JPH0219836B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel compound (antibiotic CB-104) having antibacterial activity, a salt thereof, and a method for producing the same. The present inventors isolated a large number of microorganisms from soil for the purpose of searching for new antibiotics, and conducted a separate search for the antibiotics produced by them, and found that certain microorganisms produce novel compounds. that it belongs to the Pseudomonas genus, and that by culturing the microorganism in an appropriate medium, compounds that exhibit antibacterial activity against Gram-positive bacteria, Gram-negative bacteria, filamentous fungi, and protozoa can be accumulated in the medium. We learned that this compound was isolated, and based on its physicochemical and biological properties, we confirmed that it was a new antibiotic, and decided to name it antibiotic CB-104. That is, the present invention provides novel compounds (antibiotic CB
-104) and a salt thereof, and an antibiotic CB-104-producing bacterium belonging to the genus Pseudomonas is cultured in a medium, and the antibiotic CB-104 is produced and accumulated in the culture, and the antibiotic CB-104 is collected.
This is the manufacturing method of CB-104. In addition, in this specification, the above compound (antibiotic
CB-104) is sometimes simply referred to as "CB-104". The antibiotic CB-104-producing microorganism used in the present invention may be any microorganism that belongs to the genus Pseudomonas and has the ability to produce antibiotic CB-104. As an example of antibiotic CB-104-producing bacteria, Pseudomonas CB-104 strain (hereinafter referred to as "CB
It is sometimes abbreviated as ``-104 shares''. ) and Pseudomonas CB collected from soil in Naruto City, Tokushima Prefecture.
-336 strain (hereinafter sometimes abbreviated as "CB-336 strain"). The mycological properties of Pseudomonas CB-104 strain and CB-336 strain are shown in Table 1.

【table】

[Table] CB-104 strain with the above mycological properties and
The CB-336 strain was purchased from Bergey's Manual of Determinative Bacteriology.
Manual of Determinative Bacteriology) No. 8
Comparing with the description in the edition, both are Gram-negative bacilli, do not form spores, have polar flagella, are obligate aerobic, catalase positive, and have no auxotrophy.
The CB-104 strain was identified as a Pseudomonas bacterium.
-104 strain (Pseudomonas sp. CB-104), CB-336
The strain was named Pseudomonas sp. CB-336. In addition, this strain CB-104 strain and CB-336 strain are
From April 23, 1980, the Ministry of International Trade and Industry, Agency of Industrial Science and Technology, Microbial Research Institute (FRI) received the accession number FERM P.
-6513 and FERM P-6514, also Showa
IFO to Fermentation Research Institute from April 7, 1957
14173 and IFO 14174, respectively. The properties of the Pseudomonas bacteria used in the present invention are generally susceptible to change, such as exposure to ultraviolet light,
CB-104, which can be easily mutated by artificial mutagenesis methods using X-rays, chemicals (e.g., nitrosoguanidine, ethyl methanesulfonic acid), etc., and any mutant strain is subject to the present invention. can be used in the present invention. When culturing CB-104-producing bacteria, carbon sources such as fructose, glucose, sucrose, maltose, blackstrap molasses, glycerol, fats and oils (e.g., soybean oil, olive oil, etc.), organic acids (e.g., citric acid, succinic acid, etc.) are recommended. A substance that can be assimilated by bacteria, such as acid, gluconic acid, etc., is used as appropriate, and fructose is particularly advantageous. Examples of nitrogen sources include organic and inorganic nitrogen compounds such as soy flour, cotton flour, corn staple liquor, dried yeast, yeast extract, meat extract, peptone, urea, ammonium sulfate, ammonium nitrate, ammonium chloride, and ammonium phosphate. compounds are available. In addition, as the inorganic salt, for example, inorganic salts normally required for culturing bacteria such as sodium chloride, potassium chloride, calcium carbonate, magnesium sulfate, monopotassium phosphate, and disodium phosphate are used alone or in appropriate combinations. . In addition, in the production method of the present invention, sulfur compounds that can be assimilated by antibiotic CB-104-producing bacteria belonging to the Pseudomonas genus, such as sulfates (e.g., ammonium sulfate, etc.), thiosulfates (e.g., ammonium thiosulfate, etc.), are added to the culture medium. , inorganic sulfur compounds such as sulfites (e.g. ammonium sulfite), organic sulfur compounds such as sulfur-containing amino acids (e.g. cystine, cysteine, L-thiazolidine-4-carboxylic acid), hypotaurine, sulfur-containing peptides (e.g. glutathione) Alternatively, it is desirable to add a mixture of these to the medium as appropriate. Further, heavy metals such as ferrous sulfate and copper sulfate, vitamins such as vitamin B ₁ and biotin, and the like are added as necessary. Furthermore, antifoaming agents and surfactants such as silicone oil and polyalkylene glycol ether may be added to the medium. Other organic or inorganic substances that aid the growth of bacteria and promote the production of CB-104 may be added as appropriate. The culturing method may be the same as a general antibiotic production method, and solid culture or liquid culture may be used. In the case of liquid culture, any of static culture, agitation culture, shaking culture, aeration culture, etc. may be carried out, but aeration agitation culture is particularly preferred. The culture temperature is preferably in the range of about 10°C to 35°C, the pH of the medium is in the range of about 5 to 8, and the culture is carried out for 8 hours to 168 hours, preferably 10 hours to 144 hours. In order to collect the target antibiotic CB-104 from the culture, separation means that are normally used to collect metabolites produced by microorganisms from the culture of the microorganisms are appropriately used. For example, CB-104 is an acidic and fat-soluble substance, so it can be collected by means that take advantage of these properties. Since CB-104 in the culture is mainly contained in the culture solution, the bacterial cells are separated, the culture solution is adjusted to pH 2-4, and extracted with a water-insoluble organic solvent such as ethyl acetate, chloroform, methyl ethyl ketone, etc. , wash the extract with water. When the extract is concentrated and left to stand, CB-104 crystals will precipitate. The mother liquor is subjected to column chromatography and thin layer chromatography using silica gel, alumina, etc., and CB-104 is eluted using an appropriate organic solvent, such as a mixed solvent such as chloroform, toluene, ethyl acetate, methanol, and acetone. urge The eluate is washed with, for example, an EDTA solution to remove impurities, concentrated, and left to stand to recover CB-104 crystals. These crystals can be recrystallized from chloroform, ethyl acetate, acetone, etc. to obtain pure CB-104 as crystals. It has been found that CB-104 obtained by the present invention is a new compound exhibiting the following physicochemical properties. (1) Molecular formula: C ₈ H ₄ O ₃ S ₂ (2) Melting point: 300°C or higher (3) Ultraviolet and visible absorption spectra: λ ^MeOH _nax 216 ± 2 nm (E1% 1 cm = 1210 ± 100) λ ^MeOH _nax 307±2nm (E1% 1cm=750±100) λ ^MeOH _nax 355±2nm (E1% 1cm=294±50) λ ^MeOH _nax 455±2nm (E1% 1cm=110±20) (4) Infrared absorption spectrum ( KBr), main peak (cm ^-1 ): 3450, 2960, 2920, 1630, 1600, 1525, 1480,
1440, 1385, 1310, 1250, 1180, 1120, 1070,
1015, 885, 840, 655, 580. (5) Solubility: Soluble: dimethyl sulfoxide, dimethyl formamide, pyridine. Slightly soluble: chloroform, ethyl acetate, acetone, methanol, ethanol. Poorly soluble: water, hexane. (6) Color reaction: Positive: ferric chloride, potassium permanganate. Negative: ninhydrin, Dragendorff, Ehrlitsu. (7) Distinction between acidic, neutral, and basic: acidic (8) Crystal appearance: yellow-orange (9) Thin layer chromatography [Silica gel F ₂₅₄
(Manufactured by Merck & Co., West Germany)] Solvent system Rf value (a) Toluene: dioxane: acetic acid (45:10:
2) 0.49±0.1 (b) Chlorofluorum:methanol (4:1)
(Contains 0.2% oxalic acid) 0.43±0.1 (c) Acetonitrile:water (4:1) 0.48±0.1 (10) High performance liquid chromatography (Waters, USA) (a) Microbondapak C ₁₃ (Water Hitachi No. 3011 (manufactured by Hitachi, Ltd., Japan) / 70 %
Methanol-0.02M acetic acid-sodium acetate buffer (PH5.5), 2ml/min; Rt (min) = 5.4±
1.0 (11) Specific rotation: [α] ²³ _D = 0゜ (C = 0.505, in dimethylformamide) (varies with time) (12) Salt formation: CB-104 is converted into equimolar amount of sodium carbonate or carbonic acid. Treatment with potassium gives the corresponding salt. Next, we will discuss the biological properties of CB-104.
The antibacterial spectrum of CB-104 against various microorganisms is shown in Tables 2, 3, and 4. As is clear from this table, CB-104 is effective against Gram-positive bacteria, Gram-negative bacteria, filamentous fungi, and protozoa.

【table】

【table】

[Table] As is clear from the above data, CB-104 obtained by the present invention exhibits a strong growth-inhibiting effect on microorganisms such as Gram-positive bacteria, Gram-negative bacteria, filamentous fungi, and protozoa. The acute toxicity of CB-104 is
LD ₅₀ 50-100 mg/Kg (mouse, oral administration). CB-104 can be used as a fungicide in bacterial and fungal infections of humans and domestic animals (eg, cattle, pigs, chickens). For example, as an ointment containing CB-104 based on vaseline, lanolin, etc., containing 2 to 50 mg, preferably 5 to 20 mg of CB-104 per gram,
It can be used to sterilize and disinfect feet, ears, etc. CB-104 is also an extremely promising compound as a synthetic intermediate or reagent for new pharmaceuticals. From the properties described above, CB-104 is clearly recognized as a new antibiotic. Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto. Percentage (%) in examples
indicates weight/volume % unless otherwise specified. Example 1 Pseudomonas grown on a nutrient agar slope
SP CB-104 (FERM P-6513, IFO
14173) strain, 1% fructose, 0.5% polypeptone (manufactured by Daigo Nutrient Chemical Co., Ltd.), and 0.5% meat extract.
%, salt 0.5% (PH 7.0) in two 2-volume Sakaguchi flasks containing 500 ml of medium, and incubated at 28°C for 48 hours.
The culture was incubated with reciprocating shaking for hours and the culture was inoculated. Next, fructose 3%, polypeptone 0.5%,
Meat extract 0.5%, NaCl 0.5%, sodium thiosulfate
Culture medium 120 consisting of 0.05% was placed in a 200-volume stainless steel fermenter, the liquid was adjusted to pH 7.0 with 30% sodium hydroxide solution, steam sterilized at 120°C for 20 minutes, and then the seed culture was inoculated. . Then temperature 24
The cells were cultured for 18 hours at a temperature of 120 rpm at an aeration rate of 180 rpm. This culture was applied to a shear press centrifuge to separate the bacterial cells, and the supernatant was
I got it. Example 2 Medium 30 consisting of 1% fructose, 0.5% polypeptone, 0.5% meat extract, and 0.5% salt was
Pour into a stainless steel fermenter, adjust the pH to 7.0 with 30% sodium hydroxide solution, and incubate at 120℃ for 20 minutes.
After steam sterilization for a minute, the seed culture shown in Example 1 was inoculated and cultured at 24° C. for 24 hours under the conditions of an aeration rate of 30/min and a stirring speed of 180 rpm, which was used as a secondary seed culture. Fructose 5%, polypeptone 0.5%, meat extract 0.5%, salt 0.5%, sodium thiosulfate 0.05
A medium consisting of 1200% of
After adjusting to 7.0 and steam sterilizing at 120℃ for 20 minutes,
A secondary inoculum was inoculated. Next, the culture was cultured for 42 hours at a temperature of 28°C, an aeration rate of 1200/min, and a stirring speed of 180 rpm.
Got 1150. Example 3 A strain of Pseudomonas sp. CB-336 (FERM P-6514, IFO 14174) grown on nutrient agar was grown with 2% glucose, 3% soluble starch,
1% raw soybean flour, 11% corn staple liquor
%, polypeptone 0.5%, salt 0.3%, calcium carbonate 0.5% (PH7.0)2 containing 500 ml of medium.
One Sakaguchi flask was inoculated and cultured with reciprocating shaking at 24°C for 24 hours, and the culture was used as a starter. Next, glucose 1.5%, soluble starch
1.5%, cotton seed flour 0.75%, defatted soybean flour 0.75%, potassium phosphate 0.025%, dipotassium phosphate 0.06%,
Medium 120 consisting of calcium carbonate 0.1% (PH7.0)
into a 200-volume stainless steel fermenter at 120℃ for 20 minutes.
After steam sterilization for a minute, the seed culture was inoculated. Then, the temperature was 24℃, the air flow rate was 120/min, and the stirring speed was
Culture was carried out for 42 hours under the condition of 180 rpm. Example 4 The culture solution obtained in Example 2 was filtered to obtain a solution (1250). After adjusting the solution to PH2.3, extract with ethyl acetate (600), wash the extract (460) with water, transfer the antibiotic with 2% sodium bicarbonate water, and adjust the transferred solution (220) to PH2. 3 and extracted with ethyl acetate. After washing the extract (110) with water, concentrating it and leaving the concentrated liquid (10) in a cold place, CB-
104 crystals (2.7 g) were precipitated. The solution was diluted with 0.2% ethylenediaminetetraacetic acid disodium salt (EDTA).
After washing with solution (5) and water, the ethyl acetate layer was concentrated to obtain CB-104 crystals (7 g). The mother liquor was concentrated to 0.2, and the concentrated solution was subjected to column chromatography on silica gel (250) g. Chlorophorum:methanol (9:1, 2.5
) was washed with EDTA solution (1), washed with water, and concentrated to obtain crystals of CB-104 (0.56 g). Example 5 The culture solution obtained in Example 3 was filtered to obtain a solution (102). Adjust the solution to PH2.3 with dilute hydrochloric acid,
Extracted with ethyl acetate (92), transferred the antibiotic in the extract (81) with 2% sodium bicarbonate water (50), adjusted the transferred solution (59) to pH 2.3, and extracted with ethyl acetate again. . Concentrate the re-extract (40),
The concentrated solution (3.5) was subjected to the above-mentioned dissolution method, and the extract was concentrated to obtain crystals (860 mg). The mother liquor was subjected to column chromatography using silica gel (250 g) and chloroform:methanol (95:5).
The fractions were concentrated to dryness, and the residue was dissolved in ethyl acetate (200
ml), washed the ethyl acetate layer with 0.2% EDTA solution (200 ml), and concentrated to obtain crystals (60 mg). The crystals obtained here matched the crystals obtained in Example 4 in melting point, TLC, HPLC, UV and IR spectra. Example 6 CB-104 crystals (160 mg) were dissolved in acetone (160 ml).
Potassium carbonate (52mg) aqueous solution (5ml)
Add water (100ml) and stir briefly.
The reaction solution was concentrated under reduced pressure to obtain the monopotassium salt of CB-104 (180 mg) in the form of crystals.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet and visible absorption spectra (in methanol) of antibiotic CB-104, and Figure 2 shows the infrared absorption spectrum (KBr) of antibiotic CB-104.
law) respectively.

Claims (1)

[Claims] 1. A compound having the following structural formula (antibiotic CB-
104) or its salt: 2 Antibiotic CB-104 belonging to the genus Pseudomonas
Cultivate the producing bacteria in a medium and add antibiotic CB to the culture.
A method for producing antibiotic CB-104, which comprises producing and accumulating -104 and collecting it.