The identification of substituted benzothiophene derivatives as PGE(2) subtype 4 receptor antagonists: From acid to non-acid

We disclose herein our preliminary SAR study on the identification of substituted benzothiophene derivatives as PGE₂ subtype 4 receptor antagonists. A potent EP₄ antagonist 6a (K_i = 1.4 nM with 10% HSA) was identified. Furthermore, we found that an acidic group was not essential for the EP₄ antagonizing activity in the series and neutral replacements were identified. This opens a new direction for future EP₄ antagonist design.     

Discovery of sodium 6-[(5-chloro-2-{[(4-chloro-2-fluorophenyl)methyl]oxy}phenyl)methyl]-2-pyridinecarboxylate (GSK269984A) an EP1 receptor antagonist for the treatment of inflammatory pain

We describe the medicinal chemistry programme that led to the identification of the EP₁ receptor antagonist GSK269984A (8h). GSK269984A was designed to overcome development issues encountered with previous EP₁ antagonists such as GW848687X and was found to display excellent activity in preclinical models of inflammatory pain. However, upon cross species pharmacokinetic profiling, GSK269984A was predicted to have suboptimal human pharmacokinetic and was thus progressed to a human microdose study.     

Discovery of a novel series of nonacidic benzofuran EP1 receptor antagonists

We describe the discovery and optimization of a novel series of benzofuran EP₁ antagonists, leading to the identification of 26d, a novel nonacidic EP₁ antagonist which demonstrated efficacy in preclinical models of chronic inflammatory pain.     

The EP1 subtype of prostaglandin E2 receptor: Role in keratinocyte differentiation and expression in non-melanoma skin cancer

We have previously demonstrated that the EP₁ subtype of PGE₂ receptor is expressed in the differentiated compartment of normal human epidermis and is coupled to intracellular calcium mobilization. We therefore hypothesized that the EP₁ receptor is coupled to keratinocyte differentiation. In in vitro studies, radioligand binding, RT-PCR, immunoblot and receptor agonist-induced second messenger studies demonstrate that the EP₁ receptor is up-regulated by high cell density in human keratinocytes and this up-regulation precedes corneocyte formation. Moreover, two different EP₁ receptor antagonists, SC51322 and AH6809, both inhibited corneocyte formation. SC51322 also inhibited the induction of differentiation-specific proteins, cytokeratin K10 and epidermal transglutaminase. We next examined the immunolocalization of the EP₁ receptor in non-melanoma skin cancer in humans. Well-differentiated SCCs exhibited significantly greater membrane staining, while spindle cell carcinomas and BCCs had significantly decreased membrane staining compared with normal epidermis. This data supports a role for the EP₁ receptor in regulating keratinocyte differentiation.     

Prostaglandin E2 inhibits the proliferation of human gingival fibroblasts via the EP2 receptor and Epac

Elevated levels of prostaglandins such as PGE₂ in inflamed gingiva play a significant role in the tissue destruction caused by periodontitis, partly by targeting local fibroblasts. Only very few studies have shown that PGE₂ inhibits the proliferation of a gingival fibroblast (GF) cell line, and we expanded this research by using primary human GFs (hGFs) and looking into the mechanisms of the PGE₂ effect. GFs derived from healthy human gingiva were treated with PGE₂ and proliferation was assessed by measuring cell number and DNA synthesis and potential signaling pathways were investigated using selective activators or inhibitors. PGE₂ inhibited the proliferation of hGFs dose‐dependently. The effect was mimicked by forskolin (adenylate cyclase stimulator) and augmented by IBMX (a cAMP‐breakdown inhibitor), pointing to involvement of cAMP. Indeed, PGE₂ and forskolin induced cAMP generation in these cells. Using selective EP receptor agonists we found that the anti‐proliferative effect of PGE₂ is mediated via the EP₂ receptor (which is coupled to adenylate cyclase activation). We also found that the effect of PGE₂ involved activation of Epac (exchange protein directly activated by cAMP), an intracellular cAMP sensor, and not PKA. While serum increased the amount of phospho‐ERK in hGFs by ～300%, PGE₂ decreased it by ～50%. Finally, the PGE₂ effect does not require endogenous production of prostaglandins since it was not abrogated by two COX‐inhibitors. In conclusion, in human gingival fibroblasts PGE₂ activates the EP₂—cAMP—Epac pathway, reducing ERK phosphorylation and inhibiting proliferation. This effect could hamper periodontal healing and provide further insights into the pathogenesis of inflammatory periodontal disease. J. Cell. Biochem. 108: 207–215, 2009. © 2009 Wiley‐Liss, Inc.     

EP3 prostanoid receptor isoforms utilize distinct mechanisms to regulate ERK 1/2 activation

Prostaglandin-E₂ (PGE₂) is a hormone derived from the metabolism of arachidonic acid whose functions include regulation of platelet aggregation, fever and smooth muscle contraction/relaxation. PGE₂ mediates its physiological and pathophysiological effects through its binding to four G-protein coupled receptor subtypes, named EP₁, EP₂, EP₃ and EP₄. The EP₃ prostanoid receptor is unique in that it has multiple isoforms generated by alternative mRNA splicing. These splice variants display differences in tissue expression, constitutive activity and regulation of signaling molecules. To date there are few reports identifying differential activities of EP₃ receptor isoforms and their effects on gene regulation. We generated HEK cell lines expressing the human EP_3-Ia, EP_3-II or EP_3-III isoforms. Using immunoblot analysis we found that nM concentrations of PGE₂ strongly stimulated the phosphorylation of ERK 1/2 by the EP_3-II and EP_3-III isoforms; whereas, ERK 1/2 phosphorylation by the EP_3-Ia isoform was minimal and only occurred at μM concentrations of PGE₂. Furthermore, the mechanisms of the PGE₂ mediated phosphorylation of ERK 1/2 by the EP_3-II and EP_3-III isoforms were different. Thus, PGE₂ stimulation of ERK 1/2 phosphorylation by the EP_3-III isoform involves activation of a Gα_i/PI3K/PKC/Src and EGFR-dependent pathway; while for the EP_3-II isoform it involves activation of a Gα_i/Src and EGFR-dependent pathway. These differences result in unique differences in the regulation of reporter plasmid activity for the downstream effectors ELK1 and AP-1 by the EP_3-II and EP_3-III prostanoid receptor isoforms.     

Discovery of a novel 2-(1H-pyrazolo[3,4-b]pyridin-1-yl)thiazole derivative as an EP1 receptor antagonist and in vivo studies in a bone fracture model

We describe a medicinal chemistry approach to the discovery of a novel EP₁ antagonist exhibiting high potency and good pharmacokinetics. Our starting point is 1, an EP₁ receptor antagonist that exhibits pharmacological efficacy in cystometry models following intravenous administration. Despite its good potency in vitro, the high lipophilicity of 1 is a concern in long-term in vivo studies. Further medicinal chemistry efforts identified 4 as an improved lead compound with good in vitro ADME profile applicable to long term in vivo studies. A rat fracture study was conducted with 4 for 4?weeks to validate its utility in bone fracture healing. The results suggest that this EP₁ receptor antagonist stimulates callus formation and thus 4 has potential for enhancing fracture healing.     

Naphthalene/quinoline amides and sulfonylureas as potent and selective antagonists of the EP4 receptor

Two new series of EP₄ antagonists based on naphthalene/quinoline scaffolds have been identified as part of our on-going efforts to develop treatments for inflammatory pain. One series contains an acidic sulfonylurea pharmacophore, whereas the other is a neutral amide. Both series show subnanomolar intrinsic binding potency towards the EP₄ receptor, and excellent selectivity towards other prostanoid receptors. While the amide series generally displays poor pharmacokinetic parameters, the sulfonylureas exhibit greatly improved profile. MF-592, the optimal compound from the sulfonylurea series, has a desirable overall preclinical profile that suggests it is suitable for further development.     

Discovery of 4-{1-[({1-[4-(trifluoromethyl)benzyl]-1H-indol-7-yl}carbonyl)amino]cyclopropyl}benzoic acid (MF-766), a highly potent and selective EP4 antagonist for treating inflammatory pain

The discovery of a highly potent and selective EP₄ antagonist MF-766 is discussed. This N-benzyl indole derivative exhibits good pharmacokinetic profile and unprecedented in vivo potency in the rat AIA model.     

SAR-based optimization of 2-(1H-pyrazol-1-yl)-thiazole derivatives as highly potent EP1 receptor antagonists

We describe a medicinal chemistry approach for generating a series of 2-(1H-pyrazol-1-yl)thiazoles as EP₁ receptor antagonists. To improve the physicochemical properties of compound 1, we investigated its structure–activity relationships (SAR). Optimization of this lead compound provided small compound 25 which exhibited the best EP₁ receptor antagonist activity and a good SAR profile.     

Prostaglandin E2 facilitates subcellular translocation of the EP4 receptor in neuroectodermal NE-4C stem cells

Prostaglandin E2 (PGE₂) is a lipid mediator released from the phospholipid membranes that mediates important physiological functions in the nervous system via activation of four EP receptors (EP1-4). There is growing evidence for the important role of the PGE₂/EP4 signaling in the nervous system. Previous studies in our lab show that the expression of the EP4 receptor is significantly higher during the neurogenesis period in the mouse. We also showed that in mouse neuroblastoma cells, the PGE₂/EP4 receptor signaling pathway plays a role in regulation of intracellular calcium via a phosphoinositide 3-kinase (PI3K)-dependent mechanism. Recent research indicates that the functional importance of the EP4 receptor depends on its subcellular localization. PGE₂-induced EP4 externalization to the plasma membrane of primary sensory neurons has been shown to play a role in the pain pathway. In the present study, we detected a novel PGE₂–dependent subcellular trafficking of the EP4 receptor in neuroectodermal (NE-4C) stem cells and differentiated NE-4C neuronal cells. We show that PGE₂ induces EP4 externalization from the Golgi apparatus to the plasma membrane in NE-4C stem cells. We also show that the EP4 receptors translocate to growth cones of differentiating NE-4C neuronal cells and that a higher level of PGE₂ enhances its growth cone localization. These results demonstrate that the EP4 receptor relocation to the plasma membrane and growth cones in NE-4C cells is PGE₂ dependent. Thus, the functional role of the PGE₂/EP4 pathway in the developing nervous system may depend on the subcellular localization of the EP4 receptor.     

The effect of estradiol on COX-2, EP2, and EP4 mRNA expression and the extracellular matrix in the cervix of the hypogonadotrophic, ovariectomized ewe

There is a degree of cervical relaxation in the ewe at estrus that is regulated by changes in prostaglandin synthesis, prostaglandin receptor expression, and changes in the cervical extracellular matrix. It is likely that these are regulated by changes in periovulatory hormones, particularly estradiol. This study determined the effect of estradiol benzoate on the mRNA expression of cyclooxygenase-2 (COX-2) and the prostaglandin E receptors EP₂ and EP₄, the concentration of cervical hyaluronan, and the proportion of smooth muscle and collagen in the cervix of the hypogonadotrophic ovariectomized ewe (Ovis aries). Ovariectomized hypogonadotrophic ewes were given 100 μg estradiol benzoate, and their cervices were collected 0, 24, and 48 h thereafter to determine the expression of cervical COX-2, EP₂, and EP₄ mRNA by in situ hybridization, the concentration of hyaluronan by ELISA, and the proportion of smooth muscle and collagen by Masson's trichrome staining. Estradiol benzoate increased the mRNA expression of COX-2 and EP₄ within 24 h after treatment (P < 0.05), whereas EP₂ mRNA, hyaluronan, and the ratio of smooth muscle to collagen did not change within 48 h after treatment. The COX-2, EP₂, and EP₄ mRNA expression were greatest in the smooth muscle layers (P < 0.05) and least in the luminal epithelium (P < 0.05). In conclusion, we inferred that estradiol regulates cervical COX-2 and EP₄ mRNA expression and may regulate cervical relaxation via the synthesis of prostaglandin E₂ and activation of the PGE₂ receptors EP₂ and EP₄.     

Lubiprostone activates CFTR, but not ClC-2, via the prostaglandin receptor (EP4)

The goal of this study was to determine the mechanism of lubiprostone activation of epithelial chloride transport. Lubiprostone is a bicyclic fatty acid approved for the treatment of constipation [1]. There is uncertainty, however, as to how lubiprostone increases epithelial chloride transport. Direct stimulation of ClC-2 and CFTR chloride channels as well as stimulation of these channels via the EP₄ receptor has been described [2], [3], [4] and [5]. To better define this mechanism, two-electrode voltage clamp was used to assay Xenopus oocytes expressing ClC-2, with or without co-expression of the EP₄ receptor or β adrenergic receptor (βAR), for changes in conductance elicited by lubiprostone. Oocytes co-expressing CFTR and either βAR or the EP₄ receptor were also studied. In oocytes co-expressing ClC-2 and βAR conductance was stimulated by hyperpolarization and acidic pH (pH = 6), but there was no response to the β adrenergic agonist, isoproterenol. Oocytes expressing ClC-2 only or co-expressing ClC-2 and EP₄ did not respond to the presence of 0.1, 1, or 10 μM lubiprostone in the superperfusate. Oocytes co-expressing CFTR and βAR did not respond to hyperpolarization, acidic pH, or 1 μM lubiprostone. However, conductance was elevated by isoproterenol and inhibited by CFTR_inh172. Co-expression of CFTR and EP₄ resulted in lubiprostone-stimulated conductance, which was also sensitive to CFTR_inh172. The EC₅₀ for lubiprostone mediated CFTR activation was ∼10 nM. These results demonstrate no direct action of lubiprostone on either ClC-2 or CFTR channels expressed in oocytes. However, the results confirm that CFTR can be activated by lubiprostone via the EP₄ receptor in oocytes.     

Synthesis and preliminary screening of novel N-{2-[4-(substituted)piperazin-1-yl]-2-oxoethyl}acetamides as potential atypical antipsychotic agents

A series of N-{2-[4-(substituted)piperazin-1-yl]-2-oxoethyl}acetamides were synthesized as prospective novel atypical antipsychotic agents. Microwave irradiation of acetyl glycine (I) with substituted piperazines in the presence of DCC in DMF for about 3-5 min gave the titled compounds (P:1-7). All the synthesized compounds were screened for their in vivo pharmacological activity in Swiss albino mice. D₂ antagonism studies were performed using the climbing mouse assay model and 5-HT_2A antagonism studies were performed using quipazine induced head twitches in mice. Among the synthesized compounds P4 was found to be the most active compound.     

Synthesis 4-[2-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamides with subnanomolar carbonic anhydrase II and XII inhibitory properties

Condensation of substituted anthranilic acids with 4-isothiocyanatoethyl-benzenesulfonamide led to series of heterocyclic benzenesulfonamides incorporating 2-mercapto-quinazolin-4-one tails. These sulfonamides were investigated as inhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I and II (cytosolic isozymes), as well as hCA XII (a transmembrane, tumor-associated enzyme also involved in glaucoma-genesis). The new sulfonamides acted as medium potency inhibitors of hCA I (K_Is of 28.5–2954 nM), being highly effective as hCA II (K_Is in the range of 0.62–12.4 nM) and XII (K_Is of 0.54–7.11 nM) inhibitors. All substitution patterns present in these compounds (e.g., halogens, methyl and methoxy moieties, in positions 6, 7 and/or 8 of the 2-mercapto-quinazolin-4-one ring) led to highly effective hCA II/XII inhibitors. These compounds should thus be of interest as preclinical candidates in pathologies in which the activity of these enzymes should be inhibited, such as glaucoma (CA II and XII as targets) or some tumors in which the activity of isoforms CA II and XII is dysregulated.     

Activation of prostaglandin E2-EP4 signaling reduces chemokine production in adipose tissue

Inflammation of adipose tissue induces metabolic derangements associated with obesity. Thus, determining ways to control or inhibit inflammation in adipose tissue is of clinical interest. The present study tested the hypothesis that in mouse adipose tissue, endogenous prostaglandin E₂ (PGE₂) negatively regulates inflammation via activation of prostaglandin E receptor 4 (EP4). PGE₂ (5–500nM) attenuated lipopolysaccharide-induced mRNA and protein expression of chemokines, including interferon-γ-inducible protein 10 and macrophage-inflammatory protein-1α in mouse adipose tissue. A selective EP4 antagonist (L161,982) reversed, and two structurally different selective EP4 agonists [CAY10580 and CAY10598] mimicked these actions of PGE₂. Adipose tissue derived from EP4-deficient mice did not display this response. These findings establish the involvement of EP4 receptors in this anti-inflammatory response. Experiments performed on adipose tissue from high-fat-fed mice demonstrated EP4-dependent attenuation of chemokine production during diet-induced obesity. The anti-inflammatory actions of EP4 became more important on a high-fat diet, in that EP4 activation suppressed a greater variety of chemokines. Furthermore, adipose tissue and systemic inflammation was enhanced in high-fat-fed EP4-deficient mice compared with wild-type littermates, and in high-fat-fed untreated C57BL/6 mice compared with mice treated with EP4 agonist. These findings provide in vivo evidence that PGE₂-EP4 signaling limits inflammation. In conclusion, PGE₂, via activation of EP4 receptors, functions as an endogenous anti-inflammatory mediator in mouse adipose tissue, and targeting EP4 may mitigate adipose tissue inflammation.     

Reactions of hybrid organotellurium ligands 1-(4-methoxyphenyl telluro)-2-[3-(6-methyl-2-pyridyl)propoxy]ethane (L) and 1-ethylthio-2-[2-thienyltelluro]ethane (L) with mercury (II) bromide: formation of complexes and their decomposition

Two tellurium ligands 1-(4-methoxyphenyltelluro)-2-[3-(6-methyl-2-pyridyl)propoxy]ethane (L¹) and 1-ethylthio-2-[2-thienyltelluro]ethane (L²) have been synthesized by reacting nucleophiles [4-MeO-C₆H₄Te⁻] and [C₄H₃S-2-Te⁻] with 2-[3-(6-methyl-2-pyridyl)propoxy]ethylchloride and chloroethyl ethyl sulfide, respectively. Both the ligands react with HgBr₂ resulting in complexes of stoichiometry [HgBr₂ · L¹/L²] (1/4), which show characteristic NMR (¹H and ¹³C{¹H}). On crystallization of 1 from acetone-hexane (2:1) mixture, the cleavage of L¹ occurs resulting in 4-MeOC₆H₄HgBr (2) and [RTe⁺→HgBr₂]Br⁻ (3) (where R = -CH₂CH₂OCH₂CH₂CH₂-(2-(6-CH₃-C₅H₃N))). The 2 is characterized by X-ray diffraction on its single crystal. It is a linear molecule and is the first such system which is fully characterized structurally. The Hg-C and Hg-Br bond lengths are 2.085(6) and2.4700(7) Å. The distance of four bromine atoms (3.4041(7)-3.546(7) Å) around Hg (cis to C) is greater than the sum of van der Waal’s radii 3.30 Å. This mercury promoted cleavage is observed for an acyclic ligand of RArTe type for the first time and is unique, as there appears to be no strong intramolecular interaction to stabilize the cleavage products. The 4 on crystallization shows the cleavage of organotellurium ligand L² and formation of a unique complex [(EtS(CH₂)₂SEt)HgBr(μ-Br)Hg(Br)(μ-Br)₂Hg(Br)(μ-Br)BrHg(EtS(CH₂)₂SEt)] · 2HgBr₂ (5), which has been characterized by single crystal structure determination and ¹H and ¹³C{¹H} NMR spectra. The elemental tellurium and [C₄H₃SCH₂]₂ are the other products of dissociation as identified by NMR (proton and carbon-13). The cleavage appears to be without any transmetalation and probably first of its kind. The centrosymmetric structure of 5 is unique as it has [HgBr₃]⁻ unit, one Hg in distorted tetrahedral geometry and one in pseudo-trigonal bipyramidal one. The molecule of 5 may also be described as having [(EtSCH₂CH₂SEt)HgBr]⁺ [HgBr₃]⁻ units, which dimerize and co-crystallize with two HgBr₂ moieties. There are very weak Hg?Br interactions between co-crystallized HgBr₂ units and rest of the molecule. [Hg(3)-Br(1)/Hg(3)-Br(4) = 3.148(1)/3.216(1) Å]. The bridging Hg?Br distances, Hg(2)-Br(4)′, Hg(2)′-Br(4) and Hg(1)-Br(2), are from 2.914(1) to 3.008(1) Å.     

Synthesis and in vitro anti-HIV activity of N-1,3-benzo[d]thiazol-2-yl-2-(2-oxo-2H-chromen-4-yl)acetamide derivatives using MTT method

A series of novel N-1,3-benzo[d]thiazol-2-yl-2-(2-oxo-2H-chromen-4-yl)acetamide derivatives has been synthesized. All the newly synthesized compounds were evaluated for their anti-HIV activity using MTT method. Most of these compounds showed moderate to potent activity against wild-type HIV-1 with an EC₅₀ ranging from >7 EC₅₀ [μg/ml] to <100 EC₅₀ [μg/ml]. Among them, N-1,3-benzo[d]thiazol-2-yl-2-(2-oxo-2H-chromen-4-yl)acetamide 6v was identified as the most promising compound (EC₅₀ = <7 μg/ml). Among all the compounds, three compounds 6m, 6v and 6u have been exhibits potent anti-HIV activity against MT-4 cells.     

Design and synthesis of novel 3-(benzo[d]oxazol-2-yl)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridin-2-amine derivatives as selective G-protein-coupled receptor kinase-2 and -5 inhibitors

G-protein-coupled receptor kinase (GRK)-2 and -5 are emerging therapeutic targets for the treatment of cardiovascular disease. In our efforts to discover novel small molecules to inhibit GRK-2 and -5, a class of compound based on 3-(benzo[d]oxazol-2-yl)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridin-2-amine was identified as a novel hit by high throughput screening campaign. Structural modification of parent benzoxazole scaffolds by introducing substituents on phenyl displayed potent inhibitory activities toward GRK-2 and -5.