JPH01199591A - Production of macrolide compound - Google Patents

Abstract

PURPOSE:To effectively obtain a macrolide compound containing a hydroxylated methyl group at the 29-position thereof with specific fungus or an enzyme produced by the funges, by treating milbemycin as one of a series of macrolides compounds. CONSTITUTION:A compound expressed by formula I (X is H, OH or oxo; a dotted line is single or double bond, with a proviso that the double bond is only the case X is H; Y is 1-8C alkyl, 2-8C alkenyl or alkynyl, 1-4C alkoxy or alkylthio 1-4C alkyl, 3-8C cycloalkyl 1-8C alkyl, etc.; Z is OH or hydroxyimino group) is treated with a fungus such as Syncephalastrum racemosum IFO 4827 or an enzyme produced by the fungus to afford a compound wherein a methyl group at the 29-position is hydroxylated, expressed by formula II.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing macrolide compounds, and more particularly to a method for producing milbemycins and their analogs.

Milbemycin is a series of macrolide compounds, and is described in Japanese Patent Application Laid-open Nos. 50-29742 and 56-32481.
This is a compound represented by the following formula (1), which is known from Japanese Patent Publication No.

JI-i In the formula, X represents a hydrogen atom, a dotted line represents a single bond, and Y
methyl and ethyl represent inglovir groups and are called milbemycin A3, milbemycin A4 and milbemycin D, respectively.

Furthermore, in the above formula (m), X is a hydrogen atom, and Y is 5
Compounds having an ee-butyl group are disclosed in Japanese Patent Application Laid-Open No. 1456-1983.
Milbemycin 1-methyl-1 described in Publication No. 99 etc.
Similar compounds which are -butenyl or 1,3-dimethyl-1-butenyl group are described in JP-A-61-10589.
Known as L-F 28249.

In the above formula (II), X is a hydrogen atom, an oxo group,
hydroxyl group or alkoxy group, Y is 1-methyl-1
-propenyl, 1-methyl-1-butenyl still 1,3
-Dimethyl-1-butenyl group is described in JP-A-61-280496. Furthermore, in the above formula (I [
C5-08 cycloalkylalkyl group which is a C5 alkyl group; C3-08 cycloalkyl or C3-C8 cycloalkenyl group (both optionally containing methylene or 1
or saturated or wholly or partially unsaturated and optionally one or more C1-C4 alkyl groups; or similar compounds which are 3- to 6-membered oxygen- or sulfur-containing heterocycles which may be substituted with halogen atoms,
It is described in No. 29590. All of these compounds are known to have insecticidal, acaricidal and anthelmintic activity.

As a result of our earnest efforts to search for analogs of these milbemycins, the present inventors discovered that the above-mentioned milbemycins,
The present invention was completed by discovering that position 29 can be efficiently hydroxylated by conversion using a microorganism or an enzyme produced by the microorganism.

JP-A-61-233686 discloses microbial conversion of 22,23-dihydroavermectin aglycone or 13-deoxy-22,23-dihydroavermectin aglycone, but the following general description and the present invention are: The microorganisms used and the position of hydroxylation also vary.

Furthermore, JP-A-62-59291 describes 22,23-dihydro-avermectin Bla, avermectin B], a
, discloses microbial conversion of avermectin Bib, but as described below, the substrate used, the microorganism, and the hydroxylated position are quite different from the present invention.

According to the present invention, a compound represented by the following general formula (I) is used as a substrate and this is cultured in a medium containing the compound represented by the following general formula (I) as a substrate, or
By contacting cultured cells or enzyme extracts of these microorganisms with the compound represented by the general formula (I), the compound represented by the general formula (I) (wherein X is a hydrogen atom,
Indicates a hydroxyl group or an oxo group. A dotted line indicates a single bond or a double bond, which can only be a double bond when X is a hydrogen atom. Y is a C1-8 alkyl group, a C2-8 fulkenyl or alkynyl group, a C1-4 alkoxy or alkylthio C1-4 alkyl group, a C3-8 cycloalkyl c1-5 alkyl group, a C3-8 cycloalkyl or id C3- B cycloalkenyl group (in both cases yodimethylene or one or more 01-4
optionally substituted with alkyl or halogen),
or saturated or unsaturated 3- to 6-membered oxygen or sulfur (8-containing heterocycle (optionally substituted with one or more 01-4 alkyl or halogen), 2 is a hydroxyl group 1 or represents a hydroxyimino group) (wherein, X, Y and 2 have the same meanings as above).

, - The method of the invention concerns the microbial hydroxylation of a compound of general formula (I[). In the method of the invention,
Only the methyl group at position 29 is hydroxylated, and the methyl group at position 4, 12, and 24
The methyl group attached to the 25th position and the Y group attached to the 25th position do not undergo hydroxylation. In addition, the double bonds at the 3rd, 8th, 10th, and 14th positions and the Y group are 1-methyl-1-propenyl groups,
1-methyl-1-butenyl group or 1,3-tmethyl-
In the case of a 1-butenyl group, the double bond at position 31 does not undergo any oxidation such as epoxidation. Among the compounds of the general formula (I) which are the starting materials for the method of the present invention, compounds in which X is a hydrogen atom and ayl, and Z is a hydroxyimino group are known from JP-A-59-1'08785. It is.

The microorganism used in the method of the present invention has the general formula (I
It is a microorganism that can convert to the compound J. The types of microorganisms used in the method of the present invention and their representative strains, which are stored in public strain distribution institutions, are as follows.

Syncephalastrum nigricans
NRRL 12479Syncephalastr
um racemosum IFO4827Sync
ephalastrum racemosum IF
O4814 racemosum IFO4827 is most amenable to the method of the present invention.

The method of the invention can be implemented in various ways.

For example, (1) a method in which a compound of formula (I) as a substrate is brought into contact with a medium in which a microorganism has been cultured; (2) bacterial cells are collected from a medium in which a microorganism has been cultured and then the compound of formula (I) is brought into contact with the microorganism. and (3) a method in which a cell-free extract prepared from the bacterial cells is brought into contact with the compound of formula (II).

Cultivation of converting bacteria is usually carried out by culturing in a medium containing nutrients that can be used by microorganisms. As the nutrient source, any known nutrient source used for culturing actinomycetes in general can be used.

For example, as the carbon source, glucose, sucrose, maltose, lactose, starch, glycerin, starch syrup, molasses, soybean oil, etc. are used.

In addition, as the nitrogen source, soybean flour, wheat germ, meat meal, fish meal, meat extract, starch, cornstarch liquor, dried yeast, ammonium salts such as ammonium nitrate, etc. are used. In addition to inorganic salts such as common salt, potassium chloride, calcium carbonate, and phosphates, additives that aid the growth of bacteria and promote the production of enzymes with hydroxylation ability, such as yeast extract and other additives, may be added as necessary. Malt extract and the like can be used in combination as appropriate. Cultivation is carried out under aerobic conditions, with a culture temperature of 20-4.0°C, preferably 26°C.
-30°C.

Method (1) is carried out by adding the compound (II) and culturing. The timing of addition varies depending on the optimal culture conditions of the converting bacteria used, especially the culture equipment, medium composition, culture temperature, etc., but the best time to add the converting bacteria is when the hydroxylation ability of the converting bacteria starts to become high. The preferred time point is 1 to 5 days after the start of culture. The amount of the raw material compound, i.e., the substrate added, is 0.01-5.0%, preferably 0.025%, based on the medium.
-2.0%.

Cultivation after addition of the raw material compound is carried out under aerobic conditions at the above-mentioned culture temperature. The culture period is 1 after the addition of the raw material compound.
- About 8 days.

Method (2) is carried out by culturing the converted bacteria in the presence of a small amount of substrate according to the method (1) above, and culturing the converted bacteria in ox where the hydroxylation ability of the converted bacteria is maximized.

That is, the hydroxylation ability varies depending on the type of medium, temperature, etc., but usually reaches its maximum 2 to 3 days after the start of culture, so the culture is terminated at this point. Bacterial collection is performed by subjecting the culture to methods such as centrifugation and filtration. It is usually preferable to wash the collected converted bacterial cells with physiological saline, a buffer solution, etc. before use. The thus obtained converted bacterial cells are brought into contact with the starting compound, usually in an aqueous medium, for example in a phosphate buffer solution at pH 5-9. The contact reaction is usually carried out at 20-45°C, preferably at 25-3°C.
Performed at 0°C. The concentration of substrate is 0 for normal medium.
．． 01-5.0%. The reaction time depends on the substrate concentration, reaction temperature, etc., but is usually about 1-5 days.

The cell-free extract in the method (3) is prepared by suspending the transformed bacterial cells obtained in the above method in an aqueous medium, for example, a phosphate buffer solution with a pH of 5-9, and For example, it can be obtained as a bacterial cell fragment by grinding, ultrasonication, etc., or as a bacterial cell lysate by organic solvent, surfactant, enzyme treatment, etc. The cell-free extract obtained in this manner is brought into contact with the raw material compound in the same manner as the method of contacting with the converted bacterial cells described above.

After the conversion reaction is completed, the target compound can be collected, separated, and purified from the product by known methods. For example, the resulting product is filtered, the resulting filtrate is extracted with a water-immiscible organic solvent such as ethyl acetate, the solvent is distilled off from the extract, and the resulting crude target compound is extracted. silica dal,
Subjected to column chromatography using alumina etc.
It can be separated and purified by elution with an appropriate eluent.

Natural milbemycins, which are the starting materials for the compound of formula (1), are fermentation products in which a large number of analogs are produced in various proportions, and each analog is isolated and then strained into a mixture. No.1! The reaction is carried out until the end of the reaction. Therefore, the formula (I
) can be either a single compound or a mixture thereof.

Therefore, the compound of formula (1) can also be produced as a single compound or a mixture thereof.

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

The compound of formula +1) can be used to target adults, larvae, and eggs of Tetranychus, Panonychus, and rust mites that parasitize fruit trees, vegetables, and flowers, as well as Ixodidae that parasitize animals.
It has excellent acaricidal activity against Ae), Dermanyssidae, Sarc-optidae, and the like.

Sara K, Oestrus, Kingfly (
Lucilja), Hypoderma
), horse flies (Gautrophlus), etc., and ectoparasites of animals and birds such as fleas and lice; cockroaches,
It is active against sanitary pests such as house flies; and various other agricultural and horticultural pests such as aphids and lepidopteran larvae. Furthermore, the nematode (Melojdo nematode) in the soil
gyne) = Bursaphe
lenchus), Nedani (Rhizoglyphus
) and others.

The compounds of formula (I) also have activity against insects that harm plants, especially insects that harm plants by ingesting them.

Furthermore, the compound of formula +1) has excellent parasitic activity as an anthelmintic for animals and humans.

It is particularly effective against nematodes that infect domestic animals such as pigs, sheep, goats, cattle, horses, dogs, cats, and chickens, poultry, and animals.

When using the compound of formula (IJ) for agricultural and horticultural purposes, powder,
It is prepared and used in formulations well known in this field, such as wettable powders and emulsions. When used diluted with water if necessary, the concentration of the active ingredient is approximately 11-1 Opp.

When the compound of formula (IJ) is used as an anthelmintic for animals, it is prepared into formulations well known in this field such as powders, tablets, capsules, injections, etc. When administered orally, the administration The amount is approximately 0.01-100m per kg of body weight.
g, preferably o, about s-5-5o.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 50' O containing 100 ml of medium with the following composition:
Synthephalastrum racemosum (S, racemosum IFO482) was added to five ml Erlenmeyer flasks.
7) was inoculated and cultured by shaking at 26°C and 22 Orpm. After 2 days, milbemycin A4 (2 TJ: The cells were cultured at 26°C and 220 rpm for days.

C17) Medium composition Glucose 1.0% Yeast extract 0.3% Malt extract 0.3% Peptone 0.5% Tap water Remaining (pH not corrected) After the cultivation is completed, the reaction solution is filtered by suction, and the bacterial cells and fermentation solution are removed. It was divided into two parts. The furnace solution was extracted three times with 300 ml of ethyl acetate, and the extract was dried over anhydrous sodium sulfate and then concentrated. The bacterial cells were extracted twice with 100 ml of 80% methanol, and after the methanol was evaporated, the extract was extracted with ethyl acetate and concentrated in the same manner as the furnace solution. The extracts from the bacterial cells and the furnace solution were combined and subjected to preparative thin layer silica gel chromatography (manufactured by Merck & Co., Ltd., Art
5717.

20 x 20 cm + 2 strips thick, developed twice with n-hexane:ethyl acetate - 1:1) and purified with 29-hydroxymilbemycin A4 (formula ■: X - hydrogen atom, Y = ethyl group,
8 mg (yield: 7.0%) of i 2 = hydroxyl group) and 30 m9 (yield: 12.0%) of milbemycin A4 were recovered.

Mass coverage (m/z): 558 (M+),
540.

430,412,195,167.151 Nuclear magnetic resonance spectrum δ(CDCt3)ppm: 0.82(d,
3H, C3oH3, J=6.5H2), 0.99(t,
3H.

C32H3,, J=7.3Hz), 1.03(d
, 3H, C28H3゜J=6.9Hz), 1.87
(S, 3H, C26H3), 3.07 (dt.

I H, C2sH+ J ”” 5.4 + 88Hz
) + 327 (m + I H+ C2H) +
3.60 (m, LH, C17H), 3.93 (d, IH
,C,H.

J-121H9), 3.95 (d, lH2C6H2J-
65Hz).

4.12 (br, s, IH, C70H), 4.27 (d
,IH,C,H.

J=12.1Hz)4.29 (br, s, IH
, C3H), 4.67 (br.

S, 2H, C27H2), 5.11 (IH, t
, C, , H, J=8. , IH7) 5.3 to 5.5 (
m, 3. H, C3) (, C11H, C,, H), 5
．． 7-5.85 Example 2 According to the method of Example 1, 29-hydroxymilbemycin A4 was obtained using milbemycin A4 as a substrate and various microorganisms listed below.

Syncephalastrum nigricans
NRRL 12479 +28yncephala
strum racemosum IFO4814+
2Syncephalastrum racemosu
m IFO4828+1, the conversion rate is + based on the following criteria: 5-5.0% +2: 5.0-10.0% Example 3
Synthephalastrum in 10 Erlenmeyer flasks with 0m1 capacity.
Racemosum (S, racemosum IFO48
27) was inoculated and cultured by shaking at 26°C and 22 Orpm. After 1 day, 5-ketomilbemycin A45-oxime (formula ■: 2 for another 7 days.
Culture was performed at 6°C and 220 rpm. After the culture was completed, the reaction solution was suction filtered and separated into bacterial cells and fermentation liquid. The furnace solution was extracted three times with 500 ml of ethyl acetate, and the extract was dried over anhydrous sodium sulfate and then concentrated. After extracting the bacterial cells three times with 200 ml of 80% methanol and evaporating the methanol,
It was extracted with ethyl acetate and concentrated in the same manner as the furnace solution. The extracts from the bacterial cells and the furnace solution were combined and purified by silica gel column chromatography to obtain 29-hydroxy-5-ketomilbemycin A45-oxime (Formula 1: X-hydrogen atom,
Y-ethyl group, 2-hydroxyimino group) 10.6m
g (yield 206%) was obtained.

Mass spectrum (r+y'z): 571 (M+)
, 553.

Nuclear magnetic resonance spectrum δ (CD(43) 0.82 (
d, 3H.

J=6.4H1,C3oH3), 1.00 (t,
3H, J=7.3Hz.

C32H3), 1.04 (d, 3H, J===6
．． 4Hz, C28H6), 1.94(s, 3
H, C26H 3 people 3.37 (m, I H, C2H)
, 3.93 (d.

LH, J = 11.7Hz, C2, H), 4.26 (d,
1'H, J=11.7 Hz, C2, H), 4.6
7 (s, IH, C, sH), 4.6-4.8 (
m, 2H, C27H), 5.13 (m, IH, C1
, H), 5.37-5.50 (m, 2H, C11H, C
1, H), 5.71-5.90 (m, 3H, C3H, C
, H, C1oH), 8.11 (br, s, I
H.

=N-OH) Example 4 500ml containing 100M of medium with the same composition as Example 1
5 Erlenmeyer flasks with 7 Rhizopus circinas (
R. circinans ATCC 1225) was inoculated and cultured at 26° C. and 20 Orpm with rotational shaking for 5 days. Two days later, milbemycin A3 (formula n: x = hydrogen atom, Y =
Methyl group, 2 = hydroxyl group) was added using a 5-functional dioxane solution to give a final concentration of 0.025, and the mixture was further cultured at 26° C. and 200 rpm for 7 days. After the culture was completed, the reaction solution was suction filtered and separated into bacterial cells and fermentation liquid. The furnace solution was extracted three times with 300 TL of ethyl acetate, and the extract was dried over anhydrous sodium sulfate and then concentrated. 80e of bacterial cells
I) Extract twice with 100 rnl of methanol, evaporate the methanol, and then extract with ethyl acetate in the same manner as the furnace solution.
Concentrated. The extract from the bacterial cells and the extract from the furnace solution were each purified by silica gel column chromatography, and 29-hydroxymilbemycin A3 (formula 1: X = hydrogen atom, Y = methyl group, 2 = hydroxyl group) 4.8q ( Yield 6
．． 2%) and milbemycin A3 with a recovery rate of 62.5■ (recovery rate 8
Section 3.4) Recovered.

Mass spectrum (m/z) 544 (M”), 5
26, 508+416.398,266.181,1
53,151 nuclear magnetic resonance spectrum δ (CDC”23
) ppm; 0.84 (d, 3H, J=6.4Hz,
C3o] (3), 1.03 (d.

3 H, J = 6.5 Hz, C28H3), 1.
15 (a, 3H, J=6.1 Hz, C31H3)
, 1.87 (d, 3H, J=1.6&.

C26H3), 3.26 (m, 2H, C2H, C,
,,5H), 3.59(m.

LH, C1, H), 3.94 (d, LH, J=12, s
Hz, C29H), 3,96 (d, I H, J
=6.4 Hz, C6H), 4.11 (S, IH,
'7 L OH), 4.28 (d, IH, J=12.5
Hz, C2, H), 4.26-4.32 (m, L
H, CsH), 4.67 (S, 2H.

Claims (1)

[Claims] (1) A microorganism belonging to the genus Syncephalastrum or Rhizopus that can convert a compound represented by the general formula (II) below into a compound represented by the general formula (I) below is used as a substrate. The compound represented by formula (II) can be cultured in a medium containing the compound represented by formula (II) as a substrate, or the cultured cells or enzyme extract of these microorganisms can be cultured with the compound represented by formula (II).
A compound represented by general formula (I) is converted into a compound represented by general formula (I) by contacting with a compound represented by general formula (I), and then the compound represented by general formula (I) is collected.
): ▲There are mathematical formulas, chemical formulas, tables, etc.▼(II) (In the formula, X represents a hydrogen atom, hydroxyl group, or oxo group. The dotted line represents a single bond or double bond, and can be a double bond only when is a hydrogen atom. Y can be 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 cycloalkenyl group (
either optionally substituted with 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 (one or more ), and Z represents a hydroxyl group or a hydroxyimino group. ): ▲Mathematical formulas, chemical formulas, tables, etc.▼(I) (In the formula, X, Y, and Z have the same meanings as above.)