Application of an activity‐based receptor bioassay to investigate the in vitro activity of selected indole‐ and indazole‐3‐carboxamide‐based synthetic cannabinoids at CB1 and CB2 receptors

Abstract

Synthetic cannabinoids (SCs) are the most chemically diverse group of new psychoactive substances. This group has been associated with several intoxications, many with fatal outcomes. Although advancements have been achieved in pharmacology, metabolism, and detection of these compounds in recent years, these aspects are still unresolved for many SCs. The aim of this study was to investigate the in vitro potency of 14 indole-and indazole-based SCs by applying a stable CB1 or CB2 receptor activation assay and correlating the activity with their structure. The half-maximal effective concentration (EC 50 ) of 5-chloropentyl, 5-bromopentyl, and 5-iodopentyl JWH-122 analogs varied from 74.1 to 283.7 nM for CB1 and 7.05 to 23.4 nM for CB2, where the addition of a chlorine atom enhanced the potency at CB1 compared with the bromo and iodo analogs. AM-2201 was the most active at CB1 within this naphthoylindole family, with an EC 50 of 23.5 nM but with the lowest efficacy (E max 98.8%). Within the indole-3-carboxamide derivatives, 5F-MDMB-PICA was the most active compound, with a CB1/CB2 EC 50 of 3.26/0.87 nM and an E max around three times higher than JWH-018. ADB-FUBINACA was the most potent tested SC overall, with a CB1/CB2 EC 50 of 0.69/0.59 nM, and an E max around 3-fold higher than that for JWH-018 at CB1. The data obtained in this study confirm how small differences in the structure of SCs might lead to large differences in their activity, especially at CB1, which may be correlated with differences in their toxic effects in humans.

The chemistry and pharmacology of putative synthetic cannabinoid receptor agonist (SCRA) new psychoactive substances (NPS) 5F‐PY‐PICA, 5F‐PY‐PINACA, and their analogs

“…The in vitro pharmacological profiles of 5F‐PY‐PICA and 5F‐PY‐PINACA indicate that these putative SCRAs have very low affinity and efficacy at CB 1 receptors, and are unlikely to be psychoactive in humans. The lack of CB 1 affinity and activity demonstrated by 5F‐PY‐PICA is concordant with inactivity of this ligand in a recently reported CB 1 nanoluciferase reporter assay . Similar to many SCRA NPS, both 5F‐PY‐PICA and 5F‐PY‐PINACA had submicromolar potency at CB 2 receptors, but this is unlikely to mediate any discernable effects on mood in people.…”

“…As with other 5‐fluoropentyl‐containing SCRAs, oxidative defluorination (and further oxidation) was a major metabolic route for 5F‐PY‐PICA in the pHLM and pHH systems, while alpha‐oxidation of the pyrrolidine nitrogen was preferred in SCRHs. In a recently reported nanoluciferase CB 1 reporter assay, 5F‐PY‐PICA demonstrated negligible agonist activity (EC 50 > 10 μM) . The pharmacological profile of 5F‐PY‐PINACA has not yet been reported.…”

Functional evaluation of carboxy metabolites of synthetic cannabinoid receptor agonists featuring scaffolds based on L‐valine or L‐tert‐leucine

“…As JWH‐018 was the first detected SCRA at the end of 2008, and the most commonly used standard SCRA in the field of toxicology, it was chosen as the reference compound. The observed EC 50 value for JWH‐018 in the bio‐assay expressing CB 1 was 36.8 nM, with a 95% confidence interval (CI) of 28.6–50.4 nM, which is in agreement with earlier published data from our research group …”

“…The generation of an HEK293T cell line stably expressing both the CB 1 receptor (C‐terminally fused to the large part of the NanoLuciferase; LgBiT) and β‐arr2 (N‐terminally fused to the small part of NanoLuciferase; SmBiT) has been described previously . This cell line can be used for structure−activity relationship determination of reference compounds, as well as for the screening of biological matrices for cannabinoid activity …”

“…Further adding to the complexity of the SCRA problem is that, in addition to the rapid and constant emergence of new SCRAs, relatively little is known about the pharmacology of these SCRAs and their metabolites. More particularly, few studies have focused on the pharmacological characteristics of hydrolysis metabolites . Recently, there has been an increased interest, since knowledge about SCRA metabolite activity data might empower forensic laboratories to elaborate on the possible contributory role of these metabolites in human intoxications and in side‐effects related to SCRAs.…”