JP2001139507A - Antibiotic blasticidin a derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a compound that has the action to inhibit filamentous fungi from producing aflatoxin and melanin, a production process therefor, and agrochemicals, medicines and reagents containing the same. SOLUTION: A variety of the derivatives that are prepared by removing the tetramic acid moiety from blasticidin A of the chemical formula by treatment with ozone have the action to inhibit filamentous fungi from producing aflatoxin and melanin.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an antibiotic blasticidin A derivative, a method for producing the same, an aflatoxin contamination controlling agent comprising the antibiotic blasticidin A derivative as an active ingredient, or melanin production upon infection. The present invention relates to an antifungal agent for pathogenic fungi.

[0002]

2. Description of the Related Art Aflatoxin
Is a potent carcinogenic substance produced by some molds belonging to the genus Aspergillus, and is known to have four types of analogous substances (B ₁ , B ₂ , G ₁ , G ₂ ). .

[0003] Molds that produce this aflatoxin include:
Because it infects agricultural products such as corn and peanuts and contaminates agricultural products with aflatoxins,
Drugs aimed at controlling aflatoxin contamination have been developed. For example, the antibiotic iturin A (Itu
The rin A) is intended to prevent aflatoxin contamination by suppressing the growth of aflatoxin-producing bacteria.

[0004] However, when the above-mentioned growth inhibitors are frequently used for fungal control, mutants that have acquired resistance to the drug are selected and eliminated by the drug itself,
Rather, the spread of drug-resistant bacteria has led to social problems. Therefore, the antibiotic iturin A (It
At present, drugs such as urin A) have not been put to practical use because they cannot prevent the spread of drug-resistant bacteria.

On the other hand, since biosynthesis of aflatoxin is not an essential biochemical requirement for the growth of aflatoxin-producing strains, the present inventors have proposed that when a drug that inhibits only the biosynthesis of aflatoxin is used, Since selection and selection of resistant bacteria to the bacterium were not performed, the spread of the resistant bacterium was considered to be suppressed, and an antibiotic that specifically inhibits aflatoxin biosynthesis was searched. As a result, a new antibiotic aflastatin A was collected (Japanese Unexamined Patent Publication No.
Further, it has been found that blasticidin A also has an activity similar to that of aflastatin A (JP-A-11-79911).

[0006] By the way, the rice fever fungus Pyriclaria
Oryze (Pyricularia oryzae) and Colletotrichum lavenarium (Col)
letotricum labenarium is a melanin (mela) that penetrates invading hyphae into plant cells.
It is known that nin) is produced, accumulates in the appressorium, and invades plant cells by the action of the melanin layer formed by the melanin. Therefore, it is known that a fungus such as a plant pathogenic fungus can be controlled by inhibiting the melanin formation, and pentachlorobenzyl alcohol and fusalide are known as compounds having the controlling action.

However, since pentachlorobenzyl alcohol and fusalide have many chlorine atoms in the molecule, there is a concern that the application thereof may cause excessive residual in the environment. Especially for pentachlorobenzyl alcohol,
When rice straw sprayed with this agent is used as compost, it causes remarkable growth inhibition on cucumber, which is caused by chemical conversion of this agent in the compost, 2, 3, 4, 6- or 2, 3, 5, Various polychlorinated benzoic acids such as 6-tetrachloro-, 2,4,6-trichloro- and pentachloro-forms are produced, and it has been found that growth inhibition is expressed by their hormonal action. It has been discontinued (Agrochemical Chemistry, Nobutaka Takahashi et al., Bunei-do, published April 20, 1984, pp. 87-88).

Therefore, a melanin formation inhibitor for filamentous fungi which does not have the above-mentioned problems has been desired.

[0009]

The above-mentioned aflastatin A
And blasticidin A have a very novel structure in which a long alkyl chain having a polyol structure is bonded to a tetramic acid skeleton, and lead to a compound having such a novel carbon skeleton. It is widely known that a more effective drug can be created by synthesizing various derivatives as compounds, and many useful drugs have been developed by this technique.

Accordingly, an object of the present invention is to provide a more effective compound having an effect of inhibiting aflatoxin biosynthesis, a method for producing the same, and an aflatoxin contamination controlling agent comprising the compound as an active ingredient.

Another object of the present invention is to provide an antifungal agent for pathogenic filamentous fungi which requires melanin production upon infection, comprising a blasticidin A derivative as an active ingredient.

[0012]

Means for Solving the Problems In order to achieve the above object, the present inventors have prepared various derivatives of blasticidin A, and have studied the growth inhibitory activity of these derivatives against aflatoxin-producing bacteria and the effect of the derivatives on aflatoxin biosynthesis. The inhibitory activity was measured.

As a result, the following general formula 1 ABC; general formula 1 (where A is the formula 2

[0014]

Embedded image Where B is the formula 3 or the formula 4

[0015]

Embedded image Or equation 4

[0016]

Embedded image And C is the equation 5

[0017]

Embedded image Wherein n is 0, 1 or 2, and R is a hydrogen atom or a linear or branched alkane containing 1 to 6 carbon atoms. The present inventors have found that the antibiotic blasticidin A derivative represented by the formula (1) specifically inhibits the biosynthesis of aflatoxin without substantially inhibiting the growth of aflatoxin-producing bacteria, thereby completing the present invention.

That is, a first aspect of the present invention is an antibiotic blasticidin A derivative represented by the above general formula 1 which has an action of inhibiting aflatoxin biosynthesis without substantially inhibiting the growth of bacteria.

Preferred examples of the above derivative are those in which B in the above general formula is represented by the formula 3 and n in the formula 3 is 1, that is, a blasticidin A derivative B24 having a structure of the following chemical formula 6 (hereinafter referred to as B24). , Derivative B24).

[0020]

Embedded image Examples of other preferred derivatives are those wherein B in the above general formula is represented by the formula 4, wherein n is 1 and R is methyl.
That is, it is a blasticidin A derivative D having a structure of the following chemical formula 7 (hereinafter, abbreviated as derivative D).

[0021]

Embedded image Another example of a preferred derivative is a blast cell having a structure represented by the following chemical formula 8, wherein B in the above general formula is represented by the formula 4, and n in the formula 4 is 1 and R is a hydrogen atom. Gin A derivative E (hereinafter abbreviated as derivative E).

[0022]

Embedded image The second aspect of the present invention is an aflatoxin contamination control agent containing the blasticidin A derivative represented by the above general formula 1 as an active ingredient.

The blasticidin A derivative of the present invention has an action of inhibiting aflatoxin production without substantially inhibiting the growth of aflatoxin-producing bacteria. Therefore, by using the blasticidin A derivative for controlling the aflatoxin contamination, not only can the agricultural product contamination of aflatoxin having a strong carcinogenic action be controlled, but resistant bacteria to the blasticidin A derivatives spread, A situation in which the effect is lost early can be avoided.

Furthermore, since the blasticidin A derivative of the present invention is uniformly dispersed and dissolved in an organic solvent such as ethanol and acetone, it is very easy to formulate it into various agricultural chemicals.

By the way, the present inventors have surprisingly discovered that the above blasticidin A derivative inhibits the production of melanin by filamentous fungi. Therefore, the third aspect of the present invention
Relates to antifungal agents against pathogenic fungi that require melanin production upon infection.

That is, the blasticidin A derivative of the present invention not only inhibits aflatoxin production as described above, but also inhibits melanin production required by certain filamentous fungi upon infection of a host. Thus, the infection of the filamentous fungus is controlled.

Next, a fourth aspect of the present invention is a method for producing an antibiotic blasticidin A derivative represented by the following chemical formula 9, wherein the antibiotic blasticidin A is treated with ozone and then reduced.

[0028]

Embedded image That blasticidin A derivative of the present invention, after the antibiotic blasticidin A was oxidized with ozone, can be easily produced by reducing the oxide with a reducing agent such as NaBH _4.

Furthermore, a derivative of blasticidin A which maintains the tetrahydropyran ring structure can be prepared by methylating and protecting the hemiacetal hydroxyl group of blasticidin A prior to the above-mentioned ozone treatment.

The blasticidin A derivative of the present invention is effective not only for controlling aflatoxin contamination but also for controlling pathogens requiring melanin production at the time of host invasion such as rice blast, and screening melanin production of filamentous fungi. It is also useful in the development of pesticides and medicines that can be obstacles, regardless of whether the production of melanin is essential for infection of the host with the filamentous fungus.

[0031]

BEST MODE FOR CARRYING OUT THE INVENTION An example of a method for producing a suitable compound in the blasticidin A derivative of the present invention is shown below.
It is characterized by reduction.

The blasticidin A as a starting material can be purified by culturing a microorganism having the ability to produce blasticidin A, and extracting from the culture with various solvents, fractional precipitation, chromatography and the like.

Examples of microorganisms having the ability to produce blasticidin A include, for example, Streptomyces griseochromogenes IFO 13413 (Strept
omyces griseochromogenes
IFO 13413 strain). This strain was obtained from Fermentation Research Institute (2-1 Jusanhoncho, Yodogawa-ku, Osaka-shi).
7-85).

Then, the above strain is cultured according to the method described in, for example, JP-A-11-79911, and the culture is purified from the culture using a purification means such as solvent extraction, precipitation, or high performance liquid chromatography. Blastcidin A can be obtained. In consideration of the derivatization reaction efficiency described later, the purity of blasticidin A is preferably higher, but it is not necessarily the same as in Japanese Patent Application Laid-Open No.
It does not need to be as high as that isolated by high performance liquid chromatography as described in JP-A-79911, but it can be obtained by solvent precipitation and precipitation (purity 67).
%) Can be efficiently derivatized.

When the target derivative is the derivative B24, the blasticidin A is oxidized by directly suspending the blasticidin A in a suitable solvent such as methanol, and passing ozone through the suspension. It is done by doing.

[0036] Then, after oxidizing the blasticidin A, after removing the excess ozone by bubbling nitrogen into the reaction mixture, adding a reducing agent such as NaBH _4, the reduction treatment by stirring for about 3 hours Do.

After the reduction treatment, the reaction solution is concentrated under reduced pressure and the solvent is removed to obtain a crude reaction product containing the derivative B24.

The derivative B24 in the crude reaction product can be purified to an appropriate purity by using a purification means such as solvent extraction or various types of chromatography. For example,
Column chromatography using Sephadex LH-20 (trade name, manufactured by Pharmacia), normal phase chromatography using silica gel, and high performance liquid chromatography using an ODS column can be used.

Further, the derivative B24 can be converted into a further suitable derivative, and after purifying the derivative, the derivative B24 can be removed to obtain a highly pure derivative B24. Specifically, for example, after drying a crude reaction product containing the above-mentioned derivative B24, a mixed solution of acetic anhydride / pyridine containing a catalytic amount of dimethylaminopyridine is added to the obtained dried product to cause a reaction with the derivative B24. And purified by subjecting the acetylated product to solvent extraction, silica gel chromatography, reversed-phase high-performance liquid chromatography, or the like. Then, the acetylated product is treated with sodium metal to remove the acetyl group, and finally, impurities in the reaction product are removed using an ion exchange method or the like, whereby a highly pure derivative B24 can be obtained.

The physicochemical properties and the results of the toxicity test of the derivative B24 obtained as described above are as follows.

Derivative B24 a) Property: amorphous b) Molecular formula: C₅₀H₁₀₂O₂₁  c) Molecular weight: 1038 FAB-MS (positive, glycerol)
matrix) m / z 1061 (M + Na)⁺  d) Nuclear magnetic resonance spectrum: CD₃During OD, 500MH
at z¹The 1 H-NMR spectrum is shown in FIG. Ma
The CD₃At 125 MHz during OD¹³C-NM
The R spectrum is shown in FIG.

E) Solubility: easily soluble in lower alcohols such as methanol and dimethylsulfoxide. Poorly soluble in water.

F) Toxicity: Male S of 6 weeks old with an average body weight of 32 g
Using lc: ddY mice, 4 groups were composed of 5 mice per group. Next, 80 mg / k was given to each group of mice.
Derivative B24 was injected intraperitoneally at doses of g, 8 mg / kg, 0.8 mg / kg and 0 mg / kg body weight, and the survival and death of the mice after 24 hours was assayed. As a result, no death was observed at any dose. In addition, necropsy was performed on the surviving cases, and the size and color of the liver, kidney, spleen, lung, and heart, the presence or absence of intestinal bleeding, and abnormalities in the genital and thymus were observed. Did not.

The following is an example of a method for producing another suitable compound of the blasticidin A derivative of the present invention, which is described below. The hemiacetal hydroxyl group of the antibiotic blasticidin A is methylated and then treated with ozone. , And reducing.

That is, when the preferred compound is the derivative D, for example, blasticidin A obtained by the above-mentioned fermentation production is reacted in hydrochloric acid-methanol for about 1 hour to methylate the hemiacetal hydroxyl group. Thus, a derivative corresponding to the methyl glycoside of blasticidin A (hereinafter abbreviated as derivative M) is obtained.

[0046] Then, the derivative M, after ozone oxidation in the same manner as derivatives B24, reduced by stirring for 2 hours at room temperature by adding a reducing agent such as NaBH _4. After the reduction treatment, the reaction solution is concentrated under reduced pressure or the like to remove the solvent, whereby a crude reaction product containing the derivative D can be obtained.

The derivative D in the crude reaction product is the derivative B2
4 Similarly, it is possible to directly purify to an appropriate purity by using a purification means such as solvent extraction or various types of chromatography, and to convert the derivative D in the crude reaction product into an appropriate further derivative. After conversion and purification of the derivative, the derivative D can be removed to obtain a highly pure derivative D. Specifically, for example, after the above-described reduction, pyridine and acetic anhydride are added, and the mixture is stirred at room temperature for 18 hours to perform acetylation. After the reaction, purification is performed by a silica gel column and reverse phase HPLC to obtain an acetylated derivative D. be able to.

Next, the acetylated derivative D is deacetylated by adding metallic sodium in anhydrous methanol and stirring at room temperature for 1 hour, and after completion of the reaction, a highly pure derivative D is obtained through a cation exchange column. Can be.

It is obvious to those skilled in the art that if a desired alcohol is used instead of methanol in the above-mentioned hydrochloric acid-methanol, the R group in the compound represented by the general formula 1 will be a group corresponding to the alcohol. Would.

Further suitable compounds can be prepared, for example, using the above-mentioned derivative D. That is, when the preferred compound is the derivative E, the derivative E can be obtained by demethylating the tetrahydropyran ring portion of the derivative D. Specifically, for example, the derivative D is 3M
The derivative E can be obtained by dissolving in acetic acid and stirring for several hours at room temperature.

The physicochemical properties of the above-mentioned derivatives D and E are shown below.

Derivative D a) Molecular formula: C₅₁H₁₀₂O₂₁  b) Molecular weight: 1050 c) High-resolution mass spectrum: HR-FABMS (po
sitive, glycerol matrix): m
/Z1073.6710 (M + Na)⁺, (Calcd
 for C₅₁H₁₀₂O₂₁Na, 1073.68
11) d) Optical rotation: [α]_D ²⁶: + 1.0 ° (c = 0.00
3, CH₃E) NMR: Table 1 shows the NMR table. Also pyr
idine-d₅At 500 MHz¹H-NM
The R spectrum is shown in Fig. 3 and in the same solvent at 125 MHz.
Put¹³FIG. 4 shows the C-NMR spectrum.

Table 1 Assignment of each signal in the NMR spectrum of derivative D ( ¹ H-NMR 500 MHz, ¹³ C-NMR 12
5 MHz, pyridine-d ₅ )

[0054]

[Table 1]Derivative E a) Molecular formula: C₅₀H₁₀₀O₂₁  b) Molecular weight: 1036 c) High-resolution mass spectrum: HR-FABMS (po
sitive, DEAmatrix): m / z 105
9.6605 (M + Na)⁺(Calcd for C
₅₀H₁₀₀O₂₁Na, 1059.6655) d) Optical rotation: [α]_D ²⁶: + 1.1 ° (c = 0.0
03, CH₃CN) e) NMR: Table 2 shows the NMR table. Also pyr
idine-d₅At 500 MHz¹H-NM
The R spectrum is shown in FIG.
Put¹³FIG. 6 shows the C-NMR spectrum. Table 2 Result of each signal in NMR spectrum of derivative E
Genus (¹H-NMR 500 MHz,¹³C-NMR 12
5MHz, pyridine-d₅)

[0055]

[Table 2] The blasticidin A derivative of the present invention effectively inhibits the production of aflatoxins and melanin by filamentous fungi, as shown in the test examples described below.

Therefore, the blasticidin A derivative of the present invention can be used as an agricultural or horticultural drug, and when used as such, it is usually used in the same manner as in the agricultural chemical formulations known in the art. Granules, powders, emulsions, wettable powders using appropriate solid carriers, liquid carriers, emulsifying dispersants, etc.
It can be formulated into any dosage form such as tablets, oils, sprays and aerosols and used.

Of these carriers, solid carriers include:
Clay, kaolin, benite, acid clay, diatomaceous earth, calcium carbonate, nitrocellulose, starch, carboxymethyl cellulose and the like are preferably used. Further, as the liquid carrier, water, methanol, ethanol, ethylene glycol, glycerin, dimethyl sulfoxide and the like are preferably used.

Further, auxiliaries generally used in the preparation, such as sulfates of higher alcohols, polyoxyethylene alkyl allyl ether, alkyl allyl polyethylene glycol ether, alkyl allyl sorbitan monolaurate, Alkyl allyl sulfonates, quaternary ammonium salts, polyalkylene oxides, desoxycholates and their esters, and the like can be appropriately blended.

The blasticidin A derivative of the present invention is very easily dissolved in lower alcohols such as methanol, so that it can be easily formulated into such agricultural and horticultural medicine.

The concentration of the blasticidin A derivative in the agricultural and horticultural drug of the present invention is adjusted in consideration of the type of the contaminating bacteria, the degree of propagation, and the like.
About 00 ppm, preferably 100 to 5,000 ppm
It is about.

The blasticidin A derivative of the present invention not only inhibits aflatoxin production as described above, but also inhibits melanin production required when certain filamentous fungi infect a host. The purpose is to control the infection of the filamentous fungus.

Examples of pathogenic filamentous fungi that require melanin production upon infection include, but are not limited to, the above-mentioned plant pathogenic bacteria such as Pyricularia oryzae and Colletotricum labenarium.

That is, if the production of melanin is an essential requirement for establishment of infection by the filamentous fungus, the host of the filamentous fungus may be an organism other than a plant. Therefore, the blasticidin A derivative of the present invention can prevent or prevent diseases caused by pathogenic fungi that require melanin production during infection.
It can also be used as an active ingredient in pharmaceuticals used for treatment.

When the compound of the present invention is used in medicines, it can be formulated into various preparations by adding pharmaceutically acceptable additives according to the administration form. As the additives at that time, various additives usually used in the field of pharmaceutical preparations can be used, for example, gelatin, lactose, sucrose, titanium oxide, starch, crystalline cellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, cornstarch, and microparticles. Crystallin wax,
White petrolatum, magnesium metasilicate aluminate, anhydrous calcium phosphate, citric acid, trisodium citrate, hydroxypropylcellulose, sorbitol, sorbitan fatty acid ester, polysorbate, sucrose fatty acid ester, polyoxyethylene hydrogenated castor oil, polyvinylpyrrolidone, magnesium stearate Light anhydrous silicic acid, talc, vegetable oil, benzyl alcohol, gum arabic, propylene glycol, polyalkylene glycol, cyclodextrin or hydroxypropylcyclodextrin.

The dosage form formulated as a mixture with these additives includes, for example, solid preparations such as tablets, capsules, granules, powders and suppositories; and, for example, syrups, elixirs and injections. And liquid preparations, which can be prepared according to the usual methods in the field of preparation. In the case of a liquid preparation, it may be in the form of being dissolved or suspended in water or another appropriate medium at the time of use. In particular, in the case of an injection, it may be dissolved or suspended in a physiological saline solution or a glucose solution as necessary,
Further, a buffer or a preservative may be added.

These preparations can contain the compound of the present invention in a proportion of 1 to 100% by weight, preferably 10 to 80% by weight of the whole drug.

When the blasticidin A derivative of the present invention is used in a clinical setting, the dosage and frequency of administration depend on the sex, age, body weight, degree of symptoms and the type and range of the intended treatment of the patient. Different, but generally for oral administration,
One to 50 mg / kg per day for an adult is divided into 1 to several times, and in the case of parenteral administration, 1 to 10 mg / kg is taken as 1
It is preferable to administer the drug in several divided doses.

The blasticidin A derivative of the present invention is effective not only for controlling aflatoxin contamination, but also for controlling pathogens requiring melanin production upon host invasion, such as rice blast, and screening melanin production of filamentous fungi. It is also useful in the development of pesticides and medicines that can be obstacles, regardless of whether the production of melanin is essential for infection of the host with the filamentous fungus.

That is, in recent years, it has the effect of increasing or decreasing the productivity of certain compounds produced by filamentous fungi, or regulating the production of certain enzymes produced by filamentous fungi. Compounds may be screened. In addition, there are cases where an inhibitor or activator of a certain enzyme produced by the filamentous fungus is screened, and cases where the enzyme itself of the filamentous fungus is screened. In such screening, if the target compound of the screening is pigmentary, the color is directly developed, if the target compound is colored by reacting with another compound or enzyme, the color is developed, and In the case of an enzyme, the color development obtained by reacting the enzyme with an appropriate substrate,
The measurement is performed using spectroscopic means such as the naked eye or a plate reader. However, when the target compound or enzyme-producing bacterium produces melanin simultaneously with the production, the melanin significantly hinders measurement of a desired coloration. Therefore, by incorporating the blasticidin A derivatives of the present invention into the above-mentioned screening, melanin production can be suppressed while minimizing the effect on the growth of filamentous fungi, so that screening can be performed very efficiently. it can.

Examples of the above-mentioned screening include, but are not limited to, screening for angiotensin converting enzyme inhibitors and protein phosphatase inhibitors.

In the above screening, the blasticidin A derivative of the present invention is added to a culture medium in which a filamentous fungus grows in a concentration of 50 to 1000 μM, preferably 100 to 20 μM.
By adding at a concentration of 0 μM, it is possible to obtain a culture solution that does not show color development based on melanin even after culturing. Therefore, the culture solution can be suitably used for the assay.

[0072]

The present invention will be specifically described below with reference to examples.

[0073]Example 1 (Production of derivative B24)  Preparation of blasticidin A According to the method described in JP-A-11-79911.
I, Streptomyces griseo chromogenes IFO
 The strain 13413 is cultured to collect the cells, and the cells are meta-
Extract, and the extract was dried to dryness.
And the butanol phase is separated into NaHCO₃Aqueous solution, then
The extract was washed with pure water. After concentrating the butanol solution under reduced pressure,
This concentrate is mixed with chloroform / methanol (3: 1)
Was added and the precipitate was collected. Further, the precipitate is
After washing with drofuran, drying and brass of about 67% purity
A crude product of tosaidin A was obtained.

Oxidation Treatment 153.5 mg of the above crude blasticidin A was suspended in 10 ml of anhydrous methanol, and 30 mg of sodium hydrogencarbonate was added thereto. Then, the suspension was cooled to -78 ° C, and blasticidin A was oxidized by passing ozone (O ₃ ) therethrough for 10 minutes.

Reduction treatment Nitrogen is bubbled through the reaction solution for 5 minutes to drive off excess ozone.
And then NaBH ₄Add 100mg and add 3mg at room temperature
The oxide was reduced by stirring for a while. Acetic acid was added to the reaction solution for 20 minutes.
The reaction is stopped by dropping, and an appropriate amount of methanol is added there.
After addition, it was concentrated under reduced pressure. Methano again in concentrate
The crude reaction containing derivative B24
I got something.

Purification Acetylation of derivative B24 After the crude reaction product was lyophilized to remove excess water,
10 ml of dry pyridine and 5 ml of acetic anhydride were added to the crude reaction product, and a catalytic amount of dimethylaminopyridine was further added.
Derivative B24 was acetylated by reacting at room temperature for 15 hours. The reaction solution was cooled to 4 ° C., and the acetylation reaction was stopped by adding 10 ml of water.
20 ml was added to obtain an ethyl acetate solution containing the acetylated derivative B24.

Purification of acetylated product 80 ml of 3 M hydrochloric acid was added to the above ethyl acetate solution, mixed well, and allowed to stand still to collect an ethyl acetate solution part separated. Then, in the same manner, extraction and washing are sequentially performed using 80 ml each of a saturated sodium hydrogen carbonate solution, a saturated saline solution, and deionized water, and an ethyl acetate solution is recovered. An appropriate amount of anhydrous sodium sulfate is added thereto and left for 3 hours. Then, the ethyl acetate solution was dehydrated.

The dehydrated ethyl acetate solution was evaporated to dryness under reduced pressure, redissolved in a small amount of ethyl acetate, and the ethyl acetate solution was equilibrated with a hexane / ethyl acetate mixture (50:50) to obtain 24 g of “Silica gel C”. −200 ”
(Trade name, manufactured by Wako Pure Chemical Industries, Ltd.)
After 50 ml of an ethyl acetate mixed solution (50:50) was passed through to elute the impurities, subsequently, 100 ml of ethyl acetate was passed through to elute the acetylated derivative B24. The fraction eluted with ethyl acetate was collected and dried under reduced pressure.

The fraction eluted with ethyl acetate, which was dried under reduced pressure, was dissolved in a small amount of acetonitrile, and subjected to high performance liquid chromatography under the following conditions. -Column: Capcell Pak _C18 UG120
(Trade name, manufactured by Shiseido Co., Ltd.), inner diameter 10 mm, length 250 mm Mobile phase: acetonitrile / water, acetonitrile concentration was linearly increased from initial 70 vol% to 90 vol% over 4 minutes, and then held as it was. -Flow rate: 3.0 ml / min-Detection wavelength: 210 nm The eluted fraction from 8 minutes to 25 minutes was collected, concentrated under reduced pressure, and lyophilized to purify the acetylated derivative B24.

Deacetylation The acetylated product of the derivative B24 obtained as described above is dissolved in 4 ml of anhydrous methanol, and 5 mm ³ of sodium metal is added thereto and reacted to obtain the derivative B24.
The acetyl group was removed from the acetylated product of 24.

Purification of the derivative B24
ex 50W × 8 ”(trade name, manufactured by Dow Chemical Company)
The column was attached to a ⁺ type column, and 50 ml of deionized water was passed therethrough.
4 were found. This fraction was concentrated under reduced pressure and freeze-dried to obtain 26.2 mg of derivative B24.

[0082]Example 2 (Production of derivative D)  ・ Preparation of derivative M The blasticidin A has a tetrahydropyran ring skeleton.
Exist. First, the hemiacetal part is converted into a ketal
Is converted to methyl glycoside of blasticidin A
A derivative M that could be prepared was prepared.

That is, the above-mentioned crude blast cydin A
50 mg was dissolved in 5% hydrochloric acid-methanol 5.0 ml,
Stirred at room temperature for 1 hour. The obtained reaction solution was prepared under the following conditions.
Purification by reverse phase HPLC gave 26.5 mg of derivative M
Was. Table 3 shows NMR spectrum data of derivative M.
Was.・ Column: Capcell Pak C₁₈UG120
(Trade name, manufactured by Shiseido), inner diameter 10 mm, length 250 mm Moving layer: 66% -90% methanol (0.5% diethyl)
Lamine), 10 min Flow rate: 3.0 ml / min Detection wavelength: 300 nm Table 3 NMR signal of derivative M
Attribution (¹H-NMR 500 MHz,¹³C-NMR 12
5MHz, DMSO-d ₆)

[0084]

[Table 3]・ Production of derivative D 140 mg of derivative M was dissolved in 5.0 ml of anhydrous methanol.
Then, 50 mg of sodium hydrogen carbonate was added, and acetone-
Pass ozone through the reaction solution for 20 minutes under dry ice cooling
Was. Nitrogen is passed through the reaction mixture to remove excess ozone and hydrogenated.
Add 50 mg of sodium boron and stir at room temperature for 2 hours.
Was. The reaction was stopped by adding 0.2 ml of acetic acid.
To remove boron from the product, add methanol and dry under reduced pressure
After repeating the operation several times, freeze-drying was performed. That
10 ml of pyridine, 5 ml of acetic anhydride and a catalytic amount of di
Add methylaminopyridine and stir at room temperature for 18 hours.
Was. After stopping the reaction by adding 10 ml of methanol under ice cooling,
Extraction was performed with 120 ml of ethyl acetate. Ethyl acetate
80 ml of 3M hydrochloric acid, saturated aqueous sodium hydrogen carbonate solution,
Wash with saturated saline and distilled water in that order, and dry with anhydrous sodium sulfate.
, And concentrated under reduced pressure. The residue obtained is silica gel
Ram (10φx120mm), hexane-acetic acid
Chill solvent ratios of 70:30 and 30:70 in the order of 5
Elution was carried out stepwise using 0 ml and the resulting 70: 3
Fraction 0 was concentrated under reduced pressure. The obtained residue was prepared under the following conditions.
Purification by reverse phase HPLC afforded the acetylated derivative D
Was. Next, the acetylated derivative D was treated with anhydrous methanol 5.
0 ml, add a small piece of metallic sodium and add
Stir for 1 hour. After the completion of the reaction, the reaction solution was added to Dowex-5.
0W (H⁺) (18φ × 40mm) column
And then concentrate the filtrate under reduced pressure and freeze.
Drying gave 45.4 mg of derivative D.・ Column: Capcell Pak C₁₈UG120
(Trade name, manufactured by Shiseido), inner diameter: 10 mm, length: 250 mm Moving bed: 70% -90% acetonitrile, 4 min Flow rate: 3.0 ml / min Detection wavelength: 210 nmExample 3 (Production of derivative E)  -Production of derivative E After dissolving 43.4 mg of derivative D in 5 ml of methanol,
3M hydrochloric acid was added, and the mixture was stirred at room temperature for 5 hours. End of reaction
Thereafter, the reaction solution was converted to Dowex-1 (OH⁻) (18φx50
mm) through the column and collect the filtered and washed fractions.
After concentration under reduced pressure, freeze-dry, 38.6 mg of derivative E
I got

[0085]Test Example 1 (A of blasticidin A derivative)
Flatoxin production inhibitory effect)  Aflatoxin production inhibitory effect of blasticidin A derivative
Fruit was measured by the agar plate dilution method shown below.
Was.

Aflatoxin producing bacteria: Aspergillus paras
iticus) NRRL2999 strain The spores were scraped using a platinum loop from the bacterial flora of the above strain cultured on a potato dextrose agar medium (manufactured by Nippon Suisan Co., Ltd.) at 27 ° C. for 14 to 21 days, and autoclaved beforehand. 0.01% surfactant (Tween
80) A spore suspension was prepared by suspending in an aqueous solution.

Agar plate: The agar plate is prepared by dissolving the blasticidin A derivative in dimethyl sulfoxide, sterilizing by filtration with a sterilizing filter “Dimex” (trade name, manufactured by Nippon Millipore Limited) to give an appropriate dilution series. After preparing a solution of each concentration and autoclaving in advance,
To 10 ml of a potato dextrose agar medium (manufactured by Nippon Suisan Kaisha, Ltd.) kept at a temperature of 10 ° C., blasticidin A derivative solutions (10 μl) of each concentration were added, mixed well, and poured into a sterile 9 cm inner diameter petri dish to prepare. A control was prepared by adding 10 μl of dimethyl sulfoxide to a potato dextrose agar medium in the same manner.

The test was conducted at one point at the center of each of the above agar plates.
10 μl of the above spore suspension (viable cell count 2.5 to 3 × 10 ⁴
Were inoculated, and cultured at 27 ° C. for 7 days.

The growth inhibitory effect was determined by measuring the diameter of the colony on each plate.

The measurement of aflatoxin is described in “J.
Assoc. Off. Anal. Chem. , 6
8,458-461 (1985) ". That is, the whole amount of the agar medium was scraped off from the petri dish together with the cells, 80 ml of chloroform was added thereto, and the mixture was vigorously stirred using a Waring blender. The liquid was recovered, and anhydrous sodium sulfate was added thereto to dehydrate. 20 ml of this extract was collected with a whole pipette, and the Florisil column “SEP-Pak FL”
The column was washed with 30 ml of a mixture of chloroform / methanol (9: 1), and then eluted with 50 ml of a mixture of acetone / water (99: 1). The eluate was collected and dried under reduced pressure to obtain an aflatoxin measurement sample. The above operations were performed under a fluorescent lamp that does not emit ultraviolet light, and brown glassware was used.

The above sample was dissolved in 5 ml of a mixture of tetrahydrofuran / water / acetic acid (20: 80: 0.1), and 25 μl thereof was subjected to high performance liquid chromatography under the following conditions. -Column: COSMOSIL5-Phenyl (trade name, manufactured by Nacalai Tesque), inner diameter 4.6 mm, length 15
0 mm Mobile phase: tetrahydrofuran / water (20:80) Flow rate: 0.8 ml / min Detection wavelength: 365 nm Aflatoxin was quantified by comparing the area of each separated peak with that of a standard product.

The results of the above test are shown in Table 4, FIGS. 7 and 8.

[0093]

[Table 4] From Table 4, it can be seen that the derivative B24 inhibits aflatoxin-producing bacteria without inhibiting the growth of the aflatoxin-producing bacteria.

FIG. 7 shows that the derivative D inhibits aflatoxin production without inhibiting the growth of the aflatoxin-producing bacteria.

FIG. 8 shows that the derivative E inhibits aflatoxin production without inhibiting the growth of the aflatoxin-producing bacteria.

[0096]Test Example 2 (method of blasticidin A derivative
Lanin production inhibitory effect)  The melanin production inhibitory activity was determined by the liquid medium dilution method.
Was. The details of the experiment are shown below.

Preparation of Test Compound Solution The blasticidin A derivative whose activity is to be measured was dissolved in dimethyl sulfoxide, diluted with the same solvent to an appropriate concentration, and sterilized by filtration with the above filter.

Preparation of Liquid Medium for Activity Measurement As a medium used for measuring melanin production inhibitory activity in a liquid medium, a 1.2 M sucrose solution, which is a melanin production induction medium, was used. After autoclaving 2.5 ml of a 1.2 M sucrose solution and allowing it to cool, 10 μl of the sterilized test compound solution was aseptically added thereto to prepare a liquid medium for activity measurement.

As a control, 10 μl of dimethyl sulfoxide alone was similarly added.

Preparation of Cell Suspension for Inoculation Melanin-producing bacteria used for measurement of melanin production inhibitory activity
Colletotricum Ravenarium
(Cum labenarium) 104T was inoculated as a cell suspension. This cell suspension was cultured by shaking the 104T strain in a PSY liquid medium at 27 ° C. for 48 hours at 126 rpm, and the culture was filtered through gauze to remove the cells.
It was prepared by suspending in 50 ml of a 2M sucrose solution. PSY medium (per liter): Yeast extract
Potato decoction of 20 g / 20 g / 200 g of sucrose Inoculation and culture of melanin-producing bacteria 2.5 ml of the cell suspension was inoculated into a liquid medium for activity measurement, and cultured with shaking at 126 rpm at 27 ° C. for 24 hours at 27 ° C.

-Measurement of activity Transfer a small amount of the culture solution to a microtube,
After centrifugation for 5 minutes, the culture supernatant was taken and the absorbance was measured at 585 nm.

The results of the above test are shown in FIGS.

FIG. 9 shows that the derivative D inhibits melanin production.

FIG. 10 shows that the derivative E inhibits melanin production.

[0105]

As described above, the blasticidin A derivative of the present invention has an action of strongly inhibiting the production of aflatoxin without substantially inhibiting the growth of aflatoxin-producing bacteria. It also inhibits melanin production by filamentous fungi.

Therefore, the blasticidin A of the present invention can be used as an agricultural and horticultural medicine.

Further, the blasticidin A derivative inhibits the production of melanin by filamentous fungi, so that it can be used as a medicine for controlling pathogenic bacteria which require the production of melanin during infection.

Further, the blasticidin A derivative of the present invention is useful in screening for physiologically active substances, enzymes and the like.

According to Blasticidin A preferred method for producing a derivative of [0109] the present invention, after the antibiotic blasticidin A was oxidized with ozone readily by reduction of the oxide with a reducing agent such as NaBH ₄ The derivative can be produced.

[Brief description of the drawings]

FIG. 1 shows the blasticidin A derivative B of the antibiotic of the present invention.
24 is a diagram showing a ¹ H-NMR spectrum of 24. FIG.

FIG. 2 Blastcidin A derivative B of the antibiotic of the present invention
It is a figure which shows the ¹³ C-NMR spectrum of 24.

FIG. 3 shows the antibiotic blasticidin A derivative D of the present invention.
It is a figure which shows the < ¹ > H-NMR spectrum of.

FIG. 4 shows the antibiotic blasticidin A derivative D of the present invention.
It is a chart showing ¹³ C-NMR spectrum of the.

FIG. 5 shows the blasticidin A derivative E of the antibiotic of the present invention.
It is a figure which shows the < ¹ > H-NMR spectrum of.

FIG. 6 shows the blasticidin A derivative E of the antibiotic of the present invention.
It is a chart showing ¹³ C-NMR spectrum of the.

FIG. 7: Antibiotics blasticidin A derivative D of the present invention
FIG. 3 is a view showing the growth and aflatoxin production inhibitory effects of A.

FIG. 8: Blasticidin A derivative E of the antibiotic of the present invention
FIG. 3 is a view showing the growth and aflatoxin production inhibitory effects of A.

FIG. 9: Antibiotics blasticidin A derivative D of the present invention
FIG. 3 is a graph showing the melanin production inhibitory effect of the present invention.

FIG. 10 shows the inhibitory effect of the antibiotic blasticidin A derivative E of the present invention on melanin production.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Akira Isogai 8916-5 Takayamacho, Ikoma City, Nara Prefecture Nara Institute of Science and Technology Bioscience Department (72) Inventor Shohei Sakuta 1-1, Yayoi Bunkyo-ku, Tokyo 1 Graduate School of Agricultural and Life Sciences, University of Tokyo (72) Inventor Hiromichi Nagasawa 1-1-1 Yayoi, Bunkyo-ku, Tokyo F-term (reference) 4C062 AA18 AA20 4H006 AA01 AB03 AB29 FE11 FG90 4H011 AA01 AA03 BA01 BB03 BB08 BC03 BC07 BC18 BC19 BC20 DA02 DA13 DA15 DA16 DH03

Claims (7)

[Claims] 1. The following general formula 1 ABC; general formula 1 (where A is a formula 2) And B is represented by Formula 3 or Formula 4 Or formula 4 And C is the formula 5 Wherein n is 0, 1 or 2, and R is a hydrogen atom or a linear or branched alkane containing 1 to 6 carbon atoms. ) An antibiotic blasticidin A derivative represented by the formula: 2. The antibiotic blasticidin A derivative according to claim 1, wherein B in the general formula 1 is represented by the formula 3, and n is 1. 3. The antibiotic blasticidin A derivative according to claim 1, wherein B in the general formula 1 is represented by the formula 4, and n is 1. 4. The antibiotic blasticidin A derivative according to claim 3, wherein R is a hydrogen atom. 5. The antibiotic blasticidin A derivative according to claim 3, wherein R is a methyl group. 6. An aflatoxin contamination control agent comprising the compound according to any one of claims 1 to 5 as an active ingredient. 7. A method for inhibiting aflatoxin production by a filamentous fungus using the compound according to any one of claims 1 to 5.