JP3474601B2 - FO-1611A, B and / or FO-1611C substances and method for producing the same - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a FO-1611A substance and / or FO-1 which is useful as a therapeutic or prophylactic agent for coccidiosis, which is an important disease in the poultry industry.
611B substance and / or FO-1611C substance and a method for producing the same.

[0002]

2. Description of the Related Art Conventionally, as anticoccidial agents, sulfa agents, quinoline agents, antithiamine agents, antibiotics, etc. have been put into practical use, and recently, polyether antibiotics such as monensin, salinomycin, lasalocid, etc. Widely used.

[0003]

However, coccidium resistant to these drugs has appeared and its effect is weakened, and an anticoccidial agent to replace them has been demanded. Therefore, an object of the present invention is to provide a drug effective not only against drug-resistant coccidium but also against coccidium that has acquired resistance to a polyether compound, from the above viewpoint. .

[0004]

In general, drug cross-resistance is established when the structure of an acting substance is similar or the mechanism of action is similar to that of a conventional anticoccidial agent. Compounds differing in are useful as therapeutic or preventive agents for coccidiosis. The present inventors continued various studies to find out a novel drug effective against coccidial parasites that have acquired resistance to conventional drugs from the natural world, using monensin-resistant coccidial protozoa as a test organism, metabolism produced by microorganisms. As a result of searching among the products, it was found that a substance effective against coccidium was produced in the culture solution of the FO-1611 strain newly isolated from soil.

Next, as a result of separating and purifying the anticoccidial active substance from the culture, the FO-1611A substance, the FO-1611B substance and the FO-1611C substance (hereinafter collectively referred to simply as FO) having the physicochemical properties described below. -1
611 substance) is a substance that has never been known.

The present invention has been completed based on the above findings, and has the physicochemical properties described below.
611A substance, FO-1611B substance and FO-16
The present invention provides an FO-1611 substance selected from the group consisting of 11C substances or a pharmaceutically acceptable salt thereof.

Furthermore, the present invention belongs to the genus Penicillium,
FO-1611A substance and / or FO-1611B
Substance and / or FO-1611C microorganism capable of producing the substance is cultivated in a medium and FO-1 in the culture
611A substance and / or FO-1611B substance and / or FO-1611C substance are accumulated, and FO-1611A substance and / or FO-16 are accumulated from the culture.
11B substance and / or FO-1611C substance are collected, and FO-1611A substance and / or
Others FO-1611b substance and / or FO-161
The present invention provides a method for producing a substance 1C or a pharmaceutically acceptable salt thereof.

FO-1611A substance of the present invention, FO-1
The physicochemical properties of the 611B substance and the FO-1611C substance are as follows. [1] FO-1611a substance (1) elemental analysis: C66.56%, H8.72% (2 ) estimated Molecular formula: C ₁₈ H ₂₈ O ₅ (by high resolution mass spectrum) (3) Molecular weight: 324 (high Measured value by resolution EI mass spectrum is as follows) Calculated value: 324.1935 Measured value: 324.1926 (4) Specific optical rotation: [α] _D ²³ -14 ° (C = 0.1, in methanol) )

(5) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in methanol is as shown in FIG. 1, and shows a characteristic absorption maximum near 263 nm. (6) Infrared absorption spectrum: by the KBr method. The measured infrared absorption spectrum is as shown in FIG.
2920, 1686, 1637, 1543, 1458,
1406, 1383, 1302, 1248, 1151,
(7) Color reaction having an absorption band at 1011 cm ^-1 : Color is developed with 50% H ₂ SO ₄ . Ninhydrin color-negative (8) Solubility in solvent: methanol, ethanol,
Acetic acid ethyl ester, soluble in chloroform, insoluble in water

(9) Basic, acidic, neutral: acidic (10) substance property: colorless powder (11) nuclear magnetic resonance spectrum: Varian XL-40
The ¹ H-NMR spectrum and ¹³ C-NMR spectrum measured in a deuterated chloroform solution using an NMR spectrometer at 0 and 400 MHz are as shown in FIGS. 3 and 4, respectively. The chemical shifts of hydrogen and carbon are as shown in Table 1 below.

[0011]

[Table 1]

[2] F O- 1611 B substance (1) Elemental analysis value: C 74.98%, H 8.36% (2) Estimated molecular formula: C ₁₈ H ₂₄ O ₃ (by high resolution mass spectrum) (3) Molecular weight: 288 (measured values by high resolution EI mass spectrum are as follows) Calculated value: 288.1724 Actual value: 288.1725 (4) Specific rotation: [α] _D ²³ + 116 ° (C = 0.
1, in methanol)

(5) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in methanol is as shown in FIG. 5, and shows a characteristic absorption maximum near 238 and 297 nm. (6) Infrared absorption spectrum: KBr. The infrared absorption spectrum measured by the method is as shown in FIG.
2922, 1660, 1624, 1545, 1454,
1379, 1306, 1259, 1151, 1063,
(7) Color reaction having an absorption band at 1022 cm ⁻¹ : Color is developed with 50% H ₂ SO ₄ . Ninhydrin color-negative (8) Solubility in solvent: methanol, ethanol,
Ethyl acetate, soluble in chloroform, insoluble in water (9) Basic, acidic, neutral: acidic

(10) Property: colorless powder (11) Nuclear magnetic resonance spectrum: Varian XL-40
The ¹ H-NMR spectrum and ¹³ C-NMR spectrum measured in a solution of deuterated methanol using an NMR spectrometer at 0 and 400 MHz are as shown in FIGS. 7 and 8, respectively. The chemical shifts of hydrogen and carbon are as shown in Table 2 below.

[0015]

[Table 2]

[3] FO-1611C substance (1) Elemental analysis value: C74.93%, H8.32% (2) Estimated molecular formula: C ₁₈ H ₂₄ O ₃ (by high resolution mass spectrum) (3) Molecular weight: 288 (measured values by high resolution EI mass spectrum are as follows) Calculated value: 288.1724 Actual value: 288.1724 (4) Specific optical rotation: [α] _D ²³ + 182 ° (C = 0.1,
(In methanol)

(5) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in methanol is as shown in FIG. 9, and has a characteristic absorption maximum near 202, 252 and 261 nm. (6) Infrared absorption spectrum The infrared absorption spectrum measured by the KBr method is as shown in FIG.
4, 2389, 1718, 1691, 1633, 145
6, 1416, 1375, 1302, 1265, 100
(7) Color reaction having an absorption band at 3 cm ⁻¹ : Color is developed with 50% H ₂ SO ₄ . Ninhydrin color-negative (8) Solubility in solvent: methanol, ethanol,
Acetic acid ethyl ester, soluble in chloroform, insoluble in water

(9) Basic, acidic and neutral: acidic (10) substance property: colorless powder (11) nuclear magnetic resonance spectrum: Varian XL-40
The ¹ H-NMR spectrum and ¹³ C-NMR spectrum measured in a deuterated methanol solution using an NMR spectrometer at 0 and 400 MHz are shown in FIG. 11 and FIG. 1, respectively.
As shown in 2. The chemical shifts of hydrogen and carbon are shown in Table 3 below.

[0019]

[Table 3]

The above-mentioned FO-1611 used in the present invention
A microorganism having the ability to produce substance A and / or FO-1611B substance and / or FO-1611C substance (hereinafter referred to as FO-1611 substance-producing bacterium) belongs to the genus Penicillium. Penicillium sp. FO-1611 belonging to the genus Penicillium
The strain is an example of the most effectively used strain of the present invention, and the bacteriological properties of the strain are as follows.

I. Morphological Properties The strain grows relatively well on potato / glucose agar medium, YpSs agar medium, Tuapec yeast agar medium and the like, and conidia are also well established. When the colonies grown on the potato-glucose agar medium are observed under a microscope, the hyphae are transparent and have septa, and the conidia peduncle is directly growing from the basal hyphae. Penicillus is a unicycle, with conidia stalks often diverging.

The tip of the conidia peduncle swells like a pouch. The infarcts are pen-tip type, and 3 to 6 clusters grow in size.
It is 5 to 10.0 × 2 to 3 μ. Initially, one filamentous conidia settles on the apical end of the infarct and becomes chain-like with the lapse of culture time, and finally this chain reaches around 170 μm. Conidia are spherical to subspherical, and the size is 2 to 2.5.
μ and its surface is smooth.

II. Various properties of the culture The case of culturing on various media at 25 ° C for 14 days was visually observed. The results are shown in Table 4.

[0024]

[Table 4]

III. Physiological and ecological properties 1) Optimal growth conditions The optimum growth conditions for this strain are pH 6 to 6 in YpSs medium.
8. The temperature is 18 to 30 ° C. 2) Range of growth The range of growth of this strain is pH 3-1 in YpSs medium.
0, the temperature is 14 to 35 ° C. 3) Distinction between aerobic and anaerobic It was clear from the results of the morphological observation and the culturing properties above aerobic that this strain belongs to the genus Penicillium. This strain is Penicillium SP FO-1611 (Penici)
Illium sp. FO-1611) has been deposited with the Institute of Biotechnology, Institute of Industrial Science and Technology (FERM).
P-13399).

Although the FO-1611 substance-producing bacterium has been described above, the mycological properties as a general property of the bacterium are extremely variable and are not constant, and the UV irradiation or mutation which is performed naturally or normally is carried out. Derivative, eg N
-Methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, etc. are known to be mutated by artificial mutagenesis means. Not only such artificial mutants but also natural mutations, penicillium Belonging to the genus FO-1611A substance and / or FO-161
All strains capable of producing 1B substance and / or FO-1611C substance can be used in the present invention. In addition, strains mutated by cell engineering such as cell fusion and gene manipulation are also included as FO-1611 substance-producing bacteria.

In the present invention, first, a FO-1611 substance-producing bacterium belonging to the genus Penicillium is cultured in a medium. In culturing the bacterium, a fungal culture method is generally used. As the medium, a nutrient medium containing a carbon source that can be assimilated by microorganisms, a nitrogen source that can be assimilated, and if necessary, inorganic salts and the like is used. That is, as the carbon source, for example, carbohydrates such as glucose, sucrose, glycerol, fructose, maltose, mannitol, xylose, galactose, ribose, dextrin, sugar condensate, starch or a hydrolyzate thereof can be used.

The concentration is 0.1 with respect to the normal medium.
% To 5% is desirable. Also, gluconic acid, pyruvic acid,
Various organic acids such as lactic acid and acetic acid, glycine, glutamic acid,
Various amino acids such as alanine, alcohols such as methanol and ethanol, non-aromatic hydrocarbons such as normal paraffin, and various plant or animal fats and oils may be added.

Examples of the assimilable nitrogen source include ammonium salts of various inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate and 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, sulfates, salt, and a trace amount of heavy metal salts are added. In addition, when using a mutant strain that exhibits auxotrophy, it is naturally necessary to add a substance that satisfies the auxotrophy to the medium, but this type of nutrient is There is a case where addition is not particularly required.

Culturing is usually carried out under aerobic conditions such as shaking or aeration stirring culture. From the industrial point of view, deep aeration stirring culture is preferred. The pH of the culture is, for example, 5.0-8.
Although it is 0, it is preferable to carry out the culture in the vicinity of neutrality. The culture temperature may be 20 to 37 ° C., but usually 26 to 32 ° C.
It is preferable to keep the temperature (preferably around 27 ° C.). When the culture time is liquid, the FO-1 is usually obtained by culturing for 2 to 5 days.
Since the 611A substance and / or the FO-1611B substance and / or the FO-1611C substance are accumulated, the culture may be terminated when the accumulated amount in the culture reaches the maximum.

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 and selected so as to obtain preferable results depending on the kind of the strain to be used and external conditions. It goes without saying that it will be done.
In liquid culture, when foaming occurs, a defoaming agent such as silicone oil, vegetable oil, or surfactant can be used as appropriate.

FO-1611A substance and / or FO-1611 accumulated in the culture thus obtained
Since the substance B and / or the FO-1611C substance is contained in the microbial cells and the culture filtrate, the culture is centrifuged to separate the culture filtrate and the microbial cells.
11A substance, FO-1611B substance and FO-161
It is advantageous to collect 1C material.

FO-1611A substance, FO-1611B
To collect the substance and the FO-1611C substance, the means usually used for collecting the metabolite from the culture of the microorganism can be used alone or in combination, or repeatedly. That is, for example, extraction 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, chromatography. Etc. are used.

From the culture solution, FO-1611A substance, FO-
In order to collect the 1611B substance and the FO-1611C substance, first, the culture filtrate is extracted with a non-hydrophilic organic solvent such as ethyl acetate, butyl acetate or benzene, or the culture filtrate or the bacterial cell extract is treated with activated carbon, alumina or a porous medium. It may be adsorbed on a water-soluble synthetic polymer resin, an ion exchange resin or the like, eluted with an elution solvent such as methanol, and the obtained extract is concentrated under reduced pressure and then extracted with an organic solvent such as ethyl acetate.

The obtained crude substance is further subjected to a known method usually used in the purification of a fat-soluble substance, for example, column chromatography using a carrier such as silica gel, alumina or reverse phase chromatography using an ODS carrier. It can be purified. Also, this FO-161
As the pharmaceutically acceptable salt of one substance, for example, salts such as sodium salt, potassium salt, ammonium salt and magnesium salt can be produced by a conventional method.

[0037]

Next, the FO-1611A substance of the present invention,
The anticoccidial activity of the FO-1611B substance and the FO-1611C substance on chickens is described. Chicken coccidium growth inhibitory activity Using the chicken coccidium and Eimeria tenella oocysts resistant to monensin, the growth inhibitory activity on the host hamster kidney-derived cells (BHK-21 cells) was determined by the method of Ohnaga et al. (Oonaga, Ishii) : Japan Veterinary Association Magazine, 31, 592-
596 (1978)). The results are shown in Table 5.

FO-1611A substance, FO-1611
The growth inhibitory concentrations of substance B and FO-1611C substance against chicken coccidium were 40 μg / ml and 10%, respectively.
μg / ml and 10 μg / ml. In addition, FO
-1611B substance 40 μg / ml and FO-1611
Cytotoxicity was observed in substance C at a concentration of 20 μg / ml, but this substance is a cell at a concentration which shows growth inhibitory activity of Eimeria tenella on hamster kidney-derived cells (BHK-21 cells) as a host. No toxicity was observed.

On the other hand, the same growth inhibition experiment was carried out for monensin which is a known chicken coccidial growth inhibitor. As a result, monensin showed no coccidial growth inhibitory activity, and cytotoxicity to main cells was observed at 0.02 μg / ml administration. In Table 5, * indicates 40
This means that no experiment was performed at a concentration of μg / ml or more.

[0040]

[Table 5]

As described above, the present FO-1611A substance,
The FO-1611B substance and the FO-1611C substance are
Since it has the activity of inhibiting the growth of coccidial parasites resistant to the polyether antibiotic monensin, it is possible to lead to the amelioration of coccidiosis. It can also be used as a concomitant drug that remarkably prolongs the effect of a known anticoccidial drug.

[0042]

【Example】

Example 1 In a 500 ml Erlenmeyer flask, glucose 2.0%, polypeptone 0.5%, yeast extract 0.2%, magnesium sulfate heptahydrate 0.05%, potassium dihydrogen phosphate 0.1.
%, Liquid medium containing 0.1% agar (pH 6.0) 100
ml was dispensed and steam sterilized at 121 ° C. for 20 minutes. Cells of Penicillium sp. FO-1611 strain (FERM P-13399) grown on an agar slant medium were aseptically inoculated with a platinum loop and cultured at 27 ° C for 3 days to give seeds. A culture solution was obtained.

Then, in a 30 L jar culture tank, sucrose 2%, glucose 1.0%, corn steep liquor 1.0%, meat extract 0.5%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 7 water. Liquid medium containing 0.05% salt, 0.3% calcium carbonate, 0.1% agar and trace metal salts such as iron, zinc, manganese, copper and cobalt (p.
H6.0) 20 L was charged and steam sterilized at 121 ° C. for 20 minutes. Inoculate this with 400 ml of the above seed culture, and
The culture was performed with aeration and stirring at 7 ° C for 3 days.

18 L of ethyl acetate was added to 18 L of this culture solution, stirred well and then centrifuged to separate the ethyl acetate layer. The ethyl acetate extract was concentrated under reduced pressure to dryness to obtain 6.54 g of a crude extract. This crude extract was washed with silica gel chromatography (chloroform 1.5 liters, chloroform: methanol (99: 1) 1.5 liters and then chloroform: methanol (98: 2) 1.5 liters) to elute the active substance, and a crudely purified product was obtained. 252 mg was obtained.

Then, the product was subjected to gel filtration (developing solvent: methanol) using a Sephadex LH20 carrier to give a crude product 8.
6 mg was obtained. Furthermore, reversed-phase (ODS) high-performance liquid chromatography (developing solvent: 0.05% phosphoric acid content 60%
Acetonitrile to 100% acetonitrile)
7.5 mg of O-1611A substance, 3.8 mg of FO-1611B substance and 5.9 mg of FO-1611C substance were obtained.

[Brief description of drawings]

FIG. 1 is an ultraviolet absorption spectrum of a FO-1611A substance.

FIG. 2 is an infrared absorption spectrum of a FO-1611A substance.

FIG. 3 is a proton nuclear magnetic resonance spectrum of a FO-1611A substance.

FIG. 4 is a ¹³ C nuclear magnetic resonance spectrum of FO-1611A substance.

FIG. 5 is an ultraviolet absorption spectrum of a FO-1611B substance.

FIG. 6 is an infrared absorption spectrum of a FO-1611B substance.

FIG. 7 is a proton nuclear magnetic resonance spectrum of a FO-1611B substance.

FIG. 8 is a ¹³ C nuclear magnetic resonance spectrum of a FO-1611B substance.

FIG. 9 is an ultraviolet absorption spectrum of a FO-1611C substance.

FIG. 10 is an infrared absorption spectrum of a FO-1611C substance.

FIG. 11 is a proton nuclear magnetic resonance spectrum of a FO-1611C substance.

FIG. 12 is a ¹³ C nuclear magnetic resonance spectrum of a FO-1611C substance.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI (C12N 1/14 C12N 1/14 C12R 1:80) C12R 1:80 (C12P 1/02 C12P 1/02 C12R 1:80) (72) Inventor Yuzuru Iwai 5-9-1, Shirokane, Minato-ku, Tokyo Incorporated Hojin Kitasato Institute (72) Inventor Katsuji Haneda 5-9-1, Shirokane, Minato-ku, Tokyo Incorporated Hojin Kitasato Institute (56 ) Reference JP 61-260888 (JP, A) JP 60-58081 (JP, A) JP 59-244691 (JP, A) The Journal of Antibiotics, 1993 Dec. , Vol. 46, No. 12, p. 1849-1853 (58) Fields investigated (Int.Cl. ⁷ , DB name) C07G 11/00 C12N 1/14 C12P 1/02 BIOSIS / WPI (DIALOG)

Claims (4)

(57) [Claims] 1. FO-161 having the following physicochemical properties:
1A substance, FO-1611B substance and FO-1611
FO-1611 substance selected from the group consisting of substance C or a pharmaceutically acceptable salt thereof. [1] FO-1611a substance (1) elemental analysis: C66.56%, H8.72% (2 ) estimated Molecular _formula: C _{18 H} 28 _{O 5} (by high resolution mass spectrum) (3) Molecular weight: 324 (4 ) Specific optical rotation: [α] _D ²³ −14 ° (C = 0.1,
(In methanol) (5) UV absorption spectrum (in methanol ): 263
(6) Infrared absorption spectrum (KBr method ): 3 402, showing a characteristic absorption maximum near nm
2920, 1686, 1637, 1543, 1458,
1406, 1383, 1302, 1248, 1151,
(7) Color reaction having an absorption band at 1011 cm ⁻¹ : Color is developed with 50% H ₂ SO ₄ . Ninhydrin color-negative (8) Solubility in solvent: methanol, ethanol,
Acetic acid ethyl ester, soluble in chloroform, insoluble in water (9) Distinction between basic, acidic and neutral: acidic (10) substance property: colorless powder [2] FO-1611B substance (1) elemental analysis value: C74. 98%, H 8.36% (2) Estimated molecular formula: C ₁₈ H ₂₄ O ₃ (according to high-resolution mass spectrum) (3) Molecular weight: 288 (4) Specific optical rotation: [α] _D ²³ + 116 ° (C = 0 ．
1, in methanol) (5) Ultraviolet absorption spectrum (in methanol ): 2 3
(6) Infrared absorption spectrum (KBr method ): 3421, which shows a characteristic absorption maximum around 8,297 nm.
2922, 1660, 1624, 1545, 1454,
1379, 1306, 1259, 1151, 1063,
(7) Color reaction having an absorption band at 1022 cm ⁻¹ : Color is developed with 50% H ₂ SO ₄ . Ninhydrin color-negative (8) Solubility in solvent: methanol, ethanol,
Ethyl acetate, soluble in chloroform, insoluble in water (9) a basic, acidic, distinguished neutral: acidic (10) substance properties: colorless powder (3) F O-1611c material (1) Elemental analysis: C74 .93%, H8.32% (2) Estimated molecular formula: C ₁₈ H ₂₄ O ₃ (according to high-resolution mass spectrum) (3) Molecular weight: 288 (4) Specific rotation: [α] _D ²³ + 182 ° (C = 0.
1, in methanol) (5) Ultraviolet absorption spectrum (in methanol ): 20
(6) Infrared absorption spectrum (KBr method ) showing characteristic absorption maxima at 2,252,261 nm: 2924,
2389, 1718, 1691, 1633, 1456,
1416, 1375, 1302, 1265, 1003c
(7) Color reaction having an absorption band at m- ¹ : Color is developed with 50% H ₂ SO ₄ . Ninhydrin color-negative (8) Solubility in solvent: methanol, ethanol,
Acetic acid ethyl ester, soluble in chloroform, insoluble in water (9) Distinguishing between basic, acidic and neutral: acidic (10) Material properties: colorless powder 2. A belongs to the genus Penicillium, claim 1 FO-1611a substance and / or description of claim 1, wherein F
O-1611B substance and / or FO according to claim 1.
A microorganism having the ability to produce -1611C substance is cultivated in a medium and FO-1611A substance and / or
Others FO-1611b substance and / or FO-161
1C substance was accumulated and FO-1611A was collected from the culture.
F and / or FO-1611B substance and / or FO-1611C substance
O-1611a substance and / or FO-1611 B product
Of qualitative and / or FO-1611C substances and their pharmaceutically acceptable salts. 3. The FO-1611A substance according to claim 1 and / or the F according to claim 1 , which belongs to the genus Penicillium.
O-1611B substance and / or FO according to claim 1.
A microorganism having the ability to produce the -1611C substance is a penicillium SP FO-1611 (Penici).
Illium sp. FO-1611 FERM P-1
3399). 4. The FO-1611A substance and the FO-1611A substance according to claim 1.
And / or the FO-1611B substance according to claim 1 and
And / or produce the FO-1611C material of claim 1.
Penicillium SP FO-16 with the ability to
11 (Penicillium sp. FO-161
1).