JPH0539265A - New substance, wk-2,955, and its production - Google Patents

Abstract

PURPOSE:To obtain a new compound useful as a coccidiostat, having growth inhibiting activity against coccidium protozoan resistant to polyether-based antibiotic monensin. CONSTITUTION:New substance, WK-2,955, shown by the formula. Streptomyces sp. WK-2,955 strain (FERM P-12,318) belonging to the genus Streptomyces is cultured in a medium under an aerobic condition and the culture mixture is extracted with an organic solvent, etc., to give the objective compound. Or, 3-indoleacetaldehyde and p-hydroxyphenylacetaldehyde are subjected to alodol addition reaction to give the compound shown by the formula. Properties of the compound shown by the formula. White solid substance. Molecular formula: C18H19NO3 (measured value: 297.1363). Soluble in methanol, ethanol, acetonitrile, dimethyl sulfoxide, etc., and insoluble in chloroform, hexance, etc.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel substance WK-2955 useful as a therapeutic agent and a preventive agent for coccidiosis, which is an important disease in the poultry industry, and a pharmaceutically acceptable salt thereof, and the production of the above substances. Concerning the law.

[0002]

2. Description of the Related Art Although coccidium is widely distributed in various livestock and poultry, the animal most affected by coccidium is chicken. As a therapeutic agent and a preventive agent for chicken coccidiosis, for example, polyether antibiotics such as monensin and salinomycin, and nicarbazine which is a synthetic agent have been known.

[0003]

However, coccidium resistant to these drugs has appeared and its effect is weakened, and a new anticoccidial agent to replace them is desired.

[0004]

In general, drug cross-resistance is established when the structures are similar or the mechanism of action is similar, and compounds that differ structurally or mechanism of action from conventional coccidiologic agents are It is useful as a therapeutic or preventive agent for coccidiosis.

The present inventors have continued various studies in order to find out from the natural world a new drug that is effective against coccidial parasites that have acquired resistance to conventional drugs. Using the monensin-resistant coccidial protozoa as a test organism, the production of microorganisms is continued. As a result of a search among the metabolites that are present, it was found that a substance effective against coccidium is produced in the culture solution of the WK-2955 strain newly isolated from the soil in Shibuya-ku, Tokyo.

Next, when the anticoccidial active substance was separated and purified from the culture, the substance having the physicochemical properties described below was not known at all in the past. ] [1- (p-hydroxyphenyl) -4- (3-indolyl) butane-]
2,3-diol] and the substance WK-2
I decided to call it 955.

The present invention has been completed based on the above findings, and is a substance WK-2955 represented by the formula [I].
Is provided.

[Chemical 1]

Further, the novel substance WK-2955 of the present invention
Is a microorganism belonging to the genus Streptomyces and having the ability to produce the substance WK-2955 represented by the formula [I], is cultured in a medium, the substance WK-2955 is produced and accumulated in the culture, and this is collected. It is manufactured by the method.

The physicochemical properties of this substance WK-2955 are as follows. (1) White solid substance (2) Molecular weight and molecular formula A molecular ion peak was observed at m / z 297 on the mass spectrum of the substance WK-2955, and the molecular formula C ₁₈ H ₁₉ NO ₃ (actual value: 297. Thirteen
63, calculated value: 297.1364).

(3) Ultraviolet absorption spectrum As shown in FIG. 1, neutral 225 nm, 280 n
The absorption maximum characteristic of m is shown.

(4) Nuclear magnetic resonance spectrum The ¹ H-NMR spectrum measured in a heavy DMSO (dimethyl sulfoxide) solution using Varian XL-400, 400 MHz, and NMR spectrometer, respectively, is as shown in FIG. .. ¹³ C-NMR measured in a heavy methanol solution is as shown in FIG.

(5) Color reaction Sulfuric acid positive Vanillin sulfate positive Phosphomolybdic acid positive Ninhydrin negative

(6) Solubility in a solvent is soluble in methanol, ethanol, acetonitrile, dimethyl sulfoxide, etc., but insoluble in chloroform, hexane, etc.

The structure represented by the above formula [I] was determined as a result of comparing the above physicochemical properties with those of known similar compounds.

As a strain used for producing the substance WK-2955, for example, Streptomyces sp. WK-2955 newly isolated from the soil of Shibuya-ku, Tokyo by the present inventors. Stocks. The mycological properties of this strain are as follows.

(I) Morphological Properties The vegetative mycelium develops well on various agar media, and no fragmentation is observed. Aerial aerial hyphae are abundantly settled on starch inorganic salt agar medium, yeast extract, malt extract agar, etc., and show pink color.

Further, when observed under a microscope, the aerial hyphae are linear or wavy, and a chain of 20 or more spores is observed. The size of the spores is 1.0 × 0.6 μm and is columnar. The surface of spores is smooth. No sclerotia, sporangia and zoospores are found.

(II) Properties on various media EB Shirrin
g) and the method of D. Gottlieb (International Journal of Cysticatic Bacteriology, 16: 313,
The following table shows the culture properties of the presently-produced bacteria, which were examined by the method of 1966).

The color tone is determined by using the Color Harmony Manual Fourth Edition (Container Corporation of America Chicago, 1958) as a standard color, and the code is put in parentheses together with the name of the color chart. I wrote it.

(III) Various properties on culture (1) Table 1, Table 2 and Table 3 show the culture findings of this strain. This finding is the result of macroscopic observation when cultured at 27 ° C. for 2 weeks on various media.

[0021]

[Table 1]

[0022]

[Table 2]

[0023]

[Table 3]

(IV) Physiological properties (1) Formation of melanin pigment (a) Tyrosine agar
Negative (b) Peptone yeast iron agar
Negative (c) Glucose / peptone / gelatin medium (21
-23 ℃) Negative (d) Tryptone / Yeast solution
negative

(2) Tyrosinase reaction
Negative (3) Hydrogen sulfide production
Negative (4) Reduction of nitrate
Negative (5) Liquefaction of gelatin (21-23 ℃)
Negative (glucose / peptone / gelatin medium)

(6) Hydrolysis of starch
Positive (7) Coagulation of skim milk (36-37 ° C)
Negative (8) Peptonization of skim milk (36-37 ° C)
Negative (9) Growth temperature range
15-37 ° C

(10) Utilization of carbon source (Pleedam-Gotrieve agar medium) Use: D-glucose, D-fructose, L-rhamnose, D-mannitol, L-arabinose, i-inositol, raffinose, D-xylose , Meribiose Not used: Sucrose (11) Cellulose decomposition Negative

(V) Cell Wall Composition The cell wall diaminopimelic acid is of the LL type.

The bacteriological characteristics of this bacterium are summarized as follows. The cell wall diaminopimelic acid is of the LL type. The aerial hyphae are linear or wavy and form long spore chains. The surface of spores is smooth. As various properties of the culture, the vegetative mycelium has a pink or wine color tone and the aerial mycelium has a pink color tone. It does not produce soluble pigment.

From these results, this strain is a strain belonging to the genus Streptomyces, and is classified into Pridham and Tresner (Virgin's Manual of Determinating Bacteriology, 8th Edition, pp. 748-829, 19).
1974) and is considered to be a bacterial species belonging to the white or red series.

Morphological characteristics of the above WK-2955 strain,
As a result of attempting comparison with known bacterial species on the basis of various properties in culture and physiological properties, this strain was identified as a strain belonging to the genus Streptomyces, and Streptomyces sp. WK-
It was named 2955. This strain is Streptomyces sp. WK-2955 (Streptomycess)
p. WK-2955) has been deposited with the Institute of Microbial Technology, National Institute of Advanced Industrial Science and Technology. (FERM P-12318
issue).

In the present invention, first, a microorganism belonging to the genus Streptomyces and having the ability to produce the substance WK-2955 [I] is cultured in an appropriate medium. Examples of microorganisms used for culture include the above-mentioned strains, and mutants thereof can be used in the present invention as long as they have the ability to produce the substance WK-2955. Other substances WK-2
Strains belonging to the genus Streptomyces having a production capacity of 955 can also be used.

The medium used in the present invention includes:
It is possible to use a synthetic medium or a natural medium that appropriately contains a carbon source, a nitrogen source, and an inorganic substance suitable for culturing ordinary actinomycetes, and optionally other nutrients. The carbon source and nitrogen source used in the medium may be any type as long as the strain used can be used.

That is, as the carbon source, various carbohydrates such as glucose, glycerol, fructose, maltose, mannitol, xylose, galactose, ribose, starch or hydrolysates thereof can be used.

The concentration is usually 0.1% to the medium.
5% is desirable. Further, various organic acids such as gluconic acid, pyruvic acid, lactic acid and acetic acid, various amino acids such as glycine, glutamic acid and alanine, further alcohols such as methanol and ethanol and non-aromatic hydrocarbons such as normal paraffin, or plants. Alternatively, various animal fats and oils can be used.

As the nitrogen source, for example, ammonium salts of various inorganic or organic acids such as ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium nitrate, urea, peptone, NZ-amine, meat extract, yeast extract, Dry yeast, corn steep liquor, casein hydrolyzate, fish meal or its digest, soybean powder or its digest, defatted soybean or its digest, nitrogen-containing organic substances such as hydrolysates, further glycine, glutamic acid, Various amino acids such as alanine can be used.

As the inorganic substance, for example, various phosphates, magnesium sulfate, sodium chloride, and trace amounts of heavy metal salts are used. In addition, when using a mutant strain showing an auxotrophy, it is naturally necessary to add a substance satisfying the auxotrophy to the medium, but this kind of nutrient is, when using a medium containing a natural product, In some cases, no addition is required.

Culturing is usually carried out under aerobic conditions such as shaking or aeration stirring culture. Industrially, deep aeration stirring culture is preferred. The pH of the medium is, for example, 5.0 to
Although it is 8.0, it is preferable to carry out the culture at around neutrality.

The culturing temperature may be, for example, 20 to 40 ° C., but it is usually preferable to keep it at 26 to 32 ° C. (preferably around 27 ° C.). The culture time is usually 3 to 6 in the case of liquid culture.
The cells may be cultured for a day, and when the amount of WK-2955 accumulated in the culture reaches the maximum, the culture may be terminated.

The culture conditions such as the composition of the medium, the liquidity of the medium, the culture temperature, the stirring speed, and the aeration rate are appropriately adjusted depending on the kind of the strain to be used, external conditions, etc. so as to obtain preferable results. To be selected. When there is foaming in the liquid culture, an antifoaming agent such as silicon oil, vegetable oil, or a surfactant can be appropriately used.

The substance WK-2955 substance of the present invention accumulated in the culture thus obtained is usually produced in the culture filtrate. To collect the substance WK-2955 from the culture filtrate, the means usually used for collecting the metabolites from the culture of the microorganism can be used alone or in any combination or repeatedly.

That is, for example, filtration, centrifugation, dialysis,
Concentration, drying, freezing, adsorption, desorption, utilizing the difference in solubility in various solvents such as precipitation, crystallization, recrystallization, phase transfer,
Countercurrent distribution method, means such as chromatography, etc. are used.

To collect the substance WK-2955 from the culture broth, the culture broth is extracted with a non-hydrophilic organic solvent such as ethyl acetate, or the culture filtrate or the microbial cell extract is treated with activated carbon, alumina or a porous synthetic solution. It may be adsorbed on a molecular resin, an ion exchange resin or the like, eluted with an elution solvent such as ethyl acetate, and the obtained extract or eluate may be concentrated under reduced pressure and then extracted with an organic solvent such as hexane.

The obtained crude substance can be further purified by a known method usually used for purification of fat-soluble substances, for example, column chromatography using a carrier such as silica gel or alumina.

As the pharmaceutically acceptable salt of this substance,
For example, hydrochlorides, sulfates, nitrates, phosphates, carbonates, acetates, tartrates, citrates, succinates, glutamate, aspartates and the like can be produced by a conventional method. Since this type of manufacturing method is well known, its description is omitted.

Further, this substance WK-2955 can be produced by organic synthesis. For example, as an example thereof, as illustrated in Examples, 3-indole ethanol is used as a raw material, and 3-indole acetaldehyde obtained by oxidizing it is reacted with p-hydroxyphenylacetaldehyde by a pinacol reaction, and then the reaction product is The desired product can be obtained by purification.

The reaction conditions are as follows: After mixing mercury chloride and magnesium in a tetrahydrofuran (THF) solution, titanium chloride is added, then 3-indoleacetaldehyde and p-hydroxyphenylacetaldehyde are added, and the mixture is stirred at 0 ° C. for 15 minutes. By doing, WK-2
955 is synthesized.

The anticoccidial activity of the substance WK-2955 described above against chickens is as follows.

Chicken Coccidiosis Growth Inhibitory Activity Using a chicken coccidium and Eimeria tenella oocysts resistant to monensin, the growth inhibitory action on the host hamster kidney-derived cells (BHK-21 cells) was examined by the method of Ohnaga et al. , Ishii: Japan Veterinary Association Magazine, 31,592-
596 (1978)), the concentration of the growth inhibitory action of this substance WK-2955 is 0.2 to 0.0.
It was 2 μg / ml.

The substance WK-2955 is a concentration that shows the growth inhibitory activity of Eimeria tenella on hamster kidney-derived cells (BHK-21 cells) which are host cells,
No cytotoxicity was observed at 0.2 to 0.02 μg / ml.

On the other hand, the same administration experiment was carried out for monensin which is a known chicken coccidial growth inhibitor. as a result,
No coccidial growth inhibitory activity was observed with monensin, and toxicity was observed with host cells at 0.02 μg / ml administration. Therefore, it can be said that this substance WK-2955 is less toxic than the known chicken coccidiosis growth inhibitor monensin.

[0052]

As described above, the substance of the present invention WK-295
Since 5 has a growth inhibitory activity against coccidial parasites resistant to the polyether antibiotic monensin, it is possible to lead to remission of coccidiosis. It can also be used as a concomitant drug that remarkably prolongs the effect of a known anticoccidial drug.

Example 1 In a 500 ml Erlenmeyer flask, glucose 0.1%, peptone 0.3%, yeast extract 0.5%, meat extract 0.3
%, Starch 2.4%, calcium carbonate 0.4%, and 100 ml of a liquid medium (pH 7.0) was dispensed and steam sterilized at 121 ° C. for 15 minutes. These were inoculated with WK-2955 cells grown on an agar plate with a platinum loop and 2
Seed mother was obtained by shaking culture at 7 ° C. for 3 days.

Next, 30 l jar fermenter 1
20 l of liquid culture medium (pH 7.0) containing 2.0% kinako, 2.0% glycerol and 0.3% sodium chloride
Was sterilized and steam sterilized at 121 ° C. for 15 minutes. The above two seeds were transplanted into each of them, and the stirring speed was 200 rp.
m, aeration rate of 15 l / min, 27 ° C, 96 hours,
The culture was performed with aeration and stirring.

Approximately 20 l of this culture broth was mixed with ethyl acetate (20 l
) Was added and stirred, and then the suspension was centrifuged (10,000 rpm) with a Sharpless centrifuge to separate into an ethyl acetate extract layer, an aqueous layer and a cell residue. About 20 l of the obtained ethyl acetate extraction layer was concentrated under reduced pressure, and then hexane (400 ml), methanol (190 ml) and water (10 ml) were added.
ml) was added and stirred, and then the suspension was separated with a separating funnel to separate into a hexane layer, a methanol layer and an aqueous layer.

About 200 ml of the obtained methanol / water layer was concentrated to dryness under reduced pressure to obtain 6.44 g of a dry solid. Silica gel chromatography (Merck, Art. 9)
385) and chloroform: methanol (20:
Elution with 1) gave 270 mg of crude active material.

Then, gel filtration using Sephadex LH-20 (Pharmacia) as a carrier was carried out and eluted with methanol to obtain 18.0 mg of a crude active substance. Further, the obtained crude active substance was fractionated by high performance liquid column chromatography.

The apparatus used was a Torirotor V (manufactured by JASCO Corporation), and the column was YMC-Pack A-343.
(ODS resin, manufactured by Yamamura Chemical Laboratory), elution solvent is 40
% Methanol, 60% water, UV280 nm was used for detection, and the flow rate was 6 ml / min. Concentrate the active portion to dryness, 15.0
9 mg of colorless oily substance WK-2955 was obtained.

Example 2 Synthesis of substance WK-2955 starting from indole Synthesis of 3-indole acetaldehyde [2] 3-indole ethanol [1] (1.0 g) was added to benzene (20 ml) and dimethyl sulfoxide (DMSO). )
It was dissolved in (20 ml), pyridine (0.5 ml), triflurooloacetic acid (0.25 ml) and dicyclocarbodiimide (DCC) (3.83 g) were added, and the mixture was stirred at room temperature for 18 hours under an argon atmosphere.

3-indole ethanol [1]
Is synthesized from indole by a conventional method. Benzene (60 ml) was added to the reaction solution, and the resulting precipitate was filtered.
The filtrate was washed with water (60 ml × 2 times), dried over anhydrous sodium sulfate, and concentrated to dryness. The residue is subjected to silica gel column chromatography (developing solvent; hexane: ethyl acetate =
The product was purified by 10: 1) to obtain 630 mg of 3-indoleacetaldehyde [2] represented by the following reaction formula. Yield 6
4%.

[0061]

[Chemical 2]

Synthesis of p-hydroxyphenylacetaldehyde [4] p-hydroxyphenethyl alcohol [3] (1.0
g) to dicyclocarbodiimide (DMSO) (15 ml)
And triethylamine (6.6 ml) was added,
Sulfur trioxide / pyridine complex (3.45 g) dissolved in DMSO (15 ml) was slowly added dropwise.

After 20 minutes, the reaction solution was poured into water (50 ml) and extracted with ethyl acetate (50 ml × 3 times). The ethyl acetate layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness. The residue is subjected to silica gel column chromatography (developing solvent; hexane: ethyl acetate =
8: 1) to obtain p-hydroxyphenylacetaldehyde [4] (260 mg) represented by the following reaction formula. Yield 26%.

[0064]

[Chemical 3]

1- (p-hydroxyphenyl) -4-
(3-Indolyl) butane-2,3-diol (Substance W
Synthesis of K-2955) Mercury chloride (60 mg) in tetrahydrofuran (THF) solution (2 ml) was added with 70-80 mesh magnesium (1
92 mg) and the resulting mixture was stirred at room temperature for 1 hour in argon.
Stir for 5 minutes. The supernatant of the turbid mixture was taken with a syringe, the remaining amalgam was washed three times with a THF solution, a THF solution (5 ml) was added, the mixture was cooled to -10 ° C, and 438 µl of titanium (IV) chloride was added dropwise. ..

The reactor wall of the reaction flask containing these was TH
After washing with F solution (2 ml), 3-indoleacetaldehyde (159 mg) and p-hydroxyphenylacetaldehyde (408 mg) in THF (4 ml) were added. The purple mixture thus obtained was stirred at 0 ° C. for 1.5 hours, then treated with 0.3 ml of saturated aqueous potassium carbonate solution and stirred at 0 ° C. for a further 15 minutes.

Diethyl ether was added to this mixture and the mixture was filtered through Celite. The filtrate was washed with saturated aqueous sodium chloride solution and concentrated under reduced pressure. The residue is subjected to silica gel column chromatography (developing solvent; chloroform:
The product was purified with methanol = 20: 1) to obtain 30 mg of the target substance WK-2955 of the present invention represented by the following reaction formula. Yield 10%.

[0068]

[Chemical 4]

Example 3 The effect on the inhibition of chicken coccidium growth was measured according to the method of Onaga et al., Using an animal cell as a host and a coccidium protozoa growing in the cell. As chicken coccidia, Eimeria tenella oocysts resistant to monensin were used.

This oocyst is a threshing device (SH
INAGAWA), chicken bile and trypsin were added, and the mixture was reacted at 41 ° C. for 2 hours. The reaction products were separated by centrifugation to obtain sporozoids.

Further, hamster kidney-derived cells (BHK-21 cells) were grown in a 96-well microplate (manufactured by Corning) so as to form a full sheet, and sporozoids already prepared were added together with a methanol solution of the substance WK-2955. The cells were cultured at 41 ° C. for 3 days in an incubator (manufactured by Hirasawa Seisakusho) under aeration of carbon dioxide.

After 3 days, the cells were fixed with methanol, stained with hematoxylin or the like, and observed with a light microscope for the presence or absence of schizonds, which is one stage of the coccidial growth process. That is, those in which sporozoites had grown to schizonds were determined to be negative for coccidial parasite growth inhibitory activity, that is, anticoccidial activity, those in which schizonds could not be evaluated as positive, and those in which BHK-21 cells died were toxic. And

This substance WK-2955 contains 0.2 to 0.0
At a concentration of 2 μg / ml, the coccidial parasite growth inhibitory activity was positive. No cytotoxicity was observed for host cells (BHK-21) at this concentration. When a similar administration test was carried out using monensin as a control, toxicity of the host cells was observed at a concentration of 0.02 μg / ml of monensin, and the coccidial parasite growth inhibitory activity was negative.

[Brief description of drawings]

FIG. 1 is an ultraviolet absorption spectrum of this substance WK-2955 measured with a methanol solution.

FIG. 2 is a proton nuclear magnetic resonance spectrum of the substance WK-2955 measured in a heavy dimethyl sulfoxide solution.

FIG. 3 is a ¹³ C nuclear magnetic resonance spectrum of the substance WK-2955 measured in a deuterated methanol solution.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Yoko Takahashi, 5-9-1, Shirokane, Minato-ku, Tokyo Within the Kitasato Research Institute (corporation corporation) (72) Toshiaki Sunazuka 5--9-1, Shirokane, Minato-ku, Tokyo Kitasato Research Institute (corporate association)

Claims (4)

[Claims] 1. The following formula: The novel substance WK-2955 represented by or a pharmaceutically acceptable salt thereof. 2. A microorganism belonging to the genus Streptomyces and having an ability to produce the substance WK-2955 represented by the formula: is cultivated in a medium, and the substance WK-2955 [I] is added to the culture. A method for producing a novel substance WK-2955 characterized by producing and accumulating and collecting this. 3. The microorganism is Streptomyces sp.
The method for producing the novel substance WK-2955 according to claim 2, which is WK-2955 (FERM P-12318). 4. 3-indole acetaldehyde and p-
A process for producing a substance WK-2955 represented by the following formula: or a pharmaceutically acceptable salt thereof, which comprises reacting hydroxyphenylacetaldehyde with a pinacol.