JPH06263784A - New antibiotic mi 481-42f4-a-related substance - Google Patents

Abstract

PURPOSE:To obtain new substances excellent in antibacterial activity. CONSTITUTION:The antibacterial subsances, MI 481-42F4-A1 (molecular weight: 1182; molecular formula: C50H50O9N14S6), MI 481-42F4-A2 (molecular weight: 1217)and MI 481-42F4-A3 (molecular weight: 1199) can be obtained by degrading a new antibiotic substance MI 481-42F4-A. Having such antibacterial activity as to highly inhibiting the growth of Gram-positive bacteria and multi-drug- resistant bacteria, these antibacterial substances are useful as antibacterial agents for medical use.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel antibiotic MI481-42F4
-MI481-42F4-A1, a new antibacterial substance obtained by decomposing -A
It relates to -A2 and -A3 substances and salts thereof, and to a method for producing these substances.

[0002]

2. Description of the Related Art A large number of antibiotics produced by microorganisms have been discovered so far, and their decomposition products or derivatives are widely used for treating infectious diseases.

[0003]

In the treatment of infectious diseases, the emergence of resistant bacteria is inevitable, and new antibacterial antibiotics effective against resistant bacteria are constantly being sought.

[0004]

As a result of the above research, the present inventors have found that a strain belonging to the genus Amycolatopsis (FERM P-127
A novel antibiotic MI481-42F4-A was obtained from the culture solution of (39) (see Japanese Patent Application No. 4-142045, filed on May 8, 1992). This time, the present inventors have developed a novel antibacterial substance MI481-42 by degrading the antibiotic MI481-42F4-A by the method described below.
We succeeded in obtaining F4-A1, MI481-42F4-A2 and MI481-42F4-A3 substances.

Therefore, according to the first aspect of the present invention, an antibiotic
MI481-42 New antibacterial substance obtained by decomposing F4-A MI481-
42F4-A1 or a salt thereof is provided.

[0006] The first antibacterial substance MI481-42F4-A1 according to the present invention obtained by decomposing the antibiotic MI481-42F4-A has the following physicochemical properties.

(1) Color and shape: colorless powder (2) Classification of acidic, neutral and basic: amphoteric substance (3) high resolution mass spectrum: HR FAB-MS m / z 1183.228
5 (M + H) (4) Molecular formula: C ₅₀ H ₅₀ O ₉ N ₁₄ S ₆

Furthermore, according to the second invention, the antibiotic MI
New antibacterial substance MI481-42 obtained by degrading 481-42F4-A
F4-A2, or a salt thereof, is provided.

Second antibacterial substance MI481-42F4-A according to the present invention
2 has the following physicochemical properties. (1) Color and shape: colorless powder (2) Classification of acidic, neutral and basic: basic substance (3) Mass spectrum: FAB-MS m / z 1218 (M + H) ⁺

Furthermore, according to the third aspect of the present invention, the antibiotic MI
New antibacterial substance MI481-42 obtained by degrading 481-42F4-A
F4-A3, or a salt thereof, is provided.

The antibacterial substance MI481-42F4-A3 has the following physicochemical properties.

(1) Color and shape: colorless powder (2) Mass spectrum: FAB-MS m / z 1200 (M + H) ⁺

The antibacterial substances MI481-42F4-A1, -A2 and -A3 of the present invention can be produced by hydrolyzing the antibiotics MI481-42F4-A, but A1 and A3 produce MI481-42F4-A substances. It is also present in the culture fluid or cells of the fungus.

The above-mentioned hydrolysis method is carried out, for example, in the dilute hydrochloric acid or hydrochloric acid-methanol solution at room temperature with the antibiotic MI481-.
It can be carried out by subjecting 42F4-A to a decomposition reaction for a predetermined time. Any known decomposition reaction can be used as long as these reactions are useful for producing the MI481-42F4-A1, -A2 and / or -A3 substance of the present invention.

The antibacterial substances MI481-42F4-A1, -A2 and -A3 of the present invention can be collected by hydrolyzing MI481-42F4-A, cooling the reaction solution and concentrating to dryness. Extraction methods using organic solvents such as alcohol and acetonitrile, adsorption chromatography, gel filtration chromatography,
Antibacterial substances MI481-42F4-A1, -A2 and -A3 can be collected purely by appropriately combining or repeating known methods such as thin layer chromatography, countercurrent partition chromatography, high performance liquid chromatography and the like. it can.

The biological properties of the antimicrobial substances MI481-42F4-A and -A1 substance are as follows. Antibacterial substance MI481-42F4-A
Table 1 shows the inhibitory concentrations (and by the agar plate dilution method) of and -A1 against various bacteria on nutrient agar medium. Table 1
As is clear, the antibacterial substances MI481-42F4-A and -A1 had strong antibacterial activity against Gram-positive bacteria. In particular
MI481-42F4-A also strongly inhibited the growth of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus and Staphylococcus aureus No. 5 (MRSA).

[0017]

The antibiotics MI481-42F4-A, -A2 and
-To evaluate the antibacterial activity of A3, antibacterial substance MI481-42F4
The inhibitory concentrations of -A, -A2 and -A3 against various bacteria (by liquid culture dilution method) in nutrient liquid medium were measured. The results are shown in Table 2 below. As is clear from Table 2, the antibacterial substances MI481-42F4-A, -A2 and -A3 had a strong antibacterial activity against Gram-positive bacteria. In particular, it also strongly inhibited the growth of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus and Staphylococcus aureus No. 5 (MRSA).

[0019]

According to an acute toxicity test carried out in mice, the LD ₅₀ of each of the antibacterial substances MI481-42F4-A and -A1 substance is 100 mg / kg or more by intraperitoneal administration, which is low toxicity.

From the above, the antibacterial substance of the present invention
MI481-42F4-A1, -A2 and -A3 may be used as antibacterial agents in the treatment of bacterial infections in humans and animals.

The physicochemical properties and biological properties of the antibiotics MI481-42F4-A1, -A2 and -A3 are as detailed above, and compounds similar to these properties have not been known so far. So the antimicrobial substance MI481-42F4-A1, -A2 and
-A3 is a new substance.

Novel antibacterial substance MI481-42F4-A1, -A2 of the present invention
And-MI3, an antibiotic used as a raw material for the manufacture of A3
481-42F4-A is a substance having the following characteristics.

(1) Color and shape: colorless powder (2) Melting point: 223-228 ° C (decomposition) (3) Elemental analysis: C 49.46%, H 4.79%, N 16.76%, O
12.23%, S 15.72% (4) Ultraviolet absorption spectrum: Lambda maximum (nm) (in methanol) 203 309 221 350 (shoulder) 245 (shoulder) (in methanol-hydrochloric acid) 205 308 222 353 (shoulder) 250 (shoulder) (In methanol-sodium hydroxide) 205 310 221 353 (shoulder) 250 (shoulder) (5) Infrared absorption spectrum (KBr method): 3300, 3120, 298
0, 1660, 1550, 1500, 1250, 990, 940, 760 cm ^-1 (6) Specific rotation: [α] _D ²⁸ = + 133 ° (c 0.745, dimethyl sulfoxide) (7) Acidic, neutral, base Classification of sex: Basic substance (8) Molecular weight: 1182.44 (9) Molecular formula: C ₅₀ H ₅₁ O ₈ N ₁₅ S ₆

This antibiotic MI481-42F4-A is obtained by culturing a MI481-42F4-A producing bacterium belonging to the genus Amycolatopsis and separating and collecting the antibiotic MI481-42F4-A from the culture. It is obtained by the method for producing the antibiotic MI481-42F4-A, which is characterized by

MI481- belonging to the genus Amycolatopsis
As an example of 42F4-A-producing bacteria, the present inventors show
Amycolatopsis sp. ( Amycolatopsis sp.) Isolated from a soil sample collected in Tokyo in June 1987
There is MI481-42F4. The mycological properties of this strain are as follows.

Mycological properties of MI481-42F4 strain 1. Well-branched basal hyphae are often zigzag,
In addition, division is recognized. The aerial hyphae are straight or irregularly curved, with fragmentation, forming cylindrical spores. The spore size is about 0.6-0.7 x 0.7-1.2 microns and the surface is smooth. No helix formation, limbus or sporangia are observed.

2. Regarding the description of the growth state color in various media, the standard shown in [] is the Container Harmony Manual (Container Corporation of America).
Color harmony manual).

(1) Sucrose / nitrate agar medium (30
(° C culture) On the development of light yellow [1 ea, Canary Yellow], white aerial hyphae were slightly settled and no soluble pigment was observed. (2) Glucose-asparagine agar medium (30 ° C culture) On the development of yellow [1 1/2 ia, Sunlight Yellow], aerial hyphae of light yellow [1 ca, Pale Yellow] are settled, and the soluble pigment is Slightly yellowish. (3) Glycerin-asparagine agar medium (ISP-medium 5, 30 ° C culture) To develop light yellow [1 1/2 ga, Butter Yellow], aerial hyphae of light yellow [1 1/2 ca, Cream] Once settled, the soluble dyes have a slight yellow tint. (4) Starch / inorganic salt agar medium (ISP-medium 4, 30 ° C culture) White aerial mycelium grows lightly on the development of light yellow [1 1/2 ic, Lt Antique Gold ~ 2gc, Bamboo]. No soluble dyes were found. (5) Tyrosine agar medium (ISP-medium 7, culture at 30 ° C) Brown [2 ca, Lt Ivory] on the growth of yellow [1 1/2 lc, Gold]
The aerial mycelia of the above are settled, and the soluble pigment becomes yellowish.

(6) Nutrient agar medium (30 ° C. culture) On the development of light yellow [2 gc, Bamboo], white aerial hyphae were slightly attached and no soluble pigment was observed. (7) Yeast-malt agar medium (ISP-medium 2, culture at 30 ° C.) White aerial hyphae grew on the development of light yellow tea [2 ne, Mastard Gold], and no soluble pigment was observed. (8) Oatmeal / agar medium (ISP-medium 3, 30 ° C culture) White aerial hyphae grew slightly on the development of light yellow [1 1/2 ic, Lt Antique Gold], and soluble dye Slightly yellowish. (9) Glycerin / nitrate agar medium (cultivated at 30 ° C) White aerial hyphae were slightly attached to the growth of light yellow [1 ga, Lt Lemon Yellow], and no soluble pigment was observed. (10) Starch agar medium (30 ° C culture) White aerial hyphae grow slightly on the development of yellow [1 la, Lemon Yellow]. No soluble dye is found.

(11) Lime malate agar medium (cultivated at 30 ° C.) On the development of light yellow [1 ga, Lt Lemon Yellow], white aerial mycelium slightly grew and no soluble pigment was observed. (12) Cellulose (synthetic solution with filter paper pieces added, cultivated at 30 ° C) Growth develops pale yellow and white aerial mycelium, and no soluble pigment is observed. (13) Gelatin stab culture In a 15% simple gelatin medium (cultured at 20 ° C), growth was pale yellow and white aerial hyphae were faintly settled, and no soluble pigment was observed. Glucose / peptone / gelatin medium (27 ℃
In the case of culture), the growth is pale yellow, aerial hyphae do not grow, and soluble pigments are not observed. (14) Skim milk (37 ℃ and 30 ℃ culture) No growth was observed at 37 ℃ culture. In the case of cultivation at 30 ° C, the growth is pale yellow, aerial hyphae do not grow, and soluble pigments are not observed.

3. Physiological properties (1) Growth temperature range Glucose / asparagine agar medium (glucose 1.0
%, L-asparagine 0.05%, dipotassium phosphate 0.05
%, String agar 3.0%, pH 7.0), 6 ℃, 20 ℃, 24
Tested at each temperature of ℃, 27 ℃, 30 ℃, 37 ℃, 50 ℃,
It grows at any temperature except 6 ° C, 37 ° C, and 50 ° C, and the optimum growth temperature seems to be around 27 ° C to 30 ° C.

(2) Liquefaction of gelatin (15% simple gelatin medium, 20 ° C. culture; glucose / peptone / gelatin medium, 27 ° C. culture) In 15% simple gelatin medium, liquefaction started about 3 days after culturing and about 3 Completed a week later. Glucose peptone
In gelatin medium, a slight liquefaction was observed from the 7th day after culturing, but the progress was extremely slow, and it was not completed in 1 month of culturing.

(3) Hydrolysis of starch (starch / inorganic salt agar medium and starch agar medium, both at 30 ° C.) Water-degradability was observed from about 6 days after culturing, and its action was moderate. It is degree. (4) Coagulation / peptonization of skim milk (skim milk, 37 ° C and 30 ° C culture) No growth was observed at 37 ° C culture. In the case of culturing at 30 ° C, peptone formation begins without coagulation around 10 days after culturing,
It was completed after about one month of culture. (5) Formation of melanin-like pigment (tryptone, yeast,
Broth, ISP-medium 1; peptone-yeast-iron agar medium, ISP-medium 6; tyrosine agar medium, ISP-medium 7; all cultures at 30 ° C) Both media are negative.

(6) Utilization of carbon source (Pridham Godleybe agar medium, ISP-medium 9; 30 ° C. culture) D-glucose, L-arabinose, D-xylose,
It grows using inositol, D-mannitol and lactose, but does not use sucrose. D-fructose, rhamnose and raffinose are likely to be utilized. (7) Dissolution of lime malate (lime malate agar medium, 30
C. Cultivation) Solubility was observed around 10 days after culturing, and its action was moderate. (8) Nitrate reduction reaction (0.1% potassium nitrate-containing peptone water, ISP-medium 8, culture at 30 ° C) Positive. (9) Decomposition of cellulose (synthesis solution with filter paper pieces added, culturing at 30 ° C) No decomposition is observed after 6 months of observation.

Summarizing the above properties, the MI481-42F4 strain often shows zigzag basal hyphae due to its morphology, and fragmentation is observed. The aerial mycelium is straight or irregularly curved, with fragmentation, forming cylindrical spores, and its surface is smooth. No helix formation, limbal branch or sporangium is observed. In various media, white to light yellow aerial hyphae grow on the development of light yellow to yellow, and the soluble pigment is slightly yellowish or not observed. The formation of melanin-like pigment is negative, the water-degradability of starch is moderate, the reduction reaction of nitrate is positive, and the proteolytic activity is moderate.

By the way, in the bacterial cell component of the MI481-42F4 strain, 2,6-diaminopimelic acid contained in the cell wall is meso-type, and the reducing sugar in all the bacterial cells is A-type containing arabinose and galactose. In addition, it contains no mycolic acid, the phospholipid is PII type (containing phosphatidylethanolamine and no phosphatidylcholine and unknown glucosamine-containing phospholipid), and the major menaquinone is MK-9 (H ₄ ).
MK-9 (H ₂ ) was also found.

From the above results, the MI481-42F4 strain was identified as Amycolatopsis (Reference, International Jo
urnal of Systematic Bacteriology, 36, 29-37,
1986) considered to belong to the genus. When searching for known bacterial species of the genus Amycolatopsis , Amycolatopsis orientalis (Reference 1, Ibid .; Reference 2, International Journal of Systematic Ba
cteriology, 37, 292-295, 1987) and Amycolatopsis medi terran.
ei , references 1 and 2, ibid.) were mentioned as closely related species. Therefore, the MI481-42F4 strain and the above-mentioned two strains preserved by this institute are currently being compared and examined. Currently MI481-42F4
Amycolatopsis sp.
) MI481-42F4.

The MI481-42F4 strain was applied for deposit at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, and on February 3, 1992, it was deposited as Microorganism Research Institute No. 12739.

The above-mentioned antibiotic MI481-42F4-A is produced by inoculating the above strain into a medium suitable for producing the antibiotic MI481-42F4-A and culturing (see Reference Example 1 below). The medium may be a nutrient-source-containing medium used for culturing ordinary actinomycetes. As its nutritional source, for example, commercially available peptone, meat extract, corn steep liquor, cottonseed flour, peanut flour, soybean flour, yeast extract, MZ-amine, casein hydrolyzate, sodium nitrate, ammonium nitrate,
Nitrogen sources such as ammonium sulfate, and commercially available glycerin, sucrose, starch, glucose, galactose, mannose, molasses and other carbohydrates, or carbon sources such as fat, and salts, phosphates, calcium carbonate, magnesium sulfate and other inorganic substances. Salt can be used. In addition, if necessary, a trace amount of a metal salt, or an antifoaming agent such as animal / vegetable / mineral oil may be added. Any of these may be used as long as it can be used by the producing strain and is useful for the production of the antibiotic MI481-42F4-A, and all known culture materials for actinomycetes can be used.

Liquid culture is preferable for large-scale production of the antibiotic MI481-42F4-A, and the culture temperature can be applied within a range where the antibiotic MI481-42F4-A can be produced. Cultivation can be performed by applying the above-mentioned conditions and selecting appropriately according to the properties of the MI481-42F4-A-producing bacterium. The antibiotic MI481-42F4-A is mainly present in the cells and can be extracted with an organic solvent such as alcohol, acetonitrile, or acetone that is miscible with water. The cell extract can be extracted with a water-immiscible organic solvent having a relatively high polarity such as butanol. In addition to the above-mentioned extraction methods, adsorption chromatography, gel filtration chromatography, thin layer chromatography, countercurrent partition chromatography, high performance liquid chromatography, etc. may be combined as appropriate or repeated to obtain a pure sample. You can

[0042]

EXAMPLES The present invention will now be described with reference to reference examples and examples, but the present invention is not limited thereto.

Reference Example 1 Production of Antibiotic MI481-42F4-A Glycerol 2.0%, Dextrin (Wako Pure Chemical Industries) 2.0
%, Polypeptone (Wako Pure Chemical Industries) 1.0%, yeast extract (Wako Pure Chemical Industries) 0.3%, ammonium sulfate 0.2%, calcium carbonate 0.2%, antifoaming agent (KM70, Shin-Etsu Chemical) 0.01% (pH 7.4) Place 110 ml in a 500 ml Erlenmeyer flask with waffles, sterilize at 120 ° C for 20 minutes, and then add MI481-42F4 belonging to the genus Amycolatopsis belonging to the genus Amycolatopsis.
One platinum loop of a strain (FERM P-12739) was inoculated, and the culture solution obtained by shaking culture at 27 ° C. for 3 days was used as an inoculum source. 2 ml of this inoculum was transplanted to the same medium as above and cultured at 27 ° C. for 4 days to accumulate the antibiotic MI481-42F4-A.

The culture broth (pH 8.2, 10.1 liter) was centrifuged to collect the cells, and this was extracted with 5 liters of methanol.
Concentrated methanol was distilled off from the extract, 5 liters of water was added, and the mixture was extracted with an equal amount of butanol. After concentration, methanol was added and the precipitate was collected to obtain 4.3 g of a dried product. This was dissolved in dimethylformamide and suspended in dimethylformamide and packed into Sephadex LH-20 (Pharmacia) (outer diameter 50φ x 100 m
It was put on a column of m) and gel-filtered with dimethylformamide.
Toyopearl HW-40 (tosaw) (outer diameter 40φ x 420 mm) packed with active fractions (fractions showing antibacterial activity against Bacillus thermophilus ) concentrated under reduced pressure, dissolved in dimethylformamide and suspended in dimethylformamide The mixture was passed through a column No. 3, and the gel was filtered through dimethylformamide, and the active fraction was concentrated and dried under reduced pressure to obtain 780 mg of a dried product.

This was dissolved in 15 ml of chloroform, 25 g of silica gel (Merck Art.7734) was suspended in this solution and charged into a packed column, and the column was washed with 200 ml of chloroform and then chloroform / methanol = 10. The active fraction was collected by elution with a mixed solution of / 1, and concentrated under reduced pressure to dryness to obtain 232 mg of pure antibiotic MI481-42F4-A colorless powder.

Example 1 Preparation of MI481-42F4-A3 and -A1 substances MI481-42F4-A 40 mg pure antibiotic obtained in Reference Example 1
Was weighed into a techno-vacuum hydrolysis tube and placed in 20 ml of 1N-H.
Cl was added and suspended, and a cap was fitted and the tube was sealed. This vacuum hydrolysis tube was kept in an oil bath kept at 110 ° C for 24 hours. After that, the reaction solution is concentrated under reduced pressure to dryness and dried product 45
to obtain mg. This was dissolved in 10 ml of acetonitrile water and purified and fractionated by high performance liquid chromatography (HPLC).

The reverse phase HPLC conditions are as follows. Column: Capsule cup (Capcell Pak C ₁₈ , Shiseido) 4.6
φ × 250 mm ;, eluent: 40% acetonitrile water → 48% acetonitrile water (17 minutes linear gradient); flow rate: 1.4 ml / min; UV detector: 254 nm.

As a result of the above analysis, the retention time was 8.8 minutes and MI
481-42F4-A3 substance and MI481-42F4-A1 substance were detected at 11.3 minutes.

Pure MI481-each according to the method described above
2 mg of 42F4-A3 substance and 8 mg of MI481-42F4-A1 substance were obtained.

Example 2 MI481-42F4-A1, -A2 and -A3
Manufacture of Substances The antibiotic MI481-42F4-A (41 mg) was weighed in an eggplant-shaped flask, 1N HCl-MeOH (10.8 ml) was added, the mixture was allowed to stand at room temperature for 1.5 hours, and the resulting reaction solution was dried under reduced pressure. The dried solid was dissolved in 10 ml of methanol water and was subjected to high performance liquid chromatography (HPLC).
Was purified and collected.

The reverse phase HPLC conditions are as follows.

Column: YMC-packed column (SH-343-5 S
-5 ODS, Yamamura Chemical Research Institute) 20 × 250 mm; Eluent: 80% MeOH
Water; Flow rate: 10.0 ml / min; Ultraviolet detector: 254 nm.

By the above-mentioned preparative separation, pure MI481-42
11 mg of F4-A2 material was obtained.

Further, active fractions other than the above were collected and subjected to thin layer chromatography (developing solvent, chloroform-methanol, 10: 1 (v / v)) to scrape the UV absorption band. By this operation, 3 mg of MI481-42F4-A1 substance and MI481-
3 mg of 42F4-A3 material was obtained.

[0055]

As described in detail above, the present invention provides a novel antibacterial substance and a method for producing the same.

[Brief description of drawings]

1 represents the ¹ H-NMR spectrum of the antimicrobial MI481-42F4-A1 measured in deuterated dimethyl sulfoxide at 400 MHz.

FIG. 2 represents the ¹³ C-NMR spectrum of the antimicrobial MI481-42F4-A1 measured in deuterated dimethylsulfoxide at 100 MHz.

FIG. 3 represents the ¹ H-NMR spectrum of the antibacterial substance MI481-42F4-A2 measured at 400 MHz in deuterated pyridine.

FIG. 4 represents the ¹³ C-NMR spectrum of the antibacterial substance MI481-42F4-A2 measured in deuterated pyridine at 100 MHz.

FIG. 5 represents the ¹ H-NMR spectrum of the antimicrobial MI481-42F4-A3 measured in deuterated pyridine at 400 MHz.

FIG. 6 represents the ¹³ C-NMR spectrum of the antibacterial substance MI481-42F4-A3 measured in deuterated pyridine at 100 MHz.

Claims (3)

[Claims] 1. The following characteristics :-( 1) Color and shape: colorless powder (2) Classification of acidic, neutral and basic: amphoteric substance (3) high resolution mass spectrum: HR FAB-MS m / z 1183.228
5 (M + H) (4) Novel antibacterial substance MI481-42F4-A1 characterized by having a molecular formula of C ₅₀ H ₅₀ O ₉ N ₁₄ S ₆
, Or its salt. 2. The following characteristics :-( 1) Color and shape: colorless powder (2) Classification of acidic, neutral and basic: basic substance (3) mass spectrum: FAB-MS m / z 1218 (M New antibacterial substance MI481-42F4-A2 characterized by having + H) ⁺
, Or its salt. 3. A novel antibacterial substance MI481- characterized by having the following characteristics :-( 1) Color and shape: colorless powder (2) Mass spectrum: FAB-MS m / z 1200 (M + H) ⁺ 42F4-A3
, Or its salt.