JPH0753359A - Lipoxygenase inhibitor - Google Patents

Abstract

PURPOSE:To provide a lipoxygenase inhibitor having excellent lipoxygenase inhibiting action and useful as an anti-inflammatory agent, anti-allergic agent, etc., by using piceatannol existing in flat-sedge belonging to the family Cyperaceae or its derivative as the active component. CONSTITUTION:Dried flat-sedge is crushed and extracted with methanol, the extracted liquid is concentrated under reduced pressure, the concentrated liquid is subjected to liquid-liquid partition with ethyl acetate-water, the obtained organic layer is concentrated under reduced pressure and partitioned with hexane-90% methanol, the 90% methanol layer is concentrated, dried and subjected to silica gel chromatography, the adsorbed material is eluted with chloroform-methanol and the eluate is collected and subjected to high- performance liquid chromatography to obtain piceatannol of the formula (R<1> to R<4> are each OH, an alkyl, alkoxy, alkanoyl, etc.) or its derivative. The compound is used as an active component, compounded with a carrier and formed in the form of a pharmaceutical preparation to obtain the objective preparation for anti-inflammatory agent, anti-allergic agent, etc., having excellent lipoxygenase inhibiting action.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a lipoxygenase inhibitor useful as an anti-inflammatory agent and an antiallergic agent.

[0002]

2. Description of the Related Art In recent years, the number of patients with allergic diseases such as bronchial asthma and hay fever has increased with the changes in eating habits and living environment, and pollution such as air pollution, which is becoming a big social problem.

Among them, asthma is a patient with high airway hypersensitivity, which causes vascular permeability increase, bronchial smooth muscle contraction, secretion increase, etc. due to external allergens or nonspecific stimulation to the airway, resulting in respiratory distress. It is a disease that causes and, in severe cases, death. Currently, as therapeutic agents for asthma, drug therapy, translocation therapy, desensitization therapy, psychotherapy, etc. are performed, but a method that produces a sufficient therapeutic effect has not yet been established. Steroids and antihistamines have been used as therapeutic agents for asthma, but since they have serious side effects, overcoming them is an important issue.

With the progress of basic research on asthma in recent years,
It has become clear that, among the arachidonic acid metabolism systems, especially the lipoxygenase metabolites play an important role in the pathological condition of asthma.

This arachidonic acid is an unsaturated fatty acid having 20 carbon atoms which is released from the cell membrane in response to various stimuli. It is further converted into prostaglandins by the cyclooxygenase system and leukotriene or HE by the lipoxygenase system.
It is known that these metabolites are metabolized to TE (hydroxyeicosatetraenoic acid) s, and that each metabolite is deeply involved in various allergic diseases and inflammatory diseases.

Psoriasis, which is a type of skin disease, is a refractory chronic inflammatory keratosis that causes proliferation of the epidermis and infiltration of inflammatory cells. Although the cause of psoriasis has not been clarified yet, the amount of lipoxygenase metabolites leukotriene and HETEs increased in the lesions, which strongly suggests the relationship between psoriasis lesion formation and abnormal arachidonic acid metabolism. Has been done.

[0007] Therefore, arachidonic acid metabolites are considered to play an extremely important role in various allergic and inflammatory diseases, and lipoxygenase, which is a metabolizing enzyme of arachidonic acid, is a major metabolic enzyme. Therefore, substances that inhibit this enzyme are considered to be useful for treating the above-mentioned various diseases.

[0008]

Therefore, an object of the present invention is to provide a lipoxygenase inhibitor useful for treating various allergic diseases and inflammatory diseases.

[0009]

In view of such circumstances, the inventors of the present invention have conducted earnest studies to obtain a substance having a lipoxygenase inhibitory action, and as a result, conventionally, antifungal activity and antibacterial activity (Chem. Pharm. Bull. .32 (1) 213
-218 (1984)), an inhibitory effect of histamine release from rat mast cells (Chem. Pharm. Bul.
l. 39 (12) 3276-3278 (1991)), which was known to have a new finding that piceatannol and its derivatives contained in Cyperus microiria of the family Cyperaceae have a lipoxygenase inhibitory action. Completed the invention.

That is, the present invention provides the following general formula (1):

[0011]

[Chemical 2]

(Wherein R ¹ , R ² , R ³ and R ⁴ may be the same or different and represent a hydroxyl group, an alkyl group, an alkoxy group, an alkanoyl group or an alkanoyloxy group) The present invention provides a lipoxygenase inhibitor containing a norl or its derivative as an active ingredient, and an antiallergic agent and an anti-inflammatory agent containing this as an active ingredient.

In the general formula (1), the alkyl group represented by R ¹ , R ² , R ³ and R ⁴ is a linear or branched alkyl group having 1 to 6 carbon atoms, such as a methyl group or an ethyl group. ,
n-propyl group, isopropyl group, n-butyl group, se
A c-butyl group, an n-pentyl group, an n-hexyl group and the like can be mentioned. As the alkoxy group, a linear or branched alkoxy group having 1 to 6 carbon atoms such as methoxy group, ethoxy group, n-propyloxy group, isopropyloxy group, n-butyloxy group, sec-butyloxy group, n
Examples include -pentyloxy group and n-hexyloxy group. Examples of the alkanoyl group include linear or branched alkanoyl groups having 1 to 6 carbon atoms, such as formyl group, acetyl group, propionyl group, n-butanoyl group, isobutanoyl group, n-pentanoyl group and n-hexanoyl group. . The alkanoyloxy group has 1 to 6 carbon atoms.
And a linear or branched alkanoyloxy group such as acetoxy group, propionyloxy group, isobutanoyloxy group, n-butanoyloxy group, n-pentanoyloxy group, n-hexanoyloxy group. R ¹
The to R ^4, a hydroxyl group, an alkoxy group, alkanoyloxy group.

The active ingredient piceatannol or its derivative (1) of the present invention can be prepared by a known method (Rev. Lati).
noamer. Quim. 18, 79-80 (198
7), J. Nat. Prod. , 50 (1), 36-4
No. 0 (1987)), but piceatannol (R ^{1 to} R ⁴ = OH in the general formula (1)) can also be extracted from cypress grass. For extraction,
First, extraction is performed from a solvent selected from diethyl ether, ethyl acetate, acetone, methanol, ethanol, hexane, ethyl acetate, and water. Then, the residue obtained by distilling off the solvent from the obtained extract is appropriately dissolved in a solvent such as methanol, ethanol and ethyl acetate, and further water, methanol, ethanol, ethyl acetate, chloroform, dichloromethane, hexane and acetone. , Benzene, etc. as elution solvents, Amberlite XAD-2, Diaion HP-
20, hydrophilic polymer such as TSK gel HW-40, Sephadex such as Sephadex LH-20, column chromatography using reversed phase silica gel, silica gel, cellulose, etc. as a carrier, and thin layer chromatography etc. The target product can be obtained by fractionating while checking the target component. In some cases, it may be purified by recrystallization using a suitable solvent such as benzene, ether, hexane, acetone, methanol, ethanol and water.

Piceatannol or its derivative (1)
Can be administered to animals and humans as is or together with conventional formulations alone. This dose may be appropriately determined depending on the age of the patient or animal, sex, degree of disease, etc.
Usually, it is preferable to set the range of 0.002 to 20 mg, especially 0.05 to 5 mg per 1 kg of body weight per day.

The dosage form of piceatannol or its derivative (1) is not particularly limited and can be appropriately selected as needed, and the dosage form is an oral preparation such as tablets, capsules, granules and powders. , Parenteral preparations such as injections, suppositories, ointments and the like.

In the case of tablets, the carrier is
A wide variety of materials known in the art can be used. This includes excipients such as starch, lactose, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, urea; water,
Binders such as ethanol, propanol, simple syrup, glucose, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone; dry starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, carbonate Disintegrators such as calcium, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch and lactose; disintegration inhibitors such as sucrose, stearin, cocoa butter and hydrogenated oils;
Absorption enhancers such as sodium lauryl sulfate and quaternary ammonium salts; moisturizers such as glycerin and starch; starch, lactose,
Adsorbents such as kaolin, bentonite, colloidal silicic acid; stearates, boric acid powder, purified talc, lubricants such as polyethylene glycol and the like. Further, the tablets are tablets coated with a usual coating as necessary, for example, sugar-coated tablets,
It may be a gelatin-coated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet, or a multi-layer tablet.

In the case of pills, a wide variety of carriers known in the art can be used, and examples thereof include starch, lactose, glucose, cocoa butter, hydrogenated vegetable oil, kaolin, talc and the like. Examples include shaping agents, gum arabic powder, tragacanth powder, binders such as gelatin and ethanol, and disintegrating agents such as laminaranthantene.

When in the form of suppositories, those widely known in the art can be widely used as the carrier, and examples thereof include cocoa butter, gelatin, polyethylene glycol, higher alcohols, esters of higher alcohols, and semisynthetic glycerides. Etc. can be mentioned.

When prepared as an injection, the solution and suspension are preferably sterilized and isotonic with blood. When these solutions, suspensions and emulsions are prepared,
As the diluent, those commonly used in this field can be used. Examples thereof include water, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylenated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In this case, the pharmaceutical preparation may contain a sufficient amount of salt, glucose, glycerin, etc. to prepare an isotonic aqueous solution, and a usual solubilizing agent, buffer, soothing agent, etc. are added. You may. Further, if necessary, a colorant, a preservative, a flavor, a flavoring agent, a sweetening agent and other pharmaceuticals may be contained in the pharmaceutical preparation.

When the present compound is in the form of a spray, those widely known in the art can be used as the dispersant and the propellant. Examples of the dispersant include soybean lecithin, egg yolk lecithins, oleic acid, Fatty acids such as linoleic acid and linolenic acid, and sorbitans such as sorbitan trioleate and sorbitan monooleate can be used. Further, as the propellant, for example, a normally incombustible liquefied gas such as Freon 11, Freon 12, Freon 114 can be used.

When the present compound is in the form of an ointment, a wide variety of compounds known in the art can be used, and examples thereof include water, ethanol, isopropyl alcohol, glycerin, polyethylene glycol, sorbitol, polyvinyl alcohol, and other polyhydric alcohols, and animal products. Oils, vegetable oils, mineral oils,
Hardened oil, waxes such as beeswax, higher hydrocarbons such as liquid paraffin and paraffin wax, fatty acids such as stearic acid, emulsifiers, surfactants such as anionic surfactants, cationic surfactants and amphoteric surfactants, xanthan gum, sodium alginate. Water-soluble polymer compounds such as methyl cellulose, hydroxyethyl cellulose and carboxyvinyl polymer can be used. Also,
Dyes, preservatives, fragrances, etc. may be added as required.

The amount of piceatannol and its derivative (1) to be blended in the pharmaceutical preparation is not particularly limited and may be appropriately selected within a wide range.
It is from 01 to 70% by weight, preferably from 0.1 to 30% by weight.

The administration method of the above-mentioned pharmaceutical preparation is not particularly limited, and various preparation forms, patient's age, sex, other conditions,
It is administered according to the degree of the patient. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are orally administered. In the case of injection, it is intravenously administered alone or in admixture with a normal replacement fluid such as glucose or amino acid, and further, if necessary, is intramuscularly, intradermally, subcutaneously or intraperitoneally administered alone. In the case of suppositories, it will be administered rectally. In the case of a spray, it is sprayed from the mouth or nose and administered to the bronchi. In the case of ointment, it is applied directly to the lesion site.

[0025]

The present invention will be described in more detail with reference to Examples, Production Examples and Test Examples, but the present invention is not limited to these.

Production Example 1 Dried cypress grass (Cyperus microiri)
a) (1 kg in weight) is ground and extracted with 1 l of methanol. The extract was concentrated under reduced pressure and then liquid-liquid partitioned with ethyl acetate-water. The obtained organic layer was concentrated under reduced pressure and further hexane-9.
Partition with 0% methanol. The 90% methanol layer was concentrated to dryness (16 g), and subjected to silica gel column chromatography (Wakogel C-200), and eluted with a chloroform-methanol system. Chloroform: methanol = 9: 1 eluate was collected and further subjected to high performance liquid chromatography (ODS; acetonitrile: 3% acetic acid water = 2: 3) to obtain 60 mg of piceatannol.

Test Example 1 Rat basophilic leukemia cell line (Rat Basophi)
lic Leukemia Cell: RBL-1) is suspended in D-PBS (+). 2 × 10 ⁵ cells of each tube were placed in a plastic tube and incubated with various concentrations of piceatannol at 37 ° C. for 15 minutes, and then calcium ionophore A23187 (final concentration 2.5 μM) was added. After incubating for another 15 minutes, EDTA (final concentration 4 mM) was added and the mixture was ice-cooled.
The mixture was centrifuged at 00 rpm for 5 minutes, and the supernatant was collected. Next, leukotriene C ₄ (LTC ₄ ) and prostaglandin D ₂ (PGD ₂ ) contained in the supernatant were quantified using a radioimmunoassay method, and their production inhibitory effect was evaluated. The results are shown in Table 1.

[0028]

[Table 1]

Test Example 2 Normal human polymorphonuclear leukocytes containing 50 mM of 1 mM EDTA and 0.1% -gelatin so as to have a concentration of 4.0 × 10 ⁷ cells / ml.
Suspend in M phosphate buffer, sonicate at 20kHz for 30 seconds to crush the cells, and at 10 000G for 10 minutes, 10
Centrifugation was performed at 5000 G for 60 minutes, and the supernatant was used as an enzyme preparation. Next, the enzyme and test compound (final concentration 10 μM) were placed in a test tube, incubated at 37 ° C. for 10 minutes, and then ¹⁴ C-labeled arachidonic acid (0.1 μC) was added.
i) was added and the reaction was continued for 10 minutes. After the reaction, add citric acid to acidify, extract with ethyl acetate, concentrate and then RI
-Subjected to HPLC (ODSC ₁₈ column 250 x 4 mm, M
eCN: MeOH: H ₂ O: AcOH = 350: 15
0: 250: 1, flow rate 1.5 ml / min), 5-HET
The radioactivity of E, 12-HETE, 15-HETE was measured. The amount of HETE is 100 when the test compound is not added.
The lipoxygenase inhibitory activity was evaluated as%. The results are shown in Table 2.

[0030]

[Table 2]

From the above results, excellent lipoxygenase inhibitory action of piceatannol or its derivative (1) was confirmed.

Test Example 3 Normal human fibroblasts were suspended in DMEM medium containing 10% FCS, seeded on a 12-well culture plate, and when confluent, replaced with serum-free DMEM medium. 24 hours later, exchange the medium with the test substance,
After incubation for 30 minutes, interleukin 1α (10 units /
ml) and incubate for another 6 hours. After 6 hours, the medium was collected, and prostaglandin E ₂ (PG
E ₂ ) was quantified by enzyme immunoassay. The results are shown in Table 3.

[0033]

[Table 3]

On the other hand, when compound a, b, c or d is administered to mice, it is orally administered at 300 mg / kg or 200 mg / kg.
None of the mice died even after intraperitoneal administration of kg.

TEST EXAMPLE 4 Hartley white guinea pigs were shaved on the back to give 0.1.
% Croton oil was applied to induce inflammation. Inflammation induction 2
Before and after 6 hours, as a sample, 25 μl / (1.5 cm ×) of a solution of piceatannol in ethanol (100 mM)
1.5 cm) was applied, and the degree of erythema was judged 24 hours after the induction of inflammation. The judgment was made according to the following Japanese Dermatological Association standards. The result is shown in FIG.

Japanese Dermatological Association Standard 0 (−) No reaction. 0.5 (±) Mild or partial erythema. 1.0 (+) Clear erythema on the whole surface. 2.0 (++) Erythema and edema. 3.0 (+++) Erythema, edema and small blisters.

[0037]

[Table 4]

From Table 4, it was confirmed that the excellent anti-inflammatory effect of piceatannol in the croton oil-induced inflammation model.

Test Example 5 Hurtley-type white guinea pigs were shaved on the back and cut to 100
Ethanol solution of mM piceatannol (sample) 25
μl / (1.5 cm × 1.5 cm) was applied. After 2 hours, the sample was wiped with 70% ethanol and UVB (1.
7 mW / cm ² × 9 minutes), and immediately after that, sample 25
μl / (1.5 cm × 1.5 cm) was applied. After 6 hours of UVB irradiation, 25 μl of the sample was applied again. At 6 hours and 24 hours after irradiation, the degree of erythema was evaluated in the same manner as in Test Example 4. The results are shown in Table 5.

[0040]

[Table 5]

From Table 5, it was confirmed that the excellent anti-inflammatory effect of piceatannol in the UV inflammation model was confirmed.

Test Example 6 Balb / c male mice aged 7 weeks were shaved on the back and
100 μl of a picryl chloride / acetone / olive oil (4. 1) solution was applied for sensitization. 6% after sensitization 1%
20 μl of a solution of picryl chloride / acetone / olive oil (4. 1) was applied to both surfaces of the right auricle to induce allergic inflammation. After 24 hours of induction, the animals were sacrificed, punched with a punch (φ = 7 mm) after cutting both auricles, the weight of the left and right auricle pieces was measured, and the difference between them was taken as the amount of edema. 20 μl of 100 mM piceatannol ethanol solution (sample) was applied to the right auricle on the day before induction, 6 hours after the induction, and only the solvent (ethanol) was applied to the target group instead of the test substance. The results are shown in Table 6.

[0043]

[Table 6]

From Table 6, the excellent anti-inflammatory effect of piceatannol in the allergic inflammation model was confirmed.

Example 1 The ingredients were uniformly mixed according to the following formulation, and the mixture was compression-molded with a tableting machine to give 200 mg tablets.

[0046]

[Table 7] Compound a (Table 2) 10 g Corn starch 30 g Starch 30 g Carboxymethyl cellulose 5 g Magnesium stearate 5 g Lactose 20 g Total 100 g

Example 2 According to the following formulation, the respective components were uniformly mixed and blended. After granulating with an extrusion granulator, it was dried and sieved to obtain granules.

[0048]

Table 8 The present compound b (Table 2) 10 g Crystalline cellulose 50 g 10% Hydroxypropyl cellulose 40 g Ethanol solution total 100 g

Example 3 A propellant was prepared by filling a cylinder with the following composition by a conventional method.

[0050]

Table 9 The present compound a (Table 2) 1 g Oleic acid 3 g Freon 11 1.2 g Freon 12 2.5 g Freon 114 1.3 g Total 9 g

Example 4 The components were uniformly mixed according to the following formulation to obtain an ointment.

[0052]

Table 10 The present compound c (Table 2) 10 g Squalane 20 g Glycerin 20 g Cetyl alcohol 5 g Magnesium stearate 3 g Propylene glycol 5 g Water 20 g Ethanol 7 g Total 90 g

[0053]

INDUSTRIAL APPLICABILITY The lipoxygenase inhibitor of the present invention has an excellent lipoxygenase inhibitory action and is useful as a medicine such as an anti-inflammatory agent and an antiallergic agent. Therefore, it can be widely used for the prevention and treatment of bronchitis, asthma, allergic rhinitis, gout, arthritis, nephritis, hepatitis, psoriasis, urticaria, contact dermatitis, atopic dermatitis and the like.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication location A61K 31/22 ADA 9454-4C (72) Inventor Yukihiro Yada 1-chome Kushita Nishi, Ninomiya Town, Haga-gun, Tochigi Prefecture 115-1 (72) Inventor Genji Imokawa 1022-89 Himurocho, Utsunomiya City, Tochigi Prefecture

Claims (3)

[Claims] 1. The following general formula (1): (Wherein R ¹ , R ² , R ³ and R ⁴ may be the same or different and represent a hydroxyl group, an alkyl group, an alkoxy group, an alkanoyl group or an alkanoyloxy group) or a piceatannol thereof. A lipoxygenase inhibitor containing a derivative as an active ingredient. 2. An anti-allergic agent comprising the piceatannol or the derivative thereof according to claim 1 as an active ingredient. 3. An anti-inflammatory agent comprising the piceatannol or the derivative thereof according to claim 1 as an active ingredient.