JPS63264484A - Novel milbemycins and production thereof - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I [V represents H, Oh or oxo; W represents H, OH, halogen atom or 4'-(alpha-L-oleandrosyl)-alpha-L- oleandrosyloxy; X represents methyl, ethyl, isoprophyl, sec-butyl, 1-methyl-1- propenyl, 1-methyl-1-butenyl, etc., when V represents H, and represents 1- methyl-1-propenyl, etc., when V represents OH or oxo; Y represents OH or hydroxyiminop Z represents H or OH]. EXAMPLE:30-Hydroxymilbemycin A4. USE:An insecticidal, miticidal and antiparasitic agent and an intermediate for producing other compounds. PREPARATION:A microorganism, belonging to genus Amycolata, Amycolatopsis, Streptomyces, Absidia, Circinella, Mucor, Syncephalastrum, Mortierella, Rhizopus or Bacillus and capable of converting a compound expressed by formula II or a substrate into a compound expressed by formula I is cultured in a medium.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel macrolide compounds and methods for producing the same, and more particularly to milbemycins and analogs thereof, and methods for producing them. Milbemycine is a series of macrolide compounds, and is known from JP-A-50-29742, JP-A-56-32481, etc., and is represented by the following formula (II).
It is a compound of

(2) In the formula, (2) and W represent a hydrogen atom, or a methyl, ethyl or isopropyl group, respectively, and each represents milbemycin A3. They are called milbemycin A4 and milbemycin D. ■ and W represent hydrogen atoms, and X is 5
The compound which is ec-butyl is disclosed in JP-A-54-14559.
This is the milbemycin cervical body described in Publication No. 9 and the like.

(2) is a hydrogen atom, W is a 4'-(α-L-oleandrosyl)-α-L-oleandrosiloxy group,
Compounds in which X is an isopropyl group or 5ec-butyl are those described in JP-A-54-61198, and are called 22,23-dihydroavermectin Bla and Blb, respectively. Further, W is a hydrogen atom, ■ is a hydroxyl group, and X is a 1-methyl-1-propenyl group, a 1-methyl-1-butenyl group, or a 1,3-dimethyl-1-butenyl group. The compound is a compound described in JP-A-61-10589 and is known as LL, -F-28249.

(2) is an oxo group, and X is a 1-methyl-1-propenyl group, 1-methyl-1-butenyl group, or
Compounds having a dimethyl-1-butenyl group are disclosed in JP-A-61
This is a compound described in JP-280496. All of these compounds are known to have insecticidal, acaricidal and anthelmintic activity.

As a result of intensive efforts to search for new analogues of these milbemycins, the present inventors have discovered that new milbemycins can be produced by converting the above-mentioned milbemycins using microorganisms or enzymes produced by microorganisms. They discovered this and completed the present invention.

JP-A-61-233686 discloses microbial conversion of 22,23-dihydroavermectin aglycone or 13-deoxy-22,23-dihydroavermectin aglycone, but as described later, the Hihara invention The microorganisms that carry out the hydroxylation and the positions at which it is hydroxylated also differ.

According to the present invention, a compound represented by the following general formula (II) is used as a substrate, and this substance can be converted into a compound represented by the following general formula (I). Cultivating a microorganism belonging to the genus Absidia, Circinella, Mucor, Syncephalastrum, Morteierella, Rhizopus or Bacillus in a medium containing a compound represented by general formula (II) as a substrate, or Alternatively, cultured cells or enzyme extracts of these microorganisms can be expressed by general formula (II).
By contacting with a compound represented by the general formula (
A compound represented by 1) can be produced.

■ (In the formula, ■ 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-oleandrosiloxy group, X is a methyl group, ethyl group, isopropyl group, 5ec-butyl group, 1-
It represents a methyl-1-propenyl group, a 1-methyl-1-butenyl group, or a 1,3-dimethyl-1-butenyl group, and when ■ is a hydroxyl group or an oxo group, it represents a 1-methyl-1-butenyl group.
It represents a 1-propenyl group, a 1-methyl-1-butenyl group, or a 1,3-dimethyl-1-butenyl group, and Y represents a hydroxyl group or a hydroxyimino group. ) (wherein U represents a hydroxymethyl group or a carboxy group, V, W,
and Y has the same meaning as above, and 2 represents a hydrogen atom or a hydroxyl group. ).

The method of the invention concerns the microbial hydroxylation and/or carboxylation of compounds of general formula (II). In the process of the invention, the methyl group at position 30, which is attached to position 24, is always hydroxylated or carboxylated.
On the other hand, the methyl group at the 26th position bonded to the 5th position is not always hydroxylated, and in some cases may not be hydroxylated. The methyl groups bonded to the 12th and 14th positions and the X group bonded to the 25th position do not undergo hydroxylation.

Among the compounds of general formula (II) which are the starting materials for the method of the present invention, compounds in which ■ is a hydrogen atom and both W and Y are hydroxyl groups are Compounds in which W is a hydrogen atom and Y is a hydroxyimino group are known from JP-A-59-108785. In addition, the compound in which ■ is a hydrogen atom, W is a hydroxyl group, and Y is a hydroxyimino group is a compound in which W is a hydrogen atom and Y is a hydroxyimino group. It can be obtained by hydroxylating W by the method described in the publication. Furthermore, ■ is a hydrogen atom, and W
A compound in which is a halogen atom and Y is a hydroxyl group is known from JP-A-61-85390.

The microorganisms used in the method of the present invention include genus Amycolata and genus Amycolatopsis.
, Streptomyces genus
yces), ganus Absid
ia), genus C1rci-ne
lla), ganus Mucor, ganus Synceph
alastrum), Morteierella genus
Mortierella), Rhizopus genus
Rh1zopus) or Bacillus genus
The present invention is a microorganism that belongs to the family Bacillus Bacillus (Bacillus 11us) and is capable of converting a compound of general formula (II) into a compound of general formula (1). The genera Amycolata and Amycolatopsis were previously classified in the genus Nocardia.

Due to differences in bacterial body components, they are now independent from the genus Nocardia and have formed new genera (Inter
atiOnal Journal of System
aticBact=riology, Vol, 36.
No, 1. p, 29.1986).

Examples of the bacteria belonging to the genus Amycolata that can be used in the method of the present invention include Amycolata and autotrophica (A, autotrophica). Representative examples thereof have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, and have been assigned deposit numbers 6181, 6182 and 6183. At the time of deposit, these deposited bacteria were Nocardia sp, 5ANK 62781,
They are called Nocardia sp, 5ANK 62881 and Nocardia sp, 5ANK 62981, respectively, and their mycological properties were described in JP-A No. 5
It is described in the publication No. 8-89191.

Examples of the fungi belonging to the genus Amycolatopsis that can be used in the method of the present invention include Amycolatopsis orientalis (A. orientalis) and Amycolatopsis mediterranei (A.

meciiterranei).

Typical of these are IFO12806 and IFO13415t, which are stored by the Fermentation Research Institute.
', and both of these strains are ISP designated strains, namely ISP 5040 and ISP 5501.

Furthermore, the bacteria used in the method of the present invention and belonging to the genus Streptomyces include, for example, Streptomyces, Flavovirens (S,
ens), Streptomyces, Griseolus (S, g
S. riseolus) and Streptomyces, S. roseus. Typical of these are ATCC3320 (Technical SP 5062) and KCC58, which are stored at public strain distribution institutions.
1 (ISP5067) and IFO12818 (ISP
5076).

In the method of the invention, preferred microorganisms are bacteria belonging to the genus Amycolata and Amycolatopsis, in particular Amycolata, Autotrophica FERM P-61.
83 and Amycolatopsis mediterranei IFO1
3415 is most preferred.

The method of the invention can be carried out in a variety of ways.

For example, (1) a method in which a compound of formula (II) 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 contacted with a compound of formula (II). and (3) a method of bringing a cell-free extract prepared from the bacterial cells into contact with the compound of formula (Technology I).

The converting bacteria are usually cultured in a medium containing nutrients that can be used by the microorganisms. As a nutrient source, it is possible to use a known one used for culturing general actinomycetes. For example, as a carbon source,
Glucose, sucrose, maltose, lactose, starch, glycerin, starch syrup, molasses, soybean oil, etc. are used.

Further, as the nitrogen source, soybean flour, wheat germ, meat meal, fish meal, meat extract, peptone, cornstarch liquor, dried yeast, ammonium salts such as ammonium nitrate, etc. are used. In addition, as necessary, in addition to inorganic salts such as table salt, potassium chloride, calcium carbonate, and phosphates, additives that support the growth of bacteria and promote the production of the enzymes having the above-mentioned hydroxylation ability may be appropriately combined. It can be used.

Cultivation was carried out under aerobic conditions, with a culture temperature of 20-40℃.
9°C, preferably 26-35°C.

Method (1) is carried out by adding the compound of formula (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 it is best to add it when the hydroxylation ability of the converting bacteria begins to increase, and usually after the start of culturing of the converting bacteria. Preferably, after 1 to 5 days have elapsed. The amount of the raw material compound, i.e., the substrate added, is 0.01-5.0%, preferably 0.0%, relative to the medium.
25-2.0%.

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

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 until the hydroxylation ability of the converted bacteria reaches its maximum.

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. To collect bacteria, centrifuge the culture.
This is done by subjecting it to a method such as 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 Z5-
Performed at 35°C. The concentration of the substrate is usually 0.01-5.0% relative to the medium. The reaction time depends on the substrate concentration, reaction temperature, etc., but is usually about 1-5 days.

The cell-free extract in method (3) is obtained by applying a physical or chemical method to the transformed bacterial cells obtained by the above method, for example,
It can be obtained as a bacterial cell fragment by grinding, ultrasonication, etc., or as a bacterial cell lysate by treatment with an organic solvent, a surfactant, an enzyme, 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 completion of the conversion reaction, the macrocompound can be collected, separated and purified from the product by known methods.

For example, the obtained product is filtered, the obtained filtrate is extracted with an organic solvent that is not easily miscible with water, such as dithyl acetate, and the solvent is distilled off from the extract. is subjected to column chromatography using silica gel, alumina, etc., and eluted with an appropriate eluent. It can be separated and purified by two methods.

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 different proportions, and each analogue is isolated or combined in a mixture. It is subjected to the reaction as is. Therefore, the formula (
The compound II) can be either a single compound or a mixture thereof.

Therefore, compounds of formula (I) can also be produced as single compounds or mixtures thereof. Among the compounds represented by the general formula (1), the compound represented by the following formula (Ia) is itself novel and forms part of the present invention: Y (in the formula, U is a hydroxymethyl group or represents a carboxyl 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 a methyl group when U is a carboxy group, Ethyl group, isopropyl group or 5
eC-butyl group, Y represents a hydroxyl group or a hydroxyimino group, 2 represents water The compound of formula (I) itself has insecticidal, acaricidal and anthelmintic activity, or has insecticidal, acaricidal and anthelmintic activity. It is useful as an intermediate for the synthesis of other compounds with

The compound of formula (I) can be used to control adults, larvae, and eggs of Tetranychus, apple spider mite, and rust mite that parasitize fruit trees, vegetables, and flowers, as well as Ixodidae that parasitize animals.
), Dermanyssidae, and Sarcoptidae.

In addition, the sheep fly (Oestrus), the golden fly (L.
ucilia), cow fly (Hypodarma),
Active against Gautrophilus and other ectoparasites of animals and birds such as fleas and lice; sanitary pests such as cockroaches and houseflies; and various agricultural and horticultural pests such as aphids and lepidopteran larvae. ing. Furthermore, Meloidog nematode (Meloidog) in the soil
yne), Bursaphal nematode (Bursaphal)
enchus), Phizoglyphus
It also has activity against.

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

Furthermore, the compounds of formula (I) have excellent parasicidal activity as anthelmintics for animals and humans. In particular, it is also effective against nematodes that infect livestock, poultry and pets, such as pigs, sheep, goats, cattle, horses, dogs, cats and chickens.

When using the compound of formula (I) for agricultural and horticultural purposes, powders,
It is prepared and used in preparations well known in this field, such as aquariums 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 (1) is used as an anthelmintic for animals, it is prepared into formulations well known in this field, such as powders, tablets, capsules, and injections. When administered orally,
Dosage is approximately 0.01-100m/kg body weight
g, preferably about 0.5-50 mg.

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

夾用忽↓ In six 500 ml Erlenmeyer flasks containing 100 ml of a medium with the following composition, amycolata, autotrophica (
A, autotrophica: Microtechnical Research Institute No. 61
No. 83) was inoculated and cultured with shaking at 28° C. and Z20 rpm. After 4 days, milbemycin A4 (Formula II:
V=W=hydrogen atom, X=ethyl group, Y=hydroxyl group) using its 5'3t dioxane solution at a final concentration of 0.05
%, and further heated at 28℃ for 1 day at 220 rpm.
Cultured at m.

Glucose 1.0% Yeast extract 0.3% Chiga-Goss 0.3% Peptone O, '5% Tap water Remaining (pH uncorrected) After culturing, dilute the reaction solution twice with 600 ml of ethyl acetate. The extract was dried over anhydrous sodium sulfate and then concentrated. The residue was purified by silica gel column chromatography,
-Hydroxymilbemycin A4 (Formula 1: U = hydroxymethyl group, V = W = Z = hydrogen atom, X = ethyl group, Y = hydroxyl group) 114.6 mg (yield 37.5 mg)
, 26,30-dihydroxymilbemycin A4 (
Formula I: U = hydroxymethyl group.

47.7 mg (yield 15.0%) of V=W=hydrogen atom, X=ethyl group, y=z=hydroxyl group, milbemycin-30-oic acid A4' (formula I: U=carboxy group, V= 30.1 o+g (yield 9.5%) of W=Z=hydrogen atom, X=ethyl group, Y=hydroxyl group) were obtained. Furthermore, 54.8 mg of milbemycin A4 (recovery rate 2
1.6%) was recovered.

Tei m12 Toji 2 Mass Spectrum (m/z) 2 558 (M+)
, 540°430.412.372.314.280
．． 211.183 nuclear magnetic resonance spectrum δ (CDC13
) ppm: 1.00 (d, 3H, C28H3, J=
7,0Hz), 1.01(t,3H,C32H3,J
=7.0Hz), 1.52(s, 3H, C29H3)
, 1.87 (br, s, 3H.

C26H3), 3.26(m, LH, C2H), 3
．． 33 (dt, LH, C25H.

J=2.8,8.5Hz), 3.45-3.65(m
, IH, C17H), 3.49 (dd, IH, C30
H, J=6.0, 10.6Hz), 3.63(dd,
IH.

C30H, J=4.4, 10.6Hz), 3.95(
d, IH, C6H, J=6.2Hz), 4.05(b
r, s, IH, C70H), 4.28 (br, s, I
H.

C5 [() 26.30-dihydroxy mass spectrum (m/z): 574 (M+),
556°538.280,211,183 Nuclear magnetic resonance spectrum δ (CDC13) ppm: 1
．． 00 (d, 3H, C28H3, J=6.5Hz),
1.02 (t, 3H, C32H3゜J=5.5Hz),
1.53 (s, 3H, C29H3), 3.49 (d
d, IH.

C30H, J=6.2, 11.0Hz), 3.63(
dd, IH, C30H, J=4.2, 11.0Hz),
3.98 (d, LH, C6H, J=6:2Hz).

4.13 (br, s, LH, C70H), 4.24 (
d, LH, C26H.

, 1=12.8Hz), 4.32(d, LH, C2,
6H, J=12.8Hz).

4.58 (br, s, ], H, C3H) 30-oic acid mass spectrum (m/z): 572 (M+),
444,426°294.225,197,151 Nuclear magnetic resonance spectrum δ (CDC13) ppm: 1
．． 00 (d, 3H, C28H3, J=6.5Hz),
1.02(t, 3')I, C32H3°J=7.2Hz
), 1.53 (s, 3H, C29H3), 1.87
(s, 3H.

C26H3), 3.28(m, IH, C2H), 3
．． 5-3.7 (m, 2H.

C17H, C25H), 3.96 (d, LH, C6H
, J=6.4Hz).

4.01 (br, s, IH, C70H) 30-Hydroxymilbemycin A4 was obtained according to the method of Example 1 using milbemycin A4 as a substrate and using the following i type of microorganism.

imitation two-core 30-hydroxy Ha, rate Am
ycolatcpsis orientalis IF
O12806+ 3Amycolatopsis me
diterranei IFO13415+4Amyc
olata autotrophica FERM P
-6481+ 1Amycolata autotro
phica FERM P-6182+IStrept
omyCes flavovirens IFO127
71+ IStreptomyces griseol
us IFO12777÷IStreptomyces
roseus IFo 12818 ÷
2Absidia reflexa IFO5874+
3Circinella umbellata Engineering F
O8093+3Mucor recyrvus engineering FO
8093+ 3Rhizopus nigricans
NRRL 45 + 2Sync
phalastrum racemosum IFO4
827+ I Mortieralla 1sabell
ina NRRL 1757 +4Baci
llus megaterium IFO12108+
2. The conversion rate is based on the following criteria: +1: 0.5-5.07 +2: 5.0-10. O% + 3: 10.0-30. + 4: 30.0-50.0% 50% medium containing 100 ml of medium with the same composition as in Example 1
Amicolata in 6 0m1 Erlenmeyer flasks.

autotrophica (A, autotrophica
: Microtechnology Research Institute No. 6183) was inoculated and incubated at 28℃ and 220℃.
Culture was performed with shaking at rpm. After 5 days, 5-ketomilbemycin A45-oxime (formula 1J: V=W: hydrogen atom, X=ethyl group, Y=hydroxyimino group) was
% dioxane solution to give a final concentration of 5%, and the cells were further cultured at 28° C. and 22 Orpm for 7 days.

After the culture was completed, the reaction solution was extracted three times with 500 ml of ethyl acetate, and the extract was dried over anhydrous sodium sulfate and then concentrated. The residue was purified by silica gel column chromatography and purified with 30-hydroxy-5-ketomilbemycin A4.
5-oxime (formula I: U=hydroxymethyl group, V=W
= Z = hydrogen atom, X = ethyl group, Y = hydroxyimino group) was obtained in an amount of 30.4 mg (yield: 9.8%).

Mass spectrum (mHz): 571 (M+),
553゜537.211,183 Nuclear magnetic resonance spectrum δ (CDC13) ppm:
L, 0O (d, 3H, C28H3, J=-7,0Hz)
, 1.03(t, 3H.

C32H3,j=7.0Hz), 1.53(s, 3H
, C29H3), 3.38 (:n.

IH, C2H), 3.40 (dt, IH, C25H,
J = 2.3, 9.5Hz).

3.45-3.65 (m, LH, C17H), 3. '
, 9 (dd, LH, C30H.

J=6.2, 10.8Hz), 3.63(dd, IH
, C30H, J=4.0°10.8Hz), 3.95
(br, s, IH, C70H), 4.67 (s, IH
.

C6H) Saniya 1 1 Containing 17 ml of medium with the same composition as Example 1 1 0
Two 0 ml Erlenmeyer flasks were inoculated with Amycolata autotrophica (A, autotrophica: FAIKEN HYUKYO NO. 6183), and cultured with shaking at 28° C. and 220 rpm. After 3 days, 13-hydroxymilbemycin A4 (Formula II: V=hydrogen atom, w=y=
hydroxyl group, X = ethyl group) was added to a final concentration of 0.05% using a 5% dioxane solution, and
The cells were cultured at 28°C and 220 rpm for days. After the culture was completed, the reaction solution was extracted three times with 50 ml of ethyl acetate, and the extract was dried over anhydrous sodium sulfate and then concentrated. The residue was purified by silica gel column chromatography.

13.30-dihydroxymilbemycin A4 (Formula 1: U = hydroxymethyl group, V = Z = hydrogen atom, W =
Y = hydroxyl group, X = ethyl group) 8.7 mg (yield 49
゜7 stone) obtained.

Mass spectrum (mHz): 574 (M+),
556°540.295,277.211,183,1
51 nuclear magnetic resonance spectrum δ (CDC13) ppm
:1.01 (t, 3H, C32H3, J=7.3Hz)
, 1.13 (d, 3H, C28H3, J=6.4Hz
), 1.58 (s, 3H, C29H3), 1.88
(s, 3H.

C26H3), 3.28(m, LH, C2H), 3
．． 35 (dt, LH, C25H.

J=3.2, 10.2Hz), 3.51(dd, LH
, C30H, J=6.2°11.0Hz), 3.5-
3.65 (m, IH, C17H), 3.68 (,:f
d.

IH, C30H, J=4.2, 11.0Hz), 3.
71 (d, LH, C13H.

,■=10. IHz), 3.96 (d, LH, C6
H, J = 6.0Hz).

3.99 (s, LH, C70H), 4.29 (br,
s, LH, C3H) Containing 100 ml of a medium with the same composition as in Example 1
Amicolata in 5 0m1 Erlenmeyer flasks.

autotrophica (A, autotrophica
: Microtechnology Research Institute No. 6183) was inoculated and incubated at 28℃ and 220℃.
Culture was performed with shaking at rpm. After 3 days, milbemycin A3 (Formula II: V=W=hydrogen atom, X=methyl group,
Y = hydroxyl group) was added using its 5% dioxane solution to a final concentration of 0.05%, and the mixture was further incubated for 4 days at 28
The cells were cultured at 220 rpm. After completion of the culture, one reaction solution was extracted twice with 500 ml of acetic acid and chill, and the extract was dried over anhydrous sodium sulfate and then concentrated. The residue was purified by silica gel column chromatography to obtain 57.5 mg of 30-hydroxymilbemycin A3 (formula I: U = hydroxymethyl group, V = W = Z = hydrogen atom, X: methyl group, Y = hydroxyl group) (yield 22.3%) and 40 mg of milbemycin A3 (recovery rate 16.0%).
Recovered.

Mass spectrum (mHz): 544 (M+),
526゜416.266.197,169,151 Nuclear magnetic resonance spectrum δ (CDC13) ppm: 1
．． 00(d,,3H,C28H3,J=6.4Hz),
1.21(d, 3H.

C31H3, LJ=6.4Hz), 1.53(s, 3
) f, C29H3), 1.87(s, 3H, C26H
3), 3.27 (mJH, C2H), 3.50 (d
d.

LH, C30H, J=6.5d1.0Hz), 3.6
5 (dd, LH, C30H.

J=4.2, 11.0Hz), 3.45-3.6(m
, 2H, C1'7H, C25H).

3.96 (d, LH, C6H, J=6.4Hz), 4
．． 07(br,'s,LH.

C70H) Example 6 50ml containing 100ml of medium with the same composition as Example 1
Amicolata in 5 0m1 Erlenmeyer flasks.

autotrophica (A)
: Microtechnology Research Institute No. 6183) was inoculated and incubated at 28℃ and 220℃.
Culture was performed with shaking at rpm. Two days later, milbemycin D (formula 1: V = W = hydrogen atom, X = isopropyl group, Y = hydroxyl group) was added using its 5% dioxane solution to a final concentration of 0.05%. , another 7 days 2
Cultured at 8°C 1220rp. After the culture was completed, the reaction solution was extracted three times with 500 ml of ethyl acetate, and the extract was dried over anhydrous sodium sulfate and then concentrated. The residue was purified by silica gel column chromatography to give 30-hydroxymilbemycin D (Formula I: U=hydroxymethyl group, V=W=Z=hydrogen atom, X=isopropyl group, Y=
22.7 mg (yield 8°8%), 26,
30-dihydroxymilbemycin D (Formula 1: U=hydroxymethyl group, V=W=hydrogen atom.

4.0 mg of X=isopropyl group, Y=Z=hydroxyl group)
(yield 1.5%), milbemycin-30-oic
Acid D (formula I: U=carboxy group, V=W=Z=hydrogen atom, X=isopropyl group, Y=hydroxyl group) is 26.4
mg (yield 10.0%) was obtained. Furthermore, 94.3 mg (recovery rate 37.7%) of milbemycin D was recovered.

42ζ5p'' mass spectrum (m/z): 572 (M+),
444°426.314,294,248,225,1
97,179,151 nuclear magnetic resonance spectrum δ (C
DC13) ppm: 0.91 (d, 3H, C32H3
, J=6.8Hz), 1.00(d, 3H.

C28H3, J=6.4Hz), 1.06(d, 3H
, C33H3, J=6.8Hz).

1.53 (s, 3H, C29H3), 1.87 (s,
3H, C26H3), 3.27(m, LH, C2H)
, 3.34 (dd, LH, C25H, J=2.0,9
．． 7Hz).

3.46 (dd, IH, C30H, J=6.0, 10.
9Hz), 3.60(m.

1) i, C17H), 3.62 (dd, LH, C30
H, J = 4.0, 10.9Hz).

3.96 (d, LH, C6H, J=6.0Hz), 4
．． 05 (br, s, LH.

C70H) Broomstick, 30-dihydroxy substance mass spectrum (m/z): 588 (M+),
572°444.426,294,225,197,1
79', 151 nuclear magnetic resonance spectrum δ (CDC1
3) ppm: 0.91 (d, 3H, C32H3, J=
6.8Hz), 1.00(d, 3H.

C28H3, J=6.4Hz), 1.06(d, 3H
, C33H3, J=6.8Hz)'.

1.53 (s, 3H, C29H3), 3.3-3.4
(m, 2H, C2H, C25H).

3.47 (dd, LH, C30H, J=6.0, 10.
9Hz), 3.56 (m, LH, C17H), 3.
62 (dd, IH, C30H, J=4.0, 10.9H
z), 3.98 (d, LH, C6H, J=6.4Hz
), 4.12 (br, s.

IH, C70H), 4.24 (d, LH, C26H,
J=13.7Hz), 4.33(d, IH, C26H
, J=13.7Hz) 30-Oi...ri Acid Mass spectrum (m/z): 586 (M+),
558°458.308,248,239,211,1
51 nuclear magnetic resonance spectrum δ (CDC13) pp
m: 0.95 (d, 3H, C32H3, J=6.8H
z), 1.00(d, 3H.

C28H3, J=6.8Hz), 1.06(d, 3H
, C33H3, J=6.8Hz).

1.53 (s, 3H, C29H3), 1.87 (s,
3H, C26H3), 3.26(m, LH, C2H)
, 3.55 (m, IH, C17H), 3.66 (d
d, LH.

C25H, J=2.0, 10.0Hz), 3.96(
d, LH, C6H) Example 7 100ml containing 20ml of medium with the same composition as Example 1
Eight m1 Erlenmeyer flasks were inoculated with Amycolata autotrophica (A, autotrophica: Kaikoken Bacteria No. 6183), and cultured with shaking at 28° C. and 220 rpm. Two days later, 13-fluoromilbemycin 4 (formula II: V = hydrogen atom, W = fluorine atom,
= ethyl group, Y = hydroxyl group) was added using a 5% dioxane solution to give a final concentration of 0.05%, and the cells were further cultured at 28°C and 220 rpm for 2 days. After the culture was completed, the reaction solution was extracted three times with 160 ml of ethyl acetate, and the extract was dried over anhydrous sodium sulfate and then concentrated.
The residue was purified by preparative thin-layer silica gel chromatography (Merck, Art 5717, 20 x 20 cm, thickness 2 mru, developed with ethyl acetate) to give 13-fluoro-0-30-hydroxymilbemycin A4 (formula I: U
= hydroxymethyl group, V = Z = hydrogen atom, W = fluorine atom, X = ethyl group, Y = hydroxyl group) and 15.7 mg (yield 19.1%) and 13-fluoro-26,30-dihydroxymilbemycin A4 9.5 mg of (Formula I: U = hydroxymethyl group, V = hydrogen atom, W = fluorine atom, X = ethyl group, y = z = hydroxyl group) (yield 11.
2%) Obtained mass spectrum (m/z): 576 (
M+), 558°448.332,266.211,1
83,151 nuclear magnetic resonance spectrum δ (CDC13)
ppm: 1.01 (t, 3H, C32H3, J=7
．． 3Hz), 1.15(d, 3H.

C28H3, J=6.0Hz), 1.61(s, 3H
, C29H3), 1.88 (s, 3H, C26H3)
, 3.49 (dd, LH, C30H, J=4.8,1
1.1Hz), 3.60 (m, LH, C17H),
3.64 (dd, LH, C30H.

J=4.8, 11.1Hz), 3.97(d, LH,
C6H, J=5.9Hz).

4.04 (s, LH, C70H), 4.29 (br,
s, LH, C3H).

4.40 (dd, LH, Cl5H, J=9.9,47
．． 9Hz) 13-fluoro-26,30-dihydroxy mass spectrum (m/z): 592 (M+),
574°556.448, 332.266, 211,
183,151 nuclear magnetic resonance spectrum δ (CDC1
3) ppm: 1.02 (t, 3H, C32H3, J=
7.3Hz), 1.16(d, 3H.

C28H3, J=6.2Hz), 1.62(s, 3H
, C29H3), 3.51(dd, IH, C30H,
J=6.6, 11.3Hz), 3.60(m, LH.

C17H), 3.65 (dd, LH, C30H, J=
4.6, 11.3Hz).

3.99 (d, IH, C6H, J=6.2Hz), 4
．． 11 (sJH, C, 70H).

4.24 (d, LH, C26H, J=13.0Hz),
4.32 (d, LH.

C26H, J=13.0Hz), 4.42(dd, L
H, C13H, J = 9.9°47.9Hz)
Amycolatopsis mediterranei (A, mediterranei: I) were placed in 22 m1 Erlenmeyer flasks.
FO13415) was inoculated and cultured with shaking at 28°C and 220 rpm.

After 2 days, 22.23-dihydroavermectin Bla
(Formula II: V=hydrogen atom, W=4'-(a-L-oleandrosyl)-α-L-oleandrosiloxy group,
= 5ec-butyl group, Y = hydroxyl group) was added using a 5% dioxane solution to give a final concentration of 0.025%, and the mixture was further cultured at 28°C and 220 rpm for 7 days. After the culture was completed, the reaction solution was centrifuged and separated into bacterial cells and supernatant. The supernatant was extracted three times with 400 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 an 80% aqueous methanol solution, and after evaporating the methanol, they were extracted with ethyl acetate in the same manner as the supernatant and concentrated.

Extracts from the bacterial cells and supernatant were purified by silica gel column chromatography, and 30-hydroxy-22,23-
29.7 mg (yield 26.5%) of dihydroavermectin Bla was obtained. Furthermore, 17.6 mg of 22,23-dihydroavermectin Bla ('-yield 16.
4%) was recovered.

Mass spectrum (m/z): 728 (M+-1
62).

602.584, 568, 323, 305.211.1
45 Nuclear magnetic resonance spectrum δ (CDC13) pp
m: 0.89 (d, 3H, C32H3, 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, C26H3), 3.41(sy3H),
3.42C5,3H), 3.35-3.55(m, 2
H, C30H2), 3.55-3.9 (m, 5H),
3.94 (br, s, IH, C13H).

3.95 (d, LH, C6H, J=6.4Hz), 4
．． lo(br, s, LH.

C70H) 100ml containing 20ml of medium with the same composition as Example 1
14 m1 Erlenmeyer flasks, Ami: Rata.

autotrophica (A, autotrophica
: Microtechnology Research Institute No. 6183) was inoculated and incubated at 28℃ and 220℃.
Culture was performed with shaking at rpm. 3 days later, LL-F282
49α (Formula: V=Y=hydroxyl group, W=hydrogen atom, X=
1,3-dimethyl-1-butenyl group) was added using a 5% dioxane solution to give a final concentration of 0.03%, and the mixture was further cultured at 28° C. and 220 rpm for 4 days. After the culture was completed, the reaction solution was extracted three times with 200 ml of ethyl acetate.

The extract was dried over anhydrous sodium sulfate and then concentrated.

The residue was purified by preparative thin layer silica gel chromatography (manufactured by Merck & Co., Art 5717.20 x 20 cm, thickness 2 mm, developed with ethyl acetate) to obtain 30-hydroxy-LL-F28249 (formula I: U=hydroxymethyl group, V=Y=water kick group, W=Z=hydrogen atom, X=1
．． 4.0 mg (yield 4.6%) of 3-dimethyl-1-butenyl group) was obtained. Furthermore, 1 LL-F28249 a
5 mg (recovery rate 17.9≦) was recovered.

4 ml ≥ Z raw mass spectrum (m/z): 628 (M+),
610°592.482,464 Nuclear magnetic resonance spectrum δ (CDC13) ppm:
3.21 (m, LH, C2H), 3.3-3.45 (
m, 3H, C25H, C30H2), 3.51 (d,
LH, C230H, J=5.6Hz), 3.5-3.
7 (m, LH, C17H), 3.75-3.78 (m
, 2H, C23H, C300H).

3.79 (s, LH, C70H), 3.95 (d, L
H, C6'H, J=6.0Hz).

4.29 (t, LH, C70H), 4.68 (br
, s, 2H, C27H2).

Claims (2)

[Claims] (1) A compound represented by the following general formula (II) is used as a substrate, and this substance can be converted into a compound represented by the following general formula (I), such as Amycolata, Amycolatopsis, Streptomyces, Absidia A microorganism belonging to the genus Circinella, Mucor, Syncephalastrum, Mortierella, Rhizopus or Bacillus is cultured in a medium containing a compound represented by formula (II) as a substrate, or Cultured cells or enzyme extracts of these microorganisms are brought into contact with a compound represented by general formula (II) to convert it into a compound represented by general formula (I), and then the compound represented by general formula (I) is converted into a compound represented by general formula (I). A method for producing a compound represented by the general formula (I), which is characterized by collecting: ▲There are mathematical formulas, chemical formulas, tables, etc.▼(II) (In the formula, 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-oleandrosiloxy group, and when V is a hydrogen atom, X represents a methyl group, ethyl group, isopropyl group, sec-butyl group, 1-
methyl-1-propenyl group, 1-methyl-1-butenyl group, or 1,3-dimethyl-1-butenyl group, and when V is a hydroxyl group or an oxo group, 1-methyl-
It represents a 1-propenyl group, a 1-methyl-1-butenyl group, or a 1,3-dimethyl-1-butenyl group, and Y represents a hydroxyl group or a hydroxyimino group. ): ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) (In the formula, U represents a hydroxymethyl group or a carboxy group, V, W, X and Y have the same meanings as above, and Z represents a hydrogen atom or (Indicates a hydroxyl group.) (2) Compound represented by the following general formula (I a): ▲
There are mathematical formulas, chemical formulas, tables, etc. ▼ (I a) (In the formula, U represents a hydroxymethyl group or a carboxy group, W represents a hydrogen atom, a hydroxyl group, or a halogen atom, and Xa represents when U is a hydroxymethyl group. represents a methyl group or an ethyl group, and when U is a carboxy group, a methyl group, an ethyl group, an isopropyl group or a se
represents a c-butyl group, Y represents a hydroxyl group or a hydroxyimino group, and Z represents a hydrogen atom or a hydroxyl group).