Ki = 1.32 vs. 29.3 nM), potency (EC50 = 55.4 vs. 497 nM), and efficacy (Emax = 207% vs. 168%) than its indole counterpart Cumyl-CBMICA. This study confirms that substitution of an indole by an indazole core tends to increase in vitro potency, which is potentially reflected by higher in vivo potency. The emergence and disappearance of SCs distributed via online shops carrying a CBM moiety once more demonstrate the "cat-and-mouse" game between manufacturers and legislation.

Dataset allowing for the identification of three new synthetic cannabimimetics featuring a norbornyl methyl side chain by spectrometric and spectroscopic techniques.

Synthetic cannabimimetics (SC) are a diverse group of new psychoactive substances with varying potency and harm potential. New SCs appear on the drug market every year, and reliable and correct identification of these new derivatives independent from the matrix relies on the availability of verified spectra. Three new synthetic cannabimimetics featuring a norbornyl methyl side chain and varying core structure elements were identified in different seizures and forms. Cumyl-BC[2.2.1]HpMeGaClone and Cumyl-BC[2.2.1]HpMINACA were laced onto herbal blends, whereas Cumyl-BC[2.2.1]HpMICA was seized as a pure solid powder. The data collection process involves a comprehensive set of orthogonal analytical techniques allowing for the unambiguous identification of the respective endo- and exo-isomers. Furthermore, the diversity of analytical techniques allows a greater number of laboratories working in the field of forensic chemistry to confidently identify the substances described in our original research article [1]. Structure elucidation and analytical characterisation were performed within the EU-project ADEBAR plus using gas chromatography-mass spectrometry (GC-MS), gas chromatography-solid state infrared spectroscopy (GC-sIR), as well as solid and neat IR spectroscopy, Raman spectroscopy, liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS), and high resolution (HR)-LC-ESI-MS, and nuclear magnetic resonance (NMR) spectroscopy. The raw analytical data files are included in the Mendeley repository alongside the individual spectra in a universally importable format. The use of the universal JCAMP format for storage of the spectra facilitates database maintenance and enables seamless integration of the verified spectra. Thus, the dataset enables other researchers worldwide to identify these three new SCs confidently.

Cumyl-CBMICA: A new synthetic cannabinoid receptor agonist containing a cyclobutyl methyl side chain.

Since the beginning of the phenomenon of new psychoactive substances (NPS), synthetic cannabinoid receptor agonists (SCRAs) have been the largest and most prevalent subclass of these drugs in Europe. Many countries implemented specific legislation scheduling classes of substances defined on the basis of their chemical structure to reduce supply. We describe the identification and analytical characterization within the EU project ADEBAR plus of 1-(cyclobutylmethyl)-N-(2-phenylpropan-2-yl)-1H-indole-3-carboxamide which resulted in the formal notification through the Early Warning System of the European Monitoring Centre for Drug and Drug Addiction (EMCDDA). This is the first identification of this new SCRA worldwide and the analytical data was distributed (inter-)nationally right after identification in 2019. First, the substance was isolated from the herbal material using preparative high-performance liquid chromatography (HPLC). Structure elucidation and analytical characterization were performed using gas chromatography-mass spectrometry (GC-MS), gas chromatography-solid state infrared spectroscopy (GC-sIR), liquid chromatography-electrospray ionization-quadrupole time of flight-mass spectrometry (LC-ESI-qToF-MS), Raman spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy. The new compound contains a cyclobutyl methyl group as a side chain and has not been described in any patent to our knowledge. Based on the semisystematic nomenclature of SCRAs, we propose Cumyl-CBMICA as a short name for the compound.