JP2000239216A - New anthraquinone derivatives - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new anthraquinone derivative having an antibacterial activity to various bacteria and useful for medicine, especially, a researching nuclei of an antimicrobial agent and medicine. SOLUTION: The compound is expressed by formula I (R is H or OH), for example, the compound of formula II. The compound of formula I is obtained by culturing a strain belonging to genus Verticillium that produces the compound of formula I, in a nutrient medium, collecting from the cultured cell accumulating the compound of formula I in a conventional process. Any of the microorganisms having the capability producing the compound of formula I can be used. A Hyphomycetes Verticillium lecanii Q57371 strain (FERM P-17197) belonging to genus Verticillium separated from soil collected at Yaku island in Kagoshima Japan is enumerated as the microorganism.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to pharmaceuticals, in particular compounds having antibacterial activity, and to a process for producing said compounds by fermentation.

[0002]

2. Description of the Related Art Conventionally, various antibiotics produced by microorganisms include β-lactam antibiotics such as penicillin, cephalosporin and carbapenem, macrolide antibiotics such as erythromycin, josamycin and rokitamicin, kanamycin, gentamicin and tobramycin. Aminoglycoside antibiotics and the like are known.

[0003]

The object of the present invention is to provide a novel compound having an antibacterial activity, to provide a production method using a microorganism capable of producing the compound, and to provide a novel microorganism. Aim.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies on compounds produced by many naturally occurring microorganisms, and as a result, have produced a compound belonging to the genus Verticillium and having an antibacterial activity. A capable microorganism was found. Further, the present invention was completed by culturing the microorganism in a medium and isolating a novel compound represented by the following formula from the culture. That is, the present invention provides a Bianthraquinone derivative represented by the following general formula or a salt thereof,

[0005]

Embedded image (In the formula, R represents a hydrogen atom or a hydroxyl group.) The medicament comprising the above-mentioned bianthraquinone derivative or a salt thereof as an active ingredient, or the above-mentioned medicament which is an antibacterial agent.

[0006]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail. The bianthraquinone derivative of the present invention or a salt thereof is obtained by a conventional method from a culture in which the compound-producing bacterium belonging to the genus Verticillium is cultured in a nutrient medium and the compound is accumulated. Any microorganism that belongs to the genus Verticillium and has the ability to produce the compound can be used as the microorganism used in the method for producing the compound. Such microorganisms include, for example, an incomplete filamentous fungus belonging to the genus Verticillium Verticillium recanii isolated from soil collected on Yakushima, Kagoshima Prefecture.
( Verticillium lecanii ) Q57371 strain can be mentioned.

The mycological properties of the strain are as follows. 1. Properties in Various Media (1) Potato / Glucose Agar Medium After culturing at 24 ° C. for 14 days, the colonies become 39 mm in diameter. On the surface of the colonies, aerial hyphae develop and loosely rise in the form of wool, taking on a white to pale pink color. Phyaro-type conidia are well formed. The back of the colony becomes light brown. (2) Malt extract agar medium The colony becomes 49 mm in diameter after culturing at 24 ° C. for 14 days. On the surface of the colonies, aerial hyphae develop and loosely rise in the form of wool, taking on a white to pale pink color. Fiaro-type conidia are sparsely formed. The back of the colony changes from light yellow to orange. (3) Tzapek agar medium After culturing at 24 ° C. for 14 days, the colony becomes 39 mm in diameter. The aerial hyphae spread on the surface of the colony in a velvety manner, but the central part gently rises like wool. The color of the colony is yellow brown and the center is light pink. Fiaro-type conidia are formed very well. The back of the colony turns yellow-brown. (4) Sabouraud agar medium Colonies become 46 mm in diameter after culturing at 24 ° C. for 14 days. The colony surface is wool-like and has a white to pale pink color. Fiaro-type conidia are sparsely formed. The back of the colony changes from light yellow to orange.

[0008] 2. Physiological properties Growth temperature: grows in the range of 10 to 32 ° C, the optimal growth temperature is 20 to 25 ° C. 3. Morphological characteristics Strains grown on potato-glucose agar medium and Tzapek agar medium were observed under a microscope. The mycelium is colorless with partitions and the surface is smooth. No sexual generation is observed and forms a conidiophore on the aerial mycelium that extends in a creeping manner. The conidiophore is colorless and elongated, has partitions, and has a smooth surface.
On a Tzapek agar medium, phialides are formed in a two- or three-ring form, and on a potato / glucose agar medium, phialides are formed in a single or two- or three-round form. Phialides vary in length (10 to 50 × 1 to 2 μm), are elongate, taper progressively towards the tip, giving rise to phyallo-conidia at the tip, later forming conidial masses. Phialo-conidia are unicellular, colorless, with a smooth surface and rounded ends,
The size (2 to 7 × 1.5 to 2.5 μm) and the shape (sub-sphere, ellipse, column, etc.) vary widely. Chlamydospores are not observed.

Based on the above mycological properties, the taxonomic position of strain Q57371 was determined by The Genera of Funjay.
Spor Rating in Pure Culture 3rd Edition
(By JA von Arx; The Genera of Fungi Sporulating i
n Pure Culture, 3rd ed., 424, Cramer, Vaduz, 1981
Year issue). As a result, the strain was determined to belong to the incomplete fungus genus Verticillium. Further, when compared with known bacterial species according to the following literatures and the like, the properties of this strain were almost identical to those described in Verticillium lecanii. References ・ W. Gams; Cephalosporium-artige Schimmelpilze
(Hyphomycetes), p. 190, G. Fischer, Stuttgart, 1971
Published by KH Domsch, W. Gams and T.-H. Anderson; Com
pendium of SoilFungi, vol. 1, p. 859, Academic Pres
s, London, 1980 Issued Therefore, this strain Verticillium potassium Rekanyi (Vertic
illiumlecanii ) and designated as Verticillium lecanii Q57371.
This strain has been deposited at the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology under the accession number FERM P-17197. In addition, since microorganisms are liable to be mutated artificially or spontaneously, the Verticillium lecanii Q57371 strain used in the present invention is:
In addition to microorganisms isolated from nature,
Included are those artificially mutated by radiation, chemical agents, and the like, and natural mutants thereof.

(Production Method) In carrying out the production method of the compound of the present invention, the compound is inoculated into a medium containing a nutrient of the strain producing the compound Verticillium lecanii Q57371 and grown aerobically. As a result, a culture containing the novel target compound of the present invention can be obtained.
As nutrients, those known as nutrients for filamentous fungi may be used. For example, commercially available peptone, meat extract, corn steep liquor, cottonseed powder, peanut powder, soybean powder, yeast extract, NZ-amine, hydrolyzate of casein,
Inorganic or organic nitrogen sources such as fish meal, sodium nitrate and ammonium nitrate, commercially available molasses, starch, dextrin, sucrose, glucose, maltose, fructose,
Carbon sources such as carbohydrates such as xylose, rhamnose, mannitol and glycerin or fats can be used. As metal salts, sulfates, hydrochlorides, nitrates, phosphates, carbonates and the like of Na, K, Mg, Ca, Zn, Fe and the like are added as required. If necessary, valine, leucine, isoleucine, threonine, phenylalanine, tryptophan, methionine, lysine, arginine, cysteine, cystine, and other commonly known amino acids, methyl oleate, lard oil, silicone oil, interface An antibiotic compound-promoting compound such as an activator or an antifoaming agent is appropriately used. In addition to these, any one can be used as long as it is used by the producing bacteria and is useful for producing the novel antibiotic compound of the present invention. The culture may be performed in the same manner as a general antibiotic compound production method, and the culture method may be solid culture or liquid culture.

In the case of liquid culture, any of stationary culture, stirring culture, shaking culture and the like may be performed, but aeration and stirring culture is particularly preferable. The culture temperature is the temperature at which the producing bacteria grow and produce the compound of the present invention, that is, 15 ° C to 37 ° C.
Can be changed as appropriate, but approximately 24 ° C. is preferable.
The pH of the medium can be appropriately changed within the range of 4 to 9,
6 to 8 are preferred. The culture time varies depending on various conditions, and is about 1 to 30 days. In order to collect the target compound from the culture, the usual extraction, separation, and purification means used for metabolites produced by microorganisms are appropriately used. The target compound in the culture can be used as it is, or as a culture filtrate obtained by centrifugation or by adding a filter aid to the culture and filtering, and adding an organic compound that is immiscible with water such as ethyl acetate, chloroform, benzene, and toluene. Add solvent and extract. The target compound can be extracted by bringing the culture filtrate into contact with an appropriate carrier, adsorbing the target compound in the filtrate, and then eluting with a suitable solvent. More specifically, for example, Amberlite XAD-2, Diaion H
The target compound is adsorbed by bringing it into contact with a porous adsorption resin such as P20, Diaion CHP20P or Diaion SP900. Next, the target substance is eluted using a mixture of water and an organic solvent such as methanol, ethanol, acetone and acetonitrile. Needless to say, the mixing ratio of the solvent at this time is a value at which the target compound can be eluted most efficiently. That is, by increasing the solvent ratio stepwise or continuously from a low concentration to a high concentration, a fraction having a higher ratio of the target compound can be obtained. Next, the target compound-containing fraction obtained using each of the above-mentioned procedures is used for a conventional adsorption carrier such as activated carbon, alumina,
Pure separation and purification can be achieved by conventional methods such as column chromatography using silica gel, cellulose, etc. as a carrier, high performance liquid chromatography using a silica gel type ODS reversed phase carrier column, or centrifugal liquid distribution chromatography. it can.

The compound of the present invention can form a salt with an acid. Suitable salts include mineral salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate and the like, formate, acetate, propionate, oxalate, malonic acid. Salt, succinate, fumarate, maleate, lactate, malate, citrate, tartrate, carbonate, picrate, methanesulfonate, ethanesulfonate,
Organic acid salts such as glutamate can be mentioned.

Hereinafter, the method of formulating and administering the compound of the present invention will be described in detail. Pharmaceutical compositions containing one or more of the compound of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient may be used in combination with commonly used carriers, excipients and other additives for pharmaceuticals. Tablets, powders, fine granules, granules,
It is prepared into capsules, pills, solutions, injections, suppositories, ointments, patches, etc., and is administered orally or parenterally. The clinical dose of the compound of the present invention for humans is appropriately determined in consideration of the patient's symptoms, weight, age, sex, and the like.
As the solid composition for oral administration according to the present invention, tablets, powders, granules and the like are used. In such solid compositions, the one or more active compounds comprise at least one inert diluent, such as lactose, mannitol, glucose, hydroxypropylcellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, metasilicate. It is mixed with magnesium aluminate. The composition is
In a conventional manner, additives other than inert diluents, such as lubricants such as magnesium stearate, disintegrants such as calcium cellulose glycolate, stabilizers such as lactose, and solubilizing agents are contained. You may. Tablets or pills may be coated with sugar or a film of a gastric or enteric compound such as sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, if necessary.

Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs and the like, and commonly used inert diluents, For example, it contains purified water and ethyl alcohol. The composition may contain, in addition to the inert diluent, auxiliary agents such as solubilizing agents, wetting agents, suspending agents, sweetening agents, flavoring agents, fragrances, and preservatives. Injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Diluents for aqueous solutions and suspensions include, for example, distilled water for injections and physiological saline. Examples of diluents for water-insoluble solutions and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethyl alcohol, and polysorbate 80 (trade name). Such compositions may further include additives such as tonicity agents, preservatives, wetting agents, emulsifiers, dispersants, stabilizers (eg, lactose), solubilizing agents. These are sterilized by, for example, filtration through a bacteria retaining filter, blending of a bactericide or irradiation. They can also be used by preparing a sterile solid composition and dissolving in sterile water or a sterile injection solvent before use. When the solubility of the compound of the present invention is low, a solubilization treatment may be performed. As a solubilization treatment, known methods applicable to pharmaceutical preparations, for example, surfactants (polyoxyethylene hydrogenated castor oils, polyoxyethylene sorbitan higher fatty acid esters, polyoxyethylene polyoxypropylene glycols, sucrose fatty acid esters) ), A drug and a solubilizer, for example, a polymer (a water-soluble polymer such as hydroxypropylmethylcellulose (HPMC), polyvinylpyrrolidone (PVP), or polyethylene glycol (PEG)), carboxymethylethylcellulose (CMEC), or hydroxy. Propyl methyl cellulose phthalate (HPMC
P), an enteric polymer such as methyl methacrylate-methacrylic acid copolymer (Eudragit L, S, trade name; manufactured by Rohm and Haas Co.) or the like. . If necessary, a method of forming a soluble salt, a method of forming an inclusion compound using cyclodextrin, or the like can also be employed. The means of solubilization can be appropriately changed depending on the target drug ["Recent formulation technology and its application I", Isamu Utsumi, Pharmaceutical Journal 157-1
59 (1983) and “Pharmaceutical Monograph No. 1, Bioavailability”, Tsuneji Nagai et al., Soft Science, 78
-82] (1988)]. Of these, preferably
A method of forming a solid dispersion of a drug and a solubilizer to improve solubility is adopted (Japanese Patent Application Laid-Open No. 56-49314, F.
R2460667).

[0015]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited thereto. Example 1 500 ml of a medium (pH 7.0) containing 10 g of glucose, 20 g of potato starch, 5 g of polypeptone, 5 g of yeast extract, 4 g of calcium carbonate, and 1 L of distilled water in 500 ml portions.
Dispense into a conical flask with a navel volume of
Sterilized for 0 minutes. Verticillium (Verticillium) grown well on potato dextrose agar
lecanii) was scraped and inoculated, and cultured with shaking at 24 ° C. and 200 rpm for 4 days. Medium 4 prepared similarly
The above culture solution was inoculated into the book at a rate of 3%, and the cells were inoculated at 24 ° C. and 200%.
Shaking culture was performed for 3 days under the condition of rotation / min to obtain a seed culture solution. Next, as a production medium, 30 g of oatmeal and 100 ml of VF mixed juice were placed in a 500 ml Erlenmeyer flask.
100 medium containing 100 ml were prepared and sterilized at 120 ° C. for 20 minutes. This medium is inoculated with a seed culture at a rate of 3%,
Stationary culture was performed at 24 ° C. for 14 days.

To 10 L of the culture thus cultured, 20 L of 80% acetone water was added and stirred for 5 hours. Thereafter, filtration was performed, and acetone was distilled off from the filtrate under reduced pressure. This concentrate (p
H7) was extracted twice with 10 L of ethyl acetate. The ethyl acetate extract was concentrated under reduced pressure to 300 ml. 200 ml of 5% aqueous sodium hydrogen carbonate was added to the ethyl acetate solution and shaken. Re-apply p to the separated ethyl acetate layer
Add 200 ml of H2.0 water and shake. The separated ethyl acetate layer was concentrated to dryness to obtain a crude extract. This crude extract is used as ODS-7515-12A (manufactured by Senshu Kagaku).
The mixture was subjected to flash chromatography using, and eluted with methanol to obtain an active fraction. The methanol fraction was concentrated to dryness, and a small amount of methanol was added to obtain 31 mg of an active fraction which precipitated. Finally, L-column ODS
(Manufactured by Japan Chemical Inspection Association) Using a column of 20φx250mm, tetrahydrofuran: acetonitrile: 0.02M
Purification was performed by HPLC using a mixed solution of sodium phosphate monobasic (10:60:20), and 9.7 mg of the active fraction YM187781 compound eluted at 18.2 minutes and 1
Active fraction YM187787 compound eluted at 9.9 minutes 7.
2 mg was isolated. Liquid chromatography (HPL
The conditions of C) are as follows.

Column: L-column ODS (manufactured by Japan Chemicals Inspection Association, 20φ × 250 mm) Elution solvent: tetrahydrofuran: acetonitrile: 0.1.
Mixed solution of 02M sodium phosphate (10:60:20) Outflow rate: 5 ml / min Detection wavelength: 210 nm The YM187781 compound extracted, separated and purified has the following physicochemical properties.

(1) Color and shape: red powder. (2) Classification of acidic, neutral and basic: neutral. (3) Solubility: soluble in dimethyl sulfoxide but in water
Hardly melts. (4) UV absorption spectrum: 202, 227, 26
The absorption maximum is shown at 1,299,501 nm, as shown in FIG.
(Solvent: methanol). (5) Molecular weight: 586.47 (6) Mass spectrum (FAB-Mass): 587 [M
+ H]⁺, 585 [MH]^-  (7) Molecular formula: C₃₀H₁₈O₁₃  (8) The infrared absorption spectrum (KBr) is as shown in FIG.
is there. (9)¹H-NMR spectrum (500 MHz, CDC
l_Three): YM187781 compound ¹The H-NMR spectrum is the third
As shown in the figure. (10)¹³C-NMR spectrum (125 MHz, CD
Cl_Three): YM187781 compound¹³The C-NMR spectrum is
As shown in FIG. From the above physicochemical properties, the chemical structural formula of YM187781 Compound
Was determined as follows.

[0019]

Embedded image

The above extracted, separated and purified YM187787 compound
The product has the following physicochemical properties: (1) Color and shape: red powder. (2) Classification of acidic, neutral and basic: neutral. (3) Solubility: soluble in dimethyl sulfoxide but in water
Hardly melts. (4) UV absorption spectrum: 201, 224, 26
It shows an absorption maximum at 4,297,467 nm, as shown in FIG.
(Solvent: methanol). (5) Molecular weight: 570.47 (6) Mass spectrum (FAB-Mass): 571 [M
+ H]⁺, 569 [MH]^-  (7) Molecular formula: C₃₀H₁₈O₁₂  (8) The infrared absorption spectrum (KBr) is as shown in FIG.
is there. (9) 1H-NMR spectrum (500 MHz, CDC
l_Three): Of YM187787 compound ¹The H-NMR spectrum is the seventh.
As shown in the figure. (10)¹³C-NMR spectrum (125 MHz, CD
Cl_Three): Of YM187787 compound¹³The C-NMR spectrum is
As shown in FIG. From the above physicochemical properties, the chemical structural formula of YM187787 Compound
Was determined as follows.

[0021]

Embedded image

Example 2 Antibacterial test example: The compound of the present invention was added to dimethyl sulfoxide in 1
After dissolving in a concentration of mg / ml, 20 μl of the solution was soaked in an antibacterial measurement disk (diameter 8 mm), and the antibacterial activity (blocking diameter: mm) was measured by the so-called paper disk method. As a result, against Staphyrococcus aureus FDA209P, YM187781 showed an inhibition circle of 9.0 mm and YM187787 showed an inhibition circle of 10.5 mm. In addition, Bacillus subtilis ATCC6633, YM1
The 87787 exhibited a stopping circle of 9.0 mm.

[0023]

INDUSTRIAL APPLICABILITY The compound of the present invention has activity against various bacteria, and is useful as a core of pharmaceuticals, especially antibacterial agents and drug search.

[Brief description of the drawings]

FIG. 1 is an ultraviolet absorption spectrum of a YM187781 compound.

FIG. 2 is an infrared absorption spectrum of a YM187781 compound.

FIG. 3 is a ¹ H-NMR spectrum of a YM187781 compound.

FIG. 4 is a ¹³ C-NMR spectrum of a YM187781 compound.

FIG. 5 is an ultraviolet absorption spectrum of a YM187787 compound.

FIG. 6 is an infrared absorption spectrum of a YM187787 compound.

FIG. 7 is a ¹ H-NMR spectrum of a YM187787 compound.

FIG. 8 is a ¹³ C-NMR spectrum of a YM187787 compound.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court (Reference) C12R 1: 045) (72) Inventor Koji Nagai 1-1-8 Shozuzawa, Itabashi-ku, Tokyo Yamanouchi Pharmaceutical Co., Ltd. (72) Inventor Nami Niimura 21 Miyukigaoka, Tsukuba City, Ibaraki Prefecture Yamanouchi Pharmaceutical Company Limited (72) Inventor Akito Yamaguchi 8-1, Mihogaoka, Ibaraki City, Osaka Prefecture F-term, Osaka University 4B064 AD94 BA06 BD02 BE19 BG04 BG09 BH02 BH04 BH05 BH06 BH07 CA05 DA02 4C206 AA01 AA02 AA03 CB29 KA05 MA01 MA04 NA14 ZB35 4H006 AA01 AA03 AB29

Claims (3)

[Claims] [Claim 1] The following general formula: (Wherein R represents a hydrogen atom or a hydroxyl group) or a salt thereof. 2. A medicament comprising the bianthraquinone derivative according to claim 1 or a salt thereof as an active ingredient. 3. The medicament according to claim 2, which is an antibacterial agent.