JPH0142960B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel antibiotic, Istamycin B, obtained from the culture solution obtained by culturing a microorganism belonging to the genus Streptomyces, and a method for producing Istamycin B. The present inventors cultivated an actinomycete strain numbered SS-939 isolated from seabed soil off the coast of Miura Peninsula, Kanagawa Prefecture, and injected with istamycin A, istamycin B, or a mixture thereof. It has been discovered that istamycin A or istamycin B can be collected from the culture solution. Istamycin A or istamycin B has a wide and strong antibacterial effect on various types of bacteria,
Since it is an aminoglycoside antibiotic with low toxicity, it is a new antibiotic used as a chemotherapeutic agent to treat bacterial infections. Istamycin A is a basic substance, and istamycin A isolated as its carbonate is a white powder that does not show a clear melting point, but decomposes at 102 to 108 °C and has a specific optical rotation of [α] ²³ _D = +155° (c0.4, water), and the elemental analysis value is the theoretical value of the molecular formula of C ₁₇ H ₃₅ N ₅ O ₅ 1/2H ₂ CO ₃ (C49.99%, H8.63%, N16 .65
%, O24.73%), and this molecular formula was further verified by high-resolution mass spectra (actual value: m/e389, 2588, theoretical value of C ₁₇ H ₃₅ N ₅ O ₅ :
389, 2635). The infrared absorption curve measured with potassium bromide tablets is as shown in FIG. The ultraviolet absorption curve measured in aqueous solution shows only the terminal absorption. ^1H nuclear magnetic resonance spectrum (TMS external standard, pD5.4) measured in heavy water, δ3.20 (s, N-
CH ₃ ), 3.57 (s, N-CH ₃ ), 3.89 (s, O-
CH ₃ ), 4.50 (s, CH ₂ ) and 5.80 (d, J=3.5
Hz, CH) showed a characteristic signal. Istamycin A is soluble in water and methanol, but sparingly soluble or insoluble in ethanol and other common organic solvents. Positive for ninhydrin and Lydon-Smith reactions. Thin layer chromatography of cellulose (Avicel) (Developing system: butanol/pyridine/acetic acid/water, 6:4:2:4 volume)
Istamycin A shows a single spot at Rf0.22, which is clearly distinguished from Rf0.25 of istamycin B described below. Formic acid/acetic acid/water (25:75:900 volume)
High-pressure paper electrophoresis (3300V, 15 minutes) using
When the mobility of alanine is 1.0, the mobilities of istamycin A and istamycin B are both
2.15 and not differentiated. Istamycin B is very similar to istamycin A in its properties. Istamycin B is also a basic substance, and its carbonate is a white powder with no clear melting point, but it decomposes at 112-124°C, and its specific rotation is [α] ²³ _D = +165° (c, 0.4 , water), and _the elemental analysis _values are the theoretical molecular formula _{of C17H35N5O5}・1/ _2H2CO3 (C49.99%, H8.63%, _N16.65
%, O24.73%), and this molecular formula was further verified by high-resolution mass spectra (actual value: m/e389, 2625, theoretical value of C ₁₇ H ₃₅ N ₅ O ₅ :
389, 2635). The infrared absorption curve measured with potassium bromide tablets is as shown in FIG. The ultraviolet absorption curve measured in aqueous solution shows only the terminal absorption. The ^1H nuclear magnetic resonance spectrum (TMS external standard, pD5.4) measured in heavy water showed δ3.26 (s, N-
CH ₃ ), 3.59 (s, N-CH ₃ ), 3.95 (s, O-
CH ₃ ), 4.57 (s, CH ₂ ) and 5.96 (d, J=3.5
Hz, CH) showed a characteristic signal. Istamycin B is soluble in water and methanol, but sparingly soluble or insoluble in ethanol and other common organic solvents. Positive for ninhydrin and Lydon-Smith reactions. As mentioned above, it is distinguished from istamycin A by cellulose thin layer chromatography. The minimum inhibitory concentrations of istamycin A and istamycin B against various test bacteria on nutrient agar plates are shown in Table 1, and both strongly inhibit the growth of Gram-positive and Gram-negative bacteria.

【table】

[Table] Regarding the acute toxicity of istamycin A and istamycin B, when a dose of 100 mg/Kg was administered intravenously to mice, both mice survived for more than 7 days and no side effects were observed. Based on the above properties, istamycin A and istamycin B are horteimycin A (R.OK
-achi et al., Journal of Antibiotics, Vol. 30, p. 541, 1977) and spolarisin A (T. Deushi et al., Journal of Antibiotics, Vol. 32, p. 173, 1979);
They were clearly distinguished from these antibiotics in that they did not have a C-methyl group and had two N-methyl groups, and were confirmed to be new antibiotics.
However, istamycin A is
It is recognized that it is the same substance as the compound KA-7038I described in Publication No. 141701 (published on November 5, 1978, after the filing of this application). Furthermore, istamycin A and istamycin B were determined as follows as a result of structural studies. Therefore, according to the first aspect of the present invention, istamycin B or an acid addition salt thereof is provided as a new substance. According to a second aspect of the present invention, there is provided a method for producing istamycin B, which comprises culturing istamycin B-producing bacteria belonging to the genus Streptomyces aerobically and collecting istamycin B from the culture solution. provided. As an example of the istamycin B-producing bacteria (hereinafter simply referred to as istamycin-producing bacteria) used in the second method of the present invention, the present inventors discovered that Strain SS-939 is an actinomycete isolated from seabed soil. The mycological properties of this strain are as shown below. (a) Morphology Strain SS-939, which grows well on agar medium, simply branches and extends straight or slightly curved aerial hyphae from well-elongated basal hyphae, and when mature, there are 10 to 50 hyphae at the tip. Forms a chain of long cylindrical spores (1 micron wide and 4-5 microns long). No spirals or axle branches are shown. Under electron microscopy, the spore surface is smooth, with no spine-like or hair-like structures visible. It is a typical Streptomyces without flagella or spore cells. (b) Characteristics on various media Seuucrose/nitrate agar medium (cultured at 27°C): White aerial mycelia appear on weak colorless growth, gradually turning blue-greenish gray (17ec aqua
blue, according to the Color Harmony Manual; the same applies hereafter). It does not produce significant diffusible pigments in the medium. Glycerin-asparagine agar medium (27℃
Culture): Almost the same, but aerial mycelia are difficult to attach to. Starch agar medium (cultured at 27°C): Almost the same, but aerial mycelium grows on it, and the starch around the bacterial growth becomes transparent. Tyrosine agar medium (cultured at 37°C): The findings are almost the same as those of , and a melanin-like pigment is produced. Nutrient agar medium (cultured at 27°C): White aerial mycelium grows on colorless growth, and the medium becomes brownish-brown. Yeast/malt agar medium (cultured at 27°C): White aerial mycelia grow on colorless growth, gradually turning blue-greenish gray (19dc aqua green). The medium appears brownish in color. Oatmeal agar medium (cultured at 27°C): White to blue-gray (17ec aqua) on colorless growth.
Aerial mycelia with a blue color are attached. (c) Physiological properties Can grow at 20-41℃. Hydrolyze starch on starch agar medium. Hardly any coagulation or peptonization of skim milk is observed. Melanin-like pigments are produced on tyrosine agar and peptone yeast iron agar. (d) Assimilation of carbon sources (on Pridham-Gottlieb agar medium) Only glucose and inositol are assimilated, and arabinose, D-xylose, sucrose,
Rhamnose, raffinose, and D-mannite are not assimilated. Assimilation of D-fructose is complicated. The above characteristics indicate that it belongs to typical Streptomyces, the aerial mycelium does not form spiral threads, and it has melanin-like pigment production (chromogenicity). When searching for similar bacterial species based on the color tone and chromogenicity of aerial hyphae, we found Streptomyces viridochromogenes, but strain SS-939 does not produce spiral threads.
They are clearly distinguished by the fact that they do not utilize xylose, arabinose, rhamnose, fructose, raffinose, or mannite, and they are definitely different in that their spores do not have a spine-like surface. Non-chromogenic,
Streptomyces viridifaciens, which has a similar color tone to aerial mycelia.
It is distinguished from SS-939 by the fact that its aerial mycelium does not form a spiral, and that it does not assimilate fructose and sucrose as carbon sources. others,
There is no Streptomyces species that exhibits the characteristic carbon source assimilation properties of strain SS-939. Therefore, the bacterial species including strain SS-939 are new species, and Streptomyces
Streptomyces
tenjimariensis). This Streptomyces tenzimariensis
Strain SS-939 has been submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology for entrustment of storage, and is designated as Microbiological Research Institute No. 4932.
Streptomyces tenzimariensis is prone to mutations both artificially and naturally, and the term Streptomyces tenzimariensis referred to in the present invention includes all such mutants. In the second method of the present invention, istamycin B-producing bacteria, particularly the SS-939 strain mentioned above, are inoculated into a medium containing nutrients that can be used by ordinary microorganisms, and grown aerobically. A culture solution containing istamycin B is then obtained. However, when culturing the SS-939 strain, istamycin A is also produced and accumulated in the culture solution. As the nutrient source, any known nutrient source used as a nutrient source for actinomycetes can be used. For example, commercially available soybean flour, peanut flour, cotton powder, dried yeast,
Nitrogen sources such as peptone, meat extract, casein, corn steep, liquor, sodium nitrate, ammonium sulfate, and commercially available glucose, starch,
glycerin, maltose, dextrin, sucrose,
Carbohydrates such as lactose, or nutritional sources such as soybean oil and fat, as well as inorganic salts such as salt, calcium carbonate, magnesium sulfate, manganese chloride, and phosphates, and various amino acids can be used as necessary. Any of these materials may be used as long as they are utilized by istamycin B-producing bacteria and useful for the production of istamycin B, and all known culture materials for actinomycetes can be used. For mass production, a liquid aeration agitation culture is preferred, and the culture temperature may be within a range that allows istamycin B-producing bacteria to grow and produce istamycin B, but a temperature of 25 to 30°C is particularly preferred. Cultivation is usually continued until sufficient istamycin B has accumulated. Culture is usually carried out for 2 to 7 days. In addition, Streptomyces sp. No. KC-7038 strain (Feikoken Bacterial Serial No. 4386) described in the above-mentioned Japanese Patent Application Laid-Open No. 54-141701 contains a compound KA- which is recognized as the same substance as istamycin A. A comparison between the Streptomyces tenzimariensis SS-939 strain isolated by the present inventor and the above-mentioned KC-7038 strain, which produces the 7038I substance, is summarized in Table 2 below.

[Table] Both of the above strains belong to the genus Streptomyces, but their morphology includes the color tone of aerial hyphae, the presence or absence of spiral threads,
The spore surface properties are clearly different, and the physiological properties are also clearly different in the production of melanin-like pigments and the content of assimilable carbon dioxide sources. Note that the KC-7038 strain also differs in that it does not produce istamycin B. Therefore, the method for producing istamycin A using Streptomyces tenzimariensis is as follows:
Streptomyces sp. No. KC-7038 strain disclosed in Japanese Patent Application Laid-open No. 141701/1988 (Feikoken Bacteria No. 4388)
This method can be distinguished from the method for producing compound KC-7038I using the following method in that the bacteria used are different. Quantification of istamycin A or istamycin B was performed using Bacillus subtilis as the test bacterium.
Using the PCI219 strain, the test was carried out by a conventional cylindrical plate method used for quantifying antibiotics, and the purified istamycin A obtained in the present invention was used as a standard substance at 1000 mcg (unit)/mg. In addition, purified istamycin B showed a titer of 3170 mcg (unit)/mg. Istamycin A and Istamycin B and their salts are highly soluble in water;
In the culture solution of the istamycin B-producing bacteria, it is mainly present in the liquid part. Since istamycin A and istamycin B in liquids are substantially not extractable by organic solvents such as butanol, butyl acetate, chloroform, etc., treatment with these solvents can be used if necessary to remove contaminants. Istamycin A in culture medium or aqueous solution
Or and istamycin B can be harvested using a variety of adsorbents. When activated carbon is used as an adsorbent, the adsorbed antibiotic can be mixed with weakly acidic water, weakly acidic methanol water, propanol water,
It is eluted with acetone water, etc. Furthermore, istamycin A or istamycin B is adsorbed and eluted onto the cation exchange resin in good yield based on its basic properties. This method is the most suitable method for collecting from a large amount of culture fluid. As a cation exchanger, Amberlite IRC-50 and CG-50 have carboxylic acid as an active group.
(Manufactured by Rohm and Haas)
CNP (manufactured by Bayer), CM-Sephadex (manufactured by Pharmacia), H type, Na type, _NH4 type, etc., and their mixed types are used, and the adsorbed antibiotics are acidic water, dilute ammonia water and inorganic salts. It is eluted with good yield by an aqueous solution of 0.2
−1N hydrochloric acid and 0.2N−1N aqueous ammonia are used. Since istamycin A or istamycin B is not substantially adsorbed on anion exchange resins, it is a preferred material for neutralizing acidic solutions and removing acidic contaminants. Istamycin A and Istamycin B are
As mentioned above, it is separated by thin layer chromatography on cellulose (Avicel) using butanol, pyridine, acetic acid, and water (6:4:2:4 volume) as the developing system (A: Rf0.22, B: Rf0 .25) Based on their properties, they can be effectively separated and purified by cellulose column chromatography using the same or similar solvent system as thin layer chromatography. Istamycin A or istamycin B can be purified in a pure manner by appropriately combining or repeating the above-mentioned extraction methods and separation methods. Istamycin A and B are usually obtained as free bases or hydrates or carbonates, but
Any non-toxic acid salt can be prepared by adding a pharmaceutically acceptable acid by conventional methods. In this case, examples of pharmaceutically acceptable acids include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and nitric acid, and organic acids such as acetic acid, malic acid, citric acid, ascorbic acid, and methanesulfonic acid. It will be done. Streptomyces tenzimariensis SS-
Strain No. 939 is a useful new type of microorganism that produces the new antibiotics istamycin A and B.
Medium (maltose 1%, yeast extract 0.4%,
An actinomycete colony grown by culturing at 27℃ for 3 days on agar (1.5%, PH7.2) was harvested, and then added to a carbohydrate assimilation test medium (Pridham-Gottlieb medium) containing glycose, xylose, arabinose, rhamnose, raffinose, 1% of each inositol,
They were screened and isolated for actinomycetes that assimilate inositol and grow in a medium containing inositol. The present invention will be described below with reference to Examples, but since the properties of istamycin B have been clarified by the present invention, various methods for producing istamycin B can be devised based on those properties. . Therefore, the present invention is not limited to the examples, and can be modified by known means based on the properties of istamycin A and istamycin B revealed by the present invention. It covers all methods for producing, concentrating, extracting, and purifying istamycin B. Example 1 Streptomyces tenzimariensis strain SS-939 (Feikoken Bacterium Co., Ltd.) cultured on agar slant medium
No. 4932) in a liquid medium (110 ml in a 500 ml Erlenmeyer flask) containing 1.0% starch, 0.2% glucose, 1.0% soybean flour, 0.3% salt, 0.1% dipotassium phosphate, and 0.1% magnesium sulfate (7H ₂ O). Inoculated and incubated at 27°C.
A seed culture was obtained by rotary shaking culture for 48 hours. 220 ml of this seed culture was added to _a liquid medium (15
) was inoculated into a 30-volume jar fermenter and cultured at 27°C for 72 hours with aeration (15 aeration per minute,
300 revolutions per minute). This culture solution (for 6 jar fermenters) was collected, adjusted to pH 2.0 with hydrochloric acid, and filtered to obtain a solution of 90 (2.5 mcg/ml). This liquid was passed through a tower filled with Amberlite IRC-50 (NH ₄ type) 12 and adsorbed, washed with water (24), and eluted with 1N aqueous ammonia. The active eluent (3) was collected and concentrated to dryness under reduced pressure to obtain 3.51 g (61 mcg/mg) of a crude powder. This crude powder was dissolved in 100 ml of water and placed in a column filled with 300 ml of Dowex 1-X4 (OH type) ( inner diameter 40mm)
Sprinkle, then develop with water and add 200ml of the initial effluent.
ml was discarded, the next 880 ml was collected, and this was then adsorbed in a column (inner diameter 25 mm) filled with 150 ml of Amberlite CG-50 (NH ₄ type), and washed with water (300 ml).
Afterwards, the elution was carried out with 900 ml of 0.2N ammonia water, then 900 ml of 0.4N ammonia water, and fractionated into 18 ml portions. fraction
66 to 80 were combined and concentrated to dryness under reduced pressure to obtain 210 mg of white powder containing istamycin A and B (potency:
460 mcg/mg). (In addition, several antibiotics other than istamycin A or istamycin B were eluted in fractions 28 to 65.) Example 2 A culture solution (distillate) obtained in the same manner as described in Example 1 was used. 10 units of Yahoo Armenter (150) was passed through a column filled with Amberlite IRC-50 (NH ₄ type) 20 and adsorbed, washed with water (40), eluted with 1N ammonia water, and the active eluate was dried under reduced pressure. 4.1 g of crude powder (potency 240 mcg/mg) was obtained. This coarse powder was dissolved in 20 ml of water, poured into a column (inner diameter 30 mm) filled with 200 ml of Dowex 1-X4 (OH type), and then dissolved in water. Develop and drain 270ml of initial liquid.
Discard the liquid and collect the next 270 ml. Next, pour this into a column (inner diameter 25 mm) filled with 150 ml of Amberlite CG-50 (NH ₄ type) to adsorb it, and wash with water (150 ml).
Afterwards, the elution was carried out with 900 ml of 0.2N ammonia water, then 900 ml of 0.4N ammonia water, and fractionated into 18 ml portions. fraction
55 to 90 were combined and concentrated to dryness under reduced pressure to obtain 980 mg of white powder containing istamycin A and istamycin B (potency: 940 mcg/mg). Example 3 White powder containing istamycin A and istamycin B obtained in Example 2 (titer
940mcg/mg) 980mg of butanol, pyridine,
It was dissolved in 15 ml of a mixture of acetic acid and water (6:4:2:2 volume) and poured into a column (inner diameter 25 mm) packed with 60 g of cellulose powder (Avicel).
Mixture of pyridine, acetic acid, and water (6:4:2:2 volume)
The mixture was developed with 1200 ml and then 600 ml of a 6:4:2:4 mixture, and fractionated into 10 ml portions. Istamycin B is present in fractions 45-74, and istamycin A is present in fractions 114-154.
However, a mixture of both was eluted in fractions 75 to 113. Fractions 45 to 74 were combined, concentrated to dryness under reduced pressure, dissolved in 5 ml of water, and mixed with Amberlite CG-50 (NH ₄ type).
After passing through a 25 ml tower and adsorbing it, and washing with water (25 ml),
Elute with 0.4N aqueous ammonia, active eluent (40ml)
was concentrated to dryness under reduced pressure to obtain 15.3 mg of purified powder of istamycin B (potency 3170 mcg/mg. Similarly, fractions 114 to 154 were combined, concentrated to dryness under reduced pressure, and dissolved in 5 ml of water. , Amber Light CG−
50 (NH ₄ form) was adsorbed through a 25 ml column, washed with water (25 ml), eluted with 0.4 N ammonia water, and the active eluate (45 ml) was concentrated to dryness under reduced pressure to obtain 50 mg of purified istamycin A powder (30 ml). A titer of 1000mcg/mg) was obtained.

[Brief explanation of drawings]

FIG. 1 shows an infrared absorption curve of istamycin A carbonate measured in a potassium bromide tablet, and FIG. 2 shows an infrared absorption curve of istamycin B carbonate measured in a potassium bromide tablet.

Claims (1)

[Claims] It has a molecular formula of 1 C ₁₇ H ₃₅ N ₅ O ₅ , has two N-methyl groups and one O-methyl group, has ultraviolet absorption only at the terminal absorption, and has a molecular formula of 1 C 17 H 35 N 5 O 5. and methanol, but poorly soluble or insoluble in common organic solvents, positive for ninhydrin reaction and Lydon-Smith reaction, and thin layer chromatography of cellulose (developing system: butanol/pyridine/acetic acid/water, 6:4:
2:4 volume) shows Rf0.25, and its 1/2 carbonate is 112
A new antibiotic, istamycin B, or its acid addition salt, which is a white powder that decomposes at ~114°C and exhibits [α] ²⁵ _D +165° (c0.4, water). 2. A method for producing istamycin B, which comprises culturing istamycin B-producing bacteria belonging to the genus Streptomyces aerobically and collecting istamycin B from the culture solution.