JPS6248676A - Physiologically active substance sen-366 and production thereof - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I [Y is formula II or III [R is H, lower alkyl, lower acyl or (substituted) arylcarbonyl]; the broken line is single or double bond}. EXAMPLE:SEN-366-Fo (Y is formula III; R is H; the broken line is double bond). USE:A medicine, having low toxicity and powerful inhibitory action on blood platelet agglutination and useful as a remedy for thrombosis, etc., inhibitor for metastasis of cancer, etc. PREPARATION:A microorganism, belonging to the genus Streptomyces and capable or producing SEN-366 is cultivated in a culture medium, and the resultant culture is directly oxidized, alkylated, acylated or arylcarbonylated and isolated to give the aimed compound expressed by formula I. Alternatively, a compound expressed by formula I (Y is formula III; R is H) is separated from the culture, oxidized, acylated or arylcarbonylated and isolated to afford the aimed compound expressed by formula I.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a physiologically active substance called S[jN-366, which is useful as a pharmaceutical, and a method for producing the same. Furthermore, nT, L
The present invention is based on the following general formula [1]/II [wherein, Y is >C=0 or;C-orn (where R is
Represents hydrogen, lower aryl, lower acyl, or substituted or unsubstituted arylcarbonyl. ),
= represents a single bond or a 2m bond. ]
The present invention relates to a physiologically active substance called EN-36G and a method for producing the same.

(Technology from UL) In recent years, vascular diseases have become a major part of medical care, both in terms of their morbidity and the difficulty of treatment. In particular, acute myocardial infarction, thrombo-occlusive vasculitis, arteriosclerosis obliterans, chronic glomerulonephritis, nephrotic syndrome, cerebral arteriosclerosis, cerebral thrombosis, angina pectoris, patients undergoing surgery, and dialysis for more than 1 month. Thrombosis, such as chronic renal failure, short-term local anemia, and blood clots caused by the use of artificial heart devices (artificial heart, artificial kidney, etc.), has become a problem in adults. For these thromboses, the administration of platelet-mediated 10Ill inhibitors has been attempted for the purpose of thrombosis prevention and treatment.For example, for thromboocclusive vasculitis, patients undergoing surgery, and chronic renal failure patients undergoing dialysis. Well,
Chiku "] Piran is applied.

Furthermore, platelet aggregation inhibitors are expected to have a wide range of usefulness as pharmaceuticals, such as being expected to be substances that control cancer metastasis.

The compound of the present invention can also be used as a raw material for producing anthrazycline compounds, which are antitumor agents.

On the other hand, several kinds of platelet 4J4 inhibitors derived from microorganisms are already known, and research on their usefulness as medicines is underway.

In one row, WF-5 from eight supergillus E.
239 (TI+eJournal or Anti
biotics Vol, 37 ff1.5 4
69-474 (1984)) and Strept
SF from bacteria of the genus Omyces, N-128-B (Japanese Unexamined Patent Publication No. 59-095893), and the like.

(Problems to be Solved by the Invention) However, the above-mentioned platelet aggregation 111 inhibitors have certain limitations in their effectiveness, and their safety is not necessarily satisfactory. There was no.

The present inventors have conducted research with the aim of developing a platelet aggregation inhibitor that is more effective than the existing medicines mentioned above and is even more useful in terms of safety.
] 11 Substances that inhibit platelet aggregation are completely unrelated in terms of chemical structure, and we have discovered that there are a series of substances that are superior in terms of effectiveness and safety, and have already applied for a patent (patent application). No. 59-222740, etc.). In the process of continuing research on the compound related to the above-mentioned application, the present inventors arrived at Compound J11'', which has a pharmacological effect equivalent to or exceeding that of the above-mentioned compound, and completed the present invention.Therefore, the present invention was completed. The purpose of the invention, similar to the above patent application, is to develop a new drug that is significantly superior to the existing platelet aggregation inhibiting substances.

(Means for solving the problem) The novel physiologically active substance according to the present invention strongly suppresses platelet aggregation, but it is produced by a microorganism newly extracted from soil, or It can be obtained by chemically modifying the produced substance.

The microorganism according to the present invention is a microorganism that succumbs to Streptomyces VrJ as will be described in detail later.
Substances in which R is hydrogen and = is a single bond or a double bond) are substances directly produced by microorganisms.

In the present invention, these microbial products and their derivatives are referred to as SEN-366.

SEN-366-F is not a single substance, SEN-36
6-F is induced into an acetyl form (referred to as SEN-366-F-A) and is converted into a single i! lt
It can be made from Kn.

These four substances are known as pyran-3 due to their physical and chemical properties.
The degree of unsaturation on the -one ring and the steric coordination (α and β
By subtracting the fact that the anomers) are different, it can be confirmed that they are novel physiologically active substances having the same structure. The acetyl forms of these four substances are each SEN-366-F-81
, A2, A3 and 84. The structural distinction between them is as shown below.

In addition, SL', N-366-F was analyzed using column chroma 1, and the olefin form of SIF, N3GGF was obtained by the general formula (1), where - is a 2m bond and R is hydrogen: a substance called 5rEN-36G-Fo. , the other side OR
The compound with α coordination is 5UN-366-Fl, and the compound with β coordination of OR is 5UN-3 (referred to as i6-F3).
and a reduced form of SEN-366-F (a substance in which = is a single bond and R is hydrogen in the general formula [1]: SEN-366-
It is called Fo, and the compound in which the OR coordination is α is called SEN.
-366-F2, the compound with β coordination of OR is S1miN-
It is called 366-F4. ) can be made in minutes.

These SIF, N-366-F and their derivatives have low toxicity and strong platelet new formation inhibiting action. Immediately,
The biological properties of sea urchins are detailed below.
-366-F&C)"cO)af:, ili body is
Suppression of platelet aggregation j1 action in domestic rabbits using adenosine-5'-diphosphate (ΔDl'), arachidonic acid (AΔ), or collagen as a platelet 115 inducer. These substances are therefore useful as medicines for the treatment and prevention of the above-mentioned conditions.

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 alkyl represented by R in the definition of Y may be straight or branched alkyl having 1 to 4 carbon atoms, such as methyl, cochyl, n-pyl, isopropyl, n-butyl, isobutyl, 5ec-butyl,
Examples include tert-butyl.

The lower acyl may be straight or branched having 2 to 6 carbon atoms, such as acetyl, propionyl, n-
Examples include butyryl, isobutyryl, n-pentanoyl, n-hexanoyl, and the like.

Examples of aryl that can be mentioned as carbonyl 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 described in a more organized manner, the following compounds can be mentioned as examples of the compound of the present invention.

SEN-366-F-Δ indicates a compound in which R is acetyl in (+), SEN-366-F-BT is
In N), R is butyryl. SEN
-366-F-B represents a compound in which R in [I] is benzoyl.

SEN-366-F-CB represents a compound in which R in [] is parachlorobenzoyl. Also SEN-3
6G-F-M represents a compound in (1) in which R is methyl. Furthermore, SEN-366-F-E indicates a compound in which R in (1) is ethyl.

And 5UN-366-F-L is Y in [1]
represents a compound where >C=O, and there are four types of these compounds depending on the types of OR and - in the general formula (1). Compounds in which the OR coordination is α and the - is a double bond are respectively SEN-366-F-
81.13 Compounds in which the coordination of Bl, CBI, Ml, El, and OR are α and = single bond are each SIE
N-366-F-A2, BT2, B2, CB
Compounds in which the coordination of 2, M2, F2, and OR are β and - are 2m bonds are SEN-366-F-A3 and BT, respectively.
SEN-3G6-F-
84, BT4, B4, CB4, M4, +? , 4.

Also, compounds where Y is 〉C=0 and = is a double bond are SE
A compound in which N-3613-, F-LO, Y is >C=0 and - is a single bond is referred to as SI', N-36 (i-F-Lll).

A representative strain producing Sumi N-366-F according to the present invention was developed by the present inventors from ±1E in Sakyo Ward, Kyoto City.
i', 11, and its mycological properties are as follows. The experiment here is International Strezo 1
According to the method of Mrs. Project 1-(IsP). For culture media, those listed in ISP or industry-specific examination standards "Applied microbial industry" are used, and those other than those listed in Waxman (S, A) are used.

The Ac Linnmyce
s Vol, 219G1) was used. Color 81! , 1 is the description of container 21
- Volley Soyon Off-Americo J (Contain
er CorporaLion of eight eric11
) Color Harmony Manual (Color I
l+rmonyM;+nual).

(1) In the case of IsI), a large number of aerial hyphae were extended from the branched medium hyphae, and 1° beyond them. r++', i l; Forms a spiral thread with 15 to 6 turns.In addition, a curling-like form is sometimes observed.
It is frequently observed on asparagine agar medium. No fragmentation of hyphae, whorl formation, or formation of spores Eε was observed. According to the ISP's standard, -Hixoyon Spiral (
Section 5piral). The spore chain that has grown is I011+, and a chain of one or more spores is recognized.

Scanning electron! When observed with an IQ speculum, the spores are cylindrical and have a size of 0.7 to 0.9 μ x 1.0 to 1.717 I, and the surface is smooth.

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

(Color of the BL Colony The color of aerial mycelia fully attached to spores is grayish (Tresner Backus Co1or Wh) on yeast agar, glycerol/asparagine agar, starch inorganic salt agar, and oatmeal agar.
eels). The typical color tone is gray (3[e~5f
e).

Ic) Color of vegetative hyphae (back side of colony) Appears yellow on oatmeal agar medium. Glycerol・
Asparagine agar, Ease] and malt agar,
It is yellow in the early stage of culture, but later turns brown. On starch-free 1 ffl salt agar medium, the color changes from pale yellow to gradually grayish. These yellow pigments are pH indicators and change to purple with each addition of 0.05N sulfur/lium hydroxide.

(d) Color of medium (soluble pigment) Melanoid pigment production is positive in peptone yeast Chichichi agar medium, Chichi I:1 Shin agar medium, and tryptone yeast Pros. Other pigments produced are yellow on O-1-i-fresh agar medium and glycerol-11-
Asparagine agar, yeast/malt agar,
It is yellow at the early stage of cultivation and becomes yellowish brown later on. These yellow pigments are pl+indicators and are 0.05N
When sodium hydroxide is added, the color changes to reddish-purple to purple. No pigment production is observed on starch mineral salt agar medium.

Utilization of tel carbon sources Utilization of sugars described in ISP Utilizes D-glucose, L-arabinose, x)-xolose, D-fructose, L-rhamnose, and D-mannitol well, and weakly uses Shoota 1''-su and raffinose. Make use of it. 1-zenone 1--le is not used.

Utilization of other sugars Galactose, mannose, maltose, and glycerol are used well, and lactose, salicin, and starch are used weakly. Do not use inulin.

(2) Other properties + 8 + Properties on culture medium (27℃, 14 days culture) (see next page) fbl physiological properties ■ Liquefaction of gelatin: positive 1 (1 ■ Nitrate reduction Ni positive 41 + (weak) ■ starch Water decomposition ability: Positive ■ Milk coagulation: Negative ■ Milk peptonization; Positive ■ Hydrogen sulfide production: Negative ■ Melanoid pigment productivity: Positive) η ■ Tyrosinase double positive ■ Cellulose fraction pl? , :Negative [phase] Growth temperature range: Grows at 10-30°C, optimal temperature is 25-27°C.

O growth p11 range: Grows at p11, 0 to 9.2, and optimal pl+ is 6.0 to 7.6.

To summarize the mycological properties of SIF and N-3GG strains, they produce gray aerial hyphae, and the spore-forming hyphae are spiral-shaped.
) and the spore surface is flat? It is Fk (smooth type).

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 thorium hydroxide. Melanin is positive.

As a result of searching ISP and Wacknoman in consideration of the above mycological properties, the bacterium according to the present invention was found to be Strepjomyces galilaeus.
aeus) was identified as succumbing to Noriaki. This strain is S
Since it has the characteristic of producing EN-366-F,
Streso l-Myces sp, SEN-366 (St
repLomyces sp, SEF, N-
366) (FERMBP-557). This strain has been deposited at the National Institute of Industrial Science and Technology, Agency of Industrial Science and Technology, Tetsu 4 (Deposit number: FERM'-557).

In the present invention, strains belonging to the genus Streptomyces other than this strain that produce SEN-366-F include:
All can be used. In addition, these strains can be exposed to N? by illuminating them with ultraviolet light or by treatment with mutagens used for mutagenesis of microorganisms, such as 21.1=1 soguanidine. Among the artificially generated mutant strains and naturally occurring mutant strains that produce SEN-366-F, these mutant strains can also be used in the present invention.

In carrying out the present invention, 5EII-366-F producing bacteria can be cultured using a conventional culture method. That is, the culture medium may be either liquid or solid, but if the culture medium is oil-tight, a shaking culture using a liquid medium or a stirring culture in a stirred container is used. Any medium may be used as long as it is suitable for the growth of actinomycetes and can produce SF and N-366-F.

That is, carbon sources include glucose, sucrose, maltose, glycerin, dextrin, starch powder, etc., and nitrogen sources include soybean 1'5), Pep 1/Nino, yeast extract, meat extract, corn staple licker, Ammonium chloride etc. are used.

In addition, good results can be obtained by adding sulium chloride, potassium chloride, Calcilla carbonate J1, various phosphates, etc.
4. Further, appropriate amounts of iron, magnesium, etc. may be added.

In addition to adding vitamin C if necessary, an antifoaming agent can be added as appropriate to suppress foaming in the 1Iib.

Medium pl+, culture? l'l! Culture strip 11 such as 1 degree
can be changed as appropriate within the range that produces SEN-366-F, but for example, shaking or aeration of the liquid
In the case of agitation culture, the pH is 6-B and the culture temperature is 22-30.
'C3 Preferably at 24 to 27° C. The culture period varies depending on the scale of fermentation and other conditions, but SEN-366-F is produced in the culture solution in 1 Wl to 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. To extract SEN-3G (3-F) from the culture source 21k and the bacterial cells, a method commonly used in Jlrl can be widely used.

For example, extraction with v''!, centrifugal separation agent, various Rt organic solvents (e.g. Nishiichi acid ester, 7 alcohol luther lamp, etc.), desorption with various adsorbents such as sori gel, alumina, etc. 71 - method, Kell filtration method, partition chromatography method, etc. can be used alone or in an appropriate combination.

More specifically, after extracting the culture solution or bacterial cells with an organic solvent such as ethyl Fitate or acetone, the culture filtrate or cells are placed in a column filled with an adsorbent such as Soriki Gel (Wako Gel C-300). Adsorbed through LSI bacterial cell extract or liquid containing this substance, methanol, chloroborum,
Organic solvents such as ethyl acetate and n-hexano are eluted using a mixed solvent of 1 U alone or a mixture of these. If necessary, add this eluate to Darco
After treating with activated carbon such as G-C)O and adsorbing it again in a column packed with an adsorbent such as solica gel, organic solvents such as n-hexano and ethyl acetate are added individually or in combination or mixed. Elution is carried out using a medium containing 100 ml of chloride, and the solvent is distilled off under reduced pressure to obtain SIE N-3 (i6-F) as an oily substance.

SEN-3[;-F thus obtained can be acylated or arylcarbonylated according to a trial decision. In this acylation and arylcarbonylation, a conventional esterification reaction can be applied, for example, using an acid anhydride, an acid halide, etc. together with an appropriate solvent for f11 or ζ31. In this way, SIF. N-
3 (i If G-F is acedelylated, 4
Acedeloid of species, III et al., SEN-313 (i-F
−△, ,SIF. 11-3 (i6-F-82, SEN
-366-F-Δ3, and SIJN-366-F-84
i6 Rugoto can do it. When the lower acyl derivative or arylcarbonyl derivative obtained in this way is hydrolyzed by density, for example, S[EN-3(i6-F-Δ
1 or SP. N-3 6 6-F-83 from SEN-
3 6 6-F olefin SEN-366-Fo
(i.e., a mixture of F and F3) is S F'. N-
3 6 6-F-Δ2 or SEN-366-F-
Reduced SL from 84? ．． N-366-F11 (ie a mixture of F2 and F4) is obtained. Also SEN-3 6
G-F or F. When catalytically reduced by density, the reduced product S
Only EN-366-Fo (ie a mixture of F2 and F4) is obtained.

Further, SEN-3 6 6-F can be alkylated by a space method. This alkylation is 0-alkylation of the hydroxyl linkage of the anomeric carbon, and a general reaction commonly used in the reaction of sugars can be applied. The purpose can also 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.

Furthermore, it is possible to produce the filtrate isomer using SEN-36 (i-F) as a raw material. The production of the gen-i-n isomer is due to the oxidation of the hyalyloxyl at the 7-nome link carbon position. , for example, direct oxidation with dimethyl sulfoxide or oxidation with activated dimethyl sulfoxide using dimethyl sulfoxide and dicyclohexylcarbodiimide.

In particular, oxidation with silver compounds such as silver oxide, silver carbonate, silver occilate, etc. is preferable.

Also, SI", N-306-F, 5EEN-366-
F o and 5UN-3G 6-Fl, each preceded by a single g'1 (Japanese Patent Application No. 1982-2227/10), have the following general formula [(Ill) (IJ, - is a - double bond) or represents a double bond) (-”
5UN-3 (i 6-Do (
- is a double bond, - is a mixture of α- or β-anomers), and SUN -36(i-1)o (=111
One leaf at the terminal bond is a mixture of α- or β-anomers),
It can be made by acid decomposition.

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

[Physical and chemical properties]

■SI7. N-3 [1-F-A developing solvent system; n-hexyne/^COF, t (]:
) Elemental analysis value: SF, N-366-F-Δ1.5IEN-36(i-F
-Δ3(C+4)12oOs) Calculated value (%) C: 59.14 II: 7.0
9 Actual value (%) C: 59.43 II Near, 02
(I・-7\l)C: 59.29 II: 7
．． 013 (F-Δ3)SEN-366-F-Δ2. S
EN-366-F-△4 ((2+4 H2206) li calculation (〆((%) C: 58.73 It:
7.75 Actual value (%) C: 5B, 96 11:
7.7/I (F-Δ2)C: 5B, 76N
: 7.83 (F-Δ4)NMR(CDCl2
) δ ; SEN-366-F-Δl: 1.22
(311, d).

1.39 (311,d), 2.1(1(3
11,s).

3.6El(Ill, hr), 4.05
(ill, dq).

4.58 (IIL q), 5.25 (
Ill, d).

C1, IO(Ill, d), 6.15
(III, br).

6.87 (ill, dd) SEN-366-F-Δ2: 1.20 (311
, d).

1.28 (311,d), 2.10 (3
11,s).

3.65 (III, br), 4.03
(III, q).

4.33 (Ill, q), 5.07 (
III, t).

6.16 (ill, br).

5IEN-366-F-Δ3: 1.29 (31
1,6).

1.38 (311,d), 2.09 (311
,s).

3.58 (Ill, br), 3.79 (I
ll, dq).

4.59 (Ill, q) + 5.21 (11
1+ d) +5.71 (ill, dd),
0.013 (ill, d), 6.85 (I
II, d) SEN 36G-F-Δ4: 1
．． 27 (311, d).

1.28 (311,d), 2.11 (311
,s).

3.55 (III, br), :3.77
(Ill, tlq).

4.33 (III, q) + 5.05 (l
lI+L) +5.71 (III, r
id).

■Sr'! , N-366-F-13T Developing medium system; n-hexane/^c(lliL (2:
I) Elemental analysis value: (C+al12sOe) δ1 calculated value (%) C: 61.13 II: 13.
34■SEN-366-F-B Developing solvent system; n-hexane/Ac0F, L (2:
1) Elemental bone 11i value: 5UN-36G-F-132, 5IEN-366-F-
34 (CI911240G) il'! (+'O, (%) C: 65..50 1!
: 6.94 Real 1 rule (direct (%) C: 65.21
II: (C99(F132)
C: 65.49 II: (i, 9G (F-134
) ■SF, N-366-F-CB Developing solvent system; n-hexane/^cO1,L (2:
1) Elemental analysis (ifi: (CI9 +T230e C1) Estimated value (%) C: 59.61 H: 6.0 (i
Actual value (%) C: 59. (12II:6.07 (
F-CB2)C:59.59 II:6. (15
(F-CB4) ■SL, N-366F-M Developing solvent system; n-hexane/AcO[iL (1:
1) Elemental analysis value: SEN-366-F-M2. SEN-366-F-M4
(C1311220s) NI conversion, value (%) C: 60.44 II: 8
．． 59■5UN-366-F-E Developing solvent system -〇-hexicane/distilled:()lmit(2:l)
Elemental analysis value: SEN-366-F-B2. SEN-:3 G 6-F
-B4 (CI4 th405) Calculated value (%) C: 61.74 +1: 8. E
H(■S F?, N-3G G-'F-L developing solvent system; 1-11:1 Pol J, 7 methanol (25:1
) Genki 1 analysis: SEN-366-F-Lu (C+211+5Os) Calculation Direct (%) C: 59.4 If
: 7.49 Actual value (%) C: 5! L, 73
If: 7.51 [Biological Properties] The biological properties of the substance of the present invention are as follows.

<Platelet aggregation inhibitory effect> [November age, l and method Specimen is dimedyl sulfoxy 1; W II+7 L,
Use with. 8 times so that the final concentration of the tank is 1%? JTru.

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

AI]P at a final concentration of 5 μH as a platelet aggregation 1B inducing substance
, 150 μH hehe (arachidonic acid), and 10 μ8
/ml collagen was used.

(2) Platelet body 1122. Blood was collected from the common carotid artery of a male rabbit weighing ~2.5 kg, 1/10 volume of 3.8% citric acid was added to the blood, mixed gently, and centrifuged at 1300 x g for 2 minutes.
! 7. The supernatant was collected as platelet-rich blood cells (hereinafter referred to as r P RP J
). The precipitate was further centrifuged at 1000 x g for 10 minutes, and the resulting supernatant was purified with platelet-poor blood 'Jlt (
, hereinafter referred to as 1-PPP-1).

(3) Method for measuring aggregation ability A platelet aggregation meter was used to record changes in absorbance over time due to platelet aggregation.

That is, take 200 μl of PRP and adjust the difference between i) l) 1) to a constant value, then add 25 μl of the control solvent or sample to r'Rp and stir at 11,000 rpm. After 1 minute, add 25 μl of the agglutination-inducing substance. Changes in absorbance were recorded.

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

Inhibition rate (%) = (1 - B/A)
The inhibitory concentration) was determined. Test results of representative compounds according to the present invention are shown in Table 1.

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

As the carrier, solid, semi-solid, or liquid diluents, fillers, and other formulation auxiliaries - 1.n or more are used. Preferably, the pharmaceutical composition is administered in a dosage form. The pharmaceutical composition of the present invention can be administered orally, intracellularly,
It can be administered topically, transdermally, or rectally. Dosage forms suitable for these administration methods, such as tablets,
Of course, it can be administered in the form of granules, Ilk tablets, capsules, injections, suppositories, and the like. For example, enteric-coated tablets or capsules are particularly preferred for oral administration.

The dosage for use as a platelet aggregation inhibitor is determined by considering the patient's condition such as age, physical condition, route of administration, nature and severity of the disease, etc. Normally, the amount of the active ingredient of the present invention for adults is 0.1 to 1 per day.
A range of 0.000 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.

(See next page) Example 1 [SEN-366-F, SEN-366-
Fo, SEN-366-Fl (manufacture) (1) [Culture] From a slant culture of Streptomyces sp. SEN-366, 100 ml of seed medium (2% starch, 1% soybean flour)
, sodium chloride 0.5%, potassium chloride 0.05%,
Magnesium sulfate 0.05%, nitric acid Lium 0.2
%, calcium carbonate 0.35%, pl+7.0) in a sterilized 500 ml Erlenmeyer flask, and cultured at 27°C for 30 minutes with shaking.
A seed culture solution was obtained.

300ml of this seed culture solution costs 151! Fermentation medium (same medium as the seed medium) was inoculated into a 30β volume Jiahua Menku, and cultured with aeration at 28°C for 2 days (aeration m15
/! /n+in, jll stirring speed 500 rpm). The culture solution 70j2 thus obtained is filtered from the bacterial cells.

(2) [Extraction, Fraction F error, purification] 7 ol of the culture filtrate obtained in the above procedure was diluted with acid-resistant ethyl 706.
Extract times. The extracts were combined and the solvent was distilled off under reduced pressure to obtain 8.3 g of a residue. Add chloroborum and water (1:
Add the mixed solvent 6e of 1) and distribute. The chloroform layer was separated, and the solvent was distilled off under reduced pressure to obtain 4.7 g of a residue. This residue was subjected to silica gel column chromatography and eluted with chloroform and then with a mixed solvent of methanol and chloroform (2%-20%). A fraction containing the active substance is built up, the solvent is distilled off under reduced pressure, and the active substance is removed.
166 g of fraction n was obtained. This is activated carbon Darco G-
After the ethyl acetate was distilled off under reduced pressure, it was subjected to silica gel column chromatography again (=j), and dissolved with a mixed solvent of n-hexane and ethyl acetate (7:3). was distilled off under reduced pressure to obtain 91 mg of SEN-366-F.

(3) [Fraction, Fo, violation of Fo, purification] SEN-366-F obtained by the same method 1. Og was subjected to reverse phase column chromatography (LiCbrocrepRplB) and dissolved with a mixed solvent of acetonitrile and water (25:75) to obtain 110 mg of SEN-366-Fo and SE
720 mg of N-366-Fu was added to 1 q. The respective IH-nuclear magnetic resonance spectra are shown in FIGS. 1 and 2.

Example 2 (Production of SEN-366-F-A+ to A4) SEN-36 obtained in the same manner as in Example 1 (2) above
2.0 g of 6-F was dissolved in 8 ml of pyridine, 10 ml of acetic anhydride was added, and the mixture was stirred at room temperature for 2 hours. After the reaction is complete, add 70 ml of water and extract three times with 80 ml of ethyl acetate. The ethyl acetate layer is washed with water, dried over anhydrous sodium sulfate, and the solvent is distilled off under reduced pressure. 2.2 g of the residue was subjected to silica gel column chromatography and eluted with n-hexane/ethyl acetate (2:1) to obtain SEN-366.
-Four types of acetyl forms of F-A, I! llchi, SEN-36
6-F-8,, SEN-3f36-F A2. SEN
-366F A3. SEN-366-F-A, 139 mg, 204 mg, 163 mg, 14
I got 9mg.

Example 3 (SEN 366 F-Br3.SEN
-366-F-Br3 Production] SEN-36 obtained in the same manner as in Example 1 (3) above
1.0 g of 6-Fu was dissolved in 3 ml of pyridine, 1.1 g of n-butyl chloride was added, and the mixture was stirred at room temperature for 30 minutes ('
Do 12. After the reaction is completed, 40 ml of water is added, and the mixture is extracted three times with 40 ml of ethyl acetate. After washing the ethyl acetate layer with water,
Nothing,! ,′,, Dry over sodium sulfate and remove the solvent under reduced pressure. The residue 2.1e was subjected to silica gel column chromatography (n-hexane/ethyl acetate 2:1) and medium pressure column chromatography (n-hexane/ethyl acetate=2:1).
4: In accordance with 1), two types of n-butyryl derivatives SEN-366-F-BT2. SEN-366-F-B
52 mg and 461 mg of r3 were obtained, respectively.

Example 4 [SEN-3G GF-82, SEN-
Production of 366-F-B4] SEN-36 obtained in the same manner as in Example 1 (3) above
Dissolve 1.1 g of 6-Fo in 5 ml of pyridine, add 2 g of benzoic anhydride, and stir at 70°C for 6 hours.

After the reaction is complete, add 40 ml of water and add 20 ml of diethyl ether.
Extract 4 times with m1. After drying the ether extract, the ether was distilled off under reduced pressure to obtain 3.8 g of a syrupy residue. This residue was purified by silica gel column chromatography (n-hexane/ethyl acetate = 11) to obtain two types of hendiyl compound SEN-366-F-B2. SEN-36
429 mg of 6-'F-34 as colorless cylindrical crystals
, 536mg15.

Example 5 [SEN-366-F-CB2. SEN-3
Production of 66-F-CB4] SEN-36 obtained in the same manner as in Example 1 (3) above
6-F ++ 1. Og to pyridine 4. Dissolved in On+1, parachlorohendiyl chlorite'0. [1m
+ and stir at room temperature for 1 hour. After the reaction is completed, 30 ml of distilled water is added and extracted 4 times with 30 ml of diethyl ether. The diethyl ether layer is washed with water, dried over anhydrous sodium sulfate, and diethyl ether and pyridine are distilled off under reduced pressure. 1.2 g of the residue was subjected to silica gel column chromatography and treated with n-hexane/ethyl acetate (21). Then, medium pressure column chromatography (eluent: n-hexane/ethyl acetate = 7:
3), and each of the two parachlorobenzoyl derivatives of SEN-366-F, namely SEN-366-F-CB2 and SEN-366-F-CB4, was purified by 55 n
Ag, 364 mg was obtained.

Example 6 (SEN-366-F-M2, SEN-3
Production of 66-F-M4] SEN-36 obtained in the same manner as in Example 1 (3) above
1.0 g of 6-Fll was added to 5.0 g of anhydrous methanol. 0ml, add 500mg of anhydrous magnesium sulfate, 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. 929 mg of the residue was subjected to silica gel column chromatography (; ), and two types of methyl ethers of SEN-366-F, namely SEN-366-F-M2
, 395 mg each of SEN-366-F-M4,
100 mg was obtained.

Example 7 (SEN-366-F-B2.SEN-3
Production of 66-F-B4] SEN-36 obtained in the same manner as in Example 1 (3) above
1.09 g of 6-Fo was dissolved in 5.0 ml of absolute ethanol, 500 mg of anhydrous magnesium sulfate was added, and 1.09 g of 6-Fo was dissolved in 5.0 ml of absolute ethanol.
Reflux for an hour. After the reaction is completed, insoluble materials are removed by filtration. The filtrate is concentrated to dryness under reduced pressure. 812 mg of the residue was subjected to silica gel column chromatography and eluted with n-hexane/ethyl acetate (2:1) to obtain SEN-36.
Two ethyl ethers of 6-F, namely SEN-366-
F-B2. 35 each of SEN-366-F-B4
1mg, 103mg 1l.

Example 8 (Production of SEN-366-F-Lu) 50% of SEN-366FH obtained by the method of Example 1 (3) above
0+ng to benzene 5. Oml 8) Good, oxidized!
Add 700+ng and react at 60°C for 1 hour. After the reaction was completed, the filtrate was separated by filtration, the benzene was distilled off under reduced pressure, and the filtrate was recrystallized from ether-〇-hexane to obtain SEN-366.
-F-Lo 80 mg was obtained.

Reference Example 1 (1) (Extraction, isolation, and purification of SEN-366-D) The culture filtrate 1ON obtained in the operation of Example 1 (1) is extracted twice with 70 p of ethyl acetate. The extracts were combined and the solvent was distilled off under reduced pressure to obtain 5.7 g of a residue. A mixed solvent 4β of chloroborum and water (1:1) is added to this and distributed. The chloroform layer was separated, the solvent was distilled off under reduced pressure, and the residue 3
．． 2g was obtained. This residue was subjected to silica gel column chromatography, eluting with chloroborm and then with a mixed solvent of methanol and chloroform (2%-20%).

Fractions containing the active substance were collected, and the solvent was distilled off under reduced pressure to obtain 1.1 g of active substance 111 fractions. Add this to activated charcoal Dar
The mixture was treated with 200 ml of ethyl acetate containing 602 g of co G-60, and the ethyl acetate was distilled off under reduced pressure, then subjected to silica gel column chromatography again, and eluted with a mixed solvent of n-hexane and ethyl acetate (1:1). The solvent was distilled off under reduced pressure to obtain 200 mg of SEN-366D.

(2) [SEN-3 (Production of i6281-A4) 1 g of SEN-366-D obtained in the same manner as above was dissolved in 3 ml of pyridine, 2 ml of acetic anhydride was added, and the mixture was stirred at room temperature for 3 hours. After the reaction is complete, add 50ml of water and extract twice with 50ml of ethyl acetate.The ethyl acetate layer is washed with water, dried over anhydrous sodium sulfate, and ethyl acetate and pyridine are distilled off under reduced pressure.Residue 1 , 05g was subjected to silica gel column chromatography and eluted with n-hexane/ethyl acetate (2:1) to obtain four acetyl forms of SEN-366-D, namely, SEN-366-Al,
5IEN-366-A2. SEN-366-A3. S.E.
50 mg and 65 mg of N-366-A4, respectively.
, 120 mg, and 250 mg were obtained.

(3) (Production of olefin body of SEN-366-D) 400 of SEN-366-A3 obtained by the same method as above
mg, 0. Add to 5 ml of IN sodium hydroxide and add 50
Warm to ℃ and stir for 1 hour. After completion of the hydrolysis α'', extraction was carried out twice with 50 ml of ether, and the ether was distilled off under reduced pressure to obtain 250+ ng of a residue.The residue was subjected to silica gel column chromatography and extracted with n-hexane/ethyl acetate. (1:1) to obtain 190 mg of an olefin form of SEN-366-D (SEN-366-Do).

Example 9 SEN-366D obtained in the same manner as the reference example.

520mg of O, IN salt! ! Add 10ml and stir at room temperature for 1 hour. After the reaction, the aqueous solution is neutralized with sodium hydroxide and extracted three times with 15 ml of ethyl acetate. The solvent of the ethyl acetate layer was distilled off under reduced pressure, and the residue was subjected to medium pressure column chromatography (silica gel).
- SEN-366-F by elution with hexane (3:7)
91.4 mg of O was obtained as an oily substance.

[Brief explanation of the drawing]

FIG. 1 shows SEN-366-F according to the present invention. The IH-NMR spectrum of is shown. The measurement conditions are 200
MHz, in CDCl3, the internal standard is TMS. The second drawing shows the IH of SEN-366-FM according to the present invention.
- Shows the NMR spectrum. Measurement conditions are 200MHz
, in CDCl3, the internal standard is TMS.

Claims (2)

[Claims] (1) The following general formula [ I ] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [ I ] [In the formula, Y is ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ or ▲
There are mathematical formulas, chemical formulas, tables, etc. (here R is hydrogen,
Represents lower alkyl, lower acyl, or substituted or unsubstituted arylcarbonyl. ), and ■ represents a single bond or a double bond. ] SEN-36
A physiologically active substance called 6. (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,
There are the following general formulas [I]▲mathematical formulas, chemical formulas, tables, etc.▼[I] [wherein Y is ▲mathematical formulas, chemical formulas, There are tables etc. ▼ or ▲
There are mathematical formulas, chemical formulas, tables, etc. (here R is hydrogen,
Represents lower alkyl, lower acyl, or substituted or unsubstituted arylcarbonyl. ), and ■ represents a single bond or a double bond. ] SEN-36
A method for producing a physiologically active substance referred to as No. 6.