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

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 CB 1 and CB 2 receptors were investigated using a G-protein coupled receptor (GPCR) activation assay based on functional complementation of a split NanoLuc luciferase and EC 50 (a measure of potency) and E max (a measure of efficacy) values determined. All compounds demonstrated higher potency at the CB 2 receptor than at the CB 1 receptor and ( S )-enantiomers had an enhanced potency to both receptors over the ( R )-enantiomers. The relative potency of the enantiomers to the CB 2 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.

Adding more “spice” to the pot: A review of the chemistry and pharmacology of newly emerging heterocyclic synthetic cannabinoid receptor agonists

“…129 The spectroscopic characterization of a seized sample of MDMB-CHMCZCA (87) by Opatz and co-workers in 2016 permitted the unambiguous assignment of the chiral methyl tert-leucinate linked group as (S)-87. 130 This finding agrees with previous reports [131][132][133] which suggest that SCRAs bearing an α-amino acid-derived "linked group" typically possess (S)-enantiomeric configuration. Synthetic methods employed in SCRA manufacturing facilities for the preparation of carbazole SCRAs currently remain elusive.…”

Detection and quantitation of synthetic cannabinoid receptor agonists in infused papers from prisons in a constantly evolving illicit market

“…A ( S )‐4F‐MDMB‐BINACA (7) reference standard purchased from Chiron, Trondheim, Norway (> 98% purity) was purchased as a quality assurance check standard once it became commercially available. Reference standards for ( S )‐5F‐MDMB‐PINACA (3) (99.6% purity); ( R )‐5F‐MDMB‐PINACA (99.6% purity); and ( S )‐AMB‐FUBINACA (4) (>98% purity) were obtained via in‐house synthesis as detailed previously . In addition, ( S )‐5F‐MDMB‐PICA (5) , ( S )‐AMB‐CHMICA (6) , ( S )‐4F‐MDMB‐BINACA (7) , and ( S )‐MDMB‐4en‐PINACA (8) were synthesized in house as part of this study and characterized using GC–MS and NMR spectroscopy (see supplementary information for synthetic methods and characterization data).…”

“…To study the variability of SCRA concentrations across papers in a more controlled manner, six 5 × 5 cm (25 cm 2 ) pieces of lined 80 g/m 2 paper were prepared and pre‐gridded into 1 cm 2 sections using a pencil. 20.1 mg of a previously synthesized ( R )‐5F‐MDMB‐PINACA (3) standard was dissolved in approximately 5 mL of ethanol to give a 4.01 mg/mL solution. The solution was poured into a glass beaker and each 5 cm 2 piece of paper was laid flat and soaked in the ( R )‐5F‐MDMB‐PINACA (3) solution for approximately 10 seconds then removed carefully from the solution taking care to keep the paper flat as it was removed from the solution.…”

“…Synthetic cannabinoid receptor agonists are a structurally diverse class of compounds that interact with human cannabinoid type 1 and type 2 G‐protein coupled receptors (GPCRs), CB 1 and CB 2. They vary widely in their potency and efficacy as a result of differences in their structural conformation, including chirality . Their diversity is due, in part, to the increased online availability of published research studies and patents describing their synthesis, in vitro potency and efficacy, and biological effects; the availability of precursor materials; increasing understanding of their structure–activity relationships by producers and suppliers; and as a response to the implementation of national and international legislation designed to control their production, prevalence, and use, and in particular, their use in prisons …”

“…[15][16][17][18][19] They vary widely in their potency and efficacy [20][21][22] as a result of differences in their structural conformation, including chirality. 23 Their diversity is due, in part, to the increased online availability of published research studies and patents describing their synthesis, in vitro potency and efficacy, and biological effects; the availability of precursor materials;…”