JP2000026497A - Antitumor substance BE-60828 and method for producing the same - Google Patents

Abstract

(57) [Summary] [Constitution] The present invention provides a novel structural formula [I] With respect to the compound represented by [Effect] The compound of the present invention has a strong growth inhibitory effect on mouse and human tumor cells, and is therefore useful as a therapeutic agent for cancer in the field of medicine. .

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.]. This situation is still continuing, and 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]

[0006]

Embedded image And a method for producing the compound, its use, and a microorganism belonging to the genus Streptomyces, which has the ability to produce the compound.

The antitumor effect and the production strain of Streptomyces sp. A60828 (Streptomy)
ces sp. A60828), the compound represented by Structural Formula [I] is referred to as BE-60828.

The physicochemical properties of the novel antitumor substance BE-60828 according to the present invention are shown below. The meanings of the abbreviations in the nuclear magnetic resonance spectrum are shown below. s: singlet d: doublet dd: doublet of doublet t: triplet q: quartet m: multiplet br: broad J: coupling constant Hz: hertz

Physicochemical properties of BE-60828 ; white powder molecular formula; C ₃₈ H ₅₀ N ₆ O ₈ mass spectrometry; [HRFAB-MS] m / z found: 719.3770 (M + H) ⁺ calculated: 719 .3768 (C ₃₈ H ₅₁ N ₆ O ₈ ) specific rotation; [α] ²⁰ _D -13.2 ° (c 0.5, Me
OH) UV absorption spectrum; λ _max (MeOH) nm
(Ε): 298 (55, 100) infrared absorption spectrum; ν _max (KBr) cm ⁻¹ : 32
91,3276,2960,2937,1733,16
56, 1513, 1440, 1270, 1159, 11
43, 1110, 1010, 802, 736 Nuclear magnetic resonance spectrum; ¹ H-NMR (500 MHz,
CDCl ₃ ): δ 0.98 (3H, d, J = 6.4H)
z), 1.38 (3H, d, J = 6.4 Hz), 1.5
2 (3H, d, J = 6.7 Hz), 1.79 (1H,
m), 1.81 (3H, d, J = 6.4 Hz), 1.9
4 (2H, m), 2.06 (1H, dd, J = 12.
8,7.3 Hz), 2.17 (1H, m), 2.36
(2H, m), 2.83 (3H, s), 2.98 (2
H, d, J = 7.0 Hz), 3.08 (1H, dd, J)
= 12.2, 8.2 Hz), 3.52 (3H, s),
3.74 (1H, m), 4.45 (1H, d, J = 7.
8Hz), 4.51 (1H, t, J = 9.5Hz),
4.64 (1H, dt, J = 8.2, 7.0 Hz),
4.77 (1H, q, J = 6.7 Hz), 4.84 (1
H, br d, J = 11.6 Hz), 4.90 (1H,
m), 5.13 (1H, dd, J = 8.2, 3.1H
z), 5.90 (1H, m), 6.13 (1H, dd,
J = 15.0, 10.4 Hz), 6.25 (2H,
m), 6.49 (1H, dd, J = 15.0, 10.4)
Hz), 6.77 (1H, d, J = 9.5 Hz), 6.
97 (1H, d, J = 8.2 Hz), 7.16 (2H,
d, J = 7.8 Hz), 7.20 (1H, t, J = 7.
8Hz), 7.29 (3H, m), 8.55 (1H,
d, J = 9.8 Hz) ¹³ C-NMR (125 MHz, CDCl ₃ ): δ15.
6 (q), 17.4 (q), 18.2 (q), 18.4
(Q), 23.1 (t), 29.4 (d), 30.8
(Q), 30.8 (t), 38.6 (t), 38.7
(T), 46.4 (t), 47.7 (d), 51.1
(D), 54.3 (t), 55.2 (d), 56.2
(D), 56.3 (d), 60.0 (d), 64.8
(T), 122.8 (d), 126.7 (d), 12
7.9 (d), 128.6 (d) × 2, 129.3
(D) × 2, 131.3 (d), 133.8 (d), 1
36.1 (s), 139.7 (d), 141.3
(D), 164.6 (s), 166.1 (s), 16
9.8 (s), 169.9 (s), 171.4 (s),
172.1 (s), 172.5 (s) Solubility; easily soluble in chloroform, methanol, acetone, and dimethyl sulfoxide, and hardly soluble in water. Rf value: 0.65 [Merck Kieselgel 60F]
₂₅₄ used, developing solvent: chloroform-methanol (2
0: 1)] Color reaction; Sulfuric acid reaction positive

[0010] Biological activity of BE-60828 (antitumor
Action and antibacterial action) To determine the growth inhibitory action of the antitumor substance BE-60828 on experimental mouse tumor cells, a test was conducted in vitro. In the antitumor test for mouse leukemia cell P388, BE-60828 was dissolved in dimethylsulfoxide, then serially diluted with dimethylsulfoxide, and then diluted 10-fold with RPMI1640 medium containing 10% fetal calf serum to obtain a test solution. Assay for cell growth inhibition was performed using a cell culture medium containing 1 × 10 ³ tumor cells (RPM containing 10% fetal bovine serum).
5 μl of the above test solution was added to 100 μl of the I1640 medium (containing 20 μM of 2-mercaptoethanol). Three
After culturing at 7 ° C. for 72 hours under 5% CO ₂ , the cells were compared with a control group by MTT measurement. As a result, BE-60828
Showed strong growth inhibitory activity in mouse tumor cells, and its 50% growth inhibitory concentration was as shown in Table 1.

Furthermore, the antitumor active substance BE-60828
Tested in vitro for antitumor activity against human tumor cells
Was. An antitumor test against human lung cancer cell PC-13 was performed using B
After dissolving E-60828 in dimethyl sulfoxide,
Dilute with dimethyl sulfoxide, then 10% fetal calf serum
10-fold dilution with RPMI1640 containing
Was. Assay for tumor cell growth inhibition was 1 × 10^Three Individual tumor cells
100 μl of cell culture medium containing cells
Aliquots for 24 hours at 37 ° C., 5% CO 2 _TwoCulture under
After feeding, 5 μl of the above test solution was added. In addition, culture for 72 hours
After feeding, the cells were fixed with 16.7% trichloroacetic acid,
% Sulforhodamine B, use 10 mM Tris solution
To extract the dye from the cells. 450 nm as reference wavelength
Measure the absorbance at 550nm and compare with control group
did. As a result, BE-60828 is found on human tumor cells.
Even when it shows strong growth inhibitory activity, its 50% growth inhibitory concentration
The degree was as shown in Table 1.

[0012]

[Table 1] As described above, BE-60828 has a remarkable growth inhibitory effect on mouse and human tumor cells. Therefore,
The present invention is useful as an antitumor agent for mammals including humans.

[0013] BE-60828 is a compound having an antibacterial activity. Measured against various pathogenic microorganisms,
The minimum inhibitory concentration was determined. Table 2 shows the results.

[0014]

[Table 2] As shown in Table 2, BE-60828 exhibits remarkable antibacterial activity against various pathogenic microorganisms. Therefore, it is also useful as an antibacterial agent against diseases caused by these pathogenic microorganisms.

Next, a method for producing BE-60828 will be described. The microorganism used for producing the antitumor substance BE-60828 of the present invention is an antitumor substance BE-6082.
As long as it produces No. 8, a microorganism having the following mycological properties, for example, A60828 can be used. 1. Form A60828 forms an aerial mycelium with a base mycelium that elongates and diverges well, and there is no division of the ring vegetation and the hypha. It forms a long chain of spores (50 or more) on the aerial hyphae, and its form is spiral. Spore surface is spiny and 0.6 in size
~ 0.9 × 0.7 ~ 1.0μm spherical ~ oval,
No special organs such as spores, flagella spores and sclerotia are observed. It is observed that the colonies gradually become wet with the maturation of aerial hyphae in an yeast malt agar medium or the like. 2. Culture properties on various agar plate media The culture characteristics (cultivation at 28 ° C for 14 days) on various agar plate media are as shown in Table 3.

[0016]

[Table 3] 3. Growth temperature The growth temperature (east-malt agar medium, cultured for 14 days) was as shown in Table 4.

[0017]

[Table 4] 4. Physiological properties Physiological properties were as shown in Table 5.

[0018]

[Table 5] 5. Utilization of carbon source Utilization of carbon source is shown in Table 6.

[0019]

[Table 6] 6. Cell components LL diaminopimelic acid and glycine were detected from the cell wall.

Due to the above mycological properties, A60828 is actinomycete Streptomyces.
It is considered to belong to the genus. Therefore, the A60828 strain was replaced with Streptomyces sp. A60828 (Stre
ptomyces sp. A60828). This strain has been deposited on May 1, 1998 with the Research Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry of Japan, and the deposit number is FERM P-16788.

The Streptomyces sp A of the present invention
60828 (Streptomyces sp. A6
0828) may be subjected to irradiation treatment with, for example, X-rays or ultraviolet rays, for example, nitrogen mustard, azaserine, nitrite, 2-aminopurine or 1-methyl-
Streptomyces sp. A60828 (St) by a commonly used bacterial species conversion treatment method such as treatment with a mutagenic agent such as 3-nitro-1-nitrosoguanidine (MNNG), phage contact, transformation, transduction or conjugation.
reptomyces sp. A60828).

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION In producing BE-60828 of the present invention, a BE-60828-producing strain is inoculated into a nutrient-containing medium and grown aerobically to obtain a BE-60828.
A culture containing -60828 is obtained. 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 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, those useful for the production of BE-60828, which are used by the producing bacteria, such as 3- (N-morpholino) propanesulfonic acid (MOP)
Any of S) and sodium borate can be used.

The culturing method may be the same as the method for producing general microbial metabolites, and may be either solid culture or liquid culture. In the case of liquid culture, static culture, stirring culture,
Either shaking culture or aeration culture may be performed, but shaking culture or deep aeration stirring culture is particularly desirable. The culture temperature is suitably from 14 to 40 ° C.,
３１31 ° C. The preferred pH of the medium is in the range of 4-8, and the culture time is 168-264 hours, preferably 192.
~ 240 hours. BE-60 of interest from culture
In order to collect 828, separation means commonly used for collecting from metabolites produced by microorganisms can be appropriately used.

Since BE-60828 is present in the cells and the filtrate, conventional separation means from the cells and the filtrate, such as a solvent extraction method, an ion-exchange resin method, or an adsorption or partition chromatography method and a gel filtration method, can be used alone or in combination. Purification can be performed by performing the combination.

Preferred examples of separation and purification include the following methods. First, the culture solution is filtered to separate cells and the filtrate. The obtained cells are extracted using an organic solvent such as methanol or acetone. The filtrate is purified by an ion exchange resin method or an adsorption chromatography method. The crude extract obtained from the cells was partitioned with water-ethyl acetate,
After evaporating the ethyl acetate layer, the obtained residue and the crude product obtained from the filtrate are subjected to reverse phase column chromatography. BE-60828 can be obtained by concentrating the fraction containing BE-60828 under reduced pressure and performing reverse phase column chromatography again.

The compound BE-60828 of the present invention inhibits the growth of tumor cells and exerts an antitumor effect, but various forms can be selected as the administration form when the compound of the present invention is used as an antitumor agent. For example, tablets, capsules, powders,
An oral preparation such as a granule or a liquid preparation, or a sterile liquid parenteral preparation such as a solution or suspension can be used.

The solid preparation can be produced as it is in the form of tablets, capsules, granules or powder, but 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 aluminate or anhydrous calcium phosphate, for example polyvinylpyrrolidone or Synthetic polymers such as polyalkylene glycols; 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; , Water, gelatin,
Commonly used additives such as talc, vegetable oil, gum arabic and the like can be mentioned.

The solid preparations such as tablets, capsules, granules and powders generally contain 0.1 to 100% by weight,
It preferably contains from 5 to 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 In particular, for parenteral administration by intramuscular injection, intravenous injection or subcutaneous injection, suitable solvents include, for example, distilled water for injection, aqueous lidocaine hydrochloride (for intramuscular injection), physiological saline, aqueous glucose, 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. 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. In addition, liquid preparations such as suspensions or syrups for oral administration,
It 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 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.

[0030]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples. Example BE-Production method of 60828 Actinomyces A60828 inoculated on a sloped soft agar medium were glucose 0.1% and dextrin (MS-3600).
2.0%, fish meal 0.5%, corn gluten meal
1.0%, yeast extract 0.1%, MOPS 0.5
%, Magnesium sulfate 0.05%, sodium chloride
0.1%, calcium chloride 0.05%, ferrous sulfate
0.0002%, cupric chloride 0.00004%, manganese chloride 0.00004%, cobalt chloride 0.00
004%, zinc sulfate 0.00008%, sodium borate 0.00008% and sodium molybdate
Medium consisting of 0.00024% (pH 7.0) 220
inoculate one 500 ml Erlenmeyer flask containing 500 ml.
The cells were cultured at 28 ° C. for 72 hours on a rotary shaker (180 rotations per minute). 200 ml of this culture was inoculated into one 20 L tank containing 10 L of the above medium, and cultured at 28 ° C. for 141 hours with aeration (1.0 vvm) with stirring (300 rpm).

The culture solution obtained by the above culture (about 10
L) was separated into bacterial cells and filtrate by filtration. The cells were added with methanol (7 L), stirred, and filtered to obtain a methanol extract. After concentrating this methanol extract under reduced pressure, 1 L
This was extracted twice with ethyl acetate, and the ethyl acetate fraction was concentrated to dryness under reduced pressure. The filtrate was Diaion HP-20SS column chromatography (Mitsubishi Chemical Corporation, φ5 × 30c)
m) and eluted with methanol-water (8: 2), and the fraction containing the desired compound was concentrated to dryness under reduced pressure. After the dried product obtained from the cells and the filtrate was dissolved in methanol (10 ml), the solution was subjected to Cosmosil 75C18 column chromatography (manufactured by Nacalai Tesque, φ3 × 50 cm).
After elution with methanol-water (9: 1), the fraction containing the desired compound was concentrated to dryness under reduced pressure. Next, the dried product was dissolved in methanol (12 ml), and then subjected to a column for high performance liquid chromatography (YMC-Guardpack ODS).
G-350-10 and YMC-Pack ODS R
-355-10 column, both manufactured by YMC, φ5 × 5
cm and φ5 × 50 cm) were used for high performance liquid chromatography. As the mobile phase, methanol-water (8
5:15), the fraction containing the target compound was concentrated to dryness under reduced pressure by elution at a flow rate of 100 ml / min. Finally, the dried product was dissolved in acetone (10 ml), hexane (30 ml) was added, acetone was distilled off under reduced pressure, and BE-60828 was added by depositing powder.
35 mg were obtained.

Formulation examples of the compound of the present invention are shown below.
The preparation of the compound of the present invention is not limited to this preparation example. Formulation Example 1 10 parts of this substance (BE-60828), 15 parts of heavy magnesium oxide and 75 parts of lactose are uniformly mixed, and
m or less powder or fine-grained powder. This powder was placed in a capsule container to obtain a capsule. Formulation Example 2 45 parts of the substance (BE-60828), 15 parts of starch, 16 parts of lactose, 21 parts of crystalline cellulose, 3 parts of polyvinyl alcohol and 30 parts of distilled water are uniformly mixed, crushed and granulated and dried. And then sieved to a diameter of 355 to 1400 μm
Granules of the size 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 90 parts of granules obtained in the same manner as in Formulation Example 2 were mixed with 10 parts of crystalline cellulose and 3 parts of calcium stearate.
The resulting mixture was compression-molded to give a tablet having a diameter of 8 mm, and a syrup gelatin and a precipitated calcium carbonate mixed suspension were added thereto to prepare a sugar-coated tablet. Formulation Example 5 0.6 part of this substance (BE-60828), 2.4 parts of a nonionic surfactant and 97 parts of physiological saline are heated and mixed, then put into an ampoule, sterilized, and an injection is prepared. Produced.

[0033]

The BE-60828 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.

Continuing from the front page (72) Inventor Hisao Kondo 3rd Okubo Tsukuba City, Ibaraki Prefecture Banyu Pharmaceutical Co., Ltd. Inside Tsukuba Research Laboratories (72) Inventor Katsuhisa 2nd 2-3 Nihonbashi Honcho, Chuo-ku, Tokyo Banyu Pharmaceutical Co., Ltd. (72) Inventor Hiroyuki Suda 3 Okubo, Tsukuba, Ibaraki Pref. ZB352 4H045 AA10 AA20 AA30 BA34 CA11 EA28 EA29 FA71 FA73

Claims (6)

[Claims] (1) Structural formula [I] A compound represented by the formula: 2. Streptomyces (Streptomyc)
es) A microorganism belonging to the genus and capable of producing the compound represented by the structural formula [I] according to claim 1 is cultured, and the compound represented by the structural formula [I] is collected from the culture. A method for producing a compound represented by the structural formula [I]. 3. A microorganism capable of producing the compound represented by the structural formula [I] according to claim 1, wherein the microorganism is Streptomyces sp. A60828.
s sp. A60828) or a mutant thereof. 4. An antitumor agent comprising the compound represented by the structural formula [I] according to claim 1 as an active ingredient. A microorganism belonging to the genus Streptomyces, which has an ability to produce the compound represented by the structural formula [I] according to claim 1. 6. Streptomyces (Streptomyc)
es) A microorganism belonging to the genus Streptomyces sp. A60828 (Streptomyces sp.
A60828) or a mutant thereof.