WO2004065607A1 - Nouvelle substance physiologiquement active rs-k3574 et son procede de production - Google Patents

Nouvelle substance physiologiquement active rs-k3574 et son procede de production Download PDF

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WO2004065607A1
WO2004065607A1 PCT/JP2003/000532 JP0300532W WO2004065607A1 WO 2004065607 A1 WO2004065607 A1 WO 2004065607A1 JP 0300532 W JP0300532 W JP 0300532W WO 2004065607 A1 WO2004065607 A1 WO 2004065607A1
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physiologically active
substance
active substance
activity
cells
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PCT/JP2003/000532
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English (en)
Japanese (ja)
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Tomio Takeuchi
Hironobu Iinuma
Ryuichi Sekizawa
Susumu Matsui
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Zaidan Hojin Biseibutsu Kagaku Kenkyu Kai
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Priority to AU2003303775A priority Critical patent/AU2003303775A1/en
Priority to JP2004567132A priority patent/JP4376189B2/ja
Priority to PCT/JP2003/000532 priority patent/WO2004065607A1/fr
Publication of WO2004065607A1 publication Critical patent/WO2004065607A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
    • C07D303/02—Compounds containing oxirane rings
    • C07D303/12—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms
    • C07D303/32—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms by aldehydo- or ketonic radicals
    • 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
    • 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
    • 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 has an inhibitory activity on ubiquitin activating enzyme and an inhibitory activity on ubiquitination of intracellular proteins, has an activity of inhibiting biosynthesis of intracellular proteins, and has an antitumor or antitumor activity.
  • the present invention relates to a novel physiologically active substance having cancer activity, RS-K3574.
  • the present invention also relates to a method for producing the above-mentioned physiologically active substance, RS-K3574 substance.
  • the present invention relates to an inhibitor of ubiquitin activating enzyme consisting of a physiologically active substance; RS-K3574, and an inhibitor of ubiquitination of intracellular protein consisting of a physiologically active substance RS-K3574.
  • the present invention relates to an antitumor agent composition, an antiinflammatory agent composition and an antiviral agent composition containing the substance HS-K3574 as an active ingredient. Further, the present invention provides a novel bioactive substance RS-K3574, which is an apoptosis-inducing effect of a cell-killing antitumor agent or anticancer agent for cancer cells or radiation for cancer treatment, and inhibits protein biosynthesis in cancer cells. And an inhibitor of the activity of NF- ⁇ transcription factor.
  • Neurological diseases including Alzheimer's disease occur when cell proliferation or cell death occurs in cancer cells or immune cells, when acute or chronic inflammation occurs, or when immune responses such as immune hypersensitivity or immunodeficiency occur.
  • One of the mechanisms that play a crucial and essential role during normal differentiation such as during infection and propagation of pathogenic viruses, including HIV viruses, and also during fertilization and development of eggs.
  • Ubiquitin It is known that there is a mechanism for ubiquitination of intracellular proteins, which involves the enzyme to be converted.
  • ubiquitination is involved in the budding, maturation, and acquisition of infectivity of these HIV viruses.
  • ubiquitin activating enzyme an enzyme present in cells, is involved in ubiquitin activation.
  • the limited degradation of HIV-derived ubiquitinated proteins can be stopped, and the HIV virus can sprout, mature, and acquire infectivity. It is known that the growth can be inhibited [PNAS, Vol. 97, No. 24, 13057-: 13062 (November, 2000)].
  • Panepoxydone represented by the following formula is known: [Biochemical and Biophysical Research Communications. 226: 214-221 (1996)]. This document describes that panepoxydone has the activity to inhibit the activation of NF.KB transcription factor involved in inflammation, but panepoxydone produces intracellular protein, RNA or DNA. It is described that it does not show activity inhibiting synthesis.
  • a study of the stereochemical structure of the panepoxydone molecule is described in Helvetica Chimica Acta, 53, Fasc. 7 (1970), No. 186, 1577-, p. 1597; According to the description in Table 2 and 1579 pages pages, Panepokishidon the specific rotation [shed] D 20 - 61 ° (solvent, dichloromethane), a viscous oil of pale yellow indicating the.
  • ubiquitination of proteins in cells is an important biochemical reaction, but substances with the activity to directly inhibit ubiquitination of proteins in cells are currently not available. There is still a need for new substances with such activity, as they have not yet been discovered. If new substances with the ability to inhibit the mechanism of ubiquitination of intracellular proteins could be provided, such new substances would be known anti-tumor tumors known or used in the past.
  • the present inventors have a novel activity having an inhibitory activity on intracellular ubiquitin activating enzyme and an inhibitory activity on intracellular protein ubiquitination that can meet the above-mentioned demands, and also have an antitumor activity and an antiviral activity.
  • a strain of the genus Aratake mushroom which belongs to the genus Pleurotus sp., Produces bioactive substances with a new structural skeleton.
  • this RS-K3574 substance is represented by the following formula (I)
  • the bioactive substance RS-K3574 of the present invention is recognized as a new stereoisomer (7-epimani) of the known substance panepoxydone.
  • the inhibitory activity of the RS-K3574 substance on ubiquitin activity was measured according to the method described in J. Nat. Prod., Vol. 65, pp. 1491-1493 (2002). That is, human-derived ubiquitin activating enzyme was expressed in Escherichia coli by recombinant genetic engineering, and a ubiquitin activating enzyme sample was prepared by collecting and purifying the ubiquitin activating enzyme from the E. coli. Piotinylated ubiquitin prepared by biotinylation of ubiquitin derived from pest was used as a substrate.
  • the ubiquitin activating enzyme and the substrate were reacted with ATP (adenosine-1,3-phosphate) for 15 minutes at 3TC in the presence or absence of the test RS-K3574 substance.
  • ATP adenosine-1,3-phosphate
  • the resulting reaction solution was subjected to polyacrylamide gel electrophoresis, and the fractionated protein in the gel, which was bound to the ubiquitin bitionate, was adsorbed on the polyvinylidene difluoride membrane by elect opening plot. .
  • the amount of the enzyme protein bound to the biotinylated ubiquitin on the membrane was assayed using the ECL method (see “Clin. Chem.j, Vol. 25, pp.
  • the inhibitory activity of the physiologically active substance RS-K3574 according to the present invention on protein ubiquitination in cells completely inhibits the production of ubiquitinated proteins induced in cells by RS-K3574 substance at a concentration of 2 ⁇ g / ml or more. It has the strength to do.
  • ubiquitinated protein in the cells was detected as follows. That is, RS-K3574 substance was added in various concentrations to a medium containing human breast cancer cells MCF7 in advance. Thirty minutes after the addition, MG-132, an inhibitor of proteasomes in cancer cells, was further added to the medium at a concentration of 1 M. (The proteasome was completely added with MG-132 added to the medium.) Inhibition, which allows the accumulation of ubiquitinated proteins in the cells to be degraded by the proteasome). Then, after culturing the cancer cells in the medium for 3 hours, the cancer cells were dissolved. The obtained cell soluble fraction was subjected to polyacrylamide gel electrophoresis.
  • the fractionated protein in the gel is adsorbed onto a polyvinylidene difluoride membrane by electroprotocol, and the adsorbed ubiquitinated protein on this membrane is reacted with an anti-ubiquitin antibody. Selectively detected.
  • the ECL method see “Clin. Chem.j, Vol. 25, 1531-: p. 1546 (1979)" was used.
  • the test was performed in the same manner as described above, without adding the RS-K3574 substance to the medium containing human breast cancer cells MCF7.
  • the absence of RS-K3574 When the test was conducted under the above conditions, the ubiquitinated protein was accumulated, but when the test was performed in the presence of the RS-K3574 substance added to the above medium, the presence of the RS-K3574 substance was observed. It was confirmed that the accumulation of ubiquitinated protein was suppressed depending on the concentration. As is evident from this, HS-K3574 is effective in suppressing the production of functional intracellular proteins whose properties are directly or indirectly controlled by being ubiquitinated in cells. .
  • the physiologically active substance RS-K3574 according to the present invention has an activity of suppressing the growth of cancer or tumor cells.
  • concentration of the RS-K3574 substance that inhibits the growth of various cancer cells by 50% was measured by the MTT method (see Journal of Immunological Methods, Vol. 65, pp. 55-60 (1983)).
  • IC 50 value The concentration of the RS-K3574 substance that inhibits the growth of various cancer cells by 50%.
  • the physiologically active substance RS-K3574 according to the present invention has an antitumor activity that suppresses the growth of various cancer cells, and is therefore useful as an antitumor agent. .
  • the physiologically active substance RS-K3574 according to the present invention has an anticancer activity.
  • the RS-K3574 substance has a life-prolonging effect on mice transplanted with, for example, Ehrlich ascites cancer.
  • RS-K3574 substance was administered to these tumor-bearing mice at a dose of 250 ⁇ g / mouse per day for 9 consecutive days, the life-span effect was longer than when ES-K3574 substance was not administered. Reaches 200%. This life extension effect was determined as follows.
  • the physiologically active substance RS-K3574 has an inhibitory effect on the malignant transformation of cancer or tumor in a living body.
  • the physiologically active substance RS-K3574 enhances the apoptosis-inducing effect of the antitumor agent, the anticancer agent, or the radiation for cancer treatment on cancer cells, and is irradiated with the antitumor agent, the anticancer agent, or the radiation for cancer treatment.
  • it has an activity of increasing the antitumor activity or anticancer activity of ⁇ -rays, X-rays, and ⁇ -rays.
  • the number of viable HeLa cells can be reduced by simultaneously adding the KS-K3574 substance. Can be significantly reduced. That is, while the number of viable cells when HeLa cells were treated with 1 j glml of adriamicin for 24 hours was 100%, 5 g / ml of the RS-K3574 substance and 1 ⁇ g / ml of adriamicin were simultaneously used. When HeLa cells are treated, the number of surviving cells is reduced to 50%.
  • the physiologically active substance RS-K3574 has an activity of enhancing the apoptosis-inducing action of a cell-killing antitumor agent or an anticancer agent on cancer cells.
  • the antitumor effect of the anticancer agent is increased. It also has the effect of similarly enhancing cancer cell apoptosis due to radiation irradiated for cancer treatment.
  • RS-K3574 substance has. Therefore, it can be used as an anti-tumor agent or anti-cancer agent or as an enhancer of cancer cell apoptosis-inducing action of radiation irradiated for cancer treatment, and as a concomitant agent for enhancing the therapeutic effect of anti-tumor agent or anti-cancer agent in cancer treatment.
  • the K3574 substance is useful.
  • the RS-K3574 substance of the present invention has an activity of remarkably and specifically inhibiting intracellular protein biosynthesis. Its inhibitory activity is such that the 50% inhibitory concentration of the RS-K3574 substance is 4.5 ⁇ g / ml. The inhibitory activity was measured as follows.
  • the measured radioactivity of the resulting solution indicates the amount of protein biosynthesized in the cell.
  • the measured value of radioactivity was 180 cpm.
  • the concentration of RS-K3574 substance that inhibits protein biosynthesis in MCP cells 50% was calculated to be ⁇ ⁇ / ⁇ .
  • the physiologically active substance RS-K3574 has an activity to completely inhibit the activity of NF- ⁇ transcription factor at a concentration of 4 Ug / ml. This activity was determined as follows.
  • NF.KB transcription factor is observed when TNF acting as an inflammatory cytokine at a concentration of 10 ng / ml or Interleukin-l (IL-1) at a concentration of 2 ng / ml is added to the culture medium of human breast cancer cells MCF7. Activation of this NF.KB transcription factor is observed. Activation of this NF.KB transcription factor is caused by a specific increase in the mRNA of MAD-3 (Cell, vol. 65, pp. 1281-1289, 1991), a gene that is expressed with the activation. Can be confirmed by measuring with the RT-PCII method.
  • the RS-K3574 substance has an activity of inhibiting the activation of NF.KB transcription factor necessary for inducing acute and chronic inflammation, and is useful as an anti-inflammatory agent. Furthermore, according to the second present invention, a P.
  • a bacterium producing RS-K3574 which can be used in the second method of the present invention
  • a bacterium producing RS-K3574 which can be used in the second method of the present invention
  • oyster mushroom genus Aragueka (Emm ⁇ mdk) strain K-3574 was obtained from a fungus hypha obtained from the Fermentation Research Institute (IFO) more than 10 years ago from July 2001, and the potato 'dextrose and agar was used for more than 10 years from that time. It is a strain that has been subcultured on a culture medium at a laboratory of Calagari Co., Ltd.
  • the strain K-3574 is mutated during the passage, and the production amount of the physiologically active substance RS-K3574 is higher than that of the Agarage moss (Panus rudis) IFO 8994 strain deposited at the Institute of Fermentation (IFO). And the composition of other metabolites is also different. From this, this strain K-3574 is a different strain from Panus rudis IFO 8994 strain.
  • Allageka mushroom (Panus rudis) K-3574 strain was deposited at the National Institute of Advanced Industrial Science and Technology, National Institute of Advanced Industrial Science and Technology at 1-1 1-1 Tsukuba-Higashi, Ibaraki, Japan June 2001 An application for deposit was made on the 26th, and the deposit was received under deposit number FERM P-18397. Also, on the date of the transfer acceptance on December 25, 2002, under the provisions of the Budapest Treaty, the Aragageka mushroom K-3574 strain was deposited at the above depository under the accession number of FEEM BP-8265.
  • Aratakeka mushrooms are known mushrooms (basidiomycetes) and are distributed almost all over the world, but they occur relatively commonly in beech trees in Japan.
  • the mushroom mycological characteristics of Kiagega are as follows.
  • the umbrella of the fruiting body is 1.5-5cm in diameter, initially bun-shaped, later opened to form a slightly funnel-shaped, tough fleshy-slightly leathery, with coarse hair on the surface, initially brownish purple, and sometimes purple.
  • the edges are almost flat, the handle is generally short (0.5-2 cm), eccentric to central, rarely lateral, and the surface is almost the same as an umbrella.
  • the spore print is white.
  • the spores are 4.5 ⁇ 5.5 ⁇ 2 ⁇ 2.5 ⁇ , spheroidal, and the basidium is four spores.
  • Thick-membrane cystizia are 53-70 X 9.5-; 14 ⁇ , club-column-shaped, or spindle-shaped. There is no hyphal peg.
  • the hypha composition of the meat tissue is dimitic ("Primary Color Japanese New Fungi Guidebook (1)"), p. 32, Rokuya Imaseki, Tsuguo Hongo, Nursery, June 30, 1987.
  • the above-mentioned Aratake Kazukitake K-3574 strain was inoculated into a nutrient medium as a strain producing a physiologically active substance RS-K3574 belonging to the genus Pleurotus edulis, and cultured. I do.
  • the nutrient medium used here is assimilated by the aforementioned production strain. It contains carbon and nitrogen sources as nutrients.
  • nutrient source of the medium those commonly used as nutrient sources for basidiomycetes strains, for example, assimilable nutrient sources such as carbon source, nitrogen source and inorganic salts can be used.
  • assimilable nutrient sources such as carbon source, nitrogen source and inorganic salts
  • carbohydrates such as glucose, maltose, molasses, dextrin, glycerin, and starch
  • carbon sources such as soybean oil, peanut oil, and other fats, as well as peptone, meat extract, cottonseed powder, soybean powder, yeast extract, and casein.
  • Nitrogen sources such as corn 'steep' liqueur, NZ-amine, ammonium sulfate, ammonium nitrate, ammonium chloride, etc., as well as potassium nitrate, sodium phosphate, salt, calcium carbonate, magnesium sulfate, manganese chloride, etc.
  • Inorganic salts can be blended. If necessary, trace metals such as cobalt and iron can be added.
  • any known nutrient source can be used as long as the RS-K3574 producing strain used for producing the physiologically active substance RS-K3574 can be used.
  • the mixing ratio of the above-mentioned nutrients in the medium is not particularly limited, and can be varied over a wide range. Depending on the RS-K3574 substance producing strain used, the optimum composition of the nutrients and the mixing ratio are as follows. However, it can be easily determined by a person concerned with a simple small-scale experiment.
  • the nutrient medium consisting of the above-mentioned nutrients can be sterilized prior to cultivation, and before or after this sterilization, the pH of the medium is in the range of 5 to 7, especially in the range of pH 5.5 to 6.5. It is advantageous to adjust
  • Cultivation of the RS-K3574 substance-producing strain in such a nutrient medium can be carried out according to a method generally used in the production of a physiologically active substance using common mushrooms. Usually, it is preferable to culture under aerobic conditions. Usually, the culture can be performed with stirring and / or aeration. As the culture method, any of stationary culture, shaking culture, and liquid culture with aeration and stirring can be used, but liquid culture is suitable for mass production of RS-K3574 substance.
  • the culture temperature that can be used is not particularly limited as long as the growth of the RS-K3574 substance producing strain is not substantially inhibited and the physiologically active substance RS-K3574 can be produced.
  • the culture temperature in the range of 25 to 30 ° C. is particularly preferred. Culture can usually be continued until the RS-K3574 substance has sufficiently accumulated.
  • the culture time depends on the composition of the medium, the culture temperature,
  • the target RS-K3574 substance can usually be obtained by culturing for 14 to 30 days, depending on the temperature used, the production strain used, etc.
  • the accumulated amount of the physiologically active substance RS-K3574 during the culture can be quantified by the above-described method for measuring the inhibitory activity of ubiquitin activating enzyme.
  • the RS-K3574 substance accumulated in the culture obtained by the culture is collected from the culture. After the culture, if necessary, the bacterial cells are removed by a known separation method such as filtration or centrifugation, and the culture filtrate is subjected to solvent extraction using an organic solvent, particularly butyl acetate, etc., adsorption,
  • the RS-K3574 substance can be isolated, further purified, and collected by treating the ion-exchange chromatography, gel filtration, or chromatography using countercurrent distribution alone or in combination. .
  • Activated carbon, silica gel, porous polystyrene-divinyl benzene resin or various ion exchange resins can be used as a carrier used for adsorption or a carrier used for mouth chromatography having ion exchange ability.
  • a novel physiologically active substance RS-K3574 having the aforementioned properties can be obtained.
  • the third present invention provides an inhibitor of a ubiquitin activating enzyme, comprising the physiologically active substance represented by the above formula (I): RS-K3574.
  • an inhibitor of ubiquitination of an intracellular protein comprising the physiologically active substance RS-K3574.
  • an antitumor agent composition comprising the above-mentioned physiologically active substance HS-K3574 as an active ingredient, and being mixed therewith and containing a pharmaceutically acceptable carrier.
  • an anti-inflammatory composition comprising the above-mentioned physiologically active substance RS-K3574 as an active ingredient, and a pharmaceutically acceptable carrier mixed therewith.
  • an antiviral composition comprising the above-mentioned physiologically active substance RS-K3574 as an active ingredient, and a pharmaceutically acceptable carrier mixed therewith.
  • the third inhibitor of the ubiquitin activating enzyme according to the present invention and the fourth inhibitor of the ubiquitination of the intracellular protein according to the present invention are each prepared from the RS-K3574 substance alone. Is what can be.
  • the antitumor agent composition according to the fifth aspect of the present invention, the anti-inflammatory agent composition according to the sixth aspect of the present invention and the antiviral agent composition according to the seventh aspect of the present invention each comprise the RS-K3574 substance as an active ingredient thereof. In the form of a composition mixed with a conventional pharmaceutically acceptable solid or liquid carrier.
  • the first physiologically active substance RS-K3574 of the present invention is used as an anti-rheumatic drug, an anti-periodical drug, an immunosuppressive or immunostimulating substance, an anti-dementia drug, an anti-cardiomyopathy, an anti-obesity drug or an anti-diabetic drug. Is also expected to be available.
  • a potentiator comprising a physiologically active substance RS-K3574, which enhances a cancer cell apoptosis-inducing effect of a cell-killing antitumor agent or anticancer agent or radiation for cancer treatment on cancer cells.
  • the cell-killing antitumor agent or anticancer agent according to the eighth aspect of the present invention refers to TNF, a TNF-like apoptosis inducer, adriamycin, and other clinical anticancer agents.
  • an inhibitor of protein biosynthesis in cancer cells comprising the physiologically active substance RS-K3574.
  • a 10 consists bioactive substance RS-K3574, NF- K B inhibitors on activation of the transcription factor are provided.
  • aqueous solution of the RS-K3574 substance dissolved in water containing 10% dimethyl sulfoxide (DMS0) and 0.5% Tween 80 (surfactant) was administered at a dose of 2 mg or more per mouse to the RS-K3574 substance.
  • DMS0 dimethyl sulfoxide
  • Tween 80 surfactant
  • a RS-K3574 substance as an active ingredient is converted into a pharmaceutically acceptable conventional liquid carrier, For example, it can be mixed with ethanol, aqueous ethanol, water, physiological saline, or a solid carrier such as a crystal cell mouth, starch and the like to form a composition containing the RS-K3574 substance.
  • the RS-K3574 substance which is an active ingredient used in a pharmaceutical composition according to the present invention, can be administered orally. Alternatively, it can be administered parenterally by intravenous or intramuscular or subcutaneous injection, or intraperitoneal or rectal administration.
  • the RS-K3574 substance is mixed as an active ingredient with a conventional pharmaceutically acceptable solid or liquid carrier, and the resulting mixture is powdered. , Tablets, capsules, suspensions, syrups and the like.
  • a desirable form of the injection preparation is a sterile aqueous solution or a lyophilized agent containing the RS-K3574 substance as an active ingredient.
  • Liquid carriers used in injectable preparations include, for example, water, saline,
  • Phenol hydrated ethanol, glycerol, propylene glycol, vegetable oil and the like.
  • the ratio of the RS-K3574 substance as an active ingredient to be incorporated in the pharmaceutical composition as described above according to the present invention may vary depending on the dosage form.
  • the content ratio of the active ingredient may be about 1 to 1% by weight of the dosage unit. Can be in the range of 95%.
  • FIG. 1 shows an ultraviolet absorption spectrum of a RS-K3574 substance in a methanol solution.
  • Figure 2 shows the UV absorption spectrum of the RS-K3574 substance in methanol-HC1 solution.
  • FIG. 3 is an ultraviolet absorption spectrum of NaOH in a methanol solution of RS-K3574 substance.
  • Figure 4 shows the infrared absorption spectrum of the RS-K3574 substance measured by the KBr tablet method.
  • FIG. 5 is a proton nuclear magnetic resonance spectrum measured with a heavy chloroform solution of RS-K3574 substance (internal standard: tetramethylsilane) at 500 MHz.
  • FIG. 6 is a 13C nuclear magnetic resonance spectrum measured at 125 MHz using a heavy chloroform solution of RS-K3574 substance (internal standard: tetramethylsilane).
  • the liquid containing medium (pH unadjusted) shake flask 500ml volume) each 200ml dispensing Then, it was sterilized at 120 ° C for 20 minutes by an ordinary method.
  • the sterilized liquid medium was inoculated with a mycelium of Pleurotus edulis (Panus dis) K-3574 strain (FERM BP-8265) cultured on an agar slant medium. Thereafter, the cells were cultured in a liquid medium at 27 ° C for 3 days. Thereafter, the cells were cultured at 27 ° C. for 2 days under rotary stirring.
  • the culture obtained here was used as a seed culture.
  • the crude product was further subjected to liquid-centrifugal partition chromatography (250 mL) by an ascending method using a liquid phase consisting of chloroform-methanol-water (5: 6: 4). Fractions were collected to separate unwanted and required components.
  • the fraction containing the RS-K3574 substance was obtained as the 24th to 27th fractions when fractionated by 10 ml. These active fractions were concentrated to dryness under reduced pressure to obtain 716 mg of a purified RS-K3574 substance of the present invention.
  • the present invention provides a novel physiologically active substance, RS-K3574 substance.
  • This RS-K3574 substance is used for cell proliferation of cancer cells or immune cells or It not only has the activity of inhibiting the intracellular enzyme ubiquitin activating enzyme, which is involved in the generation of apoptosis or inflammation, but also has antitumor, anticancer and antiinflammatory activities. The activity of inhibiting the biosynthesis of proteins in cells.
  • the RS-K3574 substance according to the present invention is useful as an antitumor agent, an anticancer agent, an antiinflammatory agent or an antiviral agent.

Abstract

Par la culture d'une souche K-3574 de Pleurotacae Panus rudis (FERM BP-8265), un composé RS-K3574 représenté par la formule (I) est produit en tant que substance physiologiquement active présentant une activité d'inhibition de l'enzyme activant l'ubiquitine, une activité d'inhibition de l'ubiquitination de protéines intracellulaires, une activité antitumorale ainsi que d'autres activités biologiques diverses et présentant un nouveau squelette moléculaire, formule dans laquelle les configurations dans les positions 2-, 3-, 4- et 7 sont respectivement S, S, R et R. La substance RS-K3574 est une substance physiologiquement active ayant une activité d'inhibition de l'enzyme activant l'ubiquitine et une activité d'inhibition de l'ubiquitination de protéines intracellulaires ainsi qu'une activité d'inhibition de la biosynthèse de protéines intracellulaires, une activité antitumorale, une activité anticancéreuse, une activité anti-inflammatoire ainsi qu'une activité antivirale.
PCT/JP2003/000532 2003-01-22 2003-01-22 Nouvelle substance physiologiquement active rs-k3574 et son procede de production WO2004065607A1 (fr)

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AU2003303775A AU2003303775A1 (en) 2003-01-22 2003-01-22 Novel physiologically active substance rs-k3574 and process for producing the same
JP2004567132A JP4376189B2 (ja) 2003-01-22 2003-01-22 生理活性物質ｒｓ−ｋ３５７４から成る、ユビキチン活性化酵素の阻害剤、ならびに生理活性物質ｒｓ−ｋ３５７４から成る、細胞内タンパク質のユビキチン化の阻害剤
PCT/JP2003/000532 WO2004065607A1 (fr) 2003-01-22 2003-01-22 Nouvelle substance physiologiquement active rs-k3574 et son procede de production

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