KR20070021171A - 1,3,4-Oxadiazol-2-ones as PPAR delta modulators - Google Patents

Abstract

The present invention relates to 1,3,4-oxadiazolone, and pharmaceutically acceptable salts thereof, stereoisomers, tautomers or solvates thereof. The compounds of the present invention are modulators of PPAR delta and are therefore particularly useful as a pharmaceutical agent for the treatment of myelin disrupting diseases and for the treatment of fatty acid metabolism and glucose using diseases.

1,3,4-oxadiazolone, PPAR delta modulator, myelin destruction disease, fatty acid metabolism disease, glucose utilization disease

Description

1,3,4-Oxadiazol-2-one as PPA delta modulator {1,3,4-Oxadiazol-2-ones as PPAR delta modulators}

The present invention relates to novel compounds and pharmaceutical agents that act as selective PPAR delta ligand receptor binders, which are useful for modulating PPAR delta receptors for the treatment of diseases mediated by nuclear hormone receptors. The PPAR delta ligand receptor binders of the invention are useful as agonists or antagonists of the PPAR delta receptor.

The peroxysome proliferator-activated receptor (PPAR) consists of a subfamily of nuclear receptor superfamily. Four fairly closely related isoforms were identified, cloned, and known as PPAR alpha, PPAR gamma-1, PPAR gamma-2 and PPAR delta. Each receptor subtype has a signal DNA binding domain (DBD) and a ligand-binding domain (LBD), both of which are essential for ligand activated gene expression. PPARs bind heterodimeric with the Retinoid X receptor. See J. Berger and DE Miller, Annu. Rev. Med. , 2002, 53, 409-435.

PPAR delta (also known as PPAR beta) is expressed in a wide range of mammalian tissues, but little information is available on the full-length arrangement of genes regulated by their biological functions or receptors. However, it has recently been found that its agonists may be useful for treating dyslipidemia and certain skin diseases, and that antagonists may also be useful for treating osteoporosis or rectal cancer (see D. Sternbach, Annual Reports in Medicinal Chemistry, Volume 38, AM Doherty, ed., Elsevier Academic Press, 2003, pp. 71-80).

PPAR delta appears to be significantly expressed in the CNS; However, most of its function is still unknown. However, it is interesting to note that PPAR delta is expressed in rodent oligodendrocytes (the major lipid producing cells of the CNS) (J. Granneman et al., J. Neurosci. Res. , 1998, 51, 563-573). ). It has also been found that PPAR delta selective agonists significantly increase oligodendrocyte myelin gene expression and significantly increase myelin sheath diameter in mouse culture (see I. Saluja et al., Glia , 2001, 33, 194-204). Thus, PPAR delta actives may be useful for the treatment of myelin destruction and myelin destruction diseases.

Myelin destruction states as myelin damage, a multiple dense layer of lipids and proteins covering many nerve fibers. These layers are provided by oligodendroglia in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). In patients with myelin destruction disease, myelin destruction can be irreversible, which usually occurs with axonal degeneration, or may appear after axonal degeneration, often with cell degradation. Myelin destruction occurs due to nerve damage or damage to myelin itself, which may be due to abnormal immune responses, local damage, ischemia, metabolic diseases, toxic substances, or viral infections (Prinneas and McDonald, Demyelinating Diseases.In Greenfield's Neuropathology , 6. sup.th ed. (Edward Arnold: New York, 1997) 813-811, Beers and Berkow, eds., The Merck Manual of Diagnosis and Therapy , 17.sup.th ed. (Whitehouse Station, NJ Merck Research Laboratories, 1999) 1299, 1437, 1473-76, 1483).

Central myelin destruction (myelin destruction of the central nervous system) occurs in several states, some of which are unknown, and have become known as primary myelin destruction diseases. Of these, multiple sclerosis (MS) is the most common. Other primary myelin destruction disorders include adrenoleukodystrophy (ALD), spinal neuropathy (adrenomyeloneuropathy), AIDS-vacuolar myelopathy, HTLV-associated myelopathy, and Leber's genetic corneal dystrophy. (hereditary optic atrophy), progressive multifocal leukoencephalopathy (PML), subacute sclerosing panencephalitis, acute inflammatory demyelinating neuropathy (Guillian-Barre syndrome), tropical stiff paraplegia spastic paraparesis). In addition, acute diseases, myelin destruction may occur in the CNS, including, for example, acute disseminated encephalomyelitis (ADEM) and acute viral encephalitis. In addition, acute transverse myelitis is a syndrome that affects the gray matter in one or more adjacent thoracic regions of the spinal cord with unknown acute spinal cord transverse, resulting in myelin destruction. In addition, glial cells that form myelin are destroyed due to spinal cord injury, neuropathy and nerve damage.

Selective PPAR delta modulators may be useful for treating or preventing other disease states. See Joel Berger et al., Annu. Rev. Med. 2002, 53, 409-435; Timothy Wilson et al., J. Med. Chem., 2000, Vol. 43, No. 4, 527-550; Steven Kliewer et al., Recent Prog Horm Res. 2001; 56: 239-63; Jean-Charles Fruchart, Bart Staels and Patrick Duriez: PPARS, Metabolic Disease and Arteriosclerosis, Pharmacological Research, Vol. 44, No. 5, 345-52; 2001; Sander Kersten, Beatrice Desvergne & Walter Wahli: Roles of PPARs in health and disease, Nature, vol 405, 25 May 2000; 421-4; Ines Pineda Torra, Giulia Chinetti, Caroline Duval, Jean-Charles Fruchart and Bart Staels: Peroxisome proliferator-activated receptors: from transcriptional control to clinical practice, Curr Opin Lipidol 12: 2001, 245-254].

Compounds that can act as PPAR delta modulators can be particularly useful for the treatment or prevention of fatty acid metabolic disorders and glucose soluble disorders linked to insulin resistance.

Prophylaxis of resting diabetes associated with diabetes mellitus, especially type 2 diabetes. Particular aspects related to this are hyperglycemia, improved insulin resistance, improved glucose tolerance, pancreatic β cell protection, macrovascular and microvascular disease prevention.

Dyslipidemia and its sequelae, such as arteriosclerosis, coronary heart disease, cerebrovascular disease, etc., especially those characterized by one or more of the following factors, including, but not limited to, high plasma triglyceride levels, high postprandial plasma: Triglyceride concentration, low HDL cholesterol concentration, low ApoA lipoprotein concentration, high LDL cholesterol concentration, small and dense LDL cholesterol particles, characterized by high ApoB lipoprotein concentration).

Various other diseases that may be associated with metabolic syndrome, such as obesity (overweight), including central obesity, thrombosis, hypercoagulant prothrombin conditions (arterial and venous), high blood pressure, heart failure, e.g. Examples include, but are not limited to, myocardial infarction, hypertension heart disease or cardiomyopathy.

Other diseases or conditions associated with an inflammatory response or cell differentiation include, for example, atherosclerosis, such as coronary sclerosis, including angina or myocardial infarction, seizures, vasostenosis or re-obstruction, chronic inflammatory bowel disease (Crohn's disease, ulcerative colitis, pancreatitis), other inflammatory conditions, retinopathy, adipocyte tumors, lipomatous carcinomas, for example, liposarcoma, solid tumors, neoplasia (eg, gastrointestinal tract, liver, biliary tract) , And inflammation of the pancreas, endocrine tumors, lungs, kidneys, urinary tracts, genitals, prostate carcinoma, etc.), acute and chronic spinal proliferative diseases and lymphoma angiogenesis, neurodegenerative diseases, Alzheimer's disease Diseases, Parkinson's disease, erythematous-scale skin diseases (psoriasis, acne).

Other skin diseases and skin conditions regulated by PPAR delta: eczema and neurodermatitis, eg seborrheic dermatitis or photodermatitis; Keratitis and keratosis, e.g. seborrheic keratosis, aging keratosis, actinic keratosis, photo-induced keratosis or keratosis follicularis keloids and keratosis prevention, warts (including wet or sexual warts), human papilloma Virus (HPV) infection (including sexually transmitted papilloma), viral warts (including infectious continuous species (molluscum contagiosum)), leukoplakiapapular dermatoses (eg, Lichen planus), skin cancer (eg, basal cells) Carcinoma, melanoma or cutaneous T-cell lymphoma), focal benign epithelial tumor (keratosis, epithelial naevi) and frostbite.

Various other diseases that can be controlled by the PPAR delta include syndrome X, polycystic ovary syndrome (PCOS), asthma osteoarthritis, lupus erythematosus (LE), or inflammatory rheumatic diseases such as rheumatoid arthritis, vasculitis, and wasting diseases ( Cachexia), gout ischemia / reperfusion syndrome and acute respiratory distress syndrome (ARDS).

Summary of the Invention

The present invention relates to a compound of formula (I), or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

In Formula I,

ARYL is phenyl or pyridinyl, wherein phenyl or pyridinyl is halogen, C _1-6 alkyl, C _2-6 alkenyl, C _1-6 alkoxy, C _1-6 perfluoroalkyl; C _1-6 alkylthio, hydroxy, hydroxyC _1-6 alkyl, C _1-4 acyloxy, nitro, cyano, C _1-6 alkylsulfonyl, amino, C _1-6 alkylamino and C _1- May be substituted with one or more substituents selected from the group consisting of ₆ alkoxycarbonyl;

Z is -O (CH ₂ ) _n- , -SO ₂ (CH ₂ ) _n -,-(CH ₂ ) _n -Y- (CH ₂ ) _n -,-(CH ₂ ) _n -CO-, -O ( CH ₂ ) _n -CO- or-(CH ₂ ) _n -Y- (CH ₂ ) _n -CO-, wherein Y is NR ₃ , O or S, and R ₃ is H, C _1-6 alkyl, C _3-8 cycloalkyl, C _{1-6 alkylC 3-8} cycloalkyl and benzyl, n is independently an integer from 1 to 5;

X is NR ₃ , O or S, wherein R ₃ is as defined above;

R ₁ is H, halogen, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 perfluoroalkyl; HydroxyC _1-6 alkyl, nitro, cyano or C _1-6 alkylamino;

R ₂ is substituted or unsubstituted phenyl, pyridinyl or thienyl, wherein the substituents are halogen, C _1-6 alkyl, C _2-6 alkenyl, C _1-6 alkoxy, C _1-6 perfluoroalkyl, C _1-6 alkylthio, hydroxy, hydroxyC _1-6 alkyl, C _1-4 acyloxy, nitro, cyano, C _1-6 alkylsulfonyl, amino, C _1-6 alkylamino and C _{1- 6} alkoxycarbonyl;

Provided that when Z is -O (CH ₂ ) _n -or -SO ₂ (CH ₂ ) _n -and ARYL is phenyl, R ₂ is a group other than phenyl.

The present invention also relates to a pharmaceutical composition of formula (I) and a method of using such compounds and compositions that modulate PPAR delta by administering to the patient in need thereof a compound that modulates PPAR delta activity.

Another aspect of the invention is directed to a disease in which a therapeutically effective amount of a compound of formula (I), or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof, is modulated by PPAR delta ligand binding activity. A method of treating a disease that can be modulated by PPAR delta ligand binding activity in a mammal, including administering to a suffering mammal, is described.

Formula I

In Formula I,

And ARYL is phenyl or pyridinyl, wherein phenyl or pyridinyl are halogen, C _{1 - 6} alkyl, C _{2 - 6} alkenyl, C _{1 - 6} alkoxy, C _{1 - 6} perfluoroalkyl; C _{1 - 6} alkylthio, hydroxy, hydroxy C _1-6 alkyl, C _1-4 acyloxy, nitro, cyano, C _1-6 alkylsulfonyl, amino, C _1-6 alkylamino and C _1- May be substituted with one or more substituents selected from the group consisting of ₆ alkoxycarbonyl;

Z is -O (CH ₂ ) _n- , -SO ₂ (CH ₂ ) _n -,-(CH ₂ ) _n -Y- (CH ₂ ) _n -,-(CH ₂ ) _n -CO-, -O ( CH ₂ ) _n -CO- or-(CH ₂ ) _n -Y- (CH ₂ ) _n -CO-, wherein Y is NR ₃ , O or S, and R ₃ is H, C _1-6 alkyl, C _3-8 cycloalkyl, C _{1-6 alkylC 3-8} cycloalkyl and benzyl, n is independently an integer from 1 to 5;

X is NR ₃ , O or S, wherein R ₃ is as defined above;

R ₁ is H, halogen, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 perfluoroalkyl; HydroxyC _1-6 alkyl, nitro, cyano or C _1-6 alkylamino;

R ₂ is substituted or unsubstituted phenyl, pyridinyl or thienyl, wherein the substituents are halogen, C _1-6 alkyl, C _2-6 alkenyl, C _1-6 alkoxy, C _1-6 perfluoroalkyl, C _1-6 alkylthio, hydroxy, hydroxyC _1-6 alkyl, C _1-4 acyloxy, nitro, cyano, C _1-6 alkylsulfonyl, amino, C _1-6 alkylamino and C _{1- 6} alkoxycarbonyl.

The terms used herein have the following meanings:

As used herein, “C _1-6 alkyl” includes methyl and ethyl groups, straight or branched propyl, butyl, pentyl and hexyl groups. Particular alkyl groups are methyl, ethyl, n-propyl, isopropyl and tertiary butyl. "C _1-6 alkoxy", "C _1-6 alkoxyC _1-6 alkyl", "hydroxyC _1-6 alkyl", "C _1-6 alkylcarbonyl", "C _1-6 alkoxycarbonylC _1-6 alkyl "," C _1-6 alkoxycarbonyl "," aminoC _1-6 alkyl "," C _1-6 alkylcarbamoylC _1-6 alkyl "," C _1-6 dialkylcarbamoylC _1-6 alkyl "," mono- or di -C _1-6 alkylamino-C _1-6 alkyl ", amino-C _1-6 alkyl-carbonyl", "diphenyl-C _1-6 alkyl", "aryl C _{1- 6} alkyl "," arylcarbonyl C _1-6 alkyl "and" aryloxyC _1-6 alkyl "are also possible.

As used herein, the expression “C _2-6 alkenyl” includes ethenyl and straight or branched propenyl, butenyl, pentenyl and hexenyl groups. Similarly, "C _2-6 alkynyl" includes ethynyl and propynyl, straight or branched butynyl, pentynyl and hexynyl groups.

As used herein, "C _1-4 acyloxy" refers to an acyl radical attached to an oxygen atom, for example, acetyloxy, propionyloxy, butanoyloxy, iso-butanoyloxy, sec-butanoyloxy, t-butanoyloxy, and the like.

As used herein, “aryl” refers to a carbocycle aromatic ring system such as phenyl, biphenyl, naphthyl, anthracenyl, phenanthrenyl, fluorenyl, indenyl, pentalenyl, azurenyl, biphenylenyl Etc. are included. Aryl is one containing partially hydrogenated derivatives of the carbocycle aromatic systems listed above. Non-limiting examples of such partially hydrogenated derivatives include 1,2,3,4-tetrahydronaphthyl, 1,4-dihydronaphthyl and the like.

As used herein, "aryloxy" refers to --O-aryl, where aryl is as defined above.

As used herein, "heteroaryl" (self or optionally combined, eg, "heteroaryloxy", or "heteroaryl alkyl") is a 5-10 membered aromatic ring system wherein at least one ring is N, O and At least one heteroatom selected from the group consisting of S, for example pyrrole, pyrazole, furan, thiophene, quinoline, isoquinoline, quinazolinyl, pyridine, pyrimidine, oxazole, thiazole, thia Diazoles, tetraazoles, triazoles, imidazoles or benzimidazoles.

As used herein, “heterocyclic or heterocyclyl” (on its own or in combination with others, eg “heterocyclylalkyl”) is a saturated or partially saturated 4 to 10 membered ring system, wherein , At least one ring comprises at least one heteroatom selected from the group consisting of N, O and S, for example pyrrolidone, piperidine, piperazine, morpholine, tetrahydropyran or imidazolidine This includes, but is not limited to.

As used herein, the expression “C _1-6 perfluoroalkyl” refers to the substitution of all hydrogen atoms of an alkyl group by a fluorine atom. Examples include trifluoromethyl and pentafluoroethyl, and straight or branched heptaropropyl, nonafluorobutyl, undecafluoropentyl and tridecafluorohexyl groups. Derivative representations such as "C _1-6 perfluoroalkoxy" are also possible.

As used herein, the expression “C _3-8 cycloalkyl” refers to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

As used herein, the expression “C _3-8 cycloalkylC _1-6 alkyl” refers to the attachment of C _3-8 cycloalkyl as defined above to the C _1-6 alkyl further described above. Representative examples include cyclopropylmethyl, 1-cyclobutylethyl, 2-cyclopentylpropyl, cyclohexylmethyl, 2-cycloheptylethyl, 2-cyclooctylbutyl, and the like.

As used herein, "halogen" or "halo" means chloro, fluoro, bromo and iodo.

As used herein, in the context of the present invention “C _1-6 alkylsulfonyl” means —S (═O) ₂ C _1-6 alkyl, wherein C _1-6 alkyl is as defined above. . Representative examples include methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, isopropylsulfonyl, butylsulfonyl, iso-butylsulfonyl, secondary butylsulfonyl, tertiary butylsulfonyl, n-pentylsul Ponyls, isopentylsulfonyl, neopentylsulfonyl, tertiary pentylsulfonyl, n-hexylsulfonyl, isohexylsulfonyl, and the like.

As used herein, "arylsulfonyl" refers to an -S (= 0) ₂ aryl group, where aryl is as defined above.

As used herein, “heteroarylsulfonyl” refers to —S (═O) ₂ heteroaryl, where heteroaryl is as defined above.

The expression “stereoisomers” is a general term used to refer to all isomers of individual molecules that differ only in the direction of their atoms in space. In general, this includes enantiomers (enantiomers) formed due to at least one asymmetric center. The compounds of the present invention possess two or more asymmetric centers, which may additionally exist as diastereoisomers, and certain individual molecules may exist as geometric isomers (cis / trans). All such isomers and mixtures of any proportion thereof are also within the scope of the present invention.

"Substituted" is C _1-6 alkyl, C _1-6 perfluoroalkyl, hydroxy, -CO ₂ H, ester, amide, C ₁ -C ₆ alkoxy, C ₁ -C ₆ perfluoroalkoxy,- Substituted by 1 to 2 substituents independently selected from the group consisting of NH ₂ , Cl, Br, I, F, -NH- lower alkyl and -N (lower alkyl) ₂ .

The compounds of the present invention and salts thereof may exist in several tautomeric forms, including enol and imine forms; Keto and enamine forms, and geometric isomers and mixtures thereof. All such tautomeric forms also fall within the scope of the present invention. Tautomers exist as a mixture of tautomeric sets in solution. As a solid, generally one tautomer predominates. Although one tautomer is described, the present invention includes all tautomers of these compounds.

As used herein, the term “modulator” is used to enhance (eg, “agonist” activity) or inhibit (eg, “antagonist” activity) biological activity or functionality of a process (eg, enzyme activity or receptor binding). Chemicals that have the capacity, such enhancement or inhibition may be incidental to the occurrence of certain events, such as activation or inhibition of signal transduction pathways, and / or may be evident only in certain cell types and cause measurable biological changes. have.

As used herein, "patient" refers to warm-blooded animals such as rats, mice, dogs, cats, guinea pigs, and primates such as humans.

The expression “pharmaceutically acceptable carrier” as used herein allows the formation of a pharmaceutical composition, ie a dosage form that can be administered to a patient in admixture with a non-toxic solvent, dispersant, excipient, adjuvant or compound of the invention. Means other substances. Examples of such carriers are pharmaceutically acceptable oils commonly used for parenteral administration.

As used herein, “pharmaceutically acceptable salts” means salts of the compounds of the invention used to make medical preparations. However, other salts may also be used to prepare the compounds of the present invention or to make pharmaceutically acceptable salts thereof. Pharmaceutically acceptable salts of the compounds of the invention include acid addition salts, for example, the solution of the compounds of the invention may be hydrochloric acid, hydrobromic acid, sulfuric acid, methanesulfonic acid, 2-hydroxy ethanesulfonic acid, p-toluenesulfonic acid, fumaric acid, maleic acid, hydroxymaleic acid, malic acid, ascorbic acid, succinic acid, glutaric acid, acetic acid, salicylic acid, cinnamic acid, 2-phenoxybenzoic acid, hydroxybenzoic acid, phenylacetic acid, benzoic acid, It can be formed by mixing with a pharmaceutically acceptable acid solution such as oxalic acid, citric acid, tartaric acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, carbonic acid or phosphoric acid. Acid metal salts may also be formed, such as sodium orthophosphate and potassium hydrogen sulfate. The salts thus formed are also mono or di acid salts and may be present in hydrated form or substantially as anhydride. In addition, when a compound of the present invention has an acidic moiety, suitable pharmaceutically acceptable salts thereof include alkali metal salts such as sodium salts or potassium salts; Alkaline earth metal salts such as calcium or magnesium salts; And salts formed with suitable organic ligands, such as tetravalent ammonium salts.

As used herein, "therapeutically effective amount" refers to an amount of a compound effective for treating a listed disease or condition.

The invention also provides a pharmaceutical composition consisting of at least one compound according to the invention and a pharmaceutically acceptable carrier. Suitably such compositions are tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, or metered amounts suitable for oral, parenteral, nasal, sublingual or rectal administration or by inhalation or aeration. It may be a unit dosage form such as an aerosol or liquid spray, drop, ampoule, automatic injection device or suppository. Alternatively, the composition may be provided in a form suitable for administration once a week or once a month, for example, using an insoluble salt of the active compound, such as a decanoate salt, for depots for intramuscular injection. depot) formulations. Corrosive polymers containing the active ingredient can also be used. In order to make solid compositions such as tablets, the main active ingredient is a pharmaceutical carrier such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gum and other pharmaceuticals. Mixing with a diluent, for example water, creates a solid initial formulation containing a homogeneous mixture of the compounds of the invention or a pharmaceutically acceptable salt thereof. When the initial formulation is said to be homogeneous, it means that the composition can be evenly distributed into unit dosage forms such as tablets, pills, capsules since the active ingredients are evenly dispersed in the composition. Such solid initial formulation compositions are dispensed in unit dosage forms of the aforementioned type containing from 0.1 to about 500 mg of the active ingredient of the invention. Flavored unit dosage forms contain 1 to about 100 mg of active ingredient, for example 1, 2, 5, 10, 25, 50 or 100 mg. Tablets or pills of the novel compositions may be formulated to provide a dosage form that is coated or capable of long-term action. For example, a tablet or pill consists of an internal dosage component and an external dosage component, which form an envelope over the internal dosage component. The two components can be separated by an enteric layer, which acts to protect the stomach from decomposing, helping to delay the release of internal components to the duodenum. Various materials can be used in such enteric layers or enteric skin, including various polymeric acids and mixtures of polymeric acids with shellac, cetyl alcohol and cellulose acetate, and the like.

Liquid forms into which the novel compositions of the invention may be introduced for oral or injection administration include aqueous solutions, appropriate flavored syrups or water soluble or oil suspensions, and edible oils such as cottonseed oil, sesame oil, coconut oil or Emulsions flavored with peanut oil, as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents which can be used for the aqueous suspension include synthetic or natural rubbers such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinyl-pyrrolidone or gelatin This includes.

In the treatment of various disease states as described herein, suitable dosage levels are about 0.01 to 250 mg / kg, suitably about 0.05 to 100 mg / kg, particularly suitably about 0.05 to 20 mg / kg. The compound may be administered 1 to 4 times a day.

As used in the Examples and the following preparations, the terminology used herein has the following meanings: "kg" is kilograms, "g" is grams, "mg" is milligrams, "μg" is micro Grams, "pg" for picograms, "mol" for moles, "mmol" for millimoles, "nmole" for nanomoles, "L" for liters, "mL" or "ml" for milliliters , "μL" for microliters, "℃" for Celsius, "R _f " for persistence, "mp" or "mp" for melting point, "dec" for decomposition, "bp" or "bp" Is the boiling point, "mmHg" is the pressure in millimeters of mercury, "cm" is centimeters, "nm" is nanometers, and [[α] ²⁰ _D "is the sodium at 20 ° C obtained in a 1 decimeter cell. For a specific turn of the D line, "c" is concentration (g / mL), "THF" is tetrahydrofuran, "DMF" is dimethylformamide, "NMP" is 1-methyl-2-pyrrolidinone Where "brine" is saturated aqueous sodium chloride solution, "M" is molar, "mM" is millimolar, and "μM" is micromolar. , "nM" is nanomolar, "TLC" is thin layer chromatography, "HPLC" is high performance liquid chromatography, "HRMS" is high resolution mass spectrum, "CIMS" is chemical ionization mass spectroscopy, "ESI" Electrospray ionization mass spectrometry, where "t _R " is the retention time, "lb" is pounds, "gal" is gallons, "LOD" is loss on drying, "Ci" is microcurie, "ip "Peritoneal,""iv" means intravenous administration, respectively.

In one aspect of the invention, the novel compounds of formula (I), or their stereoisomers, tautomers, solvates, or pharmaceutically acceptable salts thereof, are described:

Formula I

In Chemical Formula I,

ARYL is phenyl or pyridinyl, wherein phenyl or pyridinyl is halogen, C _1-6 alkyl, C _2-6 alkenyl, C _1-6 alkoxy, C _1-6 perfluoroalkyl; C _1-6 alkylthio, hydroxy, hydroxyC _1-6 alkyl, C _1-4 acyloxy, nitro, cyano, C _1-6 alkylsulfonyl, amino, C _1-6 alkylamino and C _1- May be substituted with one or more substituents selected from the group consisting of ₆ alkoxycarbonyl;

Z is -O (CH ₂ ) _n- , -SO ₂ (CH ₂ ) _n -,-(CH ₂ ) _n -Y- (CH ₂ ) _n -,-(CH ₂ ) _n -CO-, -O ( CH ₂ ) _n -CO- or-(CH ₂ ) _n -Y- (CH ₂ ) _n -CO-, wherein Y is NR ₃ , O or S, and R ₃ is H, C _1-6 alkyl, C _3-8 cycloalkyl, C _{1-6 alkylC 3-8} cycloalkyl and benzyl, n is independently an integer from 1 to 5;

X is NR ₃ , O or S, wherein R ₃ is as defined above;

R ₁ is H, halogen, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 perfluoroalkyl; HydroxyC _1-6 alkyl, nitro, cyano and C _1-6 alkylamino;

R ₂ is substituted or unsubstituted phenyl, pyridinyl or thienyl, wherein the substituents are halogen, C _1-6 alkyl, C _2-6 alkenyl, C _1-6 alkoxy, C _1-6 perfluoroalkyl, C _1-6 alkylthio, hydroxy, hydroxyC _1-6 alkyl, C _1-4 acyloxy, nitro, cyano, C _1-6 alkylsulfonyl, amino, C _1-6 alkylamino and C _{1- 6} alkoxycarbonyl;

Provided that when Z is -O (CH ₂ ) _n -or -SO ₂ (CH ₂ ) _n -and ARYL is phenyl, R ₂ is a group other than phenyl.

In another aspect of this embodiment, compounds wherein ARYL is phenyl and X is O or S are described.

In another aspect of this embodiment, compounds wherein X is O are described.

Exemplary compounds of this embodiment are 5- (4- {2- [5-methyl-2- (4-trifluoromethyl-phenyl) -thiazol-4-yl] -ethoxy} -phenyl) -3 H -[1,3,4] oxadiazol-2-one.

In another aspect of this embodiment, a pharmaceutical composition consisting of an effective amount of a compound of Formula I and a pharmaceutically acceptable carrier is described.

In another embodiment of the invention, a disease in which a therapeutically effective amount of a compound of Formula (I), or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof, can be modulated by PPAR delta ligand binding activity A method of cure of a disease that can be modulated by PPAR delta ligand binding activity in a mammal, comprising administering to a mammal suffering from the present invention.

Formula I

In Formula I,

ARYL is phenyl or pyridinyl, wherein phenyl or pyridinyl is halogen, C _1-6 alkyl, C _2-6 alkenyl, C _1-6 alkoxy, C _1-6 perfluoroalkyl; C _1-6 alkylthio, hydroxy, hydroxyC _1-6 alkyl, C _1-4 acyloxy, nitro, cyano, C _1-6 alkylsulfonyl, amino, C _1-6 alkylamino and C _1- May be substituted with one or more substituents selected from the group consisting of ₆ alkoxycarbonyl;

Z is -O (CH ₂ ) _n- , -SO ₂ (CH ₂ ) _n -,-(CH ₂ ) _n -Y- (CH ₂ ) _n -,-(CH ₂ ) _n -CO-, -O ( CH ₂ ) _n -CO- or-(CH ₂ ) _n -Y- (CH ₂ ) _n -CO-, wherein Y is NR ₃ , O or S, and R ₃ is H, C _1-6 alkyl, C _3-8 cycloalkyl, C _{1-6 alkylC 3-8} cycloalkyl and benzyl, n is independently an integer from 1 to 5;

X is NR ₃ , O or S, wherein R ₃ is as defined above;

R ₁ is H, halogen, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 perfluoroalkyl; HydroxyC _1-6 alkyl, nitro, cyano or C _1-6 alkylamino;

R ₂ is substituted or unsubstituted phenyl, pyridinyl or thienyl, wherein the substituents are halogen, C _1-6 alkyl, C _2-6 alkenyl, C _1-6 alkoxy, C _1-6 perfluoroalkyl, C _1-6 alkylthio, hydroxy, hydroxyC _1-6 alkyl, C _1-4 acyloxy, nitro, cyano, C _1-6 alkylsulfonyl, amino, C _1-6 alkylamino and C _{1- 6} alkoxycarbonyl.

In another aspect of this embodiment of the process of the invention, compounds wherein ARYL is phenyl are described.

In another aspect of this embodiment of the process of the invention, a compound is described wherein ARYL is phenyl and R ₂ is phenyl.

In another aspect of this embodiment of the process of the invention, a compound is described wherein ARYL is phenyl, Z is -O (CH ₂ ) _n -and R ₂ is phenyl.

In another aspect of this embodiment of the process of the invention, a compound is described wherein ARYL is phenyl, Z is -O (CH ₂ ) _n- , X is O or S and R ₂ is phenyl.

In another aspect of this embodiment of the method of the invention, ARYL is phenyl, Z is -O (CH ₂ ) _n- , X is O or S, R ₁ is C _1-6 alkyl, R ₂ is phenyl A phosphorus compound is demonstrated.

In another aspect of this embodiment of the method of the invention, compounds wherein X is O are described.

In another aspect of this embodiment of the method of the invention, compounds wherein X is S are described.

In another aspect of this embodiment, the disease is multiple sclerosis, Charcot-Marie-Tooth disease, Felizaeus-Merzbacher disease, encephalomyelitis, neuromyelitis optica, adrenal gland Dystrophic disease, acute inflammatory demyelinating neuropathy (Guillian-Barre syndrome) and myelin-forming glial cell destruction, ie myelin destruction disease selected from diseases of the spinal cord, neuropathy and nerve damage.

In another aspect of this embodiment, a method is described in which the myelin disrupting disease is multiple sclerosis.

In another aspect of the invention, the disease is obesity, hypertriglyceridemia, hyperlipidemia, hypoalphalipoproteinemia, hypercholesterolemia, dyslipidemia, syndrome X, type II diabetes mellitus and neuropathy, nephropathy, retinopathy, And its complications selected from the group consisting of cataracts, hyperinsulinemia, impaired glucose tolerance, insulin resistance, atherosclerosis, hypertension, coronary heart disease, peripheral vascular disease and congestive heart failure. do.

The compounds described herein can be synthesized according to the following procedure, wherein the ARYL, X, Z and R substituents are as defined in Formula I, unless otherwise noted. If necessary, in the following synthesis process, the reactive functional groups present in the compounds described herein may be protected with appropriate protecting groups. The protecting group can be removed at a later stage of synthesis. For the reactive functional groups protected, and its removal process literature can be consulted for the Reference TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, Wiley and Sons, 1991].

Scheme A shows the synthesis of suitable imidazoles, oxazoles or thiazoles, intermediates of compounds of formula I, wherein X is O, S, NR ₃ . Heterocycles can be made using known methods found in the chemical literature [Katritzky, AR; Rees, CW ed., Comprehensive Heterocyclic Chemistry, Vol. 5; Pergamon Press (1984); Katritzky, AR; Rees, CW; Scriven, EFV Eds. Comprehensive Heterocyclic Chemistry II ; Vols 3 & 4, Pergamon Press (1996)]. In particular, the oxazoles, imidazoles and thiazoles are fused with an appropriate α halo-ketone (1) and an amide, amidine or thioamide (Formula 2) at about 40 to 150 ° C., respectively, to react the intermediate heterocycle (3). I can make it.

In Scheme B, the general synthesis of compounds of formula I, wherein Z is -O (CH ₂ ) _n- , is illustrated. Thus, in step B1, suitably substituted carboxylic ester 4 which can be synthesized as described in Scheme A is reduced to alcohol 5 by methods well known in the art. For example, it may be reduced in an inert solvent by aluminum hydride such as lithium aluminum hydride or diisobutylammonium hydride. In step B2, the alcohol functional group of compound (5) is converted into a leaving group to form compound (6), where Lg is a halogen or sulfonate ester such as mesylate or tosylate Is a leaving group). Conversion to leaving group is accomplished by reacting a reagent such as N-bromosuccinimide with alcohol in the presence of triphenylphosphine to obtain a compound having a leaving group bromide, or thiol chloride to obtain a compound having a leaving group chloride It can be achieved by reacting with. If a sulfonate ester is desired, the desired sulfonate ester is obtained by reacting compound (5) with the appropriate sulfonyl chloride in the presence of a suitable base. For example, when methanesulfonyl chloride is reacted with compound (5) in the presence of an organic base such as triethylamine or pyridine in an inert solvent, compound (6) having a leaving group of OSO ₂ CH ₃ is obtained.

In step B3, the appropriately substituted hydroxy aryl ester (7) is reacted with the heterocycle (6) to displace the leaving group to give the bound ester (8). Substitution reactions are carried out under conditions well known in the art. Generally this reaction is carried out in an inert solvent in the presence of a base such as sodium hydroxide or other inorganic base such as alkali carbonate or an alkali hydroxide. Although not critical, the temperature of the reaction is from 0 ° C. to the reflux temperature of the inert solvent.

Treatment of compound (8) with hydrazine in step B4 or reaction with hydrazine in an appropriate organic solvent at elevated temperature yields acid hydrazide (9). In general, the reaction is carried out in the range of 50 ° C. to the reflux temperature of the organic solvent.

In step B5, the reaction of cyclizing the acid hydrazide (9) with the targeted 1,3,4-oxadiazol-2-one (10) is carried out in the presence of an organic base such as pyridine, Strong blocked amine base such as 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) in a suitable organic solvent, such as acetonitrile, reacted with a mate and sealed at elevated temperature Is carried out by treatment. In general, the reaction can be carried out at 100 to 200 ° C. 1,3,4-oxadiazol-2-one can also be synthesized by reacting compound (9) with phosgene. Stempel, A., et al., J. Org. Chem. 1955, 20, 412].

In step B6, the synthesis of the bonded ester 8 in another way is described. Thus, alcohol (5) is hydroxyaryl in the presence of triaryl or trialkylphosphine such as triphenylphosphine or tri-n-butylphosphine and diethylazodicarboxylate in an inert solvent such as THF or dichloromethane. Reaction with ester (8) yields bound ester (8). In general, the reaction is carried out between room temperature and the reflux temperature of the inert solvent.

Reaction Scheme C is a compound of formula I (wherein, Z is - Im _{- (CH 2) n -Y- (} CH 2) n) describes the synthesis of. This scheme is most useful for synthesizing compounds, where n represents 1 or 2 in the alkylene chain attached to ARYL. In step C1, compound (5) (Y = O) is converted to compound (6), wherein Lg is chloro or bromo, as described in step B2 of Scheme B. Compound (6) was then reacted with thiourea compound (11) under an environment similar to that described by Treau, M. et al., Heterocycles, 2001, 55 (9), 1727-1735 to thiol ( Make 5a).

When compound (6) is reacted with primary amine (12), aminoalkyl heterocycle (5b) is produced. Substitution of leaving groups by amines is well known to those skilled in the art. Substitution reactions are generally carried out in polar organic solvents in the presence of organic bases which can act as acid scavengers. Although not critical, the substitution reaction takes place between room temperature and the reflux temperature of the solvent.

In step C3, compounds (5, 5a and 5b) can be reacted with compound (13) to give the bound aryl ester (14), wherein Y is O, S or NR ₃ . Thus, when compounds (5) (Y = O) and (5a) (Y = S) are reacted with compound (13) to replace the leaving group, the reaction is generally performed at DMF or DMSO at temperatures around 0 to 150 ° C. It may be carried out in the presence of a strong base such as sodium hydride in a polar aprotic solvent such as. When reacting compound 5b (Y = NR ₃ ) with compound 14, the same conditions as those described in step C2 are used for the primary amine.

Synthesis of the desired 1,3,4-oxadiazol-2-one (16) from compound (14) is carried out in two steps (C4 and C5) which are exactly the same as described in Scheme B, steps B4 and B5.

In Scheme D, compound (wherein, Z is being _{- - (CH 2) n -Y-} (CH 2) n) of formula (I) it represents another method for the synthesis of. This procedure is most useful for synthesizing compounds with n from 3 to 5 in the alkylene chain attached to ARYL.

In step D1, the terminal aldehyde compound (17), which can be synthesized using the method described in Scheme A, is converted to terminal acetylene (19) in a two step reaction. Thus, compound (17) is reacted with bromomethylenetriphenylphosphorane (stage 1) and potassium t-BuOK to produce an intermediate bromoolefin (not shown), followed by treatment with 2 equivalents of t-BuOK (2 Step) to form acetylene (19). The reaction process for the conversion is described in Pianetti, P., Tet. Letters, 1986, 48, 5853-5856, and also in Corey, E. J., et al., J. Am. Chem. Soc., 1969, 91, 4318-4320. Alternatively, as shown in step D2, an intermediate of type (19) can be made by substituting a leaving group using a nucleophile such as compound (18) in which terminal acetylene has been introduced from an intermediate such as compound (6) (see Scheme C). .

In step D3, the Sonogashira coupling of the acetylene intermediate 19 with the aryl iodide 20 is carried out in the presence of tetrakistriphenylphosphinepalladium (0), cuprous iodide and an appropriate organic base in an inert solvent. When carried out under the following, a bound terminal acetylene 21 is obtained. The reduction of acetylene 21 can then be carried out by obtaining a saturated ester 14 by catalytic hydrogenation of compound 21 in step D4. Generally, hydrogen can be reduced using a catalyst such as palladium on carbon or chlorotris (triphenylphosphine) rhodium (I) in an inert organic solvent at a pressure of 30 to 300 p.s.i. Reduction can be carried out at room temperature to 175 ° C.

Synthesis of the desired 1,3,4-oxadiazol-2-one (16) from compound (14) is carried out in the same two steps (D5 and D6) as described in Scheme B, steps B4 and B5.

Scheme E describes a specific synthesis of a compound of Formula I, wherein Z is (CH ₂ ) _n NR ₃ (CH ₂ ) _n- . In this way, the linker (Z) can make the aldehyde into a reducing amine reaction using an amine. For example, in step E1, an aldehyde compound 5b, such as 4-formyl-benzoic acid methyl ester (n = 1), compound 22, in a polar solvent, generally an alcohol or alcohol THF mixture, wherein Y = NR ₃ ) and treatment with a reducing agent such as sodium triacetoxy-borohydride to obtain the desired intermediate 14a (n = 1).

Similarly, in step E2, treating an aldehyde, such as compound (17a), with an amine, such as 4-aminoalkyl benzoic acid methyl ester (n = 1), compound (23), wherein n is 1 and , R ₃ is (CH ₂ ) _{n for} NR ₃ ). In steps E3 and E4 compound 14a is converted to 1,3,4-oxadiazol-2-one 16a as described in Scheme B, steps B4 and B5.

More generally, suitable amines (which are R'OOC-ARYL- (CH ₂ ) _n NHR ₃ ) are made by catalytic hydrogenation from the corresponding nitrile or nitro compound or by sonoga from acetylene amine and aryl iodide or aryl bromide Following Shira bonds can be prepared by catalytic hydrogenation as described in Scheme D.

Scheme F illustrates the synthesis of a compound of Formula I, wherein Z is SO ₂ (CH ₂ ) _n −. In step F1, aryl sulfonyl chloride (24) is treated with aqueous sodium sulfide to give sulfinic acid (25). In step F2, reacting compound (25) with an intermediate such as compound (6) in a polar solvent such as DMF, acetonitrile or ethanol in the presence of a base such as DBU, pyridine, sodium methoxide or sodium hydroxide gives intermediate (26) Get Intermediate 26 is converted to the corresponding 1,3,4-oxadiazol-2-one in steps F3 and F4 as described in Scheme B, steps B4 and B5.

Scheme G illustrates the synthesis of compounds of Formula I, wherein Z is —O (CH ₂ ) _n CO—. The scheme illustrates the case where n is one. The starting material 2-acyl heterocycle 29 adds the appropriate Grignard reagent to the intermediate N-methoxy N-methyl carboxamide from the corresponding carboxylic acid (prepared by the method described in Scheme A). (Khlestkin, VK et al .; Current Organic Chemistry, 2003, 7 (10), 967-993 and Singh, J. et al., Journal fur Praktische Chemie, 2000, 342, 340-347). ). The preparation of intermediate N-methoxy-N-methyl carboxamides involves the use of N-methoxy acids in the presence of EDC, DCC, peptide binders such as DMPU, and tertiary amine bases such as diisopropylethylamine or triethylamine. This is most conveniently done by reacting with -N-methyl hydroxylamine hydrochloride.

Thus, compound (29) is brominated under control to produce bromoketone (30) as shown in step G1. Bromination reactions are well known methods, for example, by reacting compound (29) with pyridium bromide against bromide in acetic acid or by reacting compound (29) with Br ₂ in an inert organic solvent such as dichloromethane. do. The bromoketone 30 produced in step G2 is reacted with an arylhydroxy ester 7 under the conditions described in Scheme B (step B3) to give the bound ester 31. Ketone functionality in compound 31 is protected as ketal 32 using methods known in the art, under conditions such as those described in step G3. Compound 32 is then converted to 1,3,4-oxadizol-2-one ketal 34 in steps G4 to G5 by the standard procedure described in Scheme B (steps B4 and B5). Finally, in step G6, the ketal functionality of compound 34 is cleaved with an inorganic acid in THF-methanol-water or cleaved by other methods known in the art to obtain target structure 35.

Using the process of Scheme G, the analog (n is the starting material of a bromoketone compound (30) of a larger bromoalkanoyl substituent (Br (CH ₂ ) _n CO −, where n is 2 to 5). It is apparent to those skilled in the art that 2 to 5) can be synthesized.

Scheme H describes the process of preparing a compound of Formula I, wherein Z is-(CH ₂ ) _n CO-. In the H1 step, a suitable methoxycarbonyl-substituted heterocycle 36 is reacted with 2 equivalents of t-butylacetate lithium enoleate in a solvent such as THF or DME in the range from -78 ° C to room temperature to give the ketoacetate intermediate ( 37). In step H2, compound (37) is treated with a base such as sodium hydride in an inert solvent at temperatures ranging from -10 ° C to room temperature and alkylated anions produced by an electrophile such as compound (13), (Advanced) The intermediate ketodiester (38) is obtained. The dicarboxylation reaction as seen in step H3 is obtained by treating compound (38) with TFA in an inert solvent such as dichloromethane and pyrolyzing at a temperature between 70 and 150 ° C. to obtain intermediate ketoester (39). Ketone functionality in compound 39 is protected by ketal 40 using methods known in the art as can be seen in the H4 step. Compound 40 is again converted to 1,3,4-oxadiazol-2-one ketal 42 in steps H5 to H6 by the standard procedure described in Scheme B (steps B4 and B5). Finally, in step H7, as described in Scheme G, step G6, the ketal functionality in compound 42 is cleaved to give the desired 1,3,4-oxadiazol-2-one compound 43.

Biological Examples:

The following test protocol can be used to confirm the biological properties of the compounds of the present invention. The following examples are provided to illustrate the present invention. However, they are not intended to limit the invention in any way.

EC in Cell Line PPAR Delta-GAL4 Assay ₅₀  Value measurement:

principle

The ability of a substance to bind to and activate it in an aggressive manner in human PPAR delta was analyzed using a stably transfected HEK cell line (HEK = h uman e mbryo k idney) (herein referred to as PPAR delta reporter cell line). The PPAR delta reporter cell line contains two genetic elements, the luciferase reporter element (pdeltaM-GAL4-Luc-Zeo) and the PPAR delta fusion protein (GR-GAL4-human PPAR delta-LBD), which is a PPAR delta Depending on the ligand, expression of the luciferase reporter element is mediated. The stable and constitutively expressed fusion protein GR-GAL4-human PPAR delta-LBD, via the GAL4 protein portion, 5 'upstream of the luciferase reporter element stably bound to the cell line genome in the cell nucleus of the PPAR delta reporter cell line. Binds to the GAL4 DNA binding motif. If fatty acid-depleted fetal calf serum (cs-FCS) was used in the assay, the luciferase reporter gene is barely expressed without the PPAR delta ligand. PPAR delta ligands bind to and activate the PPAR delta fusion protein, thus stimulating luciferase reporter gene expression. The luciferases formed can be detected by chemiluminescence through a suitable substrate.

Composition of PPAR delta reporter cell lines:

Stable PPAR delta reporter cell line production is based on stable HEK-cell clones stably transfected with luciferase reporter elements. This step has already been described in the section "Configuration of PPAR alpha reporter cell line". In the second step, PPAR delta fusion protein (GR-GAL4-human PPAR delta-LBD) is stably introduced into cell clones. For this purpose, the cDNA (Accession # P04150) encoding the N-terminal 76 amino acid of the glucocorticoid receptor is linked to the cDNA portion encoding amino acids 1-147 of the yeast transcription factor GAL4 (Accession # P04386). The cDNA of the ligand-binding domain of the human PPAR delta receptor (amino acids S139-Y441; accession number # L07592) is cloned at the 3 'end of the GR-GAL4 construct. The fusion structure (GR-GAL4-human PPAR delta-LBD) constructed in this way is cloned back into plasmid pcDNA3 (Invitrogen) and constitutively expressed using a cytomegalovirus promoter. This plasmid was linearized by treatment with restriction endonucleases and stably transfected into the cell clones described above containing luciferase reporter elements. The resulting PPAR delta reporter cell lines containing luciferase reporter elements and constitutively expressing the PPAR delta fusion protein (GR-GAL4-human PPAR delta-LBD) are zeocin (0.5 mg / ml) and G418 (0.5 mg). / ml).

Analytical Process and Evaluation:

The activity of PPAR delta agonists is determined through a three day assay described below:

first day

Incubate the PPAR delta reporter cell line with 80% confluence in DMEM (# 41965-039, Invitrogen), which contains the following additional material: 10% cs-FCS (fetal calf serum # SH-30068.03, Hyclone), 0.5 mg / ml zeocin (# R250-01, Invitrogen), 0.5 mg / ml G418 (# 10131-027, Invitrogen), 1% penicillin-streptomycin solution (# 15140-122, Invitrogen) and 2 mM L-glutamine (# 25030-024, Invitrogen). Incubate in a standard cell culture vessel (# 353112, Becton Dickinson) in a 37 ° C. cell incubator in the presence of 5% CO ₂ . 80% -conjugated cells were washed once with 15 ml PBS (# 14190-094, Invitrogen), treated with 3 ml trypsin solution (# 25300-054, Invitrogen) for 2 minutes at 37 ° C., 5 ml DMEM described above, Count at the counter. After diluting to 500.000 cells / ml, 35,000 cells are inoculated in 180 μl volume into each well of a 96-well microtiter plate (# 3610, Corning Costar) in clear plastic. Plates are incubated for 24 hours in a cell incubator at 37 ° C., 5% CO ₂ .

Second day

The PPAR delta agonist tested is dissolved in DMSO at a concentration of 10 μmM. This stock solution is diluted with DMEM (# 41965-039, Invitrogen), which is treated with 5% cs-FCS (# SH-30068.03, Hyclone), 2 mM L-glutamine (# 25030-024, Invitrogen) and the antibiotics described above. Uo, G418, penicillin and streptomycin). Test materials were tested at eleven different concentrations ranging from 10 μM to 100 pM. More potent compounds are tested at concentrations ranging from 1 μM to 10 pM or concentration ranges from 100 nM to 1 pM.

The test substance diluted with the medium is added directly to the cells with or without a complete removal of the medium inoculated on day 1 of the PPAR delta reporter cell line by aspiration. Dilution and addition of the material uses a robot (Beckman FX). The final volume of test material diluted with medium is 100 μl per 96 well microtiter plate well. To avoid the cytotoxic effects of solvents, the DMSO concentration in the assay should be less than 0.1% v / v.

Each plate is filled with a standard PPAR delta agonist, which is similarly diluted to 11 different concentrations to account for the test capacity in each individual plate. Test plates are incubated in 37 ° C. and 5% CO ₂ incubator for 24 hours.

Alternatively, 20 μl at 10 times the final concentration of test substance is added directly to 180 μl containing plated cells. Test substances are tested at eight different concentrations in triplicate on a set up test plate.

third day

PPAR delta reporter cells treated with the test substance are removed from the incubator and the medium is aspirated. Cells were lysed by pipetting 50 μl Bright Glo reagent (in Promega) into each well of a 96 well microtiter plate. After incubation in the dark at room temperature for 10 minutes, the microtiter plate roughness was measured on a light meter (Trilux, Wallac). Each well measurement time of the microtiter plate is 1 second.

evaluation:

Transfer raw data from the light meter to a Microsoft Excel file. Dose-effect plots and EC ₅₀ values of PPAR agonists are calculated using XL. Fit to the program specified by the manufacturer (IDBS).

PPAR delta EC ₅₀ values in the range from 1 nM to more than 10 μM were measured for the PPAR modulators of the present application examples. Compounds of the invention of formula I may act as agonists or antagonists. Assays to determine partial agonist or antagonist activity are described below.

Determination of efficacy of partial agonists or antagonists at PPAR delta receptors

This test determines whether the compound acts as a partial agonist or antagonist at the PPAR delta receptor.

Plating and harvesting of this test plate is as described in the first and third days above.

Second day

Partially agonists or antagonists and known selective agonists are diluted with DMEM (# 41965-039, Invitrogen) 20 times the desired final concentration, where DMEM is 10% cs-FCS (# SH-30068.03, Hyclone), 2 mM L -Glutamine (# 25030-024, Invitrogen), and the previously described antibiotics (zeosin, G418, penicillin and streptomycin) are mixed. 10 μl of a partial agonist or antagonist is added to the cell containing test plate. The test plate is incubated at 37 ° C. and 5% CO ₂ for 30 minutes. After preincubation of the partial agonists or antagonists, 10 μL of 20 × known selective agonists are then added. The test plate is incubated at 37 ° C. and 5% CO ₂ for 24 hours. The effect on the EC ₅₀ of known selective agonists is measured for each partial agonist or antagonist concentration.

SPA PPAR Delta-LBD Binding Assay

Stock:

1M Tris (pH = 8.0 or pH = 7.6) (Gene Medicine Stock Room)

2M KCl (powder at N2140)

Twin 20

100 mM DTT

13.9μM GW2331 in hot ethanol

10mM GW 2331 in cold DMSO

PPAR-alpha (various concentrations)

E.g. .884 µg / μl

Wash buffer (stored at 4 ° C., buffer is good for 1 week):

10 ml Tris (pH = 7.6 or 8)

50mM KCl 25ml

0.05% Tween 20 0.5ml

Millipore Water 964.5

Check if PH = 7.6

Binding Buffer : (Prepare new binding buffer each time)

50 ml wash buffer

10mM DTT 5.5ml

Reaction reagent preparation for one plate:

Glutathione Coated SPA Beads

Each SPA Bead Bottle contains 500 mg of beads

500 mg of SPA beads were reconstituted in 5 ml of wash buffer, which would be good for several weeks.

Store at 4 ℃

SPA beads diluted in binding buffer.

Add 1 ml of the reconstituted SPA beads to 60 ml of binding buffer.

20 μl of the diluted beads are added to each well of a 96-well plate.

2 ml of the diluted beads are used for each plate (dead volume not included).

3H GW-2331 + GST-PPAR Delta-LBD (1 96-well plate, no dance volume) 13.9 μM

40 nM / well

3.0ml / plate (including dance volume)

If 3H-GW2331 specific activity is 1 mci / ml (Amersham), dilute 17 μl of 3H GW-2331 with 3.0 ml of binding buffer = .08 μM

If the protein concentration is 1 mg / mL, 21 μl of protein is added to 3.0 ml of binding buffer.

In summary, one 96-well plate: 3000 μL of binding buffer + 17 μL of 3H-GW2331 + 21 μL of GST-PPAR-delta (1 mg / ml)

Control plate

96-well mother plate (for 2 control plates)

In column # 1:

5 μl of cold GW2331 (10 mM) is added to well E-H.

45 μl of DMSO is added to wells A-H.

In column # 12 (3-fold dilution):

10 μl of cold GW2331 (10 mM) is added to Well A.

90 μl of DMSO is then added to Well A and the solution is mixed well.

20 μl of DMSO is added to well B-H.

Take 10 μl of solution from well A, place in B, mix well,

Take 10 μl of solution from B, add to C, mix well,

Take 10µl of solution in C, add to D, mix well

Finally, take 10 μl of solution from F and add to H.

Control Plate (for 8 Reaction Plates)

Control plate is 1:10 dilution of mother plate. Dilution buffer is wash buffer.

Sample plate

To a new CPC library plate, 90 μl of DMSO is added.

Take 10 μl of DMSO dilution and add it to 90 μl of wash buffer in the sample plate.

Reaction plate:

20 μl of SPA beads, 30 μl of 3H-GW2331 and GST-PPAR-delta are added to each well of the reaction plate.

5 μl of compound from each well of the sample plate is added to columns 2-11 of the reaction plate.

5 μl of compound from column 1 and column 12 of the control plate is added to columns 1 and 12 of the reaction plate.

96-well SPA protocol:

The reaction plate is allowed to equilibrate for 20 minutes to 2 hours.

Plates are sealed and counted on a Microbeta counter (Wallac).

Calculate IC ₅₀ .

In the SPA PPAR delta-LBD binding assay, IC ₅₀ values were measured in the range of 1 nM to greater than 10 μM for the PPAR modulators of the Examples of this application. Compounds of the invention of formula I may act as agonists or antagonists.

Rat / mouse oligodendrocyte culture

Cell preparation :

1. Obtaining primary rat oligodendrocyte progenitors from newly born (2-3 days) mice or rat neocortex, by modifying the original technique described in McCarthy and de Vellis (1980). Microglia are removed and concentrated by mechanical separation from an astrocytic monolayer.

2. Remove the brain membrane from the newborn rat brain and mechanically dissociate the tissue. Plate the cells on a T75 flask and feed the cells with DMEM / F12 + 10% FBS.

3. The oligodendrocytes grown on the stellate cell bed layer are obtained using a shaking off method for 14 days from the day of preparation of raw materials. The suspension was centrifuged and the growth factor (platelet induced growth factor-AA (PDGF), fibroblast growth factor-2 (FGF) supplemented serum-free medium (SFM; 25 μg / ml transfer with DMEM, 30 nM Triyo) Cell pellets were combined with dotyronine, 20 nM hydrocortisone, 20 nM progesterone, 10 nM biotin, 1x residual element, 30 nM selenium, 1 μg / ml putrescine, 0.1% BSA, 5U / ml PenStrep, 10 μg / ml insulin) Resuspend.

4. Cells were plated in PDL-coated petri dishes and incubated at 37 ° C., 6-7% CO ₂ .

5. Change the media components every 48 hours to keep the cells in the original cell state.

Passage of progenitor cells for screening test to increase cell number :

1. Once the cultures have joined, wash the cultures with PBS, add trypsin and incubate at 37 ° C. for about 2-3 minutes.

2. Neutralize the cell suspension and centrifuge at 900 g for 5 minutes.

3. Resuspend the cell pellet in SFM + PDGF / FGF.

4. The cells are supplied with fresh growth factors every 48 hours to rapidly differentiate progenitor cells.

5. Cells are passaged 4-5 times before experimental testing.

6. All experiments involving oligodendrocyte progenitor cells were carried out using cells that were maintained continuously under these conditions. At least 95% of all cells are A2B5 immunopositive and express 2'3'-cyclic nucleotide 3'-phosphodiesterase II mRNA.

7. To produce mature oligodendrocytes, progenitor cells were plated and converted to IGF-I supplemented or unsupplemented SFM 24 hours later and grown in this state for 7 days prior to experimental testing.

8. Alternatively, concentrated murine Central Glia-4 (CG4) progenitor cell lines can be used, which are basal medium (DMEM, 2 mM glutamine, 1 mM pyruvate sodium, biotin (40 nM), insulin (1 μM) and N1) Is maintained in supplemented with 30% conditioned medium (B-104 neuroblastoma cell line. To induce differentiation, CG4 cells were supplemented with 1% fetal calf serum (removed after 2 days) and insulin (500 nM) in basal medium. A2B5 and MBP immunoreactivity were used to confirm> 95% enrichment in incomplete and mature cultures, respectively.

Rat / mouse culture compound treatment :

1.Put 10,000-15,000 cells per well into a PDL coated 24-well plate and incubate the cells overnight in the presence of mitogen (10 ng / ml).

2. In the presence of mitogen:

a. The next day, remove old media and add compounds to fresh media (including mitogen)

b. Compound dose response assessments are performed at six different concentrations (10 μM, 1 μM, 100 nM, 10 nM, 1 nM, 0.1 nM);

c. Each compound concentration is repeated in triplicate wells.

3. Without Mitogen:

a. The next day, the old medium is removed and the compound is added to fresh medium (no mitogen).

b. Compound dose response assessments are performed at 6 concentrations (10 μM, 1 μM, 100 nM, 10 nM, 1 nM, 0.1 nM);

c. Each compound concentration is repeated in triplicate wells.

4. Incubate the treated cells for 7 days prior to use in the experimental test.

Human oligodendrocyte culture

Cell preparation:

1. Human neurospheres obtained from E19.5-E22 human embryo cortex were cultured in progenitor cell medium [(DMEM / F12 + 100 μg / ml transfer, 30 nM triiodothyronine, 20 nM hydrocortisone, 20 nM progesterone, 10 nM biotin). , 1 × residual element, 30 nM selenium, 60 μM putrescine, 0.1% BSA, 5 U / ml PenStrep, 25 μg / ml in combination with insulin) supplemented with PDGF and FGF for 2 weeks.

2. Neurospheres were dissociated for 30-50 minutes at 37 ° C. using 20 U / ml papain.

3. Cells were plated at a density of 50,000-100,000 cells per well in progenitor cell medium containing PDGF / FGF on PDL coated culture dishes and incubated at 37 ° C., 5-6% CO ₂ .

4. Medium and growth factors are changed every 48 hours.

Human culture compound treatment :

1. Remove old media 24 to 48 hours after plate and add compound to fresh media (containing mitogen).

2. Compound Dose Response Evaluation is performed at 3-6 different concentrations (10 μM, 1 μM, 100 nM, 10 nM, 1 nM, 0.1 nM).

3. Repeat each compound concentration in triplicate wells.

5. Incubate the treated cells for 7 days before using them for experimental testing.

Rat / mouse / human oligodendrocyte cell specific immune staining :

After compound exposure, oligodendrocyte-specific antibodies are used to assess the ability of a compound to promote or accelerate oligodendrocyte differentiation (eg, O4, O1, or over time between compound treated and untreated cultures) Myelin-based protein immunoreactivity).

1. Cells were plated at 5 × 10 ³ to 20 × 10 ³ cells / well on poly-D-lysine treated 4-well chamber slides and grown as described above. Measurement in days without PDGF and FGF results in continuous staining of the oligodendrocyte population with increasing cell differentiation.

2. Detecting oligodendrocytes expressing specific cell surface markers (including A2B5, O4, O1) using live staining at 37 ° C for 30 minutes.

3. In succession, the cells are fixed in 4% paraformaldehyde for 10 minutes at room temperature.

4. A fixed staining procedure is used to detect oligodendrocyte states that express specific markers (including myelin basic protein, MBP).

5. Rinse with PBS.

6. Infiltrate with 0.1% Triton / 0.01% NaAz diluted in 1 × PBS for 10 minutes at room temperature.

7. Block in antibody dilution buffer (0.1% Triton-X 100, 1% IgG-free bovine serum albumin; also used to dilute antibody) with 5-10% goat serum at room temperature for 15 minutes.

8. Add primary antibody diluted with antibody dilution buffer.

9. Incubate overnight at 4 ° C with gentle shaking.

10. The next day, wash PBS 1 × 5 minutes at room temperature and then 3 × 15 minutes.

11. Incubate at room temperature for 45 minutes with the appropriate secondary antibody.

12. Cell nuclei are stained with 4,6-diimidino-2-phenylindole (DAPI) for 15 minutes at room temperature.

13. Wash several times with PBS and evaluate using fluorescence microscope.

14. The following conditions are compared over time and according to different compound doses: PDGF / FGF alone, SFM alone, SFM-IGF1 alone, PDGF / FGF and compounds, SFM and compounds.

Rat / mouse / human bromodeoxyuridine (BrdU) immunostaining:

To confirm that the compounds do not promote cell proliferation.

1. Label oligodendrocyte progenitor cells with 10 μM BrdU for 20 hours and fix with 70% ethanol or 4% paraformaldehyde.

2. Cells are subsequently incubated with biotinylated mouse anti-BrdU and streptavidin-peroxidase and washed with PBS three times.

3. Color development confirmation of BrdU immunoreactivity is printed with DAB and total cell number is assessed with counter-stained hemoxylline.

4. BrdU immune positive cells are counted by two different observers.

Mouse / mouse / human culture image analysis : fluorescence microscopy is used to quantify the degree of oligodendrocyte differentiation following compound exposure. This test demonstrates that selective agonists accelerate / promote oligodendrocyte differentiation.

1. Manual Cell Counting: Randomly select four sections for each experimental condition, counting 500-600 cells in each section. The ratio of MBP (or O4) immunopositive cells (maturation process involving cells with or without myelin sheet) and DAPI positive cells (total cell number) was compared in the control and drug treated groups.

2. Automated Cell Number Measurement: Fluorescence microscopy was used to quantify the degree of oligodendrocyte differentiation after exposure to the compound. Six portions per well were randomly selected to assess the number of oligodendrocytes that differentiated into the entire population (about 8-15 × 10 ³ cells are counted per well). Immunofluorescence images are obtained using a Zeiss AxioVision digital imaging system + a Zeiss AxioCam HRc cooled CCD camera connected to the same microscope. All microscopic image parameters were set to allow images to be obtained for cellular immunofluorescence intensity analysis. The percentages of MBP positive (differentiated) cells and total cells (DAPI nuclear stained) were compared in the control and drug-treated groups. Cell autofluorescence was not detected under imaging conditions.

3. Human oligodendrocyte differentiation test: Manually count the total number of O4 immunopositive cells / well (bipolar and multipolar).

Rat / Mice / Human Quantitative Polymerase Chain Reaction (PCR ): To assess how much a compound activates the PPAR delta pathway and induces oligodendrocyte maturation (mRNA level change).

1. Total RNA is extracted from oligodendrocytes cultured using TriZol reagent.

2. Subsequently, mRNA is treated with RNase-free DNase, rearranged and then converted to cDNA template using RT reaction (Clontech Advantage RT for PCR kit).

3. PPAR delta path member transcript expression is quantified using Sybr Green PCR Master Mix.

4. 18S ribosomal RNA primer / probe mixture (18S bp product) suspended in Taqman 2X PCR Master Mix is used as internal control.

5. Quantitative PCR is performed with a Model 7700 Sequence Detector System (Biosystems, Foster City, CA), using real-time Taqman ™ technology (Gibson, et al., 1996).

6. The results are analyzed using Sequence Detection System software version 1.91.

Rat ELISA Test: To assess whether a compound induces PPAR delta pathway activation and to what extent it induces oligodendrocyte maturation (protein level changes).

1. Wash the plate with PBS and place on ice. Lysis buffer (tris 50mM, pH7.4, MgCl ₂ 2mM, EDTA 1mM, β-mercaptoethanol 5mM, Nonidet P-40 1%, Protease Inhibition Cocktail (Roche), 1 tablet per well / 50 ml) is added to each well.

2. Pipet up and down cells using a pipette to lyse and spin the plate at 2000 rpm, 4 ° C. for 5 minutes. Prepare the supernatant for use.

3. Pipette 50 μl of standards, controls and samples into the wells.

4. Pipette 50 μl of MBP assay buffer into each well.

5. Incubate the wells with stirring at 500-700 rpm on an orbital microplate shaker for 2 hours at room temperature.

6. Add 100 μl of MBP antibody-biotin conjugate to each well.

7. Incubate the wells with stirring at 500-700 rpm on an orbital microplate shaker for 1 hour at room temperature.

8. Rinse wells five times with wash solution, invert the plate over the absorbent material and blot dry.

9. The streptavidin-enzyme conjugate is diluted in MBP Elisa assay buffer in a 1:50 ratio (it must be diluted immediately before testing).

10. Add 100 μl Streptavidin-enzyme conjugate solution to each well.

11. Incubate the wells with stirring at 500-700 rpm on an orbital microplate shaker for 30 minutes at room temperature.

12. Rinse wells five times with wash solution, invert plates over absorbent material and blot dry.

13. Add 100 μl of TMB chromogen solution to each well.

14. Incubate the wells with stirring at 500-700 rpm on an orbital microplate shaker for 10-20 minutes at room temperature. Avoid exposure to direct sunlight.

15. Add 100 μl of stop solution to each well.

The absorbance of the solution in the wells is read within 30 minutes using a microplate reader set at 450 nM.

In vivo proof of the concept model

Local lesions: (used to assess whether a compound protects myelin integrity or accelerates or enhances the rate of myelin remodeling )

1. Make the food and water freely available to the 7 week old rats and allow at least 4 days to acclimate to the experiment.

2. Weigh each animal before surgery. Rats are then anesthetized with ketamine (100 mg / ml) and silazine (20 mg / ml) (1.8: 1 ratio). Surgery is performed after injection of an anesthetic solution of 0.15ml / 180g body weight to i.p. Prepare animals for surgery using sterile conditions in accordance with IACUC regulations. All surgical instruments are steam sterilized. Whisk between the ears, rub with betadine, pour sterile saline, and finally wipe with prepackaged sterile alcohol swabs.

3. For surgery, the rat is pressed to reach the belly surface in a small animal stereotaxic instrument designed to fix the rat head. The incisor bar is always fixed at -3.9 mm to obtain the flat cranial position of the SD rats.

4. Make an incision in the pre-shaved skin on your skull.

5. Drill a small hole (0.75 mm in diameter) from the lambda onto the AP-1.8, ML-3.1 coordinate location bone.

6. Remove the bone and inject 2 μl of ethidium bromide, lysocithin, or SIN-1 into the right cerebellar cerebellar peduncle, DV-7.1 mm at a 2-minute interval with a Hamilton μL syringe and needle. do. Another injection is injected into the spinal cord, corpus callosum or white matter.

7. Leave the needle in position for two consecutive minutes.

8. Remove the needle and seal the incision.

9. Add 0.003 mg buprenorphine to i.m. Inject.

10. The rat is placed on a warm cupboard until it finds consciousness. When the time comes, we send it back to us. Make sure there are no more than two rats per cage to prevent rats from biting the sutures.

11. Perform similar procedures using mice.

Experimental allergic encephalomyelitis (rat EAE) disease model in rats:

Experimental allergic encephalomyelitis (EAE) is a T-cell mediated autoimmune neurological disease in the nervous system that develops in disease-prone animals after sensitization with whole spinal cord homogenates or components (myelin basic protein). The EAE rodent model is an appropriate tool for the study of inflammation of the brain and spinal cord observed in MS patients. Injection of whole spinal cord or spinal cord components such as myelin basic protein in rodents induces an autoimmune response based on T-lymphocyte activation. Clinical disease is usually evident 8-10 days after inoculation, with a wide range of behavioral abnormalities ranging from gait disturbance and tail relaxation to complete paralysis and death. Weight loss is common. In surviving animals, spontaneous recovery occurs and most motor nerves recover to varying degrees. Depending on the species, allergen, and method used, the animal tested by the EAE model may experience a single (acute EAE) or several attacks (chronic recurrent EAE). Several treatment courses may be used: Drugs and treatment options may be administered prior to immunization, during asymptomatic times, or during clinical disease course.

Animals :

Female Lewis Rat, 160-220 g (Charles River)

Antigen :

Whole Guinea Pig Spinal Cord (Harlan Biosciences).

Complete fluent adjuvant H37 Ra [1 mg / ml Mycobacterium tuberculosis H37 Ra] (Difco).

Additional antigens :

Mycobacterium tuberculosis (Difco).

Bordetella pertussis [lethal using heat] (Difco).

Antigen Preparation (approximately 720 animals):

1. Weigh 5 g of frozen guinea pig spinal cord.

2. Add 5 g of spinal cord to 5 ml of 0.9% saline solution (1 g / ml) in a round centrifuge tube.

3. Homogenize on ice using Tissue-tech until the tissue is completely ground (about 5 minutes).

4. Add 10 ml of complete ring fluent adjuvant H37 supplemented with 200 mg of Mycobacterium tuberculosis (20 mg per complete Fluent Adjuvant H37 Raml).

5. Extract the homogenate / adjuvant from the tube by capturing the homogenate / adjuvant with a 10 ml syringe with an 18 gauge emulsion needle.

6. Emulsify in two 30 ml glass syringes until it is difficult to continuously pass material through the needle (about 5 minutes (no separation between the oil phase and the aqueous phase)].

7. Use immediately or leave on ice until needed (no more than 30 minutes) (do not freeze).

protocol

Female Lewis Rivers must be free to eat food and water and adapt at least 3 days before use in experiments.

2. Rats weighing 160 g to 220 g are first induced with 5% isoflurane (Aerrane, Fort Dodge), 30% O _2, 70% N ₂ O for 2 to 5 minutes.

3. The rats are then placed on a circulating water heater blanket (Gaymar) (abdomen upwards), covered with a cone to spontaneously inhale anesthesia gas and reduced isoflurane to 2%.

4. Place two subcutaneous injections (0.1 ml each) of antigen or normal saline solution under the hind paw.

5. Remove the cone on your nose, weigh it, and number it.

6. Allow the rats to wake up from the anesthesia and place them in individual cages.

7. Monitor animals daily for signs of EAE induction (see control below).

Phase: 0 Normal

Steps: 1 abnormal, tail relaxation

Stage: 2 weak but obviously weakened on one or both hind limbs

Stages: 3 One or both hind limbs become severely weak or weak limbs

Stage: 4 serious paralysis and minimal hind limb movements

Stage: 5 No hind limb movement, paralysis

Stages: 6 Deaths with no spontaneous movement and difficulty breathing.

          Increased foreleg movement, urinary and incontinence may occur

Stage: 7 deaths

Treatment begins 10 days after immunization. Disease symptoms in this model usually appear 10-11 days after vaccination, which appears to be the onset of acute MS symptoms. Delayed treatment initiation is believed to mimic the clinical condition more closely than traditionally used protocols for administering the drug at the time of inoculation or even prior to inoculation. Teitelbaum D. et al. 96: 3842-3847 and Brod S. A., et al., Ann Neurol 2000; 47: 127-131.

The present invention will be described more clearly through the following examples provided herein, but such embodiments are intended to illustrate the present invention, but are not intended to be limiting.

Synthetic Example

Normal

Commercially available reagents and solvents were used. ¹ HNMR spectra are recorded with a Varian MercuryPlus-300 (300 MHz) or Varian Unity Inova (400 MHz) spectrometer. Proton chemical variations are reported in ppm relative to internal tetramethylsilane (0.0 ppm). Using a scintillation mass spectrophotometer and electrospray ionization, MS (LC-MS) data is obtained at a 5 minute data acquisition time of m / z 100 to 1000. LC (LC-MS) is a Hypersil C18 column (4.6x50 mm, 3.5 μ) mobile phase is 0.1% TFA / H ₂ O (A), 0.1% TFA / ACN (B), 5% to 100 for 3 minutes Gradient of% B, followed by 2 minutes at 100% B). Alternatively, a platform LC-MS equipped with an electrospray source can be run with an HP 1100 LC system running at 2.0 ml / min, 200 μL / min split for an ESI source with inline HP1100 DAD detection and SEDEX ELS detection. Can be used together. The Luna C18 (2) column (30 × 4.6 mm 3μ) has a 5% to 95% B gradient for 4.5 minutes, and the mobile phase uses 0.1% formic acid / H ₂ O and 0.1% formic acid / ACN (B). HPLC purification is performed on a Varian ProStar system with an ACN / H ₂ O linear gradient containing 0.1% trifluoroacetic acid. Microwave synthesis was performed using a Personal Chemistry Smithcreator microwave reaction system using a 2 or 5 mL reaction vessel.

Example 1

Intermediate: [5-Methyl-2- (4-trifluoromethyl-phenyl) -thiazol-4-yl] -acetic acid ethyl ester

Ethyl-4-bromo-3-oxo-pentanoate (2.07 g, 9 mmol) is added to a solution of 4-trifluoromethyl-benzene-thioamide (1.845 g, 9 mMol) in ethanol (15 ml, 200 proof). . The solution is sealed and the solution is Personal Chemistry ^™ . Warm to 170 ° C. in a microwave oven and stir at this temperature for 20 minutes. The resulting solution is cooled to room temperature, concentrated under reduced pressure and purified by flash chromatography (eluted with 30% ethyl acetate / 10% dichloromethane in heptane) to afford the title compound (1.4 g) as a white solid.

MS (ESI) m / z 330 (M + H); H1 NMR (CDCl ₃ ) δ 1.87 (bs, 1H), 2.49 (s, 3H), 4.86 (s, 2H), 7.67 (d, J = 8 Hz, 2H), 8.02 (d, J = 8 Hz, 2H).

Example 2

Intermediate: 4- (2-hydroxy-ethyl) -5-methyl-2- (4-trifluoromethyl-phenyl) thiazole

Lithium aluminum hydroxide (5.3 ml, 1M in THF) solution was cooled (0 ° C.) and [5-methyl-2- (4-trifluoromethyl-phenyl) -thiazol-4-yl] in THF (15 ml) Add a solution of ethyl acetate (Example 1, 1.4 g, 4.25 mmol). When the addition is complete, the cold bath is removed and stirred for 2 hours. The solution is cooled to 5 ° C., then water (0.2 ml) is added dropwise followed by NaOH solution (0.2 ml, 5M in water) and water (0.2 ml) dropwise. The resulting mixture is diluted with ethyl acetate and filtered through a pad of celite. The solid is washed with dichloromethane and the combined filtrates are concentrated under reduced pressure. The residue was purified by flash chromatography (eluted with 30% ethyl acetate / 40% dichloromethane in heptane) to afford the title compound (0.879 g) as a yellow solid. The title compound is obtained using the compound of Example 1 as a starting material.

MS (ESI) m / z 288 (M + H); H1 NMR (CDCl ₃ ) δ 2.44 (s, 3H), 2.91 (t, J = 7 Hz, 2H), 3.62 (t, J = 6 Hz, 1H), 4.01 (dt, J = 7, 6 Hz, 2H), 7.66 (d, J = 8 Hz, 2H), 7.96 (d, J = 8 Hz, 2H).

Example 3

Intermediate: 4- [5-methyl-2- (4-trifluoromethyl-phenyl) -thiazol-4-ylethoxy] -benzoic acid methyl ester

4-hydroxy in 4- (2-hydroxy-ethyl) -5-methyl-2- (4-trifluoromethyl-phenyl) thiazole (Example 3, 288 mg, 1.0 mmol) solution in THF (3 ml) Add benzoic acid methyl ester (167 mg, 1.1 mmol) and triphenylphosphine (288 mg, 1.1 mmol). To this solution, diethyl azodicarboxylate (174 mu l, 1.1 mmol) was added dropwise. Upon completion of the addition, the resulting red solution was stirred for 20 minutes, concentrated under reduced pressure and the residue was purified by flash chromatography (eluted with 15% ethyl acetate / 15% dichloromethane in heptane) to give the title compound (410 mg) as a white solid. ).

MS (ESI) m / z 422 (M + H); H1 NMR (DMSO) δ 2.51 (s, 3H), 3.19 (t, J = 7 Hz, 2H), 3.80 (s, 3H), 4.40 (t, J = 7 Hz, 2H), 7.05 (d, J = 9 Hz, 2H), 7.83 (d, J = 8 Hz, 2H), 7.88 (d, J = 8 Hz, 2H) 8.05 (d , J = 8 Hz, 2H).

Example 4

Intermediate: 4- {2- [5-Methyl-2- (4-trifluoromethyl-phenyl) -thiazol-4-yl] -ethoxy} -benzoic acid hydrazide

Anhydrous in suspension of 4- [5-methyl-2- (4-trifluoromethyl-phenyl) -thiazol-4-ylethoxy] -benzoic acid methyl ester (Example 4, 410 mg, 1 mmol) in methanol (3 ml). Add hydrazine (0.32 ml, 10 mmol). The resulting mixture is warmed to 60 ° C. and stirred at this temperature for 66 hours. The resulting solution is cooled to room temperature and 3 drops of water are added. The precipitate is filtered off and washed with ether to give the title compound (279 mg).

MS (ESI) m / z 422 (M + H); H1 NMR (DMSO) δ 2.51 (s, 3H), 3.17 (t, J = 7 Hz, 2H), 4.36 (t, J = 7 Hz, 2H), 4.38 (bs, 2H), 6.98 (d, J = 9 Hz, 2H), 7.77 (d, J = 9 Hz, 2H), 7.83 (d, J = 8 Hz, 2H) 8.06 (d , J = 8 Hz, 2H) 9.58 (bs, 1H).

Example 5

5- (4- {2- [5-methyl-2- (4-trifluoromethyl-phenyl) -thiazol-4-yl] -ethoxy} -phenyl) -3 H -[1,3,4] oxadiazol-2-one

4- {2- [5-Methyl-2- (4-trifluoromethyl-phenyl) -thiazol-4-yl] -ethoxy} -benzoic acid hydrazide in dichloromethane (4 ml) (Example 4, 276 mg, 0.65 mmol) is added pyridine (104 μl, 1.3 mmol) followed by phenylchloroformate (0.88 μl, 0.71 mmol). At this temperature, the resulting mixture is stirred until all starting material is used up (check by TLC analysis). The mixture is diluted with ethyl acetate, washed with water, brine, dried over MgSO ₄ and concentrated under reduced pressure. The residue is dissolved in acetonitrile (5 ml). To this mixture, DBU (106 μl, 0.71 mmol) is added. The resulting solution is sealed and warmed to 170 ° C. in a Personal Chemistry ^™ microwave oven and stirred at this temperature for 120 minutes. The reaction was cooled to rt, diluted with ethyl acetate, washed with 1M HCl solution (or saturated NaH ₂ PO ₄ solution), dried over MgSO ₄ and concentrated. The resulting residue was triturated several times with dichloromethane to give the title compound (137 mg) (recrystallized from ethyl acetate in a sealed tube at 140 ° C.) as a tan solid.

MS (ESI) m / z 448 (M + H); H1 NMR (DMSO) δ 2.51 (s, 3H), 3.19 (t, J = 7 Hz, 2H), 4.40 (t, J = 7 Hz, 2H), 7.10 (d, J = 8 Hz, 2H), 7.70 (d, J = 8 Hz, 2H), 7.83 (d, J = 8 Hz, 2H) 8.05 (d , J = 8 Hz, 2H) 12.41 (bs, 1H).

Claims (16)

A compound of formula (I), or a stereoisomer, tautomer or solvate thereof, or a pharmaceutically acceptable salt thereof. Formula I

In Formula I, ARYL is phenyl or pyridinyl, wherein phenyl or pyridinyl is halogen, C _1-6 alkyl, C _2-6 alkenyl, C _1-6 alkoxy, C _1-6 perfluoroalkyl; C _1-6 alkylthio, hydroxy, hydroxyC _1-6 alkyl, C _1-4 acyloxy, nitro, cyano, C _1-6 alkylsulfonyl, amino, C _1-6 alkylamino and C _1- May be substituted with one or more substituents selected from the group consisting of ₆ alkoxycarbonyl; Z is -O (CH ₂ ) _n- , -SO ₂ (CH ₂ ) _n -,-(CH ₂ ) _n -Y- (CH ₂ ) _n -,-(CH ₂ ) _n -CO-, -O ( CH ₂ ) _n -CO- or-(CH ₂ ) _n -Y- (CH ₂ ) _n -CO-, wherein Y is NR ₃ , O or S, and R ₃ is H, C _1-6 alkyl, C _3-8 cycloalkyl, C _{1-6 alkylC 3-8} cycloalkyl and benzyl, n is independently an integer from 1 to 5; X is NR ₃ , O or S, wherein R ₃ is as defined above; R ₁ is H, halogen, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 perfluoroalkyl; HydroxyC _1-6 alkyl, nitro, cyano or C _1-6 alkylamino; R ₂ is substituted or unsubstituted phenyl, pyridinyl or thienyl, wherein the substituents are halogen, C _1-6 alkyl, C _2-6 alkenyl, C _1-6 alkoxy, C _1-6 perfluoroalkyl, C _1-6 alkylthio, hydroxy, hydroxyC _1-6 alkyl, C _1-4 acyloxy, nitro, cyano, C _1-6 alkylsulfonyl, amino, C _1-6 alkylamino and C _{1- 6} alkoxycarbonyl; Provided that when Z is -O (CH ₂ ) _n -or -SO ₂ (CH ₂ ) _n -and ARYL is phenyl, R ₂ is a group other than phenyl. The compound of claim 1, wherein ARYL is phenyl and X is O or S. 5. The compound of claim 2, wherein X is O. 4. 5- (4- {2- [5-methyl-2- (4-trifluoromethyl-phenyl) -thiazol-4-yl] -ethoxy} -phenyl) -3H- [1,3,4] A compound that is oxadiazole-2-one. A pharmaceutical composition comprising an effective amount of a compound according to claim 1 and a pharmaceutically acceptable carrier. A therapeutically effective amount of a compound of claim 1, or a stereoisomer, tautomer or solvate thereof, or a pharmaceutically acceptable salt thereof, is administered to a mammal suffering from a disease modulated by PPAR delta ligand binding activity. A method for treating a disease controlled by PPAR delta ligand binding activity in a mammal. Formula I

In Formula I, ARYL is phenyl or pyridinyl, wherein phenyl or pyridinyl is halogen, C _1-6 alkyl, C _2-6 alkenyl, C _1-6 alkoxy, C _1-6 perfluoroalkyl; C _1-6 alkylthio, hydroxy, hydroxyC _1-6 alkyl, C _1-4 acyloxy, nitro, cyano, C _1-6 alkylsulfonyl, amino, C _1-6 alkylamino and C _1- May be substituted with one or more substituents selected from the group consisting of ₆ alkoxycarbonyl; Z is -O (CH ₂ ) _n- , -SO ₂ (CH ₂ ) _n -,-(CH ₂ ) _n -Y- (CH ₂ ) _n -,-(CH ₂ ) _n -CO-, -O ( CH ₂ ) _n -CO- or-(CH ₂ ) _n -Y- (CH ₂ ) _n -CO-, wherein Y is NR ₃ , O or S, and R ₃ is H, C _1-6 alkyl, C _3-8 cycloalkyl, C _1-6 alkylC _3-8 cycloalkyl, and benzyl, n is independently an integer from 1 to 5; X is NR ₃ , O or S, wherein R ₃ is as defined above; R ₁ is H, halogen, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 perfluoroalkyl; HydroxyC _1-6 alkyl, nitro, cyano or C _1-6 alkylamino; R ₂ is substituted or unsubstituted phenyl, pyridinyl or thienyl, wherein the substituents are halogen, C _1-6 alkyl, C _2-6 alkenyl, C _1-6 alkoxy, C _1-6 perfluoroalkyl, C _1-6 alkylthio, hydroxy, hydroxyC _1-6 alkyl, C _1-4 acyloxy, nitro, cyano, C _1-6 alkylsulfonyl, amino, C _1-6 alkylamino and C _{1- 6} alkoxycarbonyl. The method of claim 6, wherein ARYL is phenyl. The method of claim 6, wherein ARYL is phenyl and R ₂ is phenyl. The method of claim 6, wherein ARYL is phenyl, Z is -O (CH ₂ ) _n -and R ₂ is phenyl. The method of claim 6, wherein ARYL is phenyl, Z is -O (CH ₂ ) _n- , X is O or S and R ₂ is phenyl. The method of claim 6, wherein ARYL is phenyl, Z is -O (CH ₂ ) _n- , X is O or S, R ₁ is C _1-6 alkyl, and R ₂ is phenyl. 12. The method of claim 11, wherein X is O. 13. The method of claim 12, wherein X is S. The disease according to claim 6, wherein the disease is multiple sclerosis, Charcot-Marie-Tooth disease, Felizeus-Merzbacher disease, encephalomyelitis, neuromyelitis optica, adrenal protein A dystrophic disorder, acute inflammatory demyelinating neuropathy (Guillian-Barre syndrome) and myelin-forming glial cell damage, myelin destruction disease selected from the group consisting of diseases including spinal cord injury, neuropathy and nerve damage. The method of claim 14, wherein the myelin disrupting disease is multiple sclerosis. The method of claim 6, wherein the disease is obesity, hypertriglyceridemia, hyperlipidemia, hypoalphalipoproteinemia, hypercholesterolemia, dyslipidemia, syndrome X; Type II diabetes mellitus and its complications selected from the group consisting of neuropathy, nephropathy, retinopathy and cataracts; And hyperinsulinemia, impaired glucose tolerance, insulin resistance, atherosclerosis, hypertension, coronary heart disease, peripheral vascular disease and congestive heart failure.