JP6590658B2 - Novel labdane-type diterpene compounds and degranulation inhibitors - Google Patents

Description

  The present invention relates to a novel labdan-type diterpene compound and a degranulation inhibitor.

As a plant-derived antihistamine, there is an antiallergic agent containing at least one steviol glycoside (Patent Document 1). Steviol glycosides extracted and purified from stevia leaves are white to light yellow powders and are conventionally used as raw materials for sweeteners. When administered orally, it has an inhibitory effect on the metabolic activity and contact dermatitis of arachidonic acid, and can suppress immediate to delayed allergies. Steviol glycosides described in the patent publication, bound -COO-glc instead the 4-position of -COOH steviol, position 13 in place of the -OH ^{-O-glc 2} - 1 glc bound A compound.

  In addition, an antihistamine substance obtained by adding yeast and water to stevia plant tissue and directly fermenting, extracting the obtained fermented stevia with an ethanol aqueous solution, and freezing and drying a 90% ethanol-soluble fraction of the extract. There is also (patent document 2). An extract excellent in antihistamine effect can be obtained by directly fermenting dry stevia powder. In the examples, antihistamine activity is evaluated by Magnus method for the obtained 90% ethanol-soluble fraction. .

  On the other hand, there is a report that a novel diterpene compound extracted from a yeast fermented product of Stevia plant has a whitening effect (Patent Document 3). Stevia plant tissue is directly fermented by adding yeast and water, and the obtained fermented stevia is extracted with an aqueous ethanol solution, and a new diterpene is added to the chloroform-dissolved substance contained in the 85.5% ethanol-soluble fraction of the extract. A compound is said to be included. When the melanin production amount per B16 mouse melanoma cell was evaluated about the said novel diterpene compound, it was said that the melanin production amount became lower than kojic acid used as positive control.

Japanese Patent No. 39591717 Japanese Patent No. 4375758 International Publication No. 2014/80666

  In Patent Document 1, steviol glycosides are used as an antiallergic agent, but the glycosides are not easy to handle because, for example, the sugar moiety is released. Therefore, development of a compound having excellent antihistaminic activity without having a glycoside is desired.

  Moreover, patent document 2 uses the crude extract obtained from the yeast fermented material of a stevia plant as an antihistamine substance. No compound having antihistamine action has been identified. And as shown in patent document 3, the whitening component is also contained in the yeast fermented material of a stevia plant. Therefore, if a specific component can be separated, it can be used as a pharmaceutical with high purity.

  On the other hand, various whitening components are also contained in the fermented yeast product of Stevia plant, and it is not easy to separate or identify substances having specific effects. It is desirable to specify ingredients and anti-allergic effects with few side effects and little alteration when manufactured.

  In view of the above situation, an object of the present invention is to provide a novel labdan-type diterpene compound derived from a fermented yeast product of a stevia plant, and a degranulation inhibitor containing the compound as a main component.

  The present inventors examined Stevia plants in detail. As a result, the fermented material obtained by fermenting Stevia plants with yeast contained a new compound different from the original Stevia plant. The inventors have found that a compound having a granule inhibitory effect is contained, and that the compound can be used as a degranulation inhibitor or an antiallergic agent, thereby completing the present invention.

That is, the present invention relates to any one of the labdane-type diterpene compounds selected from the group consisting of the following formulas (I) to (V) : formulas (III) to (V) , or a medically acceptable salt thereof. Is to provide.

  The present invention also provides a degranulation inhibitor comprising a labdane diterpene compound (II) or (III) represented by the following formula, or a medically acceptable salt thereof as an active ingredient.

According to the present invention, there is provided a novel labdane diterpene compound represented by the above formulas ( III ) to (V). Labdan-type diterpene compound (II) and labdan-type diterpene compound (III) are excellent in degranulation and can be used as antiallergic agents.

It is a figure explaining the basic frame | skeleton and substituent of a labdan type diterpene compound (I)-(V). In Example 1, it is a figure explaining the isolation | separation process and yield at the time of manufacturing the compound shown by Formula (I)-(IV). In Example 1, it is a figure explaining the isolation | separation process and yield at the time of manufacturing the compound shown by compound (V).

The first aspect of the present invention is that any one of the labdan type diterpene compounds selected from the group consisting of the above formulas (I) to (V) , the formulas (III) to (V) , or a medically acceptable one thereof. Salt. “Labdan” is a diterpene having a 6-carbon side chain at the 9-position carbon of trans-decalin. Since all of the compounds (I) to (V) of the present invention are diterpenes having a side chain having 6 carbon atoms at the 9-position carbon of trans-decalin, the compounds represented by the above formulas (I) to (V) are used. It is called a labdan type diterpene compound.

  In the labdan-type diterpene compounds (I) to (V) of the present invention, two methyl groups are introduced into the 4-position carbon atom of the trans-decalin structure, and one methyl group is present at each of the 8-position and 10-position carbon atoms. It is common in that it has a basic skeleton that is introduced, one hydroxyl group is introduced into each of the 6-position, 7-position, and 8-position carbon atoms, and a 9-position carbon atom has a side chain. FIG. 1 shows the structure of this basic skeleton and the substituent X introduced into the carbon atom at the 9-position.

Compound (I), (II), (III) and Compound (IV) are all optical isomers having a molecular formula of C ₂₀ H ₃₆ O ₆ , and Compound (I) and Compound (II) are in the 13th position. Is an R-coordinate (13R), and compound (III) and compound (IV) are the same 13S. Further, in Compound (I) and Compound (III), the 14-position asymmetric carbon is in R coordination, and Compound (II) and Compound (IV) are in the same S coordination. As a result, Compound (I) becomes 13R14R and Compound (II) becomes 13R14S. Similarly, compound (III) is 13S14R and compound (IV) is 13S14S.

On the other hand, the molecular formula of the compound (V) is C ₂₀ H ₃₆ O _5, which has a double bond between carbons 12-13, and therefore the 13-position is not an asymmetric carbon and has no hydroxyl group. This is different from compound (I) and the like. In addition, 14th carbon of a compound (V) is R coordination.

  The labdane type diterpene compounds (I) to (IV) of the present invention are characterized in that they have three hydroxyl groups in the side chain introduced into the 9-position carbon atom. Conventionally, as a compound derived from a stevia plant, stubren A, sturbine B and the like are known, but a compound having three hydroxyl groups in the 9-position side chain is not known.

  Moreover, the labdane type diterpene compound (V) of the present invention is a compound having a double bond between carbons at the 12th and 13th positions of the side chain introduced into the 9th position carbon atom. Conventionally, as known compounds derived from stevia plants, stuben A, sthbene B and the like are known, but the side chain introduced into the 9-position carbon atom generally has two or more double bonds. It was. Therefore, there is conventionally no compound having one double bond and two hydroxyl groups in this side chain. These are novel compounds produced by yeast fermentation of stevia plants. Although the production route is not clear, it may be produced, for example, using austroinulin, which is a main component of a stevia plant (scientific name: Stevia rebaudiana), as a starting compound.

  It has been found that the labdan-type diterpene compounds (I) to (IV) of the present invention contain a compound having an excellent degranulation inhibitory effect. The labdane diterpene compounds (I) to (IV) are optical isomers, but have different degranulation inhibitory effects, and include compounds having a degranulation inhibitory effect equal to or higher than that of conventional antiallergic drugs.

  The said novel compound can be manufactured by extracting from the yeast fermented material of a stevia plant, or modifying an extract.

  Yeast is a microorganism that discharges alcohol and carbon dioxide from sugar, and is used to produce alcoholic beverages such as sake, beer and wine, fermented foods such as soy sauce and cheese, and bread. Examples of yeasts that can produce the labdane diterpene compounds represented by the above formulas (I) to (V) include Saccharomyces and Saccharomyces cerevisiae.

  The Stevia plant (scientific name: Stevia rebaudiana) used in the present invention is a perennial plant belonging to the genus Stevia that originates in South America. It is currently cultivated in Asia such as Japan, China and Korea. In the present invention, in particular, Stevia Rebaudiana Bertoni and its related plants can be preferably used. Stevia plants that can be used include Stevia plant stems, leaves, whole plants before buds, mature plant roots and flowers.

  Stevia plants contain steviol glycosides that can be used as sweeteners, and there are also crude steviol glycosides obtained by extracting steviol glycosides from stevia plants with water or the like at high concentrations. In the present invention, such a crude steviol glycoside prepared in advance may be fermented with yeast.

  As the stevia plant, a plant harvested at an appropriate time can be used as it is, or a plant that has been dried after harvesting can be used. When Stevia plant is cut into a predetermined size, pulverized, or otherwise chopped or crushed, water and yeast are added and stirred to start fermentation. It is preferable that a stevia plant selects and uses a leaf part and a stem part.

  The amount of water to be added is sufficient as long as it is necessary for fermentation, and an amount sufficient to wet the whole is sufficient. Stevia plants before fermentation have sweetness due to terpenoid glycosides, but the sweetness disappears as fermentation progresses. In the present invention, the disappearance of sweetness is taken as a measure of the end of fermentation. Such disappearance of sweetness is 2-3 weeks at room temperature. After completion of the reaction, the fermented stevia yeast is dried to stop the yeast activity. The drying temperature is 5 to 35 ° C.

  For example, 2 to 30 times the amount of stevia yeast fermented product, preferably 5 to 20 times the amount of 10-50 (v / v)% alcohol aqueous solution, preferably 20-40 (v / v)% alcohol aqueous solution, The residue is filtered off. As the alcohol, a monohydric alcohol having 1 to 5 carbon atoms is preferable, and ethanol is particularly preferable. Next, the filtrate is concentrated and adsorbed on a synthetic adsorbent, followed by elution with a hydrous alcohol solution. The alcohol concentration of the hydrous alcohol is preferably 30 to 99 (v / v)%, more preferably 50 to 99 (v / v)%, and particularly preferably 80 to 95 (v / v)%. The labdane type diterpene compound can be obtained using the water-containing solvent extract thus obtained.

  When isolating the labdan-type diterpene compounds (I) to (V) from the aqueous solvent extract of stevia yeast fermented product, branching with 1 to 12 carbon atoms such as methanol, ethanol, propanol, butanol, etc. in addition to water. Alcohols that may have, polyhydric alcohols such as ethylene glycol, propylene glycol, butylene glycol, ketones such as acetone and methyl ethyl ketone, esters such as methyl acetate and ethyl acetate, ethers such as tetrahydrofuran and diethyl ether , Polyethers such as polyethylene glycol, carbon halides such as dichloromethane, chloroform and carbon tetrachloride, hydrocarbons such as hexane, cyclohexane and petroleum ether, aromatic hydrocarbons such as benzene and toluene, pyridines, super Critical coal dioxide , It can be used oils and fats, waxes and other oils and the like as an extraction solvent.

  In the separation and purification, liquid-liquid distribution, column chromatography, liquid chromatography, gel filtration, activated carbon treatment, precision distillation, etc. can be performed alone or in combination. These can be appropriately selected depending on the type of extraction solvent used and the like.

  On the other hand, since the compounds (I) to (IV) of the present invention contain optical isomers, separation and purification by a solvent or column chromatography may not be easy. A derivative may be introduced by introducing a substituent into the racemate, and chiral separation may be performed with this derivative, and the introduced group may be removed by hydrolysis after isolating the optical isomer. Such derivatives include benzoyl compounds, but the preparation of the derivatives is not limited to benzoylation, provided that optical isomers can be separated.

  In addition, when separating and purifying optical isomers, a substituent is introduced for derivatization and a hydrolysis process is performed after isolation, so that the chemical structure originally contained in the stevia yeast fermented product is maintained. There may be no possibility. In any case, the compounds represented by the above formulas (I) to (V) can be produced. By using a fermented yeast product of a stevia plant as a raw material, a compound having an extremely high degranulation inhibitory effect can be produced.

  In the present invention, in addition to the labdan type diterpene compounds (I) to (V), these medically acceptable salts may be used. “Medical acceptable salt” refers to a salt form that is a compound of the invention that is pharmacologically acceptable and substantially non-toxic to an administered subject. Pharmaceutically acceptable salts of the labdane type diterpene compounds include conventional stoichiometric acid addition salts or base addition salts formed from suitable non-toxic organic or inorganic acids or bases. Suitable inorganic acids are, for example, halogen acids such as hydrochloric acid, sulfuric acid or phosphoric acid. Suitable organic acids are, for example, carboxylic acids, phosphonic acids or sulfonic acids, such as acetic acid, propionic acid, glycolic acid, lactic acid, hydroxybutyric acid, malic acid, maleic acid, malonic acid, salicylic acid, fumaric acid, 琥珀Acids, adipic acid, tartaric acid, citric acid, glutaric acid, 2- or 3-glycerophosphoric acid, and other mineral acids well known to those skilled in the art. The salt is prepared by contacting the free base form with a sufficient amount of the desired acid to produce the salt in the conventional manner. Compounds containing acidic substituents can also form salts with inorganic or organic bases. Examples of suitable bases for forming salts include, but are not limited to, inorganic bases such as alkali or alkaline earth metal (eg, sodium, potassium, lithium, calcium, or magnesium) hydroxides, and ammonium water. There are those derived from oxides (eg, quaternary ammonium hydroxides such as tetramethylammonium hydroxide). Also contemplated are salts formed with pharmaceutically acceptable amines such as ammonia, alkylamines, hydroxyalkylamines, N-methylglucamine, benzylamine, piperidine, and pyrrolidine. Certain compounds are acidic, such as compounds having a carboxyl group or a phenolic hydroxyl group. Phenol salts can be made by heating acidic compounds with any of the bases described above by procedures well known to those skilled in the art.

  The second of the present invention is a degranulation inhibitor comprising a labdane diterpene compound (II) or (III) represented by the following formula, or a medically acceptable salt thereof as an active ingredient.

  The labdan type diterpene compounds (I) to (IV) are optical isomers and have almost the same physicochemical properties. However, when β-hexosaminidase release inhibitory effect in mast cells was evaluated, 13R14S compound (II) was superior in β-hexosaminidase release inhibitory effect to terfenadine, which is an existing antiallergic drug. found. In addition, compound (III) exhibited almost the same degranulation inhibitory effect as terfenadine.

  The labdane type diterpene compound (II) is 13R14S, and is different from the compound (I) only in the configuration at the 14-position. However, almost no inhibitory effect on degranulation is observed in compound (I). Compound (III) is 13S14R, which differs from compound (IV) only in the 14-position configuration, but compound (IV) shows almost no degranulation inhibitory effect. Conventionally, it is known that a specific effect is different between optical isomers, but in a labdan type diterpene compound in which a side chain having 6 carbon atoms is introduced into the 9-position carbon atom, It has never been known at all that a degranulation inhibitory effect is different only by the structure, and a compound that exhibits a degranulation inhibitory effect equal to or higher than that of a conventional antiallergic drug is included.

  Although steroidal compounds are known as degranulation inhibitors, the labdane type diterpene compound of the present invention does not have a steroid skeleton and suppresses degranulation separately from side effects similar to steroid hormones. It is presumed to have an effect and is excellent in safety.

  Since the labdan type diterpene compound (II) and the labdane type diterpene compound (III) of the present invention are excellent in the degranulation inhibitory effect of mast cells, they can be used as a degranulation inhibitor or an antiallergic agent. In addition to transdermal administration such as application, oral administration including oral administration and sublingual administration may also be used.

  As degranulation inhibitors and antiallergic external dosage forms, powders, fine granules, granules, tablets, capsules, pills, solutions, extracts, suppositories, ointments, plasters, aerosols, poultices, injections And eye drops. The compounding amount of the above-mentioned compound in these degranulation inhibitors can be appropriately selected depending on the base material, but is 0.00001% to 20% by mass, preferably 0.0001% to 5% by mass, more preferably 0.001 to 3%. % By mass.

  In the degranulation inhibitor of the present invention, components that are blended in ordinary cosmetics, quasi drugs, pharmaceuticals, and the like can be blended as long as the effect is not impaired. Such ingredients include preservatives such as methyl paraoxybenzoate, propyl paraoxybenzoate, phenoxyethanol, thymol, antibiotics such as sodium bisulfite, ascorbic acid, tocopherol, dibutylhydroxytoluene, sodium edetate hydrate, and benzotriazole. Surfactant such as oxidizing agent, glyceryl monostearate, sorbitan monostearate, polyoxyethylene hydrogenated castor oil 60, polysorbate 60, citric acid hydrate, sodium citrate hydrate, lactic acid, diisopropanolamine, acetic acid, Examples include pH adjusters such as sodium acetate hydrate, humectants, thickeners, inorganic fillers, colorants, fragrances, ultraviolet absorbers, cell activators, various skin nutritional ingredients, and the like.

  EXAMPLES Next, although an Example is given and this invention is demonstrated concretely, these Examples do not restrict | limit this invention at all.

Example 1
(1) Preparation of Stevia Fermented Yeast Stevia Plant Stevia (Stevia rebaudiana) produced in Hokkaido, Japan was dried and crushed to about 1 cm or less. To 1.2 kg of crushed stevia plant, 27 g of yeast (Saccharomyces cerevisiae) and 2.3 liters of water were added and stirred under a temperature of 25 to 35 ° C. to initiate fermentation. This was allowed to stand at a temperature of 25 to 35 ° C. for 14 days, and the time when the sweetness of the fermented product disappeared was regarded as the end of fermentation. Drying was performed at a temperature of 25 to 35 ° C. for 10 days to stop yeast fermentation, and a stevia fermented product was obtained.

(2) Preparation of hydrous solvent extract To the total amount of the stevia fermented product, 10 mass times 30.0 (v / v)% ethanol aqueous solution was added and allowed to infiltrate at room temperature for several days with gentle stirring. . After infiltration, it was filtered with a size of about 140 mesh. The obtained filtrate was concentrated under reduced pressure (60 mmHg) at 60 ° C., adsorbed on a Diaion HP20 column, and eluted with a 90% aqueous ethanol solution to obtain a stevia fermented aqueous solvent extract.

(3) Extraction of compounds (I) to (IV) According to the separation process diagram of FIG. 2, the stevia fermented hydrous solvent extract is dissolved in 1000 mL of chloroform, and then 1000 mL of water is added to the chloroform layer and the aqueous layer. After separation, 1000 mL of ethyl acetate was added to the aqueous layer, and the mixture was separated into an ethyl acetate layer and an aqueous layer. Further, 1000 mL of n-butanol was added to the aqueous layer, the butanol layer and the aqueous layer were separated, and the butanol layer (19.2 g) was separated.

  Next, the butanol layer (19.2 g) was separated into 5 fractions by silica gel column chromatography using chloroform: methanol: water = 80: 40: 1 → 30: 15: 1 → 6: 4: 1 → methanol as an elution solvent. did. The yield of each fraction is shown in FIG.

  Fraction 1 (4.61 g) was separated into 5 fractions by silica gel column chromatography using chloroform: methanol = 10: 1 → 7: 1 → 5: 1 → 3: 1 → methanol as an elution solvent.

  Fraction 1-4 (1.37 g) was separated into five fractions by Sephadex LH-20 column chromatography using methanol as the eluting solvent.

  Fraction 1-4-2 (1.01 g) was eluted with chloroform: methanol = 30: 1 → 15: 1 → 7: 1 → 3: 1 → 1: 1 → methanol, and 5 fractions from silica gel column chromatography. Separated into fractions.

  Fraction 1-4-2-3 (58.1 mg) was separated into four fractions by ODS column chromatography using methanol: water = 50: 50 → 60: 40 as an elution solvent.

  The obtained 1-4-2-3-2 fraction (8.0 mg) is benzoylated according to the following benzoylation reaction and purification method of the reaction product, and the two kinds of benzoyl derivatives contained are isolated, After isolation, each derivative was hydrolyzed with sodium hydroxide.

Benzoylation reaction and purification method of the reaction product:
The entire amount of fraction 1-4-2-3-2 was dissolved in 2 mL of pyridine, 6 mg of DMAP and 100 μl of benzoyl chloride were added, and the mixture was reacted at room temperature for 1 hour. After completion of the reaction, benzene and water were added to the reaction solution for partition extraction, the benzene layer was dried over anhydrous sodium sulfate, and the solvent was distilled off with an evaporator. A benzoyl derivative was obtained by using an ODS column having a column inner diameter of 1.5 cm and a column height of 15.7 cm and using MeOH: H ₂ O = 4: 1 as an elution solvent. Subsequently, the benzoyl derivative was chirally separated under the following HPLC conditions. Derivatives were separated at elution times of 22 minutes and 25 minutes, respectively.

(HPLC conditions)
Pump: Senshu SSC-3461
Detector: Senshu SSC-5410
Column: Daicel CHIRALPAKIE 1cm x 25cm
Detection wavelength: 254 nm
Elution solvent: MeOH: H ₂ O = 50: 1

Next, each of these benzoyl derivatives was dissolved in 1 mL of methanol, 200 μl of 1N NaOH was added, and the mixture was reacted at room temperature for 1 hour. The solvent of the reaction product was distilled off with an evaporator as it was. Using an ODS column having a column inner diameter of 1.5 cm and a column height of 12 cm, MeOH: H ₂ O = 7: 3 was used as an elution solvent, and 15 mL was fractionated to obtain two types of hydrolysates. All of these were colorless oils.

  As for the obtained fraction 1-4-2-3-3, the benzoylation reaction and the reaction product were purified in the same manner as described above to obtain two hydrolysates. All of these were colorless oils.

(4) Extraction of Compound (V) 1000 mL of chloroform was added to the extract (60 g) obtained by distilling off the solvent of the above-mentioned stevia fermented hydrous solvent extract, and extracted at a temperature of 23 to 28 ° C. (room temperature). The residue was removed by filtration, divided into a chloroform layer (28.84 g) and a residue, and 10 times the amount of methanol was added to the residue to obtain 25.98 g of a methanol extract.

  The methanol extract (25.98 g) was separated into three fractions by silica gel column chromatography using chloroform: methanol: water = 80: 40: 1 → 30: 15: 1 → 6: 4: 1 → methanol as the eluting solvent. did. The yield of each fraction is shown in FIG.

  Fraction 1 ′ (8.38 g) was separated into 6 fractions by silica gel column chromatography using chloroform: methanol = 10: 1 → 7: 1 → 5: 1 → 3: 1 → 1: 1 → methanol as an eluting solvent. did.

  Fraction 1'-3 (1.36 g) was separated into five fractions by ODS column chromatography using methanol: water = 50: 50 → 60: 40 as an elution solvent.

  The 1'-3-3 fraction (235.4 mg) was separated into 7 fractions by silica gel column chromatography using chloroform: methanol = 15: 1 → 10: 1 → methanol as an eluting solvent.

Fraction 1′-3-3-5 (12.3 mg) was separated into 5 fractions by ODS column chromatography using methanol: water = 50: 50 → 60: 40 as an elution solvent.
The 1′-3--3-5-2 fraction (4.4 mg) was a colorless oil.

(5) Structure determination Two colorless oils isolated from fraction 1-4-2-3-2 shown in FIG. 2 and two colorless oils isolated from fraction 1-4-2-3-3 ¹ H-NMR and ¹³ C-NMR were measured for the oily substance and the colorless oily substance isolated from fraction 1′-3--3-5-2 shown in FIG. The results are shown in Tables 1 and 2. In the lower part of Table 1, the basic skeleton and position number of the labdane type diterpene compound are shown. ^From the signal patterns of ¹ H-NMR and ¹³ C-NMR, Sturpentine A, A ring, and B ring, which have 20 carbon atoms and have already been isolated from Stevia, have the same structure, and the side chain at position 9 is different. Estimated compound.

Having a C ₂₀ molecular formula of H ₃₆ H _6, two colorless oily product was isolated from fractions 1-4-2-3-2, the fraction 1-4-2-3-3 two isolated A detailed structural analysis was performed on the colorless oil.
First, ¹ H- ¹ HCOSY correlation and HMPC correlation were evaluated for one of the isolates of fraction 1-4-2-3-3. As a result, the 9-position of a hydrogen atom and the 11-position hydrogen atom, and the 11-position hydrogen atom and 12th hydrogen atom, 14 of ¹ H- ¹ HCOSY correlation hydrogen atoms and 15-position hydrogen atom, and position 16 HMPC correlation was observed between the hydrogen atom of and the 12th, 13th and 14th carbon atoms, and the labdan type diterpene compound (I) represented by the formula (I) was determined. On the other hand, since it was concluded that the result of NMR was the same as that of the compound (I) and that only the carbon atom at the 14th position was different by X-ray analysis, the 14th position of the compound (I) Were determined to be optical isomers having different configuration of carbon atoms.
Similarly, one of the fractions 1-4-2-3-2 isolated is analyzed in the same manner as described above to determine the compound (III), and the other is a compound in which the configuration of the 14th carbon atom is different. (IV) was determined. The respective yields are shown in FIG.

On the other hand, the isolate of fraction 1′-3-3-3-2 has a molecular formula of the molecular formula C ₂₀ H ₃₆ H ₅ , and ¹ H-NMR and ¹³ C-NMR are labdan type diterpene compound (I) and Analogously, the side chain at the 9-position was different and this side chain was presumed to consist of methylene, olefinic carbon and hydrogen, glycol and methyl groups. Therefore, ¹ H- ¹ HCOSY correlation and HMPC correlation were evaluated. As a result, the 9-position of a hydrogen atom and the 11-position hydrogen atom, and the 11-position hydrogen atom and 12th hydrogen atom, 14 of ¹ H- ¹ HCOSY correlation hydrogen atoms and 15-position hydrogen atom, and position 16 HMPC correlation was observed between the hydrogen atom of and the 12th, 13th and 14th carbon atoms, and the compound was determined to be compound (V).

(Example 2)
Evaluation of anti-allergic action of compounds (I), (II), (III) and (IV) obtained in Example 1 using β-hexosaminidase release inhibition using cultured cells as an indicator of degranulation reaction did.
(1) Rat basophil leukemia cell RBL-2H3 (JCRB0023) was used as a mast cell model. The medium is Dulbecco's modified Eagle medium (Gibco) supplemented with 10% fetal bovine serum (Biological Industry), 100 U / mL penicillin, 100 μg / mL streptomycin (Gibco), 37 ° C., 5% CO _2. The culture was performed under the conditions of
(2) DMSO was added to each of the compounds (I) to (IV) obtained in Example 1 to prepare a 10 mg / mL sample solution. The concentration of each compound was a final concentration of 50 μg / mL, and the DMSO concentration was 0.5%.
(3) RBL-2H3 cells were seeded in a 24-well plate at a concentration of 5 × 10 ⁵ cells / mL. Mouse monoclonal anti-DNP-IgE antibody (manufactured by Yamasa Co., Ltd.) was added to the culture solution to a final concentration of 0.45 μg / mL and cultured overnight at 37 ° C. and 5% CO ₂ conditions. The supernatant was removed and washed twice with 500 μL of Silaganian buffer (119 mM NaCl, 5 mM KCl, 0.4 mM MgCl ₂ , 25 mM PIPES, 40 mM NaOH), and 160 μL of release buffer (D-glucose 5.6 mM, (Silagadian buffer containing 1.0 mM CaCl ₂ and 0.1% BSA) was added, and the mixture was incubated at 37 ° C. for 10 minutes, and 20 μL of the sample solution (final concentration 50 μg / mL) was added, followed by further incubation for 10 minutes. Thereafter, 20 μL of 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) (manufactured by LSL) was added as an antigen solution to a final concentration of 10 μg / mL, and the mixture was further reacted at 37 ° C. for 10 minutes. Degranulation was triggered. The reaction was stopped by cooling on ice, and the reaction supernatant was collected. A substrate solution (1 mM p-nitrophenyl-2-acetamido-2-deoxy-β-D-glucopyranoside (manufactured by Nacalai), 0.1 M citrate buffer (PH 4.5)) 50 μL of the reaction supernatant was added to 50 μL and reacted at 37 ° C. for 60 minutes. Subsequently, 200 μL of 0.1 M NaHCO ₃ / Na ₂ CO ₃ buffer (pH 10.0) was added, and the absorbance at 405 nm of the reaction product was measured with a microplate reader (manufactured by Tosoh Corporation). The reaction supernatant obtained by adding only the antigen without antibody treatment to the cells was used as a control, and the same amount of terfenadine added instead of compound (I) was used as a positive control. The control ratio (%) of each compound and positive control, and β-hexosaminidase release inhibition (%) were calculated from the respective absorbances according to the following formula. The results are shown in Table 3.

Control ratio (%) = {(C−D) / (A−B)} × 100
β-hexosaminidase release inhibition (%) = {1- (C−D) / (A−B)} × 100
A: Absorbance of positive control (added with Shiraganian buffer instead of sample and stimulated with antigen)
B: Absorbance of the negative control (added with a Siraganian buffer instead of the sample and antigen)
C: Absorbance of sample (sample and antigen added)
D: Absorbance of specimen control (sample added and Siraganian buffer added instead of antigen)

(result)
Terfenadine, used as a positive control, is a second generation antihistamine with histamine H1 receptor antagonism. Compounds (I) to (IV) all showed β-hexosaminidase release inhibition from the control. In particular, compound (II) showed a statistically significant β-hexosaminidase release inhibitory effect as compared with control, and compound (III) also showed a β-hexosaminidase release inhibitory effect equivalent to terfenadine.

  The novel labdan-type diterpene compounds (II) and (III) of the present invention have an excellent degranulation inhibitory effect and are useful as antiallergic agents and the like.

Claims (2)

Any labdane diterpene compound selected from the group consisting of the following formulas ( III ) to (V), or a medically acceptable salt thereof.
A degranulation inhibitor comprising a labdane diterpene compound (II) or (III) represented by the following formula or a medically acceptable salt thereof as an active ingredient.