JP2002212137A - New antibiotics pasteurestin a and b, and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide new antibiotics exhibiting antibacterial activity capable of specifically suppressing the growth of bacteria belonging to the genus Pasteurella. SOLUTION: Antibiotics pasteurestin A and B of the compounds expressed by general formula (I) (wherein, in pasteurestin A, R1 is hydroxymethyl and R2 is H, and in pasteurestin B, R1 is methyl and R2 is hydroxy). The antibiotics and pharmaceutically permissible salts of the antibiotics have antibacterial activity against various kinds of bacteria.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to a novel antibiotic, pasteurestin A and pastelessin B, or salts thereof, which exhibit antibacterial activity. The present invention also relates to a method for producing the antibiotics pastrestin A and B. Further, the present invention relates to an antibacterial agent containing at least one of antibiotics pastrestin A and B and a salt thereof as an active ingredient. In addition, the present invention relates to Agrocybe cylindracea (DC .: F.) as a mushroom having the property of producing the novel antibiotics pastrestin A and B.
r.) Maire) · K-3793 strain.

[0002]

2. Description of the Related Art Many various antibacterial substances have been known. Among the antibacterial substances, a large number of antibacterial antibiotics produced by culturing microorganisms are known.

[0003]

The emergence of resistant bacteria is an important problem in the chemotherapy of infectious diseases in animals, and the use of therapeutic agents for human infectious diseases in animals is an important problem.
Discovery or creation of new compounds that have a different chemical structure from previously known or used known antibacterial compounds, and exhibit excellent selective antibacterial activity depending on the type of bacteria of the infectious disease Is always desired and research is being done for it.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have developed a novel antibacterial compound which is useful for preventing or treating pulmonary inflammation in cattle, which is currently causing great damage. Has promoted research on the development and commercialization of various antibiotics. In 1992, a mushroom collected in Kagoshima Prefecture and named as a strain belonging to the genus Fumio mushroom was named willow matsutake K-3793. As a result of culturing this strain, he discovered that it produced an antibiotic having a new structure, and succeeded in isolating two novel antibiotics from the culture.
It was confirmed to have a chemical structure represented by the following general formula (I). These two substances were named the antibiotics pastrestin A and B, respectively. Furthermore, the present inventors have found that these novel antibiotics exhibit selectively excellent antibacterial activity against Pasteurella, a causative bacterium of pneumonia.

Accordingly, in the first invention, the following general formula (I): (In the formula, R ¹ represents a hydroxymethyl group and R ² represents a hydrogen atom in pastorcin A, and R ¹ represents a methyl group and R ² represents a hydroxyl group in pastorastin B)
Provided are antibiotics pastrestin A and B, or pharmaceutically acceptable salts thereof, which are compounds represented by the formula:

The antibiotics pastrestin A and B are acidic substances and their pharmaceutically acceptable salts include:
There are salts with organic bases such as quaternary ammonium salts, and salts with various pharmaceutically acceptable metals, for example, salts with alkali metals such as sodium, and these salts also have the above antibacterial activity.

Next, the antibiotics pastrestin A and B
Describe the physicochemical properties of

(1) Physicochemical properties of pastrestin A A) Appearance and properties: white powder, acidic substance B) Melting point: 198-210 ° C (decomposition) C) Specific rotation [α] _D ²⁴ -65 ° (C 1.34, methanol) D) RLC value of TLC: 0.06 Toluene-acetone (1: 1) as a developing solvent in thin layer chromatography on silica gel (Art. 105715, manufactured by Merck)
1) If the measured and developed with E) FABMS mass spectrum (m / z): 265 ( MH) - F) Molecular _{Formula: C 15 H 22 O 4 (} MW 266) G) UV absorption spectrum: end absorption H) IR Absorption spectrum (KBr tablet method): shown in FIG. 1 of the accompanying drawings. The main absorption bands are as follows. νmax (cm ^-1 ); 3423, 1683, 1660, 1558, 1455, 1386,
1245, 1037, 968, 921 I) ¹ H-NMR spectrum (CD ₃ OD / TMS): shown in FIG. 2 of the accompanying drawings. J) ¹³ C-NMR spectrum (CD3OD / TMS): shown in FIG. 3 of the accompanying drawings.

(2) Physicochemical properties of pastrestin B A) Appearance and properties: white powder, acidic substance B) Melting point:> 220 ° C. (decomposition) C) Specific rotation [α] _D ²⁴ −45 ° (c B) Tf Rf value: 0.13 Toluene-acetone (1: 1) as a developing solvent in thin-layer chromatography on silica gel (Art. 105715, Merck)
In expanded when measured by E) FAB mass spectrum (m / z): 265 ( MH) - C) Molecular _{Formula: C 15 H 22 O 4 (} MW 266) F) UV absorption spectrum: end absorption G) Infrared absorption spectrum (KBr tablet method): shown in FIG. 4 of the accompanying drawings. The main absorption bands are as follows. νmax (cm ^-1 ); 3436, 2952, 1779, 1567, 1457, 1392,
1106, 1068, 788 H) ¹ H-NMR spectrum (CD ₃ OD / TMS): shown in FIG. 5 of the accompanying drawings. I) ¹³ C-NMR spectrum (CD3OD / TMS): FIG.
Shown in

[0010] Pastrestin A and B are composed of water,
It is soluble in methanol, ethanol, butyl acetate, and dimethyl sulfoxide.

Further, the biological properties of the antibiotics pastrestin A and B are described below.

Antibacterial Activity The minimum growth inhibitory concentrations of the antibiotics pastrostin A and B of the present invention against various bacteria are shown in Table 1 below. This antibacterial spectrum was measured by a multiple dilution method on an agar medium (manufactured by Difco) based on the standard method of the Japanese Society of Chemotherapy.

[Table 1]

As is clear from the results shown in Table 1, the antibiotics pastorastin A and pastorastin B according to the present invention are shown.
Has a specific antibacterial activity against bacteria belonging to the genus Pasteurella, and is therefore useful as a selective antibacterial agent for the prevention and treatment of upper respiratory tract and lung infections in mammals and birds.

According to a second aspect of the present invention, bacteria producing the above-mentioned antibiotics of the general formula (I), ie, the pastelotsin A and B, belonging to the genus Fumus mushrooms, are cultured in a nutrient medium, and The antibiotics pastrestin A and
The present invention provides a method for producing the antibiotics pastorastin A and / or pastorastin B, which comprises collecting (or) pastorastin B.

As an example of a bacterium producing the antibiotics pastrestin A and B which can be used in the second method of the present invention, Agrocybe cylindracea (DC .: Fr.) Maire.
There is K-3793 strain. This produced fungus is a mushroom collected in Kagoshima Prefecture in 1992, and is a microorganism with a strain number of K-3793. In addition, Agrocybe cylindracea K-3793 strain was applied to the National Institute of Advanced Industrial Science and Technology,
On November 30, it was commissioned as FERM P-17666.

The morphological properties of willow matsutake are as follows. The willow matsutake umbrella is 5 to 10 cm in diameter and opens almost flat from the bun shape, the surface is smooth, ocher brown / greyish brown, the periphery is pale and wrinkles are seen. The meat has a white, slightly flour-like odor except under the umbrella and under the handle.
The folds grow slightly straight, but separate from the peduncle when the umbrella is fully opened, and are brownish and dense. The pattern is 3 ~ 10 × 0.5
Approximately 1.2 cm in size, often spindle-shaped at the root, slightly fibrous on the surface but dark brown at the bottom. The brim is on the top of the handle with a film quality. The spores are oval, 8.5-11 x 5.5-7 μm in size, and the germination holes are small and unclear. The edge cistidia is club-shaped, spindle-shaped,
It is 19-30 × 7.5-14 μm such as sac. The lateral cistidia has a spindle shape, a club shape and the like, and has a size of 28 to 33 × 9 to 15 μm.

In practicing the second method of the present invention, the antibiotics Pastressin A belonging to the genus Fumichus
And B-producing bacteria are inoculated into a nutrient medium and cultured in this medium. The nutrient medium used here contains a carbon source and a nitrogen source that can be assimilated by the above-mentioned production fungus, which is a mushroom, as nutrient components.

As the nutrients, those usually used as nutrients of microorganisms, for example, assimilable nutrients such as carbon source, nitrogen source and inorganic salts can be used. For example, carbohydrates such as glucose, maltose, molasses, dextrin, glycerin, starch, and carbon sources such as soybean oil, oils such as peanut oil, and peptone, meat extract, cottonseed powder, soybean powder, yeast extract, casein, corn・ Nitrogen sources such as steep liquor, NZ-amine, ammonium sulfate, ammonium nitrate and ammonium chloride can be used, and inorganic salts such as dipotassium phosphate, sodium phosphate, salt, calcium carbonate, magnesium sulfate and manganese chloride can be used. If necessary, trace metals such as cobalt and iron can be added. Other sources of nutrition include
Any known nutrient sources can be used as long as the bacteria used can produce the antibiotics pastrestin A and B.

The mixing ratio of the above-mentioned nutrients in the medium is not particularly limited, and can be varied over a wide range. Those skilled in the art can easily determine the mixing ratio by a simple small-scale experiment. Further, the nutrient medium consisting of the above-mentioned nutrients can be sterilized prior to culturing, before or after this sterilization,
It is advantageous to adjust the pH of the medium to a range of 5 to 7, especially a pH of 5.5 to 6.5.

Cultivation of the bacteria producing the pastrestin A and B in such a nutrient medium can be carried out according to a method usually used in the production of antibiotics by general mushrooms. Usually, it is preferable to culture under aerobic conditions, and it can be usually performed with stirring and / or aeration. As the culture method, any of static culture, shaking culture, and liquid culture with aeration and stirring can be used, but liquid culture is suitable for mass production of pastrestins A and B.

The cultivation temperature that can be used is not particularly limited as long as the growth of the pastelesstin A and B producing bacteria is not substantially inhibited and the antibiotic can be produced. It can be appropriately selected according to the bacteria. Particularly preferred is a culture temperature in the range of 25 to 30 ° C. Culture can usually be continued until pastrestin A and B have accumulated sufficiently. The culturing time varies depending on the composition of the medium, the culturing temperature, the operating temperature, the used bacterial strain, and the like, but usually the desired antibiotic can be obtained by culturing for 4 to 14 days.

[0023] The novel antibiotic pastrostin A in culture
And the accumulated amount of B can be determined by the cylindrical plate method used for the usual determination of antibiotics, using Pasteurella haemolytica BBP 0102 N811 as a test bacterium.

[0024] Pastrestins A and B thus accumulated in the culture are collected from the culture. After culturing, if necessary, the cells are removed by a known separation method such as filtration or centrifugation, and then the filtrate is subjected to solvent extraction using an organic solvent, particularly butyl acetate, etc., and adsorption and ion exchange capacity. Chromatography, gel filtration,
By using the chromatography utilizing countercurrent distribution alone or in combination, it is possible to isolate and purify the pastrestins A and B from the culture and collect them. Activated carbon, silica gel, porous polystyrene / divinylbenzene resin or various ion exchange resins can be used as a chromatography carrier having adsorption and ion exchange capabilities. Thus, each of the novel antibiotics pastrestin A and B, having the above-mentioned properties, is obtained.

Further, in the third aspect of the present invention, an antibacterial agent comprising as an active ingredient at least one of pastrestins A and B represented by the above general formula (I) and a pharmaceutically acceptable salt thereof is provided. Provided.

The antibacterial agent according to the third aspect of the present invention is prepared in the form of a composition obtained by mixing a compound as an active ingredient with a conventional pharmaceutically acceptable liquid or solid carrier such as ethanol, water, starch and the like. Can be used.

In the fourth aspect of the present invention, there is provided, as a novel microorganism, a strain of Salix matsutake K-3793 having a property of producing the pastrestins A and B of the general formula (I).

[0028]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

Example 1 Production of antibiotics pastrestin A and B 1% glucose, 0.5% polypeptone, 0.3% yeast extract
%, KH ₂ PO ₄ 0.3%, and MgSO ₄ .7H ₂ O 0.1% liquid medium (without adjusting pH) was dispensed into shake flasks (500 ml volume) in 200 ml portions, and sterilized at 120 ° C. for 20 minutes by a conventional method. Willow matsutake K-3793 strain (FERM P-176) cultured on an agar slant medium
66). Then, the cells were cultured at 27 ° C. for 4 days.
Thereafter, the culture was rotated and stirred at 27 ° C. for 4 days. This culture was used as a seed culture. Glucose 1%, polypeptone 0.5
%, Yeast extract 0.3%, KH ₂ PO ₄ 0.3%, MgSO ₄・ 7H ₂ O 0.1
Liquid medium (pH unadjusted) containing a shake flask (500 ml
) Was sterilized at 120 ° C for 20 minutes by a conventional method, and the liquid culture was inoculated with 10 ml of each of the above seed cultures. Then, the cells were cultured at 27 ° C. for 5 days. Thereafter, shaking culture was performed for 6 days.

The thus obtained culture solution is filtered,
The culture filtrate was separated. After adjusting the culture filtrate (10 L) to pH 2, an equal volume of butyl acetate was added, and the mixture was shaken, stirred and extracted. The upper layer was dried over anhydrous sodium sulfate, and the upper layer was concentrated under reduced pressure to 1 L. 1 L of water was added to the obtained concentrate, and the mixture was shaken, stirred, and extracted at pH 8; the lower layer was separated;
After that, 1 L of butyl acetate was added to this layer, and the mixture was shaken, stirred and extracted. The upper layer obtained at this time was dried over anhydrous sodium sulfate and then dried under reduced pressure to obtain 422 mg of an oil.

This oil was subjected to silica gel column chromatography (35 g), and eluted with chloroform: methanol (100: 2). The eluate was concentrated under reduced pressure and dried to obtain 60 mg of a candy. This is chloroform: methanol (1:
The solution was dissolved in 3 ml of the solution of 1), and gel filtration was performed using a Sephadex LH-20 (200 ml) column packed with the same solution. The fraction containing pastrestin was concentrated to dryness under reduced pressure to obtain 25.5 mg of a crude product.

The crude product is subjected to liquid chromatography.
(Column: Shiseido Capsule Pack UG), eluted with 30% acetonitrile water-2 mM ammonium carbonate, and eluted fractions containing pastrestin A under reduced pressure to dryness.
Pasterestin A (3.7 mg) was obtained as a white powder. Similarly, pastelessin B (6.0 mg) was isolated as a white powder.

[Brief description of the drawings]

FIG. 1 is an infrared absorption spectrum of pastrestin A measured by the KBr tablet method.

FIG. 2 is a proton nuclear magnetic resonance spectrum measured with a heavy methanol solution of pastrestin A (internal standard: trimethylsilane).

FIG. 3 is a proton nuclear magnetic resonance spectrum measured with a heavy methanol solution of pastrestin A (internal standard: trimethylsilane).

FIG. 4 is an infrared absorption spectrum of pastrestin B measured by the KBr tablet method.

FIG. 5 is a proton nuclear magnetic resonance spectrum measured with a heavy methanol solution of pastrestin B (internal standard: trimethylsilane).

FIG. 6 is a C-13 nuclear magnetic resonance spectrum measured with a heavy methanol solution of pastrestin B (internal standard: trimethylsilane).

[Procedure amendment]

[Submission date] September 14, 2001 (2001.9.1)
4)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claim 3

[Correction method] Change

[Correction contents]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/14 C12N 1/14 G C12P 15/00 C12P 15/00 // (C12P 15/00 (C12P 15 / 00 C12R 1: 645) C12R 1: 645) (72) Inventor Isao Momose 3-21-12-703 Minamirokugo, Ota-ku, Tokyo Tama Riverview Mansion (72) Inventor Yu Matsui Noji-cho, Kusatsu City, Shiga Prefecture 2257 No. Takara Agri Co., Ltd.F-term (reference) 4B064 AE41 BA03 BB08 BB19 BC03 BC04 BD03 BE01 BE09 BE10 BG01 BH01 BH02 BH03 BH06 BH07 BH08 BJ01 BJ04 CA07 DA02 4B065 AA71X AC14 BA22 CA34 CA04 A03 A02 MAA 4H006 AA01 AB03 BJ30 BN10 BN20 BS20 4H011 AA02 BB06

Claims (5)

[Claims] 1. The following general formula (I): (In the formula, R ¹ represents a hydroxymethyl group and R ² represents a hydrogen atom in pastorcin A, and R ¹ represents a methyl group and R ² represents a hydroxyl group in pastorastin B)
Or the antibiotics pastrestin A and B, or a pharmaceutically acceptable salt thereof. 2. A bacterium , which belongs to the genus Agrocybe ( Fayod ), which produces the antibiotics pastrestin A and B, and is cultured in a nutrient medium. A method for producing the antibiotics pastorastin A and / or pastorastin B, wherein A and / or pastorastin B are collected. 3. The method according to claim 2, wherein the producing bacterium used is Salix mushroom K-3793. 4. An antibacterial agent comprising, as an active ingredient, at least one of the antibiotics pastrestin A and B, and pharmaceutically acceptable salts thereof. 5. A willow matsutake ( Agrocybe cylindrace) having properties to produce the antibiotics pastrestin A and B
a (DC.:Fr.)Maire) K-3793 strain.