CB1 activity of three classes of synthetic cannabinoids and their metabolites – The tail needs to know what the head is doing

Abstract

Synthetic cannabinoids (SCs) are amongst the most prevalent of the new psychoactive substances. They encompass various structural classes with a wide range of CB1 activity. Most SCs are extensively bio-transformed in vivo and in vitro. The available data on the CB1 activity of SC metabolites are limited but such knowledge is needed to evaluate SC toxicology, duration of effects and to develop bioassays for screening purposes. To evaluate any structure-activity relationships, we determined the human CB1 receptor efficacy and potency of three SC classes and their metabolites. An activity assay incorporating AequoScreen recombinant CHO-K1 cells expressing the human CB1 receptor was used to study SCs and their metabolites. Dose-response curves were prepared in triplicate in three independent experiments and GraphPad Prism was used to calculate substance efficacy and potency. Eight concentrations, 29 nM–60 μM, were used for each drug and JWH-018 was used as a reference. The three SC classes investigated were (i) JWH-018, AM2201, THJ-018 and THJ-2201 and their corresponding 4-OH-pentyl and 5OH-pentyl metabolites; (ii) MMB-4en-PICA (MMB022) and MDMB-4en-PINACA and their corresponding ester-hydrolysis and dihydrodiol metabolites; and (iii) 5F-MDMB-PINACA, 5F-MDMB-PICA, 4F-MDMB-BINACA, and 4F-MDMB-BICA and their oxidative-defluorination and ester-hydrolysis metabolites. Parent SC potency values ranged from 7-200 nM. The 4-OH-pentyl metabolites of JWH-018, AM2201, THJ-018 and THJ-2201 had similar potency to their respective parent SC. The 5-OH-pentyl-JWH-018 and 5-OH-pentyl-THJ-018 metabolites were 5–10 times less potent than their parent SCs. No CB1 activity was observed for MMB-4en-PICA ester-hydrolysis and dihydrodiol metabolites, possibly due to the lower activity of the parent and a subsequent large loss of activity, whilst the dihydrodiol and ester-hydrolysis MDMB-4en-PINACA metabolites were 53 times and 130 times less potent respectively than MDMB-4en-PINACA. 5F-MDMB-PINACA and 5F-MDMB-PICA oxidative-defluorinated metabolites were 7 and 27 times less potent than the parent SCs. The 4OH-butyl metabolites of 4F-MDMB-BINACA, and 4F-MDMB-BICA were 6 and 11 times less potent than the parent drug, respectively, and their ester-hydrolysis metabolites were 21-47 times less potent. Where hydroxylations occur at the non-terminal (4) position of the pentyl tail of JWH-018 and THJ -2201 there is no loss of CB1 activity. The CB1 potency of the JWH-018 and THJ -2201 5OH-pentyl metabolites is reduced but in the same potency range as those reported for other parent SCs. Dihydrodiol metabolites of 4en-compounds show a large reduction in CB1 activity (> 53 times) compared to the parent, as do their ester hydrolysis metabolites (> 130-times). The oxidative defluorinated metabolites of the 4F-MDMB and 5F-MDMB compounds retain some CB1 activity (7–27 times reduction) but a more significant loss of activity is observed when ester hydrolysis occurs in the head group (21–417 times reduction). The head and core moieties of SCs are most influential in determining CB1 potency. The tail, when fulfilling certain steric requirements, acts as an anchor point in the CB1 receptor, influencing the overall position and rigidity of the SC in the CB1 receptor. From a urinary bioassay perspective, where metabolites can be 10 times more abundant than parent SCs, the activity of the metabolites might be important for SC detection. We have established a structure-activity relationship for three SC classes and their metabolites. The relative change in CB1 activity caused by a biotransformation on the tail of the SC depends on the head structure present and biotransformation of the head group leads to a greater reduction in potency than single hydroxylations on the tail. So, “the tail of the SC metabolite needs to know what the head is doing” when activating the CB1 receptor. The study is part of Eurostars-2 Joint Programme NPS-REFORM.