JPH0517484A - Antitunor substance be-22, 179 - Google Patents

Abstract

PURPOSE:To obtain a new compound useful as an antitumor agent, strongly inhibiting topoisomerase, RNA synthesis and an essential enzyme for cell growth. CONSTITUTION:The compound shown by the formula. The compound. is obtained by culturing Streptomyces sp. A22,179 strain (FERM P-12,078) or a variant thereof in an agar plate medium at 28 deg.C for 14 days.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention is useful in the field of medicine, and more specifically, a novel compound that inhibits tumor cell growth and exerts an antitumor effect, a process for producing the same, a use thereof and a production of the novel substance. The present invention relates to a novel microorganism that does.

[0002]

2. Description of the Related Art In the field of cancer chemotherapy, it has been attempted to clinically apply many microbial metabolites such as bleomycin and Adriamycin, and they have been actually used in clinical practice. There is. However, its effect is not always sufficient for various types of tumors, and as the phenomenon of tumor cell resistance to these drugs is clarified clinically, its clinical applicability becomes complicated [47th. 12th Annual Meeting of the Japanese Cancer Society, pages 12 to 15 (1
(1988)]].

Under such circumstances, in the field of cancer treatment, there is a constant demand for the development of new regulated cancer substances. In particular, there is a need for a substance that overcomes resistance to existing carcinostatic substances and is effective against the types of cancer for which existing carcinostatic substances are not sufficiently effective.

[0004]

In view of the present situation as described above, the present inventors intend to find an excellent novel antitumor substance by widely screening microbial metabolites.

[0005]

[Means for Solving the Problems] The present inventors have screened a wide range of microbial metabolites with an inhibitory activity against topoisomerases I and II as an index, and thus actinomycetes isolated from the soil of a forest near Myogiyama, Gunma Prefecture. A2217
It was discovered that 9 strains produce a substance having a strong topoisomerase II inhibitory activity, and the substance was extracted and purified, isolated, and the structure was determined. As a result, a novel compound represented by the following formula (I) was obtained. The present invention has been completed by clarifying that it has an excellent antitumor effect.

That is, the present invention is

[0007]

[Chemical 2] A novel antitumor substance represented by (hereinafter, BE-2217)
9)), its production method and its use as an antitumor agent, and a novel microorganism producing BE-22179.

The physicochemical properties of the compound according to the present invention are shown below. Physicochemical properties of BE-22179 Properties: Light yellow powder Molecular formula: C ₄₆ H ₄₈ O ₁₂ N ₁₀ S ₄ Mass spectrum: High resolution FAB-MS; m / z 10
61.2410 [M + H] ⁺ UV spectrum: λ [MeOH, max, nm] 21
8,226,290,360 IR spectrum: IR spectrum by the KBr tablet method is shown in FIG.

H-NMR spectrum: The H-NMR spectrum (300 MHz) measured in deuterated chloroform is shown in FIG.

Solubility: Soluble in dimethylsulfoxide, chloroform and ethyl acetate, sparingly soluble in methanol and water Distinguishing acidic, neutral and basic substances: Neutral substance Rf value: 0.35 [Merck Kieselgel 60F
₂₅₄ used, developing solvent: chloroform / ethyl acetate / acetic acid (100: 200: 3)] Color reaction: phosphomolybdic acid reaction Positive, iodine reaction
Biological activity of positive BE-22179 The inhibitory effect on topoisomerase I and II, which are enzymes essential for cell growth, was investigated. BE-22179 has a DNA relaxation activity of 0.4 μg / ml of topoisomerase II purified from mouse tumor cells L1210.
Completely blocked. This was 125 times stronger than the effect of etoposide (VP-16) used as a control.

Topoisomerase I purified from the same tumor cells had no inhibitory effect even when 10 μg / ml BE-22179 was added.

Further, mouse tumor cells L1210 and BE-
After culturing 22179 for 3 hours, the uptake of tritium-labeled thymidine, uridine, and leucine into cells was measured.
C _50), respectively, show the 100,13,660ng / ml, BE-22179 showed strong RNA synthesis inhibitory action.

BE-22179 mouse experimental tumor cells L
In order to determine the growth inhibitory effect on 1210
The test was performed in vitro. BE-22179 was first dissolved in dimethylsulfoxide, and then 20% dimethylsulfoxide-containing cell culture medium (20% DMSO-
(RPMI-1640 medium), serially diluted to 2.5 × 10 5.
Cell culture medium containing ⁴ tumor cells (fetal calf serum 10%
Containing RPMI-1640 medium) 2 μl for 200 μl
Was added. After culturing at 37 ° C. for 72 hours under 5% CO ₂ , the number of surviving cells was counted by a Coulter counter and compared with the control group. As a result, BE-2217
9 shows a strong growth-inhibitory effect on L1210 tumor cells, and the concentration (I) that inhibits the growth of the tumor cells by 50%.
C ₅₀₎ was 5.8 ng / ml.

Next, the compound of the present invention, BE-2217.
The manufacturing method of No. 9 will be described.

The microorganism or its mutant strain used for the production of the antitumor substance BE-22179 of the present invention may be any one as long as it produces the antitumor substance BE-22179. For example, the following mycological characteristics Examples of microorganisms having

1. The Form A22179 strain forms aerial hyphae and basal hyphae that are well elongated and branched, and no division of the limbus or the basal hyphae is observed.
A spore chain is formed on the aerial mycelium, and its morphology is spiral. The surface of spores is smooth and the size is 1.0 x 0.5 ~
It has an oval shape of about 0.7 × 0.4 μm. Also, sporangium,
No special organs such as flagella spores and sclerotia are observed.

It was observed that in the oatmeal agar medium, as the aerial hyphae matured, they gradually wet and become black.

2. Table 1 shows the results of culturing at 28 ° C for 14 days using various agar plate media.

[0019]

[Table 1] 3. Growth temperature (yeast / malt agar medium, cultured for 14 days) 11 ° C: No growth 14 ° C: Growth and aerial hyphae formation failure 17 ° C: Growth and aerial hyphae formation good 20 ° C: Growth and aerial hyphae formation very good 24 ° C : Very good growth and formation of aerial mycelia 29 ° C: Very good growth and formation of aerial mycelium 34 ° C: Poor growth and poor formation of aerial mycelium 38 ° C: No growth 4. Physiological properties (1) Liquefaction of gelatin Positive (glucose / peptone / gelatin medium) (2) Hydrolysis of starch Positive (starch / inorganic salt agar medium) (3) Negative coagulation of skim milk powder (skimmed milk medium) (4) 4. Skim milk powder positive to peptone (skimmed milk medium) (5) Positive formation of melanin-like pigment (peptone-yeast-iron agar medium) (6) Salt tolerance Growing with salt content of 7% or less (yeast-malt agar medium) 5. Utilization of carbon source Using Pridham Godleybe's agar as a basal medium, the following various sugars were added and cultured at 28 ° C for 14 days. The result is the second
Shown in the table.

[0020]

[Table 2] 6. The cell wall composition LL-diaminopimelic acid was detected.

From the above-mentioned mycological properties, the A22179 strain is considered to belong to the genus Streptomyces of actinomycetes. Therefore, the A22179 strain was designated as Streptomyces sp. A22179 (Streptomyces sp.
A22179). In addition, Bergies Manual of Determinative Bacteriology Ace Edition (Bergey's
Manual of Determinative
Bacteriology, 8th Edition
n), 1974, International Journal
Of Systematic Bacteriology (Int
international Journalof System
ematic Biology), 18th
Vol. 1968 and Actinobase.
(RIKEN), and results of literature searches show that Streptomyces gangtokensis (Streptomyces g)
angtokensis) (Patent Publication Sho 40-137)
98) was estimated to be a closely related species.

This bacterial strain has been deposited at the Institute of Microbial Technology, Ministry of International Trade and Industry, and has a deposit number of Micro Engineering Research Institute, Micro Engineering Research Institute No. 12078 (FERM P-120).
78).

The antitumor substance BE-22 used in the present invention
The mutant strain of the 179-producing microorganism is, for example, irradiated with X-rays or ultraviolet rays, for example, nitrogen mustard, azaserine, nitrous acid, 2-aminopurine or N-methyl-N′-nitro-N-nitrosoguanidine ( Treatment with mutagens such as NTG), phage contact,
It is a microorganism obtained by mutating a BE-22179-producing bacterium by a commonly used bacterial species conversion treatment method such as transformation, transduction or conjugation.

In producing BE-22179 of the present invention, BE-22179 is inoculated into a nutrient-containing medium by inoculating a BE-22179 producing strain into a nutrient-source-containing medium to give BE-2221.
A culture containing 79 is obtained. As the nutrient source, those known as the nutrient source for actinomycetes can be used. For example, as the carbon source, commercially available glucose, glycerin, maltose, starch, sucrose, molasses, dextrin, etc. are used alone or as a mixture. As the nitrogen source, commercially available soybean flour, corn steep liquor, meat extract, yeast extract, cottonseed flour, peptone, wheat germ, fish meal,
Inorganic ammonium salts or sodium nitrate are used alone or as a mixture. As the inorganic salt, commercially available calcium carbonate, sodium chloride, potassium chloride,
Magnesium sulfate or various phosphates can be used. In addition, if necessary, a trace amount of a heavy metal salt such as iron, manganese, copper or zinc can be added. When foaming is remarkable, vegetable oils such as soybean oil or linseed oil, higher alcohols such as octadecanol, and various silicon compounds may be appropriately added as an antifoaming agent. In addition to these, BE-
Anything useful in the production of 22179 can be used.

The culturing method may be the same as the method for producing a general microbial metabolite, and may be solid culture or liquid culture. In the case of liquid culture, static culture, stirring culture, shaking culture, aeration culture or the like may be carried out,
Shaking culture or deep aeration stirring culture is particularly preferable. The culture temperature is appropriately 20 ° C to 37 ° C, preferably 25 ° C.
~ 30 ° C. The preferable pH of the medium is in the range of 4 to 8, and the culture time is 24 hours to 192 hours, preferably 48 hours.
Hours ~ 144 hours.

In order to collect the desired BE-22179 from the culture, a separation means usually used for collecting from the culture of the metabolite produced by the microorganism is appropriately used.
BE-22179 exists in the culture filtrate and in the bacterial cells, and therefore, the usual separation means from the culture filtrate or bacterial cells, such as the solvent extraction method, the ion exchange resin method or the adsorption or partition chromatography method and the gel filtration method, is used alone. Alternatively, the purification can be carried out in combination. High performance liquid chromatography and thin layer chromatography can also be used for extraction and purification.

Examples of preferable separation-purification include the following methods. First, cells are obtained by centrifuging or filtering the culture solution. The obtained bacterial cells are extracted with an organic solvent such as methanol or acetone. The residue obtained by distilling off the extract is dissolved in ethyl acetate, washed with water and then concentrated. The crude substance obtained here is dissolved in hydrous ethanol,
After washing with hexane, concentrate and extract with chloroform. The extract is subjected to silica gel column chromatography (eluted with chloroform / ethyl acetate / acetic acid). The fraction containing BE-22179 was concentrated to dryness, dissolved in methanol, and concentrated to give BE-22179.
Can be obtained as a pale yellow precipitate.

Further, examples of pharmaceutically acceptable salts include:
Examples thereof include inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid, and organic acids.

The compound BE-22179 of the present invention inhibits the growth of tumor cells and has an antitumor effect, but when the compound of the present invention is used as an antitumor agent, various administration forms can be selected. Examples thereof include oral agents such as tablets, capsules, powders, granules or liquids, or sterilized liquid parenteral agents such as solutions or suspensions.

The solid preparation can be produced as it is in the form of tablets, capsules, granules or powders, or can be produced using appropriate additives. Examples of such additives include sugars such as lactose or glucose, starches such as corn, wheat or rice, fatty acids such as stearic acid, inorganic salts such as magnesium aluminometasilicate or anhydrous calcium phosphate, such as polyvinyl. Synthetic polymers such as pyrrolidone or polyalkylene glycol, fatty acid salts such as calcium stearate or magnesium stearate, alcohols such as stearyl alcohol or benzyl alcohol, synthetic cellulose derivatives such as methyl cellulose, carboxymethyl cellulose, ethyl cellulose or hydroxypropyl methyl cellulose. In addition, commonly used additives such as water, gelatin, talc, vegetable oil, gum arabic, etc. may be mentioned.

These solid preparations such as tablets, capsules, granules and powders generally contain 0.1 to 100% by weight, preferably 5 to 100% by weight of the active ingredient.

The liquid preparation may be a suspension, syrup or injection using appropriate additives usually used in liquid preparations such as water, alcohols or plant-derived oils such as soybean oil, peanut oil or sesame oil. And so on.

In particular, when administered parenterally by intramuscular injection, intravenous injection or subcutaneous injection, suitable solvents include, for example, distilled water for injection, lidocaine hydrochloride aqueous solution (for intramuscular injection), physiological saline, An aqueous solution of glucose, ethanol, a liquid for intravenous injection (for example, an aqueous solution of citric acid and sodium citrate), an electrolyte solution (for intravenous drip and intravenous injection), or the like, or a mixed solution thereof can be used.

These injections are pre-dissolved ones,
It may be in the form of a powder or a powder to which appropriate additives are added and dissolved at the time of use. These injections are usually 0.1
It contains 10% by weight, preferably 1 to 5% by weight of active ingredient.

The liquid preparation such as suspension or syrup for oral administration contains 0.5 to 10% by weight of the active ingredient.

It should be noted that the actual preferred dosage of the compounds of the present invention will vary depending on the type of compound used, the type of composition formulated, the frequency of application and the particular site to be treated, the host and the tumor. Is. For example, the dose per adult per day is 10 for oral administration.
˜500 mg, preferably 10 to 100 mg per day for parenteral administration, preferably for intravenous injection. The frequency of administration is 1 to 5 times, though it varies depending on the administration method and symptoms.

Next, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the examples, and based on the properties of BE-22179 revealed by the present invention, as well as the modification means of the examples,
It includes all methods for producing, concentrating, extracting and purifying BE-22179 using known means.

[0038]

【Example】

Example 1 Actinomycete A22179 strain cultivated on a slope agar medium, glucose 0.1%, dextrin 2.0%, corn gluten meal 1.0%, fish meal 0.5%, yeast extract 0.1%,
Sodium chloride 0.1%, magnesium sulfate 0.05
%, Calcium chloride 0.05%, ferrous sulfate 0.000
2%, cupric chloride 0.00004%, manganese chloride 0.
00004%, cobalt chloride 0.00004%, zinc sulfate 0.00008%, sodium borate 0.00008
%, Ammonium molybdate 0.00024% and 3
-Inoculate 4 Erlenmeyer flasks of 500 ml volume containing 100 ml of medium (pH 6.7) consisting of 0.5% of (N-morpholino) propanesulfonic acid, and incubate at 28 ° C for 72 hours at a rotary shaker (180 rpm) Cultured above. 2 ml each of this culture solution was inoculated into 100 500 ml Erlenmeyer flasks containing 100 ml of the above-mentioned medium, and 96 hours at 28 ° C.
The culture was carried out on a rotary shaker (180 rpm).

The obtained culture solution (about 10 L) was filtered by a filtration method to obtain cells. After adding 2 L of methanol to the bacterial cells and stirring at room temperature for 1 hour, a methanol extract was obtained by a filtration method. This operation was repeated 3 times and about 6 L of the methanol extract was concentrated under reduced pressure to remove methanol. Salt solution was added to the obtained concentrated liquid (about 1 L) to make 2% to make 2 L, and then extracted twice with 2 L of ethyl acetate. The ethyl acetate layers were combined and concentrated to dryness, then dissolved in 2 L of 70% ethanol water, and impurities were extracted and removed using 2 L of hexane. The hydrous ethanol layer was concentrated to remove ethanol, and the concentrated solution (about 200 ml) was extracted three times with 200 ml of chloroform. The chloroform layers were combined and concentrated to dryness, and the resulting crude material was subjected to silica gel column chromatography (inner diameter: 4.5 cm, length: 4 cm).
0 cm), chloroform / ethyl acetate / acetic acid (1
It was eluted at 00: 200: 3). The fraction containing BE-22179 was concentrated to dryness under reduced pressure, dissolved in a small amount of methanol, and concentrated to produce a precipitate of BE-22179. Therefore, BE-22179126 was collected by filtration.
mg was obtained.

[0040]

INDUSTRIAL APPLICABILITY BE-22179 described in the present invention is an enzyme essential for cell growth, topoisomerase and RNA.
Since it strongly inhibits the synthesis and strongly suppresses the growth of cancer cells, it is useful as a therapeutic agent for cancer in the field of medicine.

[Brief description of drawings]

FIG. 1 IR of BE-22179 with potassium bromide tablets
It is a spectrum figure.

FIG. 2: 30 of BE-22179 in deuterated chloroform.
It is a 0 MHz proton-NMR spectrum figure.

Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI Technical display location C12R 1: 465) 7804-4B (C12P 17/16 C12R 1: 465) 7804-4B (72) Inventor Hajime Suzuki 2-9-3 Shimo-Meguro, Meguro-ku, Tokyo Inside the Yuri Research Co., Ltd. (72) Inventor Akira Okura 2-9-3 Shimo-Meguro, Meguro-ku, Tokyo Inside the Yuri Pharmaceutical Co., Ltd. (72) Inventor Hiroyuki Suda 2-9-3 Shimomeguro, Meguro-ku, Tokyo Manyu Pharmaceutical Co., Ltd.

Claims (6)

[Claims] 1. The formula: A compound represented by or a pharmaceutically acceptable salt thereof. 2. An antitumor agent comprising the compound according to claim 1 as an active ingredient. 3. A method for producing a compound according to claim 1, which comprises culturing a microorganism producing the compound according to claim 1 or a mutant strain thereof. 4. Streptomyces sp.
tomyces sp. ) The method according to claim 3, wherein the A22179 strain or a mutant strain thereof is cultured. 5. A microorganism belonging to the genus Streptomyces or a mutant thereof having the ability to produce the compound according to claim 1. 6. Streptomyces sp.
tomyces sp. The microorganism according to claim 5, which is the A22179 strain or a mutant strain thereof.