Enantiospecific synthesis, chiral separation and biological activity of four indazole-3-carboxamide-type synthetic cannabinoid receptor agonists and their detection in seized drug samples

Lysbeth H. Antonides, Annelies Cannaert, Caitlyn Norman, Loelia Vives, Aidan Harrison, Andrew Costello, Niamh Nic Daeid, Christophe Stove, Oliver Sutcliffe (Lead / Corresponding author), Craig McKenzie (Lead / Corresponding author)

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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.

Original languageEnglish
Article number321
Pages (from-to)1-20
Number of pages20
JournalFrontiers in Chemistry
Volume7
DOIs
Publication statusPublished - 16 May 2019

Fingerprint

Indazoles
Cannabinoid Receptor Agonists
Enantiomers
Bioactivity
Pharmaceutical Preparations
Esters
Amylose
Alarm systems
Personal digital assistants
G-Protein-Coupled Receptors
Chromatography
Photodiodes
Luciferases
Amides
Cellulose
Isomers
Amines
Mass spectrometry
Assays
Chemical activation

Keywords

  • Bioassay
  • Chiral
  • Detection
  • Pharmacology
  • Synthetic cannabinoid receptor agonists

Cite this

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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",
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{\circledR} Amylose-1 [Amylose tris(3,5-dimethylphenylcarbamate)] has the greatest selectivity for the SCRAs with a terminal methyl ester moiety and a Lux{\circledR} 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.",
keywords = "Bioassay, Chiral, Detection, Pharmacology, Synthetic cannabinoid receptor agonists",
author = "Antonides, {Lysbeth H.} and Annelies Cannaert and Caitlyn Norman and Loelia Vives and Aidan Harrison and Andrew Costello and {Nic Daeid}, Niamh and Christophe Stove and Oliver Sutcliffe and Craig McKenzie",
note = "Lysbeth Antonides is funded by the Leverhulme Trust. Loelia Vives acknowledges the support of the ERASMUS student exchange programme for her internship with the Forensic Drug Research Group at the University of Dundee. Annelies Cannaert is funded by the BRAIN project grant (NPSSAY) from The Belgian Science Policy Office and the Ghent University - Special Research Fund (BOF; PDO026-18). Christophe Stove also acknowledges the support from Ghent University - Special Research Fund (BOF; project grants no. 01N00814 and 01J15517).",
year = "2019",
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doi = "10.3389/fchem.2019.00321",
language = "English",
volume = "7",
pages = "1--20",
journal = "Frontiers in Chemistry",
issn = "2296-2646",
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TY - JOUR

T1 - Enantiospecific synthesis, chiral separation and biological activity of four indazole-3-carboxamide-type synthetic cannabinoid receptor agonists and their detection in seized drug samples

AU - Antonides, Lysbeth H.

AU - Cannaert, Annelies

AU - Norman, Caitlyn

AU - Vives, Loelia

AU - Harrison, Aidan

AU - Costello, Andrew

AU - Nic Daeid, Niamh

AU - Stove, Christophe

AU - Sutcliffe, Oliver

AU - McKenzie, Craig

N1 - Lysbeth Antonides is funded by the Leverhulme Trust. Loelia Vives acknowledges the support of the ERASMUS student exchange programme for her internship with the Forensic Drug Research Group at the University of Dundee. Annelies Cannaert is funded by the BRAIN project grant (NPSSAY) from The Belgian Science Policy Office and the Ghent University - Special Research Fund (BOF; PDO026-18). Christophe Stove also acknowledges the support from Ghent University - Special Research Fund (BOF; project grants no. 01N00814 and 01J15517).

PY - 2019/5/16

Y1 - 2019/5/16

N2 - 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.

AB - 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.

KW - Bioassay

KW - Chiral

KW - Detection

KW - Pharmacology

KW - Synthetic cannabinoid receptor agonists

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DO - 10.3389/fchem.2019.00321

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