GW627368X ((N-{2-[4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetyl} benzene sulphonamide): a novel, potent and selective prostanoid EP4 receptor antagonist.

1. N-{2-[4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetyl}benzene sulphonamide (GW627368X) is a novel, potent and selective competitive antagonist of prostanoid EP4 receptors with additional human TP receptor affinity. 2. At recombinant human prostanoid EP4 receptors expressed in HEK293 cells, GW627368X produced parallel rightward shifts of PGE2 concentration-effect (E/[A]) curves resulting in an affinity (pKb) estimate of 7.9 +/- 0.4 and a Schild slpoe not significantly different from unity. The affinity was independent of the agonist used. 4. In rings of phenylephrine precontracted piglet saphenous vein, GW627368X (30-300 nM) produced parallel rightward displacement of PGE2 E/[A] curves (pKb = 9.2 +/- 0.2; slope = 1). 4. GW627368X appears to bind to human prostanoid TP receptors but not the TP receptors of other species. In human washed platelets, GW627368X (10 microM) produced 100% inhibition of U-46619 (EC100)-induced aggregation (approximate pA2 approximately 7.0). However, in rings of rabbit and piglet saphenous vein and of guinea-pig aorta GW627368X (10 microM) did not displace U-46619 E/[A] curves indicating an affinity of < 5.0 for rabbit and guinea-pig prostanoid TP receptors. 5. In functional assays GW627368X is devoid of both agonism and antagonist affinity for prostanoid CRTH2, EP2, EP3, IP and FP receptors. At prostanoid EP1 receptors, GW627368X was an antagonist with a pA2 of 6.0, and at prostanoid IP receptors the compound increased the maximum effect of iloprost by 55%. At rabbit prostanoid EP2 receptors the pA2 of GW627368X was < 5.0. 6. In competition radioligand bioassays, GW627368X had affinity for human prostanoid EP4 and TP receptors (pKi = 7.0 +/- 0.2 (n = 10) and 6.8 (n = 2), respectively). Affinity for all other human prostanoid receptors was < 5.3. 7. GW627368X will be a valuable tool to explore the role of the prostanoid EP4 receptor in many physiological and pathological settings.

Functional pharmacology of human prostanoid EP2 and EP4 receptors.

Prostanoid EP(2) and EP(4) receptor-mediated responses are difficult to distinguish pharmacologically because of the lack of potent, selective antagonists. We describe systematic agonist fingerprints for recombinant human prostanoid EP(2) and EP(4) receptors expressed in CHO and HEK293 cells, respectively. The rank orders of potency of endogenous prostaglandins were: prostanoid EP(2) receptors: prostaglandin E(2)>>prostaglandin D(2)=prostaglandin F(2alpha)>prostaglandin I(2); prostanoid EP(4) receptors: prostaglandin E(2)>>prostaglandin I(2)>prostaglandin D(2)=prostaglandin F(2alpha). Butaprost free acid (9-oxo-11alpha,16R-dihydroxy-17-cyclobutyl-prost-13E-en-1-oic acid) behaved as a highly selective partial agonist at prostanoid EP(2) receptors while butaprost methyl ester elicited small, low potency responses. The prostanoid EP(1) and EP(3) receptor agonists misoprostol (9-oxo-11alpha,16-dihydroxy-16-methyl-prost-13E-en-1-oic acid, methyl ester), sulprostone (N-(methylsulphonyl)-9-oxo-11alpha,15R-dihydroxy-16-phenoxy-17,18,19,20-tetranor-prosta-5Z,13E-dien-1-amide), and GR63799X ([1R-[1alpha(Z),2beta(R*),3alpha]-(-)-4-benzoylamino)phenyl-7-[3-hydroxy-3-phenoxy-propoxy)-5-oxocyclopentyl]-4-heptenoate), and the prostanoid DP receptor agonist BW245C ((4S)-(3-[(3R,S)-3-cyclohexyl-3-hydropropyl]-2,5-dioxo)-4-imidazolidineheptanoic acid), activated both prostanoid EP(2) and EP(4) receptors. Prostaglandin I(2), iloprost (6,9alpha-methylene-11alpha,15S-dihydroxy-16-methyl-prosta-5E,13E-dien-18-yn-1-oic acid, trometamol salt) and cicaprost (5-[(E)-(1S, 5S, 6S, 7R)-7-hydroxy-6-[(3S, 4S)-3-hydroxy-4-methylnona-1,6-diinyl]-bicyclo[3.3.0]octan-3-yliden]-3-oxapentanoic acid; ZK96480) were full agonists at prostanoid EP(4) receptors. Key differentiating agonists are: butaprost FA, 16,16-dimethyl-prostaglandin E(2), 19-(R)-hydroxy prostaglandin E(2), misoprostol, BW245C, prostaglandin F(2alpha) and prostaglandin D(2).