Abstract
Synthetic cannabinoid receptor agonists (SCRAs) have been the largest group of illicit psychoactive substances reported to international monitoring and early warning systems for many years. Carboxamide-type SCRAs are amongst the most prevalent and potent. Enantiospecific synthesis and characterization of four indazole-3-carboxamides, AMB-FUBINACA, AB-FUBINACA, 5F-MDMB-PINACA (5F-ADB), and AB-CHMINACA is reported. The interactions of the compounds with CB1 and CB2 receptors were investigated using a G-protein coupled receptor (GPCR) activation assay based on functional complementation of a split NanoLuc luciferase and EC50 (a measure of potency) and Emax (a measure of efficacy) values determined. All compounds demonstrated higher potency at the CB2 receptor than at the CB1 receptor and (S)-enantiomers had an enhanced potency to both receptors over the (R)-enantiomers. The relative potency of the enantiomers to the CB2 receptor is affected by structural features. The difference was more pronounced for compounds with an amine moiety (AB-FUBINACA and AB-CHMINACA) than those with an ester moiety (AMB-FUBINACA and 5F-MDMB-PINACA). An HPLC method was developed to determine the prevalence of (R)-enantiomers in seized samples. Lux® Amylose-1 [Amylose tris(3,5-dimethylphenylcarbamate)] has the greatest selectivity for the SCRAs with a terminal methyl ester moiety and a Lux® i-Cellulose-5 column for SCRAs with a terminal amide moiety. Optimized isocratic separation methods yielded enantiomer resolution values (Rs) ≥ 1.99. Achiral GC-MS analysis of seized herbal materials (n = 16), found 5F-MDMB-PINACA (<1.0–91.5 mg/g herbal material) and AMB-FUBINACA (15.5–58.5 mg/g herbal material), respectively. EMB-FUBINACA, AMB-CHMICA, 5F-ADB-PINACA isomer 2, and ADB-CHMINACA were also tentatively identified. Analysis using chiral chromatography coupled to photodiode array and quadrupole time of flight mass spectrometry (chiral HPLC-PDA-QToF-MS/MS) confirmed that the (S)-enantiomer predominated in all samples (93.6–99.3% (S)-enantiomer). Small but significant differences in synthesis precursor enantiopurity may provide significant differences between synthesis batches or suppliers and warrants further study. A method to compare potency between samples containing different SCRAs at varying concentrations was developed and applied in this small preliminary study. A 10-fold difference in the “intrinsic” potency of samples in the study was noted. With the known heterogeneity of SCRA infused materials, the approach provides a simplified method for assessing and communicating the risk of their use.
Highlights
Synthetic cannabinoids (SCs) are the most chemically diverse group of new psychoactive substances (NPS) reported to national and international drug early warning systems (EMCDDA, 2018a; UNODC, 2018a)
The synthesis of AB-CHMINACA was based on the synthesis of AMB-CHMINACA with valinamide being substituted for the valine methyl ester and the synthesis optimized by the addition of another equivalent of potassium tert-butoxide and bromomethyl cyclohexane and the reaction being heated to a reflux for 18 h instead of being stirred at room temperature for 48 h as described in the original method
We have described the investigation of the effect of chirality on the potency and, for the first time, efficacy, of four indazole-3-carboxamide Synthetic cannabinoid receptor agonists (SCRAs)
Summary
Synthetic cannabinoids (SCs) are the most chemically diverse group of new psychoactive substances (NPS) reported to national and international drug early warning systems (EMCDDA, 2018a; UNODC, 2018a). Many SCRA classes and hundreds of analogs have been synthesized and studied as pharmacological targets by research groups across academia and industry These compounds have often been found to bind to and activate both receptors and, as a result of their psychoactive effects, few have reached clinical trials and have been developed as medicinal products (e.g., Huffman and Padgett, 2005; Huffman et al, 2005; Huffman, 2009; Han et al, 2013). Due to variations in their molecular structure, different SCRAs may cause different conformational changes in the receptor-ligand complex conformation and as a result give rise to different cellular responses with a bias to a particular signaling pathway (e.g., G-protein coupling vs β-arrestin recruitment) In opioid receptors, such biased signaling has been utilized to create agonists which are biased toward pathways which give rise to desired therapeutic effects rather than those which give rise to unwanted side effects (Ho et al, 2018)
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