JP5513754B2 - Novel compound and preventive or therapeutic agent for bone diseases containing the compound as an active ingredient - Google Patents

Description

  The present invention relates to a novel compound useful for the prevention and treatment of bone diseases such as osteoporosis and a preventive or therapeutic agent for bone diseases comprising the compound as an active ingredient.

  Bone repeats bone formation and bone resorption. The life cycle of such a bone is roughly as follows.

  That is, chondrocytes, osteoblasts, and osteoclasts perform functional division of labor, and each cell controls bone metabolism. Bone metabolism consists of (1) osteogenesis and growth by endochondral ossification, (2) qualitative and quantitative maintenance by remodeling consisting of the cycle of bone formation and bone resorption, and (3) damage such as fractures. It is classified into three processes of bone regeneration. Bone metabolism is controlled by regulating the function of each cell system and the interaction between cells via various calcium-metabolizing hormones and cytokines, various intracellular transmission mechanisms and transcription factors.

  For example, when the bone surface in the resting state is stimulated, information is transmitted to osteoclasts and bone resorption starts. On the other hand, when bone is being resorbed, bone repair and formation are performed by osteoblasts. Thus, in a living body in a normal state, bone resorption and bone formation are performed in parallel, and the bone is newly reborn.

  By the way, in recent years, osteoporosis of the elderly has attracted attention. Osteoporosis is caused by bone remodeling in which bone resorption exceeds bone formation in bone remodeling. Therefore, prevention or treatment of osteoporosis requires suppression of bone resorption or promotion of bone formation.

  Under such circumstances, various osteoporosis therapeutic agents and components derived from traditional Chinese medicines and foods that are expected to have an osteoporosis therapeutic effect have been proposed. Examples of the former include active vitamin D and reveromycin A derivatives described in Patent Document 1. Moreover, as the latter, Cordyceps sinensis described in Patent Document 2, water extract of eringi and five peels described in Patent Document 3, water extract of maturity yellow and osteoclastic supplement described in Patent Document 4, For example, the extract of the mushrooms described in Patent Document 5 can be used.

JP 2005-35895 A JP 2004-315454 A JP 2005-330289 A JP 2005-330290 A JP 2006-193452 A

  Similar to the inventors of the above-mentioned patent document, the present inventors have been researching and seeking compounds that have an effect of preventing and treating osteoporosis and have few side effects. And it discovered that the extract from the fruit body of the cocoon whose scientific name is Grifola gargal (Grifola gargal; Japanese name: Anninkou) shows an osteoclast formation inhibitory effect. However, in order to be ingested by humans as a preventive or therapeutic agent for bone diseases such as osteoporosis, the compound itself having an osteoclast formation inhibitory effect present in the fruit body of Griphora Gargal is more convenient. Therefore, an object of the present invention is to identify a compound having an osteoclast formation inhibitory effect present in the fruit body of Griphora galgar and to provide the compound as a preventive or therapeutic agent for bone diseases.

  The inventors of the present invention have intensively studied in order to identify a compound exhibiting an osteoclast formation inhibitory effect existing in the fruit body of Griphora Gargal, and completed the present invention.

That is, the present invention relates to compounds represented by the following formulas (1) to (4):

as well as

  More specifically, the present invention relates to a compound represented by any one of the above formulas (1) to (4) or a pharmacologically acceptable salt thereof, or the above formulas (1) to (4) by hydrolysis. The present invention relates to a compound that provides a compound represented by any of the following:

  The present invention provides a compound represented by the formula (1), a pharmacologically acceptable salt of the compound represented by the formula (1), and a compound that provides the compound represented by the formula (1) by hydrolysis. A compound that provides the compound represented by the formula (2), the pharmacologically acceptable salt of the compound represented by the formula (2), and the compound represented by the formula (2) by hydrolysis, A compound represented by the formula (3), a pharmacologically acceptable salt of the compound represented by the formula (3), a compound which provides a compound represented by the formula (3) by hydrolysis, the formula (4) ), A pharmacologically acceptable salt of the compound represented by formula (4), and a compound that provides the compound represented by formula (4) by hydrolysis. One or more pharmacologically effective In characterized in that it contains, it relates to a preventive or therapeutic agent for bone diseases.

  Examples of the bone disease include osteoporosis. The active ingredient is preferably used as an osteoclast formation inhibitor. The compound used as an active ingredient in the preventive or therapeutic agent for bone diseases may be an extract from the fruit body of Gryfora galgar (Japanese name: Anninkou).

  The compounds represented by the formulas (2) and (4) and pharmacologically acceptable salts thereof are novel compounds.

  The present invention provides a preventive or therapeutic agent for bone diseases that is highly safe and effective in the prevention and treatment of bone diseases such as osteoporosis.

It is a microscope picture of the control well which performed TRAP dyeing | staining after coculture. It is a microscope picture of the well in which formation of the osteoclast was suppressed by performing TRAP dyeing | staining after coculture. It is a figure which shows the extraction procedure of the component which shows osteoclast formation inhibitory activity from a Griphora galgar fruit body warm air dried material. It is a figure which shows the extraction procedure of the component which shows the osteoclast formation inhibitory activity performed after the procedure shown in FIG. 2 is a molecular model showing the results of X-ray crystallographic analysis of Compound 4.

  Each of the compounds represented by the formulas (1) to (4) according to the present invention is an ergosterol compound. Among these, it is preferable that the steric configuration of the compound represented by the formula (4) is represented by the following formula (4-1).

  The compounds represented by the formulas (1) to (4) may have isomers in terms of chemical structure. In the present invention, the “compound represented by the formula (x)” means all geometrical isomers generated in the structure of the compound, optical isomers based on asymmetric carbon, stereoisomers, tautomers and the like. And mixtures of isomers. However, the steric configurations of the compounds represented by the formulas (1) and (3) are as shown in the respective formulas.

  In the present invention, when there are crystal polymorphs in the compounds represented by the formulas (1) to (4), the crystal form is not limited in the present invention. It may be a mixture.

  The pharmacologically acceptable salts of these compounds are, for example, alkali or alkaline earth metal salts in which, in the above formulas (1) to (4), the hydrogen of the hydroxyl group is replaced with sodium, potassium, calcium or the like. Or an ammonium salt substituted with an ammonium group, an inorganic acid such as hydrochloric acid or sulfuric acid, an organic acid such as p-toluenesulfonic acid, or an amino acid such as glycine. However, it is not limited to these, and any may be used as long as it is pharmacologically acceptable.

  “A compound that provides a compound represented by the formula (x) by hydrolysis” means that the hydroxyl group in the above formulas (1) to (4) is protected by a protecting group, in other words, in the hydroxyl group. A compound in which a hydrogen atom is substituted with some group and becomes a hydroxyl group when hydrolyzed. Examples of such “hydroxyl groups protected by protecting groups” include methoxymethyl ether group, tetrahydropyranyl ether group, tertiary butyl ether group, allyl ether group, benzoate group, acetate group (acetyloxy group), Examples include a formate group, a crotonate group, a p-phenylbenzoic acid ester group, a trimethylacetyloxy group, a tertiary butyldimethylsilyloxy group, a tertiary butyldiphenylsilyloxy group, a trityloxy group, and a benzyloxy group.

  The compounds represented by the formulas (1) to (4) can be organically synthesized with reference to a method for synthesizing sterols.

  In addition, the compounds represented by the formulas (1) to (4) can be isolated by extraction from the fruit bodies of Gryfora galgar or Kawariharatake. Here, Griphora Gargal is a genus of Griphora genus native to South America, and in recent years, fungus bed cultivation has become possible. A fungus bed cultivation method for obtaining a fruit body of Griphora galgar is disclosed in, for example, Japanese Patent Application Laid-Open No. 2007-20560. Kawariharatake is a spider belonging to the genus Agaricus.

  Extraction of the compounds represented by the formulas (1) to (4) from the fruit body of Griphora Gargal is performed, for example, as follows. The dried fruit body is put into an organic solvent such as hexane or water, and separated into a soluble fraction and an insoluble fraction. Thereafter, extraction is performed from each fraction using ethyl acetate or the like. The extract may be subjected to column chromatography to retain useful components, and then effluent under appropriate conditions to separate and purify the compound.

  The active ingredient of the agent for preventing or treating bone disease according to the present invention comprises a compound represented by the formula (1), a pharmacologically acceptable salt of the compound represented by the formula (1), and a compound represented by the formula (1) by hydrolysis. A compound that provides a compound represented by formula (2), a compound represented by formula (2), a pharmacologically acceptable salt of the compound represented by formula (2), and a compound represented by formula (2) by hydrolysis. Provided compound, compound represented by formula (3), pharmacologically acceptable salt of compound represented by formula (3), compound providing formula (3) by hydrolysis, formula (3) 1 type selected from the group consisting of a compound represented by 4), a pharmacologically acceptable salt of a compound represented by formula (4), and a compound that provides a compound represented by formula (4) by hydrolysis That's it.

  The active ingredient of the preventive or therapeutic agent for bone diseases according to the present invention exhibits at least an action of suppressing the formation of osteoclasts. Therefore, diseases for which the preventive or therapeutic agent for bone diseases of the present invention is effective include endogenous diseases in which bone mass is reduced due to excessive formation of osteoclasts and hyperfunction, and exogenous diseases such as fractures. Is included. Specific examples of the bone disease include osteoporosis, osteoclastoma, osteopenia, osteoporosis, osteomalacia, traumatic fracture, fatigue fracture and the like.

  The dosage form, administration method, dosage and the like of the preventive or therapeutic agent for bone diseases according to the present invention can be appropriately determined in consideration of the purpose of use and the physical properties of the active ingredient. Examples of dosage forms include tablets, capsules, granules, fine granules, powders, etc. for oral administration, and injections, inhalants, suppositories, transdermal absorption for parenteral administration. Agents and the like. Examples of the additive used together with the active ingredient include an excipient, a binder, a disintegrant, and a solvent.

  The dosage varies depending on conditions such as prevention or treatment, disease state, dosage form, administration method, patient age and weight, etc., but when administered orally to an adult, any of the compounds of the present invention In the case of using, it is usually 0.1 to 200 μg / kg body weight / day, preferably 0.2 to 50 μg / kg body weight / day.

  Hereinafter, the present invention will be described specifically by way of examples.

(Example 1) Extraction and identification of active ingredient from fruit body of Griphora galgar (1) Determination method of presence or absence of active ingredient Measurement of activity and cytotoxicity by active ingredient in extract or fractionation solution is as follows This was done as follows.

(1-1) Cell culture by co-cultivation method Collected from tibia and femur of mouse (♀, 5 to 7 weeks old), 1.3x10 ⁸ cultured bone marrow cells, and collected from skull and cultured 1.0 × 10 ⁶ osteoblast-like stromal cells thus prepared were suspended in 7.2 ml of α-MEM (Minimum Essential Medium α-Medium) containing 10% fetal bovine serum. This was dispensed at 150 μl / well into a 48-well osteoclast activity assay substrate plate. Α-MEM solution (concentration: 20 ng / ml) containing 10% fetal bovine serum of 1,25-dihydroxyvitamin D ₃ (concentration: 20 ng / ml) and a test sample dissolved in dimethyl sulfoxide (DMSO) (test sample 100 mg / ml), 50 μl / well diluted with α-MEM containing 10% fetal bovine serum (preparation of a dilution series) was added to make a total volume of 250 μl / well. Using a carbon dioxide incubator, the cells were cultured for 7 days at 37 ° C. and a carbon dioxide concentration of 5%. On the third day of culture, 100 μl of the supernatant in each well was removed, and instead, 50 μl of α-MEM solution containing 10% fetal bovine serum (concentration: 40 ng / ml) of 1,25-dihydroxyvitamin D ₃ / Well and 50 μl / well of 10% fetal bovine serum-containing α-MEM solution of the test sample containing the test sample at a concentration twice that used before the start of culture.

(1-2) Measurement of osteoclast formation number by TRAP staining method As described above, when bone marrow cells and osteoblast-like stromal cells are cocultured in the presence of active vitamin D ₃ , TRAP ( Differentiate into osteoclast-like multinucleated cells (osteoclasts) with tartrate-resistant acid phosphatase). The cells are stained with TRAP stain. By counting the number of stained cells, it can be determined whether or not the formation of osteoclasts was suppressed.

  The TRAP staining solution was prepared as follows. First, a TRAP buffer solution (pH = 5.0) containing 50 mM sodium tartrate and 0.1 M sodium acetate was prepared. Next, 1.5 mg of naphthol AS-MX phosphate was dissolved in 150 ml of N, N-dimethylformamide as prepared at the time of use. To this was added 15 ml of TRAP buffer, and then 9 mg of Fast Red Violet LB salt was added and dissolved.

  Staining was performed as follows. The culture medium in the well was removed, and the cells were washed with phosphate buffered saline (PBS). Then, a PBS solution containing 10% formalin (= 3.7% formaldehyde) was added at 0.5 ml / well and allowed to stand for 10 minutes to fix the cells in the well. Thereafter, ethanol was added at 0.5 ml / well and left for 1 minute. After drying, 0.3 ml / well of TRAP staining solution was added to the wells and incubated at room temperature for 30 minutes. The wells were washed with distilled water 0.5 ml / well and then dried.

  Under the microscope, the number of multinucleated cells (TRAP-positive multinucleated cells) having two or more nuclei stained with TRAP staining solution per well was counted. This is the number of osteoclasts per well.

  When culturing the cells, 10% fetal calf serum-containing α-MEM (50 μl) was used instead of the test sample dissolved in DMSO and diluted with α-MEM containing 10% fetal calf serum (50 μl). What was added was prepared and processed in the same manner. This is the control.

  Taking the number of cells in the control well as 100, the ratio of the number of cells in the well to which the test sample was added was calculated. The smaller the value, the higher the osteoclast formation inhibitory effect.

  FIG. 1 shows a micrograph of a control well, and FIG. 2 shows a micrograph of a well in which osteoclast formation was suppressed.

(1-3) Measurement of cell viability by MTT assay method MTT refers to 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide. This compound is water-soluble, and the aqueous solution is yellow. However, when a redox reaction occurs between MTT and NADH by dehydrogenase, MTT is reduced to blue formazone. Since this dehydrogenase is an enzyme related to the mitochondrial respiratory chain, it is an indicator of cell health. That is, if the cell is fine, the enzyme activity is high and a large amount of formazone is produced, but if the cell dies, no formazone is produced. Therefore, the amount of produced formazone and thus the cell viability can be measured by measuring the absorbance at a wavelength of 570 nm.

  The MTT solution is a PBS solution containing MTT at a concentration of 1 mg / ml. After co-culture, the MTT solution was added in an amount of 125 μl / well without removing the culture medium. Subsequently, it incubated for 2 hours at 37 degreeC and the carbon dioxide gas concentration 5% using the carbon dioxide incubator. The culture solution was removed and dimethyl sulfoxide (DMSO) was added in an amount of 100 μl / well. After formazone was well dissolved, the absorbance of the DMSO solution was measured at a wavelength of 570 nm.

  When culturing the cells, 10% fetal calf serum-containing α-MEM (50 μl) was used instead of the test sample dissolved in DMSO and diluted with α-MEM containing 10% fetal calf serum (50 μl). What was added was prepared and processed in the same manner. This is the control.

  Taking the absorbance of the control as 100, the ratio of absorbance when the test sample was added was calculated. The smaller the value, the lower the cell viability or cell activity, which indicates that the test sample is more cytotoxic.

(2) Raw material In the fungus bed cultivation, griffola galgar fruit bodies were harvested about 120 days after the inoculation planting. This was dried by applying warm air of 45 ° C. all day and night and then warm air of 70 ° C. for 1 hour. The Griphora Gargal fruit body dried in warm air in this way was used as a raw material.

(3) Extraction of active ingredient The outline of the extraction method is as shown in FIG. 3 and FIG. Specifically, active ingredients were extracted as follows.

(3-1) Hexane Extraction 8.78 kg of Griphora Galgar fruiting body dried with hot air was pulverized. This was placed in 50 l of hexane and left at room temperature. Separation into hexane solution and residue by filtration.

(3-2) Extraction with ethyl acetate 50 l of ethyl acetate was added to the residue and left at room temperature. Filtration separated into an ethyl acetate solution and a residue. The ethyl acetate solution contained 101.5 g of solid content (ethyl acetate soluble fraction).

(3-3) Flash column chromatography The ethyl acetate solution obtained in (3-2) (the amount corresponding to 90.1 g in solid content) was subjected to flash column chromatography (silica gel 60N; Kanto Chemical Co., Inc.). Manufactured; packing weight: 350 g; φ4 cm × 60 cm), and the active ingredient was retained in the column. Next, 500 ml of a hexane / ethyl acetate (9: 1 (volume)) mixture, 500 ml of methylene chloride, 500 ml of a methylene chloride / acetone (8: 2 (volume)) mixture, 500 ml of acetone and 1,000 ml of 100% ethanol were used as eluents. It flowed in this order.

  The eluate was collected in 20 ml portions. Thereafter, 90% methanol was allowed to flow through the column. For each of the collected solutions, the number of osteoclasts formed was measured by the TRAP staining method by the method described in (1-1) and (1-2).

  Based on the measurement results, the eluates were grouped into 28 groups (in order, 3GA-EA-1 to 3GA-EA-28). Using each of these 28 groups as test samples, the method of (1-1), (1-2) and (1-3) was used to measure the number of osteoclasts formed by the TRAP staining method and MTT assay. went. It was 3GA-EA-11 to 3GA-EA-27 that osteoclast formation inhibitory activity was confirmed.

(3-4) Pretreatment with Sep pak and high performance liquid chromatography using reverse phase column Fraction 3GA-EA-18 (3.5892 g) obtained by the treatment described in (3-3) (Trademark) Plus C18 cartridge (Waters Co., Ltd .; ODS) was passed. A fraction of 2.7352 g that flowed out without being retained in Sep Pak (registered trademark) was taken, and this was named 3GA-EA-18-4.

  3GA-EA-18-4 (2.7352 g) was passed through a preparative reversed phase column (Nomura Chemical Co., Ltd .; ODS column 30C-UG-15 / 30) connected to a high performance liquid chromatograph. The active ingredient was retained. Elution was performed with 90% methanol to obtain fractions of 3GA-EA-18-4-1 to 3GA-EA-18-4-57.

  Using each fraction, the number of osteoclasts formed was measured by the TRAP staining method according to the method described above. As a result, osteoclast inhibitory activity was confirmed in 3GA-EA-18-4-2 to 3GA-EA-18-4-57. Based on these results, further fractionation and purification were performed on three fractions, namely 3GA-EA-18-4-33, 3GA-EA-18-4-34 and 3GA-EA-18-4-49. went.

(3-5) Fractionation and Purification of Fraction 3GA-EA-18-4-33 -Pretreatment with Sep pak and High Performance Liquid Chromatography with Normal Phase Column-
Fraction 3GA-EA-18-4-33 (amount corresponding to a solid content of 46.5 mg; chloroform solution) was passed through a Sep Pak (registered trademark) plus silica cartridge (manufactured by Waters; normal phase). The fraction that flowed out without being retained in Sep Pak (registered trademark) was named 3GA-EA-18-4-33-1. Then, what was not eluted with chloroform (adsorbed material; unsoluble part) was eluted with methanol. The solid content was 34.6 mg.

  Normal phase column (Senshu Pak AQ ss-5251; 3SH-Pak AQ ss-5251; 3GA-EA-18-4-33-1 (amount corresponding to 12.6 mg in solid content) connected to a high performance liquid chromatograph; The active ingredient was retained. Thereafter, elution was carried out with methanol, and fractions 3GA-EA-18-4-33-1-1 to 3GA-EA-18-4-3-33-1-12 were obtained.

  Using each fraction, the number of osteoclasts formed was measured by the TRAP staining method according to the method described above. As a result, the active ingredient was concentrated in 3GA-EA-18-4-3-33-1-7. The solvent was distilled off to obtain 1.9 mg of solid content. As described later, this solid content was Compound 1 represented by the formula (1).

(3-6) Fractionation and purification of fraction 3GA-EA-18-4-34-pretreatment with Sep pak and high performance liquid chromatography with normal phase column-
Fraction 3GA-EA-18-4-34 (amount corresponding to a solid content of 53.4 mg; dissolved in ethyl acetate / chloroform (1: 9 (volume)) mixture) Sep Pak® Passed through a plus silica cartridge (Waters, normal phase). The fraction that flowed out without being retained in the Sep Pak (registered trademark) was named 3GA-EA-18-4-34-1. Then, what was not eluted with ethyl acetate / chloroform (adsorbent; unsolvable part) was eluted with methanol. The solid content was 17.1 mg.

  Normal phase column (Senshu Pak AQ ss-5251; 3SH-Pak AQ ss-5251; 3GA-EA-18-4-34-1 (amount equivalent to 37.0 mg in solid content) connected to a high performance liquid chromatograph; The active ingredient was retained. Elution was performed with ethyl acetate / chloroform (1: 9 (volume)) to obtain fractions of 3GA-EA-18-4-34-1-1 to 3GA-EA-18-4-34-1-13. .

  Using each fraction, the number of osteoclasts formed was measured by the TRAP staining method according to the method described above. As a result, the active ingredient was concentrated in 3GA-EA-18-4-34-1-4. The solvent was distilled off to obtain 1.0 mg of a solid content. As described later, this solid content was Compound 1 represented by the formula (1).

(3-7) Fractionation and purification of fraction 3GA-EA-18-4-49 (3-7-1) Recrystallization of fraction 3GA-EA-18-4-49 Fraction 3GA-EA-18- The solvent of 4-49 was distilled off to obtain 75.7 mg of a solid content. This was recrystallized in a two-solvent system of chloroform (solvent in which the solid content was dissolved) and methanol (solvent in which the solid content was not dissolved). Crystallized samples and amorphous parts were obtained.

(3-7-2) Pretreatment of crystallized sample with Sep pak and high performance liquid chromatography using normal phase column The obtained crystallized sample 23.8 mg was added to ethyl acetate / chloroform (1: 9 (volume)). Dissolved. The solution was passed through a Sep Pak (registered trademark) plus silica cartridge (Waters Corp .; normal phase). The fraction that flowed out without being retained in Sep Pak (registered trademark) was named 3GA-EA-18-4-49-c-1. Then, what was not eluted with ethyl acetate / chloroform (adsorbent; unsolvable part) was eluted with methanol. The solid content was 4.4 mg.

  Normal phase column (Senshu Pak AQ ss-5251; 3GA-EA-18-4-49-c-1 (amount corresponding to 17.8 mg in solid content) connected to a high performance liquid chromatograph; The active ingredient was retained through a product of Senshu Kagaku. Elution was performed with ethyl acetate / chloroform (1: 9 (volume)).

  Using each eluted fraction, the number of osteoclasts formed was measured by the TRAP staining method according to the method described above. Based on the results, fractions in which the active ingredient was concentrated were collected. Those fractions were divided into three groups, 3GA-EA-18-4-49-c-1-1, 3GA-EA-18-4-49-c-1-2, 3GA-EA-18, respectively. It was named -4-49-c-1-others.

  The solvent was distilled off from 3GA-EA-18-4-49-c-1-1 to obtain 13.9 mg of a solid content. As will be described later, this solid content was Compound 2 represented by the formula (2).

  The solvent was distilled off from 3GA-EA-18-4-49-c-1-2 to obtain 1.0 mg of a solid content. As described later, this solid content was the compound 3 represented by the formula (3).

(3-7-3) Pretreatment of fraction 3GA-EA-18-4-49-o with Sep pak and high performance liquid chromatography using normal phase column (3-7-1) Amorphous part (Others), the adsorbate (3-7-2) obtained in (3-7-2) and 3GA-EA-18-4-49-c-1-others were collected and 3GA-EA-18-4- It was named 49-o.

  3GA-EA-18-4-49-o (amount corresponding to a solid content of 54.1 mg; ethyl acetate / chloroform (1: 9 (volume)) solution) was added to a Sep Pak (registered trademark) plus silica cartridge (Waters). Manufactured; normal phase). The soluble part that was not retained in the Sep Pak® was named 3GA-EA-18-4-49-o-1. Then, what was not eluted with ethyl acetate / chloroform (adsorbent; unsolvable part) was eluted with methanol. The solid content was 31.2 mg.

  A normal phase column (Senshu Pak AQ ss-5251; 3GA-EA-18-4-49-o-1 (amount corresponding to 22.5 mg in solid content)) connected to a high performance liquid chromatograph apparatus; The active ingredient was retained through a product of Senshu Kagaku. Elution was performed with ethyl acetate / chloroform (1: 9 (volume)), and 3GA-EA-18-4-49-o-1-1 to 3GA-EA-18-4-49-o-1-13 fractions were obtained. Got the minute.

  Using each fraction, the number of osteoclasts formed was measured by the TRAP staining method according to the method described above. As a result, 3GA-EA-18-4-49-o-1-4, 3GA-EA-18-4-49-o-1-7, 3GA-EA-18-4-49-o-1-8 And 3GA-EA-18-4-49-o-1-9 and the active ingredient was concentrated.

  The solvent was distilled off from 3GA-EA-18-4-49-o-1-4 to obtain 6.5 mg of a solid content. As will be described later, this solid content was Compound 2 represented by the formula (2).

  The solvent was distilled off from 3GA-EA-18-4-49-o-1-7 to obtain 2.3 mg of a solid content. As described later, this solid content was the compound 4 represented by the formula (4-1).

(3-7-4) Pretreatment by Sep pak and reverse phase column of fractions 3GA-EA-18-4-49-o-1-8 and 3GA-EA-18-4-49-o-1-9 High performance liquid chromatography by Fractions 3GA-EA-18-4-49-o-1-8 and 3GA-EA-18-4-49-o-1-9 were combined and 3GA-EA-18-4 It was named -49-o-1-8 / 9. This was made into a 90% methanol solution and passed through a Sep Pak (registered trademark) plus C18 cartridge (manufactured by Waters; ODS). The fraction that flowed out without being retained in the Sep Pak (registered trademark) was named 3GA-EA-18-4-49-o-1-8 / 9-1. Then, what was not eluted with 90% methanol (adsorbed material; unsoluble part) was eluted with methanol. The solid content was 0.9 mg.

  3GA-EA-18-4-49-o-1-8 / 9-1 (an amount corresponding to 8.5 mg in solid content) was mixed with a reverse phase preparative column (connected to a high performance liquid chromatograph ( The active ingredient was retained through a cholester water (manufactured by Nacalai Tesque). Elution was performed with 80% methanol, and 3GA-EA-18-4-49-o-1-8 / 9-1-1 to 3GA-EA-18-4-49-o-1-8 / 9-1- Ten fractions were obtained.

  Using each fraction, the number of osteoclasts formed was measured by the TRAP staining method according to the method described above. As a result, 3GA-EA-18-4-49-o-1-8 / 9-1-4 and 3GA-EA-18-4-49-o-1-8 / 9-1-8 contain active ingredients. It was concentrated.

  The solvent was distilled off from 3GA-EA-18-4-49-o-1-8 / 9-1-4 to obtain 1.8 mg of a solid content. As described later, this solid content was the compound 3 represented by the formula (3).

  The solvent was distilled off from 3GA-EA-18-4-49-o-1-8 / 9-1-8 to obtain 1.7 mg of a solid content. As described later, this solid content was the compound 4 represented by the formula (4-1).

(4) Identification of active ingredient (4-1) Compound 1
Solid content obtained in (3-5) and (3-6) (what was contained in 3GA-EA-18-4-3-33-1-7, 3GA-EA-18-4-34-1-4 ), respectively, dissolved in CDCl _3, and Joel (JOEL) manufactured by lambda 500 FT ¹ H-NMR spectrometer and subjected to Joel Co. lambda ^{500 FT} 13 C-NMR spectrometer. The results are shown in Table 1. HR-ESI-TOFMS m / z 441.27676 (Δ-2.6 mmu) From [M−H] ⁻ , the molecular weight of this solid content (hereinafter referred to as “Compound 1”) is 442, and the molecular formula is C ₂₈ H It was found to be ₄₂ O ₄ .

In order to specify the position of the hydroxyl group in Compound 1, a compound in which the hydroxyl group of Compound 1 was converted to p-bromobenzoate ester was prepared, dissolved in CDCl ₃ , and placed on a lambda 500FT ¹ H-NMR spectrometer manufactured by JOEL. Provided. From this result, it was confirmed that the position of the hydroxyl group of Compound 1 was the 3rd position. That is, it was confirmed that the chemical structure of Compound 1 is represented by the formula (1).

Each solid content obtained in (3-5) and (3-6) was dissolved in CDCl ₃ to obtain heteronuclear multiple-bond connectivity (HMBC) spectra. However, the chemical structure of this solid content (Compound 1) was estimated to be as shown in Formula (1).

  About the compound 1 obtained by (3-5) and (3-6), IR spectrum, optical rotation, and melting | fusing point were calculated | required by the conventional method. The results are shown in Table 2.

(4-2) Compound 2
Solids (3GA-EA-18-4-49-c-1-1 and 3GA-EA-18-4-49-o-1) obtained in (3-7-2) and (3-7-3) those) that had been contained in the -4, respectively dissolved in CDCl _3, and Joel Co. lambda 500 FT ¹ H-NMR spectrometer and subjected to Joel Co. lambda ^{500 FT} 13 C-NMR spectrometer. The results are shown in Table 3. HR-ESI-TOFMS m / z 425.3056 (Δ-2.7 mmu) From [M−H] ⁻ , the molecular weight of this solid content (hereinafter referred to as “compound 2”) is 442, and the molecular formula is C ₂₈ H It was found to be ₄₂ O ₄ .

In order to specify the position of the hydroxyl group in Compound 2, a compound in which the hydroxyl group of Compound 2 was converted to p-bromobenzoate ester was prepared, dissolved in CDCl ₃ , and placed on a lambda 500FT ¹ H-NMR spectrometer manufactured by JOEL. Provided. From this result, it was confirmed that the position of the hydroxyl group of Compound 2 was the 3rd position. That is, it was confirmed that the chemical structure of Compound 2 is represented by the formula (2).

The solid contents obtained in (3-7-2) and (3-7-3) were each dissolved in CDCl ₃ to obtain an HMBC spectrum. Also from this data, it was estimated that the chemical structure of this solid content (compound 2) is as shown in Formula (2).

  About the compound 2 obtained by (3-7-2) and (3-7-3), IR spectrum, optical rotation, and melting | fusing point were calculated | required by the conventional method. The results are shown in Table 4.

(4-3) Compound 3
Solids (3GA-EA-18-4-49-c-1-2 and 3GA-EA-18-4-49-o-1 obtained in (3-7-2) and (3-7-4) -8 / 9-1-4 that was contained in) were each dissolved in CDCl _3, Joel (JOEL) manufactured by lambda 500 FT ¹ H-NMR spectrometer and, Joel Co. lambda ^{500 FT} 13 C-NMR spectrometer It was used for. The results are shown in Table 5. From HR-ESI-TOFMS m / z 411.3263 (Δ-2.3 mmu) [M + H] ⁺ , the molecular weight of this solid (hereinafter referred to as “compound 3”) is 426, and the molecular formula is C ₂₈ H ₄₂ O. It turned out to be ₃ .

In order to specify the position of the hydroxyl group in Compound 3, a compound in which the hydroxyl group of Compound 3 was converted to p-bromobenzoate ester was prepared, dissolved in CDCl ₃ , and placed on a lambda 500FT ¹ H-NMR spectrometer manufactured by JOEL. Provided. From this result, it was confirmed that the position of the hydroxyl group of Compound 3 was the 3rd position. That is, it was confirmed that the chemical structure of Compound 3 is represented by the formula (3).

The solid contents obtained in (3-7-2) and (3-7-4) were each dissolved in CDCl ₃ to obtain an HMBC spectrum. Also from this data, it was estimated that the chemical structure of this solid content (compound 3) is as shown in Formula (3).

  About the compound 3 obtained by (3-7-2) and (3-7-4), IR spectrum, optical rotation, and melting | fusing point were calculated | required by the conventional method. The results are shown in Table 6.

(4-4) Compound 4
Solids (3GA-EA-18-4-49-o-1-7 and 3GA-EA-18-4-49-o-1 obtained in (3-7-3) and (3-7-4) -8 / 9-1-8) were dissolved in CDCl ₃ and supplied to Joel Lambda 500FT ¹ H-NMR spectrometer and Joel Lambda 500FT ¹³ C-NMR spectrometer. did. The results are shown in Table 7. HR-ESI-TOFMS m / z 451.3188 (Δ-3.3 mmu) From [M + Na] ⁺ , the molecular weight of this solid content (hereinafter referred to as “Compound 4”) is 428, and the molecular formula is C ₂₈ H ₄₄ O. It turned out to be ₃ .

In order to specify the position of the hydroxyl group in Compound 4, a compound in which the hydroxyl group of Compound 4 is converted to p-bromobenzoate ester is prepared, dissolved in CDCl ₃ , and placed on a lambda 500FT ¹ H-NMR spectrometer manufactured by JOEL. Provided. From this result, it was confirmed that the position of the hydroxyl group of Compound 4 was the 3rd and 6th positions. Moreover, it was confirmed that it has an epoxy group on the 4th and 5th positions. That is, the chemical structure of Compound 4 was estimated to be as shown in Formula (4-1).

The solid contents obtained in (3-7-3) and (3-7-4) were each dissolved in CDCl ₃ to obtain an HMBC spectrum. Also from this data, it was estimated that the chemical structure of this solid content (compound 4) is as shown in Formula (4-1).

  About the compound 4 obtained by (3-7-3) and (3-7-4), IR spectrum, optical rotation, and melting | fusing point were calculated | required by the conventional method. The results are shown in Table 8.

  Moreover, when the compound 4 was used for the X-ray crystallography, it became clear that it was a molecular structure shown in FIG.

  Since the compound according to the present invention has an osteoclast formation inhibitory effect, it is useful as a preventive or therapeutic agent for bone diseases such as osteoporosis.

Claims (9)

Formula (2)
Or a pharmacologically acceptable salt thereof, or a hydroxyl group in the formula (2) is a methoxymethyl ether group, a tetrahydropyranyl ether group, a tertiary butyl ether group, an allyl ether group, a benzoic acid ester group, an acetate group (Acetyloxy group), formate group, crotonate group, p-phenylbenzoic acid ester group, trimethylacetyloxy group, tertiary butyldimethylsilyloxy group, tertiary butyldiphenylsilyloxy group, trityloxy group or benzyloxy group compound that has been. Formula (2)
A compound represented by Formula (4)
Or a pharmacologically acceptable salt thereof, or a hydroxyl group in the formula (4) is a methoxymethyl ether group, a tetrahydropyranyl ether group, a tertiary butyl ether group, an allyl ether group, a benzoic acid ester group, an acetate group (Acetyloxy group), formate group, crotonate group, p-phenylbenzoic acid ester group, trimethylacetyloxy group, tertiary butyldimethylsilyloxy group, tertiary butyldiphenylsilyloxy group, trityloxy group or benzyloxy group compound that has been. Formula (4-1)
A compound represented by Compound represented by formula (2), pharmacologically acceptable salt of compound represented by formula (2), hydroxyl group in formula (2) is methoxymethyl ether group, tetrahydropyranyl ether group, tertiary butyl ether group , Allyl ether group, benzoate group, acetate group (acetyloxy group), formate group, crotonate group, p-phenylbenzoate group, trimethylacetyloxy group, tertiary butyldimethylsilyloxy group, tertiary butyldiphenylsilyl oxy group, compounds that have been substituted with a trityl group or benzyloxy group, the compound represented by the formula (4), pharmacologically acceptable salts of the compounds of formula (4), equation (4) The hydroxyl group of methoxymethyl ether group, tetrahydropyranyl ether group, tertiary buty Ether group, allyl ether group, benzoate group, acetate group (acetyloxy group), formate group, crotonate group, p-phenylbenzoate group, trimethylacetyloxy group, tertiary butyldimethylsilyloxy group, tertiary butyl butyldiphenylsilyloxy group, compounds that have been substituted with a trityl group or benzyloxy group, the compound represented by the formula (3), pharmaceutically acceptable salts of the compounds of formula (3), and ( 3) The hydroxyl group in methoxymethyl ether group, tetrahydropyranyl ether group, tertiary butyl ether group, allyl ether group, benzoate group, acetate group (acetyloxy group), formate group, crotonate group, p-phenylbenzoate ester Group, trimethylacetylo Shi group, tertiary butyldimethylsilyl group, tert-butyldiphenylsilyloxy group, with a trityl group or a pharmacologically effective amount of least one selected from the group consisting of not that compounds substituted with benzyloxy A preventive or therapeutic agent for bone diseases, comprising:
The compound represented by formula (4) is represented by formula (4-1).
The preventive or therapeutic agent of a bone disease of Claim 5 which is a compound represented by these. The preventive or therapeutic agent for bone disease according to claim 5 or 6, wherein the bone disease is osteoporosis. The preventive or therapeutic agent for bone diseases according to claim 7, which is an osteoclast formation inhibitor. Compound represented by formula (2), pharmacologically acceptable salt of compound represented by formula (2), hydroxyl group in formula (2) is methoxymethyl ether group, tetrahydropyranyl ether group, tertiary butyl ether group , Allyl ether group, benzoate group, acetate group (acetyloxy group), formate group, crotonate group, p-phenylbenzoate group, trimethylacetyloxy group, tertiary butyldimethylsilyloxy group, tertiary butyldiphenylsilyl oxy group, compounds that have been substituted with a trityl group or benzyloxy group, the compound represented by the formula (4), pharmacologically acceptable salts of the compounds of formula (4), equation (4) The hydroxyl group of methoxymethyl ether group, tetrahydropyranyl ether group, tertiary buty Ether group, allyl ether group, benzoate group, acetate group (acetyloxy group), formate group, crotonate group, p-phenylbenzoate group, trimethylacetyloxy group, tertiary butyldimethylsilyloxy group, tertiary butyl butyldiphenylsilyloxy group, compounds that have been substituted with a trityl group or benzyloxy group, the compound represented by the formula (3), pharmaceutically acceptable salts of the compounds of formula (3), and ( 3) The hydroxyl group in methoxymethyl ether group, tetrahydropyranyl ether group, tertiary butyl ether group, allyl ether group, benzoate group, acetate group (acetyloxy group), formate group, crotonate group, p-phenylbenzoate ester Group, trimethylacetylo Shi group, tertiary butyldimethylsilyl group, tert-butyldiphenylsilyloxy group, one or more selected from the group consisting of trityl group or benzyloxy group compounds that have been substituted with found Gurifora-Garugaru (Japanese name: The preventive or therapeutic agent for bone diseases according to any one of claims 5 to 8, which is an extract from the fruiting body of Anninkou).