JPS5995893A - Sen-128-b substance and its preparation - Google Patents

Abstract

NEW MATERIAL:SEN-128-B substance having the following characteristics: appearance, colorless acicular crystal; melting point, 119-125 deg.C; elementary analysis (%), C 63.21, H 6.63, N 11.01; molecular weight, 248; molecular formula C13H16N2O3; specific rotation, [alpha]<24>D=0 deg. (C=0.5%, chloroform); solubility, easily soluble in chloroform, etc. and insoluble in water; color reactions, positive to iodine and potassium permanganate reactions and negative to ferric chloride and ninhydrin reactions. USE:Remedy for thrombosis, etc. PROCESS:An SEN-128-B-producing microbial strain [e.g. Streptomyces melanosporofaciens SEN-128 (FERM-P No.6775)] is cultured in a medium, and the SEN- 128-B is separated from the culture liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention has significant biological activity. The present invention relates to a novel microbial metabolite 5EN-128-B substance.

5EN-128-B according to the present invention is 5EN-1 together with the substance referred to as 5EN-128-A in this specification.
They are collectively called 28. 5EN-128-A is 5EN-1
It is a substance obtained by the same method as 28-B and has similar physicochemical properties and biological activities.

5EN-128-B according to the present invention has the following physical and chemical properties.

+a+ Appearance Colorless needle crystals (bl melting point 119-125°C TC+ elemental analysis actual value c: 63.21 H: 6.63
N: 11.01 theoretical value C: 62.89 H:
6.50 N: 11.28fd) Molecular weight
248 (Mass spectrum) te1 molecular formula C1
RH16N203 (fl optical rotation (α] g=0° C
= 0.5% (in chloroborum) (g+ Ultraviolet absorption spectrum as shown in Figures 1 and 2, showing maximum absorption at 247 mμ and 315 mμ in methanol and acidic methanol. 238 mμ in alkaline methanol, 26
Maximum absorption is shown at 5 mμ (shoulder) and 290 to 310 mμ.

(hll infrared and absorption spectrum This is shown in Figure 3.

+11lllll-1-N spectrum It is shown in Figure 4.

(Jl Soluble Easily soluble in chloroform, methanol, ethyl acetate 1 acetone, dimethyl sulfoxide, insoluble in water.

(kl color reaction positive: iodine, potassium permanganate negative: ferric chloride, ninhydrin FIIRf value Thin layer chromatography using silica gel (^r t 5554) manufactured by Merck & Co., West Germany) Odor shows the following Rf value .

Developing solvent chloroform:methanol-50:10,7
8 Developing solvent Hensen: Ethyl acetate: Ri00vol-1:
1:1 0.63 ((b) Distinction between neutral, acidic, and basic basic substances The biological properties of the substances of the present invention 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 adjusted to 1%. Dimethyl sulfoxide at the same concentration was used as a control. As platelet aggregation-inducing substances, adenosine-2-phosphate (hereinafter referred to as ADP) at a final concentration of 5 μm and arachidonic acid (hereinafter referred to as AA) at a final concentration of 150 μm were used.
) and 10 μg/ml collagen were used.

(2) Preparation of platelets Collect blood from the common artery of a male rabbit weighing 2.0 to 2.5 kg, add 3.8% citric acid to 1/10 volume of blood, mix gently, and heat at 1300x for 2 minutes. The supernatant obtained by centrifugation was used as platelet-rich plasma (hereinafter referred to as PPP). The precipitate was further centrifuged at 1,600×g for 10 minutes, and the resulting supernatant was used as platelet-poor blood! II (hereinafter referred to as PPP).

(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 PPP, adjust the difference from PPP to a constant value, and then add 25 μl of control solvent or sample to PRP.
After 1 minute, 25 μl of the agglutination-inducing substance was added and the change in absorbance was recorded. The inhibition rate is the maximum agglutination rate of the control (Δ%) and the maximum agglutination rate of the specimen (B%).
), it was determined according to the quintic equation.

Inhibition rate (%) = (1-B/A)×100 The inhibition rate was determined by changing the sample concentration, and IC50 (concentration that inhibits 50%) was determined from it. The results are as follows.

From the above physical and chemical properties and biological properties, 5EN-12
It was confirmed that 8-B is a new substance that has not been described in any literature.

As mentioned above, 5EN-128-B has an important biological property of inhibiting platelet aggregation, and is expected to be used as a therapeutic agent for, for example, thrombosis.

The strain used in the present invention as a producer of the new substance 5EN-128-B (hereinafter referred to as 5EN-128 strain)
was isolated from the soil of Sangatsudo, Nara City, Nara Prefecture, but a mycological search revealed that it was Streptomyces melanosborofascens.
rofaciens), and Streptomyces melanosporofamiens 5EN-128
(Streptomyces melanosporo
faciens SEN-128),
Microbiological Research Institute No. 677 at the Institute of Microbial Technology, Agency of Industrial Science and Technology
It has been deposited as No. 5.

The 5B11-128 strain has the following mycological properties.

The experimental procedure is based on the International Streptomyces Project (IsP) experimental methods (IE, B, S
Hirling et al; Intern, J. Sy.
Stematic Bacteriol,, Vol.
16 (3) p., 313-340 (1966)
) according to In addition to the above-mentioned literature, the culture medium can be found in industry-specific examination base 4a "Applied Microbial Industry" and by Waxman (S.

8. Waksman: Tbe Actinomyc
etes Vol. 2 (1961)) were used.

The description of 1i is Container Corporation Off.
America (Container Corporation
on of America)'s Color Harmony Manual (Color Ilarmony M)
annual) and added this if necessary.

(1) Description by the ISP method (al Morphological characteristics Under the microscope, aerial hyphae arise from well-branched basal hyphae, 1
A chain of 0 or more spores is observed. The sporulation chain has a helical shape, and according to the ISP standard, it is a section spiral (S
ction Spirales). The spores are usually oval to spherical, (0.45 to 0.6) x (0
, 6 to 0.75) μ, and the spore surface structure observed with an electron microscope is a forged mold.

The above morphological characteristics make yeast/malt agar medium, glycerol/asparagine agar medium, and other aerial mycelia very good.
It is observed in the culture medium that forms.

(The color tone of aerial mycelium with sufficient spores of bl colonies is determined by yeast/malt agar medium, glycerol/asparagine agar medium, or
- Gray threads (Tres
ner Backus Color Wheels). That is, typical color tones are light brown gray (3fe) to light gray (5fe to 7fe, 85hes). Occasionally, for example, on starch/inorganic salt agar, yeast 1-malt agar, white to pale yellow (lba to ldb).
and reaches a light gray color (5fe to 7fe). In addition, in certain types of media, such as glucose-asparagine agar, oatmeal agar, and glycerol-asparagine agar, a portion of the colony becomes wet, becoming so-called ``hygroscopic'' and turning black.

(Color of C1 vegetative hyphae (back side of colony) is yellow on yeast/malt agar medium or glycerol/asparagine agar medium, and pale yellow on oatmeal agar medium.

On a starch mineral salt agar medium, it is usually colorless or yellowish at one time.

fd) Color of medium (soluble pigments) Production of melanoid pigments: Negative Production of other pigments: Produces soluble pigments that are yellow on yeast malt agar and glycerol asparagine agar and pale yellow on oatmeal agar. On starch inorganic salt agar medium, no lightning soluble pigment is produced, but a pale yellow color appears at 5 o'clock.

(Utilization of carbon sources I S l) Utilization of sugars D-glucose, L-arahynose, 1]-glucose, D-mannitol, D-fructose, sucrose, i-inositol, raffinose, rhamnose. Make use of it.

Availability of other sugars Galactose, lactose, mannose, maltose.

Use salicin and inulin.

(II) Other properties (blank below) (a) Properties on the culture medium (cultivated at 27°C for 14 days) Other properties on the culture medium Glucose-peptone-gelatin medium: Growth is good and there are no aerial mycelia. The vegetative hyphae and soluble pigment are light yellowish and light brown. Strongly liquefies gelatin.

Cellulose: Grows well in both Czapek's nitrate solution and Czapek's ammonium chloride solution.

Tryptone yeast medium: Growth is good and the soluble pigment is pale yellowish brown.

(blPhysiological properties ■ Liquefaction of gelatin: Positive ■Nitrate reduction: Positive ■Starch ice-melting ability: Positive ■ Milk coagulation: Positive ■Peptonization of milk: Positive ■Hydrogen sulfide production: Negative ■Methi 241 pigment production: Negative ■Tyrosinase: Negative ■Growth temperature range.

5℃, 10℃115℃, 20℃, 25'C, 28
°C930°C135'C137°C, 40°C, 45°C
, respectively, at 50°C.

It does not grow below 10°C and above 40°C, and does not grow at 15°C1.
Poor growth at 37°C, good growth at all other temperatures,
Approximately 28°C is the optimum temperature.

[Phase] Growth pl + range pH 4,5 to 9.0, p116.0 to pl! 7
．． 5 is optimal.

■Cellulase: One strain used in the present invention (SEN
-128 strain) belongs to the genus Slept-Mycetes. This strain has gray aerial hyphae and has spiral-shaped morphological characteristics.

The spore surface is jil! The vegetative hyphae and soluble pigments are both yellow, negative for melanin, and the main characteristics are that in some types of media, some parts of the colony become wet, become hygroscopic, and turn black.

Considering these points, three documents (Shirling,
E, B, and Gottlieb, D, Intern.
, J. Systematjc Bacteriol
, + 18 (2) 69-189 (1968)
; 18 (4) 279-392 (1968)
i 19 (4) 391-512 (1969)
; 22 (4) 265 ~ 394 (19
72).

S, 8, Waksman; The Acti
nomycetes Vcl, 2 (196
As a result of searching for 1)), it was identified as belonging to the abbreviated form of Streptomyces melanosporfascens. Furthermore, the 5EN-128 strain contains novel substances 5EN-128-B and 5EN- which are the substances of the present invention that have platelet aggregation inhibiting effects.
Since it has the useful feature of producing 128-A,
Mrs. Melanosborofascens S
EN-128 (Streptomycesmela
nosporofaciens SEN-128
) to distinguish it from known strains.

Bacteria used in the present invention include not only the above-mentioned Streptomyces melanosborofascens 5EN-128 strain and mutant strains of this strain, but also strains belonging to the genus Streptomyces that produce 5EN-128-B. Any bacteria can be used. In carrying out the present invention, Leptomyces melanosborofascens 5E
Strain N-128 can be cultured using a normal microorganism culture method. As the nutrient source for the medium, those commonly used for culturing microorganisms can be used. That is, as a carbon source, glucose, starch 15), etc. are used, and as a nitrogen source, soybean flour, Pep-I-N, meat extract, corn staple licker, etc. are used. In addition, good results can be obtained by adding appropriate amounts of sodium chloride, potassium chloride, ammonium chloride, and calcium carbonate, and trace amounts of iron, magnesium sulfate, etc. may also be added. As the culture method, static culture, shaking culture, and deep stirring aeration culture are preferable. The culture temperature is 22-30°C5 preferably 24-27°C9ρ11 is normal and if cultured at 6-8, 9 current is applied for 48-72 hours to reach 5E
N-128-B substance is produced in the culture medium. 5EII
A wide variety of commonly used methods can be used to extract and isolate -128-B.

That is, extraction methods using water-immiscible organic solvents such as acetic acid esters, alcohols, ketones, ethers, etc., adsorption methods using activated carbon, etc., chromatography methods using silica gel, alumina, etc. may be used alone or in appropriate combinations. can be used. The present invention will be explained in more detail with reference to Examples below.

Example Streptomyces melanosborofascens 5EN-
From a slant culture of 128 strains, 100 ml of seed medium (#
Flour 2%, Soy flour 1%, Sodium chloride 0.5%, Potassium chloride 0.05%1 Magnesium sulfate 0.05%, Sodium nitrate 0.2%, Calcium carbonate 0.35%, ρ1
17.0) was inoculated into a 500ml Erlenmeyer flask, and cultured with shaking at 27°C for 3 days.
A seed culture solution was obtained.

200ml of this seed culture was inoculated into a Li shear fermenter containing 61 fermentation medium (same medium as the seed medium),
Aerated agitation culture for 3 days at 213'C (aeration rate 67!/m
in, stirring speed 500 rpm). After filtration of 50 ff of the culture solution obtained in this way from the bacterial cells, n-bucnol was added without preparation of p11 and extraction was performed twice. This n-
The butanol layers were combined and concentrated to dryness under reduced pressure.

One distribution was performed by adding a solution 1β of chloroborum:water-1=1. The chloroform layer was separated and concentrated to dryness under reduced pressure to obtain 1.5 g of an oily substance. Silica gel (Wakogel C-20) filled with this oily substance with chloroborum
0, Wako Pure Chemical Industries) Column lo00ml was added to the upper end.

Chloroborm then chloroborm:methanol-100:
Elute sequentially using 1 l volume.

It was fractionated into 30 ml portions. Fractions containing the active substance were collected and concentrated to dryness under reduced pressure to obtain 370+ ng of crude powder. This was dissolved in chloroform, spotted on a thin layer of silica gel (Art 5744, manufactured by Merck & Co.), and developed using chloroform:methanol.

Both fractions containing the active substance were scraped off and extracted with chloroform. The solvent was removed under reduced pressure, and the solution was further dissolved in chloroform and transferred to Nricagel Thin N (Merck & Co., Art 5744) to give ethyl acetate: Hensen: Chloroform-1:
l: l's? Developed using medium g. 5EII-12
Both parts of 8-B were scraped off from the thin plate and extracted with chloroporm. After removing the solvent under reduced pressure.

When recrystallized using methanol, 20 mg of SE N -1,28-B was obtained as colorless plate-like crystals.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectra of the present invention substance 5EN-128-B in methanol and acidic methanol. FIG. 2 shows the ultraviolet absorption spectrum of one of the present invention substances, 5EN-128-B, in alkaline methanol. FIG. 3 shows the infrared absorption spectrum of the present invention substance 5EN-128-B in potassium bromide tablets. Figure 4 shows 1 Invention substance 5
NMR of EN-128-B in deuterated chloroform
Absorption spectrum (200Ml1z) is shown. Applicant Nippon Shinyaku Co., Ltd. Agent Patent Attorney Hiroshi KatamaContinued from page 10 Inventor Teruya Nakamura 3-4-1 Seta, Otsu City Takara Shuzo Co., Ltd. Central Research Laboratory Inventor Nao Matsunaga 3-chome Seta, Otsu City No. 4-1 Takara Shuzo Co., Ltd. Central Research Institute 0 Inventor Shuji Nishio 3-4-1 Seta, Otsu City Takara Shuzo Co., Ltd. Central Research Laboratory 0 Applicant Takara Shuzo Co., Ltd. 609 Takenaka-cho, Fushimi-ku, Kyoto Preparation Nef. Book 1, Incident Display Bu7-2o'? 272 - Patent side submitted on November 25, 1982 (2) (Application number notification not received) 2. Title of invention 5EN-128-B substance and its manufacturing method 3. Person making amendment Relationship with case Patent applicant Address: 14, Kisshoin Nishinosho Monguchi-cho, Minami-ku, Kyoto, 601 Name (415) Japan Nippon Shinyaku Co., Ltd. (1 other person) President: Hiroshi Morishita 4, Agent Address: 601 Kisshoin Nishinosho Monguchi, Minami-ku, Kyoto Column 6 of Detailed Description of the Invention of Town No. 14 Specification, Contents of Amendment (1) The following table listed on page 8 of the specification. [-Flocculant AI)F AA Collagen”. [Flocculant ADP AA Kolaken I
C5o (,i7g/ml) >50 30 3
Correct it to 0. (2) On the bottom line of page 15 of the specification, “Melanospora J
Correct that to "Melanosboropha." (3) On page 18, line 5 of the specification, the phrase ``Add In-butanol'' is corrected to ``Add In-butanol 251''. ”-529-

Claims (2)

[Claims] (1) New substance 5EN-1 with the following physical and chemical properties
28-B. +a+ Appearance Colorless sword-shaped crystal (bl Melting point 119-125°C (C1 elemental analysis actual value C: 63.21 H: 6.63 N:
11.01 Theoretical value c: 62.89 H: 6
．． 50 N: 11.2B (d1 molecule N
248 (mass spectrum) te1 molecular formula C10
1116N203 (fl Optical rotation [α1g-0° C
= 0.5% (in chloroform) (gl Ultraviolet absorption spectra 1 to 1) As shown in Figures 1 and 2, it shows maximum absorption at 247 mμ and 315 mμ in methanol and acidic methanol.In alkaline methanol 238 mμ, 26
5m, c+ (shoulder), shows maximum absorption at 290-310mμ. (The hl infrared absorption spectrum is shown in Figure 3. (The IIIH-NMR spectrum is shown in Figure 4.)
In thin layer chromatography using dimethyl sulfoxide (kl color reaction positive: iodine, potassium permanganate negative: ferric chloride, ninhydrin (1iRf value, Silica Kel (Art 5554) manufactured by Merck & Co., Germany), the following Rr values were obtained. Developing solvent chloroform:methanol-50:10,
7 8 H Opening solvent Hensen: Ethyl acetate: 1 chrome of chloron = ill O. 63 (m+ basic substance, distinguishing between neutral, acidic, and basic) (2) A bacterium belonging to the genus Streptomyces that produces the new substance S rE N-128-B having the following physical and chemical properties was cultured in a medium, and 5EN-128 was extracted from the resulting culture solution.
- Separate B. It is characterized by collecting. Method for producing 5EN-128-B. fat Appearance Colorless needle-like crystals (BL Melting point 119-125°C (C1 elemental analysis actual value C: 63.21 H: 6.63 N:
11.01 Theoretical value C: 62.89 H: 6.5
0 N: 11.2B (di molecular weight 248
(Mass spectrum) te+molecular formula C10Hls
N2 o3(fl optical rotation (α):=0' C=
The ultraviolet absorption spectrum of 0.5% (in liquid volume) ta is as shown in Figures 1 and 2, and shows maximum absorption at 247 mμ and 315 mμ in methanol and acidic methanol. 238 mμ in alkaline methanol, 26
Maximum absorption is shown at 5 mμ (shoulder), 290-310 m, and u. (The infrared and absorption spectra are shown in Figure 3.) The H-NMR spectra are shown in Figure 4. (k) Color reaction positive: Iodine, potassium permanganate negative: Ferric chloride, ninhydrin (11 Rf value, West German Merck & Co., Ltd. silica gel/Art 5554) Thin layer chromatography showed the following Rf value. m medium chloroform:methanol-50=10,7
8 Developing solvent Benzene: Ethyl acetate: Chloroporm = 1:
1. :1 0.63Fml neutral, acidic,
Basic distinction Basic substances