WO1999031127A1 - Cyclopeptolides - Google Patents

Cyclopeptolides Download PDF

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Publication number
WO1999031127A1
WO1999031127A1 PCT/JP1998/005716 JP9805716W WO9931127A1 WO 1999031127 A1 WO1999031127 A1 WO 1999031127A1 JP 9805716 W JP9805716 W JP 9805716W WO 9931127 A1 WO9931127 A1 WO 9931127A1
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Prior art keywords
substance
group
val
antifungal
chc
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PCT/JP1998/005716
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French (fr)
Japanese (ja)
Inventor
Kenichi Kaida
Toshiyuki Kameyama
Ryosuke Fudou
Toshihiko Andou
Makoto Ojika
Youji Sakagami
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Ajinomoto Co., Inc.
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Priority to AU16835/99A priority Critical patent/AU1683599A/en
Publication of WO1999031127A1 publication Critical patent/WO1999031127A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K11/00—Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K11/02—Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof cyclic, e.g. valinomycins ; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/72—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel cyclic peptide and an antifungal containing the peptide as an active ingredient.
  • bacteriosis has been easily and effectively treated with the use of a number of chemotherapeutic agents, mainly antibiotics.
  • chemotherapeutic agents mainly antibiotics.
  • due to the use of these chemotherapeutic agents deep mycosis due to infection with soybeans, yeasts, and other fungi has been taken up as a problem.
  • the present inventors have improved the fungi and improved the piles j J) to ') -filtrate, and selected antibiotics having antifungal activity to obtain antifungal agents.
  • Microorganisms were searched extensively from the field and succeeded in obtaining microorganisms suitable for this purpose. These microorganisms were filamentous fungi belonging to Clavaryopsis. The antibiotic produced by this microorganism was separated and its structure was determined. As a result, it was found to be a cyclic deb-peptide.
  • the present invention has been made based on these findings, and the cyclic depsibeptide of the present invention has the following amino acid sequence.
  • Cyclic depsipeptide FA-l cyclo (-2-hydroxy isovaleryl- Hpr-MeVa Val-MeAsp-Me 11 e-Me 11 eG 1 y-MeVal ⁇ Tyr (OMe)-), or cyclic depsipeptide FA15- 2: cyclo (-2-hydroxvisovaleryl-Hpr-Val-Val-MeAsp-MeI le- eI le-Gly- eVal- Tyr (OMe) -) c is be shown as follows the annular Depushi peptide by structure
  • FIG. 1 is UV absorption scan Bae spectrum diagram of the cyclic depsipeptide FA15-1
  • FIG. 2 IR absorption scan Bae-vector diagram
  • FIG. 3 is 1 H- NMR spectrum view
  • Fig. 4 13 FIG. 3 is a C-NMR spectrum diagram.
  • Fig. 5 shows the UV absorption spectrum of cyclic depsipeptide FA15-2
  • Fig. 6 shows I.
  • FIG. 7 is a 1 H-NMR spectrum diagram
  • FIG. 8 is a 13 C-NMR spectrum diagram.
  • Microorganisms producing the cyclic debpeptides FA15-1 and FA15-2 of the present invention include Clavariopsis aquatica AJ 117363. Strain (FERM BP-6594). AJ 117363 strain was isolated as follows. The fallen leaves in a mountain stream collected at Takaoyama, Hachioji, Tokyo were soaked in water.
  • the colony surface is light gray fluff, and the back surface is greenish black. No leaching of dye or formation of oil droplets in the medium is observed.
  • the growth on the malt extract agar medium is slow, and the diameter of the colony is 19 mm in 25 :, 14 days.
  • the colony surface is gray fluffy, and the back surface is greenish black. No leaching of dye or formation of oil droplets in the medium was observed.
  • the conidia obtained by immersing the cultured cells in water are It is a tetrapot type consisting of two cells, a main shaft and three accessory branches.
  • the main axis is in the center with one septum, club-shaped, and the size is (30-40) X (10-) ⁇ m.
  • the three appendages are radial near the tip of the main shaft, are thin thread-like, and lack a septum-the size is (50-70) X (1.5-2.5) ⁇ .
  • H is 3-10.
  • this fungus was used in accordance with the Mycobacterium Illustrative Guidebook (1978, Kodansha (Japan), Shunichi Udagawa, Keisuke Tsubaki, et al., 6 authors), incomplete mycoplasma, incomplete filamentous fungi, and Clavaryopsis.
  • the strain was identified as belonging to Aquatica (Clavariopsis aquatica), and this strain was named Clavariopsis aquatica AJ 117363 strain.
  • Bacteria producing such novel antibiotics F A15-1 and F A15-2 can be cultivated using a general method for culturing filamentous fungi.
  • a medium obtained by solidifying a food material such as oatmeal or the like, adding an additive solution to the solid layer, and sterilizing by high pressure steam can be used.
  • Substance FA - 1 and substance FA 15-2-producing bacteria it is most desirable to culture at 10 e C ⁇ 30 as possible out culturing ° C shall force particularly 20 ⁇ 25 C.
  • Culture period is usually 14 days ⁇ 21 days; can be appropriately changed depending on the culture conditions.
  • the substance FA15_1 and the substance FA12 produced by the culture accumulate in the culture.
  • a solvent extraction method using an organic solvent such as acetone or methanol an adsorption column chromatography using a silica gel or the like, a reverse phase partitioning force chromatography using an octadodecylated ( () US) silica gel as a carrier, and a column chromatography. Then, it is isolated and purified by a combination of means for purifying C.
  • the C ⁇ H group of MeAsp of FA15-1 and FA15-2 can be easily modified, and alkyl ester group, aryl ester group, aralkyl ester group, alkyl amide group, aryl It can lead to mono- and aralkyl amide groups.
  • alkyl group include an alkyl group having 1 to 12 carbon atoms, which may be linear or branched or cyclic. Further, it may contain 1 to 3 hetero atoms in the chain or the ring.
  • Examples of the alkyl group include a methyl group, an ethyl group, an isopropyl group, a tertiary butyl group, a cyclohexyl group, a methoxymethyl group, and an ethoxycarbonyloxy-2-ethyl group.
  • Examples of the aryl group include an aryl group having 1 to 12 carbon atoms, which may have a substituent. Further, it may contain 1 to 3 hetero atoms in the chain or the ring.
  • Examples of the aryl group include a phenyl group, a methoxyphenyl group, a nitrophenyl group, a naphthyl group, a 'pyridyl group and a quinolyl group.
  • the aralkyl group includes an aralkyl group having 1 to 12 carbon atoms, and the ring may have a substituent. Also, the chain or the substituent may contain 1-3 heteroatoms. Examples of the aralkyl group include a benzyl group, a methoxybenzyl group, a naphthylmethyl group, and a pyridylmethyl group.
  • Alkyl ester groups and aryl ester groups can be introduced by dissolving FA-1 and FA15-2 in solvents such as acetonitrile, chloroform, dimethylformamide, dimethylsulfoxide, and dioxane, and then dissolving them in thionyl chloride and oxychloride. It can be carried out by deriving an acid halide with phosphorus or the like and then subjecting it to dehydration condensation with an alcohol in the presence of an alcohol catalyst or an acid catalyst.Also, it can be reacted with an alkyl halide or aryl halide in the presence of a base. Often, in the case of the methyl ester, it is easily obtained by reacting with diazomethane or its equivalent.
  • Alkyl amides and aryl amides can be introduced by dissolving FA 15-1 and DO 15-2 in solvents such as acetonitrile, chlorophonolem, dimethinoleformamide, dimethylsulfoxide, and dioxane, and then adding thionyl chloride and phosphorus oxychloride.
  • the reaction can be carried out by leading to an acid halide by the method described above, and then reacting with the amine using a condensing agent such as dicyclohexyl carbamide or the like, a reaction force with the corresponding amine.
  • a condensing agent such as dicyclohexyl carbamide or the like
  • N-hydroxysuccinimide ⁇ N-hydroxybenzotriazole or the like may coexist and the active ester form may be used.
  • an excess amine may be used to condense by heating, and once an appropriate ester form is formed, it may be reacted with an amine to perform an ester-amide
  • the modified product thus obtained can be purified by ordinary purification means such as column chromatography, thin-layer chromatography, crystallization and the like.
  • An example of a method for purifying the substance FA-11 is as follows. (4) An equal amount of acetone was added to the iWiW and 'H materials and extracted. After extraction, the extract obtained by centrifugation was concentrated using a rotary evaporator to obtain an aqueous extract solution. The aqueous extract solution was extracted with ethyl acetate. This ethyl acetate layer was concentrated to dryness with a rotary-evaporator. This concentrated and dried product was dissolved in a small amount of a solution containing 5% of mouth mouth 95 ° / ⁇ methanol 5%, and subjected to silylation gel chromatography equilibrated with the same solvent to elute.
  • the antibacterial activity against Aspergillus niger was concentrated, and the solution was concentrated on reversed phase partition force column chromatography (C18), washed with a 60% aqueous solution of acetonitrile, and eluted with an 80% aqueous solution of acetonitrile.
  • the eluate was concentrated and subjected to high performance liquid chromatography (condition column, Shiseido's CAPSELL PAK C18 UG 120 10 X 250 mm developing solvent 85% acetonitrile, 0.1 /. TFA aqueous solution, flow rate 2.5 ml / min, 18- (Elution for 20 minutes) to obtain substance FA15-1.
  • An example of a purification method for the substance FA15-2 is as follows.
  • This fraction was dissolved in methanol and passed through a cartridge for ODS pretreatment (T0Y0PAK ODS M), and the adsorbed substance was eluted with chloroform-formanol. Separation of methanol-eluted fraction by high-performance liquid chromatography (Condition column, Shiseido's CAPSELL PAK C18 UG80 10 X 250 ⁇ Selution solvent ⁇ 0% methanol, 0.1% TFA- 100% methanol 0.1% TF ⁇ , 3 () min gradient, flow rate 2. ⁇ / ⁇ ⁇ ).
  • Substance F A15-1 has the following physicochemical properties.
  • the structure of this substance was analyzed.
  • the substance has the molecular formula C 59 H 95 N 9 from high-resolution mass spectrometry.
  • 1 4 has the (integer molecular weight 1153), the absorption of your Keru Ami de the IR spectrum (1681, 1648 cm-l), Ami de NH hydrogen and NCH3 hydrogen signals in 1 H NMR, 11 pieces in @ 13 C NMR Amide (or ester, carboxylic acid) was estimated to be a peptide compound from the carbonyl signal.
  • the plane structure is
  • the 2D NMR was determined by a detailed examination. That is, 10 amino acid residues (Tyr (or Tyr (OMe)) from COSY and H0HAHA, MeVal x 2, Gly, Melle x 2, MeAsp
  • Substance F A15-2 has the following physicochemical properties.
  • Solubility Soluble in chloroform, methanol, acetone, ethyl acetate, dimethyl sulfoxide, poorly soluble in water
  • the antifungal activity of substance FA15-1 was determined by the agar dilution method, and a small number of three substances F ⁇ ⁇ -1 were prepared. 25 ⁇ l of the mixture was mixed with 10 ml of the heated and dissolved sub-agar-agar medium and dispensed into a plate.c. The culture suspension of the test bacterium was spread on an agar plate containing this new antibiotic FA15-1. The cells were cultured at 25 nC for 2 to 4 days, and the growth inhibitory concentration was measured. (Rib: 25 ° C, 4 days, Yeast and bacteria: 25 ° C, 2 days) The results are shown in Table 1.
  • the antifungal activity of the substance FA15-2 was determined by the microdilution method. After dissolving the substance FA15-2 with a small amount of dimethylsulfoxide, an equal dilution series was prepared with dimethyls / rephoxide. 0.5% of RPMI was added to RPM I medium (manufactured by Nissui Pharmaceutical Co., Ltd. fh). In the RPMI medium, the suspension spore solution was added to a final spore concentration of 2 ⁇ 105 C FU / ml was added. The medium containing the diluted material and the spore solution was added to a 96-well microtiter plate in a 1-well volume. After culturing at 37 ° C for 24 hours, the minimum growth inhibitory concentration was visually determined after culturing. Table 2 shows the results.
  • the cyclic debpeptide of the present invention is excellent in antifungal activity and can provide an antifungal agent having a strong new antibacterial activity.
  • this cyclic depsipeptide can be produced by a usual microbial culture method, and since no special method is used for extraction and purification, industrial mass production is possible.

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Abstract

Cyclopeptolides, cyclo(-2-hydroxyisovaleryl-Hpr-MeVal-Val-MeAsp-MeIle-MeIle-Gly-MeVal-Tyr(OMe)-) and cyclo(-2-hydroxyisovaleryl-Hpr-Val-Val-MeAsp-MeIle-MeIle-Gly-MeVal-Tyr(OMe)-), produced by a mold of the genus Clavariopsis and having a potent antifungal activity.

Description

明 細 書 環状デブシぺプチド 技 術 分 野  Description Cyclic debut peptide technology field
本発明は新規環状デブシぺプチド及びこのぺプチドを有効成分とする抗真菌剤 に関する。  The present invention relates to a novel cyclic peptide and an antifungal containing the peptide as an active ingredient.
背 景 技 術 Background technology
近年、 抗生物質を主とする多数の化学療法剤の使用によつて細菌症は容易かつ 効果的に治療されるようになっている。 しかしながら、 これら化学療法剤の使用 により力ビ、 酵母などレ、わゆる真菌類の感染による深在性真菌症が一つの問題と して取り上げられている。  In recent years, bacteriosis has been easily and effectively treated with the use of a number of chemotherapeutic agents, mainly antibiotics. However, due to the use of these chemotherapeutic agents, deep mycosis due to infection with soybeans, yeasts, and other fungi has been taken up as a problem.
本発明の目的は寫菌に対してより強く新しレ、抗菌力を有する抗真菌剤を得るこ とにある。  It is an object of the present invention to obtain an antifungal agent which is stronger and newer against bacteria and has antibacterial activity.
発 明 の 開 示 Disclosure of the invention
本発明者達は上述の事情に鑑み、 真菌に対してより強レ、杭 j J)を ') -ろ新しし、 抗真菌剤を取得するべく、 抗真菌活性を有する抗生物質を i する微生物を 1 1然 界から広く検索し、 この目的に適合する微生物の取得に成功した この微生物は クラバリォプシスに属する糸状菌であった。 この微生物の産生する抗生物質を分 離して構造決定を行なったところ環状デブシぺプチドであつた。  In view of the above-mentioned circumstances, the present inventors have improved the fungi and improved the piles j J) to ') -filtrate, and selected antibiotics having antifungal activity to obtain antifungal agents. Microorganisms were searched extensively from the field and succeeded in obtaining microorganisms suitable for this purpose. These microorganisms were filamentous fungi belonging to Clavaryopsis. The antibiotic produced by this microorganism was separated and its structure was determined. As a result, it was found to be a cyclic deb-peptide.
本発明はこれらの知見に基づいてなされたものであり、 本発明の環状デプシベ プチドは次のァミノ酸配列を有する。  The present invention has been made based on these findings, and the cyclic depsibeptide of the present invention has the following amino acid sequence.
環状デプシぺプチ ド FA - l : cyclo (- 2- hydroxy isovaleryl- Hpr- MeVa卜 Val - MeAsp-Me 11 e-Me 11 e-G 1 y-MeVal ~Tyr (OMe) -) , 又は環状デプシペプチ ド FA15 - 2： cyclo (-2-hydroxvisovaleryl-Hpr-Val-Val-MeAsp-MeI le- eI le-Gly- eVal- Tyr(OMe)-)この環状デプシぺプチドを構造式で示せば次の通りである c Cyclic depsipeptide FA-l: cyclo (-2-hydroxy isovaleryl- Hpr-MeVa Val-MeAsp-Me 11 e-Me 11 eG 1 y-MeVal ~ Tyr (OMe)-), or cyclic depsipeptide FA15- 2: cyclo (-2-hydroxvisovaleryl-Hpr-Val-Val-MeAsp-MeI le- eI le-Gly- eVal- Tyr (OMe) -) c is be shown as follows the annular Depushi peptide by structure
Figure imgf000004_0001
又は Me 上記環状デプシぺプチドの Rがメチル基のものを F A15— 1、 Rが水素原子 のものを FA15— 2と命名した。
Figure imgf000004_0001
Or Me In the cyclic peptides described above, those in which R was a methyl group were named FA15-1, and those in which R was a hydrogen atom were named FA15-2.
図 面 の 簡 単 な 説 明 Brief explanation of drawings
第 1図は環状デプシペプチド FA15—1の UV吸収スぺクトル図、 第 2図は I R吸収スぺク トル図、 第 3図は1 H— NMRスぺク トル図、 そして、 第 4図は 13C— NMRスぺクトル図である。 Figure 1 is UV absorption scan Bae spectrum diagram of the cyclic depsipeptide FA15-1, FIG. 2 IR absorption scan Bae-vector diagram, FIG. 3 is 1 H- NMR spectrum view, and Fig. 4 13 FIG. 3 is a C-NMR spectrum diagram.
第 5図は環状デプシペプチド FA15— 2の UV吸収スペク トル図、 第 6図は I Fig. 5 shows the UV absorption spectrum of cyclic depsipeptide FA15-2, and Fig. 6 shows I.
R吸収スぺク トル図、 第 7図は1 H— NMRスぺク トル図、 そして、 第 8図は 13C— NMRスぺク トル図である。 FIG. 7 is a 1 H-NMR spectrum diagram, and FIG. 8 is a 13 C-NMR spectrum diagram.
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
本発明の環状デブシぺプチド F A15— 1、 F A15- 2を生産する微生物として は、 クラバリォプシス アクアティカ （Clavariopsis aquatica) A J 117363 株 (FERM B P— 6594)が挙げられる。 AJ 117363株は次のようにして分離された。 東京都八王子巿高尾山にて採集された渓流中の落葉を水につけた。 水面をピべ ッ トでとり、 L CA培地上 （ブドウ糖 1 g/L、 KH2P04 0.1 g/L, Mg S 04 - 7H20 0.2gZL、 KC 1 0.2g/L, N a N 03 2 g/L、 酵母エキス 0.2gZL、 寒天 15gZL、 pH6. δ) に載せた。 顕微鏡で分生子を確認後、 単 胞子分離を行なった。 2日後顕微鏡下で胞子の発芽の確認を行なった。 その後、 室温で培養した。 以下に、 このようにして分離された AJ 117363株の菌学的性質 を記す。 Microorganisms producing the cyclic debpeptides FA15-1 and FA15-2 of the present invention include Clavariopsis aquatica AJ 117363. Strain (FERM BP-6594). AJ 117363 strain was isolated as follows. The fallen leaves in a mountain stream collected at Takaoyama, Hachioji, Tokyo were soaked in water. Taking the water in Pibe Tsu bets, L CA medium on (glucose 1 g / L, KH 2 P0 4 0.1 g / L, Mg S 0 4 - 7H 2 0 0.2gZL, KC 1 0.2g / L, N a N 0 3 2 g / L, yeast extract 0.2GZL, placed on agar 15gZL, pH6. δ). After confirming conidia under a microscope, monospores were separated. Two days later, spore germination was confirmed under a microscope. Thereafter, the cells were cultured at room temperature. The bacteriological properties of the thus isolated AJ117363 strain are described below.
(a) 各種寒天培地に於ける生育形態  (a) Form of growth on various agar media
L C A寒天培地上での生育は遅く、 25=C、 14日間でコロニ一径は 10瞧である = コロニー表面は緑味灰色綿毛状で、 裏面は緑味灰色を呈する。 培地中への色素の 浸出や油滴の生成は認められなレ、。 Growth on LCA agar is slow, 25 = C, colony diameter is 10 瞧 in 14 days = colony surface is greenish gray fluffy, reverse side is greenish gray. No leaching of dye or formation of oil droplets in the medium was observed.
コーンミール寒天培地上での生育は遅く、 25eC、 14 日間でコロニ一径は 16隱 である。 コロニー表面は緑味白綿毛状で、 裏面は緑味白色を呈する。 培地中への 色素の浸出や油滴の生成は認められなレ Growth on cornmeal agar is slow, with a colony diameter of 16 secrets at 25 e C for 14 days. The colony surface is greenish fluffy, and the backside is greenish white. No leaching of dye or formation of oil droplets in the medium was observed.
バレイショ 'ブドウ糖寒天培地上での生育は遅く、 25=C、 14 日問でコロニー 径は 21隱である。 コロニー表面は明るい灰色綿毛状で、 裏面は緑味黒色を呈す る。 培地中への色素の浸出や油滴の生成は認められない。 Potato growth on glucose agar is slow, 25 = C, colony diameter is 21 hidden in 14 days. The colony surface is light gray fluff, and the back surface is greenish black. No leaching of dye or formation of oil droplets in the medium is observed.
麦芽エキス寒天培地上での生育は遅く、 25 ：、 14 日間でコロニ一径は 19mmで ある。 コロニー表面は灰色綿毛状で、 裏面は緑味黒色を呈する。 培地中への色素 の浸出や油滴の生成は認められなレ、。  The growth on the malt extract agar medium is slow, and the diameter of the colony is 19 mm in 25 :, 14 days. The colony surface is gray fluffy, and the back surface is greenish black. No leaching of dye or formation of oil droplets in the medium was observed.
これら培地上で分生子は形成されない。 培養菌体の一部を水中に浸すことによ り分生子形成が見られた。  No conidia are formed on these media. Conidia formation was observed by immersing part of the cultured cells in water.
(b) 形態的性状  (b) Morphological properties
培養菌体を水中に浸すことにより得られた分生子は、ァレゥ口型分生子、無色、 2細胞、 主軸と 3本の付属枝からなる、 テトラポット型である。 主軸は中央に 1隔壁、 棍棒状であり、 サイズは (30〜40) X (10〜 ） μ mである。 3本の付属枝 は主軸の先端近くに放射状にあり、 細い糸状で隔壁を欠く- サイズは（50〜70) X (1. 5〜2. 5) μ πιである。 The conidia obtained by immersing the cultured cells in water are It is a tetrapot type consisting of two cells, a main shaft and three accessory branches. The main axis is in the center with one septum, club-shaped, and the size is (30-40) X (10-) μm. The three appendages are radial near the tip of the main shaft, are thin thread-like, and lack a septum-the size is (50-70) X (1.5-2.5) μππι.
生育温度は、 10〜3( Cであり、 至適生育温度は 20〜25=Cである。 また生育 pThe growth temperature is 10-3 (C, and the optimal growth temperature is 20-25 = C.
Hは 3〜10である。 H is 3-10.
以上の菌学的性質から、本菌は、 「菌類図鑑」 （1978、講談社（日本）、宇田川 俊 一、 椿 啓介 他 6名著） に従い、 不完全菌亜門、 不完全糸状菌綱、 クラバリォ プシス アクアティカ（Clavariopsis aquatica)に属することが明かとなり、 本 菌株をクラバリォプシス アクアティカ（Clavariopsis aquatica) A J 117363株と 命名した。 本菌株は、 1997年 12月 8日より、 工業技術院生命工学工業技術研究 所 （郵便番号 305 筑波県つくば巿東一丁目 1番 3号） に寄託されており、 F E RM B P— 6594の受託番号が付与されている。  Based on the mycological properties described above, this fungus was used in accordance with the Mycobacterium Illustrative Guidebook (1978, Kodansha (Japan), Shunichi Udagawa, Keisuke Tsubaki, et al., 6 authors), incomplete mycoplasma, incomplete filamentous fungi, and Clavaryopsis. The strain was identified as belonging to Aquatica (Clavariopsis aquatica), and this strain was named Clavariopsis aquatica AJ 117363 strain. This strain has been deposited at the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology (Postal code: 305 Tsukuba, Higashi 1-3-1, Tsukuba, Japan) since December 8, 1997, and FE RM BP-6594 has been deposited. Numbers are given.
このような新規抗生物質 F A 15— 1、 F A15— 2生産菌は一般的な糸状菌の培 養方法を用いて培養することが可能である。  Bacteria producing such novel antibiotics F A15-1 and F A15-2 can be cultivated using a general method for culturing filamentous fungi.
液体振盪培養や液体静置培養に用いられる培地としては、 物質 F A 15— 1及び 物質 F A 15— 2生産菌が利用できる栄養源を含有するものであればよレ、c例えば、 炭素源としてグルコースゃデキストリン等が用いられ、 窒素源として大豆粉、 綿 実粕、 菜種油粕、 酵母エキスなどの各種タンパク質やアミノ酸混合物が用いられ る = この他、 例えばリン酸水素ナトリウム、 硫酸マグネシウム、 炭酸カルシウム 等の無機塩も必要に応じて用いる事が出来る。 The medium used in the liquid shaking culture or liquid stationary culture, yo les, c for example as long as it contains the nutrient sources available to substance FA 15-1 and substance FA 15-2 producing bacteria, glucose as carbon sourceゃ Dextrin etc. are used, and various proteins and amino acid mixtures such as soybean flour, cottonseed meal, rapeseed oil meal, yeast extract, etc. are used as nitrogen sources = In addition, for example, sodium hydrogen phosphate, magnesium sulfate, calcium carbonate, etc. Inorganic salts can also be used as needed.
固体培養では、 オートミール等の食品素材等を固層として、 添加液を固層に加 え高圧蒸気滅菌した培地を用いることが出来る。  In solid culture, a medium obtained by solidifying a food material such as oatmeal or the like, adding an additive solution to the solid layer, and sterilizing by high pressure steam can be used.
物質 F A — 1及び物質 F A 15— 2生産菌は 10eC〜30°Cで培養することがで きる力 特に 20 〜 25 Cで培養することが最も望ましい。 培養期間は通常 14 日 〜21 日である力;、 培養条件により適宜変更することが可能である。 Substance FA - 1 and substance FA 15-2-producing bacteria, it is most desirable to culture at 10 e C~30 as possible out culturing ° C shall force particularly 20 ~ 25 C. Culture period is usually 14 days ~ 21 days; can be appropriately changed depending on the culture conditions.
培養により生成した物質 F A 15 _ 1及び物質 F A 一 2は、 培養物中に蓄積さ れる。 この生産菌の培養物から抗生物質 F A 一 1及び物質 F Λ 1 Β- 2を得るた めには微生物の代謝産物を採集するのに用レ、られる方法によって ''·離、 ':Ί'ί·製され る。  The substance FA15_1 and the substance FA12 produced by the culture accumulate in the culture. In order to obtain the antibiotic FA-1 and the substance FΛ1Β-2 from the culture of this producing bacterium, it is necessary to collect the metabolites of the microorganism by the method used for '' ί · Made.
例えば、 アセトン、 メタノール等の有機溶媒による溶媒抽出法、 シリ力ゲル等 を使用する吸着カラムクロマトグラフィー、 ォクタドデシル化（()US)された シリカゲルを担体とする逆相分配力ラムクロマトグラフィ一及びト 1 し Cの精製 手段を組み合わせて行なうことにより単離、 精製される。 For example, a solvent extraction method using an organic solvent such as acetone or methanol, an adsorption column chromatography using a silica gel or the like, a reverse phase partitioning force chromatography using an octadodecylated ( () US) silica gel as a carrier, and a column chromatography. Then, it is isolated and purified by a combination of means for purifying C.
F A 15— 1及び F A 15— 2の MeAspの C〇〇 H基は容易に修飾可能であり、 通 常の方法により、 アルキルエステル基、 ァリールエステル基、 ァラルキルエステ ル基、 アルキルアミ ド基、 ァリ一ルアミ ド基、 ァラルキルアミ ド基に導くことが 出来る。 ここで、 アルキル基としては、 炭素数 1〜12のアルキル基が挙げられ、 直鎖でも分枝していてもよく、 環状になっていてもよい。 又、 鎖中、 若しくは環 中に 1〜3個のへテロ原子を含んでいてもよい。 アルキル基としては例えば、 メ チル基、 ェチル基、 イソプロピル基、 ターシャリーブチル基、 シクロへキシル基、 メ トキシメチル基、 エトキシカルボニルォキシー 2—ェチル基等が挙げられる。 ァリール基としては炭素数 1〜12 のァリール基が挙げられ、 これらは置換基を 有していてもよレ、。 又、 鎖中、 若しくは環中に 1〜 3個のへテロ原子を含んでい てもよい。 ァリール基としては例えば、 フエニル基、 メ トキシフエニル基、 ニト 口フエニル基、 ナフチル基、 'ピリジル基、 キノリル基、 等が挙げられる。 ァラル キル基としては炭素数 1〜12 のァラルキル基が挙げられ、 環には置換基を有し ていてもよい。 また、 鎖中、 若しくは置換基中に 1〜3個のへテロ原子を含んで いてもよレ、。 ァラルキル基としては例えば、 ベンジル基、 メ トキシベンジル基、 ナフチルメチル基、 ピリジルメチル基などが挙げられる。 これらの修飾体はその まま、 或いは投与後に F A 15— 1及び F A 15— 2に生体内で変換され、 抗真菌活 性を示すことが期待される。 以下にこれらの修飾体の調製法を示す。 The C〇〇H group of MeAsp of FA15-1 and FA15-2 can be easily modified, and alkyl ester group, aryl ester group, aralkyl ester group, alkyl amide group, aryl It can lead to mono- and aralkyl amide groups. Here, examples of the alkyl group include an alkyl group having 1 to 12 carbon atoms, which may be linear or branched or cyclic. Further, it may contain 1 to 3 hetero atoms in the chain or the ring. Examples of the alkyl group include a methyl group, an ethyl group, an isopropyl group, a tertiary butyl group, a cyclohexyl group, a methoxymethyl group, and an ethoxycarbonyloxy-2-ethyl group. Examples of the aryl group include an aryl group having 1 to 12 carbon atoms, which may have a substituent. Further, it may contain 1 to 3 hetero atoms in the chain or the ring. Examples of the aryl group include a phenyl group, a methoxyphenyl group, a nitrophenyl group, a naphthyl group, a 'pyridyl group and a quinolyl group. The aralkyl group includes an aralkyl group having 1 to 12 carbon atoms, and the ring may have a substituent. Also, the chain or the substituent may contain 1-3 heteroatoms. Examples of the aralkyl group include a benzyl group, a methoxybenzyl group, a naphthylmethyl group, and a pyridylmethyl group. These modifications are It is expected to be converted to FA15-1 and FA15-2 in vivo as it is or after administration, and to exhibit antifungal activity. The method for preparing these modified products is described below.
アルキルエステル基、 ァリールエステル基の導入は、 F A — 1及び F A 15— 2をァセトニトリル、 クロ口ホルム、 ジメチルホルムアミ ド、 ジメチルスルホキ シド、 ジォキサン等の溶媒に溶解し、 塩化チォニル、 ォキシ塩化リン等により酸 ハロゲン化物に導き、 その後、 アルコールで処理する力 \ 或いは酸触媒存在 下、 アルコールと脱水縮合させることによって行うことが出来る また、 塩基存 在下でアルキルハライド、 ァリールハラィ ドと反応させてもよく、 メチルェステ ル体の場合には、 ジァゾメタン或いはその等価 ( と反応させろことによつても、 容易に得られる。  Alkyl ester groups and aryl ester groups can be introduced by dissolving FA-1 and FA15-2 in solvents such as acetonitrile, chloroform, dimethylformamide, dimethylsulfoxide, and dioxane, and then dissolving them in thionyl chloride and oxychloride. It can be carried out by deriving an acid halide with phosphorus or the like and then subjecting it to dehydration condensation with an alcohol in the presence of an alcohol catalyst or an acid catalyst.Also, it can be reacted with an alkyl halide or aryl halide in the presence of a base. Often, in the case of the methyl ester, it is easily obtained by reacting with diazomethane or its equivalent.
アルキルァミ ド基、 ァリ一ルァミ ド基の導入は、 F A 15 - 1及びド Λ 15- 2を ァセトニトリノレ、 クロロホノレム、 ジメチノレホルムアミド、 ジメチルスルホキシド、 ジォキサン等の溶媒に溶解し、 塩化チォニル、 ォキシ塩化リン等により酸ハロゲ ン化物に導き、 その後、 対応するァミン類と反応させる力、 ジシクロへキシルカ ルポジィミ ド等の縮合剤を用いて、 ァミン類と反応させることによって行うこと が出来る。 後者の場合、 N—ヒ ドロキシスクシンイミ ドゃ N—ヒ ドロキシ ベンゾトリアゾール等を共存させ、 活性エステノレ体を経由してもよい。 また、 過 剰のアミンを用いて加熱して縮合させることによつてもよく、 一旦、 適当なエス テル体とした後、 ァミンと反応させて、 エステル一アミ ド交換反応を行っても良 い。  Alkyl amides and aryl amides can be introduced by dissolving FA 15-1 and DO 15-2 in solvents such as acetonitrile, chlorophonolem, dimethinoleformamide, dimethylsulfoxide, and dioxane, and then adding thionyl chloride and phosphorus oxychloride. The reaction can be carried out by leading to an acid halide by the method described above, and then reacting with the amine using a condensing agent such as dicyclohexyl carbamide or the like, a reaction force with the corresponding amine. In the latter case, N-hydroxysuccinimide ゃ N-hydroxybenzotriazole or the like may coexist and the active ester form may be used. Alternatively, an excess amine may be used to condense by heating, and once an appropriate ester form is formed, it may be reacted with an amine to perform an ester-amide exchange reaction. .
このようにして得られた修飾体は、 カラムクロマトグラフィー、 薄層クロマタ グラフィ一、 結晶化等の通常の精製手段により精製する事が出来る。  The modified product thus obtained can be purified by ordinary purification means such as column chromatography, thin-layer chromatography, crystallization and the like.
実 施 例 Example
培養例  Culture example
麦芽エキス寒天培地上にクラバリォプシスアクアテイク AJ117363, F E RM BP— 6594を接種し、 25°Cで 14 ョ間培養を行なった。 この麦芽ヱキス寒天 培地からァガーピースを切り出し、 以下の条件で培養を行なった。 Clavariopsis aquatake on malt extract agar medium AJ117363, FE RM BP-6594 was inoculated and cultured at 25 ° C for 14 hours. An agar piece was cut out from the malt kiss agar medium and cultured under the following conditions.
a) 固体静置培養 a) Solid stationary culture
500mlルー型フラスコ、 25。C、 14日間  500ml roux flask, 25. C, 14 days
使用培地量及び組成 Medium volume and composition used
オートミ一ノレ 20gZフラスコ  Oatmeal 20gZ flask
下記液体培地 28ml Zフラスコ  The following liquid medium 28ml Z flask
'コース 2  'Course 2
フルク ト一ス 5  Fructos 5
シユークロース 8  Sucrose 8
ΝΖァミン 2  Jamin 2
Mg S04 · 4 H20 0.5 Mg S0 4 4 H 2 0 0.5
KC 1 0. δ  KC 1 0.δ
Z n SO. 7H20 0. δ Z n SO. 7H 2 0 0.δ
KH2P04 KH 2 P0 4
( p H 6.0)  (pH 6.0)
b) 液体静置培養  b) Liquid stationary culture
500mlルー型フラスコ、 25°C、 14日間  500ml roux flask, 25 ° C, 14 days
倍地量 Double land volume
100ml/ルー型フラスコ  100ml / Lou flask
培地組成 gZL Medium composition gZL
グノレコース 25  Gunolle Course 25
エスサンミート （味の素株式会社製） 15  S Sun Meat (Ajinomoto Co., Inc.) 15
総合アミノ酸 2  Synthetic amino acids 2
粉末酵母エキス S (日本製薬株式会社製） 丄 炭酸 Powdered yeast extract S (Nippon Pharmaceutical Co., Ltd.) 丄 Carbonic acid
( Η 6. 0)  (Η 6.0)
c ) 液体振盪培養、 25°C、 14日間 c) Liquid shaking culture, 25 ° C, 14 days
三角フラスコ、 25°C、 14日間  Erlenmeyer flask, 25 ° C, 14 days
培地量 lSOmlZSOOml三角フラスコ Medium volume lSOmlZSOOml Erlenmeyer flask
培地組成 Medium composition
グノレコース 25  Gunolle Course 25
エスサンミート （味の素株式会社製） 15  S Sun Meat (Ajinomoto Co., Inc.) 15
fc"n"ノ 6¾ 2  fc "n" ノ 6¾ 2
粉末酵母エキス S (日本製薬株式会社製） 1  Powdered yeast extract S (Nippon Pharmaceutical Co., Ltd.) 1
炭酸カルシウム 5  Calcium carbonate 5
( p H 6. 0)  (pH 6.0)
分離精製例  Example of separation and purification
物質 F A 一 1の精製法の一例を示せば、 次の如くである。 ΙΪΙ iWiWひ, 'H物に 等量のァセトンを加え抽出した。 抽出後、 遠心分離することにより ί'.}.られた抽出 液をロータリーエバポレーターで濃縮し、 抽出物水溶液を得た。 この抽出物水溶 液を酢酸ェチルで抽出した。 この酢酸ェチル層をロータリ --エバボレーターで濃 縮乾固した。 この濃縮乾固物をク口口ホルム 9 5 ο/οメタノール 5 %の少量の溶液 に溶かし、 同じ溶媒で平衡化したシリ力ゲルクロマ卜グラフィ一を行し、、 溶出さ せた。 ァスペルギルス 二ガー (AJ117374)に対する抗菌活性部分を濃縮し、 逆相 分配力ラムクロマトグラフィ一（C18)に力け、 60%ァセトニトリル水溶液で洗浄 した後、 80%ァセトニトリル水溶液で溶出させた。 溶出液を濃縮し、 高速液体ク 口マトグラフィ （条件 カラム、 資生堂社製 CAPSELL PAK C18 UG 120 10 X 250mm 展開溶媒 85%ァセトニトリル、 0. 1。/。T F A水溶液、 流速 2. 5ml/min、 18-20 分溶出） を行い、 物質 F A 15— 1を得た。 物質 F A 15— 2の精製法の一例を示せば、 次の如くである。 培養物に等量のァ セトンを加え抽出した。 抽出後、 遠心分離することにより得られた抽出液を口一 タリ一エバポレ—ターで濃縮し、 抽出物水溶液を得た。 この抽出物水溶液を酢酸 ェチルで抽出した。 この酢酸ェチル層をロータリ一エバポレーターで濃縮乾固し た。 この濃縮乾固物をク口口ホルム 95%メタノール 5 %の少量の溶液に溶かし、 同じ溶媒で平衡化したシリカゲルクロマトグラフィーを行い、 溶出させた。 ァス ペルギルス ニガ一 (AJ 117374)に対する抗菌活性部分を濃縮し、 逆相分配カラム クロマトグラフィー（C18)に力 4ナ、 60%ァセトニトリル水溶液で洗浄した後、 80% ァセトニトリル水溶液で溶出させた。 溶出液を濃縮し、 高速液体ク口マトダラフ ィー （条件 カラム、 資生堂社製 CAPSELL PAK C18 UG120 10 X 250mm 溶出溶 媒 85%ァセトニトリル、 0. 1% T F A水溶液、 流速 2. 5mlZmin) を行い、 15. 0 分一 16. 0 分の画分を集めた。 この画分をメタノールに溶解させ、 O D S前処理 用カートリッジ（T0Y0PAK ODS M)を通し、 吸着物はクロ口ホルム一メタノ一ルで 溶出した。 メタノ一ル溶出画分を高速液体ク口マトグラフィ一で分離 （条件 力 ラム、 資生堂社製 CAPSELL PAK C18 UG80 10 X 250讓 溶出溶媒 《0%メタノ —ル、 0. 1% T F A— 100%メタノ一ル、 0. 1% T F Λ , 3()min グラジェント、 流速 2. δπιΐ/η π) した。 An example of a method for purifying the substance FA-11 is as follows. (4) An equal amount of acetone was added to the iWiW and 'H materials and extracted. After extraction, the extract obtained by centrifugation was concentrated using a rotary evaporator to obtain an aqueous extract solution. The aqueous extract solution was extracted with ethyl acetate. This ethyl acetate layer was concentrated to dryness with a rotary-evaporator. This concentrated and dried product was dissolved in a small amount of a solution containing 5% of mouth mouth 95 ° / οmethanol 5%, and subjected to silylation gel chromatography equilibrated with the same solvent to elute. The antibacterial activity against Aspergillus niger (AJ117374) was concentrated, and the solution was concentrated on reversed phase partition force column chromatography (C18), washed with a 60% aqueous solution of acetonitrile, and eluted with an 80% aqueous solution of acetonitrile. The eluate was concentrated and subjected to high performance liquid chromatography (condition column, Shiseido's CAPSELL PAK C18 UG 120 10 X 250 mm developing solvent 85% acetonitrile, 0.1 /. TFA aqueous solution, flow rate 2.5 ml / min, 18- (Elution for 20 minutes) to obtain substance FA15-1. An example of a purification method for the substance FA15-2 is as follows. An equal volume of acetone was added to the culture and extracted. After the extraction, the extract obtained by centrifugation was concentrated with a mouth evaporator to obtain an aqueous extract solution. This aqueous extract solution was extracted with ethyl acetate. This ethyl acetate layer was concentrated to dryness by a rotary evaporator. The concentrated and dried product was dissolved in a small amount of a solution containing 95% methanol and 5% methanol, and subjected to silica gel chromatography equilibrated with the same solvent to elute. The antibacterial activity against Aspergillus niger (AJ 117374) was concentrated, and the residue was washed with a 60% aqueous solution of 60% acetonitrile by reversed-phase partition column chromatography (C18) and eluted with an 80% aqueous solution of acetonitrile. The eluate was concentrated and subjected to high-speed liquid chromatography (Drug column, Shiseido CAPSELL PAK C18 UG120 10 X 250 mm elution solvent 85% acetonitrile, 0.1% TFA aqueous solution, flow rate 2.5 mlZmin), and 15 Fraction 0. Fractions of 16.0 min were collected. This fraction was dissolved in methanol and passed through a cartridge for ODS pretreatment (T0Y0PAK ODS M), and the adsorbed substance was eluted with chloroform-formanol. Separation of methanol-eluted fraction by high-performance liquid chromatography (Condition column, Shiseido's CAPSELL PAK C18 UG80 10 X 250 讓 Selution solvent << 0% methanol, 0.1% TFA- 100% methanol 0.1% TF Λ, 3 () min gradient, flow rate 2. δπιΐ / η π).
17. 8 分のピークを高速液体ク口マトグラフィ一で分離 （条件 力ラム 野村化 学 Develosil 0DS-HG-5 10 X 250mm 溶出溶媒 85%メタノ一ル、 0. 02M N H40 A c— 99%メタノ一ル、 0. 02M N H4O A c、 30min 直線グラジェント、 流速 2. OmlZmin) を行い、 物質 F A 15— 2を得た。 17. separated peaks 8 minutes high-performance liquid inlet Matogurafi one (condition forces the ram Nomura Chemical Develosil 0DS-HG-5 10 X 250mm eluent 85% methanol Ichiru, 0. 02M NH 4 0 A c- 99% methano Ichiru, 0. 02M NH 4 OA c, 30min linear gradient, perform flow rate 2. OmlZmin), to obtain a substance FA 15-2.
物質 F A 15— 1は以下のような理化学的性質を有する。  Substance F A15-1 has the following physicochemical properties.
a ) 外観：無色粉末  a) Appearance: colorless powder
b ) 溶解性：クロ口ホルム、 メタノール、 アセトン、 酢酸ェチル、 ジメチルスル ホキシドに可溶、 水に難溶 c) TLC : R f =0.82 (CHC 13— M e OH 9 : 1 )、 0.69 (H e x a n e — E t OA c -Me OH 9 : 9 : 2) b) Solubility: Soluble in chloroform, methanol, acetone, ethyl acetate, dimethyl sulfoxide, poorly soluble in water c) TLC: R f = 0.82 (CHC 1 3 - M e OH 9: 1), 0.69 (H exane - E t OA c -Me OH 9: 9: 2)
d) 旋光度： [a] 22D —210° (c0.37、 C H C 13) d) Optical rotation: [a] 22 D -210 ° (c0.37, CHC 1 3)
e) 紫外部吸収スぺク トル （Me〇H： 1 max 20 (end, 58800)、 222 (s h、 25700)、 277 (1900)、 288 (1700) nm (図 1参照） e) Ultraviolet absorption spectrum (Me〇H: 1 max 20 (end, 58800), 222 (sh, 25700), 277 (1900), 288 (1700) nm (see Fig. 1)
f )赤外部吸収スぺク トル（KB r ) :3700- 3100 (br)、 3385、2966、2935、2877、 1739、 1683、 1648、 16,14、 15、 1472、 1409、 1292、 1249、 1210、 1178、 1133、 1101、 1039cm-1 (図 2参照） f) Infrared absorption spectrum (KB r): 3700-3100 (br), 3385, 2966, 2935, 2877, 1739, 1683, 1648, 16,14, 15, 1472, 1409, 1292, 1249, 1210 , 1178, 1133, 1101, 1039cm- 1 (See Fig. 2)
g) 質量分析： HR FABMS (F AB十、 matrix： nrnitrobenzyl alchol、NaI 添加） ； ； Found 1176.6924 (M+Na) Calcd for C59H95N9014Na 1176.6896 h) 'H—核磁気共鳴スぺク トル （C6D6、 600MHz) ： (図 3参照） g) Mass spectrometry: HR FABMS (F AB10, matrix: nrnitrobenzyl alchol, added NaI);; Found 1176.6924 (M + Na) Calcd for C59H95N9014Na 1176.6896 h) 'H-nuclear magnetic resonance spectrum (C6D6, 600MHz) : (See Fig. 3)
0.64 (d、 J=6.7H z、 3H)、 0.71 (d、 J=6.8Hz、 3ト 1)、 0.75 (t、 J=7.4Hz、 31-1)、 0.79 (m、 1 H)、 0.80 (m、 ： - 1)、 0.81 (d、 J =6.7 Hz、 3H)、 0.85 (d、 J =6.4H z、 3H)、 0.87 (m、 1 H)、 0. HI (d、 J =6.9Hz、 3H)、 0.90 (d、 J=6.8Hz、 3H)、 0.92 (d、 .1 =6.811 z、 3Η)、 0·99 (m、 l H)、 1.06 (d、 J =6.6H z、 3 H)、 1.14 (m、 1 H)、 1. " (d、 J=6.4Hz、 3 H), 1.18 (m、 1 H)、 1.25 (m、 2H)、 1. 5 (d、 J =6.5Hz、 3H)、 1.47 (m、 1H)、 1.61 (b r d、 J =13.6Hz、 1卜 、 1.73 (m、 1H)、 2.18 (m、 1 H)、 2.22 (m、 1ト I)、 2.22 (s、 3ト 1)、 2.28 (m、 1H)、 2.39 (m、 1 H)、 2.50 (m、 1 H)、  0.64 (d, J = 6.7Hz, 3H), 0.71 (d, J = 6.8Hz, 3t1), 0.75 (t, J = 7.4Hz, 31-1), 0.79 (m, 1H), 0.80 (m,:-1), 0.81 (d, J = 6.7 Hz, 3H), 0.85 (d, J = 6.4Hz, 3H), 0.87 (m, 1H), 0.HI (d, J = 6.9) Hz, 3H), 0.90 (d, J = 6.8 Hz, 3H), 0.92 (d, .1 = 6.811 z, 3Η), 099 (m, lH), 1.06 (d, J = 6.6 Hz, 3H), 1.14 (m, 1H), 1. "(d, J = 6.4Hz, 3H), 1.18 (m, 1H), 1.25 (m, 2H), 1.5 (d, J = 6.5Hz, 3H), 1.47 (m, 1H), 1.61 (brd, J = 13.6Hz, 1 ton, 1.73 (m, 1H), 2.18 (m, 1H), 2.22 (m, 1 to I), 2.22 (s, 3 to 1), 2.28 (m, 1H), 2.39 (m, 1H), 2.50 (m, 1H),
2.88 (s、 3H)、 2.93 ( d、 ' J =10.1H z、 1H)、 2.99 (m、 1 H)、 3.07 (br d、 J =13.5Hz、 1H)、 3.09 (s、 3H)、 3.15 (m、 1H)、 3.15 (s、 3 H)、 3.154 (s、 3ト 1)、 3.48 (dd、 J =13.7、 5. OH z、 1 H)、 3.52 (dd、 J = 13.0N 11.5Hz、 1 H)、 3.54 (s、 3H)、 3.57 (dd、 J =17.2、 2.8H z、 1 H)、 3.64 (dd、 J =13.7、 12.2H z、 丄 H)、 4, 10 (dd、 J =17.2、 6. OH z、 1 H)、 4· 22 (ddd、 J = 13· 5、 13.5、 2.7H z、 ] H)、 .4.52 (d、 J =10.711 z , 1 H)、 4.77 (dd、 J =10.4、 10.4H z、 1 H)、 4.98 (d、 J =10.9H z、 1 11)、 .24 (d、 J =3.5Hz、 1 H)、 5.40 (d、 J =U. lHz、 1 H)、 5.57 (ddd、 J =12.2、 10.0、 5.0Hz、 11-1)、 5.72 (d、 J =6.5H z、 1 H)、 6.55 (dd、 J =11.5、 5.4H z、 1 I- 1)、 6.76 (d、 J =8.5Hz、 2H)、 7.07 (d、 J =8.5 Hz、 2H)、 7.25 (d、 J =10.4H z , 1 H)、 7.48 (dd、 J =6· 0、 2, 8H z、 1 H)、 7.65 (d、 J =10. OH z、 1 H) 2.88 (s, 3H), 2.93 (d, 'J = 10.1Hz, 1H), 2.99 (m, 1H), 3.07 (br d, J = 13.5Hz, 1H), 3.09 (s, 3H), 3.15 (m, 1H), 3.15 (s, 3H), 3.154 (s, 3t1), 3.48 (dd, J = 13.7, 5.OHz, 1H), 3.52 (dd, J = 13.0 N 11.5Hz , 1H), 3.54 (s, 3H), 3.57 (dd, J = 17.2, 2.8Hz, 1H), 3.64 (dd, J = 13.7, 12.2Hz, 丄 H), 4, 10 (dd, J = 17.2, 6.OH z, 1H), 4.22 (ddd, J = 13.5, 13.5, 2.7Hz,] H), .4.52 (d, J = 10.711z, 1H), 4.77 (dd, J = 10.4, 10.4H z, 1 H), 4.98 (d, J = 10.9 Hz z, 1 11), .24 (d, J = 3.5 Hz, 1 H), 5.40 (d, J = U. lHz, 1 H), 5.57 ( ddd, J = 12.2, 10.0, 5.0Hz, 11-1), 5.72 (d, J = 6.5Hz, 1H), 6.55 (dd, J = 11.5, 5.4Hz, 1I-1), 6.76 ( d, J = 8.5Hz, 2H), 7.07 (d, J = 8.5Hz, 2H), 7.25 (d, J = 10.4Hz, 1H), 7.48 (dd, J = 6.0, 2.8Hz , 1H), 7.65 (d, J = 10.OHz, 1H)
1 ) 13C—核磁気共鳴スペク トル （150MHz、 C 6 D 6) (図 4参照） 1) 13 C—nuclear magnetic resonance spectrum (150 MHz, C 6 D 6) (see Fig. 4)
9.71(q)、 10.97 (q)、 15.19 (q)、 16.34 (q)、 17.79 (q)、 18.13 (q)、 18.65 (q)、 18.85 (t)、 19.0 (q)、 19.09 (q)、 19.83 (q)、 19.93 (q)、 20.26 (q)、 24, 68 (t)、 25.27 (t)、 26.13 (t)、 26.35 (d)、 26.92 (d)、 28.19 (t)、 28.36 (q)、 28.78 (q)、 29.50 (d)、 30.24 (q)、 30.57 (d)、 30.90 (q)、 32.81 (d)、 34.08 (d)、 34.98 (t)、 35.64 (t)、 41.10 (q) ,41.60 (t)、 43.37 (t)、 46.81 (d) ,51.34 (d)、 52.72 (d)、 55.14 (q)、 55.22 (d)、 56.68 (d)、 62.41 (cl)、 67.43 (d)、 74.86 (d)、 75.09 (d)、 113.77 (d)、 130.38 (s)、 130.88 (d)、 158.78 (s)、 168.17(s)、169.10(s)、169.13 (s)、 169.79 (s)、 171.06 (s、 2C)、 171.47 (s)、 171.58 (s)、 171.78(s)、171.99(s)、172.03(s)  9.71 (q), 10.97 (q), 15.19 (q), 16.34 (q), 17.79 (q), 18.13 (q), 18.65 (q), 18.85 (t), 19.0 (q), 19.09 (q), 19.83 (q), 19.93 (q), 20.26 (q), 24, 68 (t), 25.27 (t), 26.13 (t), 26.35 (d), 26.92 (d), 28.19 (t), 28.36 (q ), 28.78 (q), 29.50 (d), 30.24 (q), 30.57 (d), 30.90 (q), 32.81 (d), 34.08 (d), 34.98 (t), 35.64 (t), 41.10 (q ), 41.60 (t), 43.37 (t), 46.81 (d), 51.34 (d), 52.72 (d), 55.14 (q), 55.22 (d), 56.68 (d), 62.41 (cl), 67.43 (d ), 74.86 (d), 75.09 (d), 113.77 (d), 130.38 (s), 130.88 (d), 158.78 (s), 168.17 (s), 169.10 (s), 169.13 (s), 169.79 (s ), 171.06 (s, 2C), 171.47 (s), 171.58 (s), 171.78 (s), 171.99 (s), 172.03 (s)
上記物性を基にこの物質の構造解析を行った。 まず、 本物質は高分解能質量 分析より分子式 C59H95N9。14 (整数分子量 1153) を有し、 I Rスぺク トルにお けるアミ ドの吸収 （1681、 1648cm-l)、 1 H NMRにおけるアミ ド NH水素と NCH3水素のシグナル、 13C NMRにおける 11個のアミド（又はエステル、 カルボン酸） カルボニルシグナルからペプチド性化合物と推定した。 平面構造はBased on the above properties, the structure of this substance was analyzed. First, the substance has the molecular formula C 59 H 95 N 9 from high-resolution mass spectrometry. 1 4 has the (integer molecular weight 1153), the absorption of your Keru Ami de the IR spectrum (1681, 1648 cm-l), Ami de NH hydrogen and NCH3 hydrogen signals in 1 H NMR, 11 pieces in @ 13 C NMR Amide (or ester, carboxylic acid) was estimated to be a peptide compound from the carbonyl signal. The plane structure is
2 D NMRを詳細に検討することにより決定した。則ち COSYと H0HAHAより 10 個のアミノ酸残基 （Tyr (又は Tyr (OMe) )， MeVal x 2， Gly, Melle x 2, MeAspThe 2D NMR was determined by a detailed examination. That is, 10 amino acid residues (Tyr (or Tyr (OMe)) from COSY and H0HAHA, MeVal x 2, Gly, Melle x 2, MeAsp
(又は MeAsp (OMe)), Val, Hpr, 2 -hydroxyisovaleric acid) の構造が明ら かとなつた。 ついで HMQCにより C— H直接結合を明らかにするとともに、 H MB Cによりこれらアミノ酸残基のつながりを決定した。 以上の結果、 本物質の 平面構造を 状デプンァカぺプチド cyclop- 2 -hydroxy isovaleryl-Hpr-Meval- Val-MeAsp-Melle-Melle-Gly-Meval-Tyr (OMe) -)と決定した r. (Or MeAsp (OMe)), Val, Hpr, 2-hydroxyisovaleric acid). Then, C—H direct binding was revealed by HMQC and H The connection of these amino acid residues was determined by MBC. As a result, the planar structure of this substance Jo Depunaka peptide cyclop- 2 -hydroxy isovaleryl-Hpr-Meval- Val-MeAsp-Melle-Melle-Gly-Meval-Tyr (OMe) -) and the determined r.
物質 F A15— 2は以下のような理化学的性質を有する。  Substance F A15-2 has the following physicochemical properties.
a ) 外観：無色粉末 a) Appearance: colorless powder
b ) 溶解性： クロ口ホルム、 メタノール、 アセ トン、 酢酸ェチル、 ジメチルスル ホキシドに可溶、 水に難溶 b) Solubility: Soluble in chloroform, methanol, acetone, ethyl acetate, dimethyl sulfoxide, poorly soluble in water
c ) T L C： R f =0.64 (CHC】 3— M e OH 9 ： 1 )、 0· 49 (H e x a n e - E t OA c -Me OH 9 : 9 : 2) c) TLC: R f = 0.64 (CHC) 3 — MeOH 9: 1), 0 · 49 (Hexane-EtOAc-MeOH 9: 9: 2)
d ) 旋光度： [a] 24D —200。 （c 0.078、 C H C 1 3) d) Optical rotation: [a] 24 D -200. (C 0.078, CHC 1 3)
e ) 紫外部吸収スぺク トル （CH3CN) ： λ max222 ( ξ 20700)、 277 (1120)、 283 (970) nm (図 5参照） e) Ultraviolet absorption spectrum (CH 3 CN): λ max222 (ξ20700), 277 (1120), 283 (970) nm (see Fig. 5)
f ) 赤外部吸収スぺク トル （CHC 1 3)： 3389、3183、2969、1731、1681、1645、1514、 1468、 1408、 1292、 1247、 1178cm-1 (図 6参照） f) Infrared absorption scan Bae-vector (CHC 1 3): 3389,3183,2969,1731,1681,1645,1514, 1468, 1408, 1292, 1247, 1178cm- 1 ( see FIG. 6)
g) 質量分析：  g) Mass spectrometry:
F ABMS (neg. , matrix： m-nitrobenzyl alchol ) ： m/ z 11 '-Ί  F ABMS (neg., Matrix: m-nitrobenzyl alchol): m / z 11 '-Ί
[M— H]— HR F ABMS (posit
Figure imgf000014_0001
1 alchol - N a I ) ： F o u n d m / z 1162.6740 [ M + N a ] , Calcd lor C58H93N9014Nall62.6740 [M—H] —HR F ABMS (positive
Figure imgf000014_0001
1 alchol-N aI): Foundm / z 1162.6740 [M + Na], Calcd lor C58H93N9014Nall62.6740
h) 'H—核磁気共鳴スペク トル （C 6 D 6、 600MH z ) (図 7参照）  h) 'H—nuclear magnetic resonance spectrum (C 6 D 6, 600 MHz) (see Figure 7)
δ 0.75 ( t、 J =7. OH z； 3 H)、 0.76 (m、 1 1-1)、 0.77 ( d、 J =6.8H z、 3 H)、 0.83 ( t、 J =6.8H z、 3 H)、 0.84 ( d、 J =6.7H z , 3 H)、 0.97 (d、 J =8.0H z、 3 H)、 0.97 (m、 1 H)、 0.99 ( d、 J =6.7H z、 3 H)、 1.06 (d、 J =6.8H z、 3 H)、 1.06 (m、 1 H)、 1. 10 (d、 J =6.6H z、 3 H)、 1. 15 ( d、 J =6.7H z、 3ト 1)、 1. 17 ( d、 J =6.6H z、 3 H)、 1.20 (m、 2H)、 1.21 (d、 J =6.6Hz、 3 H) δ 0.75 (t, J = 7.OHz; 3H), 0.76 (m, 11-1), 0.77 (d, J = 6.8Hz, 3H), 0.83 (t, J = 6.8Hz, 3H), 0.84 (d, J = 6.7Hz, 3H), 0.97 (d, J = 8.0Hz, 3H), 0.97 (m, 1H), 0.99 (d, J = 6.7Hz, 3H), 1.06 (d, J = 6.8Hz, 3H), 1.06 (m, 1H), 1.10 (d, J = 6.6Hz, 3H), 1.15 (d, J = 6.7 Hz, 3 to 1), 1.17 (d, J = 6.6 Hz, 3 H), 1.20 (m, 2H), 1.21 (d, J = 6.6Hz, 3H)
1.24 (m、 1 H)、 1.46 (m、 1ト 1)、 1.50 (m、 2 H)、 1.81 (m、 1 H)ゝ 2.27 (m、 1 H)、 2.31 (m、 1 H)ゝ 2.37 (s、 3 H)、 2.42 (m、 1ト 1)、 2.47 (m、 1.24 (m, 1 H), 1.46 (m, 1 to 1), 1.50 (m, 2 H), 1.81 (m, 1 H) ゝ 2.27 (m, 1 H), 2.31 (m, 1 H) ゝ 2.37 (s, 3H), 2.42 (m, 1 to 1), 2.47 (m,
1 H)、 2.60 (br. d、 J = 13. OH z、 1ト I)、 2.80 ( s、 3 H)、 2.89 ( d、 J =10. lHz、 1 H)、 2.93 (m、 1 H)ゝ 3.04 (m、 1 H)、 3.06 ( s、 3H)、 3.08 (m、 1ト 1)、 3.10 (m、 1 H)、 3.13 (s、 3 H)ゝ 3.14 (m、 1 H)ゝ 3.39 (s、1H), 2.60 (br.d, J = 13.OHz, 1t I), 2.80 (s, 3H), 2.89 (d, J = 10.lHz, 1H), 2.93 (m, 1H ) ゝ 3.04 (m, 1 H), 3.06 (s, 3H), 3.08 (m, 1 to 1), 3.10 (m, 1 H), 3.13 (s, 3 H) ゝ 3.14 (m, 1 H) ゝ3.39 (s,
3H)ゝ 3.50 (dd、 J =17.3Hz、 3.2H z、 1 H)、 3.62 (m、 1 H)、 4.13 (dd、3H) ゝ 3.50 (dd, J = 17.3Hz, 3.2Hz, 1H), 3.62 (m, 1H), 4.13 (dd,
J =17.3Hz、 5.9Hz、 1 H)、 4.49 (d、 J=5.3Hz、 1 H)、 5.04 (m、 1J = 17.3Hz, 5.9Hz, 1H), 4.49 (d, J = 5.3Hz, 1H), 5.04 (m, 1
H)、 5.06 (m、 1 H)、 5.27 (d、 J =10.9H z , 1 H)、 5.39 (d、 J =11.0 Hz、 1 H)、 5.58 (ddd、 J =10.3Hz、 U).3H z、 6.1Hz, 1ト I)、 5,75 (br. d、 J=4.6Hz、 1ト 1)、 6.43 (d、 J =7.6H z s 1 H)、 6.48 (dd、 J =11.9H), 5.06 (m, 1 H), 5.27 (d, J = 10.9 Hz, 1 H), 5.39 (d, J = 11.0 Hz, 1 H), 5.58 (ddd, J = 10.3 Hz, U). 3 Hz, 6.1 Hz, 1 to I), 5,75 (br.d, J = 4.6 Hz, 1 to 1), 6.43 (d, J = 7.6 Hz s 1 H), 6.48 (dd, J = 11.9
Hz、 3.9Hz、 1H)、 6.71 (d、 J =6· 8H z、 1H)ゝ Hz, 3.9Hz, 1H), 6.71 (d, J = 6.8Hz, 1H) ゝ
6.73 ( d、 J =8.5H z、 2 H)、 7.15 ( d、 J =8.5H z、 2 H)、 7.32 ( b r . d、 J=5.8Hz、 1 H)、 7.40 (d、 J =9.7H z、 1 H)  6.73 (d, J = 8.5Hz, 2H), 7.15 (d, J = 8.5Hz, 2H), 7.32 (br.d, J = 5.8Hz, 1H), 7.40 (d, J = 9.7 (H z, 1 H)
i ) '3C—核磁気共鳴スペク トル （100MHz、 C 6 D 6) (図 8参照） i) '3 C-Nuclear magnetic resonance spectrum (100MHz, C 6 D 6) ( see Figure 8)
δ 10.2 (q)、 11.0 (q)、 15.4 (q)、 17. δ (q)、 18.0 (q)、 18.7 (q)、 18.8 (q)、 19.2 (q)、 19.4 (q)、 19.6 (q)、 20.2 (q)、 20.7 (t)、 24.2 (t)、 25.2 (t)、 25.6 ( t)、 26.1 (t)、 27.8 (d)、 28.6 (q)、 30.1 (q)、 30.2 (d)、 31.0 (q)、 31.3 (d)、 32.5 (d)、 32.8 (d)、 34.0 (d)、 34.1 (t)、 34.3 (t)、 40.7 (q)、 41.7 (t)、 44.1 (t)、 51.4 (d)、 52.6 (d)、 54.0 (d)、 54.6 (d)、 55.0 (q)、 57.1(d) 61.1(d)、61.8(d)、74.8(d)、76.1 (d)、 113.7 (d、 2C)、 129.6 (s)、 131.0 (d、 2C)、 159.0(s)  δ 10.2 (q), 11.0 (q), 15.4 (q), 17.δ (q), 18.0 (q), 18.7 (q), 18.8 (q), 19.2 (q), 19.4 (q), 19.6 ( q), 20.2 (q), 20.7 (t), 24.2 (t), 25.2 (t), 25.6 (t), 26.1 (t), 27.8 (d), 28.6 (q), 30.1 (q), 30.2 ( d), 31.0 (q), 31.3 (d), 32.5 (d), 32.8 (d), 34.0 (d), 34.1 (t), 34.3 (t), 40.7 (q), 41.7 (t), 44.1 ( t), 51.4 (d), 52.6 (d), 54.0 (d), 54.6 (d), 55.0 (q), 57.1 (d) 61.1 (d), 61.8 (d), 74.8 (d), 76.1 (d ), 113.7 (d, 2C), 129.6 (s), 131.0 (d, 2C), 159.0 (s)
168.0 (s)、 168.6 (s)、 169.1 (s)、 169.5 (s)、 169.6 (s)、 170.2 (s)、 170· 5 (s)、 170.8 (s)、 172.3 (s), 173.4 (s)  168.0 (s), 168.6 (s), 169.1 (s), 169.5 (s), 169.6 (s), 170.2 (s), 1705 (s), 170.8 (s), 172.3 (s), 173.4 (s )
上記物性を基に構造解析を行い、 このものは | 記構造式の ¾状デフシべツチド cyclo ι,-2-hydroxyisovaleryl-Hpr-Val-Val-MeA.sp-MeI Je- eJ 1 e-Ci I v- uVal- Tyr (O e) -)である事を確認した。 Structural analysis was performed on the basis of the above physical properties, and the structure was determined as follows: ¾-shaped decibetatide cyclo ι, -2-hydroxyisovaleryl-Hpr-Val-Val-MeA.sp-MeI Je- eJ v- uVal- Tyr (O e)-).
試験例  Test example
物質 F A 15— 1の抗真菌活性  Antifungal activity of substance F A 15-1
物質 F A15— 1の抗真菌活性を寒天希釈法により求めた 3 物質 F Λΐπ- 1を少 を作成した。 その 25μ1 を加熱溶解したサブ口一寒天培地 10ml と混合しシヤー レに分注した c; この新規抗生物質 FA 15— 1が入った寒天プレートに被検菌の培 養懸濁液を塗抹して 25nCで 2〜 4日間培養し生育阻害濃度を測定した。 （力ビ： 25°C、 4日問、 酵母及び細菌： 25°C、 2日間） これらの結果を表 1に示す。 The antifungal activity of substance FA15-1 was determined by the agar dilution method, and a small number of three substances F 物質 π-1 were prepared. 25 μl of the mixture was mixed with 10 ml of the heated and dissolved sub-agar-agar medium and dispensed into a plate.c. The culture suspension of the test bacterium was spread on an agar plate containing this new antibiotic FA15-1. The cells were cultured at 25 nC for 2 to 4 days, and the growth inhibitory concentration was measured. (Rib: 25 ° C, 4 days, Yeast and bacteria: 25 ° C, 2 days) The results are shown in Table 1.
表 1 寒天希釈法 被検菌 M I C (Mg/ml) Table 1 Agar dilution method Test bacteria M I C (Mg / ml)
Escherichia coli NIHJ 〉 1 00 Escherichia coli NIHJ〉 1 00
Staphylococcus aureus AJ 12510 〉 1 00  Staphylococcus aureus AJ 12510〉 1 00
Candida albicans IFO 0583 1 00  Candida albicans IFO 0583 1 00
Aspergillus niger AJ 117374 1 6  Aspergillus niger AJ 117374 1 6
Aspergillus fumigatus AJ 117190 0. 2 5  Aspergillus fumigatus AJ 117190 0.2 5
Aspergillus umigatus JCM 1739 0. 5  Aspergillus umigatus JCM 1739 0.5
Trichoderma ressei AJ 117127 > 1 00  Trichoderma ressei AJ 117127> 1 00
Curvularia lunata AJ 117375 > 1 00  Curvularia lunata AJ 117375> 1 00
Alternaria alternata AJ 117376 8  Alternaria alternata AJ 117376 8
Pestalotiopsis distinca AJ 117377 1  Pestalotiopsis distinca AJ 117377 1
Absidia glauca A J 117378 〉 1 00  Absidia glauca A J 117378〉 1 00
Mucor hiemails A J 117379 > 1 00  Mucor hiemails A J 117379> 1 00
*MI C：最小生育阻止濃度 * MIC: minimum inhibitory concentration
表 1から明かなように物質 F A 1 5 _ 1は、 ァスペルギルス フミガタス (AJ-117190)等に対して強レ、抗真菌活性を有する事が分かった  As is clear from Table 1, the substance F A 15 _1 was found to have strong antifungal activity against Aspergillus fumigatus (AJ-117190), etc.
物質 F A 15— 2の抗真菌活性  Antifungal activity of substance F A 15-2
物質 FA15— 2の抗真菌活性をマイクロダイリューション法により *めた.； 物 質 FA15— 2を少量のジメチルスルホキシドで溶解した後、 ジメチルス/レホキシ ドで等倍希釈系列を作成した。 RPM I培地 （日水製薬株式会 fh製） に^釈液を 0.5%添加した。 なお、 RPMI培地には、 懸濁胞子液を胞子終濃度 2 X 105 C F U/ml となるよう添加した。 この物質希釈液と胞子液が入った培地を 96穴マ イクロタイタ一プレートに 1穴 lOOul 入れた。 37°C、 24 時間培養を行い、 培養 後、 肉眼で最小生育阻止濃度を測定した。 これらの結果を表 2に示す。 The antifungal activity of the substance FA15-2 was determined by the microdilution method. After dissolving the substance FA15-2 with a small amount of dimethylsulfoxide, an equal dilution series was prepared with dimethyls / rephoxide. 0.5% of RPMI was added to RPM I medium (manufactured by Nissui Pharmaceutical Co., Ltd. fh). In the RPMI medium, the suspension spore solution was added to a final spore concentration of 2 × 105 C FU / ml was added. The medium containing the diluted material and the spore solution was added to a 96-well microtiter plate in a 1-well volume. After culturing at 37 ° C for 24 hours, the minimum growth inhibitory concentration was visually determined after culturing. Table 2 shows the results.
表 2 マイクロダイリューション法 ヽ Table 2 Microdilution method ヽ
被検菌 M I C ( μ g/ mlj  Test bacteria M I C (μg / mlj
FA15-1 FA . -2 アンホテリシン B ミコナソ" - -ル FA15-1 FA. -2 Amphotericin B Mikonazo "--
Candida albicans IFO 0583 8 8 1 0. 2 5Candida albicans IFO 0583 8 8 1 0.25
Candida albicans TIMM 1529 8 8 2 0. 2 5Candida albicans TIMM 1529 8 8 2 0.25
Candida albicans ATCC 10231 8 8 0. 5 0 . 5Candida albicans ATCC 10231 8 8 0.5 0.5 .5
Aspergillus niger AJ 117374 1 6 1 6 1 0 . 5Aspergillus niger AJ 117374 1 6 1 6 1 0 .5
Aspergillus fumigatus AJ 1丄 190 2 4 1 1Aspergillus fumigatus AJ 1 丄 190 2 4 1 1
Aspergillus fumigatus JCM 1739 4 4 0. 2 5 2 Aspergillus fumigatus JCM 1739 4 4 0.2 2 5 2
*M I C：最小生育阻止濃度 * M I C: Minimum inhibitory concentration
表 5から明かなように物質 F A 15— 2は、 ァスペルギルス フミガタス（JCM 1739)等に対して FA15— 1と同様に強い抗真菌活性を有する事が分かった。 産業上の利用可能性  As is clear from Table 5, it was found that the substance F A15-2 has a strong antifungal activity against Aspergillus fumigatus (JCM 1739) and the like as well as FA15-1. Industrial applicability
本発明の環状デブシぺプチドは抗真菌活性に優れており、 強く新しい抗菌力を 有する抗真菌剤を提供することができる。 また、 この環状デプシペプチドは通常 の微生物培養法で生産することができ、 抽出、 精製にも特別の方法を用いないの で工業的な大量生産が可能である。  INDUSTRIAL APPLICABILITY The cyclic debpeptide of the present invention is excellent in antifungal activity and can provide an antifungal agent having a strong new antibacterial activity. In addition, this cyclic depsipeptide can be produced by a usual microbial culture method, and since no special method is used for extraction and purification, industrial mass production is possible.

Claims

請 求 の 範 囲 The scope of the claims
1. 下記式で示される環状デブシぺプチド 1. Cyclic debutpeptide represented by the following formula
cyclo、_2 - hydroxyisovaleryl - Hpr-MeVal - Val-MeAsp - Melie— Melle— Gly— MeVal-Tyr (OMe) -) 、 又 は cyclo (-2-hydroxyisovaleryl-Hpr-Val-Val-MeAsp- き a  cyclo, _2-hydroxyisovaleryl-Hpr-MeVal-Val-MeAsp-Melie— Melle— Gly— MeVal-Tyr (OMe)-) or cyclo (-2-hydroxyisovaleryl-Hpr-Val-Val-Val-MeAsp-)
Melle- Melle- Gly- MeVal- Tyr (OM正e)-) Melle- Melle- Gly- MeVal- Tyr (OM 正 e)-)
2. 下記の理化学的性質を有する物質 F A15— 1、 FA15-2 2. Substances with the following physicochemical properties: FA15-1, FA15-2
F A15- 1  F A15- 1
溶解性：クロロホノレム、 メタノーノレ、 アセトン、 酢酸ェチノレ、 ジメチノレスルホ キシドに可溶、 水に難溶  Solubility: Soluble in chlorophonolem, methanol, acetone, ethinole acetate, dimethinolesulfoxide, poorly soluble in water
TLC ： R f =0.82 (CHC 13~Me OH 9 ： 1 )、 0.69 (Hexane-EtoAc- Me OH 9 : 9 : 2) TLC: R f = 0.82 (CHC 1 3 ~ Me OH 9: 1), 0.69 (Hexane-EtoAc- Me OH 9: 9: 2)
旋光度： [a] 22D — 210° (c0.37、 CHC 13) Optical rotation: [a] 22 D - 210 ° (c0.37, CHC 1 3)
紫外部吸収スぺク トル (Me OH) ： Imax 20 (end, ミ 58800)、 222 (s h、 25700)、 277 (1900)、 288 (1700) nm  Ultraviolet absorption spectrum (MeOH): Imax 20 (end, mi 58800), 222 (sh, 25700), 277 (1900), 288 (1700) nm
赤外部吸収スぺク トル (KB r ) ： 3700 - 3100 (br)、 3385、 2966、 2935、 2877、 1739、 1683、 1648、 1614、 15 、 1472、 1409、 1292、 1249、 1210、 1178、 1133、 1101、 1039cnT' 抗真菌活性：ァスペルギス フミガタス及びぺスタ口チォプシス デイスティ ンクに対しェッシェリシァ コリの 10倍以上の生育阻害活性を示す。  Infrared absorption spectrum (KBr): 3700-3100 (br), 3385, 2966, 2935, 2877, 1739, 1683, 1648, 1614, 15, 1472, 1409, 1292, 1249, 1210, 1178, 1133 , 1101, 1039cnT 'Antifungal activity: exhibits 10 times or more the growth inhibitory activity against Aspergillus fumigatus and Pestia typhosis dustin compared to Escherichia coli.
F A15- 2  F A15-2
溶解性： クロロホルム、 メタノール、 ァセトン、 酢酸ェチル、 ジメチルスルホ キシドに可溶、 水に難溶  Solubility: Soluble in chloroform, methanol, acetone, ethyl acetate, dimethyl sulfoxide, poorly soluble in water
T L C ： R f =0.64 (CHC 13— Me OH 9 ： 1)、 0.49 (Hexane-EtoAc- Me OH 9 : 9 : 2)  TLC: Rf = 0.64 (CHC13—MeOH9: 1), 0.49 (Hexane-EtoAc-MeOH 9: 9: 2)
れた用紙 旋光度： [ α ] 2D 一 200° ( c 0.078、 CHC 13) Paper Optical rotation: [α] 2 D one 200 ° (c 0.078, CHC 13 )
紫外部吸収スぺク トル （CH3CN) ： λ max222 ( 20700)、 277 (1120)、 283 (970) nm Ultraviolet absorption spectrum (CH 3 CN): λmax222 (20700), 277 (1120), 283 (970) nm
赤外部吸収スぺク トル （CHC 13) ： 3389、 3183、 2969、 1731、 1681、1645、 14、 1468、 1408、 1292、 1247、 1178cm-1 Infrared absorption scan Bae-vector (CHC 1 3): 3389, 3183, 2969, 1731, 1681,1645, 14, 1468, 1408, 1292, 1247, 1178cm- 1
抗真菌活性：ァスペルギス フミガタスに対し強し、生育阻害活性を示す。  Antifungal activity: Strong against Aspergillus fumigatus, showing growth inhibitory activity.
3. 請求の範囲第 1項記載の環状デブシぺプチドを有効成分とする抗真菌剤3. An antifungal agent comprising the cyclic debpeptide described in claim 1 as an active ingredient.
4. 請求の範囲第 1項記載の環状デプシぺブチドの MeAspの C O〇 H基に修 飾基が導入されている化合物を有効成分とする抗真菌剤 4. An antifungal agent comprising, as an active ingredient, a compound in which a modifying group has been introduced into the COH group of MeAsp of the cyclic depsipeptide described in claim 1.
5. 修飾基が、 g換基を有してもよい、 アルキルエステル基、 ァリールエス テル基、 アルキルァミド基又はァリ一ルァミ ド基である請求の til 项^載の 化合物を有効成分とする抗真菌剤 5. An antifungal comprising, as an active ingredient, a compound according to claim あ る wherein the modifying group is an alkyl ester group, an aryl ester group, an alkylamide group or an arylamide group, which may have a g-substituted group. Agent
6. 請求の範囲第 2項記載の物質 F A 15— 1又は F Λ 15一 2を有効成分とす る抗真菌剤 7. クラバリォプシス属に属し、 請求の範囲第 2項記載の物質 F A 一 1又 は FA15— 2産生能を有する糸状菌を培養し、 培養物から物質 FA15— 1又は物 質 F A 一 2を採取することを特徴とする物質 FA15— 1又は物質 F 2の 製造方法 6. An antifungal agent comprising the substance FA15-1 or FΛ15-12 as claimed in claim 2 as an active ingredient 7. The substance FA1 or 1 belonging to the genus Clavaryopsis and according to claim 2 Is a method for producing a substance FA15-1 or a substance F2, which comprises culturing a filamentous fungus capable of producing FA15-2 and collecting the substance FA15-1 or the substance FA1-2 from the culture.
PCT/JP1998/005716 1997-12-18 1998-12-17 Cyclopeptolides WO1999031127A1 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02134399A (en) * 1988-09-23 1990-05-23 Sandoz Ag Pipecolic acid-containing peptrids, their manufacture and medicine composition
JPH08504165A (en) * 1991-12-20 1996-05-07 ノボ ノルディスク アクティーゼルスカブ Agricultural use of specific compounds, compositions containing said compounds and processes for their preparation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02134399A (en) * 1988-09-23 1990-05-23 Sandoz Ag Pipecolic acid-containing peptrids, their manufacture and medicine composition
JPH08504165A (en) * 1991-12-20 1996-05-07 ノボ ノルディスク アクティーゼルスカブ Agricultural use of specific compounds, compositions containing said compounds and processes for their preparation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
EMMER G. et al., "Derivatives of a Novel Cyclopeptolide. 1. Synthesis, Antifungal Activity and Structure-Activity Relationships", J. MED. CHEM., 1994, Vol. 37, No. 13, pp. 1908-1917. *

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