WO2009101959A1 - Nouveau composé, composé de lachnochromonin - Google Patents

Nouveau composé, composé de lachnochromonin Download PDF

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Publication number
WO2009101959A1
WO2009101959A1 PCT/JP2009/052275 JP2009052275W WO2009101959A1 WO 2009101959 A1 WO2009101959 A1 WO 2009101959A1 JP 2009052275 W JP2009052275 W JP 2009052275W WO 2009101959 A1 WO2009101959 A1 WO 2009101959A1
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measured
ppm
magnetic resonance
shown below
dimethyl sulfoxide
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PCT/JP2009/052275
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English (en)
Japanese (ja)
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Kazuki Yano
Isshin Tanaka
Takahiro Miyauchi
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Daiichi Sankyo Company, Limited
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Publication of WO2009101959A1 publication Critical patent/WO2009101959A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the present invention relates to a novel compound having an effect of treating or preventing inflammation, lifestyle-related diseases and adult diseases, a pharmaceutical containing the compound as an active ingredient, a method for producing the compound, a novel microorganism producing the compound, and the like.
  • the inventors of the present invention have conducted extensive research for the purpose of searching for substances having a lifestyle-related disease and adult disease-preventing action, and have found a novel compound isolated and purified from a microorganism culture, thereby completing the present invention.
  • the present invention includes: (1) The following general formula (I):
  • R 1 is CH 3
  • R 2 is OH
  • R 3 is H
  • R 4 is CH 3
  • R 5 is CH (CH 3 ) (CH 2 ) 2 OH, CH (CH 3 ) CH (OH) CH 3 or CH (CH 3 ) CH 2 CH 3 is shown.
  • 1 H-nuclear magnetic resonance spectrum The 1 H-nuclear magnetic resonance spectrum measured in deuterated dimethyl sulfoxide using the tetramethylsilane signal (0.00 ppm) as an internal standard is shown below.
  • a compound having the following physicochemical properties or a salt thereof 1) Material properties: colorless powder, aqueous solution is weakly acidic, 2) Solubility: soluble in acetone, methanol, dimethyl sulfoxide, insoluble in water, ethyl acetate, hexane 3) Molecular formula: C 15 H 18 O 4 4) Molecular weight: 262 (measured by ESI mass spectrometry) 5) The accurate mass [M + Na] + measured by the high-resolution ESI mass spectrum method is as follows.
  • 1 H-nuclear magnetic resonance spectrum The 1 H-nuclear magnetic resonance spectrum measured in deuterated dimethyl sulfoxide using the tetramethylsilane signal (0.00 ppm) as an internal standard is shown below.
  • 13 C-nuclear magnetic resonance spectrum The 13 C-nuclear magnetic resonance spectrum measured in deuterated dimethyl sulfoxide using the deuterated dimethyl sulfoxide signal (39.5 ppm) as an internal standard is shown below. 7.8, 9.2, 11.8, 17, 9, 27.3, 36.8, 110.4, 113.4, 114.3, 114.6, 123.2, 155.0, 159.
  • the compound according to any one of (1) to (4) is cultured from the culture product of the compound according to any one of (1) to (4) belonging to the genus Lachnum. Collecting the compound according to any one of (1) to (4), (6)
  • the production strain of the compound according to any one of (1) to (4) belonging to the genus Lachnum is Lachnum virginium SANK10207 strain (FERM BP-10913).
  • the manufacturing method described (7) A microorganism belonging to the genus Lachnum and producing the compound according to any one of (1) to (4), (8) The microorganism according to (7), which is Lachnum virusine SANK10207 strain (FERM BP-10913), (9) A pharmaceutical comprising the compound according to any one of (1) to (4) or a pharmacologically acceptable salt thereof as an active ingredient, (10) The medicament according to (9), which is a therapeutic or prophylactic agent for inflammation, lifestyle-related diseases or adult diseases, About.
  • lacnochromonin (English name: “lachnochromonin”) A in the general formula (I), R 1 is CH 3 , R 2 is OH, R 3 is H, R 4 is CH 3 , R 5 represents a compound represented by CH (CH 3 ) (CH 2 ) 2 OH, or a compound having the physicochemical properties described in (2) above.
  • “Lacnochromonin B” means that in the above general formula (I), R 1 is CH 3 , R 2 is OH, R 3 is H, R 4 is CH 3 , R 5 is CH (CH 3 ) CH (OH) CH 3 Or a compound having the physicochemical properties described in (3) above.
  • “Lachnochromonin C” means that in the above general formula (I), R 1 is CH 3 , R 2 is OH, R 3 is H, R 4 is CH 3 , R 5 is CH (CH 3 ) CH 2 CH 3
  • R 1 is CH 3
  • R 2 is OH
  • R 3 is H
  • R 4 is CH 3
  • R 5 is CH (CH 3 ) CH 2 CH 3
  • the compound represented, or the compound which has the physicochemical property as described in said (4) is shown.
  • lacnochromonins compounds These three compounds are collectively referred to as “lacnochromonins compounds” and sometimes referred to as “compounds of the present invention”.
  • the compound of the present invention can be converted into a salt by a method well known to those skilled in the art using a base.
  • the present invention also includes these salts.
  • the salt of the compound of the present invention is used as a pharmaceutical, it is not particularly limited as long as it is used medically and is pharmacologically acceptable. There is no particular limitation as long as it can be used.
  • salts include alkali metal salts such as sodium salt, potassium salt and lithium salt; alkaline earth metal salts such as calcium salt and magnesium salt; aluminum salt, iron salt, zinc salt and copper Metal salts such as salts, nickel salts and cobalt salts; inorganic salts such as ammonium salts; t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglu Camin salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt , Tris (Hydro Shimechiru) organic amine
  • the compound of the present invention and a salt thereof may be combined with water or a solvent by being left in the atmosphere or mixed with water or an organic solvent to form a hydrate or a solvate.
  • hydrates and solvates are also included in the present invention.
  • the lacnochromonins compounds of the present invention have various steric structures.
  • equal and unequal mixtures of these stereoisomers are all represented by a single structural formula, however, the compounds of the present invention are composed of these stereoisomers and two or more stereoisomers. All mixtures of isomers are also included.
  • R 1 is CH 3
  • R 2 is OH
  • R 3 is H
  • R 4 is CH 3
  • R 5 is CH (CH 3 ) (CH 2 ).
  • R 1 is CH 3
  • R 2 is OH
  • R 3 is H
  • R 4 is CH 3
  • R 5 is CH (CH 3) CH (OH ) CH 3
  • the optical rotation in methanol is [ ⁇ ] D 27 + 88.7 ° (c 1.00, methanol).
  • R 1 is CH 3
  • R 2 is OH
  • R 3 is H
  • R 4 is CH 3
  • R 5 is CH (CH 3 ) CH 2 CH 3
  • a compound in which the optical rotation in methanol is [ ⁇ ] D 27 + 58.6 ° (c 1.00, methanol).
  • the lacnochromonins compound of the present invention can be isolated and purified from a culture of a microorganism producing the substance according to a conventional method.
  • the microorganism producing the compound of the present invention is not particularly limited, but is preferably a fungus, more preferably a strain belonging to the genus Lachnum, such as Lachnum virginum SANK10207 (hereinafter referred to as Lactum virginium). Simply referred to as “SANK10207 strain”).
  • the SANK10207 strain was isolated from offspring spores collected in Nagano Prefecture. In order to observe the bacteriological properties of the bacterium, culture was performed on the following media. The composition of each medium used is described below.
  • PDA medium Growth on PDA medium is 20-24 mm at 23 ° C. for 3 weeks of culture.
  • the fungus is fluffy, often radially fluted, white, and may have exudate on the surface of the fungus.
  • the reverse side is grayish orange (5B5) to yellowish white (4A2).
  • Growth on MEA medium is 29-30 mm at 23 ° C. for 3 weeks of culture. The fungus seems to be wooly in the center, but is crushed and thin and white. There is no change on the back side.
  • the aerial hyphae in PDA and MEA media are 1 to 3 ⁇ m wide, thin-walled, smooth and colorless. Often, hyphae with oil droplets attached to the wall are observed. Concubines and conidia are not formed.
  • the mycological properties of the dried fruit body specimens used for isolation were observed.
  • the mycological properties of the dried specimen are as follows.
  • the baby's disc is scattered, superficial, has a handle, the hair is white, and the other parts are light yellow.
  • the disk surface has a diameter of 0.6 mm or less and has a bowl shape or a disk shape.
  • the handle is 0.4 mm or less in length. ⁇
  • the outer skin layer forms a short-form fungus tissue.
  • the hair is 95 ⁇ m or less in length, 3 to 4 ⁇ m in width, cylindrical, generally rough, thin walled, colorless, blunt at the tip and often slightly enlarged.
  • the baby cub is 44 to 58 ⁇ 3.5 to 5 ⁇ m, cylindrical rod-shaped, 8-spore, and its tip turns blue with the Melzer reagent.
  • the ascospore is 6 to 10.5 ⁇ 1.5 to 2 ⁇ m, cylindrical to long spindle, single cell, thin wall, smooth.
  • the side yarns extend from 40 to 64 ⁇ 3.5 to 5 ⁇ m, a die, 19 ⁇ m or less from the child's heel.
  • the above bacteriological properties of this bacterium are described by Spooner, “Lachnum virginium” (Spooner B. M. (1987), Helotiales of Australia, H., B. , Vol. 116: pp. 534-539), this strain was identified as Lachnum virginium.
  • the SANK10207 strain was internationally deposited on September 26, 2007 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, 1-1-1 East Tsukuba, Ibaraki, Japan. The deposit number is FERM BP-10913.
  • the SANK10207 strain referred to in the present invention includes all mutants thereof.
  • the mutant strain encompassed by the SANK10207 strain of the present invention is a strain characterized by producing lacnochromonins A, B and C.
  • these mutant strains include those obtained by genetic methods such as recombination, transduction, transformation, etc., but all such mutant strains are present as long as they produce a lacnochromonin compound.
  • the mutant10207 strain of the invention that is, all of the mutant strains of SANK10207 that produce a lacnochromonin compound and strains that are not clearly distinguished from them are included in the SANK10207 strain.
  • the lacnochromonins compound of the present invention can be produced by culturing a bacterium producing the substance.
  • the culture of the SANK10207 strain can be usually performed using a medium such as that used for the production of secondary metabolites of microorganisms.
  • a medium contains a carbon source, a nitrogen source and an inorganic salt that can be assimilated by the microorganism.
  • the carbon source include glucose, fructose, maltose, sucrose, mannitol, glycerin, dextrin, oats, rye, barley, brown rice, corn starch, potato, corn flour, soybean oil, cottonseed oil, molasses, citric acid, tartaric acid Etc.
  • carbon sources can be used alone or in combination.
  • the amount of the carbon source added to the medium varies depending on other components in the medium, but is usually 1 to 10% by weight of the medium amount.
  • nitrogen sources include soy flour, bran, falling raw flour, cottonseed flour, casein hydrolyzate, pharmamine, fish meal, corn steep liquor, peptone, meat extract, yeast, yeast extract, malto extract, gelatin, nitric acid Examples thereof include sodium, ammonium nitrate, ammonium sulfate, sodium L-glutamate and the like, and these nitrogen sources can be used alone or in combination.
  • the addition amount of the nitrogen source varies depending on other components in the medium, but is usually in the range of 0.2 to 10% by weight, preferably 0.2 to 6% by weight of the medium amount.
  • the nutrient inorganic salt for example, salts capable of obtaining ions such as sodium ion, potassium ion, ammonium ion, calcium ion, phosphate ion, sulfate ion, chloride ion and carbonate ion are used.
  • salts containing trace amounts of metals such as calcium, cobalt, manganese, magnesium, and molybdenum can also be used.
  • vitamin B1 which can assimilate a microbe, vitamins like biotin, a microbial growth promoting substance like thiamine, etc. as needed.
  • a carbon source, a nitrogen source, a nutrient mineral salt, a cell growth promoting substance, and the like are generally used in combination, but need not be in a pure form, and those having a lower purity may be used.
  • an antifoaming agent such as silicone oil, polyalkylene glycol ether, vegetable oil, animal oil, or surfactant may be added to the medium as necessary. It is particularly preferable to add these antifoaming agents during liquid culture.
  • the pH of the medium varies depending on the components of the medium, the growth temperature, and the like, but may be any pH that is usually used for producing a fermentation product.
  • Lactonochrominin A, B and C can be obtained by aerobically culturing the SANK10207 strain.
  • Examples of such a culture method include a culture method using a solid medium, a shaking culture method, a stirring culture method, and an aeration stirring culture method.
  • the growth temperature of the fungus varies depending on the components of the medium, pH and the like, but is usually 15 ° C. to 30 ° C., and the range of 20 to 28 ° C. is good for growth.
  • 20-26 ° C. is suitable for the production of the lacnochromonins A, B and C.
  • static culture at 20 to 26 ° C. (preferably 23 ° C.) for 10 days or longer.
  • Cultivation is started in a conical flask by a single-stage or multi-stage bacterial growth process.
  • a carbon source, a nitrogen source, a trace amount of growth factor, inorganic salts and trace metals can be used in combination.
  • After adding a sterilized preculture medium to the Erlenmeyer flask and inoculating the SANK10207 strain is it shaken at 20 to 26 ° C. (preferably 23 ° C.) for 5 to 10 days (preferably 7 days) in a constant temperature incubator? By shaking until fully grown.
  • a part or all of the grown preculture solution is used to inoculate a preculture medium or a production culture medium in the next stage.
  • Production culture is started by inoculating the culture medium for production culture in an Erlenmeyer flask containing a sterile production culture medium or a plastic bag for solid culture. Production culture is carried out by shaking the Erlenmeyer flask containing the inoculated production culture medium at a constant temperature for several days or until fully grown. Alternatively, the culture vessel containing the solid medium is allowed to stand at a certain temperature for several days or until it grows sufficiently, and production culture is performed.
  • a suitable jar or tank fermenter equipped with a stirrer and an aeration device.
  • the medium can be prepared in a jar or tank fermenter, heated to 121 ° C., sterilized, and then cooled before use.
  • Cultivation is performed by inoculating a pre-cultured medium in a production culture medium in a jar or tank fermenter and aeration and stirring at 9 to 35 ° C. (preferably 15 ° C. to 33 ° C., optimally 23 ° C.). it can.
  • This method is suitable for obtaining large amounts of compounds.
  • the target compound After completion of the culture, by extracting and purifying using the obtained culture, the filtrate or supernatant obtained by centrifugation of the culture, or filtration, and the cells, or using the whole culture, The target compound can be obtained.
  • the amount of lacnochromonin A, B and C produced as the culture progresses is determined by extracting a part of the culture solution, extracting the compound, performing high performance liquid chromatography, and measuring the compound. Can be monitored. Lactonochromin A, B and C present in the solid culture are extracted with 50 to 100% hydrous acetone or acetone, hydrous methanol or methanol, and the solid part is filtered using diatomaceous earth as a filter aid, or centrifuged. The obtained filtrate can be extracted and purified from the obtained filtrate using high-performance liquid chromatography as an index, utilizing its physicochemical properties.
  • the lacnochromonins A, B and C present in the liquid part of the culture liquid, the fungus body, or both are all cultivated products or the fungus body and other solid parts are removed from the diatomaceous earth. Separation by filtration using a filter aid or centrifugation, and extraction and purification from the obtained filtrate or supernatant and bacterial cells using high-performance liquid chromatography as an index and using their physicochemical properties it can.
  • Lactonochrominins A, B and C present in the filtrate or supernatant are organic solvents that are not miscible with water under neutral or acidic conditions, for example, ethyl acetate, chloroform, ethylene chloride, methylene chloride, butanol alone or Extraction and purification can be performed by a combination thereof.
  • an adsorbent for example, activated carbon or Amberlite XAD-2, XAD-4 (Rohm and Haas Co., Ltd.), which is an adsorption resin, Diaion HP-10, etc.
  • the impurities in the liquid containing the target compound are adsorbed on the adsorbent and removed by obtaining the passing liquid, or the target compound is adsorbed to wash away the impurities, followed by methanol water, acetone water, butanol.
  • the target compound can be extracted and purified by eluting the target compound with water or the like.
  • Lactonochrominins A, B and C present in the microbial cells are obtained by extracting with 50 to 90% aqueous acetone or aqueous methanol, removing the organic solvent by concentration, and then performing extraction and purification in the same manner as above. be able to.
  • the target compound can be extracted by adding an appropriate amount (preferably a final concentration of 50%) of acetone or methanol after completion of the culture. After completion of the extraction, the target compound can be extracted and purified by performing a filtration operation using diatomaceous earth as a filter aid, and performing the same extraction and purification operation as the filtrate.
  • the solution containing the lacnochromonins A, B and C extracted and purified by the above method is silica gel, Sephadex LH-20 (manufactured by Amersham Biosciences), Florisil, Cosmosil (manufactured by Nacalai Tesque). Partition column chromatography using a carrier; Adsorption column chromatography using a carrier such as Diaion HP-20 and CHP20P (Mitsubishi Chemical Corporation); Sephadex G-10 (Amersham Biosciences Co., Ltd.) It can be further purified by gel filtration chromatography using Toyopearl HW40F (Tosoh Co., Ltd.) and the like; and high performance liquid chromatography using normal phase and reverse phase columns.
  • the lacnochromonins A, B and C of the present invention can be separated and purified.
  • the present invention relates to a medicament containing, as an active ingredient, lacnochromonin A, B and C or a pharmaceutically acceptable salt thereof.
  • Lachnochromonins A, B and C have antioxidant activity, antitumor activity, anti-inflammatory activity, antiallergic activity, osteogenesis promoting activity and / or bone resorption inhibiting activity.
  • Reactive oxygen species is a general term for oxygen molecules with unpaired electrons (radicals) in the molecule, or related substances, and it has a very strong oxidizing power and is used to protect against bacteria that have invaded the body. Excessive reactive oxygen species are believed to damage DNA and contribute to aging, carcinogenesis, lifestyle-related diseases and the like. The function of suppressing the excessive generation of reactive oxygen species also exists in the living body, but the function decreases with aging. Antioxidants have a function to suppress the generation of reactive oxygen species, and are expected to prevent aging, carcinogenesis, lifestyle-related diseases and the like.
  • Inflammatory action is a protective immune response against harmful events such as invasion of foreign bodies and damage caused by it, but excessive expression of inflammatory mediators such as inflammatory cytokines and nitric oxide (NO)
  • inflammatory mediators such as inflammatory cytokines and nitric oxide (NO)
  • TNF- ⁇ tumor necrosis factor
  • NO nitric oxide
  • NO nitric oxide
  • NO nitric oxide
  • NO nitric oxide
  • NO nitric oxide
  • NO nitric oxide
  • TNF- ⁇ tumor necrosis factor
  • NO is used to protect against the invasion of pathogens and viruses and the damage caused by it, but excessive NO damages cells and causes carcinogenesis and worsening of inflammatory diseases.
  • NO reacts with O 2 radicals in the living body and changes to peroxynitrite (ONOO-) having stronger cytotoxic activity.
  • CSAIDs cytokine-suppressed anti-inflammatory drugs
  • An allergic reaction is also an excessive immune reaction, which is classified from type I to type V according to the mechanism of its occurrence, among which hay fever and atopic dermatitis are classified as type I.
  • type I allergy a foreign substance serving as an antigen binds to IgE, which is a kind of immunoglobulin bound to mast cells and basophils, whereby physiologically active substances such as histamine are released from these cells.
  • IgE is a kind of immunoglobulin bound to mast cells and basophils, whereby physiologically active substances such as histamine are released from these cells.
  • physiologically active substances cause vasodilation and increased permeability, and symptoms such as edema and pruritus appear. Therefore, if the release of histamine and the like from mast cells and its action can be suppressed, it can be expected that the onset of type I allergy will be alleviated / reduced.
  • Osteoporosis is a disease in which the balance of bone metabolism is lost, bone formation and bone resorption increase, bone mass and bone quality decrease, and fracture risk increases. Osteoporosis is broadly divided into primary osteoporosis caused by aging and decreased estrogen due to menopause, and secondary osteoporosis caused by rheumatoid arthritis, hyperparathyroidism, and steroids and immunosuppressive drugs.
  • the Candidate substances for osteoporosis therapeutics include substances that promote bone formation or substances that suppress enhanced bone resorption, and the compounds of the present invention have such activity.
  • the medicament containing the compound of the present invention is useful as a therapeutic or prophylactic agent for cancer, allergy, inflammation, osteoporosis, lifestyle-related diseases and / or adult diseases.
  • the present invention also relates to a method for treating or preventing cancer, allergy, osteoporosis, lifestyle-related diseases and / or adult diseases by administering the medicament.
  • the present invention relates to the use of lacnochromonin A, B and C or a pharmaceutically acceptable salt thereof for the manufacture of a therapeutic or prophylactic agent for cancer, allergy, osteoporosis, lifestyle-related diseases and / or adult diseases.
  • the lacnochromonins A, B and C of the present invention or pharmacologically acceptable salts thereof can be administered in various forms.
  • the administration form include oral administration by tablets, capsules, granules, emulsions, pills, powders, syrups (solutions), etc., or injections (intravenous, intramuscular, subcutaneous or intraperitoneal administration), Examples include parenteral administration such as instillation and suppository (rectal administration).
  • These various preparations are usually used in the pharmaceutical preparation technical field such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents, coating agents, etc. as main ingredients in accordance with conventional methods. It can be formulated with the resulting adjuvant.
  • excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid; water, ethanol, propanol, simple syrup, glucose Solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc .; dried starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid Disintegrators such as esters, sodium lauryl sulfate, monoglyceride stearate, starch, lactose; disintegrators such as sucrose, stearin, cocoa butter, hydrogenated oil; quaternary ammonium salts, sodium lauryl sulfate Moisturizers such as glycerin and starch; Adsorbents such as starch
  • the tablet which gave the normal coating for example, a sugar-coated tablet, a gelatin-encapsulated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet, and a multilayer tablet.
  • excipients such as glucose, lactose, cocoa butter, starch, hydrogenated vegetable oil, kaolin, talc; binders such as gum arabic powder, tragacanth powder, gelatin, ethanol; laminaran agar Disintegrants such as can be used.
  • a carrier conventionally known in this field can be widely used as a carrier, and examples thereof include polyethylene glycol, cocoa butter, higher alcohol, esters of higher alcohol, gelatin, semi-synthetic glyceride and the like.
  • solutions, emulsions or suspensions When used as an injection, it can be used as a solution, emulsion or suspension. These solutions, emulsions or suspensions are preferably sterilized and isotonic with blood.
  • the solution used for the production of these solutions, emulsions or suspensions is not particularly limited as long as it can be used as a diluent for medical use.
  • water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isoforms are used. Examples include stearyl alcohol and polyoxyethylene sorbitan fatty acid esters.
  • a sufficient amount of sodium chloride, glucose or glycerin may be included in the preparation to prepare an isotonic solution, and a normal solubilizing agent, buffer, soothing agent, etc. may be included. You may go out.
  • the above-mentioned preparation may contain a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetening agent, and the like as required, and may further contain other medicines.
  • the amount of the active ingredient compound contained in the preparation is not particularly limited and is appropriately selected within a wide range, but is usually 170% by weight, preferably 1 to 30% by weight, based on the total composition.
  • the amount used varies depending on symptoms, age, body weight, administration method, dosage form, etc., but is usually 2000 mg (preferably 100 mg) as an upper limit per day for adults, and 0.1 mg (preferably as the lower limit) 1 mg, more preferably 10 mg) can be administered once a day or divided into several times according to symptoms.
  • a novel compound having an antioxidant activity, an antitumor activity, an anti-inflammatory activity, an antiallergic activity, an osteogenesis promoting activity, and / or a bone resorption inhibiting activity is provided.
  • Example 1 Cultivation of SANK10207 strain (1) Preparation of frozen seed The hyphae of SANK10207 strain grown on a PDA slant medium shown in Table 3 to be described later was scraped together with agar using platinum ears, and physiological saline Suspended in The above-mentioned bacterial suspension was aseptically inoculated into a 100 mL Erlenmeyer flask containing 20 mL of the medium shown in Table 4 which will be sterilized at 121 ° C. for 30 minutes. In seed culture, the culture was shaken on a rotary shaker at 23 ° C. and 210 rpm for 6 days.
  • Glucose and agar were added to this potato boiled liquid, and finally adjusted to 1000 mL with tap water. pH: No adjustment. Sterilization: Sterilized at 121 ° C. for 20 minutes.
  • Composition of seed culture and preculture medium ⁇ Glycerin 30g Glucose 30g 20g soluble starch 10g soy flour Gelatin 2.5g Yeast extract 2.5g NH 4 NO 3 2.5g Agar 3g Antifoaming agent (CB442) 0.1g ⁇
  • Tap water was added to the above components so that the final volume was 1000 mL. pH: adjusted to about 6.5 with 25% aqueous sodium hydroxide solution.
  • the suspension was allowed to stand for 24 hours, Celite 545 (22.7 kg) was added, and the mixture was filtered with a filter press (manual press type filter press TFP-310-8, manufactured by Tokyo Engineering Industry Co., Ltd.).
  • the obtained acetone extract (990 L) was concentrated under reduced pressure, and acetone was distilled off.
  • the pH of the concentrated liquid (550 L) was adjusted to 2.9 with 75% concentrated sulfuric acid (260 mL), and then extraction was performed by adding ethyl acetate (250 L).
  • the aqueous layer after fractionation was extracted again by adding ethyl acetate (250 L), and the organic layers obtained by the two extraction operations were combined.
  • This organic layer (530 L) was washed with 0.2 M aqueous solution of disodium hydrogenphosphate (150 L), and further washed with 25% brine (100 L) to obtain a washed organic layer (500 L).
  • the washed organic layer was concentrated under reduced pressure, and anhydrous sodium sulfate (about 1 kg) was added to the resulting concentrated liquid (27.5 L), followed by dehydration for about 2 hours. After removing anhydrous sodium sulfate by filtration, the filtrate was concentrated to dryness under reduced pressure to obtain an oily substance (450 g).
  • DPPH radical elimination rate measurement test DPPH radical is a relatively stable radical, and has a maximum absorption at 517 nm. Can be evaluated. Specifically, DPPH and a test substance are mixed and reacted in a solvent, and then the absorbance at 517 nm is measured. The DPPH radical elimination rate by the test substance can be calculated by the following method, and the higher the elimination rate, the higher the test substance has a higher antioxidant effect.
  • DPPH radical scavenging rate (AB) / (CD)
  • Active oxygen removal active xanthine oxidase, its substrate hypoxanthine and test substance are mixed and reacted in a phosphate buffer, and the generated active oxygen is measured to thereby reduce the active oxygen removal effect of the test substance. Can be evaluated.
  • a luminescent method using a luminescent reagent or 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) or N-tert-butyl- ⁇ -phenylnitrone (PBN) as a radical scavenger is used.
  • DMPO 5,5-dimethyl-1-pyrroline-N-oxide
  • PBN N-tert-butyl- ⁇ -phenylnitrone
  • ESR electron spin resonance
  • Cancer cell lines used in this test are shown below, but the cells used in the test of anti-tumor activity of the novel compounds of the present invention, lacnochromonins A, B and C are limited to these. Is not to be done. Cancer cell lines used in the test: HeLa (cervical cancer), HGC-27 (gastric cancer), MCF-7 (breast cancer), HepG2 (hepatocellular carcinoma), U937 (leukemia cancer), CACO2 (colon cancer) More specifically, the anticancer activity of the novel compounds of the present invention, lacnochromonins A, B and C can be evaluated by the following method.
  • Cell growth inhibition test Various cancer cells are seeded in a 96-well microplate, and after adding a test substance, they are cultured for several hours to several days.
  • Cell growth inhibitory activity includes methods for measuring mitochondrial dehydrogenase activity such as 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazole bromide (MTT) method, tritium-labeled thymidine, etc. Can be evaluated by a method of measuring the amount of incorporation into DNA. By setting the cell growth (control) of the test substance-free group to 100%, the cell growth rate when the test substance is added can be calculated, and the cancer cell growth inhibitory activity of the test substance can be evaluated.
  • mitochondrial dehydrogenase activity such as 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazole bromide (MTT) method, tritium-labeled thymidine, etc.
  • Apoptosis-inducing activity test a) Detection of DNA ladder A test substance is added to various cancer cells and incubated at 37 ° C. for several minutes to several hours. Adherent cells are washed as they are with phosphate buffered saline (PBS), and floating cells are collected by centrifugation and then washed with PBS. Each cell lysate contains a surfactant such as Triton-X100. Lyse the cells with RNA and protein are degraded using RNase, proteinase K, etc., 5M saline and isopropanol are added, and the mixture is allowed to stand overnight at ⁇ 20 ° C. to precipitate DNA.
  • PBS phosphate buffered saline
  • the DNA precipitated by centrifugation is precipitated, redissolved with Tris-ethylenediaminetetraacetic acid buffer (TE buffer), and then subjected to agarose gel electrophoresis. After electrophoresis, DNA is developed with a fluorescent reagent, and the apoptosis-inducing activity of cancer cells by the test substance can be evaluated by evaluating the generation of a DNA ladder using an imaging analyzer or the like.
  • a test substance is added to various cancer cells and incubated at 37 ° C. for several minutes to several hours.
  • a Hoechst 33342 solution (Calbiochem; final concentration 0.1 ⁇ g / mL) is added, and the mixture is further incubated for 2 hours, and observed with a fluorescence microscope (Ultra violet: excitation light 330-380 nm, observation light 420 nm). By observing granular fluorescence accompanying chromatin aggregation, the chromatin aggregation promoting activity of the test substance can be evaluated.
  • the anti-inflammatory activity of the novel compounds of the present invention can be evaluated by inflammatory cytokine and nitric oxide (NO) production inhibition tests.
  • inflammatory cytokine and nitric oxide (NO) production inhibition tests An example of the test method is given below.
  • IL-6 production inhibitory activity of Lactonochromonin A was measured under the conditions of a DMEM medium as a medium, a cell number at the time of adjustment of 1 ⁇ 10 5 cells / mL, and a culture period of 3 days after addition of the test compound. Suppression rate was 16.6% in ml, 30% at 40 ng / ml, 38.7% at 120 ng / ml.
  • the inhibition rate of IL-6 production was calculated by a calculation formula for the inhibition rate of cytokine production described later. ⁇ 3-2. NO production suppression> Regarding NO production, the Grease method (L. J. Ignarro, et.al., (1987), Endothelium-derived relaxed factor produced and released from fragile and vein is nitride, US. C. .84: pp. 9265-9269).
  • the antiallergic activity of the novel compounds of the present invention, lacnochromonins A, B and C can be evaluated by a histamine release inhibition test or the like.
  • An example of the test method is given below. After Wistar male rats are decapitated and bled, 10 mL of Hank's solution containing 10 U / mL heparin is injected into the abdominal cavity, massaged well, and the intraperitoneal fluid is collected. The obtained intraperitoneal fluid is overlaid on a 40% Ficoll solution, left at room temperature for 30 minutes, and then centrifuged to collect mast cells on the Ficoll layer.
  • the rat peritoneal mast cells can be obtained by suspending the mast cells in PBS, repeating washing by centrifugation three or four times, and then suspending again in PBS.
  • Rat peritoneal mast cells are adjusted to 2 ⁇ 10 6 cells / mL and 1.8 mL / tube with PBS and allowed to stand at 37 ° C. for 10 minutes.
  • 0.1 mL of a test substance is added, and further 0.1 mL of 10 ⁇ g / mL compound 48/80 (manufactured by Wako Pure Chemical Industries, Ltd.) is added and reacted for 10 minutes. Stop the reaction by cooling with ice and centrifuge to prepare the supernatant and sediment.
  • the histamine release rate from rat peritoneal mast cells can be calculated by the following formula.
  • Test Example 5 Osteogenesis Promoting Activity The osteogenesis promoting activity of the novel compounds of the present invention, lacnochromonins A, B and C can be evaluated by the following osteoblast differentiation test. Alkaline phosphatase (ALP) is an osteoblast differentiation marker.
  • ALP Alkaline phosphatase
  • the osteoblast differentiation promoting activity can be evaluated by obtaining an increase rate of osteoblast ALP activity. More specifically, the rate of increase in ALP activity of osteoblasts can be evaluated by the following method.
  • a mouse bone marrow-derived osteoblast cell line, ST2 (RIKEN Cell Bank, RCB0224) was suspended in ⁇ -MEM medium (hereinafter abbreviated as ⁇ MEM) containing 10% fetal bovine serum, and this suspension was suspended in 4,000 cells / well. Then, dispense 100 ⁇ L each into a 96-well microplate and incubate in a CO 2 incubator for 24 hours.
  • ALP activity increase rate (%) absorbance value when sample is added / absorbance value when sample is not added ⁇ 100.
  • Bone resorption inhibitory activity Tartrate-resistant acid phosphatase is a marker of osteoclast differentiation, and the inhibitory activity of osteoclast differentiation is evaluated by determining the TRAP activity inhibition rate of the test substance. I can do it. More specifically, the TRAP activity inhibition rate can be evaluated by the following test. Ddy mice are euthanized by cervical dislocation and the spleen is removed. Spleen cells are collected from the removed spleen, and cell pellets are collected by centrifugation, and then erythrocytes are destroyed with NH 4 Cl-containing Tris-HCl buffer (erythrocyte disruption solution).
  • the erythrocyte destruction solution is removed by centrifugation, and the cells are suspended in ⁇ MEM. After washing with ⁇ MEM twice by centrifugation, the cells can be resuspended in ⁇ MEM to obtain a mouse spleen cell suspension.
  • the ⁇ MEM to which the test substance is added is dispensed into a 96-well plate at 100 ⁇ L / well.
  • Mouse spleen cells (4 ⁇ 10 5 cells / 50 ⁇ L / well) and ST2 cells (10 4 cells / 50 ⁇ L / well) were mixed with 2 ⁇ 10 ⁇ 8 M active vitamin D 3 , 2 ⁇ 10 ⁇ 7 M. Seed suspended in ⁇ MEM with dexamethasone.
  • TRAP activity inhibition rate (%) (1 ⁇ absorbance value when sample is added / absorbance value when sample is not added) ⁇ 100.
  • the compounds of the present invention are useful as therapeutic and / or ameliorating agents for inflammation, lifestyle-related diseases or adult diseases.

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Abstract

Cette invention concerne un nouveau composé ayant une activité prophylactique et/ou thérapeutique sur des maladies inflammatoires, des maladies en rapport avec le mode de vie des patients et des maladies touchant l'adulte. L'invention concerne en particulier : un composé de formule générale (I) [R1 représente CH3; R2 représente OH; R3 représente H; R4 représente CH3 ; et R5 représente CH(CH3)(CH2)2OH, CH(CH3)CH(OH)CH3 ou CH(CH3)CH2CH3]; entre autres.
PCT/JP2009/052275 2008-02-13 2009-02-12 Nouveau composé, composé de lachnochromonin WO2009101959A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102250778A (zh) * 2011-07-01 2011-11-23 合肥工业大学 一种粒毛盘菌及其胞外多糖和在降脂利肝药物中的用途
CN102442985A (zh) * 2010-09-29 2012-05-09 北京大学 一种具有抑制肿瘤细胞生殖生长活性的天然化合物p71及其应用
CN102746705A (zh) * 2012-06-28 2012-10-24 合肥工业大学 一种将粒毛盘菌胞外黑色素改性为水溶性黑色素的方法
WO2013111850A1 (fr) 2012-01-26 2013-08-01 第一三共株式会社 Dérivé de chromone présentant un effet favorisant l'ostéogénèse
WO2014030688A1 (fr) * 2012-08-24 2014-02-27 第一三共株式会社 Chromone à chaîne latérale linéaire
CN104561137A (zh) * 2015-01-15 2015-04-29 合肥工业大学 一种粒毛盘菌液体发酵产多酚的方法
CN116239557A (zh) * 2022-12-13 2023-06-09 广东轻工职业技术学院 含有7-羟基色原酮结构的化合物及其制备方法与应用

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WO2007018194A1 (fr) * 2005-08-09 2007-02-15 Daiichi Sankyo Company, Limited Procédé pour la production de cercosporamide

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WO2007018194A1 (fr) * 2005-08-09 2007-02-15 Daiichi Sankyo Company, Limited Procédé pour la production de cercosporamide

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102442985A (zh) * 2010-09-29 2012-05-09 北京大学 一种具有抑制肿瘤细胞生殖生长活性的天然化合物p71及其应用
CN102250778A (zh) * 2011-07-01 2011-11-23 合肥工业大学 一种粒毛盘菌及其胞外多糖和在降脂利肝药物中的用途
WO2013111850A1 (fr) 2012-01-26 2013-08-01 第一三共株式会社 Dérivé de chromone présentant un effet favorisant l'ostéogénèse
US9221811B2 (en) 2012-01-26 2015-12-29 Daiichi Sankyo Company, Limited Chromone derivative having osteogenesis promoting effect
CN102746705A (zh) * 2012-06-28 2012-10-24 合肥工业大学 一种将粒毛盘菌胞外黑色素改性为水溶性黑色素的方法
WO2014030688A1 (fr) * 2012-08-24 2014-02-27 第一三共株式会社 Chromone à chaîne latérale linéaire
CN104561137A (zh) * 2015-01-15 2015-04-29 合肥工业大学 一种粒毛盘菌液体发酵产多酚的方法
CN116239557A (zh) * 2022-12-13 2023-06-09 广东轻工职业技术学院 含有7-羟基色原酮结构的化合物及其制备方法与应用

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