JPS6230774A - Physiologically active substance sen-366 and production thereof - Google Patents

Abstract

NEW MATERIAL:A compound expressed by the formula I [Y is >CO or >CH(OR) (R is 1-4C alkyl or substituted arylcarbonyl); X is H or OH; the dotted line part is single or double bond]. USE:A blood platelet agglutination inhibitor. PREPARATION:A microorganism, belonging to the genus Streptomyces and capable of producing SEN-366 is cultivated in a culture medium, and the resul tant culture is directly oxidized, alkylated or arylcarbonylated to give the aimed compound expressed by the formula. Alternatively, a substance expressed by the formula [Y is >CH(OR)] is isolated from the culture, oxidized, alkylated or arylcarbonylated to afford the aimed compound expressed by the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a physiologically active substance called SEN-366 useful as a pharmaceutical and a method for producing the same. More specifically, the present invention relates to the following general formula (1) (wherein, Y is -C=O or C
-0R (wherein R represents lower alkyl or substituted arylcarbonyl having 1 to 4 carbon atoms, X represents hydrogen or hydroxy, = represents -m bond or double bond) The present invention relates to a physiologically active substance designated as SEN-366 and a method for producing the same.

(Prior Art) In recent years, vascular diseases have come to occupy a large part of medical care, both in terms of their morbidity and the difficulty of treatment. In particular, patients with acute myocardial infarction, thrombo-occlusive vasculitis, arteriosclerosis obliterans, chronic glomerulonephritis, nephrotic syndrome, cerebral arteriosclerosis, sequelae of cerebral thrombosis, angina pectoris, patients undergoing surgery, and patients undergoing dialysis for more than one month Chronic renal failure, short-term local anemia,? Thrombosis, such as blood clots, in patients using iR devices (artificial hearts, artificial kidneys, etc.) is becoming a problem as an adult disease. For these thromboses, attempts have been made to administer platelet aggregation inhibitors for the purpose of thrombosis prevention and treatment. , ticlopidine has been applied.

Furthermore, it has been confirmed that platelet aggregation inhibitors have a wide range of usefulness as pharmaceuticals, such as being expected to be substances that inhibit cancer metastasis.

On the other hand, several types of platelet aggregation inhibitors derived from microorganisms are already known, and research on their usefulness as medicines is underway.

For example, WF-52 from eight Spergillus Q.
39 (The Journal of Antibi
otics VOl, 37 N15 469-4
74) and SE from Streptomyces spp.
N-128-B (Japanese Unexamined Patent Publication No. 59-095893), etc.

(Problems to be Solved by the Invention) However, the above-mentioned platelet aggregation inhibitors have certain limitations in their effectiveness, and there is no solid evidence that their safety is necessarily satisfactory.

The present inventors have reluctantly pursued the development of a platelet aggregation inhibitor that is more effective than the existing medicines mentioned above and is even more useful in terms of safety. discovered the existence of a series of substances that are completely unrelated in terms of their chemical structures but are superior in terms of effectiveness and safety, and has already applied for a patent (Japanese Patent Application No. 59-222,740). In the process of continuing research on the compounds related to the above-mentioned application, the present inventors arrived at a group of compounds having pharmacological effects equivalent to or superior to those mentioned above, and completed the present invention. Therefore, the purpose of the present invention, similar to the above-mentioned patent application, is to develop a new drug that is significantly superior to the existing platelet aggregation inhibitors.

(Means for Solving the Problems) The novel physiologically active substance according to the present invention strongly suppresses platelet aggregation, but chemically modifying the substance produced by microorganisms freshly isolated from soil This can be obtained by

The microorganism according to the present invention is a microorganism that succumbs to Streptomyces (S tr≦p to*yces ) as will be described in detail later.
) in which R' and X' are both hydrogen and - is a -m bond or a dim bond), and in SEN-366-PC general formula (II), R' is hydrogen and X' is hydroxy, =
is a -m bond) is a substance directly produced by microorganisms.In the present invention, these microorganism products and their derivatives are referred to as SEN-366.

SEN-366-D is not a single substance, SEN-36
6-D is an acetyl body (referred to as SEN-366-A).
It can be isolated and purified as four substances resembling sex.

These four substances are known as pyran-3 due to their physical and chemical properties.
-A novel physiology with the same structure except for the degree of unsaturation on the on ring and the configuration (α and β anomers) of the OR of the 2-bilanol ring (R' is acetyl in general formula (II)). It can be confirmed that it is an active substance. These 4
The acetyl form of the substance is SEN-366Al and A, respectively.
2, A3 and A4. The structural distinction between them is as shown below.

1.2.3.4 are assigned to those corresponding to Al-A4, respectively. ) In the general formula (I[), R' and For example, benzoyl compound (in the general formula [■], R' is benzoyl,
SEN where X' is hydrogen, = single bond or 2m bond)
-366-B, B2, B3, and B4 pyran-3-
Four substances having different degrees of unsaturation on the on-ring and steric configurations of the OR of the 2-bilanol ring (α and β anomers) can be obtained.

These SEN-366-D and its derivatives have low toxicity and strong platelet aggregation inhibiting action. That is, as detailed below as biological properties, SEN-366
-D and its derivatives are adenosine-5'-diphosphate (
ADP), arachidonic acid (AA), or collagen is used as a platelet aggregation-inducing substance to significantly inhibit platelet aggregation in domestic rabbits. Therefore, these substances are useful as medicaments for the treatment and prevention of the aforementioned thrombosis.

Patent applications have already been filed for these.

The present inventors further studied various analogues of these useful compounds, and found that there are compounds that have pharmacological effects equal to or greater than the derivatives of the above-mentioned compounds, and have completed the present invention. It is something.

Hereinafter, the compounds according to the present invention will be explained in detail.

The compound according to the present invention is represented by the general formula (1), and the following specific examples can be given as each substituent defined herein.

The lower alkyl represented by R in the definition of Y may be linear or branched, for example, methyl,
Examples include ethyl, n-propyl, isopropyl, n-butyl, isobutyl, 5ec-butyl, tert-butyl, and the like. Examples of the aryl that can be cited as substituted arylcarbonyl include phenyl, and these phenyl groups may have the same or different substituents at any position. Examples of substitution on these phenyl groups include halogen, with chlorine or fluorine being particularly preferred.

The compound according to the present invention is represented by the above-mentioned general formula (1), but if it is described in a more organized manner, the following compounds can be mentioned as examples of the compound of the present invention. Here, SEN-366-CB represents a compound in which R in (1) is parachlorobenzoyl.

Moreover, SEN-36G-M represents a compound in which R in [1] is methyl. Furthermore, SEN-366-E is [■
] indicates a compound in which R is ethyl. And S
EN-366-L indicates a compound in (1) where Y is C=O.

SEN-366-D and SEN- according to the present invention.

A representative strain producing 366-P was isolated from soil in Sakyo Ward, Kyoto City by the present inventors, and its mycological properties are as follows. The experiment here was conducted according to the method of International Streptomyces progyecae (ISP). For culture media, those listed in ISP or industry-specific examination standards "Applied microbial industry" are used, and for other media, Waksman (S, A, Waksma) is used.
n; TbeActinomyces Vol, 2
1961) was used. Color descriptions are provided by Container Corporation in America (Cor+La1ner Corporation).
of America) Color Harmony Manual
According to

(1) Description by the ISP method (aJ-type Synthetic characteristics Under the microscope, many aerial hyphae extend from well-branched basal hyphae, and the tip forms a spiral thread with 5 to 6 turns. A curling-like morphology may sometimes be observed. This is especially frequently observed on glucose-asparagine agar media. Division of basal hyphae, whorl formation, and sporangia formation are not observed. ・ISP According to the standards, it belongs to Section 5 spiral.A mature spore chain consists of 10 or more spores.

When observed with a scanning electron V4 microscope, the spores are usually cylindrical and 0.
．． It has a size of T~0.9μ×1.0-1.4μ, and its surface is smooth.

The above morphological characteristics are observed in yeast/malt agar, glycerol/asparagine agar, oatmeal agar, starch inorganic salt agar, and other media that favorably form aerial mycelia.

(The color tone of aerial mycelia that have fully attached spores of bl colonies is grayish (T
resner Backus Co1or Whee
ls). The typical color tone is gray (3fe~5fe
).

(C) Color of vegetative hyphae (back side of colony) exhibits yellow color on oatmeal agar medium. Glycerol・
On asparagine agar medium and yeast malt agar medium, the color is yellow at the early stage of culture, but turns brown later. On starch inorganic salt agar medium, the color changes from pale yellow to gradually grayish. These yellow pigments are pH indicators and change to purple by adding 0.05N sodium hydroxide.

(d) Color of medium (soluble pigment) Melanoid pigment production is positive in peptone yeast soft agar medium, tyrosine agar medium, and tryptone yeast broth. Other pigments produced appear yellow on oatmeal agar, and on glycerol/asparagine agar or yeast/malt agar, the color becomes yellow at the early stage of culture and yellowish brown later. These yellow pigments are pH indicators, and change to reddish-purple to purple by adding 0.05N sodium hydroxide. No pigment production is observed on starch mineral salt agar medium.

(el carbon source utilization IsP description) h utilization D-glucose, L-arabinose, D-xylose,
D-fructose, rhamnose, and D-mannitol are used frequently, and sucrose and raffinose are used weakly. i-inositol is not used.

Utilization of other sugars Galactose, mannose, maltose, and glycerol are used well, and lactose, salicin, and starch are weakly used.9 Inulin is not used.

(2) Other properties (Properties on 81 culture medium (27°C, 114 days culture #) (following next page) ■ Liquefaction of gelatin: Positive ■ Reducibility of nitrate: Positive (weak) ■ Water decomposition ability of starch: Positive ■ Milk Coagulation: Negative ■ Milk peptonization: Positive ■ Hydrogen sulfide production: Negative ■ Furano 41 pigment production: Positive ■ Tyrosinase: Positive ■ Cellulose decomposition: Negative [Phase] Growth temperature range: 10-30°C The optimum temperature is 25-27°C.

■Growth pl+ range: Grows at pl+ of 4.0 to 9.2, and the optimum pH is 6.0 to 7.6.

To summarize the mycological properties of the SEN-366 strain, it produces gray aerial hyphae, the sporulation chain is spiral, and the spore surface is smooth.

The basal hyphae are yellow to brown, the soluble pigments are yellow to yellowish brown, these pigments are pl+ indicators, and 0.0
The color changes to purple by adding 5N sodium hydroxide. Melanin is positive.

As a result of searching ISP and Waxman in consideration of the above mycological properties, the bacterium according to the present invention was found to be Streptomyces galilaeus (Streptomyces galilaeus).
ilaeus). This strain has the characteristic of producing SEN-366-D and/or SEN-366-P, so it is suitable for Streptomyces sp, SEN 36G (Streptomyces sp.
s sp, SEN-366) (FERMBP
-557). This bacterial strain has been deposited at the Institute of Microbial Technology iPj, Agency of Industrial Science and Technology.

In the present invention, strains succumbing to the genus Streptomyces from other countries and SEN-366-D and/or SEN
All bacteria that produce -366-P can be used. In addition, there are artificial mutant strains obtained by irradiating these strains with ultraviolet rays or treatment with mutagenic agents used for mutagenesis of microorganisms such as nitrosoguanidine, and naturally occurring mutant strains. SEN-366-
Variants that produce D and/or SEN-366-P have been found, and these mutant strains can also be used in the present invention.

In implementing the present invention, SEN-366-D, SE
N-366-P-producing bacteria can be cultured using normal culture methods. That is, although the medium may be liquid or solid, shaking culture or aerated agitation culture using a liquid medium is usually used. The medium may be any medium as long as it allows the growth of actinomycetes and produces SEN-366-D and/or SEN-366-P.

That is, as carbon sources, glucose, sucrose, maltose, glycerin, dextrin, starch powder, etc. are used, and as nitrogen sources, soybean flour, peptone, yeast extract, meat extract, corn staple licker, ammonium chloride, etc. are used. Ru.

In addition, good results can be obtained by adding appropriate amounts of sodium chloride, potassium chloride, calcium carbonate, various phosphates, etc.
Furthermore, appropriate amounts of iron, magnesium, etc. may be added, vitamins may be added as necessary, and an antifoaming agent may be added as appropriate to suppress foaming during fermentation.

Culture conditions such as medium pH and culture temperature are as follows: SEN-36
It can be changed as appropriate within the range that produces 6-D and/or SEN-366-r', but for example, in the case of liquid shaking or aeration stirring culture, pl + 6 to 8, culture temperature 22 to 30 ° C. , preferably 24 to 27°C, and the culture period:
It varies depending on the scale of fermentation and other conditions, but usually 1~
SEN-366-D and SEN-366-P are produced in the culture solution in 7 days.

The substance according to the present invention exists in both the culture filtrate and the bacterial cells, and can be obtained from either. SEN-366-D and/or SEN- from culture filtrate and bacterial cells
A wide variety of commonly used methods can be used to extract 366-P.

For example, filtration, centrifugation, extraction using various organic solvents (e.g. acetic acid esters, alcohols, ketones, ethers, etc.), silica adsorption using various adsorbents such as silica gel, alumina, etc., chromatography, gel filtration, and distribution. Chromatography and the like can be used alone or in an appropriate combination.

More specifically, after extracting the culture filtrate or bacterial cells with a ta-rich solvent such as ethyl acetate or acetone, the culture filtrate or bacterial cells are extracted using a column filled with an adsorbent such as silica gel (Wako Gel C-300). The substance is adsorbed through a liquid or a liquid containing the substance, and eluted using an organic solvent such as methanol, chloroborum, ethyl acetate, n-hexane, etc. alone or a mixed solvent of a combination thereof. If necessary,
Furthermore, this eluate is treated with activated carbon such as Darco G-60 and adsorbed again on a column packed with an adsorbent such as silica gel, and then treated with a column containing an adsorbent such as n-hexane or ethyl acetate.
When a solvent is eluted alone or a mixed solvent is used, and the solvent is distilled off under reduced pressure, SEN-366
-D and/or SEN-366-P are obtained as an oil.

(See next page) J゛30
~120°C for 10 minutes to 18 hours, preferably 90
By heating at ~100°C for 15-60 minutes, S
It can be converted into an olefin compound of EN-366-D (a compound represented by the following formula [■[]).

(lit) In addition, the olefin form of SEN-366-D can be efficiently produced by heating a solution or culture solution containing SEN-366-P under the same conditions as above, and performing extraction and purification in the same manner as above. can do.

SEN-366-D thus obtained can be alkylated by a conventional method. This alkylation is 0-alkylation of the hydroxyl group of the anomeric carbon, and a general reaction commonly used for sugar reactions can be applied. For example, the objective can be achieved by reacting with a lower alcohol in the presence of an acid catalyst such as a mineral acid, a Lewis acid, or a cation exchange resin, but preferably a Lewis acid can be used.

When SEN-366-D alkylated as described above is purified by silica gel chromatography and then recrystallized, it is separated into four types of alkyl forms. That is, for example, from methylated SEN-366-D, SEN-366
-M,,SEN-366-M2,SEN 366
M3 and SEN-366-M4 are ethylated SE
From N-366-D, SEN-366-El,,SE
N-366-B2. SEN-366-B3 and SE
N-366-B4 is obtained respectively.

Further, SEN-366-D can be carbonylated by a conventional method. In this aryl carbonylation, in the esterification reaction, for example, acid anhydride,
A conventional esterification reaction using an acid halide or the like alone or together with a suitable catalyst can be applied. If SEN-366-D is 7-arylcarbonyl-formed in this way, four types of arylcarbonyl forms, for example, when parachlorobenzoylated, SEN-366-CB+
, SEN-366-CB2. SEN-366-CB3
, and SEN-366-CB4 are obtained.

Ketone bodies can be produced using SEN-366-D as a raw material. The production of this ketone body is by oxidation of the hydroxyl group at the anomeric carbon position, but it can be produced by ordinary oxidation reactions such as direct oxidation with dimethyl sulfoxide or oxidation with activated dimethyl sulfoxide using dimethyl sulfoxide and dicyclohexylcarbodiimide. Able to apply. In particular, oxidation with silver compounds such as silver oxide, silver carbonate, and silver nitrate is preferred.

Next, the properties of the compounds of the present invention will be described.

(See next page) [Physical and chemical properties] ■SEN-366-CB Developing solvent system; n-hexano/Ac0Et (1:1
) Elemental analysis value: SEN-366-CBI, SEN 366 CB3
(CzsH3tOBC1) God 494.5 calculated value (%)
C: 60.67 H: 6.31 Cl near, 16S
EN 366-CB2. SEN-3(i6-CB4(
C25H330s Cl) Mll 496.5 Calculated value (%) C: 60.42 H: 6.69 C1
: 7.13Rf value is V# layer chromatography Nisilica gel (Merck Art5554) Developer solvent system: n-hexane/Ac0EL (1:1
) Elemental analysis value: SEN-366-M+, SEN-366-M3(C+
91-1300 v) MW 370 calculated value (%)
C: 61. f30 1 (: 8.16 actual value (%)
C: 61.35 II: 8.31 (Mt)
C: 61.33 H: 8.2B (M3)SE
N-366-M2. SEN-366-M4 (C19H3
20) )Men372 Calculated value (%) C: G1.27 H: 8.66 Actual value (%) C: 61.09 H: 8.81 C
M2) C: C+1.20 H: 8.79 (M
4) Rf value is determined by thin layer chromatography: silica gel (
Merck Art5554) Developing solvent system; n-hexane/Ac0Et (1:1
) Elemental analysis values: SEN-366-El, SEN-366-E3(C
2oH3zC)r) MW 384 calculated value (%
) C: 62.48 8: 8.39 Actual value (%)
C: 62.60 II: 8.21 (Et
) C: 62.63 H: 8.35 (E3) SEN-3
66-E2. SEN-366-E4 (C20H3qOy
) MW 386 calculated value (%) C: 62.15
H: 8.87 Actual value (%) C: 61.98 H
:8.99 (E2)C:62.07H:8.9
2(E4) Rf value is determined by VIJii9 chromatography:
Silica gel (Merck ArL5554) Developing solvent system; ClICl3/Me011 (25:
l) Elemental analysis value; SEN-366-Ll (CIl+ 1121107) MW 354 calculated value (%') C: 61.00 H: 7.40 Actual value (%) C: et, t3H near. 26SEN-366
-L2 (ClsH2@Ot) 356 Calculated value (%) C: 60.66 H: 7.92 Actual value (%) C: GO, 42II: 7.76 [Biological properties] Biology of the substance of the present invention The properties are as follows.

<Platelet aggregation inhibitory effect> (1) Materials and methods The specimen is used after being dissolved in dimethyl sulfoxide. The final concentration of solvent is 1%.

Dimethyl sulfoxide at the same concentration was used as a control.

ADPS at a final concentration of 5 μH as a platelet aggregation-inducing substance
150μh of AA (arachidonic acid) and 1oHg/
n+l collagen was used.

(2) Preparation of platelets Collect blood from the common carotid artery of a male rabbit weighing 2.0 to 2.5 kg, add 1/10 volume of 3.8% citric acid to the blood, mix gently, and test at 1300 x g for 2 hours. The supernatant obtained by centrifugation for 1 minute was used as platelet-rich plasma (hereinafter referred to as rPRPJ), and the supernatant obtained by further centrifuging the sediment at 1600 x g for 10 minutes was used as platelet-poor plasma (hereinafter referred to as rPPPJ). And so.

(3) Aggregation light measurement method A platelet aggregation meter was used to record changes over time in absorbance due to hemostatic Fi'624A.

That is, take 200 μi of PRP, adjust the difference from PPP to a constant value, and then add 25 μi of control solvent or sample to PRP.
1 minute later, 25 μa of the agglutination-inducing substance was added, and the change in absorbance was recorded.

The inhibition rate was determined from the maximum agglutination rate (A%) of the control and the maximum agglutination rate (B%) of the specimen according to the following formula.

Inhibition rate (%) = (1-B/A) x100 Find the inhibition rate by changing the sample concentration, then I Cso (5
0% inhibition concentration) was determined. Test results of representative compounds according to the present invention are shown in Table 1.

(See next page) Table 1 I C5O (μg/ml) [Toxicity] It has been confirmed that the compounds of the present invention have extremely low toxicity. For example, LDsO for SEN-366-CB mice is 500 mg/k orally.
g or more, and by intraperitoneal injection it was more than 8 in 100 mg/g.

As shown above, the compound of the present invention has an important biological property of inhibiting platelet aggregation, and is useful as a therapeutic agent for thrombosis, etc., an agent for suppressing cancer metastasis, and the like.

When administering the compound of the present invention as a medicine to patients with thrombosis, the compound of the present invention may be administered as is or in a pharmaceutically acceptable non-toxic and inert carrier, for example, at a concentration of 0.1% to 99.5%.
%, preferably 0.5% to 90%, to animals including humans.

As carriers, one or more solid, semisolid, or liquid diluents, fillers, and other formulation auxiliaries are used.

Preferably, the pharmaceutical composition is administered in dosage unit form. The pharmaceutical composition of the present invention can be administered orally, intracellularly, locally, transdermally, or rectally. Of course, it is administered in a dosage form suitable for these administration methods, such as tablets, granules, powders, capsules, and suppositories. For example, enteric-coated tablets or capsules are particularly preferred for oral administration.

It is desirable to adjust the dose of the platelet aggregation inhibitor after considering the patient's condition such as age, weight, route of administration, nature and severity of the disease, etc. As the active ingredient ■, 0.1 to 100 per day
A range of 0 mg is common.

In some cases, a lower dose than this may be sufficient, and in other cases, a higher dose may be required. When administering a large amount, it is desirable to divide the dose into several times a day.

(Example) The present invention will be specifically described below with reference to Examples, but the present invention is not limited thereto.

Reference Example 1 (Production of SEN-366-D) (1) [Culture] From slant culture of Streptomyces sp, SEN-366, 100ml of seed medium 1 flour 2%, soybean flour 1%
, sodium chloride 0.5%, potassium chloride 0.05%,
Magnesium sulfate 0.05%, sodium nitrate 0.2%
, 0.35% calcium carbonate, pH 7.0> was inoculated into a sterilized 500 ml Erlenmeyer flask, and cultured with shaking at 27° C. for 3 days to obtain a seed culture.

300 ml of this seed culture was inoculated into a 301 volume jar fermenter containing 151 fermentation medium (same medium as the seed medium), and cultured in an aerated container at 28°C for 2 days (aeration volume 1517
m1n, stirring speed 500 rpm). The culture solution 70f thus obtained is separated from the bacterial cells by filtration.

(2) [Extraction, purification of fractionation] The culture filtrate 71 obtained in the above procedure was diluted with 70 Il of ethyl acetate.
Extract times. The extracts were combined and the solvent was distilled off under reduced pressure to obtain 5.7 g of a residue. Add chloroform and water (1:
Add the mixed solvent 41 of 1) and distribute. The chloroform layer was separated, and the solvent was distilled off under reduced pressure to obtain 3.2 g of a residue. The residue was subjected to silica gel column chromatography (=t L, eluting with chloroform and then a mixed solvent of methanol and chloroborm (2%-20%). The fractions containing the active substance were collected and the solvent was distilled off under reduced pressure. 1.1 g of the active substance phase fraction was obtained, which was coated with activated carbon Darco G.
treated with ethyl acetate 200n+ 1 containing -602 g;
After ethyl acetate was distilled off under reduced pressure, the product was subjected to silica gel column chromatography again, eluted with a mixed solvent of n-hexane and ethyl acetate (1:1), and the solvent was distilled off under reduced pressure to obtain SEN-366- 200 mg of D was obtained.

Example 1 (Production of SEN-366-CBI-CB4) 200 mg of SEN-366-D obtained in the same manner as above was dissolved in 1.0 ml of pyridine, 150 mg of parachlorobenzoyl chloride was added, and the mixture was heated at room temperature. After the reaction is complete, add 50 ml of distilled water and add 50 ml of ethyl acetate.
Extract twice. After washing the ethyl acetate with water, it is dried over anhydrous sodium sulfate, and ethyl acetate and pyridine are distilled off under reduced pressure. 300 mg of the residue was subjected to silica gel column chromatography and filtered with n-hexane/ethyl acetate (2:
1), the parachlorobenzoyl compound of SEN-366-D 44! ! , i.e., SEN-366-CB, ,
SEN-366-CB2. 10.15.1 each of SEN-366-CB3 and SEN-366-CB4
0112 mg was obtained.

Example 2 (Manufacture of SEN-366-Ml-M4) 800+sg of SEN-366-D obtained by the same method as above was dissolved in 4.0 ml of anhydrous methanol, 400 mg of anhydrous magnesium sulfate was added, and the mixture was incubated for 3 hours. Reflux.

After the reaction is completed, insoluble materials are removed by filtration. The filtrate is concentrated to dryness under reduced pressure. The residue 750B was subjected to silica gel column chromatography and eluted with n-hexane/ethyl acetate (2:1) to obtain four methyl ether forms of SEN-366-D, namely SEN-366Ml and SEN-36.
6M2, SEN-366-M3, SEN-366-M4
15mg, 26+wg, 10n+g, respectively.
, I got 10w+g.

Example 3 (Production of SEN-366-El to E4) 300+ mg of SEN-366-D obtained in the same manner as above was dissolved in 2.0 ml of absolute ethanol, and 1 ml of anhydrous magnesium sulfate was dissolved.
Add 50 mg and reflux for 2 hours.

After the reaction is completed, insoluble materials are removed by filtration. The filtrate is concentrated to dryness under reduced pressure. 290 mg of the residue was subjected to silica gel column chromatography and eluted with n-hexane/ethyl acetate (2:1) to obtain four ethyl ether forms of SEN-366-D, namely SEN-366-El, SE
N-366-B2, SEN-366-B3. SEN
-366-B4, IOB, 25 mg,
10 mg and 20 mg were obtained.

Reference Example 2 (Production of reduced product and olefin product of SEN-366-D) (1) I of SEN-366-D obtained by the same method as above
Dissolve g in 3 ml of pyridine, add 2 ml of acetic anhydride,
After the reaction was completed by stirring at room temperature for 3 hours, 50-1 of water was added, and the mixture was extracted twice with 50 ml of ethyl acetate. The ethyl acetate layer is washed with water and then dried with anhydrous sulfuric acid. Ethyl acetate and pyridine are distilled off under reduced pressure.

1.05 g of the residue was subjected to silica gel column chromatography and eluted with n-hexane/ethyl acetate (2:l) to obtain four acetyl forms of SEN-366-D, namely SE
N-3 (i6-At, SEN-366-A2.SEN
-366-A3 and SEN-366-A4 were obtained in an amount of 50.65.120.25 On+g.

(2) SEN-366-83 obtained by the same method as above
400mg, 0. Add to 5 ml of IN sodium hydroxide, warm to 50°C and stir for 1 hour. After completion of hydrolysis,
Extraction was carried out twice with 50 ml of ether, and the ether was distilled off under reduced pressure to obtain 250 mg of a residue. The residue was subjected to silica gel column chromatography and treated with n-hexane/ethyl acetate (1:1) to obtain 190 mH of an olefin compound of SEN-366-D.

(3) SEN-366-D 2 obtained by the same method as above
．． Dissolve 5 g in 25 ml of ethanol, add 300 mg of palladium on carbon (5%), and add hydrogen gas while stirring. Quantitatively, only 2.43 g of the reduced form of SEN-366-D was obtained.

Example 4 ('!A construction of SEN-366-L+) Olefin body 1 of SEN-366-D obtained by the above method
00mg in benzene 2.0ml, silver oxide 150mg
mg and reflux for 1 hour. After the reaction was completed, it was filtered and the filtrate was concentrated to dryness under reduced pressure to obtain SEN-366-Lt 8
0 mg was obtained.

Example 5 (Production of SEN-366-L2) 100 m of reduced form of SEN-366-D obtained by the above method
g to benzene2. 150 mg of silver oxide was added and refluxed for 1 hour. After the reaction was completed, it was filtered and the filtrate was distilled off to remove benzene under reduced pressure. SEN-366-L2 BO
B was obtained.

Claims (2)

[Claims] (1) The following general formula [ I ] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [ I ] (In the formula, Y is > C=O or ▲ There are mathematical formulas, chemical formulas, tables, etc. , represents lower alkyl or substituted arylcarbonyl having 1 to 4 carbon atoms.), X represents hydrogen or hydroxy, and ■ represents a single bond or double bond.)
A physiologically active substance called SEN-366 represented by (2) Streptomyces
) SEN-366-producing bacteria belonging to the genus are cultured in a medium, and the culture is used as is, or in [I], Y is ▲ mathematical formula,
There are chemical formulas, tables, etc. ▼ After separating the substances, oxidation,
The following general formula [I], which is characterized by alkylation or arylcarbonylation, ▲There are mathematical formulas, chemical formulas, tables, etc.▼[I] (wherein, Y is >C=O or ▲mathematical formula, chemical formula, There are tables, etc. ▼ (Here, (R represents lower alkyl or substituted arylcarbonyl having 1 to 4 carbon atoms), X represents hydrogen or hydroxy, and ■ represents a single bond or a single bond.)
A method for producing a physiologically active substance called SEN-366 represented by