WO2012096494A2 - Agent containing b-ring-substituted flavonoid derivatives for treating arthritis - Google Patents

Abstract

The present invention relates to flavonoid derivatives expressed by the following general formula (I), to a composition containing the pharmaceutically acceptable salts thereof as an active ingredient, and to the use of the flavonoid derivatives. More particularly, the flavonoid derivatives expressed by the following general formula (I), or the pharmaceutically acceptable salts thereof, may exhibit an activity of inhibiting the decomposition of cartilage constituents, an activity of inhibiting the decomposition of ECM, and an activity of inhibiting the expression of MMP-13, and may exhibit a superior activity of inhibiting the deterioration of joints in a chronic-arthritis animal test. Therefore, the flavonoid derivatives or the pharmaceutically acceptable salts thereof may be used in a pharmaceutical composition and a functional health food that is useful for preventing and treating arthritis.

Description

Arthritis Therapeutics Containing Cyclocyclic Substituted Flavonoid Derivatives

The present invention relates to a therapeutic agent for arthritis and the use thereof containing a B-ring substituted flavonoid derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

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Arthritis includes rheumatoid arthritis and degenerative arthritis (osteoarthritis), but these diseases are painful diseases that ultimately involve cartilage destruction of the joint cavity. In particular, degenerative arthritis suffered by most patients is a disease in which local degenerative changes occur as the cartilage of the joint loses its weight. The disease mainly affects the elderly population and significantly reduces the quality of life. It is most common in the joints of the hands, feet, spine and weight, that is, the hips and knees. Genetic causes, physicomechanical causes, and aging factors are important causes of degenerative arthritis. The causes of these arthritis are different, and not yet all are known, but finally, the factor that destroys the articular cartilage, matrix metalloproteinase (MMP) overexpressed in the joint area is important, especially collagenase-3 / MMP-13 is a joint It is known to play the most important role in destruction (Mitchell PG, Magna HA, Reeves LM, Lopresti-Morrow LL, Yocum SA, Rosner PJ, Geoghegan KF, Hambor JE. Cloning, expression, and type II collagenolytic activity of matrix metalloproteinase-13 from human osteoarthritic cartilage.J Clin Invest 1996; 97: 761-8; Takaishi H, Kimura T, Dalah S, Okada Y, DArmiento J. Joint diseases and matrix metalloproteinases: a role for MMP-13.Current Phramaceutical Biotechnology 2008; 9 : pp.47-54).

MMP-13 breaks down collagen, the most important of the extracellular matrix (ECM) of cartilage and joints, to promote joint destruction. Thus, substances that inhibit or inhibit expression of MMP-13 may inhibit and delay the destruction of articular cartilage (Johnson AR, Pavlovsky AG, Ortwine DF, Prion F, Man CF, Bornemeier DA, Banotai CA, Mueller WT, McConell P, Yan C, Baragi V, Lesch C, Roark WH, Wilson M, Datta K, Guzman R, Han HK, Dyer RD. Discovery and characterization of a novel inhibitor of matrix metalloproteinase-13 that reduces cartilage damage in vivo without joint fibroplasias side effects.J. Biol. Chem. 2007; 282; pp. 27781-91).

For the purpose of discovering these substances and using them as medicines, we studied several natural ingredients. Among them, some of the flavonoid derivatives, especially those bearing substituents on the B-ring, strongly inhibit MMP-13 expression. The cartilage destruction inhibitory activity has been confirmed in in vivo animal experiments to confirm the high possibility of development as a therapeutic and prophylactic agent for the treatment of arthritis diseases and completed the present invention.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of arthritis containing a flavonoid compound having a structure of the following general formula (I), or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a health functional food for the prevention and improvement of arthritis containing a flavonoid compound having a structure of the following general formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.

Formula 1

Where

R ₁ is one or more substituents selected from the group consisting of a halogen atom, a hydroxy group, and a C ₁ to C ₃ lower alkoxy group,

R ₂ to R ₅ are each independently one or more substituents selected from the group consisting of a hydrogen atom, a halogen atom, a hydroxy group, and a C ₁ to C ₃ lower alkoxy group.

Among the compound groups belonging to the general formula (I), preferably, R ₁ is one or more substituents selected from the group consisting of a hydroxy group and a methoxy group, more preferably a hydroxy group, and R ₂ to R ₅ are each independently a hydrogen atom or a hydroxyl group. And at least one substituent selected from the group consisting of methoxy groups, more preferably a hydroxy group.

As the most preferable compound among the compound group which belongs to the said general formula (I),

5,7,2 ', 3'-tetrahydroxyflavone, 5,7,2', 4'-tetrahydroxyflavone, 5,7,3 ', 4'-tetrahydroxyflavone or 7- Methoxy-5,3 ', 4'-trihydroxyflavone.

The compounds of the present invention represented by the above formula (I) may be prepared with pharmaceutically acceptable salts and solvates according to conventional methods in the art.

As salts are acid addition salts formed with pharmaceutically acceptable free acids. Acid addition salts are prepared by conventional methods, for example by dissolving a compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent, such as methanol, ethanol, acetone or acetonitrile. Equivalent molar amounts of the compound and acid or alcohol (eg, glycol monomethylether) in water can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered.

In this case, organic acids and inorganic acids may be used as the free acid, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc. may be used as the inorganic acid, and methanesulfonic acid, p -toluenesulfonic acid, acetic acid, trifluoroacetic acid, Citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, gluconic acid acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, and the like.

Bases can also be used to make pharmaceutically acceptable metal salts. An alkali metal or alkaline earth metal salt is obtained by, for example, dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate. In this case, as the metal salt, it is particularly suitable to prepare sodium, potassium or calcium salts, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Pharmaceutically acceptable salts of the compounds having the structure of formula (I) above include salts of acidic or basic groups which may be present in compounds having the structure of formula (I), unless otherwise indicated. . For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, dihydrogen Phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p -toluenesulfonate (tosylate) salts, and methods or processes for preparing salts known in the art It can be prepared through.

Another object of the present invention is to provide a method for preparing the compound of general formula (I), which may be prepared by a synthesis method known in the art, and may be chemically synthesized by the method shown in the following schemes. It is not limited only to these examples.

Formula 2

Dissolving a certain amount of hydroxy acetophenone (II) and potassium hydroxide in an organic solvent such as methanol, and then adding a certain amount of 4-benzyloxybenzaldehyde (III) to react at room temperature; Two steps of reacting with iodine or the like; The desired compound flavones of the present invention can be prepared by adding a saturated aqueous solution of sodium thiosulfate, followed by three steps of reaction and recrystallization.

"Arthritis" as defined herein includes one or more diseases selected from degenerative arthritis, rheumatoid arthritis or Lupus arthritis, preferably degenerative arthritis.

Since the chondrocytes of the joints produced ECM and biosynthesized MMPs, chondrocytes were cultured through SW1353 cell line cell culture, which is human joint chondrocytes, and activated by treating IL-1. The amount of expression rapidly increases and the amount of hydrolyzed collagen degradation product and proteoglycan increases. The system was treated with the compounds of the general formula (I) of the present invention obtained by the above production method and the effects on them were studied. In addition, in vivo animal model was confirmed to inhibit the joint destruction.

Therefore, the present invention provides a pharmaceutical composition for the treatment and prevention of inflammatory diseases, immune system disorders or cancer diseases containing a flavonoid compound having the structure of Formula (I) as an active ingredient and a pharmaceutically acceptable excipient or carrier. To provide a composition.

In another aspect, the present invention comprises administering a flavonoid compound having the structure of Formula (I) to a patient with an inflammatory disease, an immune system disorder or a cancer disease, a treatment for treating a patient with an inflammatory disease, an immune system disorder or a cancer disease Provide a method.

The present invention also provides the use of a flavonoid compound having the structure of Formula (I) for the preparation of a medicament for the treatment of inflammatory diseases, immune system disorders or cancer diseases.

Compositions comprising a compound of the present invention may further comprise a suitable carrier, excipient or diluent according to conventional methods.

Carriers, excipients and diluents that may be included in the compositions of the present invention include 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.

Compositions comprising the compounds of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, or sterile injectable solutions, respectively, according to conventional methods. Can be used.

Specifically, when formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, or the like. ), Lactose, gelatin and the like can be mixed. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups, and include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. 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. As a base of suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the compounds of the present invention depend on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, but may be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention can be administered in 0.0001 ~ 500 mg / kg, preferably in an amount of 0.001 ~ 100 mg / kg divided once to several times daily. The compound of the present invention in the composition should be present in an amount of 0.0001 to 10% by weight, preferably 0.001 to 1% by weight relative to the total weight of the total composition.

In addition, the pharmaceutical dosage forms of the compounds of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds, as well as in a suitable collection.

The pharmaceutical composition of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

In the pharmaceutical composition, the effective dose of the flavonoid derivative represented by the general formula (I) and the pharmaceutically acceptable salt thereof according to the present invention is 0.1 to 500 mg / kg or 0.1 to 200 mg / cm ² when applied to the skin and , Preferably 10 to 100 mg / kg or 1 to 10 mg / cm ² when applied to the skin, may be administered 1-6 times a day. However, the dosage level for a particular patient may vary depending on the weight, age, sex, health status, diet, time of administration, administration method, excretion rate, severity of the disease, and the like of the patient.

The present invention provides a health functional food for the prevention and improvement of arthritis, which contains a flavonoid derivative represented by the general formula (I) showing the prevention and improvement effect of arthritis as an active ingredient.

The composition containing the compound of the present invention can be used in various ways, such as drugs, food and beverages for the prevention and improvement of arthritis. Examples of the food to which the compound of the present invention may be added include various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, and may be used in the form of powders, granules, tablets, capsules, or beverages. have.

Since the compound of the present invention has little toxicity and side effects, it is a drug that can be used safely even when taken for a long time for the purpose of prevention.

The compound of the present invention may be added to food or beverage for the purpose of preventing and improving arthritis. At this time, the amount of the compound in the food or beverage is generally added to the health food composition of the present invention to 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 10 g, preferably based on 100 ml Can be added in a ratio of 0.3 to 1 g.

The health beverage composition of the present invention, in addition to containing the compound as an essential ingredient in the indicated ratio, is not particularly limited in the liquid component and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose, for example polysaccharides such as maltose and sucrose, and conventional sugars such as dextrin and cyclodextrin. And sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Flavonoid derivatives represented by the general formula (I) and pharmaceutically acceptable salts thereof according to the present invention were subjected to oral administration, intraperitoneal administration, and subcutaneous injection toxicity experiments. The 50% lethal dose (LD ₅₀ ) was found to be at least 800 mg / kg, and therefore the flavonoid derivatives and their pharmaceutically acceptable salts according to the invention proved to be relatively safe substances.

As described above, the flavonoid derivative represented by the general formula (I) of the present invention or a pharmaceutically acceptable salt thereof shows cartilage component degradation inhibition, ECM degradation inhibition activity, and MMP-13 expression inhibition activity. In addition, since the arthritis-induced animal experiments are excellent in inhibiting joint destruction, it can be used in pharmaceutical compositions and health functional foods useful for the prevention and treatment of arthritis.

1 is a diagram showing the inhibitory effect of MMP-13 expression of Example compounds (A: apigenin, **: P <0.01, significantly different from the IL-1-treated control group (n = 3)), the vertical axis Expression level of MMP-13 is ng / ml;

2 is a diagram showing the intracellular mechanism of action of MMP-13 expression inhibition of compounds of apigenin and synthetic flavone derivatives (5,7,2 ', 3'-tetrahydroxyflavone, 7 );

Figure 3 is a diagram showing the histological findings in the joints of the example compounds ((a) Iodoacetate-treated (b) apigenin (c) compound 7 (d) compound 8 (e) compound 9 treated group).

Hereinafter, the present invention will be described in detail by reference examples, examples and experimental examples.

However, the following Reference Examples, Examples, and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Reference Examples, Examples, and Experimental Examples.

Reference Example 1. Experiment Preparation and Devices

1-1. Analyzer

The instrument used to confirm the structure of the product obtained in this experiment is as follows. Nuclear magnetic resonance spectra ( ¹ H NMR, ¹³ C NMR) were used at 200 MHz, 300 MHz or 400 MHz, and the solvents were CDCl ₃ and DMSO-d ₆ . Coupling constants ( J ) are expressed in Hz. Mass spectra were used and expressed in m / z form.

1-2. TLC and tube chromatography

Thin layer chromatography (TLC) was used as silica gel (Merck F254) manufactured by E. Merck, and silica (Merck EM9025, 70-230 mesh) was used for column chromatography. In addition, UV lamp (= 254 nm) was used to identify the material separated on TLC.

1-3. Used reagents

The reagents used in this experiment were purchased from Sigma-Aldrich, Lancaster, and Fluka, and the solvent used in the reaction was Sigma-Aldrich, Merck. A first grade reagent from Merck, Junsei Chemical Co. was used without purification.

Example 1 Synthesis of Flavonoid Derivatives

The following flavonoid derivatives were synthesized according to the method of Dao et al. (Arch. Pharm. Res. 2003; 26: 345-350, Bioorg. Med. Chem. Lett. 2004; 14: 1165-1167).

1-1. Preparation of Flavone (1)

2-hydroxy acetophenone (1 equiv) and potassium hydroxide (3 equiv) were dissolved in methanol, and then benzaldehyde (1 equiv) was added and stirred at room temperature for 12 hours to react. After the reaction was completed, 3% by weight aqueous hydrochloric acid solution was slowly added dropwise. The yellow solid produced at this time was filtered, washed with methanol and dried. The solid obtained in the reaction was dissolved in dimethyl sulfoxide (DMSO), and then iodine (0.1 equivalent) was added based on the obtained solid, followed by stirring at 100 ° C. for 10 hours. After the solution was cooled to room temperature, a saturated aqueous solution of sodium thiosulfate was slowly added dropwise until the solution turned colorless. The solid produced at this time was filtered and recrystallized with methanol to prepare a target compound flavone having the following physical properties.

Yield: 46%

White solid, mp 95-96 ° C.

¹ H-NMR (200MHz, CDCl ₃ ): δ 8.22-8.27 (dd, 1H, J = 7.8 Hz, 1.6Hz, H5), 7.93-7.95 (dd, 2H, J = 7.6Hz, H2 ', H6') , 7.68 ~ 7.77 (t, 1H, J = 7.0Hz, 1.6Hz, H7), 7.40 ~ 7.60 (m, 7H, J = 8.4Hz, 8.8Hz, 7.8Hz, 2.6Hz, 2.4Hz, H2 ', H3' , H4 ', H5', H6 ', H6, H8), 6.85 (s, 1H, H3).

1-2. Preparation of 4'-hydroxyflavone (2)

2-hydroxy acetophenone (1 equiv) and potassium hydroxide (3 equiv) were dissolved in methanol, followed by addition of 4-benzyloxybenzaldehyde (1 equiv), followed by stirring at room temperature for 24 hours. After the reaction was completed, 3% by weight aqueous hydrochloric acid solution was slowly added dropwise. The yellow solid produced at this time was filtered, washed with methanol and dried. The solid obtained in the reaction was dissolved in dimethyl sulfoxide (DMSO), and then iodine (0.1 equivalent) was added based on the obtained solid, followed by stirring at 100 ° C. for 10 hours. After the solution was cooled to room temperature, a saturated aqueous solution of sodium thiosulfate was slowly added dropwise until the solution turned colorless. The resulting solid was filtered off, recrystallized from methanol and dried. The solid obtained in the above reaction was dissolved in acetic acid, and concentrated hydrochloric acid (2 ml / equivalent) was added on a solid basis, and the mixture was heated to reflux for 30 minutes to 1 hour. After the reaction was completed, the reaction mixture was cooled to room temperature and stirred with slowly dropwise addition of water at 0 ° C. At this time, the resulting solid was filtered and recrystallized with methanol to prepare a target compound flavone (4-hydroxyflavone) having the following physical properties.

Yield: 28%

Yellow solid, mp 264 ° C.

¹ H-NMR (200 MHz, DMSO- d ₆ ): δ 8.17-8.21 (d, 1H, J = 8.8 Hz, H5), 7.80-7.85 (d, 2H, J = 9.0 Hz, H2 ', H6') , 7.67 ~ 7.74 (t, 1H, H7), 7.38 ~ 7.66 (m, 2H, H6, H8), 6.96 ~ 7.01 (d, 2H, J = 8.8 Hz, H3 ', H5'), 6.71 (s, 1H , H3).

1-3. Preparation of 2 ', 3'-dihydroxyflavone (3)

2-hydroxy acetophenone (1 equiv) and potassium hydroxide (3 equiv) were dissolved in methanol, followed by addition of 2,3, -dimethoxybenzaldehyde (1 equiv) and stirred at room temperature for 24 hours for reaction. After the reaction was completed, 3% by weight aqueous hydrochloric acid solution was slowly added dropwise. The yellow solid produced at this time was filtered, washed with methanol and dried. The solid obtained in the reaction was dissolved in dimethyl sulfoxide (DMSO), and then iodine (0.1 equivalent) was added based on the obtained solid, followed by stirring at 100 ° C. for 10 hours. After the solution was cooled to room temperature, a saturated aqueous solution of sodium thiosulfate was slowly added dropwise until the solution turned colorless. The resulting solid was filtered off, recrystallized from methanol and dried. The solid obtained in the reaction was dissolved in CHCl ₃ , and then 1M BBr ₃ (5 equivalents) dissolved in CHCl ₃ on a solid basis was added and heated to reflux. After the reaction was completed, the reaction mixture was cooled to room temperature, a small amount of methanol was added dropwise at 0 ° C, and the organic solvent was concentrated under reduced pressure. The obtained solid was recrystallized with methanol to prepare a target compound flavone (2 ', 3'-dihydroxyflavone) having the following physical properties.

Yield: 28%

White solid, mp 238-239 캜.

¹ H-NMR (200 MHz, CDCl ₃ ): δ 8.02 to 8.06 (dd, 1H, J = 8.0 Hz, 1.2 Hz, H5), 7.70 to 7.85 (m, 2H, H6, H7), 7.44 to 7.52 (m, 3H, H5 ', H6', H8), 6.89-6.63 (d, 1H, J = 9.2 Hz, H4 '), 6.75 (s, 1H, H3).

1-4. Preparation of 3 ', 4'-dihydroxyflavone (4)

The desired compound flavone (3 ', 3), having the following physical properties by the same synthesis method as Compound (2) using 2-hydroxy acetophenone (1 equivalent) and 3,4, -dibenzyloxybenzaldehyde (1 equivalent) as starting materials 4'-dihydroxyflavone) was prepared.

Yield: 30%

Yellow solid, mp 250 ° C.

¹ H-NMR (200 MHz, DMSO- d ₆ ): δ 9.02 (s, 1H, OH), 8.15-8.19 (d, 1H, J = 8.0 Hz, H5), 7.58-7.71 (t, 1H, H7), 7.36-7.54 (m, 4H, H2 ', H6', H6, H8), 6.95-6.99 (d, 1H, J = 8.0 Hz, H5 '), 6.67 (s, 1H, H3).

1-5. Preparation of 2 ', 4'-dihydroxyflavone (5)

Target compound flavone (2 ', 4) having the following physical properties by the same synthesis method as Compound (3) using 2-hydroxy acetophenone (1 equivalent) and 2,4, -dimethoxybenzaldehyde (1 equivalent) as starting materials '-dihydroxyflavone) was prepared.

Yield: 34%

Yellow solid, mp 285-286 ° C .;

¹ H-NMR (200 MHz, DMSO- d ₆ ): δ 10.67 (s, 1H, OH), 10.27 (bs, 1H, OH), 7.98-8.03 (d, 1H, J = 9.6 Hz, H5), 7.45- 7.84 (m, 3H, H6 ', H7, H8), 7.45-7.49 (t, 1H, H6), 7.09 (s, 1H, H3), 6.42-6.50 (m, 2H, H3', H5 ')

1-6. Preparation of 7,3 ', 4'-trihydroxyflavone (6)

Synthesis same as Compound (3) using 2-dihydroxy-4-methoxy acetophenone (1 equiv) and 3,4, -dimethoxybenzaldehyde (1 equiv) as starting materials using 1M BBr ₃ (10 equiv) The desired compound flavone (7,3 ', 4'-trihydroxyflavone) having the following physical properties was prepared by the method.

Yield: 31%

Yellow solid, mp> 300 ° C.

¹ H-NMR (200 MHz, DMSO- d ₆ ): δ 10.75 (s, 1H, OH), 9.84 (s, 1H, OH), 7.83-7.88 (dd, 1H, J = 8.8 Hz, H5), 7.38 ( t, 2H, J = 8.0Hz, 1.8Hz, H2 ', H6'), 6.86 ~ 6.93 (t, 3H, J = 8.8Hz, 2.6Hz, H5 ', H6, H8), 6.60 (s, 1H, H3 ).

1-7. Preparation of 5,7,2 ', 3'-tetrahydroxyflavone (7)

The same synthetic method as Compound (6) using 2-hydroxy-4,6-dimethoxy acetophenone (1 equiv) and 2,3, -dimethoxybenzaldehyde (1 equiv) as a starting material to have the following physical properties Compound flavone (5,7,2 ', 3'-tetrahydroxyflavone) was prepared.

Yield: 26%

Yellow solid, mp> 300 ° C.

¹ H-NMR (200 MHz, DMSO- d ₆ ): δ 12.90 (bs, 1H, 5-OH), 10.86 (bs, 1H, OH), 10.02 (bs, 1H, OH), 9.63 (bs, 1H, OH ), 7.29-7.74 (d, 1H, J = 8.6 Hz, H6 '), 6.98-7.02 (d, 2H, J = 9.6 Hz, H2', H5 '), 6.81 (s, 1H, H3), 6.45 ( d, 1H, J = 1.8 Hz, H6), 6.20 (d, 1H, J = 1.6 Hz, H8).

1-8. Preparation of 5,7,3 ', 4'-tetrahydroxyflavone (8)

A compound having the following physical properties by the same synthesis method as Compound (6) using 2-hydroxy-4,6-dimethoxy acetophenone (1 equivalent) and 3,4, -dimethoxybenzaldehyde (1 equivalent) as starting materials Compound flavone (5,7,3 ', 4'-tetrahydroxyflavone) was prepared.

Yield: 24%

Yellow solid, mp> 300 ° C.

¹ H-NMR (200 MHz, DMSO- d ₆ ): δ 7.42 (dd, 1H, J = 8.3 Hz, 2.2 Hz, H6 '), 7.40 (d, 1H, J = 2.2 Hz, H2'), 6.90 (d , 1H, J = 8.3 Hz, H5 '), 6.68 (s, 1H, H3), 6.45 (d, 1H, J = 2.0 Hz, H8), 6.20 (1H, d, J = 2.0 Hz, H6).

1-9. Preparation of 5,7,2 ', 4'-tetrahydroxyflavone (9)

The same synthetic method as the compound (6) using 2-hydroxy-4,6-dimethoxy acetophenone (1 equivalent) and 2,4, -dimethoxybenzaldehyde (1 equivalent) as a starting material, having the following physical properties Compound flavone (5,7,2 ', 4'-tetrahydroxyflavone) was prepared.

Yield: 26%

Yellow solid, mp> 300 ° C.

¹ H-NMR (200 MHz, DMSO- d ₆ ): δ 13.06 (s, 1H, 5-OH), 10.74 (s, 1H, OH), 10.72 (s, 1H, OH), 10.16 (s, 1H, OH ), 7.74 ~ 7.78 (d, 1H, J = 8.4Hz, H6 '), 6.98 (s, 1H, H2'), 6.42-6.69 (m, 3H, H3, H5 ', H6), 6.16 (d, 1H , J = 2.0 Hz, H8).

1-10. Preparation of 5-hydroxy-7-methoxyflavone (10)

2-hydroxy-4,6-dimethoxy acetophenone (1 equiv) potassium hydroxide (3 equiv) was dissolved in methanol, and then benzaldehyde (1 equiv) was added and stirred at room temperature for 24 hours to react. After the reaction was completed, 3% by weight aqueous hydrochloric acid solution was slowly added dropwise. The yellow solid produced at this time was filtered, washed with methanol and dried. The solid obtained in the reaction was dissolved in dimethyl sulfoxide (DMSO), and then iodine (0.1 equivalent) was added based on the obtained solid, followed by stirring at 100 ° C. for 10 hours. After the solution was cooled to room temperature, a saturated aqueous solution of sodium thiosulfate was slowly added dropwise until the solution turned colorless. The resulting solid was filtered off, recrystallized from methanol and dried. The solid obtained in the reaction was dissolved in acetonitrile (CH ₃ CN) and cooled to about 0 ° C. with ice, then anhydrous aluminum trichloride (5 equivalents) was added and reacted for 4 hours under heating reflux conditions. After completion of the reaction, the solution was concentrated under reduced pressure to remove acetonitrile, and the obtained solid was dissolved in a solution in which chloroform and 3N hydrochloric acid aqueous solution were mixed in a volume ratio of 2: 1, and then maintained under heating and reflux for 40 minutes. Thereafter, the organic layer was separated, excess water was removed with anhydrous magnesium sulfate, and concentrated under reduced pressure. The solid obtained at this time was washed with methanol again and dried again to prepare a target compound flavone (5-hydroxy-7-methoxyflavone) having the following physical properties.

Yield: 26%

Yellow solid, mp> 300 ° C.

¹ H-NMR (200 MHz, DMSO- d ₆ + CDCl ₃ ): δ 13.02 (s, 1H, 5-OH), 8.05-8.09 (dd, 2H, J = 9.8 Hz, H2 ', H6'), 7.56- 7.60 (m, 3H, H3 ', H4', H5 '), 6.67 (s, 2H, H6, H8), 6.47 (s, 1H, H3), 3.99 (s, 3H, OCH ₃ ).

1-11. Preparation of 3 ', 4'-dihydroxy-7-methoxyflavone (11)

Compound having the following physical properties by the same synthesis method as Compound (2) using 2-hydroxy-4-methoxy acetophenone (1 equivalent) and 3,4, -dibenzyloxybenzaldehyde (1 equivalent) as starting materials Flavones (3, 4-dihydroxy-7-methoxyflavone) were prepared.

Yield: 22%

Yellow solid, mp> 240 ° C.

¹ H-NMR (200 MHz, DMSO- d ₆ + CDCl ₃ ): δ 9.12 (bs, 1H, OH), 8.01-8.05 (d, 1H, J = 8.4 Hz, H5), 7.33-7.40 (t, 2H, J = 8.6 Hz, H2 ', H6'), 6.92-7.04 (t, 2H, J = 8.2 Hz, H6, H8), 6.57 (s, 1H, H3), 3.94 (s, 3H, OCH ₃ ).

1-12. Preparation of 7-methoxy-5,3 ', 4'-trihydroxyflavone 12

Compound having the following physical properties by the same synthesis method as Compound (10) using 2-hydroxy-4,6-dimethoxy acetophenone (1 equivalent) and 3,4, -dibenzyloxybenzaldehyde (1 equivalent) as starting materials The desired compound flavone (7-methoxy-5,3 ', 4'-trihydroxy flavone) was prepared.

Yield: 17%

Yellow solid, mp 286-287 ° C.

¹ H-NMR (200 MHz, DMSO- d ₆ ): δ 8.09 (d, 1H, J = 8.9 Hz, H2 '), 6.94 (d, 1H, J = 8.9 Hz, H3'), 6.75 (d, 1H, J = 2.2 Hz, H8), 6.36 (d, 1H, J = 2.2 Hz, H6), 3.87 (s, 1H, OCH ₃ ).

1-13. Preparation of 3 ', 4'-dimethoxyflavone (13)

The desired compound flavone (3 ', 4) having the following physical properties by the same synthesis method as Compound (1) using 2-hydroxy acetophenone (1 equivalent) and 3,4, -dimethoxybenzaldehyde (1 equivalent) as starting materials '-dimethoxyflavone) was prepared.

Yield: 35%

Yellow solid, mp 155 ° C.

¹ H-NMR (200MHz, CDCl ₃ ): δ 8.22 ~ 8.26 (dd, 1H, J = 8.0Hz, 2.4Hz, H5), 7.67 ~ 7.75 (t, 1H, J = 8.4Hz, 7.4Hz, 1.6Hz, H7), 7.56 ~ 7.62 (m, 2H, H6, H8), 7.40 ~ 7.60 (m, 2H, H2 ', H6'), 6.98 ~ 7.03 (d, 1H, J = 8.6Hz, H5 '), 6.78 ( s, 1H, H 3), 4.00 (s, 3H, OCH ₃ ), 3.98 (s, 3H, OCH ₃ ).

1-14. Preparation of 3 ', 4'-dichloroflavone 14

Target compound flavone (3 ', 4') having the following physical properties by the same synthesis method as Compound (1) using 2-hydroxy acetophenone (1 equivalent) and 3,4, -dichlorobenzaldehyde (1 equivalent) as starting materials -dichloroflavone) was prepared.

Yield: 77%

Yellow solid, mp 184 ° C.

¹ H-NMR (200MHz, CDCl ₃ ): δ 8.22 ~ 8.26 (d, 1H, J = 7.4Hz, H5), 8.04 ~ 8.05 (ds, 1H, J = 2.0Hz, H2 '), 7.42 ~ 7.78 (m , 6H, J = 8.6 Hz, 2.0 Hz, 1.6 Hz, H5 ', H6', H6, H7, H8), 6.80 (s, 1H, H3).

Experimental Example 1. Effect on the amount of MMP-13

In order to assay the effect on the amount of MMP-13 of the derivatives synthesized in the Examples were tested as described in the literature (Mengshol JA, Vincenti MP, Coon CI, Barchowsky A, Brinckerhoff CE.Interleukin-1 induction) of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c-Jun N-terminal kinase and nuclear factor κB: differential regulation of collagenase 1 and collagenase 3. Arthritis Rheum 2000; 43: 801-11.)

Chondrocyte (SW1353) Culture and Activation

SW1353 (purchased from ATCC), a human chondrosarcoma cell line, was cultured using DMEM (Gibco) with 10% FBS, penicillin / strentomycin and glutamine. To confirm the expression of MMP-13 secreted into the medium, IL-1β was treated at 10 ng / ml under serum-free DMEM conditions, and supernatant was obtained after 24 hours, followed by ELISA (R & D Systems (Minneapolis, MN)). The amount of these MMP-13 was confirmed, and the results are shown in FIG. 1.

As a result, all the groups except compound 1 and 14 showed significant MMP-13 expression inhibitory ability, but in particular, the derivatives 7 , 8 , 9 , 12, etc. showed a strong decrease in the amount of MMP-13.

This result makes it possible to predict that the degradation of ECM in the joints will be suppressed.

Experimental Example 2 Intracellular Action Mechanism

Methods described in the literature for assaying the intracellular mechanism of action of derivatives synthesized in the Examples (Lim H, Kim HP. Matrix Metalloproteinase-13 Expression in IL-1β-treated Chondrocytes by Activation of the p38 MAPK / c-Fos / AP -1 and JAK / STAT Pathways.Arch. Pharm. Res. 2011 Jan. in press). Apigenin was used as a control.

In order to elucidate the mechanism of action of apigenin and 7, the most potent inhibitor, SW1353 cells were treated with 10 ng / ml of IL-1β and treated with 10 uM of flavones at the same time.

As a result, as shown in Figure 2, these derivatives were confirmed to inhibit the expression of MMP-13 as a complex mechanism that inhibits the activation of c-Fos / AP-1 and STAT1 / 2.

Experimental Example 3. Effect on glycosaminoglycan and collagen degradation products

In order to test the effect of the derivatives synthesized in Example on glycosaminoglycan and collagen degradation products, the following experiments were carried out.

3-1. Culture and activation of Rabbit articular cartilage slice

According to Baek et al. (Biol. Pharm. Bull. 2006; 29: 1423-1430), rabbit articular cartilage slices were cut out and incubated in complete DMEM medium containing 5% FBS for 1 day, followed by 40 mg of cartilage. Each was cut into 48-well plate wells, treated with IL-1, and the amount of glycosaminoglycan was measured in the supernatant cultured for 3 days, and the amount of collagen degradation product was measured in the supernatant cultured for 14 days.

3-2. Measurement of glycosaminoglycan and collagen degradation products

To determine the degradation of cartilage glycosaminoglycan, the supernatant was quantified using a bilyscan sulfated glycosaminoglycan assay kit (Biocolor).

In addition, collagen type II cleavage ELISA was used for the type II collagen in the supernatant to measure collagen degradation of the cartilage (IBEX), and the results are shown in Table 1.

Table 1 Inhibition of Glycosaminoglycan and Collagen Degradation of Flavonoids

Compounds	Concentration (uM)	% degradation of glycosaminoglycan	% idegradation of collagen
Control	-	18.2 ± 4.3	23.8 ± 5.5
IL-1-treated	-	100.0 ± 4.1	100.0 ± 5.1
Prednisolone	25	28.7 ± 5.2 *	50.2 ± 5.8 *
apigenin	10	40.5 ± 6.1 *	38.4 ± 4.9 *
	25	11.4 ± 5.2 *	18.0 ± 6.6 *
5,7,2 ', 3'-tetrahydroxyflavone ( 7 )	10	32.0 ± 5.1 *	30.8 ± 12.1 *
5,7,2 ', 4'-tetrahydroxyflavone ( 8 )	10	54.3 ± 5.3 *	33.9 ± 10.0 *
5,7,3 ', 4'-tetrahydroxyflavone ( 9 )	10	35.1 ± 4.2 *	37.8 ± 4.6 *
*: P <0.01, significant different from IL-1-treated group by unpaired Student t-test (n = 3).

In our experiments, these flavonoids showed potent inhibition at concentrations of 10-25 uM, indicating that the destruction of ECM was actually inhibited by the inhibition of MMP-13 induction.

Experimental Example 5. Inhibition of Chronic Arthritis in Animal Models

In order to assay the inhibitory ability of the derivatives synthesized in the example in a chronic articular destruction animal model, the following experiment was conducted according to the method described in the literature.

As a model of chronic degenerative arthritis, according to the method of Guingamp et al. (Arthritis Rheum. 1997; 40: 1670-1679), 30 ul of Iodoacetic acid was added to the right side of SD rat (200-220 g, Orient-Bio). The knee was administered by intramuscular injection. Flavonoid derivatives were administered orally at 100 mg / kg / day once daily for 5 days / week.

After 5 weeks of iodine acetate administration, the rats were sacrificed and the knee joints dissected, the tissues were fixed according to histological methods, stained with safranin O, and observed under a microscope. Shown in In addition, an example of histological observation of the joint is shown in FIG. 3.

TABLE 2 Inhibition of joint destruction of flavonoids

Compounds	Dose (mg / kg / day)	Histology result (degree of joint destruction)
iodoacetate-treated control group	-	+++
apigenin	100	++
5,7,2 ', 3'-tetrahydroxyflavone ( 7 )	100	+
5,7,2 ', 4'-tetrahydroxyflavone ( 8 )	100	++
5,7,3 ', 4'-tetrahydroxyflavone ( 9 )	100	++

As a result of the experiment, it was confirmed that the derivatives of the present invention strongly inhibit the symptoms of joint destruction in vivo.

Experimental Example 6. Toxicity Test

In order to assay the toxicity of the derivatives synthesized in the examples, the synthetic flavonoids ( 7 , 8 , 9 ) that showed activity were the result of acute toxicity assay conducted according to the protocol of KFDA, i.e. 800 mg / kg (po Acute toxicity was not observed.

Hereinafter, the preparation examples of the composition containing the compound of the present invention will be described, but the present invention is not intended to limit the present invention but only to explain in detail.

Formulation Example 1 Preparation of Powder

20 mg of compound 7 (Example 1)

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2 Preparation of Tablet

10 mg of compound 8 (Example 1)

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation Example 3 Preparation of Capsule

10 mg of compound 7 (Example 1)

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

10 mg of compound 9 (Example 1)

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO _4, 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

20 mg of compound 12 (Example 1)

10 g of isomerized sugar

5 g of mannitol

Purified water

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding a proper amount of lemon aroma, and then mixing the above components, adding purified water and adjusting the whole to 100 ml by adding purified water and filling into a brown bottle. The solution is prepared by sterilization.

Formulation Example 6 Preparation of Healthy Food

Compound 8 (Example 1) 1000 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

Folate 50 ㎍

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the vitamin and mineral mixture is a composition suitable for a relatively healthy food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation Example 7 Preparation of Healthy Drink

Compound 7 (Example 1) 1000 mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components in accordance with a conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

Although the composition ratio is a composition that is relatively suitable for the preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

The present invention found that some of the flavonoid derivatives having substituents on the B-ring strongly inhibit the expression of MMP-13. Such cartilage destruction inhibitory activity was also confirmed in animal experiments to treat arthritis diseases and It is highly likely to be developed as a preventive agent

Claims (10)

1. A pharmaceutical composition for the prevention and treatment of arthritis, which comprises a flavonoid compound having a structure of the following general formula (I), or a pharmaceutically acceptable salt thereof as an active ingredient:

   

   Where

   R ₁ is one or more substituents selected from the group consisting of a halogen atom, a hydroxy group, and a C ₁ to C ₃ lower alkoxy group,

   R ₂ to R ₅ are each independently one or more substituents selected from the group consisting of a hydrogen atom, a halogen atom, a hydroxy group, and a C ₁ to C ₃ lower alkoxy group.
2. The method of claim 1,

   R ₁ of the general formula (I) structure is one or more substituents selected from the group consisting of a hydroxy group and a methoxy group, and R ₂ to R ₅ are each independently one or more substituents selected from the group consisting of a hydrogen atom, a hydroxy group and a methoxy group A pharmaceutical composition that is a compound.
3. The method of claim 2,

   The compound is 5,7,2 ', 3'-tetrahydroxyflavone, 5,7,2', 4'-tetrahydroxyflavone, 5,7,3 ', 4'-tetrahydroxyflavone Or 7-methoxy-5,3 ', 4'-trihydroxyflavone.
4. The method of claim 1,

   The arthritis is a pharmaceutical composition is one or more diseases selected from degenerative arthritis, rheumatoid arthritis or Lupus arthritis.
5. A health functional food for the prevention and improvement of arthritis, which contains a flavonoid compound having the structure of general formula (I) as an active ingredient.
6. The method of claim 5,

   R ₁ of the general formula (I) structure is one or more substituents selected from the group consisting of a hydroxy group and a methoxy group, and R ₂ to R ₅ are each independently one or more substituents selected from the group consisting of a hydrogen atom, a hydroxy group and a methoxy group Functional foods that are compounds.
7. The method of claim 6,

   The compound may be 5,7,2 ', 3'-tetrahydroxyflavone, 5,7,2', 4'-tetrahydroxyflavone, 5,7,3 ', 4'-tetrahydroxyflavone or 7- A dietary supplement that is methoxy-5,3 ', 4'-trihydroxyflavone.
8. The method of claim 5,

   The arthritis is a health functional food is one or more diseases selected from degenerative arthritis, rheumatoid arthritis or lupus arthritis.
9. A method of treating a patient with an inflammatory disease, an immune system disorder or a cancer disease, comprising administering a flavonoid compound having a structure of Formula (I) to a patient with an inflammatory disease, an immune system disorder, or a cancer disease.
10. Use of a flavonoid compound having the structure of Formula (I) according to claim 1 for the manufacture of a medicament for the treatment of inflammatory diseases, immune system disorders or cancer diseases.

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