Synthesis and biological evaluation of a ring analogs of the selective CB2 inverse agonist SMM-189.

Microglia are the principle cell type driving sustained neuroinflammation in neurodegenerative diseases such as Alzheimer's, Parkinson's, and Multiple Sclerosis. Interestingly, microglia locked into a chronic M1 pro-inflammatory phenotype significantly up-regulate the cannabinoid receptor 2 (CB2) expression. Our approach to exploiting CB2 as a therapeutic target in neuroinflammatory diseases focuses on the development of selective CB2 inverse agonists to shift microglia bias to a M2 pro-wound healing phenotype. Herein we report work designed to refine the structure activity relationship of the 2,6-dihydroxy-biphenyl-aryl-methanone CB2 inverse agonist scaffold. A series of analogs of our lead compound SMM-189 were synthesized and measured for affinity/selectivity, potency, and efficacy in regulating cAMP production and ß-arrestin recruitment. In this series compound 40 demonstrated a significant increase in potency and efficacy for cAMP stimulation compared to SMM-189. Akin to our lead SMM-189, this compound was highly efficacious in biasing microglia to an M2 pro-wound healing phenotype in LPS stimulated cell lines. These results advance our understanding of the structure-activity relationship of the 2,6-dihydroxy-biphenyl-aryl-methanone scaffold and provide further support for regulating microglia activation using CB2 inverse agonists.

Cannabinoid type-2 receptor agonist, inverse agonist, and anandamide regulation of inflammatory responses in IL-1ß stimulated primary human periodontal ligament fibroblasts.

OBJECTIVE: The aim of this study is to understand the role of cannabinoid type 2 receptor (CB2R) during periodontal inflammation and to identify anti-inflammatory agents for the development of drugs to treat periodontitis (PD). BACKGROUND: Cannabinoid type 2 receptor is found in periodontal tissue at sites of inflammation/infection. Our previous study demonstrated anti-inflammatory responses in human periodontal ligament fibroblasts (hPDLFs) via CB2R ligands. METHODS: Anandamide (AEA), HU-308 (agonist), and SMM-189 (inverse agonist) were tested for effects on IL-1ß-stimulated cytokines, chemokines, and angiogenic and vascular markers expressed by hPDLFs using Mesoscale Discovery V-Plex Kits. Signal transduction pathways (p-c-Jun, p-ERK, p-p-38, p-JNK, p-CREB, and p-NF-kB) were investigated using Cisbio HTRF kits. ACTOne and Tango™ -BLA functional assays were used to measure cyclic AMP (cAMP) and ß-arrestin activity. RESULTS: IL-1ß stimulated hPDLF production of 18/39 analytes, which were downregulated by the CB2R agonist and the inverse agonist. AEA exhibited pro-inflammatory and anti-inflammatory effects. IL-1ß increased phosphoproteins within the first hour except p-JNK. CB2R ligands attenuated p-p38 and p-NFĸB, but a late rise in p-38 was seen with HU-308. As p-ERK levels declined, a significant increase in p-ERK was observed later in the time course by synthetic CB2R ligands. P-JNK was significantly affected by SMM-189 only, while p-CREB was elevated significantly by CB2R ligands at 180 minutes. HU-308 affected both cAMP and ß-arrestin pathway. SMM-189 only stimulated cAMP. CONCLUSION: The findings that CB2R agonist and inverse agonist may potentially regulate inflammation suggest that development of CB2R therapeutics could improve on current treatments for PD and other oral inflammatory pathologies.

Inverse Agonism of Cannabinoid Receptor Type 2 Confers Anti-inflammatory and Neuroprotective Effects Following Status Epileptics.

Prolonged status epilepticus (SE) in humans causes high mortality and brain inflammation-associated neuronal injury and morbidity in survivors. Currently, the only effective treatment is to terminate the seizures swiftly to prevent brain damage. However, reliance on acute therapies alone would be imprudent due to the required short response time. Follow-on therapies that can be delivered well after the SE onset are in an urgent need. Cannabinoid receptor type 2 (CB2), a G protein-coupled receptor that can be expressed by activated brain microglia, has emerged as an appealing anti-inflammatory target for brain conditions. In the current study, we reported that the CB2 inverse agonism by our current lead compound SMM-189 largely prevented the rat primary microglia-mediated inflammation and showed moderate neuroprotection against N-methyl-D-aspartic acid (NMDA) receptor-mediated excitotoxicity in rat primary hippocampal cultures containing both neurons and glia. Using a classical mouse model of epilepsy, in which SE was induced by systemic administration of kainate (30 mg/kg, i.p.) and proceeded for 1 h, we demonstrated that SE downregulated the CB1 but slightly upregulated CB2 receptor in the hippocampus. Transient treatment with SMM-189 (6 mg/kg, i.p., b.i.d.) after the SE was interrupted by diazepam (10 mg/kg, i.p.) prevented the seizure-induced cytokine surge in the brain, neuronal death, and behavioral impairments 24 h after SE. Our results suggest that CB2 inverse agonism might provide an adjunctive anti-inflammatory therapy that can be delivered hours after SE onset, together with NMDA receptor blockers and first-line anti-convulsants, to reduce brain injury and functional deficits following prolonged seizures.

Anti-inflammatory activity of cannabinoid receptor 2 ligands in primary hPDL fibroblasts.

OBJECTIVES: Approximately 65 million adults in the US have periodontitis, causing tooth loss and decreased quality of life. Cannabinoids modulate immune responses, and endocannabinoids are prevalent during oral cavity inflammation. Targets for intervention in periodontal inflammation are cannabinoid type 1 and 2 receptors (CB1R, CB2R), particularly CB2R because its levels increase during inflammation. We previously demonstrated that SMM-189 (CB2R inverse agonist) decreased pro-inflammatory cytokine production in primary microglial cells. The hypothesis of this study was that cannabinoids anandamide (AEA), HU-308 (CB2R selective agonist), and SMM-189 decrease pro-inflammatory IL-6 and MCP-1 production by primary human periodontal ligament fibroblasts (hPDLFs) stimulated with P. gingivalis LPS, TNF-α, or IL-1ß. DESIGN: Cytotoxic effects of cannabinoid compounds (10-4-10-6.5 M), LPS (1-1000 ng/ml), TNFα (10 ng/ml) and IL-1ß (1 ng/ml) were assessed by measuring effects on cellular dehydrogenase activity. IL-6 and MCP-1 production were measured using Mesoscale Discovery (MSD) Human Pro-Inflammatory IL-6 and MSD Human Chemokine MCP-1 kits and analyzed using MSD Sector 2400 machine. RESULTS: EC50 values for AEA, SMM-189, and HU-308 were 16 µM, 13 µM, and 7.3 µM respectively. LPS (1 µg/ml), TNF-α (10 ng/ml), and IL-1ß (1 ng/ml) increased IL-6 and MCP-1 production, which were inhibited by AEA, SMM-189, and HU-308. AEA alone significantly increased IL-6, but not MCP-1 levels, but the other cannabinoids alone had no effect. CONCLUSION: The effective inhibition of LPS, TNF-α, IL-1ß stimulated IL-6 and MCP-1 production by CB2R ligands in hPDLFs suggests that targeting the endocannabinoid system may lead to development of novel drugs for periodontal therapy, aiding strategies to improve oral health.

Selective Cannabinoid 2 Receptor Stimulation Reduces Tubular Epithelial Cell Damage after Renal Ischemia-Reperfusion Injury.

Ischemia-reperfusion injury (IRI) is a common cause of acute kidney injury (AKI), which is an increasing problem in the clinic and has been associated with elevated rates of mortality. Therapies to treat AKI are currently not available, so identification of new targets that can be modulated to ameliorate renal damage upon diagnosis of AKI is essential. In this study, a novel cannabinoid receptor 2 (CB2) agonist, SMM-295 [3'-methyl-4-(2-(thiophen-2-yl)propan-2-yl)biphenyl-2,6-diol], was designed, synthesized, and tested in vitro and in silico. Molecular docking of SMM-295 into a CB2 active-state homology model showed that SMM-295 interacts well with key amino acids to stabilize the active state. In human embryonic kidney 293 cells, SMM-295 was capable of reducing cAMP production with 66-fold selectivity for CB2 versus cannabinoid receptor 1 and dose-dependently increased mitogen-activated protein kinase and Akt phosphorylation. In vivo testing of the CB2 agonist was performed using a mouse model of bilateral IRI, which is a common model to mimic human AKI, where SMM-295 was immediately administered upon reperfusion of the kidneys after the ischemia episode. Histologic damage assessment 48 hours after reperfusion demonstrated reduced tubular damage in the presence of SMM-295. This was consistent with reduced plasma markers of renal dysfunction (i.e., creatinine and neutrophil gelatinase-associated lipocalin) in SMM-295-treated mice. Mechanistically, kidneys treated with SMM-295 were shown to have elevated activation of Akt with reduced terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling (TUNEL)-positive cells compared with vehicle-treated kidneys after IRI. These data suggest that selective CB2 receptor activation could be a potential therapeutic target in the treatment of AKI.

Synthesis and biological evaluation of (3',5'-dichloro-2,6-dihydroxy-biphenyl-4-yl)-aryl/alkyl-methanone selective CB2 inverse agonist.

Cannabinoid receptor 2 (CB2) selective agonists and inverse agonists possess significant potential as therapeutic agents for regulating inflammation and immune function. Although CB2 agonists have received the greatest attention, it is emerging that inverse agonists also manifest anti-inflammatory activity. In process of designing new cannabinoid ligands we discovered that the 2,6-dihydroxy-biphenyl-aryl methanone scaffold imparts inverse agonist activity at CB2 receptor without functional activity at CB1. To further explore the scaffold we synthesized a series of (3',5'-dichloro-2,6-dihydroxy-biphenyl-4-yl)-aryl/alkyl-methanone analogs and evaluated the CB1 and CB2 affinity, potency, and efficacy. The studies reveal that an aromatic C ring is required for inverse agonist activity and that substitution at the 4 position is optimum. The resorcinol moiety is required for optimum CB2 inverse agonist activity and selectivity. Antagonist studies against CP 55,940 demonstrate that the compounds 41 and 45 are noncompetitive antagonists at CB2.

Tethered yohimbine analogs as selective human alpha2C-adrenergic receptor ligands.

Yohimbine is a potent and relatively nonselective alpha(2)-adrenergic receptor (AR) antagonist. In an earlier report, we demonstrated that dimeric yohimbine analogs containing methylene and methylene-diglycine tethers were highly selective human alpha(2C)-AR ligands. Little work has been done to examine the role of the tether group or the absence of the second yohimbine pharmacophore on selectivity for human alpha(2)-AR subtypes. The goal of our study was to determine the binding affinities and functional subtype selectivities of a series of tethered yohimbine ligands in the absence of the second pharmacophore. The profiles of pharmacological activity for the yohimbine analogs on the three human alpha(2)-AR subtypes expressed in Chinese hamster ovary cells were examined using receptor binding and cAMP inhibition assays. All of the tethered yohimbine analogs exhibited higher binding affinities at the alpha(2C)- versus alpha(2A)- and alpha(2B)-AR subtypes. Notably, the benzyl carboxy alkyl amine and the carboxy alkyl amine analogs exhibited 43- and 1995-fold and 295- and 54-fold selectivities in binding to the alpha(2C)- versus alpha(2A)- and alpha(2B)-ARs, respectively. Data from luciferase reporter gene assays confirmed the functional antagonist activities and selectivity profiles of selected compounds from the tethered series. The data demonstrate that the second pharmacophore may not be essential to obtain alpha(2C)-AR subtype selectivity, previously observed with the dimers. Further changes in the nature of the tether will help in optimization of the structure-activity relationship to obtain potent and selective alpha(2C)-AR ligands. These compounds may be used as pharmacological probes and in the treatment of human disorders.

Preclinical pharmacology of a nonsteroidal ligand for androgen receptor-mediated imaging of prostate cancer.

Proper management of prostate cancer patients is highly dependent on the spread of the disease. High expression levels of the androgen receptor (AR) in prostate tumor offer a target for identifying cancer metastasis. We investigated the use of nonsteroidal AR ligands for receptor-mediated imaging as a diagnostic tool for prostate cancer staging. Compound S-26 [S-3-(4-fluorophenoxy)-2-hydroxy-2-methyl-N-(4-cyano-3-iodophenyl)-propionamide]was identified from a series of iodinated ether-linked derivatives of bicalutamide due to its high-AR binding affinity of 3.3 nM (which is similar to testosterone and approximately 25% of the binding affinity of dihydrotestosterone) in an in vitro competitive binding assay using rat prostate cytosol. Furthermore, S-26 exhibited a greater binding affinity (K(i) = 4.4 nM) in a whole-cell binding assay using COS-7 cells transfected with human AR than testosterone (K(i) = 32.9 nM) and dihydrotestosterone (K(i) = 45.4 nM). We also confirmed that sex hormone-binding globulin (SHBG), a plasma protein that binds steroids with high affinity, does not bind with S-26. Cotransfection studies with the estrogen, progesterone, and glucocorticoid receptor indicated that S-26 does not cross-react with other members of the steroid hormone receptor family. The nonsteroidal structure, high-AR binding affinity, specificity, and lack of binding to SHBG indicate that S-26 exhibits favorable properties for further development as an imaging agent for prostate cancer.

Synthesis of oxazolidinedione derived bicalutamide analogs.

The synthesis of chiral oxazolidinedione derived bicalutamide analogs has been discussed.

Synthesis and biological studies of yohimbine derivatives on human alpha2C-adrenergic receptors.

A series of yohimbine derivatives was synthesized and evaluated for binding affinity at the human alpha(2C)-adrenergic receptors expressed in Chinese hamster ovary cells. It has been found that compound 5 shows a higher affinity for alpha(2C)-AR than the parent compound yohimbine 1, thereby illustrating that the nature of the linkers affect binding potencies on these receptors.

Synthesis of irreversibly binding bicalutamide analogs for imaging studies.

A new synthetic methodology for preparing radioactive androgen receptor binding compounds in order to determine receptor-ligands interactions has been developed.