JPS6330915B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel antibiotic, more specifically, the following formula: The present invention relates to a compound represented by the above formula, a salt thereof, and a method for producing the compound. The following formula 7-oxo-1-azabicyclo[3.
2.0] Antibiotics with a hept-2-ene-2-carboxylic acid skeleton generally have high antibacterial activity and β-lactamase inhibitory activity, and have traditionally been treated by fermentation, semi-synthesis, and total synthesis methods. various 7-oxo-1-azabicyclo[3.2.0]hept-2
-ene-2-carboxylic acid derivatives have been produced [for example, thienamycin (journal of
Antibiotics, Volume 32 (1979), 1-12
Page), epithienamycins (17th Interscience Conference on Antimicrobial Agents and Chemotherapy,
Abstract No. 80 and 81 (1977)), N-acetylthienamycin (West German Patent No. 2652681 (1977)
), olivanic acids (Journal of Antibiotics, vol. 32 (1979), 287-304
p.), PS-5 (Journal of Antibiotics, Vol. 32 (1979), pp. 262-286), PS-5
6 (Public Patent Publication No. 54-59295), PS-7 (Public Patent Publication No. 54-92983), etc.]. The compound of formula () provided by the present invention is 7-oxo-1-azabicyclo[3.2.
0] 3 of hept-2-ene-2-carboxylic acid skeleton
It is a novel antibiotic that has not been described in conventional literature and has a structural feature in that it has a pantetheinyl group at the 6-position and a 1-hydroxyethyl group at the 6-position.
Hereinafter, this antibiotic will be abbreviated as "antibiotic OA-6129B". The novel antibiotic OA-6129B of formula () is unique in that it is more stable than previously proposed antibiotics with the same basic skeleton, and
As described in the above-mentioned known documents, it has strong antibacterial activity and β-lactamase inhibitory activity, and synergistically enhances the antibacterial activity of antibacterial substances such as penicillins and cephalosporins against β-lactamase producing bacteria. He also has the ability to
Useful as an antibacterial agent. Furthermore, the carboxyl group at the 2-position of the compound of formula () can also be in the form of a salt, and examples of such salts include alkali metal salts such as sodium salts, potassium salts, and lithium salts; calcium salts;
alkaline earth metal salts such as magnesium salts; other metal salts such as aluminum salts; ammonium salts; with primary, secondary or tertiary amines such as monoethylamine, dimethylamine, trimethylamine, monoethanolamine, diethanolamine. Salts include salts with organic bases such as benzathine salts and procaine salts, among which pharmaceutically acceptable salts are preferred, particularly sodium salts,
Alkali metal salts such as potassium salts are preferred. According to the invention, the antibiotic OA of the formula ()
-6129B is obtained by culturing the antibiotic OA-6129B-producing microorganism in a nutrient medium, and from the culture, β-
The above antibiotics with lactamase inhibitory activity
It can be produced by a method consisting of collecting OA-6129B. The antibiotic OA-6129B-producing bacteria used in the present invention may belong to any genus as long as it has the ability to produce antibiotic OA-6129B having the above-mentioned physicochemical and biological properties. can be used and selected from a wide range of microorganisms. Therefore, a search for a bacterial strain suitable for the purpose of the present invention can be carried out as follows, and a person skilled in the art will be able to identify the antibiotic OA-
6129B producing bacteria can be easily obtained. That is, a culture solution of soil-isolated bacteria was assayed using a bioassay agar plate containing β-lactam-sensitive bacteria as the assay bacteria and a bioassay agar plate to which various types of β-lactamases were added. We search for soil isolates that give a culture solution that gives a zone of inhibition on some of the latter plates, and whose zones of inhibition on some of the latter agar plates are smaller than those of the former. Next, the active ingredients in the culture solution of the soil-isolated bacteria are adsorbed onto activated carbon, and the eluted concentrate is developed using paper chromatography or thin layer chromatography. Antibiotic OA− by graphie
If 6129B is detected, it can be said that the bacterium is a bacterium producing antibiotic OA-6129B that can be used in the method of the present invention. This search method will be further explained using a specific example as follows. As a bioassay agar plate for β-lactam-sensitive bacteria, we used the Comamonas assay plate described below, and the Comamonas CV assay plate to which β-lactamase produced by Bacillus cereus 569 was added, and the production of Citrobacter freundei-E-9. β
- Prepare a Comamonas CM assay plate supplemented with lactamase. On the other hand, the culture solution of soil isolated bacteria was
After making pulp disks with a diameter of mm and placing them on each assay plate and incubating at 35℃ for 20 hours, give an inhibition circle on the Comamonas assay plate, and
Select soil isolates given the culture solution whose inhibition circle on the CV assay plate or Comamonas CM assay plate is smaller than the inhibition circle on the Comamonas assay plate. Next, add 2% of the solution to the culture solution of the soil-isolated bacteria.
(W/V) amount of special Shirasagi activated carbon (manufactured by Takeda Pharmaceutical Co., Ltd.) was added, stirred for 15 minutes, collected the precipitate by centrifugation, and distilled the same volume of culture solution using this precipitate. Wash with water and centrifuge again to collect the precipitate. To this precipitate was added 50% (V/V) acetone water in an amount equivalent to half the volume of the culture solution used above, stirred at room temperature for 30 minutes, and then centrifuged to obtain a supernatant. This supernatant was concentrated using a rotary evaporator at 30 to 35°C to obtain a solution 20 times as concentrated as the culture solution used above. This concentrated solution was mixed with Toyo Paper No. 50 (manufactured by Toyo Paper Co., Ltd.) using 80% acetonitrile/Tris/EDTA [120 ml of acetonitrile,
Perform descending paper chromatography for 16 hours using a solvent consisting of 30 ml of 1/10M tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (pH 7.5) and 1 ml of 1/10M ethylenediaminetetraacetic acid sodium salt aqueous solution (PH 7.5). After the ivy, Comamonas terrigena B
-996 was used as the test strain to perform pyo-autography. Then, soil isolates that showed an inhibition zone at the same migration distance (at the Rf value) as antibiotic OA-6129B are selected as antibiotic OA-6129B producing bacteria candidates. Regarding candidate bacteria selected in this way,
Furthermore, paper chromatography and thin layer chromatography will be performed to confirm the productivity of antibiotic OA-6129B. Accordingly, those skilled in the art can easily search for antibiotic OA-6129B-producing bacteria that are suitable for the purpose of the present invention. Antibiotic OA searched as above
Typical 6129B-producing bacteria include the antibiotic OA-6129B-producing bacteria belonging to the genus Streptomyces, and a suitable example is a strain isolated from soil collected near Sumiyoshi Shrine in Fukuoka City, Fukuoka Prefecture. Examples of actinomycetes include a strain that the present inventors have designated as OA-6129 strain. The mycological properties of this OA-6129 strain are as follows. 1. Morphology Under the microscope, the well-branched basal hyphae are straight to
Extends curved (Straight to Flexuous) aerial hyphae,
Whorled branches are not observed. A mature spore chain consists of 10 or more oval to cylindrical spores, and no sporangia are observed. Spore size is 0.6-1.0×0.7
Approximately 2.5 microns in diameter, the spore surface is smooth.
Flagellated spores are not observed. 2 Growth conditions in various media Cultivation was carried out at 28° to 30°C unless otherwise specified.
In addition, descriptions of color tones are mainly based on H.D. Tresner and E.G. Bakas (HDTres-ner).
and E. J. Backus), Journal of
Applied Microbiology (Journal of
Applied Microbiology) Volume 11, No. 4, (1963)
According to the method on pages 335 to 338, the code in [ ] [CHM code] is based on the Container Corporation of America's Color Harmony Manual.
America's Color Harmony Manual) was used. (1) Seuucrose/nitrate agar medium: medium growth of yellow ash [2dc] to light gray-yellow brown [3ge], and yellow ash [2dc] to gray-yellow pink [5dc]
Aerial mycelia are attached, and no soluble pigments are observed. (2) Glucose-asparagine agar medium: light yellow [2db] to bright olive brown [2ge]
Bright gray [d] aerial mycelium grows on top of the good growth. This aerial mycelium later becomes grayish-yellow-pink [5 dc]. No soluble pigments are observed. (3) Glycerin-asparagine agar medium (ISP medium-5): Gentle yellow-pink [4gc] to bright brown [4ie]
Aerial mycelium of light gray [d] to light gray reddish brown [5fe] grows on the good growth. No soluble pigments are observed. (4) Starch inorganic salt agar medium (ISP medium-4): Bright gray [d] aerial mycelium grows on light yellow [2db] to gray [2fe] growing well. No soluble dyes are produced. (5) Tyrosine agar medium (ISP medium-7): On the growth of gray-yellow [3ec] to light brown [4ie],
It grows on aerial mycelia of light gray [d] to light brown gray [3fe]. The medium will have a very slight brown tinge. (6) Nutrient agar medium: Aerial mycelium of light gray-reddish brown [5fe] grows on a well-growing medium of light yellow [2db] or bright yellow [2fb] to light olive brown [2ge]. No soluble pigments are observed. (7) Yeast extract/malt extract agar medium (ISP
Medium-2): Gentle yellow pink [4gc] to bright brown [4ie]
On top of the good growth, gray-yellow-pink [5dc] or slightly later bright gray [d] aerial mycelia are attached. No soluble pigments are observed. (8) Oatmeal agar medium (ISP medium-3): On the good growth of gray-yellow [3ec] to bright orange-yellow [3ea] or bright gray-yellow-brown [3ge],
Aerial mycelium of light brownish-gray [3fe] to light gray-reddish-brown [5fe] grows on it, and the medium around the fungal colony is slightly brown. (9) Malate lime agar medium: Aerial mycelia of light gray [d] to light gray reddish brown [5fe] grow on medium growth of dark to yellow gray [2dc], with no soluble pigments. I can't. A dissolution zone of calcium salts can be seen around the growing bacterial colonies. (10) Glucose-peptone-gelatin medium (20℃
Culture) White [B] to gray-yellow pink [5CB] aerial mycelia grow on the pale yellow [2dB] to brown good growth. When cultured for a long period of time (about 3 weeks or more), a brown soluble pigment was produced. 3 Physiological properties (1) Growth temperature range Yeast extract/malt extract agar medium (ISP
10°, 20°, 25°, 30°, 34°, using medium-2)
As a result of experiments at temperatures of 37°, 40°, 45°, and 50°C, 37
It can hardly grow at ℃. It does not grow at all above 40℃. Growth was observed at all other temperatures. The optimal growth temperature range seems to be 20-30°C. (2) Liquefaction of gelatin: Liquefaction. (3) Starch hydrolysis: decomposes. (4) Coagulation and peptonization of skim milk: It does not coagulate, but it converts into peptonization. (5) Production of melanin-like pigment: No production of melanin-like pigment was observed in peptone yeast iron agar medium (ISP medium-5) and tryptone yeast extract prosthetic medium (ISP medium-11). Although it shows a very slight brown color on tyrosine agar medium, there is only a trace of melanin production. 4 Assimilation of various carbon sources (using Pridham-Gotrib agar medium) (1) L-arabinose + (2) D-xylose + (3) D-glucose + (4) D-fructose + (5) Seuculose Doubtful. (6) Inositol - (7) L-rhamnose + (8) Raffinose - (9) D-mannite ++ is assimilated, - is not assimilated. Based on the above mycological properties, the OA-6129 strain is
It is a strain belonging to the genus Streptomyces, and the shape of the aerial hyphae is Section Rectifle (RF).
xibiles), and the spore surface was smooth.
The color of aerial mycelia is light gray on most media, such as oatmeal agar, glycerin/asparagine agar, starch/inorganic salt agar, etc.
It is a strain of the Gray series. However, depending on the culture period, a red series of gray-yellow-pink [5 dc] may be exhibited on seurose/nitrate agar, yeast extract/malt extract agar, and glucose/asparagine agar media. In addition, the color of the basal hyphae is light yellow to grayish yellow in any medium at the initial stage of culture, and as the culture continues, the color becomes yellowish brown to grayish yellowish brown or brown. Melanin pigment was not observed in peptone/yeast extract/iron agar medium or tryptone/yeast extract/pross, and other water-soluble pigments were not produced in many media. However, tyrosine agar medium,
A slight brown pigment was observed in the glucose-peptone-gelatin medium and oatmeal agar medium. The present inventors have deposited this bacterium as Streptomyces sp. OA-6129 with the National Institute of Microbial Technology, Agency of Industrial Science and Technology, as Fiber Science and Technology Research Institute No. 5714. The antibiotic OA-6129B of the present invention is an antibiotic OA-6129B.
−6129B-producing bacteria, such as the above Streptomyces
It is produced by inoculating spores or hyphae of sp.OA-6129 into a nutrient-containing medium and growing aerobically. As the nutrient source, those normally used as a nutrient source for actinomycetes, such as assimilable nutrient sources such as carbohydrates, nitrogen sources, and inorganic salts, can be used. For example, carbohydrates such as glucose, glycerin, maltose, sucrose, molasses, dextrin, and starch;
Carbon sources such as oils and fats such as soybean oil, peanut oil, and lard; peptone, meat extract, soybean flour, cottonseed flour, dried yeast, corn steep liquor, yeast extract, skim milk, casein, sodium nitrate, ammonium nitrate, Nitrogen sources such as ammonium sulfate; inorganic salts such as dipotassium phosphate, common salt, calcium carbonate, and magnesium sulfate can be used, and if necessary, trace metals such as cobalt and manganese can be added. In addition, antibiotic OA-6129B-producing bacteria can be used as a nutritional source.
Any nutrient source that produces 6129B can be used, and any known culture material for actinomycetes can be used. Further, in order to suppress foaming during heat sterilization and culturing, an antifoaming agent such as silicone or vegetable oil may be added. The blending ratio of the nutrient sources as described above is not particularly restricted and can be varied over a wide range, and the optimal nutrient source composition and blending ratio for the antibiotic OA-6129B producing bacteria to be used is A person skilled in the art can easily determine this through simple small-scale experiments. The nutrient medium can also be sterilized prior to culturing, and it is advantageous to adjust the pH of the medium to a range of 4 to 9, particularly 6 to 8, before or after this sterilization. In principle, the antibiotic OA-6129B-producing bacteria can be cultured in such a nutrient medium according to a method commonly used in the production of antibiotics using general actinomycetes. It is usually preferred to culture under aerobic conditions, usually with stirring and/or
Alternatively, it can be carried out with ventilation. In addition, as a culture method, static culture, shaking culture, and submerged culture with aeration and agitation can all be used.
Submerged culture is advantageous. The culture temperature that can be used is such that the growth of antibiotic OA-6129B-producing bacteria is not substantially inhibited.
There is no particular restriction as long as it can produce 6129B, and it can be changed depending on the production strain used, but generally the temperature is 20 to 40°C, preferably 25°C.
Temperatures in the range ˜35° C. are preferred. Moreover, in order to carry out the culture suitably, the pH of the culture can be adjusted to a range of 4 to 9, particularly 6 to 8, during the culture, if necessary. In the case of large-scale mass cultivation, it is advantageous to perform a seed culture as appropriate, inoculate it into a nutrient medium, and perform liquid culture. Cultivation can normally be continued until sufficient accumulation of antibiotic OA-6129B occurs. The culture time varies depending on the composition of the medium, culture temperature, production strain used, etc., but is usually in the range of 30 to 90 hours. As for the culture conditions to be used, those skilled in the art can easily determine the optimal conditions through simple experiments, depending on the characteristics of the production strain used. The amount of antibiotic OA-6129B accumulated during culture can be quantified by the pyoassay method and bioautography described below, and thereby the optimum amount of antibiotic OA-6129B accumulated can be easily determined. Thus, the antibiotic OA accumulated in the culture
Since −6129B is water-soluble and mainly exists outside the bacterial cells, it is advantageous to use filtration, centrifugation,
The bacterial cells are removed by a separation method known per se, such as extraction, and recovered from the resulting liquid, supernatant liquid, extract liquid, etc. Recovery can be carried out by various methods known per se, and in particular methods often used for recovery of carboxylic acid type antibiotics are advantageously applied. For example, solvent extraction with ethyl acetate, n-butanol, etc. at low pH and transfer of the solvent layer to high pH water layer; activated carbon, Amberlite XAD (manufactured by Rohm and Haas), Diaion HP-20 (manufactured by Mitsubishi Kasei Corporation), etc., and elution with methanol water, acetone water, etc.; Dowex 1x2 (manufactured by Dow Chemical Company), QAE-Sephadex A-25 (manufactured by Pharmacia), DEAE-Cellulose Watman DE- Adsorption and elution with ion exchange resins such as 32 (manufactured by Watmann) and DEAE-Sephadex A-25 (manufactured by Pharmashima); Cephadex G-
10 (manufactured by Pharmacia), Bio-Gel P-2 (manufactured by Bio-Rad), etc.; column chromatography of cellulose, Avicel SF (manufactured by American Viscose); addition of solvents such as acetone A forced precipitation method; a freeze-drying method, etc. may be used alone or in appropriate combinations, and may be used repeatedly in some cases. The behavior of antibiotic OA-6129B during the recovery and purification process can be quantitatively measured by the bioassay method and bioautography described below. Thus, antibiotic OA with the above-mentioned properties
−6129B is obtained. Antibiotic OA- produced by the above fermentation method
6129B, that is, the compound of the above formula (), generally has hydrogen atoms bonded to the 5- and 6-position carbon atoms each having a trans or cis configuration,
The formula () includes 5,6-trans form, 5,6-
Either the cis form or a mixture thereof is included. In addition, the pantetheinyl group bonded to the 3-position carbon atom and the 1-hydroxyethyl group bonded to the 6-position carbon atom each have one asymmetric carbon atom, and are S-type. and R-form, or racemic form. Since the salt form of this antibiotic OA-6129B is generally more stable than the free form, it can be used for the pharmaceutical purposes described below, or as an intermediate for further conversion into derivatives. Alternatively, when subjecting it to the above-mentioned purification process, it is preferable to treat it in the form of a salt. Conversion of the antibiotic OA-6129B into its salt form can be carried out by treating the antibiotic OA-6129B with an inorganic or organic base according to known methods. Inorganic or organic bases that can be used in this salt-forming reaction include, for example, alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, and lithium hydroxide; alkaline earth bases such as calcium hydroxide and magnesium hydroxide; Metal hydroxides include primary, secondary, or tertiary organic amines such as monoethylamine, dimethylamine, trimethylamine, monoethanolamine, diethanolamine, benzathine, and procaine. Antibiotic OA-6129B provided by the present invention
or its salts have a wide range of antibacterial activity and exhibit very strong antibacterial activity against various microorganisms, such as Gram-positive bacteria belonging to the genus Staphylococcus, Sarcina, Bacillus, etc. It also shows very strong antibacterial activity against Gram-negative bacteria belonging to the genus. Furthermore, the antibiotic OA-6129B of the present invention exhibits a fairly strong antibacterial activity against Gram-negative bacteria belonging to the genus Escherichia, Klebsiella, Proteus, and the like. In particular, the antibiotic OA-6129B of the present invention is β-
It is unique in that it exhibits fairly strong antibacterial activity against Gram-negative bacteria that are resistant to antibiotics with lactam rings, such as those belonging to the genus Citrobacter, Proteus, Enterobacter, Klebsiella, and Serratia. be. The antibacterial spectrum of the antibiotic OA-6129B of the present invention is verified by measuring the minimum inhibitory concentration against various pathogenic test bacteria as described below.

【table】

[Table] Furthermore, the antibiotic OA-6129B of the present invention is effective against other antibiotics, especially against β-lactamase producing bacteria such as Citrobacter freundii, Proteus vulgaris, Enterobacter aerogenes, and Serratia marcesans. It has the ability to enhance the antibacterial activity of β-lactam antibiotics such as penicillins and cephalosporins, and in many cases, its ability is synergistic. The acute toxicity of the antibiotic OA-6129B of the present invention is
When 500 mg/Kg was administered intravenously to ddy mice,
It was not observed at all. Furthermore, as mentioned above, the antibiotic OA-6129B of the present invention is more stable than conventionally known antibiotics of the same kind, and its excellent stability is due to its short half-life in phosphate buffers at various pHs. This is clear from measurement. Its half-life can be measured as follows. That is, prepare a 0.05M phosphate buffer, add 2% caustic soda solution or 10% hydrochloric acid to adjust the pH to 4, 5, 6, 7, 8, and 9, and then add it to a test tube. Dispense 3 ml each. Next, after adding 0.05 ml of a separately prepared concentrated solution of antibiotic OA-6129B, it was left to stand in a constant temperature room at 28°C, and the concentration of antibiotic OA-6129B over time was measured by measuring ultraviolet absorption (300 nm). A reduction curve can be constructed and the half-life calculated from this diagram. The antibiotic OA-6129B and its salts mentioned above are
As mentioned above, it has strong antibacterial activity and is used as an active ingredient of antibacterial agents for the prevention, treatment, and/or treatment of infections caused by Gram-positive and Gram-negative bacteria.
Prevention of bacterial infections not only for humans but also for non-human animals such as mammals, poultry, fish, etc.
It can be effectively used for treatment, treatment, etc. The antibiotic OA-6129B or a salt thereof of the present invention is
When used as an antibacterial agent, it can be administered orally, locally, or parenterally (intravenously, intramuscularly, intraperitoneally, etc.), and depending on the method of administration, various drugs may be used as commonly used. It can be formulated and used. For example, the antibiotic of the present invention
OA-6129B or a salt thereof can be prepared in solid form (e.g., tablet, capsule, powder, granule, sugar-coated tablet, troche, powder spray, suppository, etc.), semi-solid form, together with a pharmaceutically acceptable carrier material, diluent, etc. They can be formulated in solid forms (eg, ointments, creams, semi-solid capsules, etc.) or liquid forms (eg, solutions, emulsions, suspensions, lotions, syrups, injections, liquid sprays, etc.). The unit dosage form containing the antibiotic OA-6129B or a salt thereof of the present invention, whether liquid, semi-solid or solid, is generally 0.1 to 99% by weight, preferably
It can contain 10-60% by weight of active ingredient. Typical auxiliary agents such as carrier materials, excipients, and diluents that can be used in these formulations, as well as formulation methods, will be further explained as follows. Tablets and capsules for oral administration may be in unit dosage form, with possible binders such as syrup, acacia, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone; excipients. Examples of the agent include lactose, sucrose, starch, calcium phosphate, sorbitol, or glycine; examples of the lubricant include magnesium stearate, talc, polyethylene glycol, and silica; examples of the disintegrant include yam and starch. Also, examples of wetting agents include sodium lauryl sulfate. Tablets can be coated using conventional methods. Oral solutions can be in the form of oil- or water-suspensions, solutions, emulsions, syrups, etc., or can be provided as a dry product for mixing with water or other suitable carrier for use. can do. These solutions can usually contain additives such as suspending agents such as sorbitol, syrup, methylcellulose, sugar syrup,
Gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, hydrogenated edible fats, etc.; emulsifiers, such as lecithin, sorbitan monooleate, or gum arabic; non-aqueous carriers, such as almond oil, fractionated coconut oil. , oily esters, propylene glycol, or ethyl alcohol; preservatives such as methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid, etc. Suppositories can contain conventional suppository bases such as cocoa butter or other glycerides. Injectables may be presented in unit dosage form in ampoules or in multi-dose containers with a preservative. It usually takes the form of a suspension, solution, or emulsion with an oily or aqueous carrier;
The formulation may contain such agents as suspending agents, stabilizers and/or solubilizing agents. Alternatively, the active ingredient may be in powder form for formulation by dissolution with sterile pyrogen-free water at the time of use. Antibiotic OA-6129B or its salts can also be formulated into dosage forms suitable for absorption through the mucous membranes of the nose and throat, bronchial tissues, and even powder sprays, liquid sprays, inhalants, lozenges, and throat applications. It can be made into an appropriate dosage form such as a drug. Formulations for eye and ear administration may be presented as liquid or semisolid capsules, and may also be provided as drops. Suitable topical preparations include ointments, creams, lotions, liniments,
Included are powders and the like, which can be formulated with hydrophobic or hydrophilic bases. The above-mentioned formulation may also contain components such as stabilizers, binders, antioxidants, preservatives, lubricants, suspending agents, thickening agents, flavoring agents, and buffering agents. Furthermore, when the antibiotic OA-6129B or its salt of the present invention is used as a veterinary drug for chickens, cows, sheep, pigs, etc., for example, a long-acting or short-disintegrating base may be used to treat the mammary glands. It can be formulated as an internal preparation, or as a concentrated feed additive according to known methods. Various forms of antibiotics provided by the present invention
OA-6129B or a salt thereof may be included as the only active ingredient, or other therapeutically useful active ingredients may be included. In particular, the antibiotic OA-6129B or a salt thereof of the present invention has the ability to synergistically enhance the antibacterial activity of β-lactam antibiotics against β-lactamase-producing bacteria, as described above. known β
- May be used in combination with lactam antibiotics. Examples of such β-lactam antibiotics include penicillin antibiotics such as benzylpenicillin, phenoxymethylpenicillin, carbenicillin, ampicillin, amoxicillin; cephaloridine, cephalothin, cefazolin, cephalexin, cefoxitin, cefacetril, cefamandole, cefapicin. Examples include cephalosporin antibiotics such as , cefradine, and cephaloglycine. When the antibiotic OA-6129B of the present invention or a salt thereof is used in combination with a β-lactam antibiotic as described above, the ratio of the two is not critical and can vary over a wide range, but generally the antibiotic OA-6129B or a salt thereof is
The weight ratio of 6129B or its salt to the β-lactam antibiotic can range from 20:1 to 1:150, preferably from 10:1 to 1:100. The dosage of the antibiotic OA-6129B or its salt of the present invention can vary widely depending on the subject to be treated and its symptoms, the weight of the host, the type of infection, the method of administration, and the number of administrations, but it is usually administered per day. 0.05
~500mg/Kg body weight, particularly preferably 0.5-200mg/
Advantageously, Kg body weight is administered in one or several doses. However, it is also possible to administer an amount smaller or larger than the above dosage range depending on the judgment of a specialist, individual differences, severity of symptoms, etc. The antibiotic OA-6129B or a salt thereof of the present invention can not only be used in the form of a drug as described above, but also can be directly included in animal feed or in the form of an additive for animal feed. Alternatively, it can also act as an active ingredient of a bactericide or preservative for food preservation. Next, the present invention will be further explained by examples. In addition, qualitative and quantitative analyzes of the antibacterial active substances used in the following examples were performed by the following methods. (1) Bioassay method Comamonas terrigena cultured overnight on nutrient agar.
terrigena) B-996 cells are suspended in nutrient broth, and the cells derived from the cells are suspended in nutrient broth.
Prepare a seed mother solution with an absorbance of 0.04 at 610 nm.
Kyokuto Powder Bouillon (manufactured by Kyokuto Pharmaceutical Industries Co., Ltd.) 0.8%
A 1% seed mother solution is inoculated onto a 1% agar medium made of Bacto Agar (manufactured by Difco), and 7 ml of this is dispensed into Petri dishes with a diameter of 9 cm and solidified to prepare a Comamonas assay plate. (2) Bioautography In the bioassay method described above, instead of using a 9 cm Petri dish, a dish measuring 32 cm in length x 24 cm in width is used, and 100 ml of agar medium inoculated with the test bacteria is dispensed and solidified to create a large assay plate. . The paper chromatography paper after the test solution has been developed is pasted on the agar surface of the large assay plate prepared above, removed after 15 minutes, the large assay plate is incubated at 35°C at 20:00, and the paper chromatogram is obtained from the position of the inhibition zone. It is possible to calculate the Rf value of (qualitatively) and semiquantitate from the size of the inhibition zone. When using a thin layer chromatography plate, place the plate on the agar surface with a piece of thin paper so that the component side touches the plate, remove it after 15 minutes, and perform qualitative and semi-quantitative analysis using the same procedure as above. Example 1 5,6-trans-3-pantetheinyl-6-
(1-Hydroxyethyl)-7-oxo-biazacyclo[3.2.0]hept-2-ene-2-
Sodium carboxylate (antibiotic OA-6129B)
Production by fermentation method (A) 100 ml of a seed culture medium (S-1) having the following composition was placed in a 500 ml Erlenmeyer flask and sterilized at 120° C. for 15 minutes by a conventional method. On the other hand, Streptomyces sp. OA-6129 (Streptmyces sp.
sp・OA−6129) to fully attach the spores of the strain,
A loopful of this was inoculated into the seed medium and heated at 28°C.
Shaking culture was carried out in a rotary shaker (200 rpm, amplitude 7 cm) for 48 hours. This seed culture solution 200
ml was inoculated into a 30 liter jar fermenter containing 15 liters of production medium (GM-1) with the following composition,
Aerated agitation culture was carried out for 90 hours at 28°C, stirring at 400 rpm, and aeration at 7.5 l/min. 0.07% silicone KM-75 (manufactured by Shin-Etsu Chemical Co., Ltd.) as an antifoaming agent
used. Sampling the culture fluid over time,
The antibacterial activity of the centrifuged supernatant was measured. The measurement results at each time were as shown in the table below. Culture time (hours) Antibacterial titer (μg/ml) 48 0.5 72 3.2 90 6.3 Composition of seed medium (S-1): Meat 1.5% (W/V) Yeast extract 0.5 (W/V) Potato starch 2.0 (W/V) CaCo ₃ 0.2 (W/V) PH (before sterilization) 7.0 Composition of production medium (GM-1): Glycerin 8.0% (W/V) Fishmeal 1.0 (W/V) Meat 3.0 (W/V ) CaCo ₃ 0.3 (W/V) K ₂ HPO ₄ 0.2 (W/V) MgSO ₄ 0.2 (W/V) Adjust the pH to 7.2 with NaOH Separately dissolve in 0.01M phosphate buffer at PH5.5, Additionally, 0.0005% (W/V) of vitamin B ₁₂ was added, which had been sterilized in an autoclave at 1 kg/cm ² G for 5 minutes. (B) 15 liters of the fermentation liquid obtained in (A) above after 90 hours of culture
5% (W/V) amount of Topcoperlite No.34
[manufactured by Toko Perlite Co., Ltd.] was added, and the bacterial cells were separated using a basket centrifuge to obtain 13 liters of culture solution. This is Diaion HP-20 [manufactured by Mitsubishi Kasei Corporation]
Adsorb into a packed column (10 x 100 cm) and add distilled water.
After washing with 1 liter, elution was performed with 30% (V/V) acetone water. Each section was 100 ml, and the eluate was fractionated.
A total of 1.3 L of eluate from fraction 5 to fraction 17 was collected,
This was adsorbed onto a Diaion PA306S (manufactured by Mitsubishi Kasei Corporation) packed column (8 x 60 cm), washed with 1 liter of distilled water, and then washed with 0.01M phosphate buffer (PH) containing 3.0% sodium chloride.
8.4). The eluate was fractionated into 100 ml portions, and a total of 1.5 liters of eluate from fraction 8 to fraction 17 was collected. This eluate was adsorbed onto a Diaion HP-20 packed column (4 x 70 cm), and the concentration ranged from 0 to 30.
Elute with a total of 4 l of acetone water increasing linearly to %. Each section was 15 ml, and the eluate was fractionated. By bioassaying each fraction, 400 ml of active fractions from fraction 61 to fraction 85 were obtained. This active fraction was introduced into a Biogel P-2 (manufactured by BioRad) packed column (8 x 100 cm) equilibrated with 0.01M phosphate buffer in advance, and developed with the above buffer.
1.4 l of active fraction was collected by bioassay. QAE with this active fraction equilibrated in advance with the above buffer
The eluate was adsorbed on a Cephadex A-25 (manufactured by Pharmacia) packed column (4 x 100 cm).
After carefully adjusting the pH to 8.4 with 10% NaOH,
It was adsorbed onto a Diaion HP-20 packed column (3 x 50 cm) pre-equilibrated with 0.01M phosphate buffer (PH8.4) and eluted with acetone water whose concentration increased linearly from 0 to 30%. One section was 15 ml, the eluate was fractionated, and the antibiotic OA-6129B sections from section 45 to section 50 were collected to obtain 90 ml. This was freeze-dried to obtain a pale yellow powder of 8.0. The obtained freeze-dried sample had the following characteristics. (1) Shape: Pale yellow powder (2) Specific rotation: [α] D ²⁴ 14.7° (c = 1.0, 0.01M phosphate buffer, PH8.4) However, ε of λmax 300nm in ultraviolet absorption
This is the value when it is set to 5400. (3) Molecular formula Theoretical molecular formula C ₂₀ H ₃₀ N ₃ O ₈ SNa (MW=495) (4) Ultraviolet absorption spectrum 0.01M phosphate buffer (PH8.4) λmax nm (ε) 300 (5400) (5) Main peak of infrared absorption spectrum (KBr) ν ^KBr _nax cm ^-1 : 1760 (β-lactam) 1660 (amide) 1600 (carboxylate) (6) Nuclear magnetic resonance spectrum (solvent: D ₂ O) (internal reference: DSS): Attach the chart No. 1
As shown in the figure. Further, the characteristic signals are as follows. δ (ppm) 0.87 (3H, s,

【formula】) 0.92(3H,s,

【formula】) 1.32 (3H, d, J = 7.0Hz,

[Formula]) 2.49 (2H, t, J=7.0Hz, NH−CH ₂ −CH ₂ −
CO) 3.97 (1H, s,

[Formula]) (7) Paper Chromatography Toyo Paper No.50 Developing solvent Acetonitrile/0.1M tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (PH7.5)/0.1M sodium ethylenediaminetetraacetate aqueous solution (PH7.5 ) (120/
30/1) Detection method Bioautography Rf value using Comamonas terrigena B996 0.12 (Rf value of OA-6129A showed 0.24) (8) Hydrolysis product (6NHCl, 115℃, 18 hours) PH1.8 buffer Paper electrophoresis using liquid (3000V, 20
Cysteamine (Rm value 2.26) and β-alanine (Rm value 1.53) were confirmed. (However, Rm value is based on alanine as 1.0) (9) Color reaction Reaction to ninhydrin: Negative Reaction to Ehrlich reagent: Positive (10) Elemental analysis, melting point Unmeasurable due to hygroscopicity (11) Benzine ester Behavior of antibiotic OA−2169B sodium salt (5.0)
mg was dissolved in 20 ml of dimethylformamide, 0.02 ml of triethylamine was added under ice cooling, and 0.02 ml of benzyl bromide was added while stirring. After reacting at the same temperature for 30 minutes, the reaction was continued at room temperature for 3 hours. The reaction solution was poured into 50 ml of ethyl acetate, washed with 10 ml of 0.01 molar phosphate buffer (PH8.4) saturated with sodium chloride, and the aqueous layer was further extracted again with 50 ml of methylene chloride. The extracts were combined, dried over sodium sulfate (anhydrous), and then evaporated under reduced pressure. Dissolve the residue in a small amount of methylene chloride and fill with benzene/acetone (1/1) 12 g of silica gel.
It was adsorbed onto a column of 1/2, developed sequentially with a mixed solvent of benzene/acetone (1/1) and (1/3) and acetone, and the fraction eluted with acetone was collected and concentrated to yield acetone/ethyl acetate (1/3).
In 1) silica gel TLC developed with mixed solvent,
Substances exhibiting UV absorption were observed at Rf values (0.59) and (0.52) (OA-6129A showed an Rf value of 0.68).

[Brief explanation of drawings]

FIG. 1 is a chart of the nuclear magnetic resonance spectrum of the antibiotic OA-6129B of the present invention.

Claims (1)

[Claims] 1 formula Compounds represented by and salts thereof. 2. The compound according to claim 1, wherein the salt is a sodium salt.