JPH02221292A - New substance 02-3, its use and production thereof - Google Patents

Abstract

NEW MATERIAL:The compound of formula (R is acetyl or H). The compound of formula wherein R is acetyl has the following physical and chemical properties. Appearance, yellow powder; melting point, 180-184 deg.C (decomposition); molecular formula. C55H80O25; elemental analysis (%), C 57.68, H 7.12, O 35.20; FAB mass spectrum (m/z), 1163 (M+Na)<+>; solubility, soluble in methanol and ethanol, slightly soluble in acetone, ethyl acetate and chloroform and insoluble in hexane and water; etc. USE:An antitumor agent. PREPARATION:A microbial strain belonging to genus Streptomyces and capable of producing 02-3 (e.g. FERM BP-2276) is cultured in a medium under aerobic condition preferably at 25-30 deg.C for 3 days.

Description

DETAILED DESCRIPTION OF THE INVENTION [Background of the Invention] Technical Field The present invention relates to a novel substance, and more particularly to a novel substance 02-3 having antitumor properties.

Many prior art antitumor substances have already been put into practical use as medicines. In general, the physiological activity 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 properties.

[Summary of the invention]

Abstract The present invention meets the above needs.

That is, the novel substance 02-3 according to the present invention has the following formula (I)
This is shown in .

The invention also relates to the use of this substance.

That is, the antitumor agent according to the present invention contains compound 02-3 represented by the following formula (I) as an active ingredient.

The invention furthermore relates to a method for producing this material.

That is, 02-3 represented by the following formula (I) according to the present invention
The production method is characterized by aerobically cultivating a strain belonging to the genus Streptomyces and capable of producing 02-3 in an appropriate medium, and obtaining 02-3 from the culture. .

(In the formula, R represents either an acetyl group or a hydrogen atom.) [Detailed Description of the Invention] New Substance 02-3 1) Chemical Structure The novel substance 02-3 according to the present invention has the above formula (I) It has the chemical structure shown below. This chemical structure was determined as follows.

02-3D in which R in the above formula (I) is an acetyl group
The chemical structure deduced from its proton nuclear magnetic resonance spectrum (Figure 5) and carbon-13 nuclear magnetic resonance spectrum (Figure 7) is chromomycin A3 (chromoI
lycln＾3. J, Th1es at at,.

J, C, S, Perkin ■, 1331, 1
979). When we compared the physicochemical properties of the two, we found that the molecule Jill140 obtained from the FAB mass spectrum of 02-3D does not produce more C2H20 than chromomycin A3, and that the acetyl group signal disappears in the NMR spectrum. , 02-3D was found to be a mono-deacetylated form of chromomycin A3. Furthermore, the substitution position of the remaining acetyl group is changed to N
This was determined by detailed analysis of MR data, and the chemical structure of 02-3D was determined as shown in formula (I) above.

Similarly, the structure of 02-3G in which R in formula (I) is a hydrogen atom was determined to be a di-deacetylated form of chromomycin 83.

2) Physicochemical properties (4) Elemental analysis value (calculated value) (%) (5) FAB7 spectrum (m/z) 1163 (M
+Na)” 11121 (M+Na)”(6)Rf
Value (using "Silica Gel 60F254" manufactured by Merck) Chloroform-methanol (I0:1) Chloroform-methanol-acetic acid-water (I00:10:1:1) (7) Production of Solubility 02-3 (9) Infrared Absorption spectrum (KBr disk method) shown in Figure 3 (I0) shown in Figure 4 Proton nuclear magnetic resonance spectrum (400 MHz) shown in Figure 5
(I1) Carbon-13 nuclear magnetic resonance spectrum (I00 MHz) shown in Figure 7 (Deuterated chloroform-deuterated acetic acid in heavy methanol)
Overview of Compound 02-3 shown in the figure (deuterochloroform-methanol (in deuterate acetic acid)) Currently, compound 02-3 can only be obtained by culturing microorganisms, but it can also be produced by synthetic chemical modification of related compounds. Alternatively, it could be produced completely synthetically.

In the case of culturing microorganisms, for example, strains belonging to the genus Streptomyces that have an ability to produce 02-3 are used. Specifically, the present inventors have revealed that the Streptomyces strain 02-3 isolated by the present inventors produces 02-3; It is possible to separate suitable things from nature by the conventional method of separation. Furthermore, there is still room to increase the production ability of 023 by subjecting 02-3 producing bacteria, including Streptomyces strain 02-3, to irradiation, J], J, and other mutational treatments.

Strain 02-3 The strain 02-3, which the present inventors have discovered as a Streptomyces strain capable of producing 02-3, has the following content.

1. Origin and deposit number Strain 02-3 was isolated from soil collected in Sakurajima, Kagoshima Prefecture, and was deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology on February 8, 1989. Article No. 2276)
(FERM BP-2276) number has been obtained.

2. Mycological properties The mycological properties of strain 02-3 are as follows.

1) Strain Form 02-3 grows poorly on sucrose/nitrate agar and glucose/asparagine agar, but grows moderately to well on other media. Aerial mycelium colonization was extremely poor on glucose-asparagine agar, poor on sucrose-nitrate agar and nutrient agar, but poor on starch-mineral salt agar, yeast-malt agar, and oatmeal agar. Grows abundant aerial mycelium. In other media, the results were generally moderate. Aerial hyphae produced from substratum hyphae form simple branches and elongate, and the spore chain is long, about 30 to 50, sometimes more than 50, and the spore chain is linear.

In addition, the shape of the spore is cylindrical, and the size is 0.6 to 0.8
μm X 0.7 to 1.0 μm, and its surface is smooth.

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

2) Growth status on various media The results of culturing strain 02-3 on various media at 27°C for 3 weeks are shown in Table 1.

3) Physiological properties The physiological properties of strain 02-3 are shown in Table 2.

4) Carbon source utilization The carbon source utilization of strain 02-3 (on Bridham-Gotlieb agar medium) is as shown in Table 3.

5) Analysis of diaminopimelic acid As a result of analysis of diaminopimelic acid, which is one of the amino acids constituting the cell wall, LL-diaminopimelic acid was detected.

From the above mycological properties, strain 02-3 was determined to be a strain of the genus Streptomyces, and has the following characteristics.

(I) The spore chain is linear and the surface of the spore is smooth.

(2) Aerial mycelia are yellowish white to bright olive gray to dark yellow-orange to light brown, and the color of the underside varies from yellowish gray to light brown yellow to dark yellow to yellowish brown to dark yellow. show.

(3) Melanin-like pigment is not produced on tyrosine agar medium, peptone yeast/iron agar medium, or tryptone yeast liquid medium, and no soluble pigment is observed.

(4) The types of sugars that can be assimilated are relatively limited, and sugars other than glucose, mannose, galactose, and maltose are generally not assimilated.

Based on the above properties, I5P (International Streptomyces Project) is described (E, B).

Shlrllng and D, Gottlleb:
Int, J, 5yst, Bact.

1869-189. 279-392 (I988),
19391-512 (I989).

22265-394 (I972)) and Bergey's Manual of Determinative Bacteriology.
r Determinative Bacter1o+
When searching for known related bacterial species using the 8th edition (I974) as a reference, Streptomyces avelanius (str.
eptoayces avellaneus ) (
Int.

J, 5ysL, Back,: 22.27B (I
972)) seemed to be the most similar.

Therefore, when comparing strain 02-3 and Streptomyces Avelanius, we found that they have morphological characteristics of linear spore chains and spores with smooth surfaces, and that they do not produce melanin-like pigments on peptone, yeast, or iron agar media. They match well in terms of dots and color tone of aerial mycelia. Differences include that the 02-3 strain does not assimilate fructose and sucrose.

As described above, although there are some differences, the 02-3 strain closely matches those of Streptomyces averanius in its basic characteristics, and therefore it is appropriate to identify it as Streptomyces averanius. Therefore, 02-3
Strains Streptomyce 7, -7 Verranius (Strept.
omyees avellaneus) 02-3.

Table 1 Table 2 Table 3 Table 12 Compound 02-3 produced by culture 102-3 used - 2 not used belongs to the genus Streptomyces 02-
It can be produced by aerobically culturing 3-producing bacteria in an appropriate medium and collecting the target product from the culture.

The medium can contain any nutrient source available to the 02-3 producing bacteria. Specifically, for example, glucose, sucrose, maltose, soluble starch, glycerin, and fats and oils can be used as carbon sources, and soybean flour, fish meal, cottonseed meal, dried yeast, yeast extract, meat extract, and nitrogen sources can be used. Organics such as corn staple liquor and inorganics such as ammonium salts or nitrates such as ammonium sulfate, sodium nitrate and ammonium chloride are available. Also, if necessary,
Inorganic salts such as calcium carbonate, sodium chloride, potassium chloride, phosphates, heavy metal salts, etc. can be added. In order to suppress foaming during fermentation, suitable antifoaming agents such as silicone oil can also be added according to conventional methods.

The most suitable culture method is the aerobic liquid culture method, which is the same as the commonly used antibiotic production method. The culture temperature is suitably 20 to 37°C, preferably 25 to 30°C. In this method, the production amount of 02-3 is as follows: shaking culture,
Both the aeration and agitation culture reached the maximum after 3 days of culture.

In this way an accumulated culture of 02-3 is obtained. In the culture, a part of 02-3 exists in the bacterial cells, but most of it exists in the culture filtrate.

Any convenient method can be used to harvest 02-3 from such cultures. One method is based on the principle of extraction, and specifically, 02-3 in the culture filtrate is extracted from water-immiscible 02-3.
For 02-3 in bacterial cells, collect the bacterial mass obtained by extraction with a solvent (see above) such as chloroform, or by i-filtration, centrifugation, etc., and treat it with methanol, ethanol, acetone, etc. There are methods etc.

The culture itself can also be subjected to the above extraction operation without separating the bacterial cells. Countercurrent distribution methods using suitable solvents can also be included in the scope of extraction.

Another method for collecting 02-3 from cultures is based on the principle of adsorption, in which 02-3 is already in liquid form.
2-3 Targeting the content, such as the culture filtrate or the extract obtained by performing the extraction operation as described above, use a suitable adsorbent, such as silica gel, activated carbon, [Diaion HP20J (manufactured by Mitsubishi Chemical Corporation)]. 02-3 can be obtained by adsorbing the desired 02-3 using, for example, a solvent, and then eluting it with an appropriate solution. The 02-3 solution thus obtained is concentrated to dryness under reduced pressure to obtain a 02-3 assembly product.

In order to further purify the crude sample of 02-3 obtained in this way, the extraction method and adsorption method described above may be combined with gel filtration method, high performance liquid chromatography, etc. as necessary, and the procedure may be performed as many times as necessary. .

For example, a column chromatography rYMc pack using an adsorbent such as silica gel or a gel filtration agent such as Sephadex LH-20J (manufactured by Pharmacia).
(manufactured by Yamamura Kagaku Co., Ltd.) or the like and a countercurrent distribution method can be carried out in an appropriate combination. Specifically, for example, the 02-3D rough assembly machine [
The active fraction was applied to a Sephadex LH-2OJ column, eluted with a chloroform-methanol (I: 1) mixture, and concentrated to dryness to obtain a pure product of 02-3D.

Uses of 02-3 Compound 02-3 according to the present invention is useful in that it has antitumor activity.

Biological activity 02-3 showed cytostatic activity against tumor cells. For example, 5X10' mouse P388 leukemia cells/
RPM containing 10% heat-inactivated calf serum to ml
I after being suspended in 11640 medium and cultured for 3 days at 37°C with various concentrations of 02-3D and 02-3G.
C50 values are 0.18 and 0.08 μg/m, respectively.
It was l.

Furthermore, 02-3 was used in mouse P388 leukemia.
Anticancer activity was also observed in vlvo. That is, CDF
For I mice, suspension of P388 leukemia cells lX1
06 mice/mouse were intraperitoneally transplanted, and after transplantation, 02-3G was intraperitoneally administered, and the survival rate (%) is shown based on the number of survival days of the control group (control) mice administered with physiological saline as 100. It was as follows.

Sample Dose Dose Dose Survival days
Life extension rate (after cancer cell transplantation) (II1g/kg)
(Mean±SD) C%) 02-30 1.
5th day 8011.6±0. 53 109〃
1.5th day 160 12.9±0.6912
4I〆 1-5 consecutive days 8014.4±0.
53 138 administration control 10.4±0.
52 100 As mentioned above, it was revealed that 02-3 of the present invention exhibits antitumor properties. Therefore, 02-3 of the present invention can be used as an antitumor agent.

Antitumor Agent It was thus revealed that 02-3 of the present invention exhibits antitumor properties against animal tumors, particularly malignant tumors.

Therefore, the compounds of the present invention can be used as antitumor or tumor therapeutic agents.

The compounds of the present invention as antitumor agents can be administered by any convenient route of administration and in a dosage form determined by the route of administration employed. The drug is usually in a diluted form with a pharmaceutically acceptable carrier or diluent.

When actually administering the compounds of the present invention as antitumor agents, one typical method is to dissolve them in distilled water for injection or physiological saline and inject them.

Specifically, in animals, intraperitoneal injection, subcutaneous injection, intravascular injection into a vein or artery, and local administration by injection, and in humans, by intravascular injection or injection into a vein or artery. Methods include local administration.

The dosage of the compound of the present invention is determined in consideration of the results of animal tests and various circumstances so that the total dosage does not exceed a certain amount when administered continuously or intermittently. The specific dosage depends on the administration method, the situation of the patient or treated animal,
For example, age, weight, gender, sensitivity, diet, time of administration,
Needless to say, this will vary depending on the concomitant drugs, the patient, and the severity of the disease, and the appropriate dosage and frequency of administration under certain conditions must be determined by a specialist's appropriate dosage determination test based on the above guidelines. . Specifically, it is about 0.1 to 1 g per day for an adult.

Experimental Examples Example 1) Preparation of Seed Mother The medium used was prepared by dissolving the components having the following composition in 1 liter of water.

Soluble starch 10g Polypeptone 10g Molasses 10g Meat extract 10g Dispense 100ml of the above medium into a 500ml Erlenmeyer flask, sterilize it, and add Streptomyces averanius 02-3.
One platinum loop of the following was inoculated from a slant and cultured under shaking at 27°C for 4 days, which was used as a seed mother.

2) The culture medium used was prepared by dissolving the components of the following composition in 1 liter of water.

Glycerin 8g Soluble starch 8g Soybean flour 3g Fishmeal 8g Calcium carbonate 2g 100ml of the above medium was dispensed into sterilized 500ml Erlenmeyer flasks, and 2ml of the seed mother was added thereto, followed by shaking culture at 27°C for 3 days.

3) Collection of 02-3 After culturing under the above conditions, add a culture solution (10 liters), adjust the 'a solution to pH 2 with 6N hydrochloric acid, and add a Diaion HP20 column (manufactured by Mitsubishi Kasei, 7amφX50cm).
Let it be adsorbed to. After washing the column with 5 liters of 50% methanol water, it was eluted with 5 liters of methanol. After concentrating the eluate to 1 liter and extracting with an equal volume of ethyl acetate,
Concentrate to dryness and apply to silica gel column (5cmφX50cm)
Let it be adsorbed to. After washing the column with 1 liter of chloroform-methanol-acetic acid (I00:1:1) and eluting with 1 liter of chloroform-methanol-acetic acid (I00:2:1), a crude fraction of 02-3D was obtained. Elution with 1 liter of loloform-methanol-acetic acid (I00:4:1) gives the 0236 crude fraction.

The 02-3D crude fraction was collected using a Sephadex LH-20 column (
5caIφX5cm) (manufactured by Pharmacia), developed with methanol, and concentrated the active fraction.

Furthermore, by high performance liquid chromatography using 0DS-5251-N (manufactured by Senshu Kagaku Co., Ltd.) using acetonitrile-water-acetic acid (40:60:1) as a developing solvent, 10 mg of pure 02-3D was obtained. can get.

The 02-30 crude fraction was transferred to a Sephadex LH-20 column (
Made by Pharmacia, 5cIIIφX50cm),
Develop with methanol and concentrate the active fraction.

Furthermore, 0DS-5251N (manufactured by Senshu Kagakusha)
The product is purified and fractionated by high performance liquid chromatography using acetonitrile-water-acetic acid (35:65:1) as a developing solvent to obtain 20 g of pure 02-3G product.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 depicts the ultraviolet absorption spectrum of 02-3D in methanol. FIG. 2 is a reproduction of the ultraviolet absorption spectrum of 02-3G in methanol. FIG. 3 is a reproduction of the infrared absorption spectrum of 02-3D obtained by the KBr disk method. FIG. 4 is a reproduction of the infrared absorption spectrum of 02-30 obtained by the KBr disk method. FIG. 5 is a reproduction of the 400 MHz proton nuclear magnetic resonance spectrum of 02-3D in deuterochloroform-deuterol. FIG. 6 is a reproduction of the 400 MHz proton nuclear magnetic resonance spectrum of 02-3G in diacetic acid. FIG. 7 is a reproduction of the 100 MHz carbon-13 nuclear magnetic resonance spectrum of 02-3D in deuterated chloroform-deuterated methanol. FIG. 8 is a reproduction of the 100 MHz carbon-13 nuclear magnetic resonance spectrum of 02-3G in diacetic acid.

Claims (1)

[Claims] 1. Compound 02-3 represented by the following formula (I). ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (I) (In the formula, R represents either an acetyl group or a hydrogen atom.) 2. Compound 02-3 shown by the following formula (I) is the active ingredient. Antitumor agent. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (I) 3. A strain belonging to the genus Streptomyces and capable of producing 02-3 is cultured aerobically in an appropriate medium, and the compound 02-3 is obtained from the culture. The following equation (I
) The manufacturing method of 02-3 shown in ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I)