JPH0912580A - Novel milbemycin and its production - Google Patents

Abstract

PURPOSE: To obtain a novel milbemycin which has acaricidal, insecticidal and vermifugal activities against mites, plant insect pests and animal parasites and as an intermediate for milbemycjin acaricides, insecticides and parasiticides. CONSTITUTION: This compound is represented by formula I (R<1> is ethyl, ethyl; X is OH, Y is H, and R<2> is 3-methyl-2-butenoyl; X is H, Y is OH and R<2> is H, 3-methyl-2-butenoyl), for example, 30-hydroxymilbemycin α11 . The compound of formula I is prepared by culturing a microorganism in Mucor in a culture medium containing a compound of formula II as a substrate or by bringing the cell bodies of the microorganism in Mucor cultured in the medium or an enzyme extract from the culture mixture into contact with the compound of formula II.

Description

Detailed Description of the Invention

[0001]

[Object of the invention]

[0002]

FIELD OF THE INVENTION The present invention has an excellent acaricidal, insecticidal or anthelmintic activity against mites, plant pests or animal parasites, or for developing an acaricide, insecticide or anthelmintic agent. The present invention relates to milbemycins that can be used as intermediates and a method for producing the milbemycins.

[0003]

2. Description of the Related Art A number of novel milbemycin derivatives have been produced so far, but most of them are derivatives of milbemycins obtained by fermentation production by chemical conversion, and microorganisms of milbemycins obtained by fermentation production. There are few new derivatives by conversion. No reports of production of the derivative of the present invention by microbial conversion have been found, and no reports of production by chemical conversion have been found.

[0004]

The object of the present invention is to provide novel milbemycins having excellent acaricidal, insecticidal or anthelmintic activity or intermediates in developing acaricides, insecticides or anthelmintics. Development of available milbemycins and a method for producing the milbemycins.

[0005]

Configuration of the Invention

[0006]

[Means for Solving the Problems] In order to solve the above-mentioned problems, the present inventors have conducted diligent research focusing on the production of novel milbemycins by microbial conversion of milbemycins, and as a result, excellent, The present invention has found new milbemycins having mite, insecticidal or anthelmintic activity or milbemycins that can be used as intermediates in developing acaricidal, insecticidal or anthelmintic agents and a novel method for producing the milbemycins.

That is, the present invention has the following general formula (I)

[0008]

Embedded image

[Wherein R ¹ represents a methyl group or an ethyl group, X represents a hydroxyl group, Y represents a hydrogen atom, and R ²
Represents a 3-methyl-2-butenoyl group, R ¹ represents a methyl group or an ethyl group, X represents a hydrogen atom, Y
Represents a hydroxyl group, R ² represents a hydrogen atom or 3-methyl-2.
Represents a butenoyl group. ] The novel milbemycins shown by these and its manufacturing method are provided.

The details will be described below.

The general formula (II) which is the starting material for the process of the present invention
The compound represented by the formula is disclosed in JP-A-1-193270, and in the formula, R ¹ is a methyl group.
IIa is milbemycin α ₁₁ and compound IIb in which R ¹ is an ethyl group is known as milbemycin α ₁₄ .

The method of the present invention is specifically described by the general formula (II)
The present invention relates to microbial hydroxylation, deacylation and isomerization of a compound represented by In the method of the present invention, position 30 is hydroxylated. The hydroxyl group at the 5-position may undergo isomerization. Further, the 3-methyl-2-butenoyl group bonded to the 26-position may be deacylated. In some cases, isomerization of the hydroxyl group at the 5-position and deacylation of the 3-methyl-2-butenoyl group bonded at the 26-position may be simultaneously performed.

The microorganism used in the method of the present invention is a microorganism belonging to the genus Mucor and is, for example, Mucor tuberculous porous (Muco
rtubercurisporus SANK 10995 (FERM BP-5141)). The bacterium was obtained by depositing (preserving) NRRL 3154 and re-depositing it, and both are the same strain.

As is well known, actinomycetes are naturally occurring in
Further, mutation is easily caused by artificial manipulation (for example, ultraviolet irradiation, radiation irradiation, chemical treatment, etc.), and the FERM BP-5141 strain of the present invention has the same point. The FERM BP-5141 strain referred to in the present invention includes all mutants thereof. In addition, among these mutants, genetic methods,
For example, those obtained by recombination, transduction, transformation, etc. are also included. That is, in the present invention, the general formula (II)
The strains which are converted into the compounds represented by the general formula (I) and are not clearly distinguished from the FERM BP-5141 strain and mutants thereof are all included in the FERM BP-5141 strain.

The method of the present invention can be carried out in various ways. For example, (1) a method of culturing a microorganism used in the present method in a medium containing a compound represented by the general formula (II) as a substrate, (2) a microbial cell collected from a medium in which the microorganism is cultivated, Examples thereof include a method of contacting a compound represented by the formula (II), and (3) a method of contacting a cell-free extract prepared from cells of the microorganism with a compound represented by the general formula (II).

The culturing of the converting bacterium is usually carried out by culturing in a medium containing a nutrient that can be utilized by the microorganism. As a nutrient source, a known nutrient used for cultivation of general actinomycetes can be used.

For example, as the carbon source, glucose,
Sucrose, maltose, starch, glycerin, starch syrup, molasses, etc. are used.

As the nitrogen source, soybean flour, wheat germ, meat flour, fish meal, meat extract, peptone, corn steep liquor, dry yeast, ammonium salts such as ammonium nitrate and the like are used. In addition, salt, if necessary
In addition to inorganic salts such as potassium chloride, calcium carbonate, and phosphate, additives that help the growth of bacteria and promote the production of the above-mentioned enzyme having hydroxylation ability can be used in appropriate combination.

The culture is carried out under aerobic conditions, and the culture temperature is 15 to 30 ° C, preferably 20 to 27 ° C.

The method (1) is carried out by adding the compound represented by the general formula (II) and culturing. The timing of addition is optimum culture conditions for the converting bacteria to be used, especially a culture device,
Although it depends on the medium composition, culture temperature, etc., it is preferable that the hydroxylating ability of the converting bacterium starts to increase, and usually 1 to 5 days after the start of culturing the converting bacterium is preferable. The amount of the raw material compound, ie, the substrate, added is 0.01 to 5.0 with respect to the medium
%, Preferably 0.025 to 0.05%.

The culture after the addition of the raw material compounds is carried out under the aerobic conditions at the above-mentioned culture temperature. The culturing period is usually 1 to 8 days after the addition of the starting compound.

The method (2) is carried out by culturing the converting bacterium in the presence of a small amount of the substrate by the method of the above (1) and culturing until the hydroxylating ability of the converting bacterium becomes maximum.

That is, although the conversion ability varies depending on the type of medium, temperature, etc., it usually reaches its maximum 2-3 days after the start of the culture, so the culture is terminated at this point. Bacterial collection is performed by subjecting the culture to a method such as centrifugation or filtration.
It is preferable that the collected transformed cells are usually washed with a physiological saline solution, a buffer solution or the like before use. The converted bacterial cells thus obtained are brought into contact with the starting compound, usually in an aqueous medium, for example, in a phosphate buffer of pH 5-9. The reaction by the contact is usually carried out at 20 to 33 ° C, preferably at 25 to 30 ° C. The concentration of the substrate is usually 0.01 to 1.0% with respect to the medium. The reaction time depends on the substrate concentration, reaction temperature, etc., but is usually 1 to 5 days.

The cell-free extract obtained by the method (3) is obtained by applying a physical or chemical method to the transformed bacterial cells obtained by the above method, for example, crushing the bacterial cells by grinding, sonication or the like. As a substance, or as a microbial cell solution by treatment with an organic solvent, a surfactant, an enzyme, or the like.

The cell-free extract thus obtained is brought into contact with the starting compound in the same manner as the above-mentioned method of bringing into contact with the cells of the converting bacterium.

After completion of the conversion reaction, the target compound can be collected, separated and purified from the product by a known method. For example, the obtained product is extracted with an organic solvent which is immiscible with water, such as ethyl acetate, the solvent is distilled off from the extract, and the obtained crude target compound is subjected to a column using silica gel, alumina or the like. It can be separated and purified by subjecting it to chromatography and eluting with an appropriate eluent.

Natural milbemycins, which are the starting materials for the compounds represented by the general formula (II), are fermentation products, and many analogs are produced in various ratios, and each analog is isolated. The reaction is carried out after or as is. Therefore, the compound represented by the general formula (II) may be either a single compound or a mixture thereof.
Therefore, the compound of formula (I) can also be produced as a single compound or a mixture thereof.

[0028]

The compound represented by the general formula (I) according to the present invention has excellent acaricidal, insecticidal or anthelmintic activity against mites, plant pests or animal parasites, or is useful milbemycin. It is useful as an intermediate for acaricides, insecticides or anthelmintics of the system.

The compound represented by the general formula (I) is a fruit tree,
Adults, larvae and eggs of the spider mites (Tetranychus), apple spider mites (Panonychus) and rust mites that parasitize vegetables and flowers, larvae and eggs, and ticks (Ixodidae) that parasitize animals
(Dermanyssidae) and Acarinae (Sarcoptidae), etc., with excellent acaricidal activity.

Furthermore, sheep flies (Oestrus) and fruit flies
(Lucilia), Bullfly (Hypoderma), Horsefly (Gautroph)
ilus), etc., and ectoparasites of animals and birds such as fleas and lice; sanitary pests such as cockroaches and house flies; and other agro-horticultural pests such as aphids and lepidopteran larvae . Furthermore, root-knot nematodes in soil
(Meloidogyne), pine wood nematode (Bursaphelench
us), ticks (Phizo-glyphus) and the like.

The compounds of formula (I) are also active against insects which feed plants, especially those which are harmed by ingesting the plant.

Furthermore, the compound represented by the general formula (I) has excellent parasiticidal activity as an anthelmintic agent for animals and humans. Especially pigs, sheep, goats, cows, horses,
It is also effective against nematodes that infect domestic animals such as dogs, cats and chickens, poultry and pets.

When the compound represented by the general formula (I) is used for agriculture and horticulture, it is prepared into a well-known preparation such as powders, wettable powders and emulsions in this field. When used by diluting with water as needed, the concentration of the active ingredient is about 1 to 10 ppm.

When the compound represented by the general formula (I) is used as an anthelmintic for animals, it is prepared into a preparation well known in the art such as powder, tablets, capsules, injections and the like. When administered orally, the dose is about 0.01 to 100 mg, preferably about 0.5 to 50 mg per kg body weight.

Next, the present invention will be described more specifically by way of examples.

[0036]

【Example】

Example 1 Mucor tuberculus porous SA was added to 6 500 ml Erlenmeyer flasks containing 100 ml of the medium [A] having the following composition.
Inoculated with NK 10995 (FERM BP-5141), 26 ℃, 200
The culture was carried out by shaking at rpm. Milbemycin α after 2 days
₁₁ (IIa) was added using the 5% dioxane solution to a final concentration of 0.025% (150 mg total),
It was further cultured for 7 days at 26 ° C. and 200 rpm. After the culture was completed, the culture solution was centrifuged at 5000 rpm for 10 minutes to separate the cells and the supernatant. The cells were extracted with 200 ml of 80% methanol and centrifuged to separate the solid component and the supernatant. This was repeated three times. After combining the supernatants and distilling off methanol under reduced pressure, the supernatants were combined with the supernatant of the culture broth and mixed with ethyl acetate
Extracted three times with 0 ml. The extract was washed with 200 ml of saturated saline, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain 200.6 mg of a concentrate.

The concentrate thus obtained was dissolved in 1.2 ml of methanol, and a reverse phase column, Senshupack ODS-H-5251 [φ
20 × 250 mm, Senshu Scientific Co., Ltd.]
While monitoring the ultraviolet absorption at 243 nm, a solution of acetonitrile: water = 40: 60 was added for 15 minutes at a flow rate of 8 ml / min, and further 45 by a gradient controller.
Linearly ramp to 100% acetonitrile in minutes,
It was developed and eluted as it was for 1 hour. 37.5 minutes, 44.8
The peaks eluted at minutes, 48.9 minutes, 54.3 minutes and 68.8 minutes were collected, and the eluate was concentrated under reduced pressure.

The concentrate of the peak eluted at 37.5 minutes was re-sorted (conditions, column: Senshupack ODS-H-525).
1, mobile phase: acetonitrile: water = 50:50, flow rate:
10 ml / min), the peak eluted at 28.8 min was collected, and the eluate was concentrated under reduced pressure to give 5-epi-26,30.
-Dihydroxymilbemycin A ₃ (Compound I, X = hydrogen atom, Y = hydroxyl group, R ¹ = methyl group, R ² = hydrogen atom) was obtained in an amount of 4.3 mg (yield 3.2%).

Similarly, the concentrate of the peak eluted at 44.8 minutes was re-sorted (conditions, column: Senshupack ODS-
H-5251, mobile phase: acetonitrile: water = 50:50, flow rate: 4 ml / min, elution for 56 min, then further gradient
With the controller, increase the flow rate to 8 ml / min, and
A linear ramp to 100% acetonitrile in minutes. ), Collecting the peak eluted at 67.6 minutes,
30-hydroxymilbemycin α was concentrated under reduced pressure.
₁₁ (Compound I, X = hydroxyl, Y = hydrogen atom, R ¹ = methyl, R ² = 3- methyl-2-butenoyl group) 6.2
mg (yield 4.0%) was obtained.

Similarly, the concentrate of the peak eluted at 48.9 minutes was re-sorted (conditions, column: Senshupack ODS-
H-5251, mobile phase: acetonitrile: water = 50: 50, flow rate: 10 ml / min), and the peak eluted at 42.6 minutes was collected and concentrated under reduced pressure to give 5-epi-30-hydroxymilbemycin α ₁₁ 2.3 mg (yield 1.5%) of (Compound I, X = hydrogen atom, Y = hydroxyl group, R ¹ = methyl group, R ² = 3-methyl-2-butenoyl group) were obtained.

Similarly, from the peak eluted at 54.3 minutes, 26-hydroxymilbemycin A ₃ (compounds I, X =
2.3 mg (yield 1.8%) of hydroxyl group, Y = hydrogen atom, R ¹ = methyl group, R ² = hydrogen atom) were obtained.

Similarly, from the peak eluted at 68.8 minutes, 18.3 mg (recovery rate 12.2%) of milbemycin α ₁₁ (compound IIa) was recovered.

Medium composition [A] Glucose 1.0% Yeast extract 0.3% Malt extract 0.3% Peptone 0.5% Tap water residual (pH uncorrected) 1) 5-Epi-26,30-dihydroxymilbemycin A ₃ (Compounds I, X = Hydrogen atom, Y = hydroxyl group, R ¹ =
Methyl group, R ² = hydrogen atom) Mass spectrum (FAB method) (m / z): 583 (M ⁺ + Na), 565 (M ⁺ + Na-H
₂ O) Nuclear magnetic resonance spectrum (400MHz, CDCl ₃ ) δ ppm: 6.14 (dt,
1H, J _d = 11.2Hz, J _t = 2.4Hz), 5.81 (s, 1H), 5.70 (dd, 1H, J = 1
1.2,14.7Hz), 5.48 (s, 1H), 5.37-5.53 (m, 2H), 4.90-4.99
(m, 2H), 4.73 (s, 1H), 4.58 (dd, 1H, J = 2.3,14.6Hz), 4.51
(dd, 1H, J = 2.5,4.5Hz), 4.46 (dd, 1H, J = 2.3,14.6Hz), 4.1
4 (d, 1H, J = 2.5Hz), 3.48-3.68 (m, 5H) 2) 30-Hydroxymilbemycin α ₁₁ (Compound I,
X =. Hydroxyl group, Y = hydrogen atom, R ¹ = methyl group, R ² =
3-Methyl-2-butenoyl group) Mass spectrum (FAB method) (m / z): 665 (M ⁺ + Na) Nuclear magnetic resonance spectrum (400MHz, CDCl ₃ ) δ ppm: 5.71-5.
84 (m, 4H), 5.34-5.44 (m, 2H), 4.98 (t, 1H, J = 7.6Hz), 4.8
3 (d, 1H, J = 13.3Hz), 4.64-4.75 (m, 3H), 4.49 (dt, 1H, Jd =
1.6Hz, Jt = 5.9Hz), 4.07 (s, 1H), 4.00 (d, 1H, J = 5.9Hz),
3.62-3.68 (m, 1H), 3.46-3.58 (m, 3H), 3.33 (t, 1H, J = 1.9H
z), 2.18 (s, 3H), 1.91 (s, 3H) 3) 5-epi-30-hydroxymilbemycin α
₁₁ (Compound I, X = hydrogen atom, Y = hydroxyl group, R ¹ = methyl group, R ² = 3-methyl-2-butenoyl group) Mass spectrum (EI method) (m / z): 642 (M ⁺ ) Mass Spectrum (FAB method) (m / z): 665 (M ⁺ + N
a) Nuclear magnetic resonance spectrum (400 MHz, CDCl ₃ ) δ
ppm: 5.70-5.85 (m, 4H), 5.33-5.42 (m, 2H), 4.98 (t, 1
H, J = 7.0Hz), 4.68 (dd, 1H, J = 2.0,14.7Hz), 4.62 (dd, 1H, J
= 2.0,14.7Hz), 4.12 (brs, 2H), 4.10 (d, 1H, J = 6.6Hz), 3.
64 (dd, 1H, J = 4.0,10.7Hz), 3.48-3.60 (m, 3H), 3.38 (br.
s, 1H), 2.20 (s, 3H), 1.92 (s, 3H) Example 2 Mucor tubercuri porous SANK 10995 in 6 500 ml Erlenmeyer flasks containing 100 ml of the medium [A] having the same composition as in Example 1. (FERM BP-5141) was inoculated and 26
The cells were cultivated by rotary shaking at 200 rpm at 0 ° C. Two days later, milbemycin α ₁₄ (IIb) was added using the 5% dioxane solution to a final concentration of 0.025% (total 150 mg), and the mixture was further cultured for 7 days at 26 ° C. and 200 rpm.
After the culture was completed, the culture solution was centrifuged at 5000 rpm for 10 minutes to separate the cells and the supernatant. The cells were extracted with 200 ml of 80% methanol and centrifuged to separate the solid component and the supernatant. This was repeated three times. The supernatants were combined and the methanol was distilled off under reduced pressure, then combined with the supernatant of the culture solution, and extracted with 800 ml of ethyl acetate three times. The extract is saturated saline solution 20
The extract was washed with 0 ml, dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain 143.6 mg of a concentrate.

The obtained concentrate was dissolved in 1 ml of methanol, and a reverse phase column, Senshupack ODS-H-5251 [φ20
X250 mm, Senshu Kagaku Co., Ltd.], and while monitoring the ultraviolet absorption at 243 nm, a solution of acetonitrile: water = 55: 45 at a flow rate of 8 ml / min for 25 minutes, and a gradient controller for 30 minutes The solution was linearly tilted to a solution of acetonitrile: water = 90: 10, and developed and eluted as it was for 30 minutes. 24.9 minutes,
The peaks eluted at 39.7 minutes, 43.6 minutes, 55.1 minutes, and 77.4 minutes were collected, and the eluate was concentrated under reduced pressure.

The peak concentrate eluted at 24.9 minutes was re-sorted (conditions, column: Senshupack ODS-H-525).
1, mobile phase: acetonitrile: water = 40:60, flow rate:
After elution at 8 ml / min for 12 minutes, a gradient controller was used to add acetonitrile 100 for 40 minutes.
Declined to%. ), The peak eluted at 48.0 minutes was collected, and the eluate was concentrated under reduced pressure to give 5-epi-2.
6,30-Dihydroxymilbemycin A ₄ (Compound I, X = hydrogen atom, Y = hydroxyl group, R ¹ = ethyl group, R ²
= Hydrogen atom) was obtained in an amount of 0.7 mg (yield 0.52%).

Similarly, from the peak eluted at 39.7 minutes, 30-hydroxymilbemycin α ₁₄ (compounds I, X =
3.7 mg (yield 2.4%) of hydroxyl group, Y = hydrogen atom, R ¹ = ethyl group, R ² = 3-methyl-2-butenoyl group)
Obtained.

Similarly, from the peak eluted at 43.6 minutes, 5-epi-30-hydroxymilbemycin α ₁₄ (compound I, X = hydrogen atom, Y = hydroxyl group, R ¹ = ethyl group, R
2.2 mg (yield 1.7%) of ² = hydrogen atom was obtained.

Similarly, from the peak eluted at 55.1 minutes, 26-hydroxymilbemycin A ₄ (compounds I, X =
0.6 mg (yield 0.39%) of a hydroxyl group, Y = hydrogen atom, R ¹ = ethyl group, R ² = hydrogen atom) was obtained.

Similarly, 28.9 mg (recovery rate 19.3%) of milbemycin α ₁₄ (compound IIb) was recovered from the peak eluted at 77.4 minutes.

1) 5-Epi-26,30-dihydroxymilbemycin A ₄ (Compound I, X = hydrogen atom, Y = hydroxyl group, R ¹ = ethyl group, R ² = hydrogen atom) Mass spectrum (FAB method) (m / z): 613 (M ⁺ + K), 595 (M ⁺ + KH
₂ O) Nuclear magnetic resonance spectrum (400MHz, CDCl ₃ ) δ ppm: 6.16 (dt,
1H, J _d = 2.2Hz, J _t = 11.0Hz), 5.78 (s, 1H), 5.70 (dd, 1H, J = 1
1.7,14.6Hz), 5.48 (s, 1H), 5.31-5.45 (m, 2H), 4.94 (d, 1
H, J = 6.6Hz), 4.74 (s, 1H), 4.58 (dd, 1H, J = 2.2,13.9Hz),
4.52 (br.s, 1H), 4.46 (dd, 1H, J = 2.2,13.9Hz), 4.14 (d, 1H,
J = 2.9Hz), 3.49-3.68 (m, 3H), 3.51 (dd, 1H, J = 6.4,11.0H
z) 2) 30-hydroxymilbemycin α ₁₄ (compound I,
X = hydroxyl group, Y = hydrogen atom, R ¹ = ethyl group, R ² = 3
-Methyl-2-butenoyl group) Mass spectrum (FAB method) (m / z): 695 (M ⁺ + K) Nuclear magnetic resonance spectrum (400MHz, CDCl ₃ ) δ ppm: 5.82 (d
t, 1H, J _d = 11.2Hz, J _t = 2.2Hz), 5.71-5.79 (m, 3H), 5.36-5.
43 (m, 2H), 4.96 (t, 1H, J = 7.6Hz), 4.83 (d, 1H, J = 13.5Hz),
4.69 (br.s, 2H), 4.67 (d, 1H, J = 13.5Hz), 4.49 (d, 1H, J =
5.8Hz), 4.05 (br.s, 1H), 3.99 (d, 1H, J = 5.8Hz), 2.17 (s,
3H), 1.91 (s, 3H) 3) 5-epi-30-hydroxymilbemycin α
₁₄ (Compound I, X = hydrogen atom, Y = hydroxyl group, R ¹ = ethyl group, R ² = 3-methyl-2-butenoyl group) Mass spectrum (FAB method) (m / z): 695 (M ⁺ + K ) Nuclear magnetic resonance spectrum (400MHz, CDCl ₃ ) δ ppm:
5.69-5.87 (m, 4H), 5.33-5.4
1 (m, 2H), 4.96 (t, 1H, J = 7.7H
z), 4.68 (dd, 1H, J = 2.2, 14.0)
Hz), 4.62 (dd, 1H, J = 2.2, 14.
0 Hz), 4.12 (s, 2H), 4.10 (d,
1H, J = 5.8 Hz), 3.64 (dd, 1H, J
= 3.7, 11.0 Hz), 3.58 (m, 1H),
3.51 (dt, 1H, J _d = 11.0 Hz, J _t
= 5.5 Hz), 2.20 (s, 3H), 1.91
(S, 3H)

────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] April 2, 1996

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0013

[Correction method] Change

[Correction contents]

The microorganism used in the method of the present invention is a microorganism belonging to the genus Mucor and is, for example, Mucor tuberculous porous (Muco
rtubercur l sporus SANK 10995 (FERM BP-5141)). The bacterium was obtained by depositing (preserving) NRRL 3154 and re-depositing it, and both are the same strain.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0014

[Correction method] Change

[Correction contents]

As is well known, microorganisms are naturally
Further, mutation is easily caused by artificial manipulation (for example, ultraviolet irradiation, radiation irradiation, chemical treatment, etc.), and the FERM BP-5141 strain of the present invention has the same point. The FERM BP-5141 strain referred to in the present invention includes all mutants thereof. In addition, among these mutants, genetic methods,
For example, those obtained by recombination, transduction, transformation, etc. are also included. That is, in the present invention, the general formula (II)
The strains which are converted into the compounds represented by the general formula (I) and are not clearly distinguished from the FERM BP-5141 strain and mutants thereof are all included in the FERM BP-5141 strain.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0047

[Correction method] Change

[Correction contents]

Similarly, from the peak eluted at 43.6 minutes, 5-epi-30-hydroxymilbemycin α ₁₄ (compound I, X = hydrogen atom, Y = hydroxyl group, R ¹ = ethyl group, R
0.6 mg (yield 0.39 %) of ² = hydrogen atom was obtained.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0048

[Correction method] Change

[Correction contents]

Similarly, from the peak eluted at 55.1 minutes, 26-hydroxymilbemycin A ₄ (compounds I, X =
2.2 mg (yield 1.7 %) of hydroxyl group, Y = hydrogen atom, R ¹ = ethyl group, R ² = hydrogen atom) was obtained.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display area C12R 1: 785)

Claims (8)

[Claims] 1. The following general formula (I): [In the formula, R ¹ represents a methyl group or an ethyl group, X represents a hydroxyl group, Y represents a hydrogen atom, R ² represents a 3-methyl-2-butenoyl group, or R ¹ represents a methyl group. Or an ethyl group, X represents a hydrogen atom, Y represents a hydroxyl group, and R ² represents a hydrogen atom or a 3-methyl-2-butenoyl group. ] A new milbemycin represented by 2. In the above general formula (I), R ¹ is a methyl group, X is a hydroxyl group, Y is a hydrogen atom, and R ² is 3-methyl-2.
A compound according to claim 1 which represents a -butenoyl group, 3. In the above general formula (I), R ¹ is an ethyl group, X is a hydroxyl group, Y is a hydrogen atom, and R ² is 3-methyl-2.
A compound according to claim 1 which represents a -butenoyl group, 4. The compound according to claim 1, wherein in the general formula (I), R ¹ is a methyl group, X is a hydrogen atom, Y is a hydroxyl group, and R ² is a hydrogen atom. 5. The compound according to claim 1, wherein in the general formula (I), R ¹ is an ethyl group, X is a hydrogen atom, Y is a hydroxyl group, and R ² is a hydrogen atom. 6. In the above general formula (I), R ¹ is a methyl group, X is a hydrogen atom, Y is a hydroxyl group, and R ² is 3-methyl-2.
A compound according to claim 1 which represents a -butenoyl group, 7. In the general formula (I), R ¹ is an ethyl group, X is a hydrogen atom, Y is a hydroxyl group, and R ² is 3-methyl-2.
A compound according to claim 1 which represents a -butenoyl group, 8. A microorganism belonging to the genus Mucor is represented by the following general formula:
(II) [Chemical formula 2] (Wherein R ¹ represents a methyl group or an ethyl group) is cultured in a medium containing a substrate as a substrate, or a cultured bacterial cell of the same microorganism or an enzyme extract is used in the general formula (II) The method for producing the novel milbemycins according to claim 1, which comprises contacting with a compound represented by