KR20130010708A - Composition comprising the pyrezole derivatives for prevention and treatment of respiratory diseases - Google Patents

Abstract

PURPOSE: A composition containing pyrazole derivatives for preventing or treating respiratory diseases is provided to effectively suppress inflammation caused by immune response due to infection or allergen and to inhibit MUC5AC, MIP-2alpha, MIP-3alpha, MCP-1, and TNF-alpha. CONSTITUTION: A composition for preventing or treating respiratory diseases contains a pyrazole derivative selected from the group containing compound of chemical formulas 1-4 and 1'-4' or a salt thereof. The pyrazole derivative suppresses the expression of MUC5AC, MIP-2alpha, MIP-3alpha, MCP-1, and TNF-alpha. The composition contains 3-phenyl-1-(pyridine-2-yl)-1H-pyrazole-5-ol of chemical formula 1, 3-(3-methoxyphenyl)-1-(pyridine-2-yl)-1H-pyrazole-5-ol of chemical formula 2, 3-(4-nitrophenyl)-1-(pyridine-2-yl)-1H-pyrazole-5-ol of chemical formula 3, or 3-(furane-3-yl)-1-(pyridine-2-yl)-1H-pyrazole-5-ol of chemical formula 4, a pharmaceutically acceptable salt thereof, or a tautomer thereof.

Description

Composition comprising the pyrezole derivatives for prevention and treatment of respiratory diseases}

The present invention relates to a composition for preventing or treating respiratory diseases comprising a pyrazole derivative. More specifically, the present invention relates to compositions that can effectively prevent or treat respiratory diseases due to infection or allergens.

The present invention relates to a composition for the prevention and treatment of respiratory diseases comprising pyrazole derivatives.

With the development of industry, modern people are frequently exposed to allergic bronchial diseases caused by air pollution, allergens (animal hair, pollen, etc.), smoking, cold, laryngitis, acute laryngitis, bronchiolitis, pneumonia, asthma, tonsillitis, etc. The same respiratory disease is one of the most frequent diseases in developed countries and is reported to occur more frequently in children than in adults (Sears, 1997).

Each year, about 4 million of the 15 million children under age 5 die from acute resparatory disease, and more than 90% of these deaths occur in developing countries. In addition, such respiratory diseases are frequently occurring in areas where air pollution is serious.

The respiratory disease is due to inflammation by the immune response of the mucosal epithelial cells of the respiratory system to incoming substances from the outside. When an infection or allergen, such as a bacterium or virus, comes into contact with the mucosal tissue of the respiratory tract, a number of cytokines and mucins in the respiratory mucosal epithelial cells significantly increase, causing an inflammatory response. The cytokines and mucins Can serve as an immunological indicator for respiratory diseases caused by infections or allergens.

Thus, respiratory diseases caused by infections or allergens can be effectively prevented or treated by inhibiting inflammation due to immune responses in mucosal epithelial cells of the respiratory tract.

However, current therapeutic agents mainly used for such respiratory diseases are not effective treatment of respiratory diseases caused by infections or allergens with antibiotics or steroids.

KR 10-2010-0086186 KR 10-2010-0086158 KR 10-0987557 KR 10-0942382

Am J Physiol Lung Cell Mol Physiol 297: L1112-L1119, 2009 Holck et al., Basic & Clinical Pharmacology & Toxicology, 101, 455-458

The present invention is to provide a composition for preventing or treating respiratory diseases comprising a pyrazole derivative.

The present invention relates to a composition for preventing and treating respiratory diseases comprising pyrazole derivatives.

The composition for preventing or treating respiratory diseases of the present invention includes pyrazole derivatives represented by the following Chemical Formulas 1 to 4, their pharmaceutically acceptable salts, or tautomers thereof.

≪ Formula 1 >

<Formula 2>

<Formula 3>

&Lt; Formula 4 >

The composition according to the present invention comprising the compound represented by Chemical Formulas 1 to 4, pharmaceutically acceptable salts thereof, or tautomers thereof may effectively inhibit the inflammatory response of mucosal epithelial tissue of the respiratory tract.

Various microorganisms or allergens can cause various immune responses to mucous membranes of the respiratory tract, which are always open to the outside, whereby respiratory diseases can develop as an excessive inflammatory reaction proceeds. Therefore, various cytokines and mucins that cause inflammation due to immune responses in respiratory diseases caused by infection or allergens appear in large amounts in mucosal epithelial cells of the respiratory tract.

Cytokines are soluble proteins and glycoproteins that mediate inflammatory and immune responses that cause respiratory disease, including interleukins (IL-1 to IL-32), chemokines (IP-10, Rantes (RNATES), MIP-1α, MIP- 1β, MIP-2α, MIP-3α, MCP-1, MCP-2, MCP-3, MCP-4, Eotaxin, I-TAC, and BCA-1), and Type 1 Interferon (IFN-α, IFN- β, etc.), type 2 interferon (IFN-γ, etc.), tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-β), and colony stimulating factor (CSF).

In addition, mucin is a family of glycoproteins secreted from epithelial cells such as the respiratory tract, gastrointestinal tract, and female reproductive organs, and includes mucins such as MUC5AC and MUC5B. Many respiratory diseases, such as cystic fibrosis and bacterial infections, are characterized by the mass production of mucins (E. Prescott et al., Eur. Respir. J., 8, 1333-1338, 1995), Kim. K. Kim et al., Eur. Respir. J., 10, 1438, 1997 and D. Steiger et al. Am. J. Respir. Cell Mol. Biol., 12, 307-314. , 1995]. When mucin is damaged by hypersecretion of mucin, mucus plugging is formed, which promotes chronic infection and airflow obstruction and sometimes death.

Therefore, cytokines such as macrophage inflammatory protein-2α (MIP-2α), macrophage inflammatory protein-3α (MIP-3α), monocyte chemotactic protein-1 (MCP-1), and Tumor Necrosis Factor α (TNF-α) Mucins such as MUC5AC (Mucin-5AC) can function as an immunological indicator for respiratory diseases.

Compositions comprising the compounds represented by Formulas 1 to 4, pharmaceutically acceptable salts thereof, or tautomers thereof according to the present invention are the same as the MIP-2α, MIP-3α, MCP-1, TNF-α, etc. It effectively suppresses the expression of cytokines or substances such as mucins such as MUC5AC. In particular, the composition of the present invention is not only caused by an inflammatory response (Am J Physiol Lung Cell Mol Physiol 297: L1112-L1119, 2009) caused by a microbial infection such as flagellin, but also by an allergen such as curdlan included in house dust mites. Inflammatory responses (Holck et al., Basic & Clinical Pharmacology & Toxicology, 101, 455-458) can also be effectively suppressed.

Therefore, the composition according to the present invention can effectively prevent or treat respiratory diseases caused by allergens as well as respiratory diseases caused by bacterial or microbial infection. Specifically, the composition according to the present invention can effectively prevent or treat colds, rhinitis, sinusitis, upper respiratory tract infections, allergic rhinitis, asthma and the like.

The pyrazole derivatives represented by Formulas 1 to 4 included in the composition of the present invention are not limited thereto, but may be synthesized by a cyclization reaction of hydrazine and β-ketoester, as shown in Scheme 1 below.

<Reaction Scheme 1>

The cyclization reaction is Korean Patent No. 726,672, Min-Sup Park et al. Synthetic Communications 2004, 34, 1541-1550; Hyun-Ja Park, et. al. Bioorganic & Medicinal Chemistry Letters 2005, 15, 3307-3312 can be prepared by a method known. Originally according to Korean Patent No. 726,672, the product is expressed in keto form, but by the keto-enol tautomer reaction, the product according to the present invention mainly exists in the enol form.

In Scheme 1, R ¹ is pyridine, R ² is phenyl, methoxyphenyl, nitrophenyl, furanyl. Thus, for example, 2-hydrazinopyridine may be used as the hydrazine. In addition, the β-ketoester is for example ethyl benzoyl acetate, 3- (3-methoxyphenyl) -3-oxo-propionic acid ethyl ester, 3- (4-nitrophenyl) -3-oxo-propionic acid or Ethyl 3- (3-furyl) -3-oxopropanoate may be used. As the reaction solvent, ethanol or acetic acid is preferably used. The cyclization reaction is preferably carried out for 2 to 72 hours at a temperature of 100 to 130 ℃.

When R ¹ and R ² are as defined above, the pyrazole derivative included in the composition of the present invention may be represented by Chemical Formulas 1 to 4, and the keto type corresponding to the enol compound represented by Chemical Formulas 1 to 4 Each compound may be represented by the following Formulas 1 'to 4'. Enol-type and keto-type compounds in tautomeric relationships are known to have similar properties.

<Formula 1 '>

<Formula 2 '>

<Formula 3 '>

<Formula 4 '>

The present invention also provides a composition for the prevention or treatment of respiratory diseases comprising a compound selected from the compounds listed below, pharmaceutically acceptable salts thereof or tautomers thereof:

Formula 1 (3-phenyl-1- (pyridin-2-yl) -1H-pyrazol-5-ol);

Formula 2 (3- (3-methoxyphenyl) -1- (pyridin-2-yl) -1H-pyrazol-5-ol);

Formula 3 (3- (4-nitrophenyl) -1- (pyridin-2-yl) -1H-pyrazol-5-ol); And

Formula 4 (3- (furan-3-yl) -1- (pyridin-2-yl) -1H-pyrazol-5-ol).

In the present invention, pharmaceutically acceptable salts mean salts commonly used in the pharmaceutical industry, for example, inorganic acid salts prepared with hydrochloric acid, nitric acid, phosphoric acid, bromic acid, iodic acid, perchloric acid, tartaric acid and sulfuric acid, Acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid Sulfonic acid salts prepared with organic acid salts, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, and the like, aspartic acid, ascorbic acid, carbonic acid, vanic acid, and hydroiodic acid. The number of salts used in the present invention is not limited by these salts fluoro-enumerated. Preferably the pharmaceutically acceptable salt may be a hydrochloride salt.

Since the composition of the present invention inhibits the production of MUC5AC, MIP-2α, MIP-3α, MCP-1, TNF-α and the like, it can be usefully used for the treatment of respiratory diseases.

In the present invention, "respiratory disease" may be a cold, rhinitis, sinusitis, upper respiratory tract infection, allergic rhinitis.

The composition of the present invention may include additives such as pharmaceutically acceptable diluents, binders, disintegrants, lubricants, pH adjusting agents, antioxidants, dissolution aids and the like within the scope of not impairing the effects of the present invention.

The diluent may be sugar, starch, microcrystalline cellulose, lactose (lactose monohydrate), glucose, di-mannitol, alginate, alkaline earth metal salts, clay, polyethylene glycol, anhydrous calcium hydrogen phosphate, or a mixture thereof; Binders are starch, microcrystalline cellulose, highly dispersible silica, mannitol, di-mannitol, sucrose, lactose monohydrate, polyethylene glycol, polyvinylpyrrolidone (povidone), polyvinylpyrrolidone copolymer (copovidone), hypromellose , Hydroxypropyl cellulose, natural gum, synthetic gum, copovidone, gelatin, or a mixture thereof.

The disintegrating agent may be a starch or modified starch such as sodium starch glycolate, corn starch, potato starch or pregelatinized starch; Clay such as bentonite, montmorillonite, or veegum; Celluloses such as microcrystalline cellulose, hydroxypropyl cellulose or carboxymethyl cellulose; Algins such as sodium alginate or alginic acid; Crosslinked celluloses such as croscarmellose sodium; Gums such as guar gum and xanthan gum; Crosslinked polymers such as crosslinked polyvinylpyrrolidone (crospovidone); Effervescent agents such as sodium bicarbonate, citric acid, or mixtures thereof can be used.

The lubricant may be selected from the group consisting of talc, stearic acid, magnesium stearate, calcium stearate, sodium lauryl sulfate, hydrogenated vegetable oil, sodium benzoate, sodium stearyl fumarate, glyceryl behenate, glyceryl monolate, glyceryl monostearate, Stearate, colloidal silicon dioxide, or a mixture thereof may be used.

pH adjusters include acidifying agents such as acetic acid, adipic acid, ascorbic acid, sodium ascorbate, sodium ether, malic acid, succinic acid, tartaric acid, fumaric acid, citric acid (citric acid) and precipitated calcium carbonate, ammonia water, meglumine, carbonate Basic agents, such as sodium, magnesium oxide, magnesium carbonate, sodium citrate, and calcium tribasic phosphate, etc. can be used.

As the antioxidant, dibutylhydroxytoluene, butylated hydroxyanisole, tocopheryl acetate, tocopherol, propyl gallate, sodium hydrogen sulfite, sodium pyrophosphate and the like can be used. In the prior-release compartment of the present invention, Polyoxyethylene sorbitan fatty acid esters such as sodium lauryl sulfate and polysorbate, docusate sodium, poloxamer and the like can be used.

In addition, an enteric polymer, a water-insoluble polymer, a hydrophobic compound, and a hydrophilic polymer may be included to form a delayed-release preparation.

The enteric polymer is insoluble or stable under acidic conditions of less than pH 5, and refers to a polymer that is dissolved or decomposed under specific pH conditions of pH 5 or higher, and for example, hypromellose acetate succinate, hypromellose phthalate (hydrate Hydroxypropylmethyl cellulose phthalate), hydroxymethylethyl cellulose phthalate, cellulose acetate phthalate, cellulose acetate succinate, cellulose acetate maleate, cellulose benzoate phthalate, cellulose propionate phthalate, methyl cellulose phthalate, carboxymethyl ethyl cellulose and ethyl hydrate Enteric cellulose derivatives such as oxyethyl cellulose phthalate and methyl hydroxyethyl cellulose; Styrene-acrylic acid copolymers, methyl acrylate-acrylic acid copolymers, methyl methacrylate acrylic acid copolymers (e.g., acrylics), butyl acrylate-styrene-acrylic acid copolymers, and methyl acrylate-methacrylic acid-octyl acrylate copolymers. The enteric acrylic acid copolymer; Poly (methacrylate methyl methacrylate) copolymer (e.g. Eudragit L, Eudragit S, Evonik, Germany), poly (methacrylate acrylate) copolymer (e.g. Eudragit L100- Enteric polymethacrylate copolymers such as 55); Vinyl acetate-maleic anhydride copolymer, styrene-maleic anhydride copolymer, styrene-maleic acid monoester copolymer, vinylmethyl ether-maleic anhydride copolymer, ethylene-maleic anhydride copolymer, vinylbutyl ether-maleic anhydride copolymer, acrylic Enteric maleic acid copolymers such as ronitrile-methyl methacrylate-maleic anhydride copolymer and butyl styrene-styrene-maleic anhydride copolymer; And enteric polyvinyl derivatives such as polyvinyl alcohol phthalate, polyvinyl acetal phthalate, polyvinyl butyrate phthalate and polyvinyl acetal phthalate.

The water insoluble polymer refers to a polymer that is not soluble in pharmaceutically acceptable water that controls the release of the drug. For example, the water insoluble polymer may be polyvinylacetate (e.g. colicoat SR30D), water insoluble polymethacrylate copolymer [e.g. poly (ethylacrylate-methyl methacrylate) copolymer (e.g. Eudragit NE30D) , Poly (ethylacrylate-methyl methacrylate-trimethylaminoethyl methacrylate) copolymer (e.g., Eudragit RSPO), etc., ethyl cellulose, cellulose ester, cellulose ether, cellulose acylate, cellulose dicylate, Cellulose triacylate, cellulose acetate, cellulose diacetate and cellulose triacetate.

The hydrophobic compound refers to a substance that does not dissolve in pharmaceutically acceptable water that controls the release of the drug. Fatty acids and fatty acid esters such as, for example, glyceryl palmitostearate, glyceryl stearate, glyceryl bihenate, cetyl palmitate, glyceryl monooleate and threric acid; Fatty acid alcohols such as cetostearyl alcohol, cetyl alcohol and stearyl alcohol; Waxes such as carnauba wax, beeswax, and microcrystalline wax; Inorganic materials such as talc, precipitated calcium carbonate, calcium dihydrogen phosphate, zinc oxide, titanium oxide, kaolin, bentonite, montmorillonite, and gum.

The hydrophilic polymer refers to a polymeric material that is dissolved in pharmaceutically acceptable water that controls the release of the drug. Sugars such as, for example, dextrins, polydextrins, dextrans, pectins and pectin derivatives, alginates, polygalacturonic acids, xylans, arabinoxylans, arabinogalactans, starches, hydroxypropylstarches, amylose, and amylopectins ; Cellulose derivatives such as hypromellose, hydroxypropyl cellulose , hydroxymethyl cellulose, hydroxyethyl cellulose, methyl cellulose and carboxymethyl cellulose sodium; Gums such as guar gum, locust bean gum, tragacantha, carrageenan, acacia gum, gum arabic, gellan gum, and xanthan gum; Proteins such as gelatin, casein, and zein; Polyvinyl derivatives such as polyvinyl alcohol, polyvinyl pyrrolidone, and polyvinyl acetal diethylamino acetate; Poly (butyl methacrylate- (2-dimethylaminoethyl) methacrylate-methylmethacrylate) copolymer (e.g. Eudragit E100, Evonik, Germany), poly (ethyl acrylate-methyl methacrylate- Hydrophilic polymethacrylate copolymers such as triethylaminoethyl- methacrylate chloride) copolymers (eg, Eudragit RL, RS, Evonik, Germany); Polyethylene derivatives such as polyethylene glycol, and polyethylene oxide; Carbomer and the like.

In addition, a pharmaceutically acceptable additive may be selected and used in the preparation of the present invention as various additives selected from colorants and fragrances.

In the present invention, the range of the additives is not limited to the use of the above additives, and the above-mentioned additives may be formulated by containing a dose in a normal range by selection.

The present invention also relates to a method for treating or preventing a respiratory disease by administering to a subject a composition comprising a pyrazole derivative represented by Formulas 1 to 4, a pharmaceutically acceptable salt thereof, or a tautomer thereof.

&Lt; Formula 1 >

<Formula 2>

<Formula 3>

&Lt; Formula 4 >

The method of preventing or treating respiratory diseases using the composition of the present invention effectively inhibits the inflammatory response due to infection or allergic cognition, in particular, the production of MUC5AC, MIP-2α, MIP-3α, MCP-1, TNF-α, and the like. Can be effectively cured or prevented from respiratory diseases.

As used herein, the term "administration" means introducing a composition for preventing and treating the respiratory disease of the present invention to a patient by any suitable method, and the route of administration of the composition for preventing and treating the respiratory disease of the present invention reaches a target tissue. As far as possible, administration may be via any general route. Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, pulmonary administration, rectal administration, intranasal administration, intraperitoneal administration, intradural administration, but is not limited thereto. . The composition for preventing and treating respiratory diseases according to the present invention may be administered once or may be administered twice or three times at regular time intervals.

In consideration of the type or dosage form of the respiratory disease and the therapeutic effect, it is possible to properly administer the composition for preventing and treating the respiratory disease according to the present invention according to various methods commonly known to those skilled in the art, and preferably the respiratory disease according to the present invention. The prophylactic or therapeutic composition can be administered once daily or at least twice a day at regular time intervals.

The dosage of the pyrazole derivatives represented by Formulas 1 to 4 of the present invention, pharmaceutically acceptable salts thereof, or tautomers thereof is determined by weight, age, sex, health condition, diet, time of administration, method of administration, excretion of the patient. The range may vary depending on the rate and the severity of the disease, but is preferably 0.1 to 100 mg / kg / day, more preferably 10 to 40 mg / kg / day.

A composition comprising a pyrazole derivative, a pharmaceutically acceptable salt thereof, or a tautomer thereof according to the present invention can effectively inhibit an inflammatory response caused by an infection or allergen, thereby preventing or treating a respiratory disease. As a result, the expression of MUC5AC, MIP-2α, MIP-3α, MCP-1, and TNF-α, which are largely expressed in the inflammatory response due to the immune response of the respiratory mucosal tissue, can be significantly reduced, thereby effectively treating or preventing respiratory diseases.

1 is a graph showing the degree of inhibition of the expression of MUC5AC, MIP-2α, MIP-3α, MCP-1 and TNF-α induced by flagellin in the nasal mucosal tissue.
2 is a graph showing the degree of suppression of the expression of MIP-3α induced by curldan in the composition of the present invention in nasal mucosal tissue.

The following Preparation Examples and Examples will be described in more detail the present invention. These preparation examples and examples are only for illustrating the present invention, and the scope of the present invention is not limited by the following preparation examples and examples.

In addition, the reagents and solvents mentioned below were purchased from Sigma unless otherwise stated.

Manufacturing example  1.Formula 1 (3- Phenyl -1- (pyridin-2-yl) -1H- Pyrazole -5-ol)

Ethyl benzoyl acetate (1 equivalent) and 4 ml of ethanol were added to a round bottom flask, and 2-hydrazinopyridine (1.1 equivalent) was diluted in 3 ml of ethanol, and slowly added dropwise at 0 ° C, and heated to reflux for 20 minutes. After distillation under reduced pressure to remove the solvent, the resulting solid was washed with hexane and ethyl acetate and dried in vacuo to give the title compound in 87% yield.

Manufacturing example  2. Formula 2 (3- (3- Methoxyphenyl ) -1- (pyridin-2-yl) -1H- Pyrazole -5-ol)

Into a round bottom flask, 3- (3-methoxyphenyl) -3-oxo-propionic acid ethyl ester (1 equivalent) and 4 ml of ethanol were added thereto, and 2-hydrazinopyridine (1.1 equivalent) was diluted in 3 ml of ethanol. And slowly added dropwise at 0 ° C. and heated to reflux for 20 minutes. After distillation under reduced pressure to remove the solvent, the resulting solid was washed with hexane and ethyl acetate and dried in vacuo to give the target compound in a yield of 67%.

Manufacturing example  3. Formula 3 (3- (4- Nitrophenyl ) -1- (pyridin-2-yl) -1H- Pyrazole -5-ol)

In a round bottom flask, 3- (4-nitrophenyl) -3-oxo-propionic acid ethyl ester (1 equivalent) and 4 ml of ethanol were added thereto, and 2-hydrazinopyridine (1.1 equivalent) was diluted in 3 ml of ethanol. Slowly dropwise added at 0 ° C. and heated to reflux for 20 minutes. After distillation under reduced pressure to remove the solvent, the resulting solid was washed with hexane and ethyl acetate and dried in vacuo to give the title compound in 92% yield.

Manufacturing example  4. Formula 4 (3- ( Furan -3-yl) -1- (pyridin-2-yl) -1H- Pyrazole -5-ol)

In a round bottom flask, ethyl 3- (3-furyl) -3-oxopropanoate (1.2 equiv) and 3 mL of acetic acid were added, and a solution of 2-hydrazinopyridine (1 equiv) diluted in 2 mL of acetic acid was added. After slowly dropwise addition, the mixture was heated to reflux at 130 ° C. for 72 hours. After distillation under reduced pressure to remove the solvent, the resulting solid was washed with hexane and dried to obtain the target compound in 66% yield.

Example  1: determine the effect of preventing or treating inflammatory reactions 1

In order to investigate the effect of the pyrazole derivatives of the present invention on the respiratory inflammatory response in mice, the following experiment was performed.

Twenty four 8-week-old C57BL / 6 mice were divided into three groups, and Experimental Group 1 dispersed 10 µg of flagellin in sterile distilled water and prepared a solution having a concentration of 5 µg / ml of flagellin using physiological saline solution. This was applied to both nasal mucosa.

Experimental Group 2 was prepared in Formula 1, Formula 1 (3-phenyl-1- (pyridin-2-yl) -1H-pyrazole-5-ol (3-phenyl-1- (pyridine-2-yl)- 1H-pyrizol-5-ol)) was dissolved in DMSO (dimethylsufoxide) and a solution having a concentration of 100 μM was prepared using physiological saline solution. 25 μl each was administered to both sides of the mouse nasal cavity. Thereafter, 10 μg of flagellin was dispersed in sterile distilled water, and a solution having a concentration of flagellin of 5 μg / ml was prepared using physiological saline, and then, it was instilled on the nasal mucosa of both mice of Experimental Group 2, respectively. The control group was pretreated with both nasal passages only with physiological saline 1 hour prior to dropping flagellin in the mice of Experimental Group 1 and Experimental Group 2.

After 4 hours, the rats of the control group, the experimental group 1 and the experimental group 2 were sacrificed, the nasal mucosa tissue was extracted, RNA was extracted, and the cytokine cytokine (MIP2-α, MCP1, MIP3-α, TNF-α) as an inflammatory mediator. The expression level of MUC5AC was analyzed. The extracted mucosal tissue was pulverized with a trizol solution, pulverized, chloroform, and reacted for 10 minutes. The supernatant obtained by centrifugation at 14000 rpm at 4 ° C. for 20 minutes was precipitated using isoformaldehyde to extract RNA. It was.

From the obtained RNA, primers (ramdom hexamer preimer, PerkinElmer Life Sciences, Waltham, MA, USA, and Roche Applied Science, Indianapolis, IN, USA) and reverse transcriptase Moloney murine leukemia virus reverse transcriptase (PerkinElmer Life Sciences, Waltham, MA, USA) cDNA was made and the expression level of each gene was examined by real-time PCR (RT-PCR) method.

The expression levels of the MIP-2α and MUC5AC were measured by RT-PCT using a TaqManuiversal PCR master mix and PE Biosystems ABI PRISM 7700 sequence detection system (Foster City, CA, USA). The primers and probes used were purchased from Applied Biosystems, which were listed in Table 1 below, and RT-PCR was performed under the following reaction conditions: ① 2 minutes at 50 ° C., 10 minutes at 95 ° C., and ③ 57 ° C. Repeat 1 minute 40 times at 15 seconds and 60 ° C.

In addition, the amount of amplification products by RT-PCR was quantified by the CT method (comprative threshold method) and GAPDH was used as a control.

[Table 1]

On the other hand, MIP-3α, TNF-α and MCP-1 expression levels were measured by RT-PCT using SYBR Green qPCR kit (Finnzymes, Finland) and PE Biosystems ABI PRISM 7700 sequence detection system (Foster City, CA, USA). Measured. The primers used were those listed in Table 2 below, and RT-PCR was performed under the following reaction conditions: ① 2 minutes at 50 ° C., ② 10 minutes at 95 ° C., ③ 15 seconds at 57 ° C. and 1 at 60 ° C. Repeat 40 minutes.

In addition, the amount of amplification products by RT-PCR was quantified by the CT method (comprative threshold method) and GAPDH was used as a control.

[Table 2]

RNA expression levels of MIP-2α, MUC5AC, MIP-3α, TNF-α, and MCP-1 were measured using RT-PCR.

In Figure 1, the expression level of RNA was expressed as the reference value of the expression level of MIP2-α, MCP1, MIP3-α, TNF-α, MUC5AC RNA in the control group 1, the experimental group 1 and RNA expression was expressed as a relative ratio to the RNA expression of these factors in the control group.

As shown in Figure 1, compared with the control group not administered flagellin, experimental group 1 administered flageline significantly increased the expression of RNA of cytokines and mucins, from which flagellin administered to the nasal cavity of mice induced inflammation. Could know.

On the other hand, in the experimental group 2 administered the compound prepared in Preparation Example 1 before the flagellin administration, the RNA expression of cytokines and mucins was significantly lower than the experimental group 1 despite the administration of flagellin. From this, it can be seen that the compound of Preparation Example 1 effectively inhibits the inflammatory response induced in nasal tissue by flagellin.

Therefore, it can be seen that the compounds of the present invention can effectively prevent or treat many respiratory diseases by inhibiting inflammation in the respiratory tract.

Example  2: Identify the effect of preventing or treating an inflammatory response 2

In order to investigate the effect of the pyrazole derivatives of the present invention on the respiratory inflammatory response in mice, the following experiment was performed.

Nine eight-week-old C57BL / 6 mice were divided into three groups, and Experimental Group 1 dissolve 50 μg of curdlan in physiological saline and drop the solution at a concentration of 5 μg / ml directly to both nasal mucosa.

Experimental Group 2 was prepared in Formula 1, Formula 1 (3-phenyl-1- (pyridin-2-yl) -1H-pyrazole-5-ol (3-phenyl-1- (pyridine-2-yl)- 1H-pyrizol-5-ol)) was dissolved in DMSO (dimethylsufoxide) and a solution having a concentration of 100 μM was prepared using physiological saline solution. Each mouse was administered 25 μl each to the nasal mucosa. After administration of the compound of Formula 1, 50 μg of curdlan was dissolved in saline, and a solution having a concentration of 5 μg / ml was directly added to both nasal mucosa. The control group was pretreated with the nasal cavity only with physiological saline 1 hour before dropping curdlan in the experimental group 1 and the experimental group 2 mice.

After 4 hours, all the mice of the experimental groups 1, 2 and control were sacrificed to obtain intranasal mucosal tissue, RNA was extracted, and the expression level of the cytokine MIP3-α as an inflammatory mediator was measured.

The extracted mucosal tissue was pulverized with a trizol solution, pulverized, chloroform, and reacted for 10 minutes. The supernatant obtained by centrifugation at 14000 rpm at 4 ° C. for 20 minutes was precipitated using isoformaldehyde to extract RNA. It was.

MIP3-α expression from the extracted RNA was measured by the method substantially the same as the method of measuring the gene expression of MIP3-a in Experimental Example 1, the results are shown in Figure 2 below. In FIG. 2, the expression level of RNA was expressed as the reference value of MIP3-α RNA in the control group, and the expression level of MIP3-α RNA in the experimental group 1 and the experimental group 2 was MIP3-α RNA in the control group. The ratio is expressed relative to the amount of expression.

As shown in FIG. 2, the experimental group 1, in which curdlan was administered, significantly increased the RNA expression level of MIP-3a compared to the control group in which curdlan was not administered, indicating that curdlan administered to the nasal cavity of the mouse caused inflammation. Could.

On the other hand, in the experimental group 2 administered the compound prepared in Preparation Example 1 before the curdlan administration, the mRNA expression level of MIP-3a was significantly lower than the experimental group 1 despite the curdlan administration. From this, it can be seen that the compound of Preparation Example 1 effectively inhibits inflammation in nasal tissues induced by nasal tissues by curdlan.

Therefore, it can be seen that the compounds of the present invention can effectively prevent or treat many respiratory diseases by inhibiting inflammation in the respiratory tract.

A composition comprising a pyrazole derivative, a pharmaceutically acceptable salt thereof, or a tautomer thereof according to the present invention can effectively inhibit an inflammatory response caused by an infection or allergen, thereby preventing or treating a respiratory disease. As a result, the expression of MUC5AC, MIP-2α, MIP-3α, MCP-1, and TNF-α, which are largely expressed in the inflammatory response due to the immune response of the respiratory mucosal tissue, can be significantly reduced, thereby effectively treating or preventing respiratory diseases.

Claims (4)

A composition for preventing or treating respiratory diseases comprising a pyrazole derivative selected from the group consisting of compounds represented by the following Chemical Formulas 1 to 4 and the following Chemical Formulas 1 'to 4' or salts thereof:
&Lt; Formula 1 >

(2)

(3)

&Lt; Formula 4 >

<Formula 1 '>

<Formula 2 '>

<Formula 3 '>

<Formula 4 '>

The composition for preventing or treating respiratory diseases according to claim 1, wherein the pyrazole derivatives inhibit the expression of MUC5AC, MIP-2α, MIP-3α, MCP-1, and TNF-α. A composition for preventing or treating respiratory diseases comprising at least one compound selected from the group consisting of the compounds listed below, pharmaceutically acceptable salts thereof, and tautomers thereof.
Formula 1 (3-phenyl-1- (pyridin-2-yl) -1H-pyrazol-5-ol);
Formula 2 (3- (3-methoxyphenyl) -1- (pyridin-2-yl) -1H-pyrazol-5-ol);
Formula 3 (3- (4-nitrophenyl) -1- (pyridin-2-yl) -1H-pyrazol-5-ol); And
Formula 4 (3- (furan-3-yl) -1- (pyridin-2-yl) -1H-pyrazol-5-ol). The composition for preventing or treating respiratory diseases according to any one of claims 1 to 3, wherein the respiratory disease is cold, rhinitis, sinusitis, upper respiratory tract infection, allergic rhinitis or asthma.

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