JPH06184126A - Cyclic depsipeptide and its production - Google Patents

Abstract

PURPOSE:To provide a new depsipeptide obtained by culturing a mold. CONSTITUTION:PF 1022 strain belonging to asporogenic imperfect fungi is cultured in a culture medium containing nutritive components capable of being incorporated by general microorganisms. From the resultant cultured material, the objective substance, PF 1022E can be isolated by using the solvent extraction method, the silica gel column chromatography method, etc. This substance is a new cyclic depsipeptide having a molecular formula of C52H76N4O13 and represented by the formula.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel cyclic depsipeptide and a method for producing the same. The cyclic depsipeptide PF1022E of the present invention has anthelmintic action and is expected to be applied to the fields of medicine, veterinary medicine and the like.

[0002]

BACKGROUND OF THE INVENTION Many compounds having anthelmintic activity are known, and substances such as destomycin A, hygromycin B, avermectin and the like, which are microbial products and have anthelmintic activity, are small in number. The present inventors previously discovered a cyclic depsipeptide substance (PF1022) having anthelmintic activity as a result of exploratory research using Ascaris roundum [JP-A-3-35796]. Japanese Patent Application No. 3-163085 is known as a related substance. Then, when the culture of this substance-producing bacterium was scrutinized, a new cyclic depsipeptide could be newly isolated.

[0003]

A disease called parasitic disease causes a great deal of damage to human and animal health and agriculture. From such a background, the emergence of new and useful anthelmintic active substances and their advantageous production methods are always demanded. The present inventors have paid attention to the above points, and have aimed to provide a novel substance having the possibility and to establish a manufacturing method thereof.

[0004]

Means for Solving the Problems The present inventors have searched for compounds expected to be used as novel antiparasitic agents in order to solve the above problems. As a result, the cyclic depsipeptide having the above-mentioned anthelmintic activity is found. (PF1022 substance) By discovering that the cyclic depsipeptide PF1022E substance having physicochemical properties similar to that of the PF1022 substance exists in the culture of the producing bacterium, isolating it, and clarifying its physicochemical properties The present invention has been completed. An object of the present invention is to provide a novel cyclic depsipeptide PF1022E substance and a method for producing the same. The gist of the first aspect of the present invention lies in the following formula (I):

[0005]

[Chemical 2] (I) (Ph represents a phenyl group and Me represents a methyl group.)

A cyclic depsipeptide PF1022E substance represented by Furthermore, the second invention cultivates a PF1022E substance-producing bacterium belonging to the filamentous fungus,
The method for producing a PF1022E substance is characterized in that the F1022E substance is collected. An example of the PF1022E substance-producing bacterium used in the present invention is the PF1022 strain isolated in 1988 from sugar cane leaves on Yonaguni Island, Okinawa Prefecture. This strain had been deposited at the Institute of Microbial Science and Technology of the Institute of Industrial Science and Technology as Microconc.
It has been deposited as P-2671). The characteristics of this strain are the inventions of the present inventors; “Cyclic depsipeptide substance and its production method and anthelmintic agent containing the same” (JP-A-3-3579).
It is the same as described in 6).

1. Cultivation method of PF1022E substance-producing bacterium The PF1022E substance-producing bacterium belonging to the incomplete Asporia order is cultured in a medium containing nutrients that can be used by ordinary microorganisms.
As the nutrient source, known ones conventionally used for culturing fungi can be used. For example, as the carbon source, glucose, sucrose, starch syrup, dextrin, starch, glycerol, sugar concentrate, animal / vegetable oil and the like can be used. As the nitrogen source, soybean flour, wheat germ, cone steve liquor, cottonseed meal, meat extract, peptone, yeast extract,
It is effective to add ammonium sulfate, sodium nitrate, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid and other inorganic salts capable of forming ions. It also helps the growth of the bacteria, PF10
Organic and inorganic substances which promote the production of 22E substance can be added appropriately.

As the culture method, a culture method under aerobic conditions,
Particularly, the deep culture method is most suitable. Suitable temperature for culturing
The temperature is 15 to 30 ° C, but in most cases, the culture is performed at around 26 ° C. PF
The production of 1022E substance varies depending on the medium and culture conditions, but the maximum accumulation is usually reached in 2 to 10 days in both shaking culture and tank culture. When the accumulated amount of the PF1022E substance in the culture reaches the maximum, the culture is stopped, and the target substance is isolated and purified from the culture solution.

2. Purification method of PF1022E substance In collecting from the culture solution of the PF1022E substance obtained by the present invention, a normal separation means utilizing its properties,
For example, a solvent extraction method, an ion exchange resin method, an adsorption or distribution column chromatography method, a gel filtration method, a dialysis method, a precipitation method and the like can be used alone or in combination and extracted and purified. For example, acetone-water, methanol-water, ethyl acetate or the like is extracted from the cultured cells. The PF1022E substance accumulated in the culture solution can be extracted into the organic solvent layer by extracting with an organic solvent immiscible with water, such as butanol or ethyl acetate. To further purify the PF1022E substance, silica gel (Wako-Gel C-200, manufactured by Wako Pure Chemical Industries, Ltd.), an adsorbent such as alumina, Sephadex LH-20 (manufactured by Pharmacia), Toyopar HW-40 Chromagraphy using (manufactured by Toso Co., Ltd.) or the like may be performed. The PF1022E substance with high purity can be obtained by the above method or by appropriately combining them. Obtained PF1022E
The physicochemical properties of the substance are as follows.

1. Physicochemical properties of PF1022E substance (1) Color and shape: colorless powder (2) Melting point: 142-144 ° C (3) Molecular formula: C ₅₂ H ₇₆ N ₄ O ₁₃
(4) Mass spectrum (EI-MS): m / z 964 (M ⁺ ) (5) Specific rotation: [α] D ²⁵ = -100
^. (c 1.0, MeOH) (6) Ultraviolet absorption spectrum: λ _max nm (MeOH, E ^1% _1cm ): 278 (6.5) (7) Infrared absorption spectrum: Fig. 1 3434,2959,2872,1744,1663, 1518,1470,1416,1370,1331,
1271,1194,1128,1078,1028,835,750,702,511 (KBr cm
^-1 ) (8) ¹ H NMR spectrum: Fig. 2 (9) Solubility: Soluble in chloroform, acetone, ethyl acetate, methanol and dimethyl sulfoxide, but insoluble in water. (10) Thin layer chromatography (silica gel plate 60
F ₂₅₄ , 0.25mm, manufactured by Merck & Co., Ltd. of West Germany): Chloroform: Ethyl acetate = 1: 1 Rf value 0.24 (11) Basic, acidic, neutral classification: Neutral substance As a result of further structural studies, the chemical structure of PF1022E substance Was determined as in formula (I) above.

An example of the present invention will be shown below. PF1022E
Since the properties of the substance have been clarified by the present invention, various methods for producing the PF1022E substance can be devised based on those properties. Therefore, the present invention is not limited to the examples, the modification means of the examples, of course, the known means based on the properties of the PF1022E substance revealed by the present invention to produce, concentrate and extract the PF1022E substance. , Including all methods of purification.

[0012]

[Example] As a seed medium, soluble starch 2.0%, glucose
1.0%, polypeptone 0.5%, wheat germ 0.6%, yeast extract 0.
A medium having a composition of 3%, soybean meal 0.2% and calcium carbonate 0.2% was used. As a production medium, glucose 2.0%, starch 1.0%, wheat germ 0.8%, soybean meal 1.3%, meat extract 0.38%,
A medium having a composition of 0.13% sodium chloride and 0.15% calcium carbonate was used. The pH before sterilization was adjusted to pH 7.0 before use.

A 200 ml Erlenmeyer flask into which the seed medium (40 ml) was dispensed was sterilized at 120 ° C. for 15 minutes, inoculated with 2 to 3 platinum loops of a slope agar culture of PF1022 strain, and at 26 ° C. for 48 hours. Shaking culture was used as a seed culture. Then, 50 L of the above seed medium was added to 130 L.
It was charged into a fermenter and sterilized at 120 ° C for 15 minutes, 25 seed cultures of the above flask were inoculated into the fermenter, and the mixture was shake-cultured at 26 ° C for 48 hours to obtain a tank seed culture. Then, 1 KL of the above-mentioned production medium was charged into a 2 KL fermenter and sterilized at 120 ° C. for 25 minutes, and 40 L of the above-mentioned tank seed culture was inoculated into the fermenter at 26 ° C.
The culture was performed with aeration and stirring for one day. After the culture was completed, diatomaceous earth was added as a filter aid and the mixture was filtered to obtain about 90 g of bacterial cells.

Two batches of bacterial cells (180 kg) were treated with methanol (710
L) was added and the mixture was stirred for 2 hours, and the bacterial cells were filtered off to obtain a bacterial cell extract. The bacterial cell extract was distilled under a reduced pressure to remove methanol to obtain 50 L.
The concentrated solution of Ethyl acetate (60 L) was added to this concentrated solution to extract the active ingredient, and the ethyl acetate layer was concentrated to dryness to obtain an oily substance. To this oily substance, 5 L of a mixed solvent of hexane-acetonitrile was added, and the mixture was stirred and distributed, and the acetonitrile was concentrated to dryness to obtain a powder (1.64 Kg). This powder was
Le-deionized water was dissolved in 1.64 L (50: 1), ice-cooled, stirred for 5 hours and cooled overnight for crystallization. The crystals are filtered through a No. 3 glass filter and washed with cold methanol (2.3 L). The crystal mother liquor was concentrated to dryness to obtain 260 g of powder. Of 260 g of this powder, 40 g was purified twice by silica gel column chromatography (chloroform: ethyl acetate = 1: 1, Waco-Gel C-200 400 g) (chloroform: ethyl acetate = 2: 1, Waco-gel). One purification with Gel C-200 400 g) and PF1022E substance
Got 1.77 g. This substance has the above-mentioned physicochemical properties.

[0015]

The PF1022E substance of the present invention is a novel cyclic depsipeptide and is expected to be used as an anthelmintic agent like the PF1022 substance.

[0016]

[Brief description of drawings]

[Figure 1] Infrared absorption spectrum of PF1022E substance in KBr

Figure 2: 500 MHz of PF1022E substance in heavy methanol solution
¹ H NMR spectrum

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Masayuki Takagi, 760, Shimooka-cho, Kohoku-ku, Yokohama-shi, Kanagawa Meiji Seika Co., Ltd., Pharmaceutical Research Laboratory (72) Tadaaki Okada, 760, Shimo-oka, Kohoku-ku, Yokohama-shi, Kanagawa Meiji Confectionery Co., Ltd., Pharmaceutical Research Laboratory (72) Inventor, Yasushi Murai, 788 Kajiyama, Odawara, Kanagawa Prefecture Meiji Seika Co., Ltd., Pharmaceutical Technology Research Institute (72) Inventor, Toshio Yoneda, 788, Kajiyama, Odawara, Kanagawa Prefecture Meiji Confectionary Co., Ltd. In-house (72) Inventor Katsuharu Iinuma 788 Kayayama, Odawara-shi, Kanagawa Meiji Seika Co., Ltd.

Claims (2)

[Claims] 1. The following formula (I): (I) (Ph represents a phenyl group and Me represents a methyl group.)
A cyclic depsipeptide PF1022E substance represented by: 2. A PF1022E substance-producing bacterium belonging to a non-sporeless bacterium is cultured, and PF10 shown in claim 1 is obtained from the culture.
A method for producing a PF1022E substance, which comprises collecting 22E substance.