KR100580953B1 - Novel Isoflavonone Derivatives which Inhibit the IL-5 Activity - Google Patents

Abstract

The present invention relates to novel isoflavone-based derivatives of low molecular weight and non-peptide-based substances as IL-5 inhibitors having a chronic allergic inflammatory effect, a method for preparing the same, and use as interleukin-5 (IL-5) inhibitors.

Since the novel isoflavone-based derivatives according to the present invention are low-molecular non-peptide-based substances, unlike the conventionally known allergic inflammation therapeutics, there is no nonspecific reaction to the protein, and thus may be useful as an allergic inhibitor.

Allergy, Inflammation, IL-5, Inhibitor

Description

Novel Isoflavonone Derivatives which Inhibit the IL-5 Activity}

The present invention relates to a novel isoflavone-based derivative of formula (1), a method for preparing the same, and a use thereof as an interleukin-5 (IL-5) inhibitor, and more particularly, an IL-5 inhibitor having a therapeutic effect of chronic allergic inflammation. The present invention relates to a method for designing and manufacturing a novel isoflavonone derivative, which is a low molecular weight non-peptide-based substance.

(Formula 1)

[Where R 1 = benzyloxy, benzyloxy, hydrogen or hydroxy substituted R 2 = benzyloxy, benzyloxy, hydrogen, methoxy or hydroxy substituted R 3 = hydroxy, —OCH 2 COOCH 3, —OCH 2 COOH, or Structure of formula (2)

(화학식 2)]

(Formula 2)]

Allergic diseases are known as bronchial asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, urticaria. Allergic diseases with very high incidences do not have adequate treatments, and most of them take a chronic course, causing problems such as pain and economic loss. Therefore, NIH is one of the five diseases to be overcome in the 21st century.

Allergies may cause symptoms such as itching due to early reactions, and epithelial cells of the target organs are deprived through late reactions, resulting in irritable local inflammation. The drugs used in the existing clinical use mainly cromoglycate, antihistamines, steroids, etc., developed as a membrane stabilizing agent for mast cells, and in case of asthma, adrenergic 2 agonist (salmetrol, etc.), anticholinergic drugs (ipratropium, etc.) xanthine derivatives (theophylline, etc.) are used, but they alleviate the symptoms and cannot provide fundamental treatment.

Allergy is an irritable disease and is accompanied by an inflammatory response. Inflammatory responses that occur after repeated exposure to antigens are associated with inflammation-related cells such as mast cells, eosinophils, and Th2 cells, and structures such as vascular endothelial cells, fibroblasts, and epithelial cells. It is caused by a complex interaction between sex cells.

Antigens are captured by antigen-presenting cells such as marcrophage, processed, and then released onto the surface of APC (Antigen-presenting cells). T-cells presented with antigen from APC secrete cytokine such as IL-4 to activate B cells, and B cells produce Ig E as antibodies. This Ig E is mixed with mast cells having an Ig E receptor on its surface. When combined with antigens, mast cells are activated, releasing chemicals such as histamine, prostaglandin (PG) and leukotriene (LT) to cause pathological symptoms. This leads to acute allergic inflammation.

Th2 cells exposed to antigens release cytokine such as IL-5, IL-3 and GM-CSF. In particular, IL-5 increases the differentiation of bone marrow cells into eosinophils and increases the adhesion of eosinophils to vascular endothelial cells, thereby increasing the migration of eosinophils to target organs, inhibiting apoptosis and activating eosinophils. It plays an important role in allergic reactions. Activated eosinophils release toxic proteins of major basic protein (MBP) and eosinophil cationic protein (ECP). These toxic proteins induce lipid mediators, cytokines, and chemokines, depriving allergic target organ epithelial cells of hypersensitivity. This causes chronic allergic inflammation such as bronchial asthma, which is a pathological symptom. Common inflammation is dominated by neutrophils, while allergic hypersensitivity is dominated by eosinophils. Therefore, it is well known that by inhibiting IL-5, it is possible to treat chronic allergic inflammation.

Development of a drug aimed at inhibiting eosinophils and IL-5 is being carried out by Schering-Plough to develop a single antibody of human IL-5 as an allergic agent, and Roche isothiazolone to inhibit IL-5. Derivatives (Devos et. Al., Europ. J. Biochem., 1994, 225, 635) have been synthesized, but are facing limitations in drug development due to non-specific responses to other proteins.

Therefore, the development of an allergy therapeutic agent capable of modulating the physiological activity of IL-5 requires a substance that can be orally administered as a substance having a low molecular weight, a selective and non-peptide inhibitory activity. Accordingly, an object of the present invention is a novel isoflavone-based derivative, a method for preparing the same, and an IL-5 inhibitor, in view of the fact that natural, low-molecular, non-peptide-based substance Sophoricoside, which is isolated from a painting tree, is effective as a selective IL-5 inhibitor. It is to provide a use as.

In order to achieve the object of the present invention as described above, the present invention provides a method for designing and preparing a novel isoflavone non-based derivative of Formula 1 and the use as an inhibitor by measuring the effect as an IL-5 inhibitor.

Compounds of formula (1) can be prepared according to the synthetic routes shown in formulas (1) and (2) from formula (3), and can be prepared according to scheme (3) from sophoricosides.

Compounds prepared according to Schemes 1, 2 and 3 are listed in Table 1.

Hereinafter, the present invention will be described in detail for the preparation method of each derivative and the measurement of IL-5 inhibitory effect.

(1) Synthesis of Novel Isoflavone-Based Derivatives of Chemical Formula 1-1 by Ring Closure Reaction of Chemical Formula 3

It is a method for synthesizing a novel isoflavone-based derivative of the general formula (1-1) by ring-closing a compound of the general formula (3) and thallium nitrate trihydrate in an alcohol solvent.

The alcohol solvent represents lower alcohols which are C1-C5 here. It is preferable that the usage-amount of thallium nitrate trihydrate is 2-4 equivalent normally.

The compound represented by Formula 1-1 is part of Formula 1.

(2) Preparation of benzyloxy-3- [4- (beta-di-glucopyranosyloxy) phenyl] chromen-4-one derivatives

Formula 1-1 is treated with glucopyranosyl bromo acetate in the presence of a catalytic amount of benzyl triethyl ammonium chloride and a base to give formula 1-2. The base is preferably potassium carbonate or sodium carbonate. Formula 1-2 and its derivatives may be obtained by deprotection reaction used in conventional organic chemistry.

(3) Preparation of benzyloxy-3- [4- (oxyranylmethoxy) phenyl] chromen-4-one derivative

Formula 1-1 is reacted with epichlorohydrin in the presence of a base to give an epoxymethyl derivative of the formula 1-4. The base used at this time is preferably an inorganic base such as potassium carbonate.

(4) Preparation of Derivative by Ring Opening Reaction of Compound of Formula 1-4

Formula 1-4 is reacted with anhydrous sodium acetate and titanium tetraisopropoxide in acetic acid to prepare a compound of formula 1-5. The compound of formula 1-6 and its derivatives can be obtained by the deprotection reaction used in conventional organic chemistry.

Compounds of formulas 1-4 are reacted with sodium acetate and titanium tetraisopropoxide in methanol to prepare compounds of formulas 1-7 and 1-8.

(5) Synthesis of Derivatives of Acetic Acid Derivatives 1-10 and 1-11

The compound of formula 1-1 is reacted with methyl chloroacetate in the presence of a base to prepare a compound of formula 1-9. At this time, it is preferable to use 2 equivalents of potassium carbonate as a base. It is preferable to use 1 equivalent of methyl chloro acetate.

Corresponding derivatives of Formulas 1-10 and 1-11 can be obtained by the deprotection reaction of compounds of Formulas 1-9 used in conventional organic chemistry.

(6) Preparation of Chemical Formula 1-12 from Sophoricoside

Sophoricoside is reacted with methyl iodine in the presence of a base such as potassium carbonate to give the compound of formula 1-12.

(7) Examination of inhibitory effect of IL-5

It was confirmed the inhibitory effect on the IL-5 of the compounds obtained through the above process.

Inhibitory effect was measured using Y16 cells and IL-5 of rats, and the inhibitory effect (%) and IC50 values at 50 uM concentration are shown in Table 3.

First, the sample was dissolved in 100% DMSO and dissolved to 100 mg / ml. The floating-proliferating floating cell Y16 cells were diluted to 1 × 10 ⁵ cells / mL in RPMI-8% FBS medium, and then 1 mL was dispensed into petri dish. Subsequently, 9 ml of RPMI-8% FBS medium and a final concentration of 5 U / ml of mIL-5 were added and then incubated at 5% CO 2, 37 ° C. After 48 hours, the cells were centrifuged at 1500 rpm and 4 ° C, and then suspended in 1 ml of medium and stained with trypan blue to count the number of cells. Dispense 1 × 10 ⁵ cells / ml of Y16 cells into 100 μl per well of a 96 well microplate, and serially dilute mIL-5 from 100 units to 0.003 units in 1/3, and add 100 μl per well. 5% CO2, 37 Incubated at ℃. After 48 hours, 20 μl of WST-1 solution was added to the above plate, and the absorbance was measured at 450 nm with a wavelength of 690 nm as a control using a microplate reader after 3-4 hours at 37% at 5% CO2.

The inhibitory effect of mIL-5 on the samples was evaluated using the proliferation of Y16 dependent on mIL-5.

100 μl / well of 1 × 10 ⁵ cells / ml of Y16 cells were dispensed per well, 50 μl of mIL-5 and 50 μl of sample were added. At this time, mIL-5 and the sample solution were diluted with RPMI-8% FBS medium, and the control group was added with the medium instead of the sample, and the control group was added with the IL-5 and the medium instead of the sample. Thus absorbance was measured after incubation.

The inhibition rate calculation for the sample was calculated by the following equation.

(8) Measurement of the bronchial asthma inhibitory effect of Compound 1 in Table 1

BALB / c mice were sensitized using egg yolk and Compound 1 was injected into the tail vein to a dose of 30 mg / kg. Then, airway resistance and airway hyperresponsiveness induced by inhalation of yolk in rats were measured, histological examination, yolk specific IgE antibody measurement, and blood eosinophil count. Through this experiment, it was confirmed that the compound 1 can be used as an asthma control agent and an inflammatory response inhibitor to improve airway hypersensitivity due to inhibition of the inflow of eosinophils into the airway and alleviate asthma symptoms.

As described above, the present invention provides a preparation method by designing a novel small isoflavone non-peptide derivative (Formula 1), and measures the IL-5 inhibitory effect of the substance to provide a use as an IL-5 inhibitor. .

The present invention will be described in more detail with reference to the following Examples. The following examples are merely examples for clearly describing the production method of the present invention, and the scope of the present invention is not limited or changed by the examples. In addition, it is easy for those skilled in the art to change and apply various conditions of the following examples within a reasonable range, and such modifications will be included in the technical scope of the present invention.

<Example>

Example 1 Preparation of 5-benzyloxy-3- (4-hydroxyphenyl) chromen-4-one

1.4 g (0.0036 mol) of 1- (2-benzyloxy-6-hydroxyphenyl) -3- [4- (methoxymethoxy) phenyl] propenone was dissolved in 400 ml of methyl alcohol. To this was added 3.19 g (2 equivalents) of thallium nitrate trihydrate and stirred for 24-48 hours at room temperature. The reaction mixture was depressurized without warming and removed until a solid precipitated in the solvent. Then, 25 mL of 2N hydrochloric acid (10% of methyl alcohol) was added thereto. After heating to 50 ° C. and stirring for 5 hours, the reaction was terminated by thin layer chromatography, and stirring was stopped. After filtration, the reaction mixture was removed by concentration under reduced pressure without heating. It was dissolved in chloroform and washed twice with distilled water. The chloroform layer was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford the crude product. The crude product was purified by column chromatography to give 860 mg of pure yellow 5-benzyloxy-3- (4-hydroxyphenyl) chromen-4-one (Table 1, Compound No. 1). The physical properties, nuclear magnetic resonance spectral data and infrared spectrospectral data of the resulting 5-benzyloxy-3- (4-hydroxyphenyl) chromen-4-one are listed in Tables 1 and 2.

Example 2 Preparation of 7-benzyloxy-3- (4-hydroxyphenyl) chromen-4-one

 Table 1 No. 2 using 1- (4-benzyloxy-6-hydroxyphenyl) -3- [4- (methoxymethoxy) phenyl] propenone as the corresponding starting material according to the same method as in Example 1 The compound of, 7-benzyloxy-3- (4-hydroxyphenyl) chromen-4-one was prepared. Physical properties, nuclear magnetic resonance spectral data, and infrared spectrospectral data of the resulting 7-benzyloxy-3- (4-hydroxyphenyl) chromen-4-one are shown in Tables 1 and 2.

Example 3: Preparation of 5-benzyloxy-3- [4- (beta-D-glucopyranosyloxy) phenyl] chromen-4-one derivatives

(1) 5-benzyloxy-3- (4-hydroxyphenyl) chromen-4-one obtained in Example 1 (Table 1, Compound No. 1) 78 mg (0.23 mmol) and glucopyranosylbromo acetate 3 Equivalent weight (233 mg) was dissolved in 6 ml of chloroform. To this was added 0.2 equivalent (10 mg) of benzyl triethyl ammonium chloride, 6 equivalents (188 mg) of potassium carbonate and 9 µl of distilled water corresponding to 5% of potassium carbonate, and the reaction mixture was stirred at room temperature. After the reaction for 1.5-2 days, the reaction was terminated by thin layer chromatography. The solvent was removed by concentration under reduced pressure, dissolved in chloroform, and washed with 10% aqueous sodium hydrogen carbonate solution. The chloroform layer was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford the crude product. The crude product was isolated by column chromatography and purified to give pure 5-benzyloxy-3- [4- (2 ', 3', 4 ', 6'-tetra-O-acetyl-beta-di-glucopyranosiloxy ) Phenyl] chromen-4-one (Table 1, 2 Compound No. 3) 124 mg was obtained.

(2) 5-benzyloxy-3- [4- (2 ', 3', 4 ', 6'-tetra-O-acetyl-beta-di-glucopyranosyloxy) phenyl] chromen-4 thus obtained After dissolving 359 mg (0.54 mmol) of -one in 10 ml of methyl alcohol, 5 equivalents of sodium methoxide (147 mg) was added thereto, followed by stirring at room temperature. After stirring for 1 hour, the solvent was removed under reduced pressure. The reaction mixture was separated and purified by column chromatography to purify pure 5-benzyloxy-3- [4- (beta-D-glucopyranosyloxy) phenyl] chromen-4-one (Table 1, Compound No. 4) 221 mg. Obtained.

(3) 5-benzyloxy-3- [4- (2 ', 3', 4 ', 6'-tetra-O-acetyl-beta-di-glucopyranosyloxy) phenyl] chromen-4-one ( 0.10 mmol, Table 1, Compound No. 3) 70 mg was dissolved in 50 ml of methyl alcohol. 10 mg of 10% palladium-carbon was added thereto, suspended and stirred under a hydrogen stream. After reacting for 1 hour 30 minutes, the reaction mixture was filtered through celite, and the filtrate was removed under reduced pressure. The reaction mixture was separated and purified by short column chromatography to give pure 5-hydroxy-3- [4- (2 ', 3', 4 ', 6'-tetra-O-acetyl-beta-di-glucopyranosiloxy) Phenyl] chromen-4-one (Table 1, 2 compound number 5) 46 mg was obtained.

(4) 70 mg (0.14 mmol) of 5-benzyloxy-3- [4- (beta-di-glucopyranosyloxy) phenyl] chromen-4-one were dissolved in 50 ml of methyl alcohol. 10 mg of 10% palladium-carbon was added thereto, suspended and stirred under a hydrogen stream. After reacting for 1 hour 30 minutes, the reaction mixture was filtered through celite, and the filtrate was removed under reduced pressure. The reaction mixture was separated and purified by short column chromatography to give pure 5-hydroxy-3- [4- (beta-di-glucopyranosyloxy) phenyl] chromen-4-one (Table 1, 2 Compound No. 6). 46 mg were obtained.

Physical properties, nuclear magnetic resonance spectral data, and infrared spectroscopic spectrum data of the compounds of Compound Nos. 3 to 6 are listed in Tables 1 and 2.

Example 4: Preparation of 5-hydroxy-7-methoxy-3- [4- (beta-di-glucopyranosyloxy) phenyl] chromen-4-one

500 mg (1.16 mmol) of sophoricoside was dissolved in 5 ml of acetone and 2 ml of distilled water. To this was added 3 equivalents of potassium carbonate (639 mg) and 3 equivalents of methyl iodine (215 μl). The mixture was stirred for 1 day while heating to reflux. The mixture was cooled and concentrated under reduced pressure to remove the solvent. The reaction was dissolved in ethyl acetate and washed twice with 10% aqueous sodium hydrogen carbonate solution, the ethyl acetate layer was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a crude product. The crude product was isolated by column chromatography and purified to give the pure off-white product 5-hydroxy-7-methoxy-3- [4- (beta-di-glucopyranosyloxy) phenyl] chromen-4-one ( Compound number 7) 283 mg was obtained.

Example 5 Preparation of 5-benzyloxy-3- [4- (2,3-dihydroxypropoxyphenyl] chromen-4-one derivatives

 (1) Dissolve 121 mg (0.35 mmol) of 5-benzyloxy-3- (4-hydroxyphenyl) chromen-4-one (Table 1, Compound No. 1) in 15 ml of anhydrous acetnitrile, followed by potassium carbonate 1.5. Equivalent weight (73 mg) was added and stirred. After 1 hour, 3 equivalents of epichlorohydrin was added thereto and stirred at 60 ° C. After 2 days, the reaction was terminated, and the solvent of the reaction product was concentrated under a reduced pressure, and then dissolved in dichloromethane, followed by washing with an aqueous 10% sodium bicarbonate solution. The dichloromethane layer was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product. The crude product was separated and purified by column chromatography to obtain 80 mg of pure 5-benzyloxy-3- [4- (oxyranylmethoxy) phenyl] chromen-4-one (Compound No. 8).

(2) Dissolve 100 mg (0.25 mmol) of 5-benzyloxy-3- [4- (oxyranylmethoxy) phenyl] chromen-4-one in 10 ml of acetic acid and add 5 equivalents of sodium acetate anhydrous (102 mg). One drop of tanium tetraisopropoxide was added and stirred. After reacting for 6 hours while heating to 65 ℃, the reaction was terminated by thin layer chromatography, the solvent was removed by concentration under reduced pressure, dissolved in chloroform and washed with distilled water. The chloroform layer was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford the crude product. The crude product was separated by column chromatography to obtain yellow pure 2- [4- (5-benzyloxy-4-oxo- 4H -chromen-3-yl) phenyloxy] -1-hydroxymethylethyl acetate ( 62 mg of compound No. 9 was obtained.

(3) 5 ml of methyl alcohol (62 mg, 0.13 mmol) of 2- [4- (5-benzyloxy-4-oxo- 4H -chromen-3-yl) phenyloxy] -1-hydroxymethylethyl acetate After dissolving in, 1 equivalent of sodium methoxide was added thereto and stirred. After stirring for 1 hour at room temperature, the termination was confirmed and concentrated under reduced pressure to remove the solvent. It was dissolved in chloroform and washed with distilled water. The chloroform layer was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford the crude product. The crude product was separated by column chromatography to give 53 mg of yellow pure 5-benzyloxy-3- [4- (2,3-dihydroxypropoxy) phenyl] chromen-4-one (Compound No. 10). It was.

(4) 90 mg (0.22 mmol) of 5-benzyloxy-3- [4- (oxyranylmethoxy) phenyl] chromen-4-one (Table 1, Compound No. 1) was dissolved in 10 ml of methyl alcohol, followed by 5 equivalents of sodium acetate (92 mg) and 1 drop of titanium tetraisopropoxide were added thereto, followed by stirring. After reacting for 6 hours while heating to 65 ℃, the reaction was terminated by thin layer chromatography, the solvent was removed by concentration under reduced pressure, dissolved in chloroform and washed with distilled water. The chloroform layer was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford the crude product. The crude product was separated by column chromatography, and 13.5 mg of yellow pure 5-benzyloxy-3- [4- (2-hydroxy-3-methoxy) phenyl] chromen-4-one (Compound No. 11) was used. 18 mg of 2- [4- (5-benzyloxy-4-oxo-4 H-chromen- 3-yl) phenoxy] -2-hydroxypropyl acetate (Compound No. 12) were obtained.

Physical properties, nuclear magnetic resonance spectral data, and infrared spectroscopic spectrum data of the 5-benzyloxy-3- [4- (2,3-dihydroxypropoxyphenyl] chromen-4-one derivatives obtained are shown in Tables 1 and 2. Described.

Example 6: Preparation of 7-benzyloxy-3- [4- (2,3-dihydroxypropoxyphenyl] chromen-4-one derivatives

Using the same method as in Example 5, using 7-benzyloxy-3- (4-hydroxyphenyl) chromen-4-one prepared in Example 2 as a starting material according to the same method as in Example 5. Compounds Nos. 13 to 15 of compounds 1 and 2 were prepared.

Example 7: [4- (5-hydroxy-4-oxo-4 H Preparation of -Cromen-3-yl) phenoxy] acetic acid derivatives

(1) 300 mg (0.87 mmol) of 5-benzyloxy-3- (4-hydroxyphenyl) chromen-4-one (Compound No. 1) were dissolved in 40 ml of anhydrous acetnitryl, followed by 1.2 equivalents of potassium carbonate 145 mg (1.05 mmol) was added. After stirring at 60 ° C. for 1 hour, 1.2 equivalents (114 mg, 1.05 mmol) of methyl chloroacetate were added thereto, followed by stirring at 65 ° C. for 2 hours. After the reaction was completed under reduced pressure to remove the solvent. It was dissolved in chloroform and washed with 5% aqueous sodium hydroxide solution. The chloroform layer was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford the crude product. The crude product was separated by column chromatography to give 275 mg (0.66) of yellow pure methyl [4- (5-benzyloxy-4-oxo- 4H -chromen-3-yl) phenoxy] acetate (Compound No. 16). Mmol).

(2) 100 mg (0.24 mmol) of the obtained methyl [4- (5-benzyloxy-4-oxo- 4H -chromen-3-yl) phenoxy] acetate was dissolved in 30 ml of methanol. To this was added 3 equivalents of potassium hydroxy 40 mg (0.72 mmol) and a small amount of distilled water (3 drops) and stirred at room temperature. The reaction was carried out for 12 hours, and then concentrated under reduced pressure to remove the solvent. After dissolving in 5% sodium hydroxide aqueous solution, washed with chloroform to remove the starting material. The aqueous layer was acidified with 2N aqueous hydrochloric acid solution and dissolved in chloroform. The chloroform layer was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product. The crude product was isolated by column chromatography to give 86 mg (0.21 mmol) of yellow pure [4- (5-benzyloxy-4-oxo-4-H-chromen-3-yl) phenoxy] acetic acid (Compound No. 17). ) Was obtained.

(2) 80 mg (0.20 mmol) of [4- (5-benzyloxy-4-oxo-4H-chromen-3-yl) phenoxy] acetic acid were dissolved in 30 ml of methyl alcohol. 8 mg of 10% palladium-carbon was added thereto, suspended and stirred for 2 hours under hydrogen gas. After the reaction was filtered in celite. The filtrate was removed under reduced pressure to give crude product. The crude product was filtered by short column chromatography to give yellow pure [4- (5-hydroxy-4-oxo-4H-chromen-3-yl) phenoxy] acetic acid (Compound No. 18). 55.2 mg (0.18 mmol) were obtained.

Physical properties, nuclear magnetic resonance spectral data, and infrared spectrospectral data of the resulting [4- (5-hydroxy-4-oxo-4H-chromen-3-yl) phenoxy] acetic acid are shown in Tables 1 and 2 It was.

Example 8: [4- (7-hydroxy-4-oxo-4 H Preparation of -Cromen-3-yl) phenoxy] acetic acid derivatives

 Example starting material 5-benzyloxy-3- (4-hydroxyphenyl) chromen-4-one (Compound No. 1) was replaced with 7-benzyloxy-3- (4-hydroxy according to the same method as in Example 7. The compound of Nos. 19 to 21 of the compounds of Tables 1 and 2 was prepared by replacing the phenyl) chromen-4-one (Compound No. 2).

Example 9 Measurement of Inhibitory Effect of IL-5 (Table 3)

1) Sample Dissolution and Dilution

The sample was dissolved in 100% DMSO and used to dissolve to 100 mg / ml.

2) Subculture of Cell Line Y16

The Y16 cell used in the experiment was a floating cell that floated in a medium and diluted to 1 × 10 ⁵ cells / ml with RPMI-8% FBS medium, and then dispensed in 1 ml in a petri dish and 9 ml RPMI-8%. FBS medium and final concentration of 5 U / ml of mIL-5 were added, followed by incubation for 48 hours at 5% CO2, 37 ° C. The cells were precipitated by centrifugation at 1500 rpm and 4 ° C, suspended in 1 ml of medium, and stained with trypan blue to determine the number of cells.

3) Measurement of proliferation of Y16 cells dependent on mIL-5

Dispense 1 × 10 ⁵ cells / ml of Y16 cells into 100 μl per well of a 96 well microplate, and serially dilute mIL-5 from 100 units to 0.003 units in 100 μl of each well. 5% CO2, 37 Incubated for 48 hours at ℃. After 48 hours, 20 μl of WST-1 solution was added to the above plate, and the absorbance was measured at 450 nm with a wavelength of 690 nm as a control using a microplate reader after 3-4 hours at 37% at 5% CO2.

4) Measurement of mIL-5 bioassay inhibition by sample

The inhibitory effect of mIL-5 on the samples was evaluated using the proliferation of Y16 dependent on mIL-5.

100 μl / well of 1 × 10 ⁵ cells / ml of Y16 cells were dispensed, and 50 μl of mIL-5 and 50 μl of sample were added. At this time, mIL-5 and the sample solution were diluted with RPMI-8% FBS medium, and the control group was added with the medium instead of the sample, and the control group was added with the IL-5 and the medium instead of the sample.

Thus absorbance was measured after incubation under the same conditions as 3) above.

The inhibition rate calculation for the sample was calculated by the following equation.

5) Measurement of bronchial asthma inhibitory effect of compound 1 of Tables 1 and 2

 (1) Experiment Method

A) Animal sensitization and induction test

6-8 years old, male BALB / c weighing 25-30 gr was used. Sensitization was performed yolk (10 ug / 100 ul) and Imject Alum (Pierce, USA) by mixing 100 ul 4 times (first, fifth, day 14, day 21) into the peritoneal cavity. Mice were inhaled with a 2% yolk solution for 10 minutes once daily, one week after the last intraperitoneal injection. One week after the last local sensitization, yolk bronchial induction was performed by inhalation with 2% yolk for 2 minutes.

B) classification of experimental groups

Normal control muscles (NC) consisted of five mice, systemically and locally sensitized, with no naive and physiological saline at all, and positive control muscles (PC) with egg yolk, 10 systemically and locally sensitized, and the drug-treated group with egg yolk. The sensitized compounds were divided into 10 groups of 30 mg / kg administered group (JSH II-3) and 10 groups administered 4% DMSO (dimethylsulfoxide) as a solvent. Drug administration was injected into the tail vein 1 hour prior to the challenge test.

C) Airway resistance measurement

Mice were placed in an animal lung function test chamber for 30 minutes after the antigen-induced test, and the animals were allowed to rest for 10 minutes. Thereafter, Penh (P enhance pause), which was automatically measured at 10 second intervals for 30 minutes, was calculated by calculating the arithmetic mean at 5 minute intervals for 30 minutes, and early asthmatic reactions by allergens were observed. The presence of early response was determined by the presence of Penh-2 in Penh that increased more than twice as much as before inhalation within 30 minutes of egg yolk inhalation.

D) Airway hypersensitivity measurement

Airway hyperresponsiveness, a basic pathophysiology of asthma, was measured before and after allergen bronchial induction, and then divided by whether compound 1 can suppress airway hypersensitivity aggravated by late reaction by allergen.

Nonspecific airway hypersensitivity was measured by methacholine bronchial challenge test. The concentration of methacholine was inhaled from 2.5 mg / ml, diluted 16-fold to 50 mg / ml, and penh was measured for 5 minutes after inhalation of methacholine. The concentration of Penh-2 was measured and observed before and after antigen inhalation.

E) cleaning of the alveoli

Twenty four hours after the induction test, pentobarbital sodium (30 mg / kg) was infused into the abdominal cavity for deep anesthesia, followed by a thoracic incision, blood puncture with a blood puncture, a smear sample was made, and eosinophils were measured. 1800 rpm, 4oC, 5 minutes) serum was stored at -80oC.

After ingesting the blood, the airway was cut and intubated, and 1 ml of warm saline was injected into the highway and the alveolar lavage fluid was collected. The test was repeated twice. Supernatant after centrifugation was stored at -80 ° C. The total number of cells was measured by hemocytometer, and cytospine was made and analyzed by measuring the fraction of cells including eosinophils.

F) biopsy

The lung was infused with 4% formalin dyddoir, and then dissected and stored in 10% formalin solution. Tissue sections were made and measured by an optical microscope at 10 times under 400 magnification, measuring the nasal mucosal deprivation and eosinophils infiltrated into the nasal cavity.

G) Measurement of egg yolk specific IgE antibody

It was measured by enzyme immunoassay. In summary, 100 ug (200 ul in PBS) of yolk was added to a 96 well plate and left at 4 degrees for 18 hours. After washing with PBS three times, 5% skim milk was added to block nonspecific reactions and incubated at room temperature for 1 hour. After washing, 100 ul of mouse serum was added and the reaction was performed at room temperature for 1 hour. For the color reaction, 100 μl of HRP-conjugated rat anti-mouse monoclonal IgE antibody diluted 1: 1000 was reacted at room temperature for 1 hour and then washed. 0.04% OPD (O- phenyldiamine, in After the color reaction was induced with citrate phosphate buffer and 30% hydrogen peroxide solution, the optical density (OD) was measured at 492 nm using a spectrophotometer (BEP II plus, Germany).

H) measurement of eosinophils in blood;

Staining specimens were Wright-Giemsa stained and measured a total of 100 leukocytes under 1000-fold by light microscopy, and the percentage of eosinophils was calculated and analyzed.

I) Statistical processing

SAS was used and the difference between symptom index and eosinophil count was analyzed using Wilcoxon's rank sum test. p <.05 was determined to be significant and the values were expressed as mean ± standard deviation.

(2) measurement result of effect

A) Measurement result of egg yolk specific IgE antibody

The optical density (OD) of egg yolk-specific IgE antibodies measured by enzyme-immunoassay was 0.11 ± 0.07 in the control group, whereas that in the yolk-sensitized group was significantly increased to 0.37 ± 0.11 (p <0.05). It was confirmed that the yolk was sensitized by the method.

B) Effect of compound 1 on the number of peripheral blood eosinophils

Compound 1 injected 1 hour before the allergen challenge did not affect the peripheral blood eosinophil count after 24 hours.

C) Effect of Compound 1 on eosinophil count in bronchoalveolar lavage fluid

The eosinophil counts in the bronchial alveolar lavage fluid, which occurred at 24 hours of allergen-induced test, were 33 ± 5.6 for the negative control group, while 78 ± 8.9 for the positive control group sensitized with egg yolk, and were treated with 4% DMSO. In case of pretreatment with Compound 1, it was significantly decreased to 42 ± 5.6 (p <0.05). This shows that Compound 1 inhibits the influx of eosinophils into the airways by allergens.

D) Effect of Compound 1 on Early Reaction by Allergens

In the negative control group, no response was induced by allergen inhalation, whereas the positive control group showed early asthmatic reaction, indicating that the animal model of this study is an asthma model. Pretreatment with 4% DMSO or Compound 1 did not inhibit premature response.

E. Effect of Compound 1 on Nonspecific Airway Hypersensitivity

Treatment with positive control and 4% DMSO increased nonspecific airway hypersensitivity after inhalation compared to before allergen inhalation, resulting in allergen-induced asthma exacerbation (p> 0.05). Airway hypersensitivity was reduced to prevent exacerbation of allergens (p <0.05).

In summary, the novel isoflavone-based derivatives (for example, Compound 1) according to the present invention inhibit the inflow of eosinophils, which are the main inflammatory cells of the asthma reaction, and improve airway hypersensitivity to relieve asthma symptoms. It is effective as an asthma symptom modulator and inflammatory response inhibitor.

Since the novel isoflavone-based derivatives according to the present invention are low-molecular non-peptide-based substances, unlike the conventionally known allergic inflammation therapeutics, there is no nonspecific reaction to the protein, and thus may be useful as an allergic inhibitor.

Claims (10)

Following isoflavone non derivatives of the general formula (1).

(Formula 1) [Where R ₁ = benzyloxy or hydrogen, R ₂ = benzyloxy or hydrogen and R ₁ ≠ R ₂ , R ₃ = hydroxy, -OCH ₂ COOCH ₃ , -OCH ₂ COOH, or structure of formula 2

(Formula 2)] (1) preparing an isoflavonone derivative by a ring closure reaction in which the compound of formula 3 and thallium nitrate trihydrate are closed in an alcohol solvent;

(Formula 3) [Where R ₁ = benzyloxy or hydrogen, R ₂ = benzyloxy or hydrogen and R ₁ ≠ R ₂ ] (2) treating glucopyranosyl bromo acetate in the presence of a product produced in step 1 with a catalytic amount of benzyl triethyl ammonium chloride and a base; a novel isoform of formula 1 below being R ₃ = O-acetylglucopyranosyl Method for preparing flavonone derivatives.

(Formula 1) [Where R ₁ = benzyloxy or hydrogen, R ₂ = benzyloxy or hydrogen and R ₁ ≠ R ₂ ] (1) preparing an isoflavonone derivative by a ring closure reaction in which the compound of formula 3 and thallium nitrate trihydrate are closed in an alcohol solvent;

(Formula 3) [Where R ₁ = benzyloxy or hydrogen, R ₂ = benzyloxy or hydrogen and R ₁ ≠ R ₂ ] (2) reacting the product produced in step 1 with methyl chloroacetate in the presence of a base to prepare an acetic acid derivative; (3) hydrolyzing the acetic acid derivative in the presence of a base; to prepare a novel isoflavonone derivative of the general formula 1 below, wherein R ₃ = -OCH ₂ CO ₂ H.

(Formula 1) [Where R ₁ = benzyloxy or hydrogen, R ₂ = benzyloxy or hydrogen and R ₁ ≠ R ₂ ] delete The isoflavone derivative according to claim 1, which is 5-benzyloxy-3- (4-hydroxyphenyl) chromen-4-one . delete delete delete (1) preparing an isoflavonone derivative by a ring closure reaction in which the compound of formula 3 and thallium nitrate trihydrate are closed in an alcohol solvent;

(Formula 3) [Where R ₁ = benzyloxy or hydrogen, R ₂ = benzyloxy or hydrogen and R ₁ ≠ R ₂ ] (2) reacting the product produced in step 1 with epichlorohydrin in the presence of a base to introduce an epoxide group; to prepare a novel isoflavonone derivative of the general formula 1 below, wherein R ₃ = epoxymethoxy Way.

(Formula 1) [Where R ₁ = benzyloxy or hydrogen, R ₂ = benzyloxy or hydrogen and R ₁ ≠ R ₂ ] Claim 9 to the the R ₃ = epoxymethoxy prepared by using a novel isoflavone derivative of formula 1 in acetic acid or methanol in Titanium tetra-iso-a Trojan polyepoxide catalyst by reaction of anhydrous sodium acetate and the ring-opened R ₃ =

Method for producing a novel isoflavonone derivative of the general formula 1 below.

(Formula 1) [Where R ₁ = benzyloxy or hydrogen, R ₂ = benzyloxy or hydrogen and R ₁ ≠ R ₂ ]