WO2003048373A1

WO2003048373A1 - Novel substance fki-0550 and process for producing the same

Info

Publication number: WO2003048373A1
Application number: PCT/JP2001/010578
Authority: WO
Inventors: Satoshi Omura; Kazuro Shiomi; Rokuro Masuma
Original assignee: The Kitasato Institute
Priority date: 2001-12-04
Filing date: 2001-12-04
Publication date: 2003-06-12
Also published as: AU2002222576A1

Abstract

A microorganism capable of producing a substance FKI-0550 represented by the following formula: is cultured in a medium, and then the substance FKI-0550 thus accumulated in the culture medium is collected from the culture medium. Because of having an activity of inhibiting the growth of microorganisms, nematodes and arthropods, the substance FKI-0550 thus obtained is useful as an antihelminthic agent or an insecticide.

Description

New F K 1-0 5 5 0 substances and their production

The present invention relates to a substance having NADH-fumarate reductase inhibitory activity, which is effective for pharmaceuticals, veterinary drugs, and agricultural chemicals.

Light

Fine

OH

And a method for producing the same. Background art

Parasitic diseases have been decreasing due to improvements in the sanitary environment and advances in anthelmintic drugs.In recent years, imported parasitic diseases, zoonotic parasitic diseases, opportunistic parasitic diseases, and parasites derived from fresh foods Diseases have become more prominent, and a wide variety of parasitic diseases have become a problem. Parasitic diseases also pose a significant economic burden in livestock and agriculture. Among helminth infections among parasites, many compounds are currently used, such as ivermectin, mebendazole and praziquantel.

However, currently used anti-helminthic agents such as ivermectin, mebendazole and braziquantel are not satisfactory in terms of their efficacy and toxicity, and new drugs are required.

The present inventors have identified NAs in the electron transport chain, one of the promising targets for antihelminthic agents. Focusing on DH-fumarate reductase, we continued to search for inhibitors of this enzyme in microbial cultures. As a result, we found FT-0554 substance (nafresin) and filed an international application (international publication). The number WO 9 9 244 3 9). This international application was transferred to the United States and was assigned No. 09/5099770 as the application number. The microorganism capable of producing the FT-104 substance (naphrezin) is Aspergillus niger FT-0554 belonging to a filamentous fungus, and this strain is disclosed by the Ministry of International Trade and Industry. Deposited with the National Institute of Advanced Industrial Science and Technology, National Institute of Advanced Industrial Science and Technology (new name; National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary), and assigned accession number FERM BP-6443. The outline of the mycological properties of this strain is as follows. Regarding the culture characteristics on various agar media, the growth on the Gapec® yeast extract agar medium, the wort agar medium, the Gapec® yeast extract agar medium containing 20% sucrose, the bailesho's glucose agar medium, and the Miura agar medium Good, velvety and smooth peripheral. The colony diameter is between 40 mm and 85 mm. The color tone of the colony surface is black-brown, and the color tone of the back of the colony is light yellow and white.

Morphological characteristics are as follows: Wapec containing 50% seawater (salinity: 3.4%) • Yeast extract agar medium, wort agar medium, and Wapek bisect extract agar containing 20% sucrose It grew well on media such as potato and glucose agar, and conidia abundantly formed.

For physiological properties, optimal growth conditions are pH 5-7, temperature 16-36 ° C, seawater concentration 50-100%. The growth range is pH 3 ~ 10, temperature is 12 ~ 45 ° C, seawater concentration is 0 ~ 100%. Disclosure of the invention

The present inventors have continued to search from the culture of microorganisms, and found that the FK 1-550 substance of the novel compound produced by the filamentous fungus strain FK 1-550 was NAD H-Fuma. The present inventors have found that they have an activity of inhibiting lactate reductase, and have completed the present invention based on this finding.

The present invention provides a substance having NADH-fumarate reductase inhibitory activity and which is satisfactory as a pharmaceutical, animal drug, or agricultural chemical having satisfactory efficacy, toxicity, etc. It is intended to do so.

That is, the following equation

The present invention provides a novel FK 1-550 compound which is a compound represented by the formula: and the novel FK I-550 compound has a growth inhibitory activity against microorganisms, nematodes, and arthropods, respectively. It has.

A further object of the present invention is to provide a method for producing a novel FK1-0550 substance having NADH-reductase fumarate inhibitory activity.

That is, it belongs to filamentous fungi,

A microorganism having the ability to produce the FK 1-550 substance represented by the following formula is cultured in a medium, the FK 1-550 substance is accumulated in the culture, and the FK 1-55 is obtained from the culture. It provides a manufacturing method for collecting 50 substances.

Further, the present invention relates to a method wherein the microorganism having the ability to produce FK 1-055 substance is a Paeci 1 omy cess p. FKI-0555 (FERM BP-777) belonging to a filamentous fungus. 5) FKI is a substance made from 0550 It provides a manufacturing method.

Furthermore, the present invention provides a microorganism itself which belongs to a filamentous fungus and has an ability to produce an FKI-550 substance. Further, the present invention provides Paecci mouth essp. FKI-050 (FERM BP-7785) as a microorganism.

Furthermore, the present invention provides the following formula:

An object of the present invention is to provide a NAD H-fumarate reductase inhibitor comprising an FK 1-550 substance represented by OH. The microorganism having the ability to produce the novel FK 1-550 substance represented by the above formula (hereinafter referred to as "FKI-550 substance-producing bacterium") is a filamentous fungus. The invention is not particularly limited as long as it has the ability to produce FKI-550 substance. Preferred examples of the strain used for producing the FK 1-550 substance of the present invention include, for example, the filamentous fungus FK 1 newly isolated by the present inventors from the soil of Okigami prefecture. -0550 shares. The bacteriological properties of this strain are as follows.

(1) Morphological features

This strain grew relatively well on yeast extract ■ solvable starch agar, wort agar, corn'meal agar, potato glucose agar, Miura agar, and the like. Conidia formation was good on yeast extract / sol brustarch agar, wort agar, cornmeal agar, potato • glucose agar, and slightly inhibited on Miura agar. Microscopic observation of colonies grown on corn-meal agar medium reveals that the hyphae are transparent and have septum, and the conidiophores stand upright from the basal hyphae and may branch irregularly. Its length is 30-400 x 4.5-5.5 m, sometimes reaching 800 m. The tip branches into three to six rings. The surface is smooth, but may be rough. Two to four fiaraids grow from the tip of the conidiophore. The phialid (6.5-11.0 X 1.8-3.5 / m) has a bulging base and a sharply tapered neck at the tip. Conidia are elliptical to spindle-to-lemon in size, 2.5-4.0 X 2.0-3.0 m, and are divergently linked.

(2) Culture properties on various agar media

Table 1 below shows the results of macroscopic observations when this strain was cultured at 25 ° C for 14 days on various agar media.

Table 1 Culture medium Growth state on culture medium Colony surface Colony back surface Color tone of soluble dye (diameter of colony) Color tone Stretch extract Soluble starch agar medium

Good (68-69 mm) yellow-brown to cream

Woolly magenta yellow-brown

Peripheral smooth wort agar medium

Good (49-53 mm) Red-yellow ~ Creamy ~ None Woolly Red-purple Yellow-brown

Peripheral slightly irregular Le agar medium Good (60-64mm) Yellow-brown to creamy-none Woolly to velvety reddish purple light reddish purple

Peripheral smooth potatoGlucose agar medium

Good (59-61mm) Yellow-brown to yellow-brown None Wooly to velvety reddish purple

Peripheral turbulent Miura agar medium

Good (6 lmm) yellowish brown white to none wooly to velvety yellowish brown

Peripheral irregularities

(3) Physiological properties

(1) Optimal growth conditions

Optimal growth conditions for this strain are pH 4-7, temperature 16-31 ° C.

(2) Growth range

The growth range of this strain is pH 3 ~ 10, temperature 9 ~ 35 ° C.

(3) Aerobic and anaerobic distinction

Aerobic Based on the morphological characteristics, culture characteristics and physiological characteristics of the above FK1-0550 strain, a comparison with known bacterial strains was carried out, and as a result, this strain belongs to genus Paeci 1 omyces It was identified as a strain and named Paechi-Mouth sp. FKI-0550. This strain was designated as Paecilomyces sp. FK I-0550 (Pa ecil omycess p. FK I-0550). Higashi 1-1, Higashi 1-C home Ts ukub as hi, I bar ak i CA I ST T suk υ ba C en tral 6, 1-1, Higashi 1-C -ken 305-856 6 Japan), the National Institute of Advanced Industrial Science and Technology (AIST), Patent Organism Depositary Center (International Institute of Advanced Industrial Science and Technology). It has been deposited internationally at advanced Indu strial Science and Technology. The date of deposit was October 29, 2001, and the deposit number is FERM BP-7785. In producing the FK 1-0550 substance of the present invention, first, an FKI 0550 substance-producing bacterium belonging to a filamentous fungus may be cultured in a medium, and then separated and purified from the culture. As the strains that can be used in the present invention, all the FK1-0550 substance-producing bacteria belonging to the filamentous fungi, including the above-mentioned strains and mutants thereof, can be used.

As a nutrient source suitable for the production of the FK 1-0550 substance, any nutrient source that can be used as a nutrient source for filamentous fungi may be used. For example, commercially available peptone, meat extract, corn-steep. Rica, cottonseed flour, peanut flour, soybean flour, yeast extract, NZ-amine, casein hydrate, sodium nitrate, ammonium nitrate, sulfuric acid Use of nitrogen sources such as ammonium, carbohydrates such as glycerin, starch, glucose, galactose and mannose, or carbon sources such as fat, and inorganic salts such as salt, phosphate, calcium carbonate, magnesium sulfate and the like, alone or in combination it can.

In addition, if necessary, trace amounts of metal salts and animal / vegetable / mineral oils can be added as antifoaming agents. Any of these can be used as long as they are useful for the production of FK 1-0550 substance by using the producing bacteria, and any known culture materials of filamentous fungi can be used. For large-scale cultivation of the FKI-0550 substance, liquid culture is preferable, and the culture temperature can be applied within a range in which the producing bacteria can grow and produce the FKI-0550 substance. The cultivation can be carried out by appropriately selecting according to the properties of the FK 1-0550 substance-producing bacterium using the conditions described above. The FKI-550 substance can be extracted from the culture solution with a water-immiscible organic solvent such as black form and ethyl acetate. In addition to the above-mentioned extraction methods, known methods used for collecting fat-soluble substances, such as adsorption chromatography, gel filtration chromatography, scraping from thin-layer chromatography, centrifugal countercurrent distribution chromatography, and high-performance liquid chromatography Some combinations of the first class can be collected purely by repetition. The physicochemical properties of the FK 1-0 550 substance according to the present invention are as follows.o

(1) Properties: pale yellow oil,

(2) Molecular weight: 3 1 4. 3424 (M + H: by high-resolution high-speed atomic bombardment mass spectrometry),

(3) Molecular formula: C ₂ . H ₄₃ NO,

(4) Specific rotation: [H]. ²⁵ = + 26.1. (c = 0.24, methanol)

(5) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in methanol is as follows: 203 ηπι (ε = 8250), 220 nm (shoulder, £ = 440), 246 nm (£ = 2040), having a maximum absorption at 2665 nm (shoulder, ε = 1570),

(6) Infrared absorption spectrum: Infrared absorption spectrum measured by the bromide power tablet method is 3350, 3286, 2924, 2852, 1574, 164. has an absorption maximum in a 1 3 7 7, 1 0 8 0 cm 1,

(7) Proton nuclear magnetic resonance spectrum: Table 2 shows the chemical shift (ppm) and spin coupling constant (Hz) in double-mouthed form.

(8) ¹³ C nuclear magnetic resonance spectrum: The chemical shifts (ppm) in double-mouthed form are shown in Table 2.

(9) Solubility in solvents: soluble in chloroform, ethyl acetate, methanol, acetate nitrile, poorly soluble in n-hexane,

(10) Color reaction: positive for ninhydrin and sulfuric acid. Table 2

¹³ CH

7 5.6 d 3.16 m (1H)

51.1 d2.72 dq (1H, J = 6.4,6.3)

4 4.7 t 0.88 d d d (1 H, J = 14.0, 7.

7.0), 1.20 m (1

36.9 t1.0 1 m (1H), 12.6m (1H)

34.2 t1.33 m (1 H), 1 • 46 m (1 H)

3 1.6 d 1. 40 m (1 H)

3 0.03 t1.25 m (2H)

3 0.0 1 d 1.45 m (1H)

29.8 t 1.25 m (2H)

2 9.6 9 t 1.25 m (2H)

2 9.65 t 1.25 m (2H)

29.2 t1.05 m (1H), 13.3 (1H)

26.9 t 1.26 m (2H)

25.8 t 1.33 m (1 H), 1 • 46 m (1 H)

2 0.9 q 1.09 d (3 H, J = 63)

2 0.3 q 0.82 d (3H, J = 66)

1 9.7 q 0.8 2 d (3H, J = 6 • 6)

1 1.2 q 0.84 dd (3H, J 7.4, 7.4) However, the symbols in the table, s is a single line, d is a double line, t is a triple line, and q is a quadruple line , M is the multiplet, H is the number of protons, and J is the spin coupling constant (Hz). As described above, as a result of detailed examination of various physicochemical properties and spectrum of the FK 1-0550 substance of the present invention, it was determined that the FK I-0550 substance had a chemical structure represented by the following formula. .

As described above, the various physicochemical properties of the FK 1-0550 substance of the present invention have been described in detail.However, no compound matching such properties has been reported so far, and the FKI-0550 substance is considered to be a novel substance. Were determined. Next, the NADH-fumarate reductase inhibitory activity of the FK 1-0550 substance of the present invention will be described in detail.

After homogenizing the round muscle of the pig worm in a 120 mM sodium phosphate solution (pH 7.0), the supernatant was collected by centrifugation at 300 × g for 10 minutes. 1 00 0

The precipitate was collected by centrifugation at 0 xg for 20 minutes. The precipitate was suspended in a 12 OmM sodium phosphate solution (pH 7.0) to obtain a mitochondrial fraction. 96 Add 10 l of a 50% dimethylsulfoxide solution of the FK1-0550 substance to a 6-well microplate, then add 0.35 mM NADH, 7.2 mM sodium fumarate, 18 mg Zm1 bovine serum Add 1 20 mM sodium phosphate solution (pH 7.0) 801 containing albumin and add it to a microplate reader.

(Preparation was carried out at 37 ° (5 minutes) at 37 ° C. (manufactured by Bio-Tek Industries, USA).

To this was added 10 1 (0.3 mg of protein) of the roundworm mitochondrial fraction, and the mixture was incubated at 37 ° C for 10 minutes to absorb NADH at 340 nm.

Measured every 15 seconds. The slope of the decrease in absorbance at 340 nm was quantified as NADH-fumarate reductase enzyme activity. At a concentration of 50% inhibited NA DH-fumarate reductase activity. Therefore, it is expected that the FKI-0550 substance can be used as a composition for treating or preventing helminth infection. Table 3 below shows the minimum growth inhibitory concentrations (based on the agar plate dilution method) of the FK 1-550 substance of the present invention for various microorganisms on a nutrient agar medium.

Table 3 Minimum growth inhibitory concentration (g / m1) Suphyrococcus aureus ATCC6538p

12.5 Bacillus subtilis ATCC6633 6.25 Micrococcus luteus ATCC9341 6.25 Mycobacterium smegmatis ATCC607

2 5 Escherichia colDNIHJ> 100 Escherichia coli NIHJ JC-2 (IF012734)

> 100 Pseudomonas aeruginosa IF03080

> 100 0 Xanthomonas camvestris p v.

(Xanthomonas campestris pv. Oryzae) KB88

> 100 Candida albicans KFl> 100 Saccharomyces cerevisiae KF26

> 1 0 0 Aspergillus niger ATCC 6275 100 Mucor racemosus IF04581 100 As can be seen from Table 3 above, the FK1-0550 substance of the present invention has growth inhibitory activity against several microorganisms. showed that. Next, the anti-nematode and anti-arthropod activities of the FK 1-0550 substance of the present invention will be described in detail below.

A methanol solution of the substance FK 1-0550 of the present invention was added to a 96-well plate (manufactured by Corning Incorporated, U.S.A.), and methanol was distilled off under a vacuum pump. Then, a medium for assay (lecithin 0.01%, Sodium bicarbonate (7.5 mM), potassium chloride (7.5 mM), calcium chloride dihydrate (7.5 mM), magnesium sulfate heptahydrate (7.5 mM), 250 z1 were added, and the mixture was shaken for 15 minutes.

There, agar medium for nematode growth [Pactogar (Difco, USA) 1.7 Nectopeptone (Difco, USA) 0.5%, Yeast extract (Difco, USA) 1.0%, 0.3% sodium chloride, 0.0005% cholesterol, 0.0007% calcium chloride, 0.03% magnesium sulfate, 0.34% potassium hydrogen phosphate, 0.11% dipotassium hydrogen phosphate Escherichia coli grown on a medium], add about 10 nematodes C aenorhab ditisele gans bred above, and add a buffer solution (0.24% Tris, 2.57% sodium chloride, 2.57% magnesium chloride, 0.47%, potassium chloride 0.07%, sodium carbonate 0.02%, magnesium sulfate 0.64%, calcium chloride 0.1 1%, adjusted to ρΗ7.1) Arthropod A rt hatched in 50 1 of a buffer solution containing several larvae of Emiasa 1 ina was added.

When the state of these organisms was observed under a microscope two days later, the growth of the nematode Caenorhabditisele gans was inhibited at a concentration of 20 μg / ml. In addition, the growth of the arthropod Artem iasalina was inhibited at a concentration of 5 ug / m1. Therefore, the FK 1-0550 substance of the present invention is Best mode for carrying out the invention that is effective as an insecticide or insecticide

Next, the present invention will be described with reference to examples, but the present invention is not limited thereto.

Glucose 2.0%, Polypeptone (Nippon Pharmaceutical, Japan) from Paeci 1 omy cess p. FKI-0550 strain (FERM BP-7785) cultured on an agar slant medium 0.5%, Yeast extract (Oriental Yeast Co., Ltd., Japan) 0.2%, Agar 0.1%, Potassium dihydrogen phosphate 0.1%, Magnesium sulfate heptahydrate 0.05% Of a liquid medium (pH 5.7) consisting of 100 ml was inoculated into each of 500 ml 1 Erlenmeyer flasks, and cultured with shaking at 27 ° C for 3 days. Using it as a seed culture, glycerol 2.0%, sucrose 1.0%, ammonium acetate 0.5%, dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.05%, chloride Potassium 0.05, cultivator No. 100 [(trade name) made by Yaizu Suisan Chemical Co., Ltd., Japan] 100 ml of liquid medium (pH 6.0) consisting of 0.2% and agar 0.1% 100 ml of 500 ml Erlenmeyer flasks dispensed at a time were inoculated in an amount of 1 ml each, and cultured at 27 ° C. for 9 days with shaking.

The culture was centrifuged, and the resulting cells were extracted with methanol, concentrated under reduced pressure to remove the solvent, and further extracted with ethyl acetate and concentrated under reduced pressure to obtain 46 lmg of crude substance I. . This was placed on a silica gel column (Merck Art. 7734, Merck, USA) packed with black-mouthed form, washed with black-mouthed methanol (10: 1), and then washed with black-mouthed methanol (1: 1). The mixture was eluted in 1) and concentrated under reduced pressure to obtain 12 Omg of crude substance II. This was placed on a silica gel column packed with ethyl acetate, washed with ethyl acetate, eluted with ethyl acetate-methanol-concentrated aqueous ammonia (8: 0.9: 0.1), and concentrated under reduced pressure to give a white powder. 70.1 mg of FKI-0550 substance was obtained. Industrial applications

As described above, a microorganism having the ability to produce a FK 1-550 substance belonging to a filamentous fungus is cultured in a medium, and the FK 1-550 substance is accumulated in the culture. The FKI-550 substance obtained by isolating the present FK 1-550 substance from microorganisms has a growth inhibitory activity against microorganisms, nematodes and arthropods, It is expected to be an effective substance for animal and pesticides.

Claims

1. The following formula

冃

A novel FK 1-550 substance, which is a compound represented by OH. Enclosure

2. A novel FK 1-550 substance, characterized in that the compound according to claim 1 has growth inhibitory activity against microorganisms.

3. A novel FK 1-550 substance, wherein the compound according to claim 1 has growth inhibitory activity against nematodes.

4. A novel FK 1-550 substance, wherein the compound according to claim 1 has growth inhibitory activity against arthropods.

5. The novel FK1-0 554 substance according to claim 3, which is used as an antihelminthic agent by inhibiting the growth of the above nematodes.

6. The novel FK 1-0 554 substance according to claim 4, which is used as an insecticide by inhibiting the growth of the arthropod.

7. It belongs to filamentous fungi and has the following formula

A microorganism having the ability to produce the FK I-0550 substance represented by the following formula is cultured in a medium, the FK 1-0550 substance is accumulated in the culture, and the FK 1-0555 substance is collected from the culture. A method for producing a novel FK 1 -0550 substance, characterized by the following.

8. It is confirmed that the microorganism capable of producing the FKI-055 substance is a fungus belonging to the fungi, Paesci sp. 'Espi 1 emy 1 omy cess p.) FKI-0550 (FERM BP-7785). The production method according to claim 7, which is characterized in that:

9. Microorganisms that have the ability to produce FKI-0550 substances are those of Paechi-michis ■ Paecilismyces ssp. FKI-0550.

10. The microorganism according to claim 9, wherein the microorganism is Paecchi's Mrs. SP (Paecci 1 omyces ss.) FK 1-0550 (FERM BP-7785).

A NADH-fumarate reductase inhibitor consisting of the FK 1-0550 substance represented by the formula: