WO2019189331A1 - Novel k95-5901-1 substance and method for producing same - Google Patents

Novel k95-5901-1 substance and method for producing same Download PDF

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WO2019189331A1
WO2019189331A1 PCT/JP2019/013132 JP2019013132W WO2019189331A1 WO 2019189331 A1 WO2019189331 A1 WO 2019189331A1 JP 2019013132 W JP2019013132 W JP 2019013132W WO 2019189331 A1 WO2019189331 A1 WO 2019189331A1
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substance
tuberculosis
compound
actinoplanes
microorganism
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PCT/JP2019/013132
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French (fr)
Japanese (ja)
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大村　智
塩見　和朗
美穂子 森
厚子 松本
定彦 鈴木
千絵 中島
山口　智之
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学校法人北里研究所
国立大学法人北海道大学
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Priority to JP2020509177A priority Critical patent/JPWO2019189331A1/en
Publication of WO2019189331A1 publication Critical patent/WO2019189331A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • 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

Definitions

  • the present invention relates to the field of compounds having the activity of inhibiting the growth of Mycobacterium tuberculosis, and pharmaceuticals and veterinary drugs containing the compounds.
  • Tuberculosis that is designated as a type 2 infectious disease is one of the three major infectious diseases, and as a single infectious disease, it is the second most common death in the world after HIV / AIDS. It is reported that there are 10.4 million tuberculosis patients, and 1.7 million people die from tuberculosis annually (2016, WHO). In HIV-infected persons, tuberculosis accounts for one-fifth of the cause of death.
  • rifampicin, isoniazid, pyrazinamide, streptomycin, ethambutol are used as the first-line drug, and levofloxacin, kanamycin, etionamide, enviomycin, paraaminosalicylic acid, cycloserine are used as the second-line drug, Used in combination of 3 or more agents.
  • multi-drug resistant tuberculosis resistant to these therapeutic agents has been spreading.
  • the emergence of multi-drug resistant tuberculosis and super multi-drug resistant tuberculosis, in which existing anti-tuberculosis drugs have no effect, has been seen in more than half of the world, and these measures are an urgent issue in the international community.
  • Non-patent Document 1 Cyclomarine, a kind of Clp protease disruptor, is a Streptomyces sp. Is a heptapeptide obtained from Cyclomarin acts on the ClpC1 subunit of Mycobacterium tuberculosis Clp, causing its function to run away and inducing cell death.
  • Iramycin is a deep-sea derived Streptomyces atratus It is an analog of cyclomarin isolated from SCSIO ZH16 and is also considered to be a Clp protease disruptor.
  • iramycin C is a compound represented by the following structure, but it has been reported that the activity of this compound against Mycobacterium tuberculosis H37Rv is as low as 9.6 ⁇ M ( Non-patent document 2).
  • the production method according to 3. (5) A microorganism belonging to actinomycetes and capable of producing the substance K95-5901-1. (6) Actinoplanes sp. K95-5901 (Accession No. NITE BP-02658) strain. (7) A pharmaceutical composition comprising the compound according to (1) or a salt thereof, or a hydrate or solvate thereof as an active ingredient. (8) The pharmaceutical composition according to (7), which is an antituberculosis drug. (9) The pharmaceutical composition according to (8), which is a tuberculosis growth inhibitor.
  • the substance K95-5901-1 of the present invention has tuberculosis growth inhibitory activity. Therefore, according to the present invention, the K95-5901-1 substance can be effectively used as a therapeutic or prophylactic agent for tuberculosis.
  • the K95-5901-1 substance is a compound represented by the following formula (I).
  • the salt of the substance K95-5901-1 is a salt formed by combining the substance K95-5901-1 with an inorganic or organic base or acid.
  • the salt include alkali metal and alkaline earth metal salts such as lithium, sodium, potassium, magnesium, calcium; ammonia, methylamine, dimethylamine, trimethylamine, dicyclohexylamine, tris (hydroxymethyl) aminomethane, N, N -Salts of amines such as bis (hydroxyethyl) piperazine, 2-amino-2-methyl-1-propanol, ethanolamine, N-methylglucamine, L-glucamine; or bases such as lysine, ⁇ -hydroxylysine, arginine And a salt with a functional amino acid.
  • alkali metal and alkaline earth metal salts such as lithium, sodium, potassium, magnesium, calcium
  • ammonia methylamine, dimethylamine, trimethylamine, dicyclohexylamine, tris (hydroxymethyl) aminomethane,
  • hydrates and solvates of the K95-5901-1 substance and hydrates and solvates of the salt of the K95-5901-1 substance are also encompassed by the compounds of the present invention.
  • the K95-5901-1 substance or the compound represented by the formula (I) is a salt of the K95-5901-1 substance even when it is not specified unless it is clearly not suitable. Hydrates and solvates as well as hydrates or solvates of the salts of the compound K95-5901-1 substance.
  • the compound of the present invention may have an asymmetric carbon, optical isomers may exist.
  • the compound of the present invention may be either a dextrorotatory (+) or levorotatory ( ⁇ ) compound, or a mixture of these isomers such as a racemate.
  • the compound of the present invention includes any tautomer or geometric isomer (for example, E-form, Z-form, etc.).
  • the K95-5901-1 substance of the present invention can be obtained by culturing a microorganism capable of producing the K95-5901-1 substance belonging to actinomycetes, and separating and purifying it from the culture.
  • Actinomycetes that can be used for the production of the K95-5901-1 substance of the present invention include actinoplanes sp. Actinoplanes sp. K95-5901 (Accession number NITE BP-02658) and its mutant strains. All of the K95-5901-1 substance-producing bacteria belonging to the fungus (preferably belonging to the genus Actinoplanes).
  • the nutrient source in actinomycetes culture suitable for the production of the above K95-5901-1 substance is not particularly limited and can be used as a nutrient source for actinomycetes.
  • a nutrient source for actinomycetes for example, commercially available peptone, meat extract, corn steep liquor, cottonseed flour, peanut flour, soy flour, yeast extract, NZ-amine, casein hydrate, nitrogen sources such as sodium nitrate, ammonium nitrate, ammonium sulfate, glycerin
  • carbohydrates such as starch, glucose, galactose and mannose, or carbon sources such as fat
  • inorganic salts such as sodium chloride, phosphate, calcium carbonate and magnesium sulfate can be used alone or in combination.
  • a trace amount of metal salt and animal / plant / mineral oil as an antifoaming agent may be added to the medium for actinomycete culture if necessary.
  • These additives may be any additives that can be used to produce the K95-5901-1 substance using the production bacteria, and all known actinomycete culture materials can be used.
  • the culture temperature of actinomycete culture can be applied in any range in which the producing bacteria can grow and produce the K95-5901-1 substance. Culturing can be carried out by appropriately selecting according to the properties of the K95-5901-1 substance-producing bacterium using the conditions described above.
  • the K95-5901-1 substance can be extracted from the culture solution with a water-immiscible organic solvent such as chloroform or ethyl acetate.
  • a water-immiscible organic solvent such as chloroform or ethyl acetate.
  • known methods used for collecting fat-soluble substances such as adsorption chromatography, partition chromatography, gel filtration chromatography, scraping from thin layer chromatography, centrifugal countercurrent distribution chromatography, high speed It can be purely collected by appropriately combining or repeating liquid chromatography and the like.
  • the present invention also relates to a pharmaceutical composition containing the substance K95-5901-1, particularly an antituberculous drug.
  • the invention relates to the use of the K95-5901-1 substance for the manufacture of an antituberculosis drug.
  • the present invention relates to the K95-5901-1 substance for treating or preventing tuberculosis.
  • the present invention further relates to a method for treating and preventing tuberculosis comprising administering an effective amount of a K95-5901-1 substance to a patient in need thereof.
  • the pharmaceutical composition of the present invention can be used as a therapeutic or prophylactic agent for tuberculosis.
  • the pharmaceutical composition of the present invention comprises drug-resistant tuberculosis bacteria in which existing drugs are not effective, multi-drug resistant tuberculosis bacteria having resistance to a plurality of drugs, and super multi-drugs that are resistant to all existing drugs. It can be used as a therapeutic or prophylactic agent for infection with resistant M. tuberculosis.
  • the tuberculosis bacterium to be treated and prevented according to the present invention is a resistant bacterium
  • the tuberculosis bacterium is selected from rifampicin, isoniazid, pyrazinamide, streptomycin, ethambutol, levofloxacin, kanamycin, etionamide, enbiomycin, paraaminosalicylic acid, and cycloserine.
  • Resistant to at least one selected drug and may be resistant to any type of drug between two or more and all types.
  • the M. tuberculosis subject to treatment and prevention of the present invention may be resistant to rifampicin and isoniazid.
  • the pharmaceutical composition of the present invention may be a combination of K95-5901-1 substance and one or more other antituberculous drugs.
  • the other antituberculosis drugs include rifampicin, isoniazid, pyrazinamide, streptomycin, ethambutol, levofloxacin, kanamycin, ethionamide, enbiomycin, paraaminosalicylic acid, and cycloserine.
  • These concomitant drugs may be provided as a single preparation, or may be provided as separate preparations for use in combination (for example, kits).
  • the pharmaceutical composition of the present invention can be used for oral administration or parenteral administration.
  • the dosage form of the pharmaceutical composition includes topical preparations, suspensions, ointments, creams, gels, inhalants, injections (for example, intravenous injections, subcutaneous injections, intramuscular injections). , Infusion), tablets, capsules, tablets, granules, powders, and the like.
  • the pharmaceutical composition of the present invention can be formulated by a conventional method using a normal pharmaceutically acceptable carrier.
  • a pharmaceutical composition containing the K95-5901-1 substance as an active ingredient and solid preparations such as tablets, pills, capsules, powders, granules, solutions, suspensions, It can be used orally as a liquid preparation such as an emulsion, or parenterally as an injection, intravenous, intramuscular, subcutaneous, or the like, a suppository, or a patch.
  • a solid preparation for oral administration When preparing a solid preparation for oral administration, add excipients to the active ingredient and, if necessary, binders, disintegrants, lubricants, etc., and then add solvents, granules, powders, capsules, etc. by conventional methods. And When preparing an injection, a pH adjuster, a buffer, a stabilizer, a solubilizing agent, etc. may be added to the main drug as necessary to obtain a subcutaneous or intravenous injection by a conventional method.
  • the present invention can further be used in a method of treating or preventing M. tuberculosis infection comprising administering an effective amount of K95-5901-1 substance to a patient in need thereof.
  • a pharmaceutical composition containing the K95-5901-1 substance as an active ingredient is administered orally or parenterally such as an injection or infusion. It can be administered in the form.
  • the K95-5901-1 substance is administered to mammals, it may be administered orally as tablets, powders, granules, syrups, etc., or parenterally as injections or drops.
  • the dose varies depending on symptoms, age, sex, body weight, dosage form, etc. For example, when administered orally to an adult, the daily dose is usually 0.1-1000 mg.
  • Example 1 Mycological characteristics of Actinoplanes sp. K95-5901 strain Actinoplanes sp. K95-5901 (Accession number NITE BP-02658) strain was collected in Asaka, Saitama, Japan. This strain is isolated from soil and its mycological properties are as follows. (I) Morphological properties Vegetative mycelium develops well on various agar media, and no fragmentation is observed. The aerial mycelium does not settle, but forms a spherical spore capsule of about 5 to 10 ⁇ m on the vegetative mycelium.
  • Diaminopimelic acid in the cell wall is meso-type.
  • Major menaquinone has a small amount of MK-8 in MK-9 (H 4) ( H 4) and MK-10 (H 4).
  • Example 2 Isolation and extraction, physicochemical properties, and structure of substance K95-5901-1 From Actinoplanes sp. K95-5901 (Accession No. NITE BP-02658) strain cultured in an agar slant medium , Starch 2.4%, glucose 0.1%, peptone 0.3%, meat extract 0.3%, yeast extract (produced by Oriental Yeast Co., Ltd.) 0.5%, calcium carbonate 0.4%
  • One platinum loop was inoculated into a test tube containing 10 ml of a liquid medium (pH 7.0) and cultured with shaking at 27 ° C. for 3 days.
  • the obtained seed culture solution was soluble starch 2.0%, glycerol 0.5%, wheat germ 1.0%, meat extract 0.3%, dry yeast (Fermipan, DSM Bakery Ingredients). 1 ml each was inoculated into 30 500 ml Erlenmeyer flasks containing 100 ml of a liquid medium (pH 7.5) consisting of 3% and calcium carbonate 0.3%, and cultured with shaking at 25 ° C. for 6 days.
  • a liquid medium pH 7.5
  • the K95-5901-1 substance has a structure represented by the following formula (I).
  • Example 3 Antibacterial activity of K95-5901-1 substance against Mycobacterium spp.
  • multidrug resistant (rifampicin and isoniazid resistant) clinically isolated Mycobacterium tuberculosis W-26 strain and W-114 strain was used.
  • Bacterial colonies within 8 weeks grown on Ogawa medium were suspended in mycobroth [manufactured by Kyokuto Pharmaceutical Co., Ltd.] and culture was started at 37 ° C. The culture solution was stirred every day, and turbidity was measured at a wavelength of 530 nm using a digital colorimeter Vi-spec II (manufactured by Kyokuto Pharmaceutical Co., Ltd.), and the culture was continued until the turbidity reached 0.15.
  • the antibacterial activity of the K95-5901-1 substance of the present invention against test bacteria other than the genus Mycobacterium is as follows. 10 ⁇ l each of a 1 mg / ml methanol solution of the K95-5901-1 substance was immersed in a filter paper disc (Advantech Toyo Co., Ltd., diameter 6 mm), air-dried for a certain period of time, and the solvent was removed. Attached to agar plates each containing the test bacteria and cultured at 35 ° C. for 24 hours, the diameter of the growth inhibition circle formed around the filter paper disc was measured.
  • the results are shown in Table 3.
  • the K95-5901-1 substance of the present invention showed no antibacterial activity against bacteria and fungi other than Mycobacterium smegmatis, Bacillus subtilis and Micrococcus luteus among the microorganisms shown in Table 3. Therefore, the K95-5901-1 substance of the present invention can be used as a drug such as an antituberculosis drug exhibiting antibacterial activity against Mycobacterium.

Abstract

As a result of searching for an antibiotic substance effective for multidrug-resistant Mycobacterium tuberculosis in a microorganism culture solution, the present inventors discovered a novel substance K95-5901-1 having the following structure, within products of an actinobacterium, Actinoplanes sp. K95-5901. This substance expresses a proliferation-inhibiting activity to multidrug-resistant Mycobacterium tuberculosis at a concentration lower than micromole level, and has a very weak cytotoxicity. Consequently, based on this discovery, the present invention provides a novel substance having a proliferation-inhibition activity on Mycobacterium tuberculosis.

Description

新規Ｋ９５－５９０１－１物質およびその製造方法Novel K95-5901-1 substance and method for producing the same
　本発明は、結核菌の増殖阻害活性を有する化合物、及び当該化合物を含有する医薬品及び動物薬の分野に関する。 The present invention relates to the field of compounds having the activity of inhibiting the growth of Mycobacterium tuberculosis, and pharmaceuticals and veterinary drugs containing the compounds.
　２類感染症に指定されている結核は３大感染症の１つであり、単一の感染症としてはＨＩＶ／ＡＩＤＳに続いて世界で２番目に死亡者数が多い疾患である。１，０４０万人の結核患者がいると報告され、年間１７０万人が結核により死亡している（２０１６年、ＷＨＯ）。またＨＩＶ感染者において、結核はその死因の５分の１を占めている。結核の治療に関しては、第一選択薬として、リファンピシン、イソニアジド、ピラジナミド、ストレプトマイシン、エタンブトールが、第二選択薬として、レボフロキサシン、カナマイシン、エチオナミド、エンビオマイシン、パラアミノサリチル酸、サイクロセリンが使用されており、３剤以上の併用で用いられる。 Tuberculosis that is designated as a type 2 infectious disease is one of the three major infectious diseases, and as a single infectious disease, it is the second most common death in the world after HIV / AIDS. It is reported that there are 10.4 million tuberculosis patients, and 1.7 million people die from tuberculosis annually (2016, WHO). In HIV-infected persons, tuberculosis accounts for one-fifth of the cause of death. For the treatment of tuberculosis, rifampicin, isoniazid, pyrazinamide, streptomycin, ethambutol are used as the first-line drug, and levofloxacin, kanamycin, etionamide, enviomycin, paraaminosalicylic acid, cycloserine are used as the second-line drug, Used in combination of 3 or more agents.
　近年、これらの治療薬に耐性な多剤耐性結核が、蔓延傾向を示している。第一選択薬であるリファンピシンに耐性な症例も年間６０万件に上り、そのうち４９万件は多剤耐性になっていると考えられている。さらに多剤耐性結核や既存の抗結核薬が全く効果を示さない超多剤耐性結核の出現が世界の半数以上の国々で見られ、これらの対策は国際社会において喫緊の課題となっている。 In recent years, multi-drug resistant tuberculosis resistant to these therapeutic agents has been spreading. There are 600,000 cases resistant to rifampicin, a first-line drug, and 490,000 cases are considered to be multidrug resistant. Furthermore, the emergence of multi-drug resistant tuberculosis and super multi-drug resistant tuberculosis, in which existing anti-tuberculosis drugs have no effect, has been seen in more than half of the world, and these measures are an urgent issue in the international community.
　しかし、これらの治療薬により先進国における新規発生患者数が減少したことに伴い、新規の抗結核薬の開発は減少し、４０年間で新規の抗結核薬は１剤も上市されていない。多剤耐性結核ならびに超多剤耐性結核の対策の一つとして、これらに対して有効な、従来の治療薬とは構造や作用機構の異なる新しい治療薬の開発が求められている。 However, with the decrease in the number of newly developed patients in developed countries due to these therapeutic agents, the development of new anti-tuberculosis drugs has decreased, and no new anti-tuberculosis drug has been launched on the market for 40 years. As one of the countermeasures against multi-drug resistant tuberculosis and super multi-drug resistant tuberculosis, development of a new therapeutic agent that is effective against them and has a structure and action mechanism different from that of conventional therapeutic agents is required.
　このような抗結核薬として、ニトロイミダゾール系化合物、ＣＰＺＥＮ－４５／カプラザマイシン類、及びＣｌｐプロテアーゼ攪乱物質が検討されている（非特許文献１）。Ｃｌｐプロテアーゼ攪乱物質の一種であるサイクロマリン（ｃｙｃｌｏｍａｒｉｎ）は海洋由来放線菌のＳｔｒｅｐｔｏｍｙｃｅｓ　ｓｐ．から得られたヘプタペプチドである。サイクロマリンは、結核菌ＣｌｐのＣｌｐＣ１サブユニットに作用し、機能を暴走させて細胞死を誘導する。イラマイシンは深海由来のＳｔｒｅｐｔｏｍｙｃｅｓ　ａｔｒａｔｕｓ
　ＳＣＳＩＯ　ＺＨ１６から単離されたサイクロマリンの類似化合物であり、同様にＣｌｐプロテアーゼ攪乱物質であると考えられている。イラマイシンの複数の同族体のうちイラマイシンＣは以下の構造で表される化合物であるが、本化合物の薬剤耐性結核菌（Ｍｙｃｏｂａｃｔｅｒｉａ　ｔｕｂｅｒｃｕｌｏｓｉｓ　Ｈ３７Ｒｖ）に対する活性は９．６μＭと低いことが報告されている（非特許文献２）。 As such antituberculosis drugs, nitroimidazole compounds, CPZEN-45 / caprazamicins, and Clp protease disruptors have been studied (Non-patent Document 1). Cyclomarine, a kind of Clp protease disruptor, is a Streptomyces sp. Is a heptapeptide obtained from Cyclomarin acts on the ClpC1 subunit of Mycobacterium tuberculosis Clp, causing its function to run away and inducing cell death. Iramycin is a deep-sea derived Streptomyces atratus
It is an analog of cyclomarin isolated from SCSIO ZH16 and is also considered to be a Clp protease disruptor. Of the multiple homologues of iramycin, iramycin C is a compound represented by the following structure, but it has been reported that the activity of this compound against Mycobacterium tuberculosis H37Rv is as low as 9.6 μM ( Non-patent document 2).
Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000002
　本発明者らは微生物培養液中から多剤耐性結核菌に有効な抗生物質を探索した結果、放線菌アクチノプラネス・エスピー（Ａｃｔｉｎｏｐｌａｎｅｓ　ｓｐ．）Ｋ９５－５９０１の生産する新規物質Ｋ９５－５９０１－１を発見した。本物質は従来の結核治療薬と異なる新規の構造をもち、多剤耐性結核菌に対してもマイクロモル以下の低濃度で増殖阻害活性を示した。さらに細胞毒性は非常に弱いものであった。これまで、このような物質が結核菌の増殖を阻害するという報告はなく、よって本発明はこのような知見に基づいて新規の結核菌の増殖阻害活性物質を提供するに至ったものである。
　本発明は係る知見に基づいて完成されたものであって、具体的には以下の発明に関する：
（１）　下記式（Ｉ）で表される化合物若しくはその塩、又はそれらの水和物若しくは溶媒和物。 As a result of searching for antibiotics effective against multidrug-resistant Mycobacterium tuberculosis from microbial cultures, the present inventors have obtained a novel substance K95-5901-1 produced by Actinoplanes sp. K95-5901. discovered. This substance has a novel structure different from conventional tuberculosis therapeutic agents, and showed growth inhibitory activity against multi-drug resistant tuberculosis bacteria at low concentrations of micromolar or less. Furthermore, the cytotoxicity was very weak. Until now, there has been no report that such a substance inhibits the growth of M. tuberculosis, and therefore the present invention has provided a novel M. tuberculosis growth inhibitory active substance based on such findings.
The present invention has been completed based on such findings, and specifically relates to the following inventions:
(1) A compound represented by the following formula (I) or a salt thereof, or a hydrate or solvate thereof.
Figure JPOXMLDOC01-appb-C000003
 （２）　放線菌に属する上記式（Ｉ）で表される化合物を生産する能力を有する微生物を培地で培養し、培養物中に前記化合物を蓄積せしめ、該培養物から前記化合物を採取することを含む、（１）に記載の化合物の製造方法。
（３）　放線菌に属する上記式（Ｉ）で表される化合物を生産する能力を有する微生物が、アクチノプラネス属に属する微生物である、（２）に記載の製造法。
（４）　放線菌に属する上記式（Ｉ）で表される化合物を生産する能力を有する微生物が、アクチノプラネス・エスピー（Ａｃｔｉｎｏｐｌａｎｅｓ　ｓｐ．）Ｋ９５－５９０１（受託番号ＮＩＴＥ　ＢＰ－０２６５８）である請求項３に記載の製造法。
（５）　放線菌に属し、Ｋ９５－５９０１－１物質を生産する能力を有する微生物。
（６）　アクチノプラネス・エスピー（Ａｃｔｉｎｏｐｌａｎｅｓ　ｓｐ．）Ｋ９５－５９０１（受託番号ＮＩＴＥ　ＢＰ－０２６５８）株。
（７）　（１）に記載の化合物若しくはその塩、又はそれらの水和物若しくは溶媒和物を有効成分として含有する、医薬組成物。
（８）　抗結核薬である、（７）に記載の医薬組成物。
（９）　結核菌増殖阻害剤である、（８）に記載の医薬組成物。
Figure JPOXMLDOC01-appb-C000003
 (2) culturing a microorganism having the ability to produce the compound represented by the above formula (I) belonging to actinomycetes in a medium, accumulating the compound in the culture, and collecting the compound from the culture The manufacturing method of the compound as described in (1) containing.
(3) The production method according to (2), wherein the microorganism having the ability to produce the compound represented by the above formula (I) belonging to actinomycetes is a microorganism belonging to the genus Actinoplanes.
(4) The microorganism having the ability to produce a compound represented by the above formula (I) belonging to actinomycetes is Actinoplanes sp. K95-5901 (Accession No. NITE BP-02658) 3. The production method according to 3.
(5) A microorganism belonging to actinomycetes and capable of producing the substance K95-5901-1.
(6) Actinoplanes sp. K95-5901 (Accession No. NITE BP-02658) strain.
(7) A pharmaceutical composition comprising the compound according to (1) or a salt thereof, or a hydrate or solvate thereof as an active ingredient.
(8) The pharmaceutical composition according to (7), which is an antituberculosis drug.
(9) The pharmaceutical composition according to (8), which is a tuberculosis growth inhibitor.
　本発明のＫ９５－５９０１－１物質は、結核菌増殖阻害活性を有する。よって本発明によれば、Ｋ９５－５９０１－１物質は結核に対する治療薬または予防薬として有効に使用し得る。 The substance K95-5901-1 of the present invention has tuberculosis growth inhibitory activity. Therefore, according to the present invention, the K95-5901-1 substance can be effectively used as a therapeutic or prophylactic agent for tuberculosis.
　本発明は、アクチノプラネス・エスピー（Ａｃｔｉｎｏｐｌａｎｅｓ　ｓｐ．）Ｋ９５－５９０１が生産するＫ９５－５９０１－１物質に関する。本明細書において、「Ｋ９５－５９０１－１物質」とは、以下の物性を有する化合物を意味する：
（１）性状：黄白色粉末
（２）分子量：１０５４．５２４４（Ｍ－Ｈ－、高分解能電子スプレーイオン化質量分析による）
（３）分子式：Ｃ５４Ｈ７３Ｎ９Ｏ１３
（４）比旋光度：［α］Ｄ２２＝－７２．２（ｃ０．１、メタノール）
（５）紫外部吸収極大（メタノール中、カッコ内はモル吸光係数ε）：２２１　ｎｍ　（８０７００），　２７６　ｎｍ　（１５９００），　３５６　ｎｍ　（３８５０）
（６）赤外部吸収極大（全反射測定法）：３３３１，　２９５６，　２３６１，　１６８３，　１６２９，　１５３９，　１３１７，　１２５０，　１１８３　ｃｍ－１に極大吸収を有する。
（７）プロトン核磁気共鳴スペクトル（重ジメチルスルホキシド中の化学シフト（ｐｐｍ）およびスピン結合定数（Ｈｚ、カッコ内））：９．１３（１Ｈ，ｂｒ．ｓ），８．５４（１Ｈ，ｄ，９．０），７．７４（１Ｈ，ｄ，８．０），７．７１（１Ｈ，ｄ，８．６），７．６５（１Ｈ，ｄ，９．８），７．５１（１Ｈ，ｄ，１．７），７．４０（１Ｈ，ｄ，６．３），７．１５（１Ｈ，ｄｄ，８．６，１．７），７．１１（１Ｈ，ｓ），７．０６（１Ｈ，ｄｄｄ，８．０，６．９，１．１），６．９６（１Ｈ，ｄｄ，８．０，７．４），６．９５（１Ｈ，ｄ，８．０），５．６１（１Ｈ，ｄ，４．６），５．３９（１Ｈ，ｄｑ，１４．９，６．３），５．２５（１Ｈ，ｄｔｄ，１４．９，６．９，１．７），５．０８（１Ｈ，ｄｄ，８．６，４．６），５．０４（１Ｈ，ｄｄ，９．２，４．６），４．７９（１Ｈ，ｍ），４．７８（１Ｈ，ｍ），４．６９（１Ｈ，ｄｄ，８．６，４．６），４．３３（１Ｈ，ｍ），４．３０（１Ｈ，ｔ，５．８），４．２２（１Ｈ，ｂｒ．ｄｄ，１１．４，２．３），３．２３（３Ｈ，ｓ），３．１８（１Ｈ，ｄｄ，３．５，２．８），２．９２（１Ｈ，ｂｒ．ｄｄ，１３．８，５．８），２．８５（１Ｈ，ｍ），２．８４（１Ｈ，ｍ），２．６５（１Ｈ，ｂｒ．ｄｄ，１３．８，９．２），２．６０（１Ｈ，ｍ），２．３２（１Ｈ，ｍ），２．２６（１Ｈ，ｍ），２．１９（１Ｈ，ｄｔ，１４．３，５．２），２．１２（３Ｈ，ｓ），１．９６（１Ｈ，ｄｄ，１４．３，９．２，６．３），１．９３（１Ｈ，ｄｄｄ，１４．９，９．７，４．６），１．７７（１Ｈ，ｄｄｄ，１４．９，９．７，４．６），１．５７（３Ｈ，ｓ），１．５７（１Ｈ，ｂｒ．ｄ，９．２），１．４０（３Ｈ，ｓ），１．３５（１Ｈ，ｍ），１．２８（１Ｈ，ｄｄｄ，１２．５，１１．５，４．０），１．１６（３Ｈ，ｄ，７．４），１．１５（３Ｈ，ｄ，７．５），０．８７（１Ｈ，ｍ），０．８４（３Ｈ，ｄ，６．３），０．８２（３Ｈ，ｄ，６．９），０．３０（３Ｈ，ｄ，６．９），０．１３（３Ｈ，ｄ，６．３），－０．８９（１Ｈ，ｄｄｄ，１２．５，９．０，３．０）．
（ｓは一重線、ｄは二重線、ｔは三重線、ｑは四重線、ｍは多重線、ｂｒ．は幅広線、Ｈはプロトンの数を示す。）
（８）１３Ｃ核磁気共鳴スペクトル（重ジメチルスルホキシド中の化学シフト（ｐｐｍ））：１７３．６，１７１．８，１７１．６，１６９．３，１６９．２，１６８．２，１６７．６，１６６．９，１５０．６，１３５．６，１３５．３，１３４．７，１２７．２，１２７．０，１２６．８，１２５．７，１２４．６，１２３．１，１２０．５，１２０．５，１１８．８，１１８．３，１１２．８，１１２．４，６８．２，５７．９，５７．１，５６．８，５６．６，５５．６，５４．６，５１．０，５０．４，４５．１，４４．２，３６．５，３６．５，３５．６，３５．４，３５．２，３４．１，２７．８，２７．５，２４．１，２３．６，２３．２，２２．７，２２．３，２１．７，２１．２，２０．１，１７．４，１６．７，１５．７．
（９）溶剤に対する溶解性：クロロホルム、エタノール、メタノール、ジメチルスルホキシドに易溶。水に難溶。
（１０）呈色反応：リンモリブデン硫酸に陽性。Ｅｈｌｒｉｃｈ試薬に陽性。 The present invention relates to the K95-5901-1 substance produced by Actinoplanes sp. K95-5901. In the present specification, “K95-5901-1 substance” means a compound having the following physical properties:
(1) Property: Yellowish white powder (2) Molecular weight: 1054.5244 (MH − , by high resolution electrospray ionization mass spectrometry)
(3) Molecular formula: C 54 H 73 N 9 O 13
(4) Specific rotation: [α] D 22 = −72.2 (c0.1, methanol)
(5) Ultraviolet absorption maximum (in methanol, parenthesis is molar extinction coefficient ε): 221 nm (80700), 276 nm (15900), 356 nm (3850)
(6) Infrared absorption maximum (total reflection measurement method): Maximum absorption at 3331, 2956, 2361, 1683, 1629, 1539, 1317, 1250, 1183 cm-1.
(7) Proton nuclear magnetic resonance spectrum (chemical shift (ppm) and spin coupling constant (Hz, in parentheses) in deuterated dimethyl sulfoxide): 9.13 (1H, br.s), 8.54 (1H, d, 9.0), 7.74 (1H, d, 8.0), 7.71 (1H, d, 8.6), 7.65 (1H, d, 9.8), 7.51 (1H, d, 1.7), 7.40 (1H, d, 6.3), 7.15 (1H, dd, 8.6, 1.7), 7.11 (1H, s), 7.06 ( 1H, ddd, 8.0, 6.9, 1.1), 6.96 (1H, dd, 8.0, 7.4), 6.95 (1H, d, 8.0), 5.61 (1H, d, 4.6), 5.39 (1H, dq, 14.9, 6.3), 5.25 (1H, dtd, 14.9, 6.9, 1.7), 5. 08 (1H, dd, 8.6 4.6), 5.04 (1H, dd, 9.2, 4.6), 4.79 (1H, m), 4.78 (1H, m), 4.69 (1H, dd, 8. 6, 4.6), 4.33 (1H, m), 4.30 (1H, t, 5.8), 4.22 (1H, br. Dd, 11.4, 2.3), 3. 23 (3H, s), 3.18 (1H, dd, 3.5, 2.8), 2.92 (1H, br.dd, 13.8, 5.8), 2.85 (1H, m ), 2.84 (1H, m), 2.65 (1 H, br. Dd, 13.8, 9.2), 2.60 (1 H, m), 2.32 (1 H, m), 2. 26 (1H, m), 2.19 (1H, dt, 14.3, 5.2), 2.12 (3H, s), 1.96 (1H, dd, 14.3, 9.2, 6) .3), 1.93 (1H, ddd, 14.9, 9.7, 4.6) , 1.77 (1H, ddd, 14.9, 9.7, 4.6), 1.57 (3H, s), 1.57 (1H, br.d, 9.2), 1.40 ( 3H, s), 1.35 (1H, m), 1.28 (1H, ddd, 12.5, 11.5, 4.0), 1.16 (3H, d, 7.4), 1. 15 (3H, d, 7.5), 0.87 (1H, m), 0.84 (3H, d, 6.3), 0.82 (3H, d, 6.9), 0.30 ( 3H, d, 6.9), 0.13 (3H, d, 6.3), -0.89 (1H, ddd, 12.5, 9.0, 3.0).
(S is a single line, d is a double line, t is a triple line, q is a quadruple line, m is a multiple line, br. Is a wide line, and H is the number of protons.)
(8) 13 C nuclear magnetic resonance spectrum (chemical shift (ppm) in deuterated dimethyl sulfoxide): 173.6, 171.8, 171.6, 169.3, 169.2, 168.2, 167.6, 166.9, 150.6, 135.6, 135.3, 134.7, 127.2, 127.0, 126.8, 125.7, 124.6, 123.1, 120.5, 120. 5, 118.8, 118.3, 112.8, 112.4, 68.2, 57.9, 57.1, 56.8, 56.6, 55.6, 54.6, 51.0, 50.4, 45.1, 44.2, 36.5, 36.5, 35.6, 35.4, 35.2, 34.1, 27.8, 27.5, 24.1, 23. 6, 23.2, 22.7, 22.3, 21.7, 21.2, 20.1, 17.4, 16.7, 1 .7.
(9) Solubility in solvents: Easily soluble in chloroform, ethanol, methanol, dimethyl sulfoxide. Insoluble in water.
(10) Color reaction: Positive for phosphomolybdenum sulfate. Positive for Ehlrich reagent.
　更に、本発明者らが見出したところによれば、Ｋ９５－５９０１－１物質は以下の式（Ｉ）で表される化合物である。 Furthermore, according to the finding of the present inventors, the K95-5901-1 substance is a compound represented by the following formula (I).
Figure JPOXMLDOC01-appb-C000004
Figure JPOXMLDOC01-appb-C000004
　本明細書において、Ｋ９５－５９０１－１物質の塩とは、Ｋ９５－５９０１－１物質が、無機又は有機の塩基又は酸と結合して形成した塩のことである。塩としては、例えば、リチウム、ナトリウム、カリウム、マグネシウム、カルシウム等のアルカリ金属及びアルカリ土類金属塩；アンモニア、メチルアミン、ジメチルアミン、トリメチルアミン、ジシクロヘキシルアミン、トリス（ヒドロキシメチル）アミノメタン、Ｎ，Ｎ－ビス（ヒドロキシエチル）ピペラジン、２－アミノ－２－メチル－１－プロパノール、エタノールアミン、Ｎ－メチルグルカミン、Ｌ－グルカミン等のアミンの塩；又はリジン、δ－ヒドロキシリジン、アルギニンなどの塩基性アミノ酸との塩を挙げることができる。また、Ｋ９５－５９０１－１物質の水和物及び溶媒和物、並びにＫ９５－５９０１－１物質の塩の水和物及び溶媒和物も本発明の化合物に包含される。また、本明細書においてＫ９５－５９０１－１物質又は式（Ｉ）で表される化合物は、それが明らかに適さない場合を除き、明示されていない場合にも、Ｋ９５－５９０１－１物質の塩、水和物及び溶媒和物、並びに化合物Ｋ９５－５９０１－１物質の塩の水和物又は溶媒和物をも含むと解釈されるべきである。 In this specification, the salt of the substance K95-5901-1 is a salt formed by combining the substance K95-5901-1 with an inorganic or organic base or acid. Examples of the salt include alkali metal and alkaline earth metal salts such as lithium, sodium, potassium, magnesium, calcium; ammonia, methylamine, dimethylamine, trimethylamine, dicyclohexylamine, tris (hydroxymethyl) aminomethane, N, N -Salts of amines such as bis (hydroxyethyl) piperazine, 2-amino-2-methyl-1-propanol, ethanolamine, N-methylglucamine, L-glucamine; or bases such as lysine, δ-hydroxylysine, arginine And a salt with a functional amino acid. In addition, hydrates and solvates of the K95-5901-1 substance, and hydrates and solvates of the salt of the K95-5901-1 substance are also encompassed by the compounds of the present invention. Further, in the present specification, the K95-5901-1 substance or the compound represented by the formula (I) is a salt of the K95-5901-1 substance even when it is not specified unless it is clearly not suitable. Hydrates and solvates as well as hydrates or solvates of the salts of the compound K95-5901-1 substance.
　本発明の化合物は不斉炭素を有することがあることから、光学異性体が存在することがある。本発明の化合物としては、右旋性（＋）又は左旋性（－）の何れの化合物であってもよいし、ラセミ体などのこれらの異性体の混合物であってもよい。また、本発明の化合物は、特に断らない限り、いずれの互変異性体、又は幾何異性体（例えば、Ｅ体、Ｚ体など）も含むものである。 Since the compound of the present invention may have an asymmetric carbon, optical isomers may exist. The compound of the present invention may be either a dextrorotatory (+) or levorotatory (−) compound, or a mixture of these isomers such as a racemate. In addition, unless otherwise specified, the compound of the present invention includes any tautomer or geometric isomer (for example, E-form, Z-form, etc.).
　本発明のＫ９５－５９０１－１物質は、放線菌に属するＫ９５－５９０１－１物質を生産する能力を有する微生物を培養し、その培養物から分離・精製することにより得ることができる。本発明のＫ９５－５９０１－１物質の製造に用いることのできる放線菌としては、アクチノプラネス・エスピー（Ａｃｔｉｎｏｐｌａｎｅｓ　ｓｐ．）Ｋ９５－５９０１（受託番号ＮＩＴＥ　ＢＰ－０２６５８）株及びその変異株を含む、放線菌に属する（好ましくは、アクチノプラネス属に属する）Ｋ９５－５９０１－１物質生産菌の全てである。 The K95-5901-1 substance of the present invention can be obtained by culturing a microorganism capable of producing the K95-5901-1 substance belonging to actinomycetes, and separating and purifying it from the culture. Actinomycetes that can be used for the production of the K95-5901-1 substance of the present invention include actinoplanes sp. Actinoplanes sp. K95-5901 (Accession number NITE BP-02658) and its mutant strains. All of the K95-5901-1 substance-producing bacteria belonging to the fungus (preferably belonging to the genus Actinoplanes).
　上記Ｋ９５－５９０１－１物質の生産に適した放線菌培養における栄養源としては、放線菌の栄養源として使用し得るもので特に制限されるものではない。例えば、市販のペプトン、肉エキス、コーン・スティープ・リカー、綿実粉、落花生粉、大豆粉、酵母エキス、ＮＺ－アミン、カゼインの水和物、硝酸ソーダ、硝酸アンモニウム、硫酸アンモニウム等の窒素源、グリセリン、澱粉、グルコース、ガラクトース、マンノース等の炭水化物、あるいは脂肪等の炭素源、及び食塩、リン酸塩、炭酸カルシウム、硫酸マグネシウム等の無機塩を単独あるいは組み合わせて使用できる。 The nutrient source in actinomycetes culture suitable for the production of the above K95-5901-1 substance is not particularly limited and can be used as a nutrient source for actinomycetes. For example, commercially available peptone, meat extract, corn steep liquor, cottonseed flour, peanut flour, soy flour, yeast extract, NZ-amine, casein hydrate, nitrogen sources such as sodium nitrate, ammonium nitrate, ammonium sulfate, glycerin Further, carbohydrates such as starch, glucose, galactose and mannose, or carbon sources such as fat, and inorganic salts such as sodium chloride, phosphate, calcium carbonate and magnesium sulfate can be used alone or in combination.
　放線菌培養の培地にはその他必要に応じて微量の金属塩、消泡剤として動・植・鉱物油等を添加することもできる。これらの添加物は生産菌を利用しＫ９５－５９０１－１物質の生産の役だつものであればよく、公知の放線菌の培養材料はすべて用いることができる。また放線菌培養の培養温度は、生産菌が発育しＫ９５－５９０１－１物質を生産できる任意の範囲で適用できる。培養は以上に述べた条件を使用するＫ９５－５９０１－１物質生産菌の性質に応じて適宜選択して行なうことができる。 ・ A trace amount of metal salt and animal / plant / mineral oil as an antifoaming agent may be added to the medium for actinomycete culture if necessary. These additives may be any additives that can be used to produce the K95-5901-1 substance using the production bacteria, and all known actinomycete culture materials can be used. The culture temperature of actinomycete culture can be applied in any range in which the producing bacteria can grow and produce the K95-5901-1 substance. Culturing can be carried out by appropriately selecting according to the properties of the K95-5901-1 substance-producing bacterium using the conditions described above.
　Ｋ９５－５９０１－１物質は、培養液よりクロロホルム、酢酸エチル等の水不混和性の有機溶媒で抽出することができる。上述の抽出法に加え、脂溶性物質の採取に用いられる公知の方法、例えば吸着クロマトグラフィー、分配クロマトグラフィー、ゲル濾過クロマトグラフィー、薄層クロマトグラフィーよりのかき取り、遠心向流分配クロマトグラフィー、高速液体クロマトグラフィー等を適宜組合わせあるいは繰返すことによって純粋に採取することができる。 The K95-5901-1 substance can be extracted from the culture solution with a water-immiscible organic solvent such as chloroform or ethyl acetate. In addition to the extraction methods described above, known methods used for collecting fat-soluble substances, such as adsorption chromatography, partition chromatography, gel filtration chromatography, scraping from thin layer chromatography, centrifugal countercurrent distribution chromatography, high speed It can be purely collected by appropriately combining or repeating liquid chromatography and the like.
　また、本発明は、Ｋ９５－５９０１－１物質を含有する医薬組成物、特には抗結核薬に関する。別の態様では、本発明は、抗結核薬を製造するための、Ｋ９５－５９０１－１物質の使用に関する。あるいは本発明は、結核を治療又は予防するためのＫ９５－５９０１－１物質に関する。更に、本発明は、Ｋ９５－５９０１－１物質の有効量をそれを必要とする患者に投与することを含む、結核の治療方法及び予防方法に関する。本発明の医薬組成物は，結核の治療薬又は予防薬とすることができる。特に、本発明の医薬組成物は、既存の薬剤が効果を示さない薬剤耐性結核菌、複数の薬剤への耐性を持つ多剤耐性結核菌、及び既存の全ての薬剤に耐性を示す超多剤耐性結核菌の感染症の治療薬又は予防薬とすることができる。本発明の治療及び予防の対象となる結核菌が耐性菌の場合、当該結核菌は、リファンピシン、イソニアジド、ピラジナミド、ストレプトマイシン、エタンブトール、レボフロキサシン、カナマイシン、エチオナミド、エンビオマイシン、パラアミノサリチル酸、及びサイクロセリンから選択される少なくとも１種類の薬剤に耐性であり、２種類以上～全ての種類の間の任意の種類の薬剤に耐性であってもよい。例えば、本発明の治療及び予防の対象となる結核菌は、リファンピシンとイソニアジドに耐性であってもよい。 The present invention also relates to a pharmaceutical composition containing the substance K95-5901-1, particularly an antituberculous drug. In another aspect, the invention relates to the use of the K95-5901-1 substance for the manufacture of an antituberculosis drug. Alternatively, the present invention relates to the K95-5901-1 substance for treating or preventing tuberculosis. The present invention further relates to a method for treating and preventing tuberculosis comprising administering an effective amount of a K95-5901-1 substance to a patient in need thereof. The pharmaceutical composition of the present invention can be used as a therapeutic or prophylactic agent for tuberculosis. In particular, the pharmaceutical composition of the present invention comprises drug-resistant tuberculosis bacteria in which existing drugs are not effective, multi-drug resistant tuberculosis bacteria having resistance to a plurality of drugs, and super multi-drugs that are resistant to all existing drugs. It can be used as a therapeutic or prophylactic agent for infection with resistant M. tuberculosis. When the tuberculosis bacterium to be treated and prevented according to the present invention is a resistant bacterium, the tuberculosis bacterium is selected from rifampicin, isoniazid, pyrazinamide, streptomycin, ethambutol, levofloxacin, kanamycin, etionamide, enbiomycin, paraaminosalicylic acid, and cycloserine. Resistant to at least one selected drug and may be resistant to any type of drug between two or more and all types. For example, the M. tuberculosis subject to treatment and prevention of the present invention may be resistant to rifampicin and isoniazid.
　幅広い結核菌に確実に作用させるため、本発明の医薬組成物はＫ９５－５９０１－１物質と１種類以上の他の抗結核薬を組み合わせて使用するものであってもよい。当該他の抗結核薬としては、リファンピシン、イソニアジド、ピラジナミド、ストレプトマイシン、エタンブトール、レボフロキサシン、カナマイシン、エチオナミド、エンビオマイシン、パラアミノサリチル酸、及びサイクロセリンを挙げることができる。これらの併用剤は、単一製剤として提供されていてもよいし、組み合わせて使用されるように個別の製剤として提供されていてもよい（例えば、キットなど）。 In order to ensure the action on a wide range of tuberculosis bacteria, the pharmaceutical composition of the present invention may be a combination of K95-5901-1 substance and one or more other antituberculous drugs. Examples of the other antituberculosis drugs include rifampicin, isoniazid, pyrazinamide, streptomycin, ethambutol, levofloxacin, kanamycin, ethionamide, enbiomycin, paraaminosalicylic acid, and cycloserine. These concomitant drugs may be provided as a single preparation, or may be provided as separate preparations for use in combination (for example, kits).
　本発明の医薬組成物は経口投与用又は非経口投与用とすることができる。医薬組成物の剤型としては，局所適用製剤，懸濁剤，軟膏，クリーム剤，ゲル剤，吸入剤，注射剤（例えば，静脈注射用注射剤，皮下投与用注射剤，筋肉注射用注射剤，点滴）、タブレット、カプセル、錠剤、顆粒剤、及び粉剤等が挙げられる。 The pharmaceutical composition of the present invention can be used for oral administration or parenteral administration. The dosage form of the pharmaceutical composition includes topical preparations, suspensions, ointments, creams, gels, inhalants, injections (for example, intravenous injections, subcutaneous injections, intramuscular injections). , Infusion), tablets, capsules, tablets, granules, powders, and the like.
　本発明の医薬組成物は、通常の薬学的に許容される担体を用いて、常法により製剤化することができる。Ｋ９５－５９０１－１物質を有効成分として含有する医薬組成物を製剤化するための剤型に制限はなく錠剤、丸剤、カプセル剤、散剤、顆粒剤等の固形剤、溶液、懸濁液、乳剤などの液状製剤として経口的に、あるいは、静脈内、筋肉内、皮下などの注射剤、坐剤、貼付剤などとして非経口的に使用することができる。経口用固形製剤を調製する場合は、主薬に賦形剤、更に必要に応じて、結合剤、崩壊剤、滑沢剤等を加えた後、常法により溶剤、顆粒剤、散剤、カプセル剤等とする。注射剤を調製する場合には、主薬に必要によりｐＨ調整剤、緩衝剤、安定化剤、可溶化剤等を添加し、常法により皮下又は静脈内用注射剤とすることができる。 The pharmaceutical composition of the present invention can be formulated by a conventional method using a normal pharmaceutically acceptable carrier. There is no limitation on the dosage form for formulating a pharmaceutical composition containing the K95-5901-1 substance as an active ingredient, and solid preparations such as tablets, pills, capsules, powders, granules, solutions, suspensions, It can be used orally as a liquid preparation such as an emulsion, or parenterally as an injection, intravenous, intramuscular, subcutaneous, or the like, a suppository, or a patch. When preparing a solid preparation for oral administration, add excipients to the active ingredient and, if necessary, binders, disintegrants, lubricants, etc., and then add solvents, granules, powders, capsules, etc. by conventional methods. And When preparing an injection, a pH adjuster, a buffer, a stabilizer, a solubilizing agent, etc. may be added to the main drug as necessary to obtain a subcutaneous or intravenous injection by a conventional method.
　本発明はさらに、それを必要とする患者に有効量のＫ９５－５９０１－１物質を投与することを備える、結核菌感染症の治療方法又は予防方法に使用することができる。例えば、Ｋ９５－５９０１－１物質を治療又は予防目的で使用する場合、Ｋ９５－５９０１－１物質を有効成分として含有する医薬組成物を、経口投与形態、又は注射剤、点滴剤等の非経口投与形態で投与することができる。Ｋ９５－５９０１－１物質を哺乳動物等に投与する場合、錠剤、散剤、顆粒剤、シロップ剤等として経口投与してもよいし、又は、注射剤、点滴剤として非経口的に投与してもよい。投与量は、症状、年齢、性別、体重、投与形態等により異なるが、例えば成人に経口的に投与する場合には、通常１日量は０．１－１０００ｍｇである。 The present invention can further be used in a method of treating or preventing M. tuberculosis infection comprising administering an effective amount of K95-5901-1 substance to a patient in need thereof. For example, when the K95-5901-1 substance is used for therapeutic or prophylactic purposes, a pharmaceutical composition containing the K95-5901-1 substance as an active ingredient is administered orally or parenterally such as an injection or infusion. It can be administered in the form. When the K95-5901-1 substance is administered to mammals, it may be administered orally as tablets, powders, granules, syrups, etc., or parenterally as injections or drops. Good. The dose varies depending on symptoms, age, sex, body weight, dosage form, etc. For example, when administered orally to an adult, the daily dose is usually 0.1-1000 mg.
　以下に実施例を挙げて本発明を具体的に説明するが、本発明はこれに限定されるものではない。なお、本願全体を通して引用される全文献は参照によりそのまま本願に組み込まれる。また、本願は２０１８年３月２８日に出願された日本国特許出願第２０１８－０６１３９７号の優先権を主張する。本願が優先権を主張する日本国特許出願第２０１８－０６１３９７号記載の内容は全て参照によりそのまま本願に組み込まれる。 Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto. It should be noted that all documents cited throughout this application are incorporated herein by reference in their entirety. This application claims the priority of Japanese Patent Application No. 2018-061397 filed on Mar. 28, 2018. The contents of Japanese Patent Application No. 2018-061397, to which this application claims priority, are all incorporated herein by reference.
（実施例１）アクチノプラネス・エスピーＫ９５－５９０１株の菌学的性状
　アクチノプラネス・エスピー（Ａｃｔｉｎｏｐｌａｎｅｓ　ｓｐ．）Ｋ９５－５９０１（受託番号ＮＩＴＥ　ＢＰ－０２６５８）株は、日本国埼玉県朝霞市において採取された土壌より単離された菌株であり、その菌学的性状は以下のとおりである。
（Ｉ）形態的性質
　栄養菌糸は各種寒天培地上でよく発達し、分断は観察されない。気菌糸は着生しないが、栄養菌糸上に約５～１０μｍの球形の胞子嚢を形成する。 (Example 1) Mycological characteristics of Actinoplanes sp. K95-5901 strain Actinoplanes sp. K95-5901 (Accession number NITE BP-02658) strain was collected in Asaka, Saitama, Japan. This strain is isolated from soil and its mycological properties are as follows.
(I) Morphological properties Vegetative mycelium develops well on various agar media, and no fragmentation is observed. The aerial mycelium does not settle, but forms a spherical spore capsule of about 5 to 10 μm on the vegetative mycelium.
（ＩＩ）各種培地上での性状
　イー・ビー・シャーリング（Ｅ．Ｂ．Ｓｈｉｒｌｉｎｇ）とデー・ゴットリーブ（Ｄ．Ｇｏｔｔｌｉｅｂ）の方法（インターナショナル・ジャーナル・オブ・システィマティック・バクテリオロジー、１６巻、３１３頁、１９６６年）によって調べた本生産菌の培養性状を表１に示す。色調は標準色として、カラー・ハーモニー・マニュアル第４版（コンテナー・コーポレーション・オブ・アメリカ・シカゴ、１９５８年）を用いて決定し、色票名とともに括弧内にそのコードを併せて記した。以下は特記しない限り、２７℃、２週間目の各培地における観察の結果である。 (II) Properties on various media Methods of EB Shirring and D. Gottlieb (International Journal of Systematic Bacteriology, Vol. 16, page 313) 1966), the culture properties of the production bacteria are shown in Table 1. The color tone was determined using the Color Harmony Manual 4th edition (Container Corporation of America Chicago, 1958) as a standard color, and the code was also written in parentheses along with the color chart name. The following are the results of observation in each medium at 27 ° C. for 2 weeks unless otherwise specified.
Figure JPOXMLDOC01-appb-T000005
 （ＩＩＩ）生理学的諸性質
Figure JPOXMLDOC01-appb-T000005
 (III) Physiological properties
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
（ＩＶ）細胞の化学組成
　細胞壁のジアミノピメリン酸はメソ型である。主要メナキノンはＭＫ－９　（Ｈ４）でＭＫ－８　（Ｈ４）およびＭＫ－１０　（Ｈ４）を少量有する。 (IV) Cell chemical composition Diaminopimelic acid in the cell wall is meso-type. Major menaquinone has a small amount of MK-8 in MK-9 (H 4) ( H 4) and MK-10 (H 4).
（Ｖ）１６Ｓ　ｒＲＮＡ　遺伝子解析
　１６Ｓ　ｒＲＮＡ遺伝子の部分塩基配列７２４塩基を決定し、ＤＮＡデータベースに登録され公開されている細菌と比較した結果、その配列はアクチノプラネス・ミズーリエンシス（Ａｃｔｉｎｏｐｌａｎｅｓ　ｍｉｓｓｏｕｒｉｅｎｓｉｓ）ＮＢＲＣ　１０２３６３と１００％一致したことから、本菌株はアクチノプラネス・ミズーリエンシス（Ａｃｔｉｎｏｐｌａｎｅｓ　ｍｉｓｓｏｕｒｉｅｎｓｉｓ）に最も近縁であり、アクチノプラネス属に分類することが妥当である。 (V) 16S rRNA gene analysis A partial base sequence of 724 bases of 16S rRNA gene was determined and compared with a bacterium registered in the DNA database and published. As a result, the sequence was determined as Actinoplanes missouriens NBRC 102363. Since it was 100% consistent, this strain is most closely related to Actinoplanes missouriensis, and it is appropriate to classify it into the genus Actinoplanes.
（ＶＩ）結論
　以上、本菌の菌学的性状を要約すると次のとおりである。細胞壁中のジアミノピメリン酸はメソ型、主要メナキノンはＭＫ－９（Ｈ４）である。気菌糸は着生しないが、栄養菌糸上に球形の胞子嚢を形成する。コロニーは黄色からオレンジ色を呈し、メラニン色素およびその他の可溶性色素は産生しない。
　これらの結果および１６Ｓ　ｒＲＮＡ遺伝子の解析結果から、本菌株はバージーズ・マニュアル・オブ・システマティック・オブ・アーキア・アンド・バクテリア（Ｂｅｒｇｅｙ’ｓ　Ｍａｎｕａｌ　ｏｆ　Ｓｙｓｔｅｍａｔｉｃｓ　ｏｆ　Ａｒｃｈａｅａ　ａｎｄ
　Ｂａｃｔｅｒｉａ）、ウィリ・オンライン・ライブラリ（Ｗｉｌｅｙ　Ｏｎｌｉｎｅ　Ｌｉｂｒａｒｙ）、２０１５年に基づくアクチノプラネス属に属する菌種であると考えられる。なお、本菌株はアクチノプラネス・エスピー（Ａｃｔｉｎｏｐｌａｎｅｓ　ｓｐ．）Ｋ９５－５９０１として、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に寄託されている（受託番号ＮＩＴＥ　ＢＰ－０２６５８）。 (VI) Conclusion As described above, the bacteriological properties of this bacterium are summarized as follows. Diaminopimelic acid in the cell wall is meso-type, and the main menaquinone is MK-9 (H 4 ). The aerial hyphae do not settle, but they form a spherical spore on the vegetative mycelium. The colonies are yellow to orange and do not produce melanin and other soluble pigments.
From these results and the analysis result of 16S rRNA gene, this strain was found to be Bergey's Manual of Systems of Archaea and Bactery's Manual of Systematic of Archaea and Bacteria.
Bacteria), Willy Online Library, considered to be a species belonging to the genus Actinoplanes based on 2015. This strain was deposited as Actinoplanes sp. K95-5901 at the Patent Microorganism Depositary, National Institute of Technology and Evaluation (Accession No. NITE BP-02658).
（実施例２）Ｋ９５－５９０１－１物質の単離抽出、理化学的性状、及び構造
　寒天斜面培地で培養したアクチノプラネス・エスピー（Ａｃｔｉｎｏｐｌａｎｅｓ　ｓｐ．）Ｋ９５－５９０１（受託番号ＮＩＴＥ　ＢＰ－０２６５８）株より、デンプン２．４％、グルコース０．１％、ペプトン０．３％、肉エキス０．３％、酵母エキス〔オリエンタル酵母工業（株）製〕０．５％、炭酸カルシウム０．４％からなる液体培地（ｐＨ　７．０）が１０ｍｌ入った試験管に１白金耳接種し、２７℃で３日間振盪培養した。得られた種培養液を可溶性デンプン２．０％、グリセロール０．５％、小麦胚芽１．０％、肉エキス０．３％、乾燥酵母〔フェルミパン、ＤＳＭベーカリーイングリィエンツ社製〕０．３％、炭酸カルシウム０．３％からなる液体培地（ｐＨ　７．５）が１００　ｍｌ入った５００ｍｌ容三角フラスコ３０本に各１　ｍｌずつ植菌し、２５℃で６日間振盪培養した。 (Example 2) Isolation and extraction, physicochemical properties, and structure of substance K95-5901-1 From Actinoplanes sp. K95-5901 (Accession No. NITE BP-02658) strain cultured in an agar slant medium , Starch 2.4%, glucose 0.1%, peptone 0.3%, meat extract 0.3%, yeast extract (produced by Oriental Yeast Co., Ltd.) 0.5%, calcium carbonate 0.4% One platinum loop was inoculated into a test tube containing 10 ml of a liquid medium (pH 7.0) and cultured with shaking at 27 ° C. for 3 days. The obtained seed culture solution was soluble starch 2.0%, glycerol 0.5%, wheat germ 1.0%, meat extract 0.3%, dry yeast (Fermipan, DSM Bakery Ingredients). 1 ml each was inoculated into 30 500 ml Erlenmeyer flasks containing 100 ml of a liquid medium (pH 7.5) consisting of 3% and calcium carbonate 0.3%, and cultured with shaking at 25 ° C. for 6 days.
　培養の終了した５００ｍｌ容三角フラスコ３０本にそれぞれ１００ｍｌのエタノールを加えて１時間激しく撹拌した。次にその抽出液中のエタノールを減圧留去し、得られた水溶液をガラスカラムに水で充填したダイヤイオンＨＰ２０〔三菱ケミカル（株）製〕に吸着させ、各濃度のメタノール水溶液を流し、目的とする活性の見られた画分を回収し、減圧濃縮して０．５ｇの粗物質を得た。このうちの１４ｍｇを逆相系ＯＤＳ－ＨＰＬＣカラムに吸着させ、溶出溶媒にアセトニトリル水溶液またはメタノール水溶液を用いて溶出し、目的とする活性の見られた画分を回収、濃縮乾固して８．９ｍｇのＫ９５－５９０１－１物質を黄白色粉末として得た。 100 ml of ethanol was added to each of 30 500 ml Erlenmeyer flasks that had been cultured, and stirred vigorously for 1 hour. Next, the ethanol in the extract was distilled off under reduced pressure, and the resulting aqueous solution was adsorbed on Diaion HP20 [manufactured by Mitsubishi Chemical Corporation] filled with water in a glass column. The fraction in which activity was observed was collected and concentrated under reduced pressure to obtain 0.5 g of a crude substance. Of this, 14 mg was adsorbed on a reverse phase ODS-HPLC column and eluted with an acetonitrile aqueous solution or methanol aqueous solution as an elution solvent, and the fraction showing the desired activity was recovered, concentrated and dried. 9 mg of K95-5901-1 material was obtained as a pale yellow powder.
　Ｋ９５－５９０１－１物質の理化学的性状は次の通りである。
（１）性状：黄白色粉末
（２）分子量：１０５４．５２４４（Ｍ－Ｈ－、高分解能電子スプレーイオン化質量分析による）
（３）分子式：Ｃ５４Ｈ７３Ｎ９Ｏ１３
（４）比旋光度：［α］Ｄ２２＝－７２．２（ｃ０．１、メタノール）
（５）紫外部吸収極大（メタノール中、カッコ内はモル吸光係数ε）：２２１　ｎｍ　（８０７００），　２７６　ｎｍ　（１５９００），　３５６　ｎｍ　（３８５０）
（６）赤外部吸収極大（全反射測定法）：３３３１，　２９５６，　２３６１，　１６８３，　１６２９，　１５３９，　１３１７，　１２５０，　１１８３　ｃｍ－１に極大吸収を有する。
（７）プロトン核磁気共鳴スペクトル（重ジメチルスルホキシド中の化学シフト（ｐｐｍ）およびスピン結合定数（Ｈｚ、カッコ内））：９．１３（１Ｈ，ｂｒ．ｓ），８．５４（１Ｈ，ｄ，９．０），７．７４（１Ｈ，ｄ，８．０），７．７１（１Ｈ，ｄ，８．６），７．６５（１Ｈ，ｄ，９．８），７．５１（１Ｈ，ｄ，１．７），７．４０（１Ｈ，ｄ，６．３），７．１５（１Ｈ，ｄｄ，８．６，１．７），７．１１（１Ｈ，ｓ），７．０６（１Ｈ，ｄｄｄ，８．０，６．９，１．１），６．９６（１Ｈ，ｄｄ，８．０，７．４），６．９５（１Ｈ，ｄ，８．０），５．６１（１Ｈ，ｄ，４．６），５．３９（１Ｈ，ｄｑ，１４．９，６．３），５．２５（１Ｈ，ｄｔｄ，１４．９，６．９，１．７），５．０８（１Ｈ，ｄｄ，８．６，４．６），５．０４（１Ｈ，ｄｄ，９．２，４．６），４．７９（１Ｈ，ｍ），４．７８（１Ｈ，ｍ），４．６９（１Ｈ，ｄｄ，８．６，４．６），４．３３（１Ｈ，ｍ），４．３０（１Ｈ，ｔ，５．８），４．２２（１Ｈ，ｂｒ．ｄｄ，１１．４，２．３），３．２３（３Ｈ，ｓ），３．１８（１Ｈ，ｄｄ，３．５，２．８），２．９２（１Ｈ，ｂｒ．ｄｄ，１３．８，５．８），２．８５（１Ｈ，ｍ），２．８４（１Ｈ，ｍ），２．６５（１Ｈ，ｂｒ．ｄｄ，１３．８，９．２），２．６０（１Ｈ，ｍ），２．３２（１Ｈ，ｍ），２．２６（１Ｈ，ｍ），２．１９（１Ｈ，ｄｔ，１４．３，５．２），２．１２（３Ｈ，ｓ），１．９６（１Ｈ，ｄｄ，１４．３，９．２，６．３），１．９３（１Ｈ，ｄｄｄ，１４．９，９．７，４．６），１．７７（１Ｈ，ｄｄｄ，１４．９，９．７，４．６），１．５７（３Ｈ，ｓ），１．５７（１Ｈ，ｂｒ．ｄ，９．２），１．４０（３Ｈ，ｓ），１．３５（１Ｈ，ｍ），１．２８（１Ｈ，ｄｄｄ，１２．５，１１．５，４．０），１．１６（３Ｈ，ｄ，７．４），１．１５（３Ｈ，ｄ，７．５），０．８７（１Ｈ，ｍ），０．８４（３Ｈ，ｄ，６．３），０．８２（３Ｈ，ｄ，６．９），０．３０（３Ｈ，ｄ，６．９），０．１３（３Ｈ，ｄ，６．３），－０．８９（１Ｈ，ｄｄｄ，１２．５，９．０，３．０）．
（ｓは一重線、ｄは二重線、ｔは三重線、ｑは四重線、ｍは多重線、ｂｒ．は幅広線、Ｈはプロトンの数を示す。）
（８）１３Ｃ核磁気共鳴スペクトル（重ジメチルスルホキシド中の化学シフト（ｐｐｍ））：１７３．６，１７１．８，１７１．６，１６９．３，１６９．２，１６８．２，１６７．６，１６６．９，１５０．６，１３５．６，１３５．３，１３４．７，１２７．２，１２７．０，１２６．８，１２５．７，１２４．６，１２３．１，１２０．５，１２０．５，１１８．８，１１８．３，１１２．８，１１２．４，６８．２，５７．９，５７．１，５６．８，５６．６，５５．６，５４．６，５１．０，５０．４，４５．１，４４．２，３６．５，３６．５，３５．６，３５．４，３５．２，３４．１，２７．８，２７．５，２４．１，２３．６，２３．２，２２．７，２２．３，２１．７，２１．２，２０．１，１７．４，１６．７，１５．７．
（９）溶剤に対する溶解性：クロロホルム、エタノール、メタノール、ジメチルスルホキシドに易溶。水に難溶。
（１０）呈色反応：リンモリブデン硫酸に陽性。Ｅｈｌｒｉｃｈ試薬に陽性。 The physicochemical properties of K95-5901-1 substance are as follows.
(1) Property: Yellowish white powder (2) Molecular weight: 1054.5244 (MH − , by high resolution electrospray ionization mass spectrometry)
(3) Molecular formula: C 54 H 73 N 9 O 13
(4) Specific rotation: [α] D 22 = −72.2 (c0.1, methanol)
(5) Ultraviolet absorption maximum (in methanol, parenthesis is molar extinction coefficient ε): 221 nm (80700), 276 nm (15900), 356 nm (3850)
(6) Infrared absorption maximum (total reflection measurement method): Maximum absorption at 3331, 2956, 2361, 1683, 1629, 1539, 1317, 1250, 1183 cm-1.
(7) Proton nuclear magnetic resonance spectrum (chemical shift (ppm) and spin coupling constant (Hz, in parentheses) in deuterated dimethyl sulfoxide): 9.13 (1H, br.s), 8.54 (1H, d, 9.0), 7.74 (1H, d, 8.0), 7.71 (1H, d, 8.6), 7.65 (1H, d, 9.8), 7.51 (1H, d, 1.7), 7.40 (1H, d, 6.3), 7.15 (1H, dd, 8.6, 1.7), 7.11 (1H, s), 7.06 ( 1H, ddd, 8.0, 6.9, 1.1), 6.96 (1H, dd, 8.0, 7.4), 6.95 (1H, d, 8.0), 5.61 (1H, d, 4.6), 5.39 (1H, dq, 14.9, 6.3), 5.25 (1H, dtd, 14.9, 6.9, 1.7), 5. 08 (1H, dd, 8.6 4.6), 5.04 (1H, dd, 9.2, 4.6), 4.79 (1H, m), 4.78 (1H, m), 4.69 (1H, dd, 8. 6, 4.6), 4.33 (1H, m), 4.30 (1H, t, 5.8), 4.22 (1H, br. Dd, 11.4, 2.3), 3. 23 (3H, s), 3.18 (1H, dd, 3.5, 2.8), 2.92 (1H, br.dd, 13.8, 5.8), 2.85 (1H, m ), 2.84 (1H, m), 2.65 (1 H, br. Dd, 13.8, 9.2), 2.60 (1 H, m), 2.32 (1 H, m), 2. 26 (1H, m), 2.19 (1H, dt, 14.3, 5.2), 2.12 (3H, s), 1.96 (1H, dd, 14.3, 9.2, 6) .3), 1.93 (1H, ddd, 14.9, 9.7, 4.6) , 1.77 (1H, ddd, 14.9, 9.7, 4.6), 1.57 (3H, s), 1.57 (1H, br.d, 9.2), 1.40 ( 3H, s), 1.35 (1H, m), 1.28 (1H, ddd, 12.5, 11.5, 4.0), 1.16 (3H, d, 7.4), 1. 15 (3H, d, 7.5), 0.87 (1H, m), 0.84 (3H, d, 6.3), 0.82 (3H, d, 6.9), 0.30 ( 3H, d, 6.9), 0.13 (3H, d, 6.3), -0.89 (1H, ddd, 12.5, 9.0, 3.0).
(S is a single line, d is a double line, t is a triple line, q is a quadruple line, m is a multiple line, br. Is a wide line, and H is the number of protons.)
(8) 13 C nuclear magnetic resonance spectrum (chemical shift (ppm) in deuterated dimethyl sulfoxide): 173.6, 171.8, 171.6, 169.3, 169.2, 168.2, 167.6, 166.9, 150.6, 135.6, 135.3, 134.7, 127.2, 127.0, 126.8, 125.7, 124.6, 123.1, 120.5, 120. 5, 118.8, 118.3, 112.8, 112.4, 68.2, 57.9, 57.1, 56.8, 56.6, 55.6, 54.6, 51.0, 50.4, 45.1, 44.2, 36.5, 36.5, 35.6, 35.4, 35.2, 34.1, 27.8, 27.5, 24.1, 23. 6, 23.2, 22.7, 22.3, 21.7, 21.2, 20.1, 17.4, 16.7, 1 .7.
(9) Solubility in solvents: Easily soluble in chloroform, ethanol, methanol, dimethyl sulfoxide. Insoluble in water.
(10) Color reaction: Positive for phosphomolybdenum sulfate. Positive for Ehlrich reagent.
　Ｋ９５－５９０１－１物質の各種理化学的性状やスペクトルデータを検討した結果、Ｋ９５－５９０１－１物質は下記式（Ｉ）で表される構造であることが決定された。 As a result of examining various physicochemical properties and spectrum data of the K95-5901-1 substance, it was determined that the K95-5901-1 substance has a structure represented by the following formula (I).
Figure JPOXMLDOC01-appb-C000007
Figure JPOXMLDOC01-appb-C000007
　以上のとおり、Ｋ９５－５９０１－１物質の各種理化学的性状について詳述したが、このような性質に一致する化合物はこれまで報告されておらず、Ｋ９５－５９０１－１物質は新規物質であると決定した。 As described above, various physicochemical properties of the K95-5901-1 substance have been described in detail. However, no compound that matches such properties has been reported so far, and the K95-5901-1 substance is a novel substance. Were determined.
（実施例３）Ｋ９５－５９０１－１物質のマイコバクテリウム属菌に対する抗菌活性
　被験菌として、多剤耐性（リファンピシンならびにイソニアジド耐性）臨床分離マイコバクテリウム・ツバクローシスＷ－２６株ならびにＷ－１１４株を用いた。小川培地上で発育した８週間以内の菌コロニーをマイコブロス〔極東製薬工業（株）製〕に懸濁して、３７℃にて培養を開始した。培養液を１日毎に撹拌しデジタル比色計Ｖｉ－ｓｐｅｃＩＩ〔極東製薬工業（株）製〕を用いて波長５３０ｎｍにおける濁度の測定を行い、濁度０．１５に達するまで培養を続けた。さらにこの培養液を、新たなマイコブロスに４０μｌ接
種し、濁度が０．１５±０．０１となるまで培養した後、マイコブロス４ｍｌに対し培養液１００μｌを加え接種菌液とした。丸底９６穴マイクロタイタープレートにあらかじめマイコブロスを用いて２倍階段希釈したＫ９５－５９０１－１物質液１００μｌを用意し、これに１００μｌの接種菌液を加えて、当該丸底９６穴マイクロタイタープレートを３７℃にて培養した。培養１０日目までに肉眼的に有意な菌発育が認められない最小の薬剤濃度を最小発育阻止濃度として決定した。Ｋ９５－５９０１－１物質のＷ－２６株およびＷ－１１４株に対する最小発育阻止濃度は、それぞれ０．０１６μｇ／ｍｌおよび０．００８μｇ／ｍｌであった。 (Example 3) Antibacterial activity of K95-5901-1 substance against Mycobacterium spp. As test bacteria, multidrug resistant (rifampicin and isoniazid resistant) clinically isolated Mycobacterium tuberculosis W-26 strain and W-114 strain Was used. Bacterial colonies within 8 weeks grown on Ogawa medium were suspended in mycobroth [manufactured by Kyokuto Pharmaceutical Co., Ltd.] and culture was started at 37 ° C. The culture solution was stirred every day, and turbidity was measured at a wavelength of 530 nm using a digital colorimeter Vi-spec II (manufactured by Kyokuto Pharmaceutical Co., Ltd.), and the culture was continued until the turbidity reached 0.15. Further, 40 μl of this culture solution was inoculated into new mycobroth and cultured until the turbidity became 0.15 ± 0.01, and then 100 μl of culture solution was added to 4 ml of mycobroth to obtain an inoculum. Prepare 100 μl of a K95-5901-1 substance solution diluted in 2-fold steps using Myco broth in advance on a round bottom 96-well microtiter plate, add 100 μl of the inoculum, and add the round bottom 96-well microtiter plate. Cultured at 37 ° C. The minimum drug concentration at which no significant bacterial growth was observed by the 10th day of culture was determined as the minimum growth inhibitory concentration. The minimum inhibitory concentrations of the K95-5901-1 substance for the W-26 and W-114 strains were 0.016 μg / ml and 0.008 μg / ml, respectively.
　本発明のＫ９５－５９０１－１物質のマイコバクテリウム属以外の試験菌に対する抗菌活性は以下のとおりである。濾紙円板〔アドバンテック東洋（株）製、直径６ｍｍ〕にＫ９５－５９０１－１物質の１ｍｇ／ｍｌのメタノール溶液をそれぞれ１０μｌ浸漬し、一定時間風乾して溶媒を除去後、表３に列挙された試験菌をそれぞれ含有する寒天平板に張り付け、３５℃で２４時間培養後、濾紙円板の周りにできた生育阻止円の直径を測定した。 The antibacterial activity of the K95-5901-1 substance of the present invention against test bacteria other than the genus Mycobacterium is as follows. 10 μl each of a 1 mg / ml methanol solution of the K95-5901-1 substance was immersed in a filter paper disc (Advantech Toyo Co., Ltd., diameter 6 mm), air-dried for a certain period of time, and the solvent was removed. Attached to agar plates each containing the test bacteria and cultured at 35 ° C. for 24 hours, the diameter of the growth inhibition circle formed around the filter paper disc was measured.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　結果を表３に示す。本発明のＫ９５－５９０１－１物質は、表３に示す微生物のうちマイコバクテリウム・スメグマチスとバチルス・サブチリス、ミクロコッカス・ルテウス以外の細菌、真菌には抗菌活性を示さなかった。したがって、本発明のＫ９５－５９０１－１物質はマイコバクテリウム属細菌に抗菌活性を示す抗結核薬などの薬剤として使用し得る。 The results are shown in Table 3. The K95-5901-1 substance of the present invention showed no antibacterial activity against bacteria and fungi other than Mycobacterium smegmatis, Bacillus subtilis and Micrococcus luteus among the microorganisms shown in Table 3. Therefore, the K95-5901-1 substance of the present invention can be used as a drug such as an antituberculosis drug exhibiting antibacterial activity against Mycobacterium.
[規則26に基づく補充 09.05.2019]　
Figure WO-DOC-TABLE-134
[Supplement under Rule 26 09.05.2019]
Figure WO-DOC-TABLE-134

Claims (9)

  1. 　下記式（Ｉ）で表される化合物若しくはその塩、又はそれらの水和物若しくは溶媒和物。
    Figure JPOXMLDOC01-appb-C000001
         A compound represented by the following formula (I) or a salt thereof, or a hydrate or solvate thereof.
    Figure JPOXMLDOC01-appb-C000001
  2. 　放線菌に属する上記式（Ｉ）で表される化合物を生産する能力を有する微生物を培地で培養し、培養物中に前記化合物を蓄積せしめ、該培養物から前記化合物を採取することを含む、請求項１に記載の化合物の製造方法。 Culturing a microorganism having the ability to produce a compound represented by the above formula (I) belonging to actinomycetes in a medium, accumulating the compound in the culture, and collecting the compound from the culture. The manufacturing method of the compound of Claim 1.
  3. 　放線菌に属する上記式（Ｉ）で表される化合物を生産する能力を有する微生物が、アクチノプラネス属に属する微生物である、請求項２に記載の製造法。 The production method according to claim 2, wherein the microorganism having the ability to produce the compound represented by the above formula (I) belonging to Actinomyces is a microorganism belonging to the genus Actinoplanes.
  4. 　放線菌に属する上記式（Ｉ）で表される化合物を生産する能力を有する微生物が、アクチノプラネス・エスピー（Ａｃｔｉｎｏｐｌａｎｅｓ　ｓｐ．）Ｋ９５－５９０１（受託番号ＮＩＴＥ　ＢＰ－０２６５８）である請求項３に記載の製造法。 The microorganism having the ability to produce a compound represented by the above formula (I) belonging to actinomycetes is Actinoplanes sp. K95-5901 (Accession Number NITE BP-02658). Manufacturing method.
  5. 　放線菌に属し、Ｋ９５－５９０１－１物質を生産する能力を有する微生物。 A microorganism that belongs to actinomycetes and has the ability to produce K95-5901-1.
  6. 　アクチノプラネス・エスピー（Ａｃｔｉｎｏｐｌａｎｅｓ　ｓｐ．）Ｋ９５－５９０１（受託番号ＮＩＴＥ　ＢＰ－０２６５８）株。 Actinoplanes sp. K95-5901 (Accession number NITE BP-02658) strain.
  7. 　請求項１に記載の化合物若しくはその塩、又はそれらの水和物若しくは溶媒和物を有効成分として含有する、医薬組成物。 A pharmaceutical composition comprising the compound according to claim 1 or a salt thereof, or a hydrate or solvate thereof as an active ingredient.
  8. 　抗結核薬である、請求項７に記載の医薬組成物。 The pharmaceutical composition according to claim 7, which is an antituberculosis drug.
  9. 　結核菌増殖阻害剤である、請求項８に記載の医薬組成物。 The pharmaceutical composition according to claim 8, which is a tuberculosis growth inhibitor.
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Publication number Priority date Publication date Assignee Title
EP4089103A1 (en) * 2021-05-12 2022-11-16 Universität Duisburg-Essen Clpcp protease-binding cyclic heptapeptides

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