JPH0637498B2 - Antibiotic TAN-876 and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel antibiotic TAN-876 (hereinafter sometimes abbreviated as “TAN-876”) which is useful as a therapeutic agent for bacterial and fungal infections, and its production. Concerning the law.

Prior Art As an antibiotic that is relatively similar to the present antibiotic, cervicarcin [cervicarcin (Cervicarcin Journal of American Chemical Society
ciety), 86, 4507 (1964)] or Nanaomycin-Journal of Antibiotics, 28,
868 (1975)] and the like, but these are compounds containing no halogen and are different from the present antibiotic.

Problems to be Solved by the Invention Diseases caused by bacteria have been considerably overcome by the development of therapeutic methods by administration of antibiotics. However, the increase of diseases due to changes in the causative bacteria and the emergence of resistant bacteria due to long-term or large-scale administration of conventional antibiotics, or the increase of fungal diseases due to weakened immunity are major problems in the current therapeutic field of infectious diseases. Has become. In order to overcome this problem, there is a need in the art for antibiotics having a novel skeleton and exhibiting excellent biological activity, or intermediate materials for synthesizing them.

Means for Solving the Problems In view of the present situation, the present inventors have repeatedly researched therapeutic agents for bacterial infections from a new viewpoint, and as a result, among a large number of microorganisms isolated from soil, The microorganism produces a new antibiotic, the microorganism belongs to the genus Streptomyces, by culturing the microorganism in an appropriate medium,
It is possible to accumulate antibiotics showing antibacterial activity against bacteria such as gram-positive and negative bacteria including resistant bacteria, acid-fast bacteria and fungi such as medicinal fungi, yeast and phytopathogenic bacteria in the soil. Knowing that, this antibiotic was isolated, and it was confirmed from the physicochemical and biological properties that it was a novel antibiotic, and it was named antibiotic TAN-876. The antibiotic TAN-876 consists of two components, which are designated as TAN-876A and B, respectively. The present inventors have completed the present invention as a result of further research based on these detections.

That is, the present invention cultivates (1) the antibiotic TAN-876 or a salt thereof, and (2) the antibiotic TAN-876-producing bacteria belonging to the genus Streptomyces in a medium to produce the antibiotic TAN-876 in the culture. The present invention relates to a method for producing an antibiotic TAN-876, which is characterized by accumulating and collecting it.

The bacterium producing the antibiotic TAN-876 used in the method of the present invention may be any microorganism as long as it belongs to the genus Streptomyces and has the ability to produce the antibiotic TAN-876. Examples thereof include C-70899 strain isolated from soil,
The mycological properties of this strain are as follows. Unless otherwise specified, the properties on various culture media were observed by culturing at 28 ° C for 14 days and observing in accordance with a conventional method. The color tone was Color Harmony Manual.
4th edition [Container Corporation of America, 1958].

1. Morphology The formation of aerial hyphae and spores is abundantly observed on mediums such as yeast / malt agar, starch inorganic salt agar, and glycerol / asparagine agar. The branch of aerial mycelium is a simple branch, no axle branch is observed, and a spiral chain of spores is observed at the tip. No special structure such as sclerotium or sporangium is observed. On observing with an electron microscope, the surface of the spores is smooth, the spores are spherical, oval or short cylinder-shaped, and their size is 0.8-1.2 x 0.8-1.0 micron,
Usually 20 or more chains.

2. Various classification Properties on culture medium (28 ° C, 14 days culture) 1) Sucrose / nitrate agar medium Growth: Medium, Light yellow (1 1 / 2ea) to Dusty yellow (1 1 / 2gc) Aerial mycelium: Poor or poor Medium, white Backside color: Butter yellow (1 1 / 2ga) to bamboo (2gc) Soluble pigment: None 2) Yeast-malt agar medium Growth: Medium to good, clove brown (3pl) Aerial mycelium: Abundant , Silver gray (3fe) Backside color: Clove brown (3pl) Soluble pigment: None 3) Oatmeal agar medium Growth: Medium, Light ivory (2ca) Aerial mycelium: Medium, Adove brown (3 lg) Back side color: Light spice brown (4 lg) Soluble pigment: Brown to yellow-brown 4) Starch inorganic salt agar medium Growth: Medium mustard brown (2ni) Aerial mycelium: Rich, natural (2dc) to silver・ Gray (3f
e) Backside color: Mustard Brown (2ni) ~ Covert Brown (2nl) Soluble pigment: None 5) Glycerol-asparagine agar medium Growth: Medium, Light mustard tan (2ie) Aerial mycelium: Rich, natural ( 2dc) -Natural (3dc) -Silver gray (3fe) Backside color: Bamboo (2gc) -Mustard tan (2 lg) Soluble pigment: None 6) Glucose-asparagine agar medium Growth: Medium, colorless Aerial mycelium: Medium, Silver Gray (3fe) to Assies (5f
e) Backside color: colorless to light mustard tan (2ie) Soluble pigment: none 7) Peptone yeast, iron agar medium Growth: Medium, wrinkled, Bayer Brown (3i
g) Aerial mycelium: Not settled Rear color: Bayer brown (3ig) Soluble pigment: Brown to clove brown (3ni) 8) Tyrosine agar growth: Medium, brown to dark brown Aerial mycelium: Poor, white Backside color: brown to dark brown Soluble pigment: light yellowish brown 9) Calcium-maleite agar medium Growth: Poor, colorless Aeromycelium: No settlement Backside color: colorless to cream (1 1 / 2ca) Soluble pigment: None 3. Physiological properties 1) Growth temperature range: 12-40 ° C Optimal temperature range: 22-37 ° C 2) Gelatin liquefaction: Negative 3) Starch hydrolysis: Positive 4) Milk peptone formation: Negative 5) Milk coagulation : Negative 6) Reduction of nitrate: Positive 7) Formation of melanin-like pigment: Positive 8) Utilization of carbon source D-sorbitol-sucrose-i-inositol + lactose + D-mannitol + raffinose + D-xylose + trehalose + L-arabinose + mannose + D-galactose + soluble starch + D-glucose + glycerol + D-fructose + melibiose + rhamnose + maltose + adonitol + esculin-erythritol-salicin + no dulutol-inulin-cellulose-ribose. -Basal medium: Bridham and Gottlieb agar 4. Analysis of diaminopimelic acid in the hydrolyzate of whole cells [Tetsu Hasegawa et al., Journal of General Applied Microbiology
ied Microbiology), 29 , 319 (1983)] LL-2,6-diaminopimelic acid was found.

To summarize the above-mentioned mycological properties, the C-70899 strain is
The aerial hyphae have a spiral shape and the spore surface is smooth. The development color tone is pale yellow, yellowish brown or brown, and the aerial mycelium is grayish. It produces a melanin-like soluble pigment. In addition, when LL-diaminopimelic acid is detected in the cells, the bacterium belongs to cell wall type I. Based on the above properties, S. Waxman, The.
The Actinomycetes Volume 2, published by The Williams and Wilkins Company,
1961 and Edited by E. Buffanan and N. Gibbons, Vergies, Manual of Determinating Bacteriology (Bergey ')
s Manual of Determinative Bacteriology) 8th Edition, 1
A search for strains was conducted according to 974. As a result, Streptomyces resist Streptomyces resist was found as a bacterial species relatively related to the C-70899 strain.
omycificus) and Streptomyces neyagawaensis. In order to carry out a more detailed study of these two bacterial strains, Streptomyces registomysficus IFO12814 and Streptomyces neyagawaensis IFO1347
As a result of examining No. 7, both of these strains were C
It was confirmed that the strain was different from the -70899 strain.

Streptomyces resist mysticus: East
On the malt agar medium, the aerial hyphae also show an open spiral, but generally show an incomplete spiral or key-like morphology.
Also, its color tone is light pink brown (fresh pink, 4c
a) to light yellow-brown (fountain, 4 ig). Use sucrose.

On Streptomyces nayagawaensis: yeast-malt agar medium, the aerial hyphae are generally poorly colonized, and the color tone is sandy (sand, 3 cb). The spore morphology is oblong. Use sucrose.

As described above, at the present time, no known bacterial species closely related to the C-70899 strain has been recognized. Therefore, C-70
The 899 strain was recognized as a new bacterial species and named Streptomyces sp. C-70899.

The C-70899 strain was assigned to the Fermentation Research Institute (IFO) on May 29, 1986 under the contract number IFO14509, and the microorganism was researched by the Ministry of International Trade and Industry, Institute of Industrial Technology, Institute of Microbiology, on June 13, 1986. (FRI) under the deposit number FERM P-8807.

Bacteria producing the antibiotic TAN-876 belonging to the genus Streptomyces, like the other genus Streptomyces, are likely to change their properties, for example, ultraviolet rays, X-rays,
Those that can be easily mutated by artificial mutagenesis means using radiation, artificial mutagen, etc., and even if such mutant strains have the ability to produce the antibiotic TAN-876, all of them can be used in the method of the present invention. Can be used.

In the method of the present invention, the medium in which the antibiotic TAN-876-producing bacterium is cultivated may be liquid or solid, but a liquid medium is more conveniently used, and surface culturing or shaking culturing may be used. The submerged culture method is more advantageously used. Carbon sources that can be assimilated by antibiotic TAN-876-producing bacteria in the medium, such as starch, glucose, dextrin, glycerin, sucrose, n-paraffin and alcohols (eg, methanol), nitrogen sources such as organic nitrogen As the source, corn steep liquor, soybean flour, cottonseed flour, peptone, meat extract and the like, and as the inorganic nitrogen source, ammonium chloride, ammonium sulfate, ammonium nitrate, urea and the like can be used. Other,
Inorganic salts such as sodium, potassium,
Salts containing magnesium or phosphorus, heavy metal salts such as salts of iron, manganese, zinc, cobalt, copper, nickel, etc., defoaming agents such as soybean oil, lard oil, chicken oil, silicon oil, ACTCOL (manufactured by Takeda Pharmaceutical Co., Ltd. ) And the like may be added as appropriate. For liquid culture, the pH of the medium
Is preferably in the vicinity of neutral, especially at a pH of about 6-8. The culture temperature is preferably about 24 ° C to 30 ° C, and the culture time is preferably about 48 to 120 hours.

The titer of the antibiotic TAN-876 produced in the culture medium as the culture progressed was Micrococcus flavus IF.
It is quantified according to a conventional method such as an agar dilution method or a paper disc method using O3242 as a test bacterium. Usually about 3-4
The maximum production of the antibiotic TAN-876 is reached in the culture for one day.

In order to collect the target antibiotic TAN-876 from the culture, a separation means usually used for collecting the metabolite produced by the microorganism from the culture of the microorganism is appropriately used. For example, TAN-876 has an acidic fat-soluble property and is contained in the cells and the filtrate. Therefore, an organic solvent immiscible with water such as chloroform, ethyl acetate,
Methyl isobutyl ketone or butanol is added to extract TAN-876. After washing the extract with water,
Concentrate the organic solvent layer.

The concentrate containing TAN-876A and B is further purified and fractionated by adsorption, partition or chromatography utilizing the property of molecular sieve. That is, a means for bringing the concentrate into contact with an appropriate carrier to adsorb the active ingredient, then desorbing the active ingredient with an appropriate solvent, and fractionating and collecting the active ingredient is advantageously used. As the carrier, silica gel, crystalline cellulose, adsorptive resin such as Diaion HP-
20 (manufactured by Mitsubishi Kasei Co., Ltd.), Amberlite XA
D-II (Rohm and Haas, USA), Sephadex LH-20 (Pharmacia Fine Chemicals, Sweden) and the like are used. The elution solvent varies depending on the type of carrier, but is, for example, n-hexane, toluene, chloroform, dichloromethane, ethyl acetate,
Acetone, alcohols, pyridine, acetic acid, water or a mixed solvent thereof may be used in an appropriate combination. TA
The eluate containing N-876 is concentrated to dryness and crystallized from an appropriate solvent such as hexane, diethyl ether, toluene, ethyl acetate or chloroform, or powdered by adding petroleum ether or hexane to the concentrated dry solid. .

When A and B are not separated by the above-mentioned chromatography, preparative reverse phase high performance liquid chromatography (HPLC) is advantageously used to isolate them. Examples of the carrier used include TSK gel (manufactured by Toyo Soda Co., Ltd., Japan), YMC gel (manufactured by Yamamura Chemical Laboratory, Japan), and the like. The mobile phase is a mixed solution of acetonitrile or methanol with a buffer solution. Is used.

In addition, TAN-876A and B form a salt by reacting with a cation in an alkaline solution, for example, sodium carbonate, a sodium ion in a potassium carbonate solution, a potassium ion and the like. Purification of these salts is carried out by an ordinary method by adsorptive chromatography, for example, chromatography using Diaion HP-20 as a carrier.

The physicochemical properties of the antibiotics TAN-876A and B obtained in Example 1 described later are as follows.

TAN-876A (1) Appearance: Light yellow solid (2) Melting point:> 300 ° C. (decomposition) (3) Measured molecular weight: (by EI-MS method) 331 (M
⁺ ) (4) Elemental analysis value (%): Actual value: C: 47.48, H: 2.83, N: 4.49, Cl: 32.57 Calculated value: C: 46.95, H: 2.42, N: 4.21, Cl: 31.98, O :
14.43 (5) Molecular formula: C ₁₃ H ₈ NCl ₃ O ₃ (6) Ultraviolet absorption (UV) spectrum: See attached Fig. 1) In methanol (shown by solid line in Fig. 1) In methanol: 1N HCl (9: 1) (shown by the broken line in Fig. 1) Methanol: 1N NaOH (9: 1) (indicated by a chain line in Fig. 1) (7) Infrared absorption (IR) spectrum (see attached Figure 2): K
The following main absorptions are shown in Br.

3430, 3210, 3150, 2980, 2950, 1615, 1590, 1570, 15
30, 1440, 1410, 1350, 1310, 1270, 1250, 1230, 117
0, 1080, 1060, 1020, 980, 900, 850, 800, 780, 77
0,750,710,645,620 (cm ^-1 ) (8) ¹ H-nuclear magnetic resonance (NMR) spectrum: 400 MHz, the following signals are observed in dimethyl sulfoxide-d ₆ .

1.17 (3H, t), 2.62 (2H, q), 7.67 (1H, s), 13.55 (1H, s) (pp
m) (however, s: singlet, t: triplet, q: quartet is shown) (9) HPLC: carrier; YMC-PAK A-312, mobile phase; 65% acetonitrile / 0.01M phosphate buffer (pH)
6.3) Flow rate; 2 ml / min. Detection method: UV (220nm) Rt = 2.75 (min.) (10) Color reaction Positive: Potassium permanganate Negative: Burton, Magnesium acetate, Dragendorff, Ninhydrin, Greg Lee Back reaction (11) Substance classification Fat-soluble acidic substance TAN-876B (1) Appearance: Light yellow solid (2) Molecular weight measurement value: (by EI-MS method) 333 (M ⁺ ) (4) Elemental analysis value (%) : Measured value: C: 46.76, H: 2.99, N: 4.33, Cl: 30.83 Calculated value: C: 46.66, H: 3.01, N: 4.19, Cl: 31.79, O: 1
4.35 (5) Molecular formula: C ₁₃ H ₁₀ NCl ₃ O ₃ (6) UV spectrum: See attached Fig. 3) In methanol (shown by solid line in Fig. 3) In methanol: 1N HCl (9: 1) (shown by the broken line in Fig. 3) Methanol: 1N NaOH (9: 1) (indicated by a chain line in Fig. 3) (7) IR spectrum (see attached FIG. 4): In KBr, the following main absorptions are shown.

3500, 3150, 2980, 2700, 2650, 1610, 1590, 1470, 14
20, 1400, 1330, 1300, 1290, 1260, 1220, 1200, 115
5, 1080, 1050, 980, 930, 875, 850, 790, 770, 750,
730, 690, 660, 620, 560 (cm ^-1 ) (8) ¹ H-NMR spectrum: 400 MHz, in dimethyl sulfoxide-d ₆ the following signals are observed.

1.10 (3H, t), 6.48 (1H, s), 7.13 (1H, s), 8.79 (1H, s), 9.
18 (1H, s), 13.2 (1H, s) (ppm) (9) HPLC: Under the same conditions as component A Rt = 5.1 (min.) (10) Color reaction Positive: Potassium permanganate, Burton, acetic acid Magnesium (yellow) reaction Negative: Dragendorff, ninhydrin, Greg-Leabach reaction (11) Substance classification Fat-soluble acidic substances The chemical structures of the antibiotics TAN-876A and B are shown by the following formula.

Next, the antibacterial activities of the antibiotics TAN-876A and B against various microorganisms are shown in Tables 1, 2 and 3.

Furthermore, the acute toxicity of TAN-876A in mice is shown in Table 4.

As is clear from the above physicochemical properties and biological properties, TAN-876A and B are novel antibiotics, exhibit strong antibacterial and antifungal actions, and have no toxicity to mammals, etc. It is a substance useful as a medicine, veterinary medicine and agricultural chemical.

When using this antibiotic, for example, TAN-87
In order to use 6 or a salt thereof as a bactericidal agent for bacterial infections, TAN-876 or a salt thereof is mixed with a pharmacologically acceptable carrier, excipient, diluent or the like, for example, TA
N-876 or a salt thereof as TAN-876 is about 0.01
~ 2w / v%, preferably about 0.1 to 1w / v% solution dissolved in alcohol or water etc. or TAN per gram
-876 as about 0.05 to 50 mg, preferably about 0.5 to 2
As an ointment containing 0 mg, it can be applied to the hands, feet, face, etc. of mammals (eg, humans, cows, horses, dogs) to sterilize and disinfect these sites.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.
The percentage (%) in the medium represents weight / volume percentage unless otherwise specified.

Example 1 Streptomyces sp. C-70899 (IFO-14) cultured in yeast extract / malt extract slope agar medium
509, FERM P-8807) in a 200 ml Erlenmeyer flask with 2% glucose, 3% soluble starch, 1% raw soybean flour, 1% corn steep liquor, 0.5 peptone.
%, NaCl 0.3%, CaCO ₃ 0.5% 40 ml seed medium (pH
7.0), and cultured at 28 ° C. for 48 hours on a rotary shaker to obtain a preculture liquid. 200 ml of 5 ml of the obtained preculture liquid
Transfer to 500 ml seed medium in a 0 ml Sakaguchi flask and
The seed culture was obtained by culturing at 8 ° C for 48 hours on a reciprocating shaker.
This seed culture solution (500 ml) was transplanted to 30 seed medium (same composition as the above seed medium) in a 50-volume stainless steel tank, aeration 30 / min, stirring 280 rpm, internal pressure 1 kg /.
It was cultured under the condition of cm ² . 200 of 5 of the obtained culture solution
Glucose in a stainless steel tank 0.5%,
Dextrin 5%, defatted soybean powder 3.5%, CoCl ₂ 0.0002
%, CaCO ₃ 0.7% in 120 main medium (pH 7.0), 28 ° C., aeration 120 / min, stirring 180 revolutions / min,
The cells were cultured for 96 hours under the condition of an internal pressure of 1 kg / cm ² .

Thus, the obtained culture solution (100) was diluted with dilute sulfuric acid to pH 4
The mixture was adjusted to, ethyl acetate (100) was added, and the mixture was stirred for 30 minutes. The mixture of the culture solution and the organic solvent was filtered using a filter aid. The aqueous layer portion of the filtrate (170) was separated and removed, and the extracted organic solvent layer (85) was washed with water. The ethyl acetate layer (77) was concentrated under reduced pressure. The concentrated solution was subjected to silica gel column chromatography (100 g), the column was washed with n-hexane, and the antibacterial activity category was chloroform (1).
Eluted at. The elution fraction was concentrated, and subjected to silica gel chromatography (50 g) again, eluting with chloroform, TA
A fraction containing N-876A, B was obtained. The concentrated solution of the fractions was further subjected to silica gel chromatography (50 g) and eluted and fractionated with toluene and a mixed solution of toluene and chloroform. The fractions were submitted for analytical HPLC and TAN-87
The contents of 6A and B were investigated. Mainly TAN-8
Fractions containing 76A were collected and concentrated to TAN-876A
Of slightly yellow crystals (120 mg) were precipitated. Mainly TAN-8
Fractions containing 76B were collected and concentrated, and N-hexane was added to the concentrated residue to obtain a powder (150 mg). This powder was dissolved in methanol and subjected to preparative HPLC [carrier: YMC-
PAK ODS-S10, mobile phase: 55% acetonitrile / 0.01 M phosphate buffer (pH 6.3)]. The content of components in each fraction was examined by analytical HPLC, and the fractions mainly containing TAN-876B were collected and concentrated. The concentrated aqueous layer was adjusted to pH 3.5, extracted with ethyl acetate, and the extract was washed with water and concentrated to give a crystalline powder of TAN-876B (85 mg).

Example 2 TAN-876A (purity about 90%, 150 mg) was dissolved in 2% sodium carbonate solution (300 ml), and a trace amount of insoluble matter was filtered off. The filtrate was subjected to Amberlite XAD-II (50 ml) column chromatography, and the column was washed with water (400 ml).
Then, the antibiotic was eluted and fractionated with 30% methanol-water (500 ml) and 50% methanol-water (500 ml). Collect effective fraction (750ml), concentrate and freeze-dry to obtain yellow powder (125m
g) was obtained.

This powder was dissolved in methanol and left to stand to precipitate crystals, which were collected by filtration and dried to obtain sodium salt of TAN-876A (95 mg) as pale yellow crystals.

Elemental analysis value (%): C ₁₃ H ₇ NCl ₃ O ₃・ Na (1.5H ₂ O) Actual value :: C: 40.11, H: 2.78, N: 3.88, Cl: 26.50, N
a: 6.8 Calculated value: ^* C: 40.92, H: 2.64, N: 3.67, Cl: 27.87,
O: 18.87, Na: 6.02 (*: Calculated assuming that it contains 1.5 mol of water) IR spectrum: KBr Main absorption 3450, 3210, 2980, 2940, 1610, 1590, 1570, 1530, 14
40, 1410, 1330, 1310, 1270, 1250, 1220, 1170, 109
0, 1060, 1015, 970, 900, 850, 800, 780, 770, 710,
640 (cm ^-1 ) Melting point:> 300 ° C. (decomposition) Effect of the invention TAN-876A and B of the present invention are novel antibiotics produced by Streptomyces, and can be applied to bacteria and fungi including resistant bacteria. Since it is effective, it is useful as a medicine, agricultural chemical or veterinary drug.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an ultraviolet absorption spectrum of the novel antibiotic TAN-876A of the present invention, FIG. 2 is its infrared absorption spectrum, FIG. 3 is an ultraviolet absorption spectrum of TAN-876B, and FIG. Figure 4 shows the infrared absorption spectrum.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI Technical display location C12N 1/20 A 7236-4B (C12P 17/10 C12R 1: 465) (C12P 17/18 C12R 1 : 465) (C12N 1/20 C12R 1: 465)

Claims (2)

[Claims] 1. A general formula Wherein either ^{R 1} is ^{R 2} represents a hydroxyl group is a hydrogen, ^{R 1}
And R ² represent an ether bond. ] The antibiotic TAN-876 or its salt represented by these. 2. An antibiotic TA belonging to the genus Streptomyces
A method for producing an antibiotic TAN-876, which comprises culturing a N-876-producing bacterium in a medium, allowing the antibiotic TAN-876 to be produced and accumulated in the culture, and collecting the TAN-876.