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 CB 1 binding affinity (K i = 0.17-39 nM), followed by indole-(K i = 0.95-160 nM) and then 7-azaindole-derived SCRAs (K i = 5.4-271 nM). Variation of the head group had the greatest effect on binding, with tert-leucine amides and methyl esters (K i = 0.17-14 nM) generally showing the greatest affinities, followed by valine derivatives (K i = 0.72-180 nM), and then phenylalanine derivatives (K i = 2.5-271 nM). Adamantyl head groups (K i = 8.8-59 nM)

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.

Cannabis-based products are increasingly being used to treat refractory childhood epilepsies such as Dravet syndrome. Cannabis contains at least 140 terpenophenolic compounds known as phytocannabinoids. These include the known anticonvulsant compound cannabidiol (CBD) and several molecules showing emergent anticonvulsant properties in animal models. Cannabichromene (CBC) is a phytocannabinoid frequently detected in artisanal cannabis oils used in the community by childhood epilepsy patients. Here we examined the brain and plasma pharmacokinetic profiles of CBC, cannabichromenic acid (CBCA), cannabichromevarin (CBCV), and cannabichromevarinic acid (CBCVA) following intraperitoneal administration in mice. The anticonvulsant potential of each was then tested against hyperthermia-induced seizures in the Scn1a +/– mouse model of Dravet syndrome. All phytocannabinoids within the CBC series were readily absorbed and showed substantial brain penetration (brain–plasma ratios ranging from 0.2 to 5.8). Anticonvulsant efficacy was evident with CBC, CBCA, and CBCVA, each significantly increasing the temperature threshold at which Scn1a +/– mice had a generalized tonic–clonic seizure. We synthesized a fluorinated derivative of CBC (5-fluoro-CBC), which showed improved brain penetration relative to the parent CBC molecule but not any greater anticonvulsant effect. Since CBC and derivatives are anticonvulsant in a model of intractable pediatric epilepsy, they may constitute part of the mechanism through which artisanal cannabis oils are anticonvulsant in patients.

Synthetic cannabinoid receptor agonists (SCRAs) represent the most rapidly expanding class of new psychoactive substances (NPSs). Despite the prevalence and potency of recent chiral indole-3-carboxamide SCRAs, few pharmacological data are available regarding the enantiomeric bias of these NPSs toward human CB1 and CB2 receptors. A series of homochiral indole-3-carboxamides derived from (S)- and (R)-α-methylbenzylamine and featuring variation of the 1-alkyl substituent were prepared, pharmacologically evaluated, and compared to related achiral congeners derived from cumyl- and benzylamine. Competitive binding assays demonstrated that all analogues derived from either enantiomer of α-methylbenzylamine (14–17) showed affinities for CB1 (K i = 47.9–813 nM) and CB2 (K i = 47.9–347 nM) that were intermediate to that of the corresponding benzylic (10–13, CB1 K i = 550 nM to >10 μM; CB2 K i = 61.7 nM to >10 μM) and cumyl derivatives (6–9, CB1 K i = 12.6–21.4 nM; CB2 K i = 2.95–24.5 nM). In a fluorometric membrane potential assay, all α-methylbenzyl analogues (excluding 17) were potent, efficacious agonists of CB1 (EC50 = 32–464 nM; E max = 89–104%) and low efficacy agonists of CB2 (EC50 = 54–500 nM; E max = 52–77%), with comparable or greater potency than the benzyl analogues and much lower potency than the cumyl derivatives, consistent with binding trends. The relatively greater affinity and potency of (S)-14–17 compared to (R)-14–17 analogues at CB1 highlighted an enantiomeric bias for this series of SCRAs. Molecular dynamics simulations provided a conformational basis for the observed differences in agonist potency at CB1 pending benzylic substitution.

The present work is the last of a three-part study investigating a panel of 30 systematically designed synthetic cannabinoid receptor agonists (SCRAs) including features such as the 4-pentenyl tail and varying head groups including amides and esters of L-valine (MMB, AB), L-tert-leucine (ADB), and L-phenylalanine (APP), as well as adamantyl (A) and cumyl moieties (CUMYL). Here, we evaluated these SCRAs for their capacity to activate the human cannabinoid receptor 1 (CB 1 ) via indirect measurement of G protein recruitment. Furthermore, we comparatively evaluated the results obtained from three in vitro assays, based on the recruitment of β-arrestin 2 (βarr2 assay) or Gα i protein (mini-Gα i assay), or binding of [ 35 S]-GTPγS. The observed efficacies (E max ) varied depending on the conducted assay. Statistical analysis suggests that the population means of the relative intrinsic activity (RA i ) significantly differ for the [ 35 S]-GTPγS assay and the other two assays, but the population means of the βarr2 and mini-Gα i assays were not statistically different. Our data suggest that differences observed between the βarr2 and mini-Gα i assays are the best The contribution to this article by Patrick Juchli is independent from and unrelated to his function at PwC Switzerland.