JP2592468B2 - Benanomycins A and B, novel antibiotics and their production - Google Patents

Description

The present invention relates to a novel antifungal antibiotic, benanomycin (Benomycin).
nanomicin) A and B or a salt thereof, and a method for producing the same.

(Prior Art) As a compound having similar physicochemical properties to the antibiotics benanomycin A and B according to the present invention, KS-619-1 [Mat
suda et al .: "J. Antibiotics" 40 , 1104-1114 (1987)] G
-2N and G-2A [Gerber et al .: ". Canad.J.Chem" 62, 28
18-2821 (1984)]. However, the antibiotics benanomycins A and B are distinct from these substances in physicochemical and biological properties and are clearly distinguishable.

(Problems to be Solved by the Invention) Conventionally, various antibiotics produced by microorganisms are known. However, since antifungal antibiotics having low toxicity have not been found so much, various infections caused by fungi have been found. In the medical field of diseases, the emergence of new antifungal antibiotics is always desired.

An object of the present invention is to provide novel and low-toxicity antifungal antibiotics, benanomycins A and B, and a method for producing the same.

(Means for Solving the Problems) That is, in the first invention, the following general formula (A): [Wherein, R ¹ is a hydroxyl group in benanomycin A and an amino group in benanomycin B, and (R) has a configuration at a carbon atom to which the symbol (R) is attached.
Or a salt thereof, which is an antibiotic benanomycin A or benanomycin B represented by the formula:

1. Details of the physicochemical properties of Benanomycin A are listed below.

(1) Color and shape: reddish brown powder (2) Molecular formula: C ₃₉ H ₄₁ NO ₁₉ (3) Mass spectrum (FD-MS): m / z827 (M ⁺ ) (4) Melting point:> 220 ° C. (5) Ratio Optical rotation: ▲ [α] ²² _D ▼ = unmeasurable (c 0.05,
DMSO) (6) UV and visible absorption spectrum λmax nm (▲ E
^1% _1cm ▼) [In methanol]: 206 (718), 230sh (600), 288 (48
2), 302sh (390), 400sh (120), 476 (197) [0.1N HCl in methanol] 207 (649), 233 (629), 298 (561), 395sh (140), 457
(223) [0.1N NaOH-in methanol] 214 (1270), 249 (637), 320 (289), 498 (287) (7) Infrared absorption spectrum (KBr cm ^-1 ): 3350, 2970, 2890, 1720,1620,1600,1485,1440,1425,139
0,1375,1330,1295,1255,1235,1205,1160,1130,1070,104
0,1000,970,900,870,830,800,750 (8) ¹ H-NMR spectrum (400 MHz, DMSO-d ₆ , 40 ° C.) δ (ppm): 1.14 (3H, d), 1.35 (3H, d), 2.34 (3H, s),
3.09 (1H, dd), 3.13 (1H, dd), 3.17 (1H, dd), 3.32 (1
H, ddd), 3.56 (1H, dd), 3.62 (2H, m), 3.72 (1H, dd),
3.74 (1H, br), 3.92 (3H, s), 4.43 (1H, d), 4.43 (1H, d
q), 4.53 (1H, d), 4.57 (1H, d), 4.65: (1H, d), 4.90
(2H, br), 5.61 (1H, br), 6.05 (1H, br), 6.86 (1H,
d), 7.21 (1H, s), 7.24 (1H, d), 8.05 (1H, s), 8.45 (1
H, d), 12.47 (1H, br), 12.77 (1H, s), 13.69 (1H, br) (9) ¹³ C-NMR spectrum (100 MHz, DMSO-d ₆ , 40 ° C.) δ (ppm): 187.3 s, 184.9s, 173.9s, 166.9s, 165.9s, 164.7
s, 156.8s, 151.1s, 147.7s, 138.1s, 137.4s, 134.2s, 131.3
s, 127.5s, 125.6s, 118.6d, 115.5s, 115.4d, 113.7s, 110.0
s, 107.5d, 106.8d, 105.2d, 104.4d, 83.0d, 81.7d, 76.0d, 7
3.6d, 71.9d, 70.3d, 70.1d, 70.1d, 69.4d, 65.6t, 56.3q, 47.
6d, 19.1q, 16.9q, 16.3q (The signals of 72.9, 81.7, 115.4, 118.6 are broad.) (10) Solubility: very slightly soluble in methanol, chloroform, ethyl acetate, acetone, dimethyl sulfoxide, Dimethylformamide, soluble in alkaline water, insoluble in water.

(11) Distinguishing basic, acidic and neutral: acidic substances As a result of the above-mentioned physicochemical properties and structural studies, benanomycin A has the following formula (I) Was determined to be a novel antibiotic having the structural formula

2. The details of the physicochemical properties of Benanomycin B hydrochloride are listed below.

(1) Color and shape: reddish brown powder (2) Molecular formula: C ₃₉ H ₄₂ N ₂ O ₁₈ .HCl (3) Mass spectrum (SI-MS): m / z 827 (M ⁺ ) (4) Melting point:> 220 ° C. (5) Specific rotation: ▲ [α] ²² _D ▼ = + 360 ゜ (c 0.05, H
₂ O) (6) UV and visible absorption spectrum λmax nm (▲ E
^1% _{▼ 1cm} ) [in methanol]: 205 (587), 233 (526), 296 (426),
390sh (100), 458 (169) [0.1N HCl-in methanol] 207 (514), 235 (530), 295 (442), 400sh (114), 457
(173) [0.1N NaOH-in methanol] 214 (1219), 247 (518), 317 (238), 496 (215), (7) Infrared absorption spectrum (KBr cm ^-1 ): 3350, 2980, 2900 , 1720,1610,1485,1350,1430,1395,138
0,1330,1300,1260,1240,1210,1160,1080,1045,970,955,
900,885,840,820 (8) ¹ H-NMR spectrum (400 MHz, DMSO-d ₆ , 40 ° C.) δ (ppm): 1.20 (3H, d), 1.36 (3H, d), 2.35 (3H, s),
3.09 (1H, dd), 3.17 (1H, m), 3.19 (1H, m), 3.34 (1H, d
dd), 3.44 (1H, br), 3.65 (1H, br), 3.75 (1H, dd), 3.9
0 (1H, br q), 3.94 (3H, s), 3.97 (1H, dd), 4.44 (1H, d
q), 4.57 (1H, d), 4.57 (1H, br d), 4.62 (1H, br d), 4.
75 (1H, d), 6.90 (1H, d), 7.27 (1H, d), 7.27 (1H, br
s), 7.99 (3H, br), 8.06 (1H, br s), 8.45 (1H, d), 8.4
5 (1H, br), 12.79 (1H, s), 13.81 (1H, br), 4.1-6.3
(5H, br) (9) ¹³ C-NMR spectrum (100 MHz, DMSO-d ₆ , 40 ° C.) δ (ppm): 187.4 s, 184.9 s, 173.9 s, 166.9 s, 165.9 s, 164.7
s, 156.8s, 151.0s, 148.0s, 137.8s, 137.3s, 134.2s, 131.2
s, 127.5s, 125.7s, 118.9d, 115.9d, 115.5s, 113.7s, 110.0
s, 107.6d, 106.8d, 104.4d, 104.1d, 81.0d, 77.4d, 75.9d, 7
3.3d, 71.5d, 69.8d, 69.4d, 67.0d, 65.7t, 56.3q, 54.2d, 47.
6d, 19.1q, 16.9q, 16.3q (The signals at 71.5, 81.0, 113.7, 115.9, 118.9, 125.7 are broad.) (10) Solubility: Slightly soluble in chloroform, ethyl acetate, acetone, methanol, dimethyl Soluble in sulfoxide, dimethylformamide and water.

(11) Basic, acidic and neutral distinctions: amphoteric substances As a result of the above physicochemical properties and structural studies, benanomycin B has the following formula (II) Was determined to be a novel antibiotic having the structural formula

The respective salts of benanomycins A and B include metal salts, especially alkali metal salts such as sodium salts, alkaline earth metal salts such as calcium salts, and pharmaceutically acceptable inorganic acids such as hydrochloric acid or sulfuric acid. Examples thereof include acid addition salts with pharmaceutically acceptable organic acids such as acetic acid and malic acid, and salts with amines as described below.

3. The antibacterial activities of Benanomycin A and B are shown below.

The minimum inhibitory concentrations of benanomycins A and B according to the present invention against various bacteria and various molds were respectively set to the first values.
The results are shown in Tables and Table 2. As shown in Table 2, benanomycins A and B exhibited antifungal activity against various molds.

4. Toxicity of Benanomycin A and B Benanomycin A and B were subjected to an acute toxicity test by intravenous administration of Jcl: ICR mice (male, body weight, 19-20 g, 5 mice per group). Benanomycin A was administered at a dose of 600 mg / Kg. There were no deaths at the dose of 100 mg / Kg of Benanomycin B.

5. Effect of Benanomycin A on infection Jcl: ICR mice (male, weight: 19-20 g, 5 mice per group) per mouse of Candida albicans
The therapeutic effect of Benanomycin A on experimental candidiasis in which subcutaneous and oral administration of Benanomycin A after inoculation of 10 ⁶ CFU (0.2 ml) intravenously is as shown in Table 3.

The gist of the second aspect of the present invention is to cultivate bacteria producing the antibiotics benanomycin A and B represented by the general formula (A) and belonging to the genus Actinomadura, and from the culture, the antibiotics benanomycin A and B and / or any one of them is a method for producing the antibiotics benanomycin A and / or benanomycin B.

An example of the benanomycin A and B producing bacteria is the actinomycete MH193-16F4 strain described below.

(B) Mycological properties of actinomycetes strain MH193-16F4 In March 1984, this strain was isolated from the soil on the premises of the Institute at the Institute of Microbial Chemistry (Actinomyce).
In te), the strain number of MH193-16F4 was assigned. The bacteriological properties of this strain are as follows.

[1] Form The MH193-16F4 strain forms aerial hyphae from a base mycelium branched under a microscope. No division of the mycelium in the base was observed. The formation of aerial hyphae was observed only in ISP-medium 2 and ISP-medium 3.
Observed around day. The aerial mycelium does not show any ring-shaped branches, helix formation, or sporangia formation, and a short spore stalk is formed almost perpendicularly to the aerial hyphae, and a chain of usually 3 to 7, rarely 2 spores is found at the tip. Can be Note that the spore chain may have a loop shape. Each spore is cylindrical (0.8 × 1.0-1.2μ)
m) to spherical (0.8-1.2 μm), and the spore surface is smooth.

[2] Growth conditions in various media Regarding color description Standards shown in [] are the color harmony manuals of Container Corporation of America (Container Corporation of Ameri).
Color Harmony Manual from ca) was used.

(1) Sucrose / nitrate agar medium (cultured at 27 ° C.) Growth is colorless, aerial mycelia do not form, and no soluble pigment is observed. In the vitamin B group supplemented medium, growth is colorless to pink ash [5ec, Dusty Peach], aerial mycelia do not form, and the soluble pigment is slightly reddish.

(2) Glucose-asparagine agar medium (cultured at 27 ° C) Growth is colorless, aerial mycelia do not form, and no soluble pigment is observed. In the vitamin B group-supplemented medium, the growth is colorless to light pink ash [4 gc, Nude Tan], aerial mycelia do not form, and the soluble pigment becomes reddish.

(3) Glycerin-asparagine agar medium (ISP-medium 5, cultured at 27 ° C.) The growth is colorless, aerial mycelia do not form, and no soluble pigment is observed. In the vitamin B group supplemented medium, ashy reddish purple [8lg, Rose
Mauve] ~ On the development of reddish purple [8le, Rose Wine], slightly faint white aerial hyphae grow, and reddish purple [8pc,
Cranberry].

(4) Starch / inorganic salt agar medium (ISP-medium 4, 27 ° C
(Culture) Growth is colorless, aerial mycelia do not form, and no soluble pigment is observed. In the vitamin B group supplemented medium, the soluble pigment is slightly reddish.

(5) Tyrosine agar medium (ISP-medium 7, cultured at 27 ° C) The growth is colorless to light yellow brown [3ic, Lt Amber], aerial mycelia do not form, and no soluble pigment is observed. In the vitamin B group supplemented medium, the growth is colorless to ash-reddish purple, aerial mycelia do not form, and the soluble pigment becomes slightly reddish.

(6) Nutrient agar medium (cultured at 27 ° C.) The growth is pale yellow [2 gc, Bamboo], no aerial mycelia form, and no soluble pigment is observed. In the case of the vitamin B group supplemented medium, the same properties were exhibited.

(7) Yeast / malt agar medium (ISP-medium 2, cultured at 27 ° C) Light yellow [2gc, Biscuit] to dark red [7pi, Dk Wine-7 1 /
2pe, Dk Red] ~ Gray red [7pg, Wine]
A gray aerial mycelium forms on the 14th day. Nibu Red [6 1/2
pe, Tomato Red] soluble pigment is found only around the development at the beginning of culture, but gradually diffuses. Both the developing pigment and the soluble pigment gradually change color with HCl, and there is no change with NaOH. Vitamin B group supplemented medium has the same properties.

(8) Oatmeal agar medium (ISP-medium 3, cultured at 27 ° C) Light pink [4gc, Nude Tan] to grayish red [6le, Cedar]
~ Dark red [7pe, Cherry Wine] ~ Purple ash [8ig, Mauve Gra
y], the grayish white [3]
cb, Sand]. The soluble pigment is reddish. Both the color of the invention and the soluble dye gradually change color with HCl, and there is no change with NaOH. The vitamin B group supplemented medium also showed the same properties.

(9) Glycerin / nitrate agar medium (cultured at 27 ° C.) Growth is colorless, aerial mycelia do not form, and no soluble pigment is observed. Vitamin B group supplemented medium has the same properties.

(10) Starch agar medium (cultured at 27 ° C) Growth is colorless, aerial mycelia do not form, and no soluble pigment is observed. In the vitamin B group supplemented medium, growth is colorless, aerial mycelia do not form, and the soluble pigment is slightly reddish.

(11) Ring acid-lime agar medium (cultured at 27 ° C) The growth is colorless, aerial mycelia do not form, and the soluble pigment is slightly reddish. In the vitamin B group supplemented medium, growth is colorless, aerial mycelia do not form, and no soluble pigment is observed.

(12) Cellulose (Synthetic solution with filter paper added, cultured at 27 ° C) No growth is observed.

(13) Gelatin puncture culture Both 15% simple gelatin medium (cultured at 20 ° C) and glucose / peptone gelatin medium (cultured at 27 ° C) have poor growth, colorless growth, no aerial mycelia, and no soluble pigment. unacceptable.

(14) Skim milk (cultured at 37 ° C) The growth is extremely poor, the growth is colorless, aerial mycelia do not form, and no soluble pigment is observed.

[3] Physiological properties (1) Growth temperature range Using starch yeast agar (soluble starch 1.0%, yeast extract 0.2%, string agar 2.0%, pH 7.0), 2
As a result of the test at each temperature of 0 ° C, 24 ° C, 27 ° C, 30 ° C, 37 ° C, 50 ° C, it grew at any temperature except 50 ° C,
The optimal growth temperature seems to be around 27 ° C to 37 ° C.

(2) Liquefaction of gelatin (15% simple gelatin medium, culture at 20 ° C: glucose / peptone / gelatin medium, 27 ° C culture) Both simple gelatin medium and glucose / peptone / gelatin medium were observed for 3 months after the start of culture, but liquefaction was observed. Did not admit.

(3) Hydrolysis of starch (starch / inorganic salt agar medium and starch agar medium are all cultivated at 27 ° C.) Neither starch / inorganic salt agar medium nor starch agar medium has hydrolytic properties.

(4) Coagulation / peptone conversion of skim milk (defatted milk, cultivation at 37 ° C.) The growth was poor, and neither coagulation nor peptone formation was observed in the observation for three months after the start of the culture.

(5) Melanin-like pigment formation (trypton yeast extract broth, ISP-medium 1; peptone yeast extract iron agar, ISP-medium 6; tyrosine agar, ISP-medium 7; all cultured at 27 ° C.) The medium was also negative.

(6) Utilization of carbon source (Pridham Götrib agar medium, ISP-medium 9, 27 ° C culture) Glucose, L-arabinose, D-xylose, D
-Grow using fructose, sucrose, rhamnose, raffinose, D-mannitol and do not use inositol.

(7) Dissolution of lime malate (lime lime agar at 27 ° C.) Negative.

(8) Nitrate reduction reaction (peptone water containing 0.1% potassium nitrate, ISP-medium 8, cultured at 27 ° C) is positive.

(9) Decomposition of cellulose (synthetic solution containing filter paper pieces, cultivation at 27 ° C) Does not grow.

In summary, the MH193-16F4 strain forms a spore chain consisting of 3-7 (rarely 2) spores, almost perpendicular to the main axis of the aerial hyphae, and forms rotifers, spirals, and spores. No formation is observed. In addition, no division of the mycelium in the base was observed. The spore surface is smooth. In two types of media, ISP-medium 2 and ISP-medium 3, gray white aerial hyphae are slightly formed on grayish red to dark red growth. In addition, the slope medium (yeast
Extract 0.2%, soluble starch 1.0%, string agar 2.0%, pH
When passaged to 7.0), pink aerial hyphae may form. It also produces a soluble red pigment in ISP-medium 2. In other various media, growth is colorless, aerial mycelia do not form, and almost no soluble pigment is observed. However, there is a medium in which the growth becomes good and the growth becomes red by adding the vitamin B group to the medium. Both developmental and soluble pigments change color gradually from red with HCl and no change with NaOH. The formation of melanin-like pigment, the proteolytic activity, and the water-decomposability of starch are all negative, and the reduction reaction of nitrate is positive. In addition, the growth may be good in the vitamin B group supplemented medium, and it is considered that the medium has a vitamin requirement.

The MH193-16F4 strain contains meso-diaminopimelic acid as a cell wall component and glucose, ribose, and majulose as sugar components in all the cells, and is used by Richevalier et al., “International Journal of Sysme
tematic Bacteriology ", vol. 20, p. 435, 1970), which is a type III B of a major component of the cell wall. The type of the phospholipid is PIV type (including phosphatidylethanolamine and an unknown glucosamine-containing phospholipid, and excluding phosphatidyl / glycerol), and the composition of menaquinone is mainly composed of MK-9 (H ₈ ). 9
_{(H 6), MK-9} (H 4), MK-9 (H 2), MK-9
(H ₁₀ ), the GC content of the DNA was 71.5%. As a result of analyzing the cells by gas chromatography, isobranched fatty acids (i-16: 0) and anteisobranched fatty acids (a-17:
0), 10 methyl fatty acid (10Me-17: 0).

Known actinomycetes with spore linkage, cell wall type III B
Are three genera of Actinomadura, Microbispora, and Microtetraspora. Table 4 shows the characteristics of the MH193-16F4 strain and the above three genera. * Of nanaquinone indicates that it is contained as a main component.

References 1) Experimental method for identification of actinic radiation, edited by Japanese Society of Actinomyces, 1985 2) J. Poschner et al .: “DNA-DNA Reassociation and C
hemotaxonomic Studies on Actinomadura, Microbispor
a, Microtetraspore, Micropolyspora and Nocardiopsis,
Systematic and Applied Microbiology, Vol. 6, 264-270
P., 1985.

3) A. Fischer et al .: "Molecular-genetic and Chemot
axonomic Studies on Actinomadura and Nocardiopsis,
Journal of General Microbiology, Vol.129, 3433-3446
1983 4) Thiemann et al .: “A New Genus of the Actinomycet”
ales: Microtetraspora gen.nov..Journal of General M
icrobiology, 50, 295-303, 1968.

5) Nonomura et al .: “Distribution of actinomycetes in soil” (No.
11) Several new species of the genus Actinomadura Lechevalier et al.
Journal of Fermentation Engineering, 49, 904-912, 1971.

As shown in Table 4, no genus corresponding to the MH193-16F4 strain was found. These genera have been very subtly converted and are described in the Bergey's Manual (Bergey's M
It is expected to be published in the next edition of the anual of Determinative Bacteriology). However, there are many species described in the genus Actinomadura, and the properties vary widely. Among them, Actinomadura spadix is different from the morphology, fatty acid composition and menaquinone composition of bacterial cells. A considerable similarity is found (1), 3), 5). As a result of conducting a comparative experiment between the MH193-16F4 strain and Actinomadura spazix, the MH193-16F4 strain is the same `` species '' as Actinomadura spargic and thus may be an actinomycete belonging to the genus Actinomadura. found.

An application for deposit of the MH193-16F4 strain was made to the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology (AIST) on August 21, 1987.
Deposited as No. 51.

(B) Culture method of MH193-16F4 strain The production of benanomycins A and B is performed by inoculating benanomycin A and B-producing bacteria belonging to actinomycetes on a nutrient-containing medium that can utilize ordinary microorganisms under aerobic conditions. This is done by developing. Benanomycins A and B are mainly produced and accumulated in the culture solution. The target substance is separated from the culture, particularly from the culture solution.

As a nutrient source in the medium to be used, a known nutrient source used as a nutrient source for actinomycetes can be used. For example, commercially available soy flour, peptone, meat extract, corn steep liquor, cottonseed flour, peanut flour, dried yeast, yeast extract, nitrogen sources such as NZ amine, casein, sodium nitrate, ammonium sulfate, ammonium nitrate, and glycerin , Sucrose, starch, glucose, galactose, maltose, dextrin, lactose, molasses, soybean oil, fat, amino acids, etc., and inorganic salts such as salt, phosphate, calcium carbonate, magnesium sulfate, cobalt chloride, manganese chloride Can be used. In addition, if necessary, a trace amount of a metal salt, a flour, a plant oil, a mineral oil, or the like can be added as an antifoaming agent. Any of these can be used as long as they are used by the benanomycin-producing bacteria and are useful for the production of benanomycins A and B, and any known actinomycete culture materials can be used.

For large-scale production of Benanomycin A and B, liquid culture is preferable.
And B can be applied in a range where it is produced, and is usually 20-40 ° C, preferably 25-37 ° C. The cultivation can be performed by appropriately selecting the above-described conditions according to the properties of the benanomycin A and B producing bacteria.

(C) Method for Purifying Benanomycins A and B In collecting Benanomycins A and B from the cultures of Benanomycins A and B obtained by the present invention, usual separation means utilizing their properties, for example, solvent extraction, ion extraction, Extraction and purification can be performed alone or in an appropriate combination of an exchange resin method, an adsorption or distribution column chromatography method, a gel filtration method, a dialysis method, a precipitation method and the like. For example, benanomycins A and B are extracted from cultured cells with acetone-water or methanol-water. Also,
Benanomycins A and B accumulated in the culture solution are adsorbed to a synthetic adsorbent such as Diaion HP-20 (manufactured by Mitsubishi Kasei Corporation). Also, organic solvents that do not mix with water, for example,
If extracted with butanol, ethyl acetate, etc., the benanomycin A and B substances are extracted into the organic solvent layer.

In order to further purify benanomycins A and B, chromatography using silica gel (Wakogel C-300, manufactured by Wako Pure Chemical Industries, Ltd.), an adsorbent such as alumina, Sephadex LH-20 (Pharmacia), or the like is performed. Good.

The benanomycins A and B thus produced in the culture can be separated in free form, ie, benanomycins A and B themselves.

Alternatively, a solution containing Benanomycin A and B or a concentrated solution thereof may be treated with a base, that is, for example, sodium hydroxide,
Operation of each step using an alkali metal compound such as potassium hydroxide, an alkaline earth metal compound such as calcium hydroxide or magnesium hydroxide, an inorganic base such as an ammonium salt, or an organic base such as ethanolamine, triethylamine or dicyclohexylamine. When processed during the operation of, for example, the extraction, separation or purification steps, benanomycins A and B change to their corresponding salt forms and are separated.

Alternatively, the salts of benanomycins A and B thus produced can be converted to the free forms, benanomycins A and B themselves, by conventional methods. Further, benanomycins A and B obtained in the free form may be converted into the corresponding salts by a conventional method using the above base. Accordingly, salts thereof as described above, as well as benanomycins A and B, are intended to be included within the scope of this invention.

Examples of the present invention are shown below. Since the properties of Benanomycins A and B have been clarified by the present invention, various methods for producing Benanomycins A and B can be devised based on those properties. Therefore, the present invention is not limited to the embodiments, and, of course, means for modifying the embodiments,
The present invention includes all methods for producing, concentrating, extracting, and purifying benanomycins A and B by applying known means based on the properties of benanomycins A and B revealed by the present invention.

Example 1 A liquid medium containing 1.0% starch and 3.0% soybean flour (80 ml in a 500 ml Sakaguchi flask, pH 7.0 before sterilization) containing MH193-16F4 strain (Nikko No. 2051) cultured on an agar slant medium. And inoculated at 28 ° C. for 3 days with shaking (135 rpm) to obtain a first seed culture. Inoculate each 3 ml of this seed culture into the same medium as above,
Under the same conditions, shaking culture was performed for 3 days to obtain a second seed culture. The second seed culture 2 was inoculated into a 100-volume culture tank containing a medium 50 of the above composition sterilized at 120 ° C. for 15 minutes, and cultured at 28 ° C. for 2 days with aeration and aeration at 200 rpm at 200 rpm to form a third culture. Seed culture was obtained. Glycerin 2.0 previously sterilized at 125 ° C for 30 minutes
%, Es San meat (trade name of soy flour) 1.5%, K ₂ HPO
₄ 0.0025%, KH ₂ PO ₄ 0.1125% CoCl ₂・ 6H ₂ O 0.0005%, KM72
(Product name of silicone oil) 0.03%, Adekanol 0.01%
The third seed culture 12 was inoculated into a 570-volume culture tank containing 300 production media consisting of: aeration rate of 150 / min at 28 ° C. for 7 days, initial 24 hours of culture, 300 / min after 24 hours, aeration at 300 rpm. Agitation culture was performed. After completion of the culture, diatomaceous earth was added as a filter aid, followed by filtration to obtain a filtrate 250 (pH 6.0).

Example 2 The culture filtrate 250 obtained in Example 1 was applied to Diaion HP
Adsorbed through 15 columns of −20, water 100 and 5
After washing with 0% methanol 45, the active substance was eluted with 70% methanol 45, then with methanol 90 and the first fraction (53
), A second fraction (38) and a third fraction (27). The first fraction containing the active substance was concentrated to 3 under reduced pressure, and the pH was adjusted to 3.5 using dilute hydrochloric acid to obtain a red precipitate. The precipitate was collected by filtration and dried under reduced pressure to obtain 152 g of a brown crude powder mainly containing benanomycin A.

Dissolve 150 g of this crude powder in 600 ml of dimethylformamide and saturate water vapor in a desiccator at room temperature for 3 days,
A crystalline precipitate precipitated. The precipitate was collected by filtration and dried under reduced pressure to obtain 29 g of benanomycin A dimethylformamide sorbate. The second fraction was treated in the same manner as the first fraction to obtain 14 g of Benanomycin A dimethylformamide sorbate.

1 g of Benanomycin A dimethylformamide sorbate obtained from the first fraction was added to dimethyl sulfoxide (5 m
l), dropped into 300 ml of methanol with stirring,
After stirring for further 10 minutes, a reddish brown precipitate was deposited. The precipitate was collected by filtration, and dried under reduced pressure to obtain 935 mg of pure red powder of Benanomycin A.

Example 3 The third fraction obtained in Example 2 was concentrated under reduced pressure to give 1.5 fractions.
When the pH was adjusted to 3.5 using dilute hydrochloric acid, a red precipitate was obtained. The precipitate was collected by filtration and dried under reduced pressure to obtain 99 g of a brown crude powder containing Benanomycin B. 1 g of this crude powder was dissolved in 10 ml of dimethylformamide by heating (40 ° C.), and Sephadex LH was filled with dimethylformamide.
It was applied to one column of -20 and developed with dimethylformamide. Fractions No. 64-72 (1 fraction, 6 ml) containing the active substance were collected and concentrated to dryness under reduced pressure to obtain 657 mg of brown crude powder containing Benanomycin B dimethylformamide sorbate. This crude powder (300 mg) was dissolved in methanol (100 ml), 1N hydrochloric acid (1 ml) was added, and the mixture was concentrated to dryness under reduced pressure. The resulting brown crude powder was dissolved in 3 ml of dimethyl sulfoxide,
The mixture was added dropwise to 200 ml of chloroform with stirring, and the mixture was further stirred for 20 minutes to precipitate a reddish brown precipitate. The precipitate was collected by filtration and dried under reduced pressure to obtain 258 mg of pure hydrochloride of benanomycin B.

Effect of the Invention As described in detail above, according to the present invention, actinomycetes MH
The novel antibiotics benanomycins A and B were isolated from the culture of the 193-16F4 strain. The present antibiotics and salts thereof have antifungal activity to inhibit the growth of various fungi.

[Brief description of the drawings]

Figure 1: Benanomycin A in methanol (20μg / ml)
2 shows the ultraviolet and visible absorption spectra of the sample. FIG. 2: Infrared absorption spectrum of potassium nanobromide tablet of benanomycin A. FIG. 3: 400 MHz hydrogen nuclear magnetic resonance spectrum of benanomycin A in a heavy dimethyl sulfoxide solution. FIG. 4: 100 MHz carbon nuclear magnetic resonance spectrum of benanomycin A in a heavy dimethyl sulfoxide solution. FIG. 5: Benanomycin B hydrochloride in methanol (20 μm)
g / ml) shows the ultraviolet and visible absorption spectra. FIG. 6 shows an infrared absorption spectrum of benanomycin B hydrochloride with a potassium bromide tablet. FIG. 7: 400 MHz hydrogen nuclear magnetic resonance spectrum of benanomycin B hydrochloride in heavy dimethyl sulfoxide solution. FIG. 8: 100 MHz carbon nuclear magnetic resonance spectrum of benanomycin B hydrochloride in heavy dimethyl sulfoxide solution.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Masaji Sezaki 1-112-16 Sangenjaya, Setagaya-ku, Tokyo (72) Inventor Haruo Yamamoto 11-16 Matsukazedai, Chigasaki-shi, Kanagawa Prefecture (72) Inventor Shuichi Gomi Tokyo 1-9-3-202 Hiramachi, Meguro-ku

Claims (5)

(57) [Claims] 1. The following general formula (A): [Wherein, R ¹ is a hydroxyl group in benanomycin A and an amino group in benanomycin B, and (R) has a configuration at a carbon atom to which the symbol (R) is attached.
Or a salt thereof, which is the antibiotic benanomycin A or benanomycin B represented by the formula: 2. The antibiotic represented by the general formula (A) is represented by the following formula (I): The compound according to claim 1, wherein the compound is benanomycin A represented by the formula: wherein (R) has the same meaning as described in claim 1. 3. The antibiotic represented by the general formula (A) is represented by the following formula (II): The compound according to claim 1, wherein the compound is benanomycin B represented by the formula: wherein (R) has the same meaning as described in claim 1. 4. A member of the genus Actinomadura which has the following general formula (A): [Wherein, R ¹ is a hydroxyl group in benanomycin A and an amino group in benanomycin B, and (R) has a configuration at a carbon atom to which the symbol (R) is attached.
A bacterium producing the antibiotics benanomycin A and benanomycin B represented by the formula: and collecting the antibiotics benanomycin A and / or benanomycin B from the culture. A method for producing the antibiotics benanomycin A and / or benanomycin B. 5. Benanomycin A and benanomycin B
MH193-16F4 strain (Microtechnical Research Co., Ltd. No. 2051)
5. The method according to claim 4, wherein the method comprises: