7-Azaindolequinuclidinones (7-AIQD): A novel class of cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptor ligands

A series of N-benzyl-7-azaindolequinuclidinone (7-AIQD) analogs have been synthesized and evaluated for affinity toward CB1 and CB2 cannabinoid receptors and identified as a novel class of cannabinoid receptor ligands. Structure–activity relationship (SAR) studies indicate that 7-AIQD analogs are dual CB1/CB2 receptor ligands exhibiting high potency with somewhat greater selectivity towards CB2 receptors compared to the previously reported indolequinuclidinone (IQD) analogs. Initial binding assays showed that 7-AIQD analogs 8b, 8d, 8f, 8g and 9b (1 μM) produced more that 50% displacement of the CB1/CB2 non-selective agonist CP-55,940 (0.1 nM). Furthermore, Ki values determined from full competition binding curves showed that analogs 8a, 8b and 8g exhibit high affinity (110, 115 and 23.7 nM, respectively) and moderate selectivity (26.3, 6.1 and 9.2-fold, respectively) for CB2 relative to CB1 receptors. Functional studies examining modulation of G-protein activity demonstrated that 8a acts as a neutral antagonist at CB1 and CB2 receptors, while 8b exhibits inverse agonist activity at these receptors. Analogs 8f and 8g exhibit different intrinsic activities, depending on the receptor examined. Molecular docking and binding free energy calculations for the most active compounds (8a, 8b, 8f, and 8g) were performed to better understand the CB2 receptor-selective mechanism at the atomic level. Compound 8g exhibited the highest predicted binding affinity at both CB1 and CB2 receptors, and all four compounds were shown to have higher predicted binding affinities with the CB2 receptor compared to their corresponding binding affinities with the CB1 receptor. Further structural optimization of 7-AIQD analogs may lead to the identification of potential clinical agents.     

Synthesis and pharmacology of 1-deoxy analogs of CP-47,497 and CP-55,940

A series of 1-deoxy analogs of CP-47,497 (8 and 13, n=0-7) and 1-deoxy analogs of CP-55,940 (9, n=0-7) have been synthesized and their affinities for the cannabinoid CB(1) and CB(2) receptors have been determined. Although the majority of these compounds exhibit selectivity for the CB(2) receptor, none have greater than modest affinity for either receptor. The interactions of these 1-deoxy nontraditional cannabinoids with the CB(2) receptor are discussed.     

Oximes short-acting CB1 receptor agonists

New oximes short-acting CB1 agonists were explored by the introduction of an internal oxime and polar groups at the C3 alkyl tail of Δ⁸-THC. The scope of the research was to drastically alter two important physicochemical properties hydrophobicity (log P) and topological surface area (tPSA) of the compound, which play a critical role in tissue distribution and sequestration (depot effect). Key synthesized analogs demonstrated sub-nanomolar affinity for CB1, marked reduction in hydrophobicity (ClogP～2.5–3.5 vs 9.09 of Δ⁸-THC-DMH), and found to function as either agonists (trans-oximes) or neutral antagonists (cis-oximes) in a cAMP functional assay. All oxime analogs showed comparable affinity at the CB2 receptor, but surprisingly they were found to function as inverse agonists for CB2. In behavioral studies (i.e. analgesia, hypothermia) trans-oxime 8a exhibited a predictable fast onset (～20?min) and short duration of pharmacological action (～180?min), in contrast to the very prolonged duration of Δ⁸-THC-DMH (>24?h), thus limiting the potential for severe psychotropic side-effects associated with persistent activation of the CB1 receptor. We have conducted 100?ns molecular dynamic (MD) simulations of CB1 complexes with AM11542 (CB1 agonist) and both trans-8a and cis-8b isomeric oximes. These studies revealed that the C3 alkyl tail of cis-8b orientated within the CB1 binding pocket in a manner that triggered a conformational change that stabilized the CB1 receptor at its inactive-state (antagonistic functional effect). In contrast, the trans-8a isomer’s conformation was coincided with that of the AM11542 CB1 agonist-bound structure, stabilizing the CB1 receptor at the active-state (agonistic functional effect). We have selected oxime trans-8a based on its potency for CB1, and favorable pharmacodynamic profile, such as fast onset and predictable duration of pharmacological action, for evaluation in pre-clinical models of anorexia nervosa.     

Synthesis and cannabinoid-1 receptor binding affinity of conformationally constrained analogs of taranabant

The design, synthesis, and binding activity of ring constrained analogs of the acyclic cannabinoid-1 receptor (CB1R) inverse agonist taranabant 1 are described. The initial inspiration for these taranabant derivatives was its conformation 1a, determined by ¹H NMR, X-ray, and molecular modeling. The constrained analogs were all much less potent than their acyclic parent structure. The results obtained are discussed in the context of a predicted binding of 1 to a homology model of CB1R.     

Interaction of vitamin D analogs with signaling pathways leading to active cell death in breast cancer cells

Induction of apoptosis is a feature of the anti-tumor effects of certain vitamin D analogs. The aim of this study was to identify if common effectors are involved in cell death mediated by serum starvation, vitamin D analogs and tumor necrosis factor (TNF) alpha in 3 human breast cancer cell lines: MCF-7, T47-D and Hs578T. Incubation of cells in serum-free medium induced apoptosis as assessed by loss of cell viability and increased DNA fragmentation. Addition of IGF-I (30 ng/ml) protected against loss of cell viability in MCF-7 cells and co-treatment with two synthetic analogs (CB1093 and EB1089, 50 nM for 4 days) prevented these anti-apoptotic effects of IGF-I. Pretreatment of MCF-7 and Hs578T cells with the vitamin D analogs substantially potentiated the cytotoxic effects of TNFalpha. This cytokine was not cytotoxic for T47-D cells but co-incubation with CB1093 led to loss of cell viability. Potentiation by CB1093 of TNFalpha-induced apoptosis in MCF-7 cells was accompanied by increased activation of cytosolic phospholipase A2 and arachidonic acid release, which was partially inhibited by AACOCF3, a specific cPLA2 inhibitor. The broad-spectrum caspase inhibitor z-VAD-fmk prevented TNFalpha but not CB1093 mediated cell death and activation of cPLA2. Serum starvation induced apoptosis was accompanied by cPLA2 activation, which was inhibited by IGF-I and by z-VAD-fmk. However, the ability of these agents to suppress cPLA2 activation was abrogated by co-treatment with CB1093, suggesting a role for arachidonic acid release in the caspase-independent mechanism by which vitamin D analogs prevent the protective effects of IGF-I on breast cancer cell survival.     

Evaluation of (Z)-2-((1-benzyl-1H-indol-3-yl)methylene)-quinuclidin-3-one analogues as novel,high affinity ligands for CB1 and CB2 cannabinoid receptors

A small library of N-benzyl indolequinuclidinone (IQD) analogs has been identified as a novel class of cannabinoid ligands. The affinity and selectivity of these IQDs for the two established cannabinoid receptor subtypes, CB1 and CB2, was evaluated. Compounds 8 (R = R² = H, R¹ = F) and 13 (R = COOCH₃, R¹ = R² = H) exhibited high affinity for CB2 receptors with K_i values of 1.33 and 2.50 nM, respectively, and had lower affinities for the CB1 receptor (K_i values of 9.23 and 85.7 nM, respectively). Compound 13 had the highest selectivity of all the compounds examined, and represents a potent cannabinoid ligand with 34-times greater selectivity for CB2R over CB1R. These findings are significant for future drug development, given recent reports demonstrating beneficial use of cannabinoid ligands in a wide variety of human disease states including drug abuse, depression, schizophrenia, inflammation, chronic pain, obesity, osteoporosis and cancer.     

Conformationally constrained analogs of BAY 59-3074 as novel cannabinoid receptor ligands

To obtain information on the pharmacophoric requirements of the CB1/CB2 partial agonist BAY 59-3074 we have synthesized a series of new conformationally constrained dibenzofuran (4a-d) and dibenzopyran analogs (5). All constrained analogs exhibited reduced binding affinity at both cannabinoid receptor subtypes, suggesting that planar conformations of these ligands are less favored by both receptors. We also found that 4c, 4d, and 5 exhibited 3- to 12-fold selectivity for hCB2 over rCB1 receptors and may serve as new chemotypes for the development of CB2-selective cannabinergics.     

Folate-dependent hydrolysis of acetyl-coenzyme A by recombinant human and rodent arylamine N-acetyltransferases

Arylamine N-acetyltransferases (NATs) are drug and xenobiotic metabolizing enzymes that catalyze the N-acetylation of arylamines and hydrazines and the O-acetylation of N-hydroxy-arylamines. Recently, studies report that human NAT1 and mouse Nat2 hydrolyze acetyl-coenzyme A (AcCoA) into acetate and coenzyme A in a folate-dependent fashion, a previously unknown function. In this study, our goal was to confirm these findings and determine the apparent Michaelis–Menten kinetic constants (V_max and K_m) of the folate-dependent AcCoA hydrolysis for human NAT1/NAT2, and the rodent analogs rat Nat1/Nat2, mouse Nat1/Nat2, and hamster Nat1/Nat2. We also compared apparent V_max values for AcCoA hydrolysis and N-acetylation of the substrate para-aminobenzoic acid (PABA). Human NAT1 and its rodent analogs rat Nat2, mouse Nat2 and hamster Nat2 catalyzed AcCoA hydrolysis in a folate-dependent manner. Rates of AcCoA hydrolysis were between 0.25–1% of the rates for N-acetylation of PABA catalyzed by human NAT1 and its rodent orthologs. In contrast to human NAT1, human NAT2 and its rodent analogs rat Nat1, mouse Nat1, and hamster Nat1 did not hydrolyze AcCoA in a folate-dependent manner. These results are consistent with the possibility that human NAT1 and its rodent analogs regulate endogenous AcCoA levels.     

Inhibition Effect of a Custom Peptide on Lung Tumors

Cecropin B is a natural antimicrobial peptide and CB1a is a custom, engineered modification of it. In vitro, CB1a can kill lung cancer cells at concentrations that do not kill normal lung cells. Furthermore, in vitro, CB1a can disrupt cancer cells from adhering together to form tumor-like spheroids. Mice were xenografted with human lung cancer cells; CB1a could significantly inhibit the growth of tumors in this in vivo model. Docetaxel is a drug in present clinical use against lung cancers; it can have serious side effects because its toxicity is not sufficiently limited to cancer cells. In our studies in mice: CB1a is more toxic to cancer cells than docetaxel, but dramatically less toxic to healthy cells.     

Chimeric analogs of human β-defensin 1 and θ-defensin disrupt pre-established bacterial biofilms

Antibiofilm activity of several human defensin analogs that have the ability to kill planktonic bacteria, against pre-established biofilms of Escherichia coli MG1655 and Staphylococcus aureus NCTC 8530 were examined. Linear and linear fatty acylated analogs did not show any activity while disulfide constrained analogs disrupted pre-established S. aureus biofilms. Chimeric analogs of human β-defensin 1 and θ-defensin, hBTD-1 and [d]hBTD-1 were highly active against S. aureus biofilms. Among the analogs tested, only the d-enantiomer [d]hBTD-1 showed activity against E. coli biofilm. Our study provides insights into the structural requirements for the eradication of pre-established biofilms in defensin analogs.     

Carbon Black Nanoparticles Promote Endothelial Activation and Lipid Accumulation in Macrophages Independently of Intracellular ROS Production

Exposure to nanoparticles (NPs) may cause vascular effects including endothelial dysfunction and foam cell formation, with oxidative stress and inflammation as supposed central mechanisms. We investigated oxidative stress, endothelial dysfunction and lipid accumulation caused by nano-sized carbon black (CB) exposure in cultured human umbilical vein endothelial cells (HUVECs), THP-1 (monocytes) and THP-1 derived macrophages (THP-1a). The proliferation of HUVECs or co-cultures of HUVECs and THP-1 cells were unaffected by CB exposure, whereas there was increased cytotoxicity, assessed by the LDH and WST-1 assays, especially in THP-1 and THP-1a cells. The CB exposure decreased the glutathione (GSH) content in THP-1 and THP-1a cells, whereas GSH was increased in HUVECs. The reactive oxygen species (ROS) production was increased in all cell types after CB exposure. A reduction of the intracellular GSH concentration by buthionine sulfoximine (BSO) pre-treatment further increased the CB-induced ROS production in THP-1 cells and HUVECs. The expression of adhesion molecules ICAM-1 and VCAM-1, but not adhesion of THP-1 to HUVECs or culture dishes, was elevated by CB exposure, whereas these effects were unaffected by BSO pre-treatment. qRT-PCR showed increased VCAM1 expression, but no change in GCLM and HMOX1 expression in CB-exposed HUVECs. Pre-exposure to CB induced lipid accumulation in THP-1a cells, which was not affected by the presence of the antioxidant N-acetylcysteine. In addition, the concentrations of CB to induce lipid accumulation were lower than the concentrations to promote intracellular ROS production in THP-1a cells. In conclusion, exposure to nano-sized CB induced endothelial dysfunction and foam cell formation, which was not dependent on intracellular ROS production.     

Involvement of the endocannabinoid system in periodontal healing

Endocannabinoids including anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are important lipid mediators for immunosuppressive effects and for appropriate homeostasis via their G-protein-coupled cannabinoid (CB) receptors in mammalian organs and tissues, and may be involved in wound healing in some organs. The physiological roles of endocannabinoids in periodontal healing remain unknown. We observed upregulation of the expression of CB1/CB2 receptors localized on fibroblasts and macrophage-like cells in granulation tissue during wound healing in a wound-healing model in rats, as well as an increase in AEA levels in gingival crevicular fluid after periodontal surgery in human patients with periodontitis. In-vitro, the proliferation of human gingival fibroblasts (HGFs) by AEA was significantly attenuated by AM251 and AM630, which are selective antagonists of CB1 and CB2, respectively. CP55940 (CB1/CB2 agonist) induced phosphorylation of the extracellular-regulated kinases (ERK) 1/2, p38 mitogen-activated protein kinase (p38MAPK), and Akt in HGFs. Wound closure by CP55940 in an in-vitro scratch assay was significantly suppressed by inhibitors of MAP kinase kinase (MEK), p38MAPK, and phosphoinositol 3-kinase (PI3-K). These findings suggest that endocannabinoid system may have an important role in periodontal healing.     

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.     

Synthesis and biological evaluation of several structural analogs of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand

2-Arachidonoylglycerol (2-AG (1)) is an endogenous ligand for the cannabinoid receptors (CB1 and CB2). There is growing evidence that 2-arachidonoylglycerol plays important physiological and pathophysiological roles in various mammalian tissues and cells, though the details remain to be clarified. In this study, we synthesized several remarkable analogs of 2-arachidonoylglycerol, closely related in chemical structure to 2-arachidonoylglycerol: an analog containing an isomer of arachidonic acid with migrated olefins (2-AGA118 (3)), an analog containing a one-carbon shortened fatty acyl moiety (2-AGA113 (4)), an analog containing an one-carbon elongated fatty acyl moiety (2-AGA114 (5)), a hydroxy group-containing analog (2-AGA105 (6)), a ketone group-containing analog (2-AGA109 (7)), and a methylene-linked analog (2-AGA104 (8)). We evaluated their biological activities as cannabinoid receptor agonists using NG108-15 cells which express the CB1 receptor and HL-60 cells which express the CB2 receptor. Notably, these structural analogs of 2-arachidonoylglycerol exhibited only weak agonistic activities toward either the CB1 receptor or the CB2 receptor, which is in good contrast to 2-arachidonoylglycerol which acted as a full agonist at these cannabinoid receptors. These results clearly indicate that the structure of 2-arachidonoylglycerol is strictly recognized by the cannabinoid receptors (CB1 and CB2) and provide further evidence that the cannabinoid receptors are primarily the intrinsic receptors for 2-arachidonoylglycerol.     

Semi-synthetic salinomycin analogs exert cytotoxic activity against human colorectal cancer stem cells

Salinomycin, a polyether antibiotic, is a well-known inhibitor of human cancer stem cells. Chemical modification of the allylic C20 hydroxyl of salinomycin has enabled access to synthetic analogs that display increased cytotoxic activity compared to the native structure. The aim of this study was to investigate the activity of a cohort of C20-O-acyl analogs of salinomycin on human colorectal cancer cell lines in vitro. Two human colorectal cancer cell lines (SW480 and SW620) were exposed to three C20-O-acylated analogs and salinomycin. The impact of salinomycin and its analogs on tumor cell number, migration, cell death, and cancer stem cell specifity was analyzed. Exposure of human colorectal cancer cells to the C20-O-acylated analogs of salinomycin resulted in reduced tumor cell number and impaired tumor cell migration at lower concentrations than salinomycin. When used at higher (micromolar) concentrations, these effects were accompanied by induction of apoptotic cell death. Salinomycin analogs further expose improved activity against cancer stem cells compared to salinomycin.     

Endocannabinoids as cardiovascular modulators

Cannabinoids, the bioactive constituents of the marijuana plant and their synthetic and endogenous analogs cause not only neurobehavioral, but also cardiovascular effects. The most important component of these effects is a profound decrease in blood pressure and heart rate. Although multiple lines of evidence indicate that the hypotensive and bradycardic effects of anandamide and other cannabinoids are mediated by peripherally located CB1 cannabinoid receptors, anandamide can also elicit vasodilation in certain vascular beds, which is independent of CB1 or CB2 receptors. Possible cellular mechanisms underlying these effects and the cellular sources of vasoactive anandamide are discussed.     

New metabolically stable fatty acid amide ligands of cannabinoid receptors: Synthesis and receptor affinity studies

We investigated the structure-activity relationships for the interactions of fatty acid amide analogs of the endocannabinoid anandamide with human recombinant cannabinoid receptors. Thirty-five novel fatty acid amides were synthesized using five different types of acyl chains and 11 different aromatic amine 'heads.' Although none of the new compounds was a more potent ligand than anandamide, we identified three amine groups capable of improving the metabolic stability of arachidonoylamides and their CB(1)/CB(2) selectivity ratio to over 20-fold, and several aromatic amines capable of improving the affinity of short chain or monosaturated fatty acids for cannabinoid CB(1) receptors. For the first time a tertiary amide of arachidonic acid was found to possess moderate affinity (K(i)=300 nM) for cannabinoid CB(1), but not CB(2), receptors.     

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.     

Thioredoxin-mimetic peptides as catalysts of S-denitrosylation and anti-nitrosative stress agents

S-nitrosylation, the coupling of a nitric oxide moiety to a reactive cysteine residue to form an S-nitrosothiol (SNO), is an important posttranslational mechanism for regulating protein activity. Growing evidence indicates that hyper-S-nitrosylation may contribute to cellular dysfunction associated with various human diseases. It is also increasingly appreciated that thioredoxin and thioredoxin reductase play significant roles in the cellular catabolism of SNO and protection from nitrosative stress. Here, we investigated the SNO reductase activity and protective effects of thioredoxin-mimetic peptides (TXMs), Ac-Cys-Pro-Cys-amide (CB3) and Ac-Cys-Gly-Pro-Cys-amide (CB4), both under cell-free conditions and in nitrosatively stressed cultured cells. In vitro biochemical analyses revealed that the TXM peptides reduced small-molecule SNO compounds, such as S-nitrosoglutathione (GSNO), and acted as general and efficient protein-denitrosylating agents. In particular, CB3 was found to be a highly potent SNO-metabolizing agent. Notably, CB3 mimicked the activity of thioredoxin by coupling with thioredoxin reductase to enhance GSNO reduction. Moreover, in a cell-free lysate system, both CB3 and CB4 synergized with an NADPH-dependent activity to denitrosylate proteins. Further investigation revealed that the TXM peptides protect the peroxiredoxin–thioredoxin system from SNO-dependent inhibition. Indeed, SNO-inhibited Prx1 was efficiently denitrosylated and reactivated by CB3 or CB4. In addition, CB3 protected thioredoxin reductase from SNO-mediated inactivation both in vitro and in intact cells. Finally, CB3 and CB4 partially rescued human neuroblastoma SH-SY5Y cells and rat insulinoma INS-1 832/13 cells from GSNO-induced growth inhibition. Collectively, the present findings indicate the efficient denitrosylation activity and protective effects of TXM peptides and suggest their potential therapeutic value in treating pathological conditions related to nitrosative stress.     

Design, synthesis, and structure-activity relationship study of bicyclic piperazine analogs of indole-3-carboxamides as novel cannabinoid CB1 receptor agonists

Bicyclic piperazine derivatives were synthesized as conformationally constrained analogs of N-alkyl piperazines and were found to be potent CB1 receptor agonists. The CB1 receptor agonist activity was dependent upon the absolute configuration of the chiral center of the bicyclic ring system. Although the conformational constraint did not protect the compounds from metabolism by N-dealkylation, several bicyclic analogs were found to be more potent than the unconstrained lead compound. Compound 8b demonstrated potent antinociceptive activity in vivo.