Serial identification and structural analysis of synthetic cannabinoids found in herbal mixtures in South Africa

Abstract

The primary objective of this study was to identify and characterise SCBs in herbal mixtures from South Africa using a serial identification approach. The analysis of seised herbal mixtures was carried out using ultra-performance liquid chromatography coupled with a high-resolution mass spectrometer (UPLC-HRMS) and gas chromatography coupled with amass selective detector (GCMSD). The study identified three SCBs associated with fatalities: N-[[1-[(4-fluorophenyl)methyl]-1Hindazol- 3-yl]carbonyl]-L-valine, methyl ester (AMB-FUBINACA), N-[[1-(5-fluoropentyl)-1H-indazol-3- yl]carbonyl]-3-methyl-D/L-valine, methyl ester (5F-ADB), N-[[1-(5-fluoropentyl)-1H-indazol-3- yl]carbonyl]-3-methyl-L-valine, ethyl ester (5F-EDMB-PINACA) and fifteen other non-SCBs compounds. The study of MS fragment ions predicted possible fragmentation pathways and found that the identified SCBs and analysed reference standards are susceptible to the amide linker group cleavage, followed by cleavage of the fluorinated side chain. These findings suggest that abundant iv fragment ions can be used to screen uncharacterised SCBs belonging to the same family or sharing similar pharmacores. Additionally, we validated isolated SCBs from herbal mixtures using NMR spectroscopy where a reference standard was not available and obtained crystal data using SC-XRD. 1H-NMR and 13C-NMR spectral fingerprinting allowed for cross-validation of the isolated 5F-ADB. 1H-NMR spectroscopy was also successfully used to cross-validate the reference standard (S)-5F-EMBICA, supplied as (S)-5FABICA. SC-XRD crystal data results of (S)-5F-EMBICA were obtained with low quality and hence crossvalidated by NMR spectroscopy. This research, for the first time, to our knowledge reports the 1HNMR spectroscopy data of (S)-5F-EMBICA and its crystal structure and crystallography data. Finally, we conducted in silico analysis to estimate the pharmacokinetics and physicochemical properties of the SCB compounds. We utilised the SwissADME web tool for this purpose, which revealed several key findings, including the lipophilicity enantiomeric discrimination, side chain fluorogroup type effects, hydrophobic nature, TPSA analysis, GI absorption levels and BBB permeation, P-gp binding, and probability for CYP3A4 inhibition. These findings suggest that the SwissADME web tool can be used as a first line of pharmacokinetic properties prediction for SCBs in the absence of bioassays and expertise in advanced computer modelling. In silico analysis was also carried out to characterise selected SCBs further through docking pose predictions on the CB1 receptor using the Maestro molecular modelling software by Schrödinger. The results showed that the indazole ring of the SCB interacts via π-π stacking with phenylalanine residues, particularly Phe 268, Phe 170, Phe 174, and His 178. The study concludes that the (S)-enantiomer has a higher CB1 affinity and more π-π stacking interactions with phenylalanine residues which are important for CB1 agonism. These results can be used to predict the properties of uncharacterised SCBs before their spread in the illicit drug market.