Novel pyridine derivatives as potent and selective CB2 cannabinoid receptor agonists

Replacement of the phenyl ring in our previous (morpholinomethyl)aniline carboxamide cannabinoid receptor ligands with a pyridine ring led to the discovery of a novel chemical series of CB₂ ligands. Compound 3, that is, 2,2-dimethyl-N-(5-methyl-4-(morpholinomethyl)pyridin-2-yl)butanamide was identified as a potent and selective CB₂ agonist exhibiting in vivo efficacy after oral administration in a rat model of neuropathic pain.     

Sulfonamide derivatives as new potent and selective CB2 cannabinoid receptor agonists

A novel series of sulfonamide derivatives 3, the CB(2) receptor agonists, was synthesized and evaluated for activity against the human CB(2) receptor. We first identified sulfonamide 3a, which was obtained by random screening of our in-house chemical library as a moderately active (CB(2) IC(50)=340nM) CB(2) receptor agonist. We then attempted to test its analogues to identify compounds with a high affinity for the CB(2) receptor. One of these, compound 3f, exhibited high affinity for the human CB(2) receptor (IC(50)=16nM) and high selectivity for CB(2) over CB(1) (CB(1) IC(50)/CB(2)IC(50)=106), and behaved as a full CB(2) receptor agonist in the [(35)S]GTPgammaS binding assay (CB(2) EC(50)=7.2nM, E(max)=100%).     

Synthesis and activity of 4,5-diarylimidazoles as human CB1 receptor inverse agonists

Structure-activity relationship studies directed toward the optimization of 4,5-diarylimidazole-2-carboxamide analogs as human CB1 receptor inverse agonists resulted in the discovery of the two amide derivatives 24a and b (hCB1 IC50 = 6.1 and 4.0 nM) which also demonstrated efficacy in overnight feeding studies in the rat for reduction in both food intake and overall body weight.     

Synthesis of quinolinyl and isoquinolinyl phenyl ketones as novel agonists for the cannabinoid CB2 receptor

A series of quinolinyl and isoquinolinyl phenyl ketones was synthesized and their CB₂ receptor-dependent G-protein activities were determined using the [³⁵S]GTPγS binding assay. Both quinoline and isoquinoline derivatives exhibited similar CB₂ receptor agonist activity, the most potent ligands being the 2-(Me₂N)-phenyl substituted derivatives, which were also full agonists at the CB₂-receptor.     

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.     

Imine derivatives as new potent and selective CB2 cannabinoid receptor agonists with an analgesic action

In this study, a novel series of CB(2) receptor agonist imine derivatives, 1-6, was synthesized and evaluated for activity against the CB(2) receptor. In a previous paper we reported the synthesis and SARs of thiazole derivative 1, a potent CB(2) receptor agonist, but we had not assessed chemical modifications of the 5-membered heteroring of 1. In the present study, we therefore tried chemically modifying the 5-membered heteroring of 1 in an attempt to further improve binding affinity for the CB(2) receptor. In the course of making the structural modifications, we discovered that a novel pyrazole derivative 6b (CBS0550) had high affinity for the CB(2) receptor (IC(50)=2.9 nM, EC(50)=1.8 nM, E(max)=85%), high selectivity for CB(2) (CB(1) IC(50)/CB(2) IC(50)=1400), and good physicochemical properties (solubility in water: 5.9 mg/100mL at 25 degrees C). Oral administration of 6b to rats at a dose of 10mg/kg resulted in significant plasma concentrations, and orally administered compound 6b significantly reversed mechanical hyperalgesia in the Randall-Selitto model of inflammatory pain in rats.     

N-Alkylidenearylcarboxamides as new potent and selective CB(2) cannabinoid receptor agonists with good oral bioavailability

A novel series of N-alkylidenearylcarboxamides 4, a CB(2) receptor agonist, were synthesized and evaluated for activity against the human CB(2) receptor. In a previous paper, we reported that sulfonamide derivative 1 acted as a potent CB(2) receptor agonist (IC(50)=65 nM, EC(50)=19 nM, E(max)=90%). However, compound 1 also exhibited poor metabolic stability in human liver microsomes. During the structural modification of 1, we found that a novel series of N-alkylidenearylcarboxamide, 4-1, had a moderate affinity for the CB(2) receptor (IC(50)=260 nM, EC(50)=86 nM, E(max)=100%) and good metabolic stability in human liver microsomes. We explored its analogues to discover compounds with a high affinity for the CB(2) receptor and with good oral bioavailability. Among them, compounds 4-9 and 4-27 had high affinities for the human CB(2) receptor (CB(2) IC(50)=13 nM and 1.2 nM) and a high selectivity for CB(2) (CB(1) IC(50)/CB(2) IC(50)=270 and 1600); furthermore, significant plasma levels were observed following oral administration in rats (C(max)=233 ng/mL and 148 ng/mL, respectively, after a dose of 10 mg/kg). Furthermore, compound 4-9 had good oral bioavailability (F=52%, 3mg/kg).     

Synthesis and SAR of 5,6-diarylpyridines as human CB1 inverse agonists

Structure-activity relationship studies for two series of 2-benzyloxy-5-(4-chlorophenyl)-6-(2,4-dichlorophenyl)pyridines having either a 3-cyano or 3-carboxamide moiety resulted in the preparation of the 2-(3,4-difluorobenzyloxy)-3-nitrile analog 10d and the 2-(3,4-difluorobenzyloxy)-3-(N-propylcarboxamide) analog 16c, (hCB1 IC(50)=1.3 and 1.7 nM, respectively) as potent and selective hCB1 inverse agonists. Their synthesis and biological activities are described herein.     

Synthesis and pharmacological evaluation of coumarin derivatives as cannabinoid receptor antagonists and inverse agonists

In the present study we synthesized 36 coumarin and 2H-chromene derivatives applying a recently developed umpoled domino reaction using substituted salicylaldehyde and α,β-unsaturated aldehyde derivatives as starting compounds. In radioligand binding studies 5-substituted 3-benzylcoumarin derivatives showed affinity to cannabinoid CB₁ and CB₂ receptors and were identified as new lead structures. In further GTPγS binding studies selected compounds were shown to be antagonists or inverse agonists.     

Opposite changes in cannabinoid CB1 and CB2 receptor expression in human gliomas

Gliomas are the most important group of malignant primary brain tumors and one of the most aggressive forms of cancer. During the last years, several studies have demonstrated that cannabinoids induce apoptosis of glioma cells and inhibit angiogenesis of gliomas in vivo. As the effects of cannabinoids rely on CB₁ and CB₂ receptors activation, the aim of the present study was to investigate both receptors protein expression in cellular membrane homogenates of human glial tumors using specific antibodies raised against these proteins. Additionally, we studied the functionality of the cannabinoid receptors in glioblastomas by using WIN 55,212-2 stimulated [³⁵S]GTPγS binding.Western blot analysis showed that CB₁ receptor immunoreactivity was significantly lower in glioblastoma multiforme (?43%, n = 10; p < 0.05) than in normal post-mortem brain tissue (n = 16). No significant differences were found for astrocytoma (n = 6) and meningioma (n = 8) samples. Conversely, CB₂ receptor immunoreactivity was significantly greater in membranes of glioblastoma multiforme (765%, n = 9; p < 0.05) and astrocytoma (471%, n = 4; p < 0.05) than in control brain tissue (n = 10). Finally, the maximal stimulation of [³⁵S]GTPγS binding by WIN 55,212-2 was significantly lower in glioblastomas (134 ± 4%) than in control membranes (183 ± 2%; p < 0.05). The basal [³⁵S]GTPγS binding and the EC₅₀ values were not significantly different between both groups.The present results demonstrate opposite changes in CB₁ and CB₂ receptor protein expression in human gliomas. These changes may be of interest for further research about the therapeutic effects of cannabinoids in glial tumors.     

Reciprocal influences of CB1 cannabinoid receptor agonists on ERK and JNK signalling in N1E-115 cells

Agonists acting at the CB₁ cannabinoid receptor in N1E-115 neuroblastoma cells were found to activate MAPK family members with reciprocal efficacies. Thus, HU 210 robustly increased phosphorylation of ERK1/2 whereas CP 55,940 was more effective in activating JNK. The use of selected kinase inhibitors confirmed that distinct signalling cascades were involved in these responses. This reciprocal control of MAPK activity was correlated with the observation that HU 210- and CP 55,940-mediated regulations of tyrosine hydroxylase gene expression were respectively impaired by MEK and JNK inhibitors. These data indicate that complex interactions of the CB₁ receptor with intracellular signalling partners controlling MAPK activities may explain the apparent disparities in cellular responses to functional selective agonists.     

Antibodies against the melanocortin-4 receptor act as inverse agonists in vitro and in vivo

Functionally active antibodies (Abs) against central G-protein-coupled receptors have not yet been reported. We selected the hypothalamic melanocortin-4 receptor (MC4-R) as a target because of its crucial role in the regulation of energy homeostasis. A 15 amino acid sequence of the N-terminal (NT) domain was used as an antigen. This peptide showed functional activity in surface plasmon resonance experiments and in studies on HEK-293 cells overexpressing the human MC4-R (hMC4-R). Rats immunized against the NT peptide produced specific antibodies, which were purified and characterized in vitro. In HEK-293 cells, rat anti-NT Abs showed specific immunofluorescence labeling of hMC4-R. They reduced the production of cAMP under basal conditions and after stimulation with a synthetic MC4-R agonist. Rats immunized against the NT peptide developed a phenotype consistent with MC4-R blockade, that is, increased food intake and body weight, increased liver and fat pad weight, and elevated plasma triglycerides. In a separate experiment in rats, an increase in food intake could be produced after injection of purified Abs into the third ventricle. Similar results were obtained in rats injected with anti-NT Abs raised in rabbits. Our data show for the first time that active immunization of rats against the NT sequence of the MC4-R results in specific Abs, which appear to stimulate food intake by acting as inverse agonists in the hypothalamus.     

Arylsulfonamide CB2 receptor agonists: SAR and optimization of CB2 selectivity

A high-throughput screening campaign resulted in the discovery of a highly potent dual cannabinoid receptor 1 (CB1) and 2 (CB2) agonist. Following a thorough SAR exploration, a series of selective CB2 full agonists were identified.     

C3-Heteroaroyl cannabinoids as photolabeling ligands for the CB2 cannabinoid receptor

A series of tricyclic cannabinoids incorporating a heteroaroyl group at C3 were prepared as probes to explore the binding site(s) of the CB1 and CB2 receptors. This relatively unexplored structural motif is shown to be CB2 selective with K(i) values at low nanomolar concentrations when the heteroaromatic group is 3-benzothiophenyl (41) or 3-indolyl (50). When photoactivated, the lead compound 41 was shown to successfully label the CB2 receptor through covalent attachment at the active site while 50 failed to label. The benzothiophenone moiety may be a photoactivatable moiety suitable for selective labeling.     

Site-selective labeling and electron paramagnetic resonance studies of human cannabinoid receptor CB2

Integral membrane G protein-coupled receptors (GPCR) regulate multiple physiological processes by transmitting signals from extracellular milieu to intracellular proteins and are major targets of pharmaceutical drug development. Since GPCR are inherently flexible proteins, their conformational dynamics can be studied by spectroscopic techniques such as electron paramagnetic resonance (EPR) which requires selective chemical labeling of the protein. Here, we developed protocols for selective chemical labeling of the recombinant human cannabinoid receptor CB₂ by judiciously replacing naturally occurring reactive cysteine residues and introducing a new single cysteine residue in selected positions. The majority of the 47 newly generated single cysteine constructs expressed well in E. coli cells, and more than half of them retained high functional activity. The reactivity of newly introduced cysteine residues was assessed by incorporating nitroxide spin label and EPR measurement. The conformational transition of the receptor between the inactive and activated form were studied by EPR of selectively labeled constructs in the presence of either a full agonist CP-55,940 or an inverse agonist SR-144,528. We observed evidence for higher mobility of labels in the center of internal loop 3 and a structural change between agonist vs. inverse agonist-bound CB₂ in the extracellular tip of transmembrane helix 6. Our results demonstrate the utility of EPR for studies of conformational dynamics of CB₂.     

The CB2 cannabinoid receptor signals apoptosis via ceramide-dependent activation of the mitochondrial intrinsic pathway

Delta9-tetrahydrocannabinol and other cannabinoids exert pro-apoptotic actions in tumor cells via the CB2 cannabinoid receptor. However, the molecular mechanism involved in this effect has remained elusive. Here we used the human leukemia cell line Jurkat-that expresses CB2 as the unique CB receptor-to investigate this mechanism. Our results show that incubation with the selective CB2 antagonist SR144528 abrogated the pro-apoptotic effect of Delta9-tetrahydrocannabinol. Cannabinoid treatment led to a CB2 receptor-dependent stimulation of ceramide biosynthesis and inhibition of this pathway prevented Delta9-tetrahydrocannabinol-induced mitochondrial hypopolarization and cytochrome c release, indicating that ceramide acts at a pre-mitochondrial level. Inhibition of ceramide synthesis de novo also prevented caspase activation and apoptosis. Caspase 8 activation-an event typically related with the extrinsic apoptotic pathway-was also evident in this model. However, activation of this protease was post-mitochondrial since (i) a pan-caspase inhibitor as well as a selective caspase 8 inhibitor were unable to prevent Delta9-tetrahydrocannabinol-induced loss of mitochondrial-membrane transmembrane potential, and (ii) cannabinoid-induced caspase 8 activation was not observed in Bcl-xL over-expressing cells. In summary, results presented here show that CB2 receptor activation signals apoptosis via a ceramide-dependent stimulation of the mitochondrial intrinsic pathway.     

2-Arylimino-5,6-dihydro-4H-1,3-thiazines as a new class of cannabinoid receptor agonists. Part 1: discovery of CB2 receptor selective compounds

2-Arylimino-5,6-dihydro-4H-1,3-thiazines have been identified as a novel class of cannabinoid agonists. A lead structure with moderate activity was discovered through a high throughput screening assay. Structure-activity relationships led to the discovery of potent agonists of CB(2) receptor. The most potent compound 13 displays K(i) values of >5000 and 9 nM to CB(1) and CB(2) receptors, respectively.     

C-terminal peptides of calcitonin gene-related peptide act as agonists at the cholecystokinin receptor

We have previously described that [Tyr⁰]CGRP(28–37) acts as a receptor antagonist of rat CGRP in guinea pig pancreatic acini. We therefore examined other C-terminal peptides of CGRP for such activity. CGRP-acetyl(28–37) acetate did act as a rat CGRP antagonist. However, C-terminal CGRP peptides of 4 to 8 amino acid residues did not antagonize the actions of rat CGRP but stimulated amylase secretion. In pancreatic acini, a maximally effective concentration of rat CGRP (100 nM) caused a 2.1-fold increase in amylase secretion. When the C-terminal peptides of CGRP were tested in at 100 μM, CGRP(34–37) caused a 1.8-fold increase in amylase secretion, CGRP(33–37) a 2.8-fold increase, CGRP(32–37) a 9.2-fold increase, CGRP(31–37) a 4.1-fold increase, and CGRP(30–37) a 5.1-fold increase. Further studies with the most effective peptide, CGRP(32–37), demonstrated that it did not cause release of lactate dehydrogenase, and thus did not cause amylase release by cell damage. Unlike rat CGRP, CGRP(32–37) did not increase cellular cyclic AMP, but did stimulate outflux of ⁴⁵Ca. CGRP(32–37)-stimulated amylase release was not inhibited by the substance P receptor antagonist, spantide, by the bombesin receptor antagonist, [D-Phe⁶]bombesin(6–13) propylamide, or by the muscarinic receptor antagonist, atropine, but was inhibited by the CCK receptor antagonist L364,718. C-terminal peptides of CGRP inhibited binding of ¹²⁵I-BH-CCK-8, with the relative potencies of the peptides being the same as their relative potencies for stimulating amylase secretion. The present data demonstrate that C-terminal peptides of CGRP, although they have only 2 amino acid residues in common with CCK(26–33), act exclusively at CCK receptors on pancreatic acini to stimulate amylase secretion.     

Cloning and molecular characterization of the rat CB2 cannabinoid receptor

The rat peripheral cannabinoid receptor (rCB2) was cloned from a Sprague-Dawley rat spleen cDNA library and when translated, encodes a protein of 410 amino acids. Alignment of rCB2 with mouse (mCB2) and human (hCB2) peripheral cannabinoid receptors reveals a high degree of homology except in the carboxy terminus where rCB2 is 50 and 63 residues longer than hCB2 and mCB2, respectively. PCR screening and sequencing of rat genomic DNA showed that rCB2 is encoded by three exons interrupted by two introns, one of which is polymorphic and contains a 209 base pair B2 (SINE) element. By Northern hybridization and ribonuclease protection assay (RPA), rCB2 mRNA was detected in rat spleen, testis, thymus and lung but not in rat brain, heart, kidney or liver. Like hCB2 and mCB2 receptors, rCB2 activates mitogen-activated protein kinase when it is stably expressed in Chinese Hamster Ovary (CHO) cells. The importance of the carboxy terminus in regulating CB2 receptor desensitization and internalization is well-established. Thus, the profound differences identified in this region of the CB2 receptor between species mandates caution when extrapolating experimental results from non-human models to the effects of chronic CB2 receptor stimulation in humans.     

Direct suppression of CNS autoimmune inflammation via the cannabinoid receptor CB1 on neurons and CB2 on autoreactive T cells

The cannabinoid system is immunomodulatory and has been targeted as a treatment for the central nervous system (CNS) autoimmune disease multiple sclerosis. Using an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we investigated the role of the CB(1) and CB(2) cannabinoid receptors in regulating CNS autoimmunity. We found that CB(1) receptor expression by neurons, but not T cells, was required for cannabinoid-mediated EAE suppression. In contrast, CB(2) receptor expression by encephalitogenic T cells was critical for controlling inflammation associated with EAE. CB(2)-deficient T cells in the CNS during EAE exhibited reduced levels of apoptosis, a higher rate of proliferation and increased production of inflammatory cytokines, resulting in severe clinical disease. Together, our results demonstrate that the cannabinoid system within the CNS plays a critical role in regulating autoimmune inflammation, with the CNS directly suppressing T-cell effector function via the CB(2) receptor.