WO2011074881A2 - Substances destinées à inhiber l'expression de gènes pour la sensibilité à des troubles allergiques - Google Patents

Substances destinées à inhiber l'expression de gènes pour la sensibilité à des troubles allergiques Download PDF

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WO2011074881A2
WO2011074881A2 PCT/KR2010/008995 KR2010008995W WO2011074881A2 WO 2011074881 A2 WO2011074881 A2 WO 2011074881A2 KR 2010008995 W KR2010008995 W KR 2010008995W WO 2011074881 A2 WO2011074881 A2 WO 2011074881A2
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gene expression
allergic disease
gene
formula
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WO2011074881A3 (fr
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후쿠이히로유키
미즈구치히로유키
타카이시요시히사
카시와다요시키
네모토히사오
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국립대학법인 토쿠시마대학
이기덕
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents

Definitions

  • 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.
  • allergens invade in vivo allergen-specific IgE antibodies are produced by B lymphocytes stimulated via macrofuges and T lymphocytes.
  • 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 (FC ⁇ RI) on the surface of the sensitized mast cells reacts with allergens.
  • 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.
  • 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.
  • 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.
  • 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 Document 1 YAKUGAKU ZASSHI, Vol. 127 (1), 15-25 (2007)
  • 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
  • the present inventors screened a compound capable of inhibiting the IL-4 gene with respect to the ginseng extract, and conducted extensive research.
  • 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.
  • 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.
  • 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.
  • the compound of the present invention classified as a pterocarphan derivative which is a kind of flavonoid represented by the general formula (I)
  • 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.
  • 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.
  • 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.
  • Example 2 is a diagram showing a scheme of overall synthesis of macchia. It is a synthesis scheme concerning the conventional method synthesize
  • Fig. 2 is a diagram showing a synthesis scheme of macchiine using sesamol bromine compound. (Example 2)
  • Example 3 is a diagram showing the effect of hot ginseng hot water extract on IL-4 gene expression in RBL-2H3 cells. (Example 1)
  • Example 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)
  • Example 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.
  • Fig. 9 shows the effect of purified macchiin on IL-5 gene expression in RBL-2H3 cells.
  • Fig. 10 shows the effects of various macchiines on H 1 R gene expression in RBL-2H3 cells.
  • FIG. 11 is a diagram showing that purified macchiine (200 ⁇ M) has almost no antioxidant activity. (Reference Example 1)
  • FIG. 12 is a diagram showing that purified macchiin (300 ⁇ M) 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the allergic disease susceptible gene expression inhibiting substance of the present invention may be extracted from natural products or obtained by synthesis.
  • 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.
  • 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.
  • 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.
  • R is particularly preferably a hydrogen atom.
  • 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.
  • R is particularly preferably a hydrogen atom.
  • the compound represented by the general formula (I) or formula (II) may also be a pharmaceutically acceptable salt.
  • pharmaceutically acceptable salts include pharmacologically and pharmaceutically acceptable general salts. Specifically as such a salt, the following is illustrated.
  • 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, benzyltriethylammoni
  • acid addition salt for example, inorganic acid salts, such as hydrochloride, sulfate, nitrate, phosphate, carbonate, hydrogencarbonate, perchlorate;
  • 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;
  • 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)).
  • 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).
  • 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.
  • Non-Patent Document 2 is a bromine compound. It can substitute by synthesize
  • 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.
  • an antiallergic agent comprising an allergic disease-sensitive gene expression inhibiting substance as an active ingredient.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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;
  • the preparation may be a solid preparation or a liquid preparation.
  • examples of the preparation include tablets, pills, powders, granules, solutions, suspensions, and capsules.
  • 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.
  • 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.
  • ethanol dimethyl sulfoxide, polyethyleneglycol, etc.
  • Preservatives such as paraoxy benzoic acid ester, sodium benzoate, salicylic acid, sorbic acid, and boric acid as needed;
  • antioxidants such as butylhydroxy anisole and dibutyl hydroxytoluene.
  • absorption promoters such as diisopropyl adipate, diethyl sebacate, ethyl caproate, and ethyl laurate.
  • absorption promoters such as diisopropyl adipate, diethyl sebacate, ethyl caproate, and ethyl laurate.
  • 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.
  • a cream agent the form of an oil-in-water cream agent is preferable in order to stabilize the compound of this invention.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • polyacrylates such as water-soluble sodium polyacrylate, calcium polyacrylate, and ammonium polyacrylate,
  • 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.
  • RBL-2H3 cells rat basophilic leukemia cell line
  • RBL-2H3 cells were cultured in a 35 mm mast, 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.
  • DNP antidinitrophenyl
  • Antigen DNP-HAS, SIGMA, St. Louis, USA
  • DNP-HAS DNP-HAS, SIGMA, St. Louis, USA
  • RNA total RNA was prepared from the scraped cells
  • cDNA was prepared as a sample by reverse transcription reaction
  • 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.
  • the high ginseng hot water extract significantly inhibited the increase of IL-4 gene expression by the DNP-HSA antigen (FIG. 3).
  • the IL-4 gene expression amount was confirmed by the same method, and the active ingredient was extracted and isolated.
  • the primers for measuring rat IL-4 mRNA 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.
  • 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 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.
  • HPLC high performance liquid chromatography
  • 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).
  • 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).
  • the target compound (4) (macchiine) (5.4 mg, 0.0144 mmol, 23.7%) was obtained.
  • IL-4 gene expression was confirmed by the same method as described in 1) above.
  • 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 dome and co-stimulated with Phenbol 12-myristate 13-acetate (20 nM) + ionomycin (1 ⁇ M) 24 hours after the addition of (-) maciaine dissolved in DMSO. Was done for 3 hours.
  • real-time RT-PCR was performed on the mRNA of IL-5 and the mRNA of GAPDH.
  • the primers for measuring human IL-5 mRNA 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.
  • sense primer 5'-CAC TGA AGA AAT CTT TCA GGG AAT-3 '
  • anti-sense primer 5'-CAG TAC CCC CTT GCA CAG TT-3 '
  • H 1 R gene expression was measured using human cervical cancer cells HeLa cells. Cells were cultured in a 35 mm canal 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.
  • the primers for measuring human H 1 R mRNA 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.
  • sense primer 5'-CAG AGG ATC AGA TGT TAG GTG ATA GC-3
  • anti-sense primer 5'-AGC GGA GCC TCT TCC AAG TAA-3 '
  • 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.
  • 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 ⁇ L of 150 ⁇ mol / 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 ⁇ mol / L 160 ⁇ L 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.
  • DPPH diphenyl-p-bicyclylhydradyl
  • the calculation method of the radical scavenging effect on DPPH is as follows.
  • 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.
  • 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
  • SP600125 WAKO
  • 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.
  • 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.
  • 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.

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Abstract

La présente invention concerne une substance destinée à inhiber l'expression de gènes pour la sensibilité aux troubles allergiques, qui cible le mécanisme d'expression de gènes pour la sensibilité aux troubles allergiques. La substance est représentée par la formule générale (1) ci-dessous. Formule : (voir formule (1) dans le corps de la description) (Dans la formule, R représente un hydrogène ou un groupe hydrocarboné aliphatique saturé ou insaturé contenant une structure linéaire ou ramifiée, ou un groupe cycloalkyle, aryle, aralkyle, alkyloxy, alcényloxy, alcynyloxy, aryloxy ou aralkyloxy éventuellement substitué, ou leurs dérivés).
PCT/KR2010/008995 2009-12-15 2010-12-15 Substances destinées à inhiber l'expression de gènes pour la sensibilité à des troubles allergiques WO2011074881A2 (fr)

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JP2009284069A JP2011126791A (ja) 2009-12-15 2009-12-15 アレルギー疾患感受性遺伝子発現抑制物質

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113604557A (zh) * 2021-08-04 2021-11-05 杭州浙大迪迅生物基因工程有限公司 一种人组胺受体HRH1 mRNA检测引物探针组、试剂盒和应用
WO2023010330A1 (fr) * 2021-08-04 2023-02-09 杭州浙大迪迅生物基因工程有限公司 Ensemble amorce-sonde, kit et procédé de détection pour détecter l'arnm du récepteur hrh1 de l'histamine humaine

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
A. L. TOKES ET AL.: 'Absolute configuration and total synthesis of (-)-cabenegrin A-I' TETRAHEDRON vol. 55, no. 30, 1999, ISSN 0040-4020 pages 9283 - 9296 *
H. ZHOU ET AL.: 'Anti-Inflammatory and antiproliferative activities of trifolirhizin, a flavonoid from Sophora flavescens roots' JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY vol. 57, no. 11, 10 June 2009, ISSN 0021-8561 pages 4580 - 4585 *
M. IINUMA ET AL.: 'Flavonoid compounds in roots of Sophora tetraptera.' PHYTOCHEMISTRY vol. 39, no. 3, 1995, ISSN 0031-9422 pages 667 - 672 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113604557A (zh) * 2021-08-04 2021-11-05 杭州浙大迪迅生物基因工程有限公司 一种人组胺受体HRH1 mRNA检测引物探针组、试剂盒和应用
WO2023010330A1 (fr) * 2021-08-04 2023-02-09 杭州浙大迪迅生物基因工程有限公司 Ensemble amorce-sonde, kit et procédé de détection pour détecter l'arnm du récepteur hrh1 de l'histamine humaine
NL2030978B1 (en) * 2021-08-04 2023-02-21 Hangzhou Zheda Dixun Biological Gene Eng Co Ltd Primer probe set for human histamine receptor hrh1 mrna detection, kit and use

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