JPS59156289A - Sen-128-a substance and its preparation - Google Patents

Abstract

NEW MATERIAL:SEN-128-A substance having the following physical and chemical properties. Appearance: colorless needle-like crystal. Melting point: 122- 127 deg.C. Elemental analysis (wt%): C 61.21, H 6.15, N 11.72. Molecular weight: 234 (mass spectrometry). Molecular formula: C12H14N2O3. Specific rotatory power: [alpha]D<24>=0 deg. (C=0.5%, chloroform). Solubility: easily soluble in chloroform, methanol, ethyl acetate, acetone, dimethyl sulfoxide, and insoluble in water. Color reaction: positive in iodine, KMnO4, negative in FeCl3, ninhydrin, etc. USE:An inhibitor for blood platelet aggregation. PREPARATION:A bacterium such as Streptomyces melanosporofaciens SEN-128 (FERM p 6775), etc. belonging to the genus Streptomyces is cultivated preferably at 24-27 deg.C at 6-8pH for 48-72hr.

Description

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

5EN-128-A according to the present invention is 5EN-1 together with the substance referred to as 5EN-128-B in this specification.
They are collectively called 28. 5EN-128-B is 5EN-1
It is a substance obtained by the same method as No. 28-8 and has similar physical and chemical properties and biological activities.

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

lal Appearance Colorless acicular crystals lbl Melting point 122-127°C IcI elemental analysis actual values C: 61.21 H: 6.15 N:
11.72 theoretical value C: 61.53 H: s:o
2N: 11.96 (d1 molecular weight 234 (mass spectrum) te1 molecular formula CI2 I14 N
203If) Optical rotation [α] g=0° C=0.5%
(In chloroform) (g) Ultraviolet absorption spectrum As shown in Figures 1 and 2, it shows maximum absorption at 246 mμ and 315 mμ in methanol and acidic methanol. 237 mμ in alkaline methanol, 265
mμ (shoulder), shows maximum absorption at 300 mμ (shoulder).

[hl infrared absorption spectrum It is shown in Figure 3.

(il'H-NMR spectrum It is shown in Figure 4.

(ji Solubility Easily soluble in chloroform, methanol, ethyl acetate, acetone, dimethyl sulfoxide, insoluble in water.

(kl color reaction positive: iodine, potassium permanganate negative: ferric chloride, ninhydrin fll Rf value The following Rf value is shown in thin layer chromatography using Silica Kel (Art 5554) manufactured by Merck & Co., West Germany.

Developing solvent Chloroform: Methanol - 50 = 1675 Developing i9 Medium Hensen: Ethyl acetate: Chloroform - 1:1:1 0.5B +m) 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 1% fl! am. Dimethyl sulfoxide at the same concentration was used as a control. Adenosine-2-phosphate (hereinafter referred to as ADP) at a final concentration of 5 μH, arachidonic acid (hereinafter referred to as AA) at 150 μH, and collagen at 10 μg/ml were used as platelet aggregation-inducing substances.

(2) Preparation of platelets Collect blood from the common carotid 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 centrifuge at 1300xg for 2 minutes. The supernatant obtained was used as a platelet-rich blood sample (hereinafter referred to as PPP). The precipitate was further centrifuged at 1600xg for 10 minutes, and the resulting supernatant was used as platelet-poor plasma (hereinafter referred to as F'PP).

(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 (A%) and the maximum agglutination rate of the specimen (B
b suppression rate (%) calculated according to the linear formula from (%) = (
1-B/A) ×100 The inhibition rate was determined by changing the sample concentration, and IC5o (concentration that inhibits 50%) was determined from it.

The results are as follows.

From the above physical and chemical properties and biological properties, 5EN-1,
It was confirmed that 28-A is a new substance that has not been described in any literature.

As mentioned above, 5EN-128-A 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 producing strain of the new substance 5EN-128-A (hereinafter referred to as 5EN-128 strain)
was isolated from the soil of Sangatsudo, Nara City, Nara Prefecture, but as a result of orchidological searches, Streptomyces melanosborofanens (Streptomyces melanospo)
Streptomyces melanosborofaamiens 5EN-128 was identified as a bacterium that succumbs to
(Streptomyces melanospor
ofaciens S E N -' 12
8), and has been deposited with the Agency of Industrial Science and Technology's Institute of Microbial Technology (D-Tokyo Research Institute) as Tekkoken Bacteria Deposit No. 6775.

The 5EN-128 strain has the following mycological properties. The experimental procedure is based on the International Streptomyces Project (ISP) experimental method (E, B, Shi
rling et al; Intern, J. Sys.
tematic'Bacteri'o1. ＋Vol,
16 (3) 9.313-340 (1966)
) according to In addition to the above-mentioned documents, the culture medium can be found in the industry-specific examination standards "Applied Microbial Industry" and by Wanksman (S.

A. Waksman: The Actinomyc
etes Vol. 2 (1961)) was used. Color descriptions are provided by Container Corporation of America.
Corporation of America)
Color Harmony Manual
If necessary, this has been added with reference to the Armony Manual.

(1) Description by the ISP method la + 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 block has a spiral shape, and according to ISP standards, it is section spiral (5
ction Spirales). The spores are concave to spherical, (9,45 to 0.6) x (0
, 6 to 0.75) μ, and the spore surface structure observed with an electron microscope is wrinkled.

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

[BL Colony Color] The color tone of aerial mycelia with sufficient spores attached can be determined by yeast/malt agar, glycerol/asparagine agar, oatmeal agar, starch inorganic salt agar, or other media that favorably form aerial hyphae. Gray thread (Tresn)
er Backus Color Wheels). That is, one typical color tone is light brown gray (3fe) to light gray (5fe to 7fe, As!Ies). Occasionally, the color ranges from white to pale yellow (1ba=ldb) to light gray (5fe to 7fe) on, for example, Scooch mineral salt agar or yeast malt agar. Also, glucose-asparagine agar medium, oatmeal agar medium,
On some types of media, such as glycerol-asparagine agar, parts of the colony become wet and turn black, taking on a so-called "hygroscopic pink" appearance.

(Color of C1 vegetative hyphae (back side of colony) is yellow on yeast/malt agar medium or glycerol/asparaquine 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.

(Color of dl medium (soluble pigment) Production of melanoid pigment: 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 Scooch's inorganic salt agar medium, soluble pigments are not normally produced, but a pale yellow color appears at 1 hour.

+e+ Utilization of carbon source Utilization of sugars listed in ISP D-Clucose, L-arabinose, D-xylose 1
D-mannitol, D-fructose, sucrose, i-inositol, raffinose 1. Use rhamnose.

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

Use salicin and inulin.

(II) Other properties (blank below) <a> Properties on the culture medium (cultured at 27°C for 14 days) Other properties on the culture medium Glucose-peptone-seratin 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: Growth is good in both Czapek's nitrate solution and Czapek's ammonium chloride/8 solution.

Tryptone yeast medium: Growth is good and the soluble pigment exhibits a light yellowish N-brown color.

fbl Physiological Properties ■ Liquefaction of Seratin: Positive ■ Reduction of Nitrate: Positive ■ Ice-melting ability of Scooch; Positive ■ Coagulation of Milk: Positive ■ Peptonization of Milk: Positive ■ Production of Hydrogen Sulfide: Negative ■ Production of Furanoid 2 Pigments : Negative■Tyrosinase: Negative■Growth temperature range ・5°C, 10°C, 15°C, 20°C, 25°C128
°C130°C335°C, 37°C940°C145°
Each experiment was conducted at 150°C.

Will not grow below 10℃ and above 40℃, 15℃53
Growth is poor at 7°C, and good growth at all other temperatures, but approximately 28°C is the optimum temperature.

[Phase] Growth pH range: Growth at pH 4, 5 to 9.0, p++e, o to pl+7
．． 5 is optimal.

■Cellulase: Based on the mycological properties of positive or higher, 9 strains used in the present invention (SEN
-128 strain) belongs to the genus Streptomyces. This strain has gray aerial hyphae and a spiral-shaped morphological feature.

The main characteristics of the spores are that the surface of the spores is cylindrical, the vegetative hyphae and soluble pigments are both yellow, and they are negative for melanin, and in some types of media, some of the colonies become wet, become hygroscopic, and turn black. It is.

Considering these points, nine documents (Shirling,
E., B. and Gottlieb, D. Inte.
rn, J. Systematic Bacteri.
ol, + 18 (2) 69-189 (1968)
, 18(4) 279-392 (1968)
; 19 (4) 391-512 (1969)
;22<4> 265-394 (1972
)5.4. Waksman; The Actino
mycetes Vol. 2 (1969)), it was identified as belonging to the family of Streptomyces melanosborofascens. Furthermore, SEN, -
The 128 strain contains novel substances S'EN-128-A and 5EN-128-, which are the substances of the present invention that have platelet aggregation inhibitory effects.
Streptomyces melanosborofascens S E N -1 has the useful property of producing B.
28 (Streptomyces melanospor
ofaciens SEN-128)
It was distinguished from known strains.

Bacteria used in the present invention include not only the above-mentioned Streptomyces melanosborofascens strain 5E11-128 and mutant strains obtained by mutating this strain, but also the Streptomyces genus that produces '5EN-128-A. Any bacteria that can be used can be used. When carrying out the present invention, Streptomyces melanosborofascens strain 5EN-128 can be cultured using a conventional microorganism culture method. As the nutrient source for the medium, those commonly used for culturing microorganisms can be used. That is, glucose, starch powder, etc. are used as the carbon source, and soybean powder 3 peptone, meat extract, corn staple licker, etc. are used as the nitrogen source. 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. The culture method is static culture,
Shaking culture and deep agitation aeration culture are preferred. The culture temperature is 2
If cultured at 2 to 30°C, preferably 24 to 27°C, ρ11 is usually 6 to 8, 5EN-1 is usually obtained in 48 to 72 hours.
28-A substance is produced in the culture medium. 5EN-12'
A wide variety of commonly used methods can be used for extraction and isolation of 8-A.

In other words, it is not miscible with water from the culture solution! a Solvent 1 For example, extraction methods using acetic acid esters, alcohols, ketone cheeks, ether cheeks, etc., adsorption methods using activated carbon, etc., silica gel,
Chromatography using alumina or the like can be used alone or in appropriate combinations. 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 (lees) 2%, soybean flour 1%, sodium chloride 0.5%, potassium chloride 0.05%.

Magnesium sulfate 0.05%, sodium nitrate 0.2%
, calcium carbonate 0.35%, pH 7.0) in a 500 ml Erlenmeyer flask.
Shaking culture was performed at 27° C. for 3 days to obtain 9 culture solutions.

200 ml of this seed culture was inoculated into a 141 shear fermenter cup containing a fermentation medium (same as the seed medium) at 6°C, and cultured with aeration and stirring using a No. 28°C trowel for 3 days (aeration volume 6β/
l1ljn + stirring speed 500 rpm). The culture 't&50p thus obtained was filtered to remove the bacterial cells, and extracted twice with iLp++ virgin product by adding n-butanol. This -n-butanol) was combined and concentrated to dryness under reduced pressure. .

A 1:l solution of chloroform:water was added to perform two distributions. The chloroform layer of this was separated and a
It was dried to a thin layer and 1.5 g of the oily substance was weighed. Silica gel filled with this oily substance in chloroform (Wakogel C-
200, Wako Tsumugi Pharmaceutical) Column 10100O was added to the upper end. Chloroform then chloroform; methanol, root
Elute sequentially using oo:i solvents.

It was fractionated into 30 ml portions. Fractions containing the active substance were collected and concentrated to dryness under reduced pressure to obtain 370 mg of crude powder. This was dissolved in chloroform, spotted on a silica gel 7# layer (manufactured by Merck & Co., Ltd., Art 5744), and developed using a solvent of chloroform:methanol-20:l. 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 poured onto silica gel 'A5WtC (Merck & Co., Art 5744) to give ethyl acetate: Hensen: chloroform = 1.
:l:1's? Developed using medium g. 5EN-12
Both portions of 8-A were scraped from the thin plate and extracted with chloroform. After removing the solvent under reduced pressure, the residue was recrystallized using 1 methanol to obtain 5On+g of 5EN-1j8-A as colorless plate-like crystals.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectra of the present substance 5EN-128-A in methanol and acidic mechnol. FIG. 2 shows the ultraviolet absorption spectrum of the two substances of the present invention, 5EN-128-A, in alkaline methanol. FIG. 3 shows the infrared absorption spectrum of the present invention substance 5EN-128-A in potassium bromide tablets. Figure 4 shows the NM of one invention substance 5EN-128-A in deuterated chloroform.
Represents R absorption spectrum (200MHz). 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 Laboratory 0 Inventor Shuji Nishio 3-4-1 Seta, Otsu City Takara Shuzo Co., Ltd. Central Research Laboratory Applicant Takara Shuzo Co., Ltd. 609 Takenaka-cho, Fushimi-ku, Kyoto City Commissioner of the Patent Office Kazuo Wakasugi 1. Indication of the case i"? 2" 27/Patent application filed on November 25, 1982 (1) (notification of application number not received) 2. Name of the invention 5EN-1'2 B-A Substances and their manufacturing methods 3, and their relationship with the case of the person making the amendment Patent applicant address: 14, Kisshoin Nishinosho Monguchi-cho, Minami-ku, Kyoto 601 Name (415) Nippon Shinyaku Co., Ltd. (1 person) President Hiroshi Morishita 4. Agent's residence Address: 14, Kisshoin Nishinosho Monguchi-cho, Minami-ku, Kyoto 601 Column 6 of the detailed description of the invention in the specification, Contents of amendments (1) The following table listed on page 8 of the specification. "" of. "Flocculant ADP AA Collagen I
C50 (, ljg/ml) >50 30 3
Correct it to 0. (2) In the second line from the bottom of page 15 of the specification, r (19
69) Correct J to r (1961) J. (3) On page 18, line 5 of the specification, the phrase ``add In-butanol'' is corrected to ``add n-butanol 251''. Above 1. Indication of the case Patent Application No. 207271 filed in 1982 2. Name of the invention 5EN-128-A Substance and its manufacturing method 3. Person making the amendment Relationship to the case Patent applicant address Kisshoin Nishi, Minami-ku, Kyoto City, 601 14 Nosho Monguchi-cho Name: Nippon Shinyaku Co., Ltd. (and 1 other person) Representative: Hiroshi Morishita 4, Agent residence: 14 Nosho-Monguchi-cho, Kisshoin, Minami-ku, Kyoto City, 601 March 27, 1980 (Procedures completed) 7. Contents of the amendment (1) Specification Box 1, Title of the Invention" shall be corrected as follows. ``rSEN-128-A substance and its manufacturing method''

Claims (2)

[Claims] (1) New substance 5EN-1 with the following physical and chemical properties
28-A. (al Appearance Colorless needle crystals (bl Melting point 122-127°C (c1 Elemental analysis actual value c: 61.21 H: 6.15
N: 11.72 theoretical value C: 61.53 H:
6.02 N: 11.96 (d1 molecular weight
234 (mass spectrum) te+molecular formula
C12N14 N20B ('') Optical rotation (α)■-0°
C=0.5% (in chloroform) ig) Ultraviolet absorption spectrum as shown in Figures 1 and 2, showing maximum absorption at 246 m/J and 315 mμ in methanol and acidic methanol. 237 mμ in alkaline methanol,
Maximum absorption is shown at 265 mμ (shoulder) and 300 mμ (shoulder). The open external absorption spectrum is shown in Figure 3. The fit' H-NMR spectrum is shown in FIG. (j) Solubility: Easily soluble in chloroform, methanol, ethyl acetate, acetone, dimethyl sulfoxide, insoluble in water. (k) Color reaction positive: iodine, potassium permanganate negative: ferric chloride, ninhydrin + 1 JRf value The following Rf value was obtained by thin layer chromatography using Silica Kel (Art 5554) manufactured by Merck & Co., West Germany. Developing solvent chloroform:methanol-50:10.7
5 Expansion 1g bX Hensen, 1! ! l [Ethyl:chloro, J (lumu 1:1:1 0.58 tm+discrimination between neutral, acidic, and basic basic substances) (2) A bacterium belonging to the genus Streptomyces that produces a new substance 5EN-128-A having the following physical and chemical properties was cultured in a medium, and 5EN-128-A was isolated from the resulting culture solution. A method for producing 5EN-128-A, which comprises collecting 5EN-128-A. Fat Appearance Colorless needle-like crystals (bl melting point 122-127℃ te1 elemental analysis actual value C: 61.21) (: 6.15 N
:11.72 Theoretical value c: 61.53 H:
6.02 N: 11.96 (d1 molecular weight
234 (mass spectrum) (e) Molecular formula CI
2 I14 N203 (') Optical rotation [α rock -0°
C=0.5% (in chloroform) (g) Ultraviolet absorption spectrum As shown in Figures 1 and 2, it shows maximum absorption at 246 mμ and 315 mμ in methanol and acidic methanol. 237 mμ in alkaline methanol, 26
Maximum absorption is shown at 5n+μ (shoulder) and 300mμ (shoulder). (hl Infrared absorption spectrum shown in Figure 3. fil' H-NMR spectrum shown in Figure 4. fjl Soluble Easily soluble in chloroform, methanol, ethyl acetate, acetone, dimethyl sulfoxide, insoluble in water. Color reaction positive: iodine, potassium permanganate negative: ferric chloride, ninhydrin Rf value The following Rf value is shown in thin layer chromatography using silica gel (Art' 5554) manufactured by Merck & Co., Germany.Developing solvent Chloroform: Methanol-50:10.7
5K open? g Medium Hensen: Ethyl acetate: Chloroform-1:1:l O, 58 ((2) Basic substances: neutral, acidic, basic