WO2007014454A1 - Ep4 receptor agonist, compositions and methods thereof - Google Patents

Ep4 receptor agonist, compositions and methods thereof Download PDF

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Publication number
WO2007014454A1
WO2007014454A1 PCT/CA2006/001243 CA2006001243W WO2007014454A1 WO 2007014454 A1 WO2007014454 A1 WO 2007014454A1 CA 2006001243 W CA2006001243 W CA 2006001243W WO 2007014454 A1 WO2007014454 A1 WO 2007014454A1
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WIPO (PCT)
Prior art keywords
oxazinan
oxo
difluoro
thiophene
propyl
Prior art date
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PCT/CA2006/001243
Other languages
French (fr)
Inventor
John Colucci
Yongxin Han
Julie A. Farand
Original Assignee
Merck Frosst Canada Ltd.
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Publication date
Application filed by Merck Frosst Canada Ltd. filed Critical Merck Frosst Canada Ltd.
Priority to US11/989,643 priority Critical patent/US20090105234A1/en
Priority to AU2006275270A priority patent/AU2006275270A1/en
Priority to JP2008524323A priority patent/JP2009502977A/en
Priority to EP06761196A priority patent/EP1912977A4/en
Priority to CA002616604A priority patent/CA2616604A1/en
Publication of WO2007014454A1 publication Critical patent/WO2007014454A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • 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
    • 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
    • A61P27/04—Artificial tears; Irrigation solutions
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

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.
  • disorders in humans and other mammals involve or are associated with abnormal or excessive bone loss.
  • Such disorders include, but are not limited to, osteoporosis, glucocorticoid induced osteoporosis, Paget's disease, abnormally increased bone turnover, periodontal disease, tooth loss, bone fractures, rheumatoid arthritis, periprosthetic osteolysis, osteogenesis imperfecta, metastatic bone disease, hypercalcemia of malignancy, and multiple myeloma.
  • osteoporosis which in its most frequent manifestation occurs in postmenopausal women.
  • Prostaglandins such as the PGE2 series are known to stimulate bone formation and increase bone mass in mammals, including man.
  • EP4 The major prostaglandin receptor in bone is EP4, which is believed to provide its effect by signaling via cyclic AMP.
  • formula I agonists of the EP4 subtype receptor are useful for stimulating bone formation.
  • WO 02/24647, WO 02/42268, EP 1114816, WO 01/46140 and WO 01/72268 disclose EP4 agonists. However, they do not disclose the compounds of the instant invention.
  • This invention relates to agonists of the EP4 subtype of prostaglandin E2 receptors and their use or a formulation thereof in the treatment of glaucoma and other conditions that are related to elevated intraocular pressure in the eye of a patient.
  • this invention relates to a series of 3,4- disubstituted l,3-oxazinan-2-one, derivatives and their use to treat ocular diseases and to provide a neuroprotective effect to the eye of mammalian species, particularly humans.
  • This invention further relates to the use of the compounds of this invention for mediating the bone modeling and remodeling processes of the osteoblasts and osteoclasts.
  • this invention relates to novel EP4 agonist having the structural formula I:
  • R and R4 independently represent H, or Ci-6 alkyl
  • Rl independently represents hydrogen, Ci_6 alkyl, halogen, CF3, aryl, said aryl optionally substituted with 1 to 3 groups of halogen, Cl -6 alkyl, CF3, or N(R4)2 ;
  • R2 represents H, or halogen;
  • R3 represents COOR or carboxylic acid isostere;
  • n represents 0-3; and
  • — represents a double or single bond.
  • therapeutically effective amount means that amount of the EP4 receptor subtype agonist of formula I, or other actives of the present invention, that will elicit the desired therapeutic effect or response or provide the desired benefit when administered in accordance with the desired treatment regimen.
  • a preferred therapeutically effective amount relating to the treatment of abnormal bone resorption is a bone formation, stimulating amount.
  • a preferred therapeutically effective amount relating to the treatment of ocular hypertension or glaucoma is an amount effective for reducing intraocular pressure and/or treating ocular hypertension and/or glaucoma.
  • “Pharmaceutically acceptable” as used herein means generally suitable for administration to a mammal, including humans, from a toxicity or safety standpoint.
  • the term “prodrug” refers to compounds which are drug precursors which, following administration and absorption, release the claimed drug in vivo via some metabolic process.
  • a non- limiting example of a prodrug of the compounds of this invention would be an ester of the carboxylic acid group, where the ester functionality is easily hydrolyzed to the active acid after administration to a patient.
  • Exemplary prodrugs include acetic acid derivatives that are non-narcotic, analgesics/non- steroidal, anti-inflammatory drugs having a free CH2COOH group (which can optionally be in the form of a pharmaceutically acceptable salt, e.g. -CH2COO-Na+), typically attached to a ring system, preferably to an aromatic or heteroaromatic ring system.
  • acetic acid derivatives that are non-narcotic, analgesics/non- steroidal, anti-inflammatory drugs having a free CH2COOH group (which can optionally be in the form of a pharmaceutically acceptable salt, e.g. -CH2COO-Na+), typically attached to a ring system, preferably to an aromatic or heteroaromatic ring system.
  • alkyl refers to a monovalent alkane (hydrocarbon) derived radical containing from 1 to 6 carbon atoms unless otherwise defined. It may be straight, branched or cyclic. Preferred alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, t-butyl, cyclopentyl and cyclohexyl. 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 6 carbon atoms, without alternating or resonating double bonds between carbon atoms.
  • Examples of cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
  • Halogen refers to chlorine, fluorine, iodine or bromine.
  • Carboxylic isostere represents tetrazole, acylsulfonamide, sulfonic acid, phosphonic acid or prodrug such as C]-6 aldehyde or C ⁇ g alcohol.
  • Aryl refers to aromatic rings e.g., phenyl, substituted phenyl and the like, as well as rings which are fused, e.g., naphthyl, phenanthrenyl and the like.
  • An aryl group thus contains at least one ring having at least 6 atoms, with up to five such rings being present, containing up to 22 atoms therein, with alternating (resonating) double bonds between adjacent carbon atoms or suitable heteroatoms.
  • aryl groups are phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl and phenanthrenyl, preferably phenyl, naphthyl or phenanthrenyl.
  • Aryl groups may likewise be substituted as defined.
  • Preferred substituted aryls include phenyl and naphthyl.
  • agonist means EP4 subtype compounds of formula I 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.
  • R3 is a carboxylic acid isostere and all other variables are as originally defined.
  • R is Ci -6 alkyl and all other variables are as originally defined.
  • a sub-embodiment of this invention is realized when R is isopropyl, ethyl or, methyl, preferably isopropyl.
  • R2 is halogen and all other variables are as originally defined.
  • a subembodiment of this invention is realized when halogen is fluorine or bromine.
  • Still another embodiment of this invention is realized when Ri is halogen and all other variables are as originally defined.
  • a sub-embodiment is realized when Ri is bromine or chlorine, preferably bromine.
  • Still another embodiment of this invention is realized when Ri is Ci -6 alkyl and all other variables are as originally defined.
  • Still another embodiment of this invention is realized when Ri is CF3 and all other variables are as originally defined.
  • R3 is COOR, R is H or isopropyl, Rl is bromine and R2 is hydrogen.
  • R3 is tetrazole, Ri is bromine and R2 is hydrogen
  • Another embodiment of this invention is reaslized when n is 0, 1, or 2 and all other variables are as originally defined. Still another embodiment of this invention is realized when n is 0 and all other variables are as originally defined
  • Another embodiment of this invention is realized when n is 1 and all other variables are as originally defined.
  • a subembodiment of this invention is realizedwhen Rj is bromine and R.2 is hydrogen.
  • Another embodiment of this invention is realized when — represents a double bond.
  • Compounds of this invention are: Isopropyl S-CS-K ⁇ H-Kl ⁇ S ⁇ -CS-bromophenylH ⁇ -difluoro-S-hydroxybut-l-en-l-y ⁇ -oxo-US- oxazinan-3-yl ⁇ propyl)thiophene-2-carboxylate;
  • Another embodiment of this invention is directed to a composition containing an EP4 agonist of Formula I and optionally a pharmaceutically acceptable carrier.
  • Yet another embodiment of this invention is directed to a method for decreasing elevated intraocular pressure or treating glaucoma by administration, preferably topical or intra-camaral administration, of a composition containing an EP4 agonist of Formula I and optionally a pharmaceutically acceptable carrier.
  • a composition containing an EP4 agonist of Formula I and optionally a pharmaceutically acceptable carrier.
  • Use of the compounds of formula I for the manufacture of a medicament for treating elevated intraocular pressure or glaucoma or a combination thereof is also included in this invention
  • This invention is further concerned with a process for making a pharmaceutical composition comprising a compound of formula I.
  • This invention is further concerned with a process for making a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula I, and a pharmaceutically acceptable carrier.
  • 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 ocular hypertension.
  • 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)).
  • Macular edema is swelling within the retina within the critically important central visual zone at the posterior pole of the eye. 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.
  • 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. As a result, 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 I.
  • compositions which may be administered to mammals, including humans, to achieve effective IOP lowering 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.
  • this invention is also concerned with compositions and methods of treating ocular hypertension, glaucoma, macular edema, macular degeneration, for increasing retinal and optic nerve head blood velocity, for increasing retinal and optic nerve oxygen tension, for providing a neuroprotective effect or for a combination thereof by administering to a patient in need thereof one of the compounds of formula I alone or in combination with one or more of the following active ingredients, a ⁇ -adrenergic blocking agent such as timolol, betaxolol, levobetaxolol, carteolol, levobunolol, a parasympathomimetic agent such as pilocarpine, a sympathomimetic agents such as epinephrine, iopidine,
  • 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 0.00001 to 5% and especially 0.00001 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.
  • For a single dose from between 0.0000001 to 0.05 mg, preferably 0.0000005 to 0.02 mg, and especially 0.000005 to 0.01 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 purpose 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.
  • Suitable subjects for the administration of the formulation of the present invention include
  • 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.
  • 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,
  • 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 compounds of the instant invention are also useful for mediating the bone modeling and remodeling processes of the osteoblasts and osteoclasts. See PCT US99/23757 filed October 12,
  • the major prostaglandin receptor in bone is EP ⁇ which is believed to provide its effect by signaling via cyclic AMP. See Dceda T, Miyaura C, Ichikawa A, Narumiya S, Yoshiki S and Suda T 1995, In situ localization of three subtypes (EP j , EP3 and EP4) of prostaglandin E receptors in embryonic and newborn mice., J Bone Miner Res 10 (sup
  • Another object of the present invention is to provide methods for stimulating bone formation, i.e. osteogenesis, in a mammal comprising administering to a mammal in need thereof a therapeutically effective amount of an EP4 receptor subtype agonist of formula I.
  • Still another object of the present invention to provide methods for stimulating bone formation in a mammal in need thereof comprising administering to said mammal a therapeutically effective amount of an EP4 receptor subtype agonist of formula I and a bisphosphonate active.
  • administering to said mammal a therapeutically effective amount of an EP4 receptor subtype agonist of formula I and a bisphosphonate active.
  • Use of the compounds of formula I for the manufacture of a medicament for stimulating bone formation is also included in this invention.
  • Yet another object of the present invention to provide pharmaceutical compositions comprising a therapeutically effective amount of an EP4 receptor subtype agonist of formula I and a bisphosphonate active.
  • Use of the compounds of formula I for the manufacture of a medicament for treating or reducing the risk of contracting a disease state or condition related to abnormal bone resorption is also included in this invention.
  • the disease states or conditions related to abnormal bone resorption include, but are not limited to, osteoporosis, glucocorticoid induced osteoporosis, Paget's disease, abnormally increased bone turnover, periodontal disease, tooth loss, bone fractures, rheumatoid arthritis, periprosthetic osteolysis, osteogenesis imperfecta, metastatic bone disease, hypercalcemia of malignancy, and multiple myeloma.
  • both concurrent and sequential administration of the EP4 receptor subtype agonist of formula I and the bisphosphonate active are deemed within the scope of the present invention.
  • the formulations are prepared containing 5 or 10 mg of a bisphosphonate active, on a bisphosphonic acid active basis.
  • the agonist and the bisphosphonate can be administered in either order.
  • the agonist and bisphosphonate are typically administered within the same 24 hour period.
  • the agonist and bisphosphonate are typically administered within about 4 hours of each other.
  • a non-limiting class of bisphosphonate actives useful in the instant invention are selected from the group consisting of alendronate, cimadronate, clodronate, tiludronate, etidronate, ibandronate, neridronate, olpandronate, risedronate, piridronate, pamidronate, zolendronate, pharmaceutically acceptable salts thereof, and mixtures thereof.
  • a non-limiting subclass of the above-mentioned class in the instant case is selected from the group consisting of alendronate, pharmaceutically acceptable salts thereof, and mixtures thereof.
  • a non-limiting example of the subclass is alendronate monosodium trihydrate.
  • the agonist is typically administered for a sufficient period of time until the desired therapeutic effect is achieved.
  • the term "until the desired therapeutic effect is achieved”, as used herein, means that the therapeutic agent or agents are continuously administered, according to the dosing schedule chosen, up to the time that the clinical or medical effect sought for the disease or condition being mediated is observed by the clinician or researcher.
  • the compounds are continuously administered until the desired change in bone mass or structure is observed, hi such instances, achieving an increase in bone mass or a replacement of abnormal bone structure with normal bone structure are the desired objectives.
  • the compounds are continuously administered for as long as necessary to prevent the undesired condition.
  • Nonlimiting examples of administration periods can range from about 2 weeks to the remaining lifespan of the mammal.
  • administration periods can range from about 2 weeks to the remaining lifespan of the human, preferably from about 2 weeks to about 20 years, more preferably from about 1 month to about 20 years, more preferably from about 6 months to about 10 years, and most preferably from about 1 year to about 10 years.
  • the instant compounds are also useful in combination with known agents useful for treating or preventing bone loss, bone fractures, osteoporosis, glucocorticoid induced osteoporosis, Paget' s disease, abnormally increased bone turnover, periodontal disease, tooth loss, osteoarthritis, rheumatoid arthritis, periprosthetic osteolysis, osteogenesis imperfecta, metastatic bone disease, hypercalcemia of malignancy, and multiple myeloma.
  • Combinations of the presently disclosed compounds with other agents useful in treating or preventing osteoporosis or other bone disorders are within the scope of the invention.
  • a person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the disease involved.
  • Such agents include the following: an organic bisphosphonate; a cathepsin K inhibitor; an estrogen or an estrogen receptor modulator; an androgen receptor modulator; an inhibitor of osteoclast proton ATPase; an inhibitor of HMG-CoA reductase; an integrin receptor antagonist; an osteoblast anabolic agent, such as PTH; calcitonin; Vitamin D or a synthetic Vitamin D analogue; and the pharmaceutically acceptable salts and mixtures thereof.
  • a preferred combination is a compound of the present invention and an organic bisphosphonate.
  • Another preferred combination is a compound of the present invention and an estrogen receptor modulator.
  • Another preferred combination is a compound of the present invention and an estrogen.
  • Another preferred combination is a compound of the present invention and an androgen receptor modulator.
  • Another preferred combination is a compound of the present invention and an osteoblast anabolic agent.
  • the formula I 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.
  • the agonists have an EC5 Q value of from about 0.0001 microM to about 10 microM.
  • the agonists have an EC50 value of from about 0.0001 microM to about 1 microM.
  • EC ⁇ Q 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.
  • the compounds of this invention can be made, with some modification, in accordance with US Patent No. 6,043,275, EP0855389, WO 03/047417 (USSN 60/337228), WO 03/047513 (USSN 60/338,117), USSN 60/406,530 (Merck Docket No. MC060), WO 2004/085430 and WO 01/46140, all of which are incorporated herein by reference in their entirety.
  • the following non-limiting schemes and examples given by way of illustration is demonstrative of the present invention.
  • Step 1 isopropyl 5- ⁇ 3-[(4R)-4-( ⁇ [/er/-butyl(dimethyl)silyl]oxy ⁇ methyl)-2-oxo-l,3-oxazinan-3- yl]propyl ⁇ thiophene-2-carboxylate
  • Step 3 isopropyl 5 - ⁇ 3 - [(4i?)-4-formy 1-2-oxo- 1 ,3 -oxazinan-3 -y 1] propyl ⁇ thi ophene-2-carboxy late
  • Step 4 isopropyl 5-(3- ⁇ (4R)-4-[(l£)-4-(3-bromophenyl)-4,4-difluoro-3-oxobut-l-en-l-yl]-2-oxo-l,3- oxazinan-3-yl ⁇ propyl)thiophene-2-carboxylate (11)
  • the cloudy solution was diluted with IN HCl/water and extracted with ethyl acetate (EA) (2x). The EA layers were washed with water and dried over MgSO 4 . The crude was purified by flash (50-80% EA/hexane) to give desired product 11 which was contaminated with the phosphonate reagent by NMR.
  • EA ethyl acetate
  • Step 5 Preparation of catalyst 14
  • the catalyst was prepared by mixing lmol equiv of [RuCl 2 (p-cymene) 2 ], 2mol equiv
  • the catalyst could also be generated in situ by mixing 0.02 mol equiv of [RuCl 2 (f>-cymene) 2 ] and 0.04 mol equiv of the (i?,/?)-N-Tosyl-l,2-diphenylethylene-l,2-diamine in DCM (dichloromethane) in the presence of 0.04 mol equiv of IM solution KOtBu in THF. After aging for 10 min at RT(room temperature), Et 3 N was added followed by HCO 2 H and a solution of the enone in DCM.
  • Example 1 isopropyl 5-(3- ⁇ (4R)-4-[(l£,3R)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2- OXO- 1 ,3-oxazinan-3-yl ⁇ propyl)thiophene-2-carboxylate
  • Example 1 To a solution of Example 1 (1 10 mg, 0.2 mmol) in EtOAc (10 mL) and acetone (1OmL) was added PtO 2 (5 mg). The resulting black reaction mixture was subjected to H 2 (1 atm (atmosphere)) for 18h. The solution was filtered over a pad of celite and the organic solvent was removed in vacuo. The crude product was purified by flash column chromatography to afford the ester which was hydrolyzed in the usual manner to the title compound. MS(+ESI) m/z 532.2, 534.2.
  • Step 1 To a solution of 3-bromo-iodobenzene (14.1g, 50 mmol) and ethyl bromo- ⁇ , ⁇ - difluoroacetate (10. Ig, 50 mmol) in DMSO (40 mL) was added copper bronze (7g, 110 mmol) and the suspension was heated to 55 0 C for 2.5 days and cooled to rt. The mixture was quenched with KH 2 PO 4 and filtered. The solid was washed with EA/water and the filtrate was separated. The aqueous layer was extracted with ether (2x) and the organic phases were combined, washed with water and brine.
  • Step 1 5-[(l£)-3-te ⁇ butoxy-3-oxoprop-l-en-l-yl]thiophene-2-carboxylic acid (16)
  • ester 17 (45g, 152 mmol) in 50OmL of dichloromethane was added 45 mL of TFA (trifluoroacetic acid) slowly and the solution was stirred at rt. for 12 hours. To the solution was added 30OmL of PhMe and followed by concentration in vacuo. The resultant solid was collected and washed with hexanes to yield 18.
  • TFA trifluoroacetic acid
  • Step 4 3-[5-(isopropoxycarbonyl)-2-thienyl]propanoic acid (19) To a solution of acid 18 (45g, 152 mmol) in 20OmL of isopropanol was added 5.32 g of
  • Step 5 isopropyl 5-(3-hydroxypropyl)thiophene-2-carboxylate (20)
  • Example 5 isopropyl 5-(3- ⁇ (4i?)-4-[(l£ ' ,3R)-4-(3,5-dichlorophenyl)-4,4-difluoro-3-hydroxybut-l-en-l- yl]-2-oxo- 1 ,3 -oxazinan-3-yl ⁇ propyl)thiophene-2-carboxylate
  • Example 6 5-(3- ⁇ (4/?)-4-[(l J E,3i2)-4-(3,5-dichlorophenyl)-4 5 4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo- 1 ,3-oxazinan-3-yl ⁇ propyl)thiophene-2-carboxylic acid
  • Example 7 isopropyl 5-(3- ⁇ (4S)-4-[(3R)-4-(3,5-dichlorophenyl)-4,4-difluoro-3-hydroxybutyl]-2-oxo-l,3- oxazinan-3 -yl ⁇ propyl)thiophene-2-carboxylate
  • Example 9 isopropyl 5-(3- ⁇ (4R)-4-[(l£,3R)-4-(3,5-dimethylphenyl)-4,4-difluoro-3-hydroxybut-l-en-l- y 1] -2-oxo- 1 ,3 -oxazinan-3 -y 1 ⁇ propy l)thiophene-2-carboxylate
  • Example 10 5-(3- ⁇ (4R)-4-[(l J E,3R)-4-(3,5-dimethylphenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo- 1 ,3-oxazinan-3-yl ⁇ propyl)thiophene-2-carboxylic acid
  • Example 1 5-(3- ⁇ (4S)-4-[(3R)-4-(3,5-dimethylphenyl)-4,4-difluoro-3-hydroxybutyl]-2-oxo-l,3- oxazinan-3 -yl ⁇ propyl)thiophene-2-carboxylic acid
  • Example 12 isopropyl 5-[3-((4R)-4- ⁇ (l£,3R)-4,4-difluoro-3-hydroxy-4-[3-(trifluoromethyl)phenyl]but-l- en-l-yl ⁇ -2-oxo-l,3-oxazinan-3-yl)propyl]thiophene-2-carboxylate
  • Example 13 5-[3-((4R)-4- ⁇ ( l£,3R)-4,4-difluoro-3-hydroxy-4-[3-(trifluoromethyl)phenyl]but- 1 -en- 1 -yl ⁇ - 2-oxo- 1 ,3-oxazinan-3-yl)propyl]thiophene-2-carboxylic acid
  • Example 14 isopropyl 5-[3-((4S)-4- ⁇ (3R)-4,4-difluoro-3-hydroxy-4-[3-(trifluoromethyl)phenyl]butyl ⁇ -2- oxo-1, 3-oxazinan-3-yl)propyl]thiophene-2-carboxylate
  • Example 15 5-[3-((4S)-4- ⁇ (3R)-4,4-difluoro-3-hydroxy-4-[3-(trifluoromethyl)phenyl]butyl ⁇ -2-oxo-l,3- oxazinan-3-yl)propyl]thiophene-2-carboxylic acid
  • Step 2 ethyl difluoro [3 -( 1 -hydroxy- 1 -methyl ethyl)pheny 1] acetate
  • Step 3 ethyl ⁇ , ⁇ -difluoro(3-isopropylphenyl)acetate
  • Step 4 dimethyl [3,3-difluoro-3-(3-isopropylphenyl)-2-oxopropyl]phosphonate To a solution of dimethyl methylphosphonate (3.1g, 25 mmol) in THF ( 100 mL) at -78
  • Example 16 5-(3- ⁇ (4R)-4-[(l£,3R)-4,4-difluoro-3-hydroxy-4-(3-isopropylphenyl)but-l-en-l-yl]-2-oxo- l,3-oxazinan-3-yl ⁇ propyl)thiophene-2-carboxylic acid
  • Example 18 Example 19 Step 1 : Isopropyl 4-bromo-5- ⁇ 3-[(4i?)-4-(bromomethyl)-2-oxo-l,3-oxazinan-3-yl]propyl ⁇ thiophene-2- carboxylate (21)
  • Step 2 Isopropyl 5-(3- ⁇ (4R)-4-[(acetyloxy)methyl]-2-oxo-l,3-oxazinan-3-yl ⁇ propyl)-4-bromothiophene- 2-carboxylate (22)
  • Step 3 Isopropyl 4-bromo-5- ⁇ 3-[(4R)-4-(hydroxymethyl)-2-oxo-l,3-oxazinan-3-yl]propyl ⁇ thiophene-2- carboxylate (23)
  • Example 18 methyl 4-bromo-5-(3- ⁇ (4R)-4-[(l£,3i?)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybut-l-en- 1 -yl]-2-oxo- 1 ,3 -oxazinan-3 -yl ⁇ propyl)thiophene-2-carboxylate
  • Example 19 4-bromo-5-(3- ⁇ (4R)-4-[( l£,3R)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybut- 1 -en- 1 -yl]-2- OXO- 1 ,3-oxazinan-3-yl ⁇ propyl)thiophene-2-carboxylic acid
  • Example 20 5-(3- ⁇ (4R)-4-[(l J E,3R)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo-l,3- oxazinan-3-yl ⁇ propyl)-3-fluorothiophene-2-carboxylic acid
  • Drug concentrations are expressed in terms of the active ingredient (base).
  • the compounds of this invention are dissolved in a suitable ophthalmic solution (e.g., 0.5% polysorbate-80, 0.1% EDTA, 0.02% benzalkonium chloride, 4.5% mannitol in 5 niM citrate buffer at pH 5-7) at 0.00063, 0.00013 % for rabbit studies and 0.0063, 0.0019%, 0.00063 and 0.000063% for monkey studies.
  • Drug or vehicle aliquots (25 ul) are administered topically unilaterally or bilaterally.
  • the contralateral eyes receive an equal volume of vehicle (0.5% polysorbate-80, 0.1% EDTA, 0.02% benzalkonium chloride, 4.5% mannitol in 5 mM citrate buffer at pH 5-7).
  • 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.
  • Unilateral ocular hypertension of the right eye is 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 are kept in a sitting position in restraint chairs for the duration of the experiment. Animals are 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 is measured using a pneumatic tonometer (Alcon Applanation Tonometer) or a Digilab pneumatonometer (Bio-Rad Ophthalmic Division, Cambridge, MA, USA). IOP is measured before treatment and generally at 30, 60, 124, 180, 300, and 360 minutes after treatment. Baseline values are also obtained at these time points generally two or three days prior to treatment.
  • Treatment consists of instilling one drop of 25 ul of the compounds of this invention (0.000063 to 0.0063 %) or vehicle. At least one-week washout period is employed before testing on the same animal.
  • the normotensive (contralateral to the hypertensive) eye is treated in an exactly similar manner to the hypertensive eye.
  • IOP measurements for both eyes are compared to the corresponding baseline values at the same time point. Results are expressed as mean plus-or-minus standard deviation in mm Hg.
  • the activity range of the compounds of this invention for ocular use is between 0.01 and 100,000 nM.
  • Example 1 Compounds from the current invention (i.e., Example 1) showed improved ocular tolerability in animal species such as rabbits and cynomolgus monkeys compared to compounds disclosed in WO 2004/085430 (i.e., Example 2).
  • Example 2 Compounds from the current invention
  • Example 3 showed improved ocular tolerability in animal species such as rabbits and cynomolgus monkeys compared to compounds disclosed in WO 2004/085430 (i.e., Example 2).
  • Example 2 Compounds from the current invention
  • Example 2 of WO 2004/085430 demonstrated treatment related findings of very slight bilateral hyperemia, very slight conjunctival chemosis and slight ocular discharge at the same dose. Similarly a 5-fold improvement in eye closure was observed with the present compound in New Zealand White rabbits after ocular dosing. The present compound also demonstrated a 3-5-fold improvement in ocular adverse effects (conjuctival hyperemia, partial eye closure, etc.) in Cynomolgus Monkeys.
  • Prostanoid receptor (PG) cDNAs corresponding to full length coding sequences were subcloned into the appropriate 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 LipofectAMINE@ (GIBCO-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 Penicillin-G, 100 ⁇ g/ml Streptomycin sulphate, 250 ⁇ g/ml active GENETICINTM (G418) (all from Life Technologies, Inc/BRL) and 200 ⁇ g/ml hygromycin (Calbiochem). Individual colonies were isolated after 2-3 weeks of growth under selection using the cloning ring method and subsequently expanded into clonal cell lines. Expression of the receptor cDNA was assessed by receptor binding assays.
  • DMEM Dulbecco's Modified Eagle Medium
  • 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 centrifugation (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 phenylmethylsulfonylfluoride, 10 ⁇ M E-64, 100 ⁇ M leupeptin and 0.05 mg/ml pepstatin).
  • protease inhibitors 2 mM phenylmethylsulfonylfluoride, 10 ⁇ M E-64, 100 ⁇ M leupeptin and 0.05 mg/ml pepstatin.
  • the 160,000 x g pellets were resuspended in 10 mM HEPES/KOH (pH 7.4) containing 1 mM EDTA at approximately 5-10 mg/ml protein by Dounce homogenisation (Dounce A; 10 strokes), frozen in liquid nitrogen and stored at -80 0 C.
  • 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 EP), 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 (1 15 Ci/mmol) for DP, 0.95 nM [ 3 H]PGF 2n (170 Ci/mmol) for FP, 5 nM [ 3 H]iloprost (16 Ci/mmol) for IP and 1.8 nM [ 3 H]SQ 29548 (46 Ci/mmol) for TP].
  • EP 3 assays also contained 100 ⁇ M GTPDS.
  • 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 % (v/v) in all incubations. Non-specific binding was determined in the presence of 1 ⁇ M of the corresponding nonradioactive prostanoid. Incubations were conducted for 60 min (EP subtypes, FP and IP) or 30 min (DP and TP) at 3O 0 C (EP subtypes, DP, FP and TP) or room temperature (IP) and terminated by rapid
  • the activity range of the compounds of this invention for bone use is between 0.01 and 100,000 nM.
  • RP-I periosteal cells are spontaneously immortalized from primary cultures of periosteal cells from tibae of 4-week old Sprague-Dawley rats and are cultured in DMEM (BRL, Gaithersburg, MD) with 10 % fetal bovine serum (JRH Biosciences, Lenexa, KS). These cells do not express 50 osteoblastic phenotypic markers in early culture, but upon confluence, express type I collagen, alkaline phosphatase and osteocalcin and produce mineralized extracellular matrix.
  • RCT-I and RCT-3 are clonal cell lines immortalized by SV-40 large T antigen from cells released from fetal rat calvair by a cmbination collagenase/hyaluronidase digestion.
  • RCT-I cells derived from cells released during the first 10 minutes of digestion (fraction I), are cultured in RPMI 1640 medium (BRL) with 10% fetal bovine serum and 0.4 mg/ml G418 (BRL). These cells differentiate and express osteoblastic features upon retinoic acid treatment.
  • RCT-3 cells immortalized from osteoblast- enriched fraction EU cells, are cultured in F- 12 medium (BRL) with 5% Fetal bovine serum and 0.4 mg/ml G418.
  • TRAB-11 cells are also immortalized by SV40 large T antigen from adult rat tibia and are
  • P815 mouse mastocytoma cells, cultured in Eagles MEM with 10% FBS, and NRK
  • RNA is extracted from the tibial metaphysis or diaphysis and calvaria using a guanidinium isothiocyanate-phenol-chloroform method after pulverizing frozen bone samples by a tissue homogenizer. See P. Chomczynski et al., Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction., Analyt Biochem, 162, 156-159 (1987), which is incorporated by reference herein in its entirety. RNA samples (20 mg) are separated on 0.9% agarose/formaldehyde
  • Membranes are prehybridized in Hybrisol I (Oncor, Gaithersburg, MD) and 0.5 mg/ml sonicated salmon sperm DNA (Boehringer) at 42 0 C for 3 hours and are hybridized at 42 0 C with rat EP2 and mouse EP4 cDNA probes labeled with p2p]-dCTP (Amersham, Buckinghamshire, UK) by random priming using the rediprime kit (Amersham).
  • membranes are washed 4 times in 2xSSC + 0.1% SDS at room 50 temperature for a total of 1 hour and once with 0.2xSSC + 0.1% SDS at 55 0 C for 1 hour and then exposed to Kodak XAR 2 film at -70 0 C using intensifying screens. After developing the films, bound probes are removed twice with 0.1% SDS at 8O 0 C and membranes are hybridized with a human GAPDH (Glyceraldehyde 3 -Phosphate Dehydrogenase) cDNA probe (purchased from Clontech, Palo Alto, CA) for loading control. 4. In-Situ Hybridization:
  • Frozen tibiae are sectioned coronally at 7 mm thickness and sections are mounted on charged slides (Probe On Plus, Fisher Scientific, Springfield, NJ) and are kept at -70 C until 5 hybridization.
  • cRNA probes are labeled with 35 ⁇ -UTPgS (ICN, Costa Mesa, CA) using a Riboprobe ⁇ kit (Promega Madison, WT). Hybridization is performed overnight at 50 C. See M. Weinreb et al, Different pattern of alkaline phosphatase, osteopontin and osteocalcin expression in developing rat bone visualized by in-situ hybridization, J. Bone Miner Res., 5, 831-842 (1990) and D.
  • EP4 and EP2 mRNA are examined in various bone derived cells including osteoblast-enriched primary rat calvaria cells, immortalized osteoblastic cell lines from fetal rat
  • osteoblastic osteosarcoma ZO calvaria or from adult rat tibia and an osteoblastic osteosarcoma cell line. Most of the osteoblastic cells and cell lines show significant amounts of 3.8 kb EP4 mRNA, except for the rat osteosarcoma cell line
  • ROS 17/2.8 Consistent with this finding, in ROS 17/2.8 cells PGE 2 has no effect on intracellular cAMP, which is markedly induced in RCT-3 and TRAB-11 cells. Treatment of RCT-I cells with retinoic acid, which promotes their differentiation, reduces the levels of EP4 mRNA. NRK fibroblasts do
  • EP4 mRNA 25 not express EP4 mRNA, while P815 mastocytoma cells, used as positive controls, express large amounts of EP4 mRNA. In contrast to EP4 mRNA, none of the osteoblastic cells and cell lines express detectable amounts OfEP 2 mRA in total RNA samples. Expression of EP4 mRNA in osteoblastic cells, EP4 is also expressed in total RNA isolated from tibiae and calvariae of 5-week-old rats. In contrast, no EP 2 mRNA is found in RNA from tibial shafts.
  • PGE 2 Induces The Expression OfEPz 1 mRNA in RP-I Periosteal Cells And In Adult Rat Tibiae
  • PGE 2 enhances its own production via upregulation of cyclooxygenase 2 expression in osteoblasts and in bone tissue thus autoamplifying its own effects. PGE 2 also increases the levels of EP4 mRNA.
  • RP- 1 cells are immortalized from a primary culture of adult rat tibia periosteum is examined. These cells express osteoblast phenotypic markers upon confluence and form mineralized bone matrix when implanted in nude mice. Similar to the other osteoblastic cells examined, RP-I periosteal cells
  • EP2 mRNA is not expressed in RP-I cells before or after treatment with PGE2.
  • PGE2 regulates EP4 mRNA levels in vivo in bone tissue
  • five-week-old male rats are injected with PGE2 (3 - 6 mg/Kg).
  • Systemic administration of PGE2 rapidly increased EP4 mRNA levels in the tibial diaphysis peaking at 2 h after injection.
  • a similar effect of PGE2 on EP4 0 mRNA is observed in the tibial metaphysis and in calvaria.
  • PGE2 induces EP4 mRNA levels in vitro in osteogenic periosteal cells and in vivo in bone tissue in a cell type-specific and tissue-specific manner.
  • PGE2 does not induce EP2 mRNA in RP-I cells nor in bone tissue.
  • EP4 is expressed in osteoblastic cells in vitro and in bone marrow cells in vivo, and is upregulated by its ligand, PGE2.

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Abstract

This invention relates to potent selective agonists of the EP4 subtype of prostaglandin E2 receptors, their use or a formulation thereof in the treatment of glaucoma and other conditions, which are related to elevated intraocular pressure in the eye of a patient. This invention further relates to the use of the compounds of this invention for mediating the bone modeling and remodeling processes of the osteoblasts and osteoclasts.

Description

TITLE OF THE INVENTION
EP4 RECEPTOR AGONIST, COMPOSITIONS AND METHODS THEREOF
BACKGROUND OF THE INVENTION 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.
Many of the drugs formerly used to treat glaucoma proved unsatisfactory. Current methods of treating glaucoma include using therapeutic agents such as pilocarpine, carbonic anhydrase inhibitors, beta-blockers, prostaglandins and the like. However, these therapies often produce undesirable local effects. As can be seen, there are several current 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 exists the need for new and effective therapies with little or no side effects.
A variety of disorders in humans and other mammals involve or are associated with abnormal or excessive bone loss. Such disorders include, but are not limited to, osteoporosis, glucocorticoid induced osteoporosis, Paget's disease, abnormally increased bone turnover, periodontal disease, tooth loss, bone fractures, rheumatoid arthritis, periprosthetic osteolysis, osteogenesis imperfecta, metastatic bone disease, hypercalcemia of malignancy, and multiple myeloma. One of the most common of these disorders is osteoporosis, which in its most frequent manifestation occurs in postmenopausal women. Prostaglandins such as the PGE2 series are known to stimulate bone formation and increase bone mass in mammals, including man. It is believed that the four different receptor subtypes, designated EP γ, EP2, EP3, and EP4 are involved in mediating the bone modeling and remodeling processes of the osteoblasts and osteoclasts. The major prostaglandin receptor in bone is EP4, which is believed to provide its effect by signaling via cyclic AMP. In the present invention it is found that the formula I agonists of the EP4 subtype receptor are useful for stimulating bone formation. WO 02/24647, WO 02/42268, EP 1114816, WO 01/46140 and WO 01/72268 disclose EP4 agonists. However, they do not disclose the compounds of the instant invention. SUMMARY OF THE INVENTION
This invention relates to agonists of the EP4 subtype of prostaglandin E2 receptors and their use or a formulation thereof in the treatment of glaucoma and other conditions that are related to elevated intraocular pressure in the eye of a patient. In particular, this invention relates to a series of 3,4- disubstituted l,3-oxazinan-2-one, derivatives and their use to treat ocular diseases and to provide a neuroprotective effect to the eye of mammalian species, particularly humans. This invention further relates to the use of the compounds of this invention for mediating the bone modeling and remodeling processes of the osteoblasts and osteoclasts.
More particularly, this invention relates to novel EP4 agonist having the structural formula I:
Figure imgf000003_0001
FORMULA I or a pharmaceutically acceptable salt, enantiomer, diastereomer, prodrug or mixture thereof, wherein,
R and R4 independently represent H, or Ci-6 alkyl;
Rl independently represents hydrogen, Ci_6 alkyl, halogen, CF3, aryl, said aryl optionally substituted with 1 to 3 groups of halogen, Cl -6 alkyl, CF3, or N(R4)2 ; R2 represents H, or halogen; R3 represents COOR or carboxylic acid isostere; n represents 0-3; and — represents a double or single bond.
This and other aspects of the invention will be realized upon inspection of the invention as a whole.
DETAILED DESCRIPTION OF THE INVENTION
The invention is described herein in detail using the terms defined below unless otherwise specified. The term "therapeutically effective amount", as used herein, means that amount of the EP4 receptor subtype agonist of formula I, or other actives of the present invention, that will elicit the desired therapeutic effect or response or provide the desired benefit when administered in accordance with the desired treatment regimen. A preferred therapeutically effective amount relating to the treatment of abnormal bone resorption is a bone formation, stimulating amount. Likewise, a preferred therapeutically effective amount relating to the treatment of ocular hypertension or glaucoma is an amount effective for reducing intraocular pressure and/or treating ocular hypertension and/or glaucoma.
"Pharmaceutically acceptable" as used herein, means generally suitable for administration to a mammal, including humans, from a toxicity or safety standpoint. The term "prodrug" refers to compounds which are drug precursors which, following administration and absorption, release the claimed drug in vivo via some metabolic process. A non- limiting example of a prodrug of the compounds of this invention would be an ester of the carboxylic acid group, where the ester functionality is easily hydrolyzed to the active acid after administration to a patient. Exemplary prodrugs include acetic acid derivatives that are non-narcotic, analgesics/non- steroidal, anti-inflammatory drugs having a free CH2COOH group (which can optionally be in the form of a pharmaceutically acceptable salt, e.g. -CH2COO-Na+), typically attached to a ring system, preferably to an aromatic or heteroaromatic ring system.
The term "alkyl" refers to a monovalent alkane (hydrocarbon) derived radical containing from 1 to 6 carbon atoms unless otherwise defined. It may be straight, branched or cyclic. Preferred alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, t-butyl, cyclopentyl and cyclohexyl. 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 6 carbon atoms, without alternating or resonating double bonds between carbon atoms. Examples of cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
Halogen (halo) refers to chlorine, fluorine, iodine or bromine. Carboxylic isostere represents tetrazole, acylsulfonamide, sulfonic acid, phosphonic acid or prodrug such as C]-6 aldehyde or Cμg alcohol.
Aryl refers to aromatic rings e.g., phenyl, substituted phenyl and the like, as well as rings which are fused, e.g., naphthyl, phenanthrenyl and the like. An aryl group thus contains at least one ring having at least 6 atoms, with up to five such rings being present, containing up to 22 atoms therein, with alternating (resonating) double bonds between adjacent carbon atoms or suitable heteroatoms. Examples of aryl groups are phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl and phenanthrenyl, preferably phenyl, naphthyl or phenanthrenyl. Aryl groups may likewise be substituted as defined. Preferred substituted aryls include phenyl and naphthyl.
The term "agonist" as used herein means EP4 subtype compounds of formula I 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, 9th edition, 1996, chapter 2.
One embodiment of this invention is realized when R3 is COOR and all other variables are as originally desfined.
Another embodiment of this invention is realized when R3 is a carboxylic acid isostere and all other variables are as originally defined.
Another embodiment of this invention is realized when R is H and all other variables are as originally defined.
Another embodiment of this invention is realized when R is Ci -6 alkyl and all other variables are as originally defined. A sub-embodiment of this invention is realized when R is isopropyl, ethyl or, methyl, preferably isopropyl.
Another embodiment of this invention is realized when R2 is hydrogen and all other variables are as originally defined.
Another embodiment of this invention is realized when R2 is halogen and all other variables are as originally defined. A subembodiment of this invention is realized when halogen is fluorine or bromine.
Still another embodiment of this invention is realized when Ri is halogen and all other variables are as originally defined. A sub-embodiment is realized when Ri is bromine or chlorine, preferably bromine.
Still another embodiment of this invention is realized when Ri is Ci -6 alkyl and all other variables are as originally defined.
Still another embodiment of this invention is realized when Ri is CF3 and all other variables are as originally defined.
Another embodiment of this invention is realized when R3 is COOR, R is H or isopropyl, Rl is bromine and R2 is hydrogen. Another embodiment of this invention is realized when R3 is tetrazole, Ri is bromine and R2 is hydrogen
Another embodiment of this invention is reaslized when n is 0, 1, or 2 and all other variables are as originally defined. Still another embodiment of this invention is realized when n is 0 and all other variables are as originally defined
Another embodiment of this invention is realized when n is 1 and all other variables are as originally defined. A subembodiment of this invention is realizedwhen Rj is bromine and R.2 is hydrogen.
Another embodiment of this invention is realized when n is 2 and all other variables are as originally defined.
Another embodiment of this invention is realized when — represents a double bond.
Compounds of this invention are: Isopropyl S-CS-K^H-Kl^S^^-CS-bromophenylH^-difluoro-S-hydroxybut-l-en-l-yη^-oxo-US- oxazinan-3-yl } propyl)thiophene-2-carboxylate;
Isopropy 1 5 -(3 - { (4R)-4- [( 1 £)-4-(3 -bromopheny l)-4,4-difluoro-3 -oxobut- 1 -en- 1 -y 1] -2-oxo- 1 ,3 -oxazinan-3 - yl}propyl)thiophene-2-carboxylate;
Isopropy 1 5 -(3 - { (4R)-4- [( 1 £)-4-(3 -bromophenyl)-4,4-difluoro-3 -hydroxybut- 1 -en- 1 -y 1] -2-oxo- 1,3- oxazinan-3-yl}propyl)thiophene-2-carboxylate;
5-(3 - {(47?)-4-[( 1 E,3R)-4-(3 -bromophenyl)-4,4-difluoro-3 -hydroxybut- 1 -en- 1 -y l]-2-oxo- 1 ,3 -oxazinan-3 - yl}propyl)thiophene-2-carboxylic acid;
Isopropy 1 5 -(3 - { (4S)-4- [(3R)-4-(3 -bromopheny l)-4,4-difluoro-3 -hydroxy butyl] -2-oxo- 1 ,3 -oxazinan-3 - yl } propyl)thioρhene-2-carboxylate; 5-(3-{(45)-4-[(3R)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybutyl]-2-oxo-l,3-oxazinan-3- yl}propyl)thiophene-2-carboxylic acid;
5-(3-{(4R)-4-[(lJE)-4,4-difluoro-3-hydroxy-4-phenylbut-l-en-l-yl]-2-oxo-l,3-oxazinan-3- yl} propyl)thiophene-2-carboxylic acid;
Isopropyl 5-(3-{(4R)-4-[(l£,3i?)-4-(3,5-dichlorophenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo-l,3- oxazinan-3-yl } propyl)thiophene-2-carboxylate;
5-(3-{(4R)-4-[(l£,3R)-4-(3,5-dichlorophenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo-l,3-oxazinan-
3-yl}propyl)thiophene-2-carboxylic acid;
Isopropy 1 5 -(3 - { (45)-4- [(3R)-4-(3 , 5-dichloropheny l)-4,4-difluoro-3 -hy droxybuty 1] -2-oxo- 1 ,3 -oxazinan-3 - y 1 } propyl)thiophene-2-carboxylate; 5-(3-{(4S)-4-[(3R)-4-(3,5-dichlorophenyl)-4,4-difluoro-3-hydroxybutyl]-2-oxo-l,3-oxazinan-3- yl}propyl)thiophene-2-carboxylic acid;
Isopropyl 5-(3-{(4R)-4-[(l£,3R)-4-(3,5-dimethylphenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo-l,3- oxazinan-3 -yl}propyl)thiophene-2-carboxylate; 5 -(3- {(4R)-4-[( l£,3R)-4-(3,5-dimethylphenyl)-4,4-difluoro-3-hydroxybut- 1 -en- 1 -yl]-2-oxo- 1 ,3-oxazinan-
3-yl}propyl)thiophene-2-carboxylic acid;
Isopropyl 5-(3-{(45)-4-[(3R)-4-(3,5-dimethylphenyl)-4,4-difluoro-3-hydroxybutyl]-2-oxo-l,3-oxazinan-3- yl}propyl)thiophene-2-carboxylate; 5-(3-{(45r)-4-[(3R)-4-(3,5-dimethylphenyl)-4,4-difluoro-3-hydroxybutyl]-2-oxo-l,3-oxazinan-3- yl } propyl)thiophene-2-carboxylic acid;
Isopropyl 5-[3-((4R)-4- {( l£',3i?)-4,4-difluoro-3-hydroxy-4-[3-(trifluoroniethyl)phenyl]but- 1 -en- 1 -yl} -2- oxo- 1 ,3-oxazinan-3-yl)propyl]thiophene-2-carboxylate;
5-[3-((4R)-4-{(l£,3R)-4,4-difluoro-3-hydroxy-4-[3-(trifluoromethyl)phenyl]but-l-en-l-yl}-2-oxo-l,3- oxazinan-3-yl)propyl]thiophene-2-carboxylic acid;
Isopropyl 5-[3-((45)-4-{(3R)-4,4-difluoro-3-hydroxy-4-[3-(trifluoromethyl)phenyl]butyl}-2-oxo-l,3- oxazinan-3-yl)propyl]thiophene-2-carboxylate;
5-[3-((4S)-4-{(3R)-4,4-difluoro-3-hydroxy-4-[3-(trifluoromethyl)phenyl]butyl}-2-oxo-l ,3-oxazinan-3- yl)propyl]thiophene-2-carboxylic acid; 5 -(3 - {(4R)-4-[( 1 £,3-ft)-4,4-difluoro-3 -hydroxy-4-(3 -isopropylphenyl)but- 1 -en- 1 -yl] -2-oxo- 1 ,3 -oxazinan-3 - yl}propyl)thiophene-2-carboxylic acid;
5-(3-{(4iS)-4-[(3i?)-4,4-difluoro-3-hydroxy-4-(3-isopropylphenyl)butyl]-2-oxo-l,3-oxazinan-3- yl } propyl )thiophene-2-carboxylic acid;
Methyl 4-bromo-5-(3-{(4R)-4-[(l£',3R)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo- l,3-oxazinan-3-yl}propyl)thiophene-2-carboxylate;
4-Bromo-5-(3 - {(4R)-4-[( 1 E,3R)-4-(3 -bromophenyl)-4,4-difluoro-3 -hydroxybut- 1 -en- 1 -yl]-2-oxo- 1,3- oxazinan-3 -yl } propyl)thiophene-2-carboxylic acid;
5-(3-{(4R)-4-[(l£',3R)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo-l,3-oxazinan-3- yl}propyl)-3-fluorothiophene-2-carboxylic acid; 5-(3-{(4S)-4-[(3R)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybutyl]-2-oxo-l,3-oxazinan-3-yl}propyl)-3- fluorothiophene-2-carboxylic acid;
5 -(3 - { (4S)-4-[(3R)-3 -(4'-chloro-2'-methylbiphenyl-3 -yl)-3 -hydroxypropyl]-2-oxo- 1 ,3 -oxazinan-3 - yl} propyl )thiophene-2-carboxylic acid;
Isopropyl 5-(3-{(4R)-4-[(l£,3R)-4-(3-bromo-5-iodophenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo- l,3-oxazinan-3-yl}propyl)thiophene-2-carboxylate; and
5-(3-{(4R)-4-[(l£,3R)-4-(3-bromo-5-iodophenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo-l,3- oxazinan-3 -yl } propyl)thiophene-2-carboxylic acid; or a pharmaceutically acceptable salt, enantiomer, diastereomer, prodrug or mixture thereof. Another embodiment of this invention is directed to a composition containing an EP4 agonist of Formula I and optionally a pharmaceutically acceptable carrier.
Yet another embodiment of this invention is directed to a method for decreasing elevated intraocular pressure or treating glaucoma by administration, preferably topical or intra-camaral administration, of a composition containing an EP4 agonist of Formula I and optionally a pharmaceutically acceptable carrier. Use of the compounds of formula I for the manufacture of a medicament for treating elevated intraocular pressure or glaucoma or a combination thereof is also included in this invention
This invention is further concerned with a process for making a pharmaceutical composition comprising a compound of formula I.
This invention is further concerned with a process for making a pharmaceutical composition comprising a compound of formula I, and a pharmaceutically acceptable carrier.
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 ocular hypertension.
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 Nation Eye Institute/Industry Workshop on Clinical Trials in Dry Eyes, The CLAO Journel, 21(4):221-231 (1995)). Functional EP4 receptors have been found in human conjuctival epithelial cells (see US Patent 6,344,477, incorporated by reference in its entirey) and it is appreciated that both human corneal epithelial cells (Progess in Retinal and Eye Research, 16:81-98(1997)) and conjuctival cells (Dartt et al. Localization of nerves adjacent to goblet cells in rat conjucntiva. Current Eye Research, 14:993-1000 (1995)) are capable of secreting mucins. Thus, the compounds of formula I are useful for treating dry eye.
Macular edema is swelling within the retina within the critically important central visual zone at the posterior pole of the eye. It is believed that EP4 agonist which lower IOP are useful for treating diseases of the macular such as macular edema or macular degeneration. Thus, 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. As a result, 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 I.
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. Thus, this invention is also concerned with compositions and methods of treating ocular hypertension, glaucoma, macular edema, macular degeneration, for increasing retinal and optic nerve head blood velocity, for increasing retinal and optic nerve oxygen tension, for providing a neuroprotective effect or for a combination thereof by administering to a patient in need thereof one of the compounds of formula I alone or in combination with one or more of the following active ingredients, a β-adrenergic blocking agent such as timolol, betaxolol, levobetaxolol, carteolol, levobunolol, a parasympathomimetic agent such as pilocarpine, a sympathomimetic agents such as epinephrine, iopidine, brimonidine, clonidine, para-aminoclonidine, a carbonic anhydrase inhibitor such as dorzolamide, acetazolamide, metazolamide or brinzolamide; COSOPT®, a Maxi-K channel blocker such as Penitrem A, paspalicine, charybdotoxin, iberiotoxin, Paxicillan, Aflitram, Verroculogen, and as disclosed in WO 03/105868 (USSN 60/389,205), WO 03/105724 (60/389,222), WO 03/105847 (60/458,981), 60/424790, filed November 8, 2002 (Attorney docket 21260PV), 60/424808, filed November 8, 2002 (Attorney docket 2128 IPV), 09/765716, filed January 17, 2001, 09/764738, filed January 17, 2001 and PCT publications WO 02/077168 and WO 02/02060863, all incorporated by reference in their entirety herein, and in particular Maxi-K channel blockers such as l-(l-isobutyl-6-methoxy-lH-indazol-3-yl)-2-methylpropan-l-one; 1 -[I -(2,2- dimethylpropyl)-6-methoxy-lH-indazol-3-yl]-2-methylpropan-l-one; l-[l-(cyclohexylmethyl)-6- methoxy- 1 H-indazol-3 -yl]-2-methylpropan- 1 -one; 1 -( 1 -hexyl-6-methoxy- 1 H-indazol-3 -yl)-2- methylpropan- 1 -one; 1 -[ 1 -(2-ethylhexyl)-6-methoxy- 1 H-indazol-3-yl]-2-methylpropan- 1 -one; 1 -(3- isobutyryl-6-methoxy-lH-indazol-l-yl)buan-2-one; l-(3-isobutyryl-6-methoxy-lH-indazol-l-yl)-3,3- dimethylbutan-2-one; l-(3-cyclopentylcarbonyl)-6-methoxy-lH-indazol-l-yl)-3,3-dimethylbutan-2-one; l-(3,3-dimethyl-2-oxobutyl) -6-methoxy-lH-indazole-3-carboxylic acid; and l-[3-(3-hydroxypropanoyl) - 6-methoxy-lH-indazol-l-yl]-3,3-dimethylbutan-2-one, a prostaglandin such as latanoprost, travaprost, unoprostone, rescula, S1033 (compounds set forth in US Patent Nos. 5,889,052; 5,296,504; 5,422,368; and 5,151,444); a hypotensive lipid such as lumigan and the compounds set forth in US Patent No. 5,352,708; a neuroprotectant disclosed in US Patent No. 4,690,931, particularly eliprodil and R-eliprodil as set forth in WO 94/13275, including memantine; and/or an agonist of 5-HT2 receptors as set forth in PCTVUSOO/31247, particularly l-(2-aminopropyl)-3-methyl-lH-imdazol-6-ol fumarate and 2-(3-chloro-6- methoxy-indazol- 1 -yl)- 1 -methyl-ethylamine.
Use of the compounds of formula I for the manufacture of a medicament for treating ocular hypertension, glaucoma, macular edema, macular degeneration, for increasing retinal and optic nerve head blood velocity, for increasing retinal and optic nerve oxygen tension, for providing a neuroprotective effect or for a combination thereof is also included in this invention.
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 0.00001 to 5% and especially 0.00001 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. For a single dose, from between 0.0000001 to 0.05 mg, preferably 0.0000005 to 0.02 mg, and especially 0.000005 to 0.01 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. Typical of 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. Additionally, suitable ophthalmic vehicles can be used as carrier media for the present purpose 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. 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.
For topical ocular administration the 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 compounds of the instant invention are also useful for mediating the bone modeling and remodeling processes of the osteoblasts and osteoclasts. See PCT US99/23757 filed October 12,
1999 and incorporated herein by reference in its entirety. The major prostaglandin receptor in bone is EPφ which is believed to provide its effect by signaling via cyclic AMP. See Dceda T, Miyaura C, Ichikawa A, Narumiya S, Yoshiki S and Suda T 1995, In situ localization of three subtypes (EPj, EP3 and EP4) of prostaglandin E receptors in embryonic and newborn mice., J Bone Miner Res 10 (sup
1):S172, which is incorporated by reference herein in its entirety. Use of the compounds of formula I for the manufacture of a medicament for mediating the bone modeling and remodeling processes are also included in this invention. Thus, another object of the present invention is to provide methods for stimulating bone formation, i.e. osteogenesis, in a mammal comprising administering to a mammal in need thereof a therapeutically effective amount of an EP4 receptor subtype agonist of formula I.
Still another object of the present invention to provide methods for stimulating bone formation in a mammal in need thereof comprising administering to said mammal a therapeutically effective amount of an EP4 receptor subtype agonist of formula I and a bisphosphonate active. Use of the compounds of formula I for the manufacture of a medicament for stimulating bone formation is also included in this invention.
Yet another object of the present invention to provide pharmaceutical compositions comprising a therapeutically effective amount of an EP4 receptor subtype agonist of formula I and a bisphosphonate active.
It is another object of the present invention to provide methods for treating or reducing the risk of contracting a disease state or condition related to abnormal bone resorption in a mammal in need of such treatment or prevention, comprising administering to said mammal a therapeutically effective amount of an EP4 receptor subtype agonist of formula I. Use of the compounds of formula I for the manufacture of a medicament for treating or reducing the risk of contracting a disease state or condition related to abnormal bone resorption is also included in this invention.
The disease states or conditions related to abnormal bone resorption include, but are not limited to, osteoporosis, glucocorticoid induced osteoporosis, Paget's disease, abnormally increased bone turnover, periodontal disease, tooth loss, bone fractures, rheumatoid arthritis, periprosthetic osteolysis, osteogenesis imperfecta, metastatic bone disease, hypercalcemia of malignancy, and multiple myeloma.
Within the method comprising administering a therapeutically effective amount of an EP4 receptor subtype agonist of formula I and a bisphosphonate active, both concurrent and sequential administration of the EP4 receptor subtype agonist of formula I and the bisphosphonate active are deemed within the scope of the present invention. Generally, the formulations are prepared containing 5 or 10 mg of a bisphosphonate active, on a bisphosphonic acid active basis. With sequential administration, the agonist and the bisphosphonate can be administered in either order. In a subclass of sequential administration the agonist and bisphosphonate are typically administered within the same 24 hour period. In yet a further subclass, the agonist and bisphosphonate are typically administered within about 4 hours of each other.
A non-limiting class of bisphosphonate actives useful in the instant invention are selected from the group consisting of alendronate, cimadronate, clodronate, tiludronate, etidronate, ibandronate, neridronate, olpandronate, risedronate, piridronate, pamidronate, zolendronate, pharmaceutically acceptable salts thereof, and mixtures thereof. A non-limiting subclass of the above-mentioned class in the instant case is selected from the group consisting of alendronate, pharmaceutically acceptable salts thereof, and mixtures thereof. A non-limiting example of the subclass is alendronate monosodium trihydrate. In the present invention, as it relates to bone stimulation, the agonist is typically administered for a sufficient period of time until the desired therapeutic effect is achieved. The term "until the desired therapeutic effect is achieved", as used herein, means that the therapeutic agent or agents are continuously administered, according to the dosing schedule chosen, up to the time that the clinical or medical effect sought for the disease or condition being mediated is observed by the clinician or researcher. For methods of treatment of the present invention, the compounds are continuously administered until the desired change in bone mass or structure is observed, hi such instances, achieving an increase in bone mass or a replacement of abnormal bone structure with normal bone structure are the desired objectives. For methods of reducing the risk of a disease state or condition, the compounds are continuously administered for as long as necessary to prevent the undesired condition. Pn such instances, maintenance of bone mass density is often the objective. Nonlimiting examples of administration periods can range from about 2 weeks to the remaining lifespan of the mammal. For humans, administration periods can range from about 2 weeks to the remaining lifespan of the human, preferably from about 2 weeks to about 20 years, more preferably from about 1 month to about 20 years, more preferably from about 6 months to about 10 years, and most preferably from about 1 year to about 10 years. The instant compounds are also useful in combination with known agents useful for treating or preventing bone loss, bone fractures, osteoporosis, glucocorticoid induced osteoporosis, Paget' s disease, abnormally increased bone turnover, periodontal disease, tooth loss, osteoarthritis, rheumatoid arthritis, periprosthetic osteolysis, osteogenesis imperfecta, metastatic bone disease, hypercalcemia of malignancy, and multiple myeloma. Combinations of the presently disclosed compounds with other agents useful in treating or preventing osteoporosis or other bone disorders are within the scope of the invention. A person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the disease involved. Such agents include the following: an organic bisphosphonate; a cathepsin K inhibitor; an estrogen or an estrogen receptor modulator; an androgen receptor modulator; an inhibitor of osteoclast proton ATPase; an inhibitor of HMG-CoA reductase; an integrin receptor antagonist; an osteoblast anabolic agent, such as PTH; calcitonin; Vitamin D or a synthetic Vitamin D analogue; and the pharmaceutically acceptable salts and mixtures thereof. A preferred combination is a compound of the present invention and an organic bisphosphonate. Another preferred combination is a compound of the present invention and an estrogen receptor modulator. Another preferred combination is a compound of the present invention and an estrogen. Another preferred combination is a compound of the present invention and an androgen receptor modulator. Another preferred combination is a compound of the present invention and an osteoblast anabolic agent.
Regarding treatment of abnormal bone resorption and ocular disorders, the formula I 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 EC5Q value of from about 0.0001 microM to about 10 microM. In a further subclass of the present invention, the agonists have an EC50 value of from about 0.0001 microM to about 1 microM. EC^Q 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, and PCT US99/23757, filed October 12, 1999, which are incoroporated by reference herein in their entirety. The herein examples illustrate but do not limit the claimed invention. Each of the claimed compounds are EP4 agonists and are useful for a number of physiological ocular and bone disorders.
The compounds of this invention can be made, with some modification, in accordance with US Patent No. 6,043,275, EP0855389, WO 03/047417 (USSN 60/337228), WO 03/047513 (USSN 60/338,117), USSN 60/406,530 (Merck Docket No. MC060), WO 2004/085430 and WO 01/46140, all of which are incorporated herein by reference in their entirety. The following non-limiting schemes and examples given by way of illustration is demonstrative of the present invention.
The preparation of compounds from the current invention can be accomplished according to general schemes 1 and 2, and is further illustrated in the experimental section. Scheme 1 : General synthetic scheme using advanced intermediate 1.
Figure imgf000015_0001
(4/?)-4-({[/eλ-/-butyl(dimethyl)silyl]oxy}methyl)-l,3-oxazinan-2-one (1) (WO 2004/085430) was first treated with a strong base such as potassium hexamethyldisilazide (KHMDS) or NaH followed by treatment with reagent 2 (L = I5 MeSO2O or Br, etc.) in either THF (tetrahydrofuran) or DMF (dimethylformamide) to give the alkylation product 3. Deprotection of the t-butyldimethylsilyl (TBS) group with IN aqueous HCl (hydrochloride) or with TBAF (tetrabutylammonium fluoride) followed by oxidation of the resultant alcohol with a suitable oxidant gave aldehyde 4. 4 was then reacted with reagent 5 using NaH as the base and ZnCl2 as the Lewis acid to give ketone 6. Reduction of 6 with suitable reducing reagents afforded I.
Scheme 2: General synthetic scheme using amino alcohol (3 R)-3-ammo-4- {[tei-t- butyl(dimethyl)silyl]oxy}butan-l-ol (7)
Figure imgf000015_0002
Reductive amination of amino alcohol 7 (WO 2004/085430) with a suitable aldehyde 8 followed by cyclization with phosgene using pyridine as the base gave 1,3-oxazinane intermediate 3. The intermediate can be processed to the desired product according to Scheme 1.
Scheme 3: Preparation of compounds shown in Example 1, 2 and 3.
Figure imgf000016_0001
Step 1 : isopropyl 5-{3-[(4R)-4-({[/er/-butyl(dimethyl)silyl]oxy}methyl)-2-oxo-l,3-oxazinan-3- yl]propyl}thiophene-2-carboxylate
To a solution of (4R)-4-({ [tert-butyl(dimethyl)silyl]oxy} methyl)- l,3-oxazinan-2 -one (1) (WO 2004/085430) ( 11 g, 44.8 mmol) in THF (400 mL) at rt (room temperature) under N2 was added
KHMDS (99 mL, 0.5M in toluene) dropwise (gel like ppt formation) and the mixture was stirred at rt for 0.5h (hour). Mesylate 12 (20 g, 65.3 mmol) in THF (150 mL) was then added via a cannula and the mixture was heated to 60 0C overnight and cooled to rt. The mixture was quenched with saturated NH4Cl and then extracted with ethyl acetate. The crude product was purified by flash (10-50% ethyl acetate/hexanes) to give a yellow oil. Step 2: isopropyl 5-{3-[(4R)-4-(hydroxymethyl)-2-oxo-l,3-oxazinan-3-yl]propyl}thiophene-2- carboxylate
To a solution of alkylation product from above (2Og, 43.9 mmol) in THF (200 mL) was added HCl (50 mL, IN) and the mixture was stirred at rt for 4h. The volatiles were removed in vacuo and the residue purified by flash (30-50% acetone/toluene or 50-100% EA (ethyl acetate)/hexanes) to give the desired product 10 as a light yellow solid. 1H NMR (Acetone-d6): 57.64 (1 H, d, J = 3.7 Hz), 7.00 (1 H, d, J = 3.7 Hz), 4.39-4.31 (1 H, m), 4.16-4.12 (2 H, m), 3.83 (3 H, s), 3.72-3.70 (2 H, m), 3.60-3.50 (2 H, m), 3.29-3.21 (1 H, m), 2.92 (2 H, t, J = 7.7 Hz), 2.15-1.99 (4 H, m).
Step 3 : isopropyl 5 - { 3 - [(4i?)-4-formy 1-2-oxo- 1 ,3 -oxazinan-3 -y 1] propyl } thi ophene-2-carboxy late
To a solution of DMSO (dimethyl sulfoxide) in DCM (dichloromethane) at -78 0C was added oxalyl chloride (141 uL, 1.615 mmol, 1.1 eq) dropwise and the mixture was stirred for 30 min. To this solution was added alcohol 10 (460 mg, 1.468 mmol) in DCM (8 mL) via a cannula and the mixture stirred for 15 min followed by the addition of triethylamine (495 uL, 3.52 mmol, 2.4 eq). The mixture was stirred at -78 0C for 30 min (minutes) and allowed to warm to rt. The DCM was evaporated in vacuo and the residue resuspended in EtOAc and filtered. The solid was washed with EtOAc thoroughly and the filtrate and washing solutions were concentrated to give the desired product. 1H NMR (ppm)(CDCl3): 9.65 (1 H, s), 7.64 (1 H, d, J = 3.7 Hz), 6.84 (1 H, d, J = 3.6 Hz), 4.34-4.22 (1 H, m), 4.09-4.01 (2 H, m), 3.87 (3 H, s), 3.82-3.74 (1 H, m), 3.13-3.03 (1 H, m), 2.94-2.86 (2 H, m), 2.31-2.19 (2 H, m), 2.07-1.99 (2 H, m). This reaction works equally well with sulfurtrioxide/pyridine complex.
Step 4: isopropyl 5-(3-{(4R)-4-[(l£)-4-(3-bromophenyl)-4,4-difluoro-3-oxobut-l-en-l-yl]-2-oxo-l,3- oxazinan-3-yl } propyl)thiophene-2-carboxylate (11)
To a solution of aldehyde isopropyl 5-{3-[(4R)-4-formyl-2-oxo-l,3-oxazinan-3- yl]propyl}thiophene-2-carboxylate from above (265 mg, 0.802 mmol) in THF (3 ml) was added zinc chloride 0.5M THF (1.9 mL, 0.936 mmol, 1.1 eq) followed by reagent 13a (sodium salt of dimethyl [3- (3-bromophenyl)-3,3-difluoro-2-oxopropyl]phosphonate) (420 mg, 1.106 mmol, 1.3 eq ) as a solid and the mixture was heated to 70 0C overnight and cooled to rt. The cloudy solution was diluted with IN HCl/water and extracted with ethyl acetate (EA) (2x). The EA layers were washed with water and dried over MgSO4. The crude was purified by flash (50-80% EA/hexane) to give desired product 11 which was contaminated with the phosphonate reagent by NMR. 1H NMR (ppm) (Acetone-d6): 7.81 (2 H, s), 7.75- 7.53 (2 H, m), 7.20-7.12 (1 H, m), 6.89-6.81 (1 H, m), 4.99-4.91 (1 H, m), 4.46 (1 H, m), 3.80-3.72 (3 H, m), 3.61-3.49 (1 H, m), 2.84-2.76 (1 H, m), 2.36-2.22 (3 H, m), 1.60-1.48 (4 H, m), 1.37-1.23 (4 H, m), 1.20 (6 H, d, J = 6.3 Hz).
Step 5: Preparation of catalyst 14 The catalyst was prepared by mixing lmol equiv of [RuCl2(p-cymene)2], 2mol equiv
(R,R)-N-Tosyl-l,2-diphenylethylene-l,2-diamine and 4.2 mol equiv OfEt3N in iPrOH at 80 0C for Ih. After solvent removal, the solid was washed with cold H2O and the recrystallized from MeOH to give the catalyst as an orange solid.
The catalyst could also be generated in situ by mixing 0.02 mol equiv of [RuCl2(f>-cymene)2] and 0.04 mol equiv of the (i?,/?)-N-Tosyl-l,2-diphenylethylene-l,2-diamine in DCM (dichloromethane) in the presence of 0.04 mol equiv of IM solution KOtBu in THF. After aging for 10 min at RT(room temperature), Et3N was added followed by HCO2H and a solution of the enone in DCM.
Example 1 : isopropyl 5-(3-{(4R)-4-[(l£,3R)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2- OXO- 1 ,3-oxazinan-3-yl} propyl)thiophene-2-carboxylate
To a solution of ketone 11 (500mg, 0.877 mmol) in DCM (5 ml) was added formic acid (52 μl, 1.31 mmol) and triethylamine (148 μl, 1.05 mmol) and the mixture was stirred at rt for 10 min. To it was then added Ru catalyst 14 (28 mg, 0.044 mmol) and the mixture was stirred at rt until all starting material was consumed by TLC analysis. The mixture was diluted with EA and washed with IN HCl. The organic layer was dried, filtered and concentrated in vacuo and the crude was purified by flash chromatography (25-75% EA/hexanes) to give the desired product in a diastereomeric ratio of 10-40/1 in favor of the desired isomer. The diastereomeric mixture was further purified by chiral AD (30% i- PrOH/hexanes) to give the title compound. 1H NMR δ (ppm)(Acetone-d6): 7.69 (2 H, d, J = 6.4 Hz), 7.62 (1 H, d, J = 3.7 Hz), 7.55 (1 H, d, J = 7.8 Hz), 7.45 (1 H, t, J = 8.2 Hz), 6.97 (1 H, d, J = 3.7 Hz), 5.86 (1 H, dd, J = 6.7, 15.5 Hz), 5.74 (1 H, dd, J = 5.5, 15.5 Hz), 5.23 (1 H, d, J = 5.8 Hz), 5.18-5.10 (1 H, m), 4.76-4.68 (1 H, m), 4.19-4.04 (3 H, m), 3.65-3.57 (1 H, m), 2.90-2.84 (1 H, m), 2.22-2.14 (1 H, m), 2.02- 1.94 (2 H, m), 1.84-1.78 (1 H, m), 1.32 (6 H, d, J = 6.7 Hz). MS (+APCI): 572.0, 574.0.
Example 2: 5-(3-{(4R)-4-[(l£',3R)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo-l,3- oxazinan-3-yl}propyl)thiophene-2-carboxylic acid
The isopropyl ester in Example 1 was treated with LiOH in Methanol/water to give the corresponding acid. 1H NMR (ρpm)(Acetone-d6): 57.69 (2 H, d, J = 7.0 Hz), 7.64 (1 H, d, J = 3.7 Hz), 7.55 (1 H, d, J = 7.7 Hz), 7.45 (1 H, t, J = 8.1 Hz), 6.98 (1 H, d, J = 3.6 Hz), 5.91-5.83 (1 H, m), 5.76-5.68 (1 H, m), 5.23 (1 H, s), 4.75-4.69 (1 H, m), 4.19-4.04 (4 H, m), 3.66-3.58 (1 H, m), 2.91-2.83 (3 H, m), 2.22-2.14 (1 H, m), 2.03-1.95 (3 H, m), 1.86-1.78 (1 H, m); MS (-ESI): m/z 527.9, 529.9.
Example 3: 5-(3-{(45)-4-[(3Λ)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybutyl]-2-oxo-l,3-oxazinan-3- yl}propyl)thiophene-2-carboxylic acid
To a solution of Example 1 (1 10 mg, 0.2 mmol) in EtOAc (10 mL) and acetone (1OmL) was added PtO2 (5 mg). The resulting black reaction mixture was subjected to H2 (1 atm (atmosphere)) for 18h. The solution was filtered over a pad of celite and the organic solvent was removed in vacuo. The crude product was purified by flash column chromatography to afford the ester which was hydrolyzed in the usual manner to the title compound. MS(+ESI) m/z 532.2, 534.2.
Scheme 4: Preparation of Horner-Wordsworth-Emmons reagent 13.
Figure imgf000019_0001
13a R1 = 3-Br, n = 1 13b R1 = 3,5-Cl, n = 2 13C R1=S1S-CH3, n = 2 13Cl R1 = S-CF31 H = I
Preparation of reagent 13a
Step 1 : To a solution of 3-bromo-iodobenzene (14.1g, 50 mmol) and ethyl bromo-α,α- difluoroacetate (10. Ig, 50 mmol) in DMSO (40 mL) was added copper bronze (7g, 110 mmol) and the suspension was heated to 55 0C for 2.5 days and cooled to rt. The mixture was quenched with KH2PO4 and filtered. The solid was washed with EA/water and the filtrate was separated. The aqueous layer was extracted with ether (2x) and the organic phases were combined, washed with water and brine. The crude was purified by flash (5-10% EA/hex) to give 10.7g of the desired product as a colorless oil. To a solution of dimethyl methylphosphonate (4.1g, 33 mmol) in THF (100 mL) at -78 0C was added n-BuLi (12.6 mL, 2.5M (minutes) in hexanes) dropwise and the mixture was stirred at -78 0C for Ih. To this solution was then added ethyl α,α-difluoro-3-bromophenylacetate (8.37g, 30 mmol) in THF via a cannula and the mixture was stirred at -78 0C for 2h and quenched with 2.2 mL AcOH and water. After warming to rt, the mixture was extracted with EA (3x). The organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo to give the desired product dimethyl [3-(3- bromophenyl)-3,3-difluoro-2-oxopropyl]phosphonate as a colorless oil. To a solution of this oil (8.28 g, 23.19 mmol) in ether at rt was added sodium hydride 60% (974 mg, 24.35 mmol, 1.05 eq) portionwise and the white suspension stirred at rt for Ih. The mixture was filtered and the white solid washed with ether/hexane. The solid thus obtained was dried under high vacuum to give 13a (white powder). Reagents 13b-13d were prepared in a similar manner.
Scheme 5: Preparation of mesylate 12.
'xyL -
Figure imgf000020_0001
-λ frtu,
15 16 17
O O O
H2 O BH3 O
HO T/ A J HO T/ A I HO \J A ^o
18 19 20
MsCI O I
MsO Y > -A JL
12
Step 1 : 5-[(l£)-3-te^butoxy-3-oxoprop-l-en-l-yl]thiophene-2-carboxylic acid (16)
To a degassed solution Of PPh3 (3.2g, 12.2 mmol), NEt3 (171 mL, 1218 mmol), t-butyl acrylate (118 mL, 812 mmol), and acid 15 (42 g, 203 mmol) in 10OmL CH3CN was added Pd(OAc)2 (911 mg, 4.1 mmol) and the solution was heated to reflux for 12 hours. The solution was cooled to rt and dilute HCl was added and the product was extracted with CH2Cl2. The organic layer was dried over Na2SO4, filtered and concentrated to yield the crude product which immediately solidified. The resultant solid was collected and washed with hexanes to yield 16. Step 2: isopropyl 5-[(l£)-3-?e^-butoxy-3-oxoprop-l-en-l-yl]thiophene-2-carboxylate (17)
A solution of acid 16 (44g, 173 mmol) and N,N-Dimethylformamide diisopropyl acetal (6Og, 342 mmol) in 50OmL of 1 ,2-Dichloroethane was heated to reflux for 12 hours. The solution was cooled to rt. and concentrated to yield the crude product which was purified by column chromotography to yield 17.
Step 3: (2£)-3-[5-(isopropoxycarbonyl)-2-thienyl]acrylic acid (18)
To a solution of ester 17 (45g, 152 mmol) in 50OmL of dichloromethane was added 45 mL of TFA (trifluoroacetic acid) slowly and the solution was stirred at rt. for 12 hours. To the solution was added 30OmL of PhMe and followed by concentration in vacuo. The resultant solid was collected and washed with hexanes to yield 18.
Step 4: 3-[5-(isopropoxycarbonyl)-2-thienyl]propanoic acid (19) To a solution of acid 18 (45g, 152 mmol) in 20OmL of isopropanol was added 5.32 g of
10% Pd on carbon and the mixture was subjected to 40psi of hydrogen for 12 hours. The solution was filtered over celite followed by concentration in vacuo. The resultant solid was collected and washed with hexanes to yield 19.
Step 5: isopropyl 5-(3-hydroxypropyl)thiophene-2-carboxylate (20)
To a solution of acid 19 (17.3 g, 71.4 mmol) in 20OmL THF at -10 C was added borane dimethyl sulfide complex (71.5 mL of a 2M solution in THF, 143 mmol) slowly. The solution was stirred for 3 hours followed by slow quenching with 1OmL of MeOH. The solution was stirred for another 2 hours and concentrated. Further addition of 10OmL of MeOH followed by concentration was repeated twice to yield the crude alcohol which was purified by column chromotography to yield 20.
Step 6: Mesylate isopropyl 5-{3-[(methylsulfonyl)oxy]propyl}thiophene-2-carboxylate (12)
To a solution of alcohol 20 (15 g, 65.7 mmol) in 20OmL dichloromethane at -20 0C was added triethylamine (18.4 mL, 131 mmol) followed by mesyl chloride (6.2 mL, 79 mmol) slowly. The solution was stirred for 3 hours followed by quenching water. The product was extracted with CH2Cl2 and the organic layer was dried over Na2SO4, filtered and concentrated to yield the crude product. The crude mesylate was purified by column chromatography to yield 12. Example 4: 5-(3-{(4R)-4-[(l£)-4,4-difluoro-3-hydroxy-4-phenylbut-l-en-l-yl]-2-oxo-l,3-oxazinan-3- yl}propyl)thiophene-2-carboxylic acid
Figure imgf000022_0001
This compound was prepared from intermediate 10 and dimethyl (3-phenyl-3,3-difluoro- 2-oxopropyl)phosphonate (WO 2004/085430) according to Scheme 3. 1H NMR δ(ppm)(Acetone-d6): 7.64 (1 H, d, J = 3.7 Hz), 7.55-7.51 (2 H, m), 7.47 (3 H, d, J = 6.1 Hz), 6.98 (1 H, d, J = 3.7 Hz), 5.83 (1 H, dd, J = 6.2, 15.5 Hz), 5.73-5.67 (1 H, m), 5.10 (1 H, s), 4.72-4.66 (1 H, m), 4.16-4.04 (4 H, m), 3.65- 3.55 (1 H, m), 2.90-2.82 (1 H, m), 2.20-2.12 (1 H, m), 1.97 (2 H, t, J = 7.5 Hz), 1.82-1.76 (1 H, m). MS (- APCI): m/z 450.3 (M-I)".
Example 5: isopropyl 5-(3-{(4i?)-4-[(l£',3R)-4-(3,5-dichlorophenyl)-4,4-difluoro-3-hydroxybut-l-en-l- yl]-2-oxo- 1 ,3 -oxazinan-3-yl } propyl)thiophene-2-carboxylate
Figure imgf000022_0002
1H NMR δ(ppm)(Acetone-d6): 7.63-7.61 (2H, m), 7.52 (2H, s), 6.96 (IH, d, J=3.2 Hz), 5.89 (IH, dd, J=15.5 Hz, J=6.6 Hz), 5.78 (IH, dd, J=15.4 Hz, J=5.0 Hz), 5.37 (IH, d, J=5.8 Hz), 5.17-5.11 (IH, m), 4.79-4.71 (IH, m), 4.15-4.19 (3H, m), 3.67-3.60 (IH, m), 2.87-2.95 (3H, m), 2.24-2.15 (IH, m), 2.02- 1.80 ( 2H, m), 1.82 (IH, dd, J=14.0 Hz, J=3.3 Hz), 1.32 (9H, d, J=6.2 Hz). Example 6: 5-(3-{(4/?)-4-[(lJE,3i2)-4-(3,5-dichlorophenyl)-454-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo- 1 ,3-oxazinan-3-yl}propyl)thiophene-2-carboxylic acid
Figure imgf000023_0001
The isopropyl ester from above was treated with LiOH in Methanol/water to give the corresponding acid MS (+ESI): m/z 520.2 (M+l)+.
Example 7: isopropyl 5-(3-{(4S)-4-[(3R)-4-(3,5-dichlorophenyl)-4,4-difluoro-3-hydroxybutyl]-2-oxo-l,3- oxazinan-3 -yl } propyl)thiophene-2-carboxylate
Figure imgf000023_0002
The double bond in Example 4 was processed to the title compound by reduction with
PtC>2 in ethyl acetate/acetone under hydrogen (1 atm) to give the saturated compound as discussed in Scheme 1. 1H NMR δ(ppm)(Acetone-d6): 7.66 (IH, s), 7.61 (IH, d, J=3.7 Hz), 7.54 (2H, d, 1.7 Hz), 6.98 (IH, d, J=3.7 Hz), 5.17-5.12 (m, IH), 4.95 (IH, d, J=6.7 Hz), 4.27 (IH, dt, J=I Ll Hz, J=3.0 Hz), 4.17- 4.06 (2H, m), 3.64-3.55 (2H, m), 3.18-3.12 (IH, m), 2.90 (2H, t, J=7.8 Hz), 2.12-1.91 (5H, m), 1.87-1.80 (IH, m), 1.75-1.69 (IH, m), 1.50-1.42 (IH, m), 13.3 (6H, d, J=6.3 Hz).
Example 8: 5-(3-{(4S)-4-[(3R)-4-(3,5-dichlorophenyI)-4,4-difluoro-3-hydroxybutyl]-2-oxo-l,3-oxazinan-
3-yl}propyl)thiophene-2-carboxylic acid
Figure imgf000024_0001
The isopropyl ester from above was treated with LiOH in Methanol/water to give the corresponding acid. 1H NMR δ(ppm)(Acetone-d6): 7.65 (IH, s), 7.63 (IH, d, 3.7 Hz), 7.54 (2H, d, J=I .6Hz), 6.98 (IH, d, J=3.4Hz), 4.27 (IH, dt, J=I 1. OHz, 2.9 Hz), 4.17-4.11 (2H, m), 3.64-3.51 (2H, m), 3.19-3.13 (IH, m), 2.90 (2H, t, J=7.8 Hz), 2.12-1.91 (3H, m), 1.87-1.81 (IH, m), 1.80-1.69 (IH, m), 1.50- 1.42 (IH, m), 1.39-1.31 (IH, m).
Example 9: isopropyl 5-(3-{(4R)-4-[(l£,3R)-4-(3,5-dimethylphenyl)-4,4-difluoro-3-hydroxybut-l-en-l- y 1] -2-oxo- 1 ,3 -oxazinan-3 -y 1 } propy l)thiophene-2-carboxylate
Figure imgf000024_0002
This compound was prepared from intermediate 10 and reagent 13c according to Scheme 3. 1H NMR δ(ppm)(CDCl3): 7.61 (IH, d, J=3.7 Hz), 7.07 (3H, s), 6.81 (IH, d, J=3.6 Hz), 5.74 (IH, dd, J=15.7 Hz, 5.9 Hz), 5.64 (IH, dd, J=15.6 Hz, 5.0 Hz), 5.22-5.16 (IH, m), 4.59-4.53 (IH, m), 4.15-4.10 (2H, m), 3.97- 3.96 (IH, m), 3.69-3.61 (IH, m), 2.90-2.80 (3H, m), 2.35 (6H, s), 2.19-2.08 (IH, m), 2.00-1.93 (2H, m), 1.79-1.75 (IH, m), 1.35 (6H, d, J=6.2 Hz).
Example 10: 5-(3-{(4R)-4-[(lJE,3R)-4-(3,5-dimethylphenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo- 1 ,3-oxazinan-3-yl}propyl)thiophene-2-carboxylic acid
Figure imgf000025_0001
The isopropyl ester from above was treated with LiOH in Methanol/water to give the corresponding acid. 1H NMR 5(PPm)(CDCl3): 7.70 (IH, d, J=3.3 Hz), 7.07 (3H, s), 6.86 (IH, d, J=2.9 Hz), 5.75 (IH, dd, J=15.6 Hz, 5.9 Hz), 5.66 (IH, dd, J=15.6 Hz, 5.0 Hz), 4.60-4.54 (IH, m), 4.15-4.13 (2H, m), 4.00-3.95 (IH, m), 3.70-3.63 (IH, m), 2.89-2.81 (3H, m), 2.35 (6H, s), 2.22-1.94 (3H, m), 1.80- 1.77 (IH, m).
Example 1 1: 5-(3-{(4S)-4-[(3R)-4-(3,5-dimethylphenyl)-4,4-difluoro-3-hydroxybutyl]-2-oxo-l,3- oxazinan-3 -yl } propyl)thiophene-2-carboxylic acid
Figure imgf000025_0002
The acid from above was treated with PtO2 in ethyl acetate/acetone under hydrogen (1 atm) to give corresponding acid. MS (-ESI): 480.2 (M-I)".
Example 12: isopropyl 5-[3-((4R)-4-{(l£,3R)-4,4-difluoro-3-hydroxy-4-[3-(trifluoromethyl)phenyl]but-l- en-l-yl}-2-oxo-l,3-oxazinan-3-yl)propyl]thiophene-2-carboxylate
Figure imgf000025_0003
The compound was prepared from intermediate 10 and reagent 13d as described in Scheme 3. 1H NMR δ(ppm)(Acetone-d6): 7.88-7.84 (3H, m), 7.77-7.73 (IH, m), 7.61 (IH, d, J=3.7 Hz), 6.97 (IH, d, J=3.7 Hz), 5.88 (IH, dd, J=15.6 Hz, 6.6 Hz), 5.77 (IH, dd, J=15.4 Hz, 5.4 Hz), 5.27 (IH, d, J=5.8 Hz), 5.19-5.09 (IH, m), 4.81-4.74 (IH, m), 4.20-4.12 (3H, m), 3.65-3.58 (IH, m), 2.93-2.85 (3H, m), 2.22-2.13 (IH, m), 2.08-1.21 (2H, m), 1.83-1.76 (IH, m), 1.32 (6H, d, J=6.2 Hz).
Example 13 : 5-[3-((4R)-4- {( l£,3R)-4,4-difluoro-3-hydroxy-4-[3-(trifluoromethyl)phenyl]but- 1 -en- 1 -yl } - 2-oxo- 1 ,3-oxazinan-3-yl)propyl]thiophene-2-carboxylic acid
Figure imgf000026_0001
The isopropyl ester from above was treated with LiOH in Methano I/water to give the corresponding acid. MS (-ESI): m/z 518.5 (M-H)".
Example 14: isopropyl 5-[3-((4S)-4-{(3R)-4,4-difluoro-3-hydroxy-4-[3-(trifluoromethyl)phenyl]butyl} -2- oxo-1, 3-oxazinan-3-yl)propyl]thiophene-2-carboxylate
Figure imgf000026_0002
The above intermediate was processed to the title compound by reduction with PtO2 in ethyl acetate/acetone under hydrogen (1 atm) as discussed in Scheme 3.
1H NMR δ(ppm)(Acetone-d6) 7.90-7.85 (3H, m), 7.80-7.73 (IH, m), 7.61 (IH, d, J=3.7 Hz), 6.97 (IH, d, J=3.7 Hz), 5.17-5.11 (IH, m), 4.95 ( IH, d, 7.3 Hz), 4.31-4.23 (IH, m), 4.18-4.10 (2H, m), 3.65-3.51 (2H, m), 3.18-3.12 (IH, m), 2,89-2.86 (2H, t, J=8.0 Hz), 2.14-1.68 (7H, m), 1.51-1.40 (IH, m), 1.33 (6H, d, J=6.3 Hz).
Example 15: 5-[3-((4S)-4-{(3R)-4,4-difluoro-3-hydroxy-4-[3-(trifluoromethyl)phenyl]butyl}-2-oxo-l,3- oxazinan-3-yl)propyl]thiophene-2-carboxylic acid
Figure imgf000027_0001
The isopropyl ester from above was treated with LiOH in Methanol/water to give the corresponding acid. MS (-ESI): m/z 520.0 (M-H)'.
Scheme 6: Preparation of Horner-Wordsworth-Emmons reagent 13e.
Figure imgf000027_0002
13e
Step 1: 2-(3-iodophenyl)propan-2-ol
To a solution of 3-iodo-acetophenone (15g, 61 mmol) in 10OmL THF at -78 0C was slowly added MeMgBr (6OmL of a 1.4M solution in THF, 84 mmol) and the solution was stirred for 1 hour as it warmed to 0 0C. The reaction was quenched with saturated NH4Cl and the crude product was extracted with ether (2x) and the organic phases were dried over Na2SO4, filtered and concentrated in vacuo. The crude was purified by flash (5-10% EA/hex) to give the desired product.
Step 2 : ethyl difluoro [3 -( 1 -hydroxy- 1 -methyl ethyl)pheny 1] acetate
To a solution of the 2-(3-iodophenyl)propan-2-ol (1Og, 38.2 mmol) in 5OmL DMSO was added ethyl bromo-α,α-difluoroacetate (7.75g, 38.2 mmol) and copper bronze (5.34g, 84 mmol) and the suspension was heated to 55 0C for 2.5 days and cooled to rt. The mixture was quenched with KH2PO4 and filtered. The solid was washed with EA/water and the filtrated was separated. The aqueous layer was extracted with ether (2x) and the organic phases were combined, washed with water and brine and then dried over Na2SO4, filtered and concentrated in vacuo. The crude was purified by flash (5-10% EA/hex) to give the desired product.
Step 3: ethyl α,α-difluoro(3-isopropylphenyl)acetate
To a solution of the tertiary alcohol ethyl difluoro[3 -(I -hydroxy- 1- methylethyl)phenyl] acetate (7g, 27.1 mmol) in 5OmL dichloromethane at -78 0C was added triethylsilane (21.7mL, 136 mmol) followed by borontrifluoride diethyl etherate (16.7mL, 136 mmol) and the solution stirred for 30 minutes. The mixture was quenched with solid NaHCθ3 and warmed to rt. Water was added an the aqueous layer was extracted with ether (2x) and the organic phases were combined, washed with water and brine and then dried over Na2SC^, filtered and concentrated in vacuo. The crude was purified by flash (5-10% EA/hex) to give the desired product.
Step 4: dimethyl [3,3-difluoro-3-(3-isopropylphenyl)-2-oxopropyl]phosphonate To a solution of dimethyl methylphosphonate (3.1g, 25 mmol) in THF ( 100 mL) at -78
0C was added n-BuLi (14.2 mL of a 1.6M solution in hexanes, 22.7mmol) dropwise and the mixture was stirred at the temperature for Ih. To this solution was then added ethyl α,α-difluoro(3- isopropylphenyl)acetate (5.5g, 22.7 mmol) in THF via a cannula and the mixture was stirred at -78 0C for 2h and quenched with 2.2 mL AcOH and water. After warming to rt, the mixture was extracted with ethyl acetate (3x). The organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo to give the desired product as a colorless oil.
Step 5: Preparation of 13e
To a solution of this oil (3g, 9.4 mmol) in ether/hexanes (1: 1, 4OmL) at 0 0C was added sodium hydride 60% (375mg, 9.37 mmol) portionwise and the white suspension was stirred at rt for Ih. The mixture was filtered and the white solid washed with ether/hexanes. The solid thus obtained was dried under high vacuum to give 13e (white powder).
Example 16: 5-(3-{(4R)-4-[(l£,3R)-4,4-difluoro-3-hydroxy-4-(3-isopropylphenyl)but-l-en-l-yl]-2-oxo- l,3-oxazinan-3-yl}propyl)thiophene-2-carboxylic acid
Figure imgf000029_0001
The title compound was prepared according to Scheme 3 using intermediate 10 and reagent 13e. MS (+ESI): m/z 494.0 (M+ 1)+.
Example 17: 5-(3-{(4S)-4-[(3R)-4,4-difIuoro-3-hydroxy-4-(3-isopropylphenyl)butyl]-2-oxo-l,3-oxazinan- S-ylJpropy^thiophene^-carboxylic acid
Figure imgf000029_0002
The title compound was prepared by hydrogenation of using PtO2 as the catalyst. MS (+ESI): m/z 496.1 (M+ 1)+.
The preparation of compounds shown Examples 18 and 19 are shown in Scheme 7.
Figure imgf000029_0003
Example 18 Example 19 Step 1 : Isopropyl 4-bromo-5-{3-[(4i?)-4-(bromomethyl)-2-oxo-l,3-oxazinan-3-yl]propyl}thiophene-2- carboxylate (21)
To a solution of thiophene 10 (0.700 g, 2.05 mmol) in AcOH (2 mL) was added bromine (0.140 mL, 2.67 mmol) dropwise. The solution was stirred for 18h. The reaction mixture was diluted with 10% Na2S2O3 (aq) and the aqueous layer was extracted with CH2CI2 (3x). The combined organic was dried over Na2SO4 and the organic solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (100% EtOAc) to afford 21 as a yellow oil. 1H NMR (ppm)(Acetone- Cl6): 7.61 (IH, s), 5.20-5.11 (IH, m), 4.37 (IH, dt, J=I Ll Hz, 3.1 Hz), 4.29-4.18 (3H, m), 3.82-3.77 (IH, m), 3.68-3.61 (IH, m), 3.33-3.27 (IH, m), 2.89 (2H, t, J=7.8 Hz), 2.25-2.16 (IH, m), 2.10-1.82 (3H, m), 1.34 (6H, d, J=6.3 Hz).
Step 2: Isopropyl 5-(3-{(4R)-4-[(acetyloxy)methyl]-2-oxo-l,3-oxazinan-3-yl}propyl)-4-bromothiophene- 2-carboxylate (22)
To a stirred suspension of 18-crown-6 (2.10 g) and potassium acetate (1.19 g, 12.1 mmol) in dry DMF (75 mL) at 105 0C was added a solution of bromide 21 (0.782 g, 1.62 mmol) in DMF (15 mL). The reaction mixture was heated at 105 0C for 18h. The reaction was cooled to room temperature and was diluted with H2O. The aqueous layer was extracted with 1 : 1 EtO Ac/ether (3x) and the combined organic layer was washed with brine and dried over Na2SO4. The organic solvent was concentrated in vacuo. The crude product was purified by flash column chromatography (100% EtOAc) to afford 22 as a yellow oil. MS (+ESI): m/z 462.1 , 464.1.
Step 3: Isopropyl 4-bromo-5-{3-[(4R)-4-(hydroxymethyl)-2-oxo-l,3-oxazinan-3-yl]propyl}thiophene-2- carboxylate (23)
To a solution of acetate 22 (0.729 g, 1.58 mmol) in dry MeOH (10 mL) was added NaOMe (340 mg, 6.31 mmol) as a solid. After stirring for 2h, the reaction was quenched with saturated NH4Cl (aq) and the aqueous layer was extracted with EtOAc (3x). The combined organic layer was dried over Na2SO4 and concentrated. The crude product was purified by flash column chromatography (100% EtOAc) to afford 23 as a yellow oil. MS (+ESI): m/z 392.2, 394.2.
Example 18: methyl 4-bromo-5-(3-{(4R)-4-[(l£,3i?)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybut-l-en- 1 -yl]-2-oxo- 1 ,3 -oxazinan-3 -yl} propyl)thiophene-2-carboxylate
Figure imgf000031_0001
The above intermediate 23 was processed to the title compound as discussed. MS (+ESI): m/z 622.1, 624.1, 626.1.
Example 19: 4-bromo-5-(3- {(4R)-4-[( l£,3R)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybut- 1 -en- 1 -yl]-2- OXO- 1 ,3-oxazinan-3-yl}propyl)thiophene-2-carboxylic acid
Figure imgf000031_0002
The methyl ester from above was treated with LiOH in Methanol/water to give the corresponding acid. MS (+ESI): m/z 610.2, 612.2.
Example 20: 5-(3-{(4R)-4-[(lJE,3R)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo-l,3- oxazinan-3-yl}propyl)-3-fluorothiophene-2-carboxylic acid
Figure imgf000031_0003
This compound was prepared according to Scheme 3 and 4. 1H NMR δ (ppm)(Acetone): 7.70 (2 H, m), 7.56 (1 H, d, J = 7.8 Hz), 7.49-7.43 (1 H, m), 6.90 (1 H, s), 5.88 (1 H, dd, J = 6.6, 15.6 Hz), 5.76 (1 H, dd, J = 5.5, 15.5 Hz), 5.23 (1 H, s), 4.74 (1 H, s), 4.20-4.13 (3 H, m), 3.65-3.57 (1 H, m), 2.93-2.81 (3 H, m), 2.23-2.16 (1 H, m), 2.01-1.95 (2 H, m), 1.85-1.79 (1 H, m). Example 21 : S-CS-K^^-^SR^^S-bromophenyl^^-difluoro-S-hydroxybutylj-Z-oxo-l^-oxazinan-S- yl } propy I)-3 -fluorothiophene-2-carboxylic acid
Figure imgf000032_0001
MS (-ESI):m/z 550.1, 548.1.
Exampel 22: 5-(3-{(45)-4-[(3i?)-3-(4'-chloro-2'-methylbiphenyl-3-yl)-3-hydroxypropyl]-2-oxo-l,3- oxazinan-3-yl}propyl)thiophene-2-carboxylic acid
Figure imgf000032_0002
Scheme 8: Preparation of Homer- Wordsworth-Emmons reagent 24.
Figure imgf000032_0003
To a nitrogen purged solution of boronic acid 25 (8g, 47mmol), 3-iodo-ethylbenzoate (7g, 25.4 mmol) in 10OmL dimethoxyethane at rt was added tetrakis(triphenylphosphine)palladium(0) (Ig, 0.865 mmol). To the solution was then added sodium carbonate (47 mL of a IM solution in water, 47 mmol) and the resultant solution was heated to 85 0C for 3 hours. The reaction was cooled to rt and water was added. The crude product was extracted with ethyl acetate (2x) and the organic phases were dried over Na2SO4, filtered and concentrated in vacuo. The crude was purified by flash chromatography (5-10% ethyl acetate/hexanes) to give desired product 26.
To a solution of dimethyl methylphosphonate (3.73 mL, 35 mmol) in THF (100 mL) at - 78 0C was added LHMDS (76 mL of a IM solutin in THF, 76mmol) dropwise and the mixture was stirred -78 0C for Ih. To this solution was then added the ethyl ester 26 (8g, 29.1 mmol) in THF via a cannula and the mixture was stirred for 2h as it warmed to 0 0C and quenched with IN HCl (80 mL). After warming to rt, the mixture was extracted with ethyl acetate (3x). The organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo to give the crude product which was purified by flash chromatography (5-10% ethyl acetate /hexanes) to yield of the desired product 27. To a solution of the phosphonate 27 (8.96 g, 25.4 mmol) in ether/hexanes (1 :1, 20OmL) at 0 0C was added sodium hydride 60% (1040 mg, 26 mmol) portionwise and the white suspension stirred at rt for Ih. The mixture was filtered and the white solid washed with ether/hexanes. The solid obtained was dried under high vacuum to yield the desired sodium salt 24 as a pale yellow powder.
The title compound was prepared from alcohol 10 and reagent 24 using procedures described in Scheme 3. MS (ESI) m/z 528.1 (M)+.
Exampel 23: isopropyl 5-(3-{(4R)-4-[(l£',3R)-4-(3-bromo-5-iodophenyl)-4,4-difluoro-3-hydroxybut-l-en- 1 -yl]-2-oxo- 1 ,3 -oxazinan-3 -yl } propyl)thiophene-2-carboxylate
Figure imgf000033_0001
MS (ESI) m/z 697.9 (M+l)+, 700.1 (M+2)+.
I. Effects of an EP4 Agonist on Intraocular Pressure (IOP) in Rabbits and Monkeys. Animals
Drug-naϊve, male Dutch Belted rabbits and female cynomolgus monkeys are used in this study. Animal care and treatment in this investigation are in compliance with guidelines by the National Institute of Health (NIH) and the Association for Research in Vision and Ophthalmology (ARVO) resolution in the use of animals for research. All experimental procedures str approved by the Institutional Animal Care and Use Committee of Merck and Company.
Drug Preparation and Administration
Drug concentrations are expressed in terms of the active ingredient (base). The compounds of this invention are dissolved in a suitable ophthalmic solution (e.g., 0.5% polysorbate-80, 0.1% EDTA, 0.02% benzalkonium chloride, 4.5% mannitol in 5 niM citrate buffer at pH 5-7) at 0.00063, 0.00013 % for rabbit studies and 0.0063, 0.0019%, 0.00063 and 0.000063% 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 vehicle (0.5% polysorbate-80, 0.1% EDTA, 0.02% benzalkonium chloride, 4.5% mannitol in 5 mM citrate buffer at pH 5-7). 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.
Analysis
The 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.
A. Intraocular Pressure Measurement in Rabbits Male Dutch Belted rabbits weighing 2.5-4.0 kg are maintained on a 12- hour light/dark cycle and rabbit chow. All experiments are performed at the same time of day to minimize variability related to diurnal rhythm. IOP is measured before treatment then the compounds of this invention or vehicle are instilled (one drop of 25 ul) into one or both eyes and IOP is 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 are evaluated and compared to drug treated animals as parallel controls.
B. Intraocular Pressure Measurements in Monkeys.
Unilateral ocular hypertension of the right eye is 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). The prolonged increase in intraocular pressure (IOP) results in changes to the optic nerve head that are similar to those found in glaucoma patients.
For IOP measurements, the monkeys are kept in a sitting position in restraint chairs for the duration of the experiment. Animals are 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 is measured using a pneumatic tonometer (Alcon Applanation Tonometer) or a Digilab pneumatonometer (Bio-Rad Ophthalmic Division, Cambridge, MA, USA). IOP is measured before treatment and generally at 30, 60, 124, 180, 300, and 360 minutes after treatment. Baseline values are also obtained at these time points generally two or three days prior to treatment. Treatment consists of instilling one drop of 25 ul of the compounds of this invention (0.000063 to 0.0063 %) or vehicle. At least one-week washout period is employed before testing on the same animal. The normotensive (contralateral to the hypertensive) eye is treated in an exactly similar manner to the hypertensive eye. IOP measurements for both eyes are compared to the corresponding baseline values at the same time point. Results are expressed as mean plus-or-minus standard deviation in mm Hg. The activity range of the compounds of this invention for ocular use is between 0.01 and 100,000 nM.
Compounds from the current invention (i.e., Example 1) showed improved ocular tolerability in animal species such as rabbits and cynomolgus monkeys compared to compounds disclosed in WO 2004/085430 (i.e., Example 2). For example, in a vehicle panel-controlled study in Dutch-Belted rabbits for 5 days (topical, bilateral, b.i.d.) with either 2.2 μM (25 μL) of an ophthalmic solution of Example 1 or vehicle (0.5% Polysorbate-80, 0.02% benzalkonium chloride, 0.1% EDTA, 4.5% mannitol in 5 mM citrate), no drug treatment related findings were noted for the present compound. In comparison, Example 2 of WO 2004/085430 demonstrated treatment related findings of very slight bilateral hyperemia, very slight conjunctival chemosis and slight ocular discharge at the same dose. Similarly a 5-fold improvement in eye closure was observed with the present compound in New Zealand White rabbits after ocular dosing. The present compound also demonstrated a 3-5-fold improvement in ocular adverse effects (conjuctival hyperemia, partial eye closure, etc.) in Cynomolgus Monkeys.
II. Radioligand binding assays:
The assays used to test these compounds were performed essentially as described in: Abramovitz M, Adam M, Boie Y, Carriere M, Denis D, Godbout C, Lamontagne S, Rochette C, Sawyer N, Tremblay NM, Belley M, Gallant M, Dufresne C, Gareau Y, Ruel R, Juteau H, Labelle M, Ouimet N, Metters KM. The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs. Biochim Biophys Acta 2000 Jan 17;1483(2):285-293 and discussed below:
Stable expression of prostanoid receptors in the human embryonic kidney (HEK) 293(EBNA) cell line Prostanoid receptor (PG) cDNAs corresponding to full length coding sequences were subcloned into the appropriate 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 LipofectAMINE@ (GIBCO-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 Penicillin-G, 100 μg/ml Streptomycin sulphate, 250 μg/ml active GENETICIN™ (G418) (all from Life Technologies, Inc/BRL) and 200 μg/ml hygromycin (Calbiochem). Individual colonies were isolated after 2-3 weeks of growth under selection using the cloning ring method and subsequently expanded into clonal cell lines. Expression of the receptor cDNA was assessed by receptor binding assays.
HEK 293(EBNA) cells were grown in supplemented DMEM complete medium at 37°C in a humidified atmosphere of 6 % CO2 in air, then harvested and membranes prepared by differential centrifugation (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 phenylmethylsulfonylfluoride, 10 μM E-64, 100 μM leupeptin and 0.05 mg/ml pepstatin). The 160,000 x g pellets were resuspended in 10 mM HEPES/KOH (pH 7.4) containing 1 mM EDTA at approximately 5-10 mg/ml protein by Dounce homogenisation (Dounce A; 10 strokes), frozen in liquid nitrogen and stored at -800C.
Prostanoid receptor binding assays
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 EP), containing 1 mM EDTA, 10 mM MgCl2 (EP subtypes) or 10 mM MnCl2 (DP, FP, IP and TP) and radioligand [0.5-1.0 nM [3H]PGE2 (181 Ci/mmol) for EP subtypes, 0.7 nM [3H]PGD2 (1 15 Ci/mmol) for DP, 0.95 nM [3H]PGF2n (170 Ci/mmol) for FP, 5 nM [3H]iloprost (16 Ci/mmol) for IP and 1.8 nM [3H]SQ 29548 (46 Ci/mmol) for TP]. EP3 assays also contained 100 μM GTPDS. The reaction was initiated by addition of membrane protein (approximately 30 μg for EPi, 20 μg for EP2, 2 μg for EP3, 10 μg for EP4, 60 μg for FP, 30 μg for DP, 10 μg for IP and 10 μg for TP) from the 160,000 x g fraction. Ligands were added in dimethylsulfoxide (Me2SO) which was kept constant at 1 % (v/v) in all incubations. Non-specific binding was determined in the presence of 1 μM of the corresponding nonradioactive prostanoid. Incubations were conducted for 60 min (EP subtypes, FP and IP) or 30 min (DP and TP) at 3O0C (EP subtypes, DP, FP and TP) or room temperature (IP) and terminated by rapid
5 filtration through a 96-well Unifϊlter GF/C (Canberra Packard) prewetted in assay incubation buffer without EDTA (at 4°C) and using a Tomtec Mach HI 96-well semi-automated cell harvester. The filters were washed with 3-4 ml of the same buffer, dried for 90 min at 550C and the residual radioactivity bound to the individual filters determined by scintillation counting with addition of 50 μl of Ultima Gold F (Canberra Packard) using a 1450 MicroBeta (Wallac). Specific binding was calculated by subtracting
10 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 invention for bone use is between 0.01 and 100,000 nM.
15
Bone Resorption Assays:
1. Animal Procedures:
For mRNA localization experiments, 5-week old Sprague-Dawley rats (Charles River) are euthanized by CO2, their tibiae and calvariae are excised, cleaned of soft tissues and frozen
10 immediately in liquid nitrogen. For EP4 regulation experiments, 6-week old rats are given a single injection of either vehicle (7% ethanol in sterile water) or an anabolic dose of PGE2 (Cayman Chemical,
Ann Arbor, MI), 3-6 mg/kg in the same vehicle) intraperitoneally. Animals are euthanized at several time points post-injection and their tibiae and calvariae, as well as samples from lung and kidney tissues are frozen in liquid nitrogen. >5
2. Cell Cultures
RP-I periosteal cells are spontaneously immortalized from primary cultures of periosteal cells from tibae of 4-week old Sprague-Dawley rats and are cultured in DMEM (BRL, Gaithersburg, MD) with 10 % fetal bovine serum (JRH Biosciences, Lenexa, KS). These cells do not express 50 osteoblastic phenotypic markers in early culture, but upon confluence, express type I collagen, alkaline phosphatase and osteocalcin and produce mineralized extracellular matrix.
RCT-I and RCT-3 are clonal cell lines immortalized by SV-40 large T antigen from cells released from fetal rat calvair by a cmbination collagenase/hyaluronidase digestion. RCT-I cells, derived from cells released during the first 10 minutes of digestion (fraction I), are cultured in RPMI 1640 medium (BRL) with 10% fetal bovine serum and 0.4 mg/ml G418 (BRL). These cells differentiate and express osteoblastic features upon retinoic acid treatment. RCT-3 cells, immortalized from osteoblast- enriched fraction EU cells, are cultured in F- 12 medium (BRL) with 5% Fetal bovine serum and 0.4 mg/ml G418. TRAB-11 cells are also immortalized by SV40 large T antigen from adult rat tibia and are
5 cultured in RPMI 1640 medium with 10% FBS and 0.4 mg/ml G418. ROS 17/2.8 rat osteosarcoma cells are cultured in F-12 containing 5% FBS. Osteoblast-enriched (fraction EEf) primary fetal rat calvaria cells are obtained by collagenase/hyaluronidase digestion of calvariae of 19 day-old rat fetuses. See Rodan et al., Growth stimulation of rat calvaria osteoblastic cells by acidic FGF, Endocrinology, 121, 1919-1923 (1987), which is incorporated by reference herein in its entirety. Cells are released during 30-50 minutes
10 digestion (fraction EEI) and are cultured in F-12 medium containing 5% FBS.
P815 (mouse mastocytoma) cells, cultured in Eagles MEM with 10% FBS, and NRK
(normal rat kidney fibroblasts) cells, cultured in DMEM with 10% FBS, are used as positive and negative controls for the expression of EPφ respectively. See Abramovitz et al., Human prostanoid receptors: cloning and characterization. In: Samulesson B. et al. ed) Advances in prostaglandin, Thrombosznes and 15 leukotriene research, vol. 23, pp. 499-504 (1995) and de Larco et al., Epithelioid and fibroblastic rat kidney cell clones: EGF receptors and the effect of mouse sarcoma virus transformation, Cell Physiol, 94, 335-342 (1978), which are both incorporated by reference herein in their entirety.
3. Northern Blot Analysis:
>0 Total RNA is extracted from the tibial metaphysis or diaphysis and calvaria using a guanidinium isothiocyanate-phenol-chloroform method after pulverizing frozen bone samples by a tissue homogenizer. See P. Chomczynski et al., Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction., Analyt Biochem, 162, 156-159 (1987), which is incorporated by reference herein in its entirety. RNA samples (20 mg) are separated on 0.9% agarose/formaldehyde
25 gels and transferred onto nylon membranes (Boehringer Mannheim, Germany). Membranes are prehybridized in Hybrisol I (Oncor, Gaithersburg, MD) and 0.5 mg/ml sonicated salmon sperm DNA (Boehringer) at 420C for 3 hours and are hybridized at 420C with rat EP2 and mouse EP4 cDNA probes labeled with p2p]-dCTP (Amersham, Buckinghamshire, UK) by random priming using the rediprime kit (Amersham). After hybridization, membranes are washed 4 times in 2xSSC + 0.1% SDS at room 50 temperature for a total of 1 hour and once with 0.2xSSC + 0.1% SDS at 550C for 1 hour and then exposed to Kodak XAR 2 film at -700C using intensifying screens. After developing the films, bound probes are removed twice with 0.1% SDS at 8O0C and membranes are hybridized with a human GAPDH (Glyceraldehyde 3 -Phosphate Dehydrogenase) cDNA probe (purchased from Clontech, Palo Alto, CA) for loading control. 4. In-Situ Hybridization:
Frozen tibiae are sectioned coronally at 7 mm thickness and sections are mounted on charged slides (Probe On Plus, Fisher Scientific, Springfield, NJ) and are kept at -70 C until 5 hybridization. cRNA probes are labeled with 35§-UTPgS (ICN, Costa Mesa, CA) using a Riboprobe π kit (Promega Madison, WT). Hybridization is performed overnight at 50 C. See M. Weinreb et al, Different pattern of alkaline phosphatase, osteopontin and osteocalcin expression in developing rat bone visualized by in-situ hybridization, J. Bone Miner Res., 5, 831-842 (1990) and D. Shinar et al., Expression ofalphav and beta3 integrin subunits in rat osteoclasts in situ, J. Bone Miner. Res., 8, 403- 10 414 (1993), which are both incorporated by reference herein in their entirety. Following hybridization and washing, sections are dipped in Ilford K5 emulsion diluted 2:1 with 6% glycerol in water at 42 C o and exposed in darkness at 4 C for 12-14 days. Slides are developed in Kodak D-19 diluted 1 :1 with water at 15°, fixed, washed in distilled water and mounted with glycerol-gelatin (Sigma) after hematoxylin staining. Stained sections are viewed under the microscope (Olympus, Hamburg, 15 Germany), using either bright-field or dark-field optics.
5. Expression Of EP /| In Osteoblastic Cell Lines And In Bone Tissue.
The expression of EP4 and EP2 mRNA is examined in various bone derived cells including osteoblast-enriched primary rat calvaria cells, immortalized osteoblastic cell lines from fetal rat
ZO calvaria or from adult rat tibia and an osteoblastic osteosarcoma cell line. Most of the osteoblastic cells and cell lines show significant amounts of 3.8 kb EP4 mRNA, except for the rat osteosarcoma cell line
ROS 17/2.8. Consistent with this finding, in ROS 17/2.8 cells PGE2 has no effect on intracellular cAMP, which is markedly induced in RCT-3 and TRAB-11 cells. Treatment of RCT-I cells with retinoic acid, which promotes their differentiation, reduces the levels of EP4 mRNA. NRK fibroblasts do
25 not express EP4 mRNA, while P815 mastocytoma cells, used as positive controls, express large amounts of EP4 mRNA. In contrast to EP4 mRNA, none of the osteoblastic cells and cell lines express detectable amounts OfEP2 mRA in total RNA samples. Expression of EP4 mRNA in osteoblastic cells, EP4 is also expressed in total RNA isolated from tibiae and calvariae of 5-week-old rats. In contrast, no EP2 mRNA is found in RNA from tibial shafts.
$0
6. PGE2 Induces The Expression OfEPz1 mRNA in RP-I Periosteal Cells And In Adult Rat Tibiae
PGE2 enhances its own production via upregulation of cyclooxygenase 2 expression in osteoblasts and in bone tissue thus autoamplifying its own effects. PGE2 also increases the levels of EP4 mRNA. RP- 1 cells are immortalized from a primary culture of adult rat tibia periosteum is examined. These cells express osteoblast phenotypic markers upon confluence and form mineralized bone matrix when implanted in nude mice. Similar to the other osteoblastic cells examined, RP-I periosteal cells
.6 express a 3.8 kb EP4 transcript. Treatment with PGE2 (10 M) rapidly increases EP4 mRNA levels peaking at 2 hours after treatment. PGE2 has no effect on EP4 mRNA levels in the more differentiated 5 RCT-3 cells pointing to cell-type specific regulation of EP4 expression by PGE2. EP2 mRNA is not expressed in RP-I cells before or after treatment with PGE2.
To examine if PGE2 regulates EP4 mRNA levels in vivo in bone tissue, five-week-old male rats are injected with PGE2 (3 - 6 mg/Kg). Systemic administration of PGE2 rapidly increased EP4 mRNA levels in the tibial diaphysis peaking at 2 h after injection. A similar effect of PGE2 on EP4 0 mRNA is observed in the tibial metaphysis and in calvaria. PGE2 induces EP4 mRNA levels in vitro in osteogenic periosteal cells and in vivo in bone tissue in a cell type-specific and tissue-specific manner. PGE2 does not induce EP2 mRNA in RP-I cells nor in bone tissue.
7. Localization of EP/| mRNA expression in bone tissue
5 In situ hybridization is used in order to localize cells expressing EP4 in bone. In control experiment (vehicle-injected) rats, low expression of EP4 is detected in bone marrow cells. Administration of a single anabolic dose of PGE2 increased the expression of EP4 in bone marrow cells.
The distribution of silver grains over the bone marrow is not uniform and occurs in clumps or patches in many areas of the metaphysis. Within the tibial metaphysis, EP4 expression is restricted to the secondary
'0 spongiosa area and is not seen in the primary spongiosa. Hybridization of similar sections with a sense probe (negative control) does not show any signal.
EP4 is expressed in osteoblastic cells in vitro and in bone marrow cells in vivo, and is upregulated by its ligand, PGE2.
'.5 8. Agonists Of the Present Invention
Using standard methods for measuring agonist activity, the following compounds are evaluated in cell cultures and in EP4 receptor cell-free systems to determine the agonist activity of the compounds in terms of their EC50 value.

Claims

WHAT IS CLAIMED IS:
1. A compound having ; the structural formula I:
Figure imgf000041_0001
FORMULA I or a pharmaceutically acceptable salt, enantiomer, diastereomer, prodrug or mixture thereof, wherein,
R and R4 independently represent H, or Cj.6 alkyl;
Rl independently represents hydrogen, Ci-6 alkyl, halogen, CF3, aryl, said aryl optionally substituted with 1 to 3 groups of halogen, Q -6 alkyl, CF3, or N(R4)2 ;
R2 represents H, or halogen;
R3 represents COOR or carboxylic acid isotere; n represents 0-3; and
— represents a double or single bond.
2. A compound according to claim 1 wherein R3 is COOR or tetrazole.
3. A compound according to claim 2 wherein R3 is COOR.
4. A compound according to claim 3 wherein R is H, isopropyl, ethyl or, methyl.
5. A compound according to claim 1 wherein Ri is halogen, Ci_6 alkyl or CF3.
6. A compound according to claim 3 wherein R is H or isopropyl, Ri is bromine and R2 is hydrogen.
7. A compound according to claim 5 wherein n is 0, 1 or 2.
8. A compound which is:
Isopropyl 5-(3-{(4R)-4-[(l£',3R)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo-l,3- oxazinan-3 -yl } propyl)thiophene-2-carboxylate;
Isopropyl 5 -(3 - { (4i?)-4-[( 1 £)-4-(3 -bromophenyl)-4,4-difluoro-3 -oxobut- 1 -en- 1 -yl] -2-oxo- 1 ,3 -oxazinan-3 - yl}propyl)thiophene-2-carboxylate;
Isopropyl 5-(3- {(4R)-4-[( l£)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybut- 1 -en- 1 -yl]-2-oxo- 1 ,3- oxazinan-3-yl}propyl)thiophene-2-carboxylate;
5-(3-{(4R)-4-[(l£',3R)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo-l,3-oxazinan-3- yl}propyl)thiophene-2-carboxylic acid; Isopropyl 5-(3-{(41S)-4-[(3R)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybutyl]-2-oxo-l,3-oxazinan-3- yl}propyl)thiophene-2-carboxylate;
5-(3-{(4S)-4-[(3R)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybutyl]-2-oxo-l,3-oxazinan-3- yl}propyl)thiophene-2-carboxylic acid;
5 -(3 - { (4R)-4- [( 1 £}-4,4-difluoro-3 -hydroxy-4-pheny lbut- 1 -en- 1 -yl] -2-oxo- 1 ,3 -oxazinan-3 - yl } proρyl)thiophene-2-carboxylic acid;
Isopropyl 5-(3-{(4R)-4-[(l£,3R)-4-(3,5-dichlorophenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo-l,3- oxazinan-3-yl}propyl)thiophene-2-carboxylate;
5-(3-{(4R)-4-[(l£',3R)-4-(3,5-dichlorophenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo-l,3-oxazinan-
3-yl}propyl)thiophene-2-carboxylic acid; Isopropyl 5-(3-{(4S)-4-[(3R)-4-(3,5-dichlorophenyl)-4,4-difluoro-3-hydroxybutyl]-2-oxo-l,3-oxazinan-3- y 1 } propy l)thiophene-2-carboxy late;
5-(3-{(4S)-4-[(3R)-4-(3,5-dichlorophenyl)-4,4-difluoro-3-hydroxybutyl]-2-oxo-l,3-oxazinan-3- yl}propyl)thiophene-2-carboxylic acid;
Isopropyl 5-(3-{(4R)-4-[(l£,3R)-4-(3,5-dimethylphenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo-l,3- oxazinan-3-yl}propyl)thiophene-2-carboxylate;
5-(3-{(4R)-4-[(l£',3R)-4-(3,5-dimethylphenyl)-4!4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo-l,3-oxazinan-
3 -yl } propyl)thiophene-2-carboxylic acid;
Isopropyl 5-(3-{(4S)-4-[(3i?)-4-(3,5-dimethylphenyl)-4,4-difluoro-3-hydroxybutyl]-2-oxo-l,3-oxazinan-3- yl } propyl)thiophene-2-carboxylate; 5-(3-{(4S)-4-[(3R)-4-(3,5-dimethylphenyl)-4,4-difluoro-3-hydroxybutyl]-2-oxo-l,3-oxazinan-3- yl}propyl)thiophene-2-carboxylic acid;
Isopropyl 5-[3-((4R)-4- {( lJE,3R)-4,4-difluoro-3-hydroxy-4-[3-(trifluoromethyl)phenyl]but- 1 -en- 1 -yl} -2-
OXO- 1 ,3-oxazinan-3-yl)propyl]thiophene-2-carboxylate; 5-[3-((4R)-4-{(l£',3R)-4,4-difluoro-3-hydroxy-4-[3-(trifluoromethyl)phenyl]but-l-en-l-yl}-2-oxo-l,3- oxazinan-3-yl)propyl]thiophene-2-carboxylic acid;
Isopropyl 5-[3-((4S)-4-{(3R)-4,4-difluoro-3-hydroxy-4-[3-(trifluoromethyl)phenyl]butyl}-2-oxo-l,3- oxazinan-3-yl)propyl]thiophene-2-carboxylate; 5 - [3 -((4S)-4- { (3 R)-4,4-difluoro-3 -hydroxy-4-[3 -(trifluoromethyl)pheny 1] butyl } -2-oxo- 1 ,3 -oxazinan-3 - yl)propyl]thiophene-2-carboxylic acid;
5-(3- {(4R)-4-[( l£',3^)-4,4-difluoro-3-hydroxy-4-(3-isopropylphenyl)but- 1-en- 1 -yl]-2-oxo- 1 ,3-oxazinan-3- yl}propyl)thiophene-2-carboxylic acid;
5-(3-{(4S)-4-[(3R)-4,4-difluoro-3-hydroxy-4-(3-isopropylphenyl)butyl]-2-oxo-l,3-oxazinan-3- yl}propyl)thiophene-2-carboxylic acid;
Methyl 4-bromo-5-(3 - { (4R)-4-[( 1 E,3R)-4-(3 -bromophenyl)-4,4-difluoro-3-hydroxybut- 1 -en- 1 -yl]-2-oxo- l,3-oxazinan-3-yl}propyl)thiophene-2-carboxylate;
4-Bromo-5-(3-{(4R)-4-[(l£,3R)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo-l,3- oxazinan-3-yl}propyl)thiophene-2-carboxylic acid; 5-(3-{(4R)-4-[(l£',3R)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo-l,3-oxazinan-3- yl } propyl)-3 -fluorothiophene-2-carboxylic acid;
5 -(3 - { (4S)-4- [(3R)-4-(3 -bromopheny l)-4,4-difluoro-3 -hydroxybutyl] -2-oxo- 1 ,3 -oxazinan-3 -y 1 } propy l)-3 - fluorothiophene-2-carboxylic acid; and
5-(3-{(45)-4-[(3R)-3-(4'-chloro-2'-methylbiphenyl-3-yl)-3-hydroxypropyl]-2-oxo-l,3-oxazinan-3- yl}propyl)thiophene-2-carboxylic acid;
Isopropy 1 5 -(3 - { (4i?)-4- [( 1 E,3R)-4-(3 -bromo-5 -iodopheny l)-4,4-difluoro-3 -hydroxybut- 1 -en- 1 -y 1] -2-oxo-
1 ,3-oxazinan-3-yl}propyl)thiophene-2-carboxylate; ancj
5-(3-{(4R)-4-[(l£,3R)-4-(3-bromo-5-iodophenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo-l,3- oxazinan-3 -yl } propyl)thiophene-2-carboxylic acid; or a pharmaceutically acceptable salt, enantiomer, diastereomer, prodrug or mixture thereof.
9. A compound according to claim 7 which is: isopropyl 5-(3- { (4R)-4-[( 1 £)-4-(3 -bromophenyl)-4,4-difluoro-3-oxobut- 1 -en- 1 -yl]-2-oxo- 1 ,3 -oxazinan-3 - yl}propyl)thiophene-2-carboxylate; isopropyl 5-(3-{(4R)-4-[( l£)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybut- 1 -en- 1 -yl]-2-oxo- 1 ,3- oxazinan-3-yl}propyl)thiophene-2-carboxylate; isopropyl 5-(3-{(4R)-4-[(l£,3R)-4-(3-bromophenyl)-4,4-difluoro-3-hydroxybut-l-en-l-yl]-2-oxo-l,3- oxazinan-3-yl}propyl)thiophene-2-carboxylate; S-CS-K^^-Kl^S^H-CB-bromophenylH^-difluoro-S-hydroxybut-l-en-l-ylJ-Z-oxo-US-oxazinan-S- ylJpropyOthiophene^-carboxylic acid;
5.(3. { (4S)-4-[(3R)-4-(3 -bromophenyl)-4,4-difluoro-3 -hydroxybutyl]-2-oxo- 1 ,3 -oxazinan-3 - yl}propyl)thiophene-2-carboxylic acid; 5-(3-{(4R)-4-[(l£)-4,4-difluoro-3-hydroxy-4-phenylbut-l-en-l-yl]-2-oxo-l,3-oxazinan-3- yl}propyl)thiophene-2-carboxylic acid; or a pharmaceutically acceptable salt, enantiomer, diastereomer, prodrug or mixture thereof.
10. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of formula I, as recited in claim 1.
11. Use of a compound of claim 1 for the manufacture of a medicament for treating ocular hypertension or glaucoma.
12. The composition according to claim 10 wherein one or more active ingredients belonging to the group consisting of: β-adrenergic blocking agent, parasympatho-mimetic agent, sympathomimetic agent, carbonic anhydrase inhibitor, Maxi-K channel blocker, and a prostaglandin, hypotensive lipid, neuroprotectant, and 5-HT2 receptor agonist is optionally added.
13. The composition according to claim 12 wherein the β-adrenergic blocking agent is timolol, betaxolol, levobetaxolol, carteolol, or levobunolol; the parasympathomimetic agent is pilocarpine; the sympathomimetic agent is epinephrine, brimonidine, iopidine, clonidine, or para- aminoclonidine, the carbonic anhydrase inhibitor is dorzolamide, acetazolamide, metazolamide or brinzolamide; COSOPT®, the Maxi-K is Penitrem A, paspalicine, charybdotoxin, iberiotoxin, Paxicillan, Aflitram, Verroculogen, l-(l-isobutyl-6-methoxy-lH-indazol-3-yl)-2-methylpropan-l-one; l-[l-(2,2- dimethylpropyl)-6-methoxy- lH-indazol-3-yl]-2-methylpropan- 1 -one; 1 -[ 1 -(cyclohexylmethyl)-6- methoxy- 1 H-indazol-3-yl]-2-methylpropan- 1 -one; 1 -( 1 -hexyl-6-methoxy- 1 H-indazol-3 -yl)-2- methylpropan- 1 -one; 1 -[ 1 -(2-ethylhexyl)-6-methoxy- 1 H-indazol-3 -yl]-2-methylpropan- 1 -one; 1 -(3- isobutyryl-6-methoxy- 1 H-indazol- 1 -yl)buan-2-one; 1 -(3-isobutyryl-6-methoxy- 1 H-indazol- 1 -yl)-3 ,3- dimethylbutan-2-one; 1 -(3 -cyclopentylcarbonyl)-6-methoxy- 1 H-indazol- 1 -yl)-3 ,3-dimethylbutan-2-one; l-(3,3-dimethyl-2-oxobutyl) -6-methoxy-lH-indazole-3-carboxylic acid; and l-[3-(3-hydroxypropanoyl) - 6-methoxy-lH-indazol-l-yl]-3,3-dimethylbutan-2-one, the prostaglandin is latanoprost, travaprost, unoprostone, rescula, or S 1033, the hypotensive lipid is lumigan, the neuroprotectant is eliprodil, R- eliprodil or memantine; and the 5-HT2 receptor agonist is l-(2-aminopropyl)-3-methyl-lH-imdazol-6-ol fumarate or 2-(3-chloro-6-methoxy-indazol-l-yl)-l-methyl-ethylamine.
14. The composition according to claim 10 which is a topical formulation in the form of a solution or suspension, said composition optionally containing xanthan gum or gellan gum.
15. Use of a compound of claim 1 for the manufacture of a medicament for treating macular edema or macular degeneration, treating dry eye, increasing retinal and optic nerve head blood velocity, increasing retinal and optic nerve oxygen tension or providing a neuroprotection.
16. Use of a compound of claim 1 for the manufacture of a medicament for stimulating bone formation, treating or reducing the risk of contracting a disease state or condition related to abnormal bone resorption, in a mammal in need thereof.
PCT/CA2006/001243 2005-08-03 2006-07-28 Ep4 receptor agonist, compositions and methods thereof WO2007014454A1 (en)

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WO2008149965A1 (en) 2007-06-07 2008-12-11 Astellas Pharma Inc. Pyridone compound
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