JPH09241226A - New derivative from antibiotic ab5366 and their production and agricultural/horticultural germicide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new antibiotic AB5366 derivative (salt, alkyl ester thereof) exhibiting effective germicidal activity at low concentrations against plant pathogenic fungi, esp. filamentous fungi, causing no chemical injuries on plants, and low in toxicity to mammals. SOLUTION: This new antibiotic AB5366 derivative [salt, alkyl ester (carboxylate) thereof] is expressed by formula I(R1 is OH; R2 is H; R1 and R2 are joined together into hydroxyimino, oxo or an alkoxyimino; R3 is H or sulfonic acid group; R4 is H or an acyl) (e.g. antibiotic AB5366-1 of formula II). One of the compounds of formula I, i.e., the illustrated compound of formula II, is obtained by reducing an antibiotic AB5366 of formula III. The antibiotic AB5366 of formula III can be produced from AB5366 strain (FERM P-14662). This compound of formula I is improved in antifungal activity as compared to that of the compound of formula III. Besides, the new derivatives include antibiotic AB5366-2 of formula IV, antibiotic AB5366-3 of formula V, other antibiotics AB5366-4, AB5366-5, AB5366-6, and antibiotic AB5366-3 methyl ester.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel derivative of antibiotic AB5366 having antifungal activity and salts or alkyl esters thereof, and a process for producing them. The present invention also relates to an agricultural / horticultural fungicide containing, as an active ingredient, a novel derivative of the antibiotic AB5366 or at least one of salts or alkyl esters thereof.

[0002]

BACKGROUND OF THE INVENTION The antibiotic AB5366 is a novel antibiotic (Japanese Patent Application No. 7-
No. 47555), which has antifungal activity and is useful as a fungicide for agriculture and horticulture.

[0003]

The object of the present invention is that it can be used in the field of agricultural and horticultural fungicides and shows effective bactericidal activity at low concentrations against phytopathogenic fungi, especially filamentous fungi, and shows phytotoxicity to plants. And to provide a novel substance having low toxicity to mammals.

[0004]

Means for Solving the Problems For the purpose of solving the above problems, the present inventors have conducted extensive studies on antibiotics produced by microorganisms isolated from various soils.
As a result, they are newly separated from the natural world.
It was first discovered that antibiotics with antifungal activity were produced in the culture fluid of a new strain belonging to the genus Pychnidiella with strain number of AB5366 strain (deposited to NIKEN as FERM P-14662). did. Therefore, the active substance was separated and purified from the culture of the strain, and it was recognized as a novel substance and named AB5366 substance, and it was found that it exhibits the above-mentioned useful antifungal activity. The chemical structure of the above was clarified (Japanese Patent Application No. 7-47555; filed on March 7, 1995). The AB5366 substance shows a high control effect at a low concentration in a plant disease control test using a plant, but it shows no phytotoxicity on the plant and has low toxicity to mammals.

The antibiotic, AB5366, has the following formula (A) It is a compound represented by these. AB5366 substance has a specific rotation [α]
It is a pale yellow powder showing _D ²³ + 2.0 ° (c 1.0, methanol).

The present inventors have found that the antibiotic of formula (A) above
Research was continued with the aim of improving the antifungal activity of AB5366. As a result, the AB5366 substance was acid-hydrolyzed or reduced or oxime- or acetylated or O-alkyloxime-oxidized or a combination thereof to produce 6 kinds of amino acid type derivatives having antifungal activity. As a result of the structural analysis of these derivatives, they are novel substances, and they are substances AB5366-1, AB5366-2,
AB5366-3, AB5366-4, AB5366-5 and AB5366-6
It was named. In addition, the alkyl ester in the carboxylic acid group of these 6 substances, AB5366-5 and AB5366-6
We also succeeded in synthesizing with the homologue of. The novel substances newly synthesized by the present inventors can be represented generically by the general formula (I) described below.

That is, in the first aspect of the present invention, the following general formula (I) (In the formula, R ₁ represents a hydroxyl group, R ₂ represents a hydrogen atom, or R ₁ and R ₂ together form a hydroxyimino group, an oxo group or an alkoxyimino group, and R ₃ represents a hydrogen atom or indicates a sulfonic acid group, R ₄ is to provide an antibiotic AB5366 derivatives or their salts or alkyl esters represented by a hydrogen atom or an acyl group) (carboxylate).

The alkyl moiety of the alkoxyimino group formed by R ₁ and R ₂ represented by formula (I) in the derivative of formula (I) preferably has 1 to 2 carbon atoms. Examples of the alkoxyimino group include lower alkoxyimino groups such as methyloxyimino group and ethyloxyimino group, and ethyloxyimino group is preferable. Further, the acyl group represented by R ₄ is preferably an alkylcarbonyl group, and the alkyl portion thereof has 1 to 10 carbon atoms, preferably 1 to 2 carbon atoms.
Examples of the acyl group include an acetyl group, a propionyl group and a butyryl group, and an acetyl group is preferable.

Examples of salts of the antibiotic AB5366 derivative of the present invention include salts of organic bases such as quaternary ammonium with various metals such as alkali metals, particularly sodium or potassium salts, and alkaline earth metals. Salt with
Especially calcium salts. The alkyl moiety of the alkyl ester (carboxylate) of the antibiotic AB5366 derivative of the present invention can be an alkyl group having 1 to 10 carbon atoms, and the alkyl group includes a methyl group, an ethyl group, a propyl group, and the like. A methyl group is preferred.

The antibiotic AB5366 derivative of the general formula (I) according to the first aspect of the present invention is AB5366-1 substance, AB5366-, which will be described later.
2 substances, AB5366-3 substance, AB5366-4 substance, AB5366-5
Substances and AB5366-6 substances, as well as the methyl ester of AB5366-3 substances.

The AB5366-1 substance according to the first aspect of the present invention is represented by the following structural formula (Ia) and has the following physicochemical properties. (1) Appearance: White powder (2) Molecular formula: C ₁₉ H ₃₉ NO ₈ S (3) Molecular weight: 441 (4) Red external absorption spectrum (KBr): Shown in FIG. 1 of the attached drawings.

(5) Solubility: Soluble in methanol and water, insoluble in acetone. (6) Thin layer chromatography: Rf value = 0.46 adsorbent: Merck Kieselgel 60F ₂₅₄ developing solvent: chloroform - methanol - formic acid (10: 5:
1) (7) Color reaction: positive for vanillin sulfate and iodine reaction. (8) Basic, acidic, neutral: acidic substance.

The AB5366-2 substance according to the first aspect of the present invention is represented by the following structural formula (Ib) and has the following physicochemical properties. (1) Appearance: White powder (2) Molecular formula: C ₁₉ H ₃₈ N ₂ O ₈ S (3) Molecular weight: 454 (4) Red external absorption spectrum (KBr): Shown in FIG. 2 of the attached drawings.

(5) Solubility: Soluble in methanol and water, insoluble in acetone. (6) Thin layer chromatography: Rf value = 0.51 adsorbent: Merck Kieselgel 60F ₂₅₄ developing solvent: chloroform - methanol - formic acid (10: 5:
1) (7) Color reaction: positive for vanillin sulfate and iodine reaction. (8) Basic, acidic, neutral: acidic substance.

The AB5366-3 substance according to the first aspect of the present invention is represented by the following structural formula (Ic) and has the following physicochemical properties. (1) Appearance: White powder (2) Molecular formula: C ₁₉ H ₃₇ NO ₅ (3) Molecular weight: 359 (4) Red external absorption spectrum (KBr): Shown in FIG. 3 of the attached drawings.

(5) Solubility: Hardly soluble in methanol, water and acetone. (6) Thin layer chromatography: Rf value = 0.75 adsorbent: Merck Kieselgel 60F ₂₅₄ developing solvent: chloroform - methanol - formic acid (10: 5:
1) (7) Color reaction: positive for vanillin sulfate and iodine reaction. (8) Basic, acidic, neutral: amphoteric substance.

The AB5366-4 substance according to the first aspect of the present invention is represented by the following structural formula (Id) and has the following physicochemical properties. (1) Appearance: White powder (2) Molecular formula: C ₁₉ H ₃₉ NO ₅ (3) Molecular weight: 361 (4) Red external absorption spectrum (KBr): Shown in FIG. 4 of the attached drawings.

(5) Solubility: It is hardly soluble in methanol, water and acetone. (6) Thin layer chromatography: Rf value = 0.68 adsorbent: Merck Kieselgel 60F ₂₅₄ developing solvent: chloroform - methanol - formic acid (10: 5:
1) (7) Color reaction: positive for vanillin sulfate and iodine reaction. (8) Basic, acidic, neutral: amphoteric substance.

The AB5366-5 substance according to the first aspect of the present invention is represented by the following structural formula (Ie) and has the following physicochemical properties. (1) Appearance: White powder (2) Molecular formula: C ₂₁ H ₃₉ NO ₆ (3) Molecular weight: 401 (4) Red external absorption spectrum (KBr): Shown in FIG. 5 of the attached drawings.

(5) Solubility: Poorly soluble in water, soluble in methanol and acetone. (6) Thin layer chromatography: Rf value = 0.93 adsorbent: Merck Kieselgel 60F ₂₅₄ developing solvent: chloroform - methanol - formic acid (10: 5:
1) (7) Color reaction: positive for vanillin sulfate and iodine reaction. (8) Basic, acidic, neutral: acidic substance.

The AB5366-6 substance according to the first aspect of the present invention is represented by the following structural formula (If) and has the following physicochemical properties. (1) Appearance: White powder (2) Molecular formula: C ₂₁ H ₄₂ N ₂ O ₈ S (3) Molecular weight: 486 (4) Red external absorption spectrum (KBr): Shown in FIG. 6 of the attached drawings.

(5) Solubility: Soluble in water, hardly soluble in methanol and acetone. (6) Thin layer chromatography: Rf value = 0.50 adsorbent: Merck Kieselgel 60F ₂₅₄ developing solvent: chloroform - methanol - formic acid (10: 5:
1) (7) Color reaction: positive for vanillin sulfate and iodine reaction. (8) Basic, acidic, neutral: acidic substance.

The methyl ester of the AB5366-3 substance according to the first aspect of the present invention is represented by the following structural formula (Ig) and has the following physicochemical properties. (1) Appearance: White powder (2) Molecular formula: C ₂₀ H ₃₉ NO ₅ (3) Molecular weight: 373 (4) Red external absorption spectrum (KBr): Shown in FIG. 7 of the attached drawings.

(5) Solubility: Poorly soluble in water, soluble in methanol and acetone. (6) Thin layer chromatography: Rf value = 0.86 adsorbent: Merck Kieselgel 60F ₂₅₄ developing solvent: chloroform - methanol - formic acid (10: 5:
1) (7) Color reaction: positive for vanillin sulfate and iodine reaction. (8) Basic, acidic, neutral: basic substance.

According to the second aspect of the present invention, the antibiotic AB5366 of the formula (A) is reduced to obtain the substance AB of the formula (Ia).
Substance of formula (Ia) characterized in that it produces 5366-1
A method of making AB5366-1 is provided. According to a third aspect of the present invention, there is provided a substance of formula (Ib), AB5366-2, which is characterized in that said substance of formula (A), AB5366, is oxime-ized to produce substance of formula (Ib), AB5366-2. A manufacturing method is provided. According to a fourth aspect of the present invention, the antibiotic AB5366 of formula (A) above is acid hydrolyzed to produce the substance AB5366-of formula (Ic).
AB5366, a substance of formula (Ic), characterized in that it produces 3
-3 is provided.

According to a fifth aspect of the present invention, the above formula (I
The substance AB5367-3 of c) is reduced to form the substance AB53 of formula (Id)
66-4 substance AB of formula (Id) characterized by producing 66-4
A method of making 5366-4 is provided. According to a sixth aspect of the present invention, the substance AB5366-5 of formula (Ie) is characterized in that the substance AB5366-3 of formula (Ic) is acetylated to produce the substance AB5366-5 of formula (Ie). A method of manufacturing the same is provided. According to a seventh aspect of the present invention, the antibiotic of formula (A) AB5366 is converted to O-ethyloxime to form a substance of formula (If).
There is provided a process for the preparation of the substance AB5366-6 of formula (If), characterized in that it produces AB5366-6.

According to an eighth aspect of the present invention, the above formula (I
A process for the preparation of substance AB5366-3 methyl ester of formula (Ig) is provided, characterized in that the carboxyl group of substance AB5366-3 of c) is methylated to form substance AB5366-3 methyl ester of formula (Ig). It

Next, the above-mentioned substance AB5366- according to the present invention
1, -2, -3, -4, -5 and -6 and substance AB53
The method for producing each substance of 66-3 methyl ester will be described.

In order to carry out the second method of the present invention, the AB5366-1 substance can be produced by dissolving the antibiotic AB5366 as a raw material in a suitable solvent and reducing it with a suitable reducing agent. . Any reducing agent may be used as long as it can reduce the ketone group (oxo group) of the raw material to form a secondary alcohol (hydroxyl group), and sodium borohydride is preferable. The amount of reducing agent added is 2 to 3 times, and preferably 2 times the molar amount of the raw material in terms of molar ratio. The solvent used is a solvent in which the raw materials or reaction products are dissolved, and preferred solvents are lower alcohols such as methanol and ethanol. The reaction is carried out under room temperature conditions, and the disappearance of the starting materials or an appropriate amount of the reaction product is used as a guideline for the end point of the reaction, and thereafter, appropriate post-treatment and purification steps are performed to obtain the desired product. AB5366 of formula (Ia)
-1 substance is obtained.

To carry out the third method of the present invention, the antibiotic AB5366 as a raw material is dissolved in a suitable solvent and oxime-ized with a suitable oxime-forming agent.
-2 substances can be produced. The oxime-forming agent used in this case may be any one as long as the ketone group (oxo group) of the raw material can be dehydrated and condensed to form an oxime group. For example, hydroxyammonium chloride is preferable. The addition amount of the oxime-forming agent is 1.1 to 1.5 times, and preferably 1.2 times, the amount of the raw material in molar ratio. The solvent used is a solvent in which the raw materials or reaction products are dissolved, and preferred solvents are lower alcohols such as methanol and ethanol. The reaction is carried out at room temperature or under reflux conditions, and the disappearance of the starting materials or an appropriate amount of the reaction product is used as a guideline for the end point of the reaction, and thereafter, appropriate post-treatment and purification steps are performed to obtain the objective. The AB5366-2 substance of formula (Ib) is obtained as a product.

To carry out the fourth method of the present invention, the antibiotic AB5366 as a raw material is dissolved in a suitable solvent and hydrolyzed by acid treatment with a suitable mineral acid to obtain AB5366-3.
The substance can be manufactured. Any mineral acid may be used as long as it can hydrolyze the sulfate ester portion of the raw material to form an alcohol, and hydrochloric acid or sulfuric acid is preferable. The concentration of mineral acid is 0.
It is 5 to 6 normals, preferably 1 normal concentration. The solvent used is a solvent in which the raw materials or reaction products are dissolved, and the preferred solvent is methanol,
Lower alcohols such as ethanol or water are used. The reaction is carried out at room temperature or under reflux conditions, and the disappearance of the starting materials or an appropriate amount of the reaction product is used as a guideline for the end point of the reaction, and thereafter, after appropriate treatment and purification steps, the desired product is obtained. As a result, AB5366-3 substance of formula (Ic) is obtained.

To carry out the fifth method of the present invention, the above-mentioned antibiotic AB5366-3 as a raw material is dissolved in a suitable solvent and treated with a suitable reducing agent to reduce AB53.
66-4 substances can be produced. Any reducing agent may be used as long as it can reduce the ketone group (oxo group) of the raw material to form a secondary alcohol (hydroxyl group), and sodium borohydride is preferable. The reducing agent is added in a molar ratio of 2 of the raw material.
˜3 times the amount, preferably twice the amount. The solvent used is a solvent in which the raw materials or reaction products are dissolved, and preferred solvents are lower alcohols such as methanol and ethanol. The reaction is carried out under room temperature conditions, and the disappearance of the starting materials or an appropriate amount of the reaction product is used as a guideline for the end point of the reaction, and thereafter, appropriate post-treatment and purification steps are performed to obtain the desired product. AB5366-4 material of formula (Id) is obtained.

To carry out the sixth method of the present invention, the antibiotic AB5366-3 as a raw material is dissolved in a suitable solvent and treated with a suitable acetylating agent to acetylate the AB5366-5 material. It can be manufactured. Any acetylating agent may be used as long as it can acetylate the amino group of the raw material to form an N-acetyl group, and acetic anhydride is preferable. The amount of the acetylating agent added is 1.1 to 1.5 times, and preferably 1.2 times the molar amount of the raw material in terms of molar ratio. The solvent used is a solvent in which the raw materials or reaction products are dissolved,
Lower alcohols such as methanol and ethanol are used as a preferable solvent. The reaction is carried out at room temperature, and the disappearance of the starting materials or an appropriate amount of the reaction product is used as a guideline for the end point of the reaction. (I
AB5366-5 substance of e) is obtained.

The compound of the formula (I) in which R ⁴ is an acyl group can be generally prepared by using a suitable acid anhydride in place of the acetic anhydride.

To carry out the seventh method of the present invention, the antibiotic AB5366 as a raw material is dissolved in a suitable solvent and converted to O-ethyloxime with a suitable O-ethyloxime-forming agent. Six substances can be produced. The O-ethyl oxime-forming agent used at this time may be any as long as the ketone group (oxo group) of the raw material can be dehydrated and condensed to form O-ethyl oxime, for example, O-ethyl hydroxyl ammonium. Chloride is preferred. The amount of the O-ethyl oxime-forming agent added is 1.1 to 1.5 times, and preferably 1.2 times the molar amount of the raw material in molar ratio. The solvent used at this time is a solvent in which the raw materials or reaction products are dissolved, and preferred solvents are lower alcohols such as methanol and ethanol. The reaction is carried out at room temperature or under reflux conditions, and the disappearance of the starting materials or an appropriate amount of the reaction product is used as a guideline for the end point of the reaction. As the formula (I
AB5366-6 substance of f) is obtained.

The compound of the formula (I) in which R ₁ is an alkoxyimino group generally has the above-mentioned O content.
-O instead of ethylhydroxylammonium chloride
It can be prepared by using an alkylhydroxylammonium chloride.

To carry out the eighth method of the present invention, the above-mentioned antibiotic AB5366-3 as a raw material is dissolved in a suitable solvent, treated with a suitable methylating agent and methylated to obtain AB5366-. It is possible to produce methyl esters of three substances. The methylating agent used in this case may be any one as long as it can methylate the carboxyl group of the raw material to form a methyl ester, and for example, diazomethane is preferable. The amount of the methylating agent added is 1.1 to 1.5 times the molar amount of the raw material, and preferably 1.2 times the molar amount. The solvent used at this time is a solvent in which the raw materials or reaction products are dissolved, and preferred solvents are lower alcohols such as methanol and ethanol. The reaction temperature is room temperature, and the disappearance of the starting materials or an appropriate amount of the reaction product is used as a guide for the end point of the reaction, and after that, appropriate post-treatment and purification steps are performed to obtain the desired product. The methyl ester of AB5366-3 substance of formula (Ig) is obtained.

The alkyl ester of the derivative of the general formula (I) according to the present invention is generally obtained by reacting the compound of the general formula (I) in the form of the corresponding carboxylic acid used as a starting material with a diazoalkane. It can be produced by carrying out an esterification reaction similar to the methyl esterification reaction.

The above-mentioned substances AB5366-1, -2, -3, -4, which are included in the derivative of the general formula (I) according to the first invention,
Methyl esters of -5, -6, and AB5366-3 have phytopathogenic growth inhibitory activity as shown in the test examples described below.

Next, AB5366 of the general formula (I) of the first invention is
The fact that the derivative has a growth inhibitory activity against phytopathogenic bacteria and is useful as a fungicide will be explained by the following test examples.

Test Example 1 AB5366-1, -2, -3,-
Growth-inhibiting effect of 4, -5 and -6 substances and AB5366-3 substance methyl ester on plant pathogens AB5366-1 (Na salt), -2 (Na salt), -3 (HCl
Salt), -4 (Na salt), -5, and -6 (Na salt) and the minimum growth inhibitory concentration (MIC) (μg / m) of AB5366-3 substance methyl ester against various plant pathogens
l) was measured by agar plate dilution method using potato-sucrose agar medium. Similarly, antibiotic AB5366
(Na salt) minimum inhibitory concentration (MIC) (μg / ml)
Was also measured for comparison. The results are shown in Table 1 below.

[0042]

[0043]

Therefore, according to the ninth aspect of the present invention, there is provided an agricultural / horticultural fungicide containing as an active ingredient at least one of the antibiotic AB5366 derivative represented by the general formula (I) or a salt or alkyl ester thereof. To be done.

[0045]

BEST MODE FOR CARRYING OUT THE INVENTION The first general formula (I) according to the present invention
When using at least one of the AB5366 derivative or the salt or alkyl ester thereof as an agricultural and horticultural fungicide, it can be used alone depending on the purpose of use, but in order to promote or stabilize the biological effect. In addition, it is mixed with a suitable solid or liquid simple substance which is conventionally used, and if necessary, an auxiliary agent is added to form a composition, which is directly used or diluted as necessary. To use.

[0046]

The present invention will be described in more detail with reference to the following examples, which are merely examples and the present invention is not limited thereto. Needless to say, it is possible to use many modified methods or modification means not illustrated here, and it can be an effective means. In the examples, “parts” means “parts by weight”.

Example 1 Preparation of substance AB5366-1 50 mg (0.11 mmol) of the antibiotic AB5366 sodium salt was added to 1 part.
It was dissolved in ml of methanol. After adding sodium borohydride to the solution, it was stirred at room temperature for 1 hour. After the reaction was completed, 100 ml of distilled water was added, and the mixture was applied to a column of Diaion HP20 (registered trademark) (10 ml) equilibrated with water in advance, washed with 100 ml of distilled water, and then 50 ml of 80% hydrous methanol was used. It eluted. Concentrate the eluate to dryness and use AB53
As a sodium salt of substance 66-1, 37.0 mg (0.
08 mmol) was obtained. The recovery rate at this time was 72.7%.

Example 2 Preparation of AB5366-2 substance 140 mg (0.30 mmol) of the antibiotic AB5366 sodium salt was added.
It was dissolved in 950 μl of an aqueous sodium carbonate solution (18 mg / ml). Hydroxylamine chloride (10 mg / m
After adding 2450 μl of the ethanol solution of l), the mixture was stirred at room temperature for 7 hours. After the reaction was completed, 100 ml of water was added, and Diaion HP20 (registered trademark) previously equilibrated with distilled water was added.
The mixture was applied to a (10 ml) column, washed with 100 ml of distilled water, and then eluted with 50 ml of 80% hydrous methanol. The eluate was concentrated to dryness to obtain 100 mg (0.21 mmol) of white powder as a sodium salt of AB5366-2 substance. The recovery rate at this time was 70.0%.

Example 3 Production of AB5366-3 substance 201 mg (0.44 mmol) of the antibiotic AB5366 sodium salt was dissolved in 5 ml of 5N hydrochloric acid and reacted at 110 ° C. for 3 hours. After the completion of the reaction, the mixture was allowed to stand at room temperature, the generated precipitate was collected by filtration using a glass filter, and sufficiently washed with distilled water until the pH of the filtrate showed no acidity on pH test paper. After that, the solid was dried under reduced pressure to obtain 128 mg (0.32 mmol) of a white powder as a hydrochloride of AB5366-3 substance. The recovery rate at this time was 72.7%.

Example 4 Preparation of substance AB5366-4 Substance 40.8 mg (0.10 mmol) of the hydrochloride salt of substance AB5366-3 obtained in Example 3 was dissolved in 1 ml of methanol. Sodium borohydride was added to the solution and then AB5366-3 on TLC.
Stir at room temperature until the material has completely disappeared. After the reaction is completed, the reaction solution is neutralized with 1N sodium hydroxide,
Concentrated under reduced pressure. The formed precipitate was collected by filtration, washed thoroughly with water, and dried under reduced pressure to obtain 32.3 mg (0.08 mmol) of sodium salt of substance AB5366-4 as a white powder. The recovery rate at this time was 80.0%.

Example 5 Preparation of substance AB5366-5 Substance 40.5 mg (0.10 mmol) of the hydrochloride salt of substance AB5366-3 obtained in Example 3 was dissolved in 2 ml of methanol. After adding 4 ml of acetic anhydride to the solution, it was stirred at room temperature for 18 hours. After completion of the reaction, 50 ml of distilled water was added under ice cooling, the mixture was transferred to a 100 ml separating funnel, 50 ml of ethyl acetate was added, and the mixture was vigorously shaken to separate the layers. The ethyl acetate phase was separated, 20 ml of distilled water was added, and the above operation was repeated 3 times to wash the ethyl acetate phase with water. The ethyl acetate phase washed with water was concentrated to dryness under reduced pressure, dissolved in 2 ml of methanol, and applied to a column of Sephadex LH20 (registered trademark) (200 ml) equilibrated with methanol in advance, and developed with methanol. The obtained fraction was concentrated to dryness to obtain 23.3 mg (0.062 mmol) of white powder of AB5366-5 substance. The recovery rate at this time was 62.0%.

Example 6 Preparation of AB5366-6 substance 103 mg (0.22 mmol) of the antibiotic AB5366 sodium salt was added.
It was dissolved in 0.65 ml of an aqueous sodium carbonate solution (18 mg / ml). After adding 1.75 ml of an ethanol solution of O-ethylhydroxylamine chloride (15 mg / ml) to the solution, at 37 ° C.
Stir for 17 hours. After completion of the reaction, add 100 ml of distilled water,
It was applied to a column of Diaion HP20 (registered trademark) (10 ml) that had been equilibrated with distilled water in advance. After washing with 100 ml of distilled water, elution with 50 ml of 80% hydrous methanol,
After concentration to dryness, 83.3 mg (0.16 mmol) of white powder was obtained as a sodium salt of AB5366-6 substance. The recovery rate at this time was 72.7%.

Example 7 Production of methyl ester of AB5366-3 substance 41.2 mg (0.10 mmol) of the hydrochloride salt of AB5366-3 substance obtained in Example 3 was dissolved in 2 ml of methanol. After adding 4 ml of ethereal diazomethane to the solution, the mixture was stirred at room temperature for 2 hours. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure, dissolved in 2 ml of methanol, applied to a column of Sephadex LH20 (registered trademark) (200 ml) equilibrated with methanol in advance, and developed using methanol. The obtained fraction was concentrated to dryness to obtain 15.0 mg (0.04 mmol) of methyl ester of AB5366-3 substance as a white powder. The recovery rate at this time is
It was 40.0%.

Example 8 (Wettable powder) Substance according to the invention AB5366-1 or -2 or -3 or -4 or -5 or -6 or AB5366-3 substance 20 parts of methyl ester, potassium alkylbenzene sulfonate 3 Parts, polyoxyethylene nonyl phenyl ether 5
Parts and 72 parts of white clay were uniformly mixed and ground to obtain a wettable powder containing 20% of the active ingredient.

Example 9 (powder) substance AB5366-1 or -2 or -3 or -4 or -5 or -6 or AB5366-3 substance 2 parts of methyl ester, 1 part of PAP (physical property improver) and clay 97 parts were uniformly mixed and ground to obtain a dust containing 2% of active ingredient.

Example 10 (Emulsion) Substance AB5366-1 or -2 or -3 or -4 or -5 or -6 or AB5366-3 Substance of methyl ester
30 parts of methyl ethyl ketone and 40 parts of polyoxyethylene nonyl phenyl ether are mixed to give 30 parts of active ingredient.
An emulsion containing 10% was obtained.

Furthermore, the test effects of the plant diseases used in the agricultural and horticultural fungicides according to the ninth aspect of the present invention are Test Examples 2, 3, 4 and 5.
To illustrate.

Test Example 2 Control of Gray Mold Disease A cotyledonary cucumber seedling (cultivar: Sagamihanjiro) cultivated in a greenhouse in a pot made of vinyl resin having a diameter of 6 cm was cultivated.
A chemical solution prepared by diluting a wettable powder prepared according to Example 8 to a predetermined concentration was sprayed at 10 ml per pot. One day after spraying the drug solution, Botrytis ci bacterium ( Botrytis ci
nerea : Botrytis cinerea) spores concentration 5 ×
A spore suspension prepared by suspending in a 1% glucose aqueous solution so that the number of spores was 10 5 / ml was added dropwise to the center of the cotyledon. Then keep the cotyledons in an inoculation box at 20 ° C and 100% humidity,
I got sick. Three days after the inoculation, the microflora diameter (mm) per leaf was measured, and then the control value (%) was calculated by the following formula.

[0059]

This test was carried out in duplicate for each chemical solution concentration group, the average control value (%) was determined, and then the control effect was determined according to the following criteria. Further, the chemical damage to cucumber (variety: Sagamihanjiro) was also investigated according to the following standard of chemical damage investigation. Average control value (%) Evaluation value of control effect 100% 5 90 to less than 100% 4 70 to 90% 〃 3 40 to 70% 〃 2 40% 〃 1 (Criteria for investigation of chemical damage) 5: Severe 4: Severe 3: Many 2: Some 1: Only 0: None

For comparison, the antibiotic AB5366 was also tested in the same manner as above. In the non-dispersed plots, the test plants were not treated with the chemical solution containing the active ingredient. The above results are shown in Table 2.

[0062]

Test Example 3 Test for control effect of kidney bean sclerotium disease According to Example 8, a cotyledon bean seedling (cultivar: Himezashi) was cultivated in a greenhouse in a vinyl resin pot having a diameter of 6 cm. The wettable powder prepared as described above was diluted to a predetermined concentration and sprayed with 10 ml per pot. One day after spraying the drug solution, Sclerotinia (Sclerotinia), which had been grown in advance on PSA medium,
sclerotiorum : sclerotinia )
The bacterium-containing disc of was placed on the center of the cotyledon. And 20
It was kept warm in the inoculation box at 100 ° C and 100% humidity to cause the disease. Inoculation 3
After the day, the microflora diameter per leaf (mm) was measured, and then the control value (%) was calculated by the calculation formula shown in Test Example 2.

This test was carried out in duplicate for each chemical solution concentration group, the average control value (%) was determined, and then the control effect was determined according to the same criteria as in Test Example 2. Also,
Based on the same phytotoxicity investigation criteria as in Test Example 2, phytotoxicity to kidney bean (variety: Himeshin) was also investigated. For comparison, antibiotic AB5366 was also tested in the same manner as above. The results are shown in Table 3.

[0065]

Test Example 4 Barley Powdery Mildew Control Effect Test No. 1 of barley (variety: Azuma Golden) cultivated in soil in a greenhouse made of a vinyl resin pot having a diameter of 6 cm.
At the leaf stage, 10 ml per pot was sprayed with a drug solution prepared by diluting the wettable powder prepared according to Example 8 to a predetermined concentration. Chemical spray 1
After a day, conidiospores of barley powdery mildew ( Erysiphe graminis ) formed on barley leaves in advance were diluted with a 50 ppm aqueous solution of a spreading agent (polyoxyethylene alkyl ether) to give a spore concentration of 5 × 10 ⁵ spores (pieces) / ml were prepared, and this was spray-inoculated on the foliage of barley using a spray gun. And 20 ℃, humidity 100
It was kept warm and sick in a 100% inoculation box. Seven days after the inoculation, the lesion length (mm) of barley powdery mildew per leaf was investigated. From these data, the control value (%) was calculated by the following formula.

[0067]

This test was carried out in duplicate for each chemical concentration group, the average control value (%) was determined, and then the control effect was determined according to the same criteria as in Test Example 2. Also,
The phytotoxicity to barley (variety: Azuma Golden) was also investigated according to the same phytotoxicity investigation criteria as in Test Example 2. For comparison, antibiotic AB5366 was also tested in the same manner as above. Table 4 shows the results.

[0069]

Test Example 5 Cucumber downy mildew control effect test At the second leaf stage of a cucumber (cultivar: Sagamihanjiro) cultivated in a greenhouse in a pot made of a vinyl resin having a diameter of 6 cm,
A chemical solution prepared by diluting a wettable powder prepared according to Example 8 to a predetermined concentration was sprayed at 10 ml per pot. One day after spraying the chemical solution, the cucumber downy mildew ( Pseudo
pernospora cubensis : spores of Sudopernospora cubensis 5 diluted with a 50ppm aqueous solution of a spreading agent (polyoxyethylene alkyl ether)
It was adjusted to 10 5 spores (pieces) / ml, and this was spray-inoculated into cucumber using a spray gun. And 20 ℃, humidity
It was kept warm and sick in a 100% inoculation box. 6 days after vaccination 1
The disease area ratio (%) of barley powdery mildew per leaf was investigated, and the average disease area ratio was calculated. The control value (%) was calculated from these data by the calculation formula shown in the following formula.

[0071]

This test was carried out in duplicate for each chemical solution concentration group, the average control value (%) was determined, and then the control effect was determined according to the same criteria as in Test Example 2. Also,
The phytotoxicity to cucumber (variety: Sagamihanjiro) was also examined according to the same phytotoxicity investigation criteria as in Test Example 2. For comparison and control, AB5366 substance was also tested in the same manner as above. Table 5 shows the results.

[0073]

Since the antibiotic AB5366 used as a raw material for producing the antibiotic AB5366 derivative of the general formula (I) according to the first aspect of the present invention is a novel substance, its production will be described below.

For the production of the antibiotic AB5366, the antibiotic AB5366-producing bacteria belonging to the genus Pychnidiella is cultivated in a medium containing nutrients that can be assimilated by ordinary microorganisms, preferably a liquid medium. For the cultivation of the present producing bacteria, the usual culturing method used for culturing microorganisms is applied. As a nutrient source, either a natural medium or a synthetic medium can be used as long as it is a medium containing a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the microorganism used.

The AB5366 strain, which is an example of the AB5366-producing bacterium, has been applied for deposit at the Institute of Biotechnology, Institute of Industrial Science and Technology, and has been deposited as FERM P-14662 on November 25, 1994. The mycological properties of the AB5366 strain are described in detail in Japanese Patent Application No. 7-47555.

As the assimilable carbon source contained in the medium used for culturing the AB5366-producing bacterium, glucose, sucrose, galactose, dextrin, glycerol, starch, starch syrup, molasses, animal / vegetable oil and the like can be used. As the nitrogen source, fish meal, soybean meal, wheat germ, corn steep liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, ammonium nitrate, sodium nitrate, urea and the like can be used. In addition, if necessary, it is effective to add inorganic salts capable of producing sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid and other ions to the medium. Further, an inorganic substance and / or an organic substance which assists the growth of the antibiotic strain AB5366 producing strain and promotes the production of the AB5366 substance can be appropriately added. When a metal ion or base is present in the medium, the antibiotic AB5366 may be obtained in the form of a salt with a corresponding metal cation or anion.

As a method for culturing the antibiotic AB5366-producing bacterium, a culture method under aerobic conditions, particularly a deep liquid culture method under aeration is most suitable. A suitable temperature for culturing is 15 to 30 ° C, but in many cases, it is preferable to culture at 24 to 30 ° C. Antibiotics
The production of AB5366 varies depending on the type of culture medium used and the culture conditions, but the accumulated amount reaches its maximum after 3 to 10 days of culture in both shaking culture and tank culture. Isolation of antibiotic AB5366 substance according to a general method of stopping the culture at the time when the accumulated amount of antibiotic AB5366 produced in the culture reaches a maximum and collecting the fermentation product from the culture Good to do. The culture conditions for efficiently producing the antibiotic AB5366 are:
The composition of the above-mentioned medium components, culture temperature, stirring speed, pH, aeration amount, seed culture time, seed inoculation amount, etc. are appropriately adjusted depending on the type of production strain to be used and external conditions, or Select and set. In the case where foaming occurs in the liquid culture method, silicone oil, vegetable oil, and a defoaming agent such as a surfactant are used alone or as a mixture, and appropriately mixed in the medium.

The antibiotic AB5366 accumulated in the culture thus obtained by culturing the bacteria producing the antibiotic AB5366 can be efficiently separated and purified by the following method. In collecting the antibiotic AB5366 accumulated in the culture from the culture, a usual method for isolating a physiologically active substance from the culture is applied. That is, by extraction with butanol from the culture broth, extraction with microbial cells by an organic solvent such as methanol, or by countercurrent distribution using water or two or more organic solvent systems, or ion exchange resin, synthetic adsorption resin. , Silica gel, silanized silica gel, alumina, cellulose, diatomaceous earth, gel filter, activated carbon, etc. column chromatography or thin layer chromatography for adsorption and desorption of the active substance, or high performance liquid chromatography using a reverse layer column. The antibiotic AB5366 substance can be isolated by, for example,

Reference Example 1 This example illustrates the fermentative production of the antibiotic AB5366. The production medium used for culturing the bacterium was glucose 1.0%, dextrin.
1.5%, yeast extract 1.0%, malto extract 1.0%, KH
_{2 PO 4 0.5%, MgSO 4} · 7H 2 O 0.5%, consisting of calcium carbonate 0.3% Composition (pH 6.5). Medium of the above composition
Ninety-one baffled Erlenmeyer flasks with baffles of 110 ml volume (total volume of 10 liters of medium) were sterilized at 121 ° C for 20 minutes. For each sterilized medium,
One to two platinum loops of slope agar culture cells of S.P. AB5366 strain (FERM P-14662) were inoculated.

Then, a rotary shaker (180 rpm)
The cells were cultivated with shaking at 25 ° C. for 5 days. After cultivation,
The culture solution was filtered to obtain 8 liters of the culture filtrate and bacterial cells. The active substance in the filtered bacterial cells was extracted from the bacterial cells with 4 liters of methanol, and the bacterial cells were removed by filtration to obtain a bacterial cell extract. Methanol was distilled off from this cell extract under reduced pressure, and 1 liter of the obtained concentrated liquid was mixed with the above-mentioned culture filtrate. 9 liters of the resulting mixed solution was adsorbed on a column (capacity: 750 ml) packed with a synthetic adsorption resin, Diaion HP-20 (registered trademark), and then, with 2 liters of water and 40% methanol water, respectively. The column was washed. Then, the fraction was eluted with 100% methanol, and the active fractions were collected and concentrated to dryness under reduced pressure to obtain 875 mg of a brown candy-like substance.

875 mg of this brown candy-like substance was dissolved in a small amount of 50% methanol water, and then the same solvent was used to prepare a column of Sephadex LH-20 (registered trademark) (volume: 150 ml).
It was placed on top and subsequently fractionated and eluted using the same solvent. The active fractions thus obtained were collected and concentrated to dryness under reduced pressure, and the solid was dissolved in a small amount of a mixed solvent having a composition of chloroform-methanol-formic acid (20: 5: 1), followed by silica gel (Merck). Column (capacity: 20 ml) packed with
Chromatography was carried out by adsorbing to and adsorbing to and developing and eluting with the same solvent. The active fractions were collected and concentrated to dryness under reduced pressure to obtain 185 mg of yellow powder.

185 mg of this yellow powder was subjected to column chromatography again in exactly the same manner as in the above Sephadex LH-20 column to isolate the desired active ingredient. By the above method, 62.1 mg of a pure antibiotic AB5366 was obtained as a yellow powder from the culture solution containing 10 liters of bacterial cells.

[0084]

The novel antibiotic AB5366 derivative of the present invention has antibacterial activity against plant pathogens as described above,
Since it has a controlling effect against actual plant diseases, it is expected as a novel fungicide for agricultural and horticultural use.

[Brief description of drawings]

1 is an infrared absorption spectrum of substance AB5366-1 of the present invention measured in a potassium bromide tablet.

FIG. 2 is an infrared absorption spectrum diagram of substance AB5366-2 of the present invention measured in a potassium bromide tablet.

FIG. 3 is an infrared absorption spectrum diagram of substance AB5366-3 of the present invention measured in a potassium bromide tablet.

FIG. 4 is an infrared absorption spectrum of substance AB5366-4 of the present invention measured in a potassium bromide tablet.

FIG. 5 is an infrared absorption spectrum of the substance AB5366-5 of the present invention measured in a potassium bromide tablet.

FIG. 6 is an infrared absorption spectrum of substance AB5366-6 of the present invention measured in a potassium bromide tablet.

FIG. 7 is an infrared absorption spectrum measured in a potassium bromide tablet of the substance AB5366-3 methyl ester of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C07C 227/18 9450-4H C07C 227/18 231/02 9547-4H 231/02 233/47 9547- 4H 233/47 303/24 7419-4H 303/24 305/10 7419-4H 305/10 (72) Inventor Yasuyuki Tezuka 2-24-7 Morinosato, Atsugi City, Kanagawa Prefecture (72) Inventor St. Sato Kanagawa Prefecture 200 Higashidawara, Hadano 96 (72) Inventor Seiichi Kuzuma 1-5-9, Tsumadahigashi, Atsugi-shi, Kanagawa-901 (72) Inventor Tomio Takeuchi 5-11, Higashigotanda, Shinagawa-ku, Tokyo 701

Claims (16)

[Claims] 1. The following general formula (I): (In the formula, R ₁ represents a hydroxyl group, R ₂ represents a hydrogen atom, or R ₁ and R ₂ together form a hydroxyimino group, an oxo group or an alkoxyimino group, and R ₃ represents a hydrogen atom or An antibiotic AB5366 derivative represented by a sulfonic acid group and R ₄ represents a hydrogen atom or an acyl group, or a salt or alkyl ester (carboxylate) thereof. 2. An antibiotic AB5366 derivative of general formula (I) is represented by the following formula (Ia) The substance according to claim 1, which is a substance AB5366-1 represented by or a salt or alkyl ester thereof. 3. An antibiotic AB5366 derivative of general formula (I) is represented by the following formula (Ib) The derivative or a salt or alkyl ester thereof according to claim 1, which is a substance AB5366-2 represented by: 4. An antibiotic AB5366 derivative of general formula (I) is represented by the following formula (Ic) The derivative or salt or alkyl ester thereof according to claim 1, which is a substance AB5366-3 represented by: 5. An antibiotic AB5366 derivative of the general formula (I) is represented by the following formula (Id) The derivative or a salt or alkyl ester thereof according to claim 1, which is a substance AB5366-4 represented by: 6. An antibiotic AB5366 derivative of general formula (I) is represented by the following formula (Ie) The derivative or salt or alkyl ester thereof according to claim 1, which is a substance AB5366-5 represented by 7. An antibiotic AB5366 derivative of general formula (I) is represented by the following formula (If): The derivative or salt or alkyl ester thereof according to claim 1, which is a substance AB5366-6 represented by: 8. An alkyl ester of an antibiotic AB5366 derivative of the general formula (I) is represented by the following formula (Ig) A methyl ester of the derivative according to claim 1, which is a methyl ester of the substance AB5366-3 [formula (Ic)] represented by: 9. The following formula (A) The substance of formula (Ia) obtained by reducing the antibiotic AB5366 represented by
The method for producing substance AB5366-1 according to claim 2, characterized in that AB5366-1 is produced. 10. Process for the preparation of the substance AB5366-2 according to claim 3, characterized in that the antibiotic AB5366 of formula (A) is oxime-ized to produce the substance AB5366-2 of formula (Ib). 11. Process for the preparation of substance AB5366-3 according to claim 4, characterized in that the antibiotic AB5366 of formula (A) is acid hydrolyzed to form substance AB5366-3 of formula (Ic). . 12. The substance AB of formula (Ic) according to claim 4.
Substance AB5366- according to claim 5, characterized in that 5366-3 is reduced to produce substance AB5366-4 of formula (Id).
4 manufacturing method. 13. The substance AB of formula (Ic) according to claim 4.
5366-3 is acetylated to obtain the substance of formula (Ie) AB5366-5
The substance AB according to claim 1, characterized in that
Manufacturing method of 5366-5. 14. Substance AB5366-6 according to claim 7, characterized in that the antibiotic AB5366 of formula (A) is O-ethyloxime-ized to produce substance AB5366-6 of formula (If).
Manufacturing method. 15. The carboxyl group of the antibiotic AB5366-3 of formula (Ic) according to claim 4 is methylated to form formula (I
Process for the production of the methyl ester of substance AB5366-3 according to claim 4, characterized in that the substance AB5366-3 methyl ester of g) is produced. 16. The general formula (I) [In the formula, R ₁ represents a hydroxyl group, R ₂ represents a hydrogen atom, or R ₁ and R ₂ together form a hydroxyimino group, an oxo group or an alkoxyimino group, and R ₃ represents a hydrogen atom or A sulfonic acid group and R ₄ represents a hydrogen atom or an acyl group), and contains at least one of the antibiotic AB5366 derivative or a salt thereof or an alkyl ester (carboxylate) thereof as an active ingredient. A fungicide for agricultural and horticultural use.