JPH0138799B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel antibiotic complex and its preparation, recovery and separation into two biologically active components. Various aminoglycoside antibiotics, such as kanamycin, dientamicin, streptomycin,
Neomycin, tobramycin, amikacin and paromomycin are well known in the art. However, there is a need for a broader range of antibiotics, especially those with activity against organisms resistant to aminoglycosides. Based on the currently used structural formula, the component Bu-2349A of the present invention has the structural formula published in U.S. Pat. No. 4,007,167: Somewhat related to the antibiotics BM-123γ ₁ and BM-123γ ₂ with The present invention provides Bu-2349, a novel water-soluble basic glycopeptide antibiotic conjugate. The above compound is a Bu-2349 producing species of the genus Bacillus,
Especially Bacillus ATCC31429 or Bacillus
ATCC31430 species or mutants thereof are cultivated under aerobic immersion conditions in an aqueous nutrient medium, thereby producing a substantial amount of Bu-2349 complex and optionally recovering the Bu-2349 complex from the culture medium. Manufactured by The present invention also provides a method for producing the bioactive antibiotic components of the Bu-2349 complex, Bu-2349A and B, as separate substances. This method involves adsorbing the Bu-2349 complex onto a cationic ion exchange resin and removing the Bu-2349 from the adsorbent.
2349A and B are differentially eluted to recover the desired separate components. Within its scope, the present invention includes diluted, crudely concentrated, and purified Bu-2349 complexes and the bioactive component Bu-2349.
Includes 2349A and B. The new antibiotics are provided as free bases or as acid addition salts with pharmaceutically acceptable organic or inorganic acids. Figure 1 shows the infrared absorption spectrum of Bu-2349A hydrochloride when pelletized in potassium bromide. Figure 2 shows the infrared absorption spectrum of Bu-2349B hydrochloride when pelletized in potassium bromide. Attached Figure 3 shows the diol using TMS as an external standard.
60MHz NMR spectrometer (TNM-C-
Bu− dissolved in D ₂ O when measured with 60HL type)
Shows the proton magnetic resonance spectrum of 2349A hydrochloride. Attached Figure 4 shows the diol using TMS as an external standard.
Figure 3 shows the proton magnetic resonance spectrum of Bu-2349B hydrochloride dissolved in _D2O as measured with a 60MHz NMR spectrometer (model TNM-C-60HL). The following Table A shows the structural properties of Bu-2349A.

TABLE The present invention relates herein to a novel aminoglycoside antibiotic conjugate designated Bu-2349. This complex is produced by fermentation of microorganisms belonging to the genus Bacillus. Any species belonging to the genus Bacillus and capable of producing Bu-2349 in the culture medium can be used. Preferred Bu-2349-producing microorganisms are Bacillus species F-173-B61 and Bacillus species F262-B54 in the Culture Collection of Bristol-Myers Laboratories. The above species were deposited at the American Type Culture Collection (ATCC), Rockville, Maryland, USA, and were collected from soil samples in West Germany and India, respectively. (ATCC31429 and Deposit 31430 are Fiber Science and Technology Research Institute Nos. 5197 and 5198, respectively) The novel aminoglycoside complex of the present invention
It consists of two bioactive aminoglycoside components called Bu-2349A and B. This complex and the components listed above exhibit a broad spectrum of antimicrobial activity and are therefore valuable as antimicrobial agents, as nutritional supplements in animal food, and as therapeutic agents for animals. In particular, this new antibiotic is useful in the treatment of infectious diseases in mammals (including humans) caused by Gram-positive and Gram-negative bacteria, such as infectious diseases caused by bacteria resistant to aminoglycosides. The antibiotic is also useful in cleaning and sterilizing laboratory glassware and surgical tools, and can be used with soaps, cleaning agents, and cleaning solutions for hygiene purposes. Microorganisms producing Bu-2349
The preferred microorganism producing Bacillus-2349 is an aerobic, Gram-positive, spore-producing rod-shaped bacterium and is therefore classified as belonging to the genus Bacillus. For the classification of the above microorganisms, Mr. Bergey's method,
The method described in 8th edition, 529-551 (1974) was used.
As seen in Table 1, F173−B61 and F262−
The morphological characteristics of B54 species are similar to those of Bacillus cereus or Bacillus megaterium. These four microorganisms have the following characteristics in common: (1) Positive Gram species: (2) Growing cells larger than those of Bacillus subtilis or related species: (3) Spores that are not swollen at endospore locations. capsule: and (4) the presence of intracellular globules that do not stain with fuchsin. The morphological characteristics of B. serlingiensis and B. anthracis (Table 1) are very similar to those of B. cereus megaterium. B. cerlingiensis is infective to Lepidoptera larvae and also produces intracellular protein crystals. B. anthracis is the microorganism that causes anthrax in humans and animals and requires thiamine and many amino acids for growth;
It also produces non-motile cells. These properties distinguish species F173-B61 and F262-B54 from B. serlingiensis or B. anthracis.

[Table] Intracellular protein crystals None None
None None

As can be seen in Table 2, the F173-B61 species differs from the F262-B54 species in growth under anaerobic conditions, positive yolk reaction, nitrate reduction reaction, and Boges-Proskauer (VP) reaction. Therefore
Species F173−B61 can be classified as Bacillus cereus, while species F262−B54 were classified by BC Knight et al. in J. Gen.
Microbial, 4:580-538, (1950).

[Table] Ri Ri

[Table] Amino acid requirements
Regarding the production of Bu-2349, especially Bacillus
Although the F173-B61 and F262-B54 strains have been described in detail, the invention is not limited to these microorganisms or to the microorganisms whose culture characteristics are described in detail herein. The present invention also describes other methods that can be produced by methods known in the art, such as X-ray or ultraviolet irradiation of the microorganisms, nitrogen mustard, phage exposure, etc.
Species that produce Bu-2349 or mutants thereof are also considered to be included. Production of antibiotics Bu-2349 antibiotics are produced by Bacillus Bu-2349-producing species under aerobic immersion conditions in an aqueous nutrient medium.
Particularly preferably Bacillus ATCC31429 or
It is produced by conventional fermentation methods by culturing ATCC31430 species or their mutants. The microorganism grows in a nutrient medium containing assimilable carbon sources, such as assimilable carbohydrates. Examples of preferred carbon sources include glucose, fructose, mannose, glycerol, and the like. The nutrient medium should also contain an assimilable nitrogen source, such as fishmeal, soybean meal, peptones. Nutrient mineral salts can also be conveniently mixed into the culture medium. These salts may include commonly used salts capable of providing sodium, potassium, ammonium, calcium phosphate, sulfate, chloride, bromide, nitrate, carbonate ions, and the like. The Bu-2349 composite can be produced at a temperature that allows this microorganism to grow sufficiently, for example 20 to 45°C, but approximately
Most preferably, it is carried out at a temperature of 28-30°C.
Optimum production typically takes about 4-6 days. The initial pH of the medium is preferably neutral or close to neutral. While shake flask and surface culture can be used for relatively small production, aerobic immersion culture in sterile tanks is preferred for large scale production. When performing tank fermentation, the soup culture is inoculated with spores from the microorganisms, and once a young, actively growing inoculum is obtained, the inoculum is transferred under aseptic conditions to the fermentation tank medium to produce an inoculum that grows into a nutrient soup. It is desirable to do so. Aeration in tanks and bottles can be by directing sterile air through or over the fermentation medium surface. Furthermore, stirring in the tank can be done by mechanical blades. If necessary, an antifoaming agent such as lard oil can be added. The production of Bu-2349 in the fermentation medium can be easily checked by the paper disc-agar diffusion method using Bacillus subtilis PCI 219 as the test organism during the fermentation process. Isolation and Purification of Bu-2349 Once the optimum potency of the soup is achieved (as determined by the test method described above), the soup is made neutral or slightly acidic (PH~6.5) and the bacterial system and undissolved residues are removed by filtration or centrifugation. Separate from the soup in the usual way, such as by separating. The antibiotic activity is in the liquid and can be recovered from it by conventional adsorption methods used for water-soluble basic antibiotics. Adsorbents that can be conveniently used are cation exchange resins, such as weakly acidic cation exchange resins of the carboxylic acid type (eg, the type sold under the trade name "Amberlite IRC-50"). The liquid is made of resin, such as ammonium type Amberlite.
Water after passing through a column packed with IRC-50, 0.1N
Develop the column with NH ₄ OH and 1N NH ₄ OH sequentially. The antibacterial fraction eluted with 1N NH ₄ OH is collected and concentrated in vacuo to obtain the Bu-2349 complex. Separation of components Bu-2349A and B The Bu-2349 complex is prepared using a cation exchange resin such as ammonium type chromatographic grade resin "Amberlite CG-50" (manufactured by Rohm and Haas, trade name). Can be separated into 2349A and B. The complex is dissolved in water and treated with the resin and then gradually eluted with 0.2N, 0.5N and 1N NH ₄ OH. Bu-2349B is eluted first with 0.5N NH ₄ OH, after which Bu-2349 exits the column with 1N NH ₄ OH. If two preferred Bacillus spp.
Most of it is Bu-2349A and a small amount of Bu-2349B. The separated antibiotic component is further treated with a cation exchange resin (e.g. ammonium type Amberlite-CG-
50) or can be purified by chromatography using a gelatinizer. A preferred gelling agent is "Sephadex LH-20" manufactured by Pharmacia Fine Chemicals AB, Uppsala, Sweden.
It is a cross-linked dextran gel. Properties of Bu-2349 Antibiotic Component Bu-2349A and B hydrochloride salts are both white amorphous solids. Both are soluble in water, slightly soluble in methanol, ethanol, dimethyl sulfoxide and dimethyl formamide, but virtually insoluble in other common organic solvents. They react positively with ninhydrin, Anthrone, Moritshu, Fehring, and Remini reagents, but negatively with FeCl ₃ and Sakaguchi's reagents. Bu-2349A hydrochloride does not have a sharp melting point, but decomposes above 215°C. Similarly Bu−2349B
Although it does not have a high melting point, it decomposes above 224℃. Bu-2349A and B hydrochlorides have the following elemental composition (%): Bu-2349A hydrochloride: C, 44.48; H, 7.34; N,
8.37; Cl, 6.44; O, (from difference) 33.37, Bu-2349B hydrochloride: C, 43.55; H, 6.92; N,
8.06; Cl, 6.64; O, (from difference) 34.83 The molecular weight of the N-pentaacetyl derivative of Bu-2349A was determined to be 1100 by vapor pressure permeation method.
Based on the molecular weight and elemental analysis _, the molecular formula _of Bu-2349A free base appears to be _C44 - _{H85-87N7O24-25} . The specific rotations of hydrochlorides are as follows: [α] ²² _D = +109° (C1.0, H 2 O) for Bu-2349A [α] ²² _D = +115° (C1.0, H ₂ O) for Bu-2349B _H2O ) The hydrochloride salts of Bu-2349A and B can be distinguished from each other by their respective Rf values in silica gel thin layer chromatography using the following solvent system: Solvent system Bu-2349A Bu-2349B Chloroform: Methanol: 28 % ammonium hydroxide:water (1:4:2:1V/V)
Rf 0.27 0.50 Methanol: 10% ammonium acetate (1:1V/
V) 0.14 0.31 Methyl acetate: n-propanol: 28% NH ₄ OH
(45:105:60V/V) 0.06 0.16 The UV absorption spectrum of Bu-2349A in water shows a single absorption maximum at 247 nm (E1% 1 cm131), which does not migrate even in acidic or alkaline media. Bu-2349B also exhibits an absorption maximum at 247 nm (E1% 1 cm148) and does not migrate depending on pH. The infrared absorption spectra (measured in KBr) of the hydrochloride salts of Bu-2349A and B are shown in Figures 1 and 2, respectively. At 1630 cm ^-1 amide carbonyl is also
Polyhydroxy groups are shown near 3400 cm ^-1 and 1040 cm ^-1 . The NMR spectra of the hydrochloride salts of Bu-2349A and B are shown in Figures 3 and 4, respectively. Bu-2349A and B are basic substances that can be formed from acids and salts, and pharmaceutically acceptable acid addition salts of these antibiotics are included within the scope of the present invention. "Pharmaceutically acceptable" salts are salts in which the toxicity of the compound as a whole is not increased in warm-blooded animals compared to the non-salt form. Examples of suitable pharmaceutically acceptable acid addition salts include organic and inorganic acids such as hydrochloric acid, sulfuric acid,
Phosphoric acid, acetic acid, succinic acid, citric acid, lactic acid, maleic acid, fumaric acid, palmitic acid, formic acid, stearic acid, propionic acid, tartaric acid, benzoic acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid, cinnamic acid, etc. These are salts produced by the standard reaction of An example of salt formation is dissolution of the free base compound in water, treatment with the desired acid, and subsequent lyophilization in vacuum. For purposes of this invention, the free base form of an antibiotic is equivalent to a pharmaceutically acceptable acid addition salt. Structural characteristics of Bu-2349 component The ^13C nuclear magnetic resonance spectrum of Bu-2349A is 2
It showed the presence of 44 carbons, including methyl and 2 carbonyl carbons. Both methyl groups appeared as doublets (δ1.27 and 1.49 ppm) in the proton NMR spectrum (Figure 3). NMR also shows 4 aromatic protons (AB quadruplet, δ7.10 and 7.70ppm) and δ4.7~
Five anomeric or double-bonded protons were shown around 5.9 ppm. When Bu−2349A is heated to reflux with 0.5N methanolic hydrogen chloride, it is divided into three parts, and _A
It was broken down into The moiety was identified as methyl D-riboside. alkaline hydrolysis (0.1N Ba
(OH) ₂ , 100° C., 18 hours), a disactucharide () having L-alanine and two primary amino groups was obtained. Hydrolysis of the N-diacetyl derivative of the compound with methanolic HCl yielded methyl galactoside and an unidentified basic sugar moiety, the latter of which was identified by mass spectrometry and NMR spectra as 2,4-diamino-2,4,6- It was assumed to be trideoxyhexose. From the NMR spectrum of the compound, it was thought that the alanine carbonyl group must be bonded to the C-2 amino group of the diamino sugar moiety of the compound. The basic _A moiety was isolated as the crystalline hydrochloride salt. Melting point 252℃.
Analysis calculation value for C ₁₈ H ₃₂ N ₄ O ₂・3HCl: C,
48.49; H, 7.91; N, 12.57; Cl, 23.86. Measured value: C, 48.48; H, 7.96; N, 12.46; Cl,
23.97. Mass: m/e336 (M ⁺ ), 278, 247, 207,
186, 121, etc. λ ^H2O _nax 252nm (ε13960), λ ^{0.1N HCl} _n
ax
252nm (ε13960) and λ ^{0.1N NaOH} _nax 289nm
(ε20380) When _A is refluxed with 6N HCl, p-hydroxybenzoic acid and a new amino compound,
Decomposed into C ₁₁ H ₂₈ N ₄ . The latter structure is N-(δ-aminobutyl)-N′-(γ-aminopropyl)-1,
It was determined to be 4-diaminobutane (referred to as isohomospermine). Mass and NMR analysis of _A showed that the terminal amino function of the aminopropyl moiety of the isohomospermine moiety was acylated with p-hydroxybenzoic acid. Therefore, the structure of _A is It is. From the above data, a partial structure of Bu-2349A can be expressed as follows. : Acid hydrolysis of Bu-2349B yielded the moieties and _B. The and parts were the same as those isolated from Bu-2349A. Part _B has the same physicochemical properties as _A and has a molecular formula of C ₁₄ H ₂₃ N ₃ O ₂
It was decided that. Acid hydrolysis of _B with 6N HCl decomposed it into p-hydroxybenzoic acid and spermidine (C ₇ H ₁₉ N ₃ ). Therefore, part _B has the structure: have. As mentioned above, ^{the 13} CNMR spectrum of Bu-2349A showed the presence of carbon 44 in the antibiotic molecule.
The three moieties isolated from acid hydrolysis of Bu-2349A (and _A ) demonstrated carbon 38. Therefore, Bu-2349A must contain another component with carbon 6, which is ¹³ C
It was inferred to be a sugar or related substance by proton NMR analysis. The structural features of Bu-2349A are shown in Figure A. From the above considerations, Bu−2349A and Bu−
2349B was found to have the following structure. However, R is (Bu−2349A) or (Bu-2349B). Biological properties Minimum inhibitory concentration (MIC) of Bu-2349A and B
Measurements were made on various bacteria by the double agar dilution method. Gonocottoccus agar medium (GC agar, Tokyo Nitsusui Co., Ltd.) was used for MIC measurements of Streptococcus, Neisseria, and Haemophilus species, whereas Mueller-Hinton agar medium was generally used for MIC measurements of other bacteria. Giff anaerobic agar medium (GAM agar, Aiken, Tokyo) was used for anaerobic bacteria. 18 for Streptococcus, Neisseria and Haemophilus
MICs were determined after overnight incubation at 37°C using an initial inoculum corresponding to a 10 ^-2 dilution of the time culture.
For aerobic and anaerobic bacteria other than those listed above,
The same procedure was carried out using a 10 ^-4 dilution. As shown in Table 3, Bu-2349A and B have many different types of aminoglycoside-denaturing enzymes, including those that produce various types of aminoglycoside-denaturing enzymes.
Active against positive and gram-negative bacteria.
Bu-2349A was inactive against most of the anaerobic organisms tested. (Table 4)

【table】

[Table] * Produced aminoglycoside denaturing enzymes ☆ Lot 113-1-62
Lot 113-1-2242

Table: The effect of inoculum on MIC was tested on Mueller-Hinton agar using inocula of 10 ⁰ , 10 ^-2 or 10 ^-4 dilutions of overnight cultures of test organisms. Table 5
As can be seen, the change in inoculum size is Bu−
It did not significantly affect the antibiotic activity of 2349A.

[Table] PH of medium using Mueller-Hinton cold
The effect of Bu-2349A on MIC was determined by 3PH concentration (6,
7 and 8) were tested. As shown in Table 6, the activity of Bu-2349A at PH6 was much smaller than the activity obtained at PH7. At PH8, PH
The activity was approximately twice that of No. 7.

【table】

[Table] Four test media: Mueller-Hinton Agar (MHA): Nutrient Agar (NA), Heart Infusion Agar (HIA) and Brain Heart
Bu− using infusion agar (BHIA)
The influence of the medium on the MIC of 2349A was examined. The pH was adjusted to 7 and a 10 ⁻⁴ dilution of an overnight culture was used for the inoculum. As shown in Table 7, Bu-2349A is MHA
or showed higher activity in NA and HIA than in BHIA.

【table】

[Table] The in vivo antibacterial activity of Bu-2349A was tested by experimentally infectious disease in mice. 5% of pig stomach mucus
A suspension (American Laboratories, Omaha, Nebraska) of ₁₀₀ x LD of medium pathogen was administered intraperitoneally to rats. Bu immediately after bacterial administration
-2349A was administered intramuscularly. Animals were observed for 5 days to determine the median protective dose (PD ₅₀ ) using groups of 5 mice for each dose. As shown in Table 8, S. aureus, E. coli, K. pneumoniae, P.
Bu-2349A showed good protection against systemic fatal infections caused by P. vulgaris and P. mirabilis.
However, in an in vivo test, it was inactive against S. pneumoniae infection.

[Table] The blood concentration of rats was examined after intramuscular administration of Bu-2349A at a drug dose of 50 mg/Kg. A blood sample was taken from the orbital sinus and the test organism B.
Tests were conducted using paper Jiscou agar plate method using PCI219. The results are shown in Table 9. Maximum blood concentrations of Bu-2349A were obtained after 15 minutes, after which it rapidly disappeared from the blood stream. No antibiotic activity was detected after 2 hours. Table 9 Concentration of Bu-2349A in rat blood (im, 50
mg/Kg) Time after administration Blood concentration 15 minutes 15 mcg/ml 30 minutes 12 60 minutes 2.8 120 minutes < 1.0 The apparent intramuscular and intravenous LD ₅₀ of Bu-2349A in mice was 315 mg/ml, respectively. Kg and 35mg/Kg. Antibiotic Usage As seen from the biological data above, Bu-
The 2349 complex and its active components Bu-2349A and B have significant inhibitory activity against Gram-positive and Gram-negative bacteria (including bacteria resistant to aminoglycosides) both in vitro and in vivo. It is useful as an antibacterial agent in human and household medicine. According to one form of the invention, Bu-2349A or Bu-2349B or a mixture thereof or a pharmaceutically acceptable acid thereof is administered to an animal (including in particular humans and other mammals) suffering from a bacterial infectious disease. A method of treating an animal patient as described above is provided which comprises administering an antimicrobially effective amount of an addition salt. Another aspect of the invention provides a pharmaceutical composition comprising a pharmaceutical carrier or diluent together with a therapeutically effective antimicrobial amount of Bu-2349A or B or a mixture thereof or a pharmaceutically acceptable acid addition salt thereof. Ru.
Preferably, the composition is administered by injection, although other methods of administration can be used if desired. Antibiotic dosages vary depending on the method of administration and the specific substance chosen. Furthermore, it will vary depending on the particular location, patient and disease being treated. Many factors that modify drug action must be considered by the physician or veterinarian, such as age, weight, sex, food, time of administration, method of administration, rate of excretion, patient condition, drug combination, response sensitivity, and severity of the disease. will be done. At the beginning of treatment, a parenteral dosage of about 2 to 15 mg per kg of body weight divided into several doses (eg, 2 to 3 doses) per day is generally convenient. The optimum dosage for a particular situation will be readily determined by one skilled in the art. 0.1 to 10 of this substance for cleaning and disinfection purposes.
% aqueous solution can be used. The following examples are for illustrative purposes and are not intended to limit the scope of the invention. "Amberlite IRC-50" and "CG-50" in the examples are the trade names of carboxylic acid-type weakly acidic cation exchange resins. "Cephadex LH-20" is the trade name of a modified alkylated dextran gel filtration agent. Example 1 Fermentation of a complex A well-grown culture of Bacillus sp., strain F262-B54 was inoculated into a growth nutrient medium containing 1% glucose, 0.5% yeast extract and 1% polypeptone with a pre-sterilization pH of 7.2. A slant was used.The seed culture was incubated at 28℃ for 24 hours on a rotary shaker (250 rpm), and 5 ml of the culture solution was mixed with 3% glycerol, 0.5% soybean flour,
Fishmeal 1%, (NH ₄ ) ₂ SO ₄ 0.1%, NaCl 0.3%,
Fermentation medium containing 0.6% CaCO ₃ with a pH of 7.2 before sterilization
Transferred to a 500 ml Erlenyer flask containing 100 ml. Fermentation was carried out for 5-6 days at 28°C on a rotary shaker. Bacillus subtilis as the test organism
The activity of the antibiotic complex in the fermented soup was investigated by paper Giscou agar diffusion test using PCI219. Antibiotic production is up to 100% after 4-6 days
~150mcg/ml was reached. Example 2 Separation and purification Fermented soup (45 L.) was adjusted to pH 6.5 with oxalic acid.
The mixture was stirred for a minute and filtered with a supernatant. After passing the liquid through a column of "Amberlite IRC-50" (NH ⁺ ₄ type, 4.5L), the column was filled with water 45, 0.1N.
Developed sequentially with NH ₄ OH and 1N NH ₄ OH solutions.
The antibacterial portions eluted with 1N NH ₄ OH were combined and concentrated in vacuo to yield 6.0 g of a mixture of Bu-2349A and B. Example 3 Separation of components To separate and purify the A and B components of Bu-2349, 5 g of the crude composite was mixed with “Amberlite CG-50” (NH ⁺ ₄ type,
1) Apply 0.2N, 0.5N and 1N to the column in sequence.
Eluted with _NH4OH . First, Bu− with 0.5N NH ₄ OH
After eluting 150mg of 2349B, the main component Bu-2349A was 1N
Elution with NH ₄ OH gave 900 mg. The Bu-
Purify 2349A to obtain 800 mg of white carbonate of Bu-2349A.
I got it. Similarly, 110 mg of carbonate of Bu-2349B was obtained by chromatography. Dissolve the carbonate in water, adjust the pH to 4.0 with 0.1N HCl, freeze and vacuum dry.
Bu-2349A hydrochloride was produced. Example 4 Well-grown agar slants of Bacillus species F173-B61 (ATCC 31429) were used to inoculate a growth nutrient medium containing 1% glucose, 0.5% yeast extract and 1% polypeptone with a pre-sterilized pH of 7.2. Seed culture at 28 °C on a rotary shaker (250 rpm)
Cultured for 24 hours with 2% glycerol, 1% corn soaking liquid, 1% cotton seed flour,
( _NH4 ) _{2SO40.3 %} , _ZnSO4・_7H2O0.003 %,
Fermentation medium containing 0.4% CaCO ₃ and adjusted to pH 7.2 before sterilization
5 ml of culture was added to a 500 ml Erlenmeyer flask containing 100 ml. When fermented for 5 days at 28° C. on a rotary shaker, antibiotic testing of this soup showed an efficacy of approximately 20 mcg/ml. The fermented soup was then separated and purified and separated into Bu-2349A and Bu-2349B hydrochloride salts by the methods of Examples 2 and 3. Bu-2349A and B acid addition salts are prepared by conventional methods,
For example, they can be converted to the corresponding free bases by ion exchange chromatography or neutralization of their solutions. The invention can be used in industrial applications.

[Brief explanation of drawings]

FIG. 1 is a diagram of an infrared absorption spectrum of Bu-2349A hydrochloride of the present invention. The vertical axis shows transmittance (%), and the horizontal axis shows wavelength (microns) and wave number (cm ^-1 ).
I'm taking it. FIG. 2 is a diagram of an infrared absorption spectrum of Bu-2349B hydrochloride of the present invention. The vertical and horizontal axes are the same as in Figure 1. FIG. 3 is a diagram of the nuclear magnetic resonance spectrum in D ₂ O of Bu-2349A hydrochloride of the present invention, and the horizontal axis shows ppm. Figure 4 shows Bu-
FIG. 2 is a nuclear magnetic resonance spectrum in D ₂ O of 2349B hydrochloride. The horizontal axis shows ppm.

Claims (1)

[Claims] 1. General formula However, R is or An antibiotic represented by or a pharmaceutically acceptable acid addition salt thereof. 2. Culture the Bu-2349A-producing strain or Bu-2349B-producing strain belonging to the genus Bacillus, and extract the general formula from the culture. However, R is (This antibiotic is called Bu-2349A) or (This antibiotic is referred to as Bu-2349B) A method for producing antibiotic Bu-2349A or Bu-2349B, which is characterized by collecting an antibiotic represented by: