Pharmacological insights emerging from the characterization of a large collection of synthetic cannabinoid receptor agonists designer drugs

Biomedicine & Pharmacotherapy 164 (2023) 114934 Available online 24 May 2023 0753-3322/© 2023 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Pharmacological insights emerging from the characterization of a large collection of synthetic cannabinoid receptor agonists designer drugs Claudia Gio´ e-Gallo a , b , Sandra Ortigueira a , b , Jos´ e Brea c , * , Iu Raïch d , e , Jhonny Azuaje a , b , M. Rita Paleo a , Maria Majellaro a , b , María Isabel Loza c , Cristian O. Salas f , Xerardo García-Mera b , Gemma Navarro d , e , ** , Eddy Sotelo a , b , *** a Centro Singular de Investigaci´ on en Química Biol´ oxica e Materiais Moleculares (CiQUS), Universidade de Santiago de Compostela, Santiago de Compostela 15782, Spain b Departamento de Química Org´ anica, Facultad de Farmacia, Universidade de Santiago de Compostela, Santiago de Compostela 15782, Spain c Centro Singular de Investigaci´ on en Medicina Molecular y Enfermedades Cr´ onicas (CiMUS), Universidade de Santiago de Compostela, Santiago de Compostela 15782, Spain d Department of Biochemistry and Physiology, School of Pharmacy and Food Science, Universitat de Barcelona, Barcelona 08028, Spain e Institute of Neurosciences (NeuroUB), Campus Mundet, University of Barcelona, Barcelona 08035, Spain f Department of Organic Chemistry, Faculty of Chemistry and Pharmacy, Pontificia Universidad Cat´ olica de Chile, Vicu˜ na Mackenna 4860, Macul, Santiago 7820436, Chile ARTICLE INFO Keywords: Synthetic cannabinoid receptor agonists SCRAs Synthetic cannabinoids NPS Abuse drugs Designer drugs CB 1 CB 2 Cannabinoids Indole Indazole ABSTRACT Synthetic cannabinoid receptor agonists (SCRAs) constitute the largest and most defiant group of abuse designer drugs. These new psychoactive substances (NPS), developed as unregulated alternatives to cannabis, have potent cannabimimetic effects and their use is usually associated with episodes of psychosis, seizures, dependence, organ toxicity and death. Due to their ever-changing structure, very limited or nil structural, pharmacological, and toxicological information is available to the scientific community and the law enforcement offices. Here we report the synthesis and pharmacological evaluation (binding and functional) of the largest and most diverse collection of enantiopure SCRAs published to date. Our results revealed novel SCRAs that could be (or may currently be) used as illegal psychoactive substances. We also report, for the first time, the cannabimimetic data of 32 novel SCRAs containing an (R) configuration at the stereogenic center. The systematic pharmacological profiling of the library enabled the identification of emerging Structure-Activity Relationship (SAR) and Structure-Selectivity Relationship (SSR) trends, the detection of ligands exhibiting incipient cannabinoid receptor type 2 (CB 2 R) subtype selectivity and highlights the significant neurotoxicity of representative SCRAs on mouse primary neuronal cells. Several of the new emerging SCRAs are currently expected to have a rather limited potential for harm, as the evaluation of their pharmacological profiles revealed lower potencies and/or efficacies. Conceived as a resource to foster collaborative investigation of the physiological effects of SCRAs, the library obtained can contribute to addressing the challenge posed by recreational designer drugs. 1. Introduction New psychoactive substances (NPS), also known as “designer drugs” or “legal highs” are a broad class of abuse drugs that have emerged on the illicit drug market [1,2]. They are usually conceived by performing structural modifications of existing controlled substances, thereby Abbreviations: NPS, New psychoactive substances; SCRAs, Synthetic cannabinoid receptor agonists; EMCDDA, European Monitoring Centre for Drugs and Drug Addiction; SAR, Structure-activity relationship; SSR, structure-selectivity relationship; GPCRs, G-protein-coupled receptors; CBRs, Cannabinoid receptors; CB1Rs, Cannabinoid 1 receptors; CB2Rs, Cannabinoid 2 receptors; CD, Circular dichroism. * Corresponding author. ** Corresponding author at: Department of Biochemistry and Physiology, School of Pharmacy and Food Science, Universitat de Barcelona, Barcelona 08028, Spain. *** Corresponding author at: Centro Singular de Investigaci´ on en Química Biol´ oxica e Materiais Moleculares (CiQUS), Universidade de Santiago de Compostela, Santiago de Compostela 15782, Spain. E-mail addresses: [email protected] (J. Brea), [email protected] (G. Navarro), [email protected] (E. Sotelo). Contents lists available at ScienceDirect Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha https://doi.org/10.1016/j.biopha.2023.114934 Received 28 December 2022; Received in revised form 1 April 2023; Accepted 22 May 2023 Biomedicine & Pharmacotherapy 164 (2023) 114934 2 mimicking their pharmacological effects, and circumventing governmental legislation [1–3]. NPS are generally grouped into four somewhat overlapping functional categories (according to their chemical structure and pharmacological effects): stimulants, hallucinogens, depressants, and cannabinoids [1,3,4]. NPS provide users with novel alternatives to traditional and well-characterized drugs of abuse (e.g., amphetamines, heroin, cocaine, and cannabis) [4]. They are undergoing a period of proliferation and diversification, with a significant increase in the challenges faced by emergency and critical care physicians, toxicologists, and regulatory governmental authorities [5,6]. Synthetic cannabinoid receptor agonists (SCRAs) have proliferated during the last decade, emerging as the largest and most defiant group of NPS controlled by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) [1,4,7]. Initially conceived for therapeutic purposes, SCRAs have become a major public health concern due to their adverse effects, limited safety profile, evolving structure and diversity (Fig. 1) [6–8]. In the middle of the 2000 s, SCRAs entered the abuse drug market in the form of herbal mixtures (e.g., “Spice”, “K2′′, “AK-47 24 Karat Gold”). Their use was popularized thanks to their image as a "legal" inexpensive alternative to cannabis (e.g., psychoactive and analgesic effects), which, at the time, was undetectable to routine drug testing [6,7,9]. SCRAs and the main psychoactive component of cannabis [Δ 9 -tetrahydrocannabinol (Δ 9 -THC)] do not differ only in their structure, but also qualitatively and quantitatively in their pharmacological profile [10,11]. [(the former usually being more potent and efficacious as cannabinoid receptor agonists than Δ 9 -THC which is a partial agonist of both Cannabinoid receptors (CBRs)]. However, in vivo studies have evidenced that SCRAs produce most of the characteristic effects of cannabis consumption in rodents (e.g., hypothermia, bradycardia, catalepsy, hypo-locomotion and antinociception) [4]. It is well documented that SCRAs exert their psychoactive effects through their agonism on the central cannabinoid receptor 1 (CB 1 R) [4,5,8] although they often exhibit a more pronounced agonist effect on cannabinoid receptor 2 (CB 2 R) [4,5,8]. The exception to this trend is a newly emerged SCRA that has been reported to have CB 1 R but not CB 2 R activation potential [12]. CBRs are classical G-protein-coupled receptors (GPCRs), that belong to the large rhodopsin-like class A family [13]. Both CBRs are coupled to G i proteins, inhibiting adenylate cyclase and inducing a reduction in the intracellular cyclic adenosine monophosphate (cAMP) levels [14]. CB 1 Rs are one of the most abundant GPCRs in the brain [14,15], also being highly expressed in the peripheral nervous system and other peripheral tissues. In contrast, CB 2 Rs are poorly expressed in brain tissue, being mainly restricted to microglial cells [16]. CB 2 Rs are predominantly expressed in immune cells [17], and are actively implicated in inflammation [18]. The paucity of thorough studies delineating the pharmacology, toxicity, and abuse liability of new SCRAs poses a threat to public health [6,19]. SCRAs consumption has been unequivocally associated with Fig. 1. Structure of Δ 9 -THC and representative SCRAs [7,20,24,25]. C. Gio´ e-Gallo et al. Biomedicine & Pharmacotherapy 164 (2023) 114934 3 severe adverse effects (e.g., psychosis, delirium, cardiotoxicity, seizures, acute kidney injury, hypothermia), being implicated in numerous deaths in several countries [4,5,9,20,21]. Because many abused SCRAs (Fig. 1) are unknown before they are detected by forensic scientists, their effects on humans are poorly or not known at all. [2,19] Likewise, clinical and forensic toxicology laboratories are confronted by analytical challenges when dealing with this family of abuse substances. The huge number and diversity of compounds, their evolving structure and the limited availability of SCRA standards, metabolites and analytical methods complicates these tasks [19,22,23]. Since their early detection on the illicit drug market, [1] SCRAs have undergone continuous structural evolution and diversification. Representative structures can be found in Fig. 1. Structural modifications not only affect the central heterobicyclic core, but also involve functional group replacements (ketones, esters, and amides) and diversification of the alkyl chains [5,7,8,21]. The most prevalent chemotypes in SCRAs are indole-3-carboxamides and indazole-3-carboxamides featuring pendant amino acid derivatives [5,7,8]. Inspired by early Pfizer patents [26], which claimed synthetic CBR agonists as analgesics, the stereocenter within the pendant amino acid in these compounds is always in the (S) configuration [4,21,24,27–29]. Although a recent study explored the role of the stereodisposition of the iso-propyl and tert-butyl side chains of representative SCRAs (Fig. 1), [24] this issue remains unresolved [30]. The evolving structure and limited availability of large SCRAs collections by the scientific community has impeded their in-depth characterization from a pharmacological, pharmacokinetic, and toxicological point of view [22]. Several academic groups have generated valuable structural and pharmacological data that sheds light on the complex physiological actions elicited by the SCRAs [31,32]. These studies generally focus on the most frequently abused compounds (and their analogues) and usually exclusively evaluate the (S) enantiomers, while the corresponding (R) stereoisomers remain almost unexplored [7,8,21,24,31]. Moreover, published available pharmacological data of SCRAs are usually incomplete. It is well accepted that both the affinity of a drug for its receptor and its ability to produce an effect are important features during ligand characterization and should be determined. Accordingly, there is a demand for large and stereochemically diverse collections of SCRAs for detailed characterization to be shared within the scientific community, in a collaborative manner, to unravel the molecular basis of the SCRAs physio/toxicological actions. Furthermore, these libraries provide reference standards to forensic laboratories and regulatory authorities [22] thus enabling the fight against abuse substances by anticipating drugs that might enter the illegal market. In addition, comprehensive SAR data would allow a better understanding of the structural features that govern CBRs activation and their translation into new therapeutic opportunities. Herein we report the synthesis and pharmacological characterization (at CB 1 R and CB 2 R) of the largest published collection of SCRAs (Fig. 2, 64 ligands). The library, which exhibits skeletal, stereochemical and functional diversity, as well as the most frequent alkyl groups at positions R 1 and R 2 , was conceived as a collaborative resource to foster the investigation of the physiological effects of SCRAs. The pharmacological profiling (binding affinities and functional data) enabled both the analysis of the most salient features emerging from the structure-activity relationship (SAR) and structure-selectivity relationship (SSR) in these series, and the identification of some CB 2 R selective ligands. We also examined the neurotoxicity of representative SCRAs on mouse primary neuronal cells, preliminarily demonstrating a significant neurotoxic effect for some of the frequently abused ligands. 2. Materials and methods 2.1. Chemistry. General procedure for the synthesis of SCRAs 22-25 A mixture of the 1-alkyl-1H-indole (or indazole)−3-carboxylic acid (1 mmol), the corresponding amino acid derivative (30 or 31) (1,5 mmol), HATU (1,5 mmol) and DIPEA (4 mmol) in CH 2 Cl 2 (2 mL) was stirred with orbital stirring at room temperature for 24 h. After completion of the reaction, water was added, and the mixture was extracted with CH 2 Cl 2 . The organic phase was dried over MgSO 4 , filtered, and concentrated. The resulting product was purified by column chromatography on silica gel. The purity of all prepared compounds was established by high-performance liquid chromatography (HPLC) and showed to be >95%. A detailed description of the experimental protocols, equipment and techniques used in the synthesis of targeted ligands, as well as the structural and spectroscopic data obtained for all the compounds described is given in the Supplementary Information. 2.2. Circular dichroism (CD) CD spectra of ligands (>98%) were recorded on a Jasco-815 system equipped with a Peltier-type thermostatic accessory (CDF-426S, Jasco). Measurements were carried out at 20 ºC using a 1-mm quartz cell in a volume of 300–350 μ L. Compounds (0.1 mg) were dissolved in MeOH Fig. 2. General structure of the library and diversity elements included. C. Gio´ e-Gallo et al. Biomedicine & Pharmacotherapy 164 (2023) 114934 4 (1.0 mL). The instrument settings were bandwidth, 1.0 nm; data pitch, 1.0 nm; speed, 500 nm/min; accumulation, 10; wavelengths, 400–190 nm. 2.3. Binding assays Radioligand binding competition assays of CB 1 receptors were carried out in polypropylene 96-well plates by incubating 5 µg of membranes from Chinese hamster ovary (CHO)-CB 1 C3 cell line (PerkinElmer) with 1 nM [ 3 H]-CP55940 (Perkin Elmer) and test compounds in binding buffer (50 mM Tris-HCl, 5 mM MgCl 2 , 1 mM EDTA, 0.5% BSA. pH:7,4). Non-specific binding was determined in the presence of 10 µM Surinabant. The reaction mixture was incubated at 30 ◦C for 60 min, then 200 μ L were transferred to GF/C 96-well plate (Millipore, Madrid, Spain) and washed four times with 250 μ L wash buffer (50 mM Tris-HCl, 5 mM MgCl 2 , 1 mM EDTA, 0.5% BSA. pH:7,4). Radioactivity was detected in a microplate beta scintillation counter (Microbeta Trilux, PerkinElmer, Madrid, Spain). Radioligand binding competition assays of CB 2 receptors were carried out in polypropylene 96-well plates by incubating 5 µg of membranes from Human embryonic kidney 293 cells (HEK)-CB 2 cell line with 0.2 nM [ 3 H]-CP55940 (Perkin Elmer) and test compounds in binding buffer (50 mM Tris-HCl, 5 mM MgCl 2 , 2.5 mM EGTA, 0.1% BSA. pH: 7,4). Non-specific binding was determined in the presence of 10 µM GW405833. The reaction mixture was incubated at 30 ◦C for 90 min, then 200 μ L were transferred to GF/C 96-well plate (Millipore, Madrid, Spain) and washed four times with 250 μ L wash buffer (50 mM Tris-HCl, 5 mM MgCl 2 , 2.5 mM EGTA, 1% BSA. pH: 7,4). Data was fitted to 4-parameter logistic equation by employing GraphPad Prism software (V7.0) and K i data was calculated with the equation: K i =IC 50 /(1+(F/K D )) where IC 50 was the concentration of the ligand that displaced the specific binding of the radioligand in a 50%; K D is the dissociation constant of the radioligand, and F is the concentration of the radioligand employed in the assay. 2.4. Functional experiments. Cell Culture and transient transfection HEK-293 T cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) medium (Gibco, Paisley, Scotland, United Kingdom) supplemented with 2 mM L-glutamine, 100 U/mL penicillin/streptomycin, MEM Non-Essential Amino Acids Solution (1/100) and 5% (v/v) heat inactivated Fetal Bovine Serum (FBS) (Invitrogen, Paisley, Scotland, United Kingdom). Cells were maintained in a humid atmosphere of 5% CO 2 at 37ºC. Cells were transiently transfected with the PEI (Polyethyleneimine, Sigma, St. Louis, MO, United States) method as previously described [33] and used for functional assays 48 h later. 2.5. Neuronal primary cultures To prepare primary neurons, brains from fetuses of pregnant CD1 mice were removed (gestational age: 19 days). Neurons were isolated as described in Hradsky et al. [34] Briefly, after removal of the meninges, samples were dissected and digested with 0.25% trypsin (20 min at 37ºC). The effect of trypsin was stopped by adding an equal volume of culture medium (supplemented DMEM). A single-cell suspension was obtained by repeated pipetting followed by passage through a 100 µm-pore mesh. Pelleted (7 min, 200g) cells were resuspended in 2 mL of supplemented DMEM and seeded at a density of 3.5 ×10 5 cells/mL in 6-well plates. After 24 h, the medium was replaced by neurobasal medium supplemented with 2 mM L-glutamine, 100 U/mL penicillin/streptomycin and 2% (v/v) B27 medium (GIBCO, Waltham, MA, USA). Primary neurons were assayed after 14 days in culture. Using NeuN as a marker, the percentage of neurons in the culture was >90%. 2.6. cAMP Determination Signaling experiments have been performed as previously described. [35] Two hours before initiating the experiment, HEK-293 T cell-culture medium was replaced by serum-starved DMEM medium. Then, cells were detached, resuspended in growing medium containing 50 mM zardaverine (Tocris, Bristol, UK) and placed in 384-well microplates (2500 cells/well). Cells were pretreated (15 min) with cannabinoid compounds (1 nM to 100 µM) -or vehiclebefore adding 0.5 mM forskolin (Tocris, Bristol, UK) to induce cAMP accumulation. Readings were performed after 15 min incubation at 25ºC. Homogeneous Time Resolved Fluorescence (HTRF) energy transfer measures were performed using the Lance Ultra cAMP kit (PerkinElmer, Waltham, MA, United States). Fluorescence at 665 nm was analyzed in a PHERA star Flagship microplate reader equipped with an HTRF optical module (BMG Lab Technologies, Offenburg, Germany). 2.7. Viability assay Primary cultures of striatal neurons were treated for 24 h with some representative SCRAs (100 nM). Afterwards, cells were scrapped from the plate and resuspended in neurobasal medium supplemented with 2 mM L-glutamine, 100 U/mL penicillin/streptomycin and 2% (v/v) B27 (GIBCO). Trypan blue staining was performed mixing 1 part of 0.4% trypan blue and 1 part of cell suspension in a plastic tube. After ~3 min of incubation at room temperature, 10 μ L of the mixture were sampled in a Neubauer chamber and counted with a Countess II FL (Life Technologies, California, CA, USA). The unstained (viable) and stained (nonviable) cells were counted separately, and the percentage of viability was calculated as: total number of viable cells/total number of cells x 100. 2.8. Data and statistical analysis K i and EC 50 values were obtained by fitting the data with nonlinear regression using Prism 9 software (GraphPad, San Diego, CA). Results are the mean of four experiments (n =4), each performed in duplicate. Data are represented as mean ±standard error of mean (SEM) with statistical significance set at P<0.05. The number of samples (n) in each experimental condition is indicated in the corresponding figure legend. Outliers were assessed by the ROUT method,[ 57 ] thus any sample was excluded assuming a Q value of 1% in GraphPad Prism 9. Comparisons among experimental groups were performed by Student’s t test or one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons post-hoc test using GraphPad Prism 9, as indicated. 3. Results and discussion 3.1. Design The obtained library (Fig. 2), which contains five diversity points (R 1 , R 2 , R 3 , X, and the stereodisposition of R 2 ), was designed to systematically explore the structural determinants governing the SCRAs’ bioactivity profile. We conceived a 64 members collection (Fig. 2) containing skeletal (indole and indazole), functional (esters and amides), alkylic (iso-propyl and tert-butyl) and stereochemical [(S) stereoisomers and (R) stereoisomers] diversity. The obtained library also features the four most frequent residues present at position 1 (n-pentyl, 5-fluoropentyl, 4-fluorobenzyl and cyclohexylmethyl) of the SCRA´s heterocyclic cores (Fig. 2). To properly compile and evaluate SAR trends, the library contains 32 representative knon SCRAs. Notably, 32 of the 64 cannabinoid receptor agonists herein documented had not been previously reported (e.g., derivatives featuring the (R) C. Gio´ e-Gallo et al. Biomedicine & Pharmacotherapy 164 (2023) 114934 5 configuration within the pendant aminoacid residue). Therefore, to our knowledge, this is the largest SCRA library ever published to date. For the sake of simplicity, and to allow a more direct appreciation of emerging SAR trends, the 64-membered collection was divided into four subsets, representing functional (esters or amides) and skeletal (indoles or indazoles) diversity (Scheme 1). 3.2. Chemistry The targeted SCRAs collection (Scheme 1, ligands 22a-p, 23a-p, 24ap and 25a-p) was assembled following robust and well-established amide synthesis protocols (Scheme 1). The required carboxylic acids (26a-h) were prepared (Scheme 1, Method C), by saponification of the N-alkylated esters 28a-h. Esters 28 were obtained by alkylation of precursors 27 [methyl 3-indolecarboxylate (27a) and methyl 3-indazolecarboxylate (27b)] with four alkyl halides (29a-d) under basic conditions (Scheme 1, Methods A or B). The two regioisomers obtained during the alkylation of methyl 3-indazolecarboxylate (27b) were separated by chromatographic techniques (Scheme 1, Method B). The unambiguous identity of the major N 1 -alkylated compound (3:1 ratio) (28e-h) was confirmed by NMR experiments. Commercially available (Sigma Aldrich) enantiopure (ee ≥99%) amino acid derivatives [methyl glycinate derivatives (30a-d) and glycinamide derivatives (31a-d)] of both the (R) and (S) series, bearing either iso-propyl or tert-butyl groups at R 1 , were used for the synthesis of targeted compounds (Scheme 1, Method D). The amide coupling protocol (HATU) afforded the targeted derivatives (22ap−25ap) in moderate (47%) to excellent yields (96%) ensuring retention of the enantiomeric fidelity. [36]. All reactions were monitored by thin-layer chromatography (TLC) employing UV light, iodine, or a dissolution of phosphomolybdic acid for the compound’s detection. After completion of the reaction, the solvent was evaporated to dryness and the isolated solid was purified by column chromatography on silica gel. A detailed description of the synthetic methods and the complete structural, spectroscopic, and analytical data for all compounds is provided in the experimental part and Supporting Information. Although the carboxylic acids 26 are non-chiral, and racemization is mechanistically unlikely under the employed amide coupling conditions, [36,37] all members of the library were routinely controlled by analytical chiral HPLC to confirm that the stereocenter remained intact in the final compounds (see experimental part and Supporting Information). A selection of the HPLC traces obtained for representative enantiomeric pairs is presented in Fig. 3. Circular dichroism (CD) spectroscopy [38,39] is an orthogonal and versatile biophysical technique that provides valuable structural information [40,41] of proteins, peptides, small molecules, and functional macromolecules. As part of the characterization of herein described SCRAs, it was envisioned that they contain the two structural requirements to interact with circularly polarized light used in CD spectroscopy: a chromophore (indole or indazole cores) and an enantiopure aminoacidic residue (chirality). Scheme 1. Synthesis of the SCRAs 22-25. Method A) NaH, DMF, rt, 24 h. Method B) NaH, DMF, rt, 24 h and then chromatographic separation to isolate the regioisomers. Method C) NaOH, dioxane, 120ºC, 12 h. Method D) HATU, DIPEA, CH 2 Cl 2 , rt, 24 h. C. Gio´ e-Gallo et al. Biomedicine & Pharmacotherapy 164 (2023) 114934 6 Accordingly, it was decided to study the circular dichroism (CD) spectra of representative SCRAs pairs, all experiments were performed employing enantiopure derivatives eliciting ee ≥98%. As observed (Fig. 3), irrespectively of the functional group within the amino acid residue (ester or amide), each single stereoisomer of the enantiomer pairs elicited symmetrical CD spectra, mirrored at the x-axis and with opposite sign. The absorbance of circularly polarized light by the chiral center in the SCRAs gives rise to a characteristic CD signal at low wavelength (far-UV: 240 nm) that provides information about the secondary structure (stereochemistry). On the other hand, the heterocyclic chromophore, despite being non-chiral, gives rise to CD signals in the near-UV (250–330 nm), because they are affected by the adjacent chiral environment. The characteristic CD signal around 240 nm allowed the unambiguous assignment of the absolute configuration of each enantiomer (Fig. 3). At this wavelength, the (S) enantiomer showed a negative Cotton effect (blue line), while the (R) stereoisomer gave a positive Cotton effect (red line). These additional experiments confirmed that all the obtained ligands contained a stereo-defined Fig. 3. HPLC traces of representative enantiopure esters and amides SCRAs (left) and circular dichroism spectra of enantiomer pairs (right). At 240 nm the (S) enantiomer showed a negative Cotton effect (blue line), while the (R) stereoisomer gave a positive Cotton effect (red line). C. Gio´ e-Gallo et al. Biomedicine & Pharmacotherapy 164 (2023) 114934 7 configuration and were pharmacologically evaluated as enantiopure compounds (ee >98%). In view of its robustness, performance and minimal sample preparation requirements, circular dichroism spectroscopy offers unique insights to rapidly assess the configuration of SCRA enantiopure samples. 3.3. Biological evaluation One of the aims of this study was to address the limited availability of large SCRA collections and, consequently, the lack of homogeneous and reliable pharmacological data of these drugs of abuse. Thus, the cannabimimetic profile of the 64 SCRAs was studied in vitro by evaluating its functional activity (EC 50 and E max ) and binding affinity (K i ) for human CB 1 Rs and CB 2 Rs according to established experimental protocols. All the studied indole and indazole derivatives bind and activated both CB 1 R and CB 2 R. The pharmacological data (Tables 1–4) are expressed as pEC 50 ±SEM or pK i ±SEM [and EC 50 and K i (nM)]. The data obtained for Δ 9 -THC, Surinabant, CP55,940 and GW405833, using described experimental protocols, were reported in tables for the sake of comparison. The in vitro affinity data was acquired via competitive radioligand binding assays using membrane preparations of human CB 1 Rs and CB 2 Rs transfected in CHO-CB1 C3 cells and HEK-293 T cells respectively and a well-characterized tritiated ligand ([ 3 H]CP55,940). As cannabinoid receptors are GPCRs coupled to the Gi protein, the functional activity of synthesized SCRAs at CB 1 Rs and CB 2 Rs was assessed by determining the decreases in cAMP levels induced by forskolin treatment. During functional experiments, the maximum effects of ligands 22–25 and Δ 9 -THC were normalized to a maximal efficacious concentration of CP 55,940. Data for each experiment were normalized to the change in fluorescence produced by a maximally effective Table 1 Structure, affinity, and functional data obtained for the indole esters 22a-p. Compound R 1 R 2 CB 1 R CB 2 R SI K i CB 1 R/ K i CB 2 R pEC 50 ±SEM a) [EC 50 nM)] E max ±SEM b) (% CP55940) pK i ±SEM c) [K i nM)] pEC 50 ±SEM a) [EC 50 nM)] E max ±SEM b) (% CP55940) pK i ±SEM d) [K i nM)] 22a (AMBICA) 21 Pentyl (S) i-Pr 7.27 ±0.05 [53.7] 80% 7.58 ±0.07 [26.6] 7.34 ±0.05 [45.2] 77% 7.79 ±0.09 [16.4] 1.62 22b (MDMB-PICA) 21 Pentyl (S) t-Bu 7.55 ±0.03 [28.4] 74% 8.14 ±0.05 [7.20] 8.94 ±0.07 [1.14] 95% 8.54 ±0.03 [2.90] 2.48 22c (5 F-AMBICA) 21 5-F-Pentyl (S) i-Pr 8.34 ±0.06 [4.62] 134% 8.09 ±0.08 [8.10] 8.98 ±0.06 [1.05] 74% 8.38 ±0.07 [4.23] 1.91 22d (5 F-MDMB-PICA) 21 5-F-Pentyl (S) t-Bu 9.15 ±0.08 [0.70] 88% 8.59 ±0.09 [2.61] 9.29 ±0.07 [0.51] 103% 9.15 ±0.05 [0.70] 3.73 22e (MMB-CHMICA) 21 CH 2 -Cy (S) i-Pr 7.74 ±0.09 [18.1] 79% 7.98 ±0.06 [10.5] 8.64 ±0.08 [2.27] 68% 8.47 ±0.07 [3.44] 3.05 22 f (MDBM-CHMICA) 21 CH 2 -Cy (S) t-Bu 8.82 ±0.07 [1.50] 58% 10.05 ±0.12 [0.09] 9.22 ±0.09 [0.60] 61% 9.74 ±0.08 [0.18] 0.50 22 g (MMB-FUBICA) 21 4-F-Bn (S) i-Pr 7.94 ±0.05 [11.4] 88% 7.32 ±0.03 [47.8] 7.32 ±0.03 [48.2] 101% 7.70 ±0.05 [20.0] 2.39 22 h (MDMB-FUBICA) 21 4-F-Bn (S) t-Bu 8.16 ±0.07 [6.99] 75% 8.42 ±0.08 [3.83] 8.67 ±0.06 [2.12] 82% 8.95 ±0.07 [1.12] 3.42 22i* Pentyl (R) i-Pr 7.14 ±0.06 [72.3] 66% 6.20 ±0.04 [632] 8.34 ±0.07 [4.60] 94% 7.43 ±0.09 [37.1] 17.1 22j* Pentyl (R) t-Bu 7.72 ±0.08 [19.0] 126% 6.60 ±0.02 [251] 7.24 ±0.04 [57.0] 66% 6.69 ±0.03 [110] 2.28 22k* 5-F-Pentyl (R) i-Pr 8.16 ±0.09 [6.92] 137% 7.22 ±0.05 [60.2] 8.11 ±0.07 [7.79] 104% 7.72 ±0.05 [19.2] 3.14 22 l* 5-F-Pentyl (R) t-Bu 7.83 ±0.05 [14.9] 137% 6.89 ±0.09 [127] 9.60 ±0.06 [0.25] 126% 8.21 ±0.04 [6.11] 20.8 22 m* CH 2 -Cy (R) i-Pr 7.86 ±0.06 [13.9] 87% 7.12 ±0.06 [76.4] 7.21 ±0.08 [61.6] 107% 7.92 ±0.08 [12.1] 6.31 22 n* CH 2 -Cy (R) t-Bu 7.75 ±0.04 [17.6] 89% 6.82 ±0.07 [151] 7.64 ±0.03 [22.8] 64% 7.27 ±0.03 [53.5] 2.82 22o* 4-F-Bn (R) i-Pr 7.71 ±0.06 [19.7] 109% 6.84 ±0.04 [145] 8.30 ±0.07 [5.01] 91% 8.10 ±0.07 [8.21] 17.6 22p* 4-F-Bn (R) t-Bu 7.88 ±0.07 [13.2] 120% 6.75 ±0.06 [179] 8.03 ±0.06 [9.43] 49% 7.64 ±0.05 [22.8] 7.85 Δ 9 -THC - - 7.47 ±0.16 [33.8] 51% - 6.62 ±0.25 [238] 21% - - Surinabant - - - - 8.52 ±0.08 [3.00] - - 6.39 ±0.06 [400] 0.0075 GW405833 - - - - 5.32 ±0.05 [4772] - - 8.23 ±0.07 [5.80] 822 CP55,940 - - - - 8.99 ±0.11 [1.01] - - 10.21 ±0.32 [0.13] 7.76 a ) Functional activity assessed in transfected HEK-293 T cells by determining cAMP levels after forskolin stimulation. b ) E max values were normalized to a maximal efficacious concentration of CP55,940. c ) Displacement of specific [ 3 H]CP 55,940 binding in membrane preparations of human CB 1 Rs transfected in CHO-CB1 C3 cell line expressed as K i in nM (n =3) or percentage displacement of specific binding at a concentration of 1 μ M (n =2). d ) Displacement of specific [ 3 H]CP 55,940 binding in membrane preparations of human CB 2 Rs transfected in HEK-293 T cells expressed as K i in nM (n =3) or percentage displacement of specific binding at a concentration of 1 μ M (n =2). * These SCRAs have not been previously described. C. Gio´ e-Gallo et al. Biomedicine & Pharmacotherapy 164 (2023) 114934 8 concentration of CP 55,940. Representative dose-response curves obtained for selected ligands during the functional studies and binding affinity determinations at CB 1 R and CB 2 R, are presented in Fig. 4. A detailed description of the protocols is reported in the experimental part. The dose-response curves obtained for all the ligands documented here are given in the Supporting Information. The pharmacological data obtained for the 64-membered collection are presented in Tables 1–4, each one containing 16 derivatives [the first 8 bearing the (S) configuration at the pendant amino acid residue and the other 8 the (R) configuration]. Tables 1 and 2 present the pharmacological data of esters (indoles and indazoles respectively) while Tables 3 and 4 report the data obtained for amides (indole and indazoles respectively). Novel compounds are indicated with an asterisk (Tables 1–4) and already described SCRAs with their common acronym in the illicit drug market (Tables 1–4). The original articles describing the known compounds are listed in tables. [7,21,24] To facilitate the identification of the most salient features of the structure-selectivity relationship throughout the series, the selectivity index (SI), calculated using the affinity data (K i CB 2 R/K i CB 1 R), was also reported in Tables 1–4. 3.4. Structure-Activity Relationship analysis The binding affinities (pK i ), potencies (pEC 50 ) and efficacies (E max ) obtained during the pharmacological evaluation of the library (22ap−25ap) at CB 1 R and CB 2 R are presented in Table 1−4. The affinity and functional data herein determined for known SCRAs (22–25ah) are in the same range as reported (see Table 1S, supporting information). [5,7,8,10,20] For comparative purposes the phytocannabinoid Table 2 Structure, affinity, and functional data obtained for the indazole esters 23a-p. Compound R 1 R 2 CB 1 R CB 2 R SI K i CB 1 R/ K i CB 2 R pEC 50 ±SEM a) [EC 50 nM)] E max ±SEM b) (% CP55940) pK i ±SEM c) [K i nM)] pEC 50 ±SEM a) [EC 50 nM)] E max ±SEM b) (% CP55940) pK i ±SEM d) [K i nM)] 23a (AMB) 21 Pentyl (S) i-Pr 8.56 ±0.08 [2.73] 72% 8.39 ±0.09 [4.17] 8.79 ±0.08 [1.61] 62% 8.89 ±0.06 [1.30] 3.21 23b (MDMB-PINACA) 21 Pentyl (S) t-Bu 8.59 ±0.07 [2.60] 66% 8.68 ±0.09 [2.10] 9.89 ±0.09 [0.13] 68% 9.70 ±0.04 [0.26] 8.08 23c (5 F-AMB) 21 5-F-Pentyl (S) i-Pr 8.90 ±0.04 [1.27] 90% 8.77 ±0.06 [1.74] 9.24 ±0.06 [0.58] 81% 9.30 ±0.07 [0.52] 3.35 23d (5FMDMBPINACA) 21 5-F-Pentyl (S) t-Bu 9.24 ±0.06 [0.58] 75% 9.10 ±0.09 [0.80] 10.0 ±0.09 [0.10] 88% 10.0 ±0.08 [0.10] 8.00 23e (AMB-CHMINACA) 21 CH 2 -Cy (S) i-Pr 8.69 ±0.07 [2.02] 104% 8.72 ±0.05 [1.90] 8.05 ±0.05 [8.82] 83% 9.30 ±0.07 [0.51] 3.73 23 f (MDMB-CHMINACA) 21 CH 2 -Cy (S) t-Bu 9.12 ±0.05 [0.76] 84% 9.00 ±0.07 [1.08] 8.72 ±0.06 [1.92] 76% 9.52 ±0.08 [0.33] 3.27 23 g (AMB-FUBINACA) 21 4-F-Bn (S) i-Pr 8.64 ±0.09 [2.29] 115% 8.14 ±0.03 [3.90] 9.92 ±0.08 [0.12] 80% 9.22 ±0.05 [0.62] 6.29 23 h (MDMBFUBINACA) 21 4-F-Bn (S) t-Bu 9.04 ±0.07 [0.92] 81% 9.10 ±0.02 [0.83] 9.77 ±0.06 [0.17] 82% 9.52 ±0.07 [0.30] 2.77 23i* Pentyl (R) i-Pr 8.04 ±0.06 [9.02] 58% 7.04 ±0.04 [91.6] 8.59 ±0.09 [2.6] 98% 8.18 ±0.06 [6.61] 13.8 23j* Pentyl (R) t-Bu 7.67 ±0.04 [21.6] 100% 6.36 ±0.07 [435] 8.38 ±0.07 [4.2] 104% 7.85 ±0.04 [14.7] 29.6 23k* 5-F-Pentyl (R) i-Pr 8.10 ±0.06 [7.91] 67% 7.63 ±0.08 [23.5] 8.94 ±0.05 [1.14] 70% 8.59 ±0.09 [2.60] 9.04 23 l* 5-F-Pentyl (R) t-Bu 7.29 ±0.03 [51.3] 66% 7.09 ±0.08 [81.1] 9.27 ±0.07 [0.54] 87% 7.86 ±0.05 [13.3] 6.10 23 m* CH 2 -Cy (R) i-Pr 8.18 ±0.05 [6.59] 130% 7.61 ±0.05 [24.4] 8.66 ±0.06 [2.17] 92% 8.85 ±0.07 [1.40] 17.4 23 n* CH 2 -Cy (R) t-Bu 8.28 ±0.07 [5.26] 138% 7.42 ±0.03 [38.4] 8.75 ±0.06 [1.79] 106% 8.10 ±0.06 [8.05] 4.77 23o* 4-F-Bn (R) i-Pr 8.28 ±0.04 [5.22] 134% 7.56 ±0.07 [29.7] 8.83 ±0.08 [1.49] 118% 9.00 ±0.07 [1.20] 24.7 23p* 4-F-Bn (R) t-Bu 8.36 ±0.09 [4.35] 152% 7.16 ±0.03 [68.4] 7.88 ±0.04 [13.2] 91% 8.17 ±0.09 [6.70] 10.2 Δ 9 -THC - - 7.47 ±0.16 [33.8] 51% - 6.62 ±0.25 [238] 21% - - Surinabant - - - - 8.52 ±0.08 [3.00] - - 6.39 ±0.06 [400] 0.0075 GW405833 - - - - 5.32 ±0.05 [4772] - - 8.23 ±0.07 [5.80] 822 CP55,940 - - - - 8.99 ±0.11 [1.01] - - 10.21 ±0.32 [0.13] 7.76 a ) Functional activity assessed in transfected HEK-293 T cells by determining cAMP levels after forskolin stimulation. b ) E max values were normalized to a maximal efficacious concentration of CP55,940. c ) Displacement of specific [ 3 H]CP 55,940 binding in membrane preparations of human CB 1 Rs transfected in CHO-CB1 C3 cell line expressed as K i in nM (n =3) or percentage displacement of specific binding at a concentration of 1 μ M (n =2). d ) Displacement of specific [ 3 H]CP 55,940 binding in membrane preparations of human CB 2 Rs transfected in HEK-293 T cells expressed as K i in nM (n =3) or percentage displacement of specific binding at a concentration of 1 μ M (n =2). * These SCRAs have not been previously described. C. Gio´ e-Gallo et al. Biomedicine & Pharmacotherapy 164 (2023) 114934 9 Table 3 Structure, affinity, and functional data obtained for the indole amides 24a-p. Compound R 1 R 2 CB 1 R CB 2 R SI K i CB 1 R/ K i CB 2 R pEC 50 ±SEM a) [EC 50 nM)] E max ±SEM b) (% CP55940) pK i ±SEM c) [K i nM)] pEC 50 ±SEM a) [EC 50 nM)] E max ±SEM b) (% CP55940) pK i ±SEM d) [K i nM)] 24a (AB-PICA) 24 Pentyl (S) i-Pr 6.92 ±0.02 [120] 85% 7.76 ±0.03 [17.5] 8.36 ±0.07 [4.32] 120% 7.95 ±0.04 [11.2] 1.56 24b (AD-BICA) 24 Pentyl (S) t-Bu 8.79 ±0.07 [1.64] 76% 9.10 ±0.08 [0.80] 9.00 ±0.09 [1.00] 105% 9.30 ±0.09 [0.52] 1.54 24c (5 F-AB-PICA) 24 5-FPentyl (S) i-Pr 8.31 ±0.07 [4.95] 109% 7.83 ±0.07 [14.8] 8.67 ±0.09 [2.15] 123% 8.07 ±0.05 [8.60] 1.72 24d (5 F-AD-BICA) 24 5-FPentyl (S) t-Bu 7.88 ±0.03 [13.1] 109% 9.05 ±0.09 [0.91] 9.62 ±0.05 [0.24] 122% 9.15 ±0.09 [0.74] 1.23 24e (AB-CHMICA) 7 CH 2 -Cy (S) i-Pr 9.60 ±0.07 [0.25] 95% 8.62 ±0.09 [2.43] 8.70 ±0.07 [1.98] 86% 7.45 ±0.04 [35.3] 0.07 24 f (ADBCHMICA) 7 CH 2 -Cy (S) t-Bu 6.66 ±0.05 [221] 70% 8.96 ±0.07 [1.10] 8.09 ±0.03 [8.20] 73% 9.12 ±0.06 [0.76] 1.45 24 g (AB-FUBICA) 24 4-F-Bn (S) i-Pr 7.94 ±0.04 [11.5] 89% 7.45 ±0.04 [35.2] 8.04 ±0.07 [9.15] 86% 6.22 ±0.06 [83.2] 0.42 24 h (ADBFUBICA) 24 4-F-Bn (S) t-Bu 7.19 ±0.06 [64.1] 69% 7.27 ±0.04 [53.8] 8.95 ±0.09 [1.11] 77% 8.29 ±0.07 [5.15] 10.4 24i* Pentyl (R) i-Pr 8.30 ±0.09 [4.96] 95% 6.17 ±0.03 [672] 7.52 ±0.04 [29.9] 117% 6.48 ±0.05 [328] 2.05 24j* Pentyl (R) t-Bu 8.60 ±0.08 [2.54] 92% 7.19 ±0.07 [65] 8.88 ±0.09 [1.32] 102% 7.69 ±0.04 [20.3] 3.20 24k* 5-FPentyl (R) i-Pr 6.59 ±0.04 [258] 72% 6.14 ±0.08 [720] 6.15 ±0.08 [703] 42% 6.37 ±0.07 [425] 1.69 24 l* 5-FPentyl (R) t-Bu 7.20 ±0.06 [62.7] 59% 7.04 ±0.06 [90.6] 8.88 ±0.07 [1.33] 77% 7.82 ±0.09 [15.1] 6.00 24 m* CH 2 -Cy (R) i-Pr 7.08 ±0.04 [82.5] 47% 6.51 ±0.03 [311] 6.65 ±0.05 [225] 48% 6.96 ±0.05 [109] 2.85 24 n* CH 2 -Cy (R) t-Bu 8.31 ±0.07 [4.90] 81% 7.18 ±0.05 [66.1] 8.60 ±0.05 [2.49] 54% 7.66 ±0.06 [21.8] 3.03 24o* 4-F-Bn (R) i-Pr 7.41 ±0.03 [39.1] 85% 6.00 ±0.07 [588] 8.08 ±0.08 [8.38] 82% 6.78 ±0.08 [167] 3.52 24p* 4 F-Bn (R) t-Bu 7.83 ±0.04 [14.8] 127% 7.14 ±0.06 [72.8] 8.49 ±0.07 [3.24] 79% 7.63 ±0.03 [23.3] 3.12 Δ 9 -THC - - 7.47 ±0.16 [33.8] 51% - 6.62 ±0.25 [238] 21% - - Surinabant - - - - 8.52 ±0.08 [3.00] - - 6.39 ±0.06 [400] 0.0075 GW405833 - - - - 5.32 ±0.05 [4772] - - 8.23 ±0.07 [5.80] 822 CP55,940 - - - - 8.99 ±0.11 [1.01] - - 10.21 ±0.32 [0.13] 7.76 a ) Functional activity assessed in transfected HEK-293 T cells by determining cAMP levels after forskolin stimulation. b ) E max values were normalized to a maximal efficacious concentration of CP55,940. c ) Displacement of specific [ 3 H]CP 55,940 binding in membrane preparations of human CB 1 Rs transfected in CHO-CB1 C3 cell line expressed as K i in nM (n =3) or percentage displacement of specific binding at a concentration of 1 μ M (n =2). d ) Displacement of specific [ 3 H]CP 55,940 binding in membrane preparations of human CB 2 Rs transfected in HEK-293 T cells expressed as K i in nM (n =3) or percentage displacement of specific binding at a concentration of 1 μ M (n =2). * These SCRAs have not been previously described. C. Gio´ e-Gallo et al. Biomedicine & Pharmacotherapy 164 (2023) 114934 16 Among these derivatives, 22 l and 22i emerge as model ligands that show excellent efficacy, moderate and consistent sub-type selectivity towards CB 2 R in functional and affinity experiments. These ligands are currently being used in our programs to explore the significant, yet unrealized, therapeutic potential of CB 2 R activation. In contrast to previous studies, usually focusing on the characterization of drugs of abuse and small subsets, the library approach herein adopted has been instrumental in discovering CB 2 R agonists with incipient selectivity and identifying SAR trends that will inspire our ongoing program to develop CB 2 R agonists as anti-inflammatory, analgesic, and immunomodulatory therapeutics. [45,46] It must be noted that even though the ligands shown in Fig. 10 have modest selectivity and reveal structural features (R configuration) that inspire the design of new CB 2 R ligands, the inherent metabolic instability of SCRA esters and suboptimal physicochemical properties preclude their direct exploitation in drug discovery programs. Accordingly, functional derivatization using different pharmacomodulation strategies and physicochemical parameters optimization are currently being pursued in our laboratories. 3.5. Preliminary neurotoxicity study The ever-changing structure of ligands in the illegal market makes it challenging to monitor abused SCRAs as well as to undertake systematic studies on their toxicological effects. Their high potency and excellent efficacy lead to lower doses being sufficient to obtain psychoactive effects. However, due to their unpredictable biological effects and unregulated and variable dosage and uses, overdose, intoxication and severe toxicity are more likely to occur. [47] With reports of intoxications and deaths following SCRAs use rapidly increasing, [48,49] toxicity studies are not only helpful in assessing the health threats associated with SCRAs abuse, but also in proposing appropriate medical management for cases of intoxication arising from SCRA consumption. [50–52] Although case reports and retrospective studies of acute intoxication by SCRAs confirm that they cause severe effects (e.g., seizures, cardiovascular damage, kidney damage, stroke psychosis, paranoia, anxiety attacks), [47,50,52,53] little is currently known about the mechanisms by which these structures exert toxic effects. [53] Several reports have assessed the toxicity of commonly abused SCRAs evidencing remarkable harmful effects. Noteworthily, growing scientific evidence highlights that SCRAs consume alters psychoactive and cognitive responses. [11,54] However, the cytotoxicity of known SCRAs (and emerging derivatives) in mammalian neuronal cells remains scarcely explored. [55]. In the context of this study, we decided to preliminarily examine the neurotoxicity signatures of a subset of herein prepared synthetic cannabinoid agonists in primary mouse neuronal cells. The selected ligands included well-known SCRAs [22c (5 F-MDMB-PICA), 22e (MMBCHMICA), 22 f (MDBM-CHMICA), 22 h (MDMB-FUBICA), 23e (AMBCHMINACA), 23 f (MDMB-CHMINACA), 23 h (MDMB-FUBINACA), 24d (5-F-AD-BICA), 24e (AB-CHMICA), 24 f (ADB-CHMICA), 24 g (ABFUBICA), 25d (5-FADB-PINACA), 25e (AD-CHMINACA), 25 f (ABDCHMINACA), 25 h (ADB-FUBINACA)] as well as four hitherto untapped derivatives [(R) stereoisomers, 22 m, 23 m, 24 n, 25 n] all of them being representatives of the structural diversity explored within the study. As the enantiomers (R) showed generally lower potency and efficacy compared to their congeners (S), for this preliminary exploration it was decided to prioritize the latter subset (S). Fig. 11 represents the results of the neuronal toxicity of selected ligands, evaluated at 100 nM in mouse primary neuronal cells. A first inspection of the generated data (Fig. 11) reveals that, in a clear contrast to the effect measured for Δ 9 -THC and CBD, all ligands significantly reduced cell viability (typically from 30% to 70%). The reduction in cell viability seems to be functional and scaffold dependent, with amides derived of the indazole core (Fig. 11, compounds 25) eliciting superior neurotoxicity. Whereas the observed toxicity profiles could be consequence of different features (e.g., pharmacological, and pharmacokinetic), a first analysis of the structural relationship among the selected ligands enables the ascertaining ofsimilarities and/or differences that could be potentially affecting the observed neurotoxicity signatures. Thus, indole ester derivatives seem to be slightly less cytotoxic than their indazole analogues (Fig. 11, compare 22e and 23e, 22 f and 23 f, 22 h and 23 h). Such a trend is considerably more pronounced in the amide series, with indole amides being clearly less cytotoxic than their corresponding analogues in the indazole series (Fig. 11, compare 24d and 25d, 24e and 25e, 24 f and 25 f). Although the limited number of derivatives with stereochemistry (R) in the pendant amino acid chain studied precludes reaching definitive conclusions, the obtained data suggest that, except for 24 n, these derivatives generally exhibit superior neurotoxicity. Indeed, the ligand exhibiting superior neuronal toxicity (25 n) is an indazole amide bearing a tert-butyl group with (R) configuration on the pendant aminoacid chain. These preliminary results suggest that special attention and control should be paid to the possible emergence on the illicit market of SCRAs based on an indazole core and containing an amide group. Further studies are currently being conducted in our laboratories to complete the pharmacological characterization and broaden the toxicity analysis of this large collection of SCRAs. 4. Conclusions SCRAs are designer drugs that mimic the effects of Δ 9 -THC and pose a serious threat to public health and require effective collaboration between the scientific and law enforcement communities. Herein we reported the functional and binding data of the largest and most diverse Fig. 10. Novel synthetic cannabinoid agonists eliciting incipient CB 2 R selective profile. C. Gio´ e-Gallo et al. Biomedicine & Pharmacotherapy 164 (2023) 114934 17 collection of SCRAs published to date. Our results revealed the cannabinomimetic profile of 14 novel derivatives that could be (or may currently be) used as illegal psychoactive substances. Comparative analysis of the affinity data provided emerging SAR and SSR trends and identified some CB 2 R selective ligands, while a preliminary study in primary neuronal cells evidenced an eventual neurotoxicity of SCRAs. The library constitutes a collaborative tool to address some of the challenges posed by the market for new psychoactive substances. CRediT authorship contribution statement Claudia Gioe-Gallo: Investigation, Validation, Writing – original draft. Sandra Ortigueira: Investigation, Validation. Jose Brea: Investigation, Validation. Iu Rai¨ch: Investigation, Validation. Jhonny Azuaje: Methodology, Investigation, Validation. M. Rita Paleo: Methodology, Investigation, Validation, Supervision. Maria Majellaro: Investigation, Validation, Writing – original draft. Maria Isabel Loza: Validation, Funding acquisition. Cristian O. Salas: Methodology, Validation. Xerardo Garcia-Mera: Methodology, Investigation, Validation. Gemma Navarro: Methodology, Investigation, Validation. Eddy Sotelo: Conceptualization, Supervision, Funding acquisition, Writing – review & editing. Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: EDDY SOTELO reports was provided by Government of Galicia. Acknowledgments This work was financially supported by the Consellería de Cultura, Educaci´ on e Ordenaci´ on Universitaria of the Galician Government: (grant: ED431B 2020/43), Centro Singular de Investigaci´ on de Galicia accreditation 2019–2022 (ED431G 2019/03), Ministerio de Ciencia e Innovaci´ on (PID2020-113430RB-I00) and the European Regional Development Fund (ERDF). Appendix A. 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