WO2007148412A1

WO2007148412A1 - Novel fki-3389 substance and method for producing the same

Info

Publication number: WO2007148412A1
Application number: PCT/JP2006/312946
Authority: WO
Inventors: Satoshi Omura; Kazuro Shiomi; Rokuro Masuma
Original assignee: The Kitasato Institute
Priority date: 2006-06-22
Filing date: 2006-06-22
Publication date: 2007-12-27

Abstract

An FKI-3389 substance is produced by culturing a microorganism (NITE ABP-244), which is a filamentous bacterium and has an ability to produce the FKI-3389 substance, in a medium, accumulating the FKI-3389 substance in the culture and isolating the FKI-3389 substance from the culture. Since the thus obtained FKI-3389 substance of the invention inhibits an NADH-fumarate reductase activity, it can be used as a composition for treating or preventing helminthic infection, therefore, it is expected to be a substance effective as a pharmaceutical product, an animal drug or an agricultural chemical.

Description

Description New FK 1-3 3 8 9 Substances and production methods Technical field

The present invention relates to a novel FK 1-3 3 8 9 substance effective as a pharmaceutical, an animal drug, and an agrochemical and a method for producing the same because it has NADH-reductase inhibitory activity. Background art and conventional technology

Parasitic diseases have decreased due to improvements in the hygienic environment and advances in anthelmintic drugs. However, in recent years, imported parasitic diseases, zoonotic parasitic diseases, opportunistic parasitic diseases, parasitic diseases derived from ginger foods This has become a problem again. In pastoral farming and agriculture, parasitic diseases also bring great economic burden. Among the parasites, many compounds are currently used for worm infections, such as ivermectin, mebendazol, and praziquantel. Problems to be solved by the invention

However, it is difficult to say that the currently used anti-helminthic agents are satisfactory in terms of their effectiveness and toxicity, and new drugs are being sought. Means for solving the problem

Therefore, the present inventors focused on NADH-fumarate reductase in the electron transport system, which is one of the promising targets of anti-helminths, and searched for inhibitors of this enzyme from microbial cultures. FT— 0 5 5 4 (Nafredin) was found (W 09 9/24 4 3 9. US Patent No. 64 8 6 1 9 7 B 1; European Patent No. 1 0 3 3 3 7 0). As a result of continuing the search, it was found that the novel compound FK 1-3 3 8 9 produced by the filamentous fungus FK 1-3 3 8 9 has NAD H -fumarate reductase inhibitory activity. The present invention has been completed based on this finding. That is, the present invention has the following formula:

It provides a novel FK 1 -338 9 substance characterized by being represented by the formula:

The present invention further belongs to a filamentous fungus, and has the following formula:

The microorganism having the ability to produce the FK 1-3389 substance represented by the medium is cultured in the medium, and the FK 1-3389 substance is accumulated in the culture, and the FK 1-3338 substance is collected from the culture. It provides a process for the production of the novel FK 1-3389 features.

The present invention further relates to a microorganism belonging to a filamentous fungus and capable of producing FK I-3389 substance, Penicillium sp. (Penici 1 1 i um sp,) FKI-3389 (NI TE ABP— 244) is provided according to claim 2.

The present invention also provides Penicillium SP (Penici 11 ium sp.) FK I-3389 (N I TE ABP-244).

The FK I-3389 substance-producing bacterium is not particularly limited as long as it is a filamentous fungus and has the ability to produce the FK I-3389 substance. As a suitable example of the strain used for producing the FK I-3389 substance of the present invention, the filamentous fungus FK 1-3389 newly isolated from the soil by the present inventors can be mentioned. The bacteriological properties of this strain are as follows:

1. Morphological features

This strain grows well on lapec yeast extract agar, 25% glycerin 'nitrate agar, wort agar, potato, carrot agar, Miura agar, etc. Was good.

Microscopic observation of the co-α grown on the lap peek extract agar medium shows that the mycelium is colorless and has a septum, and a conidial pattern (7 0 to 1 7 0 X 1. 0 to 2.5 / am) Occurred upright from the aerial hyphae and never branched. Benicillus is formed at the tip of the conidia pattern. The Penicillus is a bicyclist consisting of a fiary and a metre, consisting of 3 to 5 metres, and 2 to 4 fierides. The meter is cylindrical and large? It was ~ 10 x 2.5 to 3.5 m. The filarid was an ampoule type with a size of 7 ~ 12.5 X 2.0 ~ 3. The mouth-mouth conidia are formed from the tip of the filament and become chained as the culture time elapses. The conidia are spherical to sub-spherical and have a size of 2.3 to 2.5 X 2.5 to 2.7 mm.

2. Incubation

Table 1 shows the results of macroscopic observation when this strain was cultured on various agar media at 25 ° C for 7 days.

Table 1 Appearance on the medium Color on the surface Color on the back Soluble pigments Wapek Yeast Extract Agar

Colony diameter: 2 3 to 2 4 mm

Pattern: Velvet, Radial Center

Raised state: slightly raised centrally pale yellowish green

Peripheral part: All edges Leachate: Yes Light yellow Wort agar

Colony diameter: 1 8

Pattern: Wool-like Pale yellow-orange None Raised protrusion: Central raised area

Peripheral part: mycelia ¾ yellow

Exudate: Yes

25% glycerin nitrate agar

Colony diameter: 1 o ~ l 1 mm

Pattern: Velvet center

Raised protuberance: Central raised yellow

Peripheral area: All edges Surroundings Light yellow None Exudate: None White Potato Ninjin Agar

Colony diameter: 2 2-3 mm

Pattern: Velvet center

Raised state: Slightly raised centrally Green Grayish green None Peripheral part: All edges Surrounding

Exudate: Yes Colorless Miura Agar

Colony diameter: 1 3 1 4 mm

Pattern: Velvet center

Raised state: Flat Green Gray green None Peripheral edge: All edges Surrounding

Exudate: None Colorless 3. Physiological properties

(1) Optimal growth conditions

The optimal growth conditions for this strain are pH 4-7, temperature 26-35 ° C.

(2) Growth range

The growth range of this strain is pH 2-8, temperature 13-38 ° C

(3) Distinguishing between aerobic and anaerobic

Aerobic

4. Conclusion

Based on the morphological characteristics, culture characteristics and physiological characteristics of the above FK 1-3389 strain, as a result of comparison with known bacterial species, this strain was identified as belonging to the genus Penicillium (Penici 1 1 ium) Penicillium SP FK I-3389. International deposit

This strain is named Penicillium 'SP FK I-3389 (Penici 1 1 i urn s p. FK I-3389) and is based on the Budapest Best Treaty on the International Approval of Deposit of Microorganisms in Patent Procedures. Kisarazu, Kazusa, Kamasa, Chima, Chiba Prefecture

Incorporated Administrative Agency National Institute of Technol¾gy and Evalution NITE Patent Microorganisms Depositary (NPMD) in Shirokane, Minato-ku, Tokyo, Japan Deposited by Kitasato Institute, Inc., 5-chome 9-1 1. The deposit date was June 1, 2006, and the receipt number was NI TE ABP— 2

44. In producing the FK 1-3389 substance of the present invention, first, a microorganism having the ability to produce the FK I-3389 substance that kills filamentous fungi is cultured in a medium, and the culture is performed. Separation and purification from products. The strains that can be used in the present invention include the above-mentioned strains and mutants thereof, and all of the FK1-3389 substance-producing bacteria belonging to the filamentous fungi.

Nutrient sources suitable for the production of the above FK 1-3389 substance may be those that can be used as nutrient sources for filamentous fungi. For example, commercially available peptone, meat extract, corn steep liquor, cottonseed powder, peanut powder, soy flour, yeast extract, NZ-amine, casein hydrate, sodium nitrate, ammonium nitrate, ammonium sulfate, etc. Nitrogen source, glycerin, starch, glucose, galactose, mannose and other carbohydrates, fat and other carbon sources, and salt, phosphate, carbonated carbonate, magnesium nitrate, etc., used alone or in combination it can.

If necessary, trace amounts of metal salts, vegetation, planting, mineral oil, etc. can be added as antifoaming agents. These can be used as long as they are used by the producing bacteria and are useful for the production of the FK 1-3389 substance, and all known filamentous fungal culture materials can be used. Mass culture of FK I-3389 substance can be carried out in solid culture or liquid culture, and the culture temperature can be applied within the range where FK I-3389 substance can be produced. Culturing can be carried out by selecting as appropriate according to the properties of the FK 1-3389 substance-producing bacteria using the conditions described above.

The FK I-3389 substance can be extracted from the culture medium with a water-immiscible organic solvent such as chloroform or ethyl acetate. In addition to the extraction methods described above, known methods used for collecting fat-soluble substances such as adsorption chromatography, gel filtration chromatography, scraping from thin layer chromatography, centrifugal countercurrent distribution chromatography, high performance liquid chromatography It can be collected purely by combining or repeating the first class as appropriate. The physicochemical properties of the FK 1-3389 substance of the present invention are as follows.

(1) Property: Yellow cocoon

(2) Molecular weight: 4 66. 2722 (M +, by high resolution fast atom bombardment mass spectrometry

)

(3) Molecular formula: C ₂₉ H ₃₈ 0 ₅ (4) Specific rotation: [hi]. ²⁵ = — 8 1.4 ° (c = l. 0, methanol)

(5) Ultraviolet absorption spectrum (in methanol): As shown in Fig. 1, 2 4 3 η m (shoulder, ε = 24 2 0), 3 0 8 ηηι (ε = 1 9 7 6 0) 32 5 nm

(ε = 4 0 1 2 0), 3400 nm (e = 6 0 9 5 0), 3 5 8 (£ = 5 7 7 4 0) have maximum absorption

(6) Red external absorption spectrum (KB r lock): As shown in Fig. 2, 3 5 7 5, 3 5 0 2, 2 9 7 5, 2 92 9, "28 5 9, 1 7 9 5, 1 74 7 cm—has a maximum absorption at ¹

(7) Proton nuclear magnetic resonance spectrum: Table 2 shows the chemical shift (p pm) and spin coupling constant (Hz).

(8) ¹³ C nuclear magnetic resonance spectrum: chemical shift (p pm) in heavy-mouthed form is shown in Table 2.

(9) Solubility in solvents: Black mouth form, soluble in methanol, hardly soluble in water

(1 0) Color reaction: Positive to sulfuric acid

Table 2 Chemical shift of carbon (p pm) Chemical shift of hydrogen (p pm)

2 0 7. 6 s

1 7 1. 4 s

1 3 8. 8 S

1 3 6. 3 s X 2

1 3 6. 1 d 6. 1 2 d (J = 1 4. 4)

1 3 4. 3 d 6. 3 6 d d (J = 10.9, 14.4)

1 3 3.5 d 6. 2 9 d d (J = 10.9, 14.4)

1 3 2. 7 d 6. 2 4 d d (J = 10.9, 14.4)

1 3 0. 8 d 5. 6 8 b r .s

1 3 0. 5 d 6. 1 6 d (J = 1 0. 9)

1 2 9.5 d 6. 2 8 d d (J = 10.9, 14.4)

1 2 9. 1 d 6. 4 6 dd (J = 10.9, 14.4) 1 27.7 d 5. 2 1 s

8 5. 2 2 d 4. 3 7 s

8 5. 1 6 d 5. 1 5 d

7 7. 2 d 3. 4 7 q

7 0. 1 s

6 7. 0 s

5 7. 9 s

4 4. 3 d 2. 6 6 d q (J = 2.2, 7.4)

2 3.5 q 1. 1 4 s

1 8.5 q 1. 4 7 s

1 6. 8 q 1. 4 0 s

1 4. 3 q 1. 8 9 s

II

1 2. 0 q 1. 7 0 s

1 1, 6 q 1. 1 8 d (J = 7.4 CO)

4. 8 q 2 0 s

Is a single line, d is a single line, q is a quadruple line, b r. Is a wide line, J

As a result of detailed examination of various physicochemical properties and spectrum data of FK 1-3 3 8 9 substance of the present invention showing constant (Hz), FK I-3 3 8 9 substance is expressed by the following formula. The chemical structure was determined. .

As described above, the various physicochemical properties of FK I _ 3 3 8 9 were described in detail. No compound matching this property has been reported so far, and it was determined that this FK I-3 389 substance is a novel substance. The NAD H-reductase inhibitory activity of the substance FK 1-3389 of the present invention is as follows.

Hog roundworm muscle was homogenized in 1 2 OmM sodium phosphate solution (pH 7.0) and centrifuged at 3000 xg for 10 minutes, and the supernatant was collected. collected. The precipitate was suspended in 1 2 OmM sodium phosphate solution (pH 7.0) to obtain 0 T of mitcon. 9 Add 20% 50% dimethyl sulfoxide solution of this substance to 6-well mic plate and add 0.35 mM NADH, 7.2 mM disodium fumarate 1 20 mM sodium phosphate solution (PH 7.0) 80/1 was added, and pre-incubation was performed at 37 ° C for 3 minutes using a microplate reader ELX808 (Bio-Tek Industries). Add the swineworm mitochondria fraction (10 mg, protein amount: 0.3 mg), incubate at 37 for 3 minutes, and incubate for 10 minutes to absorb NADH at 340 nm for 15 seconds. Measured every time. As a result of quantifying the slope of decrease in absorbance at 340 nm as NADH-fumarate reductase enzyme activity, FKI-3389 substance inhibited NADH-fumarate reductase activity by 50% at a concentration of 2.4 nM. Therefore, it is expected that FK I-3389 substance can be used as a composition for treating or preventing helminth infections. Brief Description of Drawings

FIG. 1 is an ultraviolet absorption spectrum of a methanol solution (10 gZml) of the FK 1-3389 substance of the present invention.

Fig. 2 shows the best mode for carrying out the invention in the infrared absorption spectrum (KBr tablet) of the substance FK 1-3389 of the present invention. Next, the present invention will be specifically described with reference to examples, but the present invention is not limited to this example.

From Benicillium sp. FK I-3389 strain (NITE ABP-244) cultured on an agar slant medium, glucose 2.0%, polypeptone (Nippon Pharmaceutical Co., Ltd., Japan) 0.5%, yeast extract (Oriental Yeast Co., Ltd.) Japan) 0.2%, agar 0.1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.05% liquid medium (p FT6.0.0) is 1 0 Om 1 A 500-ml triangular flask was inoculated with 1 platinum loop and incubated at 2'7 ° C for 2 days. Inoculate 1 ml each of 59 500 ml Erlenmeyer flasks containing 50 g of Italian rice 50 g, 10 OmgZmi medicinal preparation Tm Γ medium (pH unadjusted) And static culture for 13 days.

After extraction by adding 10 L of ethyl acetate to the culture, the ethyl acetate extract was filtered and concentrated under reduced pressure to obtain 14 g of crude substance I. Place this on a silica gel column packed with black mouth form (Ar t. 7734, Merck, Germany), wash with black mouth form, elute with black mouth form-methanol (100: 1), and concentrate under reduced pressure. 2. 7 g of crude material II was obtained. This was further applied to a silica gel column (Pega sil Prep SIL—75 1 5-12 A, Senshu Issei Co., Ltd., Japan) which was filled with black mouth form, eluted with black mouth form and concentrated under reduced pressure. 1.9 g of crude substance III was obtained. This was subjected to high performance liquid chromatography (P egasi 1 S i 1 ica, ø 20 x 25 Omm, Senshu Issei Co., Ltd., Japan), and the peak was fractionated using black mouth formuaceton (1 00: 1) as the mobile phase. By concentrating it under reduced pressure, 5 1 5 mg of yellow candy-like FK 1-3389 substance was obtained. Industrial application fields

As described above, microorganisms belonging to filamentous fungi and capable of producing FK I-3389 substance are cultured in a medium, and FK 1-3389 substance is accumulated in the culture, and FK 1-3389 is accumulated from the culture. The FK1-3389 substance of the present invention obtained and obtained by isolating the substance inhibits NAD H-reductase activity of NAD H-fumarate and is used as a composition for treating or preventing helminth infections. From getting medicine □ P, expected to be an effective substance for animal medicine and pesticides <

Claims

1. Following formula

Contract

A novel FK 1-3389 substance characterized by being represented by the formula: Surrounding

2. Belongs to filamentous fungi, the following formula

The microorganism having the ability to produce FK I-3389 represented by the medium is cultured in a culture medium, and FK 1-33389 substance is accumulated in the culture, and the FK1-3389 substance is collected from the culture. Process for the production of new FK 1-3389 substances characterized by:

3. A microorganism belonging to the filamentous fungus and capable of producing FK I-3389 is Penicillium sp. FK I-3389 (NI TE ABP—244) Manufacturing method according to claim 2.

4. Penicillium SP (Penici 1 1 i um sp.) FK I-338 9 (N I TE ABP— 244).