JPH0615555B2 - Antibiotic Zofimarin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a new antibiotic, Zofimarin, a method for producing the same, and an antibacterial agent containing the same as an active ingredient.

The present inventors have found that the ascomycete fungus Zofuiera marina SANK 21274 strain isolated from the seabed mud of 120 m undersea in the East China Sea produces the new antibiotic zofimarin, and completed the present invention.

The antibiotic zofimarin of the present invention has the following structural formula.

Zofuimarin has antibacterial activity against fungi including deep fungal pathogens such as Cajundeida albicans, Trichophyton mentaglophytes, and so Zofuimarin is used for the prevention or treatment of human, animal or plant diseases caused by these fungi. Used for.

Zofuiera Marina SANK 2 producing Zofui Marine
The 1274 strain was published in Kohei Furuya and Shunichi Udagawa, "Plant Research Magazine", Vol. 50, No. 8, 1975, and has been deposited as CBS155.77. The zofimarin-producing bacterium in the present invention is not limited to the strains described in the document, but includes all strains that produce zofimarin belonging to the genus Zophieella.

The culturing in the present invention is carried out according to the culturing method in general fungi, and is carried out by shaking culturing in a liquid medium or by aeration and stirring culturing, which is characterized by using artificial seawater instead of tap water. To do. The medium components include, for example, glucose, maltose, sucrose, sugar concentrate, glycerin, dextrin, starch, soybean oil, cottonseed oil and potato as carbon sources, and soybean flour, peanut flour, cottonseed flour, pharmamine, fish meal as nitrogen sources. , Corn steep liquor, peptone, meat extract, yeast, yeast
Extract, casamino acid, sodium nitrate, ammonium nitrate,
Ammonium sulfate and the like, and salt, phosphate, calcium carbonate, magnesium sulfate, trace metal salt and the like as an inorganic salt are appropriately added as necessary. In liquid culture, silicone oil, vegetable oil, surfactant, etc. are appropriately used as an antifoaming agent. The pH of the medium is weakly alkaline or close to neutral, and the culture temperature is preferably 20 ° C to 30 ° C, particularly around 26 ° C. The time course of the titer of zofimarin produced in the culture solution with the progress of the culture was as follows: Candida albicanas SANK 50157 strain was used as a test bacterium, and the solution obtained by overnight culture was treated with yeast extract 0.4%. , 1.0% malt extract, 0.4% glucose, 1.5% agar, and a plate prepared by adding to the medium and measured by a paper disc (Toyo Kagaku Sangyo Co., Ltd., diameter 8 mm) assay method. Usually, the maximum production of zofimarin is reached after 60 to 80 hours of culture. After the completion of the culture, the zofimarin, which is mainly present in the liquid part of the culture solution, is removed by the overoperation of the bacterial cells and other solid parts with diatomaceous earth or the like as an auxiliary agent, or by centrifugation, and the solution or supernatant. It is obtained by extracting and purifying from the inside. By taking advantage of its physicochemical properties, zofimarin can be collected using, for example, an adsorbent. Examples of the adsorbent include activated carbon or adsorbent resins such as Amberlite XAD-2, XAD-4, XAD-7 (made by Rohm and Haas) and Diaion HP10, HP20, HP20AG, HP50 (Mitsubishi Kasei Kogyo Co., Ltd.) is used, and a liquid containing zofimarin is passed through a layer of an adsorbent as described above to adsorb impurities contained in the liquid containing zofimarin, or after adsorbing zofimarin. , Methanol water, n-butanol water, acetone water, etc. to elute it.

Alternatively, a method of collecting a neutral fat-soluble substance from a culture solution, for example, extraction from a culture solution or an aqueous solution by an organic solvent immiscible with water, such as dichloromethane, chloroform, ethyl acetate, n-butanol, etc., alone or in combination thereof. Can also be obtained by In order to purify the zofimarin thus obtained, reverse phase partition chromatography using a carrier having an octadecyl group, an octyl group, etc .: Avicel (Asahi Kasei Co., Ltd.)
Partition column chromatography using cellulose such as Cefaddex LH-20 (manufactured by Pharmacia): Adsorption column chromatography using a carrier such as silica gel, alumina, florisil: Or against a solvent with impurities mixed with zofimarin An extraction method that utilizes the difference in distribution ratio is a useful method. Zofimarin can be purified by using the above-mentioned purification means alone or in appropriate combination and repeatedly used. Zofimarin, like general fat-soluble antibiotics, is present in the bacterial cell portion in the culture medium depending on the culture conditions. in this case,
Extraction with a hydrophilic organic solvent such as alcohols and acetone, removal of the solvent from the extract, and subsequent formation of an aqueous solution can be followed by extraction and purification in the same manner as in culture.

The zofimarin thus obtained has the following characteristics.

1) Color and shape of substance: colorless oily substance 2) Specific irradiance [α] ^22.5 _D = -13.2 ° (c0.5, methanol) 3) Molecular formula: C ₃₃ H ₄₆ O ₉ (molecular weight 586) 4) Mass spectrometry (FAB-MS method, molecular ion peak): m / z586 5) UV absorption spectrum: The ultraviolet absorption spectrum measured in methanol shows the first
As shown in the figure.

6) Infrared absorption spectrum: The infrared absorption spectrum measured in ν _max cm ^-1 KBr is shown in FIG.

7) Proton-Nuclear Magnetic Resonance Spectra: δ: ppm Proton-Nuclear Magnetic Resonance Spectra (270M
Hz) is as shown in FIG.

8) Carbon-13-nuclear magnetic resonance spectrum: δ: ppm Carbon-13-nuclear magnetic resonance spectrum (22.5 ppm) measured using tetramethylsilane as an internal standard in deuterated chloroform.
MHz) is as shown in FIG. 4, and shows the following chemical shift (ppm) and multiplicity.

9) Solubility in solvent: Soluble in methanol, ethanol, acetone, ethyl acetate and chloroform, and sparingly soluble in water and n-hexane.

10) Color reaction: Positive for iodine and sulfuric acid.

11) High performance liquid chromatography: It has a retention time of 4.6 minutes under the following conditions.

Column: Waters, Novapack inner diameter 8 mm Solvent: Acetonitrile: 0.2% triethylamine-phosphate buffer pH 3.5 (65:35) Flow rate: 2.5 ml / min Detection: UV210nm 12) Thin layer chromatography: Rf value 0.25 adsorbent Merck silica gel plate No.5715 developing solvent; n-hexane: ethyl acetate (1: 1) The zofimarin thus obtained exhibits activity against various fungi. The minimum inhibitory concentration (MIC) of zofimarin for various fungi is shown in Table 1. M
The IC was measured after culturing at 26 ° C. for 48 to 72 hours by a 2-fold dilution method using Sabouraud-dextrose liquid medium. Moreover, no antibacterial activity was observed at 100 μg / ml against bacteria.

When the above properties were compared with those of known antibiotics, there was no agreement, and zofimarin was judged to be a new antibiotic.

From the above, zofimarin is used as an antibacterial agent for controlling various bacterial infectious diseases. Examples of the dosage form include subcutaneous injection, intravenous injection, intramuscular injection, parenteral administration method such as suppository, and oral administration method such as tablet, capsule, powder, granule and the like. The dose varies depending on the disease to be treated, the route of administration, the number of administrations and the like, but for example, it is usually preferable to administer 1 mg to 50 mg daily to an adult once or in several divided doses.

Next, the present invention will be described in more detail with reference to examples.
The invention is not limited to this example.

Example 1 80 ml of A medium having the following medium composition is contained from a slope culture of Zofuimarina SANK 21274 strain, a Zofuimarin producing bacterium.
One platinum loop was inoculated into a 500 ml Erlenmeyer flask and cultured at 26 ° C for 65 hours using a rotary shaker at 140 rpm. 4 ml of this culture solution was inoculated into a 500 ml Erlenmeyer flask containing 80 ml of the same medium at 140 rpm at 26 ° C.
The cells were cultivated for 71 hours using the rotary shaking incubator. Under this condition, the amount of zofimarin produced was about 1 μg / ml. After the culture was completed, 4% (W / V) Celite was added to the culture and allowed to pass to obtain a culture medium 12. Adsorb the culture solution to 500 ml of Diaion HP20, wash with water, then wash with 40% methanol,
The active substance was then eluted with 60% methanol. The eluate was concentrated and methanol was removed, followed by extraction with ethyl acetate, and the obtained ethyl acetate layer was concentrated and dried under reduced pressure,
320 mg of the substance on oil were obtained. On the other hand, the bacterial cells obtained in excess of 3
Acetone was added and left overnight. After removing the cells by filtration, the liquid was concentrated and the concentrate was purified with 300 ml of Diaion HP20. 20 by the same method as the culture solution
0 mg of oily substance was obtained. This substance was combined with the oily substance extracted from the culture solution, dissolved in a small amount of methanol,
Reversed-phase partition chromatography was performed using a Rover column (RP-8 size B) manufactured by Merck. Acetonitrile:
2% triethylamine-phosphate buffer (TEAP buffer) pH
Elution was carried out at 3.5 (70:30), and the active fraction was examined by an agar plate production assay of Candida albicans. The active fraction was concentrated and then extracted with ethyl acetate, and the obtained ethyl acetate layer was concentrated and dried to obtain 120 mg of an oily substance. This substance was dissolved in a small amount of methanol, divided into several times, and subjected to reverse phase partition chromatography using a Novapack C-18 column manufactured by Waters. Acetonitrile: 0.2
% TEAP buffer (65:35) was used as a developing solution, and UV26
The peak detected at 0 nm near 4.6 minutes was collected. The separated solution was concentrated and then extracted with ethyl acetate. The obtained ethyl acetate layer was concentrated and dried to obtain about 13 mg of a single zofimarin, as an oily substance.

[Brief description of drawings]

Figure 1 shows the UV absorption spectrum of zofimarin.
FIG. 2 shows the infrared absorption spectrum of the same substance, FIG. 3 shows the proton-nuclear magnetic resonance spectrum of the same substance, and FIG. 4 shows the carbon-13-nuclear magnetic resonance spectrum of the same substance.

Claims (4)

[Claims] 1. An antibiotic, zofimarin, having the following structural formula: 2. A method for producing zofimarin, which comprises isolating zofimarin by culturing a zofimarin-producing bacterium belonging to the genus Zofuiera of the ascomycetes. 3. The method according to claim 2, wherein the zofimarin-producing bacterium belonging to the genus Zophieella of the ascomycete genus is Zophieella marina. 4. The method according to claim 3, wherein the zofimarin-producing bacterium belonging to the genus Zophieella of the ascomycete genus is Zofuiera marina SANK 21274.