Selective CB2 agonists with anti-pruritic activity: Discovery of potent and orally available bicyclic 2-pyridones

The CB2 receptor has emerged as a potential target for the treatment of pruritus as well as pain without CB1-mediated side effects. We previously identified 2-pyridone derivatives 1 and 2 as potent CB2 agonists; however, this series of compounds was found to have unacceptable pharmacokinetic profiles with no significant effect in vivo. To improve these profiles, we performed further structural optimization of 1 and 2, which led to the discovery of bicyclic 2-pyridone 18e with improved CB2 affinity and selectivity over CB1. In a mouse pruritus model, 18e inhibited compound 48/80 induced scratching behavior at a dose of 100 mg/kg. In addition, the docking model of 18e with an active-state CB2 homology model indicated the structural basis of its high affinity and selectivity over CB1.     

7-Azaindolequinuclidinones (7-AIQD): A novel class of cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptor ligands

A series of N-benzyl-7-azaindolequinuclidinone (7-AIQD) analogs have been synthesized and evaluated for affinity toward CB1 and CB2 cannabinoid receptors and identified as a novel class of cannabinoid receptor ligands. Structure–activity relationship (SAR) studies indicate that 7-AIQD analogs are dual CB1/CB2 receptor ligands exhibiting high potency with somewhat greater selectivity towards CB2 receptors compared to the previously reported indolequinuclidinone (IQD) analogs. Initial binding assays showed that 7-AIQD analogs 8b, 8d, 8f, 8g and 9b (1 μM) produced more that 50% displacement of the CB1/CB2 non-selective agonist CP-55,940 (0.1 nM). Furthermore, Ki values determined from full competition binding curves showed that analogs 8a, 8b and 8g exhibit high affinity (110, 115 and 23.7 nM, respectively) and moderate selectivity (26.3, 6.1 and 9.2-fold, respectively) for CB2 relative to CB1 receptors. Functional studies examining modulation of G-protein activity demonstrated that 8a acts as a neutral antagonist at CB1 and CB2 receptors, while 8b exhibits inverse agonist activity at these receptors. Analogs 8f and 8g exhibit different intrinsic activities, depending on the receptor examined. Molecular docking and binding free energy calculations for the most active compounds (8a, 8b, 8f, and 8g) were performed to better understand the CB2 receptor-selective mechanism at the atomic level. Compound 8g exhibited the highest predicted binding affinity at both CB1 and CB2 receptors, and all four compounds were shown to have higher predicted binding affinities with the CB2 receptor compared to their corresponding binding affinities with the CB1 receptor. Further structural optimization of 7-AIQD analogs may lead to the identification of potential clinical agents.     

Design,synthesis, and binding mode prediction of 2-pyridone-based selective CB2 receptor agonists

Selective CB2 agonists have the potential for treating pain without central CB1-mediated adverse effects. Screening efforts identified 1,2-dihydro-3-isoquinolone 1; however, this compound has the drawbacks of being difficult to synthesize with two asymmetric carbons on an isoquinolone scaffold and of having a highly lipophilic physicochemical property. To address these two major problems, we designed the 2-pyridone-based lead 15a, which showed moderate affinity for CB2. Optimization of 15a led to identification of 39f with high affinity for CB2 and selectivity over CB1. Prediction of the binding mode of 39f in complex with an active-state CB2 homology model provided structural insights into its high affinity for CB2.     

Oximes short-acting CB1 receptor agonists

New oximes short-acting CB1 agonists were explored by the introduction of an internal oxime and polar groups at the C3 alkyl tail of Δ⁸-THC. The scope of the research was to drastically alter two important physicochemical properties hydrophobicity (log P) and topological surface area (tPSA) of the compound, which play a critical role in tissue distribution and sequestration (depot effect). Key synthesized analogs demonstrated sub-nanomolar affinity for CB1, marked reduction in hydrophobicity (ClogP～2.5–3.5 vs 9.09 of Δ⁸-THC-DMH), and found to function as either agonists (trans-oximes) or neutral antagonists (cis-oximes) in a cAMP functional assay. All oxime analogs showed comparable affinity at the CB2 receptor, but surprisingly they were found to function as inverse agonists for CB2. In behavioral studies (i.e. analgesia, hypothermia) trans-oxime 8a exhibited a predictable fast onset (～20?min) and short duration of pharmacological action (～180?min), in contrast to the very prolonged duration of Δ⁸-THC-DMH (>24?h), thus limiting the potential for severe psychotropic side-effects associated with persistent activation of the CB1 receptor. We have conducted 100?ns molecular dynamic (MD) simulations of CB1 complexes with AM11542 (CB1 agonist) and both trans-8a and cis-8b isomeric oximes. These studies revealed that the C3 alkyl tail of cis-8b orientated within the CB1 binding pocket in a manner that triggered a conformational change that stabilized the CB1 receptor at its inactive-state (antagonistic functional effect). In contrast, the trans-8a isomer’s conformation was coincided with that of the AM11542 CB1 agonist-bound structure, stabilizing the CB1 receptor at the active-state (agonistic functional effect). We have selected oxime trans-8a based on its potency for CB1, and favorable pharmacodynamic profile, such as fast onset and predictable duration of pharmacological action, for evaluation in pre-clinical models of anorexia nervosa.     

Synthesis and preliminary evaluation of a new fluorine-18 labelled triazine derivative for PET imaging of cannabinoid CB2 receptor

Cannabinoid CB2 PET tracers are considered as a promising alternative to PBR/TSPO tracers for the in-vivo imaging of neuroinflammation. We describe here the synthesis and characterization of compound 3, a new potent and brain penetrating CB2 ligand based on an original triazine template. The PET tracer [¹⁸F]-dideutero-3 was prepared in a three steps radiosynthesis, and demonstrated significant uptake in rhesus macaque and baboon brain with a maximum SUV of about 0.7–0.9 g/mL, followed by a moderate washout over time.     

Optimisation of a novel series of selective CNS penetrant CB(2) agonists

A series of benzimidazole CB₂ receptor agonists were prepared and their properties investigated. Optimisation of the three benzimidazole substituents led to the identification of compound 23, a potent CB₂ full agonist (EC₅₀ 2.7 nM) with excellent selectivity over the CB₁ receptor (>3000-fold). Compound 23 demonstrated good CNS penetration in rat. Further optimisation led to the identification of compound 34 with improved selectivity over hERG and excellent CNS penetration in rat.     

Fluorine-18 isotope labeling for positron emission tomography imaging. Direct evidence for DBPR211 as a peripherally restricted CB1 inverse agonist

The [¹⁸F] isotope-labelled CB1 inverse agonist 3 was elaborated and synthesized for positron emission tomography scanning studies. After immediate purification and calibration with its unlabeled counterpart, compound 3 was intravenously injected in mice and revealed that its distribution percentage in brain over 90-min scans among five region of interests, including brain, liver, heart, thigh muscle and kidney was lower than 1%, thus providing direct evidence to justify itself as a peripherally restricted CB1 antagonist.     

Functionalized 6-(piperidin-1-yl)-8,9-diphenyl purines as inverse agonists of the CB1 receptor – SAR efforts towards selectivity and peripheralization

Antagonists of type 1 cannabinoid receptors (CB1) may be useful in treating diabetes, hepatic disorders, and fibrosis. Otenabant (1) is a potent and selective CB1 inverse agonist that was under investigation as an anti-obesity agent, but its development was halted once adverse effects associated with another marketed inverse agonist rimonabant (2) became known. Non-tissue selective antagonists of CB1 that have high levels of brain penetration produce adverse effects in a small subset of patients including anxiety, depression and suicidal ideation. Currently, efforts are underway to produce compounds that have limited brain penetration. In this report, novel analogs of 1 are explored to develop and test strategies for peripheralization. The piperidine of 1 is studied as a linker, which is functionalized with alkyl, heteroalkyl, aryl and heteroaryl groups using a connector in the form of an amine, amide, sulfonamide, sulfamide, carbamate, oxime, amidine, or guanidine. We also report more polar replacements for the 4-chlorophenyl group in the 9-position of the purine core, which improve calculated physical properties of the molecules. These studies resulted in compounds such as 75 that are potent inverse agonists of hCB1 with exceptional selectivity for hCB1 over hCB2. SAR studies revealed ways to adjust physical properties to limit brain exposure.     

Identification and hit-to-lead optimization of a novel class of CB1 antagonists

The discovery, synthesis and preliminary structure–activity relationships (SARs) of a novel class of CB1 antagonists is described. Initial optimization of benzimidazole-based screening hit 4 led to the identification of ‘inverted’ indole-based lead compound 18c with improved properties versus compound 4 including reduced A log P, improved microsomal stability and improved aqueous solubility. Compound 18c demonstrates in vivo CB1 antagonist efficacy (CB1 agonist induced hypothermia model) and is orally bioavailable in rat.     

Synthesis and pharmacological characterization of functionalized 6-piperazin-1-yl-purines as cannabinoid receptor 1 (CB1) inverse agonists

Antagonists of peripheral type 1 cannabinoid receptors (CB1) may have utility in the treatment of obesity, liver disease, metabolic syndrome and dyslipidemias. We have targeted analogues of the purine inverse agonist otenabant (1) for this purpose. The non-tissue selective CB1 antagonist rimonabant (2) was approved as a weight-loss agent in Europe but produced centrally mediated adverse effects in some patients including dysphoria and suicidal ideation leading to its withdrawal. Efforts are now underway to produce compounds with limited brain exposure. While many structure-activity relationship (SAR) studies of 2 have been reported, along with peripheralized compounds, 1 remains relatively less studied. In this report, we pursued analogues of 1 in which the 4-aminopiperidine group was switched to piperazine group to enable a better understanding of SAR to eventually produce compounds with limited brain penetration. To access a binding pocket and modulate physical properties, the piperazine was functionalized with alkyl, heteroalkyl, aryl and heteroaryl groups using a variety of connectors, including amides, sulfonamides, carbamates and ureas. These studies resulted in compounds that are potent antagonists of hCB1 with high selectivity for hCB1 over hCB2. The SAR obtained led to the discovery of 65 (Ki?=?4?nM, >1,000-fold selective for hCB1 over hCB2), an orally bioavailable aryl urea with reduced brain penetration, and provides direction for discovering peripherally restricted compounds with good in vitro and in vivo properties.     

Synthesis and evaluation of potent and selective MGL inhibitors as a glaucoma treatment

Monoacylglycerol lipase (MGL) inhibition provides a potential treatment approach to glaucoma through the regulation of ocular 2-arachidonoylglycerol (2-AG) levels and the activation of CB1 receptors. Herein, we report the discovery of new series of carbamates as highly potent and selective MGL inhibitors. The new inhibitors showed potent nanomolar inhibitory activity against recombinant human and purified rat MGL, were selective (>1000-fold) against serine hydrolases FAAH and ABHD6 and lacked any affinity for the cannabinoid receptors CB1 and CB2. Protein-based ¹H NMR experiments indicated that inhibitor 2 rapidly formed a covalent adduct with MGL with a residence time of about 6?h. This interconversion process “intrinsic reversibility” was exploited by modifications of the ligand’s size (length and bulkiness) to generate analogs with “tunable’ adduct residence time (τ). Inhibitor 2 was evaluated in a normotensive murine model for assessing intraocular pressure (IOP), which could lead to glaucoma, a major cause of blindness. Inhibitor 2 was found to decrease ocular pressure by ～4.5?mmHg in a sustained manner for at least 12?h after a single ocular application, underscoring the potential for topically-administered MGL inhibitors as a novel therapeutic target for the treatment of glaucoma.     

Discovery of potent selective bioavailable phosphodiesterase 2 (PDE2) inhibitors active in an osteoarthritis pain model. Part II: Optimization studies and demonstration of in vivo efficacy

Selective phosphodiesterase 2 (PDE2) inhibitors are shown to have efficacy in a rat model of osteoarthritis (OA) pain. We identified potent, selective PDE2 inhibitors by optimizing residual PDE2 activity in a series of phosphodiesterase 4 (PDE4) inhibitors, while minimizing PDE4 inhibitory activity. These newly designed PDE2 inhibitors bind to the PDE2 enzyme in a cGMP-like binding mode orthogonal to the cAMP-like binding mode found in PDE4. Extensive structure activity relationship studies ultimately led to identification of pyrazolodiazepinone, 22, which was >1000-fold selective for PDE2 over recombinant, full length PDEs 1B, 3A, 3B, 4A, 4B, 4C, 7A, 7B, 8A, 8B, 9, 10 and 11. Compound 22 also retained excellent PDE2 selectivity (241-fold to 419-fold) over the remaining recombinant, full length PDEs, 1A, 4D, 5, and 6. Compound 22 exhibited good pharmacokinetic properties and excellent oral bioavailability (F = 78%, rat). In an in vivo rat model of OA pain, compound 22 had significant analgesic activity 1 and 3 h after a single, 10 mg/kg, subcutaneous dose.     

Facile synthesis, ex-vivo and in vitro screening of 3-sulfonamide derivative of 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid piperidin-1-ylamide (SR141716) a potent CB1 receptor antagonist

Facile synthesis of biaryl pyrazole sulfonamide derivative of 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid piperidin-1-ylamide (SR141716, 1) and an investigation of the effect of replacement of the –CO group in the compound 1 by the –SO₂ group in the aminopiperidine region is reported. Primary ex-vivo pharmacological testing and in vitro screening of sulfonamide derivative 2 showed the loss of CB1 receptor antagonism.     

Design, synthesis and structure-activity relationships of (indo-3-yl) heterocyclic derivatives as agonists of the CB1 receptor. Discovery of a clinical candidate

We report an expansion of the structure-activity relationship (SAR) of a novel series of indole-3-heterocyclic CB1 receptor agonists. Starting from the potent but poorly soluble lead, 1, a rational approach was taken in order to balance solubility, hERG activity and potency while retaining the desired long duration of action within the mouse tail flick test. This led to the discovery of compound 38 which successfully progressed into clinical development.     

Discovery of indole alkaloids with cannabinoid CB1 receptor antagonistic activity

Three indole alkaloids, voacamine (1), 3,6-oxidovoacangine (2), and a new alkaloid, 5-hydroxy-3,6-oxidovoacangine (3), isolated from Voacanga africana were found to exhibit potent cannabinoid CB1 receptor antagonistic activity. This is the first example of CB1 antagonists derived from natural alkaloids.     

Pyrazole antagonists of the CB1 receptor with reduced brain penetration

Type 1 cannabinoid receptor (CB1) antagonists might be useful for treating obesity, liver disease, metabolic syndrome, and dyslipidemias. Unfortunately, inhibition of CB1 in the central nervous system (CNS) produces adverse effects, including depression, anxiety and suicidal ideation in some patients, which led to withdrawal of the pyrazole inverse agonist rimonabant (SR141716A) from European markets. Efforts are underway to produce peripherally selective CB1 antagonists to circumvent CNS-associated adverse effects. In this study, novel analogs of rimonabant (1) were explored in which the 1-aminopiperidine group was switched to a 4-aminopiperidine, attached at the 4-amino position (5). The piperidine nitrogen was functionalized with carbamates, amides, and sulfonamides, providing compounds that are potent inverse agonists of hCB1 with good selectivity for hCB1 over hCB2. Select compounds were further studied using in vitro models of brain penetration, oral absorption and metabolic stability. Several compounds were identified with predicted minimal brain penetration and good metabolic stability. In vivo pharmacokinetic testing revealed that inverse agonist 8c is orally bioavailable and has vastly reduced brain penetration compared to rimonabant.     

Discovery of potent and orally bioavailable heterocycle-based cannabinoid CB1 receptor agonists

Novel 3-(1H-indol-3-yl)-1,2,4-oxadiazoles and -thiadiazoles were synthesized and found to be potent CB1 cannabinoid receptor agonists. The oral bioavailability of these compounds could be dramatically improved by optimization studies of the side chains attached to the indole and oxadiazole cores, leading to identification of a CB1 receptor agonist with good oral activity in a range of preclinical models of antinociception and antihyperalgesia.     

Synthetic bioactive olivetol-related amides: The influence of the phenolic group in cannabinoid receptor activity

Focusing on the importance of the free phenolic hydroxyl moiety, a family of 23 alkylresorcinol-based compounds were developed and evaluated for their cannabinoid receptor binding properties. The non-symmetrical hexylresorcinol derivative 29 turned out to be a CB2-selective competitive antagonist/inverse agonist endowed with good potency. Both the olivetol- and 5-(2-methyloctan-2-yl)resorcinol-based derivatives 23 and 24 exhibited a significant antinociceptive activity. Interestingly, compound 24 proved to be able to activate both cannabinoid and TRPV1 receptors. Even if cannabinoid receptor subtype selectivity remained a goal only partially achieved, results confirm the validity of the alkylresorcinol nucleus as skeleton for the identification of potent cannabinoid receptor modulators.     

Cannabinoid CB2 Receptors Regulate Central Sensitization and Pain Responses Associated with Osteoarthritis of the Knee Joint

Osteoarthritis (OA) of the joint is a prevalent disease accompanied by chronic, debilitating pain. Recent clinical evidence has demonstrated that central sensitization contributes to OA pain. An improved understanding of how OA joint pathology impacts upon the central processing of pain is crucial for the identification of novel analgesic targets/new therapeutic strategies.Inhibitory cannabinoid 2 (CB₂) receptors attenuate peripheral immune cell function and modulate central neuro-immune responses in models of neurodegeneration. Systemic administration of the CB₂ receptor agonist JWH133 attenuated OA-induced pain behaviour, and the changes in circulating pro- and anti-inflammatory cytokines exhibited in this model. Electrophysiological studies revealed that spinal administration of JWH133 inhibited noxious-evoked responses of spinal neurones in the model of OA pain, but not in control rats, indicating a novel spinal role of this target. We further demonstrate dynamic changes in spinal CB₂ receptor mRNA and protein expression in an OA pain model. The expression of CB₂ receptor protein by both neurones and microglia in the spinal cord was significantly increased in the model of OA. Hallmarks of central sensitization, significant spinal astrogliosis and increases in activity of metalloproteases MMP-2 and MMP-9 in the spinal cord were evident in the model of OA pain. Systemic administration of JWH133 attenuated these markers of central sensitization, providing a neurobiological basis for analgesic effects of the CB₂ receptor in this model of OA pain. Analysis of human spinal cord revealed a negative correlation between spinal cord CB₂ receptor mRNA and macroscopic knee chondropathy.These data provide new clinically relevant evidence that joint damage and spinal CB₂ receptor expression are correlated combined with converging pre-clinical evidence that activation of CB₂ receptors inhibits central sensitization and its contribution to the manifestation of chronic OA pain. These findings suggest that targeting CB₂ receptors may have therapeutic potential for treating OA pain.     

Discovery of novel tetrahydroisoquinoline derivatives as orally active N-type calcium channel blockers with high selectivity for hERG potassium channels

N-type calcium channels represent a promising target for the treatment of neuropathic pain. The selective N-type calcium channel blocker ziconotide ameliorates severe chronic pain but has a narrow therapeutic window and requires intrathecal administration. We identified tetrahydroisoquinoline derivative 1a as a novel potent N-type calcium channel blocker. However, this compound also exhibited potent inhibitory activity against hERG channels. Structural optimizations led to identification of (1S)-(1-cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-{[(1-hydroxycyclohexyl)methyl]amino}ethanone ((S)-1h), which exhibited high selectivity for hERG channels while retaining potency for N-type calcium channel inhibition. (S)-1h went on to demonstrate in vivo efficacy as an orally available N-type calcium channel blocker in a rat spinal nerve ligation model of neuropathic pain.