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Purpose: AMB-FUBINACA is a synthetic cannabinoid receptor agonist (SCRA) which is primarily metabolised by hepatic enzymes producing AMB-FUBINACA carboxylic acid. The metabolising enzymes associated with this biotransformation remain unknown. This study aimed to determine if AMB-FUBINACA metabolism could be reduced in the presence of carboxylesterase (CES) inhibitors and recreational drugs commonly consumed with it. The affinity and activity of the AMB-FUBINACA acid metabolite at the cannabinoid type-1 receptor (CB1) was investigated to determine the activity of the metabolite. Methods: The effect of CES1 and CES2 inhibitors, and delta-9-tetrahydrocannabinol (Δ9-THC) on AMB-FUBINACA metabolism were determined using both human liver microsomes (HLM) and recombinant carboxylesterases. Radioligand binding and cAMP assays comparing AMB-FUBINACA and AMB-FUBINACA acid were carried out in HEK293 cells expressing human CB1. Results: AMB-FUBINACA was rapidly metabolised by HLM in the presence and absence of NADPH. Additionally, CES1 and CES2 inhibitors both significantly reduced AMB-FUBINACA metabolism. Furthermore, digitonin (100 µM) significantly inhibited CES1-mediated metabolism of AMB-FUBINACA by ~ 56%, while the effects elicited by Δ9-THC were not statistically significant. AMB-FUBINACA acid produced only 26% radioligand displacement consistent with low affinity binding. In cAMP assays, the potency of AMB-FUBINACA was ~ 3000-fold greater at CB1 as compared to the acid metabolite. Conclusions: CES1A1 was identified as the main hepatic enzyme responsible for the metabolism of AMB-FUBINACA to its less potent carboxylic acid metabolite. This biotransformation was significantly inhibited by digitonin. Since other xenobiotics may also inhibit similar SCRA metabolic pathways, understanding these interactions may elucidate why some users experience high levels of harm following SCRA use. [ABSTRACT FROM AUTHOR]

Synthetic cannabinoid receptor agonists (SCRAs) are one of the largest and most structurally diverse classes of new psychoactive substances (NPS). Despite this, pharmacological data are often lacking following the identification of a new SCRA in drug markets. In this first of a three‐part series, we describe the synthesis, analytical characterization, and binding affinity of a proactively generated, systematic library of 30 indole, indazole, and 7‐azaindole SCRAs related to MMB‐4en‐PICA, MDMB‐4en‐PINACA, ADB‐4en‐PINACA, and MMB‐4CN‐BUTINACA featuring a 4‐pentenyl (4en‐P), butyl (B/BUT), or 4‐cyanobutyl (4CN‐B/BUT) tail and a methyl l‐valinate (MMB), methyl l‐tert‐leucinate (MDMB), methyl l‐phenylalaninate (MPP), l‐valinamide (AB), l‐tert‐leucinamide (ADB), l‐phenylalaninamide (APP), adamantyl (A), or cumyl head group. Competitive radioligand binding assays demonstrated that the indazole core conferred the highest CB1 binding affinity (Ki = 0.17–39 nM), followed by indole‐ (Ki = 0.95–160 nM) and then 7‐azaindole‐derived SCRAs (Ki = 5.4–271 nM). Variation of the head group had the greatest effect on binding, with tert‐leucine amides and methyl esters (Ki = 0.17–14 nM) generally showing the greatest affinities, followed by valine derivatives (Ki = 0.72–180 nM), and then phenylalanine derivatives (Ki = 2.5–271 nM). Adamantyl head groups (Ki = 8.8–59 nM) were suboptimal for binding, whereas the cumyl analogues consistently conferred high affinity (Ki = 0.62–36 nM). Finally, both butyl (Ki = 3.1–163 nM) and 4‐cyanobutyl (Ki = 5.5–44 nM) tail groups were less favorable for CB1 binding than their corresponding 4‐pentenyl counterparts (Ki = 0.72–25 nM). [ABSTRACT FROM AUTHOR]

Synthetic cannabinoid receptor agonists (SCRAs) are the second largest class of new psychoactive substances (NPS) and are associated with serious adverse effects and even death. Despite this, little pharmacological data are available for many of the most recent SCRAs. This study consists of three different parts, aiming to systematically evaluate a panel of 30 SCRAs using binding and different in vitro human cannabinoid 1 receptor (CB1) activation assays. The present Part II investigated the SCRA analogs for their CB1 activation via a β‐arrestin recruitment assay. The panel was systematically designed to include key structural sub‐features of recent SCRAs. Thus, the 4‐pentenyl tail of MMB‐4en‐PICA and MDMB‐4en‐PINACA was retained while incorporating varying head groups from other prevalent SCRAs, including amides and esters of L‐valine, L‐tert‐leucine, and L‐phenylalanine, and adamantyl and cumyl moieties. All 30 SCRAs activated CB1, with indazoles generally showing the greatest potency (EC50 = 1.88–281 nM), followed by indoles (EC50 = 11.5–2293 nM), and the corresponding 7‐azaindoles (EC50 = 62.4–9251 nM). Several subunit‐linked structure–activity relationships were identified: (i) tert‐leucine‐functionalized SCRAs were more potent than the corresponding valine derivatives; (ii) no major difference in potency or efficacy was observed between tert‐leucine/valine‐derived amides and the corresponding methyl esters; however, phenylalanine analogs were affected by this change; and (iii) minor structural changes to the 4‐pentenyl substituent had little influence on activity. These findings elucidate structural features that modulate the CB1 activation potential of currently prevalent SCRAs and a systematic panel of analogs, some of which may appear in NPS markets in future. [ABSTRACT FROM AUTHOR]

Synthetic cannabinoid receptor agonists (SCRAs) are a diverse class of new psychoactive substances (NPS). They commonly comprise N -alkylated indole, indazole, or 7-azaindole scaffolds with amide-linked pendant amino acid groups. To explore the contribution of the amino acid side chain to the cannabinoid pharmacology of SCRA NPS, a systematic library of side chain-modified SCRAs was prepared based on the recent detections of amino acid derivatives 17 (5F-AB-PINACA), 18 (5F-ADB-PINACA), 15 (PX-1), 19 (PX-2), and 20 (NNL-1). In vitro binding affinities and functional activities at cannabinoid type 1 and 2 receptors (CB 1 and CB 2 , respectively) were determined for all the library members using radioligand competition experiments and a fluorescence-based membrane potential assay. Binding affinities and functional activities varied widely across compounds ( K i = 0.32 to >10 000 nM, EC 50 = 0.24-1259 nM), with several clear structure-activity relationships (SARs) emerging. Affinity and potency at CB 1 changed as a function of the heterocyclic core (indazole > indole > 7-azaindole) and the pendant amino acid side chain ( tert -butyl > iso -propyl > iso -butyl > benzyl > ethyl > methyl > hydrogen). Ensemble docking at CB 1 revealed a clear steric basis for observed SAR trends. Interestingly, although 15 (PX-1) and 19 (PX-2) have been detected in recreational drug markets, they failed to induce centrally CB 1 -mediated effects (e.g., hypothermia) in mice using radiobiotelemetry. Together, these data provide insights regarding structural contributions to the cannabimimetic profiles of 17 (5F-AB-PINACA), 18 (5F-ADB-PINACA), 15 (PX-1), 19 (PX-2), 20 (NNL-1), and other SCRA NPS.

Synthetic cannabinoid receptor agonists (SCRAs) remain one the most prevalent classes of new psychoactive substances (NPS) worldwide, and examples are generally poorly characterised at the time of first detection. We have synthesised a systematic library of amino acid-derived indole-, indazole-, and 7-azaindole-3-carboxamides related to recently detected drugs ADB-BUTINACA, APP-BUTINACA and ADB-P7AICA, and characterised these ligands for in vitro binding and agonist activity at cannabinoid receptor subtypes 1 and 2 (CB 1 and CB 2 ), and in vivo cannabimimetic activity. All compounds showed high affinity for CB 1 ( K i 0.299-538 nM) and most at CB 2 ( K i = 0.912-2190 nM), and most functioned as high efficacy agonists of CB 1 and CB 2 in a fluorescence-based membrane potential assay and a βarr2 recruitment assay (NanoBiT®), with some compounds being partial agonists in the NanoBiT® assay. Key structure-activity relationships (SARs) were identified for CB 1 /CB 2 binding and CB 1 /CB 2 functional activities; (1) for a given core, affinities and potencies for tert -leucinamides (ADB-) > valinamides (AB-) ≫ phenylalaninamides (APP-); (2) for a given amino acid side-chain, affinities and potencies for indazoles > indoles ≫ 7-azaindoles. Radiobiotelemetric evaluation of ADB-BUTINACA, APP-BUTINACA and ADB-P7AICA in mice demonstrated that ADB-BUTINACA and ADB-P7AICA were cannabimimetic at 0.1 mg kg -1 and 10 mg kg -1 doses, respectively, as measured by pronounced decreases in core body temperature. APP-BUTINACA failed to elicit any hypothermic response up to the maximally tested 10 mg kg -1 dose, yielding an in vivo potency ranking of ADB-BUTINACA > ADB-P7AICA > APP-BUTINACA., Competing Interests: None., (This journal is © The Royal Society of Chemistry.)

Synthetic cannabinoid receptor agonists (SCRAs) are the second largest class of new psychoactive substances (NPS) and are associated with serious adverse effects and even death. Despite this, little pharmacological data are available for many of the most recent SCRAs. This study consists of three different parts, aiming to systematically evaluate a panel of 30 SCRAs using binding and different in vitro human cannabinoid 1 receptor (CB 1 ) activation assays. The present Part II investigated the SCRA analogs for their CB 1 activation via a β-arrestin recruitment assay. The panel was systematically designed to include key structural sub-features of recent SCRAs. Thus, the 4-pentenyl tail of MMB-4en-PICA and MDMB-4en-PINACA was retained while incorporating varying head groups from other prevalent SCRAs, including amides and esters of L-valine, L-tert-leucine, and L-phenylalanine, and adamantyl and cumyl moieties. All 30 SCRAs activated CB 1 , with indazoles generally showing the greatest potency (EC 50  = 1.88-281 nM), followed by indoles (EC 50  = 11.5-2293 nM), and the corresponding 7-azaindoles (EC 50  = 62.4-9251 nM). Several subunit-linked structure-activity relationships were identified: (i) tert-leucine-functionalized SCRAs were more potent than the corresponding valine derivatives; (ii) no major difference in potency or efficacy was observed between tert-leucine/valine-derived amides and the corresponding methyl esters; however, phenylalanine analogs were affected by this change; and (iii) minor structural changes to the 4-pentenyl substituent had little influence on activity. These findings elucidate structural features that modulate the CB 1 activation potential of currently prevalent SCRAs and a systematic panel of analogs, some of which may appear in NPS markets in future., (© 2021 John Wiley & Sons, Ltd.)