JPH0158197B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention describes the new antibiotic phosphonochlorin (fos-
Fonochlorin) and its production method. The present inventors isolated a soil-isolated filamentous fungus SANK15680 strain,
SANK10182 stock, SANK10282 stock and
We found that strain SANK10382 produces a novel antibiotic, phosphonochlorin, that is effective against Gram-positive and Gram-negative bacteria. Therefore, phosphonochlorin is used for the prevention or treatment of human, animal, or plant diseases caused by these bacteria. The present invention also includes pharmaceutically acceptable salts of phosphonochlorin. Examples of such salts include alkali metal salts such as sodium and potassium, alkaline earth metal salts such as calcium and magnesium, and aluminum salts. The mycological properties of the phosphonochlorin-producing strain are as follows. 1 Talaromyces flavus (Talaromyces
flavus) SANK15680 is an ascomycete isolated from soil collected by the present inventors in Taga-cho, Inuyama-gun, Shiga Prefecture. Growth on CYA medium is very good at 25°C.
The colony diameter reaches 33-35 mm in 7 days. The surface of the colony is woolly and pale orange-yellow, and the underside is gray-yellow. It forms many ascocarps (gymnothecia) but few conidia. It also produces a pale yellow soluble pigment. The ascus is made of a yellow mycelial network, spherical, has no opening, and has a diameter of 200 to 500 μm.
It is. The ascocarp primordium consists of a thick, long ospore and a thin sperm wrapped around it. Most of the cysts are club-shaped, with a length of 30 to 200 μm and a diameter of 3 to 200 μm.
It is 5μm. The asci are almost spherical, colorless, octosporous, 8-13 μm in diameter, and effacement. Ascospores yellow, oval, 3.5-5.5 x 2.5-3.0 μm
It has thorns all over. The conidiophore is smooth and develops from the basal hyphal layer, 20
~70 x 1.5-2.0 μm. Penicilli are unilobed or bilobed. Metole grows in clusters of 2 to 3, and is 10 to 15 x 2 to 2.3 μm. Fireride is pen-shaped with 2 to 5 whorls per metre, 7.5
~15 x 2.0~2.5μm. Most of the conidia are oval, 2.0-4.0 x 1.5-2.5 μm, smooth and chained. On CYA medium, growth is slightly slower at 37°C than at 25°C, and the colony diameter is 32 to 33 mm.
Asci formation is very low and no soluble pigments are produced. The underside is grayish-yellow to orange-brown. Spores and conidia do not germinate on CYA medium at 5°C. On MEA medium, ascocarp formation approximates but is better than on CYA medium;
Does not produce soluble pigments. Growth was extremely poor on G25N medium, with colony diameter being 4 to 5 mm in 7 days, forming conidia but not asci. As a result of searching for strains with the above-mentioned characteristics, I found a copy of “The genus Penicillium” by JIPitt.
andits teleomorphic states Eupenicillium
and Telaromyces” (Academic Press, 1979)
The results were in good agreement with the mycological characteristics of Talaromyces flavus as described in detail in the publication. Therefore, the SANK15680 strain was transformed into Talaromyces flavus (Klo¨cker) Stolk.
et Samson). The composition and preparation method of the medium used here are described in the above-mentioned document. 2 Fusarium oxysporan (Fusarium
oxysporum) SANK10182 is a deficient bacterium isolated from soil collected in Tazawako-cho, Semboku-gun, Akita Prefecture. Potato sucrose agar medium (hereinafter referred to as
PSA (abbreviated as PSA) has good growth on 26
℃, the colony diameter reaches 4.5-5.0 cm on the 4th day. The center of the colony is pale purple, the periphery is almost white and velvety, and the underside is cream-colored. Although complete generations are not observed, large numbers of large and small conidia are formed. Large conidia are translucent and sickle-shaped, tapering on both sides. It is also often hooked inward at one or both ends and has 2 to 6 septa. Its size is 20 to 28 x 4.5 with two partitions.
5.5μm, 25~35×4.5~ with 3 partitions
5.0μm, 25~40×4.5~ with 4 partitions
5.5μm, 35~50×4.5~ with 5 partitions
5.5μm, 40~50×4.5~ with 6 partitions
It is 5.5 μm. Large conidiophores form metres or direct phialides on the hyphae and are 10-30 x 3-5 μm. The small conidia are colorless, oblong and slightly curved, have no septa, and are 5-10 x 3-5 μm in size and are not linked together. Small conidiophores form simple phialides on aerial hyphae and are 5-30 x 2.5-4.5 μm. It forms numerous chlamydospores, subglobular, smooth or rough, and 8-12 μm in size. Chlamydospores are mostly apical and usually solitary, but in rare cases they may form chains of 2 or 3. On the other hand, it does not grow at 37℃. As a result of searching for strains with the above characteristics, I found "The genus Fusarium" by C. Booth.
pp. 130-154, published 1971, Commonwealth
The mycological characteristics of Fusarium oxysporum were in good agreement with those described in detail in Mycolo-gical Institute, Kew, England. Therefore,
The SANK10182 strain was identified as Fusarium oxysporum. 3 Fusarium avenaceum (Fusarium
avenaceum) SANK10282 is a deficient bacterium isolated from persimmon seeds collected in Fuchu City, Tokyo. Growth on PSA was good, with colonies reaching approximately 5 cm in diameter on the 4th day at 26°C. Colonies are pale reddish-purple, woolly, and have a reddish-purple underside. Large conidia are often formed in the center, but no small conidia are observed. Large conidia are translucent, long spindle-shaped, and the tip is often inwardly curved and pointed, 3 to 5 (6)
It has a partition wall. Its size is 28 with 3 partitions.
~45×3.5~4.5μm, 40~50×4 with 4 partitions
~5μm, 45~63×4~ with 5 partitions
It is 5μm. Large conidiophores are of the polyblastic type and phialide type, and some are formed directly from aerial hyphae, while others are formed directly from aerial hyphae (4.5 to 4.5 in diameter).
7.0 μm). No chlamydospores are formed, nor are full generations formed. On the other hand, it does not grow at 37℃. As a result of searching for strains with the above characteristics, I found "The genus Fusarium" by C. Booth.
pages 91-94, published 1971, Commonwealth
The mycological characteristics of Fusarium avenaceum were in good agreement with those described in detail in the Mycological Institute, Kew, England. Accordingly
The SANK10282 strain was identified as Fusarium avenaceum. 4 Fusarium tricintum (Fusarium
tricinctum) SANK10382 is a deficient bacterium isolated from persimmon stems collected in Fuchu City, Tokyo. Growth on PSA was good, with colonies reaching approximately 4 cm in diameter on the 4th day at 26°C. The colony is woolly, pale purple-red in the center, almost white on the periphery, and reddish-purple on the underside. Large conidia are formed in large numbers especially in the center, but only a small number of small conidia are formed. Large conidia are translucent, sickle-shaped, and have (2)3-4(5) septa. The size is the one with two partitions.
15-25 x 2.5-4.5μm, 20 with 3 partitions
〜25×2.5～4.5μm, 26 with 4 partitions
35×3.0~4.5μm, 26~35 with 5 partitions
×2.5 to 4.5 μm. Large conidiophores consist of phialides directly on the hyphae or formed into metres, 10-30 x 3-
It is 5μm. The small conidia are colorless, smooth, unchained, 7.5-15.0 x 5.0-7.5 μm, and are mostly pear-shaped, although elliptical or subglobular ones are also present. Small conidiophores form phialides on hyphae and are 5-20 x 2.5-4.5 μm. Neither chlamydospores nor complete generations are observed. On the other hand, it does not grow at 37℃. As a result of searching for strains with the above characteristics, I found "The genus Fasarium" by C. Booth.
pp. 83-84, published 1971, Commonwealth
The mycological characteristics of Fusarium tricintum were in good agreement with those described in detail in the Mycological Institute, Kew, England. Therefore,
The SANK10382 strain was identified as Fusarium tricinctum. And SANK15680 stock, SANK10182 stock,
SANK10282 strain and SANK10382 strain have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and their microbial deposit numbers are No. 6534 and 6535, respectively.
No. 6536 and No. 6537. Although the phosphonochlorin-producing bacteria have been explained above, it is well known that the properties of these fungi are not constant and can easily change naturally or artificially, and the strains that can be used in the present invention are of the genus Talaromyces. It also includes all strains that produce phosphonochlorin belonging to the genus Fusarium. Furthermore, the incomplete generation of Talaromyces flavus is known as Penicillium dangeldei;
Furthermore, since the complete generation of Fusarium avenaceum is well known as Gibberella avenacea, these strains are naturally capable of producing phosphonochlorin and are included in the phosphonochlorin-producing bacteria of the present invention. Cultivation in the present invention is carried out in accordance with the cultivation method for general microorganisms, and is preferably carried out by shaking culture in a liquid medium or aerated agitation culture. Examples of medium components include carbon sources such as glucose, maltose, sucrose, mannitol, molasses, glycerin, dextrin, starch, soybean oil, and cottonseed oil, and nitrogen sources such as soybean flour, peanut flour, cottonseed flour, casamino acids, and firmamine. , fishmeal, corn
Steep liquor, peptone, meat extract, yeast, yeast extract, sodium nitrate, ammonium nitrate, ammonium sulfate, etc., and inorganic salts such as common salt, phosphate, calcium carbonate, trace metal salts, etc. are added as appropriate. During liquid culture, silicone oil, vegetable oil, surfactant, etc. are appropriately used as antifoaming agents. The pH of the medium is weakly acidic to near neutral, and the culture temperature is preferably 20°C to 30°C, particularly around 24°C. Time-dependent changes in the titer of phosphonochlorin produced as the culture progresses Paper disc with Proteus mirabilis SANK70461 as the test bacterium (Toyo Kagaku Sangyo)
It was measured using the 8mm diameter test method. The production of phosphonochlorin usually reaches its maximum value after 120 to 130 hours of culture. Phosphonochlorin, which mainly exists in the liquid part of the culture solution, can be removed by removing the bacterial cells and other solid parts after the culture is over by over-operation using diatomaceous earth as an aid, or by centrifugation. Alternatively, it is extracted and purified from the supernatant. Phosphonochlorin can be collected by utilizing its physicochemical properties, for example, using an adsorbent. Examples of adsorbents include activated carbon or adsorption resin Amberlite.
XAD-2, XAD-4, XAD-7, etc. (ROHM
and Haas), Diaion HP10,
HP20, HP20AG, HP50, etc. (manufactured by Mitsubishi Chemical Industries, Ltd.)
The solution containing phosphonochlorin is passed through a layer of adsorbent as mentioned above to adsorb and remove the contained impurities, or after adsorbing phosphonochlorin, methanol water, acetone water, n-butanol is used. Elute using water etc. Utilizing the fact that it behaves as an acidic substance, it can be purified by adsorption and desorption onto an anion or cation exchanger. Examples of anion exchangers include DEAE-cellulose (manufactured by Braun), DEAE-cephadex, QAE-cephadex (manufactured by Pharmacia), Duolite A-
2. A-2 (manufactured by Diamond Shamrock Chemical Co.), Amberlite IRA-68 (ROHM
& Haas), Dowex 1×4,
21K (manufactured by Dow Chemical Company) etc. can be used. or,
Amberlite IRC as a cation exchanger
50, Dowex 50W, etc. can be used. In addition, by utilizing the properties of acidic substances as mentioned above, phosphonochlorin can be extracted from an aqueous solution by dissolving a quaternary ammonium salt such as dimethyl benzyl cetyl ammonium chloride in a solvent that is immiscible with water, such as dichloromethane. A method of extraction as a salt is also used. To further purify phosphonochlorin, use cellulose such as Avicel (manufactured by Asahi Kasei Corporation) or Cephadex LH-20 (manufactured by Pharmacia).
partition column chromatography using a carrier such as silica gel, alumina, or adsorption column chromatography using a carrier such as Florisil, or reverse phase column chromatography using a carrier for reversed phase,
Effective methods include an extraction method that utilizes the difference in the distribution ratio of phosphonochlorin and mixed impurities to the solvent, or a countercurrent distribution method. Phosphonochlorin can be purified by repeatedly using the above purification means alone or in appropriate combinations. The phosphonochlorin thus obtained can be converted into a pharmacologically acceptable salt according to a conventional method, if desired. The phosphonochlorin thus obtained has the following physicochemical properties. 1. Properties of the substance: acidic, water-soluble colorless oily substance 2. Specific optical rotation: [α] ²⁵ _D O (C, 0.56, water) 3. Molecular formula: C ₂ H ₄ O ₄ PCl (high-resolution mass spectrometry of trimethylsilyl derivatives) 4 Molecular weight: 158 (measured by mass spectrometry of trimethylsilyl derivatives) 5 Ultraviolet absorption spectrum: λ _nax nm (E1% 1cm) The ultraviolet absorption spectrum measured in an aqueous solution was 276nm (E1%) as shown in Figure 1. Maximum absorption is shown at 1 cm = 19.7). 6 Infrared absorption spectrum: ν ^KBr _nax cm ^-1 The infrared absorption spectrum of the sodium salt measured with a KBr disk is shown in FIG. 3150, 1690, 1620, 1400, 1200 - 1050, 910 7 Nuclear magnetic resonance absorption spectrum: δ: ppm Nuclear magnetic resonance absorption spectrum (90MHz) measured using TMS (tetramethylsilane) as an internal standard in DMSO- _d6 ) is as shown in Figure 3. 3.7 (2H, d) 8 Solubility: Soluble in water and methanol, insoluble in acetone, chloroform, and ethyl acetate. 9 Color reaction: 1% ammonium molybdate solution - positive for 50% sulfuric acid, iodine, bromo cresol green. 10 Thin layer chromatography: R _f value 0.46 Adsorbent: Merck silica gel plate No. 5715 Developing solvent: 70% n-propanol-water11 Antibacterial activity: Minimum inhibitory concentration (MIC) of phosphonochlorin against various microorganisms is shown in Table 1. The MIC values for each microorganism were measured after culturing at 37°C for 24 hours using the agar dilution method using an ordinary agar medium manufactured by Difco.

[Table] As a result of comparing the properties of phosphonochlorin with those of known antibiotics, it was determined to be a new antibiotic. Phosphonochlorins are used for infections caused by Gram-positive and Gram-negative bacteria, such as sepsis, respiratory infections, and urinary tract infections. The mode of administration is oral or parenteral. For oral administration, for example, tablets, capsules, powders,
It is a granule, and for parenteral administration, for example, an injection. When preparing injections, it is preferable to sterilize using a Millipore filter or the like rather than sterilizing with an autoclave. The dosage varies depending on symptoms, age, weight, etc., but is usually about 1 day per day for adults.
It is preferable to administer 200 mg to 2000 mg once or in divided doses. This dosage can be further increased or decreased as necessary. Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples. Example 1 Fusarium oxysporum SANK10182 strain was grown in a medium consisting of 2.5% pine potato, 2.0% sucrose, 1.0% casamino acid, 0.5% potassium phosphate, and 0.25% magnesium sulfate (no PH adjustment before sterilization).
One platinum loopful was inoculated into ten 500 ml Erlenmeyer flasks (with navels) each containing 80 ml, and cultured at 24°C for 120 hours. Add 600ml of this culture solution to Dowex 1x.
4 (CH ₃ COO ^- form) was adsorbed onto a 40 ml column.
After washing the column and 2.5% acetic acid, 2.5% acetic acid at PH5.0;
Elution was carried out with pyridine buffer to obtain 200 ml of active fraction.
After concentrating this active fraction under reduced pressure and adjusting the pH to 3.0, it was adsorbed on a 100 ml column of Amberlite CG-50 (H ⁺ type) and developed with water to obtain 45 ml of an active fraction. The obtained active fraction was diluted to pH7.0 with dilute aqueous sodium hydroxide solution.
After adjustment, 55 mg of crude powder containing 7 mg of phosphonochlorin was obtained by freeze-drying. Example 2 Talaromyces flavus SANK15680 strain was grown in a medium 80 containing 2.5% mash potato, 2.0% sucrose, 1.0% casamino acid, 0.5% potassium phosphate, and 0.25% magnesium sulfate (no PH adjustment before sterilization).
One platinum loopful was inoculated into ten 500 ml Erlenmeyer flasks (with navels) each containing 500 ml, and cultured at 24°C for 96 hours in a rotary shaking incubator at 220 rpm. This culture solution was inoculated at 5% into 125 500 ml Erlenmeyer flasks (with navel attachments) containing 80 ml of the same medium, and cultured at 24°C for 120 hours in a rotary shaking incubator at 220 rpm. Combine the obtained culture solutions, add celite and filter.
Liquid 10 was obtained. Activated carbon for chromatography
column and dowex the passing liquid.
It was adsorbed onto a 600 ml column of 21K (CH ₃ COO ^- type).
After washing the column with 2.5% acetic acid, wash the column with 2.5% acetic acid at pH 5.0.
About 1700 ml of active fraction was obtained by elution with pyridine buffer. This active fraction was concentrated under reduced pressure, and after distilling off the acetic acid/pyridine buffer, Dowex 50W x 4 (H ⁺
It was adsorbed onto a 200 ml column (type) and developed with water to obtain 140 ml of active fraction. Further, this active fraction was concentrated under reduced pressure, adsorbed onto a 400 ml column of Amberlite CG-50 (H ⁺ type), and developed with water to obtain 150 ml of an active fraction.
The active fraction was further concentrated under reduced pressure and
The active fraction (80 ml) was adsorbed on a 50 W x 4 (H ⁺ type) column and developed with water, and the resulting active fraction was concentrated to dryness under reduced pressure to obtain 450 mg of phosphonochlorin. Example 3 Talaromyces flavus SANK15680 strain was cultured in the same manner as in Example 1, and the resulting culture solution 30 was adsorbed on Dowex 21K (CH ₃ COO ^- type) 1.8, washed with 2.5% acetic acid water, and then adjusted to pH 5.0. 15 was obtained as an active fraction by elution with 2.5% acetic acid/pyridine buffer. This active fraction was concentrated under reduced pressure to remove the acetic acid/pyridine buffer. Concentrate to 500ml and adsorb onto a 1000ml column of Dowex 50W x 4 (H ⁺ type).
It was developed with water to obtain 327 ml of active fraction. This active fraction was concentrated under reduced pressure to form Amberlite CG-50 (H ⁺ type).
After adsorption on a 1000ml column, develop with water and separate the active fraction.
Obtained 445ml. DEAE buffered this active fraction with triethylamine/acetate buffer at 0.05M and pH5.0.
-Sephadex A-25 It was adsorbed onto a 150ml column and sequentially eluted with 0.05M to 0.1M triethylamine/acetic acid buffer at pH 5.0 to obtain active fraction 5. Further, this active fraction was concentrated under reduced pressure, adsorbed onto a column of Dowex 50W x 4 (H ⁺ type) and developed with water to obtain 300 ml of an active fraction. Furthermore, this active fraction was concentrated under reduced pressure, and Amberlite CG-50
(H ⁺ form) was adsorbed onto a column of 1 and eluted with water.
420 ml of the obtained active fraction was concentrated to dryness under reduced pressure to obtain 1.4 g of an oil. 185 mg of the obtained oil was transferred to Merck's silica gel plate No. 5715 (20 x 20 cm) 10
Adsorb it onto a sheet and add 70% n-propanol-water for 15 minutes.
After cm development, the positive part was scraped off by color reaction with iodine and eluted with 80% methanol-water. After concentrating the eluate under reduced pressure to remove methanol, it was adsorbed onto a column of Dowex 50W x 4 (H ⁺ type) and eluted with water to obtain fractions 33 to 38 (fraction 10 ml).
60 ml of the active fraction was collected. This active fraction is 1/25N
The pH was adjusted to 7.0 with NaOH, and then concentrated under reduced pressure and freeze-dried to obtain 71.2 mg of the sodium salt of phosphonochlorin as a white powder.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectrum of phosphonochlorin. In the figure, 1 is 0.01N hydrochloric acid aqueous solution, 2 is
PH7.0, 3 indicates 0.01N sodium hydroxide aqueous solution. FIG. 2 shows the infrared absorption spectrum of the same material, and FIG. 3 shows the nuclear magnetic resonance absorption spectrum of the same material.

Claims (1)

[Claims] 1. Antibiotic phosphonochlorin and its pharmacologically acceptable salts. Here, phosphonochlorin has the following properties. 1. Properties of substance: acidic, water-soluble colorless oily substance 2. Specific rotation: [α] ²⁵ _D O (C, 0.56, water) 3. Molecular formula: C ₂ H ₄ O ₄ PCl (high-resolution mass spectrometry of trimethylsilyl derivatives) 4 Molecular weight: 158 (measured by mass spectrometry of trimethylsilyl derivatives) 5 Ultraviolet absorption spectrum: λ _nax nm (E1% 1cm) The ultraviolet absorption spectrum measured in an aqueous solution is
It exhibits maximum absorption at 276 nm (E1% 1 cm = 19.7). 6 Infrared absorption spectrum: ν ^KBr _nax cm ^-1 The infrared absorption spectrum of the sodium salt measured with a KBr disk is as follows. 3150, 1690, 1620, 1400, 1200 - 1050, 910 7 Nuclear magnetic resonance absorption spectrum: δ: ppm Nuclear magnetic resonance absorption spectrum (90MHz) measured using TMS (tetramethylsilane) as an internal standard in DMSO- _d6 ) is as follows. 3.7 (2H, d) 8 Solubility: Soluble in water and methanol, insoluble in acetone, chloroform, and ethyl acetate. 9 Color reaction: 1% ammonium molybdate solution - positive for 50% sulfuric acid, iodine, bromo cresol green. 10 Thin layer chromatography: R _f value 0.46 Adsorbent: Merck silica gel plate No. 5715 Developing solvent: 70% n-propanol-water 2 Phosphonochlorin-producing bacteria belonging to the genus Talaromyces or Fusarium were cultured. A method for producing phosphonochlorin, which comprises collecting phosphonochlorin from a culture. Here, phosphonochlorin has the following physical and chemical properties. 1. Properties of substance: acidic, water-soluble colorless oily substance 2. Specific rotation: [α] ²⁵ _D O (C, 0.56, water) 3. Molecular formula: C ₂ H ₄ O ₄ PCl (high-resolution mass spectrometry of trimethylsilyl derivatives) 4 Molecular weight: 158 (measured by mass spectrometry of trimethylsilyl derivatives) 5 Ultraviolet absorption spectrum: λ _nax nm (E1% 1cm) The ultraviolet absorption spectrum measured in an aqueous solution is
It exhibits maximum absorption at 276 nm (E1% 1 cm = 19.7). 6 Infrared absorption spectrum: ν ^KBr _nax cm ^-1 The infrared absorption spectrum of the sodium salt measured with a KBr disk is as follows. 3150, 1690, 1620, 1400, 1200 - 1050, 910 7 Nuclear magnetic resonance absorption spectrum: δ: ppm Nuclear magnetic resonance absorption spectrum (90MHz) measured using TMS (tetramethylsilane) as an internal standard in DMSO- _d6 ) is as follows. 3.7 (2H, d) 8 Solubility: Soluble in water and methanol, insoluble in acetone, chloroform, and ethyl acetate. 9 Color reaction: 1% ammonium molybdate solution - positive for 50% sulfuric acid, iodine, bromo cresol green. 10 Thin layer chromatography: R _f value 0.46 Adsorbent: Merck silica gel plate No. 5715 Developing solvent: 70% n-propanol-water 3 Phosphonochlorin-producing bacteria belonging to the genus Talaromyces or the genus Fusarium are Talaromyces.
Flavus SANK15680 strain (Feikoken Bacteria No. 6534
), Fusarium oxysporum
SANK10182 strain (Feikoken Bacillus No. 6535), Fusarium avenaceum SANK10282 strain (Feikoku Kenkyori No. 6536), and Fusarium tricincumum
The production method according to claim 2, which is the SANK10382 strain (Feikoken Bibori No. 6537).