PROSTAGLANDIN E1 AND E2 ANALOGS FOR THE TREATMENT OF VARIOUS MEDICAL CONDITIONS

Abstract

A prostaglandin analog with selectivity to EP receptors and demonstrating EP agonist activity that may be used to expand hematopoietic stem cell populations or to treat or prevent influenza, bone fracture, bone disease, glaucoma, ocular hypertension, dysmenorrhoea, pre-term labor, immune disorders, osteoporosis, asthma, allergy, male sexual dysfunction, female sexual dysfunction, periodontal disease, gastric ulcer, renal disease, or other EP receptor-mediated conditions.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention claims priority from U.S. Provisional Application No. 60/987,859 filed Nov. 14, 2007 entitled “Prostaglandin E₁ and E₂ Derivatives as Selective E₂ Agonists for Medicinal Treatment,” and U.S. Provisional Application No. 61/037,493 filed Mar. 18, 2008 entitled “Prostaglandin E₁ and E₂ Derivatives as Selective E₂ Agonists for Medicinal Treatment.”

FIELD OF THE INVENTION

The present invention relates to pharmaceutically active compounds and more particularly to prostaglandin analogs with selectivity for prostaglandin E (EP) receptors and demonstrating EP agonist activity, and the use of such compounds and compositions thereof for the treatment of various medical conditions.

BACKGROUND OF THE INVENTION

Prostanoids are ubiquitous lipid mediator biomolecules involved in numerous physiological processes, such as the contraction and relaxation of smooth muscle, vasodilation, vasoconstriction, pain, regulation of blood pressure, and modulation of inflammation. Prostanoids are a family of eicosanoids that comprise prostaglandins (PGs), prostacyclins (PGIs), and thromboxanes (Txs). Their receptors belong to the G-protein coupled receptor (GPCR) superfamily of receptors and may be grouped into five classes, namely, prostaglandin D (DP), prostaglandin E (EP), prostaglandin F (FP), prostaglandin I (IP), and Thromboxane A (TP) based on their sensitivity to five naturally occurring prostanoids, PGD₂, PGE₂, PGF_2α, PGI₂, and TxA₂, respectively (Coleman, R. A., Prostanoid Receptors. IUPHAR compendium of receptor characterization and classification, 2^nd edition, 338-353, 2000). EP receptors have been characterized into four subtypes EP₁, EP₂, EP₃, and EP₄. Each subtype has been cloned and is distinct at both a molecular and pharmacological level.

Prostanoids are synthesized from essential fatty acids comprising twenty carbon atoms, such as arachidonic acid and 8,11,14-eicosatrienoic acid. Prostanoids are synthesized in response to both extracellular and intracellular stimuli and are then rapidly released from the cells. In general, the short half-lives of most prostanoids ensure they act near the sites of their biosynthesis.

Prostaglandin E₂ (PGE₂) is a potent endogenous EP receptor agonist derived from arachidonic acid, shown below, and possesses two carbon-carbon double bonds, one in each the α-chain and co-chain, and is thus called a “Series 2” prostaglandin.

Prostaglandin E₁ (PGE₁) is derived from 8,11,14-eicosatrienoic acid and possesses only one carbon-carbon double bond, located in the co-chain, and is thus called a “Series 1” prostaglandin.

Both prostanoid and non-prostanoid EP receptor agonists are known. EP receptor agonists may have a number of utilities. These include, but are not limited to treatment of influenza (WO 2008/058766), bone fracture healing (Li, M., et al., J. Bone Miner. Res., 18(11), 2003, 2033-2042; Paralkar, V. M., PNAS, 100(11), 2003, 6736-6740; WO 2002/24647; WO 1998/27976), bone disease (WO 2002/24647), glaucoma (WO 2008/015517; WO 2007/027468; WO 2003/040126), ocular hypertension (WO 2003/040126), dysmenorrhoea (WO 2003/037433), pre-term labor (GB 2 293 101), immune disorders (WO 2003/037433), osteoporosis (WO 1998/27976; WO 2001/46140), asthma (WO 2003/037433), allergy (WO 2003/037433), fertility (Breyer, R. M., et al., Ann. N.Y. Acad. Sci., 905, 2000, 221-231), male sexual dysfunction (WO 2000/40248), female sexual dysfunction (U.S. Pat. No. 6,562,868), periodontal disease (WO 2000/31084), gastric ulcer (U.S. Pat. No. 5,576,347), and renal disease (WO 1998/34916). EP receptor agonists may also be useful for expansion of hematopoietic stem cell populations (WO 2008/073748; North, T. E., et al., Nature, 447, 200 7, 1007-1011).

SUMMARY OF THE INVENTION

The exemplary embodiments may be directed to compounds of structural formula (I) that may be used to expand hematopoietic stem cell populations or to treat or prevent influenza, bone fracture, bone disease, glaucoma, ocular hypertension, dysmenorrhoea, pre-term labor, immune disorders, osteoporosis, asthma, allergy, male sexual dysfunction, female sexual dysfunction, periodontal disease, gastric ulcer, renal disease, or other EP receptor-mediated conditions wherein C⁹, C¹¹, R¹, Z¹, Z², Z³, Z⁴, Z¹, Z⁶, and Z⁷ are defined herein:

Another aspect of the embodiment is a pharmaceutical composition comprising a pharmaceutically effective amount of a compound according to formula (I), any stereoisomer or geometric isomer thereof, or a prodrug thereof, or a hydrate or solvate thereof, or a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable carrier.

Another aspect of the embodiment is directed to a method of expanding hematopoietic stem cell populations in a culture or patient in need thereof by administering to the culture or patient a compound according to formula (I), any stereoisomer or geometric isomer thereof, or a prodrug thereof, or a hydrate or solvate thereof, or a pharmaceutically acceptable salt thereof.

Another aspect of the embodiment is directed to a method of treating or preventing influenza, bone fracture, bone disease, glaucoma, ocular hypertension, dysmenorrhoea, pre-term labor, immune disorders, osteoporosis, asthma, allergy, male sexual dysfunction, female sexual dysfunction, periodontal disease, gastric ulcer, renal disease, or other EP receptor-mediated conditions in a patient in need thereof by administering to the patient a compound according to formula (I), any stereoisomer or geometric isomer thereof, or a prodrug thereof, or a hydrate or solvate thereof, or a pharmaceutically acceptable salt thereof.

Other exemplary embodiments of the invention will become apparent from the detailed description provided hereinafter. It should be understood that the detailed description and specific examples, while disclosing exemplary embodiments of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The exemplary embodiments are directed to a compound of formula (I), their preparation, pharmaceutical compositions comprising these compounds, and their pharmaceutical use in the prevention and treatment of EP receptor-mediated diseases or conditions. The compounds of formula (I) are shown below:

wherein:
dashed bonds may each independently represent a second carbon-carbon bond in order to give a carbon-carbon double bond with either (E) or (Z) geometry or may be ignored in order to give a carbon-carbon single bond;
C⁹ and C¹¹ each is independently C═CH₂, C═O, CF₂, CHF (any stereoisomer), or C(H)OH (any stereoisomer) with the proviso that C⁹ does not equal C¹¹, and also with the proviso that when one of either C⁹ or C¹¹ is C═O, and the other is C(H)OH, at least one of Z², Z³, Z⁴, and Z⁵ is fluorine, and also with the proviso that when one of either C⁹ or C¹¹ is CHF, the other is not C(H)OH;
R¹ is CO₂R³, CH₂OR³, CONR⁴R⁵, COCH₂OH, CONR⁴SO₂R⁵, P(O)(OR⁴)₂, or

R³ is hydrogen or (C₁-C₆)-alkyl;
R⁴ and R⁵ each is independently hydrogen or (C₁-C₆)-alkyl;
Z¹ are hydrogen or fluorine;
Z² and Z³ each is independently hydrogen or fluorine;
Z⁴ and Z⁵ each is independently hydrogen, fluorine, hydroxy, or methyl, or together are an oxygen atom that form a carbonyl group with the adjoining carbon atom of the ω chain;
Z⁶ and Z⁷ each is independently hydrogen, fluorine, hydroxy, or methyl, or together are an oxygen atom that form a carbonyl group with the adjoining carbon atom of the ω chain;

The exemplary embodiment above may also include any stereoisomer or geometric isomer thereof, or an equivalent thereof, or a prodrug thereof, or a hydrate or solvate thereof, or a pharmaceutically acceptable salt thereof.

Another exemplary embodiment may be directed to a compound of formula (I) wherein C⁹ is C═O and C¹¹ is C═CH₂.

Another exemplary embodiment may be directed to a compound of formula (I) wherein C⁹ is C(H)OH and C¹¹ is C═CH₂.

Another exemplary embodiment may be directed to a compound of formula (I) wherein C⁹ is C═CH₂ and C¹¹ is C═O.

Another exemplary embodiment may be directed to a compound of formula (I) wherein C⁹ is C═O and C¹¹ is CF₂.

Another exemplary embodiment may be directed to a compound of formula (I) wherein C⁹ is C(H)OH and C¹¹ is CF₂.

Another exemplary embodiment may be directed to a compound of formula (I) wherein C⁹ is CF₂ and C¹¹ is C═O.

Another exemplary embodiment may be directed to a compound of formula (I) wherein C⁹ is CF₂ and C¹¹ is C(H)OH.

Another exemplary embodiment may be directed to a compound of formula (I) wherein C⁹ is C═O and C¹¹ is CHF.

Another exemplary embodiment may be directed to a compound of formula (I) wherein C⁹ is CHF and C¹¹ is C═O.

Another exemplary embodiment may be directed to a compound of formula (I) wherein R¹ is CO₂H.

Another exemplary embodiment may be directed to a compound of formula (I) wherein R¹ is CO₂ⁱPr.

Another exemplary embodiment may be directed to a compound of formula (I) wherein R¹ is CON(H)Et.

Another exemplary embodiment may be directed to a compound of formula (I) wherein R¹ is CON(H)SO₂Me.

Another exemplary embodiment may be directed to a compound of formula (I) wherein R¹ is CH₂OH.

Another exemplary embodiment may be directed to a compound of formula (I) wherein R¹ is

Another exemplary embodiment may be directed to a compound of formula (I) wherein Z¹ is hydrogen.

Another exemplary embodiment may be directed to a compound of formula (I) wherein Z¹ is fluorine.

Another exemplary embodiment may be directed to a compound of formula (I) wherein Z² is fluorine and Z³ is hydrogen.

Another exemplary embodiment may be directed to a compound of formula (I) wherein Z² is hydrogen and Z³ is fluorine.

Another exemplary embodiment may be directed to a compound of formula (I) wherein each Z⁴ and Z⁵ is fluorine.

Another exemplary embodiment may be directed to a compound of formula (I) wherein each Z⁴ and Z⁵ is methyl.

Another exemplary embodiment may be directed to a compound of formula (I) wherein Z⁴ is hydroxy and Z⁵ is methyl.

Another exemplary embodiment may be directed to a compound of formula (I) wherein Z⁴ and Z⁵ together is an oxygen atom that form a carbonyl with the adjoining carbon atom.

Another exemplary embodiment may be directed to a compound of formula (I) wherein each Z⁶ and Z⁷ is hydrogen.

Another exemplary embodiment may be directed to a compound of formula (I) wherein each Z⁶ and Z⁷ is fluorine.

Another exemplary embodiment may be directed to a compound of formula (I) wherein each Z⁶ and Z⁷ is methyl.

Another exemplary embodiment may be directed to a compound of formula (I) wherein Z⁶ is hydroxy and Z⁷ is hydrogen.

Another exemplary embodiment may be directed to a compound of formula (I) wherein Z⁶ is hydroxy and Z⁷ is methyl.

Another exemplary embodiment may be directed to a compound of formula (I) wherein Z⁶ is methyl and Z⁷ is hydrogen.

Another exemplary embodiment may be directed to a compound of formula (I) wherein Z⁶ and Z⁷ together is an oxygen atom that form a carbonyl with the adjoining carbon atom.

Another exemplary embodiment may be directed to a more specific embodiment of the compound of formula (I), namely to a compound of formula (I):

or an equivalent thereof, or a hydrate or solvate thereof, or a pharmaceutically acceptable salt thereof.

Another exemplary embodiment may be directed to a more specific embodiment of the compound of formula (I), namely to a compound of formula (III):

or an equivalent thereof, or a hydrate or solvate thereof, or a pharmaceutically acceptable salt thereof.

Another exemplary embodiment may be directed to a more specific embodiment of the compound of formula (I), namely to a compound of formula (IV):

or an equivalent thereof, or a hydrate or solvate thereof, or a pharmaceutically acceptable salt thereof.

Another exemplary embodiment may be directed to a more specific embodiment of the compound of formula (I), namely to a compound of formula (V):

or an equivalent thereof, or a hydrate or solvate thereof, or a pharmaceutically acceptable salt thereof.

Another exemplary embodiment may be a compound selected from the group consisting of: (Z)-2,2-difluoro-7-((1R,2R)-2-((S,E)-3-hydroxyoct-1-enyl)-3-methylene-5-oxocyclopentyl)hept-5-enoic acid; (Z)-7-((1R,2R)-2-((1R,3S)-1-fluoro-3-hydroxyoctyl)-3-methylene-5-oxocyclopentyl)hept-5-enoic acid; (Z)-7-((1R,2R)-2-((1S,3S)-2-fluoro-3-hydroxyoctyl)-3-methylene-5-oxocyclopentyl)hept-5-enoic acid; (Z)-7-((1R,2R)-2-((2R,3S)-2-fluoro-3-hydroxyoctyl)-3-methylene-5-oxocyclopentyl)hept-5-enoic acid; (Z)-7-((1R,2R)-2-((2S,3S)-2-fluoro-3-hydroxyoctyl)-3-methylene-5-oxocyclopentyl)hept-5-enoic acid; (Z)-7-((1R,2R)-2-((E)-3,3-difluorooct-1-enyl)-3-methylene-5-oxocyclopentyl)hept-5-enoic acid; (Z)-7-((1R,2R)-2-((R,E)-3-hydroxy-3-methyloct-1-enyl)-3-methylene-5-oxocyclopentyl)hept-5-enoic acid; (Z)-7-((1R,2R)-2-((R,E)-3-hydroxy-4,4-dimethyloct-1-enyl)-3-methylene-5-oxocyclopentyl)hept-5-enoic acid; (Z)-7-((1R,2R)-2-((R,E)-4,4-difluoro-3-hydroxyoct-1-enyl)-3-methylene-5-oxocyclopentyl)hept-5-enoic acid; and (Z)-7-((1R,2R)-2-((S,E)-3-hydroxy-3-methyloct-1-enyl)-3-methylene-5-oxocyclopentyl)hept-5-enoic acid; or an equivalent thereof, or a (C₁-C₆)-alkyl ester thereof, or an N—(C₁-C₆)-alkyl amide thereof, or an N-methylsulfonyl amide thereof, or a hydrate, solvate, or a pharmaceutically acceptable salt thereof.

Another exemplary embodiment may be a compound selected from the group consisting of: (Z)-7-((1R,2R)-2-((E)-3,3-difluorooct-1-enyl)-5-methylene-3-oxocyclopentyl)hept-5-enoic acid; (Z)-7-((1R,2R)-3,3-difluoro-2-((S,E)-3-hydroxy-3-methyloct-1-enyl)-5-oxocyclopentyl)hept-5-enoic acid; (Z)-7-((1R,2R,3R)-2-((E)-3,3-difluorooct-1-enyl)-3-hydroxy-5-methylenecyclopentyl)hept-5-enoic acid; (Z)-7-((1R,2R,3R)-3-fluoro-2-((R,E)-3-hydroxy-4,4-dimethyloct-1-enyl)-5-oxocyclopentyl)hept-5-enoic acid; (Z)-7-((1R,2R,3S)-3-fluoro-2-((R,E)-3-hydroxy-4,4-dimethyloct-1-enyl)-5-oxocyclopentyl)hept-5-enoic acid; (Z)-7-((1R,2R,5R)-5-fluoro-2-((R,E)-3-hydroxy-4,4-dimethyloct-1-enyl)-3-oxocyclopentyl)hept-5-enoic acid; (Z)-7-((1R,2R,5S)-2-((E)-3,3-difluorooct-1-enyl)-5-hydroxy-3-methylenecyclopentyl)hept-5-enoic acid; (Z)-7-((1R,2R,5S)-3,3-difluoro-5-hydroxy-2-((R,E)-3-hydroxy-4,4-dimethyloct-1-enyl)cyclopentyl)hept-5-enoic acid; (Z)-7-((1R,2R,5S)-5-fluoro-2-((R,E)-3-hydroxy-4,4-dimethyloct-1-enyl)-3-oxocyclopentyl)hept-5-enoic acid; (Z)-7-((1R,4R,5R)-2,2-difluoro-4-hydroxy-5-((R,E)-3-hydroxy-4,4-dimethyloct-1-enyl)cyclopentyl)hept-5-enoic acid; and (Z)-7-((1R,5R)-2,2-difluoro-5-((S,E)-3-hydroxy-3-methyloct-1-enyl)-4-oxocyclopentyl)hept-5-enoic acid; or an equivalent thereof, or a (C₁-C₆)-alkyl ester thereof, or an N—(C₁-C₆)-alkyl amide thereof, or an N-methylsulfonyl amide thereof, or a hydrate, solvate, or a pharmaceutically acceptable salt thereof.

The exemplary embodiments may also be directed to a method of preventing or treating a disease or condition mediated at least in part by agonism of an EP receptor, in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of a compound of any exemplary embodiment of formula (I), or a pharmaceutically acceptable salt, hydrate, or solvate thereof; the use of a compound of any exemplary embodiment of formula (I), or a pharmaceutically acceptable salt, hydrate, or solvate thereof, for the manufacture of a medicament for preventing or treating a disease or condition mediated at least in part by agonism of an EP receptor; a compound of any exemplary embodiment of formula (I), or a pharmaceutically acceptable salt, hydrate, or solvate thereof, for use as a medicament; a compound of any exemplary embodiment of formula (I), or a pharmaceutically acceptable salt, hydrate, or solvate thereof, for use in the prevention or treatment of a disease or condition mediated at least in part by agonism of an EP receptor; a pharmaceutical composition comprising a compound of any exemplary embodiment of formula (I), or a pharmaceutically acceptable salt, hydrate, or solvate thereof, and a pharmaceutically acceptable excipient; a pharmaceutical composition for the prevention and treatment of a disease or condition mediated at least in part by agonism of an EP receptor comprising a compound of any exemplary embodiment of formula (I), or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

The diseases and conditions mediated at least in part by agonism of an EP receptor may include allergy and allergic inflammation. Diseases and conditions of this kind may be allergic respiratory conditions such as allergic rhinitis, nasal congestion, rhinorrhea, perennial rhinitis, nasal inflammation, asthma of all types, chronic obstructive pulmonary disease (COPD), chronic or acute bronchoconstriction, chronic bronchitis, small airways obstruction, emphysema, chronic eosinophilic pneumonia, adult respiratory distress syndrome, exacerbation of airways hyper-reactivity consequent to other drug therapy, airways disease that may be associated with pulmonary hypertension, acute lung injury, bronchiectasis, sinusitis, allergic conjunctivitis, or atopic dermatitis, particularly asthma or chronic obstructive pulmonary disease.

Types of asthma may include atopic asthma, non-atopic asthma, allergic asthma, atopic bronchial IgE-mediated asthma, bronchial asthma, essential asthma, true asthma, intrinsic asthma caused by pathophysiologic disturbances, extrinsic asthma caused by environmental factors, essential asthma of unknown or inapparent cause, bronchitic asthma, emphysematous asthma, exercise-induced asthma, exertion asthma, allergen-induced asthma, cold air induced asthma, occupational asthma, infective asthma caused by bacterial, fungal, protozoal, or viral infection, non-allergic asthma, incipient asthma, wheezy infant syndrome, and bronchiolytis.

Included in the use of the compounds of any exemplary embodiment of formula (I) for the treatment of asthma, may be palliative treatment for the symptoms and conditions of asthma such as wheezing, coughing, shortness of breath, tightness in the chest, shallow or fast breathing, nasal flaring (nostril size increases with breathing), retractions (neck area and between or below the ribs moves inward with breathing), cyanosis (gray or bluish tint to skin, beginning around the mouth), runny or stuffy nose, and headache.

The exemplary embodiments may also be directed to any of the uses, methods, or compositions as defined above wherein the compound of any exemplary embodiment of formula (I), or a pharmaceutically acceptable salt, hydrate, or solvate thereof, may be used in combination with another pharmacologically active compound. Specific combinations useful for the treatment of allergy or asthma may include combinations comprising a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, or solvate thereof, and (i) a glucocorticosteroid or DAGR (dissociated agonist of the corticoid receptor); (ii) a β₂ agonist, an example of which is a long-acting β₂ agonist; (iii) a muscarinic M3 receptor antagonist or anticholinergic agent; (iv) a histamine receptor antagonist or inverse agonist, which may be an H1 or an H3 antagonist or inverse agonist; (v) a 5-lipoxygenase inhibitor; (vi) a thromboxane inhibitor; (vii) an LTD₄ inhibitor; (viii) a kinase inhibitor; or (ix) a vaccine. Generally, the compounds of the combination may be administered together as a formulation in association with one or more pharmaceutically acceptable excipients.

Other diseases and conditions that may be mediated, at least in part, by agonism of an EP receptor are influenza, bone fracture healing, bone disease, glaucoma, ocular hypertension, dysmenorrhoea, pre-term labor, immune disorders, osteoporosis, asthma, allergy, fertility, male sexual dysfunction, female sexual dysfunction, periodontal disease, gastric ulcer, and renal disease. EP receptor agonists may also be useful for expansion of hematopoietic stem cell populations.

Besides being useful for human treatment, compounds of formula (I) may also be useful for veterinary treatment of companion animals, exotic animals, and farm animals.

When used in the present application, the following abbreviations have the meaning set out below: Ac is acetyl; ACN is acetonitrile; BBr₃ is boron tribromide; Bn is benzyl; BnNH₂ is benzylamine; BSA is bovine serum albumin; CH₂Cl₂ is dichloromethane; CHCl₃ is chloroform; CDCl₃ is deuterochloroform; DAST is diethylaminosulfur trifluoride; DCC is N,N′-dicyclohexylcarbodiimide; DCM is dichloromethane; DIBAL-His diisobutylaluminum hydride; DME is 1,2-dimethoxyethane; DMEM is Dulbecco's minimal essential medium; DMF is N,N-dimethylformamide; DMSO is dimethyl sulfoxide; DBU is 1,8-diazabicyclo[5.4.0]undec-7-ene; EDC/EDAC is N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride; EDTA is ethylenediaminetetraacetic acid; EIA is enzyme immunoassay; Et is ethyl; Et₃N is triethylamine; HOBt is 1-hydroxybenzotriazole; HBSS is Hank's balanced salt solution; IBMX is isobutylmethylxanthine; ⁱPr is isopropyl; MCS is multiple cloning site; Me is methyl; MES is 2-(N-morpholino)ethanesulfonic acid; NaHMDS is sodium hexamethyldisilazane, also known as sodium bis(trimethylsilyl)amide; NMP is 1-methyl-2-pyrrolidinone; PCR is polymerase chain reaction; Ph is phenyl; Pd(PPh₃)₄ is tetrakis(triphenylphosphine)palladium; PhB(OH)₂ is benzeneboronic acid, also known as phenylboronic acid; PhMe is toluene; rt is room temperature; TBAF is tetrabutylammonium fluoride; t-Bu is tert-butyl; TCA is trichloroacetic acid; THF is tetrahydrofuran; and Tris-HCl is 2-amino-2-(hydroxymethyl)-1,3-propanediol hydrochloride.

Unless otherwise defined herein, scientific and technical terms used in connection with the exemplary embodiments shall have the meanings that are commonly understood by those of ordinary skill in the art.

Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclature used in connection with, and techniques of chemistry and molecular biology described herein are those well known and commonly used in the art.

The phrase “therapeutically effective” is intended to qualify the amount of compound or pharmaceutical composition, or the combined amount of active ingredients in the case of combination therapy. This amount or combined amount may achieve the goal of treating the relevant condition.

The term “treatment,” as used herein to describe the exemplary embodiments and unless otherwise qualified, means administration of the compound, pharmaceutical composition, or combination to effect preventative, palliative, supportive, restorative, or curative treatment. The term treatment encompasses any objective or subjective improvement in a subject with respect to a relevant condition or disease.

The term “preventative treatment,” as used herein to describe the exemplary embodiments, means that the compound, pharmaceutical composition, or combination may be administered to a subject to inhibit or stop the relevant condition from occurring in a subject, particularly in a subject or member of a population that may be significantly predisposed to the relevant condition.

The term “palliative treatment,” as used herein to describe the exemplary embodiments, means that the compound, pharmaceutical composition, or combination may be administered to a subject to remedy signs and/or symptoms of a condition, without necessarily modifying the progression of, or underlying etiology of, the relevant condition.

The term “supportive treatment,” as used herein to describe the exemplary embodiments, means that the compound, pharmaceutical composition, or combination may be administered to a subject as part of a regimen of therapy, but that such therapy is not limited to administration of the compound, pharmaceutical composition, or combination. Unless otherwise expressly stated, supportive treatment may embrace preventative, palliative, restorative, or curative treatment, particularly when the compounds or pharmaceutical compositions are combined with another component of supportive therapy.

The term “restorative treatment,” as used herein to describe the exemplary embodiments, means that the compound, pharmaceutical composition, or combination may be administered to a subject to modify the underlying progression or etiology of a condition. Non-limiting examples include an increase in forced expiratory volume in one second (FEV 1) for lung disorders, inhibition of progressive nerve destruction, reduction of biomarkers associated and correlated with diseases or disorders, a reduction in relapses, improvement in quality of life, and the like.

The term “curative treatment,” as used herein to describe the exemplary embodiments, means that the compound, pharmaceutical composition, or combination may be administered to a subject for the purpose of bringing the disease or disorder into complete remission, or that the disease or disorder in undetectable after such treatment.

The term “alkyl,” alone or in combination, means an acyclic radical, linear or branched, preferably containing from 1 to about 6 carbon atoms. Examples of such radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, heptyl, octyl, and the like. Where no specific substitution is specified, alkyl radicals may be optionally substituted with groups consisting of hydroxy, sulfhydryl, methoxy, ethoxy, amino, cyano, chloro, and fluoro.

The carbon atom content of various hydrocarbon-containing moieties is indicated by a prefix designating a lower and upper number of carbon atoms in the moiety, that is, the prefix C_i-C_j indicates a moiety of the integer “i” to the integer “j” carbon atoms, inclusive. Thus, for example, ‘(C₁-C₈)-alkyl’ refers to alkyl of one to eight carbon atoms, inclusive.

The terms “hydroxy” and “hydroxyl,” as used herein, mean an OH radical.

The term “sulfhydryl,” as used herein, means an SH radical.

The term “oxo” means a doubly bonded oxygen.

The term “alkoxy” means a radical comprising an alkyl radical that is bonded to an oxygen atom, such as a methoxy radical.

The term “aryl” means a fully unsaturated mono- or multi-ring cycloalkyl having a cyclic array of p-orbitals containing 4n+2 electrons, including, but not limited to, substituted or unsubstituted phenyl, naphthyl, or anthracenyl optionally fused to a carbocyclic radical wherein aryl may be optionally substituted with one or more substituents from the group consisting of halo, methoxy, ethoxy, (C₁-C₆)-alkyl, phenyl, O-phenyl, cyano, nitro, hydroxyl, sulfhydryl, or trifluoromethyl.

The term “halo,” as used herein, means one of the following group consisting of fluoro, chloro, bromo, or iodo.

The terms “heterocycle”, “heterocyclic ring system,” and “heterocyclyl” refer to a saturated or unsaturated mono- or multi-ring cycloalkyl wherein one or more carbon atoms is replaced by N, S, or O. The terms “heterocycle”, “heterocyclic ring system,” and “heterocyclyl” include fully saturated ring structures such as piperazinyl, dioxanyl, tetrahydrofuranyl, oxiranyl, aziridinyl, morpholinyl, pyrrolidinyl, piperidinyl, thiazolidinyl, and others. The terms “heterocycle”, “heterocyclic ring system,” and “heterocyclyl” also include partially unsaturated ring structures such as dihydrofuranyl, pyrazolinyl, imidazolinyl, pyrrolinyl, chromanyl, dihydrothiphenyl, and others.

The term “heteroaryl” refers to an aromatic heterocyclic group. Heteroaryl is preferably: (a) a five-membered aromatic heterocyclic group containing either (i) 1-4 nitrogen atoms or (ii) 0-3 nitrogen atoms and 1 oxygen or 1 sulfur atom; (b) a six-membered aromatic heterocyclic group containing 1-3 nitrogen atoms; (c) a nine-membered bicyclic heterocyclic group containing either (i) 1-5 nitrogen atoms or (ii) 0-4 nitrogen atoms and 1 oxygen or 1 sulfur atom; or (d) a ten-membered bicyclic aromatic heterocyclic group containing 1-6 nitrogen atoms; each of said groups (a)-(d) being optionally substituted by one or more of (C₁-C₆)-alkyl, (C₁-C₆)-fluoroalkyl, (C₃-C₆)-cycloalkyl, hydroxy(C₃-C₆)-cycloalkyl, (C₂-C₆)-alkenyl, (C₂-C₆)-alkynyl, halo, oxo, hydroxyl, (C₁-C₆)-alkoxy, sulfhydryl, —SMe, or cyano. Examples of “heteroaryl” include pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, thionyl, furanyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, and tetrazolyl, optionally substituted as specified above.

In “heterocycle” or “heteroaryl,” the point of attachment to the molecule of interest may be at a heteroatom or elsewhere within the ring.

The term “cycloalkyl” means a mono- or multi-ringed cycloalkyl wherein each ring contains three to ten carbon atoms, preferably three to six carbon atoms. “Cycloalkyl” is preferably a monocyclic cycloalkyl containing from three to six carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

The term “excipient” is used herein to describe any ingredient other than a compound of formula (I). The choice of excipient will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. The term “excipient” encompasses diluents, carrier, or adjuvant.

Pharmaceutically acceptable salts of the compounds of formula (I) include the acid addition and base salts thereof.

Suitable acid addition salts are formed by acids which form non-toxic salts. Examples include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, propionate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate, naphthalene-1,5-disulfonic acid, and xinofoate salts.

Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc salts.

Hemisalts of acids and bases may also be formed, for example, hemisulfate and hemicalcium salts. For a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use, by Stahl and Wermuth (Wiley-VCH, 2002).

The compounds of any exemplary embodiment of formula (I) may also exist in unsolvated and solvated forms. The term “solvate” is used herein to describe a molecular complex comprising the compound of any exemplary embodiment of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term “hydrate” is employed when said solvent is water.

Also included herein are multi-component complexes other than salts and solvates wherein the compound of formula (I) and at least one other component are present in stoichiometric or non-stoichiometric amounts.

The compounds of any exemplary embodiment of formula (I) may exist in a continuum of solid states ranging from fully amorphous to fully crystalline.

The compounds of any exemplary embodiment of formula (I) may also exist in a mesomorphic state (mesophase or liquid crystal) when subjected to suitable conditions. The mesomorphic state is intermediate between the true crystalline state and the true liquid state (either melt or solution).

Hereinafter all references to compounds of any exemplary embodiment of formula (I) include references to salts, solvates, multi-component complexes, and liquid crystals thereof and to solvates, multi-component complexes, and liquid crystals of salts thereof.

Also included herein are all polymorphs and crystal habits of compounds of any exemplary embodiment of formula (I), prodrugs, and isomers thereof (including optical, geometric, and tautomeric isomers) and isotopically-labeled forms thereof.

Compounds of any exemplary embodiment of formula (I) may be administered orally, topically, transdermally, intranasally, by inhalation, directly into the bloodstream, into muscle, into an internal organ, into the eye, into the ear, into the rectum, or by other means.

The compounds herein, their methods or preparation and their biological activity will appear more clearly from the examination of the following examples that are presented as an illustration only and are not to be considered as limiting the invention in its scope. Compounds herein are identified, for example, by the following analytical methods.

Mass spectra (MS) methods include positive electrospray ionization (ESI⁺), negative electrospray ionization (ESI⁻), positive atmospheric pressure chemical ionization (APCI⁺), or negative atmospheric pressure chemical ionization (APCI⁻).

300 MHz proton nuclear magnetic resonance spectra (¹H NMR) are recorded at ambient temperature using a Bruker (300 MHz) spectrometer. In the ¹H NMR chemical shifts (δ) are indicated in parts per million (ppm) with reference to tetramethylsilane (TMS) as the internal standard.

The above description of embodiments of the invention is merely exemplary in nature and, thus, variations thereof are not to be regarded as a departure from the spirit and scope of the invention.

EXAMPLES

Example 1

Preparation of (Z)-7-((1R,2R)-2-((E)-3-hydroxy-3-methyloct-1-enyl)-3-methyl-5-oxocyclopent-3-enyl)hept-5-enoic Acid

Step A: Preparation of (3aR,4R,5R,6aS)-2-oxo-4-((E)-3-oxooct-1-enyl)hexahydro-2H-cyclopenta[b]furan-5-yl Benzoate

A reactor equipped with a mechanical stirrer, under nitrogen, was charged with (3aR,4R,5R,6aS)-4-formyl-2-oxohexahydro-2H-cyclopenta[b]furan-5-yl benzoate (Corey lactone aldehyde benzoate, Cayman Chemical Catalog No. 70030, 99.2 g, 0.362 mol) in DCM and lithium chloride (1 molar equivalent) dissolved in THF. Some lithium chloride precipitated from solution when the THF and DCM solutions were mixed. Dimethyl 2-oxoheptylphosphonate (1 molar equivalent) was subsequently added to the reactor NEAT and the reagent was rinsed down into the reactor with DCM. The mixture was stirred under nitrogen and cooled to −20° C. The lithium chloride precipitate dissolved as the stirring and cooling continued. After stirring for 2.5 hours, triethylamine (1 molar equivalent) was added NEAT via addition funnel and the temperature was maintained at −5° C. with stirring for 19 hours. The reaction mixture was warmed to 0° C. and treated with 5% aqueous citric acid. The layers were separated and the organic layer was dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified on silica gel, eluted with hexanes-ethyl acetates (1:1) to afford the title intermediate.

Step B: Preparation of (3aR,4R,5R,6aS)-4-((E)-3-hydroxy-3-methyloct-1-enyl)-2-oxohexahydro-2H-cyclopenta[b]furan-5-yl Benzoate

To a stirring mixture consisting of (3aR,4R,5R,6aS)-2-oxo-4-((E)-3-oxooct-1-enyl)hexahydro-2H-cyclopenta[b]furan-5-yl benzoate (limiting reagent, prepared in Step A) in THF (0.1 M) under a nitrogen atmosphere cooled to −78° C. was added methyl magnesium bromide (1 M solution in THF, 1 molar equivalent) dropwise. The reaction mixture was stirred at −78° C. until reaction progress stopped. Upon completion, brine was added to the crude reaction mixture and the product was extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and evaporated. The product was purified by flash chromatography on regular silica gel, eluted with hexanes-ethyl acetate to afford the title intermediate.

Step C: Preparation of (3aR,4R,5R,6aS)-4-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-2-oxohexahydro-2H-cyclopenta[b]furan-5-yl Benzoate

To a stirring mixture consisting of (3aR,4R,5R,6aS)-4-((E)-3-hydroxy-3-methyloct-1-enyl)-2-oxohexahydro-2H-cyclopenta[b]furan-5-yl benzoate (limiting reagent, prepared in Step B) and imidazole (1.1. molar equivalents) in DMF (5 M in limiting reagent) cooled to 0° C. under a nitrogen atmosphere was slowly added a solution consisting of TBDPSCl (1.1 molar equivalent) in DMF. Upon completion of the reaction, as judged by TLC, the reaction mixture was diluted with ethyl acetate and washed with water and brine. The organic layer was dried over sodium sulfate, filtered, and evaporated. The crude product was purified by flash chromatography on regular silica gel eluted with hexanes-ethyl acetate to afford the title intermediate.

Step D: Preparation of (3aR,4R,5R,6aS)-4-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-5-hydroxyhexahydro-2H-cyclopenta[b]furan-2-one

To a stirring mixture consisting of (3aR,4R,5R,6aS)-4-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-2-oxohexahydro-2H-cyclopenta[b]furan-5-yl benzoate (limiting reagent, prepared in Step C) in methanol (0.2 M) was added potassium carbonate (0.6 molar equivalent). The reaction mixture was stirred at room temperature and the progress was monitored by TLC every 30 minutes. After complete consumption of starting material, the reaction mixture was acidified with 5% KHSO₄ and diluted with brine. The product was extracted with ethyl acetate twice. The combined organic layers were dried over sodium sulfate, filtered, and evaporated. The crude product was purified by flash chromatography on regular silica gel eluted with hexanes-ethyl acetate (1:1) to afford the title intermediate.

Step E: Preparation of (3aR,4R,5R,6aS)-4-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-5-(tetrahydro-2H-pyran-2-yloxy)hexahydro-2H-cyclopenta[b]furan-2-one

To a stirring mixture consisting of (3aR,4R,5R,6aS)-4-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-5-hydroxyhexahydro-2H-cyclopenta[b]furan-2-one (limiting reagent, prepared in Step D) in DCM (0.1 M) under a nitrogen atmosphere was added dihydropyran (1.1 molar equivalent) followed by a catalytic amount of p-toluenesulfonic acid. The reaction mixture was stirred at room temperature under a nitrogen atmosphere and the reaction progress was monitored by TLC. Upon completion, brine was added to the reaction mixture and the layers were separated. The organic phase was dried over sodium sulfate, filtered, and the solvent was evaporated. The crude product was purified by flash chromatography on regular silica gel eluted with hexanes-ethyl acetate to afford the title intermediate.

Step F: Preparation of (3aR,4R,5R,6aS)-4-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-5-(tetrahydro-2H-pyran-2-yloxy)hexahydro-2H-cyclopenta[b]furan-2-ol

A stirring mixture consisting of (3aR,4R,5R,6aS)-4-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-5-(tetrahydro-2H-pyran-2-yloxy)hexahydro-2H-cyclopenta[b]furan-2-one (limiting reagent, prepared in Step E) in anhydrous THF (0.5 M) under a nitrogen atmosphere was cooled to −78° C. A solution consisting of DIBAL-H (1 M in toluene, 2 molar equivalents) was added to the reaction mixture dropwise and stirred for 3 hours. Ethyl acetate (20 mL) was added and the mixture was stirred for an additional 5 minutes. The mixture was subsequently treated with 30% aqueous K, Na tartrate and stirred vigorously overnight. The layers were separated and the organic phase was dried, filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography on regular silica gel eluted with hexanes-ethyl acetate to afford the title intermediate.

Step G: Preparation of (Z)-7-((1R,2R,3R,5S)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-5-hydroxy-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoic Acid

To a white suspension consisting of 4-carboxybutyltriphenylphosphonium bromide (3.5 molar equivalents) in anhydrous THF under nitrogen atmosphere was added dropwise a solution consisting of 1 M potassium tert-butoxide (7 molar equivalents) in THF. The reaction mixture became bright red over the course of the addition and was stirred for 30 minutes at room temperature and subsequently cooled to −15° C. with a ice/NaCl bath. The lactol (3aR,4R,5R,6aS)-4-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-5-(tetrahydro-2H-pyran-2-yloxy)hexahydro-2H-cyclopenta[b]furan-2-ol (limiting reagent, prepared in Step F) was dissolved in THF and added dropwise to the reaction mixture containing the ylide. The reaction mixture became lighter orange in color and was stirred for 2 hours at −15° C. and was subsequently allowed to warm to room temperature and stir overnight. The reaction mixture became dark red and TLC analysis indicated no remaining starting material. The reaction mixture was acidified with 5% KHSO₄, diluted with brine (250 mL), and extracted with ethyl acetate (200 mL). The aqueous layer was extracted with another portion of ethyl acetate (50 mL) and the combined organic extracts were washed twice with brine (2×250 mL), dried over sodium sulfate, and evaporated. The crude product was purified by flash chromatography on regular silica gel using hexanes-ethyl acetate with 0.4% acetic acid as eluent to afford the title intermediate.

Step H: Preparation of (Z)-methyl 7-((1R,2R,3R,5S)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-5-hydroxy-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoate

A stirring mixture consisting of (Z)-7-((1R,2R,3R,5S)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-5-hydroxy-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoic acid (prepared in Step G) in diethyl ether (0.1 M) was cooled to 0° C. under a nitrogen atmosphere. Diazomethane (freshly prepared solution in diethyl ether) was added to the stirring mixture until a light-yellow color persisted. The completion of the reaction was confirmed by the absence of starting material as judged by TLC. Upon completion, the solvents were evaporated and the product was purified by flash chromatography using hexanes-ethyl acetate as eluent to afford the title intermediate.

Step I: Preparation of (Z)-methyl 7-((1R,2R,3R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoate

To a stirring mixture consisting of (Z)-methyl 7-((1R,2R,3R,5S)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-5-hydroxy-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoate (limiting reagent, prepared in Step H) in DMF (5 M) and imidazole (1.1 molar equivalents) cooled to 0° C. under a nitrogen atmosphere was slowly added a solution consisting of TBDPSCl (1.1 molar equivalent) in DMF. Upon completion of the reaction, as judged by TLC, the reaction mixture was diluted with ethyl acetate and was washed with water and brine. The organic layer was dried over sodium sulfate, filtered, and evaporated. The crude product was purified by flash chromatography on regular silica gel eluted with hexanes-ethyl acetate to afford the title intermediate.

Step J: Preparation of (Z)-methyl 7-((1R,2R,3R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-3-hydroxycyclopentyl)hept-5-enoate

A mixture consisting of (Z)-methyl 7-((1R,2R,3R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3-(tert-butydiphenylsilyloxy)-3-methyloct-1-enyl)-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoate (prepared in Step I) in 4:2:1 acetic acid-water-THF (0.5 M) was stirred for several days at room temperature until the reaction was complete, as judged by TLC. The solvents were removed by evaporation and the crude product was purified by flash chromatography on regular silica gel using hexanes-ethyl acetate with 0.4% acetic acid as eluent to afford the title intermediate.

Step K: Preparation of (Z)-methyl 7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-3-oxocyclopentyl)hept-5-enoate

To a stirring mixture consisting of (Z)-methyl 7-((1R,2R,3R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-3-hydroxycyclopentyl)hept-5-enoate (limiting reagent, prepared in Step J) in acetone (0.1 M) cooled to −25° C. was added Jones reagent (1 molar equivalent) dropwise. Upon completion, as judged by TLC, the reaction was quenched with isopropyl alcohol and the crude reaction mixture was diluted with ethyl acetate, washed three times with brine, and dried over magnesium sulfate. After filtration and solvent evaporation the product was purified by flash chromatography using hexanes-ethyl acetate as eluent to afford the title intermediate.

Step L: Preparation of (Z)-methyl 7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-3-methylenecyclopentyl)hept-5-enoate

A stirring mixture consisting of (Z)-methyl 7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-3-oxocyclopentyl)hept-5-enoate (limiting reagent, prepared in Step K) in DCM (0.05 M) under a nitrogen atmosphere was cooled to 0° C. A zinc methylenedibromide titanium tetrachloride solution was prepared by combining stirring zinc dust (2.3 g) in THF (40 mL) with methylene dibromide (0.81 mL) at −40° C. under a nitrogen atmosphere. To the suspension was slowly added TiCl₄ (0.92 mL). Portions (2 mL) of zinc methylenedibromide titanium tetrachloride solution were added to the stirring mixture containing the limiting reagent until the reaction was complete as judged by TLC. Upon completion, the reaction mixture was diluted with ethyl acetate and filtered twice through a bed of Celite. The filtrate was washed with a saturated aqueous solution of sodium bicarbonate and subsequently with a 50% aqueous solution of brine. The organic phase was dried over sodium sulfate, filtered, and the solvent was evaporated. The crude product was purified by flash chromatography on regular silica gel eluted with hexanes-ethyl acetate to afford the title intermediate.

Step M: Preparation of (Z)-7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-3-methylenecyclopentyl)hept-5-enoic Acid

A mixture consisting of (Z)-methyl 7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3-(tert-butyldiphenylsilyloxy)-3-methyloct-1-enyl)-3-methylenecyclopentyl)hept-5-enoate (limiting reagent, prepared in Step L) in a 3:1 solution of methanol and 1 N LiOH (0.01 M) was stirred at 4° C. Upon completion of the reaction, as judged by TLC, the reaction mixture was diluted with ethyl acetate, washed with 5% KHSO₄, and brine. The organic phase was dried over sodium sulfate, filtered, and the solvent was evaporated. The crude product was purified by flash chromatography on regular silica gel eluted with hexanes-ethyl acetate to afford the title intermediate.

Step N: Preparation of (Z)-7-((1R,2R,5S)-5-hydroxy-2-((E)-3-hydroxy-3-methyloct-1-enyl)-3-methylenecyclopentyl)hept-5-enoic Acid

To a stirring mixture consisting of (Z)-7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3-(tert-butydiphenylsilyloxy)-3-methyloct-1-enyl)-3-methylenecyclopentyl)hept-5-enoic acid (limiting reagent, prepared in Step M) in THF (0.3 M) under a nitrogen atmosphere was added a solution of TBAF (1.2 molar equivalents; 1 M in THF) and the reaction mixture was stirred at room temperature. Upon completion, as judged by TLC, water was added and most of the THF was removed under reduced pressure. The remaining aqueous solution was extracted with ethyl acetate and was washed with water and brine. The organic phase was dried over sodium sulfate, filtered, and the solvent was evaporated. The crude product was purified by flash chromatography on regular silica gel eluted with hexanes-ethyl acetate with 0.4% acetic acid to afford the title compound.

Step O: Preparation of (Z)-7-((1R,2R)-2-((E)-3-hydroxy-3-methyloct-1-enyl)-3-methyl-5-oxocyclopent-3-enyl)hept-5-enoic Acid

To a stirring mixture consisting of (Z)-7-((1R,2R,5S)-5-hydroxy-2-((E)-3-hydroxy-3-methyloct-1-enyl)-3-methylenecyclopentyl)hept-5-enoic acid (limiting reagent, prepared in Step N) in acetone (0.1 M) cooled to −25° C. was added Jones reagent (1 molar equivalent) dropwise. Upon completion, as judged by TLC, the reaction was quenched with isopropyl alcohol and the crude reaction mixture was diluted with ethyl acetate, washed three times with brine, and dried over magnesium sulfate. After filtration and solvent evaporation the product was purified by flash chromatography using hexanes-ethyl acetate as eluent to afford an epimeric mixture of the title compound. The 15-hydroxy and 15-methyl epimers may be resolved or used as an epimeric mixture.

Example 2

Preparation of (Z)-7-((1R,2R)-2-((E)-3,3-difluorooct-1-enyl)-3-methyl-5-oxocyclopent-3-enyl)hept-5-enoic Acid

Step A: Preparation of (3aR,4R,5R,6aS)-4-((E)-3,3-difluorooct-1-enyl)-2-oxohexahydro-2H-cyclopenta[b]furan-5-yl Benzoate

To a stirring mixture consisting of (3aR,4R,5R,6aS)-2-oxo-4-((E)-3-oxooct-1-enyl)hexahydro-2H-cyclopenta[b]furan-5-yl benzoate (limiting reagent, prepared in Example 1, Step A) and a catalytic amount of ethanol (25 mole %) in anhydrous DCM (0.5 M in limiting reagent) cooled to 0° C. under nitrogen a atmosphere is slowly added DAST (5 molar equivalents). The reaction mixture is allowed to slowly warm to room temperature overnight. Stirring is continued for several days until the reaction is complete as judged by TLC. Upon completion the reaction is cooled to 0° C. and quenched by the slow addition of a saturated aqueous solution of sodium bicarbonate. The layers are separated and the aqueous phase is extracted with ethyl acetate. The organic layers are combined and dried over magnesium sulfate. The solvents are evaporated and the crude material is purified on regular silica gel eluted with hexanes-ethyl acetate to afford the title intermediate.

Step B: Preparation of (3aR,4R,5R,6aS)-4-((E)-3,3-difluorooct-1-enyl)-5-hydroxyhexahydro-2H-cyclopenta[b]furan-2-one

The title intermediate may be prepared from (3aR,4R,5R,6aS)-4-((E)-3,3-difluorooct-1-enyl)-2-oxohexahydro-2H-cyclopenta[b]furan-5-yl benzoate (prepared in Step A) using a procedure described in Example 1, Step D for the removal of the benzoate group.

Step C: Preparation of (3aR,4R,5R,6aS)-4-((E)-3,3-difluorooct-1-enyl)-5-(tetrahydro-2H-pyran-2-yloxy)hexahydro-2H-cyclopenta[b]furan-2-one

The title intermediate may be prepared from (3aR,4R,5R,6aS)-4-((E)-3,3-difluorooct-1-enyl)-5-hydroxyhexahydro-2H-cyclopenta[b]furan-2-one (prepared in Step B) using the THP-protection procedure described in Example 1, Step E.

Step D: Preparation of (3aR,4R,5R,6aS)-4-((E)-3,3-difluorooct-1-enyl)-5-(tetrahydro-2H-pyran-2-yloxy)hexahydro-2H-cyclopenta[b]furan-2-ol

The title intermediate may be prepared from (3aR,4R,5R,6aS)-4-((E)-3,3-difluorooct-1-enyl)-5-(tetrahydro-2H-pyran-2-yloxy)hexahydro-2H-cyclopenta[b]furan-2-one (prepared in Step C) using the procedure described in Example 1, Step F.

Step E: (Z)-7-((1R,2R,3R,5S)-2-((E)-3,3-difluorooct-1-enyl)-5-hydroxy-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoic Acid

The title intermediate may be prepared from the lactol (3aR,4R,5R,6aS)-4-((E)-3,3-difluorooct-1-enyl)-5-(tetrahydro-2H-pyran-2-yloxy)hexahydro-2H-cyclopenta[b]furan-2-ol (prepared in Step D) using the procedure described in Example 1, Step G.

Step F: Preparation of (Z)-methyl 7-((1R,2R,3R,5S)-2-((E)-3,3-difluorooct-1-enyl)-5-hydroxy-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoate

The title intermediate may be prepared from (Z)-7-((1R,2R,3R,5S)-2-((E)-3,3-difluorooct-1-enyl)-5-hydroxy-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoic acid (prepared in Step E) using the diazomethane procedure described in Example 1, Step H.

Step G: Preparation of (Z)-methyl 7-((1R,2R,3R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluorooct-1-enyl)-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoate

The title intermediate may be prepared from (Z)-methyl 7-((1R,2R,3R,5S)-2-((E)-3,3-difluorooct-1-enyl)-5-hydroxy-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoate (prepared in Step F) using the silylation procedure described in Example 1, Step I.

Step H: Preparation of (Z)-methyl 7-((1R,2R,3R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluorooct-1-enyl)-3-hydroxycyclopentyl)hept-5-enoate

The title intermediate may be prepared form (Z)-methyl 7-((1R,2R,3R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluorooct-1-enyl)-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl)hept-5-enoate (prepared in Step G) using the THP-deprotection procedure described in Example 1, Step J.

Step I: Preparation of (Z)-methyl 7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluorooct-1-enyl)-3-oxocyclopentyl)hept-5-enoate

The title intermediate may be prepared from (Z)-methyl 7-((1R,2R,3R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluorooct-1-enyl)-3-hydroxycyclopentyl)hept-5-enoate (prepared in Step H) using the Jones oxidation procedure described in Example 1, Step K.

Step J: Preparation of (Z)-methyl 7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluorooct-1-enyl)-3-methylenecyclopentyl)hept-5-enoate

The title intermediate may be prepared from (Z)-methyl 7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluorooct-1-enyl)-3-oxocyclopentyl)hept-5-enoate (prepared in Step I) using the procedure described in Example 1, Step L.

Step K: Preparation of (Z)-7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluorooct-1-enyl)-3-methylenecyclopentyl)hept-5-enoic Acid

The title intermediate may be prepared from (Z)-methyl 7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluorooct-1-enyl)-3-methylenecyclopentyl)hept-5-enoate (prepared in Step J) using the ester hydrolysis procedure described in Example 1, Step M.

Step L: Preparation of (Z)-7-((1R,2R,5S)-2-((E)-3,3-difluorooct-1-enyl)-5-hydroxy-3-methylenecyclopentyl)hept-5-enoic Acid

The title compound may be prepared from (Z)-7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluorooct-1-enyl)-3-methylenecyclopentyl)hept-5-enoic acid (prepared in Step K) using the silyl-deprotection procedure described in Example 1, Step N.

Step M: Preparation of (Z)-7-((1R,2R)-2-((E)-3,3-difluorooct-1-enyl)-3-methyl-5-oxocyclopent-3-enyl)hept-5-enoic Acid

The title compound may be prepared from (Z)-7-((1R,2R,5S)-2-((E)-3,3-difluorooct-1-enyl)-5-hydroxy-3-methylenecyclopentyl)hept-5-enoic acid (prepared in Step L) using the Jones oxidation procedure described in Example 1, Step O.

Example 3

Preparation of (Z)-7-((1R,2R,3R)-2-((E)-3,3-difluoro-4-hydroxyoct-1-enyl)-3-hydroxy-5-oxocyclopentyl)hept-5-enoic Acid

Step A: Preparation of dimethyl 2-oxo-3-(tetrahydro-2H-pyran-2-yloxy)heptylphosphonate

To a stirring mixture consisting of methyl 2-hydroxyhexanoate (limiting reagent, available from Sinochemexper) in DCM (0.1 M) under a nitrogen atmosphere is added dihydropyran (1.1 molar equivalents) followed by a catalytic amount of p-toluenesulfonic acid. The reaction mixture is stirred at room temperature and the reaction progress is monitored by TLC. Upon completion, brine is added to the reaction mixture and the layers are separated. The organic phase is dried over sodium sulfate, filtered, and the solvent is evaporated. The crude product is purified by flash chromatography on regular silica gel eluted with hexanes-ethyl acetate to give methyl 2-(tetrahydro-2H-pyran-2-yloxy)hexanoate. Methyl 2-(tetrahydro-2H-pyran-2-yloxy)hexanoate is converted to the title intermediate by treating with diethyl methyphosphonate as previously described in the Journal of Organic Chemistry, 73 (12), 200 8, 4568-4574.

Step B: Preparation of (3aR,4R,5R,6aS)-2-oxo-4-((E)-3-oxo-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)hexahydro-2H-cyclopenta[b]furan-5-yl Benzoate

A reactor equipped with a mechanical stirrer purged with nitrogen gas is charged with (3aR,4R,5R,6aS)-4-formyl-2-oxohexahydro-2H-cyclopenta[b]furan-5-yl benzoate (99.2 g, 0.362 mol) dissolved in DCM and lithium chloride (0.362 mol) dissolved in THF. Some lithium chloride precipitates from solution when the THF and DCM solutions are mixed. Dimethyl 2-oxo-3-(tetrahydro-2H-pyran-2-yloxy)heptylphosphonate (0.362 mol, prepared in Step A) is added NEAT and rinsed into the reaction vessel with DCM. The mixture is stirred under nitrogen and cooled to −20° C. and the lithium chloride precipitate dissolves. After stirring for 2.5 hours, triethylamine (0.362 mol) is added NEAT via addition funnel and the temperature is maintained at −5° C. and stirring is continued for 19 hours. The temperature is adjusted to 0° C. and the reaction mixture treated with 5% aqueous citric acid. The layers are separated and the organic layer is dried over magnesium sulfate, filtered, and evaporated. The crude product is purified on silica gel. Elution with hexanes-ethyl acetate affords the title intermediate.

Step C: Preparation of (3aR,4R,5R,6aS)-4-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-2-oxohexahydro-2H-cyclopenta[b]furan-5-yl Benzoate

The title intermediate may be prepared from (3aR,4R,5R,6aS)-4-((E)-4-(tert-butyldimethylsilyloxy)-3-oxooct-1-enyl)-2-oxohexahydro-2H-cyclopenta[b]furan-5-yl benzoate (prepared in Step B) using the DAST fluorination procedure described in Example 2, Step A.

Step D: Preparation of (3aR,4R,5R,6aS)-4-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxyhexahydro-2H-cyclopenta[b]furan-2-one

The title intermediate may be prepared from (3aR,4R,5R,6aS)-4-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-2-oxohexahydro-2H-cyclopenta[b]furan-5-yl benzoate (prepared in Step C) using a procedure described in Example 1, Step D for the removal of the benzoate group.

Step E: Preparation of (3aR,4R,5R,6aS)-4-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)hexahydro-2H-cyclopenta[b]furan-2,5-diol

The title intermediate may be prepared from (3aR,4R,5R,6aS)-4-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxyhexahydro-2H-cyclopenta[b]furan-2-one (prepared in Step D) using the reduction procedure described in Example 1, Step F.

Step F: Preparation of (Z)-7-((1R,2R,3R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-3,5-dihydroxycyclopentyl)hept-5-enoic Acid

The title intermediate may be prepared from (3aR,4R,5R,6aS)-4-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)hexahydro-2H-cyclopenta[b]furan-2,5-diol (prepared in Step E) using the procedure described in Example 1, Step G.

Step G: Preparation of (Z)-7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxy-3-oxocyclopentyl hept-5-enoic acid and (Z)-7-((1R,2R,3R)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-3-hydroxy-5-oxocyclopentyl)hept-5-enoic Acid

To a stirring mixture consisting of (Z)-7-((1R,2R,3R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-3,5-dihydroxycyclopentyl)hept-5-enoic acid (limiting reagent, prepared in Step F) in acetone (0.1 M) cooled to −25° C. is added Jones reagent (1 molar equivalent) dropwise. Upon completion, as judged by TLC, the reaction is quenched with isopropyl alcohol and the crude reaction mixture is diluted with ethyl acetate, washed three times with brine, and dried over magnesium sulfate. After filtration and solvent evaporation, the product mixture is separated by flash chromatography using ethyl acetate-hexane as eluent to afford the separated regioisomers (Z)-7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxy-3-oxocyclopentyl)hept-5-enoic acid and (Z)-7-((1R,2R,3R)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-3-hydroxy-5-oxocyclopentyl)hept-5-enoic acid.

Step H: Preparation of (Z)-7-((1R,2R,3R)-2-((E)-3,3-difluoro-4-hydroxyoct-1-enyl)-3-hydroxy-5-oxocyclopentyl)hept-5-enoic Acid

The title compound may be prepared from (Z)-7-((1R,2R,3R)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-3-hydroxy-5-oxocyclopentyl)hept-5-enoic acid (prepared in Step G) using the THP-deprotection procedure described in Example 1, Step J.

Example 4

Preparation of (Z)-7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-hydroxyoct-1-enyl)-5-hydroxy-3-oxocyclopentyl)hept-5-enoic Acid

The title compound may be prepared from (Z)-7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxy-3-oxocyclopentyl)hept-5-enoic acid (prepared in Example 3, Step G) using the THP-deprotection procedure described in Example 1, Step J.

Example 5

Preparation of (Z)-7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-hydroxyoct-1-enyl)-5-hydroxy-3-methylenecyclopentyl)hept-5-enoic Acid

Step A: Preparation of (Z)-methyl 7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxy-3-oxocyclopentyl)hept-5-enoate

The title intermediate may be prepared from (Z)-7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxy-3-oxocyclopentyl)hept-5-enoic acid (prepared in Example 3, Step G) using the diazomethane procedure described in Example 1, Step H.

Step B: Preparation of (Z)-methyl 7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-3-oxocyclopentyl)hept-5-enoate

The title intermediate may be prepared from (Z)-methyl 7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxy-3-oxocyclopentyl)hept-5-enoate (prepared in Step A) using the silylation procedure described in Example 1, Step I.

Step C: Preparation of (Z)-methyl 7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-3-methylenecyclopentyl)hept-5-enoate

The title intermediate may be prepared from (Z)-methyl 7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-3-oxocyclopentyl)hept-5-enoate (prepared in Step B) using the procedure described in Example 1, Step L.

Step D: Preparation of (Z)-methyl 7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxy-3-methylenecyclopentyl)hept-5-enoate

The title intermediate may be prepared from (Z)-methyl 7-((1R,2R,5S)-5-(tert-butyldiphenylsilyloxy)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-3-methylenecyclopentyl)hept-5-enoate (prepared in Step C) using the TBDPS-deprotection procedure described in Example 1, Step N.

Step E: Preparation of (Z)-7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxy-3-methylenecyclopentyl)hept-5-enoic Acid

The title intermediate may be prepared from (Z)-methyl 7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxy-3-methylenecyclopentyl)hept-5-enoate (prepared in Step D) using the ester hydrolysis procedure described in Example 1, Step M.

Step F: Preparation of (Z)-7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-hydroxyoct-1-enyl)-5-hydroxy-3-methylenecyclopentyl)hept-5-enoic Acid

The title compound may be prepared from (Z)-7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxy-3-methylenecyclopentyl)hept-5-enoic acid (prepared in Step E) using the THP-deprotection procedure described in Example 1, Step J.

Example 6

Preparation of (Z)-7-((1R,2R)-2-((E)-3,3-difluoro-4-hydroxyoct-1-enyl)-3-methyl-5-oxocyclopent-3-enyl)hept-5-enoic Acid

Step A: Preparation of (Z)-7-((1R,2R)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-3-methyl-5-oxocyclopent-3-enyl)hept-5-enoic Acid

The title intermediate may be prepared from (Z)-7-((1R,2R,5S)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-5-hydroxy-3-methylenecyclopentyl)hept-5-enoic acid (prepared in Example 5, Step E) using the Jones oxidation procedure described in Example 1, Step O.

Step B: Preparation of (Z)-7-((1R,2R)-2-((E)-3,3-difluoro-4-hydroxyoct-1-enyl)-3-methyl-5-oxocyclopent-3-enyl)hept-5-enoic Acid

The title compound may be prepared from (Z)-7-((1R,2R)-2-((E)-3,3-difluoro-4-(tetrahydro-2H-pyran-2-yloxy)oct-1-enyl)-3-methyl-5-oxocyclopent-3-enyl)hept-5-enoic acid (prepared in Step A) using the THP-deprotection procedure described in Example 1, Step J.

EP Receptor Binding and Agonism

The ability of compounds to bind the EP receptors and their selectivity for each receptor can be demonstrated in radioligand competition displacement binding experiments using the cell lines described above which stably overexpress the human EP receptors. The ability of compounds to activate the receptors can be demonstrated in second messenger functional assays, measuring changes in intracellular calcium for EP₁ and changes in cAMP formation for EP₂, EP₃ and EP₄.

Test Details

Binding Ability to Human EP Receptors

Membranes are prepared from cells stably transfected with human EP receptor DNA. In brief, cells are cultured to confluence, scraped from culture flasks and centrifuged to pellet (800×g, 5 minutes, 4° C.). Cells are washed twice with ice-cold homogenization buffer containing 10 mM Tris-HCl, 1 mM EDTA, 250 mM sucrose, 1 mM PMSF, 300 μM indomethacin, pH 7.4, homogenized by sonication and centrifuged as before. The supernatant is stored on ice; the pellets are rehomogenized and respun. Supernatants are pooled and centrifuged at 100,000×g for 10 minutes at 4° C. The resultant membrane pellet is stored at −80° C. until use.

For assays, membranes from cells expressing human EP₁, EP₂, EP₃ or EP₄ receptors are added to assay buffer (10 mM MES, pH 6.0, 10 mM MgCl₂, 1 mM EDTA, 3 μM indomethacin) containing 5 nM [³H]-PGE₂ (GE Healthcare) and 0.1 to 10,000 nM concentrations of compounds to be tested. Incubations are performed at suitable temperatures and times to allow equilibration to be reached. Non-specific binding is determined in the presence of 10 μM PGE₂. Reactions are terminated by the addition of ice-cold buffer followed by rapid filtration through Whatman GF/B filters. The filters are dried after washing, and membrane-bound radioactivity is quantified by scintillation counting.

The affinity or pK_i of each compound for each receptor is calculated from the concentration causing 50% radioligand displacement (IC₅₀) using the Cheng-Prosoff equation:

K_i=IC₅₀/[1+(radioligand concentration/radioligand K_d)]

Functional Assays: Effect of Compounds on Second Messenger Generation

The following sections describe in vitro assays to determine the effect of compounds on calcium mobilization, and on the induction or inhibition of cAMP generation, that is, to determine the functional efficacy of compounds at the EP₁ (calcium mobilization), EP₂ (induction of cAMP), EP₃ (inhibition of forskolin-induced cAMP) or EP₄ (induction of cAMP) receptor.

EP₁ Receptor Agonism Assay (Intracellular Calcium Assay)

Functional Assay #1AGi

To test the ability of compounds to activate the EP₁ receptor, calcium mobilization experiments are performed. Cells expressing the EP₁ receptor are plated in clear-bottom black 96-well plates in normal growth medium and grown to confluence. When the cells have reached confluence, the culture medium is replaced with 50 μl of Fluo-4 NW dye mix (Invitrogen) that is dissolved in Hank's balanced salt solution containing 20 mM HEPES, pH 7.4 and 2.5 mM probenecid. Experiments are initiated by the addition of 50 μl/well of vehicle or compound to be tested diluted in this same buffer. Plates are incubated for 30 minutes at 37° C. and then at room temperature for an additional 30 minutes. Calcium fluorescence is measured using an Analyst AD (Molecular Devices) with an excitation wavelength of 485 nm, emission wavelength of 560 nm, and emission cutoff of 505 nm. Responses are quantified as peak fluorescence intensity minus basal fluorescence intensity.

Alternative EP₁ Receptor Agonism Assay

Functional Assay #1AGii

(Cerep, Catalog reference 722-55a; UNGRIN, M. D., SINGH L. M. R., STOCCO, R., SAS, D. E. and ABRAMOVITZ, M. (1999), An automated aequorin luminescence-based functional calcium assay for G-Protein-Coupled Receptors. Analytical Biochem., 272, 34.)

Evaluation of the agonist activity of compounds at the human EP₁ receptor in transfected HEK-293 cells, determined by measuring their effect on cytosolic Ca²⁺ ion mobilization using a fluorimetric detection method.

The cells are suspended in DMEM buffer (Invitrogen), then distributed in microplates at a density of 3·10⁴ cells/well. The fluorescent probe (Fluo4 NW, Invitrogen) mixed with probenicid in HBSS buffer (Invitrogen) complemented with 20 mM Hepes (Invitrogen) (pH 7.4) is then added into each well and equilibrated with the cells for 30 minutes at 37° C. then 30 minutes at 22° C. Thereafter, the assay plates are positioned in a microplate reader (CellLux, PerkinElmer) which is used for the addition of the test compound, reference agonist or HBSS buffer (basal control), and the measurements of changes in fluorescence intensity which varies proportionally to the free cytosolic Ca²⁺ ion concentration. For stimulated control measurements, PGE₂ at 100 nM is added in separate assay wells.

The results are expressed as a percent of the control response to 100 nM PGE₂. The standard reference agonist is PGE₂, which is tested in each experiment at several concentrations to generate a concentration-response curve from which its EC₅₀ value is calculated.

EP₁ Receptor Antagonism Assay

Functional Assay #1ANT

(Cerep, Catalog Reference 722-55B; Ungrin, M. D., et al., Ibid.)

Evaluation of the antagonist activity of compounds at the human EP₁ receptor in transfected HEK-293 cells, determined by measuring their effect on agonist-induced cytosolic Ca²⁺ ion mobilization using a fluorimetric detection method.

The cells are suspended in DMEM buffer (Invitrogen), then distributed in microplates at a density of 3·10⁴ cells/well. The fluorescent probe (Fluo4 NW, Invitrogen) mixed with probenicid in HBSS buffer (Invitrogen) complemented with 20 mM Hepes (Invitrogen) (pH 7.4) is then added into each well and equilibrated with the cells for 30 minutes at 37° C. then 30 minutes at 22° C. Thereafter, the assay plates are positioned in a microplate reader (CellLux, PerkinElmer) which is used for the addition of the test compound, reference antagonist or HBSS buffer (basal control), then 5 minutes later 3 nM PGE₂, and the measurements of changes in fluorescence intensity which varies proportionally to the free cytosolic Ca²⁺ ion concentration. The results are expressed as a percent inhibition of the control response to 3 nM PGE₂. The standard reference antagonist is SC 51322, which is tested in each experiment at several concentrations to generate a concentration-response curve from which its IC₅₀ value is calculated.

EP₂ and EP₄ Receptor Agonism Assay (Cyclic AMP Induction Assay)

Functional Assay #2AGi and Functional Assay #4AGi, Respectively

To test the ability of compounds to activate the EP₂ and EP₄ receptors, accumulation of cAMP following treatment with these compounds is measured. Cells expressing the EP₂ or EP₄ receptor are plated in 24-well plates in normal growth medium and grown to confluence. When the cells have reached confluence, the medium is replaced with 450 l of serum-free medium containing 0.25 mM IBMX and 20 μM indomethacin. Cells are incubated in this medium for one hour. Fifty microliters of this same buffer containing various concentrations of PGE₂ or compounds to be tested is subsequently added to the cells and the cells are incubated for fifteen to thirty minutes to allow the accumulation of cAMP. Reactions are terminated by the addition of 500 μl of 10% TCA. cAMP measurements of the cell lysates are performed using Cayman Chemical's commercially available cAMP EIA Kit following the instructions provided in the kit booklet.

Alternative EP₂ Receptor Agonism Assay

Functional Assay #2AGii

(Cerep, Catalog reference 758-54a; Wilson, R. J., Rhodes, S. A., Wood, R. L., Shield, V. J., Noel, L. S., Gray, D. W. and Giles H. (2004), Functional pharmacology of human prostanoid EP₂ and EP4 receptors, Eur. J. Pharmacol., 501, 49.)

Evaluation of the agonist activity of compounds at the human EP₂ receptor in transfected CHO cells, determined by measuring their effects on cAMP production using the HTRF detection method.

The cells are suspended in HBSS buffer (Invitrogen) complemented with HEPES 20 mM (pH 7.4) and 500 μM IBMX, then distributed in microplates at a density of 10⁴ cells/well and incubated for 30 minutes at 37° C. in the absence (control) or presence of the test compound or the reference agonist. For stimulated control measurements, separate assay wells contain 10 μM PGE₂. Following incubation, the cells are lysed and the fluorescence acceptor (D2-labeled cAMP) and fluorescence donor (anti-cAMP antibody labeled with europium cryptate) are added. After 60 minutes at room temperature, the fluorescence transfer is measured at λex=337 nm and λem=620 and 665 nm using a microplate reader (Rubystar, BMG). The cAMP concentration is determined by dividing the signal measured at 665 nm by that measured at 620 nm (ratio). The results are expressed as a percent of the control response to 10 μM PGE₂. The standard reference agonist is PGE₂, which is tested in each experiment at several concentrations to generate a concentration-response curve from which its EC₅₀ value is calculated.

EP₂ Receptor Antagonism Assay

Functional Assay #2ANT

(Cerep, Catalog Reference 758-54B; Wilson, R. J., et al., Ibid.)

Evaluation of the antagonist activity of compounds at the human EP₂ receptor in transfected CHO cells, determined by measuring their effects on agonist-induced cAMP production using the HTRF detection method.

The cells are suspended in HBSS buffer (Invitrogen) complemented with HEPES 20 mM (pH 7.4) and 500 μM IBMX, then distributed in microplates at a density of 10⁴ cells/well and preincubated for 5 minutes at room temperature in the absence (control) or presence of the test compound or the reference antagonist. Thereafter, the reference agonist PGE₂ is added at a final concentration of 300 nM. For basal control measurements, separate assay wells do not contain PGE₂. Following 30 minutes incubation at 37° C., the cells are lysed and the fluorescence acceptor (D2-labeled cAMP) and fluorescence donor (anti-cAMP antibody labeled with europium cryptate) are added. After 60 minutes at room temperature, the fluorescence transfer is measured at λex=337 nm and λem=620 and 665 nm using a microplate reader (Rubystar, BMG). The cAMP concentration is determined by dividing the signal measured at 665 nm by that measured at 620 nm (ratio). The results are expressed as a percent inhibition of the control response to 300 nM PGE₂. The standard reference antagonist is AH 6809, which is tested in each experiment at several concentrations to generate a concentration-response curve from which its IC₅₀ value is calculated.

EP₃ Receptor Agonism Assay (Inhibition of Forskolin-induced cAMP Generation Assay)

Functional Assay #3AG

To test the ability of compounds to activate the EP₃ receptor, the decrease in cAMP accumulation induced by forskolin following treatment with compounds is measured. Cells expressing the EP₃ receptor are plated in 24-well plates in normal growth medium and allowed to come to confluence. When the cells have come to confluence, the medium is replaced with 450 μl of serum-free medium containing 0.25 mM IBMX and 20 μM indomethacin. Cells are incubated in this medium for one hour. Fifty microliters of this same buffer containing 3 μM forskolin and various concentrations of PGE₂ or compounds to be tested are subsequently added to the cells. After incubation at 37° C. for 10 minutes, reactions are terminated by the addition of 500 μl of 10% TCA. cAMP measurements of the cell lysates are performed using Cayman Chemical's cAMP EIA Kit following the instructions provided in the kit booklet.

Alternative EP₄ Receptor Agonism Assay

Functional Assay #4AGii

(Cerep, Catalog Reference 758-49a; Wilson, R. J., et al., Ibid.)

Evaluation of the agonist activity of compounds at the human EP₄ receptor in transfected CHO cells, determined by measuring their effects on cAMP production using the HTRF detection method.

The cells are suspended in HBSS buffer (Invitrogen) complemented with HEPES 20 mM (pH 7.4) and 500 μM IBMX, then distributed in microplates at a density of 2·10⁴ cells/well and incubated for 10 minutes at room temperature in the absence (control) or presence of the test compound or the reference agonist. For stimulated control measurements, separate assay wells contain 1 μM PGE₂. Following incubation, the cells are lysed and the fluorescence acceptor (D2-labeled cAMP) and fluorescence donor (anti-cAMP antibody labeled with europium cryptate) are added. After 60 minutes at room temperature, the fluorescence transfer is measured at λex=337 nm and λem=620 and 665 nm using a microplate reader (Rubystar, BMG). The cAMP concentration is determined by dividing the signal measured at 665 nm by that measured at 620 nm (ratio). The results are expressed as a percent of the control response to 1 μM PGE₂. The standard reference agonist is PGE₂, which is tested in each experiment at several concentrations to generate a concentration-response curve from which its EC₅₀ value is calculated.

EP₄ Receptor Antagonism Assay

Functional Assay #4ANT

(Cerep, Catalog reference 758-49b; Wilson, R. J., et al., Ibid.)

Evaluation of the antagonist activity of compounds at the human EP₄ receptor in transfected CHO cells, determined by measuring their effects on agonist-induced cAMP production using the HTRF detection method.

The cells are suspended in HBSS buffer (Invitrogen) complemented with HEPES 20 mM (pH 7.4) and 500 μM IBMX, then distributed in microplates at a density of 2·10⁴ cells/well and preincubated for 5 minutes at room temperature in the absence (control) or presence of the test compound or the reference antagonist.

Thereafter, the reference agonist PGE₂ is added at a final concentration of 10 nM. For basal control measurements, separate assay wells do not contain PGE₂. Following 10 minutes incubation at room temperature, the cells are lysed and the fluorescence acceptor (D2-labeled cAMP) and fluorescence donor (anti-cAMP antibody labeled with europium cryptate) are added. After 60 minutes at room temperature, the fluorescence transfer is measured at λex=337 nm and λem=620 and 665 nm using a microplate reader (Rubystar, BMG). The cAMP concentration is determined by dividing the signal measured at 665 nm by that measured at 620 nm (ratio). The results are expressed as a percent inhibition of the control response to 10 nM PGE₂. There is no standard reference antagonist for this assay.

Claims

1. A compound of the general formula (I):

wherein dashed bonds may each independently represent a second carbon-carbon bond in order to give a carbon-carbon double bond with either (E) or (Z) geometry or may be ignored in order to give a carbon-carbon single bond;

wherein C9 and C11 each is independently C═CH2, C═O, CF2, CHF (any stereoisomer), or C(H)OH (any stereoisomer) with the proviso that C9 does not equal C11, and also with the proviso that when one of either C9 or C11 is C═O, and the other is C(H)OH, at least one of Z2, Z3, Z4, and Z5 is fluorine, and also with the proviso that when one of either C9 or C11 is CHF, the other is not C(H)OH;

wherein R1 is CO2R3, CH2OR3, CONR4R5, COCH2OH, CONR4SO2R5, P(O)(OR4)2, or

wherein R3 is hydrogen or (C1-C6)-alkyl:

wherein R4 and R5 each is independently hydrogen or (C1-C6)-alkyl;

wherein Z1 are hydrogen or fluorine;

wherein Z2 and Z3 each is independently hydrogen or fluorine;

wherein Z4 and Z5 each is independently hydrogen, fluorine, hydroxy, or methyl, or together are an oxygen atom that form a carbonyl group with the adjoining carbon atom of the ω chain; and

wherein Z6 and Z7 each is independently hydrogen, fluorine, hydroxy, or methyl, or together are an oxygen atom that form a carbonyl group with the adjoining carbon atom of the ω chain;

or any stereoisomer of the compound of the general formula (I), or any geometric isomer of the compound of the general formula (I), or an equivalent of the compound of the general formula (I), or a prodrug of the compound of the general formula (I), or a hydrate of the compound of the general formula (I), or a solvate of the compound of the general formula (I), or a pharmaceutically acceptable salt of the compound of the general formula (I).

2. The compound of claim 1, wherein the compound of the general formula (I) comprises the compound of general formula (II):

3. The compound of claim 1, wherein the compound of the general formula (I) comprises the compound of general formula (III):

4. The compound of claim 1, wherein the compound of the general formula (I) comprises the compound of general formula (IV):

5. The compound of claim 1, wherein the compound of the general formula (I) comprises the compound of general formula (V):

6. A pharmaceutical composition comprising a pharmaceutically effective amount of a compound of claim 1 in admixture with a pharmaceutically acceptable carrier.

7. A method of expanding hematopoietic stem cell populations in a culture or patient in need thereof comprising administering to the culture or the patient a compound according to claim 1.

8. A method of treatment for a patient comprising administering to the patient a compound according to claim 1.

9. The method of claim 8, wherein said compound further comprises a pharmaceutically acceptable carrier.

10. A method for treating or preventing EP receptor-mediated conditions in a subject, comprising the step of administering to the subject a compound according to claim 1.