WO2018230936A1 - NOVEL COMPOUND HAVING PPARα/γ DUAL ACTIVITY AND COMPOSITION CONTAINING SAME AS ACTIVE INGREDIENT FOR PREVENTING OR TREATING METABOLIC DISEASE - Google Patents

NOVEL COMPOUND HAVING PPARα/γ DUAL ACTIVITY AND COMPOSITION CONTAINING SAME AS ACTIVE INGREDIENT FOR PREVENTING OR TREATING METABOLIC DISEASE Download PDF

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WO2018230936A1
WO2018230936A1 PCT/KR2018/006649 KR2018006649W WO2018230936A1 WO 2018230936 A1 WO2018230936 A1 WO 2018230936A1 KR 2018006649 W KR2018006649 W KR 2018006649W WO 2018230936 A1 WO2018230936 A1 WO 2018230936A1
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pparα
compound
formula
preventing
active ingredient
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PCT/KR2018/006649
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French (fr)
Korean (ko)
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박상규
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아주대학교산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/3262—Foods, ingredients or supplements having a functional effect on health having an effect on blood cholesterol

Definitions

  • the present invention relates to a composition for treating metabolic diseases containing a novel compound having a PPAR ⁇ / ⁇ dual action activity or a pharmaceutically acceptable salt thereof as an active ingredient.
  • Obesity is a disease that causes a variety of diseases, 80% of diabetics, 21% of heart disease patients are caused by obesity, nonalcoholic fatty liver disease is also reported as the main cause of obesity.
  • Non-alcoholic fatty liver disease refers to a disease in which triglycerides accumulate in the liver regardless of drinking, and are known as steatosis and non-alcoholic steatohepatitis (NASH). ) Is included. Simple fatty liver is considered to be a benign disease with a good prognosis, but NASH with inflammation or fibrosis along with fatty liver has been reported as a proliferative disease that causes cirrhosis or liver cancer as a progressive liver disease.
  • Obesity and insulin resistance are risk factors for representative non-alcoholic fatty liver disease. 69-100% of non-alcoholic fatty liver patients are obese, 20-40% of obese patients have non-alcoholic fatty liver, and the prevalence of liver disease among male obese patients is higher than that of female obese. In society, 3-30% of normal weight adults as well as obese patients are reported to have non-alcoholic fatty liver lesions.
  • NAFDL nonalcoholic fatty liver disease
  • the first is to improve the fatty liver by correcting risk factors such as obesity treatment (Zenical), insulin resistance treatment (Metformin, pioglitazone, rosiglitazone), hyperlipidemia treatment (clofibrate, gemfibrozil, bezafibrate, atorvastatin, simvastatin) Drugs that treat and improve it belong here.
  • metformin increases the oxidation of fatty acids, decreases fat-forming enzymes, improves insulinosis and insulin resistance.
  • Chiazolidinedione, rosiglitazone, and pioglitazone activate PPAR ⁇ , a nuclear hormone receptor. Promotes absorption.
  • the second type of treatment is a drug that repairs hepatocellular damage independent of risk factor correction for non-alcoholic fatty liver.
  • Hepatoprotective agents ursodioxycholic acid and taurine
  • antioxidants vitamins E and C
  • nutrition Supplements lecithin, betaine, N-acetylcysteine, etc. belong to this, but to date, there is no ideal drug that is effective in treatment without side effects.
  • the present invention has been confirmed that the problem that the PPAR agonist that has been used as a conventional diabetes treatment for worsening non-alcoholic fatty liver, solve the side effects of the PPAR agonist to exhibit a safe and excellent non-alcoholic fatty liver treatment effect to the human body
  • One compound and a composition for treating metabolic diseases containing the same as an active ingredient are provided.
  • the present invention provides a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.
  • R 1 to R 4 may be the same or different, respectively, hydrogen; C1 to C4 alkyl; Any one selected from the group consisting of C1 to C4 alkoxy.
  • the present invention provides a pharmaceutical composition for preventing or treating metabolic diseases, which comprises the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a health food for preventing or improving metabolic diseases containing the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the compound of the present invention solves the side effects of the PPAR ⁇ agonist and is excellent in treating non-alcoholic fatty liver and hyperlipidemia.
  • the composition containing the novel compound as an active ingredient can be used as a non-alcoholic fatty liver and hyperlipidemia treatment that can replace the conventional treatment.
  • Figure 2 is a result of identifying the target genes induced expression by PPAR ⁇ and PPAR ⁇ activated in MD001
  • Figure 2A and 2B is a human HA-PPAR ⁇ (A) and HA-PPAR ⁇ (B) expression vector, reporter in HEK293 cells Temporary co-transfection with plasmid (PPRE-pk-Luc) or control reporter plasmid (pk-Luc) and Renilla vector for 24 hours, followed by MD001 (10 ⁇ M), rosiglitazone (rosi (10 ⁇ M) or WY14643 (WY; 10 ⁇ M) Was treated with cells for 24 hours, and luciferase activity was confirmed.
  • FIG. 2C shows the relative expression level of qp-PCR after synthesis of cDNA by treating MD001 (10 ⁇ M) with HepG2 cells for 24 hours, and separating total RNAs.
  • Figure 2D is HepG2 cells transfected with control si-RNA, PPAR ⁇ si-RNA (D) or PPAR ⁇ si-RNA (E) for 48 hours and WY14643 (10 ⁇ M), Treatment with rosiglitazone (10 ⁇ M) or MD001 (10 ⁇ M) for 24 hours followed by cell collection RNA was isolated and qRT-PCR confirmed the relative expression level of the target gene (#, vs. control si-RNA; ⁇ , vs. control si-RNA / vehicle; ⁇ , vs.
  • Figure 3 shows that MD001 induces GLUT expression to promote glucose consumption.
  • HepG2 A
  • differentiated 3T3-L1 adipocytes B
  • differentiated C2C12 root canal cells C
  • MD001 10 , 50 ⁇ M
  • rosi rosiglitazone
  • Figure 4 is a result of confirming the fatty acid oxidation promoting effect by MD001 in vitro
  • Figures 4A, 4B and 4C are HepG2 (A), differentiated 3T3-L1 adipocytes (B) and differentiated C2C12 myotubes (C) Treatment of vehicle or MD001 (10 ⁇ M) for 24 hours, and the total RNA was isolated to synthesize cDNA and qRT-PCR to determine the relative gene expression
  • Figure 4D is HepG2 (D), differentiated 3T3-L1 fat Treatment of cells (E) and differentiated C2C12 root canal cells (F) with vehicle, MD001 (10 ⁇ M) or WY14643 (WY; 10 ⁇ M) for 24 hours, and confirmed the fatty acid oxidation rate, and whether the fatty acid oxidation rate is improved by MD001
  • HepG2 (G), differentiated 3T3-L1 (H), and differentiated C2C12 myotubes (I) were transfected with control or PP
  • WY14643 (WY; 10 ⁇ M) as a positive control for 24 hours
  • FIG. 5 is a result confirming the weight loss caused by MD001 in db / db mice
  • FIG. 6 is a result of confirming the effect of improving the fatty liver by MD001 in db / db mice
  • Figure 6A is ingested vehicle, rosiglitazone (rosi; 20mg / kg) or MD001 (5 or 20mg / kg) for 4 weeks in db / db mice
  • FIG. 6B is liver triglycerol (TG).
  • FIG. 6C is cholesterol
  • FIG. 6D is a result of quantitative analysis of free fatty acid levels.
  • FIG. 6E is performed by qRT-PCR. This is the result of confirming the relative expression level of each gene in db / db mice (*, **, vs. vehicle.The data represent the mean ⁇ SD * P ⁇ 0.01, ** P ⁇ 0.05).
  • FIG. 7 is a result of confirming the effect of reducing the size and macrophage infiltration of fat cells by MD001
  • Figure 7A is a result of performing the H & E staining by collecting the adipose tissue of db / db mice
  • Figure 7B is a high power HPF ( The average number of fat cells per field) is calculated
  • FIG. 7C is a result of confirming relative gene expression by qRT-PCR.
  • FIGS. 7D and 7E are CLS (Crown-like structures) of fat cells in white adipose tissue. (*, **, vs. vehicle; ⁇ , vs. rosi.The data represent the mean ⁇ SD * P ⁇ 0.05, ** P ⁇ 0.01, and ⁇ P ⁇ 0.01).
  • the present invention can provide a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.
  • R 1 to R 4 may be the same or different, respectively, hydrogen; C1 to C4 alkyl; Alkoxy of C1 to C4; It may be any one selected from the group consisting of halogen.
  • the compound represented by Formula 1 or a pharmaceutically useful salt thereof may be 3-cinnamoyl-5,7-dihydroxy-8-methyl-4-phenyl-2H-chromen-2-one.
  • the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof may be a PPAR ⁇ / ⁇ dual agonist.
  • HEK293 cells are transient for 24 hours with human HA-PPAR ⁇ and HA-PPAR ⁇ expression vectors, reporter plasmids (PPRE-pk-Luc) or control reporter plasmids (pk-Luc) and Renilla vectors.
  • reporter plasmids PPRE-pk-Luc
  • pk-Luc control reporter plasmids
  • Renilla vectors As a result of treatment of the compounds with co-transfected HEK293 cells at 20 ⁇ M for 24 hours and luciferase activity, compound 8a (MD001) simultaneously confirmed the transcriptional activity of PPAR ⁇ and PPAR ⁇ as shown in FIG. 1.
  • the compound represented by Formula 1 may be used as a PPAR ⁇ / ⁇ dual agonist.
  • the present invention can provide a pharmaceutical composition for preventing or treating metabolic diseases containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the compound represented by Formula 1 or a pharmaceutically useful salt thereof may be 3-cinnamoyl-5,7-dihydroxy-8-methyl-4-phenyl-2H-chromen-2-one, and Formula 1
  • the compound represented by or a pharmaceutically acceptable salt thereof may be a PPAR ⁇ / ⁇ dual agonist.
  • the metabolic disease may be selected from the group consisting of nonalcoholic fatty liver and hyperlipidemia.
  • Peros Peroxisome proliferator-activated receptors
  • PPAR ⁇ Peroxisome proliferator-activated receptors
  • PPAR ⁇ / ⁇ and PPAR ⁇ one of the nuclear hormone receptor family
  • PPAR ⁇ or PPAR ⁇ / ⁇ Activation of PPAR ⁇ or PPAR ⁇ / ⁇ is known to induce the expression of genes that regulate fatty acid oxidation to promote lipid consumption by promoting lipid consumption, whereas PPAR ⁇ induces the expression of lipid carrier genes including adipocyte fatty acid binding protein and CD36.
  • PPAR ⁇ agonists cannot be used to treat fatty liver or hyperlipidemia because they promote lipid production in adipocytes.
  • rosiglitazone a conventional PPAR ⁇ agonist, exacerbated hepatic steatosis, while MD001 was treated, MD001 dose-dependent liver fat.
  • the compound MD001 is found to improve fatty liver, and thus, the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof may be used as a metabolic disease treatment agent.
  • the pharmaceutical composition containing the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient is an injection, granules, powders, tablets, pills, capsules, Any formulation selected from the group consisting of suppositories, gels, suspensions, emulsions, drops or solutions may be used.
  • the pharmaceutical composition containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient is a suitable carrier, excipient, disintegrant, sweetener commonly used in the preparation of the pharmaceutical composition. It may further comprise one or more additives selected from the group consisting of coatings, expanding agents, lubricants, lubricants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders and lubricants.
  • the carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules, capsules.
  • solid preparations may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like in the composition.
  • excipients such as starch, calcium carbonate, sucrose or lactose, gelatin and the like
  • lubricants such as magnesium styrate and talc may also be used.
  • Oral liquid preparations include suspensions, solvents, emulsions, syrups, and the like, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like.
  • non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used.
  • Witsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like may be used as the base material of the suppository.
  • the pharmaceutical composition is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, nasal, inhaled, topical, rectal, oral, intraocular or intradermal Via the route can be administered to the subject in a conventional manner.
  • the preferred dosage of the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof may vary depending on the condition and weight of the subject, the type and severity of the disease, the form of the drug, the route and duration of administration, and may be appropriately selected by those skilled in the art. Can be. According to one embodiment of the present invention, but not limited thereto, the daily dosage may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, more specifically 0.1 to 100 mg / kg. Administration may be administered once a day or divided into several times, thereby not limiting the scope of the invention.
  • the 'subject' may be a mammal including a human, but is not limited thereto.
  • the present invention may be provided as a health food for preventing or improving metabolic diseases containing a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the compound represented by Formula 1 or a pharmaceutically useful salt thereof may be 3-cinnamoyl-5,7-dihydroxy-8-methyl-4-phenyl-2H-chromen-2-one, and Formula 1
  • the compound represented by or a pharmaceutically acceptable salt thereof may be a PPAR ⁇ / ⁇ dual agonist.
  • the metabolic disease may be selected from the group consisting of nonalcoholic fatty liver and hyperlipidemia.
  • the health food is used with other foods or food additives in addition to the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof, and may be appropriately used according to a conventional method.
  • the mixed amount of the active ingredient may be appropriately determined depending on the purpose of use thereof, for example, prophylactic, health or therapeutic treatment.
  • the effective dose of the compound contained in the health food may be used in accordance with the effective dose of the therapeutic agent, but may be less than the above range in the case of long-term intake for health and hygiene purposes or health control purposes It is evident that the component can be used in an amount above the range because there is no problem in terms of safety.
  • Methyl phloroglucinol (2.0 g, 14.3 mmol), ethyl benzoyl acetate (g, 28.6 mmol, 2.0 eq.), Trifluoroacetic acid (2 mL) and AcOH (40 mL ) was placed in a round bottom flask and heated for 12 hours to reflux the mixture.
  • Methyl phloroglucinol (4.0 g, 24.6 mmol), ethyl benzoyl acetate (9.90 mL, 57.2 mmol, 2.0 eq.), Trifluoroacetic acid (4 mL) and AcOH (100 mL) were placed in a round bottom flask for 12 hours. After refluxing with heat, the reaction mixture was concentrated under reduced pressure.
  • Acetone was removed under reduced pressure, and the organic layer was separated by dissolving the residue in water (50 mL) / EtOAc (50 mL).
  • the aqueous layer was extracted with EtOAc (50 mL ⁇ 2) and the organic layer was washed twice with water (10 mL ⁇ 2), dried over MgSO 4 , concentrated under reduced pressure and adsorbed onto silica gel.
  • the first reaction compound exhibited high insolubility in a general purification system and was obtained in significantly smaller amounts, so instead of purifying the mixture, the crude mixture was concentrated under reduced pressure and volatiles were removed under high vacuum conditions.
  • the washed organic layer was dried over MgSO 4 and concentrated under reduced pressure.
  • the washed organic layer was dried over MgSO 4 and concentrated under reduced pressure.
  • Acetone was removed under reduced pressure, and the residue was dissolved in water (50 mL) / EtOAc (50 mL) and the organic layer was separated.
  • the cells were maintained at 37 ° C. in a humidification chamber containing 5% CO 2 .
  • HepG2 cells were cultured in minimum Eagle's medium (MEM) medium containing 10% FBS and 1% penicillin and streptomycin.
  • MEM minimum Eagle's medium
  • HEK293, C2C12 and 3T3-L1 cells were maintained in DMEM (Dulbecco's modified Eagle's medium) medium containing 10% FBS and 1% penicillin and streptomycin.
  • DMEM Dulbecco's modified Eagle's medium
  • Plasmid DNA constructs were transfected into cells with polyethyleneimine (EI, Polysciences, Inc., PA, USA) according to the manufacturer's instructions.
  • HA-PPAR ⁇ , HA-PPAR ⁇ , PPRE reporter plasmid (PPRE-pk-Luc) or control reporter plasmid (pk-Luc) and Renilla vector were co-coated for 24 hours.
  • cells were treated with Int B, rosiglitazone (Sigma-Aldrich, USA) or WY14643 (Sigma-Aldrich, USA) at various concentrations for 24 hours.
  • Luciferase activity was confirmed by the Dual-Luciferase Reporter Assay System (Promega, WI, USA), and quantitated according to the manufacturer's protocol using GloMax® (Promega, WI, USA).
  • PPAR ⁇ and PPAR ⁇ targeting si-RNA were transfected into HepG2, 3T3-L1 or C2C12 cells using Lipofectamine 2000 (Invitrogen, USA) for 48 hours.
  • GAPDH was used as an internal control, and primers of the sequences shown in Tables 1 and 2 were used.
  • primer sequence species PPAR ⁇ F 5'-CGGTGACTTATCCTGTGGTCC-3 Human R, 5'-CCGCAGATTCTACATTCGATGTT-3 ' PPAR ⁇ F, 5'-TACTGTCGGTTTCAGAAATGCC-3 ' R, 5'-GTCAGCGGACTCTGGATTCAG-3 ' RXR F, 5'-GACGGAGCTTGTGTCCAAGAT-3 ' R, 5’-AGTCAGGGTTAAAGAGGACGAT-3 ’ ACOX F, 5'-ACTCGCAGCCAGCGTTATG-3 ' R, 5'-AGGGTCAGCGATGCCAAAC-3 ' CPT F, 5'-TCCAGTTGGCTTATCGTGGTG-3 ' R, 5'-TCCAGAGTCCGATTGATTTTTGC-3 ' MLYCD F, 5'-ACGTCCGGGAAATGAATGGG-3 ' R, 5'-GTAACCCGTTCTAGGTTCAGGA-3 ' FATP F, 5'-CTTCGA
  • primer sequence species PPAR ⁇ F 5'-TCTTCACGATGCTGTCCTCCT-3 ' mouse R, 5'-CTATGTTTAGAAGGCCAGGC-3 ' GLUT2 F, 5'-TTCCAGTTCGGCTATGACATCG-3 ' R, 5'-CTGGTGTGACTGTAAGTGGGG-3 ' GK F, 5'-TGAACCTGAGGATTTGTCAGC-3 ' R, 5'-CCATGTGGAGTAACGGATTTCG-3 ' CD36 F, 5'-ATGGGCTGTGATCGGAACTG-3 ' R, 5'-GTCTTCCCAATAAGCATGTCTCC-3 ' LPL F, 5'-TTGCCCTAAGGACCCCTGAA-3 ' R, 5'-TTGAAGTGGCAGTTAGACACAG-3 ' GLUT4 F, 5'-ACACTGGTCCTAGCTGTATTCT-3 ' R, 5'-CCAGCCACGTTGCATTGTA-3 ' ACOX F, 5'-TGTTAAGA
  • fatty acid oxidation analysis cells were cultured with 0.1 mM palmitate (9, 10- [ 3 H] palmitate, 5 ⁇ ci / ml; Perkin Elmer Life, Boston, Mass.) And a-MEM (Hyclone) containing 1% bovine serum albumin. , USA) for 24 hours.
  • the medium was collected, precipitated with 10% trichloroacetic acid (Sigma-Aldrich, USA), mixed vigorously, incubated at room temperature for 20 minutes, and centrifuged at 4 ° C. and 16,000 g for 10 minutes.
  • 10% trichloroacetic acid Sigma-Aldrich, USA
  • the supernatant was transferred to a 1.5 ml tube, placed in a scintillation vial containing 0.5 ml water and incubated at 60 ° C. for 12 hours.
  • radioactivity 3 H 2 O in water was quantified using a liquid scintillation counter (LKB, USA).
  • Interruptin B and rosiglitazone were treated with the cells for 3 days, and the cells cultured in conditioned medium were collected and analyzed for glucose content using Glucose Colorimetric Assay Kit II (BioVision Inc., CA, USA).
  • SPR Surface plasmon resonance
  • Int B was treated at 31.25, 62.5, 125, 250, and 500 ⁇ M concentrations and KD values were determined using the Scrubber 2 program (Informer Technologies, Inc., TX, USA).
  • mice were fasted for 12 hours, sterile glucose (1 g / kg, Sigma-Aldrich, USA) was taken and blood glucose levels were checked at the designated times using OneTouch Ultra (LifeScan Inc., USA).
  • Serum and liver TG, FFA, and total cholesterol were measured using Hitachi clinical analyzer 7180 (HITACHI, JAPAN) and WAKO reagent (WAKO, USA).
  • Serum LDL and HDL contents were measured using SEKISUI reagent (SEKISUI, JAPAN).
  • ALT alanine aminotransferase
  • AST aspartic aminotransferase
  • Tissues such as liver, peripheral adipose tissue, skeletal muscle, spleen, kidney and heart were collected from each group of mice, fixed in 10% formalanine and embedded in paraffin.
  • Tissues cut at 5 ⁇ m were stained with hematoxylin and eosin.
  • the MD001 compound chemically synthesized from the above results was assumed to be bound to PPAR ⁇ , PPAR ⁇ / ⁇ , and PPAR ⁇ , and as a result, the compound MD001 binds to PPAR ⁇ and PPAR ⁇ and not to PPAR ⁇ / ⁇ .
  • luciferase activity analysis was performed on HEK293 cells using PPRE.
  • MD001 significantly increased the transcription level through PPAR ⁇ and PPAR ⁇ activity as shown in FIGS. 2A and 2B, whereas the levels of WY14643 and rosiglitazone were lower than MD001.
  • MD001 is an ACOX (acyl-CoA oxidase), CPT (carnitine-palmitoyl transferase), which are PPAR ⁇ target genes related to ⁇ -oxidation in cells that have reduced expression using PPAR ⁇ si-RNA, And increased expression of middlechain acyl-CoA dehydrogenase (mCAD).
  • ACOX acyl-CoA oxidase
  • CPT carnitine-palmitoyl transferase
  • mCAD middlechain acyl-CoA dehydrogenase
  • GPAR glycol kinase
  • CD36 fatty acid binding protein 1
  • FABP1 fatty acid binding protein 1
  • MD001 may be proposed to modulate metabolism through the specific activity of PPAR ⁇ and PPAR ⁇ as dual agents.
  • PPAR ⁇ is recognized as a traditional molecular target for the development of antidiabetic drugs that enhance insulin sensitivity and glucose tolerance.
  • MD001 increased glucose consumption dose-dependently, as shown in Figures 3A, 3B and 3C.
  • quantitative RT-PCR analysis showed that MD001 significantly increased the expression of GLUT2 (HepG2) and GLUT4 (3T3-L1 and C2C12), as shown in FIGS. 3D, 3E and 3F.
  • MD001 like 4A, 4B and 4C, expressed ACOX, CPT, malonyl-CoA decarboxylase (MLYCD) and fatty acid transporter (FATP) in HepG2 and ACOX and CPT expression in 3T3-L1 and C2C12 cells. Increased significantly.
  • MD001 stimulates fatty acid oxidation, as shown in Figure 4D MD001 significantly increased the ⁇ -oxidation rate in HepG2 cells, even in 3T3-L1 and C2C12 root canal cells differentiated as 4E and 4F Similar results were confirmed.
  • MD001 was treated once a day in normal C57BL / 6J mice and diabetic db / db mice.
  • IPGTT intraperitoneal glucose tolerance test
  • FIG. 5B and Supplementary Fig. 3A
  • IPITT insulin tolerance test
  • TZDs including rosiglitazone, pioglitazone and troglitazone, are known to have side effects such as severe weight loss in humans as well as animals, and MD001 has also been a cause of side effects such as weight loss.
  • Rosiglitazone was used as a positive control to confirm the effect of hyperglycemia and lipid profile improvement in db / db mice.
  • MD001 a PPAR ⁇ / ⁇ dual agonist as shown in FIGS. 5D-5E and 5K, significantly reduced blood glucose, triglycerol (TG) and free fatty acids in db / db mice.
  • TG triglycerol
  • MD001 did not affect the levels of plasma lipids and blood glucose compared to the control normal C57BL / 6J mice.
  • rosiglitazone significantly increased alanine transferase (ALT) and aspartate aminotransferase (AST) levels in the blood of db / db mice, whereas MD001 treated db / db mice.
  • ALT and AST levels in blood were greatly reduced, and no change was seen in normal C57BL / 6 mice.
  • MD001 significantly decreased low density lipoprotein (LDL) levels and increased high density lipoprotein levels.
  • LDL low density lipoprotein
  • MD001 can be suggested to restore cholesterol metabolism in obese animals.
  • rosiglitazone induced a significant increase in liver weight (40%) and fat mass (50%) in db / db mice, whereas MD001 did not induce an increase in liver weight or adipose tissue mass.
  • MD001 was found to significantly reduce blood glucose and lipid content without weight loss and hepatotoxicity.
  • Fatty liver is a common complication of obesity, insulin resistance and type 2 diabetes, while PPAR ⁇ activity is known to stimulate fat consumption and reduced lipogenesis to improve fatty liver, whereas activated PPAR ⁇ has an opposite effect on fatty liver.
  • MD001 was identified as a PPAR ⁇ / ⁇ dual agonist, so that MD001 could relieve fatty liver in db / db mice.
  • rosiglitazone as known, exacerbated hepatic steatosis, whereas when MD001 was treated, the size and number of hepatic fat droplets decreased depending on the MD001 dose.
  • MD001 decreased liver TG and FFA, but did not reduce cholesterol, whereas rosiglitazone did not reduce liver TG.
  • MD001 significantly increased the expression of PPAR ⁇ target genes ACOX, CPT and MLYCD and the expression of PPAR ⁇ target genes GLUT2, GK, and CD36 as shown in FIG. 6E.
  • MD001 promotes ⁇ -oxidation to reduce hepatic steatosis or, in part, to reduce blood glucose levels by activating PPAR ⁇ .
  • MD001 was found to not only increase the absorption of fatty acids and glucose, but also partially promote ⁇ -oxidation in adipose tissue as shown in FIG. 7C.
  • Rosiglitazone was found to significantly increase the number of CLS in adipose tissue as shown in Figures 7D and 7E, while MD001 did not stimulate the inflammatory response of adipocytes.
  • H & E staining of skeletal muscle showed no difference between vehicle control, rosiglitazone and MD001 treatment groups in db / db mice.
  • MD001 significantly increased the expression of CPT, GLUT4, and lipoprotein lipase (LPL) in qRT-PCR analysis using skeletal muscle.
  • MD001 may be suggested to increase glucose and fatty acid metabolism in skeletal muscle.
  • MD001 may contribute to the improvement of hyperglycemia and hyperglycemia as a dual agonist of PPAR ⁇ / ⁇ .

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Abstract

The present invention relates to a composition for treating metabolic diseases containing a novel compound or a pharmaceutically acceptable salt thereof as an active ingredient. In particular, unlike the PPARγ agonist rosiglitazone, which has been conventionally used as a therapeutic agent for diabetes and exhibits side effects such as hepatotoxicity, fatty liver deterioration and weight loss, the compound of the present invention has been confirmed to exert excellent effects in the treatment of nonalcoholic fatty liver and hyperlipidemia. Accordingly, a composition containing as an active ingredient the novel compound can be used as a therapeutic agent, replacing conventional therapeutic agents, for non-alcoholic fatty liver and hyperlipidemia.

Description

PPARα/γ 이중 작용 활성을 갖는 신규 화합물 및 이를 유효성분으로 함유하는 대사질환 예방 또는 치료용 조성물Novel compounds having PPARα / γ dual action activity and compositions for preventing or treating metabolic diseases containing the same as active ingredients
본 발명은 PPARα/γ 이중 작용 활성을 갖는 신규한 화합물 또는 이의 약제학적으로 허용가능한 염을 유효성분으로 함유하는 대사질환 치료용 조성물에 관한 것이다.The present invention relates to a composition for treating metabolic diseases containing a novel compound having a PPARα / γ dual action activity or a pharmaceutically acceptable salt thereof as an active ingredient.
비만은 다양한 질환의 원인이 되는 질환으로, 당뇨병 환자의 80%, 심장질환환자의 21%가 비만이 원인이 되고 있으며, 비알콜성 지방간 질환 역시 비만이 주요원인으로 보고되어 있다.Obesity is a disease that causes a variety of diseases, 80% of diabetics, 21% of heart disease patients are caused by obesity, nonalcoholic fatty liver disease is also reported as the main cause of obesity.
비알콜성 지방간질환(non-alcoholic fatty liver disease; NAFLD)은 음주와 관계없이 간 내에 중성지방이 축적되는 질환을 의미하고, 단순지방간(steatosis)과 비알콜성 지방간염(non-alcoholic steatohepatitis, NASH)이 포함된다. 단순 지방간은 임상적으로 예후가 양호한 양성질환으로 생각되고 있으나, 지방간과 함께 염증 또는 섬유화를 동반하는 NASH는 진행성 간질환으로 간경변 또는 간암을 유발하는 전구 질환으로 보고되어 있다.Non-alcoholic fatty liver disease (NAFLD) refers to a disease in which triglycerides accumulate in the liver regardless of drinking, and are known as steatosis and non-alcoholic steatohepatitis (NASH). ) Is included. Simple fatty liver is considered to be a benign disease with a good prognosis, but NASH with inflammation or fibrosis along with fatty liver has been reported as a proliferative disease that causes cirrhosis or liver cancer as a progressive liver disease.
이러한 비만과 인슐린저항성은 대표적인 비알콜성 지방간질환의 위험인자이다. 비알콜성 지방간환자의 69 내지 100%는 비만환자이고, 비만환자의 20 내지 40%는 비알콜성 지방간을 동반하며, 특히 남성 비만환자의 간질환 유병율이 여성 비만자에 비해 더 높게 나타나고 있으며, 서구사회에서는 비만환자뿐만 아니라 정상체중 성인의 3 내지 30%가 비알콜성 지방간 병변을 나타내고 있는 것으로 보고되고 있다. Obesity and insulin resistance are risk factors for representative non-alcoholic fatty liver disease. 69-100% of non-alcoholic fatty liver patients are obese, 20-40% of obese patients have non-alcoholic fatty liver, and the prevalence of liver disease among male obese patients is higher than that of female obese. In society, 3-30% of normal weight adults as well as obese patients are reported to have non-alcoholic fatty liver lesions.
비알콜성 지방간질환(NAFDL)을 치료하기 위해 가장 좋은 방법은 운동과 같은 생활방식의 변화를 통한 체중 감소이다. 그러나 운동만으로 치유가 어려운 경우 약물을 이용한 치료가 병행되어 선택될 수 있으며, 현재까지 비알콜성 지방간 환자에게 사용되고 있는 치료제는 크게 두 가지 개념으로 분류된다. The best way to treat nonalcoholic fatty liver disease (NAFDL) is weight loss through lifestyle changes such as exercise. However, if exercise alone is difficult to cure, treatment with drugs may be selected in parallel, and the therapeutic agents used in non-alcoholic fatty liver patients so far are classified into two concepts.
첫 번째는 비만치료제(제니칼), 인슐린저항성치료제(메트포르민, 피오글리타존, 로시글리타존), 고지혈증치료제(클로피브레이트, 젬피브로질, 베자피브레이트, 아토바스타틴, 심바스타틴)와 같이 위험인자의 교정을 통해 지방간을 치료 및 개선하는 약제가 여기에 속한다. 즉, 메트포르민은 지방산의 산화를 증가시키고 지방형성 효소를 감소시키며, 인슐린혈증과 인슐린저항성을 개선하는 효과가 있으며, 치아졸리디네디온, 로시글리타존 및 피오글리타존은 핵 호르몬 수용체인 PPARγ를 활성화시켜 근육에서의 당 흡수를 촉진시킨다. The first is to improve the fatty liver by correcting risk factors such as obesity treatment (Zenical), insulin resistance treatment (Metformin, pioglitazone, rosiglitazone), hyperlipidemia treatment (clofibrate, gemfibrozil, bezafibrate, atorvastatin, simvastatin) Drugs that treat and improve it belong here. In other words, metformin increases the oxidation of fatty acids, decreases fat-forming enzymes, improves insulinosis and insulin resistance. Chiazolidinedione, rosiglitazone, and pioglitazone activate PPARγ, a nuclear hormone receptor. Promotes absorption.
두 번째 유형의 치료제는 비알콜성 지방간의 위험인자 교정과는 독립적으로 간세포 손상을 회복시키는 기능을 담당하는 약물로서 간세포보호제(우르소디옥시콜산 및 타우린), 항산화제(비타민 E 및 C) 및 영양 보조제(레시틴, 베타인, N-아세틸시스테인) 등이 여기에 속하나, 현재까지 부작용이 없으면서도 치료에 효과가 좋은 이상적인 약제는 없는 실정이다.The second type of treatment is a drug that repairs hepatocellular damage independent of risk factor correction for non-alcoholic fatty liver. Hepatoprotective agents (ursodioxycholic acid and taurine), antioxidants (vitamins E and C) and nutrition Supplements (lecithin, betaine, N-acetylcysteine), etc. belong to this, but to date, there is no ideal drug that is effective in treatment without side effects.
본 발명은 종래 당뇨병 치료제로 사용되어 왔던 PPAR 작용제가 비알콜성 지방간을 악화시키는 문제점이 확인됨에 따라, 상기 PPAR 작용제의 부작용을 해결하여 인체에 안전하고 우수한 비알콜성 지방간 치료 효과를 나타낼 수 있는 신규한 화합물 및 이를 유효성분으로 함유하는 대사질환 치료용 조성물을 제공하고자 한다.The present invention has been confirmed that the problem that the PPAR agonist that has been used as a conventional diabetes treatment for worsening non-alcoholic fatty liver, solve the side effects of the PPAR agonist to exhibit a safe and excellent non-alcoholic fatty liver treatment effect to the human body One compound and a composition for treating metabolic diseases containing the same as an active ingredient are provided.
본 발명은 하기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용가능한 염을 제공한다.The present invention provides a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.
[화학식 1][Formula 1]
Figure PCTKR2018006649-appb-I000001
Figure PCTKR2018006649-appb-I000001
상기 화학식 1에 있어서, In Chemical Formula 1,
상기 R1 내지 R4는 각각 동일하거나 다를 수 있으며, 수소; C1 내지 C4의 알킬; C1 내지 C4의 알콕시로 이루어진 군에서 선택된 어느 하나임.R 1 to R 4 may be the same or different, respectively, hydrogen; C1 to C4 alkyl; Any one selected from the group consisting of C1 to C4 alkoxy.
본 발명은 상기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용가능한 염을 유효성분으로 함유하는 대사질환 예방 또는 치료용 약학조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating metabolic diseases, which comprises the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
또한, 본 발명은 상기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용가능한 염을 유효성분으로 함유하는 대사질환 예방 또는 개선용 건강식품을 제공한다.In another aspect, the present invention provides a health food for preventing or improving metabolic diseases containing the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
본 발명에 따르면 종래 당뇨병 치료제로 사용되었던 PPARγ 작용제 로시글리타존이 간독성, 지방간 악화 및 몸무게 감소와 같은 부작용을 나타내는 것과 다르게 본 발명의 화합물은 상기 PPARγ 작용제의 부작용을 해결하고 비알콜성 지방간 및 고지혈증 치료에 우수한 효과를 나타내는 것이 확인됨에 따라, 상기 신규 화합물을 유효성분으로 함유하는 조성물은 종래의 치료제를 대체할 수 있는 비알콜성 지방간 및 고지혈증 치료제로 사용될 수 있다.According to the present invention, unlike the PPARγ agonist rosiglitazone, which has been used as a conventional diabetes treatment, exhibits side effects such as hepatotoxicity, fatty liver deterioration and weight loss, the compound of the present invention solves the side effects of the PPARγ agonist and is excellent in treating non-alcoholic fatty liver and hyperlipidemia. As it is confirmed to exhibit the effect, the composition containing the novel compound as an active ingredient can be used as a non-alcoholic fatty liver and hyperlipidemia treatment that can replace the conventional treatment.
도 1은 HEK293 세포에 사람 HA-PPARα(A) 및 HA-PPARγ(B) 발현 벡터, 리포터 플라스미드(PPRE-pk-Luc) 또는 대조군 리포터 플라스미드(pk-Luc)와 레닐라 벡터로 24시간 동안 일시적으로 공동 형질주입한 후 합성된 화합물을 각각 20 μM로 24시간 동안 처리하고 PPARα(A) 및 PPARγ(B) 루시페라제 활성을 확인한 결과이다.1 is transient for 24 hours with human HA-PPARα (A) and HA-PPARγ (B) expression vectors, reporter plasmid (PPRE-pk-Luc) or control reporter plasmid (pk-Luc) and Renilla vector in HEK293 cells. After co-transfection, the synthesized compounds were treated with 20 μM for 24 hours and PPARα (A) and PPARγ (B) luciferase activity was confirmed.
도 2는 MD001에 활성화된 PPARα 및 PPARγ에 의해 발현이 유도된 타겟 유전자를 확인한 결과로, 도 2A 및 도 2B는 HEK293 세포에 사람 HA-PPARα(A) 및 HA-PPARγ(B) 발현 벡터, 리포터 플라스미드(PPRE-pk-Luc) 또는 대조군 리포터 플라스미드(pk-Luc)와 레닐라 벡터로 24시간 동안 일시적으로 공동 형질주입한 후 MD001 (10μM), 로시글리타존(rosi; 10μM) 또는 WY14643(WY; 10μM)를 24시간 동안 세포에 처리하고 루시퍼레이즈 활성을 확인한 결과이며, 도 2C는 HepG2 세포에 MD001 (10μM)를 24시간 처리하고 전체 RNAs를 분리하여 cDNA를 합성한 후 qRT-PCR를 수행하여 상대적 발현량을 확인한 결과이며(*, vs. vehicle), 도 2D는 HepG2 세포를 대조군 si-RNA, PPARα si-RNA (D) 또는 PPARγ si-RNA (E)로 48시간 동안 형질주입하고 WY14643(10μM), 로시글리타존(10μM) 또는 MD001(10μM)를 24시간 동안 처리한 후 세포를 수집하여 전체 RNA를 분리하고 qRT-PCR를 이용하여 타겟 유전자의 상대적 발현량을 확인한 결과이다(#, vs. control si-RNA; ‡, vs. control si-RNA/vehicle; §, vs. control si-RNA/WY; **, vs. control si-RNA/MD001. Data represent the mean ± SD of three independent experiments. *P < 0.01, **P < 0.05, #P < 0.01, ‡P < 0.01, 및 §P < 0.01).Figure 2 is a result of identifying the target genes induced expression by PPARα and PPARγ activated in MD001, Figure 2A and 2B is a human HA-PPARα (A) and HA-PPARγ (B) expression vector, reporter in HEK293 cells Temporary co-transfection with plasmid (PPRE-pk-Luc) or control reporter plasmid (pk-Luc) and Renilla vector for 24 hours, followed by MD001 (10 μM), rosiglitazone (rosi (10 μM) or WY14643 (WY; 10 μM) Was treated with cells for 24 hours, and luciferase activity was confirmed. FIG. 2C shows the relative expression level of qp-PCR after synthesis of cDNA by treating MD001 (10 μM) with HepG2 cells for 24 hours, and separating total RNAs. (*, Vs. vehicle), Figure 2D is HepG2 cells transfected with control si-RNA, PPARα si-RNA (D) or PPARγ si-RNA (E) for 48 hours and WY14643 (10μM), Treatment with rosiglitazone (10 μM) or MD001 (10 μM) for 24 hours followed by cell collection RNA was isolated and qRT-PCR confirmed the relative expression level of the target gene (#, vs. control si-RNA; ‡, vs. control si-RNA / vehicle; §, vs. control si-RNA / WY; **, vs. control si-RNA / MD001.Data represent the mean ± SD of three independent experiments. * P <0.01, ** P <0.05, #P <0.01, ‡ P <0.01, and §P < 0.01).
도 3은 MD001가 GLUT 발현을 유도하여 글루코스 소비를 촉진시키는 것을 확인한 결과로, HepG2(A), 분화된 3T3-L1 지방세포 (B) 및 분화된 C2C12 근관세포 (C)에 vehicle, MD001 (10, 50μM) 또는 로시글리타존 (rosi; 10μM)을 24시간 동안 처리한 후 글루코스 소비를 확인한 결과이며, 추가적으로 세포를 수집하여 전체 RNA를 분리하여 qRT-PCR를 수행하여 GLUT2 (D) 및 GLUT4 (E&F)의 상대적 발현량을 확인한 결과이다 (*, **, and † vs. vehicle. Data represent the mean ± SD of three independent experiments. *P < 0.01, **P < 0.05, 및 †P < 0.01).Figure 3 shows that MD001 induces GLUT expression to promote glucose consumption. HepG2 (A), differentiated 3T3-L1 adipocytes (B) and differentiated C2C12 root canal cells (C) vehicle, MD001 (10 , 50μM) or rosiglitazone (rosi; 10μM) treatment after 24 hours to confirm the glucose consumption, additional cells were collected to isolate the total RNA to perform qRT-PCR of GLUT2 (D) and GLUT4 (E & F) This is the result of confirming the relative expression level (*, **, and † vs. vehicle.Data represent the mean ± SD of three independent experiments. * P <0.01, ** P <0.05, and † P <0.01).
도 4는 in vitro 에서 MD001에 의한 지방산 산화 촉진 효과를 확인한 결과로, 도 4A, 4B 및 4C는 HepG2(A), 분화된 3T3-L1 지방세포(B) 및 분화된 C2C12 근관세포(C)에 vehicle 또는 MD001(10μM)를 24시간 처리하고, 전체 RNA를 분리하여 cDNA를 합성하고 qRT-PCR를 수행하여 상대적 유전자 발현량을 확인한 결과이며, 도 4D는 HepG2(D), 분화된 3T3-L1 지방세포(E) 및 분화된 C2C12 근관세포(F)에 vehicle, MD001 (10μM) 또는 WY14643 (WY; 10μM)을 24시간 동안 처리하고 지방산 산화율을 확인한 결과이며, 지방산 산화율이 MD001에 의해 향상되는 여부를 확인하기 위해, HepG2 (G), 분화된 3T3-L1 (H), 및 분화된 C2C12 근관세포 (I)에 대조군 또는 PPARα si-RNA (20 nM)를 48시간 형질주입한 후 vehicle, MD001 (10μM) 또는 양성 대조군으로 WY14643 (WY; 10μM)를 24시간 동안 처리한 결과이다(*, **, vs. vehicle; †, ‡, vs. vehicle/control siRNA; §, vs. WY/control si-RNA; ¶, vs. MD001/control si-RNA. Data represent the mean ± SD of three independent experiments. *P < 0.01, **P < 0.05, †P < 0.01, ‡P < 0.05, §P < 0.01, and ¶P < 0.05).4 is a result of confirming the fatty acid oxidation promoting effect by MD001 in vitro , Figures 4A, 4B and 4C are HepG2 (A), differentiated 3T3-L1 adipocytes (B) and differentiated C2C12 myotubes (C) Treatment of vehicle or MD001 (10 μM) for 24 hours, and the total RNA was isolated to synthesize cDNA and qRT-PCR to determine the relative gene expression, Figure 4D is HepG2 (D), differentiated 3T3-L1 fat Treatment of cells (E) and differentiated C2C12 root canal cells (F) with vehicle, MD001 (10 μM) or WY14643 (WY; 10 μM) for 24 hours, and confirmed the fatty acid oxidation rate, and whether the fatty acid oxidation rate is improved by MD001 To confirm, HepG2 (G), differentiated 3T3-L1 (H), and differentiated C2C12 myotubes (I) were transfected with control or PPARα si-RNA (20 nM) for 48 hours before vehicle, MD001 (10 μM). ) Or WY14643 (WY; 10 μM) as a positive control for 24 hours (*, **, vs. vehicle; †, ‡, vs. vehicle / control siRNA §, vs. WY / control si-RNA; ¶, vs. MD001 / control si-RNA.Data represent the mean ± SD of three independent experiments. * P <0.01, ** P <0.05, † P <0.01, ‡ P <0.05, § P <0.01, and ¶ P <0.05).
도 5는 db/db 생쥐에서 MD001에 의한 몸무게 감소 여부를 확인한 결과로, 도 5A는 db/db 생쥐에 하루에 한 번씩 vehicle, 로시글리타존(rosi)(20mg/kg) 또는 MD001 (n = 5-6 per group)을 섭취시키고 혈액 글루코스 수준을 확인한 결과이며, 도 5(B)는 글루코스 경구부하시험(Oral glucose tolerance test; OGTT)을 수행한 결과로, 약물 섭취 후 5주째에 무균 글루코스(1g/kg)를 처리한 결과이며(n = 5-6 그룹당), 도 5C는 약물을 섭취시킨 30일 동안 몸무게 변화를 확인한 결과이며(n = 5-6 그룹당), 도 5D 내지 5K는 db/db 생쥐에서 하루에 한 번씩 vehicle, rosiglitazone (rosi) 또는 MD001 (n = 5-6 per group)를 섭취시킨 후 다양한 혈액 대사물질의변화를 확인한 결과이다(*, **, #, vs. vehicle; †, vs. vehicle, MD001(5mg/kg), and MD001(20mg/kg). The data represent the mean ± S.D. *P < 0.05, **P < 0.01, #P < 0.001, and †P < 0.01).5 is a result confirming the weight loss caused by MD001 in db / db mice, Figure 5A is once a day in the db / db mice vehicle, rosiglitazone (rosi) (20mg / kg) or MD001 (n = 5-6 per group) and the blood glucose level was confirmed, Figure 5 (B) is a result of performing the Oral glucose tolerance test (OGTT), sterile glucose (1 g / kg 5 weeks after drug intake) ), (N = 5-6 group), Figure 5C is the result of the weight change for 30 days of drug intake (n = 5-6 group), Figures 5D to 5K in db / db mice Intake of vehicle, rosiglitazone (rosi) or MD001 (n = 5-6 per group) once a day confirmed the changes in various blood metabolites (*, **, #, vs. vehicle; †, vs vehicle, MD001 (5 mg / kg), and MD001 (20 mg / kg) .The data represent the mean ± SD * P <0.05, ** P <0.01, # P <0.001, and † P <0.01).
도 6은 db/db 생쥐에서 MD001에 의한 지방간 개선 효과를 확인한 결과로, 도6A는 db/db 생쥐에 vehicle, 로시글리타존(rosi; 20mg/kg) 또는 MD001(5 또는 20mg/kg)를 4주간 섭취시킨 후 간을 수집하여 H&E 염색을 수행한 결과이며, 도 6B는 간 트리글리세롤(TG) 도 6C는 콜레스테롤, 및 도 6D는 유리지방산 수준을 정량분석한 결과이며, 도 6E는 qRT-PCR를 수행하여 db/db 생쥐에서 각 유전자의 상대적 발현량을 확인한 결과이다(*, **, vs. vehicle. The data represent the mean ± S.D. *P < 0.01, **P < 0.05).6 is a result of confirming the effect of improving the fatty liver by MD001 in db / db mice, Figure 6A is ingested vehicle, rosiglitazone (rosi; 20mg / kg) or MD001 (5 or 20mg / kg) for 4 weeks in db / db mice After the liver was collected and H & E staining was performed. FIG. 6B is liver triglycerol (TG). FIG. 6C is cholesterol, and FIG. 6D is a result of quantitative analysis of free fatty acid levels. FIG. 6E is performed by qRT-PCR. This is the result of confirming the relative expression level of each gene in db / db mice (*, **, vs. vehicle.The data represent the mean ± SD * P <0.01, ** P <0.05).
도 7은 MD001에 의해 지방세포의 크기 및 대식세포 침투 감소효과를 확인한 결과로, 도 7A는 db/db 생쥐의 지방 조직을 수집하여 H&E 염색을 수행한 결과이며, 도 도 7B는 HPF(high power field)당 지방세포의 평균수를 계산한 결과이며, 도 7C는 qRT-PCR를 수행하여 상대적 유전자 발현량을 확인한 결과이며, 도 7D 및 7E는 백색 지방 조직내 지방 세포의 CLS(Crown-like structures)를 확인한 결과이다(*, **, vs. vehicle; †, vs. rosi. The data represent the mean ± S.D. *P < 0.05, **P < 0.01, and †P < 0.01).7 is a result of confirming the effect of reducing the size and macrophage infiltration of fat cells by MD001, Figure 7A is a result of performing the H & E staining by collecting the adipose tissue of db / db mice, Figure 7B is a high power HPF ( The average number of fat cells per field) is calculated, and FIG. 7C is a result of confirming relative gene expression by qRT-PCR. FIGS. 7D and 7E are CLS (Crown-like structures) of fat cells in white adipose tissue. (*, **, vs. vehicle; †, vs. rosi.The data represent the mean ± SD * P <0.05, ** P <0.01, and † P <0.01).
본 발명은 하기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용가능한 염을 제공할 수 있다.The present invention can provide a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.
[화학식 1][Formula 1]
Figure PCTKR2018006649-appb-I000002
Figure PCTKR2018006649-appb-I000002
상기 화학식 1에 있어서, In Chemical Formula 1,
상기 R1 내지 R4는 각각 동일하거나 다를 수 있으며, 수소; C1 내지 C4의 알킬; C1 내지 C4의 알콕시; 할로겐으로 이루어진 군에서 선택된 어느 하나일 수 있다.R 1 to R 4 may be the same or different, respectively, hydrogen; C1 to C4 alkyl; Alkoxy of C1 to C4; It may be any one selected from the group consisting of halogen.
보다 상세하게는 상기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 유용한 염은 3-신나모일-5,7-디하이드록시-8-메틸-4-페닐-2H-크로멘-2-온일 수 있으며, 상기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용가능한 염은 PPARα/γ 이중 작용자(Dual agonist)일 수 있다.More specifically, the compound represented by Formula 1 or a pharmaceutically useful salt thereof may be 3-cinnamoyl-5,7-dihydroxy-8-methyl-4-phenyl-2H-chromen-2-one. The compound represented by Formula 1 or a pharmaceutically acceptable salt thereof may be a PPARα / γ dual agonist.
본 발명의 일실시예에 따르면, HEK293 세포에 사람 HA-PPARα 및 HA-PPARγ 발현 벡터, 리포터 플라스미드(PPRE-pk-Luc) 또는 대조군 리포터 플라스미드(pk-Luc)와 레닐라 벡터로 24시간 동안 일시적으로 공동 형질주입한 HEK293 세포에 상기 화합물을 각각 20 μM로 24시간 동안 처리하고 루시페라제 활성을 확인한 결과, 도 1과 같이 화합물 8a(MD001)가 PPARα 및 PPARγ의 전사 활성을 동시에 증가시키는 것이 확인됨에 따라, 화학식 1로 표시되는 화합물은 PPARα/γ 이중 작용자(Dual agonist)로 사용될 수 있다.According to one embodiment of the present invention, HEK293 cells are transient for 24 hours with human HA-PPARα and HA-PPARγ expression vectors, reporter plasmids (PPRE-pk-Luc) or control reporter plasmids (pk-Luc) and Renilla vectors. As a result of treatment of the compounds with co-transfected HEK293 cells at 20 μM for 24 hours and luciferase activity, compound 8a (MD001) simultaneously confirmed the transcriptional activity of PPARα and PPARγ as shown in FIG. 1. As such, the compound represented by Formula 1 may be used as a PPARα / γ dual agonist.
이에 따라, 본 발명은 상기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용가능한 염을 유효성분으로 함유하는 대사질환 예방 또는 치료용 약학조성물을 제공할 수 있다.Accordingly, the present invention can provide a pharmaceutical composition for preventing or treating metabolic diseases containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
상기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 유용한 염은 3-신나모일-5,7-디하이드록시-8-메틸-4-페닐-2H-크로멘-2-온일 수 있으며, 상기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용가능한 염은 PPARα/γ 이중 작용자(Dual agonist)일 수 있다.The compound represented by Formula 1 or a pharmaceutically useful salt thereof may be 3-cinnamoyl-5,7-dihydroxy-8-methyl-4-phenyl-2H-chromen-2-one, and Formula 1 The compound represented by or a pharmaceutically acceptable salt thereof may be a PPARα / γ dual agonist.
상기 대사질환은 비알콜성 지방간 및 고지혈증으로 이루어진 군에서 선택될 수 있다.The metabolic disease may be selected from the group consisting of nonalcoholic fatty liver and hyperlipidemia.
보다 상세하게 PPARs(Peroxisome proliferator-activated receptors)는 PPARα, PPARβ/δ 및 PPARγ로 구성되며, 핵 호르몬 수용체 패밀리 중 하나로, 지질 대사를 조절하는 것으로 알려져있다.More specifically, Peros (Peroxisome proliferator-activated receptors) composed of PPARα, PPARβ / δ and PPARγ, one of the nuclear hormone receptor family, is known to regulate lipid metabolism.
PPARα 또는 PPARβ/δ의 활성화는 지방산 산화를 조절하는 유전자 발현을 유도하여 지질 소비를 촉진시켜 고지혈증을 개선시키는 것으로 알려진 반면, PPARγ는 지방세포 지방산 결합 단백질 및 CD36을 포함한 지질 운반 유전자의 발현을 유도를 통하여 지방세포에서 지질 생성을 촉진시키므로, PPARγ 작용제는 지방간 또는 고지혈증 치료에 사용될 수 없다.Activation of PPARα or PPARβ / δ is known to induce the expression of genes that regulate fatty acid oxidation to promote lipid consumption by promoting lipid consumption, whereas PPARγ induces the expression of lipid carrier genes including adipocyte fatty acid binding protein and CD36. PPARγ agonists cannot be used to treat fatty liver or hyperlipidemia because they promote lipid production in adipocytes.
본 발명의 다른 일실시예에 따르면, PPARα/γ 이중 작용자로 확인된 화합물 MD001의 지방간을 개선 효과를 확인하기 위해, db/db 생쥐에 vehicle, 로시글리타존(rosi; 20mg/kg) 또는 MD001(5 또는 20mg/kg)를 4주간 섭취시킨 후 간을 수집하여 H&E 염색을 수행한 결과, 도 6A와 같이 종래 PPARγ 작용제인 로시글리타존은 간 지방증을 악화시킨 반면, MD001가 처리된 경우, MD001 용량의존적으로 간 지방 방울의 크기와 수의 감소가 나타남에 따라, 화합물 MD001은 지방간을 개선하는 것이 확인됨에 따라, 상기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용가능한 염은 대사질환 치료제로 사용될 수 있다.According to another embodiment of the present invention, PPARα / γ dual action to verify the fatty liver improving effect of the identified compound MD001 character, vehicle, rosiglitazone (rosi; 20mg / kg) in db / db mice or MD001 (5 or 20 mg / kg) for 4 weeks, and liver was collected and H & E staining was performed. As shown in FIG. 6A, rosiglitazone, a conventional PPARγ agonist, exacerbated hepatic steatosis, while MD001 was treated, MD001 dose-dependent liver fat. As the size and number of drops are reduced, the compound MD001 is found to improve fatty liver, and thus, the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof may be used as a metabolic disease treatment agent.
본 발명의 한 구체예에서, 상기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용가능한 염을 유효성분으로 함유하는 약학조성물은 통상적인 방법에 따라 주사제, 과립제, 산제, 정제, 환제, 캡슐제, 좌제, 겔, 현탁제, 유제, 점적제 또는 액제로 이루어진 군에서 선택된 어느 하나의 제형을 사용할 수 있다.In one embodiment of the present invention, the pharmaceutical composition containing the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient is an injection, granules, powders, tablets, pills, capsules, Any formulation selected from the group consisting of suppositories, gels, suspensions, emulsions, drops or solutions may be used.
본 발명의 다른 구체예에서, 상기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용가능한 염을 유효성분으로 함유하는 약학조성물은 약학조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제, 붕해제, 감미제, 피복제, 팽창제, 윤활제, 활택제, 향미제, 항산화제, 완충액, 정균제, 희석제, 분산제, 계면활성제, 결합제 및 윤활제로 이루어진 군에서 선택되는 하나 이상의 첨가제를 추가로 포함할 수 있다.In another embodiment of the present invention, the pharmaceutical composition containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient is a suitable carrier, excipient, disintegrant, sweetener commonly used in the preparation of the pharmaceutical composition. It may further comprise one or more additives selected from the group consisting of coatings, expanding agents, lubricants, lubricants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders and lubricants.
구체적으로 담체, 부형제 및 희석제는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 사용할 수 있으며, 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 조성물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트, 수크로스 또는 락토오스, 젤라틴 등을 섞어 조제할 수 있다. 또한 단순한 부형제 이외에 마그네슘 스티레이트, 탈크 같은 윤활제들도 사용할 수 있다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 있으며 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제 등이 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기재로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.Specifically, the carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules, capsules. And the like, and such solid preparations may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like in the composition. In addition to simple excipients, lubricants such as magnesium styrate and talc may also be used. Oral liquid preparations include suspensions, solvents, emulsions, syrups, and the like, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. Witsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like may be used as the base material of the suppository.
본 발명의 일실시예에 따르면 상기 약학 조성물은 정맥내, 동맥내, 복강내, 근육내, 동맥내, 복강내, 흉골내, 경피, 비측내, 흡입, 국소, 직장, 경구, 안구내 또는 피내 경로를 통해 통상적인 방식으로 대상체로 투여할 수 있다.According to one embodiment of the invention the pharmaceutical composition is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, nasal, inhaled, topical, rectal, oral, intraocular or intradermal Via the route can be administered to the subject in a conventional manner.
상기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용가능한 염의 바람직한 투여량은 대상체의 상태 및 체중, 질환의 종류 및 정도, 약물 형태, 투여경로 및 기간에 따라 달라질 수 있으며 당업자에 의해 적절하게 선택될 수 있다. 본 발명의 일실시예에 따르면 이에 제한되는 것은 아니지만 1일 투여량이 0.01 내지 200 mg/kg, 구체적으로는 0.1 내지 200 mg/kg, 보다 구체적으로는 0.1 내지 100 mg/kg 일 수 있다. 투여는 하루에 한 번 투여할 수도 있고 수회로 나누어 투여할 수도 있으며, 이에 의해 본 발명의 범위가 제한되는 것은 아니다.The preferred dosage of the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof may vary depending on the condition and weight of the subject, the type and severity of the disease, the form of the drug, the route and duration of administration, and may be appropriately selected by those skilled in the art. Can be. According to one embodiment of the present invention, but not limited thereto, the daily dosage may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, more specifically 0.1 to 100 mg / kg. Administration may be administered once a day or divided into several times, thereby not limiting the scope of the invention.
본 발명에 있어서, 상기 '대상체'는 인간을 포함하는 포유동물일 수 있으나, 이들 예에 한정되는 것은 아니다.In the present invention, the 'subject' may be a mammal including a human, but is not limited thereto.
또한, 본 발명은 상기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용가능한 염을 유효성분으로 함유하는 대사질환 예방 또는 개선용 건강식품으로 제공될 수 있다.In addition, the present invention may be provided as a health food for preventing or improving metabolic diseases containing a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
상기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 유용한 염은 3-신나모일-5,7-디하이드록시-8-메틸-4-페닐-2H-크로멘-2-온일 수 있으며, 상기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용가능한 염은 PPARα/γ 이중 작용자(Dual agonist)일 수 있다.The compound represented by Formula 1 or a pharmaceutically useful salt thereof may be 3-cinnamoyl-5,7-dihydroxy-8-methyl-4-phenyl-2H-chromen-2-one, and Formula 1 The compound represented by or a pharmaceutically acceptable salt thereof may be a PPARα / γ dual agonist.
상기 대사질환은 비알콜성 지방간 및 고지혈증으로 이루어진 군에서 선택될 수 있다.The metabolic disease may be selected from the group consisting of nonalcoholic fatty liver and hyperlipidemia.
상기 건강식품은 상기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용가능한 염 이외에 다른 식품 또는 식품 첨가물과 함께 사용되고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합양은 그의 사용 목적 예를들어 예방, 건강 또는 치료적 처치에 따라 적합하게 결정될 수 있다.The health food is used with other foods or food additives in addition to the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use thereof, for example, prophylactic, health or therapeutic treatment.
상기 건강식품에 함유된 화합물의 유효용량은 상기 치료제의 유효용량에 준해서 사용할 수 있으나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며, 유효성분은 안전성 면에서 아무런 문제가 없기 때문에 상기 범위 이상의 양으로도 사용될 수 있음은 확실하다.The effective dose of the compound contained in the health food may be used in accordance with the effective dose of the therapeutic agent, but may be less than the above range in the case of long-term intake for health and hygiene purposes or health control purposes It is evident that the component can be used in an amount above the range because there is no problem in terms of safety.
상기 건강식품의 종류에는 특별한 제한이 없고, 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제등을 들 수 있다.There is no particular limitation on the kind of the health food, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinks, tea, Drinks, alcoholic drinks, vitamin complexes, etc. are mentioned.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.
<< 합성예Synthesis Example > MD001 화합물 합성MD001 Compound Synthesis
하기 반응식 1과 같은 과정으로 화합물을 합성하였다.Compounds were synthesized in the same manner as in Scheme 1 below.
[반응식 1]Scheme 1
Figure PCTKR2018006649-appb-I000003
Figure PCTKR2018006649-appb-I000003
1. 화합물 5a 및 5b 합성1.Synthesis of Compounds 5a and 5b
메틸 플로로글루시놀(Methyl phloroglucinol; 2.0 g, 14.3 mmol), 에틸 벤조일 아세테이트(ethyl benzoyl acetate; g, 28.6mmol, 2.0 eq.), 트리플루오로아세트산(trifluoroacetic acid; 2 mL) 및 AcOH (40 mL)를 둥근 바닥 플라스크에 넣고 12시간 동안 열을 가하여 혼합물을 환류시켰다. Methyl phloroglucinol (2.0 g, 14.3 mmol), ethyl benzoyl acetate (g, 28.6 mmol, 2.0 eq.), Trifluoroacetic acid (2 mL) and AcOH (40 mL ) Was placed in a round bottom flask and heated for 12 hours to reflux the mixture.
감압하에서 반응 혼합물을 증발시킨 후 물 20 mL를 첨가하고 EtOAc (50 mL)로 수분층을 세 번 추출하였다. 유기 결합층을 물(10 mL) 두 번 세척하고, 무수 황산마그네슘(MgSO4)으로 건조시키고 실라카 겔에 흡착시켰다. After evaporation of the reaction mixture under reduced pressure, 20 mL of water was added and the aqueous layer was extracted three times with EtOAc (50 mL). The organic combined layer was washed twice with water (10 mL), dried over anhydrous magnesium sulfate (MgSO 4) and adsorbed onto silica gel.
혼합물을 실리카 겔 컬럼 크로마토그래피(EtOAc : hexane = 1 : 2)로 정제하여 두 개의 구조 이성질체 5a 및 5b 화합물을 얻었다.The mixture was purified by silica gel column chromatography (EtOAc: hexane = 1: 2) to give two structural isomers 5a and 5b compounds.
Figure PCTKR2018006649-appb-I000004
Figure PCTKR2018006649-appb-I000004
5,7- 디하이드록시 -8- 메틸 -4-페닐-2H- 크로멘 -2-온 (5,7- Dihydroxy -8-methyl-4-phenyl-2H-chromen-2-one; 5a) 수율: 19%; 노란색 고체. m.p. 210-212 ℃; 1H-NMR (500MHz, DMSO-d6): 10.32 (s, 1H), 9.81 (s, 1H), 7.37-7.29 (m, 5H), 6.29 (s, 1H), 5.72 (s, 1H), 2.08 (s, 3H); 13C-NMR (75MHz, DMSO-d6): 160.0, 159.6, 156.3, 154.3, 154.1, 139.9, 127.6, 127.3, 127.2, 109.6, 102.1, 100.6, 98.6, 7.7; HRMS (ESI) m/z calcd for C16H13O4 (M+H)+ 269.0814, found 269.0798 5, 7-dihydroxy-8-methyl-4-phenyl -2H- chromen-2-one (5,7- Dihydroxy -8-methyl-4 -phenyl-2H-chromen-2-one; 5a) yields : 19%; Yellow solid. mp 210-212 ° C; 1 H-NMR (500 MHz, DMSO-d 6 ): 10.32 (s, 1H), 9.81 (s, 1H), 7.37-7.29 (m, 5H), 6.29 (s, 1H), 5.72 (s, 1H), 2.08 (s, 3 H); 13 C-NMR (75 MHz, DMSO-d 6 ): 160.0, 159.6, 156.3, 154.3, 154.1, 139.9, 127.6, 127.3, 127.2, 109.6, 102.1, 100.6, 98.6, 7.7; HRMS (ESI) m / z calcd for C 16 H 13 O 4 (M + H) + 269.0814, found 269.0798
Figure PCTKR2018006649-appb-I000005
Figure PCTKR2018006649-appb-I000005
5,7- 디하이드록시 -6- 메틸 -4-페닐-2H- 크로멘 -2-온 (5,7- Dihydroxy -6-methyl-4-phenyl-2H-chromen-2-one; 5b) 수율: 14%; 노란색 고체; m.p. 274-276 ℃; 1H-NMR (300MHz, DMSO-d6): 10.46 (s, 1H), 8.87 (s, 1H), 7.37-7.31 (m, 5H), 6.44 (s, 1H), 5.74 (s, 1H), 1.98 (s, 3H); 13C-NMR (75MHz, DMSO-d6): 159.9, 159.8, 156.3, 154.0, 153.9, 140.3, 127.6, 127.4, 127.3, 110.7, 107.7, 101.6, 94.7, 8.6; HRMS (ESI) m/z calcd for C16H13O4 (M+H)+ 269.0814, found 269.0815 5, 7-dihydroxy-6-methyl-4-phenyl -2H- chromen-2-one (5,7- Dihydroxy -6-methyl-4 -phenyl-2H-chromen-2-one; 5b) Yield : 14%; Yellow solid; mp 274-276 ° C; 1 H-NMR (300 MHz, DMSO-d 6 ): 10.46 (s, 1H), 8.87 (s, 1H), 7.37-7.31 (m, 5H), 6.44 (s, 1H), 5.74 (s, 1H), 1.98 (s, 3 H); 13 C-NMR (75 MHz, DMSO-d 6 ): 159.9, 159.8, 156.3, 154.0, 153.9, 140.3, 127.6, 127.4, 127.3, 110.7, 107.7, 101.6, 94.7, 8.6; HRMS (ESI) m / z calcd for C 16 H 13 O 4 (M + H) + 269.0814, found 269.0815
2. 화합물 6a 및 6b 합성2. Synthesis of Compounds 6a and 6b
메틸 플로로글루시놀(4.0 g, 24.6 mmol), 에틸 벤조일 아세테이트(9.90 mL, 57.2mmol, 2.0 eq.), 트리플루오로아세트산(4 mL) 및 AcOH (100 mL)를 둥근바닥 플라스크에 넣고 12시간 동안 열을 가하여 환류시킨 후 반응혼합물을 감압하에서 농축시켰다. Methyl phloroglucinol (4.0 g, 24.6 mmol), ethyl benzoyl acetate (9.90 mL, 57.2 mmol, 2.0 eq.), Trifluoroacetic acid (4 mL) and AcOH (100 mL) were placed in a round bottom flask for 12 hours. After refluxing with heat, the reaction mixture was concentrated under reduced pressure.
정제되지 않은 반응혼합물을 아세톤(150 mL)에 용해시키고, K2CO3 (11.7 g, 86.1 mmol, 3.5 eq.) 및 디메틸 설페이드(dimethyl sulfate; 5.84 mL, 61.5 mmol, 2.5 eq.)을 0℃에서 소량 첨가한 후 실온에서 교반하였다.The crude reaction mixture was dissolved in acetone (150 mL) and K 2 CO 3 (11.7 g, 86.1 mmol, 3.5 eq.) And dimethyl sulfate (5.84 mL, 61.5 mmol, 2.5 eq.) Were 0 A small amount was added at ° C and then stirred at room temperature.
시작 물질이 관찰되지 않을 때까지 시약을 추가적으로 첨가하였다.Additional reagent was added until no starting material was observed.
감압하에서 아세톤을 제거하고, 물(50 mL)/EtOAc (50 mL)에 잔사를 용해시켜 유기층을 분리시켰다.Acetone was removed under reduced pressure, and the organic layer was separated by dissolving the residue in water (50 mL) / EtOAc (50 mL).
수분층은 EtOAc (50 mL × 2)로 추출하고 유기결합층을 물(10 mL × 2)로 두번 세척한 후 MgSO4로 건조시키고 감압하에서 농축한 후 실리카 겔에 흡착시켰다.The aqueous layer was extracted with EtOAc (50 mL × 2) and the organic layer was washed twice with water (10 mL × 2), dried over MgSO 4 , concentrated under reduced pressure and adsorbed onto silica gel.
반응 혼합물을 실리카겔 컬럼 크로마토그래피(EtOAc : hexane = 1 : 4, 5% DCM)로 정제하여 두 개의 구조 이성질체 6a 및 6b를 얻었다.The reaction mixture was purified by silica gel column chromatography (EtOAc: hexane = 1: 4, 5% DCM) to give two structural isomers 6a and 6b.
Figure PCTKR2018006649-appb-I000006
Figure PCTKR2018006649-appb-I000006
5,7- 디메톡시 -8- 메틸 -4-페닐-2H- 크로멘 -2-온 (5,7- Dimethoxy -8-methyl-4-phenyl-2H-chromen-2-one; 6a) Yield: 31%; Yellow solid m.p. 162-163℃; 1H-NMR (800MHz, CDCl3): 7.35-7.33 (m, 3H), 7.22-7.21 (m, 2H), 6.23 (s, 1H), 5.94 (s, 1H), 3.88 (s, 3H), 3.41 (s, 3H) 2.21 (s, 3H); 13C-NMR (200MHz, CDCl3): 161.0, 160.9, 156.2, 155.7, 153.9, 140.1, 127.7, 127.3, 126.9, 112.2, 106.6, 103.2, 91.4, 55.8, 55.5, 7.7; HRMS (ESI) m/z calcd for C18H17O4 (M+H)+ 297.1127, found 297.1113 5,7-dimethoxy-8-methyl-4-phenyl -2H- chromen-2-one (5,7- Dimethoxy -8-methyl-4 -phenyl-2H-chromen-2-one; 6a) Yield: 31%; Yellow solid mp 162-163 ° C .; 1 H-NMR (800 MHz, CDCl 3 ): 7.35-7.33 (m, 3H), 7.22-7.21 (m, 2H), 6.23 (s, 1H), 5.94 (s, 1H), 3.88 (s, 3H), 3.41 (s, 3 H) 2.21 (s, 3 H); 13 C-NMR (200 MHz, CDCl 3 ): 161.0, 160.9, 156.2, 155.7, 153.9, 140.1, 127.7, 127.3, 126.9, 112.2, 106.6, 103.2, 91.4, 55.8, 55.5, 7.7; HRMS (ESI) m / z calcd for C 18 H 17 O 4 (M + H) + 297.1127, found 297.1113
Figure PCTKR2018006649-appb-I000007
Figure PCTKR2018006649-appb-I000007
5,7- 디메톡시 -6- 메틸 -4-페닐-2H- 크로멘 -2-온 (5,7- Dimethoxy -6-methyl-4-phenyl-2H-chromen-2-one; 6b) Yield: 20%; Yellow solid m.p. 164-165℃; 1H-NMR (400MHz, CDCl3): 7.39-7.35 (m, 5H), 6.68 (s, 1H), 6.04 (s, 1H), 3.87 (s, 3H), 2.93 (s, 3H) 2.05 (s, 3H); 13C-NMR (150MHz, CDCl3): 161.6, 160.8, 156.2, 155.2, 154.7, 138.7, 128.3, 127.8, 127.4, 117.4, 113.9, 106.6, 95.5, 60.9, 55.9, 8.7; HRMS (ESI) m/z calcd for C18H17O4 (M+H)+ 297.1127, found 297.1125 5,7-dimethoxy-6-methyl-4-phenyl -2H- chromen-2-one (5,7- Dimethoxy -6-methyl-4 -phenyl-2H-chromen-2-one; 6b) Yield: 20%; Yellow solid mp 164-165 ° C .; 1 H-NMR (400 MHz, CDCl 3 ): 7.39-7.35 (m, 5H), 6.68 (s, 1H), 6.04 (s, 1H), 3.87 (s, 3H), 2.93 (s, 3H) 2.05 (s , 3H); 13 C-NMR (150 MHz, CDCl 3 ): 161.6, 160.8, 156.2, 155.2, 154.7, 138.7, 128.3, 127.8, 127.4, 117.4, 113.9, 106.6, 95.5, 60.9, 55.9, 8.7; HRMS (ESI) m / z calcd for C 18 H 17 O 4 (M + H) + 297.1127, found 297.1125
첫 번째 반응 화합물은 일반적인 정제 시스템에서 높은 불용성을 나타내어 상당히 적은량으로 수득됨에 따라, 혼합물을 정제하지 않는 대신 정제되지 않은 혼합물을 감압하에서 농축시키고 높은 진공 조건하에서 휘발성분을 제거하였다.The first reaction compound exhibited high insolubility in a general purification system and was obtained in significantly smaller amounts, so instead of purifying the mixture, the crude mixture was concentrated under reduced pressure and volatiles were removed under high vacuum conditions.
그 후 하기와 같은 과정으로 메틸화시켰다. Thereafter, methylation was carried out in the following manner.
3. 화합물 7a 및 7b 합성3. Synthesis of Compounds 7a and 7b
앞서 합성된 화합물 6b (2.50 g, 8.44 mmol)과 신나모일 클로라이드(cinnamoyl chloride; 3.50 g, 21.1 mmol, 2.5 eq.)를 디클로로메탄(DCM; 120 mL)에 용해시키고, 0℃에서 교반하면서 TiCl4 (5.09 mL, 46.4 mmol, 5.5 eq.)를 천천히 첨가한 후 48시간 동안 반응 혼합물에 열을 가하여 환류시켰다.The previously synthesized compound 6b (2.50 g, 8.44 mmol) and cinnamoyl chloride (cinnamoyl chloride; 3.50 g, 21.1 mmol, 2.5 eq.) Was dissolved in dichloromethane (DCM; 120 mL) and stirred at 0 ° C. with TiCl 4 (5.09 mL, 46.4 mmol, 5.5 eq.) Was added slowly, and the reaction mixture was heated to reflux for 48 hours.
메탄올(MeOH)에 냉각시킨 TiCl4를 0℃에서 매우 조심스럽게 첨가한 후 얼음같이 차가운 물을 첨가하였다.TiCl 4 cooled in methanol (MeOH) was added very carefully at 0 ° C. followed by ice cold water.
유기층을 분리하고 EtOAc (50 mL × 3)로 수분층을 추출한 후 유기결합층을 물(10 mL × 2)로 세척하였다.The organic layer was separated, the aqueous layer was extracted with EtOAc (50 mL × 3), and the organic binding layer was washed with water (10 mL × 2).
MgSO4로 유기층을 건조시키고 감압하에서 농축하였다. 반응 혼합물을 실리카겔 컬럼크로마토 그래피(EtOAc: hexane = 1: 3, 5% DCM)로 정제하여 화합물 7b (2.44 g, 5.74 mmol)를 얻었다.The organic layer was dried over MgSO 4 and concentrated under reduced pressure. The reaction mixture was purified by silica gel column chromatography (EtOAc: hexane = 1: 3, 5% DCM) to give compound 7b (2.44 g, 5.74 mmol).
Figure PCTKR2018006649-appb-I000008
Figure PCTKR2018006649-appb-I000008
3- 신나모일 -5,7- 디메톡시 -6- 메틸 -4-페닐-2H- 크로멘 -2-온 (3- Cinnamoyl -5,7-dimethoxy-6-methyl-4-phenyl-2H-chromen-2-one; 7b) 38%; Yellow solid m.p. 197-198 ℃; 1H-NMR (300MHz, CDCl3): 7.37-7.21 (m, 11H), 6.72 (s, 1H), 6.53 (d, J = 16.3 Hz, 1H), 3.91 (s, 3H), 2.95 (s, 3H), 2.06 (s, 3H); 13C-NMR (100MHz, CDCl3): 192.0, 162.4, 158.7, 157.0, 154.2, 151.9, 145.5, 135.8, 134.4, 130.7, 128.8, 128.4, 128.1, 127.4, 126.8, 124.2, 118.1, 106.7, 95.5, 61.1, 56.1, 8.9; HRMS (ESI) m/z calcd for C27H23O5 (M+H)+ 427.1545, found 427.1525 3-cinnamoyl-5,7-dimethoxy-6-methyl-4-phenyl -2H- chromen-2-one (3- Cinnamoyl -5,7-dimethoxy-6 -methyl-4-phenyl-2H-chromen -2-one; 7b) 38%; Yellow solid mp 197-198 ° C; 1 H-NMR (300 MHz, CDCl 3 ): 7.37-7.21 (m, 11 H), 6.72 (s, 1 H), 6.53 (d, J = 16.3 Hz, 1 H), 3.91 (s, 3H), 2.95 (s, 3H), 2.06 (s, 3H); 13 C-NMR (100 MHz, CDCl 3 ): 192.0, 162.4, 158.7, 157.0, 154.2, 151.9, 145.5, 135.8, 134.4, 130.7, 128.8, 128.4, 128.1, 127.4, 126.8, 124.2, 118.1, 106.7, 95.5, 61.1 , 56.1, 8.9; HRMS (ESI) m / z calcd for C 27 H 23 O 5 (M + H) + 427.1545, found 427.1525
앞서 합성된 화합물 6a (2.50 g, 8.44 mmol)과 신나모일 클로라이드(cinnamoyl chloride; 3.50 g, 21.1 mmol, 2.5 eq.)를 DCM (120 mL)에 용해시키고, 0℃에서 교반하면서 TiCl4 (5.09 mL, 46.4 mmol, 5.5 eq.)를 천천히 첨가한 후 48시간 동안 반응 혼합물에 열을 가하여 환류시켰다.The previously synthesized compound 6a (2.50 g, 8.44 mmol) and cinnamoyl chloride (3.50 g, 21.1 mmol, 2.5 eq.) Was dissolved in DCM (120 mL) and stirred at 0 ° C. with TiCl 4 (5.09 mL , 46.4 mmol, 5.5 eq.) Was slowly added, and the reaction mixture was heated to reflux for 48 hours.
0℃에서 냉각시킨 TiCl4를 메탄올에 매우 조심스럽게 첨가한 후 얼음물을 첨가하였다. 유기층을 분리시키고 EtOAc (50 mL × 3)로 수분층을 추출한 후 유기결합층을 물(10 mL × 2)로 세척하고 MgSO4로 유기층을 건조한 후 감압조건하에서 농축시켰다.TiCl 4 cooled at 0 ° C. was added very carefully to methanol followed by ice water. The organic layer was separated and the aqueous layer was extracted with EtOAc (50 mL × 3). The organic layer was washed with water (10 mL × 2), dried over MgSO 4 and concentrated under reduced pressure.
반응 혼합물을 실리카 겔 컬럼크로마토그래피(EtOAc: hexane = 1: 3, 5% DCM)로 정제하여 화합물 7a(2.44 g, 5.74 mmol)를 얻었다.The reaction mixture was purified by silica gel column chromatography (EtOAc: hexane = 1: 3, 5% DCM) to give compound 7a (2.44 g, 5.74 mmol).
Figure PCTKR2018006649-appb-I000009
Figure PCTKR2018006649-appb-I000009
3- 신나모일 -5,7- 디메톡시 -8- 메틸 -4-페닐-2H- 크로멘 -2-온 (3- cinnamoyl -5,7-dimethoxy-8-methyl-4-phenyl-2H-chromen-2-one; 7a) Yield: 68%; Yellow solid m.p. 224-226 ℃; 1H-NMR (300MHz, CDCl3): 7.36-7.08 (m, 11H), 6.72 (s, 1H), 6.53 (d, J = 16.3 Hz, 1H), 6.18 (s, 1H), 3.86 (s, 3H), 3.30 (s, 3H), 2.21 (s, 3H); 13C-NMR (100MHz, CDCl3): 192.1, 161.7, 159.0, 157.4, 153.4, 152.8, 145.2, 137.1, 134.4, 130.6, 128.8, 128.4, 127.8, 127.4, 127.3, 127.1, 122.5, 106.7, 103.1, 91.8, 55.9, 55.8, 7.8; HRMS (ESI) m/z calcd for C27H23O5 (M+H)+ 427.1545, found 427.1533. 3-cinnamoyl-5,7-dimethoxy-8-methyl-4-phenyl -2H- chromen-2-one (3- cinnamoyl -5,7-dimethoxy-8 -methyl-4-phenyl-2H-chromen 7-) Yield: 68%; Yellow solid mp 224-226 ° C; 1 H-NMR (300 MHz, CDCl 3 ): 7.36-7.08 (m, 11 H), 6.72 (s, 1 H), 6.53 (d, J = 16.3 Hz, 1 H), 6.18 (s, 1 H), 3.86 (s, 3H), 3.30 (s, 3H), 2.21 (s, 3H); 13 C-NMR (100 MHz, CDCl 3 ): 192.1, 161.7, 159.0, 157.4, 153.4, 152.8, 145.2, 137.1, 134.4, 130.6, 128.8, 128.4, 127.8, 127.4, 127.3, 127.1, 122.5, 106.7, 103.1, 91.8 , 55.9, 55.8, 7.8; HRMS (ESI) m / z calcd for C 27 H 23 O 5 (M + H) + 427.1545, found 427.1533.
4. 화합물 8a 및 8b 합성4. Synthesis of Compounds 8a and 8b
디클로로에틸렌(DCE; 30 mL)에 앞서 합성된 화합물 7b (0.29 g, 0.68 mmol)를 용해시킨 용액을 0℃에서 교반하면서 BBr3 (1.29 mL, 13.6 mmol, 20 eq.)을 천천히 첨가한 후 24시간 동안 반응 혼합물에 열을 가하여 환류시켰다.A solution of the compound 7b (0.29 g, 0.68 mmol) dissolved before dichloroethylene (DCE; 30 mL) was slowly added BBr 3 (1.29 mL, 13.6 mmol, 20 eq.) With stirring at 0 ° C., followed by 24 The reaction mixture was heated to reflux for a period of time.
메탄올과 차가운 얼음물의 반응이 완료된 후 냉각된 BBr3을 0℃에서 조심스럽게 첨가하였다. After completion of the reaction with methanol and cold ice water, cooled BBr 3 was carefully added at 0 ° C.
감압하에서 DCE를 제거하고 EtOAc를 첨가하여 유기층을 분리시키고 EtOAc (50 mL × 2)로 수분층을 추출한 후 유기결합층을 물(10 mL × 2)로 세척하였다. DCE was removed under reduced pressure, EtOAc was added to separate the organic layer, the aqueous layer was extracted with EtOAc (50 mL × 2), and the organic layer was washed with water (10 mL × 2).
세척된 유기층을 MgSO4로 건조시키고 감압하에서 농축시켰다.The washed organic layer was dried over MgSO 4 and concentrated under reduced pressure.
반응 혼합물을 실리카 겔 컬럼크로마토그래피(EtOAc: hexane = 1: 2)로 정제하여 화합물 8b(0.11 g, 0.276 mmol)를 얻었다.The reaction mixture was purified by silica gel column chromatography (EtOAc: hexane = 1: 2) to give compound 8b (0.11 g, 0.276 mmol).
Figure PCTKR2018006649-appb-I000010
Figure PCTKR2018006649-appb-I000010
3- 신나모일 -5,7- 디하이드록시 -6- 메틸 -4-페닐-2H- 크로멘 -2-올 (3- Cinnamoyl -5,7-dihydroxy-6-methyl-4-phenyl-2H-chromen-2-one; 8b); 노란색 고체, m.p. 110-114 ℃; 1H-NMR (800MHz, CDCl3): 8.00 (s, 1H), 7.46-7.44 (m, 3H), 7.41-7.38 (m, 5H), 7.31-7.30 (m, 4H), 6.89 (s, 1H), 6.62 (d, J = 16.1 Hz, 1H), 5.35 (s, 1H), 1.94 (s, 3H); 13CNMR (200MHz, CDCl3): 191.7, 160.5, 159.8, 153.8, 153.2, 152.0, 146.2, 134.0, 133.5, 130.9, 130.5, 129.8, 128.8, 128.6, 127.9, 126.7, 121.8, 109.6, 100.8, 96.5, 7.9; HRMS (ESI) m/z calcd for C25H19O5 (M+H)+ 399.1232, found 399.1213 3-cinnamoyl-5,7-dihydroxy-6-methyl-4-phenyl -2H- chromen-2-ol (3- Cinnamoyl -5,7-dihydroxy-6 -methyl-4-phenyl-2H- chromen-2-one; 8b); Yellow solid, mp 110-114 ° C .; 1 H-NMR (800 MHz, CDCl 3 ): 8.00 (s, 1H), 7.46-7.44 (m, 3H), 7.41-7.38 (m, 5H), 7.31-7.30 (m, 4H), 6.89 (s, 1H ), 6.62 (d, J = 16.1 Hz, 1H), 5.35 (s, 1H), 1.94 (s, 3H); 13 CNMR (200 MHz, CDCl 3 ): 191.7, 160.5, 159.8, 153.8, 153.2, 152.0, 146.2, 134.0, 133.5, 130.9, 130.5, 129.8, 128.8, 128.6, 127.9, 126.7, 121.8, 109.6, 100.8, 96.5, 7.9 ; HRMS (ESI) m / z calcd for C 25 H 19 O 5 (M + H) + 399.1232, found 399.1213
DCE (30 mL)에 앞서 합성된 화합물 7a (0.29 g, 0.68 mmol)를 용해시킨 용액을 0℃에서 교반하면서 BBr3 (1.29 mL, 13.6 mmol, 20 eq.)을 천천히 첨가한 후 24시간 동안 반응 혼합물에 열을 가하여 환류시켰다.A solution of Compound 7a (0.29 g, 0.68 mmol), synthesized before DCE (30 mL), was added slowly with stirring at 0 ° C. while BBr 3 (1.29 mL, 13.6 mmol, 20 eq.) Was reacted for 24 hours. The mixture was heated to reflux.
메탄올과 차가운 얼음물의 반응이 완료된 후 냉각된 BBr3을 0℃에서 조심스럽게 첨가하였다. After completion of the reaction with methanol and cold ice water, cooled BBr 3 was carefully added at 0 ° C.
감압하에서 DCE를 제거하고 EtOAc를 첨가하여 유기층을 분리시켰다. EtOAc (50 mL × 2)로 수분층을 추출한 후 유기결합층을 물(10 mL × 2)로 세척하였다. DCE was removed under reduced pressure and EtOAc was added to separate the organic layer. The aqueous layer was extracted with EtOAc (50 mL × 2) and the organic combined layer was washed with water (10 mL × 2).
세척된 유기층을 MgSO4로 건조시키고 감압하에서 농축시켰다.The washed organic layer was dried over MgSO 4 and concentrated under reduced pressure.
반응 혼합물을 실리카 겔 컬럼 크로마토그래피(EtOAc : hexane = 1 : 2)로 정제하여 화합물 8a(0.11 g, 0.276 mmol)를 얻었다.The reaction mixture was purified by silica gel column chromatography (EtOAc: hexane = 1: 2) to give compound 8a (0.11 g, 0.276 mmol).
Figure PCTKR2018006649-appb-I000011
Figure PCTKR2018006649-appb-I000011
3- 신나모일 -5,7- 디하이드록시 -8- 메틸 -4-페닐-2H- 크로멘 -2-온 (3- Cinnamoyl -5,7-dihydroxy-8-methyl-4-phenyl-2H-chromen-2-one; 8a; MD001) Yield: 41%; 노란색 고체 m.p. 274-278℃; 1H-NMR (600MHz, acetone-d6): 9.31 (s, 1H), 8.58 (s, 1H), 7.55-7.54 (m, 2H), 7.53 (d, J = 16.3 Hz, 1H), 7.34-7.31 (m, 3H), 7.24-7.20 (m, 2H), 7.20-7.14 (m, 3H), 6.72 (s, 1H), 6.59 (d, J = 16.3 Hz, 1H), 6.30 (s, 3H), 2.16 (s, 3H); 1H-NMR (400MHz, DMSO-d6 10.46 (s, 1H), 9.82 (s, 1H), 7.63 (d, J = 6.0 Hz, 2H), 7.49 (d, J = 16.3 Hz, 2H), 7.39-7.37 (m, 3H), 7.22-7.16 (m, 5H), 6.63 (d, J = 16.3 Hz, 1H), 6.27 (s, 3H), 2.11 (s, 3H); 13C-NMR (150MHz, acetone-d6): 193.5, 161.2, 159.9, 156.6, 155.4, 153.8, 146.6, 138.6, 136.1, 131.8, 131.1, 129.7, 129.2, 128.9, 128.8, 128.5, 122.9, 104.6, 102.8, 100.4, 8.3; HRMS (ESI) m/z calcd for C25H19O5 (M+H)+ 399.1232, found 399.1223 3-cinnamoyl-5,7-dihydroxy-8-methyl-4-phenyl -2H- chromen-2-one (3- Cinnamoyl -5,7-dihydroxy-8 -methyl-4-phenyl-2H- chromen-2-one; 8a; MD001) Yield: 41%; Yellow solid mp 274-278 ° C .; 1 H-NMR (600 MHz, acetone-d 6 ): 9.31 (s, 1H), 8.58 (s, 1H), 7.55-7.54 (m, 2H), 7.53 (d, J = 16.3 Hz, 1H), 7.34- 7.31 (m, 3H), 7.24-7.20 (m, 2H), 7.20-7.14 (m, 3H), 6.72 (s, 1H), 6.59 (d, J = 16.3 Hz, 1H), 6.30 (s, 3H) , 2.16 (s, 3 H); 1 H-NMR (400 MHz, DMSO-d 6 10.46 (s, 1H), 9.82 (s, 1H), 7.63 (d, J = 6.0 Hz, 2H), 7.49 (d, J = 16.3 Hz, 2H), 7.39 -7.37 (m, 3H), 7.22-7.16 (m, 5H), 6.63 (d, J = 16.3 Hz, 1H), 6.27 (s, 3H), 2.11 (s, 3H); 13 C-NMR (150 MHz, acetone-d 6 ): 193.5, 161.2, 159.9, 156.6, 155.4, 153.8, 146.6, 138.6, 136.1, 131.8, 131.1, 129.7, 129.2, 128.9, 128.8, 128.5, 122.9, 104.6, 102.8, 100.4, 8.3; HRMS ( ESI) m / z calcd for C 25 H 19 O 5 (M + H) + 399.1232, found 399.1223
아세톤(20 mL)에 화합물 8a (53 mg, 0.133 mmol)를 용해시킨 용액을 0℃에서 교반시키면서 K2CO3 (64 mg, 0.466 mmol, 3.5 eq.), 디메틸 설페이트(31 μL, 0.333 mmol. 2.5 eq.)를 소량 첨가하고 실온에서 시작 물질이 발견되지 않을 때까지 교반하였다. A solution of Compound 8a (53 mg, 0.133 mmol) in acetone (20 mL) was stirred at 0 ° C. with stirring at K ° C 3 (64 mg, 0.466 mmol, 3.5 eq.), Dimethyl sulfate (31 μL, 0.333 mmol. 2.5 eq. ) Was added in small portions and stirred at room temperature until no starting material was found.
감압하에서 아세톤을 제거하고 잔사를 물(50 mL)/EtOAc (50 mL)에 용해시키고 유기층을 분리하였다.Acetone was removed under reduced pressure, and the residue was dissolved in water (50 mL) / EtOAc (50 mL) and the organic layer was separated.
수분층을 EtOAc (50 mL)로 추출하고 유기결합층을 물(10 mL)로 두 번 세척하고 MgSO4로 건조시킨 후 감압하에서 농축하였다.The aqueous layer was extracted with EtOAc (50 mL) and the organic combined layer was washed twice with water (10 mL), dried over MgSO 4 and concentrated under reduced pressure.
반응 혼합물을 실리카 겔 컬럼 크로마토그래피(EtOAc:hexane = 1:3, 5% DCM)로 정제하여 화합물 7a(35 mg, 0.082 mmol)를 62% 수율로 얻었다.The reaction mixture was purified by silica gel column chromatography (EtOAc: hexane = 1: 3, 5% DCM) to give compound 7a (35 mg, 0.082 mmol) in 62% yield.
합성된 화합물의 NMR 스펙트럼을 화합물 7a와 맞춰보았다.NMR spectra of the synthesized compounds were matched with compound 7a.
<< 실험예Experimental Example >> 화합물 MD001 활성 확인Confirmation of Compound MD001 Activity
1. 세포 배양1. Cell Culture
세포를 5% CO2가 포함된 가습 챔버에서 37℃ 조건으로 세포를 유지시켰다.The cells were maintained at 37 ° C. in a humidification chamber containing 5% CO 2 .
HepG2 세포를 10% FBS 및 1% 페니실린 및 스트렙토마이신이 포함된 MEM( minimum Eagle’s medium) 배지에서 배양하였다.HepG2 cells were cultured in minimum Eagle's medium (MEM) medium containing 10% FBS and 1% penicillin and streptomycin.
HEK293, C2C12 및 3T3-L1 세포는 10% FBS 및 1% 페니실린 및 스트렙토마이신이 포함된 DMEM(Dulbecco’s modified Eagle’s medium) 배지에서 유지시켰다.HEK293, C2C12 and 3T3-L1 cells were maintained in DMEM (Dulbecco's modified Eagle's medium) medium containing 10% FBS and 1% penicillin and streptomycin.
C2C12 세포 및 지방세포 분화는 이미 보고되어진 방법(Lee KW, Cho JG, Kim CM, Kang AY, Kim M, Ahn BY, et al., 2013; Kim M, Ahn BY, Lee JS, Chung SS, Lim S, Park SG, et al., 2009)으로 유도되었다.C2C12 cell and adipocyte differentiation methods have already been reported (Lee KW, Cho JG, Kim CM, Kang AY, Kim M, Ahn BY, et al., 2013; Kim M, Ahn BY, Lee JS, Chung SS, Lim S , Park SG, et al., 2009).
2. 일시적인 형질주입 및 루시페라제 활성 분석2. Transient Transfection and Luciferase Activity Assay
일시적인 형질주입을 위해, 플라스미드 DNA 구성물을 폴리에틸렌이민(EI, Polysciences, Inc., PA, USA)으로 제조사의 설명서에 따라, 세포에 형질주입하였다. For transient transfection, plasmid DNA constructs were transfected into cells with polyethyleneimine (EI, Polysciences, Inc., PA, USA) according to the manufacturer's instructions.
HEK293 세포에 PPARα 및 PPARγ의 전사 활성 분석을 수행하기 위해, HA-PPARα, HA-PPARγ, PPRE 리포터 플라스미드 (PPRE-pk-Luc) 또는 대조 리포터 플라스미드 (pk-Luc)와 Renilla 벡터를 24 시간 동안 공동 형질 감염시킨 후, 세포에 Int B, 로시글리타존(Sigma-Aldrich, USA) 또는 WY14643 (Sigma-Aldrich, USA)를 다양한 농도로 24시간 동안 처리하였다.To perform transcriptional activity analysis of PPARα and PPARγ on HEK293 cells, the HA-PPARα, HA-PPARγ, PPRE reporter plasmid (PPRE-pk-Luc) or control reporter plasmid (pk-Luc) and Renilla vector were co-coated for 24 hours. After transfection, cells were treated with Int B, rosiglitazone (Sigma-Aldrich, USA) or WY14643 (Sigma-Aldrich, USA) at various concentrations for 24 hours.
루시페라제 활성은 Dual-Luciferase Reporter Assay System (Promega, WI, USA)으로 확인되었으며, GloMax® (Promega, WI, USA)를 이용하여 제조사의 프로토콜에 따라 정량분석하였다.Luciferase activity was confirmed by the Dual-Luciferase Reporter Assay System (Promega, WI, USA), and quantitated according to the manufacturer's protocol using GloMax® (Promega, WI, USA).
발현 억제 실험을 위해, 20 nM의 PPARα 및 PPARγ 타겟팅 si-RNA(Invitrogen, USA)를 리포펙타민 2000 (Invitrogen, USA)을 이용하여 HepG2, 3T3-L1 or C2C12 세포에 48시간 동안 형질주입시켰다.For expression inhibition experiments, 20 nM of PPARα and PPARγ targeting si-RNA (Invitrogen, USA) were transfected into HepG2, 3T3-L1 or C2C12 cells using Lipofectamine 2000 (Invitrogen, USA) for 48 hours.
그 후, 세포에 vehicle, 로시글리타존, WY14643 또는 Int B를 24시간 동안 처리하였다.Cells were then treated with vehicle, rosiglitazone, WY14643 or Int B for 24 hours.
3. 정량적인 RT-PCR 분석3. Quantitative RT-PCR Analysis
RNeasy kit (Qiagen, USA)를 이용하여 제조사의 설명서에 따라, 전체 RNA를 분리하였다. cDNA는 전체 RNA 0.5 μg으로 역전사하여 합성하였으며, 정량적 RT-PCR은 Power SYBR Green PCR Master Mix® (ABI, USA) 및 stepOne 48 well real time PCR system (ABI, USA)으로 수행되었다.Total RNA was isolated using the RNeasy kit (Qiagen, USA) according to the manufacturer's instructions. cDNA was synthesized by reverse transcription with 0.5 μg total RNA, and quantitative RT-PCR was performed with Power SYBR Green PCR Master Mix® (ABI, USA) and stepOne 48 well real time PCR system (ABI, USA).
GAPDH를 내부 대조군으로 사용하였으며, 하기 표 1 및 표 2와 같은 서열의 프라이머를 사용하였다. GAPDH was used as an internal control, and primers of the sequences shown in Tables 1 and 2 were used.
primerprimer sequencesequence speciesspecies
PPARαPPARα F, 5‘-CGGTGACTTATCCTGTGGTCC-3F, 5'-CGGTGACTTATCCTGTGGTCC-3 HumanHuman
R, 5‘-CCGCAGATTCTACATTCGATGTT-3’R, 5'-CCGCAGATTCTACATTCGATGTT-3 '
PPARγPPARγ F, 5‘-TACTGTCGGTTTCAGAAATGCC-3’F, 5'-TACTGTCGGTTTCAGAAATGCC-3 '
R, 5‘-GTCAGCGGACTCTGGATTCAG-3’R, 5'-GTCAGCGGACTCTGGATTCAG-3 '
RXRRXR F, 5‘-GACGGAGCTTGTGTCCAAGAT-3’F, 5'-GACGGAGCTTGTGTCCAAGAT-3 '
R, 5‘-AGTCAGGGTTAAAGAGGACGAT-3’R, 5’-AGTCAGGGTTAAAGAGGACGAT-3 ’
ACOXACOX F, 5‘-ACTCGCAGCCAGCGTTATG-3’F, 5'-ACTCGCAGCCAGCGTTATG-3 '
R, 5‘-AGGGTCAGCGATGCCAAAC-3’R, 5'-AGGGTCAGCGATGCCAAAC-3 '
CPTCPT F, 5‘-TCCAGTTGGCTTATCGTGGTG-3’F, 5'-TCCAGTTGGCTTATCGTGGTG-3 '
R, 5‘-TCCAGAGTCCGATTGATTTTTGC-3’R, 5'-TCCAGAGTCCGATTGATTTTTGC-3 '
MLYCDMLYCD F, 5‘-ACGTCCGGGAAATGAATGGG-3’F, 5'-ACGTCCGGGAAATGAATGGG-3 '
R, 5‘-GTAACCCGTTCTAGGTTCAGGA-3’R, 5'-GTAACCCGTTCTAGGTTCAGGA-3 '
FATPFATP F, 5‘-CTTCGATGGCTATGTCAGCGA-3’F, 5'-CTTCGATGGCTATGTCAGCGA-3 '
R, 5‘-AGCACGTCACCTGAGAGGTAG-3’R, 5’-AGCACGTCACCTGAGAGGTAG-3 ’
mCADmCAD F, 5‘-GCCACGGTAGAAACATTGGCT-3’F, 5'-GCCACGGTAGAAACATTGGCT-3 '
R, 5‘-CTTTTGCAGTACCCAGCACCT-3’R, 5'-CTTTTGCAGTACCCAGCACCT-3 '
GKGK F, 5‘-CTGGGACAAGATAACTGGAGAGC-3’F, 5'-CTGGGACAAGATAACTGGAGAGC-3 '
R, 5’-TCAACGGTAGACTGGGTTCTTA-3’R, 5’-TCAACGGTAGACTGGGTTCTTA-3 ’
PEPCKPEPCK F, 5‘-AAAACGGCCTGAACCTCTCG-3’F, 5'-AAAACGGCCTGAACCTCTCG-3 '
R, 5‘-ACACAGCTCAGCGTTATTCTC-3’R, 5'-ACACAGCTCAGCGTTATTCTC-3 '
CD36CD36 F, 5‘-AAGCCAGGTATTGCAGTTCTTT-3’F, 5'-AAGCCAGGTATTGCAGTTCTTT-3 '
R, 5‘-GCATTTGCTGATGTCTAGCACA-3’R, 5'-GCATTTGCTGATGTCTAGCACA-3 '
FABP1FABP1 F, 5‘-GTGTCGGAAATCGTGCAGAAT-3’F, 5'-GTGTCGGAAATCGTGCAGAAT-3 '
R, 5‘-GACTTTCTCCCCTGTCATTGTC-3’R, 5'-GACTTTCTCCCCTGTCATTGTC-3 '
GLUT2GLUT2 F, 5‘-CCATCTTCCTCTTTGTCAGCTT-3’F, 5'-CCATCTTCCTCTTTGTCAGCTT-3 '
R, 5‘-AAATTGCAGGTCCAATTGCT-3’R, 5'-AAATTGCAGGTCCAATTGCT-3 '
GAPDHGAPDH F, 5‘-AAGGTGAAGGTCGGAGTCAAC-3’F, 5'-AAGGTGAAGGTCGGAGTCAAC-3 '
R, 5‘-GGGGTCATTGATGGCAACAATA-3’R, 5'-GGGGTCATTGATGGCAACAATA-3 '
primerprimer sequencesequence speciesspecies
PPARαPPARα F, 5‘-TCTTCACGATGCTGTCCTCCT-3’F, 5'-TCTTCACGATGCTGTCCTCCT-3 ' mousemouse
R, 5‘-CTATGTTTAGAAGGCCAGGC-3’R, 5'-CTATGTTTAGAAGGCCAGGC-3 '
GLUT2GLUT2 F, 5‘-TTCCAGTTCGGCTATGACATCG-3’F, 5'-TTCCAGTTCGGCTATGACATCG-3 '
R, 5‘-CTGGTGTGACTGTAAGTGGGG-3’R, 5'-CTGGTGTGACTGTAAGTGGGG-3 '
GKGK F, 5‘-TGAACCTGAGGATTTGTCAGC-3’F, 5'-TGAACCTGAGGATTTGTCAGC-3 '
R, 5‘-CCATGTGGAGTAACGGATTTCG-3’R, 5'-CCATGTGGAGTAACGGATTTCG-3 '
CD36CD36 F, 5‘-ATGGGCTGTGATCGGAACTG-3’F, 5'-ATGGGCTGTGATCGGAACTG-3 '
R, 5‘-GTCTTCCCAATAAGCATGTCTCC-3’R, 5'-GTCTTCCCAATAAGCATGTCTCC-3 '
LPLLPL F, 5‘-TTGCCCTAAGGACCCCTGAA-3’F, 5'-TTGCCCTAAGGACCCCTGAA-3 '
R, 5‘-TTGAAGTGGCAGTTAGACACAG-3’R, 5'-TTGAAGTGGCAGTTAGACACAG-3 '
GLUT4GLUT4 F, 5‘-ACACTGGTCCTAGCTGTATTCT-3’F, 5'-ACACTGGTCCTAGCTGTATTCT-3 '
R, 5‘-CCAGCCACGTTGCATTGTA-3’R, 5'-CCAGCCACGTTGCATTGTA-3 '
ACOXACOX F, 5‘-TGTTAAGAAGAGTGCCACCAT-3’F, 5'-TGTTAAGAAGAGTGCCACCAT-3 '
R, 5‘-ATCCATCTCTTCATAACCAAATTT-3’R, 5'-ATCCATCTCTTCATAACCAAATTT-3 '
CPTCPT F, 5‘-ACTCCTGGAAGAAGAAGTTCAT-3’F, 5'-ACTCCTGGAAGAAGAAGTTCAT-3 '
R, 5‘-AGTATCTTTGACAGCTGGGAC-3’R, 5'-AGTATCTTTGACAGCTGGGAC-3 '
MLYCDMLYCD F, 5‘-GCACGTCCGGGAAATGAAC-3’F, 5'-GCACGTCCGGGAAATGAAC-3 '
R, 5‘-GCCTCACACTCGCTGATCTT-3’R, 5'-GCCTCACACTCGCTGATCTT-3 '
GAPDHGAPDH F, 5‘-ACCCCAGCAAGGACACTGAGCAAG-3’F, 5'-ACCCCAGCAAGGACACTGAGCAAG-3 '
R, 5‘-GGCTCCCTAGGCCCCTCCTGTTATT-3’R, 5’-GGCTCCCTAGGCCCCTCCTGTTATT-3 ’
4. 대사 분석4. Metabolic Analysis
지방산 산화작용 분석을 위해, 세포를 0.1 mM 팔미테이트(9, 10-[3H]palmitate, 5μci/ml; Perkin Elmer Life, Boston, MA) 및 1% 소혈청알부민이 포함된 a-MEM (Hyclone, USA)에서 24시간 동안 배양하였다.For fatty acid oxidation analysis, cells were cultured with 0.1 mM palmitate (9, 10- [ 3 H] palmitate, 5 μci / ml; Perkin Elmer Life, Boston, Mass.) And a-MEM (Hyclone) containing 1% bovine serum albumin. , USA) for 24 hours.
그 후, 배지를 수거한 후, 10% 트리클로로아세틱산 (Sigma-Aldrich, USA)로 침전시키고 격렬히 혼합하여 실온에서 20분간 인큐베이트하고 4℃, 16,000g로 10분간 원심분리하였다.Thereafter, the medium was collected, precipitated with 10% trichloroacetic acid (Sigma-Aldrich, USA), mixed vigorously, incubated at room temperature for 20 minutes, and centrifuged at 4 ° C. and 16,000 g for 10 minutes.
상층액을 1.5ml 튜브에 옮겨담고, 0.5ml 물이 포함된 신틸레이션 바이알에 담은 후 60℃에서 12시간 동안 인큐베이션하였다.The supernatant was transferred to a 1.5 ml tube, placed in a scintillation vial containing 0.5 ml water and incubated at 60 ° C. for 12 hours.
튜브를 제거한 후 액체 신틸레이션 계수기(LKB, USA)를 이용하여 방사능(3H2O in water)을 정량하였다.After the tube was removed, radioactivity ( 3 H 2 O in water) was quantified using a liquid scintillation counter (LKB, USA).
5. 5. 세포외에서Extracellularly 글루코스Glucose 소비 분석 Consumption analysis
인터럽틴 B(interruptin B) 및 로시글리타존을 3일간 세포에 처리하고, 조건 배지에서 배양된 세포를 수집한 후 Glucose Colorimetric Assay Kit II (BioVision Inc., CA, USA)를 이용하여 글루코스 함량을 분석하였다.Interruptin B and rosiglitazone were treated with the cells for 3 days, and the cells cultured in conditioned medium were collected and analyzed for glucose content using Glucose Colorimetric Assay Kit II (BioVision Inc., CA, USA).
6. 표면 6. Surface 플라즈몬Plasmon 공명 분석 Resonance analysis
SR7500DC (Reichert, NY, USA)을 이용하여, 표면 플라즈몬 공명(SPR) 분석을 수행하였다. 이미 공개되어진 방법으로 HA-PPARα, HA-PPARβ/δ 및 HA-PPARγ를 정제하고, CMDH chips(Reichert, NY, USA)을 이용하여 제조사의 설명서에 따라, 고정화시켰다.Surface plasmon resonance (SPR) analysis was performed using SR7500DC (Reichert, NY, USA). HA-PPARα, HA-PPARβ / δ and HA-PPARγ were purified by previously published methods and immobilized using CMDH chips (Reichert, NY, USA) according to the manufacturer's instructions.
SPR 분석을 위해, Int B를 31.25, 62.5, 125, 250, 및 500μM 농도로 처리하고 Scrubber 2 program (Informer Technologies, Inc., TX, USA)을 이용하여 KD 값을 확인하였다.For SPR analysis, Int B was treated at 31.25, 62.5, 125, 250, and 500 μM concentrations and KD values were determined using the Scrubber 2 program (Informer Technologies, Inc., TX, USA).
7. In 7. In vivovivo 실험 Experiment
모든 동물 연구(IACUC2015-0001)를 아주 대학교 동물 관리 및 사용위원회에서 승인받았으며, 미국 국립 보건원(NIH)에서 발행한 실험실 동물의 관리 및 사용 안내서에 따라 수행되었다.All animal studies (IACUC2015-0001) were approved by the Ajou University Animal Care and Use Committee and were conducted in accordance with the Care and Use Guide for Laboratory Animals published by the National Institutes of Health (NIH).
6주령 C57BLKS/J-Leprdb/Leprdb 또는 정상 6 주령 C57BL/6J 수컷 생쥐를 Orient Bio (Sungnam, Korea)에서 구입하여, 1주일간 안정화시켰다.Six-week-old C57BLKS / J-Leprdb / Leprdb or normal six-week-old C57BL / 6J male mice were purchased from Orient Bio (Sungnam, Korea) and stabilized for one week.
C57BLKS/JLeprdb/Leprdb 또는 정상 6 주령 C57BL/6J 수컷 생쥐를 무작위로 그룹화하고 vehicle, 로시클리타존(20 mg/kg), Int B(5mg/kg 또는 20mg/kg)를 하루에 한 번씩 1개월간 섭취시켰다. Randomly group C57BLKS / JLeprdb / Leprdb or normal 6-week-old C57BL / 6J male mice and receive vehicle, rosiclittazone (20 mg / kg), Int B (5mg / kg or 20mg / kg) once daily I was.
글루코스 내성 실험을 위해, 12시간 동안 생쥐를 금식시키고 무균 포도당 (1 g/kg, Sigma-Aldrich, USA)을 섭취시키고 OneTouch Ultra (LifeScan Inc., USA)를 사용하여 지정된 시간에서 혈당치를 확인하였다.For glucose tolerance experiments, mice were fasted for 12 hours, sterile glucose (1 g / kg, Sigma-Aldrich, USA) was taken and blood glucose levels were checked at the designated times using OneTouch Ultra (LifeScan Inc., USA).
8. 생화학 분석8. Biochemical Analysis
각 그룹의 생쥐로부터 혈액 시료 및 간을 수집하였다. Blood samples and livers were collected from each group of mice.
Hitachi clinical analyzer 7180 (HITACHI, JAPAN) 및 WAKO 시약(WAKO, USA)을 이용하여 혈청 및 간 TG, FFA, 및 전체 콜레스테롤을 측정하였다.Serum and liver TG, FFA, and total cholesterol were measured using Hitachi clinical analyzer 7180 (HITACHI, JAPAN) and WAKO reagent (WAKO, USA).
혈청내 LDL 및 HDL의 함량을 SEKISUI 시약(SEKISUI, JAPAN)을 이용하여 측정하였다. Serum LDL and HDL contents were measured using SEKISUI reagent (SEKISUI, JAPAN).
간독성은 WAKO 시약(WAKO, USA)을 이용하여 혈청에서 알라닌 아미노 전달효소(ALT) 및 아스파르트 아미노 전달효소(AST) 수준을 측정하여 확인하였다.Hepatotoxicity was confirmed by measuring alanine aminotransferase (ALT) and aspartic aminotransferase (AST) levels in serum using WAKO reagent (WAKO, USA).
9. 조직 절단 및 염색9. Tissue cutting and staining
간, 목주변 지방 조직, 골격근, 비장, 신장 및 심장과 같은 조직을 각 그룹의 생쥐로부터 수집하고 10% 포름알라닌에 고정시키 후 파라핀에 박았다.Tissues such as liver, peripheral adipose tissue, skeletal muscle, spleen, kidney and heart were collected from each group of mice, fixed in 10% formalanine and embedded in paraffin.
5 μm로 절개된 조직을 헤마톡실린 및 에오신으로 염색하였다.Tissues cut at 5 μm were stained with hematoxylin and eosin.
<실시예 1> 화합물 MD001에 의한 PPARα 및 PPARγ 활성 확인Example 1 Confirmation of PPARα and PPARγ Activity by Compound MD001
상기 합성예와 같이 합성된 화합물들이 PPARα 또는 PPARγ의 전사 활성을 증가시킬 것으로 예상하고 이를 확인하였다.Compounds synthesized as in Synthesis Example were expected to increase the transcriptional activity of PPARα or PPARγ was confirmed.
HEK293 세포에 사람 HA-PPARα 및 HA-PPARγ 발현 벡터, 리포터 플라스미드(PPRE-pk-Luc) 또는 대조군 리포터 플라스미드(pk-Luc)와 레닐라 벡터로 24시간 동안 일시적으로 공동 형질주입한 후 합성된 화합물을 각각 20 μM로 24시간 동안 처리하고 루시페라제 활성을 확인하였다.Compounds synthesized after transient co-transfection of HEK293 cells with human HA-PPARα and HA-PPARγ expression vectors, reporter plasmids (PPRE-pk-Luc) or control reporter plasmids (pk-Luc) and Renilla vectors for 24 hours Were treated with 20 μM each for 24 hours and luciferase activity was checked.
그 결과, 도 1과 같이 합성된 화합물 중 화합물 8a(MD001)가 PPARα 및 PPARγ의 전사 활성을 동시에 증가시키는 것을 확인할 수 있었다.As a result, it was confirmed that Compound 8a (MD001) in the compound synthesized as shown in FIG. 1 simultaneously increased the transcriptional activities of PPARα and PPARγ.
상기 결과로부터 화학적으로 합성된 MD001 화합물은 PPARα, PPARβ/δ, 및 PPARγ와 결합될 것으로 가정하고 이를 확인한 결과, 화합물 MD001은 PPARα 및 PPARγ와 결합하고 PPARβ/δ와는 결합하지 않았다.The MD001 compound chemically synthesized from the above results was assumed to be bound to PPARα, PPARβ / δ, and PPARγ, and as a result, the compound MD001 binds to PPARα and PPARγ and not to PPARβ / δ.
한편, MD001 화합물의 PPARα 및 PPARγ 자극 활성을 WY14643 및 로시글리타존(rosiglitazone)과 비교하기 위해, PPRE를 이용하여 HEK293 세포에서 루시페라제 활성 분석을 수행하였다. Meanwhile, in order to compare PPARα and PPARγ stimulating activity of MD001 compound with WY14643 and rosiglitazone, luciferase activity analysis was performed on HEK293 cells using PPRE.
그 결과, 도 2A 및 2B와 같이 MD001은 PPARα 및 PPARγ 활성을 통해 전사 수준을 상당히 증가시킨 반면, WY14643 및 로시글리타존의 수준은 MD001보다 낮은 것으로 확인되었다.As a result, MD001 significantly increased the transcription level through PPARα and PPARγ activity as shown in FIGS. 2A and 2B, whereas the levels of WY14643 and rosiglitazone were lower than MD001.
다음으로, MD001 화합물이 PPARα 및 PPARγ의 활성 또는 발현 증가를 유도할 수 있는지 HepG2 세포에서 확인한 결과, 도 2C와 같이 화합물 MD001은 PPARα, PPARγ 및 RXR 유전자의 발현을 매우 증가시켰다. Next, as a result of confirming in HepG2 cells that MD001 compound can induce increased activity or expression of PPARα and PPARγ, as shown in FIG. 2C, compound MD001 significantly increased expression of PPARα, PPARγ, and RXR genes.
화합물 MD001이 간세포에서 PPARα와 PPARγ를 활성화시키는지를 확인하기 위해 HepG2 세포에서 표적 유전자의 발현 수준을 확인하였다. To determine whether compound MD001 activates PPARα and PPARγ in hepatocytes, expression levels of target genes in HepG2 cells were checked.
그 결과, 도 2D 및 도 2E와 같이 MD001는 PPARα si-RNA를 사용하여 발현을 감소시킨 세포에서 β-산화와 관련된 PPARα 타겟 유전자인 ACOX(acyl-CoA oxidase), CPT(carnitine-palmitoyl transferase), 및 mCAD(middlechain acyl-CoA dehydrogenase)의 발현을 증가시켰다.As a result, as shown in Fig. 2D and 2E, MD001 is an ACOX (acyl-CoA oxidase), CPT (carnitine-palmitoyl transferase), which are PPARα target genes related to β-oxidation in cells that have reduced expression using PPARα si-RNA, And increased expression of middlechain acyl-CoA dehydrogenase (mCAD).
또한, PPARγ의 타겟 유전자인 GK(glycerol kinase), CD36 및 FABP1(fatty acid binding protein 1)의 발현을 증가시켰다.In addition, the expression of GPAR (glycerol kinase), CD36 and FABP1 (fatty acid binding protein 1) of PPARγ was increased.
상기 결과로부터 MD001는 이중 작용자로서 PPARα 및 PPARγ의 특이적인 활성을 통하여 대사작용을 조절하는 것으로 제안될 수 있다. From these results, MD001 may be proposed to modulate metabolism through the specific activity of PPARα and PPARγ as dual agents.
PPARγ는 인슐린 감수성과 포도당 내성을 향상시키는 항 당뇨병 약물의 개발을 위한 전통적인 분자 표적으로 인정받고 있다.PPARγ is recognized as a traditional molecular target for the development of antidiabetic drugs that enhance insulin sensitivity and glucose tolerance.
이에 따라, MD001이 글루코스 대사작용을 향상시킬 수 있는지를 확인하기 위해, HepG2, 분화된 3T3-L1 및 C2C12 근관세포에서 로시글리타존과 비교하였다.Accordingly, to determine if MD001 can improve glucose metabolism, it was compared with rosiglitazone in HepG2, differentiated 3T3-L1 and C2C12 myotubes.
그 결과, 도 3A, 3B 및 3C와 같이 MD001은 용량의존적으로 글루코스 소비량을 증가시켰다. 또한, 정량적인 RT-PCR 분석결과, 도 3D, 3E 및 3F와 같이 MD001은 GLUT2 (HepG2) 및 GLUT4 (3T3-L1 및 C2C12)의 발현을 유의하게 증가시켰다.As a result, MD001 increased glucose consumption dose-dependently, as shown in Figures 3A, 3B and 3C. In addition, quantitative RT-PCR analysis showed that MD001 significantly increased the expression of GLUT2 (HepG2) and GLUT4 (3T3-L1 and C2C12), as shown in FIGS. 3D, 3E and 3F.
상기 결과로부터 MD001은 부분적으로 글루코오스 운반자의 발현을 증가시킴으로써 글루코스 대사를 자극한다는 것을 시사한다.These results suggest that MD001 stimulates glucose metabolism by partially increasing the expression of glucose carriers.
MD001가 PPARα 활성을 통하여 β-산화를 증가시킬 수 있는지를 확인하기 위해, HepG2, 분화된 3T3-L1, 및 C2C12 근관세포에서 β-산화와 관련된 PPARα 타겟 유전자의 발현을 확인하였다.To confirm whether MD001 can increase β-oxidation through PPARα activity, expression of PPARα target genes related to β-oxidation in HepG2, differentiated 3T3-L1, and C2C12 root canal cells was confirmed.
그 결과, 4A, 4B 및 4C와 같이 MD001은 HepG2에서 ACOX, CPT, 말로닐-CoA 디카르복실라아제(MLYCD) 및 지방산 수송체(FATP)와 3T3-L1 및 C2C12 세포에서는 ACOX 및 CPT의 발현을 유의하게 증가시켰다.As a result, MD001, like 4A, 4B and 4C, expressed ACOX, CPT, malonyl-CoA decarboxylase (MLYCD) and fatty acid transporter (FATP) in HepG2 and ACOX and CPT expression in 3T3-L1 and C2C12 cells. Increased significantly.
상기 결과에 따라, MD001이 지방산 산화를 자극하는지 확인한 결과, 도 4D와 같이 MD001은 HepG2 세포에서 β-산화 비율을 유의하게 증가시켰으며, 4E 및 4F와 같이 분화된 3T3-L1 및 C2C12 근관세포에서도 유사한 결과가 확인되었다.According to the results, it was confirmed that MD001 stimulates fatty acid oxidation, as shown in Figure 4D MD001 significantly increased the β-oxidation rate in HepG2 cells, even in 3T3-L1 and C2C12 root canal cells differentiated as 4E and 4F Similar results were confirmed.
PPARα 표적 유전자의 발현 증가와 β-산화의 증가가 PPARα에 의존적인지 여부를 알아보기 위해, PPARα 특이적 si-RNA를 이용하여 PPARα의 발현을 억제 한 결과, 도 4G 내지 4I를 참고하면, MD001에 의해 증가된 β-산화는 PPARα 발현의 감소에 의해 폐기되었다.In order to determine whether the expression of PPARα target gene and the increase of β-oxidation are dependent on PPARα, the expression of PPARα was inhibited using PPARα-specific si-RNA. Referring to FIGS. 4G to 4I, MD001 Increased β-oxidation was discarded by a decrease in PPARα expression.
상기 결과로부터 MD001은 PPARα 활성화를 통해 β-산화를 강화시킨다는 것이 확인되었다.From the above results, it was confirmed that MD001 enhances β-oxidation through PPARα activation.
<< 실시예Example 2>  2> db/dbdb / db 마우스 동물모델에서 화합물 MD001에 의해 개선된 플라스마 지질 및  Plasma Lipids Improved by Compound MD001 in Mouse Animal Models and 글루코스Glucose 프로파일 확인 Profile Check
생체 내(in vivo)에서 MD001의 효과를 확인하기 위해, 정상 C57BL/6J 생쥐 및 당뇨병 db/db 생쥐에게 하루 한 번 MD001를 처리하였다.To verify the effect of MD001 in vivo, MD001 was treated once a day in normal C57BL / 6J mice and diabetic db / db mice.
그 결과, 도 5A와 같이 MD001 용량 의존적으로 db/db 생쥐의 혈당 수준이 유의하게 감소한 것을 확인할 수 있었다.As a result, as shown in FIG. 5A, it was confirmed that blood glucose levels of db / db mice were significantly reduced in MD001 dose-dependent manner.
또한, 복강 내 당부하시험(Intraperitoneal glucose tolerance test; IPGTT) 결과, 도 5B (and Supplementary Fig. 3A)와 같이 MD001에 의해 혈액 내 글루코스 수준이 감소된 것을 확인할 수 있었으며, 복강 내 인슐린 저항성 시험(intraperitoneal insulin tolerance test; IPITT) 결과에서도 MD001는 인슐린 민감성 증가를 통해 혈액 글루코스 수준을 낮춘 것을 확인할 수 있었다.In addition, as a result of the intraperitoneal glucose tolerance test (IPGTT), as shown in FIG. 5B (and Supplementary Fig. 3A), the blood glucose level was reduced by MD001, and the intraperitoneal insulin resistance test was performed. In the results of the insulin tolerance test (IPITT), MD001 lowered blood glucose levels by increasing insulin sensitivity.
로시글리타존(rosiglitazone), 피오글리타존(pioglitazone) 및 트로글리타존(troglitazone)을 포함하는 TZDs는 동물뿐만 아니라 사람에게서 심각한 몸무게 감소와 같은 부작용이 있는 것으로 알려짐에 따라, MD001 역시 몸무게 감소와 같은 부작용이 나타나지는 화인하였다.TZDs, including rosiglitazone, pioglitazone and troglitazone, are known to have side effects such as severe weight loss in humans as well as animals, and MD001 has also been a cause of side effects such as weight loss.
그 결과, 도 5C와 같이 로시글리타존을 처리한 db/db 생쥐에게서는 유의한 몸무게 감소가 나타난 반면, MD001는 정상 C57BL/6J 및 db/db 생쥐 모두에게서 음식 섭취 변화없이 몸무게 감소를 유도하지 않는 것을 확인할 수 있었다.As a result, as shown in Figure 5C rosiglitazone-treated db / db mice showed a significant weight loss, while MD001 did not induce weight loss without changes in food intake in both normal C57BL / 6J and db / db mice there was.
PPARα 또는 PPARγ의 작용제에 의한 활성화가 당뇨병 환자의 혈당 및 지질 함량을 낮추는 것으로 알려져 있으므로 MD001이 다양한 지질 함량을 낮출 수 있는지 확인하였다.Since activation by agonists of PPARα or PPARγ is known to lower blood sugar and lipid content in diabetic patients, it was confirmed that MD001 could lower various lipid content.
로시글리타존을 양성 대조군으로 사용하여 db/db 생쥐에서 고혈당증 및 지질 프로파일 개선 효과를 확인하였다. Rosiglitazone was used as a positive control to confirm the effect of hyperglycemia and lipid profile improvement in db / db mice.
그 결과, 도 5D-5E, 및 5K와 같이 PPARα/γ 이중 작용자인 MD001은 db/db 생쥐에서 혈액 글루코스, 트리글리세롤(TG) 및 유리 지방산을 유의하게 감소시키는 것을 확인할 수 있었다.As a result, MD001, a PPARα / γ dual agonist as shown in FIGS. 5D-5E and 5K, significantly reduced blood glucose, triglycerol (TG) and free fatty acids in db / db mice.
그러나, MD001은 대조군인 정상 C57BL/6J 생쥐와 비교하여 혈장 지질 및 혈액 글루코스의 수준에 영향을 주지 못하였다.However, MD001 did not affect the levels of plasma lipids and blood glucose compared to the control normal C57BL / 6J mice.
또한, 도 5I 및 5J를 참고하면, 로시글리타존은 db/db 생쥐의 혈액에서 알라닌 전달효소(ALT) 및 아스파르테이트아미노전달효소(AST) 수준을 상당히 증가시킨 반면, MD001가 처리된 db/db 생쥐에게서는 혈액 내 ALT 및 AST 수준이 매우 감소하였으며, 정상 C57BL/6 생쥐에게서는 어떠한 변화도 나타나지 않았다.5I and 5J, rosiglitazone significantly increased alanine transferase (ALT) and aspartate aminotransferase (AST) levels in the blood of db / db mice, whereas MD001 treated db / db mice. In ALT and AST levels in blood were greatly reduced, and no change was seen in normal C57BL / 6 mice.
상기 결과로부터 MD001은 순수한 PPARγ 작용자와 다르게 간 독성을 유도하지 않는 것이 확인되었다.From the above results, it was confirmed that MD001 does not induce liver toxicity unlike pure PPARγ agonists.
또한, 도 5G 및 5H와 같이 MD001은 낮은 밀도 지질단백질(LDL) 수준을 유의하게 감소시켰으며, 고밀도 지질단백질 수준은 증가시키는 것을 확인할 수 있었다.5G and 5H, MD001 significantly decreased low density lipoprotein (LDL) levels and increased high density lipoprotein levels.
상기 결과로부터 MD001은 비만 동물에서 콜레스테롤 대사를 회복시키는 것으로 제안될 수 있다.From these results MD001 can be suggested to restore cholesterol metabolism in obese animals.
한편, 로시글리타존은 db/db 생쥐에서 대조군보다 유의적으로 간 중량 (40 %) 및 지방량 (50 %) 증가를 유도한 반면, MD001은 간 무게 또는 지방 조직량 증가를 유도하지 않았다.On the other hand, rosiglitazone induced a significant increase in liver weight (40%) and fat mass (50%) in db / db mice, whereas MD001 did not induce an increase in liver weight or adipose tissue mass.
상기 결과로부터 MD001은 몸무게 감소 및 간독성 없이 혈액 글루코스 및 지질 함량을 유의적으로 감소시키는 것이 확인되었다.From these results, MD001 was found to significantly reduce blood glucose and lipid content without weight loss and hepatotoxicity.
<실시예 3> 화합물 MD001에 의한 대사장애 개선 효과 확인Example 3 Confirmation of Improvement of Metabolic Disorders by Compound MD001
지방간은 비만, 인슐린 저항성 및 제2형 당뇨병의 일반적인 합병증으로, PPARα 활성은 지방 소비 및 지방생성 감소를 자극하여 지방간을 개선하는 것으로 알려져있는 반면, 활성화된 PPARγ는 지방간에 반대 효과를 나타낸다.Fatty liver is a common complication of obesity, insulin resistance and type 2 diabetes, while PPARα activity is known to stimulate fat consumption and reduced lipogenesis to improve fatty liver, whereas activated PPARγ has an opposite effect on fatty liver.
앞선 실험에서 MD001은 PPARα/γ 이중 작용제로 확인됨에 따라, MD001이 db/db 마우스에서 지방간을 완화시킬 수 있는지 확인하였다.In the previous experiments, MD001 was identified as a PPARα / γ dual agonist, so that MD001 could relieve fatty liver in db / db mice.
도 6A를 참고하면, 이미 알려진 바와 같이 로시글리타존은 간 지방증을 악화시킨 반면, MD001가 처리된 경우, MD001 용량의존적으로 간 지방 방울의 크기와 수가 감소되었다.Referring to FIG. 6A, rosiglitazone, as known, exacerbated hepatic steatosis, whereas when MD001 was treated, the size and number of hepatic fat droplets decreased depending on the MD001 dose.
상기 결과로부터 MD001은 지방간을 개선하는 것이 확인되었다.From the above results, it was confirmed that MD001 improves fatty liver.
또한, 도 6B 및 6D와 같이 MD001은 간의 TG, FFA를 감소시켰으나, 콜레스테롤은 감소시키지 않은 반면, 로시글리타존은 간의 TG를 감소시키지 못하였다.6B and 6D, MD001 decreased liver TG and FFA, but did not reduce cholesterol, whereas rosiglitazone did not reduce liver TG.
정량적인 RT-PCR 분석 결과, 도 6E와 같이 MD001은 PPARα 타겟 유전자인 ACOX, CPT 및 MLYCD의 발현과 PPARγ 타겟 유전자인 GLUT2, GK, 및 CD36의 발현을 유의하게 증가시켰다.As a result of quantitative RT-PCR analysis, MD001 significantly increased the expression of PPARα target genes ACOX, CPT and MLYCD and the expression of PPARγ target genes GLUT2, GK, and CD36 as shown in FIG. 6E.
상기 결과로부터 MD001은 β-산화를 촉진시켜 간 지방증을 감소시키거나 부분적으로 PPARγ를 활성화시킴으로써 혈당 수치를 감소시키는 것이 확인되었다.From these results, it was confirmed that MD001 promotes β-oxidation to reduce hepatic steatosis or, in part, to reduce blood glucose levels by activating PPARγ.
다음으로, db/db 생쥐에서 지방 세포 크기에 대한 MD001의 효과를 확인하였다. 비만의 진행에 따라 지방 세포의 크기뿐만 아니라, 지방 세포의 수가 증가하고 지방 세포 크기는 인슐린 내성, 염증 및 고지혈증을 포함하는 대사성 스트레스의 지시자 중 하나이기 때문에 db/db 마우스의 지방 세포 크기에 대한 MD001의 영향을 확인하였다.Next, the effect of MD001 on fat cell size in db / db mice was confirmed. As the progression of obesity increases the number of fat cells, as well as the number of fat cells and MD001 for fat cell size in db / db mice, because fat cell size is one of the indicators of metabolic stress including insulin resistance, inflammation and hyperlipidemia The influence of the was confirmed.
도 7A 및 7B를 참고하면, 백색 지방 조직에서 지방 세포 크기가 MD001에 의해 유의하게 감소하였다. 또한 정량적 RT-PCR 분석 결과, MD001은 CPT, GLUT4, 및 CD36의 발현을 유의하게 증가시켰다.7A and 7B, fat cell size in white adipose tissue was significantly reduced by MD001. In addition, quantitative RT-PCR analysis showed that MD001 significantly increased the expression of CPT, GLUT4, and CD36.
상기 결과로부터 MD001은 지방산과 글루코스의 흡수를 증가시킬 뿐만 아니라, 도 7C와 같이 부분적으로 지방 조직 내 β-산화를 촉진시키는 것을 확인할 수 있었다.From the above results, MD001 was found to not only increase the absorption of fatty acids and glucose, but also partially promote β-oxidation in adipose tissue as shown in FIG. 7C.
한편, 식이에 의해 비만이 유도된 생쥐 및 비만인 사람의 지방 조직에서는 지방 세포의 죽음이 현저하게 증가하고, 그 후 대식세포가 죽은 지방 세포에 침투하여 지방 세포 잔여물을 제거하기 위해 크라운 형 구조(CLS)를 형성하여, 만성 염증의 특징인 다중 핵 거대 세포를 형성한다고 보고되어졌다.On the other hand, in fat tissues of mice and obese humans induced by obesity, fat cell death is markedly increased, and then macrophages penetrate into dead fat cells to remove the fat cell residues. CLS), which has been reported to form multiple nuclear giant cells that are characteristic of chronic inflammation.
로시글리타존은 도 7D 및 7E와 같이 지방 조직에서 CLS 수를 현저하게 증가시키는 것이 확인된 반면, MD001은 지방세포의 염증반응을 자극하지 않았다. Rosiglitazone was found to significantly increase the number of CLS in adipose tissue as shown in Figures 7D and 7E, while MD001 did not stimulate the inflammatory response of adipocytes.
또한, 골격근의 H & E 염색 결과, db/db 마우스에서 비히클 대조군, 로시글리타존 및 MD001 처리 그룹 간에 차이가 나타나지 않았다.In addition, H & E staining of skeletal muscle showed no difference between vehicle control, rosiglitazone and MD001 treatment groups in db / db mice.
흥미롭게도, 골격근을 이용한 qRT-PCR 분석 결과에서 MD001는 CPT, GLUT4, 및 지질단백질 리파아제(LPL)의 발현을 현저하게 증가시켰다.Interestingly, MD001 significantly increased the expression of CPT, GLUT4, and lipoprotein lipase (LPL) in qRT-PCR analysis using skeletal muscle.
상기 결과로부터 MD001는 골격근에서 글루코스와 지방산 대사작용을 증가시키는 것으로 제안될 수 있다.From these results, MD001 may be suggested to increase glucose and fatty acid metabolism in skeletal muscle.
또한, vehicle, 로시글리타존 또는 MD001가 처리된 정상 C57BL/6J 생쥐의 간, 골격근 및 지방 조직의 H&E 염색 결과에서는 그룹간의 차이가 나타나지 않았다.In addition, there was no difference in the H & E staining of liver, skeletal muscle and adipose tissue of normal C57BL / 6J mice treated with vehicle, rosiglitazone or MD001.
상기 결과들로부터 MD001은 PPARα/γ의 이중 작용자로서 고혈당증 및 고질혈증 개선에 기여할 수 있을 것이다.From these results MD001 may contribute to the improvement of hyperglycemia and hyperglycemia as a dual agonist of PPARα / γ.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (11)

  1. 하기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용가능한 염:A compound represented by formula (1) or a pharmaceutically acceptable salt thereof:
    [화학식 1][Formula 1]
    Figure PCTKR2018006649-appb-I000012
    Figure PCTKR2018006649-appb-I000012
    상기 화학식 1에 있어서, In Chemical Formula 1,
    상기 R1 내지 R4는 각각 동일하거나 다를 수 있으며, 수소; C1 내지 C4의 알킬; C1 내지 C4의 알콕시; 할로겐으로 이루어진 군에서 선택된 어느 하나임.R 1 to R 4 may be the same or different, respectively, hydrogen; C1 to C4 alkyl; Alkoxy of C1 to C4; Any one selected from the group consisting of halogen.
  2. 청구항 1에 있어서, 상기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 유용한 염은 3-신나모일-5,7-디하이드록시-8-메틸-4-페닐-2H-크로멘-2-온인 것을 특징으로 하는 화합물 또는 이의 염.The method of claim 1, wherein the compound represented by Formula 1 or a pharmaceutically useful salt thereof is 3-cinnamoyl-5,7-dihydroxy-8-methyl-4-phenyl-2H-chromen-2-one Characterized by a compound or a salt thereof.
  3. 청구항 1에 있어서, 상기 화합물은 PPARα/γ 이중 작용자(Dual agonist)인 것을 특징으로 하는 화합물 또는 이의 염.The compound of claim 1, or a salt thereof, wherein the compound is a PPARα / γ dual agonist.
  4. 하기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용가능한 염을 유효성분으로 함유하는 대사질환 예방 또는 치료용 약학조성물.A pharmaceutical composition for preventing or treating metabolic diseases containing a compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
    [화학식 1][Formula 1]
    Figure PCTKR2018006649-appb-I000013
    Figure PCTKR2018006649-appb-I000013
    상기 화학식 1에 있어서, In Chemical Formula 1,
    상기 R1 내지 R4는 각각 동일하거나 다를 수 있으며, 수소; C1 내지 C4의 알킬; C1 내지 C4의 알콕시; 할로겐으로 이루어진 군에서 선택된 어느 하나임.R 1 to R 4 may be the same or different, respectively, hydrogen; C1 to C4 alkyl; Alkoxy of C1 to C4; Any one selected from the group consisting of halogen.
  5. 청구항 4에 있어서, 상기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 유용한 염은 3-신나모일-5,7-디하이드록시-8-메틸-4-페닐-2H-크로멘-2-온인 것을 특징으로 하는 대사질환 예방 또는 치료용 약학조성물.The method according to claim 4, wherein the compound represented by Formula 1 or a pharmaceutically useful salt thereof is 3-cinnamoyl-5,7-dihydroxy-8-methyl-4-phenyl-2H-chromen-2-one A pharmaceutical composition for preventing or treating metabolic diseases.
  6. 청구항 4에 있어서, 상기 화합물은 PPARα/γ 이중 작용자(Dual agonist)인 것을 특징으로 하는 대사질환 예방 또는 치료용 약학조성물.The pharmaceutical composition for preventing or treating metabolic diseases according to claim 4, wherein the compound is a PPARα / γ dual agonist.
  7. 청구항 4에 있어서, 상기 대사질환은 비알콜성 지방간 및 고지혈증으로 이루어진 군에서 선택되는 것을 특징으로 하는 대사질환 예방 또는 치료용 약학조성물.The pharmaceutical composition for preventing or treating metabolic diseases according to claim 4, wherein the metabolic disease is selected from the group consisting of nonalcoholic fatty liver and hyperlipidemia.
  8. 하기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용가능한 염을 유효성분으로 함유하는 대사질환 예방 또는 개선용 건강식품.A health food for preventing or improving metabolic diseases containing a compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
    [화학식 1][Formula 1]
    Figure PCTKR2018006649-appb-I000014
    Figure PCTKR2018006649-appb-I000014
    상기 화학식 1에 있어서, In Chemical Formula 1,
    상기 R1 내지 R4는 각각 동일하거나 다를 수 있으며, 수소; C1 내지 C4의 알킬; C1 내지 C4의 알콕시; 할로겐으로 이루어진 군에서 선택된 어느 하나임.R 1 to R 4 may be the same or different, respectively, hydrogen; C1 to C4 alkyl; Alkoxy of C1 to C4; Any one selected from the group consisting of halogen.
  9. 청구항 8에 있어서, 상기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 유용한 염은 3-신나모일-5,7-디하이드록시-8-메틸-4-페닐-2H-크로멘-2-온인 것을 특징으로 하는 대사질환 예방 또는 개선용 건강식품.The method according to claim 8, wherein the compound represented by Formula 1 or a pharmaceutically useful salt thereof is 3-cinnamoyl-5,7-dihydroxy-8-methyl-4-phenyl-2H-chromen-2-one Health foods for preventing or improving metabolic diseases.
  10. 청구항 8에 있어서, 상기 화합물은 PPARα/γ 이중 작용자(Dual agonist)인 것을 특징으로 하는 대사질환 예방 또는 개선용 건강식품.The method of claim 8, wherein the compound is a PPARα / γ dual agonist (Dual agonist), characterized in that for preventing or improving metabolic diseases health food.
  11. 청구항 8에 있어서, 상기 대사질환은 비알콜성 지방간 및 고지혈증으로 이루어진 군에서 선택되는 것을 특징으로 하는 대사질환 예방 또는 개선용 건강식품.The method of claim 8, wherein the metabolic disease is non-alcoholic fatty liver and hyperlipidemia health foods for preventing or improving metabolic disease, characterized in that selected from the group consisting of.
PCT/KR2018/006649 2017-06-12 2018-06-12 NOVEL COMPOUND HAVING PPARα/γ DUAL ACTIVITY AND COMPOSITION CONTAINING SAME AS ACTIVE INGREDIENT FOR PREVENTING OR TREATING METABOLIC DISEASE WO2018230936A1 (en)

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