JPH10168054A - Anti-tumor substance, be-41926 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new substance with excellent anti-tumor action, useful in the medical and pharmaceutical fields by innoculating a BE-41926 producing bacterium strain in a medium containing nutritional sources and culturing it aerobically. SOLUTION: This substance, BE-41926, of the formula, is obtained by culturing microorganisms belonging to the genus Streptomyces sp. A41926 (FERM P-15780). As for nutritional sources, glucose or sugar is used as a carbon source and soybean meal or fish meal is used as a nitrogen source. The bacteria are cultured according to the same method as general methods for producing metabolites from bacteria at a culturing temperature of suitably 12-39 deg.C, preferably, 21-28 deg.C at a pH range of 4-8 for 48-240hrs, preferably, 72-192hrs. The objective BE-41926 is collected from the cultured product according to conventional separating means.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention is useful in the field of medicine, and more specifically, a novel compound having an antitumor effect by inhibiting the growth of tumor cells, a method for producing the compound, its use, and production of the compound. Pertaining to microorganisms that do.

[0002]

2. Description of the Related Art In the field of cancer chemotherapy, many compounds have already been put into practical use as pharmaceuticals. However, its effect on various types of tumors is not always sufficient, and its clinical applicability is complicated as the phenomenon of resistance of tumor cells to these drugs is clinically revealed [47] Article of the General Meeting of the Japanese Cancer Society, 12
Pp. 15-15 (1988) etc.]. Under such circumstances, the development of new antitumor substances is always required in the field of cancer treatment.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel antitumor substance which can satisfy the above demand. That is, an object of the present invention is to provide a compound that exhibits an antitumor effect even on various tumors in which existing antitumor substances cannot sufficiently exert an effect.

[0004]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors conducted a wide screening of secondary metabolites of microorganisms for substances having antitumor activity, and as a result, they were represented by the following structural formula [I]. The present invention has been completed by finding out that the compounds exhibit excellent antitumor activity. That is, the present invention provides a novel structural formula [I]

[0005]

Embedded image And a method for producing and using the same, and a microorganism belonging to the genus Streptomyces having the ability to produce the compound of structural formula [I]. The physicochemical properties of the novel antitumor substance BE-41926 according to the present invention are shown below. The meanings of the abbreviations in NMR measurement are shown below. s: singlet d: doublet t: triplet q: quartet m: multiplet br: broad J: coupling constant Hz: physicochemical properties of Hertz BE-41926 ; dark brown amorphous solid or crystal molecular formula; C ₄₀ H ₃₀ O ₉ S mass spectrometry; [high resolution FAB-MS] (M + H) ⁺ : Found 687.1665 Calculated 687.1689 Ultraviolet absorption spectrum; λ _max (0.2% DMSO / M
OH, nm (ε)): 230 (105, 600), 2
45sh (80,900), 286sh (52,80)
0), 360sh (18,500) infrared absorption spectrum; (KBr, cm ^-1 ): 344
4,1655,1583,1464,1269,111
7,1074,1030,606 ¹ H-NMR spectrum (500 MHz, DMSO-d
_6, δ ppm): 10.5 (1H, s), 7.82 (1
H, t, J = 7.6 Hz), 7.60 (1H, s),
7.56 (1H, d, J = 7.6 Hz), 7.52 (1
H, d, J = 7.6 Hz), 7.50 (1H, t, J =
7.6 Hz), 7.44 (1H, s), 7.41 (1
H, d, J = 7.6 Hz), 7.27 (1H, d, J =
7.6 Hz), 6.85 (1H, brs), 6.80
(1H, brs), 6.31 (1H, d, J = 6.7H)
z), 5.79 (1H, d, J = 6.7 Hz), 4.9
4 (1H, s), 3.94 (3H, s), 3.78 (3
H, s), 2.90 (1H, d, J = 16.2 Hz),
2.86 (2H, brs), 2.60 (1H, d, J =
16.2 Hz), 2.28 (3H, s), 1.19 (3
H, s) ¹³ C-NMR spectrum (125 MHz, DMSO-d
_6, δ ppm): 196.8 (s), 186.5 (s),
186.4 (s), 181.9 (s), 158.6
(S), 156.5 (s), 154.4 (s), 14
4.1 (s), 142.6 (s), 141.1 (s),
139.2 (s), 139.1 (d), 137.1
(S), 136.6 (s), 135.9 (s), 13
5.8 (s), 135.4 (s), 135.4 (d),
135.3 (s), 134.7 (s), 132.7
(D), 131.4 (s), 130.4 (s), 12
9.6 (d), 120.3 (s), 117.8 (d),
117.6 (d), 117.6 (d), 117.5
(D), 116.5 (s), 115.2 (d), 11
4.9 (d), 69.5 (s), 58.8 (d), 5
6.3 (q), 55.8 (q), 53.2 (t), 4
2.6 (t), 28.6 (q), 21.1 (q) Solubility; easily soluble in organic solvents such as chloroform and hardly soluble in water. Acid, neutral, basic substance; weakly acidic substance color reaction; sulfuric acid reaction positive Rf value: 0.64 [Merck Kieselgel 60F
₂₅₄ used, developing solvent: chloroform-methanol (1
0: 1)] Biological activity of BE-41926 (anti-tumor effect) A test was conducted in vitro to determine the anti-proliferative effect of the anti-tumor substance BE-41926 on experimental mouse tumor cells. In an antitumor test for mouse leukemia cell P388, BE-41926 was dissolved in dimethylsulfoxide, and then serially diluted with dimethylsulfoxide. The test solution was used.
50 μl of a cell culture medium containing 1 × 10 ³ tumor cells (RPMI1640 medium containing 10% fetal bovine serum, containing 20 mM 2-mercaptoethanol) was dispensed into a 96-well microplate, and was placed at 37 ° C. for 24 hours at 5% CO 2. After culturing under ₂ , the above test solution (50 µl) was added,
After culturing for 5 hours under 5% CO ₂ , the cells were compared with the control group by MTT measurement. In an antitumor test for mouse colon cancer cell colon26, BE-41926 was dissolved in dimethylsulfoxide, then serially diluted with dimethylsulfoxide, and added to RPMI1640 medium containing 10% fetal calf serum to prepare a test solution. Cell culture medium containing 1 × 10 ³ tumor cells (RPMI 1640 medium containing 10% fetal bovine serum) 1
After dispensing 00 μl into a 96-well microplate and culturing at 37 ° C. for 24 hours under 5% CO ₂ , the above test solution 10
After adding 0 μl and culturing at 37 ° C. for 72 hours under 5% CO ₂ , the cells were fixed with 50% trichloroacetic acid, stained with 0.4% sulforhodamine B, and the dye was extracted from the cells using a 10 mM Tris solution. . 550 with 450 nm as control wavelength
The absorbance in nm was measured and compared with the control group. As a result, BE-41926 showed a strong growth inhibitory activity on both tumor cells, and the 50% growth inhibitory concentration (IC ₅₀ ) was the first.
It was as shown in the table. In addition, the antitumor activity of BE-41926 on human tumor cells was tested in vitro. Cells are human colon cancer cell DLD-1, human lung cancer cell PC-1
3 and human gastric cancer cells MKN-45, and the cell culture medium was RP containing 10% fetal calf serum for all tumor cells.
Using the MI1640 medium, the above mouse colon cancer cell co
It measured using the same method as lon26. as a result,
BE-41926 also showed strong growth inhibitory activity against human tumor cells, and its 50% growth inhibitory concentration (IC ₅₀ ) is as shown in Table 1.

[0006]

[Table 1] As described above, BE-41926 has a significant growth inhibitory effect on mouse and human tumor cells. Therefore, the present invention is useful as an antitumor agent for mammals including humans. Next, a method for producing BE-41926 will be described. The microorganism or a mutant thereof used for producing the antitumor substance BE-41926 of the present invention is an antitumor substance B
As long as it produces E-41926, any microorganism may be used, and examples thereof include microorganisms having the following mycological properties. 1. Form A41926 strain forms an aerial mycelium with a base mycelium that elongates and diverges well, and no division of a vegetative branch and a mycelium is observed. It forms long chains of spores (30-50) on aerial hyphae, which are straight and wavy. The surface of the spores is smooth and cylindrical with a size of about 1.0 to 0.8 × 1.5 to 1.2 μm, and no special organs such as spores, flagella spores and sclerotia are observed. 2. Cultural properties on various agar plate media (28 ° C, 14
Days culture)

[0007]

[Table 2] 3. Growth temperature (yeast / malt agar medium, cultured for 14 days) 4 ° C; non-growth 9 ° C; little growth, no aerial mycelium formation 12 ° C; good growth, no aerial mycelium formation 16 ° C; very good growth, air Very low mycelium formation 18 ° C; very good growth, good aerial mycelium formation 21 ° C; very good growth and aerial mycelium formation 25 ° C; very good growth and aerial mycelium formation 28 ° C; very good growth and aerial mycelium formation 30 ° C Very good growth, little formation of aerial hyphae 33 ° C; very good growth, little formation of aerial hyphae 36 ° C; very good growth, no formation of aerial hyphae 39 ° C; good growth, no formation of aerial hyphae 42 ° C; growth No 4 Physiological properties (1) Liquefaction of gelatin positive (glucose / peptone / gelatin medium) (2) Starch hydrolysis positive (starch / inorganic salt agar medium) (3) Coagulation of skim milk powder positive (Skim milk medium) (4) 4. Peptone conversion of skim milk powder positive (Skim milk medium) (5) Melanin-like pigment formation negative (6) Salt tolerance Growing at a salt content of 4% or less (Yeast / malt agar medium) Utilization ability of carbon source Prideham-Godribe agar was used as a basal medium, and the following various sugars were added thereto and cultured at 28 ° C for 14 days.

[0008]

[Table 3] 6. Cell wall composition LL diaminopimelic acid and glycine were detected. Based on the above mycological properties, the A41926 strain is considered to belong to the genus Streptomyces. Therefore, A4
1926 strains Streptomyces sp. A4192
6 (Streptomyces sp. A4192)
6). This strain has been deposited with the Research Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry, and the deposit number is FERM P-15780. The mutant strain of the microorganism producing the antitumor substance BE-41926 used in the present invention may be, for example, irradiated with X-rays or ultraviolet rays,
For example, treatment with a mutagen such as nitrogen mustard, azaserine, nitrite, 2-aminopurine or N-methyl-N'-nitro-N-nitrosoguanidine (NTG), phage contact, transformation, transduction or conjugation, etc. BE-
It is a microorganism obtained by mutating 41926 producing bacteria. In producing BE-41926 of the present invention, BE-4192
A culture containing BE-41926 can be obtained by inoculating the production strain No. 6 into a nutrient source-containing medium and growing it aerobically. As a nutrient source, a known nutrient source for actinomycetes can be used. For example, as a carbon source, commercially available glucose, maltose, starch, sucrose, molasses, dextrin, or the like is used alone or as a mixture. Nitrogen sources include commercially available soy flour, corn steep liquor, gluten meal, meat extract, yeast extract, dried yeast, cottonseed powder, peptone, wheat germ, fish meal, meat meal, defatted rice bran, defatted meat-and-bone meal , Inorganic ammonium salts or sodium nitrate are used alone or as a mixture. As inorganic salts, commercially available calcium carbonate,
Sodium chloride, potassium chloride, magnesium sulfate, sodium bromide, sodium borate or various phosphates can be used. In addition, if necessary, trace amounts of heavy metal salts such as iron, manganese, zinc, cobalt, and molybdic acid can be added. In the case of severe foaming, for example, vegetable oils such as soybean oil or linseed oil, higher alcohols such as octadecanol, various silicon compounds, and the like may be appropriately added as antifoaming agents. Other than these, any of those utilized by the producing bacteria and useful for the production of BE-41926, such as 3- (N-morpholino) propanesulfonic acid or sodium borate, can be used. The culturing method may be the same as the method for producing a general microbial metabolite, and may be a solid culture or a liquid culture. In the case of liquid culture, any of static culture, stirring culture, shaking culture, and aeration culture may be performed, but shaking culture or deep aeration stirring culture is particularly desirable. The culture temperature is suitably from 12 to 39 ° C, preferably from 21 to 28 ° C. The preferred pH of the medium is 4-8.
The culture time is 48 hours to 240 hours, preferably 72 hours to 192 hours. In order to collect the target BE-41926 from the culture, a separation means usually used for collecting from a metabolite produced by a microorganism can be appropriately used. Since BE-41926 is present in the culture filtrate and the cells, it can be separated from the culture solution or the cells by a usual separation means such as a solvent extraction method, an ion exchange resin method or an adsorption or partition chromatography method and a gel filtration method. Alternatively, it can be purified by performing the combination. Preferred examples of separation and purification include the following methods. First, the culture solution is filtered, and the obtained cells are extracted with an organic solvent such as methanol or acetone. The crude extract of the bacterial cells and the culture filtrate are partitioned with a water-ethyl acetate system, and the residue obtained after evaporating the ethyl acetate layer is subjected to silica gel chromatography (eluted with chloroform / methanol) to obtain a BE. -4192
6 can be obtained as a powder or a solid. The compound BE-41926 of the present invention inhibits the growth of tumor cells,
Although exerting an antitumor effect, various forms can be selected as a dosage form when the compound of the present invention is used as an antitumor agent, for example, tablets, capsules, powders, oral preparations such as granules or liquids, or A sterilized liquid parenteral preparation such as a solution or suspension can be used. Solid formulations are
Although it can be produced as it is in the form of tablets, capsules, granules or powder, it can also be produced using appropriate additives. Such additives include, for example, sugars such as lactose or glucose, for example, starches such as corn, wheat or rice, fatty acids such as stearic acid, inorganic salts such as magnesium metasilicate aluminate or anhydrous calcium phosphate, such as polyvinyl chloride Synthetic polymers such as pyrrolidone or polyalkylene glycol, for example, fatty acid salts such as calcium stearate or magnesium stearate,
Examples thereof include alcohols such as stearyl alcohol and benzyl alcohol, synthetic cellulose derivatives such as methyl cellulose, carboxymethyl cellulose, ethyl cellulose and hydroxypropyl methyl cellulose, and other commonly used additives such as water, gelatin, talc, vegetable oil, and gum arabic. These solid preparations such as tablets, capsules, granules and powders generally have a concentration of 0.1 to 100% by weight, preferably 5 to 100% by weight.
Contains 100% by weight of active ingredient. Liquid preparations use suitable additives usually used in liquid preparations such as water, alcohols or plant-derived oils such as soybean oil, peanut oil or sesame oil, and are used in the form of suspensions, syrups or injections. Manufactured as Particularly suitable solvents for parenteral administration by intramuscular injection, intravenous injection or subcutaneous injection include, for example, distilled water for injection, aqueous lidocaine hydrochloride (for intramuscular injection), physiological saline, aqueous dextrose,
Examples thereof include ethanol, an intravenous injection liquid (for example, an aqueous solution such as citric acid and sodium citrate) or an electrolyte solution (for intravenous injection and intravenous injection), or a mixed solution thereof. These injections may be dissolved in advance, or may be in the form of powder or dissolved in a suitable additive before use. These injections are usually 0.1 to 10% by weight, preferably 1 to 5% by weight.
Containing active ingredients. Liquid preparations such as suspensions or syrups for oral administration contain 0.5 to 10% by weight of active ingredient. It should be noted that the actual preferred dosage of the compounds of the present invention will vary with 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. For example, the daily adult dosage is 10-500 mg for oral administration and 10-100 mg per day for parenteral administration, preferably intravenous injection. The number of administrations varies depending on the administration method and symptoms, but is 1 to 5 times.
Hereinafter, the present invention will be described specifically with reference to Examples.
The present invention is not limited to only these examples.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION

[0010]

【Example】

Example 1 Production method of BE-41926 An actinomycete A41926 strain inoculated on a soft slope agar medium was prepared using 0.1% glucose, 2% dextrin, 0.5% fish meal, 1% corn gluten meal, 0.1% yeast extract, Magnesium sulfate 0.05%, sodium chloride 0.1%, calcium chloride 0.05%, ferrous sulfate 0.0002%, cupric chloride 0.00004%, manganese chloride 0.000%
4%, cobalt chloride 0.00004%, zinc sulfate 0.0
Inoculate three 500 ml Erlenmeyer flasks containing 110 ml of a medium (pH 7.0) consisting of 0008%, sodium borate 0.00008% and sodium molybdate 0.00024%, and shake at 48 ° C. for 48 hours at 28 ° C. (180 rotations per minute). 2 m of this culture
100 l of a 500 ml Erlenmeyer flask containing 110 ml of the above-mentioned medium was inoculated in each l and cultured at 28 ° C. for 168 hours on a rotary shaker (180 rotations per minute). The cells were separated from the thus obtained culture solution (about 10 L) by filtration, 5 L of methanol was added, and after stirring for several hours, the cells were filtered off to obtain a methanol extract. The methanol in this extract was distilled off under reduced pressure, 1.4 L of deionized water was added to the residue, and 3 L of ethyl acetate was added to perform solvent extraction. Further, 10 L of ethyl acetate was added to the culture filtrate for extraction. The ethyl acetate extract of the cells and the filtrate was concentrated under reduced pressure, and the residue was applied to a silica gel chromatography column (2.4 φ × 28 cm), and a mixed solvent of methanol / chloroform (30: 1) was used.
Eluted. The eluted active fraction was concentrated to dryness under reduced pressure,
340 mg of a crude product containing BE-41926 was obtained. Then, it was applied to a chromatography column of Sephadex LH-20 (3φ × 65 cm) and eluted with methanol. The desired fraction is concentrated under reduced pressure, and then cooled at a low temperature (4 ° C., 2
(Days) to obtain 89 mg of dark brown BE-41926. Formulation examples of the compound of the present invention are shown below, but the formulation of the compound of the present invention is not limited to this formulation example. Formulation Example 1 Is uniformly mixed to obtain a powdery or fine-grained powder having a particle size of 350 μm or less. This powder was placed in a capsule container to obtain a capsule. Formulation Example 2 Was uniformly mixed, crushed and granulated, dried, and then sieved to obtain granules having a diameter of 1410 to 177 μm. Formulation Example 3 After a granule was prepared in the same manner as in Formulation Example 2, 3 parts of calcium stearate was added to 96 parts of the granule, followed by compression molding to prepare a tablet having a diameter of 10 mm. Formulation Example 4 To 90 parts of the granules obtained by the method of Formulation Example 2, 10 parts of crystalline cellulose and 3 parts of calcium stearate were added and compression-molded to obtain a tablet having a diameter of 8 mm. Sugar-coated tablets were prepared by adding a mixed calcium carbonate suspension.

[0011]

The BE-41926 described in the present invention is:
Since it has a strong growth inhibitory effect on mouse and human tumor cells, it is useful as a therapeutic agent for cancer in the field of medicine.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Masao Nagashima 3 Okubo Tsukuba, Ibaraki Pref. Manyu Pharmaceutical Co., Ltd. Inside Tsukuba Research Laboratories (72) Katsuhisa Ojiri 3 Okubo Tsukuba, Ibaraki Pref. Inside Tsukuba Research Laboratories (72) Inventor Hiroyuki Suda 3rd Okubo Tsukuba City, Ibaraki Pref.

Claims (7)

[Claims] (1) Structural formula [I] A compound represented by the formula: 2. A method comprising culturing a microorganism capable of producing the compound of claim 1 represented by the formula [I] or a mutant thereof, and collecting the compound represented by the formula [I]. A method for producing a compound represented by the structural formula [I]. 3. The method according to claim 2, wherein the microorganism or a mutant thereof is Streptomyces sp. 4. The method according to claim 1, wherein the microorganism is Streptomyces sp. A.
41926 (Streptomyces sp. A4
1926) or a mutant thereof. 5. A Streptomyces sp. Capable of producing the compound according to claim 1.
ces sp. ) Or a mutant thereof. 6. Streptomyces sp. A4192
6 (Streptomyces sp. A4192)
6) or a mutant thereof. 7. An antitumor agent comprising the compound according to claim 1 represented by the structural formula [I] as an active ingredient.