WO2014030597A1 - Novel compound and method for producing same, novel microorganism, pharmaceutical composition, and anti-tumor agent - Google Patents

Novel compound and method for producing same, novel microorganism, pharmaceutical composition, and anti-tumor agent Download PDF

Info

Publication number
WO2014030597A1
WO2014030597A1 PCT/JP2013/072011 JP2013072011W WO2014030597A1 WO 2014030597 A1 WO2014030597 A1 WO 2014030597A1 JP 2013072011 W JP2013072011 W JP 2013072011W WO 2014030597 A1 WO2014030597 A1 WO 2014030597A1
Authority
WO
WIPO (PCT)
Prior art keywords
general formula
compound
culture
compound represented
agent
Prior art date
Application number
PCT/JP2013/072011
Other languages
French (fr)
Japanese (ja)
Inventor
洋子 百瀬
百瀬　功
哲也 染野
Original Assignee
公益財団法人微生物化学研究会
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 公益財団法人微生物化学研究会 filed Critical 公益財団法人微生物化学研究会
Priority to JP2014531609A priority Critical patent/JP6154382B2/en
Publication of WO2014030597A1 publication Critical patent/WO2014030597A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
    • 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
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial 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
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces

Definitions

  • the present invention relates to a novel compound, a method for producing the same, a novel microorganism capable of producing the novel compound, a pharmaceutical composition containing the novel compound, and an antitumor agent containing the pharmaceutical composition.
  • a novel compound having high anti-tumor activity against cancers including prostate cancer and high safety a method for producing the novel compound, a novel microorganism that is a bacterium producing the novel compound, and the novel compound are used.
  • a pharmaceutical composition and an antitumor agent it is strongly desired to provide a pharmaceutical composition and an antitumor agent.
  • the present invention relates to a novel compound having antitumor activity and high safety, a method for producing the novel compound, a novel microorganism capable of producing the novel compound, and a pharmaceutical composition containing the novel compound And an antitumor agent containing the pharmaceutical composition.
  • the compound of the present invention as a means for solving the above problems is a compound represented by the following general formula (I).
  • X represents group represented by either the following general formula (IA) or the following general formula (IB).
  • * represents a bond.
  • the method for producing the compound of the present invention comprises culturing Streptomyces sp. Strain MK932-CF8 (accession number: NITE BP-00838) having the ability to produce the compound represented by the general formula (I). Thus, a compound represented by the following general formula (I) is produced.
  • the microorganism of the present invention is a microorganism having the ability to produce the compound represented by the general formula (I), and the microorganism is Streptomyces sp. MK932-CF8 strain (Accession number: NITE BP). -00838).
  • the pharmaceutical composition of the present invention contains the compound represented by the general formula (I).
  • the antitumor agent of the present invention contains the pharmaceutical composition.
  • the conventional problems can be solved, the object can be achieved, the antitumor action has a high safety, a method for producing the novel compound, and the novel compound
  • a novel microorganism having the ability to produce a pharmaceutical composition, a pharmaceutical composition containing the novel compound, and an antitumor agent containing the pharmaceutical composition can be provided.
  • FIG. 1 is a graph showing androgen-dependent growth inhibitory action and cytotoxicity of androprostamine A on LNCaP cells.
  • FIG. 2 is a graph showing the androgen-dependent growth inhibitory action and cytotoxicity of androprostamine B on LNCaP cells.
  • FIG. 3 is a graph showing androgen-dependent growth inhibitory action and cytotoxicity of androprostamine A on VCaP cells.
  • FIG. 4 is a graph showing the androgen-dependent growth inhibitory action and cytotoxicity of androprostamine B on VCaP cells.
  • FIG. 5 is a graph showing the effect of androprostamine A on the expression of prostate specific antigen (PSA) in LNCaP cells.
  • FIG. 6 is a graph showing the effect of androprostamine A on prostate-specific antigen (PSA) expression in VCaP cells.
  • PSA prostate specific antigen
  • the compound of the present invention is a compound represented by the following general formula (I), and is a novel compound isolated by the present inventors.
  • the compound represented by the following general formula (I) is a novel compound that is clearly distinguished from known compounds by physicochemical properties and structural characteristics described later.
  • X represents group represented by either the following general formula (IA) or the following general formula (IB).
  • * represents a bond.
  • the compound represented by the general formula (I) includes a compound represented by the following structural formula (I) (hereinafter sometimes referred to as “androprostamine A”) and the following structural formula. It is preferably any one of the compounds represented by (II) (hereinafter sometimes referred to as “Androprosamine B”).
  • the compound has the structure represented by the general formula (I) can be confirmed by various analysis methods selected as appropriate. For example, the mass spectrometry, the ultraviolet spectroscopy, and the like are analyzed. In addition, analysis methods such as infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, and carbon-13 nuclear magnetic resonance spectroscopy can be used.
  • the compound may be a salt of the compound represented by the general formula (I).
  • the salt is not particularly limited as long as it is a pharmacologically acceptable salt, and can be appropriately selected according to the purpose. Examples thereof include organic salts such as acetates and citrates, hydrochlorides and carbonates. Examples include salt.
  • the compound represented by the general formula (I) may be obtained from a microorganism that produces the compound represented by the general formula (I), or may be obtained by chemical synthesis. However, it is preferably obtained by the method for producing the compound of the present invention described later.
  • the compound represented by the general formula (I) has a superior antitumor action and is a highly safe compound. Therefore, the compound represented by the general formula (I) can be suitably used, for example, as an active ingredient of the pharmaceutical composition of the present invention described later and the antitumor agent of the present invention.
  • Streptomyces sp. Strain MK932-CF8 (accession number: NITE BP-00838) having the ability to produce the compound represented by the general formula (I) is cultured. It includes at least a process (hereinafter sometimes referred to as “culturing process”), and further includes other processes as necessary.
  • Examples of the method for analyzing that the microorganism has the ability to produce the compound represented by the general formula (I) include, for example, a culture of the microorganism, preferably a culture supernatant after liquid culture or a solid culture. Examples include a method for analyzing the antitumor action of the components in the solid medium later, a method for detecting the compound represented by the general formula (I) by various analysis methods, and the like.
  • the culture of the microorganism when the culture of the microorganism has an antitumor effect, it can be determined that the microorganism has an ability to produce the compound represented by the general formula (I). .
  • the culture is added to cultured cancer cells such as humans, and the growth inhibitory action of the cultured cancer cells is analyzed by a known method. When the growth of the cultured cancer cells is suppressed, It can be determined that the culture has an antitumor effect.
  • a producing bacterium that produces the compound represented by the general formula (I) may be referred to as a nutrient medium (hereinafter simply referred to as “medium”). Inoculated in) and culturing at a good temperature for the production of the compound represented by the general formula (I).
  • the nutrient source added to the nutrient medium is not particularly limited and can be appropriately selected according to the purpose.
  • inorganic salts such as sodium chloride and calcium carbonate can be added to the medium for use, and in addition, a trace amount of metal salt can be added to the medium for use. Any of these materials may be used as long as they are useful for the production of the compound represented by the general formula (I) by the compound-producing bacteria, and all known culture materials can be used.
  • a seed mother for production of the compound represented by the said general formula (I) there is no restriction
  • the culture method is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably cultured under aerobic conditions.
  • the temperature of the culture is not particularly limited as long as it is within a range in which the compound represented by the general formula (I) can be produced without substantially inhibiting the growth of the compound-producing bacteria. Although it can be appropriately selected depending on the producing bacteria, it is preferably 25 ° C to 35 ° C.
  • the collecting step is a step of collecting the compound represented by the general formula (I) from the culture obtained in the culturing step. Since the compound represented by the general formula (I) has the physicochemical properties described above, it can be collected from the culture according to its properties.
  • collecting means separating and / or purifying the compound represented by the general formula (I) from the culture.
  • the culture is not particularly limited as long as it contains the compound represented by the general formula (I) obtained in the culture step, and can be appropriately selected according to the purpose.
  • Examples include a culture supernatant after liquid culture, a solid medium after solid culture, and a mixture thereof.
  • the use of a culture supernatant after liquid culture and / or a solid medium after solid culture as the culture enables the compound represented by the general formula (I) to be obtained efficiently. Is preferable.
  • the compound represented by the general formula (I) is isolated from the cells by an extraction method using an appropriate organic solvent or an elution method by disrupting the cells. It may be extracted and subjected to separation and / or purification.
  • the collection method is not particularly limited, and a method used for collecting a metabolite produced by a microorganism can be appropriately selected.
  • a solvent extraction method a method using a difference in adsorption affinity for various adsorbents, a chromatographic method, and the like can be mentioned.
  • the compounds represented by the general formula (I) can be separated and / or purified by using these methods singly or in appropriate combination and, if necessary, repeatedly.
  • the solvent used in the solvent extraction method is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include ethanol, methanol, acetone, butanol, and acetonitrile.
  • adsorbent there is no restriction
  • the chromatographic method is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a thin layer chromatographic method, a high-performance liquid chromatograph for fractionation using a normal phase or reverse phase column (preparation). HPLC) method and the like. There is no restriction
  • ion exchange resins such as Amberlite (registered trademark) CG50 (manufactured by Sigma Aldrich); Toyopearl (registered trademark) HW-40F (manufactured by Tosoh Corporation) Gel filtration such as Sephadex (registered trademark) LH-20 (manufactured by GE Healthcare); silica gel such as CAPCELL PAK SG120 (manufactured by Shiseido Co., Ltd.), and the like.
  • Amberlite registered trademark
  • HW-40F manufactured by Tosoh Corporation
  • Gel filtration such as Sephadex (registered trademark) LH-20 (manufactured by GE Healthcare)
  • silica gel such as CAPCELL PAK SG120 (manufactured by Shiseido Co., Ltd.), and the like.
  • the method for eluting the compound represented by the general formula (I) from the adsorbent or the carrier in the chromatographic method is not particularly limited, and is appropriately selected depending on the kind and properties of the adsorbent and the carrier. can do.
  • a method of elution using water-containing alcohol, water-containing acetone or the like as an elution solvent can be mentioned.
  • the microorganism of the present invention is a microorganism having the ability to produce a compound represented by the following general formula (I), and the microorganism is Streptomyces sp. MK932-CF8 strain (Accession number: NITE BP). -00838).
  • X represents group represented by either the following general formula (IA) or the following general formula (IB).
  • * represents a bond.
  • the compound represented by the general formula (I) is preferably a compound represented by any one of the following structural formula (I) and the following structural formula (II).
  • the Streptomyces sp. Strain MK932-CF8 (accession number: NITE BP-00838) is a strain isolated by the present inventors from the soil of Yokohama, Kanagawa Prefecture.
  • the mycological properties of the MK932-CF8 strain are as follows.
  • the aerial hyphae having spiral formation are elongated from the branched basic hyphae.
  • a mature spore chain links 10-50 oval spore.
  • the spore has a width of 0.4 ⁇ m to 0.5 ⁇ m and a length of 0.8 ⁇ m to 0.9 ⁇ m, and the spore surface is smooth. No roticular branches, mycelial bundles, spore capsules or motile spores are observed.
  • Growth temperature range Yeast starch agar medium [1.0% by mass soluble starch (starch (soluble), manufactured by Koso Chemical Co., Ltd.), 0.2% by mass yeast extract (powdered yeast extract S, Manufactured by Nippon Pharmaceutical Co., Ltd.), 2.6 mass% string agar (Gifu Hogar Agar, Kitahara Sangyo Co., Ltd.), pH 7.0], 10 ° C., 20 ° C., 24 ° C., 27 ° C., 30 ° C., 37 As a result of testing at each temperature of 0 ° C. or 45 ° C., growth at 10 ° C. and 45 ° C. was not observed, and growth occurred in the range of 20 ° C.
  • Starch hydrolysis (starch / inorganic salt agar medium, ISP-medium 4, 27 ° C. culture) On the third day after culturing, starch hydrolysis is observed and its action is moderate.
  • Cellular component 2,6-diaminopimelic acid in the cell wall is LL-type.
  • the bacterial cell component was analyzed by thin layer chromatography.
  • the strain MK932-CF8 extends from its well-branched basic mycelium to aerial hyphae that form a spiral, and its tip links oval spores.
  • light gray to gray aerial hyphae grow on light yellow to light yellow tea.
  • the optimum temperature for growth is around 30 ° C.
  • the water disintegration of starch is moderate.
  • 2,6-Diaminopimelic acid in the cell wall of MK932-CF8 strain is LL-type.
  • MK932-CF8 strain was considered to belong to the genus Streptomyces . Therefore, the strain MK932-CF8 was designated as Streptomyces sp. MK932-CF8.
  • the MK932-CF8 strain was filed for deposit at the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamashichi, Kisarazu, Chiba 292-0818), November 13, 2009. After that, on July 17, 2013, a request was made to transfer to an international deposit under the Budapest Treaty, and the deposit was made internationally under the deposit number NITE BP-00838.
  • the MK932-CF8 strain is susceptible to changes in properties.
  • mutant strains derived from the MK932-CF8 strain for example, ultraviolet rays, X-rays, radiation, drugs, etc.
  • Even artificial mutants, natural mutants), zygotes, recombinants, etc. that can be obtained by the mutation treatment of the above are those having the ability to produce the compound represented by the general formula (I). Included in the microorganism of the invention.
  • the pharmaceutical composition of the present invention contains at least the compound of the present invention, and further contains other components as necessary.
  • the pharmaceutical composition may be the compound itself represented by the general formula (I).
  • the additive or the adjuvant is not particularly limited and may be appropriately selected depending on the intended purpose.
  • examples thereof include bactericides, preservatives, binders, thickeners, fixing agents, binders, and coloring agents. , Stabilizers, pH adjusters, buffers, isotonic agents, solvents, antioxidants, UV inhibitors, crystal precipitation inhibitors, antifoaming agents, physical property improvers, preservatives, and the like.
  • the bactericidal agent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include cationic surfactants such as benzalkonium chloride, benzethonium chloride and cetylpyridinium chloride.
  • the preservative is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include p-hydroxybenzoates, chlorobutanol, and cresol.
  • the binder, thickener and fixing agent are not particularly limited and may be appropriately selected depending on the intended purpose.
  • the binder is not particularly limited and may be appropriately selected depending on the intended purpose.
  • examples include propyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate, and polyvinyl pyrrolidone.
  • the colorant is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include titanium oxide and iron oxide.
  • the stabilizer is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include tragacanth, gum arabic, gelatin, sodium pyrosulfite, ethylenediaminetetraacetic acid (EDTA), thioglycolic acid, and thiolactic acid. Is mentioned.
  • the pH adjusting agent or the buffering agent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include sodium citrate, sodium acetate, and sodium phosphate.
  • the isotonic agent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include sodium chloride and glucose.
  • the said pharmaceutical composition contains the compound represented by the said general formula (I), it has the outstanding anti-tumor action and high safety
  • the antitumor agent of the present invention contains at least the pharmaceutical composition of the present invention, and further contains other components as necessary.
  • ⁇ Pharmaceutical composition> There is no restriction
  • the antitumor agent may be the pharmaceutical composition itself.
  • the said antitumor agent may be used individually by 1 type, and may be used together with the pharmaceutical and chemical
  • the method for examining the antitumor action of the antitumor agent is not particularly limited and may be appropriately selected from known methods according to the purpose. Examples thereof include the methods described in Test Examples described later. .
  • ⁇ Dosage form> There is no restriction
  • the solid preparation is not particularly limited and may be appropriately selected depending on the intended purpose.
  • a suppository, a poultice, a plaster agent etc. are mentioned, for example.
  • liquid agent a syrup agent, a drink agent, a suspension agent, an alcoholic agent etc. are mentioned, for example.
  • a liquid agent, eye drops, an aerosol agent, a spray agent etc. are mentioned, for example.
  • administering There is no restriction
  • the administration method include a local administration method, an enteral administration method, a parenteral administration method and the like.
  • the dose is not particularly limited, and is appropriately selected in consideration of various factors such as the age, weight, constitution, symptom, and presence / absence of administration of a drug or drug containing other ingredients as active ingredients. be able to.
  • the animal species to be administered is not particularly limited and can be appropriately selected according to the purpose. For example, human, monkey, pig, cow, sheep, goat, dog, cat, mouse, rat, bird, etc. Among these, it is preferably used for humans.
  • the antitumor agent has an excellent antitumor action and high safety, it can be suitably used as a preventive or therapeutic agent for cancer. Since the antitumor agent has a growth inhibitory action against androgen-dependent growth among tumors, it can be preferably used as an anti-prostate cancer agent, and can also be suitably used as a prophylactic or therapeutic agent for prostate cancer. is there. Furthermore, it can be used as a prophylactic or therapeutic agent for other tumors that proliferate depending on the androgen receptor (for example, breast cancer, uterine cancer, etc.).
  • the sterilized liquid medium in these flasks was inoculated with the strain MK932-CF8 cultured on an agar slant medium, and cultured with shaking at 27 ° C. for 3 days. This culture was used as a seed culture.
  • the culture preparation solution 8L thus obtained was centrifuged to separate the cells and culture filtrate.
  • the culture filtrate was concentrated under reduced pressure, ethanol was removed, and deionized water was added to obtain a total 2 L aqueous solution.
  • This aqueous solution was applied to 500 mL of a synthetic adsorbent (Diaion (registered trademark) HP20, manufactured by Mitsubishi Chemical Corporation), washed with 2 L of deionized water, and then eluted with 2 L of 50 vol% acetone. This elution fraction was concentrated under reduced pressure to remove acetone and obtain 200 mL of an aqueous solution.
  • a synthetic adsorbent Diaion (registered trademark) HP20, manufactured by Mitsubishi Chemical Corporation
  • aqueous solution was further applied to an ion exchange resin (Amberlite (registered trademark) CG50, manufactured by Sigma Aldrich Co., Ltd.) to obtain a passing liquid.
  • the passing liquid was concentrated under reduced pressure to obtain 30 mL of an aqueous solution.
  • this aqueous solution was subjected to gel filtration chromatography using a resin for size exclusion chromatography (Toyopearl (registered trademark) HW-40, manufactured by Tosoh Corporation) and eluted with deionized water.
  • the obtained elution fraction was concentrated to dryness under reduced pressure to obtain 1.066 g of a crude product.
  • This crude product was subjected to chromatography using 1,000 mL of a gel filtration chromatographic carrier (Sephadex (registered trademark) LH-20, manufactured by GE Healthcare) and eluted with 50% by volume methanol water. The obtained fraction was concentrated to dryness to obtain 87.2 mg of a crude product.
  • a gel filtration chromatographic carrier Sephadex (registered trademark) LH-20, manufactured by GE Healthcare
  • the obtained elution fraction was concentrated to dryness under reduced pressure to obtain 20.9 mg of a crude product.
  • this crude product was dissolved in 2 mL of deionized water, and subjected to chromatography by 200 mL of gel filtration chromatography (Sephadex (registered trademark) LH-20, manufactured by GE Healthcare), and 66 volume% aqueous methanol Eluted with.
  • the obtained elution fraction was concentrated to dryness under reduced pressure to obtain 9.2 mg of a crude product.
  • Test Example 1 The androgen-dependent growth inhibitory action of androprostamine A and androprostamine B on prostate cancer cells was examined by the following method.
  • LNCaP cells A human prostate cancer cell line LNCaP cell (obtained from ATCC) that shows proliferation in response to androgen is used with RPMI medium (manufactured by Life Technologies Japan Co., Ltd.) containing 2% by volume of cFBS (charcol-treated FBS) without phenol red.
  • RPMI medium manufactured by Life Technologies Japan Co., Ltd.
  • cFBS charcol-treated FBS
  • the cells were seeded in a 96-well plate at 5 ⁇ 10 4 cells / mL, and cultured in an atmosphere of 37 ° C. and 5% CO 2 for 24 hours to completely adhere the LNCaP cells.
  • the cFBS was added with 0.5% by mass activated carbon to FBS (manufactured by MP Biomedicals) and stirred for 60 minutes, and then a 0.45 ⁇ m filter. The one prepared by sterilization and removal of activated carbon by passing through was used.
  • the final concentrations of androprostamine A or androprostamine B are 0.002 ⁇ g / mL, 0.008 ⁇ g / mL, 0.03 ⁇ g / mL, 0.13 ⁇ g / mL. , 0.52 ⁇ g / mL, 2.08 ⁇ g / mL, 8.33 ⁇ g / mL, or 33.3 ⁇ g / mL, and synthetic androgen (R1881, manufactured by Waterstone Technology) to a final concentration of 1 nM was added as follows. Subsequently, the cells were cultured for 5 days at 37 ° C. in a 5% CO 2 atmosphere.
  • Cell growth inhibition rate (%) ⁇ (BA) / B ⁇ ⁇ 100 Formula (1)
  • concentration (IC 50 value) at which the cell growth of LNCaP cells was inhibited by 50% was determined from the cell growth inhibition rate. The results are shown in Table 2 below and FIG. 1 and FIG.
  • Test Example 2 In order to confirm that the androgen-dependent growth inhibitory action in Test Example 1 was specific, cytotoxicity against LNCaP cells and VCaP cells was examined by the following method.
  • LNCaP cells or VCaP cells (both obtained from ATCC) were added at 5 ⁇ 10 4 cells / without using phenol red-added RPMI medium (Life Technologies Japan) containing 2 liquid% FBS or 10 liquid% FBS. were seeded in 96-well plates in mL, 37 ° C., it was cultured for 24 hours in an atmosphere of 5% CO 2 the cells were completely adhered.
  • Androprostamine A or Androprostamine B adjusted to 2.08 ⁇ g / mL, 8.33 ⁇ g / mL, or 33.3 ⁇ g / mL was added. Subsequently, the cells were cultured for 5 days at 37 ° C. in a 5% CO 2 atmosphere. As a comparative control in the system to which 10% by volume FBS was added, in the 96-well plate to which the LNCaP cells or VCaP cells were adhered, androprostamine A or androprostamine B was not added for 5 days at 37 ° C., 5 ° C. Culturing was performed in a% CO 2 atmosphere.
  • cell proliferation of LNCaP cells and VCaP cells was measured using the MTT method. Assuming that the absorbance of the system to which androprostamine A or androprostamine B is added is “A” and the absorbance of the comparative control is “B”, the cell growth inhibition rate (%) is calculated according to the equation (1) in Test Example 1. Calculated. Next, the concentration of androprostamine A or androprostamine B exhibiting cytotoxicity (IC 50 value) was determined from the cell growth inhibition rate. The results are shown in Table 3 below and FIGS.
  • Test Example 3 Human prostate cancer cell line LNCaP cells (obtained from ATCC) are seeded in 3 cm dishes at 5 ⁇ 10 5 cells / mL using RPMI medium (manufactured by Life Technologies Japan) containing 2% by volume of cFBS and without phenol red. The cells were cultured in an atmosphere of 37 ° C. and 5% CO 2 for 24 hours to completely adhere the cells.
  • the cFBS used was the same as in Test Example 1.
  • PSA prostate specific antigen
  • actin mRNA The expression level of prostate specific antigen (PSA: Prostate Specific Antigen) and actin mRNA was measured, and the expression level of PSA mRNA (PSA / actin) relative to the expression level of actin mRNA was calculated.
  • PSA and actin primers were purchased from Takara Bio Inc. The results are shown in FIG.
  • VCaP cells were prepared in the same manner as in “confirmation of expression of prostate-specific antigen in LNCaP cells” except that LNCaP cells were changed to VCaP cells.
  • expression level of PSA mRNA relative to the expression level of actin mRNA was calculated in the same manner as the confirmation of expression of prostate-specific antigen. The results are shown in FIG.
  • Examples of the aspect of the present invention include the following.
  • ⁇ 1> A compound represented by the following general formula (I).
  • X represents group represented by either the following general formula (IA) or the following general formula (IB).
  • * represents a bond.
  • ⁇ 2> The compound according to ⁇ 1>, which is represented by any one of the following structural formula (I) and the following structural formula (II).
  • MK932-CF8 strain having the ability to produce a compound represented by the following general formula (I) is cultured, and the following general formula ( A method for producing a compound, comprising producing the compound represented by I).
  • X represents group represented by either the following general formula (IA) or the following general formula (IB).
  • * represents a bond.
  • MK932-CF8 strain (Accession number: NITE BP-00838) It is a microorganism characterized by being.
  • X represents group represented by either the following general formula (IA) or the following general formula (IB).
  • * represents a bond.
  • a pharmaceutical composition comprising the compound according to any one of ⁇ 1> to ⁇ 2>.
  • An antitumor agent comprising the pharmaceutical composition according to ⁇ 5> and having an antitumor action.
  • a cancer preventive or therapeutic agent comprising the antitumor agent according to ⁇ 6> and used for the prevention or treatment of cancer.
  • An anti-prostate cancer agent comprising the pharmaceutical composition according to the above ⁇ 5> and having a prostate cancer growth inhibitory action.
  • a prophylactic or prophylactic agent for prostate cancer comprising the anti-prostatic cancer agent according to ⁇ 8> and used for the prevention or treatment of prostate cancer.
  • the compound of the present invention Since the compound of the present invention has excellent antitumor activity and is highly safe, it can be suitably used as an active ingredient such as pharmaceutical compositions and antitumor agents. Since the pharmaceutical composition of the present invention contains the compound of the present invention, it has excellent antitumor activity and high safety, and can be suitably used for antitumor agents and the like.
  • the antitumor agent of the present invention has an excellent antitumor action, particularly a growth inhibitory action against androgen-dependent growth, and is highly safe. Therefore, it is a prophylactic or therapeutic agent for cancer, preferably a prostatic cancer prophylactic agent. Or it can utilize suitably as a therapeutic agent.

Abstract

A compound represented by general formula (I); a method for producing the compound; a microorganism capable of producing the compound; a pharmaceutical composition containing the compound; an anti-tumor agent comprising the pharmaceutical composition; and others. In general formula (I), X represents a group represented by general formula (IA) or general formula (IB). In general formula (IA) and general formula (IB), * represents a bond.

Description

新規化合物及びその製造方法、並びに、新規微生物、医薬組成物、及び抗腫瘍剤NOVEL COMPOUND AND METHOD FOR PRODUCING THE SAME, NOVEL MICROORGANISM, PHARMACEUTICAL COMPOSITION, AND ANTI-TUMOR
　本発明は、新規化合物及びその製造方法、並びに、前記新規化合物を産生する能力を有する新規微生物、前記新規化合物を含有する医薬組成物、及び前記医薬組成物を含有する抗腫瘍剤に関する。 The present invention relates to a novel compound, a method for producing the same, a novel microorganism capable of producing the novel compound, a pharmaceutical composition containing the novel compound, and an antitumor agent containing the pharmaceutical composition.
　癌患者に対する治療としては、様々な治療が行われているが、中でも化学療法剤による治療が広く行われている。癌細胞は、正常細胞と比べて細胞増殖が頻繁に行われていることから、従来から、抗腫瘍剤として細胞の増殖を抑制し停止させる薬剤が臨床で多く用いられてきた。しかしながら、従来の薬剤は、癌細胞の増殖のみを選択的に抑制することができないため、骨髄抑制などの副作用がおこることが問題となっていた。このため癌細胞に特異的に作用する薬剤の開発が行われ、現在では癌細胞に特異的に作用する薬剤が臨床で用いられるようになってきた。しかしながら、これらの薬剤でも臨床での効果は満足できるものではなく、正常細胞、正常組織に対する毒性が低く、かつ効果が十分に期待される薬剤が望まれている（特許文献１～４参照）。 Various treatments are performed for cancer patients, and among them, treatment with chemotherapeutic agents is widely performed. Since cancer cells are frequently proliferated as compared with normal cells, conventionally, drugs that suppress and stop cell growth have been used in clinical practice as antitumor agents. However, since conventional drugs cannot selectively suppress only the proliferation of cancer cells, side effects such as bone marrow suppression have been a problem. For this reason, drugs that specifically act on cancer cells have been developed, and now drugs that act specifically on cancer cells have come into clinical use. However, even these drugs are not satisfactory in clinical effects, and drugs that have low toxicity to normal cells and normal tissues and are expected to be sufficiently effective are desired (see Patent Documents 1 to 4).
　近年、癌の中でも前立腺癌の罹患数が増加しており、全世界では年間約４０万人と報告され、これは全ての男性癌の９．２％を占め、４番目に多い。日本での前立腺癌の年齢調整罹患率は、胃癌、肺癌、結腸癌、肝臓癌、直腸癌に次いで６番目に高い。前立腺癌罹患数は、今後も更に増加することが見込まれており、２０２０年には男性癌の中で肺癌に次いで２番目になると予測されている。また、今後の前立腺癌による死亡率は、２０２０年には２０００年の２．８倍になると予測されている。（非特許文献１参照）。 In recent years, the number of prostate cancers among cancers has increased, and it is reported that about 400,000 people worldwide annually, accounting for 9.2% of all male cancers, the fourth most common. Age-adjusted prevalence of prostate cancer in Japan is the sixth highest after gastric cancer, lung cancer, colon cancer, liver cancer, and rectal cancer. The number of prostate cancer cases is expected to increase further in the future, and by 2020, it is predicted that it will be the second male cancer after lung cancer. Moreover, the death rate from prostate cancer in the future is predicted to increase 2.8 times in 2000 to 2000. (Refer nonpatent literature 1).
　初期の前立腺癌は、アンドロゲン感受性癌であり、進行病期に癌が発見されてもその大多数は、外科的あるいは薬物的アンドロゲン除去に反応し、症状は軽快する。しかしながら、数年後には、その半数以上がアンドロゲン依存性喪失、即ち、内分泌療法抵抗性増殖を示すようになり、更にその後、１年間～２年間で癌死に至ることが知られている。 初期 Early prostate cancer is androgen-sensitive cancer, and even if cancer is discovered in an advanced stage, the majority responds to surgical or drug-induced androgen removal and symptoms are relieved. However, after several years, it is known that more than half of them show androgen-dependent loss, that is, endocrine therapy-resistant growth, and cancer death occurs in 1 to 2 years thereafter.
　現在、前立腺癌の治療に臨床で使用されている抗アンドロゲン剤は、アンドロゲンとアンドロゲン受容体との結合を阻害するアンタゴニストとして作用する薬剤のみである。近年の研究から、アンドロゲン非依存性を獲得した前立腺癌に対しても、異なった機序によるアンドロゲン受容体機能阻害物質は、抗癌作用を発揮することが示唆されている（非特許文献２参照）。しかしながら、アンタゴニスト以外の作用機序を持つ薬剤は未だ開発されていない。 Currently, the only anti-androgen agents clinically used for the treatment of prostate cancer are drugs that act as antagonists that inhibit the binding of androgens and androgen receptors. Recent research suggests that androgen receptor function inhibitory substances with different mechanisms exert anti-cancer effects even on prostate cancer that has acquired androgen independence (see Non-Patent Document 2). ). However, drugs having a mechanism of action other than antagonists have not been developed yet.
　そこで、前立腺癌をはじめとする癌に対する抗腫瘍作用を有し、安全性の高い新規化合物及び該新規化合物の製造方法、並びに、前記新規化合物の生産菌である新規微生物、前記新規化合物を利用した医薬組成物及び抗腫瘍剤の提供が強く望まれているのが現状である。 Therefore, a novel compound having high anti-tumor activity against cancers including prostate cancer and high safety, a method for producing the novel compound, a novel microorganism that is a bacterium producing the novel compound, and the novel compound are used. At present, it is strongly desired to provide a pharmaceutical composition and an antitumor agent.
特開平８－１４３５６９号公報Japanese Patent Laid-Open No. 8-14369 特開平８－２３９３７９号公報JP-A-8-239379 特開平９－２４９６４７号公報JP-A-9-249647 特開平１１－２１２６３号公報JP-A-11-212263
　本発明は、従来における前記諸問題を解決し、以下の目的を達成することを課題とする。即ち、本発明は、抗腫瘍作用を有し、安全性の高い新規化合物及び該新規化合物の製造方法、並びに、前記新規化合物を産生する能力を有する新規微生物、前記新規化合物を含有する医薬組成物、及び前記医薬組成物を含有する抗腫瘍剤を提供することを目的とする。 This invention makes it a subject to solve the said various problems in the past and to achieve the following objectives. That is, the present invention relates to a novel compound having antitumor activity and high safety, a method for producing the novel compound, a novel microorganism capable of producing the novel compound, and a pharmaceutical composition containing the novel compound And an antitumor agent containing the pharmaceutical composition.
　前記課題を解決するための手段としての本発明の化合物は、下記一般式（Ｉ）で表される化合物である。
Figure JPOXMLDOC01-appb-C000009
 　ただし、前記一般式（Ｉ）において、Ｘは、下記一般式（ＩＡ）及び下記一般式（ＩＢ）のいずれかで表される基を表す。
Figure JPOXMLDOC01-appb-C000010
 　ただし、前記一般式（ＩＡ）及び前記一般式（ＩＢ）において、＊は、結合手を表す。 The compound of the present invention as a means for solving the above problems is a compound represented by the following general formula (I).
Figure JPOXMLDOC01-appb-C000009
 However, in the said general formula (I), X represents group represented by either the following general formula (IA) or the following general formula (IB).
Figure JPOXMLDOC01-appb-C000010
 However, in the general formula (IA) and the general formula (IB), * represents a bond.
　本発明の化合物の製造方法は、前記一般式（Ｉ）で表される化合物を生産する能力を有するストレプトミセス　エスピー（Ｓｔｒｅｐｔｏｍｙｃｅｓ　ｓｐ．）ＭＫ９３２－ＣＦ８株（受託番号：ＮＩＴＥ　ＢＰ－００８３８）を培養して、下記一般式（Ｉ）で表される化合物を製造する。 The method for producing the compound of the present invention comprises culturing Streptomyces sp. Strain MK932-CF8 (accession number: NITE BP-00838) having the ability to produce the compound represented by the general formula (I). Thus, a compound represented by the following general formula (I) is produced.
　本発明の微生物は、前記一般式（Ｉ）で表される化合物を生産する能力を有する微生物であって、前記微生物が、ストレプトミセス　エスピー（Ｓｔｒｅｐｔｏｍｙｃｅｓ　ｓｐ．）ＭＫ９３２－ＣＦ８株（受託番号：ＮＩＴＥ　ＢＰ－００８３８）である。 The microorganism of the present invention is a microorganism having the ability to produce the compound represented by the general formula (I), and the microorganism is Streptomyces sp. MK932-CF8 strain (Accession number: NITE BP). -00838).
　本発明の医薬組成物は、前記一般式（Ｉ）で表される化合物を含有する。 The pharmaceutical composition of the present invention contains the compound represented by the general formula (I).
　本発明の抗腫瘍剤は、前記医薬組成物を含有する。 The antitumor agent of the present invention contains the pharmaceutical composition.
　本発明によれば、従来における前記諸問題を解決し、前記目的を達成することができ、抗腫瘍作用を有し、安全性の高い新規化合物及び該新規化合物の製造方法、並びに、前記新規化合物を産生する能力を有する新規微生物、前記新規化合物を含有する医薬組成物、及び前記医薬組成物を含有する抗腫瘍剤を提供することができる。 According to the present invention, the conventional problems can be solved, the object can be achieved, the antitumor action has a high safety, a method for producing the novel compound, and the novel compound A novel microorganism having the ability to produce a pharmaceutical composition, a pharmaceutical composition containing the novel compound, and an antitumor agent containing the pharmaceutical composition can be provided.
図１は、アンドロプロスタミンＡのＬＮＣａＰ細胞に対するアンドロゲン依存増殖阻害作用及び細胞毒性を示すグラフである。FIG. 1 is a graph showing androgen-dependent growth inhibitory action and cytotoxicity of androprostamine A on LNCaP cells. 図２は、アンドロプロスタミンＢのＬＮＣａＰ細胞に対するアンドロゲン依存増殖阻害作用及び細胞毒性を示すグラフである。FIG. 2 is a graph showing the androgen-dependent growth inhibitory action and cytotoxicity of androprostamine B on LNCaP cells. 図３は、アンドロプロスタミンＡのＶＣａＰ細胞に対するアンドロゲン依存増殖阻害作用及び細胞毒性を示すグラフである。FIG. 3 is a graph showing androgen-dependent growth inhibitory action and cytotoxicity of androprostamine A on VCaP cells. 図４は、アンドロプロスタミンＢのＶＣａＰ細胞に対するアンドロゲン依存増殖阻害作用及び細胞毒性を示すグラフである。FIG. 4 is a graph showing the androgen-dependent growth inhibitory action and cytotoxicity of androprostamine B on VCaP cells. 図５は、アンドロプロスタミンＡのＬＮＣａＰ細胞における前立腺特異的抗原（ＰＳＡ）の発現に対する影響を示すグラフである。FIG. 5 is a graph showing the effect of androprostamine A on the expression of prostate specific antigen (PSA) in LNCaP cells. 図６は、アンドロプロスタミンＡのＶＣａＰ細胞における前立腺特異的抗原（ＰＳＡ）の発現に対する影響を示すグラフである。FIG. 6 is a graph showing the effect of androprostamine A on prostate-specific antigen (PSA) expression in VCaP cells.
（新規化合物）
　本発明の化合物は、下記一般式（Ｉ）で表される化合物であり、本発明者らが分離した新規化合物である。
　下記一般式（Ｉ）で表される化合物は、後述する物理化学的性質及び構造上の特徴によって、既知の化合物と明確に区別される新規化合物である。
Figure JPOXMLDOC01-appb-C000011
 　ただし、前記一般式（Ｉ）において、Ｘは、下記一般式（ＩＡ）及び下記一般式（ＩＢ）のいずれかで表される基を表す。
Figure JPOXMLDOC01-appb-C000012
 　ただし、前記一般式（ＩＡ）及び前記一般式（ＩＢ）において、＊は、結合手を表す。 (New compound)
The compound of the present invention is a compound represented by the following general formula (I), and is a novel compound isolated by the present inventors.
The compound represented by the following general formula (I) is a novel compound that is clearly distinguished from known compounds by physicochemical properties and structural characteristics described later.
Figure JPOXMLDOC01-appb-C000011
 However, in the said general formula (I), X represents group represented by either the following general formula (IA) or the following general formula (IB).
Figure JPOXMLDOC01-appb-C000012
 However, in the general formula (IA) and the general formula (IB), * represents a bond.
　これらの中でも、前記一般式（Ｉ）で表される化合物は、下記構造式（Ｉ）で表される化合物（以下、「アンドロプロスタミン（Ａｎｄｒｏｐｒｏｓｔａｍｉｎｅ）Ａ」と称することがある）及び下記構造式（ＩＩ）で表される化合物（以下、「アンドロプロスタミン（Ａｎｄｒｏｐｒｏｓｔａｍｉｎｅ）Ｂ」と称することがある）のいずれかであることが好ましい。
Figure JPOXMLDOC01-appb-C000013
Figure JPOXMLDOC01-appb-C000014
 Among these, the compound represented by the general formula (I) includes a compound represented by the following structural formula (I) (hereinafter sometimes referred to as “androprostamine A”) and the following structural formula. It is preferably any one of the compounds represented by (II) (hereinafter sometimes referred to as “Androprosamine B”).
Figure JPOXMLDOC01-appb-C000013
Figure JPOXMLDOC01-appb-C000014
＜アンドロプロスタミンＡの物理化学的性質＞
（１）　外観は、白色の粉状である。
（２）　分子式は、Ｃ２６Ｈ３８Ｎ５Ｏ１０Ｃｌで表され、分子量は、６１５である。
（３）　高分解能質量分析（ＨＲＥＳＩＭＳ：正イオンモード）による、実験値は、ｍ／ｚ　６１６．２３８６（Ｍ＋Ｈ）＋であり、計算値は、ｍ／ｚ　６１６．２３８０（Ｃ２６Ｈ３９Ｎ５Ｏ１０Ｃｌとして）である。
（４）　比旋光度は、［α］Ｄ ２３＝＋７６．８°（ｃ０．１，メタノール）である。
（５）　メタノール溶液で測定した紫外吸収スペクトルは、下記表１に示すとおりである。
　　　　λｍａｘ　ｎｍ（ε）　：２２７（１３，０００，ｓｈ），２８９（１１，０００）
（６）　ＴＬＣプレート（ＴＬＣ　Ｓｉｌｉｃａ　ｇｅｌ　６０Ｆ２５４、メルク製）の薄層クロマトグラフィーにおいて、展開溶媒〔ブタノール：メタノール：水（４：１：２、体積比）〕で展開して測定したＲＦ値は、０．２２であり、展開溶媒〔ブタノール：酢酸：水（４：１：２、体積比）〕で展開して測定したＲＦ値は、０．２７である。 <Physicochemical properties of androprostamine A>
(1) The appearance is white powder.
(2) The molecular formula is represented by C 26 H 38 N 5 O 10 Cl, and the molecular weight is 615.
(3) The experimental value by high resolution mass spectrometry (HRESIMS: positive ion mode) is m / z 616.2386 (M + H) + , and the calculated value is m / z 616.2380 (C 26 H 39 N 5 As O 10 Cl).
(4) The specific rotation is [α] D 23 = + 76.8 ° (c0.1, methanol).
(5) The ultraviolet absorption spectrum measured with a methanol solution is as shown in Table 1 below.
λ max nm (ε): 227 (13,000, sh), 289 (11,000)
(6) In a thin layer chromatography of a TLC plate (TLC Silica gel 60F254, manufactured by Merck), an RF value measured by developing with a developing solvent [butanol: methanol: water (4: 1: 2, volume ratio)] is The RF value measured by developing with a developing solvent [butanol: acetic acid: water (4: 1: 2, volume ratio)] is 0.27.
＜アンドロプロスタミンＢの物理化学的性質＞
（１）　外観は、白色の粉状である。
（２）　分子式は、Ｃ３１Ｈ４５Ｎ６Ｏ１１Ｃｌで表され、分子量は、７１２である。
（３）　高分解能質量分析（ＨＲＥＳＩＭＳ：正イオンモード）による、実験値は、ｍ／ｚ　７１３．２９０３（Ｍ＋Ｈ）＋であり、計算値は、ｍ／ｚ　７１３．２９０８（Ｃ３１Ｈ４６Ｎ６Ｏ１１Ｃｌとして）である。
（４）　比旋光度は、［α］Ｄ ２３＝＋５６．８°（ｃ０．２、メタノール）である。
（５）　メタノール溶液で測定した紫外吸収スペクトルは、下記表１に示すとおりである。
　　　　λｍａｘ　ｎｍ（ε）　：２２７（１５，１００，ｓｈ），２８９（１２，８００）
（６）　ＴＬＣプレート（ＴＬＣ　Ｓｉｌｉｃａ　ｇｅｌ　６０Ｆ２５４、メルク製）の薄層クロマトグラフィーにおいて、展開溶媒〔ブタノール：メタノール：水（４：１：２、体積比）〕で展開して測定したＲＦ値は、０．１８であり、展開溶媒〔ブタノール：酢酸：水（４：１：２、体積比）〕で展開して測定したＲＦ値は、０．２６である。 <Physicochemical properties of androprostamine B>
(1) The appearance is white powder.
(2) The molecular formula is represented by C 31 H 45 N 6 O 11 Cl, and the molecular weight is 712.
(3) The experimental value by high resolution mass spectrometry (HRESIMS: positive ion mode) is m / z 713.2903 (M + H) + , and the calculated value is m / z 713.2908 (C 31 H 46 N 6 As O 11 Cl).
(4) The specific rotation is [α] D 23 = + 56.8 ° (c0.2, methanol).
(5) The ultraviolet absorption spectrum measured with a methanol solution is as shown in Table 1 below.
λ max nm (ε): 227 (15, 100, sh), 289 (12, 800)
(6) In a thin layer chromatography of a TLC plate (TLC Silica gel 60F254, manufactured by Merck), an RF value measured by developing with a developing solvent [butanol: methanol: water (4: 1: 2, volume ratio)] is The RF value measured by developing with a developing solvent [butanol: acetic acid: water (4: 1: 2, volume ratio)] is 0.26.
Figure JPOXMLDOC01-appb-T000015
Figure JPOXMLDOC01-appb-T000015
　前記化合物が、前記一般式（Ｉ）で表される構造を有するか否かは、適宜選択した各種の分析方法により確認することができ、例えば、前記質量分析法、前記紫外分光法等の分析の他、赤外分光法、プロトン核磁気共鳴分光法、炭素１３核磁気共鳴分光法等の分析方法などが挙げられる。 Whether or not the compound has the structure represented by the general formula (I) can be confirmed by various analysis methods selected as appropriate. For example, the mass spectrometry, the ultraviolet spectroscopy, and the like are analyzed. In addition, analysis methods such as infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, and carbon-13 nuclear magnetic resonance spectroscopy can be used.
　前記化合物は、前記一般式（Ｉ）で表される化合物の塩であってもよい。
　前記塩としては、薬理学的に許容され得る塩であれば、特に制限はなく、目的に応じて適宜選択することができ、例えば、酢酸塩、クエン酸塩等の有機塩、塩酸塩、炭酸塩などが挙げられる。 The compound may be a salt of the compound represented by the general formula (I).
The salt is not particularly limited as long as it is a pharmacologically acceptable salt, and can be appropriately selected according to the purpose. Examples thereof include organic salts such as acetates and citrates, hydrochlorides and carbonates. Examples include salt.
　前記一般式（Ｉ）で表される化合物は、前記一般式（Ｉ）で表される化合物を生産する微生物から得られたものであってもよいし、化学合成により得られたものであってもよいが、後述する本発明の化合物の製造方法により得られることが好ましい。 The compound represented by the general formula (I) may be obtained from a microorganism that produces the compound represented by the general formula (I), or may be obtained by chemical synthesis. However, it is preferably obtained by the method for producing the compound of the present invention described later.
＜用途＞
　前記一般式（Ｉ）で表される化合物は、優れた抗腫瘍作用を有し、安全性の高い化合物である。そのため、前記一般式（Ｉ）で表される化合物は、例えば、後述する本発明の医薬組成物や、本発明の抗腫瘍剤等の有効成分として好適に利用可能である。 <Application>
The compound represented by the general formula (I) has a superior antitumor action and is a highly safe compound. Therefore, the compound represented by the general formula (I) can be suitably used, for example, as an active ingredient of the pharmaceutical composition of the present invention described later and the antitumor agent of the present invention.
（化合物の製造方法）
　本発明の化合物の製造方法は、前記一般式（Ｉ）で表される化合物を生産する能力を有するストレプトミセス　エスピー（Ｓｔｒｅｐｔｏｍｙｃｅｓ　ｓｐ．）ＭＫ９３２－ＣＦ８株（受託番号：ＮＩＴＥ　ＢＰ－００８３８）を培養する工程（以下、「培養工程」と称することがある）を少なくとも含み、必要に応じて、更にその他の工程を含む。 (Method for producing compound)
In the method for producing the compound of the present invention, Streptomyces sp. Strain MK932-CF8 (accession number: NITE BP-00838) having the ability to produce the compound represented by the general formula (I) is cultured. It includes at least a process (hereinafter sometimes referred to as “culturing process”), and further includes other processes as necessary.
＜培養工程＞
　前記培養工程は、前記一般式（Ｉ）で表される化合物を生産する能力を有するストレプトミセス　エスピー（Ｓｔｒｅｐｔｏｍｙｃｅｓ　ｓｐ．）ＭＫ９３２－ＣＦ８株（受託番号：ＮＩＴＥ　ＢＰ－００８３８）を培養して、下記一般式（Ｉ）で表される化合物を製造する工程である。
　なお、前記ＭＫ９３２－ＣＦ８株を、放射線照射やその他の変異処理に供することにより、前記一般式（Ｉ）で表される化合物の生産能を高めることも可能である。更に、遺伝子工学的手法による前記一般式（Ｉ）で表される化合物の生産も可能である。 <Culture process>
In the culturing step, Streptomyces sp. MK932-CF8 strain (accession number: NITE BP-00838) having the ability to produce the compound represented by the general formula (I) is cultured, This is a process for producing a compound represented by formula (I).
The production ability of the compound represented by the general formula (I) can be enhanced by subjecting the MK932-CF8 strain to irradiation or other mutation treatment. Furthermore, production of the compound represented by the general formula (I) by genetic engineering techniques is also possible.
　前記微生物が前記一般式（Ｉ）で表される化合物を生産する能力を有することを分析する方法としては、例えば、該微生物の培養物、好ましくは、液体培養後の培養上清中又は固体培養後の固体培地中の成分の、抗腫瘍作用を分析する方法、各種分析法により前記一般式（Ｉ）で表される化合物を検出する方法などが挙げられる。 Examples of the method for analyzing that the microorganism has the ability to produce the compound represented by the general formula (I) include, for example, a culture of the microorganism, preferably a culture supernatant after liquid culture or a solid culture. Examples include a method for analyzing the antitumor action of the components in the solid medium later, a method for detecting the compound represented by the general formula (I) by various analysis methods, and the like.
　前記抗腫瘍作用を分析する方法においては、前記微生物の培養物が抗腫瘍作用を有する場合、前記微生物は前記一般式（Ｉ）で表される化合物を生産する能力を有すると判断することができる。
　具体的には、ヒト等の培養癌細胞に前記培養物を添加し、公知の方法で該培養癌細胞の増殖抑制作用を分析し、前記培養癌細胞の増殖が抑制された場合、前記微生物の培養物が抗腫瘍作用を有すると判断することができる。 In the method for analyzing an antitumor effect, when the culture of the microorganism has an antitumor effect, it can be determined that the microorganism has an ability to produce the compound represented by the general formula (I). .
Specifically, the culture is added to cultured cancer cells such as humans, and the growth inhibitory action of the cultured cancer cells is analyzed by a known method. When the growth of the cultured cancer cells is suppressed, It can be determined that the culture has an antitumor effect.
　前記培養は、前記一般式（Ｉ）で表される化合物を生産する生産菌（以下、単に「化合物類生産菌」と称することがある）を栄養培地（以下、単に「培地」と称することがある）中に接種し、前記一般式（Ｉ）で表される化合物の生産に良好な温度で培養することによって行われる。 In the culture, a producing bacterium that produces the compound represented by the general formula (I) (hereinafter sometimes simply referred to as “compound producing bacterium”) may be referred to as a nutrient medium (hereinafter simply referred to as “medium”). Inoculated in) and culturing at a good temperature for the production of the compound represented by the general formula (I).
　前記栄養培地としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、従来放線菌の培養に利用されている公知のものを使用することができ、液体培地であってもよく、固体（寒天）培地であってもよい。
　前記栄養培地に添加する栄養源としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、市販されている大豆粉、小麦胚芽、押し麦、ペプトン、綿実粕、酵母エキス、肉エキス、コーン・スティープ・リカー、硫酸アンモニウム、硝酸ナトリウム、尿素等の窒素源；トマトペースト、グリセリン、デンプン、グルコース、ガラクトース、デキストリン、バクトソイトン等の炭水化物、脂肪等の炭素源；などが挙げられる。
　更に、食塩、炭酸カルシウム等の無機塩を培地に添加して使用することもでき、その他、必要に応じて微量の金属塩を培地に添加して使用することもできる。
　これらの材料は、前記化合物類生産菌が利用し、前記一般式（Ｉ）で表される化合物の生産に役立つものであればよく、公知の培養材料は全て用いることができる。 There is no restriction | limiting in particular as said nutrient medium, According to the objective, it can select suitably, For example, the well-known thing utilized conventionally for culture | cultivation of actinomycetes can be used, even if it is a liquid medium. It may be a solid (agar) medium.
The nutrient source added to the nutrient medium is not particularly limited and can be appropriately selected according to the purpose.For example, commercially available soybean flour, wheat germ, pressed wheat, peptone, cottonseed meal, yeast extract, Nitrogen sources such as meat extract, corn steep liquor, ammonium sulfate, sodium nitrate, urea, etc .; carbohydrate sources such as tomato paste, glycerin, starch, glucose, galactose, dextrin, bacto-soyton; carbon sources such as fat;
Furthermore, inorganic salts such as sodium chloride and calcium carbonate can be added to the medium for use, and in addition, a trace amount of metal salt can be added to the medium for use.
Any of these materials may be used as long as they are useful for the production of the compound represented by the general formula (I) by the compound-producing bacteria, and all known culture materials can be used.
　前記一般式（Ｉ）で表される化合物の生産のための種母としては、特に制限はなく、目的に応じて適宜選択することができ、平板培地、斜面培地、半斜面培地等の培地上で前記化合物類生産菌を培養した生育物などを使用することができる。 There is no restriction | limiting in particular as a seed mother for production of the compound represented by the said general formula (I), According to the objective, it can select suitably, It is on culture media, such as a plate culture medium, a slope culture medium, and a half slope culture medium. A growth product obtained by culturing the above-mentioned compound-producing bacteria can be used.
　前記培養の方法としては、特に制限はなく、目的に応じて適宜選択することができるが、好気的条件で培養することが好ましい。
　前記培養の温度としては、前記化合物類生産菌の発育が実質的に阻害されずに、前記一般式（Ｉ）で表される化合物を生産し得る範囲であれば、特に制限はなく、使用する生産菌に応じて適宜選択することができるが、２５℃～３５℃が好ましい。
　前記培養の期間としては、特に制限はなく、前記一般式（Ｉ）で表される化合物の蓄積に合わせて適宜選択することができる。 The culture method is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably cultured under aerobic conditions.
The temperature of the culture is not particularly limited as long as it is within a range in which the compound represented by the general formula (I) can be produced without substantially inhibiting the growth of the compound-producing bacteria. Although it can be appropriately selected depending on the producing bacteria, it is preferably 25 ° C to 35 ° C.
There is no restriction | limiting in particular as the period of the said culture | cultivation, According to accumulation | storage of the compound represented by the said general formula (I), it can select suitably.
＜その他の工程＞
　前記その他の工程としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記培養工程で得られた培養物から前記一般式（Ｉ）で表される化合物を採取する採取工程、前記培養工程で得られた培養物又は前記採取工程で得られた前記一般式（Ｉ）で表される化合物を洗浄する洗浄工程、前記採取工程で得られた前記一般式（Ｉ）で表される化合物を更に精製する精製工程などが挙げられる。前記洗浄工程や前記精製工程は、公知の方法で適宜行われる。 <Other processes>
There is no restriction | limiting in particular as said other process, According to the objective, it can select suitably, For example, collection | collection which extract | collects the compound represented by the said general formula (I) from the culture obtained by the said culture | cultivation process. A washing step for washing the compound represented by the general formula (I) obtained in the step, the culture obtained in the culturing step, or the general formula (I) obtained in the collecting step. Examples include a purification step for further purification of the represented compound. The washing step and the purification step are appropriately performed by a known method.
＜＜採取工程＞＞
　前記採取工程は、前記培養工程で得られた培養物から前記一般式（Ｉ）で表される化合物を採取する工程である。前記一般式（Ｉ）で表される化合物は、上述した物理化学的性質を有するので、その性状に従って培養物から採取することができる。ここで、本発明において、採取とは、前記一般式（Ｉ）で表される化合物を、前記培養物から分離及び／又は精製することを意味する。 << collecting process >>
The collecting step is a step of collecting the compound represented by the general formula (I) from the culture obtained in the culturing step. Since the compound represented by the general formula (I) has the physicochemical properties described above, it can be collected from the culture according to its properties. Here, in the present invention, collecting means separating and / or purifying the compound represented by the general formula (I) from the culture.
　前記培養物としては、前記培養工程で得られ、前記一般式（Ｉ）で表される化合物を含むものであれば、特に制限はなく、目的に応じて適宜選択することができ、菌体、液体培養後の培養上清、固体培養後の固体培地、及びこれらの混合物などが挙げられる。これらの中でも、前記培養物としては、液体培養後の培養上清及び／又は固体培養後の固体培地を用いることが、前記一般式（Ｉ）で表される化合物を効率よく得ることができる点で好ましい。
　なお、前記培養物として、前記菌体を用いる場合は、適当な有機溶媒を用いた抽出方法や、菌体破砕による溶出方法などにより、前記一般式（Ｉ）で表される化合物を菌体から抽出し、これを分離及び／又は精製に供してもよい。 The culture is not particularly limited as long as it contains the compound represented by the general formula (I) obtained in the culture step, and can be appropriately selected according to the purpose. Examples include a culture supernatant after liquid culture, a solid medium after solid culture, and a mixture thereof. Among these, the use of a culture supernatant after liquid culture and / or a solid medium after solid culture as the culture enables the compound represented by the general formula (I) to be obtained efficiently. Is preferable.
When the cells are used as the culture, the compound represented by the general formula (I) is isolated from the cells by an extraction method using an appropriate organic solvent or an elution method by disrupting the cells. It may be extracted and subjected to separation and / or purification.
　前記採取の方法としては、特に制限はなく、微生物の生産する代謝物を採取するのに用いられる方法を適宜選択することができる。例えば、溶媒抽出法、各種吸着剤に対する吸着親和性の差を利用する方法、クロマトグラフ法などが挙げられる。これらの方法を単独又は適宜組み合せて、場合によっては反復使用することにより、前記一般式（Ｉ）で表される化合物を分離及び／又は精製することができる。 The collection method is not particularly limited, and a method used for collecting a metabolite produced by a microorganism can be appropriately selected. For example, a solvent extraction method, a method using a difference in adsorption affinity for various adsorbents, a chromatographic method, and the like can be mentioned. The compounds represented by the general formula (I) can be separated and / or purified by using these methods singly or in appropriate combination and, if necessary, repeatedly.
　前記溶媒抽出法に用いる溶媒としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、エタノール、メタノール、アセトン、ブタノール、アセトニトリルなどが挙げられる。 The solvent used in the solvent extraction method is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include ethanol, methanol, acetone, butanol, and acetonitrile.
　前記吸着剤としては、特に制限はなく、公知の吸着剤の中から目的に応じて適宜選択することができ、例えば、ポリスチレン系吸着樹脂などが挙げられる。
　前記吸着剤の市販品の具体例としては、アンバーライトＸＡＤ（ローム・アンド・ハース社製）、ダイヤイオン（登録商標）ＨＰ－２０（三菱化学株式会社製）などが挙げられる。 There is no restriction | limiting in particular as said adsorbent, According to the objective, it can select suitably from well-known adsorbents, For example, a polystyrene type adsorption resin etc. are mentioned.
Specific examples of commercially available adsorbents include Amberlite XAD (Rohm and Haas), Diaion (registered trademark) HP-20 (Mitsubishi Chemical Corporation), and the like.
　前記クロマトグラフ法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、薄層クロマトグラフ法、順相あるいは逆相カラムを用いた分取用高速液体クロマトグラフ（分取用ＨＰＬＣ）法などが挙げられる。
　前記クロマトグラフ法に用いる担体としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、イオン交換樹脂、ゲル濾過、シリカゲル、アルミナ、活性炭などが挙げられる。
　前記クロマトグラフ法に用いる担体の市販品の具体例としては、アンバーライト（登録商標）ＣＧ５０（シグマアルドリッチ株式会社製）等のイオン交換樹脂；トヨパール（登録商標）ＨＷ－４０Ｆ（東ソー株式会社製）、セファデックス（登録商標）ＬＨ－２０（ＧＥヘルスケア社製）等のゲル濾過；ＣＡＰＣＥＬＬ　ＰＡＫ　ＳＧ１２０（資生堂株式会社製）等のシリカゲル；などが挙げられる。 The chromatographic method is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a thin layer chromatographic method, a high-performance liquid chromatograph for fractionation using a normal phase or reverse phase column (preparation). HPLC) method and the like.
There is no restriction | limiting in particular as a support | carrier used for the said chromatographic method, According to the objective, it can select suitably, For example, ion exchange resin, gel filtration, a silica gel, an alumina, activated carbon etc. are mentioned.
Specific examples of commercially available carriers used in the chromatographic method include ion exchange resins such as Amberlite (registered trademark) CG50 (manufactured by Sigma Aldrich); Toyopearl (registered trademark) HW-40F (manufactured by Tosoh Corporation) Gel filtration such as Sephadex (registered trademark) LH-20 (manufactured by GE Healthcare); silica gel such as CAPCELL PAK SG120 (manufactured by Shiseido Co., Ltd.), and the like.
　前記吸着剤や前記クロマトグラフ法における担体から前記一般式（Ｉ）で表される化合物を溶出させる方法としては、特に制限はなく、該吸着剤や該担体の種類や性質等に応じて適宜選択することができる。例えば、ポリスチレン系吸着樹脂の場合には、溶出溶媒として、含水アルコール、含水アセトン等を用いて溶出する方法などが挙げられる。 The method for eluting the compound represented by the general formula (I) from the adsorbent or the carrier in the chromatographic method is not particularly limited, and is appropriately selected depending on the kind and properties of the adsorbent and the carrier. can do. For example, in the case of a polystyrene-based adsorption resin, a method of elution using water-containing alcohol, water-containing acetone or the like as an elution solvent can be mentioned.
　以上のようにして前記一般式（Ｉ）で表される化合物を製造することができる。 Thus, the compound represented by the general formula (I) can be produced.
（微生物）
　本発明の微生物は、下記一般式（Ｉ）で表される化合物を生産する能力を有する微生物であって、前記微生物が、ストレプトミセス　エスピー（Ｓｔｒｅｐｔｏｍｙｃｅｓ　ｓｐ．）ＭＫ９３２－ＣＦ８株（受託番号：ＮＩＴＥ　ＢＰ－００８３８）である。
Figure JPOXMLDOC01-appb-C000016
 　ただし、前記一般式（Ｉ）において、Ｘは、下記一般式（ＩＡ）及び下記一般式（ＩＢ）のいずれかで表される基を表す。
Figure JPOXMLDOC01-appb-C000017
 　ただし、前記一般式（ＩＡ）及び前記一般式（ＩＢ）において、＊は、結合手を表す。 (Microorganism)
The microorganism of the present invention is a microorganism having the ability to produce a compound represented by the following general formula (I), and the microorganism is Streptomyces sp. MK932-CF8 strain (Accession number: NITE BP). -00838).
Figure JPOXMLDOC01-appb-C000016
 However, in the said general formula (I), X represents group represented by either the following general formula (IA) or the following general formula (IB).
Figure JPOXMLDOC01-appb-C000017
 However, in the general formula (IA) and the general formula (IB), * represents a bond.
　前記一般式（Ｉ）で表される化合物は、下記構造式（Ｉ）及び下記構造式（ＩＩ）のいずれかで表される化合物であることが好ましい
Figure JPOXMLDOC01-appb-C000018
Figure JPOXMLDOC01-appb-C000019
 The compound represented by the general formula (I) is preferably a compound represented by any one of the following structural formula (I) and the following structural formula (II).
Figure JPOXMLDOC01-appb-C000018
Figure JPOXMLDOC01-appb-C000019
　前記ストレプトミセス　エスピー（Ｓｔｒｅｐｔｏｍｙｃｅｓ　ｓｐ．）ＭＫ９３２－ＣＦ８株（受託番号：ＮＩＴＥ　ＢＰ－００８３８）は、本発明者らが神奈川県横浜市の土壌より分離した菌株である。前記ＭＫ９３２－ＣＦ８株の菌学的性状は、以下の通りである。 The Streptomyces sp. Strain MK932-CF8 (accession number: NITE BP-00838) is a strain isolated by the present inventors from the soil of Yokohama, Kanagawa Prefecture. The mycological properties of the MK932-CF8 strain are as follows.
１．形態
　分枝した基生菌糸より、らせん形成を有する気菌糸を伸長する。成熟した胞子鎖は、１０個～５０個の卵円形の胞子を連鎖する。胞子の大きさは、幅が０．４μｍ～０．５μｍ、長さが０．８μｍ～０．９μｍで、胞子の表面は平滑である。輪生枝、菌糸束、胞子のう及び運動性胞子は認められない。 1. Morphology The aerial hyphae having spiral formation are elongated from the branched basic hyphae. A mature spore chain links 10-50 oval spore. The spore has a width of 0.4 μm to 0.5 μm and a length of 0.8 μm to 0.9 μm, and the spore surface is smooth. No roticular branches, mycelial bundles, spore capsules or motile spores are observed.
２．各種培地における生育状態
　色の記載について［　］内に示す標準は、コンティナー・コーポレーション・オブ・アメリカのカラー・ハーモニー・マニュアル（Ｃｏｎｔａｉｎｅｒ　Ｃｏｒｐｏｒａｔｉｏｎ　ｏｆ　Ａｍｅｒｉｃａ　の　ｃｏｌｏｒ　ｈａｒｍｏｎｙｍａｎｕａｌ）を用いた。
（１）イースト・麦芽寒天培地（ＩＳＰ－培地２、２７℃培養）
　うす黄茶［２ｎｇ，Ｄｕｌｌ　Ｇｏｌｄ］の発育上に、ピンク白［７ｂａ，Ｐｉｎｋ　Ｔｉｎｔ］～明るい灰［ｅ，Ｇｒａｙ］の気菌糸を着生し、可溶性色素は認められない。
（２）オートミール寒天培地（ＩＳＰ－培地３、２７℃培養）
　うす黄［２ｂａ，Ｐｅａｒｌ］の発育上に、明るい灰［３ｆｅ，Ｓｉｌｖｅｒ　Ｇｒａｙ］～灰［３ｍｌ，Ｂｅａｖｅｒ　Ｇｒａｙ］の気菌糸を着生し、培養１０日目より気菌糸の湿潤化が観察された。可溶性色素は認められない。
（３）スターチ・無機塩寒天培地（ＩＳＰ－培地４、２７℃培養）
　うす黄［２ｂａ，Ｐｅａｒｌ］の発育上に、明るい灰［２ｆｅ，Ｃｏｖｅｒｔ　Ｇｒａｙ］の気菌糸を着生し、可溶性色素はうすピンクを帯びていた。０．１モル塩酸及び０．１モル水酸化ナトリウムの添加による発育の色及び可溶性色素の変化は認められない。
（４）グリセリン・アスパラギン寒天培地（ＩＳＰ－培地５、２７℃培養）
　うす黄［２ｂａ，Ｐｅａｒｌ］～うすピンク［７　１／２ｇｃ，Ｄｕｓｔｙ　Ｒｏｓｅ］の発育上に、明るい灰［３ｆｅ，Ｓｉｌｖｅｒ　Ｇｒａｙ］の気菌糸を着生し、培養１０日目より気菌糸の湿潤化が観察される。可溶性色素は認められない。
（５）シュクロース・硝酸塩寒天培地（２７℃培養）
　うす黄茶［２ｎｇ，Ｄｕｌｌ　Ｇｏｌｄ］の発育上に、うす黄［１　１／２ｅａ，Ｌｔ　Ｙｅｌｌｏｗ］の気菌糸を着生し、可溶性色素は茶色味を帯びた。０．１モル塩酸及び０．１モル水酸化ナトリウムの添加による発育の色及び可溶性色素の変化は認められない。 2. About the description of the growth state color in various culture media The standard shown in [] was the color harmony manual of Container Corporation of America (color harmony manual of Container Container Corporation of America).
(1) Yeast / malt agar medium (ISP-medium 2, 27 ° C. culture)
On the growth of light yellow tea [2 ng, Dull Gold], aerial mycelia of pink white [7ba, Pink Tint] to bright ash [e, Gray] are formed, and no soluble pigment is observed.
(2) Oatmeal agar medium (ISP-medium 3, 27 ° C culture)
On the growth of light yellow [2ba, Pearl], aerial hyphae of bright ash [3fe, Silver Gray] to ash [3 ml, Beaver Gray] grew, and wetting of the aerial hyphae was observed from the 10th day of culture. . No soluble pigment is observed.
(3) Starch / inorganic salt agar medium (ISP-medium 4, 27 ° C. culture)
On the development of light yellow [2ba, Pearl], aerial mycelium of bright ash [2fe, Covert Gray] was formed, and the soluble pigment was light pink. There is no change in growth color or soluble pigment due to the addition of 0.1 molar hydrochloric acid and 0.1 molar sodium hydroxide.
(4) Glycerin / asparagine agar medium (ISP-medium 5, 27 ° C. culture)
On the growth of light yellow [2ba, Pearl] to light pink [7 1 / 2gc, Duty Rose], aerial mycelium of bright ash [3fe, Silver Gray] was formed, and the aerial mycelium was moistened from the 10th day of culture. Is observed. No soluble pigment is observed.
(5) Sucrose / Nitrate agar medium (27 ° C culture)
On the growth of light yellow tea [2 ng, Dull Gold], aerial mycelium of light yellow [1 1/2 ea, Lt Yellow] was formed, and the soluble pigment was brownish. There is no change in growth color or soluble pigment due to the addition of 0.1 molar hydrochloric acid and 0.1 molar sodium hydroxide.
３．生育温度範囲
（１）生育温度範囲
　イースト・スターチ寒天培地〔１．０質量％溶性デンプン（でんぷん（溶性）、小宗化学薬品株式会社製）、０．２質量％イーストエキス（粉末酵母エキスＳ、日本製薬株式会社製）、２．６質量％ひも寒天（岐阜細寒天、北原産業株式会社製）、；ｐＨ７．０〕を用い、１０℃、２０℃、２４℃、２７℃、３０℃、３７℃、又は４５℃の各温度で試験した結果、１０℃及び４５℃での生育は認められず、２０℃～３７℃の範囲で生育した。生育至適温度は３０℃付近である。
（２）スターチの加水分解（スターチ・無機塩寒天培地、ＩＳＰ－培地４、２７℃培養）
　培養後３日目にはスターチの加水分解が認められ、その作用は中等度である。 3. Growth temperature range (1) Growth temperature range Yeast starch agar medium [1.0% by mass soluble starch (starch (soluble), manufactured by Koso Chemical Co., Ltd.), 0.2% by mass yeast extract (powdered yeast extract S, Manufactured by Nippon Pharmaceutical Co., Ltd.), 2.6 mass% string agar (Gifu Hogar Agar, Kitahara Sangyo Co., Ltd.), pH 7.0], 10 ° C., 20 ° C., 24 ° C., 27 ° C., 30 ° C., 37 As a result of testing at each temperature of 0 ° C. or 45 ° C., growth at 10 ° C. and 45 ° C. was not observed, and growth occurred in the range of 20 ° C. to 37 ° C. The optimum temperature for growth is around 30 ° C.
(2) Starch hydrolysis (starch / inorganic salt agar medium, ISP-medium 4, 27 ° C. culture)
On the third day after culturing, starch hydrolysis is observed and its action is moderate.
４．菌体成分
　細胞壁中の２，６－ジアミノピメリン酸は、ＬＬ－型である。
　なお、前記菌体成分は、薄層クロマトグラフィーにより分析した。 4). Cellular component 2,6-diaminopimelic acid in the cell wall is LL-type.
The bacterial cell component was analyzed by thin layer chromatography.
５．１６Ｓ　ｒＲＮＡ遺伝子解析
　１６Ｓ　ｒＲＮＡ遺伝子の部分塩基配列（１，４３２ｂｐ）を決定し、ＤＮＡデータベース（ＤＮＡ　Ｄａｔａ　Ｂａｎｋ　ｏｆ　Ｊａｐａｎ）に登録された公知菌株のデータと比較した。
　その結果、ＭＫ９３２－ＣＦ８株の塩基配列は、以下に示すように、ストレプトミセス（Ｓｔｒｅｐｔｏｍｙｃｅｓ）属放線菌の１６Ｓ　ｒＲＮＡ遺伝子と高い相同性を示した。即ち、Ｓｔｒｅｐｔｏｍｙｃｅｓ　ｐｌａｔｅｎｓｉｓ（９９％）、Ｓ．ｈｙｇｒｏｓｃｏｐｉｃｕｓ　ｓｕｂｓｐ．　ｇｌｅｂｏｓｕｓ（９９％）、Ｓ．ｌｉｂａｎｉ　ｓｕｂｓｐ．　ｒｕｆｕｓ（９９％）、Ｓ．ｃａｎｉｆｅｒｕｓ（９９％）、Ｓ．ｎｉｇｒｅｓｃｅｎｓ（９８％）、Ｓ．ｃａｔｅｎｕｌａｅ（９８％）などである。なお、前記括弧内の数値（％）は、塩基配列の相同値を示す。 5. Analysis of 16S rRNA gene A partial base sequence (1,432 bp) of 16S rRNA gene was determined and compared with data of known strains registered in a DNA database (DNA Data Bank of Japan).
As a result, the base sequence of the MK932-CF8 strain showed high homology with the 16S rRNA gene of Streptomyces genus Streptomyces as shown below. Streptomyces platensis (99%), S. hygroscopicus subsp. glebosus (99%), S. libani subsp. rufus (99%), S. caniferus (99%), S. nigrescens (98%), S. catenulee (98%). In addition, the numerical value (%) in the parenthesis indicates the homology value of the base sequence.
　以上の菌学的性状を要約すると、ＭＫ９３２－ＣＦ８株は、その形態上、よく分枝した基生菌糸より、らせん形成を有する気菌糸を伸長し、その先端は卵円形の胞子を連鎖する。種々の培地で、うす黄～うす黄茶の発育上に明るい灰～灰の気菌糸を着生する。生育至適温度は３０℃付近である。スターチの水解性は中等度である。
　ＭＫ９３２－ＣＦ８株の細胞壁中の２，６－ジアミノピメリン酸はＬＬ－型である。
　ＭＫ９３２－ＣＦ８株の１６ＳｒＲＮＡ遺伝子の部分塩基配列を解析し、公知菌株のデータと比較したところ、ストレプトミセス属放線菌と高い相同性を示した。 Summarizing the above bacteriological properties, the strain MK932-CF8 extends from its well-branched basic mycelium to aerial hyphae that form a spiral, and its tip links oval spores. In various media, light gray to gray aerial hyphae grow on light yellow to light yellow tea. The optimum temperature for growth is around 30 ° C. The water disintegration of starch is moderate.
2,6-Diaminopimelic acid in the cell wall of MK932-CF8 strain is LL-type.
When the partial base sequence of 16S rRNA gene of MK932-CF8 strain was analyzed and compared with the data of known strains, it showed high homology with Streptomyces genus actinomycetes.
　以上の結果より、ＭＫ９３２－ＣＦ８株は、ストレプトミセス（Ｓｔｒｅｐｔｏｍｙｃｅｓ）属に属するものと考えられた。そこで、ＭＫ９３２－ＣＦ８株をストレプトミセス・エスピー（Ｓｔｒｅｐｔｏｍｙｃｅｓ　ｓｐ．）ＭＫ９３２－ＣＦ８株とした。
　なお、前記ＭＫ９３２－ＣＦ８株は、独立行政法人製品評価技術基盤機構　特許微生物寄託センター（〒２９２－０８１８　千葉県木更津市かずさ鎌足２－５－８）に寄託申請し、２００９年１１月１３日に国内寄託され、その後、２０１３年７月１７日にブダペスト条約に基づく国際寄託への移管請求がされ、受託番号ＮＩＴＥ　ＢＰ－００８３８として国際寄託されている。 From the above results, MK932-CF8 strain was considered to belong to the genus Streptomyces . Therefore, the strain MK932-CF8 was designated as Streptomyces sp. MK932-CF8.
The MK932-CF8 strain was filed for deposit at the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamashichi, Kisarazu, Chiba 292-0818), November 13, 2009. After that, on July 17, 2013, a request was made to transfer to an international deposit under the Budapest Treaty, and the deposit was made internationally under the deposit number NITE BP-00838.
　なお、他の菌にも見られるように、前記ＭＫ９３２－ＣＦ８株は、性状が変化し易いが、例えば、前記ＭＫ９３２－ＣＦ８株に由来する突然変異株（例えば、紫外線、エックス線、放射線、薬品等の変異処理により取得できる人工変異株や、自然変異株）、形質接合体、遺伝子組換体などであっても、前記一般式（Ｉ）で表される化合物を生産する能力を有するものは、本発明の微生物に含まれる。 As seen in other bacteria, the MK932-CF8 strain is susceptible to changes in properties. For example, mutant strains derived from the MK932-CF8 strain (for example, ultraviolet rays, X-rays, radiation, drugs, etc.) Even artificial mutants, natural mutants), zygotes, recombinants, etc. that can be obtained by the mutation treatment of the above are those having the ability to produce the compound represented by the general formula (I). Included in the microorganism of the invention.
（医薬組成物）
　本発明の医薬組成物は、本発明の前記化合物を少なくとも含有し、必要に応じて、更にその他の成分を含有する。 (Pharmaceutical composition)
The pharmaceutical composition of the present invention contains at least the compound of the present invention, and further contains other components as necessary.
＜一般式（Ｉ）で表される化合物＞
　前記医薬組成物中の前記一般式（Ｉ）で表される化合物の含有量としては、特に制限はなく、目的に応じて適宜選択することができる。また、前記医薬組成物は、前記一般式（Ｉ）で表される化合物そのものであってもよい。 <Compound represented by formula (I)>
There is no restriction | limiting in particular as content of the compound represented by the said general formula (I) in the said pharmaceutical composition, According to the objective, it can select suitably. The pharmaceutical composition may be the compound itself represented by the general formula (I).
＜その他の成分＞
　前記その他の成分としては、特に制限はなく、例えば、薬理学的に許容され得る担体の中から目的に応じて適宜選択することができ、例えば、添加剤、補助剤、水などが挙げられる。これらは、１種単独で使用してもよく、２種以上を併用してもよい。 <Other ingredients>
There is no restriction | limiting in particular as said other component, For example, it can select suitably according to the objective from the support | carrier accept | permitted pharmacologically, For example, an additive, an adjuvant, water, etc. are mentioned. These may be used alone or in combination of two or more.
　前記添加剤又は前記補助剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、殺菌剤、保存剤、粘結剤、増粘剤、固着剤、結合剤、着色剤、安定化剤、ｐＨ調整剤、緩衝剤、等張化剤、溶剤、酸化防止剤、紫外線防止剤、結晶析出防止剤、消泡剤、物性向上剤、防腐剤などが挙げられる。 The additive or the adjuvant is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include bactericides, preservatives, binders, thickeners, fixing agents, binders, and coloring agents. , Stabilizers, pH adjusters, buffers, isotonic agents, solvents, antioxidants, UV inhibitors, crystal precipitation inhibitors, antifoaming agents, physical property improvers, preservatives, and the like.
　前記殺菌剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、塩化ベンザルコニウム、塩化ベンゼトニウム、塩化セチルピリジニウム等のカチオン性界面活性剤などが挙げられる。 The bactericidal agent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include cationic surfactants such as benzalkonium chloride, benzethonium chloride and cetylpyridinium chloride.
　前記保存剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、パラオキシ安息香酸エステル類、クロロブタノール、クレゾールなどが挙げられる。 The preservative is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include p-hydroxybenzoates, chlorobutanol, and cresol.
　前記粘結剤、増粘剤、固着剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、デンプン、デキストリン、セルロース、メチルセルロース、エチルセルロース、カルボキシメチルセルロース、ヒドロキシエチルセルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、カルボキシメチルデンプン、プルラン、アルギン酸ナトリウム、アルギン酸アンモニウム、アルギン酸プロピレングリコールエステル、グアーガム、ローカストビーンガム、アラビアゴム、キサンタンガム、ゼラチン、カゼイン、ポリビニルアルコール、ポリエチレンオキサイド、ポリエチレングリコール、エチレン・プロピレンブロックポリマー、ポリアクリル酸ナトリウム、ポリビニルピロリドンなどが挙げられる。 The binder, thickener and fixing agent are not particularly limited and may be appropriately selected depending on the intended purpose. For example, starch, dextrin, cellulose, methylcellulose, ethylcellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropyl Cellulose, hydroxypropylmethylcellulose, carboxymethyl starch, pullulan, sodium alginate, ammonium alginate, propylene glycol ester alginate, guar gum, locust bean gum, gum arabic, xanthan gum, gelatin, casein, polyvinyl alcohol, polyethylene oxide, polyethylene glycol, ethylene propylene Block polymers, sodium polyacrylate, polyvinylpyrrolidone, etc. .
　前記結合剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、水、エタノール、プロパノール、単シロップ、ブドウ糖液、デンプン液、ゼラチン液、カルボキシメチルセルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルスターチ、メチルセルロース、エチルセルロース、シェラック、リン酸カルシウム、ポリビニルピロリドンなどが挙げられる。 The binder is not particularly limited and may be appropriately selected depending on the intended purpose. For example, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxy Examples include propyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate, and polyvinyl pyrrolidone.
　前記着色剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、酸化チタン、酸化鉄などが挙げられる。 The colorant is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include titanium oxide and iron oxide.
　前記安定化剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、トラガント、アラビアゴム、ゼラチン、ピロ亜硫酸ナトリウム、エチレンジアミン四酢酸（ＥＤＴＡ）、チオグリコール酸、チオ乳酸などが挙げられる。 The stabilizer is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include tragacanth, gum arabic, gelatin, sodium pyrosulfite, ethylenediaminetetraacetic acid (EDTA), thioglycolic acid, and thiolactic acid. Is mentioned.
　前記ｐＨ調整剤又は前記緩衝剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、クエン酸ナトリウム、酢酸ナトリウム、リン酸ナトリウムなどが挙げられる。 The pH adjusting agent or the buffering agent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include sodium citrate, sodium acetate, and sodium phosphate.
　前記等張化剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、塩化ナトリウム、ブドウ糖などが挙げられる。 The isotonic agent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include sodium chloride and glucose.
　前記医薬組成物中の、前記その他の成分の含有量としては、特に制限はなく、前記一般式（Ｉ）で表される化合物の効果を損なわない範囲内で、目的に応じて適宜選択することができる。 There is no restriction | limiting in particular as content of the said other component in the said pharmaceutical composition, In the range which does not impair the effect of the compound represented by the said general formula (I), it selects suitably according to the objective. Can do.
＜用途＞
　前記医薬組成物は、前記一般式（Ｉ）で表される化合物を含むため、優れた抗腫瘍作用を有し、安全性の高いものであり、後述する本発明の抗腫瘍剤などに好適に利用可能である。 <Application>
Since the said pharmaceutical composition contains the compound represented by the said general formula (I), it has the outstanding anti-tumor action and high safety | security, and is suitable for the antitumor agent of this invention mentioned later etc. Is available.
（抗腫瘍剤）
　本発明の抗腫瘍剤は、本発明の前記医薬組成物を少なくとも含有し、必要に応じて、更にその他の成分を含有する。 (Anti-tumor agent)
The antitumor agent of the present invention contains at least the pharmaceutical composition of the present invention, and further contains other components as necessary.
＜医薬組成物＞
　前記抗腫瘍剤中の前記医薬組成物の含有量としては、特に制限はなく、目的に応じて適宜選択することができる。また、前記抗腫瘍剤は、前記医薬組成物そのものであってもよい。 <Pharmaceutical composition>
There is no restriction | limiting in particular as content of the said pharmaceutical composition in the said antitumor agent, According to the objective, it can select suitably. The antitumor agent may be the pharmaceutical composition itself.
＜その他の成分＞
　前記その他の成分としては、特に制限はなく、例えば、前記医薬組成物中のその他の成分と同様のものなどが挙げられる。
　前記抗腫瘍剤中の、前記その他の成分の含有量としては、特に制限はなく、前記一般式（Ｉ）で表される化合物の効果を損なわない範囲内で、目的に応じて適宜選択することができる。 <Other ingredients>
There is no restriction | limiting in particular as said other component, For example, the same thing as the other component in the said pharmaceutical composition etc. are mentioned.
There is no restriction | limiting in particular as content of the said other component in the said antitumor agent, In the range which does not impair the effect of the compound represented by the said general formula (I), it selects suitably according to the objective. Can do.
　なお、前記抗腫瘍剤は、１種単独で使用してもよいし、他の成分を有効成分とする医薬や薬剤と併せて使用してもよい。また、前記抗腫瘍剤は、他の成分を有効成分とする医薬や薬剤中に配合された状態で使用してもよい。 In addition, the said antitumor agent may be used individually by 1 type, and may be used together with the pharmaceutical and chemical | medical agent which use another component as an active ingredient. Moreover, you may use the said antitumor agent in the state mix | blended in the medicine and chemical | medical agent which use another component as an active ingredient.
＜抗腫瘍作用＞
　前記抗腫瘍剤の抗腫瘍作用を調べる方法としては、特に制限はなく、公知の方法の中から目的に応じて適宜選択することができ、例えば、後述する試験例に記載の方法などが挙げられる。 <Anti-tumor action>
The method for examining the antitumor action of the antitumor agent is not particularly limited and may be appropriately selected from known methods according to the purpose. Examples thereof include the methods described in Test Examples described later. .
＜剤型＞
　前記抗腫瘍剤の剤型としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、固形剤、半固形剤、液剤などが挙げられる。 <Dosage form>
There is no restriction | limiting in particular as a dosage form of the said antitumor agent, According to the objective, it can select suitably, For example, a solid agent, a semi-solid agent, a liquid agent etc. are mentioned.
－固形剤－
　前記固形剤としては、特に制限はなく、目的に応じて適宜選択することができるが、内用剤として用いられる場合、例えば、錠剤、チュアブル錠、発泡錠、口腔内崩壊錠、トローチ剤、ドロップ剤、硬カプセル剤、軟カプセル剤、顆粒剤、散剤、丸剤、ドライシロップ剤、浸剤などが挙げられる。
　前記固形剤が、外用剤として用いられる場合、例えば、坐剤、パップ剤、プラスター剤などが挙げられる。 -Solid agent-
The solid preparation is not particularly limited and may be appropriately selected depending on the intended purpose. However, when used as an internal preparation, for example, a tablet, chewable tablet, effervescent tablet, orally disintegrating tablet, troche, drop Agents, hard capsules, soft capsules, granules, powders, pills, dry syrups, soaking agents and the like.
When the said solid agent is used as an external preparation, a suppository, a poultice, a plaster agent etc. are mentioned, for example.
－半固形剤－
　前記半固形剤としては、特に制限はなく、目的に応じて適宜選択することができるが、内用剤として用いられる場合、例えば、舐剤、チューインガム剤、ホイップ剤、ゼリー剤などが挙げられる。
　前記半固形剤が、外用剤として用いられる場合、例えば、軟膏剤、クリーム剤、ムース剤、インヘラー剤、ナザールジェル剤などが挙げられる。 -Semi-solid agent-
There is no restriction | limiting in particular as said semi-solid preparation, Although it can select suitably according to the objective, When using as an internal preparation, a licking agent, a chewing gum agent, a whipping agent, a jelly agent etc. are mentioned, for example.
When the semi-solid preparation is used as an external preparation, examples thereof include an ointment, a cream, a mousse, an inhaler, and a nazar gel.
－液剤－
　前記液剤としては、特に制限はなく、目的に応じて適宜選択することができるが、内用剤として用いられる場合、例えば、シロップ剤、ドリンク剤、懸濁剤、酒精剤などが挙げられる。
　前記液剤が、外用剤として用いられる場合、例えば、液剤、点眼剤、エアゾール剤、噴霧剤などが挙げられる。 -Liquid-
There is no restriction | limiting in particular as said liquid agent, Although it can select suitably according to the objective, When used as an internal preparation, a syrup agent, a drink agent, a suspension agent, an alcoholic agent etc. are mentioned, for example.
When the said liquid agent is used as an external preparation, a liquid agent, eye drops, an aerosol agent, a spray agent etc. are mentioned, for example.
＜投与＞
　前記抗腫瘍剤の投与方法、投与量、投与時期、及び投与対象としては、特に制限はなく、目的に応じて適宜選択することができる。
　前記投与方法としては、例えば、局所投与法、経腸投与法、非経口投与法などが挙げられる。
　前記投与量としては、特に制限はなく、投与対象個体の年齢、体重、体質、症状、他の成分を有効成分とする医薬や薬剤の投与の有無など、様々な要因を考慮して適宜選択することができる。
　前記投与対象となる動物種としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ヒト、サル、ブタ、ウシ、ヒツジ、ヤギ、イヌ、ネコ、マウス、ラット、トリなどが挙げられるが、これらの中でもヒトに好適に用いられる。 <Administration>
There is no restriction | limiting in particular as an administration method of the said anti-tumor agent, dosage amount, administration time, and administration object, According to the objective, it can select suitably.
Examples of the administration method include a local administration method, an enteral administration method, a parenteral administration method and the like.
The dose is not particularly limited, and is appropriately selected in consideration of various factors such as the age, weight, constitution, symptom, and presence / absence of administration of a drug or drug containing other ingredients as active ingredients. be able to.
The animal species to be administered is not particularly limited and can be appropriately selected according to the purpose. For example, human, monkey, pig, cow, sheep, goat, dog, cat, mouse, rat, bird, etc. Among these, it is preferably used for humans.
＜用途＞
　前記抗腫瘍剤は、優れた抗腫瘍作用を有し、安全性が高いため、癌の予防剤又は治療剤として好適に利用可能である。前記抗腫瘍剤は、腫瘍の中でも、アンドロゲン依存性の増殖に対する増殖抑制作用を有するため、好ましくは抗前立腺癌剤として利用でき、また、前立腺癌の予防剤又は治療剤としても好適に利用可能である。更に、アンドロゲンレセプターに依存して増殖をする他の腫瘍（例えば、乳癌、子宮癌など）に対しても予防又は治療剤として利用可能である。 <Application>
Since the antitumor agent has an excellent antitumor action and high safety, it can be suitably used as a preventive or therapeutic agent for cancer. Since the antitumor agent has a growth inhibitory action against androgen-dependent growth among tumors, it can be preferably used as an anti-prostate cancer agent, and can also be suitably used as a prophylactic or therapeutic agent for prostate cancer. is there. Furthermore, it can be used as a prophylactic or therapeutic agent for other tumors that proliferate depending on the androgen receptor (for example, breast cancer, uterine cancer, etc.).
　以下に本発明の実施例を挙げて本発明を具体的に説明するが、本発明はこれらの実施例に何ら限定されるものではない。 Hereinafter, the present invention will be specifically described with reference to examples of the present invention, but the present invention is not limited to these examples.
（製造例１）
＜アンドロプロスタミンＡ及びアンドロプロスタミンＢの製造＞
－種母培養液の調製－
　２．０質量％ガラクトース（Ｄ－（＋）－ガラクトース、和光純薬工業株式会社製）、２．０質量％デキストリン（デキストリン水和物、和光純薬工業株式会社製）、１．０質量％バクトソイトン（日本ベクトン・ディッキンソン株式会社製）、０．５質量％コーン・スティープ・リカー（向後スターチ株式会社製）、０．２質量％硫酸アンモニウム（和光純薬工業株式会社製）、０．２質量％炭酸カルシウム（和光純薬工業株式会社製）、シリコーン消泡剤〔シリコーン消泡剤（ＫＭ－７０、信越化学工業株式会社製）と、大豆油（和光純薬工業株式会社製）とを１：１（体積比）で混合した溶液〕３０μＬを含む液体培地（ｐＨ７．４）を震盪フラスコ（５００ｍＬ容）に１１０ｍＬずつ分注し、常法により１２０℃で２０分間滅菌した。これらのフラスコ内の滅菌された液体培地に、寒天斜面培地に培養したＭＫ９３２－ＣＦ８株を接種し、２７℃で３日間震盪培養し、この培養液を種母培養液とした。 (Production Example 1)
<Production of Androprostamine A and Androprostamine B>
-Preparation of seed culture-
2.0% by mass galactose (D-(+)-galactose, manufactured by Wako Pure Chemical Industries, Ltd.), 2.0% by mass dextrin (dextrin hydrate, manufactured by Wako Pure Chemical Industries, Ltd.), 1.0% by mass Bact soyton (Nippon Becton Dickinson Co., Ltd.), 0.5% by mass Corn steep liquor (Mukago Starch Co., Ltd.), 0.2% by mass ammonium sulfate (Wako Pure Chemical Industries, Ltd.), 0.2% by mass Calcium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.), silicone antifoaming agent (silicone defoaming agent (KM-70, manufactured by Shin-Etsu Chemical Co., Ltd.)) and soybean oil (manufactured by Wako Pure Chemical Industries, Ltd.) Solution mixed at 1 (volume ratio)] Liquid medium (pH 7.4) containing 30 μL was dispensed 110 mL each into a shake flask (500 mL volume) and sterilized at 120 ° C. for 20 minutes by a conventional method. The sterilized liquid medium in these flasks was inoculated with the strain MK932-CF8 cultured on an agar slant medium, and cultured with shaking at 27 ° C. for 3 days. This culture was used as a seed culture.
－培養工程（固体培養）－
　次に、１０枚のバット（２，１００ｍＬ容）に、１バット当たり１５０ｇの押し麦（胚芽押し麦、ムソー株式会社製）及び２５０ｍＬの脱イオン水を含む固体培地を添加し、これを常法により１２０℃で２０分間滅菌した。この固体培地上に前記種母培養液７０ｍＬを接種し、３０℃で１４日間静置培養した。次いで、各バットの固体培地を１００体積％エタノール４００ｍＬで抽出後、再度７０体積％エタノール水溶液４００ｍＬで抽出し、培養調整液を得た。 -Culture process (solid culture)-
Next, a solid medium containing 150 g of pressed barley (germ pressed barley, manufactured by Muso Co., Ltd.) and 250 mL of deionized water was added to 10 vats (2,100 mL), and this was added by a conventional method. Sterilized at 20 ° C. for 20 minutes. On this solid medium, 70 mL of the seed culture solution was inoculated, followed by stationary culture at 30 ° C. for 14 days. Subsequently, after extracting the solid medium of each vat with 400 mL of 100 volume% ethanol, it extracted again with 400 mL of 70 volume% ethanol aqueous solution, and the culture adjustment liquid was obtained.
－洗浄工程－
　このようにして得られた培養調整液８Ｌを遠心分離し、菌体と培養濾液に分離した。この培養濾液は、減圧下で濃縮し、エタノールを取り除き、脱イオン水を加えることにより、合計２Ｌの水溶液を得た。この水溶液を５００ｍＬの合成吸着剤（ダイヤイオン（登録商標）ＨＰ２０、三菱化学株式会社製）に付し、２Ｌの脱イオン水で洗浄した後、２Ｌの５０体積％アセトンで溶出した。この溶出画分を減圧下で濃縮しアセトンを取り除き２００ｍＬの水溶液を得た。 -Washing process-
The culture preparation solution 8L thus obtained was centrifuged to separate the cells and culture filtrate. The culture filtrate was concentrated under reduced pressure, ethanol was removed, and deionized water was added to obtain a total 2 L aqueous solution. This aqueous solution was applied to 500 mL of a synthetic adsorbent (Diaion (registered trademark) HP20, manufactured by Mitsubishi Chemical Corporation), washed with 2 L of deionized water, and then eluted with 2 L of 50 vol% acetone. This elution fraction was concentrated under reduced pressure to remove acetone and obtain 200 mL of an aqueous solution.
－採取工程－
　前記水溶液を、更にイオン交換樹脂（アンバーライト（登録商標）ＣＧ５０、シグマアルドリッチ株式会社製）に付し、通過液を得た。この通過液を減圧下で濃縮し、３０ｍＬの水溶液を得た。次いで、この水溶液をサイズ排除クロマトグラフ用樹脂（トヨパール（登録商標）ＨＷ－４０、東ソー株式会社製）によるゲル濾過クロマトグラフィーに供し、脱イオン水で溶出した。得られた溶出画分を減圧下で濃縮乾固し、１．０６６ｇの粗精製物を得た。この粗精製物を、１，０００ｍＬのゲル濾過クロマトグラフ用担体（セファデックス（登録商標）ＬＨ－２０、ＧＥヘルスケア社製）によるクロマトグラフィーに供し、５０体積％メタノール水で溶出した。得られた画分を濃縮乾固し８７．２ｍｇの粗精製物を得た。 -Sampling process-
The aqueous solution was further applied to an ion exchange resin (Amberlite (registered trademark) CG50, manufactured by Sigma Aldrich Co., Ltd.) to obtain a passing liquid. The passing liquid was concentrated under reduced pressure to obtain 30 mL of an aqueous solution. Next, this aqueous solution was subjected to gel filtration chromatography using a resin for size exclusion chromatography (Toyopearl (registered trademark) HW-40, manufactured by Tosoh Corporation) and eluted with deionized water. The obtained elution fraction was concentrated to dryness under reduced pressure to obtain 1.066 g of a crude product. This crude product was subjected to chromatography using 1,000 mL of a gel filtration chromatographic carrier (Sephadex (registered trademark) LH-20, manufactured by GE Healthcare) and eluted with 50% by volume methanol water. The obtained fraction was concentrated to dryness to obtain 87.2 mg of a crude product.
－精製工程－
　得られた粗精製物８７．２ｍｇを高速液体クロマトグラフィー（カラム：ＣＡＰＣＥＬＬ　ＰＡＫ　ＳＧ１２０、資生堂株式会社製）に供し、１体積％トリフルオロ酢酸を含む１０体積％アセトニトリル水溶液で溶出した。得られた溶出画分を濃縮乾固し、白色粉末のアンドロプロスタミンＡを２３．９ｍｇ、及び白色粉末のアンドロプロスタミンＢを１２．０ｍｇ、をそれぞれ単離精製した。 -Purification process-
The obtained crude product (87.2 mg) was subjected to high performance liquid chromatography (column: CAPCELL PAK SG120, manufactured by Shiseido Co., Ltd.) and eluted with 10 volume% aqueous acetonitrile containing 1 volume% trifluoroacetic acid. The elution fraction thus obtained was concentrated to dryness, and 23.9 mg of white powder androprostamine A and 12.0 mg of white powder androprostamine B were isolated and purified.
（製造例２）
＜アンドロプロスタミンＡの製造＞
－培養工程（液体培養）－
　１．５質量％ポテトスターチ（バレイショデンプン、吉田製薬株式会社製）、０．７５質量％トーストソーヤ（脱脂大豆、日清オイリオグループ株式会社製）、０．２５質量％コーン・スティープ・リカー（向後スターチ株式会社製）、０．１質量％酵母エキス（オリエンタル酵母工業株式会社製）、０．１５質量％塩化ナトリウム（国産化学株式会社製）、０．０２５質量％硫酸マグネシウム七水和物（国産化学株式会社製）、０．１５質量％炭酸カルシウム（国産化学株式会社製）、０．０００５質量％塩化コバルト（ＩＩ）六水和物（和光純薬株式会社製）を含む液体培地（ｐＨ７．２）を震盪フラスコ（５００ｍＬ容）１１０ｍＬずつ分注し、常法により１２０℃で２０分間滅菌した。
　これらのラスコ内の滅菌された液体培地に、製造例１で調製した種母培養液を、それぞれ３ｍＬ接種し、２７℃で５日間震盪培養した。 (Production Example 2)
<Production of Androprostamine A>
-Culture process (liquid culture)-
1.5% by mass potato starch (potato starch, manufactured by Yoshida Pharmaceutical Co., Ltd.), 0.75% by mass toasted soya (defatted soybeans, manufactured by Nisshin Oillio Group Co., Ltd.), 0.25% by mass corn steep liquor (Okogo) Starch Co., Ltd.), 0.1% by mass yeast extract (Oriental Yeast Co., Ltd.), 0.15% by mass sodium chloride (Kokusan Chemical Co., Ltd.), 0.025% by mass magnesium sulfate heptahydrate (Domestic product) Chemical Co., Ltd.), 0.15 mass% calcium carbonate (produced by Kokusan Chemical Co., Ltd.), 0.0005 mass% cobalt chloride (II) hexahydrate (produced by Wako Pure Chemical Industries, Ltd.) (pH 7. 110 ml of shake flasks (500 mL volume) were dispensed 2) and sterilized at 120 ° C. for 20 minutes by a conventional method.
These sterilized liquid media in Lasco were inoculated with 3 mL each of the seed mother culture solution prepared in Production Example 1 and cultured with shaking at 27 ° C. for 5 days.
－洗浄工程－
　得られた培養液２Ｌを遠心分離し、培養濾液１．６Ｌを得た。この培養濾液を４００ｍＬの合成吸着剤（ダイヤイオン（登録商標）ＨＰ２０、三菱化学株式会社製）に付し、８００ｍＬの脱イオン水で洗浄した後、４００ｍＬの１０体積％アセトン水で溶出した。この溶出画分を減圧下で濃縮乾固し、１．３０５ｇの粗精製物を得た。 -Washing process-
2 L of the obtained culture solution was centrifuged to obtain 1.6 L of a culture filtrate. This culture filtrate was applied to 400 mL of a synthetic adsorbent (Diaion (registered trademark) HP20, manufactured by Mitsubishi Chemical Corporation), washed with 800 mL of deionized water, and then eluted with 400 mL of 10% by volume acetone water. This elution fraction was concentrated to dryness under reduced pressure to obtain 1.305 g of a crude product.
－採取工程－
　前記粗精製物を２０ｍＬの５０体積％メタノール水に溶解し、９００ｍＬのゲル濾過クロマトグラフィー（セファデックス（登録商標）ＬＨ－２０、ＧＥヘルスケア社製）によるクロマトグラフィーに供し、５０体積％メタノール水で溶出した。得られた溶出画分を減圧下で濃縮乾固し、４２６．１ｍｇの粗精製物を得た。次いで、この粗精製物を２０ｍＬの脱イオン水に溶解し、５００ｍＬのサイズ排除クロマトグラフ用樹脂（トヨパール（登録商標）ＨＷ－４０、東ソー株式会社製）によるゲル濾過クロマトグラフィーに供し、脱イオン水で溶出した。得られた溶出画分を減圧下で濃縮乾固し、２０．９ｍｇの粗精製物を得た。次いで、この粗精製物を２ｍＬの脱イオン水に溶解し、２００ｍＬのゲル濾過クロマトグラフィー（セファデックス（登録商標）ＬＨ－２０、ＧＥヘルスケア社製）によるクロマトグラフィーに供し、６６体積％メタノール水で溶出した。得られた溶出画分を減圧下で濃縮乾固し、９．２ｍｇの粗精製物を得た。 -Sampling process-
The crude product is dissolved in 20 mL of 50% by volume methanol water and subjected to chromatography by 900 mL gel filtration chromatography (Sephadex (registered trademark) LH-20, manufactured by GE Healthcare), and 50% by volume methanol aqueous solution. Eluted with. The obtained elution fraction was concentrated to dryness under reduced pressure to obtain 426.1 mg of a crude product. The crude product was then dissolved in 20 mL of deionized water and subjected to gel filtration chromatography using 500 mL of a size exclusion chromatography resin (Toyopearl (registered trademark) HW-40, manufactured by Tosoh Corporation). Eluted with. The obtained elution fraction was concentrated to dryness under reduced pressure to obtain 20.9 mg of a crude product. Next, this crude product was dissolved in 2 mL of deionized water, and subjected to chromatography by 200 mL of gel filtration chromatography (Sephadex (registered trademark) LH-20, manufactured by GE Healthcare), and 66 volume% aqueous methanol Eluted with. The obtained elution fraction was concentrated to dryness under reduced pressure to obtain 9.2 mg of a crude product.
－精製工程－
　得られた粗精製物を１ｍＬの脱イオン水に溶解し、１０ｍＬの逆層クロマトグラフィー（ダイヤイオン（登録商標）ＣＨＰ２０Ｐ、三菱化学株式会社製）に付し、脱イオン水－メタノールの濃度勾配溶出を行った。得られた溶出画分を濃縮乾固し、アンドロプロスタミンＡ　２．４ｍｇを得た。
　なお、製造例２の方法では、アンドロプロスタミンＢは、得られなかった。 -Purification process-
The obtained crude product is dissolved in 1 mL of deionized water, and subjected to 10 mL of reverse layer chromatography (Diaion (registered trademark) CHP20P, manufactured by Mitsubishi Chemical Corporation) to elute the gradient of deionized water-methanol. Went. The obtained elution fraction was concentrated to dryness to obtain 2.4 mg of androprostamine A.
In addition, in the method of Production Example 2, androprostamine B was not obtained.
＜アンドロプロスタミンＡ及びアンドロプロスタミンＢの理化学的性状の分析＞
－アンドロプロスタミンＡの理化学的性状－
　製造例１及び２で得られたアンドロプロスタミンＡの理化学的性状は、以下のとおりであった。
（１）　外観は、白色の粉状であった。
（２）　分子式は、Ｃ２６Ｈ３８Ｎ５Ｏ１０Ｃｌで表され、分子量は、６１５であった。
（３）　高分解能質量分析（ＨＲＥＳＩＭＳ：正イオンモード）による、実験値は、ｍ／ｚ　６１６．２３８６（Ｍ＋Ｈ）＋であり、計算値は、ｍ／ｚ　６１６．２３８０（Ｃ２６Ｈ３９Ｎ５Ｏ１０Ｃｌとして）であった。
（４）　比旋光度は、［α］Ｄ ２３＝＋７６．８°（ｃ０．１，メタノール）であった。
（５）　メタノール溶液で測定した紫外吸収スペクトルは、前記表１に示すとおりであった。
　　　　λｍａｘ　ｎｍ（ε）　：２２７（１３，０００，ｓｈ），２８９（１１，０００）
（６）　ＴＬＣプレート（ＴＬＣ　Ｓｉｌｉｃａ　ｇｅｌ　６０Ｆ２５４、メルク製）の薄層クロマトグラフィーにおいて、展開溶媒〔ブタノール：メタノール：水（４：１：２、体積比）〕で展開して測定したＲＦ値は、０．２２であり、展開溶媒〔ブタノール：酢酸：水（４：１：２、体積比）〕で展開して測定したＲＦ値は、０．２７であった。 <Analysis of physicochemical properties of androprostamine A and androprostamine B>
-Physicochemical properties of androprostamine A-
The physicochemical properties of androprostamine A obtained in Production Examples 1 and 2 were as follows.
(1) The appearance was a white powder.
(2) The molecular formula was represented by C 26 H 38 N 5 O 10 Cl, and the molecular weight was 615.
(3) The experimental value by high resolution mass spectrometry (HRESIMS: positive ion mode) is m / z 616.2386 (M + H) + , and the calculated value is m / z 616.2380 (C 26 H 39 N 5 As O 10 Cl).
(4) The specific rotation was [α] D 23 = + 76.8 ° (c0.1, methanol).
(5) The ultraviolet absorption spectrum measured with a methanol solution was as shown in Table 1 above.
λ max nm (ε): 227 (13,000, sh), 289 (11,000)
(6) In a thin layer chromatography of a TLC plate (TLC Silica gel 60F254, manufactured by Merck), an RF value measured by developing with a developing solvent [butanol: methanol: water (4: 1: 2, volume ratio)] is The RF value measured by developing with a developing solvent [butanol: acetic acid: water (4: 1: 2, volume ratio)] was 0.27.
－アンドロプロスタミンＢの理化学的性状－
　製造例１で得られたアンドロプロスタミンＢの理化学的性状は、以下のとおりであった。
（１）　外観は、白色の粉状であった。
（２）　分子式は、Ｃ３１Ｈ４５Ｎ６Ｏ１１Ｃｌで表され、分子量は、７１２であった。
（３）　高分解能質量分析（ＨＲＥＳＩＭＳ：正イオンモード）による、実験値は、ｍ／ｚ　７１３．２９０３（Ｍ＋Ｈ）＋であり、計算値は、ｍ／ｚ　７１３．２９０８（Ｃ３１Ｈ４６Ｎ６Ｏ１１Ｃｌとして）であった。
（４）　比旋光度は、［α］Ｄ ２３＝＋５６．８°（ｃ０．２、メタノール）であった。
（５）　メタノール溶液で測定した紫外吸収スペクトルは、前記表１に示すとおりであった。
　　　　λｍａｘ　ｎｍ（ε）　：２２７（１５，１００，ｓｈ），２８９（１２，８００）
（６）　ＴＬＣプレート（ＴＬＣ　Ｓｉｌｉｃａ　ｇｅｌ　６０Ｆ２５４、メルク製）の薄層クロマトグラフィーにおいて、展開溶媒〔ブタノール：メタノール：水（４：１：２、体積比）〕で展開して測定したＲＦ値は、０．１８であり、展開溶媒〔ブタノール：酢酸：水（４：１：２、体積比）〕で展開して測定したＲＦ値は、０．２６であった。 -Physicochemical properties of androprostamine B-
The physicochemical properties of androprostamine B obtained in Production Example 1 were as follows.
(1) The appearance was a white powder.
(2) The molecular formula was represented by C 31 H 45 N 6 O 11 Cl, and the molecular weight was 712.
(3) The experimental value by high resolution mass spectrometry (HRESIMS: positive ion mode) is m / z 713.2903 (M + H) + , and the calculated value is m / z 713.2908 (C 31 H 46 N 6 As O 11 Cl).
(4) The specific rotation was [α] D 23 = + 56.8 ° (c0.2, methanol).
(5) The ultraviolet absorption spectrum measured with a methanol solution was as shown in Table 1 above.
λ max nm (ε): 227 (15, 100, sh), 289 (12, 800)
(6) In a thin layer chromatography of a TLC plate (TLC Silica gel 60F254, manufactured by Merck), an RF value measured by developing with a developing solvent [butanol: methanol: water (4: 1: 2, volume ratio)] is The RF value measured by developing with a developing solvent [butanol: acetic acid: water (4: 1: 2, volume ratio)] was 0.26.
（試験例１）
　アンドロプロスタミンＡ及びアンドロプロスタミンＢの前立腺癌細胞に対するアンドロゲン依存増殖阻害作用を以下の方法で検討した。 (Test Example 1)
The androgen-dependent growth inhibitory action of androprostamine A and androprostamine B on prostate cancer cells was examined by the following method.
－ＬＮＣａＰ細胞を用いた検討－
　アンドロゲンに応答し増殖を示すヒト前立腺癌細胞株ＬＮＣａＰ細胞（ＡＴＣＣより入手）を２体積％ｃＦＢＳ（ｃｈａｒｃｏｌ－ｔｒｅａｔｅｄ　ＦＢＳ）を含むフェノールレッド非添加のＲＰＭＩ培地（ライフテクノロジーズジャパン株式会社製）を用いて５×１０４ｃｅｌｌｓ／ｍＬで９６ウェルプレートに播種し、３７℃、５％ＣＯ２の雰囲気下で２４時間培養してＬＮＣａＰ細胞を完全に接着させた。
　なお、前記ｃＦＢＳは、ＦＢＳ中に含まれるアンドロゲンを除去するために、ＦＢＳ（ＭＰバイオメディカルズ社製）に０．５質量％の活性炭を添加して６０分間撹拌した後、０．４５μｍのフィルターに通すことにより滅菌と活性炭の除去を行い調製したものを用いた。 -Examination using LNCaP cells-
A human prostate cancer cell line LNCaP cell (obtained from ATCC) that shows proliferation in response to androgen is used with RPMI medium (manufactured by Life Technologies Japan Co., Ltd.) containing 2% by volume of cFBS (charcol-treated FBS) without phenol red. The cells were seeded in a 96-well plate at 5 × 10 4 cells / mL, and cultured in an atmosphere of 37 ° C. and 5% CO 2 for 24 hours to completely adhere the LNCaP cells.
In addition, in order to remove the androgen contained in FBS, the cFBS was added with 0.5% by mass activated carbon to FBS (manufactured by MP Biomedicals) and stirred for 60 minutes, and then a 0.45 μm filter. The one prepared by sterilization and removal of activated carbon by passing through was used.
　前記ＬＮＣａＰ細胞を接着させた９６ウェルプレートにおいて、アンドロプロスタミンＡ又はアンドロプロスタミンＢを、終濃度が、０．００２μｇ／ｍＬ、０．００８μｇ／ｍＬ、０．０３μｇ／ｍＬ、０．１３μｇ／ｍＬ、０．５２μｇ／ｍＬ、２．０８μｇ／ｍＬ、８．３３μｇ／ｍＬ、又は３３．３μｇ／ｍＬとなるように添加し、更に合成アンドロゲン（Ｒ１８８１、ＷａｔｅｒＳｔｏｎｅ　Ｔｅｃｈｎｏｌｏｇｙ社製)を終濃度が１ｎＭとなるように添加した。次いで、５日間、３７℃、５％ＣＯ２雰囲気下で培養した。
　また、比較対照としては、前記ＬＮＣａＰ細胞を接着させた９６ウェルプレートにおいて、アンドロプロスタミンＡ又はアンドロプロスタミンＢを添加せず、合成アンドロゲン（Ｒ１８８１）を終濃度が１ｎＭとなるように添加し、５日間、３７℃、５％ＣＯ２雰囲気下で培養した。
　５日間培養後、ＭＴＴ法を用いてＬＮＣａＰ細胞の細胞増殖を測定した。アンドロプロスタミンＡ又はアンドロプロスタミンＢを添加した系の吸光度を「Ａ」とし、比較対照の系の吸光度を「Ｂ」として、下記式（１）により、細胞増殖阻害率（％）を算出した。
　細胞増殖阻害率（％）＝｛（Ｂ－Ａ）／Ｂ｝×１００　・・・式（１）
　次に、前記細胞増殖阻害率より、ＬＮＣａＰ細胞の細胞増殖を５０％抑制する濃度（ＩＣ５０値）を判定した。結果を下記表２、並びに、図１及び図２に示す。 In the 96-well plate to which the LNCaP cells have been adhered, the final concentrations of androprostamine A or androprostamine B are 0.002 μg / mL, 0.008 μg / mL, 0.03 μg / mL, 0.13 μg / mL. , 0.52 μg / mL, 2.08 μg / mL, 8.33 μg / mL, or 33.3 μg / mL, and synthetic androgen (R1881, manufactured by Waterstone Technology) to a final concentration of 1 nM Was added as follows. Subsequently, the cells were cultured for 5 days at 37 ° C. in a 5% CO 2 atmosphere.
Further, as a comparative control, in the 96-well plate to which the LNCaP cells were adhered, without adding androprostamine A or androprostamine B, synthetic androgen (R1881) was added to a final concentration of 1 nM, The cells were cultured for 5 days at 37 ° C. in a 5% CO 2 atmosphere.
After culturing for 5 days, cell proliferation of LNCaP cells was measured using the MTT method. The absorbance of the system to which androprostamine A or androprostamine B was added was “A”, and the absorbance of the comparative control system was “B”, and the cell growth inhibition rate (%) was calculated by the following formula (1). .
Cell growth inhibition rate (%) = {(BA) / B} × 100 Formula (1)
Next, the concentration (IC 50 value) at which the cell growth of LNCaP cells was inhibited by 50% was determined from the cell growth inhibition rate. The results are shown in Table 2 below and FIG. 1 and FIG.
－ＶＣａＰ細胞を用いた検討－
　前記「ＬＮＣａＰ細胞を用いた検討」において、ＬＮＣａＰ細胞を、アンドロゲン低依存性のヒト前立腺癌細胞株ＶＣａＰ（ＡＴＣＣより入手）に変えたこと以外は、前記「ＬＮＣａＰ細胞を用いた検討」と同様の方法でＶＣａＰ細胞を培養した。
　５日間培養後、ＭＴＴ法を用いてＶＣａＰ細胞の細胞増殖を測定し、前記同様の方法で５０％抑制する濃度（ＩＣ５０値）を判定した。結果を下記表２、並びに、図３及び図４に示す。 -Examination using VCaP cells-
In the above “examination using LNCaP cells”, the same as the above “examination using LNCaP cells” except that LNCaP cells were changed to an androgen-independent human prostate cancer cell line VCaP (obtained from ATCC). VCaP cells were cultured by this method.
After culturing for 5 days, the cell proliferation of VCaP cells was measured using the MTT method, and the concentration (IC 50 value) at which 50% inhibition was achieved by the same method as described above was determined. The results are shown in Table 2 below and FIGS. 3 and 4.
Figure JPOXMLDOC01-appb-T000020
Figure JPOXMLDOC01-appb-T000020
（試験例２）
　試験例１における、アンドロゲン依存増殖阻害作用が特異的であることを確認するため、ＬＮＣａＰ細胞及びＶＣａＰ細胞に対する細胞毒性を以下の方法で検討した。 (Test Example 2)
In order to confirm that the androgen-dependent growth inhibitory action in Test Example 1 was specific, cytotoxicity against LNCaP cells and VCaP cells was examined by the following method.
　ＬＮＣａＰ細胞又はＶＣａＰ細胞（共に、ＡＴＣＣより入手）を２液量％ＦＢＳ又は１０液量％ＦＢＳを含むフェノールレッド非添加のＲＰＭＩ培地（ライフテクノロジーズジャパン株式会社製）を用いて５×１０４ｃｅｌｌｓ／ｍＬで９６ウェルプレートに播種し、３７℃、５％ＣＯ２の雰囲気下で２４時間培養して細胞を完全に接着させた。 LNCaP cells or VCaP cells (both obtained from ATCC) were added at 5 × 10 4 cells / without using phenol red-added RPMI medium (Life Technologies Japan) containing 2 liquid% FBS or 10 liquid% FBS. were seeded in 96-well plates in mL, 37 ° C., it was cultured for 24 hours in an atmosphere of 5% CO 2 the cells were completely adhered.
　前記９６ウェルプレートの２体積％ＦＢＳを添加した系において、アンドロプロスタミンＡ又はアンドロプロスタミンＢを、終濃度が、０．００２μｇ／ｍＬ、０．００８μｇ／ｍＬ、０．０３μｇ／ｍＬ、０．１３μｇ／ｍＬ、０．５２μｇ／ｍＬ、２．０８μｇ／ｍＬ、８．３３μｇ／ｍＬ、又は３３．３μｇ／ｍＬとなるように添加した。
　２体積％ＦＢＳを添加した系における比較対照としては、前記ＬＮＣａＰ細胞又はＶＣａＰ細胞を接着させた９６ウェルプレートにおいて、アンドロプロスタミンＡ又はアンドロプロスタミンＢを添加せず、５日間、３７℃、５％ＣＯ２雰囲気下で培養した。
　また、前記９６ウェルプレートの１０体積％ＦＢＳを添加した系において、終濃度が、０．００２μｇ／ｍＬ、０．００８μｇ／ｍＬ、０．０３μｇ／ｍＬ、０．１３μｇ／ｍＬ、０．５２μｇ／ｍＬ、２．０８μｇ／ｍＬ、８．３３μｇ／ｍＬ、又は３３．３μｇ／ｍＬとなるように調整したアンドロプロスタミンＡ又はアンドロプロスタミンＢを添加した。次いで、５日間、３７℃、５％ＣＯ２雰囲気下で培養した。
　１０体積％ＦＢＳを添加した系における比較対照としては、前記ＬＮＣａＰ細胞又はＶＣａＰ細胞を接着させた９６ウェルプレートにおいて、アンドロプロスタミンＡ又はアンドロプロスタミンＢを添加せず、５日間、３７℃、５％ＣＯ２雰囲気下で培養した。
　５日間培養後、ＭＴＴ法を用いてＬＮＣａＰ細胞及びＶＣａＰ細胞の細胞増殖を測定した。アンドロプロスタミンＡ又はアンドロプロスタミンＢを添加した系の吸光度を「Ａ」とし、比較対照の吸光度を「Ｂ」として、前記試験例１における式（１）により、細胞増殖阻害率（％）を算出した。
　次に、前記細胞増殖阻害率より、細胞毒性を示すアンドロプロスタミンＡ又はアンドロプロスタミンＢの濃度（ＩＣ５０値）を判定した。結果を下記表３、並びに、図１～４に示す。 In the 96-well plate to which 2 vol% FBS was added, androprostamine A or androprostamine B had final concentrations of 0.002 μg / mL, 0.008 μg / mL, 0.03 μg / mL, 0. It added so that it might become 13 microgram / mL, 0.52 microgram / mL, 2.08 microgram / mL, 8.33 microgram / mL, or 33.3 microgram / mL.
As a comparative control in the system to which 2% by volume FBS was added, in the 96-well plate to which the LNCaP cells or VCaP cells were adhered, androprostamine A or androprostamine B was not added for 5 days at 37 ° C., 5 ° C. Culturing was performed in a% CO 2 atmosphere.
In the 96-well plate to which 10 vol% FBS is added, the final concentrations are 0.002 μg / mL, 0.008 μg / mL, 0.03 μg / mL, 0.13 μg / mL, 0.52 μg / mL. Androprostamine A or Androprostamine B adjusted to 2.08 μg / mL, 8.33 μg / mL, or 33.3 μg / mL was added. Subsequently, the cells were cultured for 5 days at 37 ° C. in a 5% CO 2 atmosphere.
As a comparative control in the system to which 10% by volume FBS was added, in the 96-well plate to which the LNCaP cells or VCaP cells were adhered, androprostamine A or androprostamine B was not added for 5 days at 37 ° C., 5 ° C. Culturing was performed in a% CO 2 atmosphere.
After culturing for 5 days, cell proliferation of LNCaP cells and VCaP cells was measured using the MTT method. Assuming that the absorbance of the system to which androprostamine A or androprostamine B is added is “A” and the absorbance of the comparative control is “B”, the cell growth inhibition rate (%) is calculated according to the equation (1) in Test Example 1. Calculated.
Next, the concentration of androprostamine A or androprostamine B exhibiting cytotoxicity (IC 50 value) was determined from the cell growth inhibition rate. The results are shown in Table 3 below and FIGS.
Figure JPOXMLDOC01-appb-T000021
Figure JPOXMLDOC01-appb-T000021
　試験例１及び２の結果から明らかなように、アンドロプロスタミンＡ及びアンドロプロスタミンＢは、前立腺癌のアンドロゲン依存増殖に対し特異的な阻害作用を有していることがわかった。 As is clear from the results of Test Examples 1 and 2, androprostamine A and androprostamine B were found to have a specific inhibitory action on androgen-dependent growth of prostate cancer.
（試験例３）
－ＬＮＣａＰ細胞における前立腺特異的抗原の発現抑制作用の確認－
　ヒト前立腺癌細胞株ＬＮＣａＰ細胞（ＡＴＣＣより入手）を、２体積％ｃＦＢＳを含むフェノールレッド非添加のＲＰＭＩ培地（ライフテクノロジーズジャパン株式会社製）を用いて５×１０５ｃｅｌｌｓ／ｍＬで３ｃｍシャーレに播種し、３７℃、５％ＣＯ２の雰囲気下で２４時間培養して細胞を完全に接着させた。なお、前記ｃＦＢＳは、試験例１と同様のものを用いた。 (Test Example 3)
-Confirmation of prostate-specific antigen expression suppression in LNCaP cells-
Human prostate cancer cell line LNCaP cells (obtained from ATCC) are seeded in 3 cm dishes at 5 × 10 5 cells / mL using RPMI medium (manufactured by Life Technologies Japan) containing 2% by volume of cFBS and without phenol red. The cells were cultured in an atmosphere of 37 ° C. and 5% CO 2 for 24 hours to completely adhere the cells. The cFBS used was the same as in Test Example 1.
　次に、アンドロプロスタミンＡを、終濃度が、０．０５μｇ／ｍＬ、０．１μｇ／ｍＬ、０．５μｇ／ｍＬ、１μｇ／ｍＬ、５μｇ／ｍＬ、又は１０μｇ／ｍＬとなるように添加し、更に合成アンドロゲン（Ｒ１８８１、ＷａｔｅｒＳｔｏｎｅ　Ｔｅｃｈｎｏｌｏｇｙ社製）を終濃度が１ｎＭとなるように添加した。次いで、４８時間、３７℃、５％ＣＯ２雰囲気下で培養した。 Next, androprostamine A is added so that the final concentration is 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, or 10 μg / mL, Further, synthetic androgen (R1881, manufactured by Waterstone Technology) was added so that the final concentration was 1 nM. Subsequently, the cells were cultured for 48 hours at 37 ° C. in a 5% CO 2 atmosphere.
　前記４８時間培養後のＬＮＣａＰ細胞から、ＲＮＡ抽出キット（ＲＮｅａｓｙ　（登録商標）　キアゲン社製）を用いて総ＲＮＡを抽出した。得られた総ＲＮＡより１μｇを用いて逆転写反応キット（Ｒｅｖｅｒｓｅ　Ｔｒａｎｓｃｒｉｐｔｉｏｎ　Ｓｙｓｔｅｍ　（登録商標）　プロメガ社製）を用いてｃＤＮＡを合成した。次いで、インターカレーター法によるリアルタイムＰＣＲ専用試薬（ＳＹＢＲ　Ｐｒｅｍｉｘ　Ｅｘ　Ｔａｑ　ＩＩ　（登録商標）　タカラバイオ株式会社製）を用いてリアルタイムＰＣＲを行った。前立腺特異的抗原（ＰＳＡ：Ｐｒｏｓｔａｔｅ　Ｓｐｅｃｉｆｉｃ　Ａｎｔｉｇｅｎ）及びアクチンのｍＲＮＡの発現量を測定し、アクチンのｍＲＮＡの発現量に対するＰＳＡのｍＲＮＡの発現量（ＰＳＡ／アクチン）を算出した。なお、ＰＳＡ及びアクチンのプライマーはタカラバイオ株式会社から購入した。結果を図５に示す。 Total RNA was extracted from the LNCaP cells after 48 hours of culture using an RNA extraction kit (RNeasy (registered trademark) manufactured by Qiagen). CDNA was synthesized using 1 μg of the total RNA obtained using a reverse transcription reaction kit (Reverse Transcription System (registered trademark), Promega). Subsequently, real-time PCR was performed using a reagent dedicated to real-time PCR by the intercalator method (SYBR Premix Ex Taq II (registered trademark) manufactured by Takara Bio Inc.). The expression level of prostate specific antigen (PSA: Prostate Specific Antigen) and actin mRNA was measured, and the expression level of PSA mRNA (PSA / actin) relative to the expression level of actin mRNA was calculated. PSA and actin primers were purchased from Takara Bio Inc. The results are shown in FIG.
－ＶＣａＰ細胞における前立腺特異的抗原の発現の確認－
　前記「ＬＮＣａＰ細胞における前立腺特異的抗原の発現の確認」において、ＬＮＣａＰ細胞を、ＶＣａＰ細胞に変えたこと以外は、前記「ＬＮＣａＰ細胞における前立腺特異的抗原の発現の確認」と同様の方法でＶＣａＰ細胞を培養し、前立腺特異的抗原の発現の確認と同様の方法でアクチンのｍＲＮＡの発現量に対するＰＳＡのｍＲＮＡの発現量（ＰＳＡ／アクチン）を算出した。結果を図６に示す。 -Confirmation of expression of prostate specific antigen in VCaP cells-
In the “confirmation of expression of prostate-specific antigen in LNCaP cells”, VCaP cells were prepared in the same manner as in “confirmation of expression of prostate-specific antigen in LNCaP cells” except that LNCaP cells were changed to VCaP cells. And the expression level of PSA mRNA relative to the expression level of actin mRNA (PSA / actin) was calculated in the same manner as the confirmation of expression of prostate-specific antigen. The results are shown in FIG.
　図５及び図６の結果より、試験例１の試験系において、合成アンドロゲン（Ｒ１８８１）によりアンドロゲンレセプターの標的遺伝子である前立腺特異抗原のｍＲＮＡの発現が誘導され、ここにアンドロプロスタミンＡを添加することにより前立腺特異抗原のｍＲＮＡの発現が抑制されることがわかった。 From the results of FIG. 5 and FIG. 6, in the test system of Test Example 1, the expression of prostate specific antigen mRNA, which is the target gene of the androgen receptor, was induced by synthetic androgen (R1881), and androprostamine A was added thereto. It was found that the expression of prostate specific antigen mRNA was suppressed.
　本発明の態様としては、例えば、以下のものなどが挙げられる。
　＜１＞　下記一般式（Ｉ）で表されることを特徴とする化合物である。
Figure JPOXMLDOC01-appb-C000022
 　ただし、前記一般式（Ｉ）において、Ｘは、下記一般式（ＩＡ）及び下記一般式（ＩＢ）のいずれかで表される基を表す。
Figure JPOXMLDOC01-appb-C000023
 　ただし、前記一般式（ＩＡ）及び前記一般式（ＩＢ）において、＊は、結合手を表す。
　＜２＞　下記構造式（Ｉ）及び下記構造式（ＩＩ）のいずれかで表される前記＜１＞に記載の化合物である。
Figure JPOXMLDOC01-appb-C000024
Figure JPOXMLDOC01-appb-C000025
 　＜３＞　下記一般式（Ｉ）で表される化合物を生産する能力を有するストレプトミセス　エスピー（Ｓｔｒｅｐｔｏｍｙｃｅｓ　ｓｐ．）ＭＫ９３２－ＣＦ８株（受託番号：ＮＩＴＥ　ＢＰ－００８３８）を培養して、下記一般式（Ｉ）で表される化合物を製造することを特徴とする化合物の製造方法である。
Figure JPOXMLDOC01-appb-C000026
 　ただし、前記一般式（Ｉ）において、Ｘは、下記一般式（ＩＡ）及び下記一般式（ＩＢ）のいずれかで表される基を表す。
Figure JPOXMLDOC01-appb-C000027
 　ただし、前記一般式（ＩＡ）及び前記一般式（ＩＢ）において、＊は、結合手を表す。
　＜４＞　下記一般式（Ｉ）で表される化合物を生産する能力を有する微生物であって、前記微生物が、ストレプトミセス　エスピー（Ｓｔｒｅｐｔｏｍｙｃｅｓ　ｓｐ．）ＭＫ９３２－ＣＦ８株（受託番号：ＮＩＴＥ　ＢＰ－００８３８）であることを特徴とする微生物である。
Figure JPOXMLDOC01-appb-C000028
 　ただし、前記一般式（Ｉ）において、Ｘは、下記一般式（ＩＡ）及び下記一般式（ＩＢ）のいずれかで表される基を表す。
Figure JPOXMLDOC01-appb-C000029
 　ただし、前記一般式（ＩＡ）及び前記一般式（ＩＢ）において、＊は、結合手を表す。
　＜５＞　前記＜１＞から＜２＞のいずれかに記載の化合物を含有することを特徴とする医薬組成物である。
　＜６＞　前記＜５＞に記載の医薬組成物を含有し、抗腫瘍作用を有することを特徴とする抗腫瘍剤である。
　＜７＞　前記＜６＞に記載の抗腫瘍剤を含有し、癌の予防又は治療に用いられることを特徴とする癌の予防剤又は治療剤である。
　＜８＞　前記＜５＞に記載の医薬組成物を含有し、前立腺癌の増殖抑制作用を有することを特徴とする抗前立腺癌剤である。
　＜９＞　前記＜８＞に記載の抗前立腺癌剤を含有し、前立腺癌の予防又は治療に用いられることを特徴とする前立腺癌の予防剤又は治療剤である。 Examples of the aspect of the present invention include the following.
<1> A compound represented by the following general formula (I).
Figure JPOXMLDOC01-appb-C000022
 However, in the said general formula (I), X represents group represented by either the following general formula (IA) or the following general formula (IB).
Figure JPOXMLDOC01-appb-C000023
 However, in the general formula (IA) and the general formula (IB), * represents a bond.
<2> The compound according to <1>, which is represented by any one of the following structural formula (I) and the following structural formula (II).
Figure JPOXMLDOC01-appb-C000024
Figure JPOXMLDOC01-appb-C000025
 <3> Streptomyces sp. MK932-CF8 strain (accession number: NITE BP-00838) having the ability to produce a compound represented by the following general formula (I) is cultured, and the following general formula ( A method for producing a compound, comprising producing the compound represented by I).
Figure JPOXMLDOC01-appb-C000026
 However, in the said general formula (I), X represents group represented by either the following general formula (IA) or the following general formula (IB).
Figure JPOXMLDOC01-appb-C000027
 However, in the general formula (IA) and the general formula (IB), * represents a bond.
<4> A microorganism having the ability to produce a compound represented by the following general formula (I), wherein the microorganism is Streptomyces sp. MK932-CF8 strain (Accession number: NITE BP-00838) It is a microorganism characterized by being.
Figure JPOXMLDOC01-appb-C000028
 However, in the said general formula (I), X represents group represented by either the following general formula (IA) or the following general formula (IB).
Figure JPOXMLDOC01-appb-C000029
 However, in the general formula (IA) and the general formula (IB), * represents a bond.
<5> A pharmaceutical composition comprising the compound according to any one of <1> to <2>.
<6> An antitumor agent comprising the pharmaceutical composition according to <5> and having an antitumor action.
<7> A cancer preventive or therapeutic agent comprising the antitumor agent according to <6> and used for the prevention or treatment of cancer.
<8> An anti-prostate cancer agent comprising the pharmaceutical composition according to the above <5> and having a prostate cancer growth inhibitory action.
<9> A prophylactic or prophylactic agent for prostate cancer comprising the anti-prostatic cancer agent according to <8> and used for the prevention or treatment of prostate cancer.
　ＮＩＴＥ　ＢＰ－００８３８ NITE BP-00838
　本発明の化合物は、優れた抗腫瘍作用を有し、安全性の高い化合物であるため、医薬組成物、抗腫瘍剤等の有効成分として好適に利用可能である。
　本発明の医薬組成物は、前記本発明の化合物を含むため、優れた抗腫瘍作用を有し、安全性の高いものであり、抗腫瘍剤などに好適に利用可能である。
　本発明の抗腫瘍剤は、優れた抗腫瘍作用、特にアンドロゲン依存性の増殖に対する増殖抑制作用を有し、安全性が高いため、癌の予防剤又は治療剤、好ましくは、前立腺癌の予防剤又は治療剤として好適に利用可能である。 Since the compound of the present invention has excellent antitumor activity and is highly safe, it can be suitably used as an active ingredient such as pharmaceutical compositions and antitumor agents.
Since the pharmaceutical composition of the present invention contains the compound of the present invention, it has excellent antitumor activity and high safety, and can be suitably used for antitumor agents and the like.
The antitumor agent of the present invention has an excellent antitumor action, particularly a growth inhibitory action against androgen-dependent growth, and is highly safe. Therefore, it is a prophylactic or therapeutic agent for cancer, preferably a prostatic cancer prophylactic agent. Or it can utilize suitably as a therapeutic agent.

Claims (6)

  1. 　下記一般式（Ｉ）で表されることを特徴とする化合物。
    Figure JPOXMLDOC01-appb-C000001
     　ただし、前記一般式（Ｉ）において、Ｘは、下記一般式（ＩＡ）及び下記一般式（ＩＢ）のいずれかで表される基を表す。
    Figure JPOXMLDOC01-appb-C000002
     　ただし、前記一般式（ＩＡ）及び前記一般式（ＩＢ）において、＊は、結合手を表す。     A compound represented by the following general formula (I):
    Figure JPOXMLDOC01-appb-C000001
     However, in the said general formula (I), X represents group represented by either the following general formula (IA) or the following general formula (IB).
    Figure JPOXMLDOC01-appb-C000002
     However, in the general formula (IA) and the general formula (IB), * represents a bond.
  2. 　下記構造式（Ｉ）及び下記構造式（ＩＩ）のいずれかで表される請求項１に記載の化合物。
    Figure JPOXMLDOC01-appb-C000003
    Figure JPOXMLDOC01-appb-C000004
         The compound of Claim 1 represented by either the following structural formula (I) or the following structural formula (II).
    Figure JPOXMLDOC01-appb-C000003
    Figure JPOXMLDOC01-appb-C000004
  3. 　下記一般式（Ｉ）で表される化合物を生産する能力を有するストレプトミセス　エスピー（Ｓｔｒｅｐｔｏｍｙｃｅｓ　ｓｐ．）ＭＫ９３２－ＣＦ８株（受託番号：ＮＩＴＥ　ＢＰ－００８３８）を培養して、下記一般式（Ｉ）で表される化合物を製造することを特徴とする化合物の製造方法。
    Figure JPOXMLDOC01-appb-C000005
     　ただし、前記一般式（Ｉ）において、Ｘは、下記一般式（ＩＡ）及び下記一般式（ＩＢ）のいずれかで表される基を表す。
    Figure JPOXMLDOC01-appb-C000006
     　ただし、前記一般式（ＩＡ）及び前記一般式（ＩＢ）において、＊は、結合手を表す。     Streptomyces sp. MK932-CF8 strain (accession number: NITE BP-00838) having the ability to produce a compound represented by the following general formula (I) is cultured, and the following general formula (I) The manufacturing method of the compound characterized by manufacturing the compound represented.
    Figure JPOXMLDOC01-appb-C000005
     However, in the said general formula (I), X represents group represented by either the following general formula (IA) or the following general formula (IB).
    Figure JPOXMLDOC01-appb-C000006
     However, in the general formula (IA) and the general formula (IB), * represents a bond.
  4. 　下記一般式（Ｉ）で表される化合物を生産する能力を有する微生物であって、前記微生物が、ストレプトミセス　エスピー（Ｓｔｒｅｐｔｏｍｙｃｅｓ　ｓｐ．）ＭＫ９３２－ＣＦ８株（受託番号：ＮＩＴＥ　ＢＰ－００８３８）であることを特徴とする微生物。
    Figure JPOXMLDOC01-appb-C000007
     　ただし、前記一般式（Ｉ）において、Ｘは、下記一般式（ＩＡ）及び下記一般式（ＩＢ）のいずれかで表される基を表す。
    Figure JPOXMLDOC01-appb-C000008
     　ただし、前記一般式（ＩＡ）及び前記一般式（ＩＢ）において、＊は、結合手を表す。     A microorganism having the ability to produce a compound represented by the following general formula (I), wherein the microorganism is Streptomyces sp. MK932-CF8 strain (Accession Number: NITE BP-00838) Microorganism characterized by
    Figure JPOXMLDOC01-appb-C000007
     However, in the said general formula (I), X represents group represented by either the following general formula (IA) or the following general formula (IB).
    Figure JPOXMLDOC01-appb-C000008
     However, in the general formula (IA) and the general formula (IB), * represents a bond.
  5. 　請求項１から２のいずれかに記載の化合物を含有することを特徴とする医薬組成物。 A pharmaceutical composition comprising the compound according to any one of claims 1 to 2.
  6. 　請求項５に記載の医薬組成物を含有し、抗腫瘍作用を有することを特徴とする抗腫瘍剤。 An antitumor agent comprising the pharmaceutical composition according to claim 5 and having an antitumor action.
PCT/JP2013/072011 2012-08-21 2013-08-16 Novel compound and method for producing same, novel microorganism, pharmaceutical composition, and anti-tumor agent WO2014030597A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2014531609A JP6154382B2 (en) 2012-08-21 2013-08-16 NOVEL COMPOUND, PROCESS FOR PRODUCING THE SAME, PHARMACEUTICAL COMPOSITION, AND ANTI-TUMOR

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2012-182144 2012-08-21
JP2012182144 2012-08-21

Publications (1)

Publication Number Publication Date
WO2014030597A1 true WO2014030597A1 (en) 2014-02-27

Family

ID=50149906

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2013/072011 WO2014030597A1 (en) 2012-08-21 2013-08-16 Novel compound and method for producing same, novel microorganism, pharmaceutical composition, and anti-tumor agent

Country Status (2)

Country Link
JP (1) JP6154382B2 (en)
WO (1) WO2014030597A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016190383A1 (en) * 2015-05-27 2016-12-01 公益財団法人微生物化学研究会 Novel compound coccoquinone, method for producing same, use of same, and novel microorganism

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CLEGG N. J. ET AL.: "ARN-509: A Novel Antiandrogen for Prostate Cancer Treatment.", CANCER RES., vol. 72, no. 6, January 2012 (2012-01-01), pages 1494 - 1503 *
DANQUAH M. ET AL.: "Combination Therapy of Antiandrogen and XIAP Inhibitor for Treating Advanced Prostate Cancer.", PHARM. RES., vol. 29, March 2012 (2012-03-01), pages 2079 - 2091 *
DATABASE GENBANK 20 December 2011 (2011-12-20), "DEFINITION: Streptomyces sp. MK932-CF8 gene for 16S ribosomal RNA, partial sequence", retrieved from http://www.ncbi.nlm.nih.gov/ nuccore/363987278?sat=3&satkey=20102528 accession no. B688983 *
ISAO MOMOSE: "Shin Senryaku ni Motozuku Koganzai no Kaihatsu ni Kansuru Kenkyu Biseibutsu Taisha Sanbutsu kara no Koganzai no Tansaku", SHIN SENRYAKU NI MOTOZUKU KOGANZAI NO KAIHATSU NI KANSURU KENKYU HEISEI 18 NENDO SOKATSU BUNTAN KENKYU HOKOKUSHO, 2007, pages 16 - 19 *
KAWATA H. ET AL.: "Biological Properties of Androgen Receptor Pure Antagonist for Treatment of Castration-Resistant Prostate Cancer: Optimization From Lead Compound to CH5137291.", THE PROSTATE, vol. 71, 2011, pages 1344 - 1356 *
TRAN C. ET AL.: "Development of a Second- Generation Antiandrogen for Treatment of Advanced Prostate Cancer.", SCIENCE, vol. 324, May 2009 (2009-05-01), pages 787 - 790 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016190383A1 (en) * 2015-05-27 2016-12-01 公益財団法人微生物化学研究会 Novel compound coccoquinone, method for producing same, use of same, and novel microorganism
JP2021008498A (en) * 2015-05-27 2021-01-28 公益財団法人微生物化学研究会 Novel compound coccoquinones, method for producing the same, use of the same, and novel microorganism
JP7026185B2 (en) 2015-05-27 2022-02-25 公益財団法人微生物化学研究会 New compounds Coccoquinones, their production methods, their uses, and new microorganisms

Also Published As

Publication number Publication date
JP6154382B2 (en) 2017-06-28
JPWO2014030597A1 (en) 2016-07-28

Similar Documents

Publication Publication Date Title
JP6130224B2 (en) Novel compound lenteztrehalose, method for producing the same, use thereof, and novel microorganism
EP1814902B1 (en) Cyclosporine analogue
JP6154382B2 (en) NOVEL COMPOUND, PROCESS FOR PRODUCING THE SAME, PHARMACEUTICAL COMPOSITION, AND ANTI-TUMOR
WO2009113661A1 (en) Cyclic compound and salt thereof
JP6130248B2 (en) Novel compound quinofuracins, process for producing the same, use thereof, and novel microorganism
EP2423319B1 (en) Amycolamicin, method for production thereof, and use thereof
JP6238956B2 (en) NOVEL COMPOUND, PROCESS FOR PRODUCING THE SAME AND USE THEREOF
EP2264179A1 (en) Aminosugar compound and process for production thereof
WO2016052656A1 (en) Novel compound, method for producing same, and use thereof
WO2018056470A1 (en) Epithelial-mesenchymal transition induced cell inhibitor
JP7026185B2 (en) New compounds Coccoquinones, their production methods, their uses, and new microorganisms
JP4836783B2 (en) Antitumor agent, method for producing antitumor agent, pharmaceutical composition containing antitumor agent, and antitumor agent-producing bacterium
JP7334927B2 (en) Anti-cancer agent
CN114540376B (en) Antitumor polyketide and preparation method and application thereof
JP5718450B2 (en) Anti-neoplastic agent and its production method and use
JP2012240974A (en) Method for producing didemnin b
EP2340251B1 (en) Pyrrolo[4,3,2-de]quinolin-8-amine compounds and methods of their preparation and use
JP5641716B2 (en) Novel compounds produced by microorganisms belonging to the genus Streptomyces, microorganisms producing the compounds, and pharmaceutical preparations containing the compounds as active ingredients
US9399656B2 (en) Compound, method for producing same, and use of same
WO2011136174A1 (en) Depsipeptide compound
JP2006213703A (en) New fermentation product
JP2006008563A (en) Compound having anticancer activity, method for producing the same and anticancer agent

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13831179

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2014531609

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13831179

Country of ref document: EP

Kind code of ref document: A1