3,4-Disubstituted indole acylsulfonamides: A novel series of potent and selective human EP3 receptor antagonists

A series of potent and selective EP₃ receptor antagonists are described. Utilizing a pharmacophore model developed for the EP₃ receptor, a series of 3,4-disubstituted indoles were shown to be high affinity ligands for this target. These compounds showed high selectivity over IP, FP and other EP receptors and are potent antagonists in functional assays.     

Discovery of novel aminothiadiazole amides as selective EP3 receptor antagonists

This Letter discloses a series of 2-aminothiadiazole amides as selective EP₃ receptor antagonists. SAR optimization resulted in compounds with excellent functional activity in vitro. In addition, efforts to optimize DMPK properties in the rat are discussed. These efforts have resulted in the identification of potent, selective EP₃ receptor antagonists with excellent DMPK properties suitable for in vivo studies.     

1,7-Disubstituted oxyindoles are potent and selective EP3 receptor antagonists

A series of novel 1,7-disubstituted oxyindoles were shown to be potent and selective EP₃ receptor antagonists. Variation of substitution pattern at the C-3 position of indole enhanced in vitro metabolic stability of the resulting derivatives. Series 27a–c showed >1000-fold selectivity over a panel of prostanoid receptors including IP, FP, EP₁, EP₂ and EP₄. These agents also featured low CYP inhibition and good activity in the functional rat platelet aggregation assay.     

3-Acrylamide-4-aryloxyindoles: Synthesis,biological evaluation and metabolic stability of potent and selective EP3 receptor antagonists

A series of potent and selective EP₃ receptor antagonists are described. Utilizing a pharmacophore model developed for the EP₃ receptor, a series of 3,4-disubstituted indoles were found to be efficient ligands for this target. These compounds showed high selectivity over IP, FP and other EP receptors. An optimized molecule 7c featured a sound profile and potency in the functional rat and human platelet aggregation assays.     

Hit-to-lead optimization of 2-(1H-pyrazol-1-yl)-thiazole derivatives as a novel class of EP1 receptor antagonists

We describe a medicinal chemistry approach to generate a series of 2-(1H-pyrazol-1-yl)thiazole compounds that act as selective EP₁ receptor antagonists. The obtained results suggest that compound 12 provides the best EP₁ receptor antagonist activity and demonstrates good oral pharmacokinetics.     

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.     

Prostanoid Receptors Involved in Regulation of the Beating Rate of Neonatal Rat Cardiomyocytes

Although prostanoids are known to be involved in regulation of the spontaneous beating rate of cultured neonatal rat cardiomyocytes, the various subtypes of prostanoid receptors have not been investigated in detail. In our experiments, prostaglandin (PG)F_2α and prostanoid FP receptor agonists (fluprostenol, latanoprost and cloprostenol) produced a decrease in the beating rate. Two prostanoid IP receptor agonists (iloprost and beraprost) induced first a marked drop in the beating rate and then definitive abrogation of beating. In contrast, the prostanoid DP receptor agonists (PGD₂ and BW245C) and TP receptor agonists (U-46619) produced increases in the beating rate. Sulprostone (a prostanoid EP₁ and EP₃ receptor agonist) induced marked increases in the beating rate, which were suppressed by SC-19220 (a selective prostanoid EP₁ antagonist). Butaprost (a selective prostanoid EP₂ receptor agonist), misoprostol (a prostanoid EP₂ and EP₃ receptor agonist), 11-deoxy-PGE₁ (a prostanoid EP₂, EP₃ and EP₄ receptor agonist) did not alter the beating rate. Our results strongly suggest that prostanoid EP₁ receptors are involved in positive regulation of the beating rate. Prostanoid EP₁ receptor expression was confirmed by western blotting with a selective antibody. Hence, neonatal rat cardiomyocytes express both prostanoid IP and FP receptors (which negatively regulate the spontaneous beating rate) and prostanoid TP, DP₁ and EP₁ receptors (which positively regulate the spontaneous beating rate).     

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.     

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.     

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.     

Peri-substituted hexahydro-indolones as novel,potent and selective human EP3 receptor antagonists

A series of peri-substituted [4.3.0] bicyclic non-aromatic heterocycles have been identified as potent and selective hEP₃ receptor antagonists. These molecules adopt a hair-pin conformation that overlaps with the endogenous ligand PGE₂ and fits into an internally generated EP₃ pharmacophore model. Optimized compounds show good metabolic stability and improved solubility over their corresponding bicyclic aromatic analogs.     

Effects of selective PGE2 receptor antagonists in esophageal adenocarcinoma cells derived from Barrett's esophagus

Accumulating evidence suggests that COX-2-derived prostaglandin E₂ (PGE₂) plays an important role in esophageal adenocarcinogenesis. Recently, PGE₂ receptors (EP) have been shown to be involved in colon cancer development. Since it is not known which receptors regulate PGE₂ signals in esophageal adenocarcinoma, we investigated the role of EP receptors using a human Barrett's-derived esophageal adenocarcinoma cell line (OE33). OE33 cells expressed COX-1, COX-2, EP₁, EP₂ and EP₄ but not EP₃ receptors as determined by real time RT-PCR and Western-blot. Treatment with 5-aza-dC restored expression, suggesting that hypermethylation is involved in EP₃ downregulation. Endogenous PGE₂ production was mainly due to COX-2, since this was significantly suppressed with COX-2 inhibitors (NS-398 and SC-58125), but not COX-1 inhibitors (SC-560). Cell proliferation (³H-thymidine uptake) was significantly inhibited by NS-398 and SC-58125, the EP₁ antagonist SC-51322, AH6809 (EP₁/EP₂ antagonist), and the EP₄ antagonist AH23848B, but was not affected by exogenous PGE₂. However, treatment with the selective EP₂ agonist Butaprost or 16,16-dimethylPGE₂ significantly inhibited butyrate-induced apoptosis and stimulated OE33 cell migration. The effect of exogenous PGE₂ on migration was attenuated when cells were first treated with EP₁ and EP₄ antagonists. These findings suggest a potential role for EP selective antagonists in the treatment of esophageal adenocarcinoma.     

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.     

Nature of the slow relaxation of smooth muscle induced by a EP2 receptor agonist with a non-prostanoid structure

The remarkably slow onset/offset of relaxation of guinea-pig isolated trachea induced by a ‘non-prostanoid’ EP₂ receptor agonist, (o-(o-benzyloxy)-cinnamyl)-cinnamic acid (coded (L)-9), was investigated. (L)-9 kinetics was slightly faster on mouse trachea and considerably faster on rabbit vena cava. In each case, reversal of (L)-9 relaxation by the selective EP₂ antagonist ACA-23 was rapid and similar to other EP₂ agonists (e.g. ONO-AE1-259). On guinea-pig aorta, in the presence of extensive EP₂ receptor blockade, (L)-9 inhibited TP agonist-induced contraction more slowly than TP antagonists of similar affinity. The slower kinetics of (L)-9 appear to correlate with greater adventitial/submucosal barriers and thicker smooth muscle layers in the tissues examined. It is proposed that interactions of (L)-9 with EP₂ and TP receptors are not rate-limiting, rather diffusion to and from the centre of the muscle mass is retarded by the high lipophilicity of (L)-9 (logP=6.69; ONO-AE1-259=3.95).     

Investigation of the pronounced synergism between prostaglandin E2 and other constrictor agents on rat femoral artery

This study investigates the pronounced synergism between the weak contractile action of prostaglandin E₂ (PGE₂) and strong actions of phenylephrine, U-46619 and K⁺ on rat isolated femoral artery. The potency ranking for synergism was SC-46275 (prostanoid receptor agonist selectivity: EP₃EP₁)=sulprostone (EP₃>EP₁)>17-phenyl PGE₂ (EP₁>EP₃). The novel EP₃ antagonist L-798106 (0.2–1 μM) blocked the enhanced action of sulprostone (pA₂=7.35–8.10), while the EP₁ antagonist SC-51322 (1 μM) did not (pA₂<6.0). Matching responses to priming agent and priming agent/sulprostone were similarly suppressed by nifedipine (300 nM) and the selective Rho-kinase inhibitors H-1152 (0.1–1 μM) and Y-27632 (1–10 μM). Our findings implicate an EP₃ receptor in the prostanoid component of contractile synergism. While the synergism predominantly operates through a Ca²⁺ influx–Rho-kinase pathway, the EP₃ receptor does not necessarily transduce via Rho-kinase.     

Characterization and ontogeny of PGE2 and PGF2α receptors on the retinal vasculature of the pig

The vasoconstrictor effects of PGE₂ and PGF_2α are less pronounced on retinal vessels of the newborn than of the adult pig. We tested the hypothesis that the decreased vasomotor response to these prostaglandins might be due to relatively fewer receptors and/or different receptor subtypes (in the case of PGE₂) on retinal vessels of the newborn animal. Binding studies using [³H]PGE₂ and [³H]PGF_2α revealed that PGE₂ (EP) and PGF_2α (FP) receptor densities in retinal microvessel membrane preparations from newborn animals were approximately 25% of those found in vessels from the adult. The K_d for PGF_2α did not differ; however, the K_d for PGE₂ was less in newborn than in adult vessels. Competition binding studies using AH 6809 (EP₁ antagonist), butaprost (EP₂ agonist), M&B 28,767 (EP₃ agonist), and AH 23848B (EP₄ antagonist) suggested that the retinal vessels of the newborn contained approximately equal number of EP₁ and EP₂ receptor subtypes whereas the main receptor subtype in the adult vessels was EP₁. In addition, PGE₂ and butaprost produced comparable increases in adenosine 3′,5′-cyclic monophosphate synthesis in newborn and adult vessels. PGE₂, 17-phenyl trinor PGE₂ (EP₁agonist) and PGF_2α caused a 2.5 to 3-fold greater increase in inositol1,4,5-triphosphate (IP₃) formation in adult than in newborn preparations. It is concluded that fewer PGF_2α receptors and an associated decrease in receptor-coupled IP₃ formation in the retinal vessels of the newborn could lead to weaker vasoconstrictor effects of PGF_2α on retinal vessels of the newborn than of adult pigs; fewer EP₁ receptors (associated with vasoconstriction) and a relatively greater proportion of EP₂ receptors (associated with vasodilation) might be responsible for the reduced retinal vasoconstrictor effects of PGE₂ in the newborn.     

Novel pyridone EP4 agonists featuring allylic alcohol ω-chains

Novel prostaglandin E2 receptor 4 (EP₄) agonists featuring a pyridone core and an allylic alcohol ω-chain were discovered. These agonists were shown to be selective over EP₁, EP₂ and EP₃. Analogs harboring a 4-carboxylic acid phenethyl α-chain displayed improved potency over those containing an n-heptanoic acid chain. Key SAR relationships were also identified.     

Upregulation of the S1P3 receptor in metastatic breast cancer cells increases migration and invasion by induction of PGE2 and EP2/EP4 activation

Breast cancer is one of the most common and devastating malignancies among women worldwide. Recent evidence suggests that malignant progression is also driven by processes involving the sphingolipid molecule sphingosine 1-phosphate (S1P) and its binding to cognate receptor subtypes on the cell surface. To investigate the effect of this interaction on the metastatic phenotype, we used the breast cancer cell line MDA-MB-231 and the sublines 4175 and 1833 derived from lung and bone metastases in nude mice, respectively. In both metastatic cell lines expression of the S1P₃ receptor was strongly upregulated compared to the parental cells and correlated with higher S1P-induced intracellular calcium ([Ca^2 +]_i), higher cyclooxygenase (COX)-2 and microsomal prostaglandin (PG) E₂ synthase expression, and consequently with increased PGE₂ synthesis. PGE₂ synthesis was decreased by antagonists and siRNA against S1P₃ and S1P₂. Moreover, in parental MDA-MB-231 cells overexpression of S1P₃ by cDNA transfection also increased PGE₂ synthesis, but only after treatment with the DNA methyltransferase inhibitor 5-aza-2-deoxycytidine, indicating reversible silencing of the COX-2 promoter. Functionally, the metastatic sublines showed enhanced migration and Matrigel invasion in adapted Boyden chamber assays, which further increased by S1P stimulation. This response was abrogated by either S1P₃ antagonism, COX-2 inhibition or PGE₂ receptor 2 (EP₂) and 4 (EP₄) antagonism, but not by S1P₂ antagonism. Our data demonstrate that in breast cancer cells overexpression of S1P₃ and its activation by S1P has pro-inflammatory and pro-metastatic potential by inducing COX-2 expression and PGE₂ signaling via EP₂ and EP₄.