WO2006067862A1 - Active substances k03-0132 and method of producing the same - Google Patents

Abstract

A microorganism (Streptomyces sp. K03-0132, FERM ABP-10148), which belongs to Streptomyces sp. and is capable of producing active substances K03-0132A and/or K03-0132B, is cultured in a medium to thereby accumulate the active substances K03-0132A and/or K03-0132B in the culture medium. Then the active substances K03-0132A and/or K03-0132B are collected from the culture medium. Because of having an effect of elevating the activity of azole-type fungicides, the obtained active substances act on a large number of fungal infections such as deep-seated mycosis in a low dose within a short period of time and are useful in lowering the frequency of the appearance of resistant strains. It is also expected that these substances are useful in overcoming resistance.

Description

 Description Active substance K 03-0 1 32 and its manufacturing method Technical field

 The present invention relates to an active substance を 03-0 1 32 having an activity enhancing action of an azole antifungal agent and a method for producing the same. This active substance Κ 03-0 1 32 is a general term for the active substance Κ 03-0 1 32 Α and the active substance Κ 03-0 1 32 B. Background art

Examples of azole compounds used for the treatment of fungal infections, especially deep mycosis, include 1— [2— (2, 4-dichloro benzoyl oxy)-2-(2, 4-di ch lor ophe ny l) et hy l] imi da zole (generic name: miconazole, manufactured by Sigma, USA), 2, 4-difluoro--, α-bis (1 H-1, 2, 4-- triazol— l _y lme t hy l) benzy 1 alc oho l (generic name: fluconazol, Icyen Fermas-Tikals, USA), and (sat) — 1— sec— bu tyl— 4— [p 1 [4 — [P-[[(2R, 4 S)-2-(2, 4-di ch lor oph en y 1)-2-(1 H- 1, 2, 4— triazo 1— 1— y 1 methy 1 )-1, 3-di oxo l an-4-yl] me t hoxy] phen l] — 1— piperazi ny l] pheny l] — 厶² — 1, 2, 4— triazoli η- 5-on e ( General name: Itraconazole, manufactured by Kyowa Hakko, Japan), etc. were known.

These azole compounds miconazole, fluconazole and itraconazole are safer than the polyene amphotericin B used in the disease (An aissie E. 5, Clinical Infexius Dies, 23, 964—972, 1 996), the most frequently used drug. Recently, however, the emergence of resistant bacteria due to long-term or repeated administration of these azole antibacterial agents has become a problem. The development of drugs is a social urgent task.

Disclosure of the invention

 Diseases with reduced immunity, such as HIV infection and blood diseases, cause an easily infectious state and increase the incidence of fungal infections as opportunistic infections. In addition, many of these diseases accompanied by decreased immunity are serious and require a long treatment period. For this reason, chemotherapy for fungal infections often lasts for a long period of time, and the most frequently used azole type antifungal agents are considered to be in a state where induction of drug resistance is extremely likely to occur.

 As a mechanism of resistance to azolic antifungal agents, the target enzyme in Cand i da a 1 bic an s is P-450 1 4-—demethylase over-expression. Decreased sex (Vand en Boss che H. et al., Anti-microbiological agent and chemotherapeutics, 36, 2602— 26 1 0, 1 992; Sang lard D. et al. 42, 24 1-253, 1 998), multi-drug-excluding transport cells such as MSF (Major Facilitator Sup erf am i y) and ABC Decrease in internal drug concentration (Fing MF et al., Mol Gen Ge net, 227, 3 1 8—329, 1 995; Sang lard D. et al., Microbiology 1, 14: 405—4 1 6 1997) but in Sa cc ha r omyc escer ev isiae, MD R (Ma 丄 tip 1 e Dr ug R esistant) It has been reported that the genes PDR 16 and PDR 17 have altered lipid metabolism and acquired resistance to azole compounds (H. Bart van de Ha zel et al., Journal ' Ob. Biological Chemistry, 274, 1 934— 1 94 1, 1 9 9 9).

For these reasons, drugs that increase the activity of azole antifungal agents reduce the frequency of emergence of resistant bacteria by reducing the dose of azole antifungal agents and shortening the administration period. It is expected that At the same time, combined use of two drugs with different skeletons is expected to overcome resistance to azolic antifungal agents. In such a situation, providing a drug having an activity enhancing activity of an azolic antifungal agent is effective for many fungal infections including deep mycosis and azool resistant fungal infections. It is considered therapeutically useful. ,

 As a result of continuing various studies on metabolites produced by microorganisms, the present inventors have found that a substance having an action-enhancing action of an azole antifungal agent is found in a culture of K 03-0132 strain newly isolated from soil. I found out that it was produced. Next, as a result of separating and purifying the active substance from the culture, a substance having a chemical structure represented by the following formula [I] and / or [I] was found. Since the substances represented by these formulas [I] and [I] have not been known at all, this active substance will be referred to as K03-01 32 A and K 03-0132 B, and the generic name is the active substance. I decided to call it K 03-01 32.

 The present invention has been completed based on such findings, and has the following formula [I]

で表される活性物質 K 03 - 01 32 Aを提供するものである。

An active substance represented by the formula K 03-01 32 A is provided.

 The present invention also provides the following formula [I]

The active substance represented by K 03-01 32 B is provided,

The present invention further provides the following formula [I]:

で表される活性物質 K O 3— 0 1 32Α、 及び下記式 [H]

KO 3— 0 1 32Α and the following formula [H]

The composition of the active substance represented by the formula Κ 0 3— 0 1 32 Β is provided.

 The present invention further belongs to Streptomyces sp. A microorganism having the ability to produce an active substance Κ 0 3-0 1 3 1 示 represented by the formula [I] is cultured in a culture medium, and the culture product contains Method for producing active substance K 0 3-0 1 3 1 A comprising accumulating active substance K 0 3-0 1 3 1 A and collecting active substance K 0 3-0 1 3 1 A from the culture Is provided.

 The present invention further belongs to Streptomyces sp. A microorganism having the ability to produce an active substance K 0 3-0 1 3 1 B represented by the formula [H] is cultured in a culture medium, and the cultured product is Method for producing active substance K 0 3-0 1 3 1 B, comprising accumulating active substance K 0 3-0 1 3 1 B and collecting active substance K 0 3-0 1 3 1 B from the culture Is provided.

The present invention further belongs to Streptomyces sp and is an active substance K 0 3-0 1 3 1 A represented by the formula [I] and an active substance K 0 3-0 1 3 2 B represented by the formula [I]. A microorganism having the ability to produce sucrose is cultured in a medium, and active substance K 0 3-0 1 32 and 1: () 3-0 1 32 B is accumulated in the culture, and active substance K 0 is accumulated from the culture. 3—Provides a method for producing a composition for collecting 0 1 3 2 A and K 0 3-0 1 3 2 B is there.

 The present invention further produces an active substance K 03-0 1 32 A represented by the formula [I] and / or an active substance K 03-0 1 32 B represented by the formula [I], which belongs to Streptomyces sp. A method for producing an active substance K 03-0 1 32 in which the active microorganism is S ス ト rep t omy cess p. K03-0 1 32 (FERM AB P-1 0 1 48) It is to provide.

 The present invention further provides Streptomyces sp. K03-0 1 32 (FERM ABP-1 0 1 48).

 A microorganism having the ability to produce an active substance K 03-0 1 32 A represented by the formula [I] and an active substance K 03-0 1 32 B represented by the formula [It] (hereinafter referred to as “K 03- 01 32 substance-producing bacteria j) belong to the genus Streptomyces, but any substance having the ability to produce the substance of the present invention may be used and is not particularly limited.

 As a preferred example of the strain used for producing the active substance K 03-0 1 32 of the present invention, Streptomyces sp (Streptomyces sp.) Newly isolated from Hokkaido soil by the present inventors is used. cessp,) K 03-0 1 32 strains.

 The bacteriological properties of this strain are as follows.

 1. Morphological features

 Vegetative mycelium develops well on various agar media and no fragmentation is observed. The aerial hyphae are abundantly grown on yeast-malt extract agar and starch ■ inorganic salt agar and have a brown to gray color. Under the microscope, more than 20 spore chains were found on the aerial hyphae, the shape of which was a spiral, and the size of the spore was a cylinder of about 0.7 X 1.2 m. The surface of the spore is smooth. No sclerotia, spore or zoospores are found.

 2. Properties on various media

The method of EB Shir 1 ing and the method of D. Go ttli eb (International Journal of Stymatic ■ Bacteriology, 1 6 Volume 3, 1 3 pages, 1 96 6 years) Therefore, the culture properties of the production bacteria examined are shown below. The color tone is determined using the color, harmony, manual 4th edition (container, corporation, ob-america, Chicago, 1958) as the standard color. Is also described. The following are the results of observations in each medium during the second week, unless otherwise specified.

 Culture properties Schwuct, nitrate agar

 Growth Good growth, pearl (2 b a)

 Back side Silver gray (3 f e)

 Qi mycelia poorly settled, beige (3 g e)

 Glucose-asparagine agar that does not produce soluble pigment

 Growth Medium growth, Pearl (2 b a)

 Back side pearl (2 b a)

 Qi mycelium does not settle

 Soluble pigment not producing roll ■ Asparagine agar (ISP)

 Growth Good growth, camel (3 i e)

 Back side dove brown (31 g)

 Aerial mycelium abundantly grown, beige gray (3 i h) soluble pigment slightly produced, mustard (21 e) chi-mineral salt agar (I SP)

 Growth Good growth, mustard (21 e)

 Back light master dotan (2 i e)

Aerial mycelium abundance, cobalt gray (2 fe) Soluble pigment Slightly produced, Light Mustard Tan (2 ie) Tyrosine Agar (I SP)

 Growth Good growth, light mustard tongue (2 i e) Back side mustard tongue (21 g)

 Aerial mycelium moderately grown, silver gray (3 f e) soluble pigment slightly produced, light mustard tongue (2 i e) oatmeal agar (I SP)

 Growth Good growth, mustard (21 e)

 Back side dark cobalt gray (2 i h)

 Aerial mycelia Mediumly grown, beige brown (3 i g) Yeast malt extract agar not producing soluble pigment

 Growth Good growth, yellow maple (3ng) back side clove brown (3p 1)

 Aerial mycelium abundance, dark cobalt gray (2 i h) soluble pigment slightly produced, golden brown (3 pg) nutrient agar

 Growth Good growth, cream (1 c a) Back side cream (1 c a)

 Qi fungus thread poorly settled, natural (2 d c)

 Soluble pigment not produced Peptone Yeast Iron Agar (I SP)

 Growth Good growth, bamboo (2 g c)

Backside Honey Gold (2 ic) Qi mycelium does not settle

 Soluble pigment Slightly produced, Honey Gold (2 i c) Glucose / Nitrate Agar

 Growth Medium growth, Pearl (2 b a)

 Back side pearl (2 b a)

 Qi mycelia poorly settled, beige (3 g e)

 Glucerol that does not produce soluble pigmentCalcium malate calcium agar

 Growth Good growth, pearl pink (3 c a) Back side pearl pink (3 c a)

 Qi mycelium does not settle

 No soluble pigment production Glucose peptone agar

 Growth Good growth, Ivory (2 db) Back side Master must (21 e)

 Qi mycelium does not settle

 No soluble pigment production. Physiological properties

 (1) Formation of melanin pigment

 (Ii) Tyrosine agar medium positive

(Mouth) Peptone Yeast 'Iron Agar Medium Negative

(C) Tryptone yeast solution negative

(2) Simple gelatin medium (2 1-23 ° C) Negative

(2) Positive reduction of nitrate

(3) Liquefaction of gelatin (21-23 ° C) (Simple gelatin medium) (4) Starch hydrolysis positive

 (5) Coagulation of skim milk (37 ° C) Negative

 (6) Peptone conversion of skim milk (37 ° C) Negative

 (7) Growth temperature range 7 ~ 3 8 ° C

(8) Utilization of carbon source (Pridham Gotrib Agar)

 Use: D-glucose, melibiose, L-rhamnose Some use: D-xylose, raffinose, D-fructose, sucrose

 Not used: L—arabinose, D—mannitol, my 0—inositol

 (9) Degradation of cellulose Negative

4. Cell wall composition

The cell wall diaminopimelic acid is LL type, and the main menaquinones are MK-9 (H _s ) and MK-9 (H ₈ ).

5. Conclusion

The bacteriological properties of this bacterium are summarized as follows. Diamide Nopimerin acid LL type in the cell wall, the main menaquinone is MK-9 (the He) and MK- 9 (H _8). The spore chain is helical, forms long spore chains, and the spore surface is smooth. As culturing properties, vegetative mycelium has a brown color, and aerial mycelium has a brown to gray color. Produces melanin pigment on tyrosine agar.

 From these results, it is considered that the fungus belongs to the genus Streptomyces, based on Buzzy's Manual 'Ob' Systematic, Bacteriophage Kichiichi, Volume 4, 1898.

 6. International deposit of microorganisms

Based on the morphological characteristics, culture characteristics, and physiological characteristics of the above 0 3-0 1 32 strain, as a result of an attempt to compare it with known bacterial species, this strain was identified as a strain belonging to the genus Streptomyces, It was named as Streptomy cessp. K 0 3-0 1 3 2 This strain is designated as Streptomyces sp. K 0 3— 0 1 3 2 Tsukupa east 1-chome, Castle Prefecture 1 Central No. 6 (postal code 305—8566) [AI ST Tsukub a Cen tral 6, 1—1, H i ga shi 1— c home Ts ukub a— shi, I bar ak i—ken, 305-856 6 Jap an] National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center [Int erna ti ona l Pat en t Or gan i sm D epositary Na ti ona l Deposited to Institute of Advanced Industrial Science and Technology. The date of entrustment is 2004 (04) 10 21 October, and the acceptance number is FERM ABP— 1 0 1 48.

 Examples of the K 03-0 1 32 substance-producing bacterium used in the present invention include the aforementioned Streptomyces sp. K 03-01 32 strain, but as a general property of the bacterium, the mycological property is extremely mutated. Easy, non-constant, natural or normal UV irradiation, X-ray irradiation or mutant derivatives, such as N-methyl-N'-nitro-1 N-nitrosoguanidine, 2-aminopurine, etc. Artificial mutants that can be obtained by the artificial mutation treatment used, as well as cell fusion strains and genetically engineered strains, belong to Streptomyces sp. And represented by the above formula [I] All the strains producing the active substance K03-0 1 32 A and the active substance K 03-0 1 32 B represented by the above [IT] can be used in the present invention.

 In practicing the present invention, first, K 03-0 1 32 substance-producing bacteria belonging to Streptomyces sp. Are cultured in a medium. Nutrient sources suitable for the production of the above active substances K 03-0 1 32 include nutrient sources such as carbon sources that can be assimilated by microorganisms, nitrogen sources that can be digested, and mineral salts and vitamins as necessary. Is used. As the assimilable carbon source, glucose, practose, maltose, lactose, galactose, dextrin, saccharides such as starch and vegetable oils such as soybean oil are used alone or in combination.

Digestible nitrogen sources include peptone, yeast extract, meat extract, soy flour, cottonseed flour, corn steep liquor, malt extract, casein, amino acids, urea, ammonium salts, nitrates, etc. alone or in combination Used. Other must As needed, phosphates, magnesium salts, calcium salts, sodium salts, potassium salts, and other salts, iron salts, manganese salts, copper salts, cobalt salts, zinc salts, and other heavy metal salts, bibutamines, and other activities Substances suitable for production of the substance K 0 3-0 1 3 2 are added as appropriate.

 In culturing, when foaming is intense, an antifoaming agent such as a surfactant such as liquid paraffin, animal oil, vegetable oil, or silicone may be added as necessary. In the above culture, the medium may be liquid or solid as long as the nutrient source is contained, but it is usually preferable to use a liquid medium for culture. In the case of small-scale production, culture using a flask is preferable. In order to industrially produce the target substance in large quantities, it is preferable to carry out aeration and agitation culture in the same manner as other fermentation products.

 When culturing in a large tank, in order to prevent the growth of bacteria in the production process, first inoculate and culture the produced bacteria in a relatively small amount of medium, then transfer the culture to a large tank and produce it there. It is preferable to culture. In this case, the composition of the medium used for the pre-culture and the medium used for the production culture may be the same, or both may be changed if necessary.

 When the culture is performed under aeration and agitation conditions, known methods such as agitation with a propeller or other machine, rotation or shaking of a meter, pumping, air blowing, etc. are appropriately used. Use sterilized air for ventilation.

 The culture temperature can be appropriately changed within the range in which the present K 0 3-0 1 3 2 substance-producing bacterium produces this active substance K 0 3-0 1 3 2, but usually 20 to 30 ° C, The culture is preferably performed at around 27 ° C. The culture pH is usually 5 to 8, preferably about 7. The culture time varies depending on the culture conditions, but is usually about 4-7 days

 The active substance K 0 3-0 1 3 2 thus obtained is present in cultured cells and in the culture filtrate. To collect the desired active substance K 0 3-0 1 3 2 from the culture, the whole culture is extracted with a water-miscible organic solvent such as acetone, and the extract is distilled off with an organic solvent under reduced pressure. Subsequently, the residue is extracted with a water-immiscible organic solvent such as ethyl acetate.

In addition to the above extraction methods, known methods used for collecting fat-soluble substances, such as absorption Active substances K 0 3-0 1 3 2 persons and 1: by appropriately combining or repeating ring chromatography, gel filtration chromatography, thin layer chromatography, centrifugal countercurrent distribution chromatography, high performance liquid chromatography, etc. It can be separated into 0 3-0 1 3 2 B components and purified.

 Next, the physicochemical properties of the active substances K 0 3-0 1 3 2 A and K 0 3-0 1 3 2 B of the present invention will be described.

Active substance K 0 3-0 1 3 2 A

 (1) Property: White needle crystal

 (2) Molecular weight: 2 9 3 (by fast atom bombardment mass spectrometry)

(3) Molecular formula: C ₁₅ H ₁₉ N0 ₅

(4) Specific rotation: [<¾]. ²⁶ = + 2.5 ° (c = 0.1, methanol)

 (5) UV absorption spectrum: UV absorption spectra measured in methanol are 2 1 7 ηπι (ε = 1 2 5 0 0), 2 6 2 ηπι (ε = 6 7 0 0), 3 4 5 (maximum absorption near ε = 2 3 0 0)

(6) Red external absorption spectrum: Infrared absorption spectra measured by the bromide-powered Rum tablet method are 3 4 40, 3 2 1 2, 3 1 3 7, 1 7 1 2, 1 6 6 0, has a characteristic absorption maximum in 1 6 3 3 cm one ^1,

 (7) Proton Nuclear Magnetic Resonance Spectrum: Chemical shift (p pm) of proton nuclear magnetic resonance spectrum (measured in heavy chloroform), measured using a nuclear magnetic resonance spectrometer, manufactured by Varian Japan ) Is 7. 0 9 (1 H, br. S)

7.43 (1 H, br.s), 3.12 (2 H, d, J = 6.5 Hz), 2.86 (1 H, ddd, J = 6.7, 6. 7, 6.5 Hz), 2.45 (2 H, d, J = 6.7 Hz), 2.3 3 (2 H, d, J = 6.7 Hz), 2.14 (3 H , S), 2.20 (3H, s), 5.64, 6.39 (2H, br.s), 12.30 (1H, s)

(8) ¹³ C nuclear magnetic resonance spectrum: Chemical shift of nuclear magnetic resonance spectrum (measured in a heavy π mouth form) measured by a nuclear magnetic resonance spectrometer, manufactured by Varian Japan (P pm) is 1 5 9. 0, 1 2 7. 2, 1 3 8. 6, 1 2 7. 5, 1 2 7. 3, 1 1 8. 5, 2 0 6. 0, 4 2 0, 2 9. 2, 3 8. 1, 1 74. 2, 39.3, 1 74.4, 1 5. 3, 20. 4

 (9) Solubility in solvents: Soluble in methanol, chloroform, and ethyl acetate. Insoluble in water and hexane

 (1 0) Color reaction: positive for phosphomolybdic acid

 (1 1) Distinguishing between acidic, neutral and basic: neutral substances

 As a result of detailed examination of various physicochemical properties and spectrum of the active substance K 03-0 1 32 A, the active substance K 03-0 1 32 A is represented by the formula [I]. It was determined to be an academic structure.

Active substance K 03-0 1 32 B

 (1) Property: White needle crystal

 (2) Molecular weight: 292 (by fast atom bombardment mass spectrometry)

(3) Molecular formula: C ₁₅ H ₁₆ O _e

(4) Specific rotation: [] _D ²⁶ =-2.2 ° (c = 0.1, methanol)

(5) Ultraviolet absorption spectrum: Ultraviolet absorption spectra measured in methanol are maximum around 217 nm (£ = 8900), 267 nm (£ = 6 100), and 355 (ε = 1 900). Have absorption

(6) Red external absorption spectrum: Infrared absorption spectra measured by the brominated power Ryumu tablet method are the maximum absorption characteristic of 3 100, 3037, 1 78 1, 1 708, 1 637 cm ¹ Having

 (7) Proton nuclear magnetic resonance spectrum: Chemical shift of proton nuclear magnetic resonance spectrum (measured in heavy chloroform) measured by Nuclear Magnetic Resonance Spectrometer, manufactured by Varian Japan (Ppm) is 7.20 (1 H, br. S)

7.52 (1 H, br.s), 5.70 (1 H, d, J = 3.0 Hz), 3.07 (1 H, m), 2.52 (1 H, dd, J = l .0, 1 7.0 Hz), 2.65 (1 H, dd, J = 8. 0, 1 7.0 Hz), 2.3 1 (2 H, dd, J = 3.5, 5 7. 5Hz), 2.82 (2H, dd, J = 8.5, 17.5 Hz)

2.19 (3H, s), 2.25 (3H, s), 1 1.80 (1H, s)

(8) ¹³ C Nuclear Magnetic Resonance Spectrum: Nuclear magnetic resonance spectrum measured by a nuclear magnetic resonance spectrometer (manufactured by Varian Japan Ltd.) The chemical shift (ppm) measured in 15.9.9, 1 28.0, 1 39.8, 1 29.0, 1 26.9, 1 14.0, 1 98.5, 80.7, 34.7, 37.0, 1 72.2, 39.0, 1 75.0, 1 5. 4, 20. 4

 (9) Solubility in solvents: Soluble in methanol, black mouth form, ethyl acetate, poorly soluble in water, hexane

 (1 0) Color reaction: positive for phosphomolybdic acid

 (1 1) Distinguishing between acidic, neutral and basic: neutral substances.

 As described above, as a result of examining various physicochemical properties and spectrum of active substance K 03-0 1 32 B in detail, this active substance K 03-0 1 32 B is represented by the formula [H]. It was determined to be a structure.

Biological properties

 Next, the biological properties of the active substances K 03-0 1 32 A and K 03-0 1 32 B of the present invention are described below.

 (1) Activity enhancement effect of azole antifungal agents

 The activity enhancing action of the azolic antifungal agent was measured as follows.

Cand i da albic an s KF 1 is used as a test bacterium, and C and idaalbic an s KF 1 is "W aksman broth (G 1 ucose 2. 0%, Peptone 0.5%, Dr yyeast 0 3 Me at Ex tract 0.5%, NaC 1 0. 5%, CaC0 ₃ 0. 3%, pH 7.0, 27, 40 hours seed culture, GY agar medium (G 1 ucose 1. 0%, Ye ast Ex tract 0.5%, Agar r 0.8%, pH 6.0), 0.3% inoculation, this plate as plate A, and Miconazo (SIGMA, USA), and the concentration added to the GY agar medium was 0.06 / M (final concentration), which does not affect the growth of the test bacteria. Disc method (thick, 8 mm: Evaluated by ADVANTEC, and the inhibition circle was measured after incubation for 24 hours at 27 ° C. The results are shown in Table 1 below. Table 1 Compound block diameter mm

 in g / d i s k) Plate A Plate B

K 03-0 1 32 A 1 0 ― ―

 twenty five -

 50 ― 1 2

K 03-0 1 32 B 1 0 One ―

 25 1 1 1

50 ― 14

(2) Antibacterial action against various test bacteria

Test bacteria include B aci 1 l_u ssubti 1 is PCI 2 1 9, t aphy lococ cu s aur eu s FDA209 P-, Mi crocoecuslut eu s PC I 1 00 1, My cob acteri um sme gm atis ATC C 607. E s_c_h erichiaco 1 i NIHJ, P seud omona saer ug i no sa P— 3, Xan t hom ona sor yz ae KB 88 ^ Ba cteroidesfra 1 is ATCC 23745, Acho l ep la sma 1 aid 1 aii PG 8, Pyriculariaor yz ae KF 1 80 As pergi 1 1 usni ge r ATCC 6275, Mu corrac emo sus IF 0458 K C_ndidaalbic an s ATCC 64548, Saccharomy cescer ev isiae 14 types were used. The activity was evaluated by the paper disk method (thin, 6 mm: manufactured by ADVANTECH), and the inhibition circle after incubation for 24 or 48 hours at 27 ° C or 37 ° C was measured. The results are shown in Table 2 below. 4019720 Table 2 Blocking circle diameter (mm)

 Test bacteria K03-0 1 32A K 03-0 1 32 B

Bacillus subtilis PCI 219 ― 1 0

 Staphylococcus aureus FDA 209P One 8

 Micrococcus luteus PCI 1001 ―

 Mycobacterium smegmatis ATCC607 ― ―

 Escherichia coli 匪 J ―

 Pseudomonas aeruginosa P-3

 Xanthomonas oryzae KB 88 ― ―

 Bacteroides fraglis ATCC 23745 ― 1 3

 Acholeplasma laidlawii PG 8 ― 1 2

 Pyricularia oryzae KF 180 ―

 Aspergillus niger ATCC 6275 ― One

 Mucor racemosus IFO 4581 ―

 Candida albicans ATCC 64548 ― ―

 Saccharomyces cerevisiae ― ― best mode for carrying out the invention

 Next, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

Example

50 Om 1 volume Erlenmeyer flask 0.1% glucose, starch 2.4%, peptone 0.3%, meat extract 0.3%, yeast extract 0.5%, calcium carbonate 0.4% ( Streptomyces sp. K 03-0 1 32 strain (Streptomyces sp. K03— 0 adjusted to pH 7.0) was prepared, and after cotton plugging, steam-sterilized and grown on an agar medium 1 32, F ERM ABP—1 0 1 4 8) was aseptically inoculated with a platinum loop and cultured with shaking at 27 ° C. for 96 hours.

 And using it as a seed culture, glucose 0.1%, starch 2.4%, peptone 0.3%, meat extract 0.3%, yeast extract 0.5%, calcium carbonate 0.4 %, Trace 5 ml (adjusted to ρΗ 7.0) 5 0 0 m 1 volume Erlenmeyer flask 10 ml, 10 bottles prepared, after steam sterilization, aseptically transplant 1 ml of the seed culture. 27 The cells were cultured at ° C for 6 days. 1 L of ethanol was added to the whole culture broth, and the mixture was stirred well and concentrated under reduced pressure. This was extracted with ethyl acetate and then concentrated under reduced pressure to obtain 2 l mg of crude material 3 6.

 Next, 26.1 mg of this crude substance was charged into a column chromatography using silica gel and subjected to column chromatography eluting with chloroform and methanol. The fraction eluting with black mouthform: methanol = 1: 1 was dried under reduced pressure to obtain 40.7 mg of a crude material. Next, 40.7 mg of the crude material was separated and purified by high performance liquid chromatography. The instrument is SSC 34 6 1 (Sensyu Kagaku Co., Japan), the column is PEGAS I L-ODS (ODS resin, Sensyu Kagaku Co., Japan), and the solvent system is 0. A 30-minute gradient of 10% to 50% acetonitrile containing% TFA was used, and detection was performed at UV 2 10 η m and a flow rate of 1.0 ml.

 As a result, the fraction eluted at 20.0 minutes was dried under reduced pressure, and 4. Omg of K 0 3— 0 1 3 2A was isolated. The fraction eluting with black mouth form: methanol = 5: 1 was evaporated to dryness under reduced pressure to obtain 4 mg of crude substance. Next, this 41. Omg crude material was separated and purified by high performance liquid chromatography. The equipment is SSC 34 6 1 (Sensyu Kagaku Co., Japan), the column is PEGAS I L-ODS (0 DS resin, Sensiyu Kagaku Co., Japan), and the solvent system is 0.05% A 30-minute gradient of acetonitrile containing 10% to 50% containing TFA was used, and detection was performed at a UV of 20 nm and a flow rate of 1.0 m 1 / min. As a result, the fraction eluted at 28.0 minutes was dried under reduced pressure, and 4. Omg of K 0 3-0 1 32 B was isolated.

Industrial application fields

From the above, the active substance according to the present invention K 0 3-0 1 3? Yaori Pi or Ri Since 3-0 1 3 2 B has an activity-increasing action of azole antifungal agents, it acts against many fungal infections, including deep mycosis, at low concentrations and in a short period of time. This is useful for reducing the frequency of the appearance of sex bacteria. Also useful for overcoming resistance.

Claims

1. Following formula [I] Blue  of

Active substance represented by K 03-0132 Ac  2. Following formula [I]

Active substance represented by K 03-0132 B,  3. The following formula [I] and

KO 3— 0132 活性, and the following formula [mutual]

An active substance represented by K 03-0 1 32 4. A microorganism having the ability to produce an active substance ピ ー 03-0 1 3 1 属 し belonging to Streptomyces sp. And represented by the formula [I] is cultured in a medium, and the active substance K 03- A method for producing an active substance K 03-0 1 3 1 A by accumulating 0 1 31 A and collecting the active substance K 03-0 1 3 1 A from the culture. 5. A microorganism belonging to Streptomyces sp and having the ability to produce an active substance K03-0 1 3 1 B represented by the formula [I] is cultured in a medium, and the active substance K 03-0 is added to the culture. A method for producing an active substance K 03-0 1 31 B, which accumulates 1 31 B and collects the active substance K 03-0 1 3 1 B from the culture. 6. Streptomyces sp. Has the ability to produce active substance K 03-0 1 3 1 A represented by formula [I] and active substance K 03-0 1 32 B represented by formula [I] Microorganisms are cultured in a medium, and active substances K 03-0 1 32 A and K 03-0 1 32 B are accumulated in the culture, and active substances K 03- 0 1 3 2 A and K 0 3 are accumulated from the culture. -0 1 32 A method for producing a composition from which 32 B is collected. , 7. The ability to produce the active substance K 03-0 1 32 A represented by the formula [I] and the active substance K 03-0 1 32 B represented by the formula [I] belonging to Streptomyces sp. A method for producing an active substance K 03-0 1 32, wherein the microorganism possessed is Streptomyces sp. K03-01 32 (FERM ABP— 1 0 1 48). 8. Streptomyces sp. (Streptomy cs s sp.) K 03-0 1 32 (FERM ABP— 1 0 1 48).