WO2011016351A1 - Dna synthase inhibitors - Google Patents
Dna synthase inhibitors Download PDFInfo
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- WO2011016351A1 WO2011016351A1 PCT/JP2010/062439 JP2010062439W WO2011016351A1 WO 2011016351 A1 WO2011016351 A1 WO 2011016351A1 JP 2010062439 W JP2010062439 W JP 2010062439W WO 2011016351 A1 WO2011016351 A1 WO 2011016351A1
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- WIPO (PCT)
- Prior art keywords
- compound
- dna
- dna synthase
- compounds
- acid
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Images
Classifications
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- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/10—Spiro-condensed systems
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/03—Organic compounds
- A23L29/035—Organic compounds containing oxygen as heteroatom
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/336—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having three-membered rings, e.g. oxirane, fumagillin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D313/00—Heterocyclic compounds containing rings of more than six members having one oxygen atom as the only ring hetero atom
- C07D313/02—Seven-membered rings
- C07D313/06—Seven-membered rings condensed with carbocyclic rings or ring systems
- C07D313/08—Seven-membered rings condensed with carbocyclic rings or ring systems condensed with one six-membered ring
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/07—Nucleotidyltransferases (2.7.7)
- C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Definitions
- the present invention relates to a compound having a DNA synthase inhibitory action and use thereof.
- This compound can be used as a DNA synthase inhibitor, for example, as a biochemical reagent, or as an anticancer agent, an anti-inflammatory agent, or a lead compound thereof.
- DNA polymerases are ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , and TdT (terminal deoxynucleotidyl transferase). ), 15 molecular species of Rev1 are known. These DNA synthase groups are involved in cell growth, division, differentiation, etc., but ⁇ type is DNA replication, ⁇ type, ⁇ type and TdT are repair and recombination, and ⁇ type and ⁇ type are replication. Both repairs are known to have different functions depending on the type, such as ⁇ to ⁇ are responsible for repair.
- DNA synthase Since DNA synthase is involved in cell growth and the like in this way, a DNA synthase inhibitor that inhibits the enzyme activity, for example, exhibits cancer cell growth inhibitory action against cancer and HIV against AIDS. It is considered that it exhibits an inhibitory effect on the derived reverse transcriptase and an immunosuppressive action that suppresses specific antibody production against an antigen against an immune disease. For this reason, development of pharmaceuticals effective for prevention and treatment of cancer diseases, viral diseases such as AIDS, and immune diseases using a DNA synthase inhibitor is expected.
- glycolipids having DNA synthase inhibitory activity are useful as anticancer agents, HIV-derived reverse transcriptase inhibitors, and immunosuppressants (see Patent Document 1 below).
- ddNTP dideoxyNTP
- N-ethylmaleimide N-ethylmaleimide
- butylphenyl-dGTP butylphenyl-dGTP
- sulfosynovosyl acylglycerides which are plant-derived glycolipids, have been found to inhibit DNA synthase (see Patent Document 2 below).
- initiator and “promoter” are related in the step of canceration of normal cells, and canceration of normal cells is “(1) normal cells. Carcinogenesis that occurs through two steps: “2) the chromosomes of 2 are damaged by the initiator at the DNA level and are changed into potential tumor cells. (2) Promoters act on the potential tumor cells and change into tumor cells.” The two-stage theory is becoming common.
- TPA (12-O-Tetradecanoylphorbol-13-acetate) is known as a representative compound having an action as an oncogenic promoter, and carcinogenic experiments using such an oncogenic promoter are conducted in the field of cancer research.
- carcinogenesis prevention and anti-carcinogenic agents the search for anti-tumor promoters that inhibit the action of such tumor promoters is ongoing.
- An object of the present invention is to find a new compound having a DNA synthase inhibitory action and to provide a new pharmaceutical composition (DNA synthase inhibitor, anticancer agent and anti-inflammatory agent) using the compound.
- the present inventors have investigated and examined the relationship between these compounds and DNA synthetase inhibitory action, anti-inflammatory action, and anti-carcinogenic action. These compounds are DNA synthase among DNA metabolic enzymes. Was confirmed to be selectively inhibited. The inventors have also found that these compounds have an anti-inflammatory effect. As a result of further research based on this knowledge, the present invention has been completed.
- the present invention provides the following compound, a DNA synthase inhibitor, an anticancer agent and an anti-inflammatory agent containing the compound as an active ingredient.
- Item 1 A compound represented by the general formula (1) or (2) or a pharmaceutically acceptable salt thereof.
- Item 2 A pharmaceutical composition comprising the compound represented by the general formula (1) or (2) according to Item 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
- a DNA synthase inhibitor comprising as an active ingredient the compound represented by the general formula (1) or (2) according to Item 1, or a pharmaceutically acceptable salt thereof.
- An anticancer agent comprising, as an active ingredient, the compound represented by the general formula (2) according to Item 1 or a pharmaceutically acceptable salt thereof.
- An anti-inflammatory agent comprising, as an active ingredient, the compound represented by the general formula (1) or (2) according to Item 1 or a pharmaceutically acceptable salt thereof.
- Item 6 An edible composition comprising a compound represented by the general formula (1) or (2) according to Item 1 or a pharmaceutically acceptable salt thereof in a food.
- novel compound 1 (trichoderonic acid A), compound 2 (trichoderonic acid B), and derivatives thereof of the present invention have a DNA synthase inhibitory action.
- Compound 2 selectively inhibits DNA synthases ⁇ , ⁇ and TdT belonging to the X family among mammalian DNA synthases.
- Compound 2 has anti-tumor promoter activity that suppresses the action of tumor promoter TPA because it suppresses inflammation induced by TPA in a dose-dependent manner.
- these compounds can be used as pharmaceutical compositions, for example, DNA synthase inhibitors, anticancer agents, anti-inflammatory agents, and the like.
- These compounds can also be used as biochemical reagents by utilizing the above-mentioned activity. Furthermore, these compounds can be blended into foods and used as edible compositions.
- 2 is a chemical structure of Compound 1.
- 2 is a chemical structure of Compound 2.
- the present invention relates to a compound represented by the general formula (1) or (2) or a pharmaceutically acceptable salt thereof.
- R represents H or an alkyl group.
- examples of the alkyl group represented by R include a chain, branched or cyclic alkyl group having 1 to 10 carbon atoms. Specific examples include alkyl groups having 1 to 6 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, pentyl, cyclopentyl, hexyl and cyclohexyl.
- the compounds represented by the general formulas (1) and (2) have a plurality of asymmetric carbons (sp 3 carbons), and the compound has a stereostructure of a plurality of asymmetric carbons of either R or S.
- sp 3 carbons asymmetric carbons
- R or S asymmetric carbons
- any of enantiomers, diastereomers, and mixtures thereof are included.
- compound 1 trichoderonic acid A in which R is H is preferable.
- compound 2 trichoderonic acid B in which R is H is preferable.
- Examples of the salt of “pharmaceutically acceptable salt thereof” in the present invention include alkali metal salts such as sodium salt and potassium salt; ammonium salt; primary, secondary or tertiary amine salt; quaternary ammonium salt; amino acid Examples include salt.
- the compound represented by the general formulas (1) and (2) or a pharmaceutically acceptable salt thereof may be a solvate such as water.
- Trichoderma virulence is statically cultured in a PDB medium (potato dextrose liquid medium), and the filtrate obtained by removing the cells from the culture is extracted with a solvent.
- PDB medium potato dextrose liquid medium
- the extraction solvent examples include alcohols such as methanol, ethanol, propanol and butanol, glycols such as 1,3-butylene glycol, glycerin and propylene glycol, esters such as ethyl acetate and butyl acetate, ethyl ether, propyl ether and isopropyl ether.
- Polar organic solvents such as ethers such as tetrahydrofuran and dioxane, halogenated hydrocarbons such as methylene chloride and chloroform; nonpolar organic solvents such as hexane, cyclohexane and petroleum ether, and the like. These solvents can be used alone or as a mixed solvent of two or more.
- At least one extraction solvent selected from the group consisting of halogenated hydrocarbons such as methylene chloride, alcohols such as methanol and ethanol, and esters such as ethyl acetate.
- halogenated hydrocarbons such as methylene chloride
- alcohols such as methanol and ethanol
- esters such as ethyl acetate.
- the active fraction (compounds 1 and 2) can be separated from the extracted liquid. Furthermore, it can also be purified by combining one or more suitable separation and purification means such as alumina column chromatography, silica gel chromatography, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, and high performance liquid chromatography. . In addition, isolation and identification of the active compound from the culture solution can be performed specifically as described in Example 1.
- R alkyl among the compounds represented by the general formulas (1) and (2) is obtained by esterifying the carboxylic acid moiety of the isolated compound 1 or 2 by a known method using a predetermined alcohol. Can be obtained.
- the compound represented by the general formula (1) or (2) of the present invention has a DNA synthase selective inhibitory action, and thus can be used for pharmaceuticals.
- it is useful as a pharmaceutical composition such as a DNA synthase selective inhibitor, an anticancer agent, and an anti-inflammatory agent.
- the compound of the present invention can also be used as a lead compound in the pharmaceutical development process.
- parenteral administration is preferable to oral administration, and it is preferable to encapsulate and administer it in a carrier such as a liposome.
- a carrier such as a liposome.
- the anticancer agent and anti-inflammatory agent containing the compound of the present invention as an active ingredient can be administered to animals and humans as they are or as a pharmaceutical composition together with a conventional pharmaceutical preparation carrier.
- the dosage form of the pharmaceutical composition is not particularly limited and may be appropriately selected as necessary.
- oral preparations such as tablets, capsules, granules, fine granules, powders, injections, Non-oral preparations such as suppositories are mentioned, and preferred examples include parenteral preparations.
- oral preparations such as tablets, capsules, granules, fine granules, and powders are produced according to a conventional method using, for example, starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, inorganic salts, and the like.
- the compounding amount of the compound of the present invention in these preparations is not particularly limited and can be appropriately designed.
- a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a corrigent, a colorant, a fragrance and the like can be appropriately used for this type of preparation.
- binder examples include starch, dextrin, gum arabic powder, gelatin, hydroxypropyl starch, sodium methylcellulose, hydroxypropylcellulose, crystalline cellulose, ethylcellulose, polyvinylpyrrolidone, macrogol and the like.
- disintegrant examples include starch, hydroxypropyl starch, carboxymethylcellulose sodium, carboxymethylcellulose calcium, carboxymethylcellulose, and low-substituted hydroxypropylcellulose.
- surfactant examples include sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polyoxyethylene sorbitan fatty acid ester and the like.
- lubricants include talc, waxes, hydrogenated vegetable oils, sucrose fatty acid esters, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol and the like.
- fluidity promoter include light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate and the like.
- the compounds of the present invention can also be administered as suspensions, emulsions, syrups, and elixirs, and these various dosage forms may contain flavoring agents and colorants.
- parenteral preparation In order to achieve the desired effect of the present invention as a parenteral preparation, it varies depending on the age, body weight, and degree of disease of the patient, but is usually intravenous in an amount of 1 to 60 mg per day as the weight of the compound of the present invention in an adult. Intravenous infusion, subcutaneous injection, and intramuscular injection are suitable.
- This parenteral preparation is produced according to a conventional method, and generally used as diluent is distilled water for injection, physiological saline, aqueous glucose solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, etc. it can. Furthermore, you may add a disinfectant, antiseptic
- this parenteral preparation can be frozen after filling into a vial or the like, the water can be removed by ordinary freeze-drying treatment, and the liquid preparation can be re-prepared from the freeze-dried product immediately before use.
- the compounding quantity of the compound of this invention in these formulations is not specifically limited, It can set arbitrarily.
- parenteral agents include liquid preparations for external use, coating agents such as ointments, suppositories for rectal administration, etc., and these are also produced according to conventional methods.
- the compound of the present invention can be used for foods as well as for pharmaceuticals.
- it can be used as an edible composition (for example, health food) having an anti-cancer effect, an anti-carcinogenic effect, or an anti-inflammatory effect by adding and blending with food and drink.
- the compound of the present invention is used as it is as a liquid, gel or solid food, for example, juice, soft drink, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, sprinkle, infant formula, cake.
- a liquid, gel or solid food for example, juice, soft drink, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, sprinkle, infant formula, cake.
- DNA synthase inhibitors of the present invention can be used as DNA synthase inhibitors derived from mammals other than humans.
- the present invention includes such a screening method.
- the method for examining the inhibitory activity against DNA synthase is not limited to the method described in the above-mentioned Examples, and a suitable method may be selected from known test methods.
- Example 1 Isolation and culture of strains Petals collected at Inage Seaside Park were cultured on PDA medium (potato dextrose agar medium) to isolate this strain (referred to as IG34HB strain for convenience). As a result of strain identification, this strain was identified as Trichoderma virens (FERM S-10, ATCC 9645, CBS 430.54, IFO 6355, IMI 45553ii, QM 365, NBRC 6347). This strain was statically cultured for 26 days in six 2 L Erlenmeyer flasks (total 6 L) containing 1 L of PDB medium (potato dextrose liquid medium).
- Fr. D (566.7 mg) eluted with chloroform-methanol (80: 1-40: 1) was obtained by column chromatography using silica gel as a carrier and dichloromethane-methanol (100: 0 ⁇ 0: 100) as a developing solvent. Fractions were fractionated into 4 fractions (Fr.DA to Fr.DD).
- Fr.DD (40.4 mg) eluted with dichloromethane-methanol (0: 100) is a column using silica gel as a carrier and toluene-ethyl acetate (8: 1 to 2: 1) containing 1% acetic acid as a developing solvent. It was fractionated into 7 fractions (Fr.DDA to Fr.DDGD) by chromatography. Then, compound 1 (4.8 mg) was eluted from toluene-ethyl acetate (4: 1) containing 1% acetic acid from Fr. DDC eluted with toluene-ethyl acetate (6: 1) containing 1% acetic acid. Compound 2 (10.5 mg) was obtained from Fr.DDF.
- FIG. 3 shows 1 H-NMR and 13 C-NMR data of Compound 1 measured in CDCl 3 .
- 4A shows the carbon number of Compound 2
- FIG. 4B shows the 1 H- 1 H COZY and HMBC correlation of Compound 2.
- FIG. 5 shows 1 H-NMR and 13 C-NMR data of Compound 2 measured in CDCl 3 .
- compound 1 is 9-hydroxy-6-isopropyl-9- (methoxymethyl) -1-oxo-1,3,5a, 6,7,8,9,9a-octahydro-2-benzoxepin-4-carbon Acid (9-hydroxy-6-isopropyl-9- (methoxymethyl) -1-oxo-1,3,5a, 6,7,8,9,9a-octahydro-2-benzoxepine-4-carboxylic acid) (Trichoderonic acid A; named trichoderonic acid A).
- Compound 2 MS, 1 H-NMR, 13 C-NMR and DEPT were shown to have the molecular formula C 30 H 40 O 10 .
- the NMR data of Compound 2 was similar to that of trichoderonic acid A (Compound 1) except for C-7, C16, C-20, C-21 and C-22.
- the HHM ⁇ C-16 HMBC correlation suggested a structure in which a similar structure of tricoderonic acid A was dimerized.
- compound 2 was obtained by using 9-hydroxy-6-isopropyl-9 ((6-isopropyl-1-oxo-1,3,5a, 6,7,9a-hexahydro-3H-spiro [2-benzoxepin-9,2 '-Oxirane] -4-ylcarbonyloxy) methyl) -1-oxo-1,3,5a, 6,7,8,9,9a-octahydro-2-benzoxepin-4-carboxylic acid (9-hydroxy-6 -isopropyl-9 ((6-isopropyl-1-oxo-1,3,5a, 6,7,9a-hexahydro-3H-spiro [2-benzoxepine-9,2'-oxirane] -4-ylcarbonyloxy) methyl) -1-oxo-1,3,5a, 6,7,8,9,9a-octahydro-2-benzoxepine-4-carboxylic acid) (name
- Example 2 (Verification of DNA synthase inhibitory activity) The activity of the compound 1 (trichoderonic acid A) and compound 2 (trichoderonic acid B) obtained in the above examples against the DNA synthase group was measured by the following method.
- DNA synthase ⁇ is a sample extracted and purified from bovine thymus by a conventional method
- DNA synthase ⁇ is a rat-derived gene
- DNA synthase ⁇ is a human-derived gene
- a conventional gene recombination method is used.
- the preparations incorporated into Escherichia coli and produced were used.
- TdT was purchased from Takara Bio Inc. and used as a reagent (product code: 2230A) incorporated into Escherichia coli by a genetic recombination method derived from calves.
- a general DNA synthase reaction system (edited by the Japanese Biochemical Society, Shinsei Chemistry Laboratory 2, Nucleic Acid IV, Tokyo Kagaku Dojin, pages 63-66) was used. That is, a DNA synthesis reaction is carried out in a system containing [ 3 H] -TTP labeled with a radioisotope, and the radioactivity is used as an index of the amount of product (synthetic DNA chain).
- FIG. 6 shows inhibition curves of Compound 2 against DNA synthases ⁇ , ⁇ and TdT.
- Compound 2 was found to most strongly inhibit DNA synthase ⁇ , secondly ⁇ -type, and thirdly TdT. As a result of calculating the inhibition mode of Compound 2 against DNA synthetase ⁇ from the reciprocal plot of Lineweaver-Burk, it was non-competitive inhibition with respect to two substrates, template DNA and nucleotide (dNTP) (data not shown).
- DNA synthase has a high enzyme activity against cells and tissues such as cancer cells that have a high cell division. Therefore, compounds 1 and 2 are expected to have an action as an anticancer agent.
- mammalian DNA synthases are classified into four families of A, B, X, and Y based on the homology of their gene sequences and differences in activity functions.
- Compound 2 selectively inhibited DNA synthases ⁇ , ⁇ and TdT belonging to the X family.
- DNA synthase ⁇ belonging to the A family, DNA synthase ⁇ , ⁇ , ⁇ belonging to the B family, and DNA synthases ⁇ , ⁇ , ⁇ belonging to the Y family were not inhibited.
- Example 3 (Verification of cell growth inhibitory activity) The cell growth inhibitory effect of compound 1 (trichoderonic acid A) and compound 2 (trichoderonic acid B) was evaluated using the following method.
- human cervical cancer-derived HeLa cells and human breast cancer-derived MCF-7 cells were used as cancer cells, and human skin epithelial cells (HDF) and human umbilical vein endothelial cells (HUBEC) were used as normal cells.
- the medium used was a DMEM medium (Nippon Pharmaceutical Co., Ltd.) supplemented with fetal bovine serum 10% (v / v) and penicillin-streptomycin 5% (v / v). Culturing was performed at 37 ° C. with 5% CO 2 incubation.
- the culture for this experiment was performed in a 96-well microplate. Each well was implanted with 5.0 ⁇ 10 3 cells and given 3 wells for one test concentration. Each cell is cultured in a plate for 24 hours. After adding each concentration of Compound 1 and 2, each cell is cultured in a 5% CO 2 incubation at 37 ° C for 48 hours for cancer cells and 72 hours for normal cells. The cell viability of the section was determined. Viability was determined using the living cell count reagent SF (performed by the method of Talanta 1997, 44).
- the novel tetrazolium salt WST-8 contained in the living cell measurement reagent SF is reduced by intracellular dehydrogenase to produce a highly sensitive water-soluble formazan, which is easily produced by directly measuring the absorbance of this formazan at 450 nm.
- the number of cells can be measured. Therefore, in this experiment, after culturing for 48 hours or 72 hours, add the live cell count reagent SF, measure the absorbance (OD) at 450 nm after further culturing for 2 hours, and calculate the cell viability using the following formula did.
- FIGS. 8 and 9 The results obtained with human cancer cells are shown in FIGS.
- the data shown in FIGS. 8 and 9 is an average of 3 wells.
- Compound 1 did not inhibit the growth of each cancer cell, but Compound 2 inhibited the growth of each cancer cell. From the results of FIGS. 8 and 9, the 50% growth inhibitory concentration (IC 50 ) of Compound 2 was 15.6 ⁇ M in HeLa cells and 8.7 ⁇ M in MCF-7 cells. From this result, it was shown that Compound 2, which specifically inhibits DNA synthase, actually exhibits a cell growth inhibitory effect on human cancer cell lines and can be used as an anticancer agent.
- Table 2 shows the 50% growth inhibitory concentration (IC 50 ) of Compound 1 and Compound 2 when normal human cells were used.
- both Compound 1 and Compound 2 had a 50% growth inhibitory concentration (IC 50 ) of 100 ⁇ M or more. From this experimental result, it can be said that Compound 1 and Compound 2 do not inhibit the growth of each normal cell. From this, it was suggested that Compound 1 and Compound 2 could be used as anticancer agents with no adverse effects on human normal cells.
- TPA (12-O-tetradecanoylphorbol-13-acetate) not only induces chronic inflammation but also promotes cell growth in mammals as an oncogenic promoter.
- the present inventors have no relationship between DNA synthase inhibitory activity (particularly DNA synthase ⁇ inhibitory activity involved in DNA repair / recombination), anti-inflammatory activity and anti-tumor promoter activity. I thought. Therefore, it was examined whether Compound 1 (trichoderonic acid A) and Compound 2 (trichoderonic acid B) have anti-inflammatory activity. The results are shown in Table 3 below.
- each inhibitory effect was calculated by measuring the weight of inflammatory edema induced by TPA after applying a predetermined amount of Compound 1 or Compound 2 to the mouse ear in advance.
- the test was basically carried out according to the method described in Cancer Lett. 1984, 25, 177-85, and 250 ⁇ g, 500 ⁇ g or 750 ⁇ g of the compound was applied to one ear of the mouse, and after 30 minutes, the same site and the opposite side were applied. Apply 0.5 ⁇ g of TPA to the ear. After 7 hours, the weight of inflammatory edema induced by TPA was measured, and each inhibitory effect was calculated by comparing with the control (opposite ear).
- Compound 2 inhibited inflammation depending on the coating amount of 250 ⁇ g, 500 ⁇ g and 750 ⁇ g. From this experimental result, Compound 2 was recognized as having an anti-inflammatory effect. Further, from this experimental result, it was recognized that Compound 2 has an anti-carcinogenic promoter activity (in other words, an anti-carcinogenic effect) that suppresses the action of the oncogenic promoter TPA.
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Abstract
The purpose of the present invention is to find compounds having a DNA synthase inhibitory effect and provide novel medicinal compositions (DNA synthase inhibitors, anticancer agents and antiinflammatory agents) using said compounds. Disclosed are compounds represented by general formula (1) or (2) or pharmaceutically acceptable salts of the same and medicinal compositions (DNA synthase inhibitors, anticancer agents, antiinflammatory agents and so on) comprising these compounds as the active ingredient.
In general formulae (1) and (2), R represents H or an alkyl group.
Description
本発明は、ＤＮＡ合成酵素阻害作用を有する化合物とその利用に関する。この化合物は、ＤＮＡ合成酵素阻害剤として、例えば生化学試薬などに利用できるほか、抗癌剤、抗炎症剤、又はこれらのリード化合物として利用し得る。
The present invention relates to a compound having a DNA synthase inhibitory action and use thereof. This compound can be used as a DNA synthase inhibitor, for example, as a biochemical reagent, or as an anticancer agent, an anti-inflammatory agent, or a lead compound thereof.
真核生物のＤＮＡ合成酵素（ＤＮＡポリメラーゼ）は、これまでα、β、γ、δ、ε、ζ、η、θ、ι、κ、λ、μ、ν及びTdT（ターミナル・デオキシヌクレオチジル・トランスフェラーゼ）、Rev1の１５種類の分子種が知られている。これらのＤＮＡ合成酵素群は、細胞の増殖、分裂、分化などに関与しているが、α型はＤＮＡ複製、β型、λ型及びTdTは修復と組換え、δ型及びε型は複製と修復の双方、ζ～κ型は修復を担うといった具合にタイプによって異なる機能を有することが知られている。
Eukaryotic DNA synthases (DNA polymerases) are α, β, γ, δ, ε, ζ, η, θ, ι, κ, λ, μ, ν, and TdT (terminal deoxynucleotidyl transferase). ), 15 molecular species of Rev1 are known. These DNA synthase groups are involved in cell growth, division, differentiation, etc., but α type is DNA replication, β type, λ type and TdT are repair and recombination, and δ type and ε type are replication. Both repairs are known to have different functions depending on the type, such as ζ to κ are responsible for repair.
このようにＤＮＡ合成酵素は細胞の増殖等に関与することから、その酵素活性を阻害するＤＮＡ合成酵素阻害剤は、例えば、癌に対して癌細胞の増殖抑制作用を示し、エイズに対してＨＩＶ由来逆転写酵素に対する阻害作用を示し、また、免疫疾患に対して抗原に対する特異的抗体産生を抑制する免疫抑制作用を示すことが考えられる。このため、ＤＮＡ合成酵素阻害剤を用いた癌、エイズ等のウイルス疾患、免疫疾患の予防・治療に効果のある医薬品の開発が期待されている。
Since DNA synthase is involved in cell growth and the like in this way, a DNA synthase inhibitor that inhibits the enzyme activity, for example, exhibits cancer cell growth inhibitory action against cancer and HIV against AIDS. It is considered that it exhibits an inhibitory effect on the derived reverse transcriptase and an immunosuppressive action that suppresses specific antibody production against an antigen against an immune disease. For this reason, development of pharmaceuticals effective for prevention and treatment of cancer diseases, viral diseases such as AIDS, and immune diseases using a DNA synthase inhibitor is expected.
例えば、ＤＮＡ合成酵素阻害活性を有する糖脂質が、制癌剤、ＨＩＶ由来逆転写酵素阻害剤、免疫抑制剤として有用であることが報告されている（下記特許文献１参照）。現在、ＤＮＡ合成酵素阻害剤として、ジデオキシＮＴＰ(ｄｄＮＴＰ)、Ｎ-エチルマレイミド、ブチルフェニル-ｄＧＴＰなどが知られている（下記非特許文献１参照）。また植物由来の糖脂質であるスルホキノボシルアシルグリセリドにもＤＮＡ合成酵素阻害作用が見出されている（下記特許文献２参照）。
For example, it has been reported that glycolipids having DNA synthase inhibitory activity are useful as anticancer agents, HIV-derived reverse transcriptase inhibitors, and immunosuppressants (see Patent Document 1 below). Currently, dideoxyNTP (ddNTP), N-ethylmaleimide, butylphenyl-dGTP, and the like are known as DNA synthetase inhibitors (see Non-patent Document 1 below). In addition, sulfosynovosyl acylglycerides, which are plant-derived glycolipids, have been found to inhibit DNA synthase (see Patent Document 2 below).
ところで、従来の抗癌剤は、その多くが発生した癌細胞を殺すこと並びに癌細胞の増殖抑制作用を有するものであったが、近年、発癌防止を目的とした抗発癌剤の開発も進められている。
By the way, conventional anticancer agents have killed many cancer cells and have cancer cell growth-inhibiting action, but in recent years, development of anticancer agents for the purpose of preventing carcinogenesis has also been promoted. .
この点について、特許文献３に記載されるように、正常細胞の癌化のステップにおいて、「イニシエーター」と「プロモーター」とが関係しており、正常細胞の癌化は「（１）正常細胞の染色体がイニシエーターによりＤＮＡレベルでの障害を受けて潜在的腫瘍細胞に変化する。（２）潜在的腫瘍細胞にプロモーターが作用して腫瘍細胞に変化させる。」の２段階を経て起こるという発癌２段階説が一般化しつつある。
In this regard, as described in Patent Document 3, “initiator” and “promoter” are related in the step of canceration of normal cells, and canceration of normal cells is “(1) normal cells. Carcinogenesis that occurs through two steps: "2) the chromosomes of 2 are damaged by the initiator at the DNA level and are changed into potential tumor cells. (2) Promoters act on the potential tumor cells and change into tumor cells." The two-stage theory is becoming common.
この発癌プロモーターとしての作用を持つ化合物の代表としてＴＰＡ（12－O－Tetradecanoylphorbol－13－acetate）等が知られており、癌研究の場でこのような発癌プロモーターを用いた発癌実験が行われると共に、発癌予防及び抗発癌剤の探索においてこのような発癌プロモーターの作用を阻害する抗発癌プロモーターの探索が進められている。
TPA (12-O-Tetradecanoylphorbol-13-acetate) is known as a representative compound having an action as an oncogenic promoter, and carcinogenic experiments using such an oncogenic promoter are conducted in the field of cancer research. In the search for carcinogenesis prevention and anti-carcinogenic agents, the search for anti-tumor promoters that inhibit the action of such tumor promoters is ongoing.
本発明は、ＤＮＡ合成酵素阻害作用を有する新規な化合物を見出すと共に、同化合物を利用した新たな医薬組成物（ＤＮＡ合成酵素阻害剤、抗癌剤及び抗炎症剤）を提供することを目的とする。
An object of the present invention is to find a new compound having a DNA synthase inhibitory action and to provide a new pharmaceutical composition (DNA synthase inhibitor, anticancer agent and anti-inflammatory agent) using the compound.
本発明者らは、上記課題を達成すべく鋭意研究を重ねた結果、Trichoderma virensをPDB培地(ポテトデキストロース液体培地)で培養して得られた培養液から、新規物質である化合物１（「trichoderonic acid A」と命名した。）と化合物２（「trichoderonic acid B」と命名した。）を単離した（実施例１を参照）。
As a result of intensive studies to achieve the above-mentioned problems, the present inventors have obtained a novel compound 1 (“trichoderonic” from a culture solution obtained by culturing Trichoderma virens in a PDB medium (potato dextrose liquid medium). acid A ”) and compound 2 (named“ trichoderonic acid B ”) were isolated (see Example 1).
本発明者らは、これらの化合物と、ＤＮＡ合成酵素阻害作用、抗炎症作用および抗発癌作用の関連性について調査・検討を行ったところ、これらの化合物は、ＤＮＡ代謝系酵素のうちＤＮＡ合成酵素を選択的に阻害することを確認した。また、本発明者らは、これらの化合物は抗炎症作用を有することも見いだした。かかる知見に基づきさらに研究を重ねた結果、本発明を完成するに至った。
The present inventors have investigated and examined the relationship between these compounds and DNA synthetase inhibitory action, anti-inflammatory action, and anti-carcinogenic action. These compounds are DNA synthase among DNA metabolic enzymes. Was confirmed to be selectively inhibited. The inventors have also found that these compounds have an anti-inflammatory effect. As a result of further research based on this knowledge, the present invention has been completed.
即ち、本発明は下記化合物、該化合物を有効成分として含むＤＮＡ合成酵素阻害剤、抗癌剤及び抗炎症剤を提供する。
That is, the present invention provides the following compound, a DNA synthase inhibitor, an anticancer agent and an anti-inflammatory agent containing the compound as an active ingredient.
項１ 一般式（１）若しくは（２）で表される化合物又はその薬学的に許容し得る塩。
Item 1. A compound represented by the general formula (1) or (2) or a pharmaceutically acceptable salt thereof.
（式中、ＲはＨ又はアルキル基を示す。）
項２ 項１に記載の一般式（１）若しくは（２）で表される化合物又はその薬学的に許容し得る塩を有効成分として含有する医薬組成物。 (In the formula, R represents H or an alkyl group.)
Item 2 A pharmaceutical composition comprising the compound represented by the general formula (1) or (2) according toItem 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
項２ 項１に記載の一般式（１）若しくは（２）で表される化合物又はその薬学的に許容し得る塩を有効成分として含有する医薬組成物。 (In the formula, R represents H or an alkyl group.)
Item 2 A pharmaceutical composition comprising the compound represented by the general formula (1) or (2) according to
項３ 項１に記載の一般式（１）若しくは（２）で表される化合物又はその薬学的に許容し得る塩を有効成分として含有するＤＮＡ合成酵素阻害剤。
Item 3. A DNA synthase inhibitor comprising as an active ingredient the compound represented by the general formula (1) or (2) according to Item 1, or a pharmaceutically acceptable salt thereof.
項４ 項１に記載の一般式（２）で表される化合物又はその薬学的に許容し得る塩を有効成分として含有する抗癌剤。
Item 4. An anticancer agent comprising, as an active ingredient, the compound represented by the general formula (2) according to Item 1 or a pharmaceutically acceptable salt thereof.
項５ 項１に記載の一般式（１）若しくは（２）で表される化合物又はその薬学的に許容し得る塩を有効成分として含有する抗炎症剤。
Item 5. An anti-inflammatory agent comprising, as an active ingredient, the compound represented by the general formula (1) or (2) according to Item 1 or a pharmaceutically acceptable salt thereof.
項６ 項１に記載の一般式（１）若しくは（２）で表される化合物又はその薬学的に許容し得る塩を食品に配合してなる食用組成物。
Item 6. An edible composition comprising a compound represented by the general formula (1) or (2) according to Item 1 or a pharmaceutically acceptable salt thereof in a food.
本発明の新規な化合物１（trichoderonic acid A）、化合物２（trichoderonic acid B）、及びそれらの誘導体化合物は、ＤＮＡ合成酵素阻害作用を有する。特に、化合物２は哺乳類のＤＮＡ合成酵素のうちＸファミリーに属するＤＮＡ合成酵素β、λ及びTdTを選択的に阻害する。
The novel compound 1 (trichoderonic acid A), compound 2 (trichoderonic acid B), and derivatives thereof of the present invention have a DNA synthase inhibitory action. In particular, Compound 2 selectively inhibits DNA synthases β, λ and TdT belonging to the X family among mammalian DNA synthases.
また、化合物２は、用量依存的にＴＰＡによって誘発される炎症を抑制することから、発癌プロモーターＴＰＡの働きを抑制する抗発癌プロモーター活性を有する。
Compound 2 has anti-tumor promoter activity that suppresses the action of tumor promoter TPA because it suppresses inflammation induced by TPA in a dose-dependent manner.
そのため、これらの化合物は、医薬組成物、例えば、ＤＮＡ合成酵素阻害剤、抗癌剤、抗炎症剤等として利用できる。
Therefore, these compounds can be used as pharmaceutical compositions, for example, DNA synthase inhibitors, anticancer agents, anti-inflammatory agents, and the like.
また、これらの化合物は、上記の活性を有することを利用して生化学試薬として用いることもできる。さらに、これらの化合物は、食品に配合して食用組成物として利用できる。
These compounds can also be used as biochemical reagents by utilizing the above-mentioned activity. Furthermore, these compounds can be blended into foods and used as edible compositions.
以下、本発明を詳細に説明する。
Hereinafter, the present invention will be described in detail.
１ 本発明の化合物及びその単離・精製
本発明は、一般式（１）若しくは（２）で表される化合物又はその薬学的に許容し得る塩に関する。 1. Compound of the present invention and isolation / purification thereof The present invention relates to a compound represented by the general formula (1) or (2) or a pharmaceutically acceptable salt thereof.
本発明は、一般式（１）若しくは（２）で表される化合物又はその薬学的に許容し得る塩に関する。 1. Compound of the present invention and isolation / purification thereof The present invention relates to a compound represented by the general formula (1) or (2) or a pharmaceutically acceptable salt thereof.
（式中、ＲはＨ又はアルキル基を示す。）
一般式（１）及び（２）で表される化合物において、Ｒで示されるアルキル基としては、例えば、鎖状、分岐状又は環状の炭素数１～１０のアルキル基が挙げられる。具体的には、メチル、エチル、ｎ－プロピル、イソプロピル、ｎ－ブチル、イソブチル、ｓｅｃ－ブチル、ペンチル、シクロペンチル、ヘキシル、シクロヘキシル等の炭素数１～６のアルキル基が例示される。 (In the formula, R represents H or an alkyl group.)
In the compounds represented by the general formulas (1) and (2), examples of the alkyl group represented by R include a chain, branched or cyclic alkyl group having 1 to 10 carbon atoms. Specific examples include alkyl groups having 1 to 6 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, pentyl, cyclopentyl, hexyl and cyclohexyl.
一般式（１）及び（２）で表される化合物において、Ｒで示されるアルキル基としては、例えば、鎖状、分岐状又は環状の炭素数１～１０のアルキル基が挙げられる。具体的には、メチル、エチル、ｎ－プロピル、イソプロピル、ｎ－ブチル、イソブチル、ｓｅｃ－ブチル、ペンチル、シクロペンチル、ヘキシル、シクロヘキシル等の炭素数１～６のアルキル基が例示される。 (In the formula, R represents H or an alkyl group.)
In the compounds represented by the general formulas (1) and (2), examples of the alkyl group represented by R include a chain, branched or cyclic alkyl group having 1 to 10 carbon atoms. Specific examples include alkyl groups having 1 to 6 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, pentyl, cyclopentyl, hexyl and cyclohexyl.
一般式（１）及び（２）で表される化合物は、複数の不斉炭素（ｓｐ３炭素）を有しており、該化合物は複数の不斉炭素の立体構造がＲ又はＳのいずれのものも包含される。例えば、エナンチオマー、ジアステレオマー、これらの混合物のいずれも包含される。
The compounds represented by the general formulas (1) and (2) have a plurality of asymmetric carbons (sp 3 carbons), and the compound has a stereostructure of a plurality of asymmetric carbons of either R or S. Are also included. For example, any of enantiomers, diastereomers, and mixtures thereof are included.
一般式（１）で表される化合物のうち好ましくは、ＲがＨである化合物１（trichoderonic acid A）である。また、一般式（２）で表される化合物のうち好ましくは、ＲがＨである化合物２（trichoderonic acid B）である。
Of the compounds represented by the general formula (1), compound 1 (trichoderonic acid A) in which R is H is preferable. Of the compounds represented by general formula (2), compound 2 (trichoderonic acid B) in which R is H is preferable.
本発明における「その薬学的に許容し得る塩」の塩としては、ナトリウム塩、カリウム塩などのアルカリ金属塩；アンモニウム塩；１級、２級又は３級アミンの塩；４級アンモニウム塩；アミノ酸塩などが挙げられる。なお、一般式（１）及び（２）で表される化合物又はその薬学的に許容し得る塩は、水等の溶媒和物であってもよい。
Examples of the salt of “pharmaceutically acceptable salt thereof” in the present invention include alkali metal salts such as sodium salt and potassium salt; ammonium salt; primary, secondary or tertiary amine salt; quaternary ammonium salt; amino acid Examples include salt. The compound represented by the general formulas (1) and (2) or a pharmaceutically acceptable salt thereof may be a solvate such as water.
一般式（１）及び（２）で表される化合物のうちＲ＝Ｈで示される化合物は、菌株トリコデルマ ビレンス(Trichoderma virens；FERM S-10, ATCC 9645, CBS 430.54, IFO 6355, IMI 45553ii, QM 365、NBRC 6347)を培養して得られる培養物から単離・精製することができる。具体的には、トリコデルマ ビレンスをＰＤＢ培地（ポテトデキストロース液体培地）で静置培養し、培養液から菌体を除去して得られる濾液を溶媒で抽出する。抽出溶媒としては、例えば、メタノール、エタノール、プロパノール、ブタノール等のアルコール、１，３－ブチレングリコール、グリセリン、プロピレングリコール等のグリコール、酢酸エチル、酢酸ブチル等のエステル、エチルエーテル、プロピルエーテル、イソプロピルエーテル，テトラヒドロフラン、ジオキサン等のエーテル、塩化メチレン、クロロホルム等のハロゲン化炭化水素等の極性有機溶媒；ヘキサン、シクロヘキサン、石油エーテル等の無極性有機溶媒等を用いることができる。これらの溶媒を単独で又は２種以上の混合溶媒として用いることもできる。
Among the compounds represented by the general formulas (1) and (2), the compound represented by R = H is a strain of Trichoderma virens; 365, NBRC 6347) can be isolated and purified from the culture obtained. Specifically, Trichoderma virulence is statically cultured in a PDB medium (potato dextrose liquid medium), and the filtrate obtained by removing the cells from the culture is extracted with a solvent. Examples of the extraction solvent include alcohols such as methanol, ethanol, propanol and butanol, glycols such as 1,3-butylene glycol, glycerin and propylene glycol, esters such as ethyl acetate and butyl acetate, ethyl ether, propyl ether and isopropyl ether. , Polar organic solvents such as ethers such as tetrahydrofuran and dioxane, halogenated hydrocarbons such as methylene chloride and chloroform; nonpolar organic solvents such as hexane, cyclohexane and petroleum ether, and the like. These solvents can be used alone or as a mixed solvent of two or more.
これらの内で、塩化メチレン等のハロゲン化炭化水素、メタノール、エタノール等のアルコール、酢酸エチル等のエステルからなる群から選ばれる少なくとも一種の抽出溶媒を用いることが好ましい。溶媒を混合して用いる場合には、各溶媒の混合比は、溶媒の種類に応じて適宜調整すればよい。
Among these, it is preferable to use at least one extraction solvent selected from the group consisting of halogenated hydrocarbons such as methylene chloride, alcohols such as methanol and ethanol, and esters such as ethyl acetate. When mixing and using a solvent, what is necessary is just to adjust the mixing ratio of each solvent suitably according to the kind of solvent.
上記した方法によって抽出物を得た後、メタノール、エタノール、プロパノール、ブタノール、クロロホルム、酢酸エチル、トルエン、ヘキサン、ベンゼン等の有機溶媒を１種又は２種以上用いた溶媒分画操作によって、得られた抽出液から活性画分（化合物１及び２）を分取することができる。更に、アルミナカラムクロマトグラフィーやシリカゲルクロマトグラフィー、ゲルろ過クロマトグラフィー、イオン交換クロマトグラフィー、疎水クロマトグラフィー、高速液体クロマトグラフィー等の適当な分離精製手段を１種若しくは２種以上組み合わせて精製することもできる。なお、培養液からの活性化合物の単離及び同定は、具体的には実施例１の記載に従い行うことができる。
After obtaining the extract by the above method, it is obtained by solvent fractionation operation using one or more organic solvents such as methanol, ethanol, propanol, butanol, chloroform, ethyl acetate, toluene, hexane, benzene and the like. The active fraction (compounds 1 and 2) can be separated from the extracted liquid. Furthermore, it can also be purified by combining one or more suitable separation and purification means such as alumina column chromatography, silica gel chromatography, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, and high performance liquid chromatography. . In addition, isolation and identification of the active compound from the culture solution can be performed specifically as described in Example 1.
さらに、単離された化合物１又は２のカルボン酸部位を所定のアルコールを用いて公知の方法でエステル化することにより、一般式（１）及び（２）で表される化合物のうちＲ＝アルキルで示される化合物を得ることができる。
Further, R = alkyl among the compounds represented by the general formulas (1) and (2) is obtained by esterifying the carboxylic acid moiety of the isolated compound 1 or 2 by a known method using a predetermined alcohol. Can be obtained.
２ 本発明の化合物の用途
本発明の一般式（１）又は（２）で表される化合物は、ＤＮＡ合成酵素選択的阻害作用を有することから、医薬品への利用が可能である。例えば、ＤＮＡ合成酵素選択的阻害剤、抗癌剤、抗炎症剤等の医薬組成物として有用である。本発明の化合物は、さらに医薬品開発過程におけるリード化合物として利用することもできる。 2. Use of the compound of the present invention The compound represented by the general formula (1) or (2) of the present invention has a DNA synthase selective inhibitory action, and thus can be used for pharmaceuticals. For example, it is useful as a pharmaceutical composition such as a DNA synthase selective inhibitor, an anticancer agent, and an anti-inflammatory agent. The compound of the present invention can also be used as a lead compound in the pharmaceutical development process.
本発明の一般式（１）又は（２）で表される化合物は、ＤＮＡ合成酵素選択的阻害作用を有することから、医薬品への利用が可能である。例えば、ＤＮＡ合成酵素選択的阻害剤、抗癌剤、抗炎症剤等の医薬組成物として有用である。本発明の化合物は、さらに医薬品開発過程におけるリード化合物として利用することもできる。 2. Use of the compound of the present invention The compound represented by the general formula (1) or (2) of the present invention has a DNA synthase selective inhibitory action, and thus can be used for pharmaceuticals. For example, it is useful as a pharmaceutical composition such as a DNA synthase selective inhibitor, an anticancer agent, and an anti-inflammatory agent. The compound of the present invention can also be used as a lead compound in the pharmaceutical development process.
本発明の化合物を体内投与する際は経口投与よりも非経口投与が好ましく、またリポソームなどの運搬体に封入して投与することが好ましい。このとき癌細胞を特異的に認識する運搬体などを利用すれば、標的部位（病変部位）に本発明の化合物を効率よく運ぶことができ効果的である。
When the compound of the present invention is administered into the body, parenteral administration is preferable to oral administration, and it is preferable to encapsulate and administer it in a carrier such as a liposome. At this time, if a carrier that specifically recognizes cancer cells is used, the compound of the present invention can be efficiently transported to the target site (lesion site).
次に、本発明の化合物を配合してなる医薬用組成物について説明する。本発明の化合物を有効成分とする抗癌剤および抗炎症剤は、これをそのまま、あるいは慣用の医薬製剤担体とともに医薬用組成物となし、動物およびヒトに投与することができる。医薬用組成物の剤形としては特に制限されるものではなく必要に応じて適宜選択すればよいが、例えば、錠剤、カプセル剤、顆粒剤、細粒剤、散剤等の経口剤、注射剤、坐剤等の非経口剤が挙げられ、好適には非経口剤を挙げることができる。
Next, a pharmaceutical composition comprising the compound of the present invention will be described. The anticancer agent and anti-inflammatory agent containing the compound of the present invention as an active ingredient can be administered to animals and humans as they are or as a pharmaceutical composition together with a conventional pharmaceutical preparation carrier. The dosage form of the pharmaceutical composition is not particularly limited and may be appropriately selected as necessary.For example, oral preparations such as tablets, capsules, granules, fine granules, powders, injections, Non-oral preparations such as suppositories are mentioned, and preferred examples include parenteral preparations.
本発明において錠剤、カプセル剤、顆粒剤、細粒剤、散剤としての経口剤は、例えば、デンプン、乳糖、白糖、マンニット、カルボキシメチルセルロース、コーンスターチ、無機塩類等を用いて常法に従って製造される。これらの製剤中の本発明の化合物の配合量は特に限定されるものではなく適宜設計できる。この種の製剤には本発明の化合物の他に、結合剤、崩壊剤、界面活性剤、滑沢剤、流動性促進剤、矯味剤、着色剤、香料等を適宜に使用することができる。
In the present invention, oral preparations such as tablets, capsules, granules, fine granules, and powders are produced according to a conventional method using, for example, starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, inorganic salts, and the like. . The compounding amount of the compound of the present invention in these preparations is not particularly limited and can be appropriately designed. In addition to the compound of the present invention, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a corrigent, a colorant, a fragrance and the like can be appropriately used for this type of preparation.
ここに、結合剤としてデンプン、デキストリン、アラビアゴム末、ゼラチン、ヒドロキシプロピルスターチ、メチルセルロースナトリウム、ヒドロキシプロピルセルロース、結晶セルロース、エチルセルロース、ポリビニルピロリドン、マクロゴール等を例示できる。崩壊剤としてはデンプン、ヒドロキシプロピルスターチ、カルボキシメチルセルロースナトリウム、カルボキシメチルセルロースカルシウム、カルボキシメチルセルロース、低置換ヒドロキシプロピルセルロース等を例として挙げることができる。界面活性剤の例としてラウリル硫酸ナトリウム、大豆レシチン、蔗糖脂肪酸エステル、ポリオキシエチレンソルビタン脂肪酸エステル等を挙げることができる。滑沢剤では、タルク、ロウ類、水素添加植物油、蔗糖脂肪酸エステル、ステアリン酸マグネシウム、ステアリン酸カルシウム、ステアリン酸アルミニウム、ポリエチレングリコール等を例示できる。流動性促進剤では、軽質無水ケイ酸、乾燥水酸化アルミニウムゲル、合成ケイ酸アルミニウム、ケイ酸マグネシウム等を例として挙げることができる。また、本発明の化合物は懸濁液、エマルション剤、シロップ剤、エリキシル剤としても投与することができ、これらの各種剤形には、矯味矯臭剤、着色剤を含有させてもよい。
Examples of the binder include starch, dextrin, gum arabic powder, gelatin, hydroxypropyl starch, sodium methylcellulose, hydroxypropylcellulose, crystalline cellulose, ethylcellulose, polyvinylpyrrolidone, macrogol and the like. Examples of the disintegrant include starch, hydroxypropyl starch, carboxymethylcellulose sodium, carboxymethylcellulose calcium, carboxymethylcellulose, and low-substituted hydroxypropylcellulose. Examples of the surfactant include sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polyoxyethylene sorbitan fatty acid ester and the like. Examples of lubricants include talc, waxes, hydrogenated vegetable oils, sucrose fatty acid esters, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol and the like. Examples of the fluidity promoter include light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate and the like. The compounds of the present invention can also be administered as suspensions, emulsions, syrups, and elixirs, and these various dosage forms may contain flavoring agents and colorants.
非経口剤として本発明の所望の効果を発現せしめるには、患者の年齢、体重、疾患の程度により異なるが、通常、成人で本発明の化合物の重量として１日あたり１～６０ｍｇの静注、点滴静注、皮下注射、筋肉注射が適当である。この非経口投与剤は常法に従って製造され、希釈剤として一般に注射用蒸留水、生理食塩水、ブドウ糖水溶液、注射用植物油、ゴマ油、ラッカセイ油、大豆油、トウモロコシ油、プロピレングリコール等を用いることができる。さらに必要に応じて、殺菌剤、防腐剤、安定剤を加えてもよい。また、この非経口剤は安定性の点から、バイアル等に充填後冷凍し、通常の凍結乾燥処理により水分を除き、使用直前に凍結乾燥物から液剤を再調製することもできる。さらに必要に応じて、等張化剤、安定剤、防腐剤、無痛化剤を加えてもよい。これら製剤中の本発明の化合物の配合量は特に限定されるものではなく任意に設定できる。その他の非経口剤の例として、外用液剤、軟膏等の塗布剤、直腸内投与のための坐剤等が挙げられ、これらも常法に従って製造される。
In order to achieve the desired effect of the present invention as a parenteral preparation, it varies depending on the age, body weight, and degree of disease of the patient, but is usually intravenous in an amount of 1 to 60 mg per day as the weight of the compound of the present invention in an adult. Intravenous infusion, subcutaneous injection, and intramuscular injection are suitable. This parenteral preparation is produced according to a conventional method, and generally used as diluent is distilled water for injection, physiological saline, aqueous glucose solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, etc. it can. Furthermore, you may add a disinfectant, antiseptic | preservative, and a stabilizer as needed. In addition, from the viewpoint of stability, this parenteral preparation can be frozen after filling into a vial or the like, the water can be removed by ordinary freeze-drying treatment, and the liquid preparation can be re-prepared from the freeze-dried product immediately before use. Furthermore, you may add an isotonic agent, a stabilizer, an antiseptic | preservative, and a soothing agent as needed. The compounding quantity of the compound of this invention in these formulations is not specifically limited, It can set arbitrarily. Examples of other parenteral agents include liquid preparations for external use, coating agents such as ointments, suppositories for rectal administration, etc., and these are also produced according to conventional methods.
また、本発明の化合物は、医薬品への利用以外に、食品への利用が可能である。例えば、飲食品へ添加・配合することにより抗癌効果、抗発癌効果、あるいは抗炎症効果をもった食用組成物（例えば、健康食品等）として利用することも可能である。
In addition, the compound of the present invention can be used for foods as well as for pharmaceuticals. For example, it can be used as an edible composition (for example, health food) having an anti-cancer effect, an anti-carcinogenic effect, or an anti-inflammatory effect by adding and blending with food and drink.
即ち、本発明の化合物は、これをそのまま液状、ゲル状あるいは固形状の食品、例えばジュース、清涼飲料、茶、スープ、豆乳、サラダ油、ドレッシング、ヨーグルト、ゼリー、プリン、ふりかけ、育児用粉乳、ケーキミックス、粉末状または液状の乳製品、パン、クッキー等に添加したり、必要に応じてデキストリン、乳糖、澱粉等の賦形剤や香料、色素等とともにペレット、錠剤、顆粒等に加工したり、またゼラチン等で被覆してカプセルに成形加工して健康食品や栄養補助食品等として利用できる。
That is, the compound of the present invention is used as it is as a liquid, gel or solid food, for example, juice, soft drink, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, sprinkle, infant formula, cake. Add to mixes, powdered or liquid dairy products, bread, cookies, etc., and if necessary, process into pellets, tablets, granules, etc. with excipients such as dextrin, lactose, starch, flavorings, pigments, etc. Further, it can be coated with gelatin or the like and molded into a capsule to be used as a health food or nutritional supplement.
なお、ヒトと他の哺乳類のＤＮＡ合成酵素の構造は殆ど同じであるため、本発明のＤＮＡ合成酵素阻害剤は、ヒト以外の哺乳類由来のＤＮＡ合成酵素阻害剤としても利用可能である。
Since the structures of DNA synthases of humans and other mammals are almost the same, the DNA synthase inhibitors of the present invention can be used as DNA synthase inhibitors derived from mammals other than humans.
また、前述のように、ＤＮＡ合成酵素阻害作用、抗炎症作用および抗発癌作用の間には互いに関連性が認められ、ＤＮＡ合成酵素を特異的に阻害する化合物２は、ＴＰＡによって誘発される慢性炎症を抑制した。この結果は、ＴＰＡによる慢性炎症の発症においてＤＮＡ合成酵素が重要な役割を担っている可能性を示すものである。また、ＴＰＡは、慢性炎症を誘発するのみならず、発癌プロモーターとして哺乳類の細胞増殖を促進する。ＤＮＡ合成酵素選択的阻害剤である化合物１及び化合物２は、このような発癌プロモーターＴＰＡの働きを抑制する抗発癌作用を示す。
Further, as described above, there is a correlation between the DNA synthase inhibitory action, the anti-inflammatory action and the anticarcinogenic action, and the compound 2 that specifically inhibits the DNA synthase is chronically induced by TPA. Inflammation was suppressed. This result indicates the possibility that DNA synthase plays an important role in the development of chronic inflammation due to TPA. TPA not only induces chronic inflammation but also promotes cell growth in mammals as an oncogenic promoter. Compound 1 and Compound 2, which are selective inhibitors of DNA synthase, exhibit an anti-carcinogenic action that suppresses the action of such tumor promoter TPA.
したがって、本発明の化合物をリードとして、ＤＮＡ合成酵素に対する阻害活性を調べることにより、抗癌剤または抗炎症剤の候補化合物の効率的なスクリーニングが期待できる。本発明には、このようなスクリーニング方法も含まれる。なお、本スクリーニング方法において、ＤＮＡ合成酵素に対する阻害活性を調べる方法は前述の実施例記載の方法に限定されるものではなく、公知の試験方法の中から適した方法を選択すればよい。
Therefore, efficient screening of candidate compounds for anticancer agents or anti-inflammatory agents can be expected by examining the inhibitory activity against DNA synthase using the compound of the present invention as a lead. The present invention includes such a screening method. In this screening method, the method for examining the inhibitory activity against DNA synthase is not limited to the method described in the above-mentioned Examples, and a suitable method may be selected from known test methods.
次に、本発明を具体的に説明するが、本発明はこれに限定されるものではない。
Next, the present invention will be specifically described, but the present invention is not limited to this.
実施例１
（１）菌株の単離と培養
稲毛海浜公園にて採取した花弁をPDA培地(ポテトデキストロース寒天培地)の上で培養し、本菌株（便宜上、IG34HB株と記載する）を単離した。本菌株は菌株同定の結果、トリコデルマ ビレンス(Trichoderma virens；FERM S-10, ATCC 9645, CBS 430.54, IFO 6355, IMI 45553ii, QM 365、NBRC 6347)と同定した。本菌株は１LのPDB培地(ポテトデキストロース液体培地)を入れた２L三角フラスコ6本（合計６L）で26日間静置培養した。 Example 1
(1) Isolation and culture of strains Petals collected at Inage Seaside Park were cultured on PDA medium (potato dextrose agar medium) to isolate this strain (referred to as IG34HB strain for convenience). As a result of strain identification, this strain was identified as Trichoderma virens (FERM S-10, ATCC 9645, CBS 430.54, IFO 6355, IMI 45553ii, QM 365, NBRC 6347). This strain was statically cultured for 26 days in six 2 L Erlenmeyer flasks (total 6 L) containing 1 L of PDB medium (potato dextrose liquid medium).
（１）菌株の単離と培養
稲毛海浜公園にて採取した花弁をPDA培地(ポテトデキストロース寒天培地)の上で培養し、本菌株（便宜上、IG34HB株と記載する）を単離した。本菌株は菌株同定の結果、トリコデルマ ビレンス(Trichoderma virens；FERM S-10, ATCC 9645, CBS 430.54, IFO 6355, IMI 45553ii, QM 365、NBRC 6347)と同定した。本菌株は１LのPDB培地(ポテトデキストロース液体培地)を入れた２L三角フラスコ6本（合計６L）で26日間静置培養した。 Example 1
(1) Isolation and culture of strains Petals collected at Inage Seaside Park were cultured on PDA medium (potato dextrose agar medium) to isolate this strain (referred to as IG34HB strain for convenience). As a result of strain identification, this strain was identified as Trichoderma virens (FERM S-10, ATCC 9645, CBS 430.54, IFO 6355, IMI 45553ii, QM 365, NBRC 6347). This strain was statically cultured for 26 days in six 2 L Erlenmeyer flasks (total 6 L) containing 1 L of PDB medium (potato dextrose liquid medium).
（２）抽出と精製
本菌株を培養した培養液はガーゼを用いて菌体を除去した。得られた濾液を塩化メチレンで抽出し、ロータリーエバポレーターを用いて溶媒を留去し、粗抽出物(932 mg)を得られた。この粗抽出物はシリカゲルを担体とし、展開溶媒としてクロロホルム-メタノール(100：0→0：100)を用いたカラムクロマトグラフィーによって図１に示すように6つのフラクション(Fr.A～Fr.F)に分画した。 (2) Extraction and purification The culture solution obtained by culturing the present strain was removed from the cells using gauze. The obtained filtrate was extracted with methylene chloride, and the solvent was distilled off using a rotary evaporator to obtain a crude extract (932 mg). This crude extract was subjected to column chromatography using silica gel as a carrier and chloroform-methanol (100: 0 → 0: 100) as a developing solvent. As shown in FIG. 1, six fractions (Fr. A to Fr. F) were obtained. It was fractionated.
本菌株を培養した培養液はガーゼを用いて菌体を除去した。得られた濾液を塩化メチレンで抽出し、ロータリーエバポレーターを用いて溶媒を留去し、粗抽出物(932 mg)を得られた。この粗抽出物はシリカゲルを担体とし、展開溶媒としてクロロホルム-メタノール(100：0→0：100)を用いたカラムクロマトグラフィーによって図１に示すように6つのフラクション(Fr.A～Fr.F)に分画した。 (2) Extraction and purification The culture solution obtained by culturing the present strain was removed from the cells using gauze. The obtained filtrate was extracted with methylene chloride, and the solvent was distilled off using a rotary evaporator to obtain a crude extract (932 mg). This crude extract was subjected to column chromatography using silica gel as a carrier and chloroform-methanol (100: 0 → 0: 100) as a developing solvent. As shown in FIG. 1, six fractions (Fr. A to Fr. F) were obtained. It was fractionated.
クロロホルム-メタノール(80：1～40：1)で溶出したFr.D (566.7 mg)はシリカゲルを担体とし、展開溶媒としてジクロロメタン-メタノール(100：0→0：100)を用いたカラムクロマトグラフィーによって4つのフラクション(Fr.D-A～Fr.D-D)に分画した。
Fr. D (566.7 mg) eluted with chloroform-methanol (80: 1-40: 1) was obtained by column chromatography using silica gel as a carrier and dichloromethane-methanol (100: 0 → 0: 100) as a developing solvent. Fractions were fractionated into 4 fractions (Fr.DA to Fr.DD).
ジクロロメタン-メタノール(0：100)で溶出したFr.D-D(40.4 mg)はシリカゲルを担体とし、展開溶媒として1％の酢酸を含むトルエン-酢酸エチル(8：1～2：1)を用いてカラムクロマトグラフィーによって7つのフラクション(Fr.D-D-A～Fr.D-D-G )に分画した。そして、1％の酢酸を含むトルエン-酢酸エチル(6：1)で溶出したFr.D-D-Cから化合物１(4.8 mg)を、1％の酢酸を含むトルエン-酢酸エチル(4：1)で溶出したFr.D-D-Fから化合物２(10.5 mg)を得た。
Fr.DD (40.4 mg) eluted with dichloromethane-methanol (0: 100) is a column using silica gel as a carrier and toluene-ethyl acetate (8: 1 to 2: 1) containing 1% acetic acid as a developing solvent. It was fractionated into 7 fractions (Fr.DDA to Fr.DDGD) by chromatography. Then, compound 1 (4.8 mg) was eluted from toluene-ethyl acetate (4: 1) containing 1% acetic acid from Fr. DDC eluted with toluene-ethyl acetate (6: 1) containing 1% acetic acid. Compound 2 (10.5 mg) was obtained from Fr.DDF.
（３）構造決定
化合物１及び化合物２はMS、IR、1H-NMR、13C-NMR、DEPT、1H-1H COSY、HMQC、HMBCによって構造決定した。図２の(A)に化合物１の炭素番号を、(B)には化合物１の1H-1H COSY、HMBC相関を示した。また、図３にはCDCl3中で測定した化合物１の1H-NMR、13C-NMRデータを示した。図４の(A)に化合物２の炭素番号を、(B)には化合物２の1H-1H COSY、HMBC相関を示した。また、図５にはCDCl3中で測定した化合物２の1H-NMR、13C-NMRデータを示した。 (3) Structure determination The structures ofCompound 1 and Compound 2 were determined by MS, IR, 1 H-NMR, 13 C-NMR, DEPT, 1 H- 1 H COZY, HMQC, and HMBC. Carbon number of the compound 1 in FIG. 2 (A), exhibited 1 H- 1 H COZY, HMBC correlations of Compound 1 in (B). FIG. 3 shows 1 H-NMR and 13 C-NMR data of Compound 1 measured in CDCl 3 . 4A shows the carbon number of Compound 2, and FIG. 4B shows the 1 H- 1 H COZY and HMBC correlation of Compound 2. FIG. 5 shows 1 H-NMR and 13 C-NMR data of Compound 2 measured in CDCl 3 .
化合物１及び化合物２はMS、IR、1H-NMR、13C-NMR、DEPT、1H-1H COSY、HMQC、HMBCによって構造決定した。図２の(A)に化合物１の炭素番号を、(B)には化合物１の1H-1H COSY、HMBC相関を示した。また、図３にはCDCl3中で測定した化合物１の1H-NMR、13C-NMRデータを示した。図４の(A)に化合物２の炭素番号を、(B)には化合物２の1H-1H COSY、HMBC相関を示した。また、図５にはCDCl3中で測定した化合物２の1H-NMR、13C-NMRデータを示した。 (3) Structure determination The structures of
化合物１の諸性質およびスペクトルデータ(NMRデータは除く)を次の通りである。無色油状、[α]D
26 +5.9 (c 0.56, CHCl3); IR νmax(film): 3434, 2958, 1705, 1241, 1104, 775 cm-1 (KBr); HR-ESIMS m/z found 335.1463 [M+Na]+ (計算値 C16H24O6Na 335.1465)
化合物２の諸性質およびスペクトルデータ(NMRデータは除く)を次の通りである。無色油状、[α]D 26 -22.3 (c 0.09, CHCl3); IR νmax(film): 2960, 1743, 1716, 1238, 1058, 756, 463 cm-1(KBr); HR-ESIMS m/z found 583.2509 [M+Na]+ (計算値 C30H40O10Na 583.2513) Various properties and spectrum data (excluding NMR data) ofCompound 1 are as follows. Colorless oil, [α] D 26 +5.9 (c 0.56, CHCl 3 ); IR ν max (film): 3434, 2958, 1705, 1241, 1104, 775 cm -1 (KBr); HR-ESIMS m / z found 335.1463 [M + Na] + (calculated value C 16 H 24 O 6 Na 335.1465)
Various properties and spectral data (excluding NMR data) ofCompound 2 are as follows. Colorless oil, [α] D 26 -22.3 (c 0.09, CHCl 3 ); IR ν max (film): 2960, 1743, 1716, 1238, 1058, 756, 463 cm -1 (KBr); HR-ESIMS m / z found 583.2509 [M + Na] + (calculated value C 30 H 40 O 10 Na 583.2513)
化合物２の諸性質およびスペクトルデータ(NMRデータは除く)を次の通りである。無色油状、[α]D 26 -22.3 (c 0.09, CHCl3); IR νmax(film): 2960, 1743, 1716, 1238, 1058, 756, 463 cm-1(KBr); HR-ESIMS m/z found 583.2509 [M+Na]+ (計算値 C30H40O10Na 583.2513) Various properties and spectrum data (excluding NMR data) of
Various properties and spectral data (excluding NMR data) of
化合物１はMS、1H-NMR、13C-NMRおよびDEPTから分子式 C16H24O6を持つことが示された。まず、1H-1H COSY相関から図２(B)の太線の部分構造の存在が示唆された。また、H-5, H-9→C-6のHMBC相関からシクロヘキサン構造の存在が示唆された。C-4の化学シフトからカルボニルの存在が示唆され、またH-3→C-2, C-4, C-13のHMBC相関とH-5→C-4, C-12, C-13のHMBC相関とH-12→C-2, C-5, C-13のHMBC相関から不飽和7員環ラクトンの存在が示唆された。Me-8の化学シフトからメトキシ基の存在が示唆され、H-8→C-7のHMBC相関とH-7→C-5, C-6, C-9の相関からC-6にメトキシメチルが結合していることが示唆された。最後にIRスペクトルの2958 cm-1とC-1の化学シフトからカルボン酸の存在が示唆され、H-3, H-13→C-1のHMBC相関からC-1のカルボン酸はC-2に結合していることが示唆された。
Compound 1, MS, 1 H-NMR, 13 C-NMR and DEPT were shown to have the molecular formula C 16 H 24 O 6 . First, the 1 H- 1 H COZY correlation suggested the existence of the partial structure shown in bold lines in FIG. In addition, the presence of cyclohexane structure was suggested by the HMBC correlation of H-5, H-9 → C-6. The chemical shift of C-4 suggests the presence of carbonyl, and the HMBC correlation of H-3 → C-2, C-4, C-13 and H-5 → C-4, C-12, C-13. HMBC correlation and H-12 → C-2, C-5, C-13 HMBC correlation suggested the existence of unsaturated 7-membered lactone. The chemical shift of Me-8 suggests the presence of a methoxy group, and the HMBC correlation from H-8 → C-7 and the correlation from H-7 → C-5, C-6, C-9 to methoxymethyl at C-6 Was suggested to be bound. Finally, the chemical shift of 2958 cm -1 and C-1 in the IR spectrum suggests the presence of carboxylic acid, and the HMBC correlation of H-3, H-13 → C-1 indicates that C-1 carboxylic acid is C-2 It was suggested that it is bound to.
以上より化合物１は、9-ヒドロキシ-6-イソプロピル-9-(メトキシメチル)-1-オキソ-1,3,5a,6,7,8,9,9a-オクタヒドロ-2-ベンズオキセピン-4-カルボン酸（9-hydroxy-6-isopropyl-9-(methoxymethyl)-1-oxo-1,3,5a,6,7,8,9,9a-octahydro-2-benzoxepine-4-carboxylic acid）(トリコデロン酸Ａ；trichoderonic acid Aと命名した)と決定した。
From the above, compound 1 is 9-hydroxy-6-isopropyl-9- (methoxymethyl) -1-oxo-1,3,5a, 6,7,8,9,9a-octahydro-2-benzoxepin-4-carbon Acid (9-hydroxy-6-isopropyl-9- (methoxymethyl) -1-oxo-1,3,5a, 6,7,8,9,9a-octahydro-2-benzoxepine-4-carboxylic acid) (Trichoderonic acid A; named trichoderonic acid A).
化合物２はMS、1H-NMR、13C-NMRおよびDEPTから分子式 C30H40O10を持つことが示された。化合物２のNMRデータはC-7, C16, C-20, C-21, C-22を除いてtrichoderonic acid A（化合物１）と似ていた。H-7→C-16のHMBC相関からtricoderonic acid Aの類似構造が二量体した構造が示唆された。また、C-21, C-22については化学シフトとカップリング定数J21,22 = 5.2 HzからC-21, C-22でエポキシドの存在が示唆された。以上より化合物２は、9-ヒドロキシ-6-イソプロピル-9((6-イソプロピル-1-オキソ-1,3,5a,6,7,9a-ヘキサヒドロ-3H-スピロ[2-ベンズオキセピン-9,2’-オキシラン]-4-イルカルボニルオキシ)メチル)-1-オキソ-1,3,5a,6,7,8,9,9a-オクタヒドロ-2-ベンズオキセピン-4-カルボン酸（9-hydroxy-6-isopropyl-9((6-isopropyl-1-oxo-1,3,5a,6,7,9a-hexahydro-3H-spiro[2-benzoxepine-9,2’-oxirane]-4-ylcarbonyloxy)methyl)-1-oxo-1,3,5a,6,7,8,9,9a-octahydro-2-benzoxepine-4-carboxylic acid） (トリコデロン酸Ｂ；tricoderonic acid Bと命名)と決定した。
Compound 2, MS, 1 H-NMR, 13 C-NMR and DEPT were shown to have the molecular formula C 30 H 40 O 10 . The NMR data of Compound 2 was similar to that of trichoderonic acid A (Compound 1) except for C-7, C16, C-20, C-21 and C-22. The HHM → C-16 HMBC correlation suggested a structure in which a similar structure of tricoderonic acid A was dimerized. For C-21 and C-22, the chemical shift and coupling constant J 21,22 = 5.2 Hz suggests the presence of epoxide at C-21 and C-22. From the above, compound 2 was obtained by using 9-hydroxy-6-isopropyl-9 ((6-isopropyl-1-oxo-1,3,5a, 6,7,9a-hexahydro-3H-spiro [2-benzoxepin-9,2 '-Oxirane] -4-ylcarbonyloxy) methyl) -1-oxo-1,3,5a, 6,7,8,9,9a-octahydro-2-benzoxepin-4-carboxylic acid (9-hydroxy-6 -isopropyl-9 ((6-isopropyl-1-oxo-1,3,5a, 6,7,9a-hexahydro-3H-spiro [2-benzoxepine-9,2'-oxirane] -4-ylcarbonyloxy) methyl) -1-oxo-1,3,5a, 6,7,8,9,9a-octahydro-2-benzoxepine-4-carboxylic acid) (named tricoderonic acid B).
実施例２（ＤＮＡ合成酵素阻害活性の検証）
上記実施例で得た化合物１（trichoderonic acid A）及び化合物２（trichoderonic acid B）のＤＮＡ合成酵素群に対する活性を以下の方法で測定した。 Example 2 (Verification of DNA synthase inhibitory activity)
The activity of the compound 1 (trichoderonic acid A) and compound 2 (trichoderonic acid B) obtained in the above examples against the DNA synthase group was measured by the following method.
上記実施例で得た化合物１（trichoderonic acid A）及び化合物２（trichoderonic acid B）のＤＮＡ合成酵素群に対する活性を以下の方法で測定した。 Example 2 (Verification of DNA synthase inhibitory activity)
The activity of the compound 1 (trichoderonic acid A) and compound 2 (trichoderonic acid B) obtained in the above examples against the DNA synthase group was measured by the following method.
ＤＮＡ合成酵素として哺乳類由来のＤＮＡ合成酵素α、βおよびλについて試験を行った。ＤＮＡ合成酵素αは、牛胸腺から常法により抽出精製した標品を、ＤＮＡ合成酵素βは、ラット由来の該当遺伝子を、ＤＮＡ合成酵素λは、ヒト由来の該当遺伝子を、通常の遺伝子組み換え法により大腸菌に組み込み、生産させた標品を用いた。TdTは子牛由来の遺伝子組み換え法により大腸菌に組み込み、生産させた試薬（製品コード：2230A）をタカラバイオ（株）から購入して使用した。
Tests were performed on mammalian DNA synthases α, β and λ as DNA synthases. DNA synthase α is a sample extracted and purified from bovine thymus by a conventional method, DNA synthase β is a rat-derived gene, DNA synthase λ is a human-derived gene, and a conventional gene recombination method is used. The preparations incorporated into Escherichia coli and produced were used. TdT was purchased from Takara Bio Inc. and used as a reagent (product code: 2230A) incorporated into Escherichia coli by a genetic recombination method derived from calves.
これらのＤＮＡ合成酵素に対する化合物１及び２の阻害作用の測定には、一般的なＤＮＡ合成酵素反応系（日本生化学会編、新生化学実験講座２、核酸IV、東京化学同人、63～66頁）を用いた。すなわち、放射性同位元素で標識した[3Ｈ]-ＴＴＰを含む系においてＤＮＡ合成反応を行い、放射比活性を生成物（合成ＤＮＡ鎖）量の指標とするものである。
For the measurement of the inhibitory action of compounds 1 and 2 on these DNA synthases, a general DNA synthase reaction system (edited by the Japanese Biochemical Society, Shinsei Chemistry Laboratory 2, Nucleic Acid IV, Tokyo Kagaku Dojin, pages 63-66) Was used. That is, a DNA synthesis reaction is carried out in a system containing [ 3 H] -TTP labeled with a radioisotope, and the radioactivity is used as an index of the amount of product (synthetic DNA chain).
阻害率は、(a)コントロールでの合成ＤＮＡ量、(b) 被検物質存在下での合成ＤＮＡ量について、
(a - b) / a × 100 = 阻害率（％）
として評価した。得られた結果は50%阻害濃度（μＭ）として表１に示した。 The inhibition rate is (a) the amount of synthetic DNA in the control, and (b) the amount of synthetic DNA in the presence of the test substance.
(a-b) / a × 100 = inhibition rate (%)
As evaluated. The results obtained are shown in Table 1 as 50% inhibitory concentration (μM).
(a - b) / a × 100 = 阻害率（％）
として評価した。得られた結果は50%阻害濃度（μＭ）として表１に示した。 The inhibition rate is (a) the amount of synthetic DNA in the control, and (b) the amount of synthetic DNA in the presence of the test substance.
(a-b) / a × 100 = inhibition rate (%)
As evaluated. The results obtained are shown in Table 1 as 50% inhibitory concentration (μM).
表１に示したように、化合物１はＤＮＡ合成酵素λを、化合物２はＤＮＡ合成酵素β、λ及びTdTを選択的に阻害した。化合物２の阻害活性は、化合物１よりも10倍以上強かった。図６に化合物２のＤＮＡ合成酵素β、λ及びTdTに対する阻害曲線を示した。
As shown in Table 1, Compound 1 selectively inhibited DNA synthase λ, and Compound 2 selectively inhibited DNA synthase β, λ and TdT. The inhibitory activity of Compound 2 was more than 10 times stronger than that of Compound 1. FIG. 6 shows inhibition curves of Compound 2 against DNA synthases β, λ and TdT.
化合物２はＤＮＡ合成酵素λを最も強く阻害し、２番目にβ型、３番目にTdTを阻害することがわかった。化合物２のＤＮＡ合成酵素λに対する阻害様式をLineweaver-Burkの両逆数プロットから算出した結果、２つの基質である鋳型ＤＮＡとヌクレオチド（ｄＮＴＰ）に対して非拮抗阻害であった（データ示さず）。
Compound 2 was found to most strongly inhibit DNA synthase λ, secondly β-type, and thirdly TdT. As a result of calculating the inhibition mode of Compound 2 against DNA synthetase λ from the reciprocal plot of Lineweaver-Burk, it was non-competitive inhibition with respect to two substrates, template DNA and nucleotide (dNTP) (data not shown).
ＤＮＡ合成酵素は、癌細胞のような細胞分裂がさかんな細胞や組織に対して本酵素活性が高い。従って、化合物１及び２は抗癌剤としての作用を有することが期待される。図７(A)に示すように、哺乳類のＤＮＡ合成酵素は、その遺伝子配列の相同性や活性機能の違いからＡ、Ｂ、Ｘ、Ｙの４つのファミリーに分類される。化合物２はＸファミリーに属するＤＮＡ合成酵素β、λ及びTdTを選択的に阻害した。一方で、Ａファミリーに属するＤＮＡ合成酵素γ、Ｂファミリーに属するＤＮＡ合成酵素α、δ、ε、Ｙファミリーに属するＤＮＡ合成酵素η、ι、κは、いずれも阻害しなかった。
DNA synthase has a high enzyme activity against cells and tissues such as cancer cells that have a high cell division. Therefore, compounds 1 and 2 are expected to have an action as an anticancer agent. As shown in FIG. 7 (A), mammalian DNA synthases are classified into four families of A, B, X, and Y based on the homology of their gene sequences and differences in activity functions. Compound 2 selectively inhibited DNA synthases β, λ and TdT belonging to the X family. On the other hand, DNA synthase γ belonging to the A family, DNA synthase α, δ, ε belonging to the B family, and DNA synthases η, ι, κ belonging to the Y family were not inhibited.
哺乳類以外の魚類、植物、原核生物由来のＤＮＡ合成酵素についても同様に調査したが、化合物２は阻害しなかった（図７(B)）。さらにＤＮＡ合成酵素以外のＤＮＡ代謝系酵素についても調査したが、化合物２は阻害作用を有しなかった（図７(C)）。これらの結果より、化合物２は哺乳類のＤＮＡ合成酵素・Ｘファミリーを選択的に阻害することが分かった。
Investigating DNA synthases derived from fish, plants, and prokaryotes other than mammals in the same manner, Compound 2 did not inhibit (FIG. 7 (B)). Furthermore, DNA metabolic enzymes other than DNA synthase were also investigated, but Compound 2 did not have an inhibitory action (FIG. 7 (C)). From these results, it was found that Compound 2 selectively inhibits mammalian DNA synthase / X family.
実施例３（細胞増殖阻害活性の検証）
化合物１（trichoderonic acid A）及び化合物２（trichoderonic acid B）の細胞増殖阻害効果を次の方法を用いて評価した。 Example 3 (Verification of cell growth inhibitory activity)
The cell growth inhibitory effect of compound 1 (trichoderonic acid A) and compound 2 (trichoderonic acid B) was evaluated using the following method.
化合物１（trichoderonic acid A）及び化合物２（trichoderonic acid B）の細胞増殖阻害効果を次の方法を用いて評価した。 Example 3 (Verification of cell growth inhibitory activity)
The cell growth inhibitory effect of compound 1 (trichoderonic acid A) and compound 2 (trichoderonic acid B) was evaluated using the following method.
本実験では、癌細胞としてヒト子宮頸癌由来HeLa細胞及びヒト乳癌由来MCF-7細胞を用い、正常細胞としてヒト皮膚上皮細胞(HDF)及びヒト臍帯静脈内皮細胞(HUBEC)を用いた。培地にはDMEM培地（日本製薬(株)製）に牛胎児血清 10％（v/v）とペニシリン-ストレプトマイシン5%（v/v）を添加したものを用いた。培養は5％ CO2インキュベートにて37℃で行った。
In this experiment, human cervical cancer-derived HeLa cells and human breast cancer-derived MCF-7 cells were used as cancer cells, and human skin epithelial cells (HDF) and human umbilical vein endothelial cells (HUBEC) were used as normal cells. The medium used was a DMEM medium (Nippon Pharmaceutical Co., Ltd.) supplemented with fetal bovine serum 10% (v / v) and penicillin-streptomycin 5% (v / v). Culturing was performed at 37 ° C. with 5% CO 2 incubation.
上記に示した培地に、あらかじめ各試験濃度になるように化合物１及び２を溶解した。ただし、化合物１及び２は水に難溶であるため、一度DMSO（ジメチルスルホキシド）に溶解し、そのものを上記の培地に溶かした。なお、培地内に存在するDMSOの終濃度は、すべての試験区で0.1％になっており、本測定例で用いた各細胞の増殖の抑制にDMSOが関わる可能性は否定できる状態である。
In the medium shown above, compounds 1 and 2 were dissolved in advance so as to obtain each test concentration. However, since compounds 1 and 2 are hardly soluble in water, they were once dissolved in DMSO (dimethyl sulfoxide) and dissolved in the above medium. The final concentration of DMSO present in the medium is 0.1% in all test groups, and the possibility that DMSO is involved in the suppression of the growth of each cell used in this measurement example can be denied.
本実験のための培養は、96穴マイクロプレートで行った。各ウェルに5.0×103個の細胞を植え込み、1つの試験濃度に対して3ウェルずつ与えた。各細胞をプレート内で24時間培養し、各濃度の化合物１, ２を添加後は、5％CO2インキュベート内、37℃で、癌細胞は48時間、正常細胞は72時間培養し、各試験区の細胞生存率の判定を行った。生存率の判定は、生細胞数測定試薬SFを用いた(Talanta 1997, 44の方法で行った)。生細胞測定試薬SFに含まれる新規テトラゾリウム塩WST-8が細胞内脱水素酵素により還元され、高感度水溶性ホルマザンを生成し、このホルマザンの450 nmの吸光度を直接測定することにより、容易に生細胞数を計測できる。よって、本実験では48時間又は72時間の培養後、生細胞数測定試薬SFを添加し、さらに2時間の培養後に450 nmの吸光度(O.D.)を測定し、以下の式で細胞生存率を算出した。
The culture for this experiment was performed in a 96-well microplate. Each well was implanted with 5.0 × 10 3 cells and given 3 wells for one test concentration. Each cell is cultured in a plate for 24 hours. After adding each concentration of Compound 1 and 2, each cell is cultured in a 5% CO 2 incubation at 37 ° C for 48 hours for cancer cells and 72 hours for normal cells. The cell viability of the section was determined. Viability was determined using the living cell count reagent SF (performed by the method of Talanta 1997, 44). The novel tetrazolium salt WST-8 contained in the living cell measurement reagent SF is reduced by intracellular dehydrogenase to produce a highly sensitive water-soluble formazan, which is easily produced by directly measuring the absorbance of this formazan at 450 nm. The number of cells can be measured. Therefore, in this experiment, after culturing for 48 hours or 72 hours, add the live cell count reagent SF, measure the absorbance (OD) at 450 nm after further culturing for 2 hours, and calculate the cell viability using the following formula did.
ヒト癌細胞で得られた結果を図８及び図９に示す。なお、図８及び図９に示したデータは3ウェルの平均である。
The results obtained with human cancer cells are shown in FIGS. The data shown in FIGS. 8 and 9 is an average of 3 wells.
化合物１は各癌細胞の増殖を阻害しなかったが、化合物２では各癌細胞の増殖を阻害した。図８及び図９の結果から、化合物２の50％増殖阻害濃度(IC50)はHeLa細胞で15.6μM、MCF-7細胞で8.7μMであった。この結果からＤＮＡ合成酵素を特異的に阻害する化合物２が、実際にヒト癌細胞株に対する細胞増殖抑制効果を発揮し、抗癌剤として利用し得ることが示された。
Compound 1 did not inhibit the growth of each cancer cell, but Compound 2 inhibited the growth of each cancer cell. From the results of FIGS. 8 and 9, the 50% growth inhibitory concentration (IC 50 ) of Compound 2 was 15.6 μM in HeLa cells and 8.7 μM in MCF-7 cells. From this result, it was shown that Compound 2, which specifically inhibits DNA synthase, actually exhibits a cell growth inhibitory effect on human cancer cell lines and can be used as an anticancer agent.
ヒト正常細胞を用いたときの化合物１及び化合物２の50％増殖阻害濃度(IC50)を下記の表２に示す。
Table 2 below shows the 50% growth inhibitory concentration (IC 50 ) of Compound 1 and Compound 2 when normal human cells were used.
表２に示すように、化合物１及び化合物２は、いずれも50％増殖阻害濃度(IC50)が100μM以上であった。この実験結果から、化合物１及び化合物２は、各正常細胞の増殖を阻害しないと言える。このことから、化合物１及び化合物２は、ヒト正常細胞への影響はなく、副作用がない抗癌剤として利用できる可能性が示唆された。
As shown in Table 2, both Compound 1 and Compound 2 had a 50% growth inhibitory concentration (IC 50 ) of 100 μM or more. From this experimental result, it can be said that Compound 1 and Compound 2 do not inhibit the growth of each normal cell. From this, it was suggested that Compound 1 and Compound 2 could be used as anticancer agents with no adverse effects on human normal cells.
実施例４（抗炎症作用および抗発癌作用の検証）
ＴＰＡ（12-O-tetradecanoylphorbol-13-acetate）は、慢性炎症を誘発するのみならず、発癌プロモーターとして哺乳類の細胞増殖を促進する。本発明者らは、ＤＮＡ合成酵素阻害作用（特にＤＮＡの修復・組換えに関与するＤＮＡ合成酵素λ阻害活性）、抗炎症作用および抗発癌プロモーター活性の間には互いに関連性があるのではないかと考えた。そこで、化合物１（trichoderonic acid A）及び化合物２（trichoderonic acid B）が抗炎症活性を有するかどうかを調べた。その結果を下記の表３に示す。 Example 4 (Verification of anti-inflammatory and anti-carcinogenic effects)
TPA (12-O-tetradecanoylphorbol-13-acetate) not only induces chronic inflammation but also promotes cell growth in mammals as an oncogenic promoter. The present inventors have no relationship between DNA synthase inhibitory activity (particularly DNA synthase λ inhibitory activity involved in DNA repair / recombination), anti-inflammatory activity and anti-tumor promoter activity. I thought. Therefore, it was examined whether Compound 1 (trichoderonic acid A) and Compound 2 (trichoderonic acid B) have anti-inflammatory activity. The results are shown in Table 3 below.
ＴＰＡ（12-O-tetradecanoylphorbol-13-acetate）は、慢性炎症を誘発するのみならず、発癌プロモーターとして哺乳類の細胞増殖を促進する。本発明者らは、ＤＮＡ合成酵素阻害作用（特にＤＮＡの修復・組換えに関与するＤＮＡ合成酵素λ阻害活性）、抗炎症作用および抗発癌プロモーター活性の間には互いに関連性があるのではないかと考えた。そこで、化合物１（trichoderonic acid A）及び化合物２（trichoderonic acid B）が抗炎症活性を有するかどうかを調べた。その結果を下記の表３に示す。 Example 4 (Verification of anti-inflammatory and anti-carcinogenic effects)
TPA (12-O-tetradecanoylphorbol-13-acetate) not only induces chronic inflammation but also promotes cell growth in mammals as an oncogenic promoter. The present inventors have no relationship between DNA synthase inhibitory activity (particularly DNA synthase λ inhibitory activity involved in DNA repair / recombination), anti-inflammatory activity and anti-tumor promoter activity. I thought. Therefore, it was examined whether Compound 1 (trichoderonic acid A) and Compound 2 (trichoderonic acid B) have anti-inflammatory activity. The results are shown in Table 3 below.
実験では、マウス耳に予め所定量の化合物１もしくは化合物２を塗布後、ＴＰＡによって誘発される炎症性浮腫の重量を測定することで各阻害効果を算出した。試験は、基本的にCancer Lett. 1984, 25, 177-85頁記載の方法にしたがって行い、マウスの一方の耳に化合物を250μｇ、500μｇもしくは750μｇ塗布し、３０分後、同じ箇所と反対側の耳にＴＰＡを0.5μｇ塗布する。７時間後、ＴＰＡによって誘発される炎症性浮腫の重量を測定し、コントロール（反対側の耳）と比較することで各阻害効果を算出した。
In the experiment, each inhibitory effect was calculated by measuring the weight of inflammatory edema induced by TPA after applying a predetermined amount of Compound 1 or Compound 2 to the mouse ear in advance. The test was basically carried out according to the method described in Cancer Lett. 1984, 25, 177-85, and 250 μg, 500 μg or 750 μg of the compound was applied to one ear of the mouse, and after 30 minutes, the same site and the opposite side were applied. Apply 0.5 μg of TPA to the ear. After 7 hours, the weight of inflammatory edema induced by TPA was measured, and each inhibitory effect was calculated by comparing with the control (opposite ear).
表３に示すように、化合物２は塗布量250μg・500μg・750μgに依存的に炎症を抑制した。この実験結果から、化合物２は抗炎症作用を有するものと認められた。また、この実験結果から、化合物２は、発癌プロモーターＴＰＡの働きを抑制する抗発癌プロモーター活性（換言すれば、抗発癌作用）を有するものと認められた。
As shown in Table 3, Compound 2 inhibited inflammation depending on the coating amount of 250 μg, 500 μg and 750 μg. From this experimental result, Compound 2 was recognized as having an anti-inflammatory effect. Further, from this experimental result, it was recognized that Compound 2 has an anti-carcinogenic promoter activity (in other words, an anti-carcinogenic effect) that suppresses the action of the oncogenic promoter TPA.
Claims (6)
- 一般式（１）若しくは（２）で表される化合物又はその薬学的に許容し得る塩。
- 請求項１に記載の一般式（１）若しくは（２）で表される化合物又はその薬学的に許容し得る塩を有効成分として含有する医薬組成物。 A pharmaceutical composition comprising the compound represented by the general formula (1) or (2) according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
- 請求項１に記載の一般式（１）若しくは（２）で表される化合物又はその薬学的に許容し得る塩を有効成分として含有するＤＮＡ合成酵素阻害剤。 A DNA synthase inhibitor comprising the compound represented by the general formula (1) or (2) according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
- 請求項１に記載の一般式（２）で表される化合物又はその薬学的に許容し得る塩を有効成分として含有する抗癌剤。 The anticancer agent which contains the compound represented by General formula (2) of Claim 1 or its pharmacologically acceptable salt as an active ingredient.
- 請求項１に記載の一般式（１）若しくは（２）で表される化合物又はその薬学的に許容し得る塩を有効成分として含有する抗炎症剤。 An anti-inflammatory agent comprising the compound represented by the general formula (1) or (2) according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
- 請求項１に記載の一般式（１）若しくは（２）で表される化合物又はその薬学的に許容し得る塩を食品に配合してなる食用組成物。 An edible composition comprising the compound represented by the general formula (1) or (2) according to claim 1 or a pharmaceutically acceptable salt thereof in a food.
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Citations (4)
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|---|---|---|---|---|
| JPS5677281A (en) * | 1979-11-29 | 1981-06-25 | Sankyo Co Ltd | Antibiotic heptelidic acid and its preparation |
| JPH07277971A (en) * | 1994-04-01 | 1995-10-24 | Kobe Steel Ltd | Antitumor agent, selective cytotoxic agent for human tumor cell, microbial strain producing heptelidic acid chlorohydrin and its production |
| KR20050093093A (en) * | 2004-03-18 | 2005-09-23 | 한국생명공학연구원 | Heptelidic acid showing apoptosis-inhibitive effect |
| WO2009013901A1 (en) * | 2007-07-23 | 2009-01-29 | Tokyo University Of Agriculture And Technology | Novel compound, method for production of the compound, pharmaceutical composition, anti-tumor agent, and use of the compound |
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| ES2096999T3 (en) * | 1989-05-10 | 1997-03-16 | Raychem Corp | APPARATUS AND PROCEDURE TO REDUCE THE CRACKING OF POLYOLEFIN INSULATION FROM CABLES. |
| CA2133815A1 (en) * | 1993-10-12 | 1995-04-13 | Jeffrey Alan Dodge | Inhibition of phosphatidylinositol 3-kinase with viridin, demethoxyviridin, viridiol, demethoxyviridiol, virone, wortmannolone, and analogs thereof |
| US6605599B1 (en) * | 1997-07-08 | 2003-08-12 | Bristol-Myers Squibb Company | Epothilone derivatives |
| JP2006131595A (en) * | 2004-11-09 | 2006-05-25 | Univ Nihon | Carcinogenesis preventing agent and method for preparing cycloartane-type triterpene compound |
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| JPS5677281A (en) * | 1979-11-29 | 1981-06-25 | Sankyo Co Ltd | Antibiotic heptelidic acid and its preparation |
| JPH07277971A (en) * | 1994-04-01 | 1995-10-24 | Kobe Steel Ltd | Antitumor agent, selective cytotoxic agent for human tumor cell, microbial strain producing heptelidic acid chlorohydrin and its production |
| KR20050093093A (en) * | 2004-03-18 | 2005-09-23 | 한국생명공학연구원 | Heptelidic acid showing apoptosis-inhibitive effect |
| WO2009013901A1 (en) * | 2007-07-23 | 2009-01-29 | Tokyo University Of Agriculture And Technology | Novel compound, method for production of the compound, pharmaceutical composition, anti-tumor agent, and use of the compound |
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| YOSHIYUKI MIZUSHINA: "Screening of Novel Bioactive Compounds from Marine Microorganisms", BIO INDUSTRY, vol. 27, no. 2, 12 February 2010 (2010-02-12), pages 54 - 60 * |
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