JPH10101663A - Antitumor substance BE-51068 and method for producing the same - Google Patents

Abstract

(57) [Summary] [Problem] To create a new antitumor agent. Kind Code: A1 Structural formula [I] A compound represented by the formula:

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 screened a wide range of secondary metabolites of microorganisms for substances having antitumor activity, and as a result, they were represented by the following structural formula [1]. 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 compound of structural formula [I] has an antitumor effect
And the producing strain Streptomyces sp. A5106
8 strains, named as anti-tumor substance BE-51068
Was. Hereinafter, a novel antitumor substance BE according to the present invention will be described.
1 shows the physicochemical properties of -51068. NMR below
The meaning of the abbreviation in measurement is shown. s: singlet d: doublet t: triplet q: quartet m: multiplet br: broad J: coupling constant Hz: physicochemical properties of Hertz BE-51068 Properties; yellow oil substance molecular formula; C₂₆H₃₀O_Four Mass spectrometry; [High-resolution FAB-MS] (MH)⁺ When
Then: Actual value: 405.2079 Calculated value: 405.2065 UV absorption spectrum; λmax (MeOH, nm (ε)): 205 (3735)
0), 234 (24360), 270 (sh), 308
(38330) λmax (0.1 N HCl-MeOH = 1: 9, nm
(Ε)): 205 (37350), 234 (2436)
0), 270 (sh), 308 (38330) λmax (0.1 N NaOH-MeOH = 1: 9, n
m (ε)): 230 (sh), 260 (24360),
 310 (27610), 385 (sh) Infrared external absorption spectrum (KBr, cm^-1): 3446,
2924, 1690, 1456, 1362, 1099¹ H-NMR spectrum (500 MHz, CDCl_Three, Δ
ppm): 1.77 (3H, s), 1.92 (3H,
s), 2.03 (3H, s), 2.37 (3H, s),
3.36 (2H, d, J = 7.3 Hz), 3.80 (3
H, s), 4.29 (2H, br d, J = 4.9H)
z), 5.70 (1H, brt, J = 7.3 Hz),
6.28 (1H, dt, J = 15.6, 5.8H
z), 6.32 (2H, m), 6.46 (1H, dd,
J = 15.6, 11.6 Hz), 6.49 (1H, d,
J = 11.6 Hz), 6.72 (1H, d, J = 1)
5.6 Hz), 7.06 (1H, brd, J = 7.
6 Hz), 7.17 (1H, d, J = 7.6 Hz),
7.33 (1H, brs)¹³ C-NMR spectrum (125 MHz, CDCl_Three,
δ ppm): 9.9 (q), 10.1 (q), 12.
6 (q), 21.1 (q), 30.6 (t), 60.1
(Q), 63.8 (t), 109.2 (s), 109.
5 (s), 124.7 (d), 125.5 (d), 12
6.7 (d), 128.0 (d), 128.5 (d),
129.5 (d), 129.9 (d), 129.9
(D), 130.9 (d), 133.0 (s), 13
5.1 (s), 136.4 (s), 136.7 (s),
138.2 (d), 156.7 (s), 165.9
(S), 168.1 (s) Solubility; easily soluble in organic solvents such as dimethyl sulfoxide
It is hardly soluble in water. Acid, neutral, basic substance; neutral substance Color reaction; sulfuric acid reaction, phosphomolybdic acid reaction Positive biological activity of BE-51068 (anti-tumor effect) Anti-tumor substance BE-51068 mouse experimental tumor On cells
Test in vitro to determine the growth inhibitory effect on
Done. Antitumor activity against mouse leukemia cell P388
Test is to convert BE-51068 to dimethyl sulfoxide.
After dissolution, serially dilute with dimethyl sulfoxide.
Et al., RPMI 1640 medium containing 10% fetal bovine serum (2
Containing 0 mM 2-mercaptoethanol)
And 1x10^ThreeCell culture medium containing single tumor cells
(RPMI 1640 medium containing 10% fetal calf serum, 20
50 μl containing 9 mM 2-mercaptoethanol)
Dispense into 6-well microplates, 24 hours at 37 ° C, 5 hours
% C O_TwoAfter culturing under 50 μl of the above test solution,
5 hours C at 37 ° C for 72 hours O_TwoMTT measurement after culturing under
The method was compared with the control group. Mouse colon cancer cell col
anti-tumor test against on 26 showed BE-51068
After dissolving in dimethyl sulfoxide
After dilution with RPMI, RPMI containing 10% fetal bovine serum
A test solution was added to the 1640 medium. 1x10^ThreeIndividual tumor cells
Cell culture medium containing vesicles (RPMI containing 10% fetal bovine serum)
1640 medium) in a 96-well microplate
Dispense, 5% CO at 37 ° C for 24 hours_TwoAfter culturing under
, 100 μl of the above test solution was added to
% CO_TwoAfter culturing under, fixed with 50% trichloroacetic acid,
After staining with 0.4% sulforhodamine B, 10 mM Tris
The dye was extracted from the cells using the solution. 450 nm as control
Measure absorbance at 550 nm as wavelength
And compared. As a result, BE-51068 was found in both tumor cells.
Shows strong growth inhibitory activity against 50% growth inhibitory concentration
Was as shown in Table 1. In addition, BE-51068
The antitumor activity against tumor cells was tested in vitro.
Cells are human colon cancer cells DLD-1, human lung cancer cells
Using PC-13 and human gastric cancer cells MKN-45,
The cell culture medium is 10 fetal bovine serum for all tumor cells.
% Of the above-mentioned mouse colon using RPMI1640 medium containing
Cancer cells were measured using the same method as colon 26.
Was. As a result, BE-51068 was found to inhibit human tumor cells.
Also shows strong growth inhibitory activity, and its 50% growth inhibitory concentration
The degree was as shown in Table 1.

[0007]

[Table 1] As described above, BE-51068 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-51068 will be described. The microorganism used for the production of the antitumor substance BE-51068 of the present invention or a mutant thereof is the antitumor substance B
Any E-51068-producing microorganism may be used, and examples include microorganisms having the following mycological properties. 1. Form A51068 strain forms an aerial mycelium with a base mycelium that elongates and diverges well. A chain of spores is formed on the aerial mycelium, and its form is spiral. The surface of the spores is wrinkled, and no special organs such as spores, flagellated spores and sclerotia are not observed. It is observed that the colonies gradually become wet with the maturation of the aerial hyphae in a yeast-malt agar medium or the like. 2. Cultural properties on various agar plate media (28 ° C, 14
Days culture)

[0008]

[Table 2] 3. Growth temperature (yeast / malt agar medium, cultivated for 14 days) 10 ° C; non-growing 15 ° C; good growth, very little aerial mycelium formation 18 ° C; very good growth and aerial mycelium formation 22 ° C; very good growth and aerial mycelium formation Good 25 ° C; very good growth and aerial mycelium formation 29 ° C; very good growth and aerial mycelium formation 32 ° C; very good growth and good aerial mycelium formation 36 ° C; good growth and little aerial mycelium formation 39 ° C; good growth 3. No formation of aerial hyphae at 42 ° C .; Physiological properties (1) Liquefaction of gelatin negative (glucose / peptone / gelatin medium) (2) Starch hydrolysis positive (starch / inorganic salt agar medium) (3) Coagulation of skim milk powder negative (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) Ability to Use Carbon Source Pridham-Godlieve Agar was used as a basal medium, and the following various sugars were added thereto and cultured at 28 ° C. for 14 days. 5. D-glucose + raffinose + D-xylose + D-mannitol + L-arabinose-inositol + L-rhamnose-salicin ± D-fructose + sucrose + D-galactose + 6. Cell wall composition LL diaminopimelic acid and glycine were detected. From the above mycological properties, the A51068 strain is considered to belong to the genus Streptomyces. Therefore A51
068 strains of Streptomyces sp. A51068
(Streptomyces sp. A51068
). This strain has been deposited with the Research Institute of Biotechnology, Industrial Technology Institute of the Ministry of International Trade and Industry, and the deposit number is FERM P-15623. A mutant strain of a microorganism producing the antitumor substance BE-51068 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, nitrous acid, 2-aminopurine or N-methyl-N'-nitro-N-nitrosoguanidine (NTG), phage contact, transformation, transduction or conjugation, etc. BE by the commonly used bacterial species conversion treatment method
This is a microorganism obtained by mutating a -51068 producing bacterium. In producing BE-51068 of the present invention, BE-510
A culture containing BE-51068 is obtained by inoculating the 68 producing strains into a nutrient-containing medium and growing 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 flour, 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 the inorganic salt, commercially available calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate, sodium bromide, sodium borate or various phosphates can be used. Other, if necessary,
Minor amounts of heavy metal salts such as iron, manganese, zinc, cobalt and molybdic acid can also 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. In addition to these, BE-510 is used by the producing bacteria.
68 useful for the production of, for example, 3- (N-morpholino)
Any such as 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 18 to 32 ° C, but preferably from 18 to 29 ° C. Preferred medium p
H ranges from 4 to 8, and the culture time is from 48 hours to 240 hours, preferably from 72 hours to 168 hours. In order to collect the desired BE-51068 from the culture, a separation means usually used for collecting from the metabolite produced by the microorganism can be appropriately used. BE-51068
Since is present in the cells, it can be purified from the cells by a usual separation means, for example, a solvent extraction method, an ion-exchange resin method or an adsorption or partition chromatography method and a gel filtration method alone or in combination. Preferred examples of separation and purification include the following methods. First, the culture solution is filtered to obtain cells. The obtained cells are extracted using an organic solvent such as methanol or acetone. The obtained crude extract is partitioned with a water-ethyl acetate system, and the residue obtained after evaporating the ethyl acetate is subjected to silica gel chromatography (eluted with chloroform / hexane). The fraction containing BE-51068 is concentrated under reduced pressure, and gel filtration (elution with methanol) is performed to obtain BE-51068 as an oily substance. The compound BE-51068 of the present invention inhibits the growth of tumor cells and exerts an antitumor effect.However, various forms can be selected as the administration form when the compound of the present invention is used as an antitumor agent. Examples include oral preparations such as capsules, powders, granules and liquids, and sterile liquid parenteral preparations such as solutions and suspensions. Solid preparations can be produced as they are in the form of tablets, capsules, granules or powders, or they can 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; fatty acid salts such as calcium stearate or magnesium stearate; alcohols such as stearyl alcohol or benzyl alcohol; synthetic cellulose derivatives such as methylcellulose, carboxymethylcellulose, ethylcellulose or hydroxypropylmethylcellulose , And other commonly used additives such as water, gelatin, talc, vegetable oil, and gum arabic. It is below.

[0009] 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. 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, lidocaine hydrochloride aqueous solution (for intramuscular injection), physiological saline, glucose aqueous solution, ethanol, Liquid for intravenous injection (for example, aqueous solution of citric acid and sodium citrate) or electrolyte solution (for intravenous drip and intravenous injection)
Or a mixed solution thereof.

[0010] These injections may be dissolved in advance, or may be in the form of powder or a solution to which an appropriate additive is added, which is dissolved at the time of use. These injections usually contain 0.1 to 10% by weight, preferably 1 to 5% by weight, of the active ingredient. 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. It is. For example, the daily adult dose is 10 to 50 for oral administration.
0 mg, and in the case of parenteral administration, preferably intravenous injection, 10 to 100 mg per day. 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, but the present invention is not limited to these examples.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION

[0013]

【Example】

Example 1 Production method of BE-51068 The actinomycete A51068 strain inoculated on a soft slope agar medium was prepared using 1.0% glucose, 2.0% dextrin, 0.2% soybean meal, 0.6% wheat germ, 0.6% polypeptone. 5
%, Yeast extract 0.3%, humic acid 0.03%, calcium carbonate 0.1% medium (pH 7.0) 110m
l inoculate three 500 ml Erlenmeyer flasks containing
The cells were cultured at 28 ° C. for 48 hours on a rotary shaker (180 rotations per minute). The above culture medium was added to 11 ml of this culture solution in 2 ml portions.
Inoculate 100 500 ml Erlenmeyer flasks containing 0 ml and rotate at 28 ° C for 120 hours with a rotary shaker (180 rotations per minute)
And cultured above.

The thus obtained culture solution (about 10
L), the cells were separated by filtration, and methanol (3 L × 2) was added to the cells for extraction.
After concentrating to 1000 ml, ethyl acetate (1000 ml x 2)
Extracted. The obtained ethyl acetate extract was concentrated under reduced pressure to obtain a crude extract. Half of the crude extract was subjected to silica gel column chromatography (Merck Kieselgel 60, 70-230 mesh, 2.5 × 35 cm, eluted with chloroform / hexane (20: 1)).
A fraction containing E-51068 was obtained. The fraction obtained by concentrating this fraction under reduced pressure was further subjected to silica gel column chromatography (Kieselgel 60, 70-70 manufactured by Merck).
230 mesh, 1.5 × 15 cm, eluting with chloroform / hexane (10: 1)) and BE-51068.
Was obtained. The fraction obtained by concentrating this fraction under reduced pressure is applied to a Sephadex LH-20 column (Pharmacia, 1.5 × 80 cm, eluted with methanol), and the fraction containing BE-51068 is concentrated to dryness. Thereby, 36.5 mg of BE-51068 was obtained. 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 This substance (BE-51068) 10 (parts) heavy magnesium oxide 15 lactose 75 is uniformly mixed to give 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 This substance (BE-51068) 45 (parts) Starch 15 Lactose 16 Crystalline cellulose 21 Polyvinyl alcohol 3 Distilled water 30 After uniformly mixed, crushed and granulated, dried, and then sieved to have a diameter of 1410 to 177 μm. m granules. 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.

[0015]

The BE-51068 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.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1: 465) (C12P 17/06 C12R 1: 465) (72) Inventor Katsuhisa Kojiri 3 Okubo, Tsukuba, Ibaraki Pref. Within Tsukuba Research Laboratories, Pharmaceutical Co., Ltd. (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.
51068 (Streptomyces sp. A5
1068) or a mutant thereof. 5. A Streptomyces sp. Capable of producing the compound according to claim 1.
yces sp. ) Or a mutant thereof. 6. Streptomyces sp. A5106
8 (Streptomyces sp. A5106)
8) or a mutant thereof. 7. An antitumor agent comprising the compound of claim 1 represented by the structural formula [I].