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=Volume 7 - 2019

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=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 characterisation 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 and Emax, 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 being 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. Optimised 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 to 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 ten-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.