Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/4015—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having oxo groups directly attached to the heterocyclic ring, e.g. piracetam, ethosuximide
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
  • A61K31/5575—Eicosanoids, e.g. leukotrienes or prostaglandins having a cyclopentane, e.g. prostaglandin E2, prostaglandin F2-alpha
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
  • A61K31/559—Eicosanoids, e.g. leukotrienes or prostaglandins having heterocyclic rings containing hetero atoms other than oxygen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
  • A61P27/06—Antiglaucoma agents or miotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0048—Eye, e.g. artificial tears

Definitions

• Glaucoma is a degenerative disease of the eye wherein the intraocular pressure is too high to permit normal eye function. As a result, damage may occur to the optic nerve head and result in irreversible loss of visual function. If untreated, glaucoma may eventually lead to blindness. Ocular hypertension, i.e., the condition of elevated intraocular pressure without optic nerve head damage or characteristic glaucomatous visual field defects, is now believed by the majority of ophthalmologists to represent merely the earliest phase in the onset of glaucoma.
• carbonic anhydrase inhibitors decrease the formation of aqueous humor by inhibiting the enzyme carbonic anhydrase. While such carbonic anhydrase inhibitors are now used to treat elevated intraocular pressure by systemic and topical routes, current therapies using these agents, particularly those using systemic routes are still not without undesirable effects. Topically effective carbonic anhydrase inhibitors are disclosed in U.S. Patent Nos. 4,386,098; 4,416,890; 4,426,388; 4,668,697; 4,863,922; 4,797,413; 5,378,703, 5,240,923 and 5,153,192.
• Prostaglandins and prostaglandin derivatives are also known to lower intraocular pressure.
• U.S. Patent 4,883,819 to Bito describes the use and synthesis of PGAs, PGBs and PGCs in reducing intraocular pressure.
• U.S. Patent 4,824,857 to Goh et al. describes the use and synthesis of PGD2 and derivatives thereof in lowering intraocular pressure including derivatives wherein C- 10 is replaced with nitrogen.
• U.S. Patent 4,599,353 describes the use of eicosanoids and eicosanoid derivatives including prostaglandins and prostaglandin inhibitors in lowering intraocular pressure. See also WO 00/38667, WO 99/32441, WO 99/02165, WO 00/38663, WO 01/46140, EP 0855389, JP 2000-1472, US Patent No. 6,043,275 and WO 00/38690.
• Prostaglandin and prostaglandin derivatives are known to lower intraocular pressure by increasing uveoscleral outflow. This is true for both the F type and A type of prostaglandins. This invention is particularly interested in those compounds that lower IOP via the uveoscleral outflow pathway and other mechanisms by which the E series prostaglandins (PGE2) may facilitate IOP reduction. While the relationship between EP receptor activation and IOP lowering effects is not well understood, there are four recognized subtypes of the EP receptor (EPi, EP2, EP3 and EP4; J. Lipid Mediators Cell Signaling, Vol. 14, pages 83-87 (1996)). See also J. Ocular Pharmacology, Vol. 4, 1, pages 13-18 (1988); J.
• prostaglandins or derivatives thereof to lower intraocular pressure is that these compounds often induce an initial increase in intraocular pressure, can change the color of eye pigmentation and cause proliferation of some tissues surrounding the eye.
• therapies for treating glaucoma and elevated intraocular pressure but the efficacy and the side effect profiles of these agents are not ideal. Therefore, there still exist the need for new and effective therapies with little or no side effects.
• This invention relates to the use of potent selective agonists of the EP4 subtype of prostaglandin E2 receptors in the treatment of glaucoma and other conditions which are related to elevated intraocular pressure in the eye of a patient.
• This invention also relates to the use of such compounds to provide a neuroprotective effect to the eye of mammalian species and/or treat dry eye in mammals, particularly humans. More particularly this invention relates to the treatment of glaucoma and/or ocular hypertension (elevated intraocular pressure) using compounds having the structural formula la, lb, Ic, Id, Ie or If:
• Rl represents COOR5, CONHR6 or tetrazol-5-yl
• R3 and R4 are each independently hydrogen, hydroxy or Ci-3 alkyl;
• R2 represents hydrogen, ⁇ -thienyl, phenyl or phenoxy, wherein said phenyl and phenoxy are optionally substituted with 1-3 substituents selected from chloro, fluoro, phenyl, methoxy, trifluoromethyl or Ci-3 alkyl;
• — represents a single or double bondj n is 0 to 3;
• R5 represents hydrogen, Ci-5 alkyl, phenyl or p-biphenyl
• R6 represents COR7 or SO2R7
• R7 represents phenyl or C 1.5 alkyl
• R8 represents C3.6 cycloalkyl or C ⁇ _6 alkyl, wherein said cycloalkyl and alkyl groups are optionally substituted with one or two C . alkyl groups
• Rib represents hydroxy, Ci-6 alkyloxy or NR6bR7b ; wherin R6b and R7b are each independently hydrogen or C ⁇ . alkyl;
• R2b represents hydrogen or hydroxy
• R3 represents a single bond or Ci-6 alkylene
• R4b represents
• Rlc represents hydroxy, C ⁇ _6 alkyloxy or NR6CR7C ; wherin R6c and R7c are each independently hydrogen or Ci-6 alkyl;
• R2c represents hydrogen or hydroxy
• R3c represents a single bond or Ci-6 alkylene
• Ci-6 alkyloxy or halogen (ii) Phenyloxy or C3-.7 cycloalkyloxy; (iii) Furyl, furyloxy, thienyl, thienyloxy, naphthyl, naphthyloxy, phthalanyl or phthalanyloxy; (iv) Phenyl, phenyloxy, C3.7 cycloalkyl or C3-7 cycloalkyloxy, wherein said phenyl, phenyloxy, cycloalkyl and cycloalkyloxy groups are optionally substituted with 1-3 substituents selected from Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, Ci-6 alkyloxy, Ci-6 alkyloxy-Ci-6 alkyl, Ci-6 alkyloxy-Ci-6 alkyloxy, -6 alkenyloxy-Ci-6 alkyl, Ci-6 alkyl substituted by 1-3 hydroxy or halogen groups, Ci-6 al
• R2d represents hydrogen or hydroxy
• R3d represents a single bond or C ⁇ . alkylene
• R4d represents (i) Ci-8 alkyl, C2-8 alkenyl, C2-8 alkynyl, phenyl or cycloalkyl, wherein said alkyl, alkenyl, alkynyl are optionally substituted with phenyl, cycloalkyl, OH,
• Ci-6 alkyloxy or halogen (ii) Phenyloxy or C3-7 cycloalkyloxy; (iii) Furyl, furyloxy, thienyl, thienyloxy, naphthyl, naphthyloxy, phthalanyl or phthalanyloxy; (iv) Phenyl, phenyloxy, C3-7 cycloalkyl or C3-7 cycloalkyloxy, wherein said phenyl, phenyloxy, cycloalkyl and cycloalkyloxy groups are optionally substituted with 1-3 substituents selected from Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, Ci-6 alkyloxy, Ci-6 alkyloxy-Ci-6 alkyl, Ci-6 alkyloxy-Ci-6 alkyloxy, Ci-6 alkenyloxy-Ci-6 alkyl, Ci-6 alkyl substituted by 1-3 hydroxy or halogen groups, Ci-6 alky
• R5d represents hydrogen, hydroxy or C 1-6 alkyl
• Rle represents carboxyl, (C3-C4)alkoxylcarbonyl or tetrazolyl
• R2e represents -Ar, or -Arl-V-Ar2, wherein V is a bond, -O-, -OCH2- or -CH2O-;
• X represents -CH2- or O
• Z represents -(CH2)3-, thienyl, thiazolyl, or phenyl;
• Ar represents a partially saturated, fully saturated or fully unsaturated five to eight membered ring optionally having one to four heteroatoms selected independently from oxygen, sulfur and nitrogen, or a bicyclic ring consisting of two fused independently partially saturated, fully saturated or fully unsaturated five or six membered rings, taken independently, optionally having one to four heteroatoms selected independently from nitrogen, sulfur and oxygen, said partially or fully saturated ring or bicyclic ring optionally having one or two oxo groups substituted on carbon or one or two oxo groups substituted on sulfur; and
• Arl and Ar2 each independently represent a partially saturated, fully saturated or fully unsaturated five to eight membered ring optionally having one to four heteroatoms selected independently from oxygen, sulfur and nitrogen, said partially or fully saturated ring optionally having one or two oxo groups substituted on carbon or one or two oxo groups substituted on sulfur;
• Ar moiety is optionally substituted on carbon or nitrogen, on one ring if the moiety is monocyclic, or on one or both rings if the moiety is bicyclic, with up to three substituents per ring each independently selected from hydroxy, halo, carboxy, (Ci-C7)alkoxy, (C ⁇ -C4)alkoxy(C ⁇ -G alkyl, (Ci-C7)alkyl, (C2-C7)alkenyl, (C3- C7)cycloalkyl, (C3-C7)cycloalkyl(C ⁇ -C4)alkyl, (C3-C7)cycloalkyl(C ⁇ -C4)alkanoyl, formyl, (C ⁇ -C8)alkanoyl, (C ⁇ -C6)alkanoyl(Ci-C6)alkyl, (C ⁇ -C4)alkanoylamino, (Ci- C4)alkoxycarbonylamino, hydroxysulfonyl, aminocarbonylamino,
• Arl and AJ2 moieties are independently optionally substituted on carbon or nitrogen with up to three substituents each independently selected from hydroxy, halo, carboxy, (C ⁇ -C7)alkoxy, (Ci-C4)alkoxy(C ⁇ -C4)alkyl, (C ⁇ -C7)alkyl, (C " 2- C7)alkenyl, (C3-C7)cycloalkyl, (C3-C7)cycloalkyl(Ci-C4)alkyl, (C3- C7)cycloalkyl(Ci-C4)alkanoyl, formyl, (C ⁇ -Cs)alkanoyl, (C ⁇ -C6)alkanoyl(C ⁇ - C6)alkyl, (C ⁇ -C4)alkanoylamino, (C ⁇ -C4)alkoxycarbonylamino, hydroxysulfonyl, aminocarbonylamino or mono-N-, di-N,N-, di-N,N- or tri-N,
• the present invention is directed to a method for decreasing elevated intraocular pressure or treating glaucoma by administration, preferably topical or intra-camaral, of a composition containing an EP4 agonist of Formula la, lb, Ic, Id, Ie or If and a pharmaceutically acceptable carrier.
• Rl is tetrazol-5-yl
• R2 is phenyl and all other variables are as originally described.
• Still another embodiment of the invention of formula lb is realized when Rib is Ci-6 alkyloxy and all other variables are as originally described. Yet another embodiment of the invention of formula lb is realized when Rib is NR6bR7b wherein R6b and R7b are independently hydrogen or Ci-6 alkyl and all other variables are as originally described.
• R3b is a single bond and R4b is Ci-8 alkyl, C2-8 alkenyl, phenyl, C3.7 cycloalkyl or C2-8 alkynyl, said alkyl, alkenyl and alkynyl optionally substituted with -6 alkyloxy or halogen.
• Another embodiment of the invention of formula lb is realized when R4b i s Ci-8 alkyl, C2-8 alkenyl, or C2-8 alkynyl, said alkyl, alkenyl and alkynyl optionally substituted with C ⁇ _6 alkyloxy or halogen.
• Another embodiment of the invention of formula lb is realized when R4b is phenyloxy or C3.7 cycloalkyloxy.
• An embodiment of the invention of formula Ic is realized when Rlc is Ci-6 alkyloxy. Another embodiment of the invention of formula Ic is realized when
• R2c is hydrogen
• R4c is phenyl which is optionally substituted with 1-3 substituents selected from Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, Ci-6 alkyloxy, C ⁇ - ⁇ alkyloxy-Ci-6 alkyl, Ci-6 alkyloxy-Ci-6 alkyloxy, Ci-6 alkenyloxy-Ci-6 alkyl, Ci-6 alkyl substituted by 1-3 hydroxy or halogen groups, Ci-6 alkylthio, C ⁇ _6 alkylthio-Ci-6 alkyl, Ci-6 alkylthio- Ci-6 alkyloxy, Ci-6 alkenylthio-Ci-6 alkyl, C ⁇ _6 alkylsulfonyl, halogen, trihalomethyl, cyano, nitro, amino, OH, C3-7 cycloalkyl, C3.7 cycloalkyloxy, C3-.7 cycloalkyl-Ci-6 al
• R2d is hydrogen
• R d is phenyl which is optionally substituted with 1-3 substituents selected from Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, Ci-6 alkyloxy, Ci-6 alkyloxy-Ci-6 alkyl, Ci-6 alkyloxy-Ci-6 alkyloxy, Ci-6 alkenyloxy-Ci-6 alkyl, Ci-6 alkyl substituted by 1-3 hydroxy or halogen groups, Ci-6 alkylthio, Ci-6 alkylthio-Ci-6 alkyl, Ci-6 alkylthio- Ci-6 alkyloxy, Ci-6 alkenylthio-Ci-6 alkyl, Ci-6 alkylsulfonyl, halogen, trihalomethyl, cyano, nitro, amino, OH, C3-7 cycloalkyl, C3-7 cycloalkyloxy, C3-7 cycloalkyl-Ci-6 alkyl, C3..7
• potent selective agonists of the EP4 subtype of prostaglandin E2 receptors useful in the treatment of glaucoma and other conditions which are related to elevated intraocular pressure in the eye of a patient.
• This invention also relates to the use of such compounds to provide a neuroprotective effect to the eye of mammalian species and/or treat dry eye in mammals, particularly humans. More particularly this invention relates to the treatment of glaucoma and/or ocular hypertension (elevated intraocular pressure) using compounds having the structural formula la or lb:
• Ri represents COOR5, CONHR6 or tetrazol-5-yl
• R3 and R4 are each independently hydrogen or hydroxy
• R2 represents ⁇ -thienyl, phenyl or phenoxy, wherein said phenyl and phenoxy are optionally substituted with 1-3 substituents selected from chloro, fluoro, phenyl, methoxy, trifluoromethyl or Ci-3 alkyl;
• — represents a single or double bond; n is 0 to 3; R5 represents hydrogen, C1.5 alkyl, phenyl or p-biphenyl; R6 represents COR7 or SO2R7; R7 represents phenyl or C 1.5 alkyl; R8 represents CH2; Rib represents hydroxy, C . alkyloxy or NR6bR7b ; wherin R6b and R7b are each independently hydrogen or Ci-6 alkyl;
• R2b represents hydrogen or hydroxy
• R3b represents a single bond or Ci-6 alkylene
• R4b represents (i) Ci-8 alkyl, C2-8 alkenyl, C2-8 alkynyl, phenyl or cycloalkyl, wherein said alkyl, alkenyl, alkynyl are optionally substituted with phenyl, cycloalkyl, OH,
• R5b represents hydrogen or C 1-6 alkyl.
• Preferred compounds of this invention are: 7-(2S-[3R-hydroxy-4-(3-phenoxy-phenyl)-butyl]-5-oxo-pyrrolidin-l-yl)-heptanoic acid;
• This is invention is also concerned with novel compounds which are useful as potent selective agonists of the EP4 subtype of prostaglandin E2 receptors in the treatment of glaucoma and other conditions which are related to elevated intraocular pressure in the eye of a patient.
• Preferred compounds are: 7- ⁇ (2R)-2-[(lE)-3-hydroxy-3-(l-phenylcyclopropyl)prop-l-enyl]-5-oxopyrrolidin-l- yljheptanoic acid;
• alkyl refers to a monovalent alkane (hydrocarbon) derived radical containing from 1 to 10 carbon atoms unless otherwise defined. It may be straight or branched. Preferred alkyl groups include methyl, ethyl, propyl, isopropyl, butyl and t-butyl. When the alkyl group is said to be substituted with an alkyl group, this is used interchangeably with “branched alkyl group”.
• Cycloalkyl is a species of alkyl containing from 3 to 15 carbon atoms, without alternating or resonating double bonds between carbon atoms. It may contain from 1 to 4 rings which are fused. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
• Alkoxy refers to C j -Cg alkyl-O-, with the alkyl group optionally substituted as described herein.
• alkoxy groups include methoxy, ethoxy, propoxy, butoxy and isomeric groups thereof.
• Alkenyl refers to alkyl groups having a double bond, including vinyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl and isomeric groups thereof.
• Alkynyl refers to alkyl groups have a triple bond, including ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl and isomers thereof.
• Halogen refers to chlorine, fluorine, iodine or bromine.
• agonist means that the EP4 subtype compounds of formula la, lb, Ic, Id, Ie or If interact with the EP4 receptor to produce maximal, super maximal or submaximal effects compared to the natural agonist, PGE2. See Goodman and Gilman, The Pharmacological Basis of Therapeutics, 9 th edition, 1996, chapter 2.
• This invention is also concerned with a method of treating ocular hypertension or glaucoma by administering to a patient in need thereof one of the compounds of formula la, lb, Ic, Id, Ie or If alone or in combination with: a ⁇ - adrenergic blocking agent, such as timolol, betaxolol, levobetaxolol, carteolol, levobunolol; a parasympathomimetic agent, such as piloca ⁇ ine; a sympathomimetic agent, such as epinephrine, iopidine, brimonidine, clonidine, para-aminoclonidine; a carbonic anhydrase inhibitor, such as dorzolamide, acetazolamide, metazolamide or brinzolamide; a prostaglandin such as latanoprost, travaprost, unoprostone, rescula, S1033 (compounds set forth in US Patent
• the claimed compounds bind strongly and act on PGE2 receptor, particularly on the EP4 subtype receptor and therefore are useful for preventing and/or treating glaucoma and/or ocular hypertension.
• Use of the compounds of formula la, lb, Ic, Id, Ie or If for the manufacture of a medicament for treating glaucoma and/or ocular hypertension is also included in this invention.
• Dry eye is a common ocular surface disease afflicting millions of people. Although it appears that dry eye may result from a number of unrelated pathogenic causes, the common end result is the breakdown of the tear film, which results in dehydration of the exposed outer surface of the eye. (Lemp, Report of the National Eye Institute/Industry Workshop on Clinical Trials in Dry Eyes, The CLAO Journel, 21(4):221-231 (1995)).
• One cause for dry eye is the decreased mucin production by the conjunctival cells and/or corneal epithelial cells of mucin, which protects and lubricates the ocular surface (Gipson and Inatomi, Mucin genes expressed by ocular surface epithelium.
• Macular edema is swelling within the retina within the critically important central visual zone at the posterior pole of the eye. An accumulation of fluid within the retina tends to detach the neural elements from one another and from their local blood supply, creating a dormancy of visual function in the area. It is believed that EP4 agonist which lower IOP are useful for treating diseases of the macular such as macular edema or macular degeneration.
• another aspect of this invention is a method for treating macular edema or macular degeneration.
• Glaucoma is characterized by progressive atrophy of the optic nerve and is frequently associated with elevated intraocular pressure (IOP). It is possible to treat glaucoma, however, without necessarily affecting IOP by using drugs that impart a neuroprotective effect. See Arch. Ophthalmol. Vol. 112, Jan 1994, pp. 37- 44; Investigative Ophthamol. & Visual Science, 32, 5, April 1991, pp. 1593-99. It is believed that EP4 agonist which lower IOP are useful for providing a neuroprotective effect. They are also believed to be effective for increasing retinal and optic nerve head blood velocity and increasing retinal and optic nerve oxygen by lowering IOP, which when coupled together benefits optic nerve health.
• IOP intraocular pressure
• this invention further relates to a method for increasing retinal and optic nerve head blood velocity, or increasing retinal and optic nerve oxygen tension or providing a neuroprotective effect or a combination thereof by using an EP4 agonist of formula la, lb, Ic, Id, Ie or
• this invention is also concerned with a method of treating ocular hypertension or glaucoma by administering to a patient in need thereof one of the compounds of formula la, lb, Ic, Id, Ie or If alone or in combination with: a ⁇ -adrenergic blocking agent, such as timolol, betaxolol, levobetaxolol, carteolol or levobunolol; a parasympathomimetic agent such as piloca ⁇ ine; a sympathomimetic agent such as, epinephrine, iopidine, brimonidine, clonidine, para-aminoclonidine; a carbonic anhydrase inhibitor, such as, dorzolamide, acetazolamide, metazolamide or brinzolamide; a prostaglandin such as latanoprost, travaprost, unoprostone, rescula, S1033 (compounds set forth in US Patent
• this invention is also concerned with a method for increasing retinal and optic nerve head blood velocity, or increasing retinal and optic nerve oxygen tension or providing a neuroprotective effect or a combination thereof by administering to a patient in need thereof one of the compounds of formula la, lb, Ic, Id, Ie or If alone or in combination with: a ⁇ -adrenergic blocking agent, such as timolol, betaxolol, levobetaxolol, carteolol or levobunolol; a parasympathomimetic agent such as piloca ⁇ ine; a sympathomimetic agent such as, epinephrine, iopidine, brimonidine, clonidine, para-aminoclonidine; a carbonic anhydrase inhibitor, such as, dorzolamide, acetazolamide, metazolamide or brinzolamide; a prostaglandin such as latanoprost, travaprost, un
• This invention is further concerned with a method for treating macular edema or macular degeneration by administering to a patient in need thereof one of the compounds of formula la, lb, Ic, Id, Ie or If alone or in combination with: a ⁇ - adrenergic blocking agent, such as timolol, betaxolol, levobetaxolol, carteolol, levobunolol; a parasympathomimetic agent, such as piloca ⁇ ine; a sympathomimetic agent, such as epinephrine, iopidine, brimonidine, clonidine, para-aminoclonidine; a carbonic anhydrase inhibitor, such as dorzolamide, acetazolamide, metazolamide or brinzolamide; a prostaglandin such as latanoprost, travaprost, unoprostone, rescula, S1033 (compounds set forth in US Patent
• Each of the claimed compounds are EP4 agonists and are useful for a number of physiological ocular disorders.
• the compounds produced in the present invention are readily combined with suitable and known pharmaceutically acceptable excipients to produce compositions which may be administered to mammals, including humans, to achieve effective IOP lowering.
• the EP4 agonist used in the instant invention can be administered in a therapeutically effective amount intravaneously, subcutaneously, topically, transdermally, parenterally or any other method known to those skilled in the art.
• Ophthalmic pharmaceutical compositions are preferably adapted for topical administration to the eye in the form of solutions, suspensions, ointments, creams or as a solid insert. Ophthalmic formulations of this compound may contain from 0.001 to 5% and especially 0.001 to 0.1% of medicament.
• Higher dosages as, for example, up to about 10% or lower dosages can be employed provided the dose is effective in reducing intraocular pressure, treating glaucoma, increasing blood flow velocity or oxygen tension.
• a single dose from between 0.001 to 5.0 mg, preferably 0.005 to 2.0 mg, and especially 0.005 to 1.0 mg of the compound can be applied to the human eye.
• the pharmaceutical preparation which contains the compound may be conveniently admixed with a non-toxic pharmaceutical organic carrier, or with a non-toxic pharmaceutical inorganic carrier.
• a non-toxic pharmaceutical organic carrier or with a non-toxic pharmaceutical inorganic carrier.
• pharmaceutically acceptable carriers are, for example, water, mixtures of water and water-miscible solvents such as lower alkanols or aralkanols, vegetable oils, peanut oil, polyalkylene glycols, petroleum based jelly, ethyl cellulose, ethyl oleate, carboxymethyl-cellulose, polyvinylpyrrolidone, isopropyl myristate and other conventionally employed acceptable carriers.
• the pharmaceutical preparation may also contain non-toxic auxiliary substances such as emulsifying, preserving, wetting agents, bodying agents and the like, as for example, polyethylene glycols 200, 300, 400 and 600, carbowaxes 1,000, 1,500, 4,000, 6,000 and 10,000, antibacterial components such as quaternary ammonium compounds, phenylmercuric salts known to have cold sterilizing properties and which are non-injurious in use, thimerosal, methyl and propyl paraben, benzyl alcohol, phenyl ethanol, buffering ingredients such as sodium borate, sodium acetates, gluconate buffers, and other conventional ingredients such as sorbitan monolaurate, triethanolamine, oleate, polyoxyethylene sorbitan monopalmitylate, dioctyl sodium sulfosuccinate, monothioglycerol, thiosorbitol, ethylenediamine tetracetic acid, and the like.
• auxiliary substances such as e
• suitable ophthalmic vehicles can be used as carrier media for the present pu ⁇ ose including conventional phosphate buffer vehicle systems, isotonic boric acid vehicles, isotonic sodium chloride vehicles, isotonic sodium borate vehicles and the like.
• the pharmaceutical preparation may also be in the form of a microparticle formulation.
• the pharmaceutical preparation may also be in the form of a solid insert. For example, one may use a solid water soluble polymer as the carrier for the medicament.
• the polymer used to form the insert may be any water soluble non-toxic polymer, for example, cellulose derivatives such as methylcellulose, sodium carboxymethyl cellulose, (hydroxyloweralkyl cellulose), hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose; acrylates such as polyacrylic acid salts, ethylacrylates, polyactylamides; natural products such as gelatin, alginates, pectins, tragacanth, karaya, chondrus, agar, acacia; the starch derivatives such as starch acetate, hydroxymethyl starch ethers, hydroxypropyl starch, as well as other synthetic derivatives such as polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl methyl ether, polyethylene oxide, neutralized carbopol and xanthan gum, gellan gum, and mixtures of said polymer.
• cellulose derivatives such as methylcellulose, sodium carboxymethyl
• Suitable subjects for the administration of the formulation of the present invention include primates, man and other animals, particularly man and domesticated animals such as cats, rabbits and dogs.
• the pharmaceutical preparation may contain non-toxic auxiliary substances such as antibacterial components which are non-injurious in use, for example, thimerosal, benzalkonium chloride, methyl and propyl paraben, benzyldodecinium bromide, benzyl alcohol, or phenylethanol; buffering ingredients such as sodium chloride, sodium borate, sodium acetate, sodium citrate, or gluconate buffers; and other conventional ingredients such as sorbitan monolaurate, triethanolamine, polyoxyethylene sorbitan monopalmitylate, ethylenediamine tetraacetic acid, and the like.
• the ophthalmic solution or suspension may be administered as often as necessary to maintain an acceptable IOP level in the eye. It is contemplated that administration to the mammalian eye will be from once up to three times daily.
• novel formulations of this invention may take the form of solutions, gels, ointments, suspensions or solid inserts, formulated so that a unit dosage comprises a therapeutically effective amount of the active component or some multiple thereof in the case of a combination therapy.
• the formula la, lb, Ic, Id and Ie agonists generally have an EC50 value from about 0.001 nM to about 100 microM, although agonists with activities outside this range can be useful depending upon the dosage and route of administration. In a subclass of the present invention, the agonists have an EC50 value of from about 0.01 microM to about 10 microM.
• the agonists have an EC50 value of from about 0.1 microM to about 10 microM.
• EC50 is a common measure of agonist activity well known to those of ordinary skill in the art and is defined as the concentration or dose of an agonist that is needed to produce half, i.e. 50%, of the maximal effect. See also, Goodman and Gilman's, The Pharmacologic Basis of Therapeutics, 9th edition, 1996, chapter 2, E. M. Ross, Pharmacodynamics, Mechanisms of Drug Action and the Relationship Between Drug Concentration and Effect,
• Step A 7 ⁇ 2R-[4-(3-Chloro-phenvl)-3-oxo-but-l- enyll-5-oxo-pyrrolidin-l-yl-heptanoic acid ethyl ester.
• reaction mixture was cooled to O°C and a solution of 7-(2R-formyl-5-oxo- pyrrolidin-l-yl)-heptanoic acid ethyl ester (assumed 10.6 mmol, prepared according to the method described in Preparation 7, but using different amounts of reagents) in THF was added and the reaction was stirred for 18 hours.
• AcOH was added and the reaction mixture was diluted with EtOAc.
• the organic solution was washed consecutively with saturated NaHCO 3 solution (2x), water (lx), and brine (lx). The organic solution was dried (MgS0 4 ), filtered and concentrated.
• Step B 7- ⁇ 2R -[4-(3-Chloro-phenyl)-3S-hydroxy-but-l-enyl]-5- oxo-pyrrolidin-1 -yllheptanoic acid ethyl ester
• Step C 7- ⁇ 2S-[4-(3-Chloro-phenyl)-3R-hydroxy-butyl]-
• Step D 7- ⁇ 2S-[4-(3-Chloro-phenyl)-3R-hydroxy-butyl]-5
• Step A 7- ⁇ 2-Oxo-5R-[3-oxo-4-(3-trifluoromethyl-phenyl)- but- 1 -enyll-pyrrolidin- 1 -yl ) - heptanoic acid ethyl ester.
• Example 1 the anion derived from [2-oxo-3-(3-trifluoromethyl-phenyl)- propyl]-phosphonic acid dimethyl ester (4.16 g, 13.40 mmol) and NaH (60% in oil, 590 mg, 14.7 mmol) was reacted with 7-(2R-formyl-5-oxo-pyrrolidin-l-yl)-heptanoic acid ethyl ester (assumed 14.74 mmol) over 24 h.
• Step B 7- ⁇ 2R-[3S-Hydroxy-4-(3-trifluoromethyl-phenyl)- but-l-enyIl-5-oxo-pyrrolidin-l-yl l-heptanoic acid ethyl ester.
• the reaction mixture was stirred at room temperature for 1 h and the layers were separated.
• the aqueous solution was washed with CH2CI2 (2x) and the combined organic layers were washed consecutively with ice-cold 0.5N NaOH and brine (two times).
• the organic solution was dried (MgSO4), filtered and concentrated.
• Step C 7- ⁇ 2S-[3R-Hydroxy-4-(3-trifluoromethyl-phenyl)-butyl]-
• Example 1 a solution of 7- ⁇ 2R-[3S-hydroxy-4-(3-trifluoromethyl-phenyl)- but-l-enyl]-5-oxo- 25 pyrrolidin-1 -yl ⁇ -heptanoic acid ethyl ester (1.18 g, 2.59 mmol) in EtOH (50 mL was hydrogenated in the presence of 10% palladium on carbon (120 mg) at 40-45 psi on a Parr shaker for 24 h.
• Step D 7- ⁇ 2S-[3R-Hydroxy-4-(3-trifluoromethyl-phenyl)- butyll-5-oxo-pyrrolidin-l-yl ⁇ - heptanoic acid
• Step E Sodium salt of 7- ⁇ 2S-[3R-Hydroxy-4-(3-trifluoromethyl-phenyl)- butyll- 5-oxo- Pyrrolidin-1-yl ⁇ -heptanoic acid
• Step A 7- ⁇ 2R-[4-(3-Chloro-phenyl)-3-oxo-but-l-enyl]-5-oxo-pyrrolidin-l - vU-heptanenitrile
• Step B 7- ⁇ 2S-[4-(3-Chloro-phenyl)-3-oxo-butyl-5-oxo-pyrrolidin-l-yl ⁇ heptanenitrile
• Step C 7- ⁇ 2S-[4-(3-Chloro-phenyl)-3-hydroxy-butyl]-5-oxo-pyrrolidin-l-yl] heptanenitrile To a solution of 7- ⁇ 2S-[4-(3-chloro-phenyl)-3-oxo-butyl]-5-oxo-
• Step D 5S-[4-(3-Chloro-phenyl)-3-hydroxy-butyl]-l-[6-(2H- tetrazol-5-yl)-hexyll- Pyrrolidin-2-one
• the residue was purified by medium pressure chromatography eluting with EtOAc to 2% MeOH in CH2CI2 to 7% MeOH in CH2CI2 to give the TMS complex.
• the residue was diluted with MeOH and 2N HCI was added and the solution was stirred for 40 minutes.
• the solution was diluted with CH2CI2 and the organic layer was washed with water followed by brine.
• Step A 7- ⁇ 2-Oxo-5R-[3-oxo-4-(3-trifluoromethyl-phenyl)- but-1-enyll-pyrrolidin-l-yl- heptanenitrile
• Step B 7- ⁇ 2R-[3S-Hydroxy-4-(3-trifluoromethyl-phenyl)- but-l-enyll-5-oxo-pyrrolidin- 1-yl) -heptanenitrile
• Step C 7- ⁇ 2S-[3R-Hydroxy-4-(3-trifluoromethyl-phenyl)- butyll-5-oxo-pyrrolidin-l -yl ⁇ heptanenitrile
• Step D 5S-[3R-Hydroxy-4-(3-trifluoromethyl- ⁇ henyl)- butvn-l-r6-(2H-tetrazol-5-yl)-Hexyll-pyrrolidin-2-one Following the procedure described for Example 3, Step D, 7
• Step E Sodium salt of 5S-[3R-Hydroxy-4-(3-trifluoromethyl-phenyl)- PutvH-l-r6-(2H-tetrazol-5-yl)-hexy ⁇ -pyrrolidin-2-one
• Step Al 7- ⁇ 2-[3-(tert-butyl-dimethyl-silanyloxy)-4-(4-fluoro-phenyl)-butyl]-5- oxo-pyrrolidin- 1 -yl I -heptanenitrile
• a solution of 5-[3-(tert-butyl-dimethyl-silanyloxy)-4-(4-fluoro- phenyl)-butyl]-pyrrolidin-2-one (150 mg, 0.41 mmol) in DMF (5 mL) was added to NaH (60% by weight in oil, 16 mg, 0.41 mmol) in DMF (5 mL) was added and the reaction mixture was stirred at 90 C for 2.5 hour.
• Step A2 7- ⁇ 2-[4-biphenyl-3-yl-3-(tert-butyl-dimethyl-silanyloxy)-butyl]-
• Step Bl 7- ⁇ 2-[4-(4-fluoro-phenyl)-3-hydroxy-butyl]-5-oxo-pyrrolidin-l-yl ⁇ - heptanenitrile
• Step B2 7- ⁇ 2-(4-Biphenyl-3-yl-3-hydroxy-butyl)-5-oxo-pyrrolidin-l - yll-heptanenitrile
• Step C 5-(4-Biphenyl-3-yl-3-hydroxy-butyl)-l -[6-(2H-tetrazol-5-yl)-hexyl]- pyrrolidin-2-one
• Step A 7- ⁇ 2-Oxo-5R-[3-oxo-4-(3-phenoxy-phenyl)-but-l-enyl]-pyrrolidin-l - yl ⁇ heptanoic acid ethyl ester
• Step C 7- ⁇ 2R-[3-Hydroxy-4-(3-phenoxy-phenyl)- but- 1 -enyll-5-oxo-pyrrolidin- 1 -yl)-heptanoic acid
• Step D 7-(2R-[3- hydroxy-4-(3-phenoxy-phenyl)-but-l -enyl]-5-oxo-pyrrolidin-l-yl)-heptanoic acid ethyl ester (29 mg, 0.060 mmol) was hydrolyzed with 2M NaOH in EtOH (4.0 mL) at room temperature over 24 h to provide the title compound.
• Step A Ethyl 7- ⁇ (2R)-2-[(l£)-4-methyl-3-oxo-4-phenylpent-l-enyl]-5- oxopyrrolidin-l-yl jheptanoate
• Step B Ethyl 7- ⁇ (2fl)-2-[(l£)-3-hydroxy-4-methyl-4-phenylpent-l-enyl]-5- oxopyrrolidin-1-yl Jheptanoate
• the residual oil was purified by flash column- chromatography on silica gel (eluent Acetone 3: Toluene 7 ) to provide both diastereoisomers of ethyl 7- ⁇ (2/?)-2-[(lE)-3-hydroxy-4-methyl-4-phenylpent-l-enyl]- 5-oxopyrrolidin-l-yl Jheptanoate in a (1 : 4.5) ratio by NMR as an oil.
• the residual oil was purified by flash column-chromatography on silica gel (gradient CH 2 C1 2 : MeOH : AcOH (100:0:0) to (95:5:0.5)) to provide both diastereoisomers of 7- ⁇ (2/?)-2-[(lE)-3-hydroxy-4-methyl-4-phenylpent-l-enyl]-5-oxopyrrolidin-l- yljheptanoic acid as an oil.
• Step A Ethyl 7- ⁇ (5#)-5-[(lE)-3-oxo-3-(l-phenylcyclopropyl)prop-l- enyllpyrrolidin-1-yl Jheptanoate
• Step B Ethyl 7- ⁇ (2 ⁇ )-2-[(l£)-3-hydroxy-3-(l-phenylcyclopropyl)prop-l- enyll-5-oxopyrrolidin- 1-yl Jheptanoate
• the residual oil was purified by flash column-chromatography on silica gel (eluent AcO ⁇ t ) to provide both diastereoisomers of ethyl 7- ⁇ (2/?)-2-[(l£)-3-hydroxy- 3-(l-phenylcyclopropyl)prop-l-enyl]-5-oxopyrrolidin-l-yl Jheptanoate in a (1 : 3.1) ratio by NMR as an oil.
• Step C 7- ⁇ (2 ⁇ )-2-[(lE)-3-hydroxy-3-(l-phenylcyclopropyl)prop-l-enyl]-5- oxopyrrolidin-l-ylj heptanoic acid
• Step A Ethyl 7- ⁇ (2R)-2-[(lE)-3-cyclohexyl-3-oxoprop-l-enyl]-5- oxopyrrolidin-l-yl Jheptanoate To a solution of dimethyl 3-methyl-2-oxo-3-phenylbutylphosphonate
• Step B Ethyl 7- ⁇ (2 ⁇ )-2-[(lE)-3-cyclohexyl-3-hydroxyprop-l-enyl]-5-o xopyrrolidin- 1 -yl J heptanoate
• Step C 7- ⁇ (2R)-2-[(lE)-3-cyclohexyl-3-hydroxyprop-l-enyl]-5-oxopyrrolidin-
• Step A 7-[2R-(tert-Butyl-dimethyl-silanyloxymethyl)-5-oxo-pyrrolidin-l-yl]-
• Step B 7-(2R-Hydroxymethyl-5-oxo-pyrrolidin- 1 -yl-heptanenitrile
• StepA 7-[2R-(tert-butyl-dimethyl-silanyloxymethyl)-5- oxo-pyrrolidin-l-yll-heptanoic acid ethyl ester
• Step B 7-(2R-Hydroxymethyl-5-oxo-pyrrolidin-l -yl)-heptanoic acid ethyl ester
• Step B 7-[2R-(tert-butyl-dimethylsilanyloxymethyl)-5-oxo-pyrrolidin-l-yl]-heptanoic acid ethyl ester (39.46 g, 102.3 mmol) was deprotected with TBAF (1 M in THF, 154.0 mL, 154.0 mmol) with a reaction time of 2.5 h.
• Step B [2-Oxo-3-(3-trifluoromethyl-phenyl)-propyl)phosphonic acid dimethyl ester
• Preparation 4 [3-(3-Chloro-phenyl)-2-oxo-propyl)-phosphonic acid dimethyl ester Substituting the appropriate starting materials, the title compound of Preparation 4 was prepared following an analogous procedure to that described for Preparation 3.
• Preparation 5 [3-(3-Chloro-phenyl)-2-oxo-propyl]-phosphonic acid dimethyl ester To a solution of dimethyl methylphosphonate (17.93 g, 144 mmol) in THF (270 mL) at -78°C was slowly added n-BuLi (2.5M, 64.2 mL, 160.6 mmol).
• Step A 5-(3-Bromo-3-oxo-butyl)-pyrrolidin-2-one
• Step B 5-(3-Bromo-3-hvdroxy-butyl)-pyrrolidin-2-one
• Step C 5-[3-Bromo-3-(tert-butyl-dimethyl-silanyloxy)-butyl1-pyrrolidin-2-one
• Step D 5-[4-Biphenyl-3-yl-3-(tert-butyl-dimethyl-silanyloxy)-butyl]- pyrrolidin-2-one
• Step A 5-[4-(3-Fluoro-phenyl)-3-oxo-butyl1-pyrrolidin-2-one
• Step B 5-[4-(3-Fluoro-phenyl)-3-hvdroxy-butyl]-pyrrolidin-2-one
• Step C 5-[3-(tert-Butyl-dimethyl-silanyloxy)-4-(3-fluoro-phenyl)-butyl]
• Step B 5-[4-(4-Fluoro-phenyl)-3-hydroxy-butyn-pyrrolidin-2-one
• Step B 5-[4-(4-fluoro-phenyl)-3-oxo-butyl]-pyrrolidin-2-one (2.64 g, mmol) was reduced with NaBH4 (400 mg, mmol) at room temperature for 1 h. Additional NaBH4 (150 mg) was added and the reaction was stirred for 20 h. Purification by medium pressure chromatography using a solvent gradient (CH2 ⁇ 2to 2% MeOH in CH2CI2 to 4%
• Step C 5-[3-(tert-Butyl-dimethyl-silanyloxy)-4-(4-fluoro-phenyl)- butyl]-pyrrolidin-2-one
• Drug concentrations are expressed in terms of the active ingredient (base).
• the compounds of this invention are dissolved in physiological saline at 0.01, 0.001, 0.0001 % for rabbit study and 0.05, 0.005% for monkey studies.
• Drug or vehicle aliquots (25 ul) are administered topically unilaterally or bilaterally. In unilateral applications, the contralateral eyes receive an equal volume of saline.
• Proparacaine (0.5%) is applied to the cornea prior to tonometry to minimize discomfort.
• Intraocular pressure (IOP) is recorded using a pneumatic tonometer (Alcon Applanation Pneumatonograph) or equivalent.
• results are expressed as the changes in IOP from the basal level measured just prior to administration of drug or vehicle and represent the mean, plus or minus standard deviation.
• Statistical comparisons are made using the Student's t- test for non-paired data between responses of drug-treated and vehicle-treated animals and for paired data between ipsilateral and contralateral eyes at comparable time intervals.
• the significance of the date is also determined as the difference from the "t- 0" value using Dunnett's "t" test. Asterisks represent a significance level of p ⁇ 0.05.
• Example 4 Male Dutch Belted rabbits weighing 2.5-4.0 kg were maintained on a 12- hour light/dark cycle and rabbit chow. All experiments were performed at the same time of day to minimize variability related to diurnal rhythm. IOP was measured before treatment then the compound of Example 4 or vehicle were instilled (one drop of 25 ul) into one or both eyes and IOP was measured at 30, 60, 120, 180, 240, 300, and 360 minutes after instillation. In some cases, equal number of animals treated bilaterally with vehicle only were evaluated and compared to drug treated animals as parallel controls.
• Unilateral ocular hypertension of the right eye was induced in female cynomolgus monkeys weighing between 2 and 3 kg by photocoagulation of the trabecular meshwork with an argon laser system (Coherent NOVUS 2000, Palo Alto, USA) using the method of Lee at al. (1985).
• IOP intraocular pressure
• IOP measurements the monkeys wee kept in a sitting position in restraint chairs for the duration of the experiment. Animals were lightly anesthetized by the intramuscular injection of ketamine hydrochloride (3-5 mg/kg) approximately five minutes before each IOP measurement and one drop of 0.5% proparacaine was instilled prior to recording IOP. IOP was measured using a pneumatic tonometer (Alcon Applanation Tonometer) or a Digilab pneumatonometer (Bio-Rad Ophthalmic Division, Cambridge, MA, USA). IOP was measured before treatment and generally at 30, 60, 124, 180,
• Prostanoid receptor (PG) cDNAs corresponding to full length coding sequences were subcloned into the approp ⁇ ate sites of the mammalian expression vector pCEP4 (Invitrogen).
• pCEP4PG plasmid DNA was prepared using the Qiagen plasmid preparation kit (QIAGEN) and transfected into HEK 293(EBNA) cells using LipofectAMTNE® (GEBCO-BRL) according to the manufacturers' instructions HEK 293(EBNA) cells expressing the cDNA together with the hygromycin resistance gene were selected in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10 % heat inactivated fetal bovine serum, 1 mM sodium pyruvate, 100 U/ml Penicilhn- G, 100 /xg/ml Streptomycin sulphate, 250 ⁇ g/ml active GENETICINTM (G418) (all from Life Technologies, Inc./BR
• HEK 293(EBNA) cells were grown in supplemented DMEM complete medium at 37°C in a humidified atmosphere of 6 % CO 2 in air, then harvested and membranes prepared by differential cent ⁇ fugation (1000 x g for 10 min, then 160,000 x g for 30 min, all at 4°C) following lysis of the cells by nitrogen cavitation at 800 psi for 30 min on ice in the presence of protease inhibitors (2 mM phenylmethylsulfonylfluo ⁇ de, 10 ⁇ M E-64, 100 ⁇ M leupeptin and 0.05 mg/ml pepstatin).
• protease inhibitors 2 mM phenylmethylsulfonylfluo ⁇ de, 10 ⁇ M E-64, 100 ⁇ M leupeptin and 0.05 mg/ml pepstatin.
• Prostanoid receptor binding assays were performed in a final incubation volume of 0.2 ml in 10 mM MES/KOH (pH 6.0) (EP subtypes, FP and TP) or 10 mM HEPES/KOH (pH 7.4) (DP and IP), containing 1 mM EDTA, 10 mM MgCl 2 (EP subtypes) or 10 mM MnCl 2 (DP, FP, IP and TP) and radioligand [0.5-1.0 nM [ 3 H]PGE 2 (181 Ci/mmol) for EP subtypes, 0.7 nM [ 3 H]PGD 2 (115 Ci/mmol) for DP, 0.95 nM [ 3 H]PGF 2 ⁇ (170 Ci/mmol) for FP, 5 nM [ 3 H]iloprost (16 Ci/mmol) for TP and 1.8 nM [ 3 H]SQ 29548 (46 Ci/mmol) for TP].
• EP 3 assays also contained 100 ⁇ M GTP ⁇ S.
• the reaction was initiated by addition of membrane protein (approximately 30 ⁇ g for EPi, 20 ⁇ g for EP 2 , 2 ⁇ g for EP 3 , 10 ⁇ g for EP 4 , 60 ⁇ g for FP, 30 ⁇ g for DP, 10 ⁇ g for IP and 10 ⁇ g for TP) from the 160,000 x g fraction.
• Ligands were added in dimethylsulfoxide (Me 2 SO) which was kept constant at 1 %
• the filters were washed with 3-4 ml of the same buffer, dried for 90 min at 55°C and the residual radioactivity bound to the individual filters determined by scintillation counting with addition of 50 ⁇ l of Ultima Gold F (Canberra Packard) using a 1450 MicroBeta (Wallac). Specific binding was calculated by subtracting non-specific binding from total binding. Specific binding represented 90-95 % of the total binding and was linear with respect to the concentrations of radioligand and protein used. Total binding represented 5-10 % of the radioligand added to the incubation media. The activity range of the compounds of this is between 0.01 and 100,000 nM.

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