JPS61263971A - Novel substance a300 - Google Patents

Abstract

NEW MATERIAL:The compound of formula. USE:Antitumor agent and antibacterial agent. PREPARATION:The objective A300 can be produced by culturing an A300- producing strain belonging to Streptomyces griseoincarnatus in a medium containing glucose, etc., as a C source, soybean flour, etc., as an N source and if necessary, inorganic salts such as sodium chloride, at 25-30 deg.C, preferably by aerobic deep-tank liquid cultivation method.

Description

DETAILED DESCRIPTION OF THE INVENTION W shellfish L11 The present invention provides novel substances, more specifically streptomy
The present invention relates to A300, a novel antibiotic having anti-pulmonary ulcer ↑1 and antibacterial properties produced by the A300 strain belonging to S. grise A incarnatus.

Many anti-inflammatory bamboo substances and antibacterial substances have already been put into practical use as medicines. In general, the physiological activity ↑11 of a chemical substance largely depends on its chemical structure, so it can be said that there is a constant desire for new compounds with antitumor and especially antibacterial properties.

W stackμ11 The present invention meets the above needs.

That is, the novel substance A300 according to the present invention has the following formula (A
).

-011 3-1) Chemical structure The novel substance A 300 according to the present invention has a chemical structure represented by the above formula (A>).

This chemical structure was determined as follows.

A300 is 1-NMR (Figure 3), 13C-NM
Analysis of R (Figure 4) revealed aquavamycin (M.

Outside of Sezaki: Tetrahedron
) -26°5171~5190 (1970) ),
2 molecules of 11 deinose (rhodinose),
It was revealed that it was composed of two molecules of cinerulose A.

Furthermore, when A300 was hydrolyzed with 2N hydrochloric acid at room temperature for 2 hours to obtain its agri-1in portion, it completely matched the standard substance of aquayamacin.

A300 was dissolved in ethyl acetate using platinum oxide as a catalyst at room temperature.
After hydrogenation for a period of time, the dihydro form of A300 is obtained. This is a related compound P-1894B (K,; 4-Yugai: Chemical and Pharmaceutical Picoletin (Chell).

Pharm, B+11. ) J? 4350-435
9 (1984)) was similarly reduced.
It was completely consistent with the hekylyhydro form of f194r3. Also, F A B (fast atmboibardn+
ent) The molecular formula of A3000 determined from the mass spectrum and elemental analysis values is C49"" 62018, which is P-18948 (C
491159018), it has 4 more hydrogen atoms.

Taking these experimental results together, the chemical structure of A300 consists of two molecules of aculose (P~1894B).
se ) was converted into two molecules of synerulose A and the structural formula (A>) was determined.

2) Physical science bamboo-like (1) Appearance: Yellow powder (2) Elemental analysis (%): Ω Ω Analysis value 62.
62 6.60 30.78 Calculated value 62.6B 6
．． 65 30.67 (C491162°18) (3) Molecular weight: 939.0 (4) Melting point: 153-158°0 (C0,1, in chloroform) (6) Ultraviolet and visible absorption spectrum 1- in methanol 219 (312>, 316 (59), 0.01N
Na0) 1-227 in methanol (3f31), 3
18 (113), 390 (36>, 553 (6
1) (7) Soluble M bamboo: Soluble in methanol, butanol, n-butanol, acetone, chloroform, ethyl acetate, and benzene. Water, to n-: insoluble in L-san.

(8) Rf value (Merckne tlsi'J7J gel 60F2
54J used) Ri[1[1form: methanol-20:1 0.71 BenU:methanol-2('):1 0.23(9)
Infrared absorption spectrum (KBr disk method) Figure 2 (10) Proton nuclear magnetic resonance spectrum (400 MHz, weight in 110 mm) Figure 3 (11) FA cable 13 nuclear magnetic resonance spectrum (100 MHz, commercial [1 [] in form] Figure 4 (12) F A B mass spectrum (71-Rix, Grilolin) m/z 961 (M+Na) + Production of A300 1) Overview Antibiotic Δ300 has so far been used in the cultivation of microorganisms. 1) -C alone cannot be obtained, but it can be produced by synthetic chemical or microbiological modification of related compounds, or by total synthetic chemical production.

In the case of microbial culture, the strain used is one belonging to the genus Streptomyces that has an A300 ability to mature cattle. Specifically, the inventors' isolated S] ~ Leptomyres glis 1! Oin Power Lunatus A300 stock is A30
Although the present inventors have revealed that antibiotic-producing strains produce 0, suitable strains can be isolated from nature using conventional methods for isolating antibiotic-producing bacteria. In addition, there is still room to increase A300 production ability by subjecting A300-producing bacteria, including the A300 strain, to irradiation or other treatments.

2) A300 strain The A300 strain, which the present inventors have discovered, is a bacterial strain of the genus Actinomaggi that has the ability to produce antibiotic A300.
The contents are as follows.

(1) Origin and deposit number Strain A300 was isolated from soil collected in Miyake Village, Tokyo, and was deposited at the Institute of Industrial Science and Technology, Agency of Industrial Science and Technology on March 30, 1985. Ter*Koken Bacteria No. 8
I have the number 171.

(2) Mycological properties and physiological properties This bacterial strain is a hypertrophic fungus that produces A300, and has the following mycological properties.

■) Morphological characteristics Long aerial hyphae extend from the branched basal mycelia, part 1
:, simple branches from the axis, forming lateral branches, forming a spiral-shaped chain (10 to 50 spores) of 3 to 6 turns at the tip.

The shape of the spore is oval or spherical (0.9 to 1.1?
0.6~0.9μ) and has thorn-like protrusions (Srl) on its surface.
inV).

Specialized forms such as sporangia, flagellated spores, and sclerotia are not observed.

■) Growth status of each rice culture medium Ten
As shown in the table.

■) Physiological properties Physiological properties are as shown in FIG.

IV) Assimilability of carbon sources (on Pridham Go-Leeve agar medium) The assimilabilities of various carbon sources are shown in Table 3.

From the above mycological properties, this strain clearly belongs to the genus Streptomyces, and its characteristics can be summarized as follows.

1) The spore chain has a spiral shape, and the spore surface is thorn-like.

2) Aerial mycelium is gray-olive to yellowish-white to light brownish-gray, and the underside is yellow-orange to brown.

3) Soluble pigment is reddish yellow to dark yellow.

4) Production of melanin-like pigment is negative.

5) Make good use of various carbon sources.

From the above properties, the description of l5P (International Streptomyces Project) (International Di17-Pull-Off Systematic Bacteriolour, Vol. 18, pp. 69-189, pp. 279-392) 1
968, and the same volume 19, pages 391-512, 19
1969 and same magazine volume 22, pages 265-394 1974
When searching for known bacterial species with reference to Streptomyces grise A inculptus (2007), B.C.
es griseoincarnatus), but NO
, is listed as the species most closely related to the A 300 strain. Therefore, No. 300 strain A and the standard strain Streptomyces griseoincarnatus IFO12871 (I
SP5274) was cultured under the same conditions and compared, as shown in Table 4, on starch-free 111I salt agar medium and O]meal agar medium, the growth state of aerial mycelia and color tone were slightly different, but other conditions were observed. The various properties and physiological properties of culture soil are very similar.

From the above results, we recognized strain No. A 300 as the most closely related strain to Streptomyces griseoinlunatus.
Streptomyces griseoine lunatus
ptomyces griseoincarnatus
) A300, distinguishing it from known strains.

Table 2 Table 3 D-glucose -I L--arabinose] D-xylose + i-inositol + D-mannyl + D-hook] Hose (-rhamnose + silucrose -1 raffinose -( :Positive - :Negative Test: False positive culture, 4 in 3
00 production antibiotic A300 is Streptomy 1? A belonging to the genus
It can be produced by culturing 300-producing bacteria aerobically in an appropriate medium and collecting the target product from the culture.

The medium can contain any nutrient source that can be utilized by the A300 producing bacteria. Specifically, for example, glucose, sucrose, maltose, starch, and fats and oils can be used as carbon sources, and soybean flour, cottonseed meal, dried yeast, yeast extract, and nitrogen sources can be used. Organic substances such as -steep liquor and inorganic substances such as ammonium salts or nitrates such as ammonium nitrate, sodium nitrate, and ammonium chloride can be used. Also,
If necessary, inorganic salts such as common salt, potassium chloride, and heavy metal salts of phosphoric acid/r can be added.

In order to suppress foaming during brewing, suitable antifoaming agents such as silicones can also be added according to conventional methods.

The most suitable culture method is the aerobic liquid deep culture method, similar to the commonly used plastic method for producing antibiotics. The appropriate temperature for cultured warm turtles is 20°C to 37°C, but 25°C
C to 30C is preferred. In this way, A300 production t1
1 reaches its peak in 3 to 4 days in both shaking culture and aeration agitation culture.

In this way a culture enriched with A300 is obtained. In culture, a portion of A300 is present in the culture solution, but most of it is present in the bacterial cells.

Any convenient method can be used to harvest A300 from such cultures. One way is to
It is based on the principle of extraction, and specifically, for example, for A300 in the culture tank solution, it is extracted with a water-immiscible solvent for A300 (see above), such as ethyl acetate, or Regarding A300 in bacterial cells, there are methods such as recovering bacterial cell aggregates obtained by filtration, centrifugation, etc., and treating them with methanol, T-ethanol, acetone, etc. The culture itself can also be subjected to the above extraction operation without separating the bacterial cells. A countercurrent fractionation method using a suitable solvent can also be included in the scope of extraction.

Another method of extracting A300 from cultures is based on the principle of adsorption, which is based on the principle of adsorption, which is based on the principle of adsorption, and is based on the principle of adsorption, in which A300, which is already in liquid form, is
00-containing substances, such as the culture medium solution or the extract obtained by performing the extraction operation as described above, an appropriate adsorbent or gel filtration agent, such as silica gel, rtFadex L, l-120,J (
Fun 7 Lumash Non 1), [Toyo Pearl HW40J (
Column chroma 1-graphy using Nucleosil 5C181 (manufactured by Nagel 1, West Germany), etc.
A300 can be removed by adsorbing the target A300 by high-speed liquid lithography using a solvent, etc., and then avoiding the solution #1. If the A300 solution obtained in this way is concentrated to dryness under reduced pressure, A3
A rough sample of 0.00 is obtained.

In order to further purify the crude sample of A300 obtained in this way, the above-mentioned extraction method and adsorption method can be carried out in the necessary number of combinations as necessary.

For example, silica gel, [Sephadex ll-120J
What kind of adsorbent can be used in conjunction with Column 1-1 adsorbent using L Gel filtering agent, high performance liquid chromatography using Nucle Asil 5C18-1, etc., and countercurrent distribution method as appropriate. It can be implemented.

Specifically, Example λ, A300 crude sample was dissolved in a small amount of methanol, [Toyopearl + 1W 4 OJ column was used, and the active ingredient was eluted by developing with an appropriate solvent [υ]]
When concentrated and dried, a yellow powder of A300 is obtained.

Uses of AC5Y The antibiotic AC5Y according to the present invention is useful in that it exhibits anticancer activity and antibacterial activity.

1) Anti-ulcer A300 showed significant growth inhibitory activity against tumor cells. For example, add 1 x 105 mouse P388 leukemia cells.
R containing 10% heat-inactivated beef tallow serum so that the amount of
The cell concentrations of cells suspended in PM11640 medium and cultured at 37° C. for 2 days with various concentrations of A300 in air containing 5% carbon dioxide were as follows.

One/ml 1.2×106 pieces/m+6
9.1 x 105 13 6.1 x 105 25 2.8 x 105 50 1.7 ) was determined by the agar plate dilution method.

(b) Kamogai Muller-Hinton (Hueller-llinton), whose test bacteria are bacteria
) Agar medium/pH 7, 3/37°C/20 hours (b) If the test bacteria is yeast, Sabouraud dex] Herose agar medium/r)■5.6/
25°C/4B hours (c) If the test bacteria is mold, 1 Novrow dextrose agar medium/pl-15i
, 6/25℃ 15 days MIC is 1C) 6 pieces/1
Prepare a cell suspension or spore suspension of the test bacteria,
The seeds were weighed in a micro planter and judged by the presence or absence of growth after culturing.

R20X2 Minimal Growth Inhibition W Maro (MIC) Harm-1-Example In the following, 1%-1 is l-w/v%-1.

Example 1) Preparation of Seed Mother The culture medium used was prepared by dissolving the following ingredients in 1 liter of water and adjusting the concentration to l')l-17.0.

Glucose 0.4% Malt Ball Kiss 1.0% Yeast J Kiss 0.4% Dispense 15ml of the medium described in Section 11 into a large 5Qml test tube.
After sterilization, Streptomyces griseolinatus A
300 was weighed with Suran 1 to J and Su1 Platinum F4, and cultured for 48 hours at 27°C, and used as seed stock.

2) Culture and Purification The medium used was prepared by dissolving the components of the following composition in 1 liter of water and adjusting it to FN-17.0.

Glucose 2.5% Otachi flour 1.5% Dry fermentation 11 0.2% Calcium fluoride 0.4% Dispense 1 (') (') ml of the above medium into Eioml Erlenmeyer flasks and sterilize them. , 2 ml of the above seeds were added, and rotational culture was performed at 27°0 using a [1-tally seeker]. After 33 days, the culture is completed, and the separated bacterial cells are extracted twice with 5001111 tons.

After concentrating the extract and removing Al-one, 200111
+ acid resistance] Extract twice with ethyl. The extract was dehydrated with anhydrous sodium sulfate, concentrated to dryness, and then dissolved in 20 ml of methanol. This was equilibrated with methanol for 1 time [
1 Heyopearl HW 40 Fj column (5cmφx5
Qcm). The eluted yellow fraction was concentrated and diluted with chloroborum-methanol (5(')
:i>Dissolved in solution 21.

This was subjected to chromatography using a silica gel ([wa]-gel C-200J Wako Pure Chemicals MA) column (3 cmφ x 3 Q cm) equilibrated with 10 ml of methanol (50:1), and the resulting fraction was concentrated. Upon drying, 75 ml of A300 yellow powder was obtained.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet/visible absorption spectrum of A300 (solid line: in methanol, broken line: 0.01NN a O1-1
- in methanol). FIG. 2 is a reproduction of the infrared absorption spectrum of A300 by the KBr disk method. Figure 3 [31, A300 heavy chlorine [in 1 form]
400 MHz' tl-NMR Spec]・
FIG. Figure 4 shows the weight of A300 [10 in 10 vol.
FIG. 3 is a reproduction of a 0 MHz 13C-NMR spectrum. Applicant's agent Kiyoshi Inomata] Procedures? =ili jTE appearance 9/θ day, 1985

Claims (1)

[Claims] New substance A300 represented by the following formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼