AUTHOR=Antonides Lysbeth H. , Cannaert Annelies , Norman Caitlyn , Vives Loelia , Harrison Aidan , Costello Andrew , Nic Daeid Niamh , Stove Christophe P. , Sutcliffe Oliver B. , McKenzie Craig 

TITLE=Enantiospecific Synthesis, Chiral Separation, and Biological Activity of Four Indazole-3-Carboxamide-Type Synthetic Cannabinoid Receptor Agonists and Their Detection in Seized Drug Samples

JOURNAL=Frontiers in Chemistry

VOLUME=7

YEAR=2019

URL=https://www.frontiersin.org/journals/chemistry/articles/10.3389/fchem.2019.00321

DOI=10.3389/fchem.2019.00321

ISSN=2296-2646

ABSTRACT=<p>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 CB<sub>1</sub> and CB<sub>2</sub> receptors were investigated using a G-protein coupled receptor (GPCR) activation assay based on functional complementation of a split NanoLuc luciferase and EC<sub>50</sub> (a measure of potency) and E<sub>max</sub> (a measure of efficacy) values determined. All compounds demonstrated higher potency at the CB<sub>2</sub> receptor than at the CB<sub>1</sub> receptor and (<italic>S</italic>)-enantiomers had an enhanced potency to both receptors over the (<italic>R</italic>)-enantiomers. The relative potency of the enantiomers to the CB<sub>2</sub> 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 (<italic>R</italic>)-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 (<italic>n</italic> = 16), found 5F-MDMB-PINACA (&lt;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 (<italic>S</italic>)-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.</p>