JPS58201990A - Antibiotic substance 80-258 and preparation thereof - Google Patents

Abstract

NEW MATERIAL:An antibiotic substance 80-258. Elementary analysis (%) C, 53.58%; H, 6.70%. Melting point: 220 deg.C (carbonization). Specific rotatory power: +226 deg. (C=0.5, methanol). Ultraviolet absorption spectrum: Shown in Fig. 1. Infrared absorption spectrum: Shown in Fig. 2. Solubility: Insoluble in water, hexane and petroleum ehter and soluble in methanol, ethanol, dichloromethane, chloroform, etc. Color reactions: Negative to the ninhydrin reaction, biuret reaction, anthrone sulfuric acid reaction and Fehling reaction. Distinction of basic, acidic and neutral: Acidic substance. Color and shape of the substance: Yellowish green powder. USE:Remedy for the Ehrlich and Sarcoma 180 tumors. PROCESS:Streptomyces No.80-258 (FERM-P No.6357) belonging to the genus Streptomyces is cultivated to accumulate antibiotic substances 80-258A, 80-258B and 80-258C, which are then collected as a simple antibiotic substance or a mixture thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic 80-258 and a method for producing the same. More specifically, the present invention is produced by culturing a bacterium that belongs to the genus Streptomyces and produces antibiotics 8O-258A, 8O-258B and/or 8O-258C, and is an anti-biotic against Gram-positive bacteria. Has activity, H
A novel antibiotic 'Q8 (1-258A, 8O-258) that has antiproliferative activity against eLtl-83 cells.
B and/or 8()-2580 or a salt thereof and a method for producing the same.

With the aim of searching for new antibiotics, the present inventors isolated a bacterial strain from the soil of the gods in November 12, and continued research on the metabolites produced by it. As a result, Nn811-258 isolated from the soil collected at Chiba Itojo It has been found that a substance having antibacterial activity and an inhibitory effect on the proliferation of HθLa-83 cells is produced in the culture of the strain. Then, as a result of separating and purifying the antibacterial substance from the culture, three substances were isolated, and the physical and chemical properties of the substance were investigated, and it was found that it had the properties described above. Ta. Since no substances with such physical and chemical properties have been found, these substances are collectively referred to as antibiotic 80-258, and are composed of chloroform-methanol-28% aqueous ammonia (340:41): 1
) Antibiotics 80-258 A, 811-2 were detected in order from the top spot by silica gel thin layer chromatography (Art, 5745 manufactured by Merck & Co., Ltd.) developed with a mixed solvent of
58B, 8 (1-258C!).The present invention was completed based on this knowledge.

The present invention is an antibiotic FlO having the physicochemical properties described below.
-258, 8O-258B and 8O-258C or salts thereof, and antibiotics 8O-258A, 8O-258B and/or 8O-258A, 8O-258B and/or 8O-258B and/or 8O-258C belonging to Streptomyces
Bacteria producing 0-2580 are cultured in a medium to accumulate antibiotics 8O-258A, 8O-258B and/or 8O-258C in the culture, and from the culture antibiotics 80=258A, 8O-258B and / Still 8O-
Novel antibiotics 8O-258A, 8O- characterized in that 258C is collected independently and as a mixture thereof.
258B and/or 8O-258C or a salt thereof.

Antibiotics 8O-258A, 8O-258B and 7/or 80-258C (hereinafter collectively referred to as Antibiotics 8)
Antibiotic 8 (hereinafter simply referred to as 1-258 producing bacteria) belongs to the genus Streptomyces, but for example, the strain belonging to the genus Streptomyces isolated by the present inventors rbso-Q5g is an example of a strain most effectively used in the present invention, and the mycological properties of this strain are as follows.

a1 Morphological characteristics 11&]80-258 strain was grown on nutrient agar plate medium (peptone 0.5%, N+CI 0.8%, glucose 1.09A
, meat extract (1.5%, agar 1.5 g) in soil at 27°C.
,,After 14 days of culture, the aerial mycelium was straight.
The spores are cylindrical, 10 in a row, the surface is smooth, and most of the spores have a long axis of 0°76~i, 21x
The short axis is 0.57 to 0.62μ.

51 Growth status in each of the following media Medium Growth Aerial mycelium Back side Soluble pigment ocher nutrient agar Medium Pink Ivory - Oatmilk agar Medium Light pink Ocher --; Melainodal pigment is formed in trace amounts or not at all .

01 Each of the following physiological properties (]) Growth temperature range: 15-39°C] Can be grown, optimally around 27°C 8 ■ Gelatin liquid (on glucose/pebut/gelatin medium): Positive ■ Hydrolysis of starch (on starch/inorganic salt agar medium)
;Positive (4) Coagulation of skim milk, Peptone Ihi (on 10% skim milk medium);Positive, Peptide/Positive both 1) Production of melanin-like pigment (Cyro 77' agar medium and Vept 7... yeast iron agar medium) ): Negative ■ Production of hydrogen sulfide (on peptone yeast iron agar medium); Negative ■ Formation of nitrite (on nitrate medium); Negative 41 Assimilation of each carbon source (on Pridham-Gotlieb Keyaki medium 27
℃ for 1 to 2 weeks) L-arabinol + i] inonthole - D-xylose + L-rhamnose + D-glucose Raffinose -
1) -Fructose +■D-ma/nit Pregnancy6) Sucrose -[Phase] Cellulose Cell wall composition Ecker et al. P [Apoll Microbi:
+111.3 +236-243 (As a result of analysis based on May 1965, LL type diaminopimelic acid was detected.

From the above mycological properties, it is clear that the present Nn8O-258 strain belongs to the genus Streptomyces.

This strain has been deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology under deposit number FIRM-PNn 6357 (FIRM-PNn 6357). As a general property of bacteria, the mycological properties are extremely easy to change and are not constant.
It is a well-known fact that mutations occur through artificial mutation methods using methyl-N-di)rho-N-nitrosoguanidine, ethyl methanesulfonate, etc., and such artificial mutant strains44 as well as natural mutant strains, Any strain that has the ability to produce antibiotic 80-258 belonging to the genus Streptomyces can be used in the present invention. The producing bacteria are cultured in an appropriate medium. In culturing this bacteria, the culture method for release bacteria is generally used. The medium contains a carbon source that can be assimilated by the microorganisms,
A nutrient medium containing a consumable nitrogen source and, if necessary, inorganic salts is used. Assimilable carbon sources include glucose, sucrose, mannitol, molasses, starch,
Dextrin/cellulose, corn staple liquor, glycerin, organic acids, etc. may be used alone or in combination. Digestible nitrogen sources include peptone, meat extract, yeast extract, dried yeast, soybean flour, co/stape,
Liquor, cottonseed meal, casein, soybean protein digests, amino acids, organic nitrogen sources such as urea, nitrates, a/moni? Inorganic substances such as salt? These elementary compounds may be used alone or in combination. Additionally, inorganic salts such as sodium salts, potassium salts, calcium salts, magnesium salts, and salts of salts are added as necessary. Furthermore, the culture medium may contain micronutrients that promote the growth of this bacterium and the production of antibiotic 80-258, as necessary.
Growth promoting substances and precursor substances may be appropriately added.

Cultivation is usually carried out under aerobic conditions such as shaking or aerated agitation culture. Industrially, deep aeration agitation culture is preferred. The pH of the medium is preferably slightly acidic to neutral. Although culturing can be carried out at a temperature of 20 to 37°C, it is usually kept at 24 to 30°C, preferably around 27°C. In the case of liquid culture, the culture is usually carried out for 4 to 6 days, and the antibiotic is produced and accumulated. Preferably, the culture may be terminated when the amount accumulated during the culture reaches the maximum. These culture conditions, such as culture composition, liquid properties of the medium, culture temperature, stirring speed, aeration 1, etc., should be adjusted and selected as appropriate to obtain favorable results depending on the type of strain used and external conditions. Needless to say. If foaming occurs in liquid culture, use a σ1 antifoaming agent such as silicone oil, vegetable oil, or surfactant as appropriate. Since it is contained in the liquid, the culture may be treated with P1 adjuvants such as celite,
Either add high-flow supercell, etc., or centrifuge to separate the culture p solution and the bacterial cells, and then culture the culture F.
It is advantageous to collect the antibiotic 80-258 from the fluid.

In order to separate and purify antibiotic 80-258 from culture p solution, antibiotic 80-258 must be insoluble in water, hexane, and petroleum ether as described below, and alcoholic solvents such as methanol and ethanol, dichloromethane, etc. , chloroform-based solvents such as chloroform, acetate/, ketone-based solvents such as methyl isobutyl ketone, ethyl acetate,
Since it is an acidic substance that can be found in many organic solvents such as ester solvents such as butyl acetate, dimethylformamide, and dimethyl sulfoxide, purification methods that take advantage of these properties are used. Usually, antibiotic 80-258 is transferred to the organic solvent layer by extracting the culture p solution with a non-hydrophilic organic solvent such as chloroform, methylinobutyl keto/, ethyl acetate, butyl acetate, etc. The above extraction procedure When performing this, prepare the culture solution in advance and adjust the TIH to 6.0 to IO.
It is preferable to adjust it within the range of .

The organic solvent layer obtained in this way is washed with a dilute aqueous solution of ethylenediaminetetraacetate to remove metal ions, etc., if necessary, and then treated with various dehydrating agents such as anhydrous sodium sulfate, anhydrous magnesium sulfate, and pease gel. It is dehydrated by adding such things as The organic solvent of the dehydrated organic solvent layer is distilled off under reduced pressure. In this concentration operation, although antibiotic 80-258 is an extremely stable substance, it is usually preferable to carry out the heating temperature 1[' at 60° C. or lower. Antibiotic η80-258 can be precipitated by adding an organic solvent such as hexane or petroleum ether to the residue.

The obtained precipitate is washed several times with hexafluoride, etc., and then subjected to suction filtration or centrifugation to collect antibiotic 80-258 as a yellow-green crude product.

- When further purifying the crude product described in F, ion exchange chromatography using synthetic ion exchange resin, ion exchange cellulose, ion exchange Sephatex, activated carbon, silica gel, alumina, hydroxyapatite, cellulose or HP-10 resin, etc. Adsorption chromatography using an adsorption resin 1-, Kel F5 perchromatography using Sephadex, biogel, etc., electrophoresis, countercurrent distribution, ultrafiltration, concentration, etc., alone or in combination in any order, or repeatedly. Antibiotics 8O-258A, 8O-258B and 8
For example, dissolve the above crude product in methanol, acetone, chloroform, etc., add a small amount of silica gel under stirring, distill off the organic solvent, and dissolve the dough into silica gel. Fill the column, perform column crawl 2 using a chloroform-methanol mixed solvent, pass the active fraction through a LH-20 Sephadex column, elute with methanol, and transfer the active fractions from 1 and 2 to silica gel (Silicagel 60). Art,
Antibiotic '18
0-258 A, 81) -258B and 80-
Since the antibiotics 80-258 thus obtained are acidic substances, salts can be formed by known methods.

Such salts include pharmaceutically acceptable non-toxic salts. Examples include alkali gold III salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, and salts with known organic amino acids.

The above antibiotics 8 (1-258A, 8O-258B and 80-2580) can be isolated individually or used as a mixture depending on the degree of separation and purification. Therefore, the present invention includes not only each active ingredient but also a mixture thereof.

Next, the scientific and biological properties of the present antibiotics 8 (1-258A, 80-25811 and 811-2580) will be described.

■Physical and chemical properties 8O-258A 8O-258B 8(1-258
0'I) Elemental analysis Carbon, hydrogen, same as left
Same as left Elemental analysis value including oxygen C58.58% 54.63%
54.50%H6,70% 6.60%
6.71%fi) Molecular weight F'DMa ea Same as left
Same as on the left, unmeasurable (3) Melting point: 22 (f' (charcoal)) 196℃ (charcoal?
) 196℃ (charcoal (E') (4) specific optical rotation +22
8′+ 331″+ 3370[α], D (c=
n, 5 methano+) <c=x, methano+) (0=
1. Methanol) ■ Ultraviolet absorption As shown in Figure 1 As shown in Figure 1 Spectrum 285.3 nm + 418. ) 284Bm (3512
) 284Bm(528)3]6nm(65,6)
316Bm (57,6) 316Bm (84fi,)4
12 to 431 nm 413 to 432 on dish 416 (12
4) (11) 0 ) (86,4) 43
2Bm (121, fi) (6) Infrared silver absorption As shown in Figure 2 Trace of Figure 3 As shown in Figure 4 Spectrum (KBn'i!:,) tyn 34211,293
+1.3420.293 (+, 3420.2930°1
720.163+), 172(1,1630,17
20,163(1゜1600.138(1,16(1(
1,1520,16011,1520°1520.12
311. 1370, 1320. 1360.138(
1°1170.112+1. 125+1.1230.
13211,126 (1°1065.90 (1,11
7 (1,1120,1230,1170°1065.9
00. 1120.1 + 165゜9 (1
Same as left Soluble Insoluble in ether, Soluble in methanol, ethanol, dichloromethane, chloroform, acetate, ethyl acetate, dimethylformamide, dimethyl sulfoxidets) Color reaction reaction Negative
Reaction A/Sulfuric acid reaction ・L Fehling reaction ・1 None of the normal color reactions showed positive results.

l9) Distinction between basic, acidic, and neutral Acidic substances Same left direction left 6φ
Color and shape of substance Yellow-green powder Same left Same left 6
Thin layer chromatography carrier; silica gel 60TLC plate (Ar
t 11798) Developing solvent; Chloroform-methanol 9:1 Rf value
0.54 0.45 0.35■Biological properties■Antibacterial spectrum CMIOμ? / m / ) test bacteria
8O-258A 811-258B 81
1-258C8 taohylococcus aure
ue FI) A209P 6.25 1.6
82 Bacitlus eubtili days PCI
219 &2 0.8
1.6 Bacillus nereus
PCI 10 (116250, 86, 25: hqr
ichiac+oliNIHJ >10(+
＞】no ＞1on8higella 5on
nei E33 > 1 (1 (>> 10
11 >10+1Shigellaflexine
ri >1011 >11111
>1101Sa1l101Sa1 sD, >”f'
00 >1011 >10 (IKlebsiel
lapneumoP(!l602 >1110 >
Hall ′″-10()Psudomona8a
eruginosaP-3>1(Ill>II
HI >H1+IProteus vu'1pri
e > 10 (1 > 10+1 \1
00Aerobacter aerogeneθ
＞10+1＞1110＞
111+ISaccharomyceesake Oandida al, bicans, Asperqllus nigerPyricul
aria oryzae'I'hichopiytOn
day p.

From the above results, it can be seen that antibiotic 80-258 exhibits antibacterial activity against certain types of Durham-positive bacteria.
Sea 83 cell proliferation inhibitory effect Antibiotic Leprosy 80-258 for each component of normal! 11 knowledge! HeLa-8 by law
As a result of measuring the growth inhibitory activity of 3 cells, the minimum concentration for complete cell death was as follows: Antibiotic 80-258
was found to have a strong He1.1a-83 cell proliferation inhibitory effect.

Antibiotic 8O-258A O, 08μt/m/8
O-258B 0.02'' 8 0-2 5 8 CQ, 0 2 -l+1 r3・Anticancer effect, -+1 Experimental method (1) 6 Mouse Ehritz ascites tumor cells @2.5xlo were added to the peritoneal cavity of Y mice. 1 day and 2 days later, one injection (Group 1) and one injection per day for 6 consecutive days (Group ■) were administered intraperitoneally to each of the two groups.

■1 x 10 sarcoma 180 cells were transplanted into the abdominal cavity of TCR mice, and 24 hours later, one injection was given to each of two groups (group 1).
, and intraperitoneal administration once a day for 9 consecutive days (■ group).

b1 Effect determination Ehritz and Sarcoma 18 using the above experimental method
For 0 tumors, the survival period of the host was observed, and the therapeutic effect was calculated using the following formula.

Intermediate survival days for untreated group <C) c1 Treatment effect The effect on Ehritzchi's ascites cancer determined by the above formula is shown in Tables 1 to 3 below, and the effect on Sarcoma 180 ascites cancer is shown in Tables 4 to 6 below. Show.

21 - Table 1 Effect of 8O-258A on prolonging survival of Ehritzchi's ascites cancer From the above-mentioned properties, antibiotic 80-258:
It is thought to be a meureoritschic acid antibiotic, but among the known antibiotics belonging to it, kelpecidin (gelbecidin),
cidine) mithramy
cinl and chromomycin group (chromomyc
It is closest to in l, but melting point, elemental analysis value, IR absorption,
They differ in solubility in chloroform, specific rotation, and biological activity (Table 7).

As shown in the table above, the U■ maximum absorption value is closest to that of the gelbescidin and chromomycin groups, but 1) the carbonization temperature is higher than that of gelbescidin, and 2) in the measurement of relative luminosity, gelbesidin has a The antibiotics of the 80-258 group were dissolved at a concentration of 1. There isn't.

a The measured value of specific optical rotation is higher than that of gelbescidin; 4. gelbescidin has antibacterial or antifungal activity against Clam-negative bacteria, negative bacteria, and molds;
Group 58 antibiotics are ineffective against Clam-negative bacteria and molds.

Due to the above four differences, the 80-258 substance group is distinguished from gelbecidin.Also, the chromomycin group is different from the antibiotics of the chromomycin group in terms of its specific rotation and its biological activity. It is distinguished from the mithramycin group by the fact that it is effective against this group of antibiotics. Therefore, this group of substances is considered to be a new group of antibiotics.

Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto.

Example 1 Curcose 2.0% in a 500 m/capacity Erlenmeyer flask,
Peptone 0.5%, meat extract 0.5%, dry yeast 0.8
Dispense a liquid medium (PH 7.0) (A medium) containing 1.5 chloride salt (1.5 chloride) and 0.8 chloride calcium carbonate, then sterilize it, and add 1% glucose, , cultured for 6 days at 27°C on an agar slant medium containing 0.5 t of meat extract, 0.8 t of salt, and 1.2 t of agar.
1 loop was inoculated from a slant culture of 857) and
72 hours of reciprocating shaking culture at 27°9 with 0 reciprocations, and aeration rate of 1
51/min, stirring speed 25 Or, p, m, 9 at 27°C
Culture was carried out with aeration and stirring for 6 hours. Obtained culture I P 06
Add 15% of ethyl acetate to ~7) and add 3% of ethyl acetate with a Chemister slurry.
After stirring at 50 r, p, m for 25 minutes, the ethyl acetate layer was separated using a chassisless centrifuge. After concentrating the ethyl acetate layer under reduced pressure to about 20 times, acetic acid was added to adjust the pH to 8.5, and the mixture was washed several times with 5001 aqueous solution of sodium diethylenediaminetetraacetate. Add the washed ethyl acetate solution to 1 round of anhydrous sodium sulfate 259
The mixture was passed through a column to dehydrate it, and then concentrated under reduced pressure. After adding 50 m/ml of hexane to the residue and stirring thoroughly, the precipitate was separated by centrifugation (200 r, p, to, l Q min). This precipitate was treated with hexane in the same manner as above to sufficiently remove oily components, and crude antibiotic 80-25
700 μ of a mixture of 8 was obtained.

Dissolve 500 μ of the above mixture in methanol 80 +++/ and add Silica Kel 60 (Merck Art 7784).
After adding 82 and thoroughly stirring, methanol was distilled off under reduced pressure. This silica gel was mixed with chloroform-methanol f
A silica gel 6° column (9:1) packed with
Add on top of 2゜5 x 40 an l, chlorophore/1
Each fraction was eluted with methanol f9:1+ and subjected to bioassay using Bacillus subtilis P ('1219) and thin layer chromatography using silica gel 60'l' L C plate (Merck Art5745). (Developing solvent: chloroform-methanol-28thiammonia water = 840:40:1)7, and the active fractions were collected and concentrated under reduced pressure to give a yellow-green powder17
0Pn9 was obtained.

Dissolve 150 μl of the above powder in 15 wLl of methanol,
This was packed with methanol and loaded onto a LH20 Sephatex (manufactured by Pharmacia) column (2,5x95-), and eluted with methanol. Each eluted fraction was followed by the same method as described above17, and the active fractions were collected and concentrated under reduced pressure [7]
A mixture of 70~ was obtained. This was dissolved in as little chloroform as possible and subjected to preparative thin layer chromatography on a 7 silica gel 60'l LC plate (manufactured by Merck & Co.). IJL, Rf value 0.54.
o. 45 *-
Antibiotic 8O-258B20■ and Antibiotic go-2
58C15■ was obtained.

[Brief explanation of the drawing]

Figure 1 shows antibiotic 8O-258A and antibiotic 8O-25.
UV-258 of 8B and antibiotic 8O-258C, respectively.
Infrared absorption spectrum of B, Figure 4 is antibiotic 8O-2
58C infrared absorption spectrum 1.

Claims (1)

[Claims] 1) Novel antibiotic 8O-25 having the following physical and chemical properties
8A or its salt. ■Elemental analysis; includes carbon, hydrogen, and oxygen, elemental analysis value;
C58,58CH H6, To% ■Melting point; 220℃ (carbonization) ■Specific optical rotation; [α):'+226° (C=0.5
, methanol) ■Ultraviolet absorption spectrum; as shown in Figure 1 φ) Infrared absorption spectrum; as shown in Figure 2 ■Solubility in solvents: Insoluble in water, hexa/, petroleum ether, methanol, ethanol, dichloromethane, chloroform, acetone, acetic acid Possible O color reaction with ethyl, dimethylformamide, dimethyl sulfoxide; negative reaction with ninhydrin reaction, Biuret reaction, Anthrone sulfuric acid reaction, Fehling reaction) Distinction between basic, acidic, and neutral; acidic substance C9, color and shape of substance ; Yellow-green powder 2) Novel antibiotic 8O-25 with the following physical and chemical properties
8B or its salt. ■Elemental analysis: Contains carbon, hydrogen, and oxygen, elemental analysis value: C
54,68chiH6,60* ■Melting point; 196℃ (carbonized) (3I specific optical rotation; [α]C'+, 881° (C=1
, methanol) ■Ultraviolet absorption spectrum; as shown in Figure 1 ■Infrared absorption spectrum; as shown in Figure 8 ■Solubility in solvents: Insoluble in water, hexane, petroleum ether, methanol, ethanol, dichloromethane, chloroform, acetone, ethyl acetate , dimethylformamide, dimethyl sulfoxide ■Color reaction; negative for ninhydrin reaction, Biuret reaction, Anthrone sulfuric acid reaction, Fehling reaction ■Distinguish between basic, acidic, and neutral; acidic substance ■Color of substance: yellow-green Powder 8) Novel antibiotic 8O-25 with the following physical and chemical properties
8C mud is that salt. (1) Elemental analysis: Contains carbon, hydrogen, and oxygen, elemental analysis values: C54,50, H6,71 (2) Melting point: 196°C (carbonized) ■ Specific optical rotation: [α)23+887° (C= 1, methanol) ■Ultraviolet absorption spectrum; as shown in Figure 1 ■Infrared absorption spectrum; as shown in Figure 4 ■Solubility in solvents: Insoluble in water, hexane, petroleum ether, methanol, ethanol, dichloromethane, li[maloform, acetone] , soluble in ethyl acetate, dimethylpormamide, and dimethyl sulfoxide ■Color reaction: Negative for ninhydrin reaction, Biuret reaction, Anthrone sulfuric acid reaction, Fehling reaction, negative for all color reactions of ordinary carbohydrates, amino acids, and peptides + =) Distinction between basic, acidic, and neutral; Acidic substances; Color and shape of Ja substances; Yellow-green powder 4) Belongs to the genus Streptomyces, antibiotics 80-25
εA, 80-258 and/or 81)-/\258C is cultured in a medium to accumulate the antibiotic t'1O-258A, 8(1-258B and/or 80-2580) in the culture. , antibiotics 8O-258A, 81)-258B and 80-25 from the culture.
A novel antibiotic Ft +1-258 A, characterized in that Ft 80 is collected singly or as a mixture thereof;
8 (1-208B and/or 80-2580 or a salt thereof, 5) The bacteria belonging to the genus Streptomyces and producing the antibiotics 8O-258A, 81)-258B and/or 80-2580 are Myces 8D Nn80-258 (Nn80-258
(FER) The manufacturing method according to claim 4.