New 1,2,3,4-tetrahydropyrrolo[3,4-b]indole derivatives as selective CB2 receptor agonists

The preparation and evaluation of a novel class of CB2 agonists based on a 1,2,3,4-tetrahydropyrrolo[3,4-b]indole moiety are reported. They showed binding affinities up to 4.2 nM toward CB2 with sub-nanomolar EC(50) values. They also showed moderate to good (>350-fold) selectivity over the CB1 receptor.     

Cannabinoid receptors CB1 and CB2 form functional heteromers in brain

Exploring the role of cannabinoid CB(2) receptors in the brain, we present evidence of CB(2) receptor molecular and functional interaction with cannabinoid CB(1) receptors. Using biophysical and biochemical approaches, we discovered that CB(2) receptors can form heteromers with CB(1) receptors in transfected neuronal cells and in rat brain pineal gland, nucleus accumbens, and globus pallidus. Within CB(1)-CB(2) receptor heteromers expressed in a neuronal cell model, agonist co-activation of CB(1) and CB(2) receptors resulted in a negative cross-talk in Akt phosphorylation and neurite outgrowth. Moreover, one specific characteristic of CB(1)-CB(2) receptor heteromers consists of both the ability of CB(1) receptor antagonists to block the effect of CB(2) receptor agonists and, conversely, the ability of CB(2) receptor antagonists to block the effect of CB(1) receptor agonists, showing a bidirectional cross-antagonism phenomenon. Taken together, these data illuminate the mechanism by which CB(2) receptors can negatively modulate CB(1) receptor function.     

Inhibition of pain responses by activation of CB(2) cannabinoid receptors

Cannabinoid receptor agonists diminish responses to painful stimuli. Extensive evidence demonstrates that CB(1) cannabinoid receptor activation inhibits pain responses. Recently, the synthesis of CB(2) cannabinoid receptor-selective agonists has allowed testing whether CB(2) receptor activation inhibits pain. CB(2) receptor activation is sufficient to inhibit acute nociception, inflammatory hyperalgesia, and the allodynia and hyperalgesia produced in a neuropathic pain model. Studies using site-specific administration of agonist and antagonist have suggested that CB(2) receptor agonists inhibit pain responses by acting at peripheral sites. CB(2) receptor activation also inhibits edema and plasma extravasation produced by inflammation. CB(2) receptor-selective agonists do not produce central nervous system (CNS) effects typical of cannabinoids retaining agonist activity at the CB(1) receptor. Peripheral antinociception without CNS effects is consistent with the peripheral distribution of CB(2) receptors. CB(2) receptor agonists may have promise for the treatment of pain and inflammation without CNS side effects.     

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%).     

Blockade of cannabinoid CB(1) receptor function protects against in vivo disseminating brain damage following NMDA-induced excitotoxicity

The ability of cannabinoid CB(1) receptors to influence glutamatergic excitatory neurotransmission has fueled interest in how these receptors and their endogenous ligands may interact in conditions of excitotoxic insults. The present study characterized the impact of stimulated and inhibited CB(1) receptor function on NMDA-induced excitotoxicity. Neonatal (6-day-old) rat pups received a systemic injection of a mixed CB(1) /CB(2) receptor agonist (WIN55,212-2) or their respective antagonists (SR141716A for CB(1) and SR144528 for CB(2) ) prior to an unilateral intrastriatal microinjection of NMDA. The NMDA-induced excitotoxic damage in the ipsilateral forebrain was not influenced by agonist-stimulated CB(1) receptor function. In contrast, blockade of CB(1), but not CB(2), receptor activity evoked a robust neuroprotective response by reducing the infarct area and the number of cortical degenerating neurons. These results suggest a critical involvement of CB(1) receptor tonus on neuronal survival following NMDA receptor-induced excitotoxicity in vivo.     

Distinct second extracellular loop structures of the brain cannabinoid CB(1) receptor: implication in ligand binding and receptor function

The G-protein-coupled receptor (GPCR) second extracellular loop (E2) is known to play an important role in receptor structure and function. The brain cannabinoid (CB(1)) receptor is unique in that it lacks the interloop E2 disulfide linkage to the transmembrane (TM) helical bundle, a characteristic of many GPCRs. Recent mutation studies of the CB(1) receptor, however, suggest the presence of an alternative intraloop disulfide bond between two E2 Cys residues. Considering the oxidation state of these Cys residues, we determine the molecular structures of the 17-residue E2 in the dithiol form (E2(dithiol)) and in the disulfide form (E2(disulfide)) of the CB(1) receptor in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer, using a combination of simulated annealing and molecular dynamics simulation approaches. We characterize the CB(1) receptor models with these two E2 forms, CB(1)(E2(dithiol)) and CB(1)(E2(disulfide)), by analyzing interaction energy, contact number, core crevice, and cross correlation. The results show that the distinct E2 structures interact differently with the TM helical bundle and uniquely modify the TM helical topology, suggesting that E2 of the CB(1) receptor plays a critical role in stabilizing receptor structure, regulating ligand binding, and ultimately modulating receptor activation. Further studies on the role of E2 of the CB(1) receptor are warranted, particularly comparisons of the ligand-bound form with the present ligand-free form.     

Stereochemical selectivity of methanandamides for the CB1 and CB2 cannabinoid receptors and their metabolic stability.

Several chiral, analogues of the endogenous cannabinoid receptor ligand, arachidonylethanolamide (anandamide), methylated at the 2,1' and 2' positions using asymmetric synthesis were evaluated in order to study (a) stereoselectivity of binding to CB1 and CB2 cannabinoid receptors; and (b) metabolic stability with regard to anandamide amidase. Enantiomerically pure 2-methyl arachidonic acids were synthesized through diastereoselective methylation of the respective chiral 2-oxazolidinone enolate derivatives and CB1 and CB2 receptor affinities of the resulting chiral anandamides were evaluated using a standard receptor binding assay. Introduction of a single 2-methyl group increased affinity for CB1, led to limited enantioselectivity and only modestly improved metabolic stability. However, a high degree of enantio- and diastereoselectivity was observed for the 2,1'-dimethyl analogues. (R)-N-(1-methyl-2-hydroxyethyl)-2-(R)-methyl-arachidonamide (4) exhibited the highest CB1 receptor affinity in this series with a K(i) of 7.42 nM, an at least 10-fold improvement on anandamide (K(i)=78.2 nM). The introduction of two methyl groups at the 2-position of anandamide led to no change in affinity for CB1 but somewhat enhanced metabolic stability. Conversely, chiral headgroup methylation in the 2-gem-dimethyl series led to chiral analogues possessing a wide range of CB1 affinities. Of these the (S)-2,2,2'-trimethyl analogue (12) had the highest affinity for CB1 almost equal to that of anandamide. In agreement with our previous anandamide structure-activity relationship work, the analogues in this study showed high selectivity for the CB1 receptor over CB2. The results are evaluated in terms of stereochemical factors affecting the ligand's affinity for CB1 using receptor-essential volume mapping as an aid. Based on the results, a partial CB1 receptor site model is proposed, that bears two hydrophobic pockets capable of accommodating 1'- and 2-methyl groups     

Structure-activity relationships for 1-alkyl-3-(1-naphthoyl)indoles at the cannabinoid CB(1) and CB(2) receptors: steric and electronic effects of naphthoyl substituents. New highly selective CB(2) receptor agonists

In an effort to improve indole-based CB(2) cannabinoid receptor ligands and also to develop SAR for both the CB(1) and CB(2) receptors, 47 indole derivatives were prepared and their CB(1) and CB(2) receptor affinities were determined. The indole derivatives include 1-propyl- and 1-pentyl-3-(1-naphthoyl)indoles both with and without a 2-methyl substituent. Naphthoyl substituents include 4- and 7-alkyl groups as well as 2-, 4-, 6-, 7-methoxy and 4-ethoxy groups. The effects of these substituents on receptor affinities are discussed and structure-activity relationships are presented. In the course of this work three new highly selective CB(2) receptor agonists were identified, 1-propyl-3-(4-methyl-1-naphthoylindole (JWH-120), 1-propyl-2-methyl-3-(6-methoxy-1-naphthoylindole (JWH-151), and 1-pentyl-3-(2-methoxy-1-naphthoylindole (JWH-267). GTPgammaS assays indicated that JWH-151 is a full agonist at CB(2), while JWH-120 and JWH-267 are partial agonists. Molecular modeling and receptor docking studies were carried out on a set of 3-(4-propyl-1-naphthoyl)indoles, a set of 3-(6-methoxy-1-naphthoyl)indoles and the pair of N-pentyl-3-(2-methoxy-1-naphthoyl)indoles. Docking studies indicated that the CB(1) receptor affinities of these compounds were consistent with their aromatic stacking interactions in the aromatic microdomain of the CB(1) receptor.     

Triaryl bis-sulfones as a new class of cannabinoid CB2 receptor inhibitors: identification of a lead and initial SAR studies

A novel class of cannabinoid CB2 receptor ligands is described. These triaryl bis-sulfones are nanomolar inhibitors of the CB2 receptor and show high selectivity over the cannabinoid CB1 receptor. One example of this new class decreases ligand-induced GTPgammaS binding to recombinant CB2 cell membranes, identifying the compound as a CB2-selective inverse agonist.     

Inverse agonism and neutral antagonism at cannabinoid CB1 receptors

There are at least two types of cannabinoid receptor, CB1 and CB2, both G protein coupled. CB1 receptors are expressed predominantly at nerve terminals and mediate inhibition of transmitter release whereas CB2 receptors are found mainly on immune cells, one of their roles being to modulate cytokine release. Endogenous cannabinoid receptor agonists also exist and these "endocannabinoids" together with their receptors constitute the "endocannabinoid system". These discoveries were followed by the development of a number of CB1- and CB2-selective antagonists that in some CB1 or CB2 receptor-containing systems also produce "inverse cannabimimetic effects", effects opposite in direction from those produced by cannabinoid receptor agonists. This review focuses on the CB1-selective antagonists, SR141716A, AM251, AM281 and LY320135, and discusses possible mechanisms by which these ligands produce their inverse effects: (1) competitive surmountable antagonism at CB1 receptors of endogenously released endocannabinoids, (2) inverse agonism resulting from negative, possibly allosteric, modulation of the constitutive activity of CB1 receptors in which CB1 receptors are shifted from a constitutively active "on" state to one or more constitutively inactive "off" states and (3) CB1 receptor-independent mechanisms, for example antagonism of endogenously released adenosine at A1 receptors. Recently developed neutral competitive CB1 receptor antagonists, which are expected to produce inverse effects through antagonism of endogenously released endocannabinoids but not by modulating CB1 receptor constitutive activity, are also discussed. So too are possible clinical consequences of the production of inverse cannabimimetic effects, there being convincing evidence that released endocannabinoids can have "autoprotective" roles.     

Tetrazole-biarylpyrazole derivatives as cannabinoid CB1 receptor antagonists

Cannabinoid CB1 receptors have been the focus of extensive studies since the first clinical results of rimonabant (SR141716) for the treatment of obesity and related metabolic disorders were reported in 2001. To further evaluate the properties of CB receptors, we have designed a new series of tetrazole-biarylpyrazoles. The various analogues were efficiently prepared and bio-assayed for binding to cannabinoid CB1 receptor. Six of the new compounds which displayed high in vitro CB1 binding affinities were assayed for binding to CB2 receptor. Noticeably, cyclopentyl-tetrazole (9a) demonstrated good binding affinity and selectivity for CB1 receptor (IC(50)=11.6nM and CB2/CB1=366).     

Synthesis and structure–activity relationship of 1,2,4-triazole-containing diarylpyrazolyl carboxamide as CB1 cannabinoid receptor–ligand

Numerous research groups have been engaged in searching for novel CB1 receptor antagonists, since SR141716A (rimonabant), a CB1 receptor antagonist, proved to be efficacious in human for the treatment of obesity. In the present study, a series of 1,2,4-triazole-containing diarylpyrazolyl carboxamides based on the 1,5-diarylpyrazole template of rimonabant, was synthesized and tested for CB1 receptor binding affinity. The structure–activity relationship studies demonstrated that incorporation of 1,2,4-triazole ring onto the pyrazole scaffold via a methylene linker led to a significant improvement for CB1 receptor binding affinity. Importantly, these analogues also exhibited excellent selectivity for CB1 receptor over CB2 receptor.     

Cannabinoid receptor interactions with the antagonists SR 141716A and SR 144528.

The G protein-coupled cannabinoid receptor subtypes CB1 and CB2 have been cloned from several species. The CB1 receptor is highly conserved across species, whereas the CB2 receptor shows considerable cross-species variations. The two human receptors share only 44% overall identity, ranging from 35% to 82% in the transmembrane regions. Despite this structural disparity, the most potent cannabinoid agonists currently available are largely undiscriminating and are therefore unsatisfactory tools for investigating the architecture of ligand binding sites. However, the availability of two highly specific antagonists, SR 141716A for the CB1 receptor and SR 144528 for the CB2 receptor, has allowed us to adopt a systematic approach to defining their respective binding sites through the use of chimeric CB1 receptor/CB2 receptor constructs, coupled with site-directed mutagenesis. We identified the region encompassed by the fourth and fifth transmembrane helices as being critical for antagonist specificity. Both the wild type human receptors overexpressed in heterologous systems are autoactivated; SR 141716A and SR 144528 exhibit classical inverse agonist properties with their respective target receptors. In addition, through its interaction with the CB1 receptor SR 141716A blocks the Gi protein-mediated activation of mitogen-activated protein kinase stimulated by insulin or insulin-like growth factor I. An in-depth analysis of this discovery has led to a modified three-state model for the CB1 receptor, one of which implicates the SR 141716A-mediated sequestration of Gi proteins, with the result that the growth factor-stimulated intracellular pathways are effectively impeded.     

Constitutive activity of the cannabinoid CB1 receptor regulates the function of co-expressed Mu opioid receptors

The human mu opioid receptor was expressed stably in Flp-In T-REx HEK293 cells. Occupancy by the agonist DAMGO (Tyr-d-Ala-Gly-N-methyl-Phe-Gly-ol) resulted in phosphorylation of the ERK1/2 MAP kinases, which was blocked by the opioid antagonist naloxone but not the cannabinoid CB1 receptor inverse agonist SR141716A. Expression of the human cannabinoid CB1 receptor in these cells from the inducible Flp-In T-REx locus did not alter expression levels of the mu opioid receptor. This allowed the cannabinoid CB1 agonist WIN55212-2 to stimulate ERK1/2 phosphorylation but resulted in a large reduction in the capacity of DAMGO to activate these kinases. Although lacking affinity for the mu opioid receptor, co-addition of SR141716A caused recovery of the effectiveness of DAMGO. In contrast co-addition of the CB1 receptor neutral antagonist O-2050 did not. Induction of the CB1 receptor also resulted in an increase of basal [(35)S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding and thereby a greatly reduced capacity of DAMGO to further stimulate [(35)S]GTPgammaS binding. CB1 inverse agonists attenuated basal [(35)S]GTPgammaS binding and restored the capacity of DAMGO to stimulate. Flp-In T-REx HEK293 cells were generated, which express the human mu opioid receptor constitutively and harbor a modified D163N cannabinoid CB1 receptor that lacks constitutive activity. Induction of expression of the modified cannabinoid CB1 receptor did not limit DAMGO-mediated ERK1/2 MAP kinase phosphorylation and did not allow SR141716A to enhance the function of DAMGO. These data indicate that it is the constitutive activity inherent in the cannabinoid CB1 receptor that reduces the capacity of co-expressed mu opioid receptor to function.     

CB1 receptor-G protein association. Subtype selectivity is determined by distinct intracellular domains.

The CB1 cannabinoid receptor in N18TG2 neuroblastoma cells inhibits adenylate cyclase, and this response can be mimicked by a peptide corresponding to the juxtamembrane C-terminal domain (CB(1)401-417). Guanosine 5'-O-(3-thio)triphosphate binding to G proteins can be stimulated by both peptide CB(1)401-417 and peptides corresponding to the third intracellular loop [Howlett, A.C., Song, C., Berglund, B.A., Wilken, G.H. & Pigg, J.J. (1998) Mol. Pharmacol. 53, 504-510; Mukhopadhyay, S., Cowsik, S.M., Welsh, W.J. & Howlett, A.C. (1999) Biochemistry 38, 3447-3455]. In Chaps-solubilized N18TG2 membranes, the CB1 receptor coimmunoprecipitated with all three Gi subtypes. Pertussis toxin significantly reduced the CB(1) receptor-G alpha(i) association and attenuated the CB(1)401-417-induced inhibition of adenylate cyclase. CB(1)401-417 significantly reduced the CB(1) receptor association with G alpha(i3), but not with G alpha(i1) or G alpha(i2). In contrast, third intracellular loop peptides significantly reduced the CB(1) receptor association with G alpha(i1) and G alpha(i2), but not G alpha(i3). These interactions are specific for the CB(1) receptor because a peptide corresponding to the juxtamembrane C-terminal domain of the CB(2) receptor failed to compete for the association of the CB1 receptor with any of the Gi alpha subtypes, and was not able to activate Gi proteins to inhibit adenylate cyclase. These studies indicate that different domains of the CB(1) receptor direct the interaction with specific G protein subtypes.     

Enantiomeric resolution of a novel chiral cannabinoid receptor ligand

The enantiomeric resolution of a racemic novel cannabinoid receptor ligand conformationally restricted at the southern aliphatic chain was accomplished using a ChiralPak AD column. Both enantiomers were tested for their competitive binding to the rat brain CB1, mouse spleen CB2 and human CB2 receptors. The levorotatory isomer showed exceptionally high affinity for the CB1 receptor with a seven-fold selectivity over CB2.     

1-Pentyl-3-phenylacetylindoles, a new class of cannabimimetic indoles

A new class of cannabimimetic indoles, with 3-phenylacetyl or substituted 3-phenylacetyl substituents, has been prepared and their affinities for the cannabinoid CB1 and CB2 receptors have been determined. In general those compounds with a 2-substituted phenylacetyl group have good affinity for both receptors. The 4-substituted analogs have little affinity for either receptor, while the 3-substituted compounds are intermediate in their affinities. Two of these compounds, 1-pentyl-3-(2-methylphenylacetyl)indole (JWH-251) and 1-pentyl-3-(3-methoxyphenylacetyl)indole (JWH-302), have 5-fold selectivity for the CB1 receptor with modest affinity for the CB2 receptor. GTPgammaS determinations indicate that both compounds are highly efficacious agonists at the CB1 receptor and partial agonists at the CB2 receptor.     

Microwave-assisted synthesis of quinoline, isoquinoline, quinoxaline and quinazoline derivatives as CB2 receptor agonists

Quinoline, isoquinoline, quinoxaline, and quinazoline derivatives were synthesized using microwave-assisted synthesis and their CB1/CB2 receptor activities were determined using the [3?S]GTPγS binding assay. Most of the prepared quinoline, isoquinoline, and quinoxalinyl phenyl amines showed low-potency partial CB2 receptor agonists activity. The most potent CB2 ligand was the 4-morpholinylmethanone derivative (compound 40e) (-log EC?? = 7.8; E(max) = 75%). The isoquinolin-1-yl(3-trifluoromethyl-phenyl)amine (compound 26c) was a high efficacy CB2 agonist (-log EC?? = 5.8; E(max) = 128%). No significant CB1 receptor activation or inactivation was shown in these studies, except 40e, which showed weak CB1 agonist activity (CB1 -log EC?? = 5.0). These ligands serve as novel templates for the development of selective CB2 receptor agonist.     

Not too excited? Thank your endocannabinoids

Endocannabinoids can mediate neuroprotection, but it is not known how. In this issue of Neuron, Monory et al. use mutant mice and localized viral targeting to produce conditional knockouts of the cannabinoid CB1 receptor. They show that protection against kainic acid-induced seizures and cell death is conferred by CB1Rs on hippocampal glutamatergic nerve terminals.     

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.