JP2577019B2 - New milbemycins and their production - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel macrolide compound and a method for producing the same, and more particularly, to milbemycins and analogs thereof and a method for producing them. Milbemycin is a series of macrolide compounds and is a compound of the following formula (III) known from JP-A-50-29742, JP-A-56-32481 and the like.

In the formula, V and W represent a hydrogen atom, X represents a methyl, ethyl or isopropyl group, which are called milbemycin A3, milbemycin A4 and milbemycin D, respectively. V and W represent a hydrogen atom, and X represents sec-
The compound that is butyl is a milbemycin analog described in JP-A-54-145699. V is a hydrogen atom, and W is 4 ′-(α-L-oleandrosyl) -α-
Compounds wherein L is oleandrosiloxy and X is isopropyl or sec-butyl are disclosed in
Compounds described in -61198, 22, 22, respectively
They are called 23-dihydroavermectins B1a and B1b. W is a hydrogen atom, V is a hydroxyl group, and X is a 1-methyl-1-propenyl group, 1-methyl-
The compound which is a 1-butenyl group or a 1,3-dimethyl-1-butenyl group is a compound described in JP-A-61-10589, which is known as LL-F28249α.
V is an oxo group and X is a 1-methyl-1-propenyl group, a 1-methyl-1-butenyl group or 1,3-
The compound which is a dimethyl-1-butenyl group is disclosed in
No. 280496. All of these compounds are known to have insecticidal, acaricidal and anthelmintic activities.

The present inventors have made intensive efforts to search for novel analogs of these milbemycins.As a result, the present inventors have found that novel milbemycins can be produced by converting the above milbemycins using a microorganism or an enzyme produced thereby. We have completed the present invention.

JP-A-61-233686 discloses the microbial conversion of 22,23-dihydroavermectin aglycone or 13-deoxy-22,23-dihydroavermectin aglycone. The different microorganisms are also hydroxylated.

According to the present invention, a compound represented by the following general formula (II) is used as a substrate, which can be converted into a compound represented by the following general formula (I), a genus Amycolata, Amycolatopsis, Streptomyces A microorganism belonging to the genus, Absididia, Mucor, Synthephalastrum, Mortierella, Rhizopus or Bacillus,
By culturing in a medium containing the compound represented by the general formula (II) as a substrate, or by contacting cultured cells of these microorganisms or an enzyme extract with the compound represented by the general formula (II), The compound represented by the general formula (I) can be produced.

(Wherein V represents a hydrogen atom, a hydroxyl group or an oxo group,
W is a hydrogen atom, a hydroxyl group, a halogen atom or 4 ′-(α
-L-oleandrosyl) -α-L-oleandrosyloxy group, and X represents a methyl group, an ethyl group, an isopropyl group, a sec-butyl group,
A methyl-1-propenyl group, a 1-methyl-1-butenyl group or a 1,3-dimethyl-1-butenyl group, and when V is a hydroxyl group or an oxo group, 1-methyl-
Y represents a 1-propenyl group, 1-methyl-1-butenyl group or 1,3-dimethyl-1-butenyl group, and Y represents a hydroxyl group or a hydroxyimino group. ) (In the formula, U represents a hydroxymethyl group or a carboxy group, V, W, X, and Y have the same meanings as described above, and Z represents a hydrogen atom or a hydroxyl group.)

The process of the invention relates to the microbial hydroxylation and / or carboxylation of the compounds of the general formula (II). In the method of the present invention, the methyl group at position 30 attached to position 24 is always hydroxylated or carboxylated. In contrast, the methyl group at the 26-position bonded to the 4-position is not always hydroxylated, and in some cases, may not be hydroxylated. And a methyl group bonded to the 12th and 14th positions and
The X group attached at position 25 does not undergo hydroxylation.

Among the compounds of the general formula (II) which are the starting materials of the process of the present invention, those in which V is a hydrogen atom and W and Y are both hydroxyl groups are described in JP-A-61-85390.
Is a hydrogen atom and Y is a hydroxyimino group, each of which is known from JP-A-59-108785. V is a hydrogen atom; W is a hydroxyl group;
Is a hydroxyimino group, the aforementioned compound wherein W is a hydrogen atom and Y is a hydroxyimino group,
According to the method described in JP-A-61-103884, W
Can be obtained by hydroxylation. Furthermore, V is a hydrogen atom, W is a halogen atom, Y
The compound wherein is a hydroxyl group is known from JP-A-61-85390.

The microorganism used in the method of the present invention may be genus Amycolata, genus Amycolatas (genus Amycolata).
us Amycolatopsis, Streptomyces (genus Stre)
ptomyces), Genus Absidia, Genus Mucor, Synsephalastrum (gen
us Syncephalastrum, Genus Mort
ierella), a microorganism belonging to the genus Rhizopus or the genus Bacillus,
A microorganism capable of converting a compound of the general formula (II) into a compound of the general formula (I). The genus Amycolata and Amycolatopsis were previously classified as Nocardia,
Due to the difference in bacterial components, they now form a new genus independently of the genus Nocardia (Internationa
l Journal of Systematic Bacteriology, Vol. 36, No. 1,
p.29,1986).

The fungi belonging to the genus Amycolata used in the method of the present invention include, for example, Amycolata. Autotrophica (A. autotrophica) can be mentioned. Representative examples are deposited at the Institute of Biotechnology and Industrial Technology, the Ministry of International Trade and Industry, and
The deposit numbers of 82 and 6183 are assigned. These deposited bacteria were Nocardia sp. SANK62781, Noc
ardia sp. SANK62881 and Nocardia sp. SANK62981, respectively, and their mycological properties are described in
No. 89191.

The fungi belonging to the genus Amycolatopsis used in the method of the present invention include, for example, Amycolatopsis. A. orientalis and Amycolatopsis. Mediterranei (A.mediterranei). Representative of them are IFO12806 and IFO13415, which are stock strains of the Fermentation Research Institute,
All of these strains are designated by ISP, namely ISP50
40 and ISP5501.

Furthermore, the bacteria used in the method of the present invention and belonging to the genus Streptomyces include, for example, Streptomyces. S. flavovirens, Streptomyces. Grizeolas (S. griseolus) and Streptomyces. Roseus (S. roseus) can be mentioned. Representative of them are ATCC3320 (ISP5062) and KCC581 (ISP50), which are stocks of public strains.
67) and IFO12818 (ISP5076).

In the method of the present invention, suitable microorganisms are bacteria belonging to the genus Amycolata and Amycolatopsis, and in particular, Amycolata. Autotrophyca FERM P-6183 and Amycolatopsis. Mediterranean IFO13415 is most preferred.

The method of the present invention can be performed in various ways. For example, (1) a method in which a compound of the formula (II) as a substrate is brought into contact with a medium in which microorganisms are cultured, and (2) cells are collected from a medium in which microorganisms are cultured, and the compound of formula (II) is contacted with the collected cells. And (3) a method in which a cell-free extract prepared from cells is contacted with a compound of the formula (II).

Culture of the transformed bacteria is usually performed by culturing in a medium containing nutrients that can be used by the microorganism. As a nutrient source, a known nutrient used for cultivation of general actinomycetes can be used.

For example, as a carbon source, glucose, sucrose, maltose, lactose, starch, glycerin, starch syrup, molasses, soybean oil and the like are used. In addition, as a nitrogen source, soybean flour, wheat germ, meat flour, fish meal, meat extract, peptone,
Corn stay liquor, dried yeast, ammonium salts such as ammonium nitrate and the like are used. In addition, if necessary, in addition to inorganic salts such as salt, potassium chloride, calcium carbonate, and phosphate, as well as additives and the like that assist the growth of bacteria and promote the production of the enzyme having the hydroxylation ability, etc. Can be used.

The culture is performed under aerobic conditions and the culture temperature is 20-40.
° C, preferably 26-35 ° C.

The method (1) is carried out by adding the compound of the formula (II) and culturing. The timing of the addition varies depending on the optimal culture conditions of the transformed bacteria used, especially the culture device, medium composition, culture temperature, etc. It is preferred that 1-5 days have elapsed. The amount of the starting compound, that is, the substrate, is 0.01-5.0%, preferably 0.025-2.0%, based on the medium.

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

The method (2) is carried out by culturing the transformed bacterium in the presence of a small amount of the substrate by the method of the above (1) and culturing until the hydroxylation ability of the transformed bacterium is maximized.

That is, the hydroxylation ability varies depending on the type of medium, temperature, etc., but usually becomes maximum 2-3 days after the start of culture.
At this point, the culture is terminated. Harvesting involves centrifuging the culture,
It is performed by subjecting to a method such as 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 thus obtained transformed bacterial cells 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 20-45 ° C, preferably at 25-35 ° C. The concentration of the substrate is usually 0.01-5.0% based on the culture medium.
It is. The reaction time depends on the substrate concentration, reaction temperature, etc.
Usually it is about 1-5 days.

The cell-free extract obtained by the method (3) is obtained by applying a physical or chemical technique to the transformed bacterial cells obtained by the above method, for example, by grinding, sonication, etc. Alternatively, it can be obtained as a cell lysate 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 raw material compound in the same manner as in the above-mentioned method of bringing into contact with the transformed bacterial cells.

After the 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 filtered, the obtained filtrate is extracted with an organic solvent which is hardly miscible with water, such as ethyl acetate, and the solvent is distilled off from the extract. The compounds can be separated and purified by column chromatography using silica gel, alumina or the like and eluting with a suitable eluent.

The natural milbemycins, which are the starting materials for the compounds of formula (II), are fermentation products in which a number of analogs are produced in various proportions and each analog is isolated or isolated after mixing. It is subjected to the reaction as it is. Therefore, the formula (I
The compound of I) can be either a single compound or a mixture thereof.

Thus, the compounds of formula (I) may also be produced as single compounds or as mixtures thereof. Of the compounds of the general formula (I), the compounds of the following formula (Ia) are novel per se and form part of the invention: (Wherein, U represents a hydroxymethyl group or a carboxy group, W represents a hydrogen atom, a hydroxyl group or a halogen atom, Xa represents a methyl group or an ethyl group when U is a hydroxymethyl group, and Is a carboxy group, methyl group, ethyl group, isopropyl group or sec
A butyl group, Y represents a hydroxyl group or a hydroxyimino group, and Z represents a hydrogen atom or a hydroxyl group).

The compounds of formula (I) have pesticidal, acaricidal and anthelmintic activity per se or are useful as synthetic intermediates for other compounds having pesticidal, acaricidal and anthelmintic activity.

The compound of the formula (I) can be used for the spider mites (Tetranychus) and the apple spider mites (Panonychu) parasitic on fruit trees, vegetables and flowers.
s) and adults such as rust mites, larvae and eggs, animals, ticks (Ixodidae), and mites (Dermanyssida)
e) and excellent acaricidal activity against Sarcoptidae and the like.

In addition, sheep flies (Oestrus) and flies (Lucili)
a), bullflies (Hypoderma), horseflies (Gautrophilu)
s), etc., and ectoparasites of animals and birds such as fleas and lice; sanitary pests such as cockroaches and house flies; and other agricultural and horticultural pests such as aphids and lepidopteran larvae . Furthermore, root-knot nematodes (Meloidogyne) and pine wood nematodes (Bursaphelen)
chus), spider mite (Phizoglyphus) and the like.

Also, the compound of formula (I) may be an insect harmful to plants,
In particular, it has activity against insects that cause harm by ingesting plants.

Furthermore, the compounds of the formula (I) have excellent parasiticidal activity as animal and human anthelmintics. It is particularly effective against nematodes that infect livestock, poultry and pets such as pigs, sheep, goats, cattle, horses, dogs, cats and chickens.

When the compound of the formula (I) is used for agricultural and horticultural purposes, it is prepared and used in formulations known in the art, such as powders, wettable powders, and emulsions. If necessary, when used after being diluted with water, the concentration of the active ingredient is about 1-10 ppm.

When the compounds of formula (I) are used in anthelmintic agents for animals,
It is prepared and used in formulations well known in the art, such as powders, tablets, capsules, and injections. When administered orally,
The dosage is about 0.01-100 mg / kg body weight, preferably
It is about 0.5-50 mg.

Next, the present invention will be described more specifically with reference to examples.

Example 1 Six 500 ml Erlenmeyer flasks containing 100 ml of a medium having the following composition were charged with Amycolata. Auto Trofica (A.autotr
ophica: Microtechnological Laboratory No. 6183), 28 ℃, 220rp
m. Four days later, Milbemycin A4 (Formula I
I: V = W = hydrogen atom, X = ethyl group, Y = hydroxyl group) was added to the final concentration of 0.05% using a 5% dioxane solution, and the cells were further cultured at 28 ° C. and 220 rpm for 1 day. .

Medium composition Glucose 1.0% Yeast extract 0.3% Malt extract 0.3% Peptone 0.5% Tap water residue (pH uncorrected) After the culture is completed, the reaction solution is extracted twice with 600 ml of ethyl acetate, and the extract is dried over anhydrous sodium sulfate. Concentrated. The residue was purified by silica gel column chromatography, and 30-hydroxymilbemycin A4 (formula I: U = hydroxymethyl group, V = W = Z = hydrogen atom, X = ethyl group,
Y = hydroxyl group (114.6 mg, yield 37.1%), 26,30-dihydroxymilbemycin A4 (formula I: U = hydroxymethyl group, V = W = hydrogen atom, X = ethyl group, Y = Z = hydroxyl group) 47.7 mg (15.0% yield), Milbemycin-30-Oic Acid A4 (Formula I: U = carboxy group, V = W =
30.1 mg of Z = hydrogen atom, X = ethyl group, Y = hydroxyl group)
(9.5% yield). In addition, Milbemycin A4 is 64.8m
g (recovery rate 21.6%).

30-hydroxy compound mass spectrum (m / z): 558 (M +), 540, 430, 412, 37
2, 314, 280, 211, 183 Nuclear magnetic resonance spectrum δ (CDCl3) ppm: 1.00 (d, 3H, C28H
3, J = 7.0Hz), 1.01 (t, 3H, C32H3, J = 7.0Hz), 1.52 (s, 3
H, C29H3), 1.87 (br.s, 3H, C26H3), 3.26 (m, 1H, C2H),
3.33 (dt, 1H, C25H, J = 2.8, 8.5Hz), 3.45-3.65 (m, 1H, C
17H), 3.49 (dd, 1H, C30H, J = 6.0, 10.6Hz), 3.63 (dd, 1
H, C30H, J = 4.4,10.6Hz), 3.95 (d, 1H, C6H, J = 6.2Hz),
4.05 (br.s, 1H, C70H), 4.28 (br.s, 1H, C5H) 26.30-dihydroxy compound Mass spectrum (m / z): 574 (M +), 556,538,280,211,
183 Nuclear magnetic resonance spectrum δ (CDCl3) ppm: 1.00 (d, 3H, C28H
3, J = 6.5Hz), 1.02 (t, 3H, C32H3, J = 6.5Hz), 1.53 (s, 3
H, C29H3), 3.49 (dd, 1H, C30H, J = 6.2,11.0Hz), 3.63 (d
d, 1H, C30H, J = 4.2,11.0Hz), 3.98 (d, 1H, C6H, J = 6.2H
z), 4.13 (br.s, 1H, C70H), 4.24 (d, 1H, C26H, J = 12.8H
z), 4.32 (d, 1H, C26H, J = 12.8Hz), 4.58 (br.s, 1H, C5
H) 30-Oic acid mass spectrum (m / z): 572 (M +), 444,426,294,225,
197,151 Nuclear magnetic resonance spectrum δ (CDCl3) ppm: 1.00 (d, 3H, C28H
3, J = 6.5Hz), 1.02 (t, 3H, C32H3, J = 7.2Hz), 1.53 (s, 3
H, C29H3), 1.87 (s, 3H, C26H3), 3.28 (m, 1H, C2H), 3.5
−3.7 (m, 2H, C17H, C25H), 3.96 (d, 1H, C6H, J = 6.4Hz),
4.01 (br.s, 1H, C70H) Example 2 According to the method of Example 1, 30-hydroxymilbemycin A4 was obtained using milbemycin A4 as a substrate and the following kinds of microorganisms. Microbial 30-hydroxy conversion ratio Amycolatopsis orientalis IFO12806 +3 Amycolatopsis mediterranei IFO13415 +4 Amycolata autotrophica FERM P-6181 +1 Amycolata autotrophica FERM P-6182 +1 Streptomyces flavovirens IFO12771 rusus +3 Streptomyces gers rusus +3 Streptomyces resp. nigricans NRRL45 +2 Syncephalastrum racemosum IFO4827 +1 Mortierella isabellina NRRL1757 +4 Bacillus megaterium IFO12108 +2 The conversion rate is based on the following criteria: +1: 0.5-5.0% +2: 5.0-10.0% +3: 10.0-30.0% +4: 30.0-50.0 Examples 3 Amycolata. Was added to six 500 ml Erlenmeyer flasks containing 100 ml of the medium having the same composition as in Example 1. Inoculated with A. autotrophica (A. autotrophica: No. 6183) and 2
The cells were cultured with shaking at 220 rpm at 8 ° C. After 5 days, 5-ketomylbemycin A45-oxime (Formula II: V = W = hydrogen atom,
(X = ethyl group, Y = hydroxyimino group) was added using the 5% dioxane solution to a final concentration of 0.05%, and the cells were further cultured at 28 ° C. and 220 ppm for 7 days.

After completion of the culture, the reaction solution was extracted three times with 500 ml of ethyl acetate, and the extract was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography, and 30-hydroxy-5-ketomylbemycin A45-oxime (formula I: U = hydroxymethyl group, V = W = Z = hydrogen atom, X = ethyl group, Y = hydroxyimino) Base) to 3
0.4 mg (9.8% yield) was obtained.

Mass spectrum (m / z): 571 (M +), 553, 537, 211, 183 Nuclear magnetic resonance spectrum δ (CDCl3) ppm: 1.00 (d, 3H, C28H)
3, J = 7.0Hz), 1.03 (t, 3H, C32H3, J = 7.0Hz), 1.53 (s, 3
H, C29H3), 3.38 (m, 1H, C2H), 3.40 (dt, 1H, C25H, J = 2.
3,9.5Hz), 3.45-3.65 (m, 1H, C17H), 3.49 (dd, 1H, C30
H, J = 6.2,10.8Hz), 3.63 (dd, 1H, C30H, J = 4.0,10.8H
z), 3.96 (br. s. 1H, C70H), 4.67 (s, 1H, C6H) Example 4 Two 100 ml Erlenmeyer flasks containing 17 ml of a medium having the same composition as in Example 1 were charged with Amycolata. Inoculated with A. autotrophica (A. autotrophica: No. 6183) and 2
The cells were cultured with shaking at 220 rpm at 8 ° C. After 3 days, 13-hydroxymilbemycin A4 (Formula II: V = hydrogen atom, W = Y = hydroxyl group, X = ethyl group) is added to a final concentration of 0.05% using a 5% dioxane solution, 28 more days
The cells were cultured at 220 ° C. at 220 ° C. After completion of the culture, the reaction solution was extracted three times with 50 ml of ethyl acetate, and the extract was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography, and 8.7 mg of 13,30-dihydroxymilbemycin A4 (formula I: U = hydroxymethyl group, V = Z = hydrogen atom, W = Y = hydroxyl group, X = ethyl group) was obtained. Rate 49.7
%)Obtained.

Mass spectrum (m / z): 574 (M +), 556, 540, 295, 277,
211,183,151 Nuclear magnetic resonance spectrum δ (CDCl3) ppm: 1.01 (t, 3H, C32H
3, J = 7.3Hz), 1.13 (d, 3H, C28H3, J = 6.4Hz), 1.58 (s, 3
H, C29H3), 1.88 (s, 3H, C26H3), 3.28 (m, 1H, C2H), 3.35
(Dt, 1H, C25H, J = 3.2,10.2Hz), 3.51 (dd, 1H, C30H, J =
6.2,11.0Hz), 3.5-3.65 (m, 1H, C17H), 3.68 (dd, 1H, C3
0H, J = 4.2,11.0Hz), 3.71 (d, 1H, C13H, J = 10.1Hz), 3.9
6 (d, 1H, C6H, J = 6.0 Hz), 3.99 (s, 1H, C70H), 4.29 (br.
s, 1H, C5H) Example 5 Amycolata. was added to five 500 ml Erlenmeyer flasks containing 100 ml of a medium having the same composition as in Example 1. Inoculated with A. autotrophica (A. autotrophica: No. 6183) and 2
The cells were cultured with shaking at 220 rpm at 8 ° C. After 3 days, milbemycin A3 (Formula II: V = W = hydrogen atom, X = ethyl group, Y = hydroxyl group) was added to its final concentration using a 5% dioxane solution.
It was added to a concentration of 0.05%, and further cultured at 28 ° C. and 220 ppm for 4 days. After completion of the culture, the reaction solution was diluted with 500 ml of ethyl acetate.
The extract was extracted twice, and the extract was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography, and 30-hydroxymilbemycin A3 (formula I: U =
57.5 mg (yield: 22.3%) of hydroxymethyl group, V = W = Z = hydrogen atom, X = ethyl group, Y = hydroxyl group were recovered, and 40 mg of milbemycin A3 was recovered (recovery rate: 16.0%).

Mass spectrum (m / z): 544 (M +), 526,416,266,197,
169,151 Nuclear magnetic resonance spectrum δ (CDCl3) ppm: 1.00 (d, 3H, C28H
3, J = 6.4Hz), 1.21 (d, 3H, C31H3, J = 6.4Hz), 1.53 (s, 3
H, C29H3), 1.87 (s, 3H, C26H3), 3.27 (m, 1H, C2H), 3.50
(Dd, 1H, C30H, J = 6.5,11.0Hz), 3.65 (dd, 1H, C30H, J =
4.2, 11.0Hz), 3.45-3.6 (m, 2H, C17H, C25H), 3.96 (d, 1
H, C6H, J = 6.4 Hz), 4.07 (br.s, 1H, C70H) Example 6 Amycolata. Was placed in five 500 ml Erlenmeyer flasks containing 100 ml of the medium having the same composition as in Example 1. Inoculated with A. autotrophica (A. autotrophica: No. 6183) and 2
The cells were cultured with shaking at 220 rpm at 8 ° C. After 2 days, Milbemycin D (Formula II: V = W = hydrogen atom, X = isopropyl group,
Y = hydroxyl group) was added thereto using a 5% dioxane solution to a final concentration of 0.05%.
The cells were cultured at 0 rpm. After the culture is completed, the reaction solution is
The extract was extracted three times with l, and the extract was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography, and 30-hydroxymilbemycin D (formula
I: U = hydroxymethyl group, V = W = Z = hydrogen atom, X
= Isopropyl group, Y = hydroxyl group) 22.7 mg (8.8 yield)
%), 26,30-dihydroxymilbemycin D (formula I: U =
4.0 mg (yield 1.5%) of hydroxymethyl group, V = W = hydrogen atom, X = isopropyl group, Y = Z = hydroxyl group, milbemycin-30-oic acid D (formula I: U = carboxy group, V = 26.4 mg (Yield 10.0%) of W = Z = hydrogen atom, X = isopropyl group, Y = hydroxyl group were obtained. Further, 94.3 mg of milbemycin D (recovery rate: 37.7%) was recovered.

30-hydroxy compound mass spectrum (m / z): 572 (M +), 444,426,314,294,
248,225,197,179,151 Nuclear magnetic resonance spectrum δ (CDCl3) ppm: 0.91 (d, 3H, C32H
3, J = 6.8Hz), 1.00 (d, 3H, C28H3, J = 6.4Hz), 1.06 (d, 3
H, C33H3, J = 6.8Hz), 1.53 (s, 3H, C29H3), 1.87 (s, 3H, C
26H3), 3.27 (m, 1H, C2H), 3.34 (dd, 1H, C25H, J = 2.0, 9.
7Hz), 3.46 (dd, 1H, C30H, J = 6.0, 10.9Hz), 3.60 (m, 1H,
C17H), 3.62 (dd, 1H, C30H, J = 4.0, 10.9Hz), 3.96 (d, 1
H, C6H, J = 6.0 Hz), 4.05 (br.s, 1H, C70H) 26,30-dihydroxy compound Mass spectrum (m / z): 588 (M +), 572,444,426,294,
225,197,179,151 Nuclear magnetic resonance spectrum δ (CDCl3) ppm: 0.91 (d, 3H, C32H
3, J = 6.8Hz), 1.00 (d, 3H, C28H3, J = 6.4Hz), 1.06 (d, 3
H, C33H3, J = 6.8Hz), 1.53 (s, 3H, C29H3), 3.3-3.4 (m,
2H, C2H, C25H), 3.47 (dd, 1H, C30H, J = 6.0,10.9Hz), 3.5
6 (m, 1H, C17H), 3.62 (dd, 1H, C30H, J = 4.0,10.9Hz), 3.
98 (d, 1H, C6H, J = 6.4Hz), 4.12 (br.s, 1H, C70H), 4.24
(D, 1H, C26H, J = 13.7Hz), 4.33 (d, 1H, C26H, J = 13.7H
z) 30-Oic Acid mass spectrum (m / z): 586 (M +), 568,458,308,248,
239,211,151 Nuclear magnetic resonance spectrum δ (CDCl3) ppm: 0.95 (d, 3H, C32H
3, J = 6.8Hz), 1.00 (d, 3H, C28H3, J = 6.8Hz), 1.06 (d, 3
H, C33H3, J = 6.8Hz), 1.53 (s, 3H, C29H3), 1.87 (s, 3H, C
26H3), 3.26 (m, 1H, C2H), 3.55 (m, 1H, C17H), 3.66 (d
d, 1H, C25H, J = 2.0,10.0Hz), 3.96 (d, 1H, C6H) Example 7 Eight 100 ml Erlenmeyer flasks containing 20 ml of the medium having the same composition as in Example 1 were charged with Amycolata. Inoculated with A. autotrophica (A. autotrophica: No. 6183) and 2
The cells were cultured with shaking at 220 rpm at 8 ° C. Two days later, 13-fluoromilbemycin A4 (Formula II: V = hydrogen atom, W = fluorine atom, X = ethyl group, Y = hydroxyl group) is adjusted to a final concentration of 0.05% using a 5% dioxane solution. Added to
The cells were further cultured at 28 ° C. and 220 rpm for 2 days. After completion of the culture, the reaction solution was extracted three times with 160 ml of ethyl acetate, and the extract was dried over anhydrous sodium sulfate and concentrated. The residue is separated by preparative thin-layer silica gel chromatography (Merck, Art5717, 20
x20 cm, thickness 2 mm, developed with ethyl acetate), 13-fluoro-30-hydroxymilbemycin A4 (formula I: U = hydroxymethyl group, V = Z = hydrogen atom, W = fluorine atom, X = ethyl group , Y = hydroxyl group) 15.7 mg (19.1 yield)
%) And 13-fluoro-26,30-dihydroxymilbemycin A4 (formula I: U = hydroxymethyl group, V = hydrogen atom,
(W = fluorine atom, X = ethyl group, Y = Z = hydroxyl group) 9.
5 mg (yield 11.2%) was obtained.

13-fluoro-30-hydroxy compound mass spectrum (m / z): 576 (M +), 558,448,332,26
6,211,183,151 Nuclear magnetic resonance spectrum δ (CDCl3) ppm: 1.01 (t, 3H, C32H
3, J = 7.3Hz), 1.15 (d, 3H, C28H3, J = 6.0Hz), 1.61 (s, 3
H, C29H3), 1.88 (s, 3H, C26H3), 3.49 (dd, 1H, C30H, J =
4.8, 11.1Hz), 3.60 (m, 1H, C17H), 3.64 (dd, 1H, C30H, J
= 4.8,11.1Hz), 3.97 (d, 1H, C6H, J = 5.9Hz), 4.04 (s, 1
H, C70H), 4.29 (br.s, 1H, C5H), 4.40 (dd, 1H, C13H, J =
9.9,47.9 Hz) 13-fluoro-26,30-dihydroxy compound mass spectrum (m / z): 592 (M +), 574,556,448,332,
266,211,183,151 Nuclear magnetic resonance spectrum δ (CDCl3) ppm: 1.02 (t, 3H, C32H
3, J = 7.3Hz), 1.16 (d, 3H, C28H3, J = 6.2Hz), 1.62 (s, 3
H, C29H3), 3.51 (dd, 1H, C30H, J = 6.6,11.3Hz), 3.60
(M, 1H, C17H), 3.65 (dd, 1H, C30H, J = 4.6,11.3Hz), 3.9
9 (d, 1H, C6H, J = 6.2Hz), 4.11 (s, 1H, C70H), 4.24 (d, 1
H, C26H, J = 13.0Hz), 4.32 (d, 1H, C26H, J = 13.0Hz), 4.4
2 (dd, 1H, C13H, J = 9.9, 47.9 Hz) Example 8 Amycolatopsis was placed in 22 100 ml Erlenmeyer flasks containing 20 ml of the medium having the same composition as in Example 1. Inoculated with mediterranea (A.mediterranei: IFO13415), 28 ℃, 220rp
m.

Two days later, 22,23-dihydroavermectin Bla (formula I
I: V = hydrogen atom, W = 4 ′-(α-L-oleandrosyl) -α-L-oleandrosyloxy group, X = sec-butyl group, Y = hydroxyl group using a 5% dioxane solution thereof To a final concentration of 0.025%, and the cells were further cultured at 28 ° C. and 220 rpm for 7 days. After the completion of the culture, the reaction solution was centrifuged to separate the cells into supernatants. Ethyl acetate 400m
The extract was extracted three times with l, and the extract was dried over anhydrous sodium sulfate and concentrated. The cells are 100 ml of 80% methanol aqueous solution
, Extracted twice with ethyl acetate in the same manner as the supernatant, and concentrated. The extract from the cells and the supernatant was purified by silica gel column chromatography,
30-hydroxy-22,23-dihydroavermectin Bla
29.7 mg (yield 26.5%) was obtained. Further, 17.6 mg (recovery rate 16.4%) of 22,23-dihydroavermectin Bla was recovered.

Mass spectrum (m / z): 728 (M + -162), 602,584,5
68,323,305,211,145 Nuclear magnetic resonance spectrum δ (CDCl3) ppm: 0.89 (d, 3H, CH32
H3, J = 6.5Hz), 0.93 (t, 3H, C34H3, J = 7.3Hz), 1.15 (d,
3H, C28H3, J = 6.8Hz), 1.24 (d, 3H, J = 6.0Hz), 1.26 (d,
3H, J = 6.0Hz), 1.49 (s, 3H, C29H3), 1.87 (s, 3H, C26H)
3), 3.41 (s, 3H), 3.42 (s, 3H), 3.35-3.55 (m, 2H, C30
H2), 3.55-3.9 (m, 5H), 3.94 (br.s, 1H, C13H), 3.95
(D, 1H, C6H, J = 6.4 Hz), 4.10 (br.s, 1H, C70H) Example 9 Amycolata. Was added to 14 100 ml Erlenmeyer flasks containing 20 ml of the medium having the same composition as in Example 1. Inoculated with A. autotrophica (A. autotrophica: No. 6183) and 2
The cells were cultured with shaking at 220 rpm at 8 ° C. Three days later, LL-F28249
α (Formula II: V = Y = hydroxyl group, W = hydrogen atom, X = 1,3-dimethyl-1-butenyl group) is added to the final concentration of 0.03% using a 5% dioxane solution. And 4 more
One day later, the cells were cultured at 28 ° C. and 220 rpm. After completion of the culture, the reaction solution was extracted three times with 200 ml of ethyl acetate, and the extract was dried over anhydrous sodium sulfate and concentrated. The residue is separated by preparative thin-layer silica gel chromatography (Merck, Art5717, 20x20c
m, thickness 2 mm, developed with ethyl acetate) and purified with 30-hydroxy-LL-F28249α (formula I: U = hydroxymethyl group, V
= Y = hydroxyl group, W = Z = hydrogen atom, X = 1,3-dimethyl-1-butenyl group) (4.0 mg, yield 4.6%). further
15 mg (recovery rate 17.9%) of LL-F28249α was recovered.

30-hydroxy compound Mass spectrum (m / z): 628 (M +), 610,592,482,464 Nuclear magnetic resonance spectrum δ (CDCl3) ppm: 3.21 (m, 1H, C2)
H), 3,3-3.45 (m, 3H, C25H, C30H2), 3.51 (d, 1H, C230H,
J = 5.6Hz), 3.5-3.7 (m, 1H, C17H), 3.75-3.78 (m, 2H,
C23H, C300H), 3.79 (s, 1H, C70H), 3.95 (d, 1H, C6H, J =
6.0Hz), 4.29 (t, 1H, C70H), 4.68 (br.s, 2H, C27H2), 4.
95 (m, 1H, C15H), 5.17 (d, 1H, C33H, J = 9.3 Hz) 5.25-5.
41 (m, 3H, C3H, C11H, C19H), 5.65-5.8 (m, 2H, C9H, C10
H)

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical indication (C12P 17/18 C12R 1: 465) (C12P 17/18 C12R 1: 785) (C12P 17/18 (C12R 1: 845) (C12P 17/18 C12R 1:11) (C12P 17/18 C12R 1:01) (C12P 17/18 C12R 1: 645)

Claims (2)

(57) [Claims] 1. A genus of the genus Amycolata, Amycolatopsis or Streptomyces which can be converted into a compound represented by the following general formula (I) using a compound represented by the following general formula (II) as a substrate: , A microorganism belonging to the genus Absidia, the genus Mucor, the genus Synthephalastrum, the genus Mortierella, the genus Rhizopus or the genus Bacillus, in a medium containing a compound represented by the general formula (II) as a substrate, The cultured cell of the microorganism is converted into a compound represented by the general formula (I) by contacting the enzyme extract with a compound represented by the general formula (II), and then the compound represented by the general formula (I) A method for producing a compound represented by the general formula (I), which comprises collecting: (Wherein V represents a hydrogen atom, a hydroxyl group or an oxo group,
W is a hydrogen atom, a hydroxyl group, a halogen atom or 4 ′-(α
-L-oleandrosyl) -α-L-oleandrosyloxy group, and X represents a methyl group, an ethyl group, an isopropyl group, a sec-butyl group,
A methyl-1-propenyl group, a 1-methyl-1-butenyl group or a 1,3-dimethyl-1-butenyl group, and when V is a hydroxyl group or an oxo group, 1-methyl-1
-Propenyl group, 1-methyl-1-butenyl group or 1,
Y represents a hydroxyl group or a hydroxyimino group, and represents a 3-dimethyl-1-butenyl group. ): (In the formula, U represents a hydroxylmethyl group or a carboxy group, V, W, X and Y have the same meanings as described above, and Z represents a hydrogen atom or a hydroxyl group.) 2. A compound represented by the following general formula (Ia): (Wherein, U represents a hydroxylmethyl group or a carboxy group, W represents a hydrogen atom, a hydroxyl group or a halogen atom, Xa represents a methyl group or an ethyl group when U is a hydroxymethyl group, and U represents a carboxy group. When it is a group, it represents a methyl group, an ethyl group, an isopropyl group or a sec-butyl group, Y represents a hydroxyl group or a hydroxyimino group, and Z represents a hydrogen atom or a hydroxyl group).