WO2011074881A2 - Substances for inhibiting expression of genes for sensitivity to allergic disorders - Google Patents

Abstract

The objective of the present invention is to provide a substance for inhibiting the expression of genes for sensitivity to allergic disorders, which targets the expression mechanism of genes for sensitivity to allergic disorders. The substance is represented by general formula (1) below. Formula: (see formula (1) in the body of the description) (In the formula, R represents a hydrogen, or a saturated or unsaturated aliphatic hydrocarbon group having a straight chain or a branched structure, or an optionally substituted cycloalkyl group, aryl group, aralkyl group, alkyloxy group, alkenyloxy group, alkynyloxy group, aryloxy group, or aralkyloxy group, or derivatives thereof.)

Description

Allergic disease-sensitive gene expression inhibitors

The present invention relates to an allergic disease susceptible gene expression inhibitor that targets the mechanism of expression of allergic disease susceptibility gene in the development of a novel therapeutic drug for allergic diseases. The present invention also relates to a novel synthesis method of an allergic disease susceptible gene expression inhibitory substance.

The immune system is a biological function that works to protect the body by excluding antigens, but this immune response may occur excessively or inappropriately, and in this case, tissue disorders, i.e., allergic diseases, may occur. Allergic diseases are caused by the following mechanisms. When allergens invade in vivo, allergen-specific IgE antibodies are produced by B lymphocytes stimulated via macrofuges and T lymphocytes. When the antibody adheres to mast cells (mast cells), the mast cells become sensitized, and when allergens invade again during this state, the high-affinity IgE receptor (FCεRI) on the surface of the sensitized mast cells reacts with allergens. As a result, mediators such as histamine and leukotrienes, which have various physiological activities, are released from the mast cells, causing smooth muscle contraction and vascular permeability, resulting in inflammation.

For example, histamine is a major mediator of allergic diseases as described above, and major symptoms are expressed through histamine H ₁ receptor (hereinafter sometimes referred to simply as "H ₁ R") of target cells. Antihistamines are drugs for the treatment of allergic diseases using blocking of signals through H ₁ R. On the other hand, allergic diseases are representative multifactorial diseases. H ₁ R gene and the like have been found as disease-related genes that cause abnormal expression in allergic diseases. The significance of the allergic disease therapeutic drug which targets such an allergic disease susceptible gene is reported by this inventor (nonpatent literature 1).

Interleukin 4 (hereinafter referred to simply as "IL-4") is a representative Th2 cytokine, which is released from activated T cells or mast cells and activates B cells, and immunologically proliferates and differentiates cells of competent cells. The IL-4 gene is thought to be an allergic disease susceptibility gene because it is a cytokine with an important effect on.

Macia (Maackiain) is a kind of flavonoid and is a natural-derived compound classified as a pterocarphan derivative, and has been confirmed to be isolated from red ginseng and other plants. Macchiane is reported as an anticancer agent (Patent Documents 1 and 2) and a Na + / glucotransporter inhibitor (Patent Document 3). However, there are no reports of anti-allergic activity of macchians.

Moreover, although macaine can be synthesize | combined, synthesis | combination using a mercury compound at that time is reported (nonpatent literature 2). However, when it is used as a pharmaceutical composition containing macchiin as an active ingredient, it is considered safe and desirable for a living body if it can be synthesized without using a mercury compound, but such a synthesis method is not particularly disclosed.

Patent Literature

Patent Document 1: Japanese Unexamined Patent Publication No. 60-178815

Patent Document 2: Japanese Patent Application Laid-Open No. 3-67045

Patent Document 3: Japanese Unexamined Patent Publication No. 2009-222622

Non-Patent Literature

[Non-Patent Document 1] YAKUGAKU ZASSHI, Vol. 127 (1), 15-25 (2007)

[Non-Patent Document 2] J. Chem. Soc. Perkin trans I, 04-1809 (1980)

An object of the present invention is to provide an allergic disease susceptible gene expression inhibitory substance which targets the expression mechanism of an allergic disease susceptible gene. Moreover, it is a subject to provide the novel synthesis | combining method of the allergic disease sensitive gene expression suppressing substance.

In order to solve the above problems, the present inventors screened a compound capable of inhibiting the IL-4 gene with respect to the ginseng extract, and conducted extensive research. As a result, allergic to macaine, which is a kind of flavonoid and classified as a pterocarphan derivative, The present invention has been completed by finding a disease-sensitive gene expression inhibitory effect.

That is, this invention is achieved by the following.

1. An allergic disease-sensitive gene expression inhibitory substance represented by the following general formula (I).

Formula (Ⅰ)

(Wherein R is a hydrogen atom or a linear or branched saturated or unsaturated aliphatic hydrocarbon group, a cycloalkyl group which may have a substituent, an aryl group, an aralkyl group, an alkyloxy group, an alkenyloxy group, an alkynyloxy group, an aryloxy group) Or an aralkyloxy group or a residue of a sugar.)

2. The allergic disease susceptible gene expression inhibitory substance as described in said 1 shown by following formula (II).

Formula (Ⅱ)

3. The allergic disease sensitive gene expression inhibitory substance according to 1 or 2, wherein the allergic disease sensitive gene is an IL-4 gene, an IL-5 gene and / or a histamine H1 receptor gene.

4. An antiallergic agent comprising the allergic disease susceptible gene expression inhibiting substance according to any one of 1 to 3 above as an active ingredient.

5. In the synthesis process of the allergic disease susceptible gene expression inhibiting substance according to any one of 1 to 3, the intermediate product 7-benzyloxy-2H-1-benzopyran (7-benzy1oxy-2H-1-benzopyran) and A method for producing an allergic disease susceptible gene expression inhibiting substance according to any one of 1 to 3, wherein the sesamol coupling step is based on a step of not using a mercury compound.

6. The manufacturing method of said 5 whose process which does not use a mercury compound synthesize | combines by the process which uses a bromine compound.

The compound of the present invention classified as a pterocarphan derivative, which is a kind of flavonoid represented by the general formula (I), inhibits the expression of IL-4 gene, IL-5 gene and H ₁ R gene, which are allergic disease susceptibility genes. From these things, the compound represented by general formula (I), ie, the allergic disease sensitive gene expression suppressing substance of this invention, shows an effect as an allergic disease sensitive gene expression suppressing substance. In addition, the allergic disease-sensitive gene expression inhibitory substance of the present invention is expected to be anti-allergic by inhibiting allergic disease-sensitive gene expression is useful in the development of new anti-allergic agents.

In addition, since the allergic disease sensitive gene expression inhibitory substance of this invention was confirmed to have low antioxidant activity and JNK inhibitory effect, it was suggested that it is a gene expression inhibitory effect based on the novel molecular basis rather than an antioxidant activity or JNK inhibitory effect. It is thus contemplated that the compounds of the present invention exhibit antiallergic action by different mechanisms of action than many other flavonoids.

1 is a diagram showing a scheme of overall synthesis of macchia. It is a synthesis scheme concerning the conventional method synthesize | combined using a mercury compound. (Example 2)

Fig. 2 is a diagram showing a synthesis scheme of macchiine using sesamol bromine compound. (Example 2)

3 is a diagram showing the effect of hot ginseng hot water extract on IL-4 gene expression in RBL-2H3 cells. (Example 1)

4 is a diagram showing the effect of acidic fractions (fraction by pH from high ginseng hydrothermal extract) on IL-4 gene expression in RBL-2H3 cells (Example 1)

Fig. 5 is a diagram showing the effect of basic fractions (fraction by pH from high ginseng hot-water extract) on IL-4 gene expression in RBL-2H3 cells. (Example 1)

Fig. 6 is a diagram showing the effect of neutral fraction (fraction by pH from high ginseng hot water extract) on IL-4 gene expression in RBL-2H3 cells. (Example 1)

7 is a diagram showing the effect of Ch1 fraction and Ch2 fraction (fraction by open column chromatography) on IL-4 gene expression in RBL-2H3 cells. (Example 1)

Fig. 8 shows the effects of various macchiines on IL-4 gene expression in RBL-2H3 cells. Experimental Example 1

Fig. 9 shows the effect of purified macchiin on IL-5 gene expression in RBL-2H3 cells. Experimental Example 2

Fig. 10 shows the effects of various macchiines on H ₁ R gene expression in RBL-2H3 cells. Experimental Example 3

FIG. 11 is a diagram showing that purified macchiine (200 μM) has almost no antioxidant activity. (Reference Example 1)

12 is a diagram showing that purified macchiin (300 μM) has little effect on jun N-teminal kinase (JNK) activity. (Reference Example 2)

The present invention relates to a novel allergic disease susceptible gene expression inhibitory substance. In the present invention, the allergic disease susceptibility gene may be a gene generally referred to as an allergic disease susceptibility gene, and the gene is not particularly limited. ₁ receptor (H ₁ R) gene, histidine decarbonase gene, and the like. The allergic disease susceptibility gene of the present invention is preferably an IL-4 gene, an IL-5 gene, or an H ₁ R gene, and particularly preferably an IL-4 gene.

The allergic disease susceptible gene expression inhibitory substance of this invention is a compound represented by the following general formula (I), and is classified into the pterocarphan derivative which is a kind of flavonoid.

Formula (Ⅰ)

(Wherein R is a hydrogen atom or a linear or branched saturated or unsaturated aliphatic hydrocarbon group, a cycloalkyl group which may have a substituent, an aryl group, an aralkyl group, an alkyloxy group, an alkenyloxy group, an alkynyloxy group, an aryloxy group) Or an aralkyloxy group or a sugar residue.)

In the above, the reactive moiety of a sugar refers to a sugar having a structure in which one hydroxyl group is separated from a sugar molecule by a reaction between a sugar and a macia derivative. Examples of the sugars include monosaccharides and disaccharides. Specifically, monosaccharides such as glucose, fructose and galactose, and disaccharides such as maltose, sucrose, lactose and trehalose are preferable.

The allergic disease-sensitive gene expression inhibitory substance of the present invention is particularly preferably a compound represented by the following formula (II), also referred to as macchiin.

Formula (Ⅱ)

The allergic disease susceptible gene expression inhibiting substance of the present invention may be extracted from natural products or obtained by synthesis. As a natural product, the natural product which can extract the pterocarphan derivative shown in General formula (I) may be used, and although it is not specifically limited, For example, red ginseng, a sapling tree, a cultivation (gallweed), a jasmine, red clover, etc. Can be mentioned. The extraction from natural products is not particularly limited, but may be carried out according to a method known per se. Moreover, the allergic disease sensitive gene expression suppressing substance of this invention can also be produced synthetically. When extracted from natural products, the compound shown by the following general formula (IV) can be obtained easily. Moreover, when it produces | generates by synthesis | combination, it is a case where it is a racemate mixed with the compound shown by the following general formula (III) and general formula (IV) in many cases. In this invention, the compound equivalent to the compound extracted from a natural product, ie, the compound represented by General formula (IV), is the most suitable.

Formula (III)

(Wherein R is a hydrogen atom or a linear or branched saturated or unsaturated aliphatic hydrocarbon group, a cycloalkyl group which may have a substituent, an aryl group, an aralkyl group, an alkyloxy group, an alkenyloxy group, an alkynyloxy group, an aryloxy group) Or an aralkyloxy group or a sugar residue.)

In the above, R is particularly preferably a hydrogen atom.

Formula (Ⅳ)

(Wherein R is a hydrogen atom or a linear or branched saturated or unsaturated aliphatic hydrocarbon group, a cycloalkyl group which may have a substituent, an aryl group, an aralkyl group, an alkyloxy group, an alkenyloxy group, an alkynyloxy group, an aryloxy group) Or an aralkyloxy group or a sugar residue.)

In the above, R is particularly preferably a hydrogen atom.

In the present invention, the compound represented by the general formula (I) or formula (II) may also be a pharmaceutically acceptable salt.

In the present invention, pharmaceutically acceptable salts include pharmacologically and pharmaceutically acceptable general salts. Specifically as such a salt, the following is illustrated. As a basic addition salt, For example, alkali metal salts, such as a sodium salt and potassium salt; Alkaline earth metal salts such as calcium salts and magnesium salts; Ammonium salts; For example, trimethylamine salt and triethylamine salt; Aliphatic amine salts such as dicyclohexylamine salt, ethanolamine salt, diethanolamine salt, triethanolamine salt and procaine salt; Aralkyl amine salts, such as N, N- dibenzyl ethylene diamine; Heterocyclic aromatic amine salts such as pyridine salt, picoline salt, quinoline salt and isoquinoline salt; Quaternary ammonium salts such as tetramethylammonium salt, tetraethylammonium salt, benzyltrimethylammonium salt, benzyltriethylammonium salt, benzyltributylammonium salt, methyltrioctylammonium salt and tetrabutylammonium salt; Arginine salts; Basic amino acid salts, such as a lysine salt, etc. are mentioned.

As acid addition salt, For example, inorganic acid salts, such as hydrochloride, sulfate, nitrate, phosphate, carbonate, hydrogencarbonate, perchlorate; For example, organic acid salts such as acetate, propionate, lactate, maleate, fumarate, tartarate, malate, citrate, ascorbate; Sulfonates such as methane sulfonate, isethionate, benzene sulfonate and p-toluene sulfonate; For example, acidic amino acids, such as aspartate and glutamate, are mentioned.

The allergic disease susceptible gene expression suppressing substance of the present invention, that is, the pterocarphan derivative represented by the general formula (I), particularly the machiane represented by the formula (II) is, for example, non-patent document 2 (J. Chem). Soc. Perkin trans I, 04-1809 (1980)). However, in the method described in Non-Patent Document 2, a mercury compound is used in the synthesis process of 2-chloromercurio-4,5-methylenedioxyphenol which is an intermediate product in the synthesis process of macchia. The method used is disclosed (see FIG. 1). In the case of a drug including an allergic disease-sensitive gene expression inhibiting substance of the present invention as an active ingredient, it is safe to synthesize the allergic disease-sensitive gene expression inhibiting substance without using a mercury compound, and it is also excellent from the viewpoint of treating synthetic waste. Do.

Specifically, the synthesis process of 2-chloromerculo-4,5-methylenedioxyphenol using a mercury compound in Non-Patent Document 2 (Fig. 1) is a bromine compound. It can substitute by synthesize | combining 2-bromo-4,5-methylenedioxyphenol from sesamol using (refer FIG. 2). As described above, a method of synthesizing macaine without using a mercury compound has not been reported. Therefore, a method for synthesizing macia, an allergic disease susceptible gene expression inhibiting substance that does not use a mercury compound, is included in the scope of the present invention.

The present invention also extends to an antiallergic agent comprising an allergic disease-sensitive gene expression inhibiting substance as an active ingredient. In the present specification, an antiallergic agent refers to a drug such as a drug having an effect on a so-called allergic disease. The antiallergic agent of the present invention may be used prophylactically or therapeutically for allergic diseases, and may be suitably used therapeutically. By allergic disease susceptibility gene expression inhibitor as an active ingredient, allergic disease susceptibility genes, ie IL-4 gene, IL-5 gene, IgE receptor gene, MHC class II gene, histamine H ₁ receptor (H ₁ R) gene, histidine It is possible to inhibit the expression of any one of the genes represented by the decarbonase gene, suitably the IL-4 gene, the IL-5 gene and / or the H ₁ R gene, more suitably the IL-4 gene, and as a result Allergic diseases can be prevented or treated.

In this specification, an allergic disease should just be what is called an allergic disease, Although it does not specifically limit, For example, type I allergy, type II allergy, type IV allergy, etc. are mentioned. Specifically, hay fever, allergic rhinitis, bronchial asthma, atopic dermatitis, plant allergy, urticaria, PIE syndrome, anaphylactic shock, etc. are mentioned, for example.

When the antiallergic agent of the present invention is used prophylactically or therapeutically, an effective amount can be administered orally or parenterally. The dosage can be appropriately determined depending on the route of administration or the method of administration. For example, in the case of oral administration, the active ingredient may be used in the range of 0.01 to 1000 mg per day for adults.

Suitable formulations for oral administration include, for example, tablets, capsules, powders, granules, granules, solutions, and syrups. Suitable formulations for parenteral administration include, for example, injections, drops, suppositories, Inhalants, eye drops, nasal drops, ointments, creams, and patches;

For oral administration, it may be a solid preparation or a liquid preparation. Examples of the preparation include tablets, pills, powders, granules, solutions, suspensions, and capsules. When preparing tablets, excipients such as lactose, starch, calcium carbonate, crystalline cellulose, or silicic acid according to a conventional method; Binders such as carboxymethyl cellulose, methyl cellulose, calcium phosphate or polyvinylpyrrolidone; Disintegrating agents such as sodium alginate, sodium bicarbonate, sodium lauryl sulfate and monoglyceride stearate; Lubricants such as glycerin; Absorbers such as kaolin and colloidal silica; Additives, such as lubricants, such as a talc and a granular boric acid, can be used.

Pills, powders or granules are also formulated according to conventional methods using additives in the same manner as above. Liquid preparations such as liquids and suspending agents are also formulated according to conventional methods. As a carrier, For example, Glycerol esters, such as a tricaplin, a triacetin, an iodide poppy oil fatty acid ester; water; Alcohols such as ethanol; Oily bases such as liquid paraffin, coconut oil, soybean oil, sesame oil and corn oil are used. The powders, granules, liquid preparations, and the like described above may be encapsulated in gelatin or the like.

The pharmaceutically acceptable carrier in the present specification may include appropriately selected auxiliaries, fragrances, isotonic agents, pH regulators, stabilizers, propellants, pressure-sensitive adhesives and preservatives, etc., which are usually used as necessary.

Examples of the formulation of the drug for transdermal administration include ointments, creams, lotions, liquids, and the like. Examples of the base of the ointment include fatty oils such as castor oil, olive oil, sesame oil and safflower oil; lanolin; White, yellow or hydrophilic petrolatum; beeswax; Higher alcohols such as oleyl alcohol, isostearyl alcohol, octyldodecanol and hexyldecanol; Glycols, such as glycerin, diglycerin, ethylene glycol, propylene glycol, sorbitol and 1,3-butanediol, etc. are mentioned. Moreover, you may use ethanol, dimethyl sulfoxide, polyethyleneglycol, etc. as a solubilizer. Preservatives, such as paraoxy benzoic acid ester, sodium benzoate, salicylic acid, sorbic acid, and boric acid as needed; You may use antioxidants, such as butylhydroxy anisole and dibutyl hydroxytoluene.

Moreover, in order to promote percutaneous absorption, you may add absorption promoters, such as diisopropyl adipate, diethyl sebacate, ethyl caproate, and ethyl laurate. Moreover, in order to stabilize, the compound of this invention can also be used, forming a clathrate compound etc. with (alpha), (beta), or (gamma)-cyclodextrin, or methylated cyclodextrin.

Ointment can be manufactured by a conventional method. As a cream agent, the form of an oil-in-water cream agent is preferable in order to stabilize the compound of this invention. As the base, as described above, fatty oil, higher alcohols, glycols, and the like are used, and emulsifiers such as diethylene glycol, propylene glycol, sorbitan mono fatty acid ester, polysorbate 80, and sodium lauryl sulfate are used. do. Moreover, you may add a preservative, antioxidant, etc. as mentioned above as needed. Moreover, it can also use as a clathrate compound of cyclodextrin and methylated cyclodextrin similarly to the case of an ointment. Creams can be prepared by conventional methods.

Examples of the lotion include suspension, emulsion and solution lotions. Suspension type lotion agents can be obtained by adding antioxidants, preservatives and the like as necessary using suspending agents such as sodium alginate, tragant and sodium carboxymethylcellulose. An emulsion lotion can be obtained by a conventional method using an emulsifier such as sorbitan mono fatty acid ester, polysorbate 80, sodium lauryl sulfate. Alcohol type lotion agent is preferable and can be obtained by a conventional method using alcohol, such as ethanol. As a liquid agent, what melt | dissolved in alcohol solutions, such as ethanol, and added antioxidant, preservative, etc. as needed is mentioned.

In addition to such formulations, formulations such as pasta, pape and aerosol can be given. Such formulations can be prepared by conventional methods.

Formulations for nasal administration are given as liquid or powdery compositions. As the base of the liquid agent, water, saline solution, phosphate buffer, acetic acid buffer, and the like may be used, and may further contain a surfactant, an antioxidant, a stabilizer, a preservative, and a viscosity imparting agent. As a base of a powdery agent, it is preferable that it is water absorbing, For example, polyacrylates, such as water-soluble sodium polyacrylate, calcium polyacrylate, and ammonium polyacrylate, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl Cellulose lower alkyl ethers such as sodium cellulose, polyethylene glycol, polyvinylpyrrolidone, amylose, and furan, and the like; Starch, carboxymethyl starch, crosslinked starch, starches such as amylose, amylopectin, pectin, proteins such as gelatin, casein, casein sodium, gums such as gum arabic, tragant gum, glucomannan, polyvinyl polypyrrolidone, crosslinked polyacrylic acid And salts thereof, crosslinked polyvinyl alcohol, poly Hydroxy ethyl methacrylate and acrylate, such as cross-linked vinyl polymers, such as, may be used by mixing them. Moreover, you may add antioxidant, a coloring agent, a preservative, a preservative, a grant agent, etc. to a powdery agent. Such liquid agents and powdery agents can be administered using, for example, a spray device.

Formulations for administration by injection are given as sterile aqueous or non-aqueous solutions, suspensions, or emulsifiers. Non-aqueous solutions or suspensions are pharmaceutically acceptable carriers of injectable organic esters, for example vegetable oils such as propylene glycol, polyethylene glycol or olive oil, ethyl oleate, iodide poppy oil fatty acid esters. do. Such formulations may also contain auxiliary agents such as preservatives, wetting agents, emulsifiers, dispersants and stabilizers, and may be sustained release. Such solutions, suspensions and emulsifiers can be sterilized by appropriately carrying out treatment such as filtration through a bacterial retention filter, blending of sterilizing agents, or irradiation. Aseptic solid preparations may also be prepared and dissolved in sterile water or sterile solvent for injection immediately before use.

EXAMPLE

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the scope of the following Examples.

Example 1 Isolation of IL-4 Gene Expression Inhibiting Substance from Ginseng Extract

1) Preparation of High Ginseng Hot Water Extract

Isolation of IL-4 gene expression inhibitory substance from the ginseng extract was tested. 60 g of red ginseng (Sophorae Radix, Oriental Medicine Kojima, Osaka) was put in 1000 ml of water, and hydrothermal extraction was performed for 90 minutes, followed by filtration at hot time. This was concentrated and adjusted to 30 mg / ml to obtain a high ginseng hot water extract.

The effect on IL-4 gene expression was confirmed by the amount of IL-4 gene expression by antigen antibody stimulation against rat basophilic leukemia cell line (RBL-2H3 cells). RBL-2H3 cells were cultured in a 35 mm chalet, and the above-mentioned hot water extracts were added to the medium so as to have a final concentration of 1.2 to 3.0 mg / ml while the cells were 70% fused, and incubated at 37 ± 0.5 ° C. for 24 hours. Antigen antibody stimulation was performed at 100 ng / ml DNP-HSA after adding 100 ng / ml of antidinitrophenyl (DNP) monoclonal antibody (SPE-7; SIGMA, St. Louis. USA) 18 hours before antigen stimulation. Antigen (DNP-HAS, SIGMA, St. Louis, USA) was added and performed. After 2 hours of antigen stimulation, cells were scraped, total RNA was prepared from the scraped cells, cDNA was prepared as a sample by reverse transcription reaction, and mRNA level was measured by real-time RT-PCR method (N = 3). ). The amount of IL-4 gene expression was confirmed by the ratio of the mRNA level of IL-4 to the mRNA level of GAPDH which is a housekeeping gene.

As a result, it was confirmed that the high ginseng hot water extract significantly inhibited the increase of IL-4 gene expression by the DNP-HSA antigen (FIG. 3). Hereinafter, the IL-4 gene expression amount was confirmed by the same method, and the active ingredient was extracted and isolated.

As the primers for measuring rat IL-4 mRNA, the oligonucleotides of the sequence shown in SEQ ID NO: 3 and the probe shown in SEQ ID NO: 3 were used. Primers and probes for rat GAPDH mRNA measurement were commercially available from TaqMan ^(R) Rodent GAPDH Control Reagents (VIC Probe) Cat No. 4308313 (Applied Biosystems) was used.

(SEQ ID NO: 1) sense primer: 5'-CAGGGTGCTTCGCAAATTTTAC-3 '

(SEQ ID NO: 2) anti-sense primer: 5'-CACCGAGAACCCCAGACTTG-3 '

(Array number 3) probe: FAM-CCCACGTGATGTACCTCCGTGCTTG-TAMRA

2) Fraction by pH from Gosam hot water extract

Fractions were performed from high ginseng hot water extract (30 mg / ml) under various pH conditions. The pH was adjusted to 3 with 1 M hydrochloric acid, extracted three times with ethyl acetate, and the solvent was removed to give an acid fraction. The residue (aqueous layer) from which the acidic fractions were extracted was adjusted to pH 10 with 1M aqueous sodium hydroxide solution, and extracted three times with ethyl acetate. The solvent of the obtained extract was removed and it was set as basic fraction. The residue (aqueous layer) from which the basic fraction was extracted was adjusted to pH 7 with an aqueous 10% citric acid solution. The solvent of the extracted residue (aqueous layer) was removed by lyophilization to obtain a neutral fraction. The distribution of alkaloids in each fraction was examined. Each fraction was developed by thin layer chromatography and sprayed with Dragendorf reagent. As a result, alkaloids were not detected in the acidic fraction, and distribution of alkaloids was observed in the basic fraction and the neutral fraction.

For these fractions, the effect on the expression of IL-4 gene was investigated using RBL-2H3 cells, respectively. IL-4 gene expression level was confirmed by the same method as described in 1) above (N = 3). As a result, the acid fraction significantly inhibited the increase of IL-4 gene expression (FIG. 4). On the other hand, basic fractions and neutral fractions did not show an inhibitory effect on IL-4 gene expression (Fig. 5, Fig. 6).

3) Fractionation by open column chromatography

The active acid fractions were separated and purified again by open column chromatography. Elution was carried out using silica gel 60N (Kanto Chemical Co., Inc, Tokyo) in the fixed phase, and chloroform: methanol (9: 1) in the moving bed, and the eluate was separated by 50 mL to obtain fractions Ch1 to Ch21. Thereafter, the column was washed with 100% methanol, and 50 mL were further separated to obtain fractions M1 to M7. For each fraction of the obtained elution fraction and washing fraction, the solvent was removed and the effect on the expression of IL-4 gene was examined. The amount of IL-4 gene expression was confirmed by the same method as described in 1) above (N = 3). Among these fractions, Ch1 showed a tendency to suppress IL-4 gene expression, and Ch2 showed a significant inhibitory effect (FIG. 7). No inhibitory effect was observed in the remaining fractions.

4) Isolation and Identification of Active Ingredients

Thin layer chromatography confirmed that there was a common spot in the Ch1 fraction and the Ch2 fraction. The spot was purified again by silica gel open column chromatography (moving layer; hexane: ethyl acetate (2: 1)), and the effect of purification fraction on the IL-4 gene expression was examined. The amount of IL-4 gene expression was confirmed by the same method as described in 1) above (N = 3). Moreover, about the fraction obtained by the refinement | purification, this was provided in nuclear magnetic resonance spectrum (NMR) and it turned out that the active ingredient is macchiin shown by Formula (II). The structure was identified by one-dimensional NMR (1H, 13C) and two-dimensional NMR (HH-COSY, DEPT, HMBC, HSQC) (Bruker Japan Co., Ltd. Kanagawa; ARX-400). Tetramethylsilane was used as an internal standard.

Formula (Ⅱ)

The fraction obtained by purification by silica gel open column chromatography was again subjected to high performance liquid chromatography (HPLC) to purify the active ingredient. HPLC was performed at 65% methanol or 55% methanol at a flow rate of 0.8 mL / min using Mightysil RP18 GP (Kanto Chemical Co., Inc., Tokyo) as a column. The fractions fractionated were recrystallized from methanol to obtain a genuine product.

Example 2 Synthesis of Machine by Heck Reaction

The synthesis of the macchiine of the present invention was partially modified by the method of the method of Non-Patent Document 2 (see FIG. 1). In the method of FIG. 1, a mercury compound is used, whereas in the present example, compound (3) (7-benzyloxy-2H-1-benzopyran) and a compound ( 1) in coupling with (Sesamol: 1,3-Benzodioxol-5-ol), JS The method of Yadav (Adv. Synth. Catal. 346: 77-82 (2004)) and the like were partially modified to use a bromine compound without using a mercury compound (see FIG. 2).

Compound (1) (300 mg, 2.17 mmol) was dissolved in methylene chloride and n-Bromosuccinimide (464 mg, 2.606 mmol) was slowly added while ice-cooling. The reaction was stopped with an aqueous sodium hydrogen carbonate solution and the ethylene chloride layer was concentrated. This was isolated by column chromatography (silica gel, hexane-chloroform = 1: 3), and compound (2) (2-bromo-4,5-methylenedioxyphenol (2-bromo-4,5-methy1enedioxyphenol)) (295.9 mg, 1.36 mmol, 62%).

Compound (3) (14.5 mg, 0.061 mmol) and Compound (2) (13.2 mg, 0.061 mol) synthesized according to the method described in Non-Patent Document 2 are dissolved in acetonitrile (5 mL), and tricyclohexylphosphine ( A mixture of tricyclohexylphosphine) (30% in toluene, 20μl, 0.0183mmol) and Pd ₂ (dba) ₃ · CHCl ₃ (63mg, 0.0061mmol) was reacted at 60 ° C. under an argon atmosphere for 4 hours. Then, the reaction solution was diluted three times with methylene chloride, filtered using floridyl ^(R) (150-250 μm), concentrated and concentrated by column chromatography (silica gel, hexane-ethyl acetate = 5: 1). The target compound (4) (macchiine) (5.4 mg, 0.0144 mmol, 23.7%) was obtained.

Experimental Example 1 Effect on the Expression of IL-4 Gene of Natural Product-derived Macaine and Synthetic Macaine

The effects on the expression of IL-4 gene were examined for the macaine derived from the natural product extracted and purified in Example 1 and the synthetic macaine synthesized in Example 2. The amount of IL-4 gene expression was confirmed by the same method as described in 1) above.

It was confirmed that each machiin significantly inhibited IL-4 gene expression by DNP-HSA antigen (FIG. 8). In addition, it was observed that the synthetic macchiin (Example 2) had a halved effect on IL-4 gene expression as compared with the natural product-derived macaine (Example 1). From this, synthetic macchiin is a racemate, and only one of the optically active agents is considered to have an IL-4 gene expression inhibitory effect. Moreover, the photoluminescence of 0.11g / 100mL (in chloroform) which is macchi obtained by the extraction and purification of Example 1 was measured, and the specific photoluminescence was computed, and [alpha] 6D = -176.4 degrees. By comparison with the literatures of the past, the natural product-derived macaine obtained in Example 1 was considered to be a (-) macaine and was considered to be an effective optically active substance.

Experimental Example 2 Effect on Expression of IL-5 Gene of (-) Macchiin

In this experimental example, the effect of (-) macchiin on IL-5 gene expression was confirmed. As the (-) macchiin, the macchiin extracted and purified in Example 1 was used.

IL-5, like IL-4, is classified as a Th2 cytokine, and is released from activated T lymphocytes and mast cells, and is a factor related to eosinophil differentiation, proliferation, and rejuvenation. have. Therefore, the IL-5 gene is an allergic disease susceptibility gene and is considered to be a factor for chronicizing and intracting allergy.

IL-5 gene expression was measured using human basophils leukemia cells KU812F cells. Cells were incubated in a 35 mm chalet and co-stimulated with Phenbol 12-myristate 13-acetate (20 nM) + ionomycin (1 μM) 24 hours after the addition of (-) maciaine dissolved in DMSO. Was done for 3 hours. By the same method as described in Example 1, real-time RT-PCR was performed on the mRNA of IL-5 and the mRNA of GAPDH.

As a result, it was confirmed that (-) maciain significantly inhibited the IL-5 gene expression increase by mitezan (FIG. 9).

As the primers for measuring human IL-5 mRNA, the oligonucleotides of the sequence shown in SEQ ID NO: 6 and the sequence shown in SEQ ID NO: 6 were used. Primers and probes for measuring human GAPDH mRNA are commercially available from Pre Devoloped TaqMan ^(R) Assay Reagent Control Kits (Human GAPDH) Cat No. 4310884E (Applied Biosystems) was used.

(SEQ ID NO: 4) sense primer: 5'-CAC TGA AGA AAT CTT TCA GGG AAT-3 '

(SEQ ID NO: 5) anti-sense primer: 5'-CAG TAC CCC CTT GCA CAG TT-3 '

(SEQ ID NO: 6) probe: FAM-ACA CTG GA-TAMRA

Experimental Example 3 Effect of (-) Maciaine on the Expression of Histamine H ₁ Receptor (H ₁ R) Gene

In this experimental example, the effect of (-) macchiin on the H ₁ R gene expression was confirmed. Histamine is a major mediator of allergic diseases, and it is well known that major symptoms are expressed through H ₁ R of target cells. Here, it has been reported by the present inventor that the H ₁ R gene is an allergic disease susceptible gene (Non-Patent Document 1).

H ₁ R gene expression was measured using human cervical cancer cells HeLa cells. Cells were cultured in a 35 mm chalet and starvation was performed by culturing in a serum-free medium for 24 hours. Simultaneously with the start of serum-free culture, (-) macaine of DMSO lysis was added to the medium, and the cultured cells were treated for 24 hours. Thereafter, stimulation with 100 nM PMA was performed for 3 hours. In the same manner as described in Example 1, real-time RT-PCR was performed on mRNA of H ₁ R and mRNA of GAPDH.

As a result, it was confirmed that (-) macaine significantly inhibited the increase in H ₁ R gene expression by PMA (FIG. 10).

As the primers for measuring human H ₁ R mRNA, the oligonucleotides of the sequences shown in SEQ ID NOs: 9 and 8 and the sequences shown in SEQ ID NO: 9 were used. Commercially available primers and probes for measuring human GAPDH mRNA were used in the same manner as in Experimental Example 2.

(SEQ ID NO: 7) sense primer: 5'-CAG AGG ATC AGA TGT TAG GTG ATA GC-3

(SEQ ID NO: 8) anti-sense primer: 5'-AGC GGA GCC TCT TCC AAG TAA-3 '

(SEQ ID NO: 9) probe: FAM-CTT CTC TCG AAC GGA CTC AGA TAC CAC C-TAMRA

Reference Example 1 Antioxidant Activity

In this reference example, the DPPH (diphenyl-p-bicryhydrhydryl) of (-) machiine extracted and purified in Example 1 based on the method of Jung (Arch Pharm Res Vol 28, No5, 534-540. 2005) ) Radical scavenging activity.

Flavonoids have been reported to have many physiological activities such as antiallergic action, anticancer action, and most of the molecular bases are known based on the antioxidant action of flavonoids. Therefore, the possibility that the macaine gene expression inhibitory effect also originates in antioxidant activity like other flavonoids could be considered. Conventionally, the antioxidant activity of macchiine has been examined as an indicator of DPPH radical scavenging activity, intracellular reactive oxygen species scavenging activity of hydrogen peroxide induction, and ONOO-erasing activity, and accordingly, machiines have different antioxidant activities. It is reported that the antioxidant activity is remarkably weak compared to the substance and does not have antioxidant activity (Arch Pharm Res Vol 28, No 5, 534-540, 2005). Therefore, in this reference example, the antioxidant activity of macaine was examined using DPPH radical scavenging activity as an index.

Ascorbic acid was used as a positive control. 40 μL of 150 μmol / L DPPH (diphenyl-p-bicyclylhydradyl) methanol solution and macchiine or ascorbic acid methanol solution with positive control adjusted to a final concentration of 50, 100, 150 or 200 μmol / L 160 μL was added and gently mixed. After standing at room temperature for 30 minutes, the absorbance at 520 nm derived from DPPH radical, which is a stable organic radical, was measured, and the ratio with the control (solution of only DPPH without containing macia and ascorbic acid) was calculated by the following formula. And DPPH radical scavenging activity. As the test solution blank, the absorbance was measured in the same manner using a methanol solution to which macchiin was not added.

The calculation method of the radical scavenging effect on DPPH is as follows.

DPPH radical scavenging activity (%) = ｛C- (St-Sb)｝ / C × 100

St: absorbance of the solution under test at a wavelength of 520 nm

Sb: the absorbance of the test solution solution blank at a wavelength of 520 nm

C: absorbance at wavelength 520 nm of control solution

As a result, it was observed that even when 200 μM reaction of (-) maciain had almost no antioxidant activity (FIG. 11), it was confirmed that macchiin had no antioxidant activity. From this point of view, it was suggested that the macaine gene expression inhibitory action is a gene expression inhibitory action based on a novel mechanism rather than an antioxidant action possessed by other flavonoids.

(Reference Example 2) Influence of (-) macchiin on jun N-terminal kinase (JNK) activity

In this reference example, the inhibitory effect of the enzyme activity of JNK was investigated on the (-) macaine extracted and purified in Example 1. JNK phosphorylates c-Jun, one of the proteins constituting the transcription factor AP-1, to promote gene expression involved in AP-1. Flavonoids often exhibit various physiological activities by inhibiting the enzyme activity of JNK.

Therefore, in this reference example, it was examined whether the inhibitory action of IL-4 gene expression of (-) maciaine was due to the inhibition of the enzyme activity of JNK. In vitro kinase assay of JNK was performed using the KinaseSTAR ^(R) JNK Activity Assay Kit (BioVision) according to the method described in the attached document. HeLa cells were first treated with 70 nM anisomycin (Sigma) for 30 minutes and JNK was activated. Cells were crushed using the JNK extraction buffer attached to the kit, JNK specific antibodies were added to the centrifugal supernatant, and activated JNK was prepared by immunoprecipitating JNK with Protein A Sepharose ^(R) attached to the kit. Substrate solution containing c-jun and ATP was added to the activated JNK and reacted at 30 ° C for 1 hour, and the resulting phosphorylated c-jun was detected by Western blotting using phosphorylated c-jun specific antibody. SP600125 (WAKO), which is known as an inhibitor of JNK, was used as a positive control.

As a result, even when 300-M reaction of (-) macaine was carried out, phosphorylation of c-Jun by JNK was not suppressed (FIG. 12).

As described in detail above, the compounds of the present invention classified as pterocarphan derivatives, a kind of flavonoid represented by the general formula (I), are IL-4 gene, IL-5 gene and H ₁ R gene which are allergic disease susceptibility genes. Suppresses expression of From this point of view, the compound represented by the general formula (I), that is, the allergic disease sensitive gene expression inhibiting substance of the present invention has an effect as an allergic disease sensitive gene expression suppressing substance. In addition, the allergic disease-sensitive gene expression inhibitory substance of the present invention is expected to be anti-allergic by inhibiting allergic disease-sensitive gene expression is useful in the development of new anti-allergic agents.

In addition, since the allergic disease sensitive gene expression inhibitory substance of this invention was confirmed to have low antioxidant activity and JNK inhibitory effect, it was suggested that it is a gene expression inhibitory effect based on the novel molecular basis rather than an antioxidant activity or JNK inhibitory effect. It is thus contemplated that the compounds of the present invention exhibit antiallergic action by different mechanisms of action than many other flavonoids.

(Array table)

SEQUENCE LISTING

<110> The University of Tokusima

<120> Suppressor for gene expression of allergic disease sensitive gene

<130> 9-1144 1734

<160> 9

<170> PatentIn version 3.1

<210> 1

<211> 22

<212> DNA

<213> Artificial

<220>

<223> Synthesized sense primer for amplifying rattus IL-4 genes

<400> 1.

cagggtgctt cgcaaatttt ac

<210> 2

<211> 20

<212> DNA

<213> Artificial

<220>

<223> Synthesized anti-sense primer for amplifying rattus IL-4 genes

<400> 2

caccgagaac cccagacttg

<210> 3

<211> 25

<212> DNA

<213> Artificial

<220>

<223> Synthesized probe for detecting rattus IL-4 genes

<400> 3

* cccacgtgat gtacctccgt gcttg

<210> 4

<211> 24

<212> DNA

<213> Artificial

<220>

<223> Synthesized sense primer for amplifying human IL-5 genes

<400> 4

cactgaagaa atctttcagg gaat

<210> 5

<211> 20

<212> DNA

<213> Artificial

<220>

<223> Synthesized anti-sense primer for amplifying human IL-5 genes

<400> 5

cagtaccccc ttgcacagtt

<210> 6

<211> 8

<212> DNA

<213> Artificial

<220>

<223> Synthesized probe for detecting human IL-5 genes

<400> 6

acactgga

<210> 7

<211> 26

<212> DNA

<213> Artificial

<220>

<223> Synthesized sense primer for amplifying human H1R genes

<400> 7

cagaggatca gatgttaggt gatagc

<210> 8

<211> 21

<212> DNA

<213> Artificial

<220>

<223> Synthesized anti-sense primer for amplifying human H1R genes

<400> 8

agcggagcct cttccaagta a

<210> 9

<211> 28

<212> DNA

<213> Artificial

<220>

<223> Synthesized probe for detecting human H1R genes

<400> 9

cttctctcga acggactcag ataccacc

Claims (6)

1. An allergic disease-sensitive gene expression inhibitory substance represented by the following general formula (I).

   Formula (Ⅰ)

   

   (Wherein R is a hydrogen atom or a linear or branched saturated or unsaturated aliphatic hydrocarbon group, a cycloalkyl group which may have a substituent, an aryl group, an aralkyl group, an alkyloxy group, an alkenyloxy group, an alkynyloxy group, an aryloxy group) Or an aralkyloxy group or a sugar residue.)
2. The method according to claim 1,

   An allergic disease-sensitive gene expression inhibitory substance represented by the following formula (II).

   Formula (Ⅱ)

   
3. The method according to claim 1 or 2,

   An allergic disease sensitive gene expression inhibitory substance, wherein the allergic disease sensitive gene is an IL-4 gene, an IL-5 gene and / or a histamine H1 receptor gene.
4. An antiallergic agent comprising the allergic disease-sensitive gene expression inhibitory substance according to any one of claims 1 to 3 as an active ingredient.
5. In the synthesis process of the allergic disease susceptible gene expression inhibiting substance according to any one of claims 1 to 3, the intermediate product 7-benzyloxy-2H-1-benzopyran (7-benzyloxy-2H-1-benzopyran) and A method for producing an allergic disease susceptible gene expression inhibiting substance according to any one of claims 1 to 3, wherein the coupling step with sesamol is based on a step that does not use a mercury compound.
6. The method according to claim 5,

   The manufacturing method which synthesize | combines by the process using a bromine compound the process which does not use a mercury compound.

Images