JP5636179B2 - Novel compound walkmycin, process for its production and use thereof - Google Patents

Description

  The present invention relates to a novel compound having an excellent antibacterial activity against a wide range of pathogenic bacteria such as drug-resistant bacteria and phytopathogenic bacteria, or an enzyme inhibitory activity against an enzyme possessed by the bacteria, a method for producing the same, and use thereof In addition, the present invention relates to a novel microorganism that is a microorganism producing the novel compound.

Conventionally, many antibacterial agents have been used as therapeutic agents for bacterial infections. Many of the antibacterial agents known so far act by inhibiting bacterial nucleic acid synthesis, protein synthesis, peptidoglycan synthesis and the like. Since these target sites are single and mainly intended to inhibit the metabolic synthesis pathway, resistant bacteria to these antibacterial agents are likely to appear, and in recent years, in particular, they have many resistances to multiple antibiotics. The emergence of drug-resistant bacteria is a problem.
For example, Staphylococcus aureus is known as a causative agent for purulent disease, pneumonia, food poisoning, etc., but the emergence of methicillin-resistant Staphylococcus aureus (MRSA) that has acquired resistance to many drugs such as the antibiotic methicillin. However, it is a big clinical problem. Currently, vancomycin, teicoplanin, arbekacin, linezolid and the like are used as typical therapeutic agents for MRSA, but it is generally considered difficult to completely eliminate MRSA. Regarding vancomycin, the appearance of vancomycin-resistant Staphylococcus aureus (VRSA) has already been reported, and it is said that sufficient caution is required for its use.
In order to overcome the problem of such drug-resistant bacteria, it is desired to develop a novel antimicrobial agent against microorganisms by a new mechanism of action different from that of conventional antibacterial agents (for example, see Non-patent Document 1).

On the other hand, bacteria are known to have receptors that accept changes in response to the environment and information transmission mechanisms that control the expression of each gene. A typical example is a two-component system. . The two-component control system is an environmentally responsive gene expression control system composed of a sensor protein exhibiting histidine kinase activity and a regulator that is a DNA-binding protein. Bacteria are used in various ways to cope with various environmental changes. It has a sensor and a regulator (for example, refer nonpatent literature 2).
As a two-component control system of bacteria, there is an information transmission system involving YycF (also referred to as WalR) and YycG (also referred to as WalK) of Gram-positive bacteria, and it is known that if this is inhibited, the bacteria will die. (For example, refer nonpatent literatures 3-6) and the antibacterial agent which shows antibacterial power to the Gram positive bacterium by inhibiting the said information transmission system is anticipated.
In addition, pathogenicity of soft rot fungi that infects crops such as Chinese cabbage and potato and causes enormous damage to agricultural production is based on three binary control systems PehS / PehR (for example, see Non-Patent Document 7), PmrB / PmrA (for example It is known that pathogenicity is regulated by ExpS / ExpA (for example, see Non-patent document 9), and control of soft rot fungi by suppressing these pathogenicity is expected. The

  Although there are the above findings, satisfactory antibacterial agents and enzyme activity inhibitors have not yet been obtained, and the development of excellent antibacterial agents and the like is desired at present.

Sievert DM, et al: Staphylococcus aureus Resistant to Vancomycin-United States, 2002. MMWR July 5, 2002; 51: 565-567. Bioscience and Industry, Vol.58, No.4 Fablet, C.I. and Hoch, A.A. A. , J. et al. Bacteriol. , 180, 6375-6383, 1998 Marti, P.M. K. Li, T .; Sun, D .; , Biek, D.M. P. and Schmid, M.M. B. , J. et al. Bacteriol. , 181, 3666-3673, 1999 Lange, R.A. , Wagner, C.I. DeSaizieu, A .; , Flint, N .; Monos, J .; , Stigger, M .; , Caspers, P .; , Kamber, M .; Keck Wolfgang, Amrein, K .; E. Gene, 237, 223-234, 1999. Beier, D.D. and Frank, R.A. , J. et al. Bacteriol. , 182, 2068-2076, 2000 Eriksson, A.D. R. B. , Andersson, R .; A. Pirhonen, M .; , And Palva, E .; T. T. et al. , Mol. Plant-Microbe Interact. , 11, 743-752, 1998 Hytiainen, H .; Sjoblom, S .; Palomaki, T .; Tuikkala, A .; , And Palva, E .; T. T. et al. , Mol. Microbiol. , 50, 795-807, 2003 Flego, D.D. , Marits, R .; Eriksson, A .; R. B. , Koiv, V .; , Karlsson, M .; -B. , Hekinheim, R .; , And Palva, E .; T. T. et al. , Mol. Plant-Microbe Interact. , 13, 447-455, 2000

  The present invention has been made in view of the above-described prior art, and an object thereof is to achieve the following object. That is, the present invention provides an excellent antibacterial activity against a wide range of pathogenic bacteria such as drug-resistant bacteria and phytopathogenic bacteria by inhibiting the two-component control system of the bacteria, or an enzyme against the enzymes possessed by the bacteria. It is an object of the present invention to provide compounds having inhibitory activity, methods for producing the same, microorganisms that produce the compounds, compound-containing compositions using the compounds, antibacterial agents, and enzyme activity inhibitors. .

In order to solve the above-mentioned problems, the present inventors have paid attention to a two-component control system which is a main information transmission mechanism of bacterial cells, and as a result of intensive studies, they belong to the genus Streptomyces as a novel microorganism. The inventors succeeded in isolating the strain and found that this strain produces a compound having a novel structural skeleton and having antibacterial activity or enzyme inhibitory activity. The inventors of the present invention have confirmed that these are novel compounds by analyzing the chemical structures of the compounds, and have completed the present invention. In addition, the present inventors named these novel compounds as Walkmycin A and Walkmycin C.
Furthermore, the present inventors have found that among known compounds produced by the strain, there are compounds having enzyme inhibitory activity, and the present invention has been completed. In addition, the present inventors named this compound Walkmycin B.

The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> It includes at least one of a compound represented by the following structural formula (A), a compound represented by the following structural formula (C), and a compound represented by the following structural formula (B). It is an enzyme activity inhibitor.
<2> The enzyme activity inhibitor according to <1>, which inhibits histidine kinase activity.
<3> A compound represented by the following structural formula (A).
<4> A compound represented by the following structural formula (C).
<5> A method for producing at least one of a compound represented by the following structural formula (A) and a compound represented by the following structural formula (C),
A culture process for culturing a microorganism belonging to the genus Streptomyces and having the ability to produce at least one of a compound represented by the following structural formula (A) and a compound represented by the following structural formula (C) When,
Collecting a compound represented by the following structural formula (A) and at least one of the compound represented by the following structural formula (C) from the culture obtained in the culturing step,
A method for producing a compound comprising adding at least one of a branched amino acid, a branched fatty acid, and a branched keto acid to the medium in the culturing step.
<6> A microorganism belonging to the genus Streptomyces and having the ability to produce at least one of a compound represented by the following structural formula (A) and a compound represented by the following structural formula (C), It is a manufacturing method of the compound as described in said <5> which is Streptomyces sp. MK632-100F11 strain of accession number NITE P-777.
<7> The method for producing a compound according to any one of <5> to <6>, wherein at least one of the branched amino acid, the branched fatty acid, and the branched keto acid is at least one of valine, leucine, and isoleucine. is there.
<8> The method for producing a compound according to any one of <5> to <7>, wherein at least one of the branched amino acid, the branched fatty acid, and the branched keto acid is isoleucine.
<9> It belongs to the genus Streptomyces and has the ability to produce at least one of a compound represented by the following structural formula (A) and a compound represented by the following structural formula (C). It is a microorganism.
<10> The microorganism according to <9>, which is a Streptomyces sp. MK632-2F11 strain having an accession number of NITE P-777.
<11> A compound-containing composition comprising at least one of a compound represented by the following structural formula (A) and a compound represented by the following structural formula (C).
<12> An antibacterial agent comprising at least one of a compound represented by the following structural formula (A) and a compound represented by the following structural formula (C).

  According to the present invention, by inhibiting the two-component control system of bacteria, excellent antibacterial activity against a wide range of pathogenic bacteria such as drug-resistant bacteria and phytopathogenic bacteria, or against the enzymes possessed by the bacteria Compounds having inhibitory activity, methods for producing the same, microorganisms that produce the compounds, compound-containing compositions utilizing the compounds, antibacterial agents, and enzyme activity inhibitors can be provided.

FIG. 1 is an infrared spectrum chart of the compound represented by the structural formula (A) (walkomycin A) measured by the KBr tablet method. Vertical axis: transmittance (%), horizontal axis: wave number (cm ⁻¹ ). FIG. 2 is a chart of an ultraviolet absorption spectrum of the compound represented by the structural formula (A) (walkomycin A) in methanol (methanol containing 0.05M HCl). Vertical axis: absorbance (Abs), horizontal axis: wavelength (nm). FIG. 3 shows a proton nuclear magnetic resonance spectrum at 600 MHz measured at 30 ° C. in a mixed solvent of deuterated chloroform: deuterated methanol = 4: 1 of the compound represented by the structural formula (A). It is a chart. Horizontal axis: ppm unit. FIG. 4 is a chart of a carbon-13 nuclear magnetic resonance spectrum at 150 MHz, measured at 30 ° C. in deuterated methanol of the compound represented by the structural formula (A) (walkomycin A). Horizontal axis: ppm unit. FIG. 5 is an infrared spectrum chart of the compound represented by the structural formula (C) (walkomycin C) measured by the KBr tablet method. Vertical axis: transmittance (%), horizontal axis: wave number (cm ⁻¹ ). FIG. 6 is a chart of an ultraviolet absorption spectrum of the compound represented by the structural formula (C) (walkomycin C) in methanol (methanol containing 0.05M HCl). Vertical axis: absorbance (Abs), horizontal axis: wavelength (nm). FIG. 7 shows a proton nuclear magnetic resonance spectrum at 600 MHz measured at 30 ° C. in a mixed solvent of deuterated chloroform: deuterated methanol = 4: 1 of the compound represented by the structural formula (C) (walkomycin C). It is a chart. Horizontal axis: ppm unit. FIG. 8 is a chart of carbon-13 nuclear magnetic resonance spectrum at 150 MHz, measured at 30 ° C. in deuterated methanol of the compound represented by the structural formula (C) (walkomycin C). Horizontal axis: ppm unit. FIG. 9 is an infrared spectrum chart of the compound represented by the structural formula (B) (walkomycin B) measured by the KBr tablet method. Vertical axis: transmittance (%), horizontal axis: wave number (cm ⁻¹ ). FIG. 10 is a chart of an ultraviolet absorption spectrum of the compound represented by the structural formula (B) (walkomycin B) in methanol (methanol containing 0.05M HCl). Vertical axis: absorbance (Abs), horizontal axis: wavelength (nm). FIG. 11 shows a proton nuclear magnetic resonance spectrum at 600 MHz measured at 30 ° C. in a mixed solvent of deuterated chloroform: deuterated methanol = 4: 1 of the compound represented by the structural formula (B) (walkomycin B). It is a chart. Horizontal axis: ppm unit. FIG. 12 is a chart of a carbon-13 nuclear magnetic resonance spectrum at 150 MHz, measured at 30 ° C. in deuterated methanol of the compound represented by the structural formula (B) (walkomycin B). Horizontal axis: ppm unit.

(Enzyme activity inhibitor)
The enzyme activity inhibitor of the present invention is at least one of a compound represented by the following structural formula (A), a compound represented by the following structural formula (C), and a compound represented by the following structural formula (B). And other components as necessary.
The enzyme activity inhibitor can suitably inhibit histidine kinase activity.

<Compound represented by Structural Formula (A)>
A compound represented by the following structural formula (A) is one of the compounds of the present invention. The compound represented by the following structural formula (A) is a novel compound separated by the present inventors (hereinafter sometimes referred to as “Walkmycin A”).

-Physicochemical properties-
The physicochemical properties of the compound represented by the structural formula (A) are as follows.
(1) The appearance is yellow powder.
(2) The molecular formula is represented by C ₄₃ H ₄₂ Cl ₂ O ₁₄ .
(3) The experimental value by high resolution mass spectrometry (HRESIMS: positive ion mode) is m / z 853.2401 (M + H) ⁺ , and the calculated value is m / z 853.2041 (C ₄₃ H ₄₃ Cl ₂ is as O _14).
(4) The specific rotation is [α] _D ²³ = −120.5 ° (c 0.025, MeOH).
(5) The infrared absorption spectrum is as shown in FIG.
ν _max (KBr) cm ⁻¹ : 3700 to 3200, 2972, 2947, 1684, 1616, 1590 (sh), 1408, 1379, 1315, 1217, 1153
(6) The ultraviolet absorption spectrum is as shown in FIG.
λ _max nm (ε):
0.005M HCl: 236 (33,700), 277 (35,400), 330 (6,600), 412 (13,600)
0.005M NaOH: 246 (33,700), 267 (sh), 340 (7,100), 429 (20,400)
(7) As a proton nuclear magnetic resonance spectrum, a proton NMR spectrum measured at 30 ° C. in a mixed solvent of deuterated chloroform: deuterated methanol = 4: 1 at 600 MHz is:
As shown in FIG. 3 and Table 1.
(8) As a carbon 13 nuclear magnetic resonance spectrum, the carbon 13 NMR spectrum measured at 30 ° C. in heavy methanol at 150 MHz is as shown in FIG.
(9) CAPCELL PAK C18 UG120 (particle diameter 5 μm, inner diameter 2.0 mm × length 150 mm, manufactured by Shiseido) column as high-speed chromatography, acetonitrile: water: trifluoroacetic acid (70: 30: 0.01, developing solvent) The retention time when developing at a flow rate of 0.2 mL / min using a volume ratio) is 7.7 minutes.

  Whether or not the compound has a structure represented by the structural formula (A) can be confirmed by various analysis methods selected as appropriate, for example, the mass spectrometry, the infrared absorption spectrum, the ultraviolet absorption spectrum. This can be confirmed by analyzing the proton nuclear magnetic resonance spectrum, the carbon 13 nuclear magnetic resonance spectrum, and the like.

  The walkmycin A may be obtained from a microorganism that produces walkmycin A or may be obtained by chemical synthesis. However, according to the method for producing a compound of the present invention described later, It is preferable to be obtained.

  The walkmycin A has excellent antibacterial activity and excellent enzyme inhibitory activity as shown in Test Examples 1 to 3 described later. Therefore, the walkmycin A can be suitably used as an active ingredient such as the enzyme activity inhibitor, the compound-containing composition of the present invention described later, and an antibacterial agent.

<Compound represented by Structural Formula (C)>
A compound represented by the following structural formula (C) is one of the compounds of the present invention. The compound represented by the following structural formula (C) is a novel compound isolated by the present inventors (hereinafter, sometimes referred to as “Walkmycin C”).

-Physicochemical properties-
The physicochemical properties of the compound represented by the structural formula (C) are as follows.
(1) The appearance is yellow powder.
(2) The molecular formula is represented by C ₄₅ H ₄₆ Cl ₂ O ₁₄ .
(3) The experimental value by high resolution mass spectrometry (HRESIMS: positive ion mode) is m / z 881.2340 (M + H) ⁺ , and the calculated value is m / z 881.2337 (C ₄₅ H ₄₇ Cl ₂ O ₁₄ ).
(4) Specific rotation is [α] _D ²³ = −39.8 ° (c 0.025, MeOH).
(5) The infrared absorption spectrum is as shown in FIG.
ν _max (KBr) cm ⁻¹ : 3700 to 3200, 2970, 2939, 1685, 1616, 1590 (sh), 1408, 1377, 1315, 1219, 1161
(6) The ultraviolet absorption spectrum is as shown in FIG.
λ _max nm (ε):
0.005M HCl: 237 (27,700), 276 (11,500), 328 (5,500), 405 (10,700)
0.005M NaOH: 245 (27,500), 271 (sh), 341 (5,500), 424 (15,000)
(7) As a proton nuclear magnetic resonance spectrum, a proton NMR spectrum measured at 30 ° C. in a mixed solvent of deuterated chloroform: deuterated methanol = 4: 1 at 600 MHz is:
As shown in FIG. 7 and Table 2.
(8) As a carbon 13 nuclear magnetic resonance spectrum, a carbon 13 NMR spectrum measured at 30 ° C. in deuterated methanol at 150 MHz is as shown in FIG.
(9) CAPCELL PAK C18 UG120 (particle diameter 5 μm, inner diameter 2.0 mm × length 150 mm, manufactured by Shiseido) column as high-speed chromatography, acetonitrile: water: trifluoroacetic acid (70: 30: 0.01, developing solvent) The retention time when developed at a flow rate of 0.2 mL / min using a volume ratio) is 10.0 minutes.

  Whether or not the compound has a structure represented by the structural formula (C) can be confirmed by various analysis methods selected as appropriate, for example, the mass spectrometry, the infrared absorption spectrum, the ultraviolet absorption spectrum. This can be confirmed by analyzing the proton nuclear magnetic resonance spectrum, the carbon 13 nuclear magnetic resonance spectrum, and the like.

  The walkmycin C may be obtained from a microorganism that produces walkmycin C or may be obtained by chemical synthesis. However, according to the method for producing a compound of the present invention described later, It is preferable to be obtained.

  The walkmycin C has excellent antibacterial activity and excellent enzyme inhibitory activity, as shown in Test Examples 1 to 3 described later. Therefore, the walkmycin C can be suitably used as an active ingredient such as, for example, the enzyme activity inhibitor, the compound-containing composition of the present invention described later, and an antibacterial agent.

<Compound represented by Structural Formula (B)>
The physicochemical properties of the compound represented by the structural formula (B) (hereinafter sometimes referred to as “Walkmycin B”) are as follows.

-Physicochemical properties-
The physicochemical properties of the compound represented by the structural formula (B) are as follows.
(1) The appearance is yellow powder.
(2) The molecular formula is represented by C ₄₄ H ₄₄ Cl ₂ O ₁₄ .
(3) The experimental value by high resolution mass spectrometry (HRESIMS: positive ion mode) is m / z 867.2180 (M + H) ⁺ and the calculated value is m / z 867.2181 (C ₄₄ H ₄₅ Cl ₂ is as O _14).
(4) Specific rotation is [α] _D ²³ = −121.7 ° (c 0.025, MeOH).
(5) The infrared absorption spectrum is as shown in FIG.
ν _max (KBr) cm ⁻¹ : 3700 to 3200, 2974, 2943, 1691, 1614, 1587 (sh), 1400, 1375, 1313, 1220, 1196, 1167
(6) The ultraviolet absorption spectrum is as shown in FIG.
λ _max nm (ε):
0.005M HCl: 235 (56,200), 279 (57,600), 331 (9,700), 425 (22,000)
0.005M NaOH: 245 (46,300), 268 (44,600), 341 (9,200), 434 (29,300)
(7) As a proton nuclear magnetic resonance spectrum, a proton NMR spectrum measured at 30 ° C. in a mixed solvent of deuterated chloroform: deuterated methanol = 4: 1 at 600 MHz is:
As shown in FIG. 11 and Table 3.
(8) As a carbon 13 nuclear magnetic resonance spectrum, the carbon 13 NMR spectrum measured at 30 ° C. in deuterated methanol at 150 MHz is as shown in FIG.
(9) CAPCELL PAK C18 UG120 (particle diameter 5 μm, inner diameter 2.0 mm × length 150 mm, manufactured by Shiseido) column as high-speed chromatography, acetonitrile: water: trifluoroacetic acid (70: 30: 0.01, developing solvent) The retention time when developed at a flow rate of 0.2 mL / min using a volume ratio) is 8.6 minutes.

  Whether or not the compound has a structure represented by the structural formula (B) can be confirmed by various analysis methods selected as appropriate, for example, the mass spectrometry, the infrared absorption spectrum, the ultraviolet absorption spectrum. This can be confirmed by analyzing the proton nuclear magnetic resonance spectrum, the carbon 13 nuclear magnetic resonance spectrum, and the like.

-Production of Walkmycin B-
There is no restriction | limiting in particular as a manufacturing method of the said walkmycin B, According to the objective, it can select suitably, For example, the method of manufacturing from the microorganisms which produce walkmycin B, the method of manufacturing by chemical synthesis, etc. are mentioned. It is done.

  There is no restriction | limiting in particular as a method to manufacture the said walk mycin B from microorganisms, According to the objective, it can select suitably, For example, it includes a culture | cultivation process and a collection process at least, and other processes as needed. The method containing is mentioned.

--Culture process--
The culturing step is a step of culturing a microorganism having an ability to produce walkmycin B.

The microorganism is not particularly limited as long as it has the ability to produce walkmycin B, and can be appropriately selected according to the purpose. For example, Streptomyces sp. MK632-100F11 strain described later (NITE P-777). Further, other strains capable of producing walkmycin B can be isolated from nature by a conventional method. In addition, the production ability of Walkmycin B is enhanced by subjecting the production bacteria producing Walkmycin B, including the Streptomyces sp. Strain MK632-100F11, to irradiation and other mutation treatments. It is also possible. Furthermore, walkmycin B can be produced by genetic engineering techniques.

The culturing is performed by inoculating a production bacterium that produces walkmycin B in a nutrient medium (hereinafter sometimes simply referred to as “medium”) and culturing at a temperature suitable for the production of walkmycin B.
There is no restriction | limiting in particular as said nutrient medium, According to the objective, it can select suitably, For example, the well-known thing utilized conventionally for culture | cultivation of actinomycetes can be used.
The nutrient source added to the nutrient medium is not particularly limited and can be appropriately selected according to the purpose. For example, as a nitrogen source, commercially available soybean flour, peptone, yeast extract, meat extract, corn Steep liquor, ammonium sulfate, and the like can be used, and as a carbon source, carbohydrates such as tomato paste, glycerin, starch, glucose, galactose, and dextrin, fats, and the like can be used. Furthermore, inorganic salts such as salt and calcium carbonate can be added to the medium for use, and in addition, a trace amount of metal salt can be added to the medium for use.
Any of these materials may be used as long as it is useful for the production of walkomycin B and can be used by production bacteria producing walkmycin B, and all known culture materials can be used.

  An amino acid may or may not be added to the medium, but it is advantageous that no amino acid is added because the amount of walkmycin B produced is large.

  There is no restriction | limiting in particular as a seed | species culture medium for the production of walkmycin B, According to the objective, it can select suitably, For example, it obtained from the slope culture of the production microbe which produces walkmycin B on an agar medium. Grows can be used.

The culture method is not particularly limited and may be appropriately selected depending on the intended purpose. A culture method under aerobic conditions is preferred.
The culture temperature is not particularly limited as long as it does not substantially inhibit the growth of production bacteria producing walkmycin B and can produce walkmycin B, depending on the production bacteria used. Although it can select suitably, 25 to 35 degreeC is preferable.
There is no restriction | limiting in particular as the period of the said culture | cultivation, According to accumulation | storage of walkmycin B, it can select suitably. Usually, the accumulation of walkmycin B is highest in 3 to 10 days of culture.

  The walkmycin B has excellent enzyme inhibitory activity as shown in Test Example 3 described later.

  The content of at least one of walkmycin A, walkmycin C, and walkmycin B in the enzyme activity inhibitor is not particularly limited and may be appropriately selected depending on the purpose. The enzyme activity inhibitor may be at least one of walkmycin A, walkmycin C, and walkmycin B.

There is no restriction | limiting in particular as said other component, For example, it can select suitably from the pharmacologically acceptable support | carrier according to the objective, For example, ethanol, water, starch, etc. are mentioned. There is no restriction | limiting in particular as content of the said other component in the said enzyme activity inhibitor, In the range which does not impair the effect of Walkmycin A, Walkmycin C, and Walkmycin B, it selects suitably according to the objective. can do.
In addition, the said enzyme activity inhibitor may be used independently and may be used in combination with the pharmaceutical which uses another component as an active ingredient. Moreover, you may use the said enzyme activity inhibitor in the state mix | blended in the pharmaceutical which uses another component as an active ingredient.

Since the enzyme activity inhibitor includes at least one of walkmycin A, walkmycin C, and walkmycin B, it includes drug-resistant bacteria, phytopathogenic bacteria, and the like as shown in Test Example 3 described later. It has excellent enzyme inhibitory activity against enzymes possessed by a wide range of Gram-positive bacteria and Gram-negative bacteria.
Therefore, the enzyme activity inhibitor can suppress the pathogenicity of a wide range of gram-positive bacteria and gram-negative bacteria including drug-resistant bacteria, and is suitably used for the prevention or treatment of infectious diseases caused by the bacteria. Is possible. Moreover, it can utilize suitably also as an active ingredient of the agricultural and horticultural fungicide.

<Dosage form, administration>
-Dosage form-
There is no restriction | limiting in particular as a dosage form of the said enzyme activity inhibitor, According to the objective, it can select suitably, For example, it can be set as dosage forms, such as a powder form, a capsule form, a tablet form, and a liquid form. These enzyme activity inhibitors of these dosage forms can be produced according to conventional methods.

The dosage form for use as the agricultural and horticultural fungicide is not particularly limited and may be appropriately selected depending on the intended purpose. Usually, it is a solid carrier, liquid carrier, surfactant, and other formulation aids. As an admixture with an agent, it can be prepared in an appropriate form such as an emulsion, a wettable powder, a liquid, a flowable (sol) agent, a powder, a granule, a fine granule, and a tablet.
Various surfactants (or emulsifiers) are used for the purpose of emulsions, wettable powders, liquids, flowable (sol) agents, powders, granules, fine granules, tablets and the like. Such surfactants include nonionic types (polyalkyl ethers, polyoxyethylene alkyl esters, polyoxyethylene sorbitan alkyl esters, etc.), anionic types (alkyl alkoxyethylene alkyl sulfates, aryl sulfonates, etc.), and cationic types. (Alkylamines, polyoxyalkylamines, etc.) and amphoteric types (sulfuric acid ester salts, etc.) are mentioned, but of course not limited to these examples. In addition to these, various adjuvants such as polyvinyl alcohol, carboxymethyl cellulose, gum arabic, polyvinyl acetate, sodium alginate, gelatin, and tragacanth gum can be used.

-Administration-
There is no restriction | limiting in particular as the administration method of the said enzyme activity inhibitor, For example, it can select suitably according to the dosage form of the said enzyme activity inhibitor, etc., It can administer orally or parenterally.
The dosage of the enzyme activity inhibitor is not particularly limited, taking into consideration various factors such as the age, weight, constitution, symptom of the administration subject individual, and the presence or absence of administration of a drug containing other ingredients as active ingredients. It can be selected appropriately.
There is no restriction | limiting in particular as an administration time of the said enzyme activity inhibitor, According to the objective, it can select suitably.
The animal species to be administered with the enzyme activity inhibitor is not particularly limited and can be appropriately selected according to the purpose. For example, human, monkey, pig, cow, sheep, goat, dog, cat, mouse , Rats, birds and the like.
Moreover, there is no restriction | limiting in particular as an administration method in the case of using as said agricultural and horticultural fungicide, dosage amount, administration time, and administration object, According to the objective, it can select suitably.

(Method for producing compound)
The production method of the compound of the present invention, that is, walkmycin A and walkmycin C includes at least a culture step and a collection step, and further includes other steps as necessary.

<Culture process>
The culturing step is a step of culturing a microorganism belonging to the genus Streptomyces and capable of producing at least one of walkmycin A and walkmycin C.
In the culturing step, at least one of a branched amino acid, a branched fatty acid, and a branched keto acid is added to the medium. Note that at least one of the branched amino acid, the branched fatty acid, and the branched keto acid may be a precursor thereof.

The microorganism is not particularly limited as long as it belongs to the genus Streptomyces and has the ability to produce at least one of walkmycin A and walkmycin C, and can be appropriately selected according to the purpose. For example, the Streptomyces sp. MK632-100F11 strain (NITE P-777, the details of which are described in the item of the microorganism of the present invention described later) isolated by the present inventors can be mentioned. In addition, other strains capable of producing at least one of walkmycin A and walkmycin C can be isolated from the natural world by conventional methods. By subjecting the producing bacteria producing at least one of walkmycin A and walkmycin C, including the Streptomyces sp. Strain MK632-100F11, to irradiation and other mutation treatments , Walkmycin A, and / or walkmycin C can also be produced. Furthermore, at least one of walkmycin A and walkmycin C can be produced by genetic engineering techniques.

In the culture, a production bacterium that produces at least one of walkmycin A and walkmycin C (hereinafter sometimes simply referred to as “walkmycin-producing bacterium”) is referred to as a nutrient medium (hereinafter simply referred to as “medium”). Inoculation) and culturing at a good temperature for the production of walkmycin A and / or walkmycin C.
There is no restriction | limiting in particular as said nutrient medium, According to the objective, it can select suitably, For example, the well-known thing utilized conventionally for culture | cultivation of actinomycetes can be used.
The nutrient source added to the nutrient medium is not particularly limited and can be appropriately selected according to the purpose. For example, as a nitrogen source, commercially available soybean flour, peptone, yeast extract, meat extract, corn Steep liquor, ammonium sulfate, and the like can be used, and as a carbon source, carbohydrates such as tomato paste, glycerin, starch, glucose, galactose, and dextrin, fats, and the like can be used. Furthermore, inorganic salts such as salt and calcium carbonate can be added to the medium for use, and in addition, a trace amount of metal salt can be added to the medium for use.
Any of these materials may be used as long as they are utilized by a walkmycin-producing bacterium and are useful for the production of at least one of walkmycin A and walkmycin C, and all known culture materials can be used.

At least one of a branched amino acid, a branched fatty acid, and a branched keto acid is added to the medium. By adding at least one of the branched amino acid, branched fatty acid, and branched keto acid, the production amount of at least one of walkmycin A and walkmycin C can be increased.
There is no restriction | limiting in particular as at least any one of the said branched amino acid, branched fatty acid, and branched keto acid, According to the objective, it can select suitably. At least one of the branched amino acids, branched fatty acids, and branched keto acids may be used alone or in combination of two or more.
Specific examples of the branched amino acid include valine, leucine and isoleucine.
Specific examples of the branched fatty acid include 3-methyl butanoic acid, 4-methyl pentanoic acid, and 3-methyl pentanoic acid.
Specific examples of the branched keto acid include 3-methyl-2-oxobutanoic acid, 4-methyl-2-oxopentanoic acid, and 3-methyl-2-oxopentanoic acid.
Among these, valine, leucine and isoleucine are preferable, and isoleucine is more preferable. The isoleucine is advantageous in that it produces a large amount of at least one of the walkmycin A and the walkmycin C.

The addition amount of at least one of the branched amino acid, branched fatty acid, and branched keto acid is not particularly limited and may be appropriately selected depending on the intended purpose. 20 mass% is preferable, 0.25 mass%-5 mass% is more preferable, 0.5 mass%-1 mass% is especially preferable.
Note that at least one of the branched amino acid, branched fatty acid, and branched keto acid is at least a medium for producing at least one of the walkmycin A and the walkmycin C (hereinafter referred to as “production medium”). As long as it is added.

  There is no restriction | limiting in particular as a seed | species culture medium for the production of at least any one of walkmycin A and walkmycin C, According to the objective, it can select suitably, For example, a walkmycin production bacteria on an agar medium The growth obtained from the slant culture can be used.

The culture method is not particularly limited and may be appropriately selected depending on the intended purpose. A culture method under aerobic conditions is preferred.
The culture temperature is not particularly limited as long as it does not substantially inhibit the growth of walkmycin-producing bacteria and can produce at least one of walkmycin A and walkmycin C. Although it can select suitably according to the production microbe to perform, 25 to 35 degreeC is preferable.
There is no restriction | limiting in particular as the period of the said culture | cultivation, It can select suitably according to accumulation | storage of at least any one of walkmycin A and walkmycin C. Usually, accumulation of at least one of walkmycin A and walkmycin C is highest in 3 days to 10 days of culture.

<Collection process>
The collecting step is a step of collecting at least one of walkmycin A and walkmycin C from the culture obtained in the culturing step.
Walkmycin A and walkmycin C have the physicochemical properties described above, and can be collected from the culture according to their properties.

The collection method is not particularly limited, and a method used for collecting a metabolite produced by a microorganism can be appropriately selected. For example, a method of extracting with a solvent that is not mixed with water, a method of using a difference in adsorption affinity for various adsorbents, a method of gel filtration, chromatography using countercurrent distribution, etc., alone or in combination can be mentioned.
From the separated cells, the walkmycin A and walkmycin C are extracted from the cells by an extraction method using an appropriate organic solvent or an elution method by disrupting the cells. It can be collected after separation and purification.

  The production method can be carried out as described above, whereby walkmycin A and walkmycin C can be obtained.

(Microorganism)
The microorganism of the present invention belongs to the genus Streptomyces , and is characterized by having the ability to produce at least one of the above-described compounds of the present invention, namely, walkmycin A and walkmycin C. The microorganism has the ability to produce at least one of walkmycin A and walkmycin C. Therefore, in the above-described method for producing a compound of the present invention, at least one of walkmycin A and walkmycin C is used. As long as it is a microorganism that can be used as a producing bacterium, there is no particular limitation, and it can be appropriately selected according to the purpose.

  Among such microorganisms, in particular, in the Microbial Chemistry Research Center of the Foundation for Microbial Chemistry, a microorganism with the strain number MK632-2F11 is used, which is an actinomycete isolated from the soil of Hokuto City, Yamanashi Prefecture. It is preferable. The mycological properties of the MK632-100F11 strain are as follows.

1. Form The strain MK632-100F11 extends a relatively long straight aerial hyphae from the branched basic hyphae. A mature spore chain links 10 to 50 oval spores. The size of the spore is about 0.5 μm to 0.6 μm × 1.2 μm to 1.7 μm, and the surface of the spore is smooth. No roticular branches, mycelial bundles, spore capsules, or motile spores are observed.

2. Regarding the description of the color of growth in various media The standard shown in [] was the color harmony manual of Container Corporation of America (color harmony manual of Container Corporation of America).
(1) Yeast / malt agar medium (ISP-medium 2, 30 ° C. culture)
On the growth of yellow [2 lc, Gold], aerial hyphae of brown [3 cb, Sand] are grown to produce a soluble pigment of yellow.
(2) Oatmeal agar medium (ISP-medium 3, 30 ° C. culture)
On the growth of light yellow [1 1/2 gc, Dusty Yellow], aerial mycelium of bright tea ash [3 dc, Natural] is grown. Soluble pigments are yellowish.
(3) Starch / inorganic salt agar medium (ISP-medium 4, 30 ° C. culture)
On the growth of yellow [1 1/2 lc, Gold], aerial mycelium of brown [5 cb] is grown. Soluble pigments are yellowish.
(4) Glycerin / asparagine agar medium (ISP-medium 5, 30 ° C. culture)
On the development of nib yellow [2 le, Mustard], aerial mycelium of brown [5 cb] is grown. Produces a soluble pigment of yellow. The growth color and soluble pigment are not affected by the addition of 0.05 molar hydrochloric acid and 0.05 molar sodium hydroxide.
(5) Sucrose / Nitrate agar medium (30 ° C culture)
On the development of light yellow [2 ca, Lt Ivory], aerial mycelia of white [3 cb, Sand] are grown. Soluble pigments are slightly yellowish.

3. Physiological properties (1) Growth temperature range Glucose / asparagine agar (glucose 1.0%, L-asparagine 0.05%, dipotassium hydrogen phosphate 0.05%, string agar 3.0%, pH 7.0) As a result of testing at 7 ° C., 16 ° C., 24 ° C., 27 ° C., 30 ° C., 37 ° C., and 45 ° C., growth at 7 ° C. and 45 ° C. was not observed, and 16 ° C. to 37 ° C. It grew in the range of ° C. The optimal growth temperature is 27 ° C to 30 ° C.
(2) Starch hydrolysis (starch / inorganic salt agar medium, ISP-medium 4, 30 ° C. culture)
On the third day after culturing, starch hydrolysis is observed and its action is moderate.

4). Cell component 2,6-diaminopimelic acid in the cell wall is LL-type.

5. 16S rRNA gene analysis The partial base sequence (1459 bp) of the 16S rRNA gene was determined and compared with the data of known strains registered in the DNA database. As a result, the base sequence of MK632-100F11 strain showed high homology with the 16S rRNA gene of Streptomyces genus Streptomyces as shown below. Streptomyces pseudovenezuelae (99%), S. novaecaesareae (99%), S. et al . canus (99%), S. canis . galilaeus (99%), S. Lavovariabilis (99%), S. regalis (99%). In addition, the homologous value of the base sequence is described in parentheses.

Summarizing the above properties, the MK632-100F11 strain extends a relatively long straight aerial hyphae from the well-branched basic hyphae and links oval spores. In various media, aerial hyphae of brown to bright brown ash are grown on light yellow to yellow growth. Produces a soluble pigment of yellow. The optimum growth temperature is around 27 ° C to 30 ° C. The water disintegration of starch is moderate.
2,6-Diaminopimelic acid in the cell wall of the MK632-100F11 strain is LL-type.
When the partial base sequence of 16S rRNA gene of MK632-2F11 strain was analyzed and compared with the data of known strains, it showed high homology with Streptomyces spp.

From the above results, it is considered that the MK632-100F11 strain belongs to the genus Streptomyces . Therefore, the strain MK632-2F11 was designated as Streptomyces sp. MK632-2F11.
The MK632-100F11 strain was applied for deposit at the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation, and was commissioned as NITE P-777 on July 1, 2009.

  As seen in other bacteria, the MK632-2100F11 strain is susceptible to changes in properties, but for example, mutant strains (naturally occurring or inducible) derived from the MK632-2100F11 strain, Even if it is a body, a recombinant, etc., what has the capability to produce at least any one of walkmycin A and walkmycin C is contained in the microorganisms of this invention.

(Compound-containing composition)
The compound-containing composition of the present invention contains at least one of the walkmycin A and the walkmycin C, and optionally contains other components as necessary.

  The content of at least one of walkmycin A and walkmycin C in the compound-containing composition is not particularly limited and may be appropriately selected depending on the purpose. The compound-containing composition may be at least one of walkmycin A and walkmycin C.

There is no restriction | limiting in particular as said other component, For example, it can select suitably from the pharmacologically acceptable support | carrier according to the objective, For example, ethanol, water, starch, etc. are mentioned. There is no restriction | limiting in particular as content of the said other component in the said compound containing composition, In the range which does not impair the effect of Walkmycin A and Walkmycin C, it can select suitably according to the objective. .
In addition, the said compound containing composition may be used independently, and may be used in combination with the pharmaceutical which uses another component as an active ingredient. Moreover, you may use the said compound containing composition in the state mix | blended in the pharmaceutical which uses another component as an active ingredient.

  Since the compound-containing composition contains at least one of walkmycin A and walkmycin C, it has at least one of an antibacterial action and an enzyme activity inhibitory action.

(Antimicrobial agent)
The antibacterial agent of the present invention includes at least one of the walkmycin A and the walkmycin C, and optionally includes other components as necessary.

  The content of at least one of walkmycin A and walkmycin C in the antibacterial agent is not particularly limited and may be appropriately selected depending on the purpose. The antibacterial agent may be at least one of walkmycin A and walkmycin C.

There is no restriction | limiting in particular as said other component, For example, it can select suitably from the pharmacologically acceptable support | carrier according to the objective, For example, ethanol, water, starch, etc. are mentioned. There is no restriction | limiting in particular as content of the said other component in the said antibacterial agent, It can select suitably according to the objective within the range which does not impair the effect of Walkmycin A and Walkmycin C.
In addition, the said antibacterial agent may be used independently and may be used in combination with the pharmaceutical which uses another component as an active ingredient. Moreover, you may use the said antibacterial agent in the state mix | blended in the pharmaceutical which uses another component as an active ingredient.

Since the antibacterial agent includes at least one of walkmycin A and walkmycin C, the antibacterial agent has excellent antibacterial activity against drug-resistant bacteria as shown in Test Examples 1 and 2 described later. .
Therefore, the antibacterial agent can be suitably used for the prevention or treatment of infectious diseases caused by drug-resistant bacteria. The antibacterial agent can also be suitably used as an agricultural and horticultural fungicide.

<Dosage form, administration>
-Dosage form-
There is no restriction | limiting in particular as a dosage form of the said compound containing composition and an antibacterial agent, According to the objective, it can select suitably, For example, it is set as dosage forms, such as a powder form, a capsule form, a tablet form, and a liquid Can do. These compound-containing compositions and antibacterial agents in these dosage forms can be produced according to conventional methods.

The dosage form for use as the agricultural and horticultural fungicide is not particularly limited and may be appropriately selected depending on the intended purpose. Usually, it is a solid carrier, liquid carrier, surfactant, and other formulation aids. As an admixture with an agent, it can be prepared in an appropriate form such as an emulsion, a wettable powder, a liquid, a flowable (sol) agent, a powder, a granule, a fine granule, and a tablet.
Various surfactants (or emulsifiers) are used for the purpose of emulsions, wettable powders, liquids, flowable (sol) agents, powders, granules, fine granules, tablets and the like. Such surfactants include nonionic types (polyalkyl ethers, polyoxyethylene alkyl esters, polyoxyethylene sorbitan alkyl esters, etc.), anionic types (alkyl alkoxyethylene alkyl sulfates, aryl sulfonates, etc.), and cationic types. (Alkylamines, polyoxyalkylamines, etc.) and amphoteric types (sulfuric acid ester salts, etc.) are mentioned, but of course not limited to these examples. In addition to these, various adjuvants such as polyvinyl alcohol, carboxymethyl cellulose, gum arabic, polyvinyl acetate, sodium alginate, gelatin, and tragacanth gum can be used.

-Administration-
The administration method of the compound-containing composition and the antibacterial agent is not particularly limited, and can be appropriately selected depending on, for example, the compound-containing composition, the dosage form of the antibacterial agent, orally or parenterally. Can be administered.
The dosage of the compound-containing composition and the antibacterial agent is not particularly limited, and includes various factors such as the age, weight, constitution, symptom of the administration target individual, and the presence / absence of administration of a drug containing other ingredients as active ingredients. Can be selected as appropriate.
There is no restriction | limiting in particular as an administration time of the said compound containing composition and an antibacterial agent, According to the objective, it can select suitably.
There is no restriction | limiting in particular as an animal species used as the administration target of the said compound containing composition and an antibacterial agent, For example, a human, a monkey, a pig, a cow, a sheep, a goat, a dog can be selected suitably , Cats, mice, rats, birds and the like.
Moreover, there is no restriction | limiting in particular as an administration method in the case of using as said agricultural and horticultural fungicide, dosage amount, administration time, and administration object, According to the objective, it can select suitably.

  EXAMPLES The present invention will be specifically described below with reference to examples, comparative examples, and test examples, but the present invention is not limited to these examples and test examples. In the following examples, comparative examples, and test examples, “%” represents “% by mass” unless otherwise specified.

(Example 1: Production of compound)
-Culture process-
Streptomyces sp. MK632-2100F11 strain (deposited as NITE P-777) cultured on an agar slant medium, galactose 2%, dextrin 2%, glycerin 1%, Bacto Soyton (manufactured by Difco) 1%, Dispense 110 mL of a liquid medium (adjusted to pH 7.0) containing 0.5% corn steep liquor, 0.2% ammonium sulfate and 0.2% calcium carbonate into an Erlenmeyer flask (500 mL volume). Inoculated into medium sterilized at 120 ° C. for 20 minutes. Thereafter, it was subjected to rotary shaking culture at 30 ° C. for 2 days to obtain a seed culture solution.

  Glycerin 2.0%, dextrin 2.0%, L-isoleucine 1.0%, yeast extract (Nippon Pharmaceutical Co., Ltd.) 0.3%, bacto soyton (Difco) 1.0%, ammonium sulfate 0.2%, carbonic acid A liquid medium containing 0.2% calcium (adjusted to pH 7.0) was dispensed 110 mL each into an Erlenmeyer flask (500 mL volume) and sterilized at 120 ° C. for 20 minutes by a conventional method to obtain a production medium. This production medium was inoculated with 2% by volume of the above-mentioned seed culture medium, and cultured at 27 ° C. for 5 days with rotary shaking (180 rpm).

-Sampling process-
The culture broth 5 liters thus obtained was centrifuged to separate it into a culture filtrate and cells. Subsequently, the cells were added with 2.7 liters of methanol and stirred well, and walkmycin A, walkmycin B, and walkmycin C were extracted from the cells with methanol, and walkmycin A, walkmycin B, and 3 liters of a bacterial cell extract containing walkmycin C was obtained. Methanol was distilled off from 3 liters of the bacterial cell extract under reduced pressure, and 2 liters of purified water was added to the extract. Purified water was combined with this extract and the pH was adjusted to 3.0 with 1 M hydrochloric acid, and then an equal amount of ethyl acetate was added to perform extraction with ethyl acetate. The ethyl acetate layer was separated, washed with water after adding water, followed by dehydration with anhydrous sodium sulfate, followed by concentration to dryness under reduced pressure, and crude extraction containing walkmycin A, walkmycin B, and walkmycin C 1.5 g of product was obtained.

The crude extract was dissolved in methanol and placed on a Sephadex LH-20 (inner diameter 36 mm × 480 mm, Pharmacia Biotech) column for chromatography. After developing 550 mL of solvent, fractions of 3 g each were fractionated, and the active fraction was eluted into fractions 3 to 21, which were collected and concentrated to dryness under reduced pressure to give 55.7 mg of walkmycin A, walkmycin. A mixture of B and Walkmycin C was obtained.
The mixture of walkmycin A, walkmycin B, and walkmycin C was dissolved in a small amount of methanol, and walkmycin A and walk were subjected to C18 reverse layer column chromatography (Capcell pak UG120, inner diameter 30 mm × length 250 mm, manufactured by Shiseido). Mycin B and walkmycin C were separated from each other. That is, when chromatography is performed using acetonitrile: water: trifluoroacetic acid = 70: 30: 0.001 as a developing solvent at a flow rate of 15 mL / min, walkmycin A is obtained in 36 to 38 minutes, and walkmycin B is obtained. From 41 minutes to 43 minutes, walkmycin C elutes from 48 minutes to 51 minutes, and these were collected and concentrated to dryness under reduced pressure to give 3.3 mg of pure walkmycin A and 6.4 mg of walkmycin B. , Walkmycin C was obtained as 3.0 mg.

When the physicochemical properties of the obtained walkmycin A were measured, it was as follows. From these facts, the walkmycin A was a novel compound having a structure represented by the following structural formula (A). Was confirmed.
(1) The appearance is yellow powder.
(2) The molecular formula is represented by C ₄₃ H ₄₂ Cl ₂ O ₁₄ .
(3) The experimental value by high resolution mass spectrometry (HRESIMS: positive ion mode) is m / z 853.2401 (M + H) ⁺ , and the calculated value is m / z 853.2041 (C ₄₃ H ₄₃ Cl ₂ is as O _14).
(4) The specific rotation is [α] _D ²³ = −120.5 ° (c 0.025, MeOH).
(5) The infrared absorption spectrum is as shown in FIG.
ν _max (KBr) cm ⁻¹ : 3700 to 3200, 2972, 2947, 1684, 1616, 1590 (sh), 1408, 1379, 1315, 1217, 1153
(6) The ultraviolet absorption spectrum is as shown in FIG.
λ _max nm (ε):
0.005M HCl: 236 (33,700), 277 (35,400), 330 (6,600), 412 (13,600)
0.005M NaOH: 246 (33,700), 267 (sh), 340 (7,100), 429 (20,400)
(7) As a proton nuclear magnetic resonance spectrum, a proton NMR spectrum measured at 30 ° C. in a mixed solvent of deuterated chloroform: deuterated methanol = 4: 1 at 600 MHz is:
As shown in FIG. 3 and Table 4.
(8) As a carbon 13 nuclear magnetic resonance spectrum, the carbon 13 NMR spectrum measured at 30 ° C. in deuterated methanol at 150 MHz is as shown in FIG.
(9) CAPCELL PAK C18 UG120 (particle diameter 5 μm, inner diameter 2.0 mm × length 150 mm, manufactured by Shiseido) column as high-speed chromatography, acetonitrile: water: trifluoroacetic acid (70: 30: 0.01, developing solvent) The retention time when developing at a flow rate of 0.2 mL / min using a volume ratio) is 7.7 minutes.

Moreover, the physicochemical properties of the obtained walkmycin C were measured, and the results were as follows. From these results, walkmycin C is a novel compound having a structure represented by the following structural formula (C). It was confirmed that there was.
(1) The appearance is yellow powder.
(2) The molecular formula is represented by C ₄₅ H ₄₆ Cl ₂ O ₁₄ .
(3) The experimental value by high resolution mass spectrometry (HRESIMS: positive ion mode) is m / z 881.2340 (M + H) ⁺ , and the calculated value is m / z 881.2337 (C ₄₅ H ₄₇ Cl ₂ O ₁₄ ).
(4) Specific rotation is [α] _D ²³ = −39.8 ° (c 0.025, MeOH).
(5) The infrared absorption spectrum is as shown in FIG.
ν _max (KBr) cm ⁻¹ : 3700 to 3200, 2970, 2939, 1685, 1616, 1590 (sh), 1408, 1377, 1315, 1219, 1161
(6) The ultraviolet absorption spectrum is as shown in FIG.
λ _max nm (ε):
0.005M HCl: 237 (27,700), 276 (11,500), 328 (5,500), 405 (10,700)
0.005M NaOH: 245 (27,500), 271 (sh), 341 (5,500), 424 (15,000)
(7) As a proton nuclear magnetic resonance spectrum, a proton NMR spectrum measured at 30 ° C. in a mixed solvent of deuterated chloroform: deuterated methanol = 4: 1 at 600 MHz is:
As shown in FIG. 7 and Table 5.
(8) As a carbon 13 nuclear magnetic resonance spectrum, a carbon 13 NMR spectrum measured at 30 ° C. in deuterated methanol at 150 MHz is as shown in FIG.
(9) CAPCELL PAK C18 UG120 (particle diameter 5 μm, inner diameter 2.0 mm × length 150 mm, manufactured by Shiseido) column as high-speed chromatography, acetonitrile: water: trifluoroacetic acid (70: 30: 0.01, developing solvent) The retention time when developed at a flow rate of 0.2 mL / min using a volume ratio) is 10.0 minutes.

Further, the physicochemical properties of the obtained walkmycin B were measured, and the results were as follows. From these, walkmycin B is a compound having a structure represented by the following structural formula (B). It was confirmed.
(1) The appearance is yellow powder.
(2) The molecular formula is represented by C ₄₄ H ₄₄ Cl ₂ O ₁₄ .
(3) The experimental value by high resolution mass spectrometry (HRESIMS: positive ion mode) is m / z 867.2180 (M + H) ⁺ and the calculated value is m / z 867.2181 (C ₄₄ H ₄₅ Cl ₂ is as O _14).
(4) Specific rotation is [α] _D ²³ = −121.7 ° (c 0.025, MeOH).
(5) The infrared absorption spectrum is as shown in FIG.
ν _max (KBr) cm ⁻¹ : 3700 to 3200, 2974, 2943, 1691, 1614, 1587 (sh), 1400, 1375, 1313, 1220, 1196, 1167
(6) The ultraviolet absorption spectrum is as shown in FIG.
λ _max nm (ε):
0.005M HCl: 235 (56,200), 279 (57,600), 331 (9,700), 425 (22,000)
0.005M NaOH: 245 (46,300), 268 (44,600), 341 (9,200), 434 (29,300)
(7) As a proton nuclear magnetic resonance spectrum, a proton NMR spectrum measured at 30 ° C. in a mixed solvent of deuterated chloroform: deuterated methanol = 4: 1 at 600 MHz is:
As shown in FIG. 11 and Table 6.
(8) As a carbon 13 nuclear magnetic resonance spectrum, a carbon 13 NMR spectrum measured at 30 ° C. in deuterated methanol at 150 MHz is as shown in FIG.
(9) CAPCELL PAK C18 UG120 (particle diameter 5 μm, inner diameter 2.0 mm × length 150 mm, manufactured by Shiseido) column as high-speed chromatography, acetonitrile: water: trifluoroacetic acid (70: 30: 0.01, developing solvent) The retention time when developed at a flow rate of 0.2 mL / min using a volume ratio) is 8.6 minutes.

(Comparative Example 1: Production of compound)
-Culture process-
In the culturing step of Example 1, the culturing step was performed in the same manner as in Example 1 except that L-isoleucine as a production medium was not used.

-Sampling process-
The culture broth 5 liters thus obtained was centrifuged to separate it into a culture filtrate and cells. Subsequently, the cells were added with 2.7 liters of methanol and stirred well, and walkmycin A, walkmycin B, and walkmycin C were extracted from the cells with methanol. 3 liters of bacterial cell extract containing A and walkmycin C was obtained. Methanol was distilled off from 3 liters of the bacterial cell extract under reduced pressure, and 2 liters of purified water was added to the extract. An equal amount of ethyl acetate was added to a solution obtained by combining purified water with purified water, followed by extraction with ethyl acetate. The ethyl acetate layer was separated, dehydrated with anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to obtain 2.0 g of a crude extract containing walkmycin B.

The crude extract was dissolved in methanol and placed on a Sephadex LH-20 (inner diameter 36 mm × 480 mm, Pharmacia Biotech) column for chromatography. After developing 550 mL of solvent, fractions of 1 g each were fractionated, and the active fraction was eluted into fractions 3 to 23. These fractions were collected and concentrated to dryness under reduced pressure, and crude fractions containing 99.7 mg of walkmycin B were collected. The product was obtained.
The crude product containing the walkmycin B was dissolved by heating in a small amount of a mixed solution of methanol: chloroform = 10: 3. When this was allowed to stand overnight at 5 ° C., pure walkmycin B was precipitated. This was collected to obtain 44.8 mg of walkmycin B.
Moreover, when the physicochemical property of the obtained walkmycin B was measured, it was the same result as the walkmycin B obtained in the Example 1. From these results, the walkmycin B has the structural formula ( It was confirmed that the compound had a structure represented by B).
In addition, walkmycin A and walkmycin C were produced in a very small amount and could not be purified.

  From the results of Example 1 and Comparative Example 1, Walkmycin B is purified in Comparative Example 1 in which at least one of a branched amino acid, a branched fatty acid, and a branched keto acid was not added to the culture medium in the culturing step. However, it was found that Walkmycin A and Walkmycin C were produced in a small amount, and a purified product could not be obtained.

  Moreover, the antibacterial activity and enzyme inhibitory activity of the obtained walkmycin A to C were confirmed in the following Test Examples 1 to 3.

(Test Example 1: Antibacterial activity-1)
The antibacterial spectrum of walkmycin A to C against vancomycin-resistant enterococci and vancomycin-sensitive enterococci was measured by a multiple dilution method on a Mura Hinton agar medium based on the standard method of the Japanese Society of Chemotherapy. Table 7 shows the measurement results of the minimum inhibitory concentration (MIC).
For reference, the same measurement was performed with vancomycin. The results are shown in Table 7.

(Test Example 2: Antibacterial activity-2)
Antibacterial spectrum of walkmycin A to C against methicillin-resistant staphylococci, vancomycin-insensitive methicillin-resistant staphylococci, and methicillin-sensitive staphylococci by multiple dilution method on Mura Hinton agar medium based on the Japanese Society of Chemotherapy Standard Method did. Table 8 shows the measurement results of the minimum inhibitory concentration (MIC).
For reference, oxacillin was also measured in the same manner. The results are shown in Table 8.

From the results of Tables 7-8, walkmycin AC has antimicrobial activity against vancomycin-resistant enterococci, vancomycin-sensitive enterococci, methicillin-resistant staphylococci, vancomycin-insensitive methicillin-resistant staphylococci, and methicillin-sensitive staphylococci. I found it.
Among walkomycins A to C, walkmycins A and C were found to have antibacterial activity superior to walkmycin B.

(Test Example 3: Enzyme inhibition activity)
-(1) YycG histidine kinase activity inhibition test-
The enzyme inhibitory activity of walkmycin AC on YycG of B. subtilis 168 strain ( B. subtilis 168) was examined.
Measurement of histidine kinase activity was performed as described in Biosci. Biotechnol. Biochem. 64, 919-923, 2000.
A region including only the kinase active domain of YycG (including amino acids 207 to 611 from the N-terminal) was prepared from the chromosomal DNA of Bacillus subtilis 168 strain by PCR, and expressed in expression vector pET-21a (+). Cloned. A protein (YycG-207-611) in which the histidine kinase active domain of YycG was expressed was purified from the culture solution of the strain obtained by transforming Escherichia coli with the plasmid pET-yycGtru thus obtained.
The composition of the reaction solution for measuring histidine kinase activity was 0.5 μM YycG-207-611, 50 mM Tris-HCl (pH 8.5), 100 mM KCl, 100 mM NH ₄ Cl, 5 mM MgCl _2. 2.5 μM ATP-10 μCi [γ- ³² P] ATP mixed solution was added to make 10 μL, the reaction was started, incubated at 30 ° C. for 10 minutes, the reaction was terminated, and SDS-polyacrylamide electrophoresis was performed. When investigating the inhibitory activity, a predetermined concentration of walkomycin A to C was added to the reaction solution before adding the ATP mixed solution, incubated at 30 ° C. for 5 minutes, and Bacillus subtilis 50% inhibitory concentration against YycG (IC ₅₀ ) The results are shown in Table 9.

-(2) VicK histidine kinase activity inhibition test-
The enzyme inhibitory activity of walkmycin A to C against VicK of Streptococcus mutans was examined.
Measurement of histidine kinase activity was performed as described in Biosci. Biotechnol. Biochem. 64, 919-923, 2000, which was modified.
A region containing only the kinase active domain of VicK (including the 31st to 450th amino acids from the N-terminal) was prepared from the cartilage chromosomal DNA by PCR and cloned into the expression vector pET22b (+). . A protein (VicK-31-450) in which only the histidine kinase active domain of VicK was expressed was purified from the culture solution of the strain obtained by transforming the plasmid pET-SMvicK31-450 thus obtained into E. coli.
The composition of the reaction solution for measuring the histidine kinase activity was 0.5 μM VicK-31-450, 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 10 mM MgCl _2. 1 μL of C was added and incubated at 25 ° C. for 5 minutes. Thereafter, 2 μL of 12.5 μM ATP containing [ ³² P] ATP is added (final concentration 2.5 μM) to start the reaction. After incubation at 25 ° C. for 20 minutes, the reaction is terminated, and SDS-polyacrylamide gel electrophoresis is performed. Thus, a 50% inhibitory concentration (IC ₅₀ ) of caries bacteria against VicK was determined. The results are shown in Table 9.

-(3) PehS histidine kinase activity inhibition test-
For PehS of soft rot MAFF301393 strain (Erwinia carotovora subsp. Carotovora MAFF301393) , it was examined enzyme inhibitory activity of the walk mycin A through C.
Measurement of histidine kinase activity was performed as described in Biosci. Biotechnol. Biochem. 64, 919-923, 2000.
A region containing only the kinase active domain of PehS (including amino acids 209 to 484 from the N-terminal) was prepared from the chromosomal DNA of the soft rot fungus MAFF301393 by the PCR method, and the expression vector pET-21a (+) was prepared. Cloned. A protein (PehS-209-484) in which only the PehS histidine kinase active domain was expressed was purified from the culture solution of the strain obtained by transforming the plasmid pET-pehScM2-2 thus obtained into E. coli.
The composition of the reaction solution for histidine kinase activity measurement, 4μM PehS-209-484,50mM Tris-HCl (pH8.5), 100mM KCl, a _{100mM NH 4 Cl, 5mM MgCl 2} , 2 in the reaction solution. 5 μM ATP-10 μCi [γ- ³² P] ATP mixed solution was added to make 10 μL, the reaction was started, incubated at 30 ° C. for 20 minutes, the reaction was terminated, and SDS-polyacrylamide electrophoresis was performed. When investigating the inhibitory activity, a predetermined concentration of walkomycin A to C was added to the reaction solution before adding the ATP mixed solution, incubated at 30 ° C. for 5 minutes, and the 50% inhibitory concentration (IC ₅₀₎ against soft rot fungus PehS. ) The results are shown in Table 9.

From the results in Table 9, it was found that walkomycins A to C have an inhibitory activity against histidine kinase possessed by gram positive bacteria and gram negative bacteria. It was found that walkomycins A to C have particularly strong inhibitory activity against YycG.
Moreover, it turned out that walkmycin A and C have the enzyme inhibitory activity superior to walkmycin B among walkmycin AC.

  Since the novel compounds (walkomycin A and walkmycin C) of the present invention have excellent antibacterial activity and excellent enzyme inhibitory activity, they can be suitably used as new antibacterial agents and enzyme activity inhibitors. Moreover, since the compound of the present invention (walkomycin B) has excellent enzyme inhibitory activity, it can be suitably used as a new enzyme activity inhibitor.

  NITE P-777

Claims (7)

Histidine kinase activity comprising at least one of a compound represented by the following structural formula (A), a compound represented by the following structural formula (C), and a compound represented by the following structural formula (B) Inhibitor.
A compound represented by the following structural formula (A):
A compound represented by the following structural formula (C):
A production method of at least one of a compound represented by the following structural formula (A) and a compound represented by the following structural formula (C),
A culture process for culturing a microorganism belonging to the genus Streptomyces and having the ability to produce at least one of a compound represented by the following structural formula (A) and a compound represented by the following structural formula (C) When,
Collecting a compound represented by the following structural formula (A) and at least one of the compound represented by the following structural formula (C) from the culture obtained in the culturing step,
A method for producing a compound, comprising adding at least one of a branched amino acid, a branched fatty acid, and a branched keto acid to the medium in the culturing step.
A microorganism belonging to the genus Streptomyces and having the ability to produce at least one of a compound represented by the following structural formula (A) and a compound represented by the following structural formula (C) is a deposit number NITE. The method for producing a compound according to claim 4, which is Streptomyces sp. MK632-100F11 strain of P-777.
A compound-containing composition comprising at least one of a compound represented by the following structural formula (A) and a compound represented by the following structural formula (C).
An antibacterial agent comprising at least one of a compound represented by the following structural formula (A) and a compound represented by the following structural formula (C).