Synthesis, Pharmacological Evaluation, and Docking Studies of Novel Pyridazinone-Based Cannabinoid Receptor Type†2 Ligands.

In recent years, cannabinoid type 2 receptors (CB2 R) have emerged as promising therapeutic targets in a wide variety of diseases. Selective ligands of CB2 R are devoid of the psychoactive effects typically observed for CB1 R ligands. Based on our recent studies on a class of pyridazinone 4-carboxamides, further structural modifications of the pyridazinone core were made to better investigate the structure-activity relationships for this promising scaffold with the aim to develop potent CB2 R ligands. In binding assays, two of the new synthesized compounds [6-(3,4-dichlorophenyl)-2-(4-fluorobenzyl)-cis-N-(4-methylcyclohexyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide (2) and 6-(4-chloro-3-methylphenyl)-cis-N-(4-methylcyclohexyl)-3-oxo-2-pentyl-2,3-dihydropyridazine-4-carboxamide (22)] showed high CB2 R affinity, with Ki values of 2.1 and 1.6 nm, respectively. In addition, functional assays of these compounds and other new active related derivatives revealed their pharmacological profiles as CB2 R inverse agonists. Compound 22 displayed the highest CB2 R selectivity and potency, presenting a favorable in silico pharmacokinetic profile. Furthermore, a molecular modeling study revealed how 22 produces inverse agonism through blocking the movement of the toggle-switch residue, W6.48.

Ribonuclease H/DNA Polymerase HIV-1 Reverse Transcriptase Dual Inhibitor: Mechanistic Studies on the Allosteric Mode of Action of Isatin-Based Compound RMNC6.

The DNA polymerase and ribonuclease H (RNase H) activities of human immunodeficiency virus type 1 (HIV-1) are needed for the replication of the viral genome and are validated drug targets. However, there are no approved drugs inhibiting RNase H and the efficiency of DNA polymerase inhibitors can be diminished by the presence of drug resistance mutations. In this context, drugs inhibiting both activities could represent a significant advance towards better anti-HIV therapies. We report on the mechanisms of allosteric inhibition of a newly synthesized isatin-based compound designated as RMNC6 that showed IC50 values of 1.4 and 9.8 µM on HIV-1 RT-associated RNase H and polymerase activities, respectively. Blind docking studies predict that RMNC6 could bind two different pockets in the RT: one in the DNA polymerase domain (partially overlapping the non-nucleoside RT inhibitor [NNRTI] binding pocket), and a second one close to the RNase H active site. Enzymatic studies showed that RMNC6 interferes with efavirenz (an approved NNRTI) in its binding to the RT polymerase domain, although NNRTI resistance-associated mutations such as K103N, Y181C and Y188L had a minor impact on RT susceptibility to RMNC6. In addition, despite being naturally resistant to NNRTIs, the polymerase activity of HIV-1 group O RT was efficiently inhibited by RMNC6. The compound was also an inhibitor of the RNase H activity of wild-type HIV-1 group O RT, although we observed a 6.5-fold increase in the IC50 in comparison with the prototypic HIV-1 group M subtype B enzyme. Mutagenesis studies showed that RT RNase H domain residues Asn474 and Tyr501, and in a lesser extent Ala502 and Ala508, are critical for RMNC6 inhibition of the endonuclease activity of the RT, without affecting its DNA polymerization activity. Our results show that RMNC6 acts as a dual inhibitor with allosteric sites in the DNA polymerase and the RNase H domains of HIV-1 RT.

Pretreatment with clonidine caused desensitization to WIN 55,212-2 in guinea pig ileum.

Considering the existence of cross-tolerance between clonidine and morphine besides the same interaction between morphine and WIN 55,212-2 persuaded us to verify this fact between WIN 55,212-2 and clonidine in guinea pig ileum, which is a well-known model to examine the mode of action of cannabinoids and α2 -adenoceptor agonists The rectangular pulses were passed to the 0.5 g stretched ileum segments that were fixed in 20-ml organ bath. PowerLab system and Graphpad Prism were applied to record twitches and analyse the data. Electrically evoked contractions were dose-dependently inhibited by WIN 55,212-2 and clonidine (pD2 = 8.56 ± 0.41 and 7.65 ± 0.15, respectively). Tolerance to this effect could be induced by 4-h incubation with WIN 55,212-2 (3 × IC50 ) (pD2  = 6.36 ± 0.26, degree of tolerance: 159.32) (P < 0.01) but not with clonidine (2 × IC50 and 4 × IC50 ) for different time courses. Dose-response curve for inhibitory action of WIN 55,212-2 was shifted to the right after 4-h incubation with clonidine (3 × 10(-10) m) comparing to the untreated tissues (pD2  = 5.26 ± 0.69, degree of tolerance: 2000) (P < 0.001). This observation provides the evidence for the cannabinoid-noradrenergic systems interaction in the enteric nervous system as a simplified representative for central nervous system.

Novelty-induced emotional arousal modulates cannabinoid effects on recognition memory and adrenocortical activity.

Although it is well established that cannabinoid drugs can influence cognitive performance, the findings-describing both enhancing and impairing effects-have been ambiguous. Here, we investigated the effects of posttraining systemic administration of the synthetic cannabinoid agonist WIN55,212-2 (0.1, 0.3, or 1.0 mg/kg) on short- and long-term retention of object recognition memory under two conditions that differed in their training-associated arousal level. In male Sprague-Dawley rats that were not previously habituated to the experimental context, WIN55,212-2 administered immediately after a 3-min training trial, biphasically impaired retention performance at a 1-h interval. In contrast, WIN55,212-2 enhanced 1-h retention of rats that had received extensive prior habituation to the experimental context. Interestingly, immediate posttraining administration of WIN55,212-2 to non-habituated rats, in doses that impaired 1-h retention, enhanced object recognition performance at a 24-h interval. Posttraining WIN55,212-2 administration to habituated rats did not significantly affect 24-h retention. In light of intimate interactions between cannabinoids and the hypothalamic-pituitary-adrenal axis, we further investigated whether cannabinoid administration might differently influence training-induced glucocorticoid activity in rats in these two habituation conditions. WIN55,212-2 administered after object recognition training elevated plasma corticosterone levels in non-habituated rats whereas it decreased corticosterone levels in habituated rats. Most importantly, following pretreatment with the corticosterone-synthesis inhibitor metyrapone, WIN55,212-2 effects on 1- and 24-h retention of non-habituated rats became similar to those seen in the low-aroused habituated animals, indicating that cannabinoid-induced regulation of adrenocortical activity contributes to the environmentally sensitive effects of systemically administered cannabinoids on short- and long-term retention of object recognition memory.

Diuretic effects of cannabinoids.

In vivo effects of cannabinoid (CB) agonists are often assessed using four well-established measures: locomotor activity, hypothermia, cataleptic-like effects, and analgesia. The present studies demonstrate that doses of CB agonists that produce these effects also reliably increase diuresis. Diuretic effects of several CB agonists were measured in female rats over 2 hours immediately after drug injection, and results were compared with hypothermic effects. Direct-acting CB1 agonists, including Δ(9)-tetrahydrocannabinol, WIN 55,212 [R-(1)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate], AM2389 [9ß-hydroxy-3-(1-hexyl-cyclobut-1-yl)-hexahydrocannabinol], and AM4054 [9ß-(hydroxymethyl)-3-(1-adamantyl)-hexahydrocannabinol], produced dose-dependent increases in diuresis and decreases in colonic temperature, with slightly lower ED(50) values for diuresis than for hypothermia. The highest doses of cannabinoid drugs yielded, on average, 26-32 g/kg urine; comparable effects were obtained with 10 mg/kg furosemide and 3.0 mg/kg trans-(-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U50-488). Methanandamide (10.0 mg/kg) had lesser effect than other CB agonists, and the CB2 agonist AM1241 [1-(methylpiperidin-2-ylmethyl)-3-(2-iodo-5-nitrobenzoyl)indole], the anandamide transport inhibitor AM404, and the CB antagonist rimonabant did not have diuretic effects. In further studies, the diuretic effects of the CB1 agonist AM4054 were similar in male and female rats, displayed a relatively rapid onset to action, and were dose-dependently antagonized by 30 minutes pretreatment with rimonabant, but not by the vanilloid receptor type I antagonist capsazepine, nor were the effects of WIN 55,212 antagonized by the CB2 antagonist AM630 [(6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl) methanone)]. These data indicate that cannabinoids have robust diuretic effects in rats that are mediated via CB1 receptor mechanisms.

WIN55212-2 attenuates amyloid-beta-induced neuroinflammation in rats through activation of cannabinoid receptors and PPAR-γ pathway.

Cannabinoids have been shown to exert neuroprotective effects in a plethora of neurodegenerative conditions. Over the past decade, some studies demonstrate that cannabinoids can interact with nuclear peroxisome proliferator-activated receptors (PPARs). We investigated protective properties of WIN55212-2 (WIN, a non-selective cannabinoid receptor agonist) in beta-amyloid (Aß)-induced neurodegeneration in rat hippocampus and possible involvement of PPAR-gamma (PPAR-γ). Aß (1-42) was injected into the hippocampus of male rats. Animals were administered by intracerebroventricular rout the following treatments on days 1, 3, 5, 7: vehicle, WIN, GW9662 (selective PPAR-γ antagonist) plus WIN, AM251 (selective CB1 receptor antagonist) plus WIN, SR144528 (selective CB2 receptor antagonist) plus WIN, each of antagonists alone. Injection of Aß-induced spatial memory impairment and a dramatic rise in hippocampal TNF-α, active caspase 3, nuclear NF-kB levels and TUNEL-positive neurons. WIN administration significantly improved memory function and diminished the elevated levels of these markers, while antagonizing either CB1 or CB2 receptor subtype partially attenuated the protective effects. Intriguingly, WIN significantly increased PPAR-γ level and transcriptional activity, the latter being partially inhibited with AM251 but not with SR144528. The enhancing effect on PPAR-γ pathway was crucial to WIN-induced neuroprotection since GW9662 partially reversed the beneficial actions of WIN. Co-administration of the three antagonists led to the complete abrogation of WIN effects. Our findings indicate that WIN exerts neuroprotective and anti-inflammatory actions against Aß damage through both CB1 and CB2 receptors. Of great note, both direct and CB1-mediated increase in PPAR-γ signaling also contributes to WIN-induced neuroprotection.

Cannabinoids prevent the development of behavioral and endocrine alterations in a rat model of intense stress.

Cannabinoids have recently emerged as a possible treatment of stress- and anxiety-related disorders such as post-traumatic stress disorder (PTSD). Here, we examined whether cannabinoid receptor activation could prevent the effects of traumatic stress on the development of behavioral and neuroendocrine measures in a rat model of PTSD, the single-prolonged stress (SPS) model. Rats were injected with the CB1/CB2 receptor agonist WIN55,212-2 (WIN) systemically or into the basolateral amygdala (BLA) at different time points following SPS exposure and were tested 1 week later for inhibitory avoidance (IA) conditioning and extinction, acoustic startle response (ASR), hypothalamic-pituitary-adrenal (HPA) axis function, and anxiety levels. Exposure to SPS enhanced conditioned avoidance and impaired extinction while enhancing ASR, negative feedback on the HPA axis, and anxiety. WIN (0.5 mg/kg) administered intraperitoneally 2 or 24 h (but not 48 h) after SPS prevented the trauma-induced alterations in IA conditioning and extinction, ASR potentiation, and HPA axis inhibition. WIN microinjected into the BLA (5 µg/side) prevented SPS-induced alterations in IA and ASR. These effects were blocked by intra-BLA co-administration of the CB1 receptor antagonist AM251 (0.3 ng/side), suggesting the involvement of CB1 receptors. These findings suggest that (i) there may be an optimal time window for intervention treatment with cannabinoids after exposure to a highly stressful event, (ii) some of the preventive effects induced by WIN are mediated by an activation of CB1 receptors in the BLA, and (iii) cannabinoids could serve as a pharmacological treatment of stress- and trauma-related disorders.

Cannabinoid/agonist WIN 55,212-2 reduces cardiac ischaemia­reperfusion injury in Zucker diabetic fatty rats: role of CB2 receptors and iNOS/eNOS.

BACKGROUND: Diabetes increases cardiac damage after myocardial ischaemia. Cannabinoids can protect against myocardial ischaemia/reperfusion injury. The aim of this study was to examine the cardioprotective effect of the cannabinoid agonist WIN 55,212-2 (WIN) against ischaemia/reperfusion injury in an experimental model of type 2 diabetes. We performed these experiments in the Zucker diabetic fatty rat, and focused on the role of cannabinoid receptors in modulation of cardiac inducible nitric oxide synthase (iNOS)/endothelial-type nitric oxide synthase (eNOS) expression. METHODS: Male 20-week-old Zucker diabetic fatty rats were treated with vehicle, WIN, the selective CB1 or CB2 receptor antagonists AM251 and AM630, respectively, AM251 + WIN or AM630 + WIN. Hearts were isolated from these rats, and the cardiac functional response to ischaemia/reperfusion injury was evaluated. In addition, cardiac iNOS and eNOS expression were determined by western blot. RESULTS: WIN significantly improved cardiac recovery after ischaemia/ reperfusion in the hearts from Zucker diabetic fatty rats by restoring coronary perfusion pressure and heart rate to preischaemic levels. Additionally, WIN decreased cardiac iNOS expression and increased eNOS expression after ischaemia/reperfusion in diabetic hearts. WIN-induced cardiac functional recovery was completely blocked by the CB2 antagonist AM630. However, changes in NOS isoenzyme expression were not affected by the CB antagonists. CONCLUSIONS: This study shows a cardioprotective effect of a cannabinoid agonist on ischaemia/reperfusion injury in an experimental model of a metabolic disorder. The activation mainly of CB2 receptors and the restoration of iNOS/eNOS cardiac equilibrium are mechanisms involved in this protective effect. These initial studies have provided the basis for future research in this field.

A novel peripherally restricted cannabinoid receptor antagonist, AM6545, reduces food intake and body weight, but does not cause malaise, in rodents.

BACKGROUND AND PURPOSE: Cannabinoid CB(1) receptor antagonists reduce food intake and body weight, but clinical use in humans is limited by effects on the CNS. We have evaluated a novel cannabinoid antagonist (AM6545) designed to have limited CNS penetration, to see if it would inhibit food intake in rodents, without aversive effects. EXPERIMENTAL APPROACH: Cannabinoid receptor binding studies, cAMP assays, brain penetration studies and gastrointestinal motility studies were carried out to assess the activity profile of AM6545. The potential for AM6545 to induce malaise in rats and the actions of AM6545 on food intake and body weight were also investigated. KEY RESULTS: AM6545 binds to CB(1) receptors with a K(i) of 1.7 nM and CB(2) receptors with a K(i) of 523 nM. AM6545 is a neutral antagonist, having no effect on cAMP levels in transfected cells and was less centrally penetrant than AM4113, a comparable CB(1) receptor antagonist. AM6545 reversed the effects of WIN55212-2 in an assay of colonic motility. In contrast to AM251, AM6545 did not produce conditioned gaping or conditioned taste avoidance in rats. In rats and mice, AM6545 dose-dependently reduced food intake and induced a sustained reduction in body weight. The effect on food intake was maintained in rats with a complete subdiaphragmatic vagotomy. AM6545 inhibited food intake in CB(1) receptor gene-deficient mice, but not in CB(1)/CB(2) receptor double knockout mice. CONCLUSIONS AND IMPLICATIONS: Peripherally active, cannabinoid receptor antagonists with limited brain penetration may be useful agents for the treatment of obesity and its complications.

WIN55212-2 ameliorates atherosclerosis associated with suppression of pro-inflammatory responses in ApoE-knockout mice.

The role of inflammation in all stages of atherosclerosis has been actively investigated, with an emphasis on the discovery of novel and innovative drugs for treatment and prevention. The anti-inflammatory and immunomodulatory capacity of cannabinoids are well established, and these agents have a broad therapeutic potential in various inflammatory diseases, including cardiovascular diseases. The aim of this study was to investigate the effect of WIN55212-2, a synthetic cannabinoid, on atherosclerosis using the apolipoprotein E-knockout (ApoE(-/-)) mouse on a cholate-containing high-fat diet. Our results showed that WIN55212-2 reduced the size of atherosclerotic lesions in the aorta root, and did not affect serum lipid levels significantly. Furthermore, alleviation of atherosclerosis by WIN55212-2 was associated with a smaller content of macrophages in plaque lesion as well as decreasing pro-inflammatory gene expression and NF-κB activation in aortic tissues. Oxidized LDL (ox-LDL) dramatically induced NF-κB activation, and enhanced pro-inflammatory mRNA and protein expression in peritoneal macrophages isolated from ApoE(-/-) mice. It is noteworthy that all of the above-mentioned effects of ox-LDL were attenuated by WIN55212-2. Moreover, WIN55212-2 also attenuated the inflammatory response that LPS induced. AM630, a cannabinoid receptor 2 (CB2) special antagonist completely abolished the protective effects of WIN55212-2 both in vivo and in vitro. Our data provide strong evidence that WIN55212-2 can potentially inhibit atherosclerosis in ApoE(-/-) mice. Importantly, all the beneficial effects of WIN55212-2 in our model were closely associated with the suppression of pro-inflammatory responses and were mediated by the CB2 receptor.

Peripherally acting CB1-receptor antagonist: the relative importance of central and peripheral CB1 receptors in adiposity control.

OBJECTIVE: To investigate whether drugs targeting peripheral cannabinoid-1 (CB1) receptor ameliorate adiposity comparable to central CB1-receptor antagonist or not. MEASUREMENTS: Receptor binding assay and functional assay in vitro. Pharmacokinetic parameters in mice, brain uptake clearance of compounds in rats and antagonism on the CB1-agonist-induced hypothermia in mice. Diet consumption, body weight changes, hepatic gene expression of sterol-regulatory element-binding protein-1 (SREBP-1) and plasma/tissue concentrations of compounds in HF diet-induced obese (HF-DIO) mice after acute and chronic treatment. RESULTS: Compound-1, an SR141716A derivative, is a peripheral CB1-receptor-selective antagonist that is 10 times less potent than SR141716A in in vitro evaluations. Although the plasma concentrations of Compound-1 are five times higher than those of SR141716A, its potency is still 10 times lower than that of SR141716A in reducing the consumption of normal or HF diet by mice. Through evaluations of brain uptake and the effect on CB1-agonist-induced hypothermia, it was verified that the blood-brain barrier (BBB) penetration of Compound-1 is much lower than that of SR141716A. In HF-DIO mice, chronic treatment by Compound-1 showed dose-dependent antiobesity activities, while its brain distribution was very low as compared with that of SR141716A. Compound-1's effective doses for antiobesity activity were just over 30 mg kg(-1). However, Compound-1 completely suppressed the elevated hepatic SREBP-1 expression even at 10 mg kg(-1). CONCLUSION: These results suggest that (1) central CB1 receptors mediate anorectic response of CB1-receptor antagonists and (2) peripheral modulations, including SREBP-1 expression, are not major mechanisms in the antiobesity effects of CB1-receptor antagonists.

Agonist-dependent cannabinoid receptor signalling in human trabecular meshwork cells.

BACKGROUND AND PURPOSE: Trabecular meshwork (TM) is an ocular tissue involved in the regulation of aqueous humour outflow and intraocular pressure (IOP). CB1 receptors (CB1) are present in TM and cannabinoid administration decreases IOP. CB1 signalling was investigated in a cell line derived from human TM (hTM). EXPERIMENTAL APPROACH: CB1 signalling was investigated using ratiometric Ca2+ imaging, western blotting and infrared In-Cell Western analysis. KEY RESULTS: WIN55212-2, a synthetic aminoalkylindole cannabinoid receptor agonist (10-100 microM) increased intracellular Ca2+ in hTM cells. WIN55,212-2-mediated Ca2+ increases were blocked by AM251, a CB1 antagonist, but were unaffected by the CB2 antagonist, AM630. The WIN55,212-2-mediated increase in [Ca2+]i was pertussis toxin (PTX)-insensitive, therefore, independent of Gi/o coupling, but was attenuated by a dominant negative Galpha(q/11) subunit, implicating a Gq/11 signalling pathway. The increase in [Ca2+]i was dependent upon PLC activation and mobilization of intracellular Ca2+ stores. A PTX-sensitive increase in extracellular signal-regulated kinase (ERK1/2) phosphorylation was also observed in response to WIN55,212-2, indicative of a Gi/o signalling pathway. CB1-Gq/11 coupling to activate PLC-dependent increases in Ca2+ appeared to be specific to WIN55,212-2 and were not observed with other CB1 agonists, including CP55,940 and methanandamide. CP55940 produced PTX-sensitive increases in [Ca2+]i at concentrations>or=15 microM, and PTX-sensitive increases in ERK1/2 phosphorylation. CONCLUSIONS AND IMPLICATIONS: This study demonstrates that endogenous CB1 couples to both Gq/11 and Gi/o in hTM cells in an agonist-dependent manner. Cannabinoid activation of multiple CB1 signalling pathways in TM tissue could lead to differential changes in aqueous humour outflow and IOP.

Rational design of inhibitors of VirA-VirG two-component signal transduction.

VirA-VirG two-component system regulates the vir (virulence) operon in response to specific host factors (xenognosins) in the plant pathogen Agrobacterium tumefaciens. Using whole cell assays, stable inhibitors inspired by the labile natural benzoxazinone inhibitor HDMBOA are developed. It is found that aromatic aldehydes represent a minimal structural unit for activity. In particular, 3-hydroxy-4,6-dimethoxy-3H-isobenzofuran-1-one (HDI) was found to have the highest activity, making it the most potent developed inhibitor of virulence gene expression in Agrobacterium.

Cannabidiol displays unexpectedly high potency as an antagonist of CB1 and CB2 receptor agonists in vitro.

BACKGROUND AND PURPOSE: A nonpsychoactive constituent of the cannabis plant, cannabidiol has been demonstrated to have low affinity for both cannabinoid CB1 and CB2 receptors. We have shown previously that cannabidiol can enhance electrically evoked contractions of the mouse vas deferens, suggestive of inverse agonism. We have also shown that cannabidiol can antagonize cannabinoid receptor agonists in this tissue with a greater potency than we would expect from its poor affinity for cannabinoid receptors. This study aimed to investigate whether these properties of cannabidiol extend to CB1 receptors expressed in mouse brain and to human CB2 receptors that have been transfected into CHO cells. EXPERIMENTAL APPROACH: The [35S]GTPS binding assay was used to determine both the efficacy of cannabidiol and the ability of cannabidiol to antagonize cannabinoid receptor agonists (CP55940 and R-(+)-WIN55212) at the mouse CB1 and the human CB2 receptor. KEY RESULTS: This paper reports firstly that cannabidiol displays inverse agonism at the human CB2 receptor. Secondly, we demonstrate that cannabidiol is a high potency antagonist of cannabinoid receptor agonists in mouse brain and in membranes from CHO cells transfected with human CB2 receptors. CONCLUSIONS AND IMPLICATIONS: This study has provided the first evidence that cannabidiol can display CB2 receptor inverse agonism, an action that appears to be responsible for its antagonism of CP55940 at the human CB2 receptor. The ability of cannabidiol to behave as a CB2 receptor inverse agonist may contribute to its documented anti-inflammatory properties.