JPH0717859A - Arachidonic acid dysbolism disease therapeutic agent - Google Patents

Abstract

(57) [Summary] (Modified) [Objective] The present invention has a 5-lipoxygenase inhibitory action and a cyclooxygenase inhibitory action, and can be applied to various pathological conditions based on abnormal arachidonic acid metabolism such as inflammation, thrombosis, allergy, and asthma. Provide a drug to deal with. [Arrangement] Arachidonic acid metabolism containing a compound of formula I or the like as an active ingredient, which is isolated from a crude drug mulberry bark, its original plant mulberry, other plants belonging to the same genus or herb medicine It is a therapeutic agent for abnormal diseases.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to compounds having 5-lipoxygenase inhibitory action and cyclooxygenase inhibitory action and useful for the treatment of diseases caused by abnormal metabolism of arachidonic acid.

[0002]

2. Description of the Related Art Arachidonic acid is an unsaturated fatty acid having 20 carbon atoms and cannot be synthesized by the body in mammals. Therefore, arachidonic acid must be supplied from the outside and is counted as one of the essential fatty acids.

The metabolic pathway of arachidonic acid is called arachidonic acid cascade, and its representative ones include cyclooxygenase system for synthesizing prostaglandin series and thromboxane series, and lipoxygenase system for synthesizing leukotriene series.

In recent years, the number of patients with allergic diseases such as bronchial asthma and hay fever has been increasing due to pollution problems and environmental changes in Japan.

With the increase in the elderly population, the number of patients with cardiovascular diseases caused by blood clots has become remarkable.
It is a big social problem.

One of the causes of these diseases is the abnormal metabolism of arachidonic acid. As described above, 5-lipoxygenase and cyclooxygenase are important enzymes of the arachidonic acid cascade. Inhibitors of the enzyme of, thrombosis including inflammation,
It is considered to be useful as a medicine for treating various pathological conditions caused by arachidonic acid metabolism abnormality such as allergy and asthma.

[0007] Therefore, the search and development of drugs using 5-lipoxygenase inhibition and cyclooxygenase inhibition as indicators have been carried out.

[0008]

[Means for Solving the Problems] The inventors of the present invention have earnestly studied for a compound having a 5-lipoxygenase inhibitory action and a cyclooxygenase inhibitory action, which is effective for treating a disease caused by arachidonic acid metabolism abnormality.

[0009] As a result, the crude mulberry bark that is clinically used, its original plant mulberry (American mulberry, Harigua, etc.),
Other homologous plants or herbal licorice (Glycyrrhizae Radi
x), found that some of the compounds isolated from the licorice, which is the original plant (Nishikita licorice, Tohoku licorice, Xinjiang licorice, etc.), and other plants of the same genus (dog licorice, etc.), have the above-mentioned inhibitory action. The present invention has been completed.

That is, the present invention is based on these findings and is as shown below.

The following formula I (However, R ₁ and R ₂ are the same or different and each represents a hydrogen atom or a 3,3-dimethylallyl group.) Formula III, Formula IV below Or the following formula V (However, R ₃ is a hydrogen atom, a 3,3-dimethylallyl group or
1,1-dimethylallyl group, R ₄ is hydrogen atom or 3,3-dimethylallyl group, R ₅ is hydroxyl group, methoxyl group or 3,3-
A dimethylallyl group, R ₆ and R ₈ are the same or different and each represents a hydrogen atom or a hydroxyl group, and R ₇ represents a hydrogen atom, a hydroxyl group or a 3,3-dimethylallyl group. ) A therapeutic agent for an arachidonic acid metabolism disorder disease comprising a compound represented by the formula (4) as an active ingredient.

Hereinafter, the compounds represented by the formulas I to V are collectively referred to as the compound of the formula.

For example, the following method can be used to obtain the compound of the formula.

[0014] Crude drug mulberry bark, its original plant mulberry (American mulberry, Harigua, etc.), other congener plants or crude drug licorice
ycyrrhizae Radix), its original plant licorice (Seihoku licorice, Tohoku licorice, Xinjiang licorice, etc.), roots or whole plants of other plants of the same genus (dog licorice, etc.), after defatting with n-hexane, if necessary, Extract with an organic solvent such as benzene, ethyl acetate, ethanol, methanol, n-hexane, methanol,
The residue obtained by removing the solvent from the extract is appropriately dissolved in a solvent such as methanol, benzene and ethyl acetate, and selected from water, methanol, ethanol, acetic acid, chloroform, ethyl acetate, n-hexane, acetone and benzene. Amberlite XAD-2, Diaion HP-20, MCI gel, porous polymers such as CHP20P, Sephadex such as Sephadex LH-20, reverse phase silica gel, silica gel, polyamide, activated carbon or cellulose with at least one as an elution solvent Column chromatography using such as a carrier,
It can be obtained by subjecting to preparative thin layer chromatography and high performance liquid chromatography at least once.

In some cases, it may be purified by recrystallization using a suitable solvent such as methanol, ethyl acetate, benzene, ethyl ether, n-hexane, acetone, ethanol and the like.

Next, specific examples of the production of the compound of the formula will be shown.

Example 1 6 kg of the above-ground portion of Tohoku licorice were extracted with ethanol, and the solvent was distilled off under reduced pressure to obtain 590 g of ethanol extract.

300 g of this ethanol extract was subjected to Amberlite XAD-2 column chromatography and eluted with a water-methanol-benzene system.

The benzene elution portion was concentrated to obtain 25 g of a benzene fraction. 25 g of this benzene fraction was subjected to silica gel column chromatography to obtain n-hexane-
Elute with benzene and fraction from the benzene elution part
Got 1, 2 and 3.

Fraction 1 was subjected to Sephadex LH-20 column chromatography and recrystallized from benzene to obtain 18 mg of colorless needles. The physicochemical properties of this colorless needle crystal are described in the literature [Phytochemistry, 30 , 1245 (1991)].
It was in agreement with that of Gankaonin S described.

Specific Example 2 Fraction 2 obtained in Specific Example 1 was subjected to high performance liquid chromatography and recrystallized from acetone-n-hexane to obtain 85 mg of colorless needle crystals. The physicochemical properties of this colorless needle crystal are described in the literature [Phytochemistry, 30 , 1245 (1991)].
It was in agreement with that of Gankaonin R described.

Example 3 Fraction 3 obtained in Example 1 was treated with Sephadex LH-2
By subjecting to 0 column chromatography and high performance liquid chromatography and recrystallizing from n-hexane-acetone, 70 mg of colorless needle crystals were obtained. The physicochemical properties of these colorless needles are described in the literature [Phytochemistry, 30 , 1245 (1991)].
Consistent with that of Gankaonin U described.

Specific Example 4 4.8 kg of northwest licorice was sequentially extracted with n-hexane, benzene and acetone. The solvent of the acetone extract was distilled off under reduced pressure to obtain 150 g of acetone extract. 150 g of this acetone extract,
Subjected to silica gel column chromatography, benzene
-Elution with an acetone system gave fractions 1 and 2 from the benzene-acetone (99: 1) eluate.

Fraction 2 was subjected to preparative thin layer chromatography and recrystallized from benzene-acetone to obtain 160 mg of colorless columnar crystals. The physicochemical properties of colorless needles was consistent with that of the literature [Heterocycles. 29 .1761 (1989) ] according Gurishiroru.

Concrete Example 5 3 kg of the underground part of dog licorice was extracted with ethanol, and the solvent was distilled off under reduced pressure to obtain 190 g of ethanol extract.

190 g of this ethanol extract was subjected to Amberlite XAD-2 column chromatography and eluted with a water-methanol-benzene system.

The methanol eluate was concentrated to obtain 60 g of methanol fraction. 60 g of this methanol fraction
Was subjected to silica gel column chromatography and eluted with a benzene-methanol system to obtain Fraction 1 from the benzene elution part.

Fraction 1 was subjected to high performance liquid chromatography to obtain 50 mg of a colorless oily substance. Physicochemical properties of this colorless oil the literature [Heterocycles. 31 .643 (199
0)] The same as that of Gankaonin J described.

Example 6 3.4 kg of root bark of American mulberry ( Morus insignis ) was sequentially extracted with n-hexane, benzene and acetone. For the n-hexane extract, the solvent was distilled off under reduced pressure to obtain 170 g of n-hexane extract, and for the benzene extract, the solvent was distilled off under reduced pressure to obtain 50 g of benzene extract.
Got

35 g of this n-hexane extract was subjected to silica gel column chromatography and eluted with an n-hexane-acetic acid ethyl ester system, and fractions 1 and 2 were extracted from the n-hexane-acetic acid ethyl ester (9: 1) elution part. Obtained. This fraction 1 was subjected to Sephadex LH-20 column chromatography and high performance liquid chromatography, and recrystallized from benzene to obtain 21 mg of yellow needle crystals. The physicochemical properties of these yellow needles are described in the literature [Tetrahedr
on, 27 , 3919 (1989), Heterocycles. 31 .1345 (1990)]
It was in agreement with that of Gartanin described.

Specific Example 7 50 g of the benzene extract obtained in Specific Example 6 was subjected to silica gel column chromatography and eluted with n-hexane-acetic acid ethyl ester system to obtain n-hexane-acetic acid ethyl ester.
(86:14) Fractions 1 and 2 were obtained from the elution part.

This fraction 1 was subjected to silica gel column chromatography, eluted with n-hexane-chloroform system, and the chloroform eluate was recrystallized from n-hexane-acetone to obtain 1.1 g of yellow needle crystals. Physicochemical properties of this yellow needles the literature [Heterocycles. 31 .1
345 (1990)] and that of molsignin A described.

Specific Example 8 10 kg of root bark of a harlequin was extracted with ethanol, and the solvent was distilled off under reduced pressure to obtain 740 g of an ethanol extract.

After 740 g of this ethanol extract was dissolved in benzene to remove insoluble matters, the solvent in the benzene-soluble portion was distilled off under reduced pressure to obtain 280 g of a benzene fraction. 180 g of this benzene fraction was subjected to silica gel column chromatography, and eluted with n-hexane-benzene-acetic acid ethyl ester system, and fractions 1 and 2 were eluted from the n-hexane-benzene (7: 3) eluate to Fraction 3 was obtained from the benzene (1: 1) eluate.

Fraction 2 was subjected to preparative thin layer chromatography and recrystallized from n-hexane-ether to obtain 60 mg of yellow needle crystals. The physicochemical properties of the yellow needle-shaped crystals were in agreement with those of kudraxanthone E described in the literature [Planta Med, 56 , 399 (1990)].

Specific Example 9 Fraction 3 obtained in Specific Example 8 was subjected to silica gel column chromatography and eluted with n-hexane-acetic acid ethyl ester system to obtain Fraction 4 from the acetic acid ethyl ester elution portion. This fraction was subjected to preparative thin layer chromatography and recrystallized from chloroform to obtain 60 mg of yellow columnar crystals. The physicochemical properties of this yellow columnar crystal were in agreement with those of kudraxanthone L described in the literature [Planta Med, 57 , 172 (1991)].

Next, experimental examples showing that the compound of the formula has excellent 5-lipoxygenase inhibitory action and cyclooxygenase inhibitory action and is useful as a drug such as antiallergic agent, antiinflammatory agent and cardiovascular drug I will give you an explanation.

Experimental Example 1 (5-lipoxygenase inhibitory effect) RBL1 cultured cells were adjusted to 5 × 10 ⁶ cells / ml with 1 mM EDTA.
And 50 mM phosphate buffer containing 10% ethylene glycol
Suspended in (pH 7.4), after ultrasonic treatment, 10,000 × G for 10 minutes,
Furthermore, the supernatant obtained by centrifugation at 105,000 × G for 60 minutes was used as a 5-lipoxygenase enzyme preparation.

As a substrate, 10 μM arachidonic acid, an enzyme preparation prepared as described above, and a dimethylsulfoxide solution of the compound obtained in the specific example were placed in a test tube, and the temperature was adjusted to 37 °
C, reacted for 10 minutes.

As an internal standard, 10 μl of 0.25 M butyl-3,5-dinitrobenzoate was added and extracted with 1.8 ml of n-hexane. The amount of 5-HETE in this is high performance liquid chromatography [column; TSK gel ODS-80TM (manufactured by TOYO SODA), mobile phase; acetonitrile: water: acetic acid (60: 40: 0.02), flow rate; 1 ml /
Min, detection; ultraviolet ray (235 nm)].

From this result, the inhibition rate (%) was calculated by the following formula.

[0042]

C: 5-HETE peak area when the compound obtained in the specific example is not included (corrected by internal standard) S: 5-HETE peak area when the compound obtained in the specific example is added (internal standard) Corrected by

The inhibitory rate of 5-lipoxygenase of the compound of the formula is shown in Table 1.

Table 1

From the above results, the excellent 5-lipoxygenase inhibitory action of the compound of the formula was confirmed.

Experimental Example 2 (Cyclooxygenase Inhibitory Action) Microsomes (100 μm) prepared from rabbit kidney medulla were placed in a test tube.
μg), potassium phosphate buffer adjusted to a final concentration of 0.1 M (p
H7.5), tryptophan adjusted to a final concentration of 10 mM, glutathione adjusted to a final concentration of 4 mM, hemoglobin adjusted to a final concentration of 0.25 mM, compounds obtained in specific examples and reaction substrate (1- ¹⁴ C) -arachidonic acid. (5 × 10 ⁴ dpm) and total 2
It was set to 00 μl.

After incubating at 37 ° C for 15 minutes, 1N
The reaction was stopped by adding 50 μl of hydrochloric acid. P as a carrier
GE ₂ was added and extracted twice with 1 ml of ether. After concentrating the extract, the reaction product was separated by thin layer chromatography (developing solvent; chloroform: methanol: acetic acid = 18: 1: 1).

Color was developed with iodine vapor, the portion corresponding to PGE ₂ was scraped off, the radioactivity was measured with a liquid scintillation counter, and the inhibition rate (%) was determined by the following formula.

[0050]

C: Radioactivity when the compound obtained in the specific example is not included S: Radioactivity when the compound obtained in the specific example is added

The results are shown in Table 2.

Table 2

From the above results, the excellent cyclooxygenase inhibitory action of the compound of the formula was confirmed.

Next, an acute toxicity test of the compound of the formula was carried out using male ICR mice, and it was a highly safe drug with no oral death at 1.0 g / kg.

As described above, the compound of the formula has extremely low toxicity and high safety.

The dosage and formulation of the compound of formula will now be described.

The compounds of formula can be administered to animals and humans either neat or together with conventional pharmaceutical carriers. The dosage form is not particularly limited and may be appropriately selected and used as needed, and examples thereof include oral preparations such as tablets, capsules, granules, fine granules and powders, parenteral preparations such as injections and suppositories. To be

In order to exert a desired effect as an oral preparation, it varies depending on the age, body weight and degree of disease of the patient, but usually 50 mg to 5 g as the weight of the compound of formula is divided into several times a day in adults. All doses seem appropriate.

Oral preparations include, for example, starch, lactose, sucrose,
Mannitol, carboxymethyl cellulose, corn starch, inorganic salts and the like are used in a conventional manner.

In this type of preparation, a binder, a disintegrating agent, a surfactant, a lubricant, a fluidity promoter, a flavoring agent, a coloring agent, a fragrance, etc. may be appropriately used in addition to the above-mentioned excipients. You can
Specific examples of each are as shown below.

[Binder] Starch, dextrin, gum arabic powder, gelatin, hydroxypropyl starch, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, crystalline cellulose,
Ethyl cellulose, polyvinylpyrrolidone, macrogol.

[Disintegrant] Starch, hydroxypropyl starch, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, carboxymethyl cellulose, low-substituted hydroxypropyl cellulose.

[Surfactant] Sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polysorbate 8
0.

[Lubricant] Talc, waxes, hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol.

[Flowability Accelerator] Light anhydrous silicic acid, dried aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate.

The compounds of the formula can also be administered as suspensions, emulsions, syrups and elixirs, and these various dosage forms may contain flavoring agents and coloring agents. .

In order to exert a desired effect as a parenteral agent, it depends on the age, body weight and degree of disease of the patient.
In general, it is considered that intravenous injection, intravenous infusion, subcutaneous injection, and intramuscular injection of 0.1 mg to 1 g per day as the weight of the compound of formula in adults are considered appropriate.

This parenteral preparation is manufactured according to a conventional method and is generally used as a diluent in distilled water for injection, physiological saline, glucose solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol. Etc. can be used. Further, if necessary, a bactericide, a preservative, and a stabilizer may be added. Further, from the viewpoint of stability, this parenteral preparation may be filled in a vial or the like, frozen, and then water may be removed by an ordinary freeze-drying technique, and a liquid preparation may be re-prepared from the freeze-dried product immediately before use. Further, an isotonicity agent, a stabilizer, a preservative, a soothing agent and the like may be added as needed.

Other parenteral agents include external preparations, coating agents such as ointments, suppositories for rectal administration, and the like.
It is manufactured according to a conventional method.

Next, the formulation examples of the present invention will be described.

[Formulation Example 1]

According to the above formulation, the ingredients (1) to (4) were uniformly mixed and compression-molded with a tableting machine to give tablets (200 mg each).

This tablet contains 20 mg of the compound obtained in Example 1, and 3 to 10 tablets for adults are to be taken in several divided doses per day.

[Formulation Example 2] Crystalline cellulose 84.5 g Magnesium stearate 0.5 g Carboxymethyl cellulose calcium 5 g Compound obtained in Example 2 10 g Total 100 g

According to the above formulation, a part of and was uniformly mixed, compression-molded, crushed, and the remaining amount of and was mixed, and the mixture was compression-molded with a tableting machine to give one tablet.
0 mg tablets were obtained.

Each tablet contains 20 mg of the compound obtained in Example 2, and 3 to 10 tablets for adults are to be taken in several divided doses per day.

[Formulation Example 3] Crystalline cellulose 79.5 g 10% Hydroxypropyl cellulose ethanol solution 50 g Carboxymethyl cellulose calcium 5 g Magnesium stearate 0.5 g Compound obtained in Example 4 10 g Total 145 g

According to the above formulation, and were uniformly mixed, and the mixture was kneaded by a conventional method, granulated by an extrusion granulator, dried and crushed, and then mixed, and compressed by a tableting machine. It was molded to obtain a tablet of 200 mg each.

Each tablet contains 20 mg of the compound obtained in Example 4, and 3 to 10 tablets for adults are to be taken in several divided doses per day.

[Formulation Example 4]

According to the above recipe, the components (1) to (4) are uniformly mixed, compression molded with a compression molding machine, and crushed with a crusher,
Sieve to obtain granules.

1 g of this granule contains 100 mg of the compound obtained in Example 4, and 1 to 2 g for adults is to be taken in divided doses.

[Formulation Example 5] Crystalline cellulose 86.5 g 10% Hydroxypropyl cellulose ethanol solution 35 g Compound obtained in Example 1 10 g Total 131.5 g

According to the above-mentioned recipe, the ingredients (1) to (4) were mixed uniformly and mixed together. After granulating with an extrusion granulator, it was dried and sieved to obtain granules.

1 g of this granule contains 100 mg of the compound obtained in Example 2, and 1 to 2 g for adults is to be taken in divided doses.

[Formulation Example 6] Corn starch 89.5 g Light anhydrous silicic acid 0.5 g Compound obtained in Example 4 10 g Total 100 g

According to the above-mentioned formulation, ~ was uniformly mixed, and 200 mg was filled in a No. 2 capsule.

20 mg of the compound obtained in Example 4 is contained in one capsule of this capsule, and 3 to 10 capsules for an adult are to be taken in several divided doses per day.

Formulation Example 7 Distilled water for injection 89.5 g Soybean oil 5 g Soybean phospholipid 2.5 g Glycerin 2 g Compound obtained in Example 1 1 g Total amount 100 g

According to the above-mentioned formulation, was dissolved in and, and a solution of and was added to this and emulsified to obtain an injection.

Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C07D 493/04 106 A // A61K 35/78 J 8217-4C ADD C 8217-4C

Claims (5)

[Claims] 1. The following formula I (However, R ₁ and R ₂ are the same or different and each represents a hydrogen atom or a 3,3-dimethylallyl group.) A therapeutic agent for an arachidonic acid metabolism disorder comprising a compound represented by the formula (I) as an active ingredient. 2. The following formula II An agent for treating arachidonic acid metabolism disorders, which comprises a compound represented by 3. The following formula III An agent for treating arachidonic acid metabolism disorders, which comprises a compound represented by 4. The following formula IV An agent for treating arachidonic acid metabolism disorders, which comprises a compound represented by 5. The following formula V (However, R ₃ is a hydrogen atom, a 3,3-dimethylallyl group or
1,1-dimethylallyl group, R ₄ is hydrogen atom or 3,3-dimethylallyl group, R ₅ is hydroxyl group, methoxyl group or 3,3-
A dimethylallyl group, R ₆ and R ₈ are the same or different and each represents a hydrogen atom or a hydroxyl group, and R ₇ represents a hydrogen atom, a hydroxyl group or a 3,3-dimethylallyl group. ) A therapeutic agent for an arachidonic acid metabolism disorder disease comprising a compound represented by the formula (4) as an active ingredient.