JPS5951795A - Novel antibiotic sf-2198 substance and its preparation - Google Patents

Abstract

PURPOSE:To prepare a novel antibiotic SF-2198 substance or its salt showing extremely high antibacterial activity against Gram-positive and Gram-negative bacteria, by cultivating a bacterium belonging to the genus Actinomadura. CONSTITUTION:A bacterium such as Actinomadura sp. SF-2198 strain (FREM-P 6650) belonging to the genus Actinomadura, capable of producing antibiotic SF- 2198 substance is cultivated at 26-36 deg.C for 5-15 days preferably in agar, antibiotic SF-2198 substance is collected mainly from a culture filtrate, and it is preferably converted into hydrochloride.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic and a method for producing the same, and more specifically, the antibiotic 8F-2198 substance and its salts, and
Antibiotic 8F-2198 substance-producing bacteria belonging to the genus P. adura were cultured in a medium, and from the obtained culture
The present invention relates to a method for producing a novel antibiotic 5F-2198 substance or a substance thereof, which is characterized by collecting the F-2198 substance or a salt thereof.

The present inventors have discovered that a substance exhibiting antibacterial activity against Gram-positive and Gram-negative bacteria is produced in the culture of certain bacterial strains, and the active substance has been isolated purely from the culture. As a result of investigating its properties, it was confirmed that it was a new substance different from known substances, and this active substance was identified as 8P-21.
It was named 98 substances.

Therefore, the first aspect of the present invention is a white to pale yellow basic substance, and its hydrochloride is a white to pale yellow powder, easily soluble in water, methanol, ethyl acetate, chloroform,
It is sparingly soluble in alkaline water, and when its molecular weight is measured by secondary ion mass spectrometry, M/Z
, 95.2.854°822.511, the decomposition point is around 150°C, the specific optical rotation is [α 5-232° (c O15, water), and the first
The ultraviolet absorption spectrum as shown in the figure and the infrared absorption spectrum as shown in Fig. 2 indicate chromatography, and the color reaction is positive for Lemieux and iodine reagents, negative for ninhydrin and Itami reagents, and the silica gel thin layer The nf value in chromatography is Rf = 0,76C chloroform-methanol; 5:1)
T'Cf=0.67 (butanol-acetic acid-water; 2:1:
1) To provide a novel antibiotic 5F-219'8 substance and its salt, characterized in that 'Rf=0.71 (butanol-isopropanol-water; 7:7:6).

Furthermore, according to the second invention, there is provided a novel method characterized in that an antibiotic 5F-2198 substance-producing bacterium belonging to the genus Actinomadeura is cultured, and an antibiotic 5F-2198 substance or a salt thereof is collected from the obtained culture. Antibiotic 5F-219
A method for producing eight substances or salts thereof is provided.

The new antibiotic 5F-2198 substance production pile may be of any type as long as it has the ability to produce sufficient 5F-2198 substance to be collected in its culture. An example of a bacterial strain is 5F, which was newly isolated by the present inventors from Kyoto-type Shimizu-mae soil.
There are -2'198 strains. The orchidological properties of the 5F-2198 strain are as follows.

(2) The morphobase hyphae are well elongated and branched, and the diameter is about 0.5 micron. Subdivision of basal hyphae is usually not observed in either agar or liquid media.

Aerial mycelium is very poor and does not adhere to most of the media commonly used for culturing actinomycetes, but white aerial mycelium grows on calcium malate agar and spore formation is good. Sometimes, a few aerial mycelia are observed on starch agar. The branches of the aerial hyphae are simple branches, and no axle branches are observed. No sporangia, flagellated spores, or sclerotia are observed. Spore chains can be linear, hook-shaped, or loop-shaped.

When observed with an electron microscope, the spores are oval to cylindrical and 0.6
It has a size of ~0.7xO08~1.3 microns, and chains of spores are often 5-10. The surface of the spores is large wrinkled or warty.

(2) Growth status on various media The growth status of SF-2198 strain on various media is as shown in the following table. The color descriptions and standards shown in [ ] are those of the Container Corporation of America.
fAmerica) Company W's ``Color Harmony Manual''
) The one described in J was used. Observations were made at 28°C, 14
This was carried out after ~21 days of culture.

■Physiological properties (1) Growth temperature range: 15℃~ in starch agar
It grows in a temperature range of 40°C, with an optimum temperature of 26°C to 36°C.

(2) Liquefaction of gelatin: Positive (cultured at 20°C, 20 days) (3) Hydrolysis of starch: Positive (cultured at 28°C, 14 days) (4) Reduction of nitrate: Positive (cultured at 28°C, 14 days) (
5) Peptonization of skim milk: Negative (28℃, 57℃, 1
4 days culture) Coagulation of skim milk: Negative (28°C, 237°C).

(14 day culture) (6) Production of melanin-like pigment: Negative IV, carbon source availability (Pridham-Gottlieb agar medium): (1) Use: D-glucose, ■, -arabinose, D-mannitol, D- xylose.

L-rhamnose, sucrose (2) usage is questionable: D-fructose (3) not used: I-inositol, raffinose■, cell wall composition: meso-diaminopimelic acid and maduroseka in whole cell hydrolyzate of 5F-2198 strain Admitted.

Based on the above properties, strain 5F-2198 is a strain belonging to the genus Actino+nadura among actinomycetes. Therefore, 1 the inventors
8 strains of Actinomadeura sp. SF-2198 (
Actinomadura sp, 5F-2198).

This strain has been deposited with the Microtech Institute, and its accession number is No. 6650.

The properties of the 8F-2198 strain are susceptible to change, as is the case with many other strains of actinomycetes, such as exposure to ultraviolet light,
Although it can be mutated by artificial mutation methods using X-rays, radiation, chemicals, etc., any mutant strain
All strains of the genus Actinomadeura that have the ability to produce the F-2198 substance can be used in the method of the present invention.

In the method of the present invention, the strain is cultured in a medium containing nutrients that can be utilized by common microorganisms. As the nutrient source, any known nutrient source conventionally used for culturing actinomycetes can be used. For example, glucose, glycerol, starch, sucrose, dextrin, starch syrup, molasses, vegetable oil, animal oil, etc. can be used as the carbon source. Also, soybean flour is used as a nitrogen source.

Wheat germ, meat extract, peptone, yeast extract, dried yeast, cornstap liquor, cottonseed meal, fish meal, ammonium sulfate, sodium nitrate, urea, etc. may be used. Calcium carbonate as needed.

Add inorganic salts such as cobalt chloride, cobalt chloride, and phosphate. -! 5F-219 helps the growth of bacteria.
Organic and inorganic substances that can promote the production of 8 substances can be appropriately added.

As a culture method, deep liquid culture is also possible, but agar culture is suitable because it has relatively good productivity. A suitable temperature for culturing is 26-56°C. The production of 5F-2198 substance is 5~
Accumulation reaches its maximum in 15 days.

For assaying the 5F-2198 substance, Bacillus subtilis A
A bioassay method using TCC6633 as the test bacterium is used. In this assay method, S, F-2198 substance is 0.625
In the concentration range from mcg/TLl to 10 mcg/ml, a linear relationship is established between its logarithm and the inhibition circle diameter, giving inhibition circle diameters of 11 to 21 L, respectively.

Since the 5F-2198 substance has the physical and chemical properties described below, it can be extracted and purified according to its properties, and can be efficiently extracted and purified by the following method. That is, the active ingredient is contained both in the aqueous layer obtained when agar is frozen and thawed and in the extract from the remaining solid matter with 50% acetone water. After distilling off the acetone, the acetone extract is combined with the aqueous layer of the former, and this is adsorbed onto a porous adsorption resin such as Diaion HP-20 (Mitsubishi Kasei), and then mixed with water such as acetone, methanol, etc. The active ingredient is eluted with a mixed solvent of water and an organic solvent that can be mixed with water. After further distilling off the organic solvent, the pi (adjusted to 9) was extracted with a solvent such as butanol or ethyl acetate, and further dissolved in water at pH 2 and lyophilized to obtain a crude powder of 8F-2198 substance. Powder, CM Sephadex (manufactured by Pharmacia).

Purification is performed by using a suitable combination of column chromatography such as Sephadex G-10 (manufactured by Pharmacia) to obtain a pure 5F-2198 substance. The 5F-2198 substance thus obtained was judged to be a pure product since it gave a single spot in thin layer chromatography using various solvent systems. Note that this substance is unstable in its free form, so it was obtained as a pure product as a hydrochloride.

The physicochemical properties of the hydrochloride of the 8F-2198 substance of the present invention are as follows.

Elemental analysis: carbon 47.64 parts, hydrogen 5.88 parts, nitrogen 4 parts
．． 24 units, sulfur 10.27 units, halogen 8.42 units.

Molecular precision: Secondary ion mass spectrometry (SI
MS), an ion peak was observed at M/Z 932.854.822.511. (Field desorption of the free base of this substance is carried out using the Kutle method (FDMS).
As a result of measurement, a peak was observed at M/Z510.

) Melting point (decomposition point): 150'C: Starts to color from around 150'C,
Browning occurred at around 16 UJ°C, and no change was observed even after the temperature was raised to 26[1'C].

Specific optical rotation: [α] 5-232° (cO05, water) Ultraviolet absorption spectrum: As shown in FIG. 1, there is no clear absorption maximum, but a shoulder near the following wavelengths.

(1) In water bath solution: 270 nm (shoulder) (B: 276).

520 nm (shoulder) (E', '432>.

(2), 0.1N sulfuric acid medium: 270 nm (shoulder) (Bicm (Ss).

520 nm (scrap) (H, #l2B).

(3) In 0.1N caustic soda: 250 nm (shoulder) (El, 120).

280 nm (shoulder) (B1., 80).

550 nm (shoulder) (El. 52).

Infrared absorption vector: Shown in Figure 2.

Color reaction Niremu, positive for iodine reagent.

Negative for ninhydrin and plate reagent.

Appearance: White to pale yellow powder.

Distinction between neutral, acidic, and basic: High pressure p paper electrophoresis (+) H
IS, 4.3000V, 15 minutes), it migrated 2.2 times as much as hairlanin on the cathode side, and it is considered to be a basic substance based on its purification behavior.

Silica gel thin layer chromatography: Rf=0.76 (chloroform-methanol; 5:1)
= 0.67 (butanol-acetic acid-water; 2:1:1) = 0
．． 71 (butanol-isopropanol-water; 7:7:
6) Solubility: Easily soluble in water and methanol.

Slightly soluble in ethyl acetate, chloroform, and basic water.

The minimum inhibitory concentration of substance 8F-2198 against various test bacteria is shown in Table 1, and it shows very strong anti-activity against Gram-positive bacteria and Gram-negative bacteria.

In addition, the acute toxicity using mice was 5 Q my, /
A test was conducted using intraperitoneal administration of 2 animals per group at 1 kg, but some died on the gold side.

Therefore, this substance is useful as a pharmaceutical, veterinary drug, bactericidal disinfectant, or a synthetic intermediate thereof.

8F having the above-mentioned physical and chemical properties and biological properties
Substance -2198 was determined to be a new substance, unlike any known substance.

Below in Table 1, examples of the method for producing 5F-2198 substance are shown.
It goes without saying that many modifications and modification means not exemplified here can be used.

Example 1 (1) Culture of SF-2198 strain Actinomadeura sp. 8F-2198 strain (Feikoken Bibori No. 665o), which had been cultured on a starch slanted agar medium at 28° C. for 14 days, was used as the inoculum. As a seed medium, soluble starch 1. (In, glucose 1.0%, polypeptone 0.5 parts, yeast extract 0.5 parts, meat extract 0.
A liquid medium (pI (no d-period adjustment)) consisting of 2'l of soybean meal, 0.2% of soybean meal, 0.1% of calcium carbonate, and tap water was used.

In advance, in a 100 ml Erlenmeyer flask, 5 to 6 platinum seeds were pressed into 2 r3 ml of the above sterilized seed medium, and 28
Culture with shaking using a rotary shaker for 3 days at °C.
The l@nutrient was used as seed culture (using 7 Erlenmeyer flasks)
.

Glucose 1.25 part, Wheat Belly V-1.0 part, Sungrain (manufactured by Zantry) 0.25 part 9 Salt 0.125 part,
After sterilization, a production medium consisting of 2.0% powdered agar and tap water (I7°0 before sterilization) 61 is dispensed into 60 large veterinary dishes (diameter 23 CIIL, height 6.5 cm) in 200 ml portions. After the agar has solidified, add the seed culture as described above)
The seeds are inoculated in dml portions into IJ dishes, and the seeds are smeared onto the entire surface of the agar using a sterilized glass rod, followed by static culture for 10 days in a culture room at 28°C.

After culturing, the agar culture in the vetri dish is taken out, transferred to a stainless steel tray, and frozen in a -20°C freezer. The frozen agar culture is then thawed using hot water (heating the outside of the tray with hot water), diatomaceous earth is added as a filter aid and immediately filtered to remove the solids containing agar and bacterial cells. It separates into liquid (5, 41). 5F-2198
The substance is mainly contained in the cultured Ffffl, but also remains in the solid material. The 8F-2198 substance in the solid was extracted using 50 thiacetate As i ).

(21S F-2198! Snostum) Extraction and Purification 50 After distilling off the acetone of the extract (5.4 g) with thiacetone water under reduced pressure, it was combined with the filtrate (51), and the Diaion HP
-20 (manufactured by Mitsubishi Kasei) was applied to a column of 40 oil to adsorb the active ingredients. The column was sequentially washed with water and 50% methanol water, and the active ingredient was eluted with 80% acetone water. After combining the active sections 11 and distilling off the acetone under reduced pressure, the pH was adjusted to 9.
Extract the active ingredients into 200 ml of ethyl acetate, and
The active ingredient was transferred to the aqueous layer with 2001 ml of H2 water.

The aqueous layer was adjusted to pH 4, concentrated to 501111 under reduced pressure, and
1 column to adsorb the active ingredients. After washing this tower with water, the active ingredient was eluted with 0.1 molar saline solution, and active ingredient 1
20m1 merged, Diamond ion)-I I)-20
15m/! The active ingredients were adsorbed in the column.

The column was sequentially washed with water and 50% methanol water, the active ingredient was eluted with 80% thiacetone water, 40ml of active fraction was combined, the pH was adjusted to 4.8, the C layer was formed under reduced pressure, and then freeze-dried. Thus, 13 my of powder with a purity of 80 was obtained.

Example 2 35 ml of powder with a purity of 80 fl) obtained by repeating the method of Example 1 was dissolved in 5 ml of 30 methanol water, and Sephadex Id-T-20 (
In a 250 m1 tower of Pharmacia (manufactured by Pharmacia), 0 methanol ('550 m1), 604 methanol (60 (
3rnl), 9CJ'ly meta/-ru (2,17')f
The active fractions (25 oil) were sequentially expanded, concentrated under reduced pressure, and then lyophilized to obtain 45 m9 of pure powder of 5F-2198 substance.

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of 5F-2198 substance, (1) is neutral water, (2) is 0.1N sulfuric acid, (
'5) is 0.1N caustic soda, both 25mcg
It was measured at a concentration of /ml. Figure 2 is 5F-2
The infrared absorption of 198 substances was measured as a potassium bromide tablet. 1. Procedural amendment (voluntary) November 15, 1980 To the Commissioner of the Japan Patent Office 1. Indication of the case 1988 Patent application No. 162291 2, Name of the invention Novel antibiotic 8F-2198 substance and its manufacturing method 3, Relationship with the person making the amendment No. 1 Patent applicant address 2-4-16 Kyobashi, Chuo-ku, Tokyo (609 ) Name Meiji Seika Co., Ltd. 4, Agent 105 Address Bussan Building Annex, 1-15 Nishi-Shinbashi, Minato-ku, Tokyo Phone: (591) 0261 Claims 11.u and invention of the specification subject to amendment Details A11
1 Explanation Column 6, Contents of Amendment (1) % The scope of the claims will be amended on a separate sheet. (-2) Delete "de" on page 3, line 12 of Meisho 1, and insert "begins to become colored and turns brown near ITO C; J." (3) From the bottom of page 14', Hiroyuki's rN/HJJ is rNI
Correct as HJ J. Claims t It is a white to pale yellow basic substance, and its hydrochloride is a white to pale yellow powder, easily soluble in water and methanol, slightly soluble in ethyl acetate, chloroform, and alkaline water, and its molecular weight When measured by secondary ion mass spectrometry, M/Z is 932. , ♂j Hiroshi,
122. It shows an ion peak at j//, and its decomposition point begins to change color around tro℃, turns brown around ito℃, and has a specific optical rotation of [α]25-co3co' (c O, evening, water). It showed an ultraviolet absorption spectrum as shown in Figure 7 and a general infrared absorption spectrum as shown in Figure 2, and the color reaction was positive for Lemieux and Yoo P reagents and ninhydrin. It was negative for the plate reagent, and the Rf value by silica gel thin layer chromatography was Rf = 0.7bu (chloroform-methanol; J-
:/) R, f=θ, t7 (butanol-acetic acid-water; λ:
/:/) A novel antibiotic 5F-2/f# substance and its salt, characterized in that f=0.71 (butanol-isozorononol-water: 7:A). 2. Cultivate bacteria producing the substance 8F-2 belonging to the genus Actinomadeura, and extract antibiotic 8F-2 from the resulting culture.
Novel (1. Method for producing biological 5isF-, ztqg substance or salt thereof) characterized by collecting F-2191 substance or salt thereof.

Claims (1)

[Scope of Claims] 1. It is a white to pale yellow basic substance, and its hydrochloride is a white to pale yellow powder, easily soluble in water and methanol, and slightly soluble in ethyl acetate, chloroform, and alkaline water. , when its molecular weight was determined by secondary ion mass spectrometry, M/Z was 952,854,
It shows an ion peak at 822,511, its decomposition point is around 150℃, and the specific optical rotation is [α几5-232°(
c O, 5, water], and showed a spectrum in the ultraviolet absorption spectrum as shown in Figure 1 and in the infrared absorption spectrum as shown in Figure 2, and the color reaction was positive for the Lemieux and iodine reagents. , ninhydrin, and the cross-grain reagent, and the nf value in silica gel thin layer chromatography is Rf = 0.
．． 76 (chloroform-methanol; s:i') Rf=
0.67 (butanol-acetic acid-water; 2:1:1) Rf
= 0.71 (butanol-isopropanol-water;
7:7:6) Novel antibiotic 5F
-2198 substance and its salt. 2. Antibiotic 8F-21 belonging to the genus Actinomadeura
1. A method for producing a novel antibiotic 8F-2198 substance or a salt thereof, which comprises culturing a 98 substance-producing bacterium and collecting the antibiotic 5F-2198 substance or a salt thereof from the resulting culture.