KR20170116586A - Composition comprising phenolic compound for treating inflammatory disease - Google Patents

Abstract

The present invention relates to a composition for treating inflammatory diseases comprising a phenolic compound. The phenolic compounds according to the present invention inhibited the expression and activity of various inflammatory mediators such as inflammation-inducing cytokines and showed an excellent anti-inflammatory effect, and the effective therapeutic effect was confirmed in an animal model of collagen-induced rheumatoid arthritis It could be used as an effective substance to replace the existing therapeutic agent for inflammatory diseases.

Description

TECHNICAL FIELD The present invention relates to a composition for treating inflammatory diseases,

The present invention relates to a composition for treating inflammatory diseases comprising a phenolic compound.

Inflammation is a series of defensive phenomena that, when a cell or tissue is damaged by some cause, minimizes the reaction and restores the damaged area to the original state, causing nerve and blood vessels, lymphatic vessels, humoral responses, Causing edema, redness, fever, and so on. The causes of inflammation are physical factors caused by trauma, bronze, burn, radioactivity, and chemical factors caused by chemical substances such as acid, and immunological factors caused by antibody reaction. In addition, they are caused by blood vessels and hormone imbalance do. Cells damaged by external stimuli secrete a variety of biological mediators such as pro-inflammatory cytokines, chemokines, interleukins, and interferons, resulting in vasodilation and increased permeability Antibodies, complement, plasma (Plasma) and phagocyte cells come into the inflammation site, which causes erythema. In order to alleviate inflammation, medicines that act to reduce inflammation-related vital reactions and symptoms, that is, substances used as anti-inflammatory drugs include Ibuprofen, Indomethacin and the like in non- (Dexamethasone). However, since their use is limited due to their side effects, development of new anti-inflammatory agents is required.

Rheumatoid arthritis (Rheumatoid arthritis), a typical inflammatory disease, is a systemic disease characterized mainly by inflammation of synovium, which causes chronic degeneration and deformity of joints. Generally, it occurs in about 1% of the population, and women have a prevalence of 2.5 times that of men. It can occur in all ages, but it is most common in ages between 40 and 70 and increases with age. The cause of rheumatoid arthritis is not yet fully understood, but it is understood to be an inflammatory reaction due to autoimmune mechanism. It is presumed that genetic predisposition and environmental factors will act on the onset. Recently, as the understanding of synovitis and the pathogenesis of rheumatoid arthritis deepens, new therapeutic agents based on it are being actively developed.

Although joint involvement by rheumatoid arthritis affects all joints of the whole body, it is characterized by swelling of the joints and pain, involving the joints of the hands and feet, especially the proximal and interphalangeal joints. Rapid diagnosis and appropriate treatment are essential because they lead to chronic progression after the onset, and when they are not treated, they cause permanent loss of joint function due to deformation and destruction of joints due to inflammation. Because rheumatoid arthritis is caused by synovial inflammation, synovitis is the most central pathologic finding of rheumatoid arthritis. Normal synovial membrane is covered with thin synoviocyte (1-2 layers), whereas synovial membrane seen in rheumatoid arthritis shows the following changes. The most obvious change is the proliferation of synovial cells, the infiltration of inflammatory cells, the formation of new blood vessels, the increased expression of adhesion molecules on the cell surface, and the expression of various cytokines and proteolytic / inhibitory substances. Inflammatory cells infiltrating the synovial membrane are usually monocytes composed of T cells, B cells, macrophages, and plasma cells. Some of the thick synovial membranes due to the proliferation and infiltration of synovial cells are characterized by local invasion by adjacent cartilage and bone, which is the most distinguishing characteristic of rheumatoid arthritis and other inflammatory arthritis. These masses of synovial tissue that pierce the surrounding tissues are called "pannus", and those that are locally destroyed by the pannus often appear as a characteristic marginal erosion in radiological examinations. T cells account for much of the inflammatory cells that invade the sublayer of the synovial membrane. T cell activation and migration into the synovial membrane occurs early in rheumatoid arthritis.

The cytokine expressed in synovial membrane of rheumatoid arthritis is mainly secreted from macrophages and synovial cells, activates / inflows inflammatory cells in the periphery, promotes synovial cell proliferation, and promotes synovitis. Tumor necrosis factor-α (TNF-α) and interleukin (TNF-α), which are produced by macrophages, are IL-6, Granulocyte-macrophage colony- stimulates inflammation by inducing the production of sub-inflammatory mediators such as stimulating factor (GM-CSF), IL-8, various chemokines and matrix metalloproteinases (MMP-1, MMP-3) And the damage of the joint is caused. The recently developed TNF-α blocker has a high effect on rheumatoid arthritis, suggesting that cytokine plays an important role in inflammation persistence and synovial hyperplasia.

In the last decade, the treatment of rheumatoid arthritis has achieved remarkable progress with the development of biological agents in addition to the existing methicrexate-based modifying anti-rheumatic drugs. However, some patients reported incomplete or unresponsive responses despite the use of these biological agents, suggesting that cells or mediators that play a central role in the pathogenesis of disease differ for each individual patient do. Therefore, studies have been made actively on drugs that can effectively regulate the production and release of physiologically active substances in synovial cells (Korean Patent Laid-Open No. 10-2015-0087552), but these are still insufficient.

DISCLOSURE OF THE INVENTION The present invention has been made in order to solve the above problems, and the present inventors have conducted intensive studies in order to discover novel substances that are likely to be developed as therapeutic agents for inflammatory diseases, and have found that the expression of various inflammatory mediators such as inflammation- The activity is inhibited, and the phenolic compound showing the effective anti-inflammatory effect is confirmed, and the present invention has been completed on the basis thereof.

Accordingly, an object of the present invention is to provide a novel use of the phenolic compound for the prevention or treatment of inflammatory diseases.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease, comprising a compound represented by the following formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient ,

[Chemical Formula 1]

In Formula 1,

R ₁ represents hydrogen, hydroxy or C ₁ -C ₅ alkoxy;

R ₂ represents hydrogen, hydroxy, or C ₁ -C ₅ alkyl;

R ₃ and R ₄ independently represent hydrogen, hydroxy, or C ₁ -C ₅ alkoxy; And

R ₅ can represent C ₁ -C ₅ alkyl, C ₁ -C ₅ alkyl alcohol, C ₁ -C ₅ alkylcarboxy, or C ₁ -C ₅ alkylaldehyde.

In one embodiment of the invention, the inflammatory disease is selected from the group consisting of atopy, psoriasis, dermatitis, allergies, arthritis, rhinitis, otitis, rhinitis, tonsillitis, cystitis, nephritis, pelvic inflammatory disease, Crohn's disease, ulcerative colitis, ankylosing spondylitis, (Such as systemic lupus erythematodes, SLE), asthma, edema, delayed allergy (type IV allergy), graft rejection, graft versus host disease, autoimmune encephalomyelitis, multiple sclerosis, inflammatory bowel disease, cystic fibrosis, diabetic retinopathy, Nephritis, < / RTI >

In another embodiment of the present invention, the composition may further comprise a pharmaceutically acceptable carrier.

In another embodiment of the present invention, the composition may be administered simultaneously, separately, or sequentially with other anti-inflammatory agents.

The present invention also provides a health functional food composition for preventing or ameliorating an inflammatory disease, which comprises the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a method of treating an inflammatory disease comprising administering to a subject a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.

The present invention also provides a therapeutic use for the inflammatory diseases of the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof.

The phenolic compounds according to the present invention inhibited the expression and activity of various inflammatory mediators such as inflammation-inducing cytokines and showed an excellent anti-inflammatory effect, and the effective therapeutic effect was confirmed in an animal model of collagen-induced rheumatoid arthritis It could be used as an effective substance to replace the existing therapeutic agent for inflammatory diseases.

1 shows the results of ¹ H NMR spectrum (250 MHz, MeOH-d ₄ ) of Compound I prepared in Preparation Example 1.
2 shows the results of ¹³ C NMR spectrum (63 MHz, MeOH-d ₄ ) of Compound I prepared in Preparation Example 1.
FIG. 3 shows the result of confirming the change in the secretion amount of TNF-.alpha. According to the treatment with Compound I in mast cells.
FIG. 4 shows the result of confirming the change in secretion amount of IL-4 according to the treatment with Compound I in mast cells.
FIG. 5 shows the results of confirming changes in the secretion amounts of TNF-a and IL-6 in the mast cells according to the treatment with the compound II-X.
FIG. 6 shows the results of confirming the change in secretion amount of β-Hexosaminidase according to the treatment with Compound I in mast cells.
FIG. 7 shows the result of confirming the change of secretion amount of histamine according to the treatment with Compound I in mast cells.
FIG. 8 shows the result of confirming the change in secretion amount of histamine according to the treatment with Compound II-X in mast cells.
FIG. 9 shows the result of confirming cytotoxicity of Compound I in synovial cells of patients with arthritis.
10 shows changes in expression levels of inflammatory cytokines (TNF-α, IL-1, IL-6) and substratease (MMP-1 and MMP-3) according to Compound I treatment in synovial cells of patients with arthritis .
Fig. 11 shows the results of confirming changes in expression levels of signal transduction agents (NF-κB, PI3K, Akt, and IKK-α / β, etc.) according to Compound I treatment in synovial cells of patients with arthritis.
FIG. 12 shows the results of confirming changes in expression levels of inflammatory cytokines (IL-6) and substratease (MMP-1) according to the synovial cells of arthritis patients treated with Compound XI to Compound XIV (10 μM).
FIG. 13 shows the results of confirming changes in expression levels of IL-6, MMP-1, and MMP-3 according to the treatment concentration (0.1, 1, 10 μM) of Compound XIV in synovial cells of patients with arthritis.
14 is a schematic diagram schematically showing a preparation process of an animal model of collagen-induced rheumatoid arthritis and an administration time of Compound I. Fig.
Fig. 15 shows the result of confirming the change in the sole thickness according to the treatment with Compound I in an animal model of collagen-induced rheumatoid arthritis.
FIG. 16 shows the results of confirming the change in arthritic progression index according to the treatment with Compound I in an animal model of collagen-induced rheumatoid arthritis.
17 is a result of confirming the change in arthritis incidence according to the treatment with Compound I in an animal model of collagen-induced rheumatoid arthritis.
Figure 18 shows the results of X-ray and PET-CT observation of ankle and peripheral joints after Compound I treatment in an animal model of collagen-induced rheumatoid arthritis.
FIG. 19 shows the results of confirming the change in the secretion amount of immunoglobulin G1 (IgG1) according to the treatment with Compound I in an animal model of collagen-induced rheumatoid arthritis.
FIG. 20 shows the result of confirming the change in the secretion amount of immunoglobulin G2a (IgG2a) according to the treatment with Compound I in an animal model of collagen-induced rheumatoid arthritis.
FIG. 21 shows the results of confirming the change of the phenotype Th1 response according to the treatment with Compound I in an animal model of collagen-induced rheumatoid arthritis.
FIG. 22 shows the results of confirming the change of phenotype Th17 response according to treatment with Compound I in an animal model of collagen-induced rheumatoid arthritis.
23 shows changes in the secretion of inflammatory cytokines (TNF-α, IL-1, IL-6) and substratease (MMP-1, MMP-3) following treatment with Compound I in an animal model of collagen-induced rheumatoid arthritis .

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition for preventing or treating an inflammatory disease, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient; The use of said compound for the prophylaxis or treatment of inflammatory diseases; And administering a therapeutically effective amount of the compound to an individual.

[Chemical Formula 1]

In the above formula (1), preferably, R ₁ represents hydrogen, hydroxy or C ₁ -C ₅ alkoxy; R ₂ represents hydrogen, hydroxy, or C ₁ -C ₅ alkyl; R ₃ and R ₄ independently represent hydrogen, hydroxy, or C ₁ -C ₅ alkoxy; And R ₅ may represent C ₁ -C ₅ alkyl, C ₁ -C ₅ alkyl alcohol, C ₁ -C ₅ alkylcarboxy, or C ₁ -C ₅ alkylaldehyde, more preferably R ₁ is hydrogen, Lt; / RTI > or methoxy; R ₂ represents hydrogen, hydroxy, methyl; R ₃ and R ₄ independently represents hydrogen, hydroxy, or methoxy; And R < ₅ > may represent methyl, methyl alcohol, carboxy, or aldehyde, and most preferably the compound represented by formula 1 is 4-methylcatechol; 3,4-dihydroxybenzyl alcohol; 4-hydroxy-benzaldehyde; 3,4-dihydroxy-benzaldehyde; 3,5-dimethoxy-4-methylbenzaldehyde; 4-hydroxy-3,5-dimethoxybenzaldehyde; 4-hydroxybenzoic acid; 3,4-dihydroxybenzoic acid; 4-hydroxy-3-methoxybenzoic acid; 3-hydroxy-2-methoxybenzoic acid; And 2-hydroxy-3-methoxybenzoic acid, but are not limited thereto.

As used herein, "alkyl" generally refers to linear and branched saturated hydrocarbon groups having the indicated number of carbon atoms (eg, 1 to 5 carbon atoms). Examples of the alkyl group include, without limitation, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl and the like. Alkyl can be attached to a parent group or substrate at any ring atom provided that attachment does not violate the atomic requirement. Likewise, alkenyl or alkynyl may contain one or more non-hydrogen substituents if the attachment does not violate the valence requirements.

"Alkoxy" refers to alkyl-O-, wherein alkyl is defined above. Examples of alkoxy groups include, without limitation, methoxy, ethoxy, and the like. Alkoxy can be attached to a parent group or substrate at any ring atom provided that attachment does not violate the atomic requirement. Likewise, an alkoxy group may include one or more non-hydrogen substituents if the attachment does not violate the valence requirements.

In addition, the compound can be produced by various methods known in the art. As an example, a strain of Pestalotiopsis sp. Is cultured to obtain a strain culture, and the obtained culture is dissolved in a solvent , Followed by purification, the compound can be prepared or obtained, but is not limited thereto.

The compound of the present invention can be used in the form of a pharmaceutically acceptable salt. The acid addition salt formed by a pharmaceutically acceptable free acid is useful as a salt.

As used herein, the term "salt" is useful as an acid addition salt formed by a pharmaceutically acceptable free acid. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- or dicarboxylate, phenyl-substituted alkanoates, hydroxyalkanoates or Alkanedioates, aromatic acids, aliphatic or aromatic sulfonic acids, and the like. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide 1-sulfo-2-naphthalene-1-sulfonate, methanesulfonate, methanesulfonate, propanesulfonate, naphthalene-1-sulfone Naphthalene-2-sulfonate, or mandelate.

The acid addition salt according to the present invention can be obtained by a conventional method, for example, by dissolving the compound represented by the formula (1) in an excess amount of an acid aqueous solution, and then mixing the salt with a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile ≪ / RTI > It is also possible to prepare the mixture by evaporating a solvent or excess acid and then drying or by filtration of the precipitated salt by suction.

In addition, the base may be used to make a pharmaceutically acceptable metal salt. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).

In addition, the compounds of the present invention include not only pharmaceutically acceptable salts, but also all salts, isomers, hydrates and solvates which can be prepared by conventional methods.

As used herein, the term "prophylactic " means any act that inhibits or delays inflammatory disease by administration of a pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to any action that improves or alters the symptoms of an inflammatory disease by the administration of the pharmaceutical composition according to the present invention.

In the present invention, "individual" means a subject in need of treatment for an inflammatory disease, and more specifically refers to mammals such as primates, mice, dogs, cats, horses and cows, which are human or non-human .

The term "inflammatory disease ", which is a disease to be prevented or treated by the composition of the present invention, refers to a disease with inflammation as a main lesion, and includes, but is not limited to, atopic, psoriasis, dermatitis, allergies, arthritis, Systemic lupus erythematodes (SLE), asthma, edema, delayed allergy (type IV allergy), transplantation, and the like in patients suffering from chronic sinusitis, sinusitis, otitis media, rhinitis, tonsillitis, cystitis, nephritis, pelvic inflammation, Crohn's disease, ulcerative colitis, Inflammatory bowel disease, cystic fibrosis, diabetic retinopathy, and glomerulonephritis, wherein the arthritis is caused by a variety of causes, such as bacteria, trauma, autoimmune diseases, etc. Refers to diseases caused by inflammatory changes in the joints, and is preferably a disease caused by osteoarthritis, degenerative arthritis, rheumatoid arthritis, Osteitis, joint ligament injury, may be a meniscus injury, misalignment of the joint, avascular necrosis, idiopathic arthritis and pediatric, may be most preferably rheumatoid arthritis.

According to one embodiment of the present invention, Pestalotiopsis sp. sp . ) (Example 1 and 2). When the mNK cells were treated with mNK cells, the TNF-a, TNF-alpha, TNF-alpha, IL-4 and IL-6, as well as the secretion of? -Hexosaminidase and histamine (see Example 1). In addition, according to another embodiment of the present invention, Compound I did not show cytotoxicity to synovial cells in arthritic patients (see Example 2), and TNF-alpha, IL-1, IL-6, And the activity of PI3K, Akt, and NF-κB, which are related factors, (see Example 3). In addition, as in the above-mentioned results, the collagen-induced rheumatoid arthritis animal model, which is a typical inflammatory disease model, exhibited excellent therapeutic effects according to the anti-inflammatory action, so that the phenolic compounds of the present invention can be effectively used for the effective treatment of inflammatory diseases (See Example 4).

The pharmaceutical composition of the present invention may contain, in addition to the active ingredient, a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers are those conventionally used at the time of formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, in addition to the above components, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like may be further included.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and the dose may vary depending on the condition and the weight of the patient, The mode of administration, the route of administration, and the time, but may be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other anti-inflammatory agents and may be administered sequentially or simultaneously with conventional anti-inflammatory agents, and may be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, body weight, the degree of absorption of the active ingredient in the body, the rate of inactivation and excretion, the type of disease, 100 to 500 mg / kg of body weight may be administered daily or every other day, or one to three times a day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

In another aspect of the present invention, there is provided a health functional food composition for preventing or ameliorating an inflammatory disease, comprising the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

As used herein, the term "improvement" means any action that at least reduces the degree of symptom associated with the condition being treated. At this time, the health functional food composition may be used either simultaneously with or separately from the medicines for treatment before or after the onset of the disease for the prevention or improvement of the inflammatory disease.

In the health functional food composition of the present invention, the active ingredient can be directly added to the food or can be used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). In general, the composition of the present invention may be added in an amount of preferably not more than 15% by weight, preferably not more than 10% by weight, based on the raw material. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range.

The health functional food composition of the present invention may contain other ingredients as essential ingredients other than those containing the above-mentioned effective ingredients. For example, various flavoring agents or natural carbohydrates may be added as an additional ingredient, such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. (Such as taurine, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (such as saccharin and aspartame) are advantageously used as flavorings other than those described above . The ratio of the natural carbohydrate can be appropriately determined by a person skilled in the art.

In addition to the above, the health functional food composition of the present invention can be used as a nutritional supplement, a vitamin, a mineral (electrolyte), a flavoring agent such as a synthetic flavoring agent and a natural flavoring agent, a coloring agent and a thickening agent (cheese, chocolate, Alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks and the like. These components can be used independently or in combination, and the proportion of such additives can also be appropriately selected by those skilled in the art.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Manufacturing Example]

Manufacturing example  1. Preparation of Compound I

1-1. Pestalotyopsis genus ( Pestalotiopsis  Identification and Culture of F12L0111 Strain

The sea surface samples obtained from sponges collected from Jeju Island were stored by freeze drying. The lyophilized sample was cut into small pieces and stamped on a glucose-yeast extract-peptone agar medium. The cells were cultured at 28 ° C for 7 days, Pure separation. Subsequently, the base sequence obtained through rRNA sequencing was searched in GenBank, and it showed 99% homology with the strain of Pestalotiopsis sp. Thereafter, the strain was transformed into a GPA solid containing 0.1% (w / v) glucose, 0.01% (w / v) yeast extract, 0.05% (w / v) peptone, 0.8% (w / The cells were cultured using a GPA liquid medium containing 0.1% (w / v) glucose, 0.01% (w / v) yeast extract, 0.05% (w / v) peptone as a medium and production medium. Specifically, the solid medium was cut and inoculated into each of 30 2-liter inoculum flasks containing 1 L of the production medium, and the cells were stationary cultured at room temperature for 18 days.

 In the following preparations and examples, the strain was designated as " Pestalotyrosis genus F12L011 ".

1-2. Separation and structural analysis of compounds

The cultured product of the Pestalothrixopsis strain F12L0111 cultured in Production Example 1-1 was extracted with ethyl acetate (60 L), and the obtained extract was concentrated by evaporation under reduced pressure using a vacuum dryer. The concentrate was adsorbed onto silica and subjected to silica vaccum column chromatography. At this time, methylene chloride / methanol (100 / 0-0 / 100, v / v) was used as a mixed solvent and eluted with increasing methanol concentration stepwise. Among them, the fraction was eluted with a mixed solvent containing methanol at a concentration of 50%. The fraction was concentrated under reduced pressure, and then eluted with a gradient of 5:95 to 20:80 using acetonitrile and water as a solvent, The compound was eluted by high performance liquid chromatography (column: Shim-pack ODS, length 250 mm, diameter 21.5 mm) under the condition of 6 ml / min to obtain a compound which exhibited a UV absorption peak at 210 nm in 32 minutes.

The molecular weight of the compound was confirmed through an ESIMS mass spectrometer and the ¹ H-NMR and ¹³ C-NMR spectra were analyzed by nuclear magnetic resonance (NMR) (Bruker 250 MHz NMR spectrometer) And the results of the ¹ H-NMR and ¹³ C-NMR spectra are shown in Table 1 below.

Based on the above experimental results, it was confirmed that the compound isolated from the culture of the strain Pestalotyopsis F12L0111 was 3,4-dihydroxybenzyl alcohol (Compound I) (Brown powder; UV (MeOH)? _Max (log?) 207 (3.9), 227 (3.7), 296 (3.5); LRESIMS m / z 163.1 [M + Na] ⁺ ).

Manufacturing example  2. Preparation and use of compounds II to X, etc.

2-1. Isolation and Structure Analysis of Compounds from Amomi Fructus

The sine (9.5 kg) was repeatedly extracted three times with methanol (15 L), and the obtained extract was evaporated under reduced pressure using a vacuum dryer to concentrate the extract (440 g). The concentrate was suspended in water and then separated into n-hexane, dichloromethane and ethyl acetate to obtain a n-hexane fraction (120 g), a dichloromethane fraction (32.7 g), an ethyl acetate fraction (9.8 g) and a water fraction ≪ / RTI > Silica column chromatography was performed with a dichloromethane fraction (32.7 g). At this time, dichloromethane / acetone (100 / 0-0 / 100, v / v) was used as a mixed solvent and eluted with increasing acetone concentration stepwise. Among them, the fraction was eluted with a mixed solvent containing 50% acetone. The fraction was concentrated under reduced pressure, and then eluted with a gradient of 30: 70-60: 40 using methanol and water as eluent, (4-hydroxy-benzaldehyde), compound III (3,4-dihydroxybenzaldehyde), which shows a UV absorption peak at 210 nm, was eluted by high performance liquid chromatography (column: Luna C18, length 250 mm, dihydroxy-benzaldehyde), compound IV (3,5-dimethoxy-4-methylbenzaldehyde) and compound V (4-hydroxy-3,5-dimethoxybenzaldehyde).

Further, the fraction eluted with 100% acetone was subjected to silica column chromatography. At this time, the fraction was divided into two fractions using dichloromethane / methanol (10/0, 9/1, v / v) as a mixed solvent, and a fraction eluted with dichloromethane / methanol (9/1, v / v) After concentration under reduced pressure, the residue was purified by high performance liquid chromatography (column: Luna C18, length 250 mm, diameter 21.2 mm) under the conditions of a gradient of 10: 90-55: 45 using methanol and water as a solvent at an elution flow rate of 6 ml / (4-hydroxybenzoic acid), compound VII (4-hydroxy-3-methoxybenzoic acid), compound X (2-hydroxy- 3-methoxybenzoic acid).

In addition, the ethyl acetate fraction (9.8 g) was subjected to silica column chromatography. At this time, dichloromethane / acetone (100 / 0-0 / 100, v / v) was used as a mixed solvent and eluted with increasing acetone concentration stepwise. The fraction eluted with 100% dichloromethane as a solvent was concentrated under reduced pressure. The residue was purified by high performance liquid chromatography (eluent: gradient of 20: 80-50: 50 using methanol and water at a flow rate of 6 ml / (Column: Luna C18, length 250 mm, diameter 21.2 mm) to obtain a compound IX (3-hydroxy-2-methoxybenzoic acid) exhibiting a UV absorption peak at 210 nm.

The ¹ H-NMR and ¹³ C-NMR spectra were analyzed by nuclear magnetic resonance (NMR) (Bruker 250 MHz NMR spectrometer) to confirm the molecular structure of the compound. The results of the ¹ H-NMR and ¹³ C- Are shown below.

2-2. Use of additional phenolic compounds

In order to compare and confirm the inflammatory response inhibitory effect of phenolic compounds having similar structures in addition to the compounds I to X, the compound XI (3-hydroxy-4-methoxybenzyl alcohol); Compound XII (4-methylcatechol); Compound XIII (4-hydroxy-3-methoxybenzyl alcohol); And compound XIV (3,4-dimethoxybenzyl alcohol) were obtained and used in the experiment.

[Example]

Example 1. Confirmation of inhibitory effect of mast cells on the secretion of inflammatory substances

1-1. Comparison of Secretion of Inflammatory Cytokines

In this Example, we examined the change in the secretion amount of inflammatory cytokine according to the treatment with the compound (Compound I-X) of the present invention in mast cells. To this end, the concentration of cytokines (TNF-α, IL-4 or IL-6) in the culture medium was calculated using enzyme-linked immunosorbant assay (San Diego, BD biosciences). Specifically, 200 μl of a sample diluted by the culture medium and the concentration was added to the wells to which the antigen was attached, and left. After 120 minutes, the cells were washed three times with a washing solution, and 100 μl of the primary antibody was added. The mixture was mixed again and left for 60 minutes. After washing three times with a washing solution, 100 μl of a mixed reagent mixed with a second antibody and an enzyme reagent was added. After the reaction was completed, the mixture was discarded, washed with the washing solution 4 times, and 100 μl of the substrate solution was added thereto. Thereafter, the mixture was allowed to stand at room temperature and dark room for 20 minutes. After the reaction, the reaction was stopped by adding 50 μl of quiescent reagent (H ₂ SO _4, 2N), and the absorbance at 450 nm was measured by an absorption analyzer (Sunnyvale, Molecular devices).

As a result, as shown in FIG. 3 or FIG. 4, the secretion amount of TNF-α, which was remarkably increased by treatment with DNP-HSA (Dinitrophenylated human serum albumin), was reduced by pretreatment of Compound I, IL-4 secretion was also decreased.

In addition, as shown in Fig. 5, the secretion amount of IL-6 was remarkably reduced by the pretreatment of Compound II-X, and the secretion amount of TNF-a was found to be significantly reduced in the group pretreated with Compound VII or X there was.

1-2. β- Hexosaminidase  Secretion comparison

In this example, we investigated the changes in the secretion of β-hexosaminidase in mast cells following treatment with the compound of the present invention (Compound I) in mast cells. First, 100 nM of the compound was pretreated with RBL-2H3 cells and sensitized with anti-DNP-HSA. Then, the cells were treated with DNP-HSA to activate the cells, and the intracellular granules were released. The amount of secreted β-hexosaminidase was measured by recovering the culture medium. Specifically, a sample of 40 μL was sampled, and the same amount of sodium citrate (0.1 M) and 4-p-nitrophenyl-N-acetyl-D-glucosamide (1 mM) were mixed and reacted at 37 ° C. for 1 hour. Thereafter _, the reaction was stopped by adding 200 μL of a mixed solution of sodium carbonate (Na ₂ CO _{3 ,} 0.1 M) and sodium bicarbonate (NaHCO _{3 ,} 0.1 M), and the absorbance at 405 nm was measured with an absorption analyzer (Sunnyvale, Molecular devices) Respectively.

As a result, as shown in FIG. 6, the secretion amount of? -Hexosaminidase, which was remarkably increased by DNP-HSA treatment, was reduced by pretreatment of Compound I, and in particular, suppression of Dexamethasone (1 μM) When compared to the activity, no significant difference was shown.

1-3. Comparison of secretion of histamine

In this example, the effect of the compound of the present invention (Compound I-X) on the mast cell was examined to determine the amount of histamine secreted in the mast cell. First, RBL-2H3 cells and RPMCs cells were pretreated with 100 nM of compound and sensitized with anti-DNP-IgE. Then, the cells were treated with DNP-HSA to activate histamine, and the histamine, which is the main ingredient of intracellular granules and the allergen inducing substance, was liberated, and the amount of histamine secreted was recovered. Specifically, a sample (200 μL) collected in an Eppendorf tube was added, and 80 μL of hydrochloric acid (0.1 N) and 20 μL of a 60% perchloric acid solution were mixed and then centrifuged (13,000 g, 20 minutes, 5415R, Eppendorf) . The resulting supernatant (200 μL) was placed in a test tube together with 100 μL of a NaOH solution (5 N), 800 L of n-butanol and 200 μL of a NaCl (5M) mixed solution, shaking it, (N-butanol) layer was obtained in the above-mentioned test tube, 200 μL of hydrochloric acid (0.1 N) and 500 μL of n-heptane were added thereto, and the mixture was shaken (1N), 10 μL of 1% o-phthalaldehyde solution (Sigma, Cat No. P1378) was added to the aqueous layer (150 μL) obtained by centrifugation (13,000 g, (Fluorescence spectrometer, GEMINIUM, manufactured by Molecular Devices) at an emission wavelength of 440 nm and an excitation wavelength of 360 nm by the addition of 20 μL of hydrochloric acid (3N). Then, .

As a result, as shown in Fig. 7 and Fig. 8, it was confirmed that the secretion amount of histamine, which was remarkably increased by DNP-HSA treatment, was reduced by pretreatment of Compound I or Compound IV-X.

The phenolic compounds of the present invention were able to effectively inhibit the secretion of inflammatory cytokines and histamine such as TNF-a, IL-4 and IL-6, It can be used as an effective material for

Example 2. Cytotoxicity test on synovial cells derived from arthritis patients

In this example, synovial cells derived from an arthritic patient were tested for MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) Respectively. Specifically, FLS cells (Kyungpook National University Hospital, Rheumatology Internal Medicine), human synovial cell line, were cultured in DMEM medium (Dulbecco's modified eagle medium (Cat No. 12800-017), Gibco Co. (USA)) supplemented with 10% 2 × 10 ⁴ cells / well. After the cells were cultured, Compound I was treated with 0.001-100 μM each and cultured again for 24 hours. Then, 20 L of MTT solution was added to each well, followed by further culturing for 2 hours. Then, the culture solution was removed, 100 L of dimethylsulfoxide was added, and the absorbance was measured at 570 nm.

As a result, as shown in Fig. 9, when the survival rate of the cells treated with the compound I was compared with that of the control group, no significant difference was observed between the treated group and the control group at both treatment concentrations of 0.001-100 μM. It was confirmed that there was almost no toxicity to the cells.

Example 3. Confirmation of Anti-inflammatory Effect on Synovial Cells Derived from Arthritis Patients

In this example, changes in inflammatory cytokines, proteolytic enzymes, and related factors were measured in synovial cells of arthritis patients to confirm the anti-inflammatory effect of Compound I treatment, and the following experiment was conducted .

3-1. Reagents and cell culture

Recombinant Human TNF-α Protein (Catalog # 210-TA) was purchased from R & D (USA). The human synovial cell line FLS cells (Kyungpook National University Hospital, Rheumatology Internal Medicine) were cultured in DMEM medium supplemented with 10% FBS (Dulbecco's modified eagle medium (Cat No. 12800-017), Gibco (USA)).

3-2. Analysis of mRNA expression of inflammatory cytokines and proteolytic enzymes

We investigated the effect of Compound I on the expression of TNF-α, IL-1, IL-6, and proteolytic enzymes MMP-1 and MMP-3, which are known to cause inflammation. First, synovial cells were treated with TNF-α (10 ng / ml) for 12 hours after treatment with Compound I (0.1, 1, 10 μM) and Maxime RT CDNA was synthesized using PreMix (Random primer, iNtRON Biotechnology). Specifically, 2 μL of cDNA, 1 μL of a sense and antisense primer solution (0.4 μM), 12.5 μL of SYBR Premix Ex Taq (Takarabio), and 9.5 μL of dH ₂ O were mixed together for each tube to give a total of 25 μL, The expression levels of TNF-α, IL-1, IL-6, MMP-1 and MMP-3 mRNA were measured by real-time PCR using TP850 software.

As a result, as shown in FIG. 10, the group treated with Compound I showed higher levels of inflammatory cytokines such as TNF-α, IL-1 and IL-6 and MMP -1, and MMP-3 was effectively inhibited, and this inhibitory effect was dependent on the concentration of Compound I.

3-3. PI3K , Akt , IKK -α / β, NF - κB , MMP1 / 3 Protein Expression Analysis

In order to elucidate the precise signal transduction pathway associated with anti-inflammatory effects, NF-κB, an important signaling substance that mediates the immune and inflammatory responses, and PI3K, Akt, To examine the effect of Compound I on the expression of IKK-alpha / beta. Inactivated NF-κB binds to the endogenous inhibitory protein IBα and remains inactive. When NF-κB is activated, IBα is liberated as it is phosphorylated and decomposed, and after transferring to the nucleus, acts as a transcription factor. The degree of decomposition was measured. In addition, Matrix metallo proteinase (MMP) is known to destroy cartilage matrix of joints, and the expression of the enzyme, which plays an important role in joint destruction, is measured. For this purpose, synovial cells were activated with TNF-α and activated with PI3K, Akt, IKK-α / β, NF-κB and MMP-1,3. Western blot analysis . Specifically, synovial cells were extracted with 0.5 mM phenylmethanesulfonyl fluoride (Sigma, Cat No. P7626) and 5 μg / mL leupeptin (Leupeptin, Sigma, Cat No. L2884) After dissolving in a buffer (PBS solution containing 0.5% triton X-100, 1 mM Na ₃ VO _4, etc.), the DNA was subjected to sonication. The amount of protein was measured using Bio-Rad's protein assay kit (cat No. 500-0002) with bovine serum albumin as a standard. 10-50 μg of total cell protein was added to 8 After electrophoresis on a 12% (w / v) polyacrylamide gel, proteins present in the gel were transferred to a nitrocellulose (NC) filter by electroblotting. Then, the nitrocellulose filter was placed in a tris-buffered saline-tween solution (TBS-tween, Sigma) containing 5% non-fat dry milk to prevent non-specific binding and allowed to react at room temperature for 1 hour. The filter was placed in a TBS-tween solution containing the antibody to the target protein and allowed to stand at 4 ° C for 12 hours, labeled with a secondary antibody labeled with HRP (horseradish peroxidase, Sigma, Cat No. P0889) Enhanced chemiluminescence, Thermo scientific Inc., Cat No. 34080).

As a result, as shown in Fig. 11, when the synovial cells were activated with TNF-α, the degradation of IB, the increase of NF-κB in the nucleus, the PI3K, Akt, IKK-α / -1, and MMP-3, whereas inhibition of IκBα degradation by TNF-α, reduction of NF-κB in the nucleus, inhibition of PI3K, In contrast, Compound I of the present invention has an inhibitory effect on the activity of NF-κB and higher signal transduction agents, as shown by the opposite effects such as reduction of Akt, IKK-α / β, MMP-1 and MMP-3 , Specifically, it is involved in gene expression of inflammatory cytokines in the nucleus.

3-4. Analysis of mRNA expression of inflammatory cytokines and substrateases of additional phenolic compounds

The effect of the compounds XI to XIV according to the present invention on IL-6, an inflammatory cytokine, and MMP-1, a substratease, was examined in the same manner as in Example 3-2. As a result, as shown in Fig. 12, unlike the compounds XI, XIII and XIV, in the group treated with the compound XII, the expression of IL-6, an inflammation-inducing cytokine and MMP-1, . In the case of MMP-1 expression, the inhibitory effect of Dexamethasone, which is a conventional therapeutic substance, was approximated.

As shown in FIG. 13, the expression of IL-6, MMP-1 and MMP-3 was observed after treatment of synovial cells with Compound XIV (0.1, 1, Dependent on the concentration of. From the above results, it was found that the inflammation-inhibiting effect of the present invention is derived from the difference in substituent and substitution position.

Example 4. Collagen-induced rheumatoid arthritis animal model to evaluate the efficacy of arthritis treatment

In this example, the therapeutic effect of Compound I on a representative inflammatory disease, rheumatoid arthritis, was investigated using a collagen-induced arthritis (CIA) mouse model of collagen-induced rheumatoid arthritis. Respectively.

4-1. Manufacture of collagen-induced rheumatoid arthritis animal model and evaluation of degree of deepening of arthritis

As a representative animal model for rheumatoid arthritis, the manufacturing process of an animal model of collagen-induced arthritis (CIA) is schematically shown in Fig. Specifically, a collagen mixture solution was prepared by mixing Type 2 collagen (bovine CII, Chondrex) and complete Freund adjuvant (Chondrex). The collagen mixture solution (100 μg) was mixed with DBA / 1J The mice were injected intracutaneously into the tail to induce primary immunity. After 21 days of primary immunization, an animal model of collagen-induced rheumatoid arthritis was prepared by mixing CII with incomplete Freund adjuvant, and then re-inoculating it for secondary immunization.

Compound I was dissolved in phosphate buffered saline (PBS) at a concentration of 2 mg / kg, 10 mg / kg or 50 mg / kg and then orally administered daily from day 28 of the first immunization to confirm the therapeutic effect of Compound I on rheumatoid arthritis . The degree of deepening of arthritis was evaluated by foot pad thickness, clinical arthritis index, incidence, and arthritis progression index was 04 (0: no edema, 1: light of one joint Edema, 2: moderate edema of 2 or more joints, 3: severe edema of most joints, 4: severe severe edema). As a comparative group, dexamethasone (Sigma), a commercially available arthritis treatment agent, was dissolved in phosphate buffered saline (PBS) at a concentration of 1 mg / kg and then orally administered.

As a result, as shown in Fig. 15 and Fig. 16, the thickness of the thickened foot and the index of arthritis progression by the induction of arthritis tended to decrease by treating Compound I, and as shown in Fig. 17, As compared with the control group, the compound I of the present invention was able to confirm the therapeutic effect of rheumatoid arthritis in vivo.

4-2. Micro positron emission tomography (PET) examination

An animal model of collagen-induced rheumatoid arthritis was anesthetized with isoflurane (1 to 2% oxygen conditions) and corrected for the imaging location. Various groups of micro PET images were obtained in tail vein via injection of [18 ^F ] FDG for 40 min (150 μl saline, 9.25 MB). The total administration time for each mouse was 20 minutes. Micro PET images were measured using an Inveon PET / CT scanner (Siemens Healthcare).

As a result of X-ray and micro PET image analysis, it was confirmed that the lesion area due to arthritis was effectively healed as shown in Fig.

4-3. Serum immunoglobulin G1 and G2a test

Collagen-induced rheumatoid arthritis animal model was inoculated and intraperitoneal blood was collected and centrifuged at 3,000 rpm for 15 minutes to separate the serum. Immunoglobulin G1 (IgG1) and G2a (IgG2a) in the serum were measured by BD Biosciences enzyme-linked immunosorbent assay (ELISA) kit. Specifically, Coating Buffer (NaHCO ₃ (8.40 g) in 1 L distilled water, Na ₂ CO ₃ (Purified rat anti-mouse IgG1, anti-mouse IgG2a) was diluted 1: 250 in each well, and 100 μL of each was dispensed into each well. Hr, and washed three times with wash buffer (0.05% Tween-20 PBS). Subsequently, 200 μL of Assay Diluent (10% FBS) was dispensed into each well, followed by reaction at 37 ° C. for 1 hour, which was then washed three times with washing buffer. Mouse serum was diluted 1: 300 in Assay Diluent, dispensed at 100 μL per well, reacted at 37 ° C for 2 hours, and washed 5 times. Detection antibody (Biotin rat-anti mouse IgG1, Bioninylatedanti-mouse IgG2a) And an enzyme reaction solution (SAv-HRP) were diluted in Assay Diluent at a ratio of 1: 250, and 100 μL of working solution was added to each well. The reaction solution was reacted at room temperature for 1 hour and then washed seven times for 1 minute each . Finally, 100 μL of the substrate solution was dispensed, and the plate was allowed to stand for 30 minutes while blocking the light. Then, 50 μL of the stop solution was treated, and the absorbance was measured at 450 nm in an ELISA reader.

As a result, as shown in Fig. 19 and Fig. 20, the concentration of IgG1 in the serum which was remarkably increased by the onset of arthritis was reduced by the treatment of Compound I, and the concentration of IgG2a was also decreased there was. In particular, when compared with the concentration of IgG1 or IgG2a in the case of treatment with the conventional anti-inflammatory drug Dexamethasone (1 mg), no significant difference was observed, and it was found that this effect tends to depend on the concentration of Compound I.

 4-4. T-cell phenotype analysis in inguinal lymph node

The collagen-induced rheumatoid arthritis animal model was opened to collect inguinal lymph nodes, and the lymph nodes were filtered with a filter. Thereafter, the RPMI (Roswell Park Memorial Institute medium) was treated to obtain cells present in the filter, and the cells were precipitated by centrifugation. Here, RPMI was added again, resuspended, and then transferred to a 6-well plate for stabilization. Then, the supernatant was separated, centrifuged to precipitate the cells, and re-suspended in phosphate buffered saline (PBS) to count the number of cells present in the 96-well. Next, the number of cells was fixed, and the cells were put into a flow cytometry tube, and the mouse phenotyping cocktail Cat. 51-9006631 was added, stabilized, and the phenotype of T cells was analyzed using FACS calibur .

As a result, as shown in Fig. 21 and Fig. 22, it was confirmed that the phenotype Th1 response and Th17 response, which were significantly increased by the onset of arthritis, were reduced by the treatment of Compound I.

4-5. Collagen induction Rheumatism  Assay inhibiting the secretion of inflammation-induced cytokines by arthritic mice

Ankle tissues were extracted from collagen-induced rheumatoid arthritis animal models. CDNA was synthesized using Maxime RT PreMix (Random primer, iNtRON Biotechnology) in order to measure the expression of inflammatory cytokines and proteolytic enzymes in ankle tissues. Specifically, 2 μL of cDNA, 1 μL of a sense and antisense primer solution (0.4 μM), 12.5 μL of SYBR Premix Ex Taq (Takarabio), and 9.5 μL of dH ₂ O were mixed together for each tube to give a total of 25 μL, The expression levels of inflammatory cytokines (TNF-α, IL-1, IL-6) and mast cell lysing enzymes (MMP-1 and MMP-3) mRNA were measured by real-time PCR using TP850 software.

As a result, as shown in FIG. 23, in the animal model of collagen-induced rheumatoid arthritis, the group treated with Compound I, like the result of Example 3-2, showed a higher level of inflammatory cytokines such as TNF- , And the expression of IL-6 and the proteolytic enzymes MMP-1 and MMP-3 were effectively inhibited, and the inhibitory effect was dependent on the concentration of Compound I.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (6)

A pharmaceutical composition for preventing or treating an inflammatory disease, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]

In Formula 1,
R ₁ represents hydrogen, hydroxy or C ₁ -C ₅ alkoxy;
R ₂ represents hydrogen, hydroxy, or C ₁ -C ₅ alkyl;
R ₃ and R ₄ independently represent hydrogen, hydroxy, or C ₁ -C ₅ alkoxy; And
R ₅ represents C ₁ -C ₅ alkyl, C ₁ -C ₅ alkyl alcohol, C ₁ -C ₅ alkylcarboxy, or C ₁ -C ₅ alkylaldehyde.
The method according to claim 1,
The compound represented by the general formula (1)
R ₁ represents hydrogen, hydroxy, or methoxy;
R ₂ represents hydrogen, hydroxy, methyl;
R ₃ and R ₄ independently represents hydrogen, hydroxy, or methoxy; And
And R ₅ represents methyl, methyl alcohol, carboxy, or aldehyde.
The method according to claim 1,
The compound represented by the general formula (1)
4-methylcatechol; 3,4-dihydroxybenzyl alcohol; 4-hydroxy-benzaldehyde; 3,4-dihydroxy-benzaldehyde; 3,5-dimethoxy-4-methylbenzaldehyde; 4-hydroxy-3,5-dimethoxybenzaldehyde; 4-hydroxybenzoic acid; 3,4-dihydroxybenzoic acid; 4-hydroxy-3-methoxybenzoic acid; 3-hydroxy-2-methoxybenzoic acid; And 2-hydroxy-3-methoxybenzoic acid. ≪ RTI ID = 0.0 > 11. < / RTI >
The method according to claim 1,
The inflammatory disease is selected from the group consisting of atopic, psoriasis, dermatitis, allergies, arthritis, rhinitis, otitis, sore throat, tonsillitis, cystitis, nephritis, pelvic inflammatory disease, Crohn's disease, ulcerative colitis, ankylosing spondylitis, systemic lupus erythematodes Wherein the disease is selected from the group consisting of asthma, edema, delayed allergy (type IV allergy), graft rejection, graft versus host disease, autoimmune encephalomyelitis, multiple sclerosis, inflammatory bowel disease, cystic fibrosis, diabetic retinopathy, ≪ / RTI >
The method according to claim 1,
Wherein said composition further comprises a pharmaceutically acceptable carrier.
The method according to claim 1,
Wherein said composition is administered simultaneously, separately, or sequentially with other anti-inflammatory agents.

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