JP2971204B2 - New substance WK-2955 and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel substance WK-2955, which is useful as a therapeutic or prophylactic agent for coccidiosis, which is an important disease in the poultry industry, a pharmaceutically acceptable salt thereof, and production of the above substance. About the law.

[0002]

2. Description of the Related Art Coccidium is widely distributed in various livestock and poultry, but chickens are the animals most frequently affected by coccidia. As therapeutic and preventive agents for chicken coccidiosis, for example, polyether antibiotics such as monensin and salinomycin, and niacarbazine as a synthetic agent have been known.

[0003]

However, the emergence of coccidium resistant to these drugs and the effects thereof are weakening, and new anticoccidial agents replacing them are demanded.

[0004]

In general, the cross-resistance of a drug is established when the structure is similar or the mechanism of action is similar, and a compound having a different structure or action from a conventional coccidiostat is not available. It is useful as a therapeutic or prophylactic agent for coccidiosis.

[0005] The present inventors have continued various studies to find a new drug effective in nature against coccidial protozoa that has acquired resistance to conventional drugs, and produced a microorganism using monensin-resistant coccidial protozoa as a test organism. As a result of searching for metabolites, it was found that a substance effective for coccidium was produced in a culture solution of the WK-2955 strain newly isolated from the soil in Shibuya-ku, Tokyo.

[0006] Next, the anticoccidial active substance was separated and purified from the culture, and since the substance having the following physicochemical properties has not been known at all, this substance is a novel substance and has the formula [I ]-(1- (p-hydroxyphenyl) -4- (3-indolyl) butane-
2,3-diol] and the substance WK-2
955.

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

Embedded image

Further, the novel substance of the present invention WK-2955
Is a microorganism belonging to the genus Streptomyces and capable of producing 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 collected. It is manufactured by the following method.

The physicochemical properties of the 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 measurement: 297. 13
63, calculated: 297.1364).

(3) Ultraviolet absorption spectrum As shown in FIG.
m shows a characteristic absorption maximum.

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

(5) Color reaction Sulfate-positive Vanillin-sulfate-positive Phosmomolybdate-positive Ninhydrin-negative

(6) The solubility in a solvent is soluble in methanol, ethanol, acetonitrile, dimethyl sulfoxide and the like, and insoluble in chloroform, hexane and the like.

The structure represented by the above formula [I] is determined by comparing the above physicochemical properties with those of a known analogous compound.

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

(I) Morphological properties Vegetative mycelia develop well on various agar media, and no fragmentation is observed. Aerial hyphae grow abundantly on a starch inorganic salt agar medium, yeast extract, malt extract agar, etc., and exhibit a pink system.

[0017] In the observation 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 cylindrical. The spore surface is smooth. Sclerotia, sporangia and zoospores are not found.

(II) Properties on Various Media E. B. Shearing (EB Shirlin)
g) and the method of D. Gottlieb (International Journal of Sistimatic Bacteriology, 16, 313,
The following Table 1 shows the culture characteristics of the microorganism produced according to the present invention.

The color tone is determined by using the Color Harmony Manual 4th edition (Container Corporation of America, Chicago, 1958) as a standard color, and the code in parentheses is added together with the color chart name. Noted.

(III) Various properties on culture (1) Tables 1, 2 and 3 show the culture findings of this strain, respectively. 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 East Iron Agar
Negative (c) Glucose / peptone / gelatin medium (21
-23 ° C) Negative (d) Trypton yeast solution
negative

(2) Tyrosinase reaction
Negative (3) Production of hydrogen sulfide
Negative (4) Reduction of nitrate
Negative (5) Liquefaction of gelatin (21-23 ° C)
Negative (glucose / peptone / gelatin medium)

(6) Starch hydrolysis
Positive (7) Coagulation of skim milk (36-37 ° C)
Negative (8) Peptone conversion of skim milk (36-37 ° C)
Negative (9) Growth temperature range
15-37 ° C

(10) Utilization of carbon source (Priedam Gottlieb agar medium) Use: D-glucose, D-fructose, L-rhamnose, D-mannitol, L-arabinose, i-inositol, raffinose, D-xylose , Melibiose Not used: sucrose (11) Cellulose degradation negative

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

The bacteriological properties of the present bacterium are summarized as follows. Diaminopimelic acid in the cell wall is of the LL type. The form of aerial hyphae is linear or wavy, forming long spore chains. The spore surface is smooth. The vegetative mycelium has a pink or wine-based color tone, and the aerial mycelium has a pink-based color tone. No soluble dye is produced.

Based on these results, this strain is a strain belonging to the genus Streptomyces, and is classified into Pridham and Tresner (Barges Manual of Deterministic Bacteriology, 8th edition, pp. 748-829, 19).
It is considered to be a fungus belonging to the white or red series according to (1974).

Morphological characteristics of the above-mentioned WK-2955 strain,
As a result of comparison with known bacterial strains based on various properties and physiological properties in culture, the strain was identified as a strain belonging to the genus Streptomyces, and Streptomyces sp. WK-
2955. This strain is Streptomyces sp. WK-2955 (Streptomyces)
p. WK-2955). (FERM P-12318
issue).

In the present invention, first, a microorganism belonging to the genus Streptomyces and capable of producing 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. In addition, other substances WK-2
A strain belonging to the genus Streptomyces having a production capacity of 955 can also be used.

[0033] The medium used in the present invention includes:
A synthetic medium or a natural medium containing a carbon source, a nitrogen source and an inorganic substance suitable for cultivation of normal actinomycetes and, if necessary, other nutrients can be used. The carbon source and nitrogen source used in the medium may be of any type as long as the strain to be used is available.

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

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

Examples of the nitrogen source include ammonium salts of various inorganic or organic acids such as ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate and ammonium nitrate, urea, peptone, NZ-amine, meat extract, yeast extract, and the like. Dried yeast, corn steep liquor, casein hydrolyzate, fishmeal or its digest, soybean flour or its digest, defatted soybean or its digest, hydrolyzate and other nitrogen-containing organic substances, further, glycine, glutamic acid, Various amino acids such as alanine can be used.

As the inorganic substance, for example, various phosphates, magnesium sulfate, salt, and a trace amount of a heavy metal salt are used. When using a mutant strain showing auxotrophy, naturally, a substance satisfying the auxotrophy must be added to the medium.However, when a medium containing natural products is used, this type of nutrient is required. In some cases, no addition is required.

The cultivation is usually carried out under aerobic conditions such as shaking or aeration and stirring. Industrially, deep aeration stirring culture is preferred. The pH of the medium is, for example, 5.0 to 5.0.
It is 8.0, but it is preferable to perform culture near neutrality.

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

The culture conditions, such as the composition of the medium, the liquid properties of the medium, the culture temperature, the stirring speed, and the aeration rate, are appropriately adjusted so as to obtain favorable results depending on the type of the strain used, external conditions, and the like. Selected. When there is foaming in the liquid culture, for example, an antifoaming agent such as silicone fat, vegetable oil, or a surfactant can be used as appropriate.

The substance WK-2955 accumulated in the culture thus obtained is usually produced in the culture filtrate. In order to collect the substance WK-2955 from the culture filtrate, means usually used for collecting metabolites from cultures of microorganisms are used alone, in combination in any order, 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,
Means such as a countercurrent distribution method and chromatography are used.

In order to collect the substance WK-2955 from the culture solution, the culture solution is extracted with a non-hydrophilic organic solvent such as ethyl acetate, or the culture filtrate or the cell extract is extracted with activated carbon, alumina, and a porous synthetic resin. 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 by adding an organic solvent such as hexane.

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

The pharmaceutically acceptable salts of this substance include:
For example, salts such as hydrochloride, sulfate, nitrate, phosphate, carbonate, acetate, tartrate, citrate, succinate, glutamate, and aspartate can be produced by a conventional method. Since this type of manufacturing method is well known, its description is omitted.

Further, the present substance WK-2955 can be produced by organic synthesis. For example, as an example, as illustrated in the examples, 3-indoleethanol is used as a raw material, and after reacting a 3-indoleacetaldehyde obtained by oxidizing the same with p-hydroxyphenylacetaldehyde, a pinacol reaction is performed. The desired product can be obtained by purification.

The reaction conditions are as follows: mercury chloride and magnesium are mixed 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 are synthesized.

The anticoccidial activity of the substance WK-2555 in chickens as described above is as follows.

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

The concentration of the substance, WK-2955, which inhibits the growth of Eimeria tenella against hamster kidney-derived cells (BHK-21 cells) as host cells,
No cytotoxicity was observed at 0.2-0.02 μg / ml.

On the other hand, a similar administration experiment was carried out for monensin, a known chicken coccidial growth inhibitor. as a result,
Monensin did not show any coccidium growth inhibitory activity, and toxicity was observed in the host cells at a dose of 0.02 μg / ml. Therefore, it can be said that the present substance WK-2955 is less toxic than the known chicken coccidiostat growth inhibitor monensin.

[0052]

As described above, the substance of the present invention WK-295
5 has the activity of inhibiting the growth of coccidial protozoa resistant to the polyether antibiotic monensin, and thus can lead to the remission of coccidial disease. In addition, it can be used as a concomitant drug that significantly maintains 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
%, 2.4% starch, and 0.4% calcium carbonate in 100 ml of a liquid medium (pH 7.0), and steam sterilized at 121 ° C. for 15 minutes. WK-2955 cells grown on an agar plate were inoculated into these with a platinum loop.
Seed mothers were obtained by shaking culture at 7 ° C for 3 days.

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

About 20 l of this culture was added to ethyl acetate (20 l).
) Was added and stirred, and the suspension was centrifuged (10,000 rpm) with a Sharpless centrifuge to separate into an ethyl acetate extract layer, an aqueous layer, and cell residue. About 20 l of the obtained ethyl acetate extract was concentrated under reduced pressure, and then hexane (400 ml), methanol (190 ml) and water (10 ml) were added.
ml), and the mixture was stirred. The suspension was separated by a separating funnel and separated 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 dried product. This was subjected to silica gel chromatography (Merck, Art. 9).
385), and chloroform: methanol (20:
Elution was performed in 1) to obtain 270 mg of a crude active substance.

Next, gel filtration was performed using Sephadex LH-20 (manufactured by Pharmacia) as a carrier, 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 Trirotor V (manufactured by JASCO Corporation), and the column was YMC-PackA-343.
(ODS resin, manufactured by Yamamura Chemical Laboratory), elution solvent is 40
% Methanol, 60% water, detection was performed at UV 280 nm, and the flow rate was 6 ml / min. The active portion was concentrated to dryness and 15.0
9 mg of a colorless oily substance WK-2955 were obtained.

Example 2 Synthesis of substance WK-2950 starting from indole Synthesis of 3-indoleacetaldehyde [2] 3-indoleethanol [1] (1.0 g) was added to benzene (20 ml) and dimethylsulfoxide (DMSO). )
(20 ml), pyridine (0.5 ml), trifluoroacetic acid (0.25 ml), and dicyclocarbodiimide (DCC) (3.83 g) were added thereto, and the mixture was stirred at room temperature for 18 hours under an argon atmosphere.

Incidentally, 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), dried over anhydrous sodium sulfate, and concentrated to dryness. The residue was subjected to silica gel column chromatography (developing solvent; hexane: ethyl acetate =
10: 1) to obtain 630 mg of 3-indoleacetaldehyde [2] represented by the following reaction formula. Yield 6
4%.

[0061]

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Synthesis of p-hydroxyphenylacetaldehyde [4] p-hydroxyphenethyl alcohol [3] (1.0
g) with dicyclocarbodiimide (DMSO) (15 ml)
And triethylamine (6.6 ml) was added.
A 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 saline, dried over anhydrous sodium sulfate, and concentrated to dryness. The residue was 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]

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1- (p-hydroxyphenyl) -4-
(3-Indolyl) butane-2,3-diol (substance W
Synthesis of mercuric chloride (60 mg) in tetrahydrofuran (THF) solution (2 ml) was carried out using 70-80 mesh magnesium (1).
92 mg) was added and the resulting mixture was cooled to room temperature under argon at room temperature.
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 wall of the reaction flask containing these was placed in TH.
After washing with an F solution (2 ml), a solution of 3-indoleacetaldehyde (159 mg) and p-hydroxyphenylacetaldehyde (408 mg) in THF (4 ml) was added. The purple mixture thus obtained was stirred at 0 ° C for 1.5 hours, then treated with 0.3 ml of a saturated aqueous solution of potassium carbonate, and further stirred at 0 ° C for 15 minutes.

Diethyl ether was added to the mixture, and the mixture was filtered using Celite. The filtrate was washed with a saturated aqueous solution of sodium chloride and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent: chloroform:
Purification was performed using 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]

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Example 3 The effect on the growth inhibition of chicken coccidia was measured using a system in which coccidia protozoa were grown in the cells using animal cells as hosts according to the method of Ohnaga et al. As the chicken coccidium, an oocyst of Eimeria tenella having resistance to monensin was used.

This oocyst was used for 20 minutes threshing (SH
INAGAWA), chicken bile and trypsin were added, and reacted at 41 ° C. for 2 hours. The reaction was separated by centrifugation to obtain sporozoids.

Further, hamster kidney-derived cells (BHK-21 cells) are grown on a 96-well microplate (manufactured by Corning) so as to form a full sheet, and the sporozoid prepared previously is added together with a methanol solution of the substance WK-2954. The cells were cultured in an incubator (manufactured by Hirasawa Seisakusho) under aeration of carbon dioxide at 41 ° C. for 3 days.

Three days later, the cells were fixed with methanol, stained with hematoxylin and the like, and observed with an optical microscope for the presence of schizond, which is one of the steps in the growth process of coccidium. That is, those in which sporozoids grew to schizonds were judged to have negative coccidial protozoan growth inhibitory activity, ie, anticoccidial activity, those in which no schizond was found were evaluated as positive, and those in which BHK-21 cells died were toxic. And

This substance WK-2955 has a content of 0.2 to 0.0
At a concentration of 2 μg / ml, the activity of inhibiting the growth of coccidial protozoa was positive. At this concentration, no cytotoxicity was observed for the host cell (BHK-21). When a similar administration test was performed using monensin as a control, toxicity of the host cells was observed at a concentration of 0.02 μg / ml of monensin, and the activity of inhibiting the growth of coccidial parasites was negative.

[Brief description of the drawings]

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

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

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

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1: 465) (72) Inventor Yoko Takahashi 5-9-1, Shirokane, Minato-ku, Tokyo Inside the Kitasato Institute (incorporated association) (72) Inventor Toshiaki Sunazuka 5-9-1, Shirogane, Minato-ku, Tokyo Kitasato Institute (corporation) (58) Field surveyed (Int. Cl. ⁶ , DB name) C07D 209/10 C12P 17 / 10 BIOSIS (DIALOG) CA (STN) REGISTRY (STN) WPI (DIALOG)

Claims (4)

(57) [Claims] [Claim 1] The following formula: Or a pharmaceutically acceptable salt thereof. 2. A microorganism belonging to the genus Streptomyces and having the ability to produce the substance WK-2955 represented by the formula: is cultured in a culture medium, and the above-mentioned substance WK-2955 [I] is added to the culture. A method for producing a novel substance WK-2955, characterized by producing and accumulating and then collecting the same. 3. The method according to claim 2, wherein the microorganism is Streptomyces sp.
The method for producing a novel substance WK-2955 according to claim 2, which is WK-2955 (FERM P-12318). 4. A method according to claim 1, wherein said 3-indoleacetaldehyde and p-
A method for producing a substance WK-2955 or a pharmaceutically acceptable salt thereof represented by the following formula, which comprises reacting hydroxyphenylacetaldehyde with a pinacol reaction.