JPH059189A - Carcinostatic substance, cytoblastine, and its production - Google Patents

Abstract

PURPOSE:To provide the subject new compound having antitumor activity, carcinostatic activity and low toxicity. CONSTITUTION:The compound of formula and its salt. The compound of formula can be produced e.g. by culturing Streptoverticillium eurocidicum MI43-37F11 strain (FERM P-10513) preferably in a liquid medium added with a nitrogen source such as peptone and meat extract, a carbon source such as glycerol and sucrose, inorganic salt such as sodium chloride and phosphate and, as necessary, a slight amount of metal salt at 27-32 deg.C under shaking, filtering the cultured product and extracting the filtrate with a water-immiscible organic solvent such as ethyl acetate.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel carcinogen, cytoblastin and its salt, and a method for producing cytoblastin.

[0002]

2. Description of the Related Art As an antibiotic having an indolactam skeleton, Teleocidin B is known as "Bull. Agr. Chem. Soc. Jap."
an ” 24 , 647 and ibid. 24 , 652 (196
2).

Cytoblastin according to the present invention is chemically treated with these antibiotics of the teleocidin group in that a substituent containing an indole ring and a 1,2-propanediol group is bonded to the 7-position of the indolactam skeleton. Structurally different,
The biological activity is also different.

[0004]

The conventionally known carcinostatic or antitumor substances are generally highly toxic, which greatly limits their clinical use. Therefore, there is a demand for new anticancer compounds with low toxicity.

[0005]

Therefore, the inventors of the present invention have conducted earnest research to find a new anticancer compound having low toxicity. Then, the present inventors acknowledged that a substance having an antitumor activity was produced and accumulated in the culture solution of a strain belonging to the genus Streptoverticillium, and isolated the substance to obtain its chemical structure. It was found that it can be represented by the formula (I) described later, that this substance has antitumor activity, and that it is a novel compound. The present invention has been completed based on the results of these studies.

That is, the first aspect of the present invention is the formula (I): The present invention relates to the anticancer substance cytoblastin represented by or its salt. Further, the second invention is characterized in that a cytoblastin-producing bacterium belonging to the genus Streptoverticillium is cultured, and the cytoblastin of the formula (I) or a salt thereof is collected from the culture. The present invention relates to a method for producing cytoblastin or a salt thereof.

The cytoblastin according to the present invention is a basic substance, and its salts include acid addition salts. Examples of acid addition salts of cytoblastine include pharmaceutically acceptable inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, etc., or pharmaceutically acceptable organic acids such as acetic acid, propionic acid,
There are addition salts with maleic acid and methanesulfonic acid.

The cytoblastin according to the present invention has the following principle.
It has a chemical property. (1) Shape: White powder (2) Elemental analysis (measured value): carbon 67.32%, hydrogen
7.57%, oxygen 13.30%, nitrogen 10.33% (3) Mass spectrometry (FAB-MS): m / z 491 (M + H)⁺                 m / z 489 (M + H)⁻   High resolution EI-MS: m / z 490.2580 (M
⁺) (Theoretical value m / z 490.2580, C₂₈H₃₄N_FourO
_Four) (4) Molecular formula: C₂₈H₃₄N_FourO_Four (5) Melting point: 177-182 ° C (decomposition) (6) Specific rotation: [α]_D ²¹= -110 ° (c= 0.
493, methanol) (7) Ultraviolet absorption spectrum: shown in FIG. 1 of the accompanying drawings.
The absorption maximum (nm) of cytoblastin in methanol is
It is as follows (the log ε value is shown in parentheses). 202 (5.06), 225 (4.96), 283
(4.33), 290 (sh, 4.31), 300 (s
h, 4.21) (8) Infrared absorption spectrum (KBr tablet): Attached drawing
As shown in FIG. 3350, 1640, 1505, 146
0,1360,1100,1070,1040,740
cm^-1Has a characteristic absorption band. (9) Nuclear magnetic resonance spectrum: 400 MHz (MH
z), cytobra measured in deuterated methanol solution at room temperature
Sutin's¹The H-nuclear spectrum is shown in Figure 3 of the accompanying drawings,
Also site blastin^ThirteenC-nuclear spectrum (100M
Hz) is shown in FIG. 4 of the accompanying drawings. (10) Color reaction: Ehrlich reagent, ferric chloride-peroxide
Positive for chloric acid reagent and anisaldehyde-sulfuric acid reagent
It (11) Solubility: methanol, dimethyl sulfoxide
Easily soluble in, soluble in chloroform, ethyl acetate, water, hexa
It is insoluble in water. (12) TLC: silica gel Art. 5715 (Merck
Chromatograph as a developing solvent in thin layer chromatography
R when developed with loform-methanol (8: 2)_fvalue
Is 0.5. (13) HPLC: retention time (Rt) under the following conditions
Is 4.1 minutes. Column: 4.6 × 150 mm, ODS moving layer: 80%
Tanol, flow rate 1 ml / min

The chemical structure of cytoblastin was analyzed as follows. That is, the ultraviolet absorption spectrum of cytoblastin has absorptions of 225 and 300 nm, which is similar to that of the teleocidin group compounds. In addition to this absorption, absorptions at 283 and 290 nm were observed, suggesting the existence of an indole ring in the cytoblastin molecule. The molecular formula of cytoblastin and its nuclear magnetic resonance spectrum were analyzed by indolactam V
[Indolactam V, "Tetrahed
oron " 42 , pp. 5905-5942, (198
It was shown that another indole ring and a propanediol moiety are bound to the skeleton of [6)]. Especially ¹ H
Heteronuclear correlation (HMBC) spectra measuring distant spin coupling between nuclei and ¹³ C nuclei were measured for the triacetylated product of cytoblastin and the spectra were analyzed in detail. As a result, as represented by the above formula (I), cytoblastine was found to have a 2,3-dihydroxy-1- (3-indolyl) propyl group [2,3-dihydroxy-1--] at the 7-position of the indolactam V skeleton. (3-ind
[Olyl) propyl group] has been attached.

Cytoblastin has the following biological properties. Cytoblastin showed no antibacterial activity against Gram-positive and negative bacteria and fungi at a concentration of 100 μg / ml.

As shown in Table 1, the cytoblastin growth inhibitory activity (IC ₅₀ value) against various animal cancer cells and human cancer cells is the IC ₅₀ value of cytoblastin except human lung cancer cell SC-6. > 100 μg / ml, thus cytoblastin has very low cytotoxicity.

[0012] The acute toxicity value of cytoblastin to mice (ICR, 6 weeks old, female) was 100 mg / kg or more after intraperitoneal administration.

The effect of cytoblastin on the proliferation of lymphocytes was examined by the following method. That is, concanavalin A was added at an amount of 5 μg / ml to nylon wool-passed Fischer rat spleen cells and cultured at 37 ° C. for 4 hours. 20 mg / ml of methyl mannoside is added and washed twice with the medium. This cell is 2 × 10 ⁵ cells / w
Cell plate was added to the microplate so that the amount of cells was 10 μl, and 10 μl of cytoblastin was added as a sample at 37 °
Cultured for 72 hours. After adding ³ H-thymidine and culturing for 18 hours, ³ H-thymidine incorporation into the cultured cells was measured by a scintillation counter. 3H taken into sites neublastin addition group when the count of ³ H- thymidine incorporated into a control group of lymphocytes and 100
-The thymidine count rate (T / C,%) is shown in Table 2.

[0014]

A therapeutic experiment of cytoblastine on a mouse bearing an Ehrlich solid tumor was carried out by the following method. That is, ICR mice (female, 6 weeks old) were transplanted with Ehrlich ascites tumor cells subcutaneously in the groin at a dose of 2 × 10 ⁶ cells / mouse, and cytoblastin was injected from 7 days to 14 days after transplantation. 8,31.25,125 or 50
Each dose of 0 mg / kg was intraperitoneally administered every day. 15 days after the transplantation of the cancer cells, the solid cancer was taken out, and the rate of decrease in the weight of the solid cancer in the cytoblastine-administered group when the weight of the solid cancer in the control group to which saline was administered was set to 100 was inhibited. The rate is shown in Table 3.

[0016]

The cytoblastin according to the present invention is obtained by culturing a cytoblastin-producing bacterium belonging to the genus Streptoverticillium, accumulating cytoblastin and / or its salt in the culture, and then collecting it. It can be manufactured by An example of a cytoblastin (or salt thereof) belonging to the genus Streptoverticillium that can be used in the method of the present invention is an actinomycete strain collected in Yokohama City, Kanagawa Prefecture, which is MI43-37F11 strain. There are strains with strain numbers. The mycological characteristics of this strain are as described below. Mycological properties of MI43-37F11 strain

1. The morphology MI43-37F11 strain extends aerial mycelia having limbus from the aerial hyphae branched under the microscope. At the tip of the aerial mycelium, a chain of 10 or more spores was found, and helix formation was not found. The size of spores is 0.7 to 0.8 × 1.
The surface of spores is smooth, showing about 1 to 1.2 microns.

2. Growth state in various media (1) Sucrose / nitrate agar medium (27 ° C. culture) Growth is colorless, aerial hyphae are white and slightly settled, and soluble dyes are not observed. (2) Glucose / asparagine agar medium (27 ° C. culture) On the development of colorless to light yellow to light yellow tea, yellowish white aerial hyphae are slightly grown. In addition, white cotton-like epiphytes were partially observed and no soluble pigment was observed. (3) Glycerin / asparagine agar medium (ISP-medium 5, 27 ° C. culture) Growth is colorless to light yellow brown, and aerial hyphae are white. Soluble dyes have a slight brown tint. (4) Starch / inorganic salt agar medium (ISP-medium 4, 2
Cultivation at 7 ° C) On the development of colorless to light yellow tea, aerial hyphae of yellowish white to bright olive ash are settled and no soluble pigment is observed. (5) Tyrosine agar medium (ISP-medium 7, 27 ° C. culture) Growth is light yellow tea to light gray tea, aerial mycelium is white to brown, and soluble pigment is not observed. (6) Nutrient agar medium (27 ° C. culture) On the development of colorless to light yellow, white aerial hyphae are slightly settled, and a blackish soluble pigment is slightly observed. (7) Yeast-malt agar medium (ISP-medium 2, 27
Culturing at ℃) On the wrinkled growth of colorless to light yellow tea, aerial hyphae of white to brownish white are settled and no soluble pigment is observed. (8) Oatmeal agar medium (ISP-medium 3, 27 ° C)
(Culture) On the development of colorless to pale yellow, white to yellowish-white aerial mycelium grows faintly and no soluble pigment is observed. (9) Glycerin / nitrate agar medium (27 ° C. culture) Growth is colorless to pale yellow, and aerial mycelium is white to yellowish white, faintly growing, and no soluble pigment is observed. (10) Starch agar medium (27 ° C. culture) On the development of colorless to light yellow to light yellow tea, white to yellowish white aerial mycelia grow and no soluble pigment is observed. (11) Lime malate agar medium (27 ° C. culture) The growth is colorless and the growth is extremely poor, white aerial hyphae are slightly settled, and soluble pigments are not observed. (12) Cellulose / filter paper piece-added synthetic solution (27 ° C. culture) No aerial hyphae are settled on colorless growth, and soluble dyes are not observed. (13) Gelatin puncture culture In a 15% simple gelatin medium (cultured at 20 ° C.), the growth was colorless, aerial hyphae did not settle, and a soluble yellow tea pigment was observed. Also, glucose-peptone-gelatin medium (27
In ℃ culture), the growth is colorless, the aerial mycelium does not settle, and the soluble pigment has a slight brownish tinge. (14) Skim milk (37 ° C. culture) In the development of colorless to light tea, aerial mycelia do not settle, and the soluble pigment has a slight light yellow to brown taste.

3. Physiological properties (1) Growth temperature range Starch / inorganic salt agar medium (ISP-medium 4, BAKU
TO-INORGANIC-SALTS STARCH
-AGAR), 20 ° C, 24 ° C, 27 ° C, 30 ° C,
As a result of the test at each temperature of 37 ° C and 50 ° C, it grew at any temperature except 50 ° C, but the optimum growth temperature was 27 ° C.
It seems to be near. (2) Liquefaction of gelatin (15% simple gelatin medium, 20
C culture: glucose / peptone / gelatin medium, 27 ° C
Cultivation) In both simple gelatin medium and glucose / peptone / gelatin medium, liquefaction starts from the 3rd day and the action is moderate to strong. (3) Hydrolysis of starch (starch / inorganic salt agar medium and starch / agar medium, both at 27 ° C.) Both starch / inorganic salt agar medium and starch agar medium were found to be hydrolyzable from about 3 days after culturing, The action is stronger. (4) Coagulation and peptone conversion of skim milk (Skim milk, 37 ° C)
Cultivation) Coagulation begins on the second day after culturing and completes on the third day, and peptone formation begins immediately. The progress of peptone formation is extremely slow and is not completed even after 3 weeks of culture. (5) Generation of dyes for melamine (tryptone, yeast,
Broth and ISP-medium 7 are both cultured at 27 ° C.) It seems to be positive on peptone yeast iron agar medium, negative on tyrosine agar medium, and positive on tryptone yeast broth medium. (6) Utilization of carbon source (Pridham-Gotrieve agar medium, ISP-medium 9, 27 ° C. culture) Glucose, fructose and inositol are used to grow, L-arabinose, D-xylose, sucrose, rhamnose and raffinose. , D-mannitol and lactose are not used. (7) Dissolution of lime malate (lime malate agar medium, 2
7 ° C culture) Negative. (8) Reduction reaction of nitrate (peptone water containing 0.1% potassium nitrate, ISP-medium 8, 27 ° C. culture) Somewhat weak but positive. (9) Decomposition of cellulose (synthetic medium with filter paper pieces added, 27 ° C)
Culture) Negative.

Summarizing the above properties, MI43-37
The aerial mycelium of strain F11 has limbus branches, no helix formation is observed, and the surface of spores is smooth. On various mediums, aerial hyphae of white to brownish-white or yellowish-white and sometimes bright olive ash grow on colorless to light yellow-brown growth. Soluble dye is not observed or is brownish. The formation of melamine-like pigment is negative on tyrosine agar and positive on tryptone yeast broth and peptone yeast iron agar. Proteolytic power is moderate to strong, and starch is also highly hydrolyzable. In addition, 2, contained in the cell wall
6-diaminopimelic acid was the LL-form.

From these properties, MI43-37F11
The strain is Streptoverticillium.
It was considered to belong to the genus Titilium ). Searching for more closely related known strains, Streptoverticillium eurosidicum ( Streptovertic)
illium eurocidicum ) and Streptoverticillium arbileticuli ( Strept
overtillium albireticul
i ) are mentioned. Therefore, the MI43-37F11 strain and these strains are compared and examined in the field, and the results are summarized in Table 4.

[0023]

As is clear from Table 4, MI43-37
The F11 strain and Streptoverticillium eurosidicum and Streptoverticillium albileticuli all have very similar properties.

When the species closely related to the MI43-37F11 strain is selected, the aerial hyphae exhibit bright olive ash, the melamine-like pigment production in ISP-medium 7 is probably negative, and fructose is probably used. From the above, Streptoverticillium eurocydicum is mentioned.

Therefore, the MI43-37F11 strain was treated with Streptoverticillium eurosidicum ( Strepto).
verticillium eurocidicu
m ) MI43-37F11 strain was identified. In addition, MI4
Application for deposit of 3-37F11 strain to Institute of Microbial Technology, National Institute of Advanced Industrial Science and Technology, January 27, 1989
Commissioned as No. 13, and transferred under the terms of the Budapest Treaty on February 28, 1990.
It has been deposited as No. 83.

Next, the production of cytoblastin and its salt will be described. A culture containing cytoblastin (and its salt) can be obtained by inoculating a nutrient-containing medium with a cytoblastin (and its salt) -producing strain belonging to the genus Streptoverticillium and aerobically growing. As a nutrient source, one that can be used as a nutrient source for actinomycetes is used. As a nutritional source, for example, commercially available peptone, meat extract, corn staple liquor,
Cottonseed flour, peanut flour, soybean flour, yeast extract, NZ-amine, hydrolyzate of casein, sodium nitrate, ammonium nitrate, nitrogen sources such as ammonium sulfate, and commercially available glycerin, sucrose, starch, glucose, galactose, mannose, Carbohydrates such as molasses or carbon sources such as fat can be used. Further, inorganic salts such as sodium chloride, phosphate, calcium carbonate and magnesium sulfate can be added and used. In addition, a trace amount of metal salt can be added if necessary. Any of these may be used as long as they are utilized by cytoblastin-producing bacteria and are useful for the production of cytoblastin (and salts thereof), and all known actinomycete culture materials can be used.

Liquid culture is preferred for large-scale production of cytoblastin (and its salt), and the culture temperature can be used within the range where the producing bacterium grows to produce cytoblastin (and its salt), usually 27 to 32 ° C. Is. Culturing can be appropriately selected and performed according to the properties of the cytoblastin (and its salt) -producing bacterium using the conditions described above. Cytoblastin (and its salts) is present in culture. The culture can be filtered and the resulting culture filtrate can be extracted with a water-immiscible organic solvent such as ethyl acetate. In addition to the above-mentioned extraction method, a known method used for collecting fat-soluble substances,
For example, it can be collected purely by appropriately combining adsorption chromatography, gel filtration chromatography, high performance liquid chromatography and the like.

[0030]

EXAMPLES The present invention will now be described with reference to examples, but the present invention is not limited thereto.

Example 1 From Streptoverticillium eurosidicum MI43-37F11 strain (Microtechnological Research Institute No. 10513) cultivated on an agar slant medium, galactose 2.0%, dextrin 2.0%, soypeptone (Difco Bact Soyton) 1.0%, corn steep liquor (Nippon Shokuhin Kako Co., Ltd.) 0.5%, ammonium sulfate 0.2%, calcium carbonate 0.2%, defoaming silicone oil (Shin-Etsu Chemical Co., Ltd. Silicon Oil KM70) 0.003
% Of the liquid medium (pH 7.4) was dispensed into two Erlenmeyer flasks with waffles, each containing 110 ml, and one platinum loop was inoculated into each of the flasks, followed by shaking culture at 30 ° C. for 2 days. Glycerin 2.0%, essan meat (Ajinomoto Co., Ltd.) 1.5%, phosphoric acid 1 potassium 0.1%, cobalt chloride hexahydrate 0.0005%, for defoaming, using it as a seed culture Silicon 0.0
2.5 ml of a liquid medium containing 03% of which was adjusted to pH 6.2 with 2 potassium phosphate was inoculated into 80 Sakaguchi flasks each having 125 ml, and cultured at 28 ° C. for 4 days with shaking.

The culture solution was filtered to separate the filtrate from the cells, and the obtained culture filtrate was extracted with an equal amount of ethyl acetate.
It was concentrated under reduced pressure to obtain 2.0 g of a brown oily substance. This was sprinkled on 10 g of silica gel and overlaid on a 60 ml silica gel column filled with chloroform for chromatography. The column was first washed with 300 ml of chloroform-methanol (10: 1) and then eluted with 300 ml of chloroform-methanol (8: 2). The active fractions were collected and concentrated under reduced pressure to obtain 0.5 g of brown oily substance.
This was dissolved in 10 ml of methanol, passed through a 10 ml carbon column filled with methanol, and washed with methanol. The passing liquids were collected and concentrated under reduced pressure to obtain 0.3 g of a brown oily substance.

This was dissolved in a small amount of methanol and subjected to high performance liquid chromatography. Column is Senshu Scientific Co., Ltd. Senshupack ODS330IN, 20φ ×
Using 300 mm, a linear concentration gradient of 50% to 100% methanol was used for elution at a flow rate of 5 ml / min, and 5 ml fractions were fractionated. The active fractions were collected and concentrated under reduced pressure to obtain 10 mg of a yellow candy-like substance. Dissolve this in a small amount of methanol and
Column chromatography of LH-20 filled with 70 cm of methanol was performed. 3g eluted with methanol
We fractionated each. The active fractions were collected and concentrated under reduced pressure to dryness.
Cytoblastin was obtained as mg of white powder.

This white substance was subjected to silica gel thin-layer chromatography (Art. 5715, manufactured by Merck & Co., Inc., developing solvent: chloroform-methanol 8: 2) to give a single UV.
It was considered to be pure cytoblastin, as it gave an absorption spot, and color development with FeCl ₃ -HClO ₄ gave a single spot of red color.

Example 2 Single purified cytoblastin was converted into hydrochloride (or sulfate) by the following method. That is, Example 1
20 mg of cytoblastin obtained in step 2 was dissolved in 20 ml of ethyl acetate, and 0.1 N hydrochloric acid (or sulfuric acid) 20 m was added to the solution.
After adding 1 and shaking well, the aqueous layer was freeze-dried to obtain the hydrochloride salt (or sulfate salt) of cytoblastine.

[0036]

INDUSTRIAL APPLICABILITY As described above in detail, the present invention provides a novel carcinostatic antibiotic cytoblastin and a salt thereof, and a method for producing cytoblastin. The antitumor antibiotic cytoblastin according to the present invention has extremely weak toxicity and exhibits antitumor activity having a remarkable characteristic in that it is effective for solid cancer.

[0037]

[Brief description of drawings]

FIG. 1 shows an ultraviolet absorption spectrum of cytoblastin (in methanol).

FIG. 2 shows an infrared absorption spectrum of cytoblastin (in KBr tablet).

FIG. 3 shows a ¹ H-NMR spectrum of cytoblastin (400 MHz in deuterated methanol).

FIG. 4 shows a ¹³ C-NMR spectrum of cytoblastin (100 MHz in deuterated methanol).

[Procedure amendment]

[Submission date] October 3, 1991

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0008

[Correction method] Change

[Correction content]

The cytoblastin according to the present invention has the following physicochemical properties.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0011

[Correction method] Change

[Correction content]

As shown in Table 1, the cytoblastin growth inhibitory activity (IC ₅₀ value) against various animal cancer cells and human cancer cells is the IC ₅₀ value of cytoblastin except human lung cancer cells LX-1. > 100 μg / ml, thus cytoblastin has very low cytotoxicity.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0012

[Correction method] Change

[Correction content]

[0012]

[Procedure amendment 4]

[Document name to be amended] Statement

[Name of item to be corrected] 0015

[Correction method] Change

[Correction content]

A therapeutic experiment of cytoblastine on a mouse bearing an Ehrlich solid tumor was carried out by the following method. That is, ICR mice (female, 6 weeks old) were subcutaneously transplanted with 2 × 10 ⁶ Ehrlich's ascites tumor cells subcutaneously in the groin, and 7 days to 14 days after transplantation with 7 cytoblastin cells. .8, 31.25, 125 or 5
Each dose of 00 μg / mouse was intraperitoneally administered every day.
15 days after the transplantation of the cancer cells, the solid cancer was taken out, and the rate of decrease in the weight of the solid cancer in the cytoblastine-administered group when the weight of the solid cancer in the control group to which saline was administered was set to 100 was inhibited. The rate is shown in Table 3.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0016

[Correction method] Change

[Correction content]

[0016]

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Sawa Riki             4-6-7 Ayanishi, Ayase City, Kanagawa Prefecture (72) Inventor Masa Hamada             26 Hidewa Residue, Naito-cho, Shinjuku-ku, Tokyo             405 (72) Inventor Kenji Maeda             2-46-11, Gobongi, Meguro-ku, Tokyo (72) Inventor Tomio Takeuchi             5-11, Higashigotanda, Shinagawa-ku, Tokyo             You Fujimansion 701

Claims (2)

[Claims] 1. The following formula (I): An anticancer substance, cytoblastin, which is a compound represented by: and a salt thereof. 2. A cytoblastin or a salt thereof, which comprises culturing a cytoblastin-producing bacterium belonging to the genus Streptoverticillium and collecting the cytoblastin or a salt thereof according to claim 1 from the culture. Manufacturing method.