JP2700028B2 - Method for producing macrolide compound - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing a macrolide compound, and more particularly to a method for producing milbemycins and analogs thereof. Milbemycin is a series of macrolide compounds disclosed in
Compounds of the following formula (III) known from JP-A-742, JP-A-56-32481 and the like.

In the formula, X represents a hydrogen atom, a dotted line represents a single bond,
Y represents a methyl, ethyl or iso-4-propyl group, and is called milbemycin A ₃ , milbemycin A ₄ and milbemycin D, respectively.

In the above formula (III), X is a hydrogen atom, and Y is
Is a sec-butyl group is a milbemycin analog described in JP-A-54-145699. In the above formula (III), X is a hydroxyl group and Y is 1
A similar compound which is a -methyl-1-propenyl, 1-methyl-1-butenyl or 1,3-dimethyl-1-butenyl group is known as LL-F28249 in JP-A-61-10589.

In the above formula (III), X is a hydrogen atom, an oxo group, a hydroxyl group or an alkoxy group, and Y is 1-methyl-1-propenyl, 1-methyl-1-butenyl or 1,
Analogous compounds which are 3-dimethyl-1-butenyl groups are described in JP-A-61-280496. Furthermore,
In the above formula (III), X is a hydrogen atom or a hydroxyl group, and Y is an alpha-branched C _3-8 alkyl, alkenyl, alkynyl, alkoxyalkyl or alkylthioalkyl group; the alkyl group is an alpha-branched C ₂ -C ₅ alkyl group A C ₅ -C ₈ cycloalkylalkyl group; C ₃ -C ₈ cycloalkyl or C ₅ -C ₈ cycloalkenyl group (both optionally methylene or one or more C ₁ -C ₄ alkyl groups or halogen atoms Or saturated or wholly or partially unsaturated and optionally substituted by one or more C ₁ -C ₄ alkyl groups or halogen atoms. Analogous compounds which are membered oxygen or sulfur containing heterocycles are described in JP-A-62-29590. All of these compounds are known to have insecticidal, acaricidal and anthelmintic activities.

The present inventors have made intensive efforts to search for analogs of these milbemycins, and have found that the milbemycins are efficiently hydroxylated at position 29 by converting the milbemycins using a microorganism or an enzyme produced thereby. Thus, the present invention has been completed.

JP-A-61-233686 discloses the microbial conversion of 22,23-dihydroavermectin aglycone or 13-deoxy-22,23-dihydroavermectin aglycone. The microbes to be hydroxylated and the locations where they are hydroxylated are also quite different.

JP-A-62-59291 discloses 22,23-dihydro-avermectin B1a, avermectin B1a and avermectin B1b.
However, as will be described later, the substrate used, the microorganism and the hydroxylation position are considerably different from those of the present invention.

According to the present invention, a microorganism belonging to the genus Synthephalastrum or the genus Rhizopus, which can convert a compound represented by the following general formula (II) into a compound represented by the following general formula (I) as a substrate: Is cultured in a medium containing the compound represented by the general formula (II) as a substrate,
Alternatively, a compound represented by the general formula (I) can be produced by contacting a cultured cell of these microorganisms or an enzyme extract with a compound represented by the general formula (II).

(In the formula, X represents a hydrogen atom, a hydroxyl group or an oxo group.
The dotted line indicates a single bond or a double bond, and may be a double bond only when X is a hydrogen atom. Y is a C _1-8 alkyl group,
C _2-8 alkenyl or alkynyl group, C _1-4 alkoxy or alkylthio C _1-4 alkyl group, C _3-8 cycloalkyl C _1-5 alkyl group, C _3-8 cycloalkyl or C _5-8
A cycloalkenyl group (both optionally substituted by methylene or one or more C _1-4 alkyl or halogen), or a saturated or unsaturated 3- to 6-membered oxygen- or sulfur-containing heterocycle ( Which may be substituted with one or more C _1-4 alkyl or halogen), and Z represents a hydroxyl group or a hydroxyimino group. ) (In the formula, X, Y and Z have the same meanings as described above.)

The process of the present invention relates to the microbial hydroxylation of a compound of general formula (II). In the method of the present invention,
Only the methyl group at position 29 is hydroxylated, and the methyl group bonded at positions 4, 12, and 24 and the Y group bonded at position 25 are not hydroxylated. Further, the double bond at the 3-, 8-, 10-, and 14-positions and the Y group are 1-methyl-1-propenyl group, 1-methyl-
In the case of a 1-butenyl group or a 1,3-dimethyl-1-butenyl group, the double bond at position 31 is not subjected to oxidation such as epoxidation. Among the compounds of the general formula (II) which are the starting materials of the process of the present invention, those wherein X is a hydrogen atom and Z is a hydroxyimino group are described in JP-A-59-10.
It is known from JP 8785.

The microorganism used in the method of the present invention is a microorganism belonging to the genus Syncephalastrum or the genus Rhizopus, and converts a compound of the general formula (II) into a compound of the general formula (I). Microorganisms to be obtained. The types of microorganisms used in the method of the present invention and the typical strains thereof and the strains stored in public strain collection agencies are as follows.

Syncephalastrum nigricans NRRL 12479 Syncephalastrum racemosum IFO 4827 Syncephalastrum racemosum IFO 4814 Syncephalastrum racemosum IFO 4828 Rhizopus circinans ATCC 1225 Among these microorganisms, Syncephalastrum racemosu
m IFO 4827 is most preferred for the method of the present invention.

The method of the present invention can be performed in various aspects. 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 (III).

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, soy 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, phosphate, etc., additives that assist the growth of bacteria and promote the production of the enzyme having the hydroxylation ability, for example, yeast extract and Malt extract and the like can be used in appropriate combination. The cultivation is performed under aerobic conditions, and the culturing temperature is 20 to 40 ° C, preferably 26 to 40 ° C.
30 ° 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 carried out by applying 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 25-30 ° 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 temperature, reaction temperature, etc.
Usually it is about 1-5 days.

The cell-free extract obtained by the method (3) is obtained by suspending the transformed bacterial cells obtained by the above method in an aqueous medium, for example, a phosphate buffer of pH 5-9, by physical, chemical or biochemical techniques. Is applied, for example, as a disrupted cell by grinding, sonication, or the like, or as a cell lysate by treating 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, and the obtained filtrate is extracted with an organic solvent that is hardly miscible with water, such as ethyl acetate, and after the solvent is distilled off from the extract, the obtained crude target compound is obtained. Separation and purification can be achieved 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 large 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.

The compounds of formula (I) themselves have insecticidal, acaricidal and anthelmintic activity, or are useful as other compounds with insecticidal, acaricidal and anthelmintic activity, synthetic intermediates.

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 (Gantrophlu)
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 (Rhizoglyphus) 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 Five 500 ml Erlenmeyer flasks each containing 100 ml of a medium having the following composition were put into a Synthefalastrum racemosam (S.ra).
cemosum IFO 4827) was inoculated and cultured with shaking at 220 rpm at 26 ° C. Two days later, milbemycin A ₄ (Formula II: X = hydrogen atom, Y = ethyl group, Z = hydroxyl group) was added using the 5% dioxane solution to a final concentration of 0.05%,
The cells were further cultured at 26 ° C. and 220 rpm for 7 days.

Medium composition Glucose 1.0% Yeast extract 0.3% Malt extract 0.3% Peptone 0.5% Remaining tap water (pH uncorrected) After completion of the culture, the reaction solution was aspirated and divided into cells and liquid. The solution was extracted three times with 300 ml of ethyl acetate, and the extract was dried over anhydrous sodium sulfate and concentrated. 80 cells
After extracting twice with 100 ml of methanol and evaporating the methanol, the mixture was extracted with ethyl acetate and concentrated in the same manner as the liquid. The extracts from the cells and the liquid were combined, and fractionated thin-layer silica gel chromatography (Merck, Art 5717, 20 × 20 cm,
Purified by 2 mm thickness, developed twice with n-hexane: ethyl acetate = 1: 1, and 18 mg of 29-hydroxymilbemycin A ₄ (formula I: X = hydrogen atom, Y = ethyl group, Z = hydroxyl group) ( (Yield 7.0%)
Milbemycin A ₄ to 30 mg (recovery rate of 12.0%) was recovered.

Mass spectrum (m / z): 558 (M ⁺ ), 540, 430, 412,195,
167,151 Nuclear magnetic resonance spectrum δ (CDCl ₃ ) ppm: 0.82 (d, 3H, C ₃₀ H ₃ , J = 6.5 Hz), 0.99 (t, 3H, C ₃₂ H ₃ , J = 7.
3Hz), 1.03 (d, 3H , C 28 H 3, J = 6.9Hz), 1.87 (s, 3H, C
_{26 H 3), 3.07 (dt} , 1H, C 25 H, J = 5.4,8.8Hz), 3.27 (m, 1H,
_{C 2 H), 3.60 (m} , 1H, C 17 H), 3.93 (d, 1H, C 29 H, J = 12.1H
z), 3.95 (d, 1H , C 6 H, J = 6.5Hz), 4.12 (br.s, 1H, C 7 O
H), 4.27 (d, 1H, C ₂₉ H, J = 12.1 Hz) 4.29 (br.s, 1H, C
_{5 H), 4.67 (br.s,} 2H, C 27 H 2), 5.11 (1H, t, C 15 H, J = 8.1H
z) 5.3~5.5 (m, 3H, C 3 H, C 11 H, C 19 H), 5.7~5.85 (m, 2H,
According C ₁₀ H, C ₉ H) the method of Example 1, a milbemycin A ₄ as a substrate, to give a 29-hydroxy milbemycin A ₄ using various microorganisms below.

The conversion rate is based on the following criteria: +1: 0.5-5.0% +2: 5.0-10.0% Example 3 Synthephalastrum racemosam (S) was placed in 10 500 ml Erlenmeyer flasks containing 100 ml of the medium having the same composition as in Example 1. .racemosum IFO 4827) was inoculated and cultured with shaking at 26 ° C. and 220 rpm. One day later, 5-ketomylbemycin A ₄
5-oxime (Formula II: X = hydrogen atom, Y = ethyl group, Z =
Hydroxyimino group) using the 5% dioxane solution to a final concentration of 0.05%, and further added for 7 days
The cells were cultured at 26 ° C. and 220 rpm. After the completion of the culture, the reaction solution was suctioned and separated into bacterial cells and the solution. Ethyl acetate 500ml
, And the extract was dried over anhydrous sodium sulfate and concentrated. The cells were extracted three times with 200 ml of 80% methanol, the methanol was evaporated, and then extracted and concentrated with ethyl acetate in the same manner as the liquid. The extracts from the cells and the liquid were combined, and purified by silica gel column chromatography.
- hydroxy-5-keto milbemycin A ₄ 5-oxime (Formula I: X = hydrogen, Y = ethyl, Z = hydroxyimino group) to give a 10.6 mg (2.06% yield).

Mass spectrum (m / z): 571 (M ⁺ ), 553,195 Nuclear magnetic resonance spectrum δ (CDCl ₃ ) 0.82 (d, 3H, J = 6.4
_{Hz, C 30 H 3),} 1.00 (t, 3H, J = 7.3Hz, C 32 H 3), 1.04 (d, 3H,
_{J = 6.4Hz, C 28 H 3} ), 1.94 (s, 3H, C 26 H 3), 3,37 (m, 1H, C
_{2 H), 3.93 (d,} 1H, J = 11.7Hz, C 29 H), 4.26 (d, 1H, J = 1
_{1.7Hz, C 29 H), 4.67} (s, 1H, C 6 H), 4.6~4.8 (m, 2H, C 27
H), 5.13 (m, 1H , C 15 H), 5.37~5.50 (m, 2H, C 11 H, C
_{19 H), 5.71~5,90 (m,} 3H, C 3 H, C 9 H, C 10 H), 8.11 (br,
s, 1H, = N-OH) Example 4 Rhizopus silcinance (R. circ.) was placed in five 500 ml Erlenmeyer flasks containing 100 ml of a medium having the same composition as in Example 1.
inans ATCC 1225), and cultivated by rotary shaking at 26 ° C. and 200 rpm. Two days later, milbemycin A ₃ (formula II: X = hydrogen atom, Y = methyl group, Z = hydroxyl group) was added using the 5% dioxane solution to a final concentration of 0.025%, and further added for 7 days The cells were cultured at 26 ° C. and 200 rpm. After the completion of the culture, the reaction solution was suctioned and separated into bacterial cells and the solution. The solution was extracted three times with 300 ml of ethyl acetate, and the extract was dried over anhydrous sodium sulfate and concentrated. The cells were extracted twice with 100 ml of 80% methanol, and the methanol was evaporated.
Extracted with ethyl acetate in the same manner as the liquid, and concentrated. The extract from the cells and the extract from the liquid were purified by silica gel column chromatography, respectively, and 29-hydroxymilbemycin A ₃ (formula I: X = hydrogen atom, Y = methyl group, Z =
Hydroxyl) 4.8 mg (yield 6.2%) milbemycin A ₃ 62.5
mg (83.4% recovery rate).

Mass spectrum (m / z) 544 (M ⁺ ), 526,508,416,398,2
66,181,153,151 Nuclear magnetic resonance spectrum δ (CDCl ₃ ) ppm; 0.84 (d, 3H, J = 6.4 Hz, C ₃₀ H ₃ ), 1.03 (d, 3H, J = 6.5 Hz, C
_{28 H 3), 1.15 (d} , 3H, J = 6.1Hz, C 31 H 3), 1.87 (d, 3H, J =
_{1.6Hz, C 26 H 3),} 3,26 (m, 2H, C 2 H, C 25 H), 3.59 (m, 1H, C
_{17 H), 3.94 (d,} 1H, J = 12.5Hz, C 29 H), 3.96 (d, 1H, J =
_{6.4Hz, C 6 H), 4.11} (s, 1H, 7-OH), 4.28 (d, 1H, J = 12.
_{5Hz, C 29 H), 4.26~4.32} (m, 1H, C 5 H), 4.67 (s, 2H, C 27
_{H 2), 5.15 (t,} 1H, J = 7.7Hz, C 15 H), 5.35~5.47 (m, 3
_{H, C 3 H, C 19} H, C 19 H, C 11 H), 5.71~5.83 (m, 2H, C 9 H, C
₁₀ H)

Claims (1)

(57) [Claims] 1. A microorganism belonging to the genus Synthephalastrum or Rhizopus, which is capable of converting a compound represented by the following general formula (II) into a compound represented by the following general formula (I) as a substrate: Culturing in a medium containing the compound represented by the general formula (II) as a substrate, or
The cultured cells or enzyme extracts of these microorganisms are brought into contact with the compound represented by the general formula (II) to convert them into the compound represented by the general formula (I), and then the compound represented by the general formula (I) General formula (I) characterized in that it is collected
Method for producing compound represented by: (In the formula, X represents a hydrogen atom, a hydroxyl group or an oxo group.
The dotted line indicates a single bond or a double bond, and may be a double bond only when X is a hydrogen atom. Y is a C _1-8 alkyl group,
C _2-8 alkenyl or alkynyl group, C _1-4 alkoxy or alkylthio C _1-4 alkyl group, C _3-8 cycloalkyl C _1-5 alkyl group, C _3-8 cycloalkyl or C _5-8
A cycloalkenyl group (both optionally substituted by methylene or one or more C _1-4 alkyl or halogen), or a saturated or unsaturated 3- to 6-membered oxygen- or sulfur-containing heterocycle ( Which may be substituted with one or more C _1-4 alkyl or halogen), and Z represents a hydroxyl group or a hydroxyimino group. ): (In the formula, X, Y and Z have the same meanings as described above.)