JPH01228483A - Antitumor agent - Google Patents

Abstract

PURPOSE:To obtain an antitumor agent effective in suppressing the proliferation of human fibroblast cell and mouse mammary cancer cell and containing a compound having specific chemical properties, by culturing Streptomyces albus A282 strain. CONSTITUTION:Streptomyces albus A282 strain (FERM P-9892) is cultured in a liquid medium under neutral pH and aerobic condition at 15-40 deg.C under shaking or aeration and agitation and the obtained culture product is treated to obtain the objective antitumor agent containing a compound having the following characteristics as an active component. Elemental analysis, C 47.99%, H 5.72%, N 14.03%, O 32.26%, melting point, 101.5-106.0 deg.C; solubility, soluble in water, methanol, ethanol, dioxane and pyridine and scarcely soluble in CHCl3 and ethyl acetate; ultraviolet absorption, infrared absorption and nuclear magnetic resonance spectra, exhibiting specific patterns; molecular weight, 267; molecular formula, C11H13N3O5.

Description

[Detailed description of the invention] (Industrial application field) TECHNICAL FIELD The present invention relates to antitumor agents.

(Prior Art and Problems to be Solved by the Invention) Conventionally, various compounds have been proposed as substances having antitumor effects, but due to the problem of tumor diversity, there are still many compounds with various antitumor effects. A compound is needed. The present inventors focused on bacteria belonging to the genus Streptomyces,
As a result of various studies to obtain compounds with antitumor effects, Streptomyces albus (Streptomyces albus)
It has been found that a compound produced by the A.sub.esalbus A.sub.2 g2 strain and having the following physicochemical properties has an antitumor effect.

(a) Molecular weight:, 267 (b) Molecular formula: CIl H13N3O5(C)
Elemental analysis value (as CIIH13N3OS ・CH3OH) (d)
Melting point: 10/,! i ~ IOA, O°C (e) Solubility: Soluble in water, methanol, ethanol, dioxane and pyridine, slightly soluble in chloroform and ethyl acetate.

(f) It has the ultraviolet absorption spectrum shown in FIG.

(g) It has the nuclear magnetic resonance spectrum shown in Figure 2.

(h) It has an infrared absorption spectrum shown in FIG.

Conventionally, it has been known that antibiotics with similar physicochemical properties to the compounds of the present invention have antibacterial activity, but there has been no description that these antibiotics have antitumor effects (Tokuko Shoda J. -20!!9 Publication).

The present inventors have recently discovered for the first time that the above compound suppresses the proliferation of human fibroblasts and mouse breast cancer cells, as shown in the Examples below, and have completed the present invention.

(Means for Solving the Problems) That is, the gist of the present invention resides in an antitumor agent containing a compound having the above-mentioned physicochemical properties as an active ingredient.

Hereinafter, to explain the present invention, the above-mentioned compound of the present invention can be obtained by culturing Streptomyces albus Aλg2 strain as described below. The mycological properties of the Streptomyces albus A232 strain are as follows.

/ Morphological Characteristics Colonies appear white to yellowish in color on synthetic media. The basal hyphae are well developed and branched.

Aerial hyphae are poorly developed and long segmented spore chains are formed on the aerial hyphae. The spore chain is wound in a loose spiral. Spore surface is smooth. The spores are short cylindrical, and their sizes are o, A~o, g x 0, Wa~/, /μm.

Growth status on various media (characteristics after 2 days of culture at 27°C): Good growth on yeast/malt agar medium (Isp, A2), white color. Strong development of aerial mycelium. Spore formation is poor. The underside of the colony is yellowish brown to yellowish white. No soluble dyes.

Oat meal agar medium (ISP/i6J) Poor growth, white color. Poor development of aerial hyphae. Poor spore settlement. The underside of the colony is white. No soluble dyes.

Poor growth on starch/inorganic salt agar medium (ISPAj), yellowish white color. Poor development of aerial hyphae.

Poor spore settlement. The back side of the colony is yellowish. No soluble dyes.

Glycerin-asparagine agar medium (ISPAj) Good growth, yellowish white. Poor development of aerial hyphae.

Poor spore settlement. The underside of the colony is yellowish white.

No soluble dyes.

Tyrosine medium (ISPA7) Good growth, yellowish brown. Poor development of aerial hyphae. Poor spore settlement. The underside of the colony is yellowish brown. No soluble dyes.

Sucrose/nitrate agar medium Poor growth, white color. Poor development of aerial hyphae. Moderate sporulation.

The underside of the colony is white. No soluble dyes.

Glucose-asparagine agar medium Good growth, yellowish white. Aerial mycelium development is poor.

Good sporulation. No soluble dyes. The underside of the colony is yellowish white.

Good growth on Benesoto agar medium, white to yellowish color. Strong development of aerial mycelium. Poor sporulation. The underside of the colony is yellowish white to yellowish brown. No soluble dyes.

Ordinary agar medium Medium growth, yellowish white. Poor development of aerial hyphae.

Poor sporulation. The underside of the colony is yellowish white. No soluble dyes.

Calcium malate medium Good growth, white color. Poor development of aerial hyphae. Poor spore settlement.

The underside of the colony is white. No soluble dyes.

3. Physiological properties/) Growth temperature range: 20 to 30% on Bennett agar medium
It grows in the temperature range of 37°C and grows well in the range of 1.27 to 30°C.

2) Liquefaction of gelatin: Positive 3) Hydrolysis of starch: Positive Reduction of nitrate: Positive Left) Peptonization of skim milk: Positive Coagulation: Positive 6) Salt tolerance: Grows in a medium containing 2% salt, but It will not grow above 5%.

7) Production of melanin-like pigment: Negative on tyrosine agar medium for melanin.

g) Assimilation of carbon sources (basal medium of Pridham gotrib) D-glucoseni+, D-xylose: +L~arabinose: +, L-rhamnoseni/17 D-fuctose: +, D-mannitol: + Raffinose: ±, Shucrosny 1-inositol: ± da Cell wall composition Method of Becker et al. [Applied Microbiology (Appl, Microbiol,
).

/3, 23&, /945) revealed that the main components of the cell wall of this strain (A2g2) were cell wall type 2 containing LL-A-2pm and glycine.

5. Taxonomic Consideration The colony color of this strain (A21:2) is white to yellowish white, and does not turn gray even after culture days have passed.

Does not produce melanin-like pigments. The sporulation mode is segmental, and the spore-bearing area shows a gentle spiral structure.

Spore surface is smooth.

Regarding these characteristics, Nonomura C Journal Opinion Technology (J, Fe
rment, Technol, ), ! r, 2.
7za-qa, /qqia] proposed by St.
A species search was performed using a search table for RSP species of the genus Reptomyces.

As a result, this strain (k2g2) was found to have Strepto-mycobacteria.
It was suggested that it is a species similar to ces albus.

So E, B, Shirling & D, Go
ttlieb, Int.

J,,5yst,Bact,,/9. pH
S, aLb described in O/ (/949)
When we compared the morphological properties, culture properties, sugar assimilation properties, etc. of S. us with various properties of this strain (A2g2), we found that this strain was S.

It matched the description of albus.

Therefore, this strain (A, 2g 2 ) is a streptomy
It was identified as cesalbus.

The above-mentioned strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERMP-9g92.

The medium used to produce the above compound using the present microorganism is a conventional liquid medium containing a carbon source, a nitrogen source, an inorganic salt, and, if necessary, organic micronutrients. Examples of carbon sources include carbohydrates such as glucose, fructose, maltose, sucrose, starch, texturin, starch hydrolysates, blackstrap molasses, and starch syrup, and organic acids such as citric acid, succinic acid, fumaric acid, acetic acid, and propionic acid. , coconut oil, glycerin, and other oils and fats are used. Examples of nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, ammonium acetate, and ammonium propionate, nitrates such as sodium nitrate and potassium nitrate, urea, aqueous ammonia, ammonia gas, amino acids, and peptone, Proteins such as soybean flakes, soybean flour and their hydrolysates, rice bran, Pharmamedia,
Sungrain etc. are used. Other inorganic salts include
For example, manganese salts, phosphates, chlorides, sulfates, carbonates, iron salts, cobalt salts, nickel salts, calcium salts, zinc salts, copper salts, etc. are used as appropriate, and organic micronutrients include amino acids, vitamins, Peptone containing these,
Meat extract, Pharmamedia, sungrain, yeast extract, cornstarch liquor, etc. are used as appropriate.

Culture conditions vary depending on medium composition and other factors, but for example,
Usually the pH is around neutral, the temperature is / -' 10℃, shaking culture,
Culture is performed under aerobic conditions such as aerated agitation culture.

The compound of the present invention is a water-soluble basic substance and is mainly contained in the culture filtrate, so for example, the culture filtrate is brought into contact with an appropriate adsorbent to adsorb the active ingredient, and the active ingredient is desorbed with an appropriate solvent. Means are used effectively. That is, column chromatography using synthetic adsorbents such as Diaion HP-20 (manufactured by Mitsubishi Kasei Corporation) and Amberlite XAD-7 (Rohm & Barr, 11), silica gel, alumina, etc. are effective.

As the elution solvent, a water-containing solution of a water-soluble organic solvent, such as water-containing methanol or water-containing dioxane, is used.

By using the above methods or by appropriately combining these methods, it is possible to obtain highly pure compounds having the above-mentioned physicochemical properties.

The method for detecting the target substance in each purification step is the activity that selectively stops the development of starfish embryos at the early blastula stage [Susumu Ikegami et al.: "From biotechnology and biochemistry to material production" edited by Bunji Maruo, p2gA, Academic Society Publication Center, /q
gs:] was used to measure the RNA synthesis inhibitory effect. That is, a fertilized egg of a starfish is suspended in a certain amount of seawater, a seawater solution containing a certain amount of the present compound is added, and culture is continued for 75 hours or more. Count the number of fertilized sea star eggs that have hatched and swim in seawater, and investigate the developmental inhibitory effect of this substance on starfish.

The antitumor agent of the present invention can be used as it is, or by converting it into a salt compound such as a hydrochloride and prescribing it as a solution for parenteral administration, to suppress tumors in living organisms.

The dosage of the above compounds of the invention will vary depending on the severity of the disease, the weight of the patient and other factors recognized by the patient, but will usually be 0.02 mg/patient for patients (animals and humans) in need of treatment. A total daily dose of ~5 orv can be administered.

(Example) The present invention will be further specifically explained below with reference to Production Examples and Examples, but the present invention is not limited to the following unless it exceeds the gist thereof.

Production example: Starch syrup 1%, soybean oil 0.3%, glucose 7%, soybean flour 2
%, Pharmamedia (Langston Buck) 7%,
Medium g 0ml containing 0.3% Sungrain (Sungrain Co., Ltd.) and 0.3% calcium carbonate (pH 7.0)
This strain (Aug: 1 strain) was inoculated and precultured in a 2001nl Erlenmeyer flask, then inoculated into a medium/critre with the above composition, and incubated at 27°C in a 3O bottle jar.
Aerated culture was carried out for 1 day. The culture containing the Jerco base was filtered through Celite to remove bacterial cells to obtain 20 liters of culture filtrate. This culture filtrate showed the activity of selectively arresting starfish embryonic development at the 37 two-cell stage immediately after blastula formation. 0.20 liter of the culture source obtained above was passed through an S liter column of Diaion HP-2θ, washed with water, and then
The active fraction was eluted with 00% methanol. After concentrating and drying, silanized silica gel (manufactured by Merck & Co., Ltd.)
After applying to a 35cIrL column and washing with water, the active fraction was eluted with 5% methanol. After concentrating and drying, it was applied to a Rover RP-g (manufactured by Merck & Co., Ltd.) 4gX, 20cm column, and the active fraction was eluted with 70% methanol. After concentration and drying, a neutral alumina column (/, /×
After eluting with methanol-water (/:/), an active fraction is obtained with methanol-water (?near). High performance liquid chromatography was performed on a Cialpac C18 column (manufactured by Waters) eluting with SO% methanol to obtain 4.4 m9 of nearly pure crystals. Its physical and chemical properties were as follows.

(a) Molecular weight: 267 (b) Molecular formula: C1l H13N3O5 (C) Elemental analysis value (d) Melting point: 10/,! i~IOA, 0℃(e)
Solubility: Soluble in water, methanol, ethanol, dioxane and pyridine, slightly soluble in chloroform and ethyl acetate.

(f) It has the ultraviolet absorption spectrum shown in FIG.

(g) It has the nuclear magnetic resonance spectrum shown in FIG.

(h) It has an infrared absorption spectrum shown in FIG.

Example/(Effect on cancerous human fibroblasts) Eagle MEM powder medium (Cell culture medium component that can be autoclaved and steam sterilized, manufactured by Tadami Seiyaku) q, + g was dissolved in distilled water, and the total volume was adjusted to /liter. Thereafter, it was autoclaved at 727°C for 75 minutes. After high-pressure steam sterilization, the medium is cooled to room temperature.
Add an appropriate amount of 0% sodium bicarbonate aqueous solution to adjust the pH of the medium.
7. I made it q. Add 0.0% sterile glutamine to this.
J9: After adding 1g, sterile fetal bovine serum (A, rm
A cell culture medium was prepared by adding 100 ml of the following product (manufactured by our company). Human cancer KMsT-4M cells (Nanba Masayoshi et al.: International J.
Our own of Cancer, 35°275-
Add 0.19g of the cell suspension at a cell concentration of 0.13g and 0xIO'm13, and aseptically dispense this cell suspension into 1ml of a 9-well microtest plate (manufactured by Falcon) in a carbon dioxide incubator. Culture was carried out for 2 q hours under conditions of carbon dioxide gas concentration s% and temperature 37°C. Add 0.0% of the above medium containing a certain amount of the present compound to this culture solution. / ml was added, and the culture was continued for another 2 days. The number of living cells was counted by cell staining method using trivan blue, and the growth inhibitory effect of this substance on KMST-6 cells was investigated. The results are shown in Table 7.

In addition, (-) in the table indicates that there is no growth inhibition.
+) indicates that all cells die.

Table 1 Concentration Growth inhibition degree of KMST-6 cells (μg/rnl) 0, OII
110, Ko +++ /, 0 + + +90
+++++ is an inhibition degree of 50-7S% +++ is an inhibition of 5-100% (influence on mouse mammary cancer cells) Mouse mammary cancer cells F M ? A cells (Koyama Katsuki 9 tissue culture, A, 2
3! ;-2'1.3,19g0). 99.7 g of ES powder medium (high-pressure steam sterilizable cell culture medium component manufactured by Hinaga Pharmaceutical Co., Ltd.) was dissolved in distilled water to make a total volume of 7 liters, followed by high-pressure steam sterilization at 727° C. for 7 S minutes.

After autoclaving, add an appropriate amount of sterile 70% sodium bicarbonate aqueous solution to the medium cooled to room temperature to adjust the pH of the medium to 7. +
Closed. Add 0.292 sterile L-glutamine to this,
! 9, and sterile fetal bovine serum (manufactured by Armor)
A cell culture medium was prepared by adding 0'rnl. 7. Add pre-cultured FMJA cells to this medium. OX/O5
/ml, and this cell suspension was placed in a 2-well microtest plate (manufactured by Falcon).
The mixture was dispensed aseptically and cultured in a carbon dioxide incubator for 3 hours at a carbon dioxide concentration of 5% and a temperature of 37°C. Add 0 of the above medium containing a certain amount of this compound to this culture solution.
．． / ml was added, the culture was continued for another 2 days, and the number of living cells was counted by a cell staining method using Trivan blue.
FM of this substance? The growth inhibitory effect on A cells was investigated. The results are shown in Table 2.

Table 2 Concentration Mouse breast cancer cell-derived FM, growth inhibition rate for 7A cells 7J &/!
tj, 22
．． 33. OA2 Otsu 3.0 9/15
θ, 099 As shown in Table 2, this compound was used in mouse breast cancer cells F
M,? The LDso for A was 27 ng/ml.

(Effects of the Invention) The above compound of the present invention suppresses the proliferation of human fibroblasts and mouse breast cancer cells, and is therefore useful as an antitumor agent.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the ultraviolet absorption spectrum of the compound of the present invention. FIG. 2 is a drawing showing the nuclear magnetic resonance spectrum of the compound of the present invention. FIG. 3 is a drawing showing the infrared absorption spectrum of the compound of the present invention.

Claims (1)

[Claims] (1) An antitumor agent containing as an active ingredient a compound obtained from Streptomyces albus A282 bacteria and having the following physicochemical properties. (a) Molecular weight: 267 (b) Molecular formula: C_1_1H_1_3N_3O_5(c
) Elemental analysis values ▲ Numerical formulas, chemical formulas, tables, etc. ▼ (as C_1_1H_1_3N_3O_5・CH_3OH) (d) Melting point: 101.5-106.0℃ (e) Solubility: Possible in water, methanol, ethanol, dioxane and pyridine It is slightly soluble in chloroform and ethyl acetate. (f) It has the ultraviolet absorption spectrum shown in FIG. (g) It has the nuclear magnetic resonance spectrum shown in FIG. (h) It has an infrared absorption spectrum shown in FIG.