Latest advances in novel cannabinoid CB₂ ligands for drug abuse and their therapeutic potential

Abstract

The field of cannabinoid (CB) drug research is experiencing a challenge as the CB₁ antagonist Rimonabant, launched in 2006 as an anorectic/anti-obesity drug, was withdrawn from the European market due to the complications of suicide and depression as side effects. There is interest in developing CB₂ drugs without CB₁ psychotropic side effects for drug-abuse treatment and therapeutic medication. The CB₁ receptor was discovered predominantly in the brain, whereas the CB₂ is mainly expressed in peripheral cells and tissues, and is involved in immune signal transduction. Conversely, the CB₂ receptor was recently detected in the CNS, for example, in the microglial cells and the neurons. While the CB₂ neurons activity remains controversial, the CB₂ receptor is an attractive therapeutic target for neuropathic pain, immune system, cancer and osteoporosis without psychoactivity. This review addresses CB drug abuse and therapeutic potential with a focus on the most recent advances on new CB₂ ligands from the literature as well as patents.

Cannabinoid drug abuse & the endocannabinoid system

Drug abuse is a concerning issue worldwide and often carries with it criminal penalties and negative physical, social and psychological effects. Drugs that are used and abused by humans for nonmedical purposes can be grouped into several major categories that include marijuana or cannabis (cannabinoids [CBs]), alcohol (ethanol), nicotine and tobacco, depressants (barbiturates and benzodiazepines), stimulants (amphetamines, cocaine), opioids (morphine, heroin and methadone), psychedelics (LSD, mescaline and ecstasy), inhalants (glue and nitrous oxide) and phencyclidine. CBs remain the most widespread drugs in use worldwide. The term ‘cannabinoid’ was first used to describe the tricyclic natural compounds from Cannabis sativa L [1]. Marijuana is the most used illicit drug in the USA, and is very often ingested with other drugs of abuse. National Institute on Drug Abuse 2009 reported that 16.4 million Americans aged 12 or older used marijuana at least once in the month prior to being surveyed [201]. Marijuana abuse and toxicities are a serious threat to human health in the USA and worldwide. It is now known that Δ⁹-tetrahydrocannabinol (Δ⁹-THC), the main psychoactive ingredient of marijuana, activates the mesocorticolimbic system, the same system responsible for the reinforcing properties of all drugs of abuse [2–4]. Δ⁹-THC acts primarily through the endocannabinoid system in the brain. This system modulates diverse physiologic functions including motor function, memory, motivation, drive, pain and emotion [5–7].

Effective treatments for the abuse of marijuana and other drugs of abuse remain elusive as evident by high rates of unpleasant withdrawal symptoms and relapse. Hence, there is a tremendous medical need for new rationally designed medications to treat drug abuse and associated diseases, an advance that likely requires the development of new research strategies and resources. There is ample evidence that most of the centrally mediated effects of many drugs of abuse, including CBs, opioids, alcohol and nicotine, occur through the endocannabinoid system [8]. The studies show that release of endocannabinoids in the ventral tegmental area can modulate the reward-related effects of dopamine and might, therefore, be an important neurobiological mechanism underlying drug addiction. There is strong evidence that the endocannabinoid system is involved in drug-seeking behavior (especially behavior that is reinforced by drugrelated cues), as well as in the mechanisms that underlie relapse to drug use [8]. Therefore, the endocannabinoid system represents a promising target for development of new treatments for drug addiction.

CB receptors & ligands

To date, at least two CB receptors have been cloned and characterized: CB₁ and CB₂, which share 48% identity at the amino acid level [9,10]. CB receptors contain an N-terminal extracellular domain that possesses glycosylation sites, a C-terminal intracellular domain coupled to a G protein complex and seven hydrophobic transmembrane segments connected by alternating extracellular and intracellular loops. Three dimensional models of the helix bundle arrangement of human, rat and mouse CB₁ and CB₂ receptors have been constructed and compared [11–13]. Both signal through activation of pertussis toxin-sensitive G proteins to inhibit adenylate cyclase, and both are positively coupled to the activation of MAPK [14].

It was initially believed that the CB₁ receptor was expressed predominantly in the brain (central receptor for CBs) [9], whereas the CB₂ receptor in peripheral cells and tissues was derived from the immune system (peripheral receptor for CBs) [10]. The CB₁ receptor was recently also found in a number of peripheral tissues, for example, the cardiovascular and reproductive systems and in the GI tract [15–17]. In addition, recent studies have indicated that the CB₂ receptor may also exist in the CNS, for example, in microglial cells as well as neurons [18–20]. Thus, CB₂ receptor biology may in the future be used to develop nonpsychotropic (or non-CB₁-mediated) approaches to manipulate endocannabinoid levels localized in the brain, offering therapeutic promise for treating CNS disorders. However, the CB₂ CNS neural activities still need to be investigated further and evaluated in greater detail.

CB₁ receptor, ligands & drug abuse

The CB₁ receptor is primarily, but not exclusively, expressed in the CNS, in particular in the hippocampus, some olfactory regions, caudate, putamen, accumbens nucleus (ventral striatum), the substantia nigra pars reticulata, globus pallidus and the horizontal limb of the diagonal band [20,21]. CB₁ mRNA is found in a lesser extent in peripheral tissues, such as the adrenal gland, heart, lung, prostate, testis, bone marrow, thymus and spleen [14]. The binding of CBs to the CB₁ receptor, which triggers the activation of this receptor, is responsible for the psychoactive effects associated with CBs, such as euphoria, drowsiness, memory lapses, disruption of motor skills, lack of concentration and disorientation [22].

Extensive evidence exists that drugs of abuse exert their reinforcing and rewarding properties through the dopaminergic mesocortical and mesolimbic associative processing and motivational pathways in the brain involving the prefrontal cortex, ventral tegmentum, amygdala and their projections to the striatum [23]. As reported, endocannabinoids are capable of indirectly enhancing dopamine outflow in the nucleus accumbens. The actions of CBs are thought to be mediated via CB₁ receptors located presynaptically on the glutamatergic fibres. In agreement with the neuroanatomical localization and function of CB receptors in reward and motivational pathways, CB₁ receptor antagonists show an ability to attenuate self-administration and/or relapse involving a variety of drugs of abuse, including nicotine [24]. Rimonabant (SR141716A) was the first selective CB₁ receptor antagonist developed [25]. Several reports indicate Rimonabant (SR141716, also inverse agonist for CB₁) can facilitate abstinence from tobacco in tobacco users and these reports have helped propel this interest in the potential applications of CB₁ antagonists as treatments for drug abuse disorders [23]. All these findings indicate that CB₁ antagonists can interfere with brain systems responsible for the expression of the acute reinforcing and motivational properties of drugs of abuse, including marijuana, cocaine and ethanol [23]. However, CB₁ agonists that penetrate the CNS result in catalepsy, sedation and undesirable psychotropic effects, which have also limited the therapeutic utility of nonselective, brain-permeable CB agonists [26]. Thus, more research is needed to confirm if the ubiquitous distribution of the CB₁ receptor in the CNS is the real reason for the adverse psychiatric effects.

CB₂ receptor, ligands & drug abuse

The CB₂ receptor was initially discovered to be widely distributed in peripheral tissues and particularly in immune tissues. Expression of the CB₂ receptor gene transcripts was found in the spleen, tonsils, thymus, mast cells and blood cells [10,27–29]. Interestingly, the CB₂ receptor was also recently detected in the CNS, for example, in the microglial cells as well as the neurons [20]. Abundant CB₂ immunoreactivity in neuronal and glial processes was detected but at a much lower level than as reported in CB₁ receptors [30]. The expression level of the CB₁ gene using RT-PCR analysis was 100-times that of the CB₂ gene expression level in the brain stem [31]. A review of the distribution of CB₁ and CB₂ receptors in the mammalian nervous system was summarized by Svizenska [20]. The most prominent staining of the CB₂ receptor was observed in the anterior olfactory nucleus, in the neurons of the piriform, orbital, visual, motor and auditory cortex, where bodies and apical dendrites of pyramidal neurons in the layers III and V were heavily stained. Moderate density of CB₂ immunopositive cell bodies was found in the periaqueductal gray, substantia nigra pars reticulata and other nuclear structures of the brain stem [20]. Data obtained in vitro and from animal models demonstrated the inducible nature of CB₂ receptors under neuroinflammatory conditions and suggests that the upregulation of CB₂ receptors is a common pattern of response against different types of chronic human brain neuropathology [32]. Mounting evidence also shows that CB₂ and its gene variants may play possible roles in neuroinflammation occurring in multiple sclerosis (MS), traumatic brain injury, HIV-induced encephalitis, Alzheimer’s, Parkinson’s and Huntington’s diseases [32]. These multifocal distributions and the presence of the CNS CB₂ receptor suggest that the CB₂ receptor may play an important role in neurotransmission.

CB₂ and their gene transcripts are expressed in the brains of naive mice and are modulated following exposure to stressors and administration of abused drugs. Onaivi found that mice preferring alcohol had reduced CB₂ gene expression in the ventral midbrain; whereas, the CB₂ gene expression was unaltered in the ventral midbrain region of mice with little or no preference for alcohol. Treatment of mice with the CB₂ agonist JWH-015 enhanced alcohol consumption in mice subjected to chronic mild stress and treatment with the CB₂ antagonist AM630 reduced the stress-induced increase in alcohol consumption. This CB₂ agonist or antagonist effect was absent in normal mice that were not subjected to chronic mild stress. Researchers also found that animals treated with cocaine or heroin showed increased CB₂ gene transcripts in comparison to controls, indicating the presence of CB₂ gene transcripts in the brain that are influenced by abused substances [33–35]. Onaivi utilized behavioral and molecular methods to study and determine whether there was a link between depression in drug/alcohol addiction and the CNS CB₂ receptor. Their studies provided the first evidence for the CNS effects of CB₂ and its possible involvement in drug addiction and neuropsychiatric disorders [33–36].

Very recently, Xi et al. found that systemic, intranasal or intra-accumbens local administration of JWH133, a selective CB₂ receptor agonist, dose-dependently inhibited intravenous cocaine self-administration, cocaine-enhanced locomotion and cocaine-enhanced accumbens extracellular dopamine in wild-type and CB₁ receptor knockout (CB₁ ^−/−, also known as Cnr1^−/−) mice, but not in CB₂ ^−/− (Cnr2^−/−) mice [37]. The result also indicated that JWH133-induced reduction in cocaine self-administration resulted from a reduction in cocaine’s rewarding efficacy, and intranasal JWH133-induced pharmacological effects are mediated by activating brain rather than peripheral CB₂ receptors. Furthermore, their findings suggested that JWH133 has no cocaine-like reinforcing or aversive effects in mice. This finding not only challenges current views that CB₂ receptors are absent from the CNS and that CB₂ receptor ligands lack CNS effects, but also suggests that brain CB₂ receptors may be a target for the pharmacotherapy of drug abuse and addiction [37].

While efforts have been devoted to develop CB₂-selective ligands for therapeutic immune intervention, emerging research and current data demonstrate that the functional expression of CB₂ receptors in brain may provide novel targets for the effects of cannabinoids in depression and drug-abuse disorders beyond neuro-immunocannabinoid activity. In addition, selective activation of CB2 receptor would not be expected to elicit undesired psychotropic effects [22]. Thus, more detailed discussions are presented below.

Therapeutic potential of CB CB₂ ligands

Nonselective CB ligands display a wide range of physiological effects including analgesic, antiinflammatory, anticonvulsive and immunosuppressive activities. Since its discovery in 1993, the CB₂ receptor has been an appealing therapeutic target for novel immunomodulators. It is now known that the CB₂ receptor is expressed in most organ systems including the cells of the brain, heart, liver, cardiovascular and gastrointestinal systems. In many cases, CB₂ receptor expression is regulated by injury or disease. Recent advances in the chemical synthesis of CB₂ receptor ligands, prompted by their potential therapeutic indications for the treatment of pain and other conditions, have led to the rapid expansion of tools for the exploration of this receptor system [38,101].

CB₂ ligands & the immune system

Most of the effects of CBs within the immune system have been attributed to the CB₂ receptor. CB₂ ligands have been demonstrated to attenuate aberrant immune responses in autoimmune disorders and, in some cases, to provide protection to the tissue that is being inappropriately targeted by the immune system. Such cases include:

• MS: an autoimmune disorder that results in the demyelination of neurons in the CNS. CB₂-selective agonist HU-308 markedly reduces the recruitment of immature myeloid and T cells, microglial and infiltrating myeloid cell proliferation, and axonal loss in the experimental autoimmune encephalomyelitis model [39];

• Allergy: CB₂ ligands were found useful in the treatment of allergic reactions. Topical administration of the CB₁/CB₂ agonist HU-210 reduces these histamine-induced responses in human skin [40]. In contrast, injection of the CB₂ receptor antagonist SR144528 exacerbates this inflammation and pruritis [41];

• Conditions associated with inflammation: CB₂ agonists have been demonstrated to attenuate inflammation in the CNS.

Administration of CB₂ agonists prevents the activation of microglia in rodent models of Alzheimer’s disease [42]. Likewise, administration of CB₂ agonists reduces the volume of infarcts by 30% in a rodent occlusion model of stroke [43].

CB₂ ligands & pain

The analgesic properties of CBs have been recognized for many years and the ability of CBs to affect pain perception has supraspinal, spinal and peripheral components [44,45]. Besides the role of CB₁ in mediating these analgesic effects, CB₂ also plays a role in mediating the analgesic effects of CBs. It is not known how CB₂ receptor- selective agonists inhibit pain. However, it is more widely accepted that CB₂ receptors could modulate pain through an indirect mechanism involving circulating cells of the immune system [46]. For example, systemic delivery of the CB₂- selective agonist AM1241 suppresses hyperalgesia induced in the carrageenan, capsaicin and formalin models of inflammatory pain in rodents [47]. Another CB₂-selective agonist GW405833 administered systemically significantly reverses hypersensitivity to mechanical stimuli in rats following the ligation of spinal nerves [48]. As for the CB₂-selective agonist O-3223, it reduced nociceptive behavior in both phases of the formalin test, reduced thermal hyperalgesia in the chronic constriction injury of the sciatic nerve model and reduced edema and thermal hyperalgesia elicited by intraplantar injection of lipopolysaccharide without affecting basal nociception or eliciting overt behavioral effects [49]. It is now clear that the CB₂ receptor plays a critical role in nociception and has been shown to modulate acute pain, chronic inflammatory pain, postsurgical pain, cancer pain and pain associated with nerve injury [46,49].

CB₂ ligands & cancer

CBs and modulators of the endocannabinoid system have recently been shown to produce antitumor actions. Guindon et al. summarized the different mechanisms and signaling pathways that CBs/CBs receptors impact proliferation, migration and apoptosis cancer cells [50]. The endocannabinoid system may be targeted to suppress the evolution and progression of the breast, prostate and bone cancer. And activation of the endocannabinoid signaling system also produces anticancer effects in other types of cancer, including skin, brain and lung [50]. For example, the CB₂ agonist JWH-133 showed good ability to decrease size and number of tumors, reduce the number and size of lung metastases, inhibit cell proliferation and decrease angiogenesis in mice injected with different breast cancer cell lines [51,52].

CB₂ ligands & osteoporosis

There is accumulating evidence to suggest that CBs and their receptors play important roles in bone metabolism by regulating bone mass, bone loss and bone cell function [53]. Osteoblasts, osteoclasts and osteocytes express CB₂ receptors at significantly higher levels than that reported for CB₁ [54–56]. Recent studies reported that bone cells also express GPR55 and TRPV1, which are known to be targeted by endocannabinoids and synthetic CB ligands [53]. The CB₂ agonist HU-308 enhances endocortical osteoblast numbers and activity while simultaneously inhibiting proliferation of osteoclast precursors in bone marrow-derived osteoblasts/stromal cells in vitro, and attenuates ovariectomy-induced bone loss and stimulates cortical thickness by stimulating endocortical bone formation and suppressing osteoclast number in vivo [54].

CB₂ ligands & other potential therapeutic uses

Endocannabinoids are also involved in the pathophysiology of acute and chronic liver disease and gastrointestinal disease [57]. Munoz-Luque et al. used the carbon tetrachloride model to induce fibrosis of the liver and treated rats chronically with the selective CB₂ receptor agonist JHW- 133. JWH-133 improved many indices of damage and markedly improved the extent of liver fibrosis leading to reduced portal pressures [58].

CB₂ receptor agonist JWH015 significantly protects retinal pigment epithelial (RPE) cells [59]. While RPE cells provide trophic support to photoreceptor cells in the eye, RPE cell death has been demonstrated to be a major contributor to age-related macular degeneration. Therefore, CB₂-selective agonists also have potential therapeutic use in preventing the onset or progression of vision loss associated with age-related macular degeneration.

CB₂ antagonists inhibit the proliferation of cultured neural stem cells and the proliferation of progenitor cells in the subventricular zone of young animals; whereas, CB₂-selective agonists stimulate progenitor cell proliferation in vivo, with this effect being more pronounced in older animals [60]. So agonists of CB₂ are useful in regenerative medicine, for example to promote the expansion of progenitor cells for the replacement of neurons lost during injury or disease, such as Alzheimer’s disease, stroke-induced damage, dementia, amyotrophic lateral sclerosis and Parkinson’s disease [101].

Novel CB₂-selective ligands

Due to the unwanted psychotropic effects resulting from activation of the CB₁ receptor, there exists much controversy surrounding the use of medicinal marijuana and the potential for abuse. CB₂-selective ligands with significantly low CB₁ affinity would not be expected to elicit undesired psychotropic effects. Increasing evidence shows that the CB₂ receptor is an attractive therapeutic target. Thus, research is currently focused on the development of CB₂-selective ligands. This review summarizes the literature, particularly patent documents, on CB₂ receptor-selective ligands developed recently and classifies them based on their chemical scaffolds as below. Readers can read other previously published articles about CB₂ ligands elsewhere [61–63].

Five-membered rings as scaffolds for CB₂ ligands

Pyrazole & pyrrolidine derivatives

Abbott Laboratories synthesized a series of selective compounds for the CB₂ receptor, among which 133 compounds were bound to CB₁ receptors, with K_i values of approximately 10–500-fold higher than that for CB₂ receptors. In a 2010 patent, pyrazole 1 (1; Figure 1) was a representative example with a very high CB₂ receptor affinity (human CB₂: K_i = 0.7 nM; rat CB₂: K_i = 1.3 nM) [102]. The in vivo activities were also tested in an incisional model of postoperative pain, a capsaicin-induced secondary mechanical hypersensitivity model and a monosodium iodoacetate-induced knee joint osteoarthritic pain model. The data indicated that certain tested compounds showed a statistical change at less than approximately 300 μM/ kg and certain measured compounds showed efficacy at less than approximately 50 μM/kg.

Figure 1.

Representative structures of pyrazole, pyrrolidine, thiazole, isothiazole and imidazole derivatives.

Over 300 exemplified compounds were claimed as CB₂ agonists in a patent application from Boehringer Ingelheim International GmbH. Among these substituted pyrrolidine derivatives, compound 2 (Figure 1) is representative and is quoted as having an EC₅₀ value of 0.015 nM against CB₂ cAMP in the binding assay [103].

Thiazole & isothiazole derivatives

Frost et al. for Abbott Laboratories disclosed a series of thiazole compounds as CB receptor ligands in 2010. The compounds were evaluated in vitro and in vivo and 38 compounds tested exhibited approximately 10–1000-times weaker binding affinity for CB₁ receptors than for CB₂. The similar result was obtained in the cyclase assays. These results showed that these compounds preferably bind to CB₂ receptors and, therefore, are selective ligands for the CB₂ receptor. From these assays, example compound 3 (Figure 1) was found to display the following values: K_i = 3.1 nM in human CB₂ binding; K_i = 1.1 nM in rat CB₂ binding, and EC₅₀ = 0.07 nM for rat CB₂ cyclase [104].

In the same year, Wang et al. at Abbott Laboratories published two patents of thiazole compounds with an azolylidenebenzamides group. In the first patent, 25 compounds tested bound to CB₂ receptors with K_i values of less than approximately 1000 mM but bound to CB₁ receptors with K_i values 10–1000-times higher than that for CB₂. The example compound 4 (Figure 1) had a high CB₂ affinity (human CB₂ binding: K_i = 1.63 nM; rat CB₂ binding: K_i = 0.80 nM) [105]. In the other patent, another six compounds were claimed as selective CB₂ ligands. The binding value of the representative compound 5 (Figure 1) was K_i = 46.37 nM in human CB₂ binding and K_i = 29.09 nM in rat CB₂ binding [106].

Abbott Laboratories are continuing their interest in this area and have disclosed 267 isothiazole derivatives. Compounds tested are approximately 100-fold to approximately >10,000-fold more potent at activating rat CB₂ versus rat CB₁ receptors in the cyclase assays. Compound 6 (Figure 1) is a representative example that was found to display the following values: human CB₂ binding: K_i = 16 nM; rat CB₂ binding: K_i = 1.5 nM; rat CB₂ cyclase: EC₅₀ = 0.72 nM [107]. The in vivo activities were also tested in the incisional model of postoperative pain, the spinal nerve ligation model of neuropathic pain, the capsaicin-induced secondary mechanical hypersensitivity model and the monosodium iodoacetate-induced knee joint osteoarthritic pain model. The data indicated that certain compounds tested showed a statistical change at less than approximately 300 μM/kg and certain compounds measured showed efficacy at less than approximately 50 μM/kg.

Imidazole derivatives

Beckett et al. at Cara Therapeutics, Inc. published over 600 substituted imidazoheterocycles compounds and tested their EC₅₀ against human CB₂, rat CB₂ and human CB₁ receptors. These compounds were separated as agonist and inverse agonist. The binding affinity data (EC₅₀) were showed as four levels from 0.1 nM to 10 μM [108]. Many of these compounds have high affinity and good selectivity to CB₂ receptor. Compound 7 (Figure 1) is a representative compound with the following values: human CB₂ binding: EC₅₀ < 0.1–10 nM; rat CB₂ binding: EC₅₀ < 0.1–10 nM; human CB₁ binding: EC₅₀ >10 μM [108]. The in vivo activities, such as antihyperalgesia and acute inflammation were also tested in the inflammatory pain model, the carrageenan model of acute inflammation and the spinal nerve ligation model. The results indicated that some compounds showed a statistical effect. No side effects were observed during the course of the experiment.

In 2010, Lange and coworkers produced a SAR study of imidazole. They found a novel imidazole compound 8 (Figure 1), which exhibited the highest CB₂ receptor affinity (K_i = 1.03 nM) in this series, as well as the highest CB₂/CB₁ subtype selectivity (>9708- fold) [64]. This represents a novel chemotype of potent and selective CB₂ receptor antagonists/ inverse agonists.

Six-membered rings as scaffolds for CB₂ ligands

Pyridine & pyrazine derivatives

Bartolozzi et al. prepared over 150 pyridinebased compounds, of which 126 compounds are preferred CB₂ agonists. One exemplified compound 9 (Figure 2) is said to have an EC₅₀ value at the CB₂ receptor of 0.093 nM [109]. They also claimed these compounds are useful for treating inflammation or pain.

Figure 2.

Representative structures of pyridine, pyrazine, pyridazine and morpholine derivatives.

Chu and co-workers replaced the phenyl ring with a pyridine ring when they further explored carboxamide CB ligands, and they found a potent and selective CB₂ agonist compound 10 (Figure 2), which displayed good affinity at the CB₂ receptor (Ki = 24 nM), 160-fold selectivity versus CB₁ (CB₁: K_i = 3800 nM) and moderate metabolic stability in rat and human liver microsomes. Importantly, compound 10 exhibited in vivo efficacy after oral administration in a rat model of neuropathic pain [65].

A series of pyrazine-2-carboxamides compounds has been claimed as CB₂ receptor ligands by F Hoffmann-La Roche AG. Three compounds are selective for the CB₂ receptor, with affinities K_i = 43–63 nM, and all of them exhibit at least tenfold selectivity against the CB₁ receptor. Compound 11 (Figure 2) has the highest affinity with K_i = 43 nM [110].

Pyridazine derivatives

Chen et al. have published a series of pyridazine derivative as therapeutic CB₂ receptor agonists. Compound 12 (Figure 2) shows high CB₂ affinity and good selectivity against CB₁ (CB₁ binding: IC₅₀ = 1028 nM; CB₂ binding: IC₅₀ = 5.4 nM; CB₂ selectivity = 189) [111].

Morpholine derivatives

By both reducing the entropy of the molecule, and incorporating a linker between the aryl rings to reduce potential for unproductive protein binding, Zindell et al. found two compounds 13 and 14 (13: CB₂ cAMP EC₅₀ = 6 nM; CB₁/CB₂: EC₅₀ = 960; 14: CB₂ cAMP EC₅₀ = 10 nM, CB₁/ CB₂ EC₅₀ >2000; Figure 2) [66]. Each of the compounds is a very potent CB₂ agonist based on the functional data with very good selectivity over CB₁.

Seven-membered rings as scaffolds for CB₂ ligands

Diazepane derivatives

A high-throughput screening campaign identified aryl 1,4-diazepane compounds as potent and selective CB₂ agonists as compared with CB₁. Cirillo et al. have synthesized 279 diazepane compounds as CB₂ receptor modulators for treating inflammation, pain and disease. Among these, over a quarter compounds showed high affinity to CB₂ receptor and exhibited agonistic activity. The representative compound 15 (Figure 3) has an EC₅₀ (concentration at which 50% of forskolin-stimulated cAMP synthesis was inhibited) of 0.7 nM or an agonist efficacy of 95% in a human CB₂ receptor binding assay [112].

Figure 3.

Representative structures of diazepane derivatives.

Cirillo et al. reported another series of diazepane compounds as CB₂ receptor modulators and claimed that these compounds were useful for treating inflammation and pain. Fifty three compounds were preferred CB₂ agonists. As a representative sample, compound 16 (Figure 3) exhibited the EC₅₀ value of 0.017 nM [113].

However, many compounds of this class suffered from poor drug-like parameters as well as low microsomal stability and poor solubility. In 2011, Zindell et al. further described the SARs with a focus on improving the drug-like parameters, resulting in compounds with improved solubility and permeability. They found incorporation of heteroalkyl offered little change to the CB₂ potency but greatly enhanced the selectivity profile, while significantly improving the aqueous solubility of the molecule. The representative compound 17 (Figure 3) showed high CB₂ affinity and selectivity with good drug-like properties (CB₂ cAMP EC₅₀ = 1 nM, CB₁ EC₅₀/ CB₂ EC₅₀ = 1770; solubility > 96 μg/ml; clog p = 2.23) [67].

Bicyclic scaffolds for CB₂ ligands

Imidazopyridine derivatives

Scientists at Acadia Pharmaceuticals Inc. recently reported the synthesis of new class ligands with high affinity to native CB₂ receptors. All these compounds have an imidazopyridine structure and the pKi value range is from 4.9 to 8.6. Among 120 analogues, 18 (Figure 4) is a representative compound, with a pK_i of 4.9 [114]. In the same year, they described another 136 imidazopyridine compounds and claimed these compounds had high affinity to native CB₂ receptors. The pK_i value range is from 4.9 to 8.6 and the representative sample is compound 19 (Figure 4), with a pK_i of 5.2 [115].

Figure 4.

Representative structures of imidazopyridine, indole and azaindole derivatives.

In 2009, scientists at Merck & Co., Inc. reported 12 imidazopyridine compounds, with two substituents at the 7- and 9-position, of which three compounds are quaternary ammonium salts. In the cAMP assay, these compounds have IC₅₀ value ranging from 1 to >17000 nM. The representative compound 20 (Figure 4) has IC₅₀ = 17 nM [116].

In 2011, Trotter et al. described a new series of imidazopyridine CB₂ agonists. They found a directly attached morpholine substituent displayed improved CB₂/CB₁ selectivity. Hydroxymethyl-containing amide 21 (Figure 4) was a potent CB₂ agonist that displayed no CB₁ agonism in vitro (hCB₂ cAMP IC₅₀ = 33 nM, hCB₁ cAMP IC₅₀ > 17000; Rat CB₂ cAMP IC₅₀ = 58 nM, Rat CB₁ cAMP IC₅₀ > 17000 nM) [68].

Indole & Azaindole derivatives

Liu et al. at Bristol-Myers Squibb Company synthesized 11 indanyl indole amide compounds as CB₂ agonists. All compounds were tested in filtration binding assays and/or GTPgS binding assays and have shown activity as an agonist of CB₂. For example, exemplified compound 22 (Figure 4) had a K_i value of 3 nM in the CB₂ binding assay and an EC₅₀ value of 2.4 ± 0.55 nM in the CB₂ GTPgS binding assay [117].

Srivastava et al. reported 69 indolecarboxylic acid trimethylbicycloheptylamides as CB₂ receptor modulators. The representative compound 23 (Figure 4) showed an IC₅₀ value of 0.027 nM in an in vitro cAMP assay [118]. Meanwhile, the in vivo activities of this compound were also tested in an CFA-induced hyperalgsia model, a chronic constriction injury of the sciatic nerve-induced neuropathic pain model and a formalin-induced nociception model. The results indicated that this compound showed a statistical effect.

In 2009, Giblin et al. published a series of novel anaindole CB₂ agonists. The representative compound 24 (Figure 4) is a highly potent CB₂ agonist with over 1200-fold selectivity for the human CB₁ receptor (CB₂: EC₅₀ = 5 nM; CB₁: EC₅₀ = 6300 nM) [69]. Furthermore, compound 24 is a potent full agonist at the human CB₂ receptor expressed in Chinese hamster ovary cells using a forskolin-induced cAMP readout (EC₅₀ = 8 nM, efficacy 100%).

Benzo-fused heterocyclic derivatives

Gahman et al. synthesized 345 aminoquinazoline CB receptor modulators [119]. All the invention compounds were evaluated for their CB receptor modulatory activity. The CB₂ ligand-binding data were given as EC₅₀ <1 μM or ≥1 μM and the selectivity of CB₂ versus CB₁ was given as >tenfold or ≤tenfold. Compound 25 (Figure 5) is an example.

Figure 5.

Representative structures of benzo-fused and pyrazole-fused heterocyclic derivatives.

Newcom et al. synthesized 110 benzo-fused heterocycles analogues and tested their EC₅₀ against human CB₂, rat CB₂ and human CB₁ receptors. These compounds were separated as agonist and inverse agonist. The binding EC₅₀ data were showed as five ranges from 0.1 nM to 10 μM. Compound 26 (Figure 5) is an example [120].

In 2010, a series of 3-substituted oxindole derivatives as CB₂ agonists were synthesized by Dollings et al. [121]. Among 490 analogues, compound 27 (Figure 5) was the representative compound with high CB₂ binding affinity (CB₂: K_i = 1 nM; EC₅₀ = 0.002 nM). Then, another series of substituted oxindole CB₂ agonists (695 analogues) was synthesized by Zhang et al. for Wyeth LLC. The representative compound 28 (Figure 5) showed high CB₂ binding affinity (CB₂: K_i = 1 nM; EC₅₀ = 0.002 nM) [122].

Pasquini et al. recently synthesized a series of quinolone-3-carboxamides and described the SAR study. Except for six compounds exhibiting K_i >100 nM, all the quinolone-3-carboxamides proved to be high-affinity CB₂ ligands, with K_i values ranging from 73.2 to 0.7 nM and selectivity (CB₁/CB₂) varying from >14,285 to 1.9. Compound 29 (Figure 5) in particular has very high CB₂ receptor affinity (K_i = 0.7 nM) and good selectivity of 14,285-fold for this receptor [70]. Recently, in their continuing effort to explore SAR for quinolones binding at CB receptors, they discovered the 8-methoxy derivative 30 (Figure 5) endowed with the higher affinity and selectivity (CB₂: K_i = 0.6 nM; CB₁: K_i >10,000 nM; selectivity >16,666), which behaved as an inverse agonist [71].

Pyrazole-fused heterocyclic derivatives

In 2009, Xia et al. reported a series of new structure CB receptor ligands. All these compounds have a cyclooctanopyrazole core structure. Compound 31 (Figure 5) is a representative compound with high CB₂ affinity and selectivity (CB₂: IC₅₀ = 0.1 nM; 400-fold CB₁/CB₂ selectivity). But the binding to CB₁ receptor of this compound is also very high (CB₁: IC₅₀ = 40 nM) [123].

Recently, Jones et al. in Arena Pharmaceuticals, Inc. synthesized a series of pyrazole-fused heterocyclic analogues (931 compounds) and tested their binding activity against CB₁/CB₂ receptors. Parts of the binding data were given, and some compounds have high affinity to CB₁ and CB₂ receptor. For example, compound 32 (Figure 5) has high affinity to both CB₁ receptor and CB₂ receptor (EC₅₀: hCB₁ = 1.1 nM; EC₅₀: hCB₂ = 0.17 nM), while some other analogues such as compound 33 (Figure 5), show high affinity against CB₂ receptor and good selectivity (EC₅₀: hCB₁ = no response; EC₅₀: hCB₂ = 6.28 nM) [101]. In the meanwhile, the in vivo activities of some compounds were also tested in eight models, such as the osteoarthritis pain model, the skin-incision model, the Freund’s complete adjuvant (FCA)-induced hyperalgesia model, the paclitaxel-induced allodynia model and so on. The compounds tested exhibited therapeutic efficacy in these models.

THC derivatives

By a concise and efficient procedure for converting a phenol to the corresponding aryl bromide, Huffman et al. modified a series of traditional THC ligands and got corresponding bromo CB analogues. All of these compounds showed selectivity for the CB₂ receptor and one of them, compound 34 (Figure 6), exhibits 52-fold selectivity for CB₂ receptor with good affinity (CB₁: K_i = 1444 nM; CB₂: K_i = 28 nM) [72].

Figure 6.

Representative structures of tetrahydrocannabinol derivatives.

Burdick and colleagues published the SAR study of substitutions at the C-1 position of Δ⁹-THC. They focused on conversion of the phenol of Δ⁹-THC to other functionality and found two analogues with sub-100 nM affinity for the CB₁ and CB₂ receptors, of which the representative compound 35 (CB₁: Ki = 67.8 nM; CB₂: Ki = 5.3 nM; Figure 6) shows a 13-fold selectivity for CB₂ over the CB₁ receptor, representing a significant improvement over Δ⁹-THC [73].

Sulfone & sulfonamide derivatives

In 2008, Berry et al. found 192 sulfonyl carboxamide compounds and all the invention compounds were evaluated for their CB₂ receptor modulatory activity. Over 80 compounds showed good CB₂ agonist activity and the representative compound 36 (Figure 7) showed high CB₂ binding affinity (CB₂: EC₅₀ = 0.04 nM) [124].

Figure 7.

Representative structures of sulfone derivatives.

Regan et al. published a series heterocyclic sulfone compounds as modulators of the CB₂ receptor for treating inflammation, pain and disease. Many compounds are preferred CB₂ agonists and the exemplified compound 37 (Figure 7) showed K_i of 25 nM in CB₂ binding assay [125].

Triaryl bis-sulfone is a core structure of a series of selective CB₂ ligands. Gilbert et al. modified this core structure by converting the aryl A-ring to a piperidine ring, and further replaced the piperidine ring with a spirocyclopropyl piperidine, then got a new selective CB₂ ligand 38 (CB₂: K_i = 0.9 nM; ratio CB₁/ CB₂ >1000; Figure 7) [74]. The further SAR studies on triaryl bis-sulfone CB₂ receptor ligands by Tong et al. led to another potent and selective compound 39 (CB₂: K_i = 0.4 nM; ratio CB₁/CB₂ = 3500; Figure 7) [75].

Sulfonamide derivatives

After sulfamoyl benzamide was identified by high-throughput screening as novel CB receptor ligands, Goodman et al. further explored the SAR around the sylfonamide core and found compound 40 (Figure 8) with high CB₂ affinity and selectivity (CB₁: K_i = 3400 nM; CB₂: K_i = 23 nM; ratio CB₁/CB₂ = 147) [76]. This compound exhibited robust antiallodynic activity in a rodent pain model when administered intraperitoneally. However, this compound displayed poor metabolic stability in rat and human liver microsomes. To improve the metabolic stability and retain potent affinity and selectivity, a novel sulfamoyl benzamide 41 (CB₁: K_i = 2500 nM; CB₂: K_i = 17 nM; ratio CB₁/CB₂ = 150; rat liver microsomes = 28%; human livermicrosomes = 17%; Figure 8) as selective CB₂ agonists with improved in vitro metabolic stability was reported by Sellitto and colleagues [77].

Figure 8.

Representative structures of sulfonamide derivatives.

Yacovan et al. synthesized a series of sulfonamide analogues. Among 110 compounds, the representative compound 42 (Figure 8) exhibited K_i values of 21,020 and 933 nM against CB₁ and CB₂, respectively [126].

Two patents have been published by Boehringer Ingelheim International GmbH and claimed many compounds are preferred CB₂ agonists. In the first patent, Cirillo et al. synthesized a series of amine and ether compounds which modulate the CB₂ receptor. Of these compounds, over 40 compounds showed good CB₂ agonist activity and the representative compound 43 (Figure 8) with sulfone structure showed high CB₂ binding affinity (CB₂: EC₅₀ = 0.13 nM) [127]. Compound 44 (Figure 8) is the representative example of the second patent with high CB₂ EC₅₀ values of 1.3 nM [128]. And both of them have sulfonamide core structures.

Miscellaneous scaffolds for CB₂ ligands

Due to the diversity of chemical structures, its always a challenge to assign a structural class of molecules to one of the categories above. The following series discussed in this section display various chemical scaffolds and bioactivities.

Through 3D-quantitative SAR studies of arylpyrazole antagonists CB receptors [78] and 3D pharmacophore database in silico screening [79], Chen et al. disclosed a novel class of CB ligands with an amidine amide core structure [129]. The representative compound 45 (Figure 9) has high CB₂ receptor affinity (K_i = 31.7 nM) and good selectivity of 132-fold over CB₁ receptor (CB₁: K_i = 4185 nM).

Figure 9.

Representative structures of miscellaneous scaffolds of CB₂ derivatives.

Recently, sulfamoyl benzamides were identified as a novel series of CB receptor ligands, Worm et al. replaced the sulfonamide functionality and reversed the original carboxamide bond and discovered compound 46 (K_i = 2.7; CB₁/CB₂ = 190; Figure 8) as a potent and selective CB₂ agonist, which displayed robust activity in a rodent model of postoperative pain [80].

In 2010, two patents of different amide CB ligands were published by the scientists at Boehringer Ingelheim International GmbH. In the first patent, all the compounds have the 2-azetidinecarboxamide core structure and were tested for the binding activity to CB₁/CB₂ receptor. Many of them are preferred CB₂ agonists, and compound 47 (Figure 9) is a representative compound with high CB₂ affinity (CB₂: EC₅₀ = 0.02 nM) [130]. They claimed these compounds are useful for treating inflammation or pain. As for the second patent, 91 compounds with N-azolyl α-aminoalkanamide core structure were synthesized and tested for their binding activity to CB₁/CB₂ receptor. Many of them are preferred CB₂ agonists, and 48 (Figure 9) is a representative compound with high CB₂ affinity and better selectivity (CB₂: EC₅₀ = 0.02 nM; CB₁: EC₅₀ > 50000 nM) [131].

Gertsch and colleagues published a novel class of CB ligands, namely dodeca-2E,4Ediene amides [81]. Among these analogues, 49 (Figure 9) is a representative compound with high CB₂ affinity and selectivity (CB₂: K_i = 60 ± 7 nM; CB₁: K_i = 1940 ± 213 nM) [132]. The results also indicated the claimed dodeca-2E,4Ediene amides inhibit AEA re-uptake and some compounds of the invention also inhibit fatty acid amide hydrolase.

Bab et al. disclosed a series of phenyl substituted pinenes compounds. Compared with the traditional selective CB₂ ligand HU-308 (CB₁: K_i >10 μM; CB₂: K_i = 22.7 nM), the representative compound 50 (HU-433; Figure 9) was found to be significantly more potent (CB₁: K_i >20 μM; CB₂: K_i = 12.2 nM) [133]. The comparative skeletal activities of HU-433 and HU-308 were also tested. The data indicated that HU-433 was a 1000-fold more active compared with HU-308 in vitro. The in vivo skeletal activity of these two compounds was analyzed in an ovariectomy (removal of ovaries) mouse model: the most widely used animal model for osteoporosis. The result showed that HU-433 was at least 100-fold more active than HU-308. They also claimed the effect of HU-433 was substantially greater than the reversal of bone volumetric density by parathyroid hormone, the only clinically approved bone anabolic agent.

In 2011, Mechoulam published a series of novel arylated camphene compounds. The representative compound 51 (HU-910; Figure 9) has high CB₂ affinity (CB₂ EC₅₀ = 26.4 nM) [134]. The in vivo data indicated HU-910 displayed a significantly greater recovery than the control group in the closed head injury model.

Conclusion

Many new selective CB₂ ligands have been emerging in the literature over the last 5 years. It is estimated from SciFinder that there are 1419 journal articles and 387 patents reported about CB₂ research and new CB₂ ligands. The recently available Web-interfaced CB molecular information database repository constructed by the Xie laboratory has over 8500 records of CB ligands [202]. As discussed here, these compounds represent a variety of different chemical classes that are distinct from chemotypes typified by the endogenous CBs. The binding affinity and selectivity of reviewed CB₂ ligands were summarized in Table 1. Among these compounds, many of them have notably high CB₂ binding affinity, for example the binding affinities of compounds 1, 2, 9 and 15 are less than 1 nM. Other compounds show good selectivity, such as compounds 8, 14, 17 and 24 with >1000. Here, the authors suggest that caution should be taken in using these data because the receptor binding data may vary as different laboratories may use different approaches. Even with the use of the same protocols, different cell lines may produce dissimilar sets of binding affinity data. Overall, this article reports on the recent advances by providing an overview of novel classes of CB₂ ligands reported in research articles and patents. The structural and bioactivity data of these novel CB₂ ligands will be valuable for scientists in industrial and academic chemistry, pharmacology and computational chemistry laboratories conducting CB₂ lead optimization/modification and SAR medicinal chemistry synthesis, pharmacological/biochemical studies and computer- aided drug design research for novel CB₂ drug-design discovery.

Future perspective

New advances in CB drug research are reviewed with a focus on the most recent development of CB₂ ligands reported in literature and patents. Overall, CBs represent an important family of large structurally diverse molecules with promising therapeutic potential. In particular, research studies involving CB₂-targeted ligands have been steadily proliferating. The quantity and the quality of this special class of molecules are expected to grow at a much faster rate in the future. These new generations of CB₂ receptor-selective compounds will overcome many of the hurdles that plague currently available pharmacological studies, including poor selectivity, low potency and/or efficacy and unsatisfactory pharmacokinetic properties. As indicated above, while such a physiological role of CB₂ receptors remains to be fully defined, several intriguing preclinical studies suggest that CB₂ ligands may be clinically useful and possible medications for chronic pain, autoimmune MS, osteoporosis and atherosclerotic lesions. Nevertheless, with all exciting points about CB₂ ligands, however, several fundamental questions still remain to be further explored in detail, in order to better understanding of physiological roles of the CB₂ receptor in immune responses. Also needed is a thorough assessment of the pharmacological properties and the relevant signaling pathways of the discovered CB₂ agonists and antagonists. As such, the newly developed CB ligands and their bioactivities will help researchers to better understand the role of CB₂ receptor played in both physiological and pathophysiological processes. With great efforts being devoted towards CB₂ drug research, we expect that highly potent and selective druggable CB₂ agents will be discovered, which will ultimately be translated in the clinic in to new CB₂ drugs that possess great therapeutic values without causing psychotropic side effects in humans.

Table 1.

Summary of binding affinity and selectivity of the CB₂ ligands reviewed.

Affinity Ki (CB2, nM)	Compound	CB1/CB2 selectivity	Compound
≤1	1, 2, 9, 15, 16, 17, 23, 27, 28, 29, 30, 31, 32, 36, 38, 39, 43, 47, 48	>1000	8, 14, 17, 24, 29, 30, 33, 38, 39, 48, 50
01–10	3, 4, 8, 12, 13, 14, 18, 19, 22, 24, 33, 35, 44, 46	100–1000	10, 12, 13, 21, 31, 40, 41, 45, 46
10–100	5, 6, 10, 11, 20, 21, 34, 37, 40, 41, 45, 49, 50, 51	10–100	34, 35, 42, 49
>100	42	1–10	32

Executive summary.

• Marijuana or cannabinoids (CBs) drug abuse and toxicities are a serious threat to human health in the USA and the world. It contains a complex mixture of compounds, including tetrahydrocannabinol, the major psychoactive constituent.

• There are no effective treatments for the abuse of marijuana today as most treatments are still relatively ineffective and have a high failure rate due to drug-addict relapse and withdrawal symptoms.

• Two CB receptors have been cloned and characterized: CB₁ and CB₂.

• Studies show that release of endocannabinoids in the ventral tegmental area can modulate the reward-related effects of dopamine and might, therefore, be an important neurobiological mechanism underlying drug addiction.

• Both CB₁ ligands and CB₂ ligands showed some good treatment effects in drug abuse. The undesirable psychotropic effects of CB₁ ligands has limited the therapeutic utility, while CB₂ ligands would not be expected to elicit such side effects.

• Selective CB₂ receptor ligands developed recently are summarized and classified in to seven types based on their chemical scaffolds.

• CB₂ ligands have therapeutic potentials, such as treatment in immune disorders, pain, cancers and osteoporosis.

• Future research will focus on finding selective and efficacious CB₂ compounds with good drug-like properties but no undesired psychotropic effects.

Key Term

Endocannabinoid system

Endocannabinoids are found in the nervous and immune systems of animals and activate cannabinoid receptors. The endocannabinoid system represents a neuromodulator system consisting of endogenous ligands, enzymes and cannabinoid receptors (subtypes CB₁ and CB₂) that are involved in a variety of physiological processes including appetite, pain sensation, mood and memory; it mediates the psychoactive effects of cannabis.