NNL-3: A Synthetic Intermediate or a New Class of Hydroxybenzotriazole Esters with Cannabinoid Receptor Activity?

Abstract

Synthetic cannabinoid receptor agonists (SCRAs) remain a prolific class of new psychoactive substances (NPS) and continue to expand rapidly. Despite the recent identification of hydroxybenzotriazole (HOBt) containing SCRAs in synthetic cannabis samples, there is currently no information regarding the pharmacological profile of these NPS with respect to human CB1 and CB2 receptors. In the current study, a series consisting of seven HOBt indole-, indazole-, and 7-azaindole-carboxylates bearing a range of N-alkyl substituents were synthesized and pharmacologically evaluated. Competitive binding assays at CB1 and CB2 demonstrated that all analogues except a 2-methyl-substituted derivative had low affinity for CB1 (K i = 3.80–43.7 μM) and CB2 (K i = 2.75–18.2 μM). A fluorometric functional assay revealed that 2-methylindole- and indole-derived HOBt carboxylates were potent and efficacious agonists of CB1 (EC50 = 12.0 and 63.7 nM; E max = 118 and 120%) and CB2 (EC50 = 10.9 and 321 nM; E max = 91 and 126%). All other analogues incorporating indazole and 7-azaindole cores and bearing a range of N1-substituents showed relatively low potency for CB1 and CB2. Additionally, a reporter assay monitoring β-arrestin 2 (βarr2) recruitment to the receptor revealed that the 2-methylindole example was the most potent and efficacious at CB1 (EC50 = 131 nM; E max = 724%) and the most potent at CB2 (EC50 = 38.2 nM; E max = 51%). As with the membrane potential assay, the indazole and other indole HOBt carboxylates were considerably less potent at both receptors, and analogues comprising a 7-azaindole core showed little activity. Taken together, these data suggest that NNL-3 demonstrates little CB1 receptor activity and is unlikely to be psychoactive in humans. NNL-3 is likely an unintended SCRA manufacturing byproduct. However, the synthesis of NNL-3 analogues proved simple and general, and some of these showed potent cannabimetic profiles in vitro, indicating that HOBt esters of this type may represent an emerging class of SCRA NPS.

Defining Steric Requirements at CB1 and CB2 Cannabinoid Receptors Using Synthetic Cannabinoid Receptor Agonists 5F-AB-PINACA, 5F-ADB-PINACA, PX-1, PX-2, NNL-1, and Their Analogues

“…In vitro cannabinoid functional activity was determined using a fluorescence-based membrane potential assay in stably transfected AtT20 cells expressing human CB 1 or CB 2 receptors, with receptor activation normalized to 1 μM CP55,940. ,,,− Several of the functional activity measurements could not be determined due to insufficient activation at the maximum concentration (see Table ). For compounds with a determinable EC 50 , the same trends were observed in their functional activity as in their binding affinity.…”

Cannabinoid receptor activation potential of the next generation, generic ban evading OXIZID synthetic cannabinoid receptor agonists

“…Looking at other SCRAs, however, this is not a consistent finding. For instance, comparing the CB 1 activity of the assumed SCRA intermediate NNL‐3 (HOBt‐5F‐P7AIC, carrying a fluoro pentyl tail), with its defluorinated (HOBt‐P7AIC) or its cyclohexyl methyl (HOBt‐CHM7AIC) analog, we noticed a dramatic decrease in activity for the HOBt‐CHM7AIC, whereas both pentyl analogs had a quite similar activation profile 61 . Furthermore, when comparing two L‐valine SCRAs at CB 1 , replacement of the fluoro pentyl tail in 5F‐AB‐PINACA (EC 50 = 55.4 nM) by a cyclohexyl methyl moiety in AB‐CHMINACA (EC 50 = 3.45 nM) did yield a more potent compound, whereas the tert ‐leucine analogs 5F‐MDMB‐PINACA (EC 50 = 0.84 nM) and MDMB‐CHMINACA (EC 50 = 0.78 nM) had essentially the same potency at CB 1.…”

Putative Synthetic Cannabinoids MEPIRAPIM, 5F-BEPIRAPIM (NNL-2), and Their Analogues Are T-Type Calcium Channel (CaV3) Inhibitors

“…A methyl group at the 3-or 5-position of the piperazine ring is reported to improve potency, 62 and our previous work has also shown that SCRAs that interact with these two residues may give rise to stronger binding affinity to the receptor. 63 The IFD score, which indicates binding affinity, shows that Org 28611 has a stronger binding affinity to both the CB 1 receptors compared to compound 7 (−9744.10 and −9726.62 kcal/mol, respectively). 2) are more critical for determining whether a compound acts on Ca V 3, CBRs, or both.…”

“…All Ca V 3.x experiments were performed using HEK293 FlpIn T-Rex cells stably transfected with pcDNA5 constructs encoding human Ca V 3.x cDNA. 63 Ca V 3.x expression was induced 24 h before membrane potential assays or electrophysiology experiments by adding 2 μg/mL tetracycline. Human CB 1 or CB 2 was stably transfected into AtT-20 FlpIn and maintained using 80 μg/mL hygromycin within the culture media.…”