JPS61216699A - Production of antibiotic substance b-41d - Google Patents

Abstract

PURPOSE:To obtain the novel antibiotic substance B-41D useful as an miticide and anthelmintic agent, by culturing a specific strain belonging to Streptomyces genus. CONSTITUTION:A microbial strain capable of producing B-41D such as Streptomyces B-41-146 (FERM-P 1438) is cultured in a conventional medium containing carbon sources, nitrogen sources, inorganic salts, etc., under aerobic condition at about 28 deg.C, and the culture product is filtered with a filter aid such as diatomaceous earth. The obtained cake is extracted with methanol to obtain the objective antibiotic substance B-41D of formula in the hydrated methanol. The extract is added with water, extracted with n-hexane, and concentrated under reduced pressure. The obtained oily substance containing the objective compound is adsorbed to an adsorbent in a column and extracted with a solvent to obtain the objective substance.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a new antibiotic B-4 with acaricidal and anthelmintic effects.
Regarding the 1Dc1 manufacturing method.

91 group of macrolide antibiotics isolated from the B-41-146 strain of the genus Streptomyces were disclosed in Japanese Patent Application Laid-open No. 50-2
It is called B-41 in Publication No. 9742, and A1*A2
9 types of *AS*A4+B1+B2+B5nCl and C2 are mentioned. Four more compounds were isolated and the structures of 13 compounds determined in a pot, published in The Journal of Antibiotics (J, Anubiozica
) 29 (3), pages 76-14 to 7G-16 and the same magazine, 29 (6), pages 76-35 to 76-42. These B-41 antibiotics were named milbemycin. The structures of these already known 7tB-41 compounds are shown in the following table.

B-41R, R2 fusion RJ R5
A5 (C1)! (HC1(! Cl75
-0HB2(C2) Fi HCH
3CH5-oCH5A4(αs) a I
(c2F15 CH5nFIBs(C4) H
Hc2F15 CH5 → CHsC+ (C9)
El Ef CJ <a2 sword 7→RC2(
α, Q) HHc2a5-cF12o knockdown → HAI
(β,) C11I5-oC111I5-ca
2oaAs(β2) c2g5 -0CH5-CH2
0Ei These B-41 compounds are known to have insecticidal and acaricidal activity in the above-mentioned literature, and also in JP-A-54-8
No. 9041 is known to have anthelmintic activity.

The present inventors analyzed B. belonging to the genus Streptomyces.
-New antibiotic B-41D from culture of strain 41-146
Heading 9. B-41D has the following formula (1).

It has also been found that B-41D has much stronger acaricidal and anthelmintic activity than the aforementioned B-41 compounds, particularly B-41As and B-41A, which have similar structures.

Streptomyces B producing antibiotic B-41D
Regarding the mycological properties of strain -41-146,
-29742, Streptomyces
Strain B-41-146 has been deposited with the National Institute of Microbial Technology, Agency of Industrial Science and Technology, and its microorganism accession number is FAIKEN Funiya No. 1438.

As is well known, actinomycetes are susceptible to mutations in the natural world and by artificial manipulation (e.g., ultraviolet irradiation, radiation irradiation, chemical treatment, etc.).1 B-4 of the present invention
The same holds true for the 1-146 strain. Tree brother BAK called B
-41-146 strain includes all its mutant strains.

That is, in the present invention, the antibiotic B-41D is produced and B-41D is produced.
Bacteria that cannot be clearly distinguished from the -41-146 strain and its mutant strains.

All are included in the B-41-146 strain.

B-41D can be obtained by culturing strain B-41-146 in an appropriate medium and harvesting it.

As the nutrient source, any known nutrient source conventionally used for culturing Streptomyces bacteria can be used. For example, glucose, sucrose, starch, glycerin, starch syrup, molasses, soybean oil, etc. can be used as carbon sources. Further, as the nitrogen source, soybean flour, wheat germ, meat extract, peptone, yeast cells, corn steep liquor, ammonium sulfate, sodium nitrate, etc. can be used.

In addition, add calcium carbonate and salt as needed.

In addition to adding inorganic salts such as potassium chloride and phosphates, organic and inorganic substances that aid the growth of bacteria and promote the production of B-41D can be appropriately added.

The most suitable barrel cultivation method is the liquid culture method, especially the deep culture method, similar to the method used to produce general antibiotics. Cultivation is carried out under aerobic conditions, and the appropriate temperature for cultivation is 22°C.
~30°C.

In most cases, the culture is carried out at around 28°C. Production of B-41D reaches its maximum value in 5 to 10 days in both shaking culture and tank culture.

The following method is used for testing B-41D.

That is, take culture 3F in a small test tube and add 10
-, shake to extract, and centrifuge. The supernatant obtained here is filled up with acetone using LoWitt. 10-20 μttl-TLC plate of this sample (
Kiesel-gel 60 F254 manufactured by Merck & Co.
) and developed it with dioxane:carbon tetrachloride (18:82) for 4 hours.
0 mμ).

The absorbed amount is compared with that of the standard substance and calculated.

When collecting B-41D from the culture, activated charcoal,
Adsorbents such as alumina and silica gel.

Synthetic adsorbents such as Diaion HP-20 (manufactured by Mitsubishi Kasei Co., Ltd.), fixatives such as Avicel (manufactured by Mukasei Co., Ltd.) and P paper, ion exchange resins, ion exchange gel filtration agents, etc. can be used. The collection method shown is the most effective.

The culture is distributed door to door using a filter aid such as diatomaceous earth,
By extracting the cake obtained here with methanol, the target substance is dissolved in methanol water. After adding water to this 1, it is extracted with n-hexane and concentrated under reduced pressure to obtain an oily substance containing the target product. This was adsorbed on a column of silica gel (Wako Gel C-200), and n-hexane:acetone (95:5) was used.
The 7-lactone containing the target product was collected and concentrated under reduced pressure to form an oil again.A small amount of methanol was added to this, and the mixture was applied to a Sephadex LH-20 (Pharmacia M) column and extracted with methanol. The 7-lactone containing the target compound was collected and concentrated under reduced pressure. The resulting residue was dissolved in a small amount of methanol, water was added, and the mixture was left at room temperature to obtain B-410 in the form of crystals. It will be done.

B-41D has the following physical and chemical properties.

Appearance: acicular crystals 1 mouth p186~1ga ℃2) Elemental analysis value (%): c; 71.4G, Mi; El, 82, O:
20.223) Chemical structural formula of formula (I) above.

4) Molecule i: 556 5) Ultraviolet absorption spectrum: Shown in FIG.

Maximum absorption; 237 mμ (shoulder, gm29400)
.

243 rna (p-30500) 6) Infrared absorption spectrum: Figure 2 shows the spectrum measured in KBr tablets.

T) NMR spectrum: Nuclear magnetic resonance absorption spectrum (Zo.

MHz) as shown in FIG.

8) Solubility: Easily soluble in n-hexane, ethyl acetate, acetone, ethanol, and methanol, slightly soluble in water.

9) Thin layer coomatography 2 Rf value 0.40 adsorbent;
Merck Kieselge160 F254 developing solvent; dioxane: Vfj carbon chloride (18282) then B-
An example of manufacturing 41D is shown.

Production Example 1 Glucose 2%, soybean flour L%, cornstarch liquor 0.5% and NaC10,
2 triangles containing 600-ml of preculture medium containing 7
1 Streptomyces B-41-146 strain in Lasco
A platinum loop was inoculated and cultured at 27° C. for 48 hours, and 2 Erlenmeyer flasks and 30 flasks were transferred to a jar ψ fermenter. Jar-Famenta contains 4% glucose, 1% large i powder, 0.5% cornstarch, 1% skim milk, 0.2% cornstarch liquor, and NaC.
Prepare 20 tons of culture medium containing 10.3% and adjust the pH to 7.
The temperature was adjusted to 2 to 7.5 and thoroughly sterilized. During the culture period, the temperature was maintained at 28° C. and the internal pressure was maintained at 0.5 Kf/−.

After culturing for 10 days, the pH of the culture was adjusted to 3 with sulfuric acid, added with 1 odor of Celite, and filtered under pressure.
4 cakes were obtained. This was extracted with 154 tons of methanol, P was separated, 5 tons of water was added to the resulting methanol solution of 15 tons, and the mixture was extracted with 20 tons of n-hexane. The obtained n-hexane layer was dehydrated with Glauber's salt and concentrated under reduced pressure in a water bath at 40 to 45°C to obtain a 22F oil.

Dissolve this in 30-hexane and preliminarily
Kf silica gel was adsorbed onto a column packed with n-hexane, and developed with n-hexane:acetone (95:5). the result.

72 tons of fractions containing the target substance were obtained.

This was concentrated under the same conditions as above to obtain 5 soq of oil. This was dissolved in methanol (1-), and the mixture was developed using methanol after applying a 200 d Sephadex LH-20 (manufactured by 7 Almasia) column to a column filled with methanol. Here, fraction 65- containing the target product was obtained. This was concentrated under reduced pressure at 45°C,
The remaining amount obtained here was dissolved in 2-methanol, and the remaining amount was dissolved in 2-methanol.
- and left at room temperature to form amorphous powder B-41D.
110 Wq was obtained.

B-41D of the present invention is suitable for adults and eggs of two-spotted spider mites (Tetranychus) that parasitize fruit trees, vegetables, and flower petals, apple spider mites, citrus spider mites (panonychus), and rust mites, and Ixodidae (Ixodidae) that parasitize animals.
odidac), red mite $ (Dermanys)
side) and Arcoptidae (5arcoptidae)
It has excellent acaricidal activity against e.g.

Furthermore, the sheep fly (Qestrua) and the golden fly (L.
u-cilia), Kushiba” (H)'pod
erma), carpenter fly (Gautrophil)
It is active against ectoparasites of animals and birds such as U.S.), chisels, and lice; sanitary pests such as cockroaches and house flies; and various agricultural and horticultural pests such as aphids and lepidopteran larvae. Furthermore, root-knot nematodes (Meloidogyn) in the soil
e).

It is also active against bed mites (Phtzoglyphus) and the like.

B-41D may be isolated and purified for use, but instead the purification may be discontinued at any stage of the purification.

A crude product can also be used as an active ingredient. When using a mixture containing two or more B-41 compounds without completely separating the various B-41 compounds obtained from purification of the culture.

It is sufficient to purify to the extent that a miticide rate of 100- is obtained at a concentration of 5 ppm. Preferable 1 B-41 in moss crude product
The D content is about 50%.

The residual amount includes contaminants from the broth.

How to use B-41D as an acaricide.

Powders, coarse powders can be prepared by diluting the active compound with a carrier and, if necessary, adding other auxiliaries.

It can be prepared and used as a dispersing agent such as granules, fine granules, irrigation agents, emulsions, and oils.

The term “carrier” used here refers to a carrier that is usually added to an insecticide to help the active ingredient reach the object to be treated, such as plants, mites, or pests, or to facilitate the storage, transport, or handling of the active ingredient. means a synthetic or natural inorganic or organic substance with which it is mixed.

Suitable solid carriers include clay and talc.

Inorganic substances such as diatomaceous earth, kaolin, bentonite, calcium carbonate and synthetic calcium silicate, coumaron resin,
Examples include natural and synthetic resins such as alkyd resins and polyvinyl chloride, waxes such as carnauba wax and paraffin wax, shells of nuts such as walnuts and nuts, and soybean flour.

Examples of suitable liquid carriers include, for example.

Water, alcohols such as ethanol, inglobanol and ethylene glycol, glycol ethers such as ethylene glycol monobutyl ether and diethylene glycol monoethyl ether, acetone, methyl isobutyl ketone, cyclohexanone, and acetate.

Ketones such as isophorone, ethers such as tetrahydrofuran and dioxa, aromatic hydrocarbons such as benzene, toluene, xylene, and methylnaphthalene, salt-accumulated hydrocarbons such as trichlorethylene and carbon tetrachloride, kerosene, and light oil. Examples include low boiling point, medium and high boiling point petroleum fractions containing aromatic hydrocarbons.

Suitable globelants include chlorofluorocarbon gas,
Examples include liquefied petroleum gas, methyl ether, and vinyl chloride monomer.

Emulsification 1 The surfactant used for the purpose of dispersion, wetting, spreading, etc. may be ionic or nonionic. Suitable anionic surfactants include, for example, sodium or calcium salts of ligninsulfonic acid, sodium or potassium salts of olenic acid, sodium laurylsulfonate, sodium or calcium salts of dodecylbenzenesulfonic acid, etc. It will be done.

Suitable cationic surfactants include glycerol of fatty acids, sucrose esters of fatty acids, ethylene oxide clamps of higher aliphatic alcohols, and ethylene oxide condensates of higher fatty acids. Examples include ethylene oxide condensates of alkylphenols or alkylnaphthols, and copolymers of ethylene oxide and propylene oxide.

The acaricides of the present invention may also contain other ingredients, such as gelatin, gum arabic, casein, polyvinyl alcohol, protective colloids such as carboxymethylcellulose, sodium polyphosphate, cicumthrobic agents such as bentonite, and the like.

The acaricide of the present invention contains other compounds having acaricidal activity, such as 2-(1-methylglobil)-4,6-sinitrophenyl-β,β-dimethylacryl), diCp-/rolfenyl )-cyclopropylcarbinol, y-(2-methyl-4-chlorophenyl)-N,N-
Dimethylformamidine, 2,4.4',5-tetrachlordiphenylsulfone #1,1-bis-(p-chlorophenyl)2,2,2-)lichloroethanol, 2-
Seconda IJ-7'tylphenyl-N-2'f-carbamate, m-to'J-N-methyl carbamate, mineral oil, etc. may be added to further increase the efficacy, and in some cases, a mutual effect may be expected. I can do it.

Of course, it can be used in combination with other fungicides, herbicides, plant growth regulators, attractants, fertilizers, etc.

Next, the effects of the acaricide of the present invention will be shown using test examples.

Test Example 1 Test Method 1) Acaricide Trial A 3-chi emulsion was prepared according to Formulation Example 3 described later, and diluted with water to a predetermined concentration to prepare a drug solution. Spray 5 cc of this chemical solution onto cowpea leaves infested with adult female two-spotted spider mites using a Mizuho-type rotary sprayer (manufactured by Mizuki Rikagaku Kikai KK). I asked for

The number of adult two-spotted spider mites tested was 60 to 70 in each treatment area.

2) Two-spotted spider mite ovicidal test The treatment was carried out in the same manner as in 1) except that one-day-old eggs of two-spotted spider mites that had been laid on cowpea leaves in advance were used, and the unhatched rate was determined after two weeks. The number of eggs tested was approximately 100 in each treatment group.

3) Orange spider mite acaricidal test: 27 leaves infested with female adult orange spider mites were used (1)
same as.

Test results are shown in Tables 1 and 2.

Table 1: Dead mites and unhatched egg rate of two-spotted spider mites Table 2 Dead mite rate of orange spider mites after 3 days From the above test example B
It can be seen that -41D has extremely superior acaricidal activity compared to B-41A3 and B-41A4.

Next, examples of formulations of the acaricide of the present invention will be shown. In the text.

All parts simply refer to parts by weight.

Formulation Example 1 10 parts of amorphous powder of B-410 obtained by culturing and extraction according to the method of Production Example 1 was uniformly mixed with 5 parts of white carbon, and 50 parts of Merck and 35 parts of clay were added to the mixture. The mixture is mixed with the powder, pulverized three times using an impact pulverizer, and mixed uniformly again to obtain a powder.

Formulation Example 2 40 parts of amorphous powder of B-41D was mixed with 20 parts of white carbon.
5 parts of dodecylbenzenesulfonic acid, 2 parts of polyvinyl alcohol, and 33 parts of clay.
and mix evenly.

Grind three times using @53 type grinder and mix uniformly again to obtain an irrigation agent.

Formulation Example 3 B-41DI7) 3 parts of amorphous powder, 3 parts of polyoxyethylene nonylphenyl ether 7m calcium dodecylbenzenesulfonate, and 87 parts of Xyleph are mixed and uniformly dissolved, and filtered to obtain an emulsion.

Formulation Example 4 10 parts of amorphous powder of B-41D is dissolved in 10 parts of xylene, and mixed with 80 parts of machine oil to obtain an oil solution.

Furthermore, the B-4LD of the present invention has excellent parasicidal activity as a stinging insect in animals and humans.

Diseases commonly described as parasitic diseases.

By infection of an animal host by a parasitic protozoan known as Helmfnth. Zoonotiasis is pigs.

It is prevalent in livestock such as sheep, goats, cattle, horses, dogs, cats, and chickens, poultry, and livestock, causing serious economic damage. Among the 3 insects, the group of parasites described as protozoa consists of various organisms.
FJlc infestations often cause serious infections. The most common genera of the eight insects that infect animals mentioned above are:

Haemonx I! 5 (Haemonchus) r Trichostrongylus! lh (Trichostrong
ylus).

Genus Qstartagia.

Nematodales! A (Nematodirus)
p Cooperia R + Ascaris r Bunostomum
ostomum) T. ostomum (Qe
sophagostomum ) r Chabeltia R (
Chabertia).

Trichuris @ (Trtchuris).

Strongylua rTrichonema.

Dictyocaurus Jii
s).

Capillaria genus.

Genus Heterakis (etBakis).

Genus Toxocara.

Ascarizoia f4 (Ascaridia).

Oxyuris F, (Oxyuris).

Ankylostoma):i (Ankylostoma)
).

Uncinaria Il+ (Uncinaria).

It belongs to the genus 'I'oxascaris' and the genus Parascaris.

Certain species of the genera Nematodeilus, Cooperia and En7Agostomum attack the intestinal tract, while those of the genera Monchus and Ostiltagia inhabit the stomach, and parasites of the genus Dictyokaurus are found in the lungs. .

Parasites of the family Filaridae and Setariidae are also found in other tissues and organs in the body, such as the heart and blood vessels, subcutaneous and lymphatic tissue.

Furthermore, it has broad spectrum activity against many endoparasites in AI animals.

For example, dogs of the genus Dirofilaria
a).

Nematos viroides, a goetta
-piroides), Chilla7asia (5yph
acia ) and Aspiculalis
uris).

B-41D again. It is also useful against parasites that infect humans. 9 are the most common parasites of the human gastrointestinal tract.

Ancylostone r 75
a) rNeat*r) r
Genus Ascaris.

Strongyloide IE (Strongyloide)
s).

Trichinella li.

Capillaria genus.

Trichuris IK (Trichuris) and engineering/
Terobics! ! 4 (Enterobia).

Other medically important parasites found in the blood or other tissues and organs outside the gastrointestinal tract are Wuchereria, Brucia HA, of the family Filariaceae.
(Brugia), Onchocerca K (0nch
In addition to parasites of the genus Loa and the genus Dra-cunculus of the family Dracunculidae, strongiroi in a special extraintestinal parasitic state of intestinal parasites. They belong to the genus Des and Trichinella.

When B-41D is used as an anthelmintic in animals and humans, it can be administered orally as a liquid beverage. Beverages are usually solutions, suspensions or dispersions in suitable non-toxic solvents or water with suspending and wetting agents such as bentonite or other excipients. Beverages generally also contain antifoaming agents. Beverage formulations generally contain about 0.0% active compound.
01-0.5% by weight, preferably 0.01-0.1% by weight
Contains.

When it is desired to administer B-41D orally in a dry, solid unit dosage form, capsules, pills, or tablets containing the desired amount of active compound are usually employed. These usage forms combine the active ingredient with a suitable finely divided diluent,
Fillers, disintegrants and/or binders 2 such as starch, lactose.

Manufactured by homogeneously mixing with Merck, magnesium stearate, vegetable gum, etc. Such unit use formulations can vary widely with respect to weight and content of anthelmintic agent depending on the type of host animal being treated, the degree of infection and type of parasite and the weight of the host.

If B-41D is administered via animal feed.

It can be homogeneously dispersed in the feed, used as a top dressing or in the form of pellets. To achieve a typical anti-parasitic effect, the final feed usually contains 0.0001-0.02% of the active compound.

Furthermore, B-41D dissolved or dispersed in a liquid carrier excipient can be administered parenterally to animals by injection into the proventriculus, intramuscularly, intratracheally, or subcutaneously. For parenteral administration, the active compound is preferably mixed with a suitable vegetable oil, such as peanut oil, cottonseed oil. Such formulations generally contain from 0.05 to 50% by weight of active compound.

B-4LD again. It may be administered topically by mixing with a suitable carrier such as dimethyl sulfoxide or a hydrocarbon solvent. This formulation is applied directly to the external surface of the animal by spray or direct injection.

The optimum amount of active compound to be used for best results will depend on the species of animal being treated and the type and extent of the parasitic infection, but will generally be about 0.01 to 1.
0011f, preferably by oral administration of O65-50.0. Such usage amount is given once or in divided usage amounts over a relatively short period of time such as 1 to 5 days.

Next, the effects of the anthelmintic agent of the present invention will be shown using test examples.

Test Example 2 Male mice of the roRFVL strain, 4 weeks old, 1 body weight, 18 to 22 were injected with Nemato-spi
roides dubia) larvae were orally infected, divided into groups of 5 animals, fed with feed supplemented with the test drug for 7 days after infection, and then changed to normal feed and reared.

The mice were dissected on the 14th day after infection, and the number of worms in the small intestine was calculated and compared with the control group. The results are shown in the table below.

B-41D 0.05%
100 Chi0.005 100 0.0005 96 (Control) Mixture of B-41Bz, Bs O,0332

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of B-41D;
The figure shows the infrared absorption spectrum of the same material, and FIG. 3 shows the nuclear magnetic resonance absorption spectrum of the same material.

Claims (1)

[Claims] B-41D-producing bacteria belonging to the genus Streptomyces are cultivated aerobically, and the resulting culture is produced as an antibiotic B-41D having the following chemical formula. A method for producing B-41D, which comprises collecting the B-41D.