Elizabeth A. Cairns

Synthetic cannabinoid receptor agonists (SCRAs) are a growing class of new psychoactive substances (NPS) commonly derived from an N-alkylated indole, indazole, or 7-azaindole scaffold. Diversification of this core (at the 3-position) with amide-linked pendant amino acid groups and modular N-alkylation (of the indole/indazole/7-azaindole core) ensures that novel SCRAs continue to enter the illicit drug market rapidly. In response to the large number of SCRAs that have been detected, pharmacological evaluation of this NPS class has become increasingly common. Adamantane-derived SCRAs have consistently appeared throughout the market since 2011, and as such, a systematic set of these derivatives was synthesized and pharmacologically evaluated. Deuterated and fluorinated adamantane derivatives were prepared to evaluate typical hydrogen bioisosteres, as well as evaluation of the newly detected AFUBIATA.

Synthetic cannabinoid receptor agonists (SCRAs) comprise the second largest class of new psychoactive substances (NPS), and typically α-amino acid moieties are incorporated as part of their design. Limited investigation has been performed into elucidating structure–activity relationships around commonly used α-amino acid-derived head groups, mainly with valine and tert-leucine-derived compounds previously described. As such, proactive synthesis, characterisation and pharmacological evaluation were performed to explore structure–activity relationships of 15 α-amino acid derivatives, with both the natural isomers and their enantiomers at CB₁ and CB₂ investigated using a fluorescence-based membrane potential assay. This library was based around the detected SCRAs MPP-5F-PICA, MMB-5F-PICA, and MDMB-5F-PICA, with the latter showing significant receptor activation at CB₁ (pEC₅₀ = 8.34 ± 0.05 M; E_max = 108 ± 3%) and CB₂ (pEC₅₀ = 8.13 ± 0.07 M; E_max = 99 ± 2%). Most valine and leucine derivatives were potent and efficacious SCRAs, while smaller derivatives generally showed reduced activity at CB₁ and CB₂, and larger derivatives also showed reduced activity. SAR trends observed were rationalised via in silico induced fit docking. Overall, while natural enantiomers showed equipotent or greater activity than the unnatural isomers in most cases, this was not universal. As such, a number of these compounds should be monitored as emerging NPS, and various substituents described herein.

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 (CB₁ and CB₂), and in vivo cannabimimetic activity. All compounds showed high affinity for CB₁ (K_i 0.299–538 nM) and most at CB₂ (K_i = 0.912–2190 nM), and most functioned as high efficacy agonists of CB₁ and CB₂ 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 CB₁/CB₂ binding and CB₁/CB₂ 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.

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 (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) were suboptimal for binding, whereas the cumyl analogues consistently conferred high affinity (K_i = 0.62–36 nM). Finally, both butyl (K_i = 3.1–163 nM) and 4‐cyanobutyl (K_i = 5.5–44 nM) tail groups were less favorable for CB₁ binding than their corresponding 4‐pentenyl counterparts (K_i = 0.72–25 nM).

Cannabinoid receptor 1 (CB1) activation has been reported to reduce transient receptor potential cation channel subfamily V member 1 (TRPV1)-induced inflammatory responses and is anti-nociceptive and anti-inflammatory in corneal injury. We examined whether allosteric ligands, can modulate CB1 signaling to reduce pain and inflammation in corneal hyperalgesia. Corneal hyperalgesia was generated by chemical cauterization of cornea in wildtype and CB2 knockout (CB2^−/−) mice. The novel racemic CB1 allosteric ligand GAT211 and its enantiomers GAT228 and GAT229 were examined alone or in combination with the orthosteric CB1 agonist Δ⁸-tetrahydrocannabinol (Δ⁸-THC). Pain responses were assessed following capsaicin (1 µM) stimulation of injured corneas at 6 h post-cauterization. Corneal neutrophil infiltration was also analyzed. GAT228, but not GAT229 or GAT211, reduced pain scores in response to capsaicin stimulation. Combination treatments of 0.5% GAT229 or 1% GAT211 with subthreshold Δ⁸-THC (0.4%) significantly reduced pain scores following capsaicin stimulation. The anti-nociceptive effects of both GAT229 and GAT228 were blocked with CB1 antagonist AM251, but remained unaffected in CB2^−/− mice. Two percent GAT228, or the combination of 0.2% Δ⁸-THC with 0.5% GAT229 also significantly reduced corneal inflammation. CB1 allosteric ligands could offer a novel approach for treating corneal pain and inflammation.

Purpose: Orthosteric cannabinoid receptor 1 (CB₁) activation leads to decreases in intraocular pressure (IOP). However, use of orthosteric CB₁ agonists chronically has several disadvantages, limiting their usefulness as clinically relevant drugs. Allosteric modulators interact with topographically distinct sites to orthosteric ligands and may be useful to circumvent some of these disadvantages. The purpose of this study was to investigate the effects of the novel CB₁-positive allosteric modulator (PAM) GAT229 on IOP. Methods: IOP was measured using rebound tonometry in anesthetized normotensive C57Bl/6 mice and in a genetic model of ocular hypertension [nose, eyes, ears (nee) mice] before drug administration, and at 1, 6, and 12 h thereafter. Results: In normotensive mice, topical administration of 5 μL GAT229 alone at either 0.2% or 2% did not reduce IOP. However, a subthreshold dose (0.25%) of the nonselective orthosteric CB₁ agonist WIN 55,212-2, when combined with 0.2% GAT229, significantly reduced IOP compared with vehicle at 6 and 12 h. Similarly, combination of subthreshold Δ⁹-tetrahydrocannabinol (a nonselective orthosteric CB₁ agonist; 1 mg/kg) with topical 0.2% GAT229 produced IOP lowering at 6 h. In nee mice, administration of topical 0.2% GAT229 or 10 mg/kg GAT229 alone was sufficient to lower IOP at 6 and 12 h, and 12 h, respectively. Conclusions: The CB₁ PAM GAT229 reduces IOP in ocular hypertensive mice and enhanced CB₁-mediated IOP reduction when combined with subthreshold CB₁ orthosteric ligands in normotensive mice. Administration of CB₁ PAMs may provide a novel approach to reduce IOP with fewer of the disadvantages associated with orthosteric CB₁ activation.