JPH0987285A - Antineoplastic material be-45653 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new antineoplastic material manifesting antineoplastic effects even in various tumors on which an ordinary antineoplastic agents can not manifest their effects. SOLUTION: This compound, BE-45653, is expressed in the formula. This compound is obtained by aerobic culture of a microorganism or its variant, such as streptomyces sp A45653, in a culturing medium like yeast, malt agar and collecting. The culturing condition is at 10-36 deg.C (preferably 21-29 deg.C), at 4-8 of pH and for 48-168 hours (preferably 72-120 hours). The applying dose is 10-500mg in oral administration and 10-100mg in pareteral administration (preferably, intravenous injection) for 1-5 times a day for adult.

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]

The present inventors extensively screened secondary microbial metabolites for substances having antitumor activity in order to solve the above-mentioned problems, and as a result, the compound represented by the formula [1] The present invention has been completed by discovering that the compound exhibits an excellent antitumor effect.

That is, the present invention has a novel formula:

[0006]

Embedded image A compound represented by the formula, a method for producing the same, and a formula [1]
Of Streptomyces (S
The present invention relates to a microorganism belonging to the genus Treptomyces. The compound of the formula [1] is BE-4 due to the production strain Streptomyces sp. (A45653 strain).
It was named 5653. The physicochemical properties of the novel antitumor substance BE-45653 according to the present invention are shown below. The abbreviations used in NMR measurement are shown below. s: singlet d: doublet t: triplet q: quartet m: multiplet br: broad J: coupling constant Hz: Hertz BE-45653 physicochemical properties; white amorphous solid or crystal molecular formula; C ₃₃ H ₅₄ O ₈ mass spectrometry; [high-resolution FAB-MS] (M + H) ⁺ : measured value 579.3919 calculated value 579.3897 specific optical rotation; [α] ²⁰ _D = + 60 ° (c 0.1,
MeOH) UV absorption spectrum; λmax [MeOH, nm]
(Ε); 200 (7,200), 270 (14,20)
0) Red external absorption spectrum: (KBr, cm ⁻¹ ) 3743, 3444, 2969, 2931, 1
693, 1550, 1515, 1459, 142
7, 1384, 1251, 1116, 1037,
970, 516 ¹ H-NMR spectrum (500 MHz, DMSO-
_{d 6) δ ppm: 7.26 (} 1H, brs),
5.60-5.40 (5H, m), 4.89 (1
H, d, J = 5.1 Hz), 4.68 (1H,
m), 4.59 (1H, d, J = 4.6Hz), 4.
19 (1H, d, J = 5.2 Hz), 4.13 (1
H, d, J = 5.8 Hz), 3.88 (2H,
m), 3.48 (1H, m), 3.23 (1
H, m), 3.19 (3H, s), 3.17 (1
H, m), 3.01 (1H, d, J = 8.5H
z), 3.00 (1H, brs), 2.88.
(1H, m), 2.42 (3H, m), 2.17
(2H, m), 2.02 (2H, m), 1.98
(3H, s), 1.92 (3H, d, J = 1.0H
z), 1.90 (1H, m), 1.79 (1
H, m), 1.46 (2H, m), 1.07 (3
H, d, J = 6.1 Hz), 1.05 (3H, d, J
= 7.3 Hz), 0.80 (3H, d, J = 6.7)
Hz), 0.79 (3H, d, J = 6.7Hz),
0.67 (3H, d, J = 7.0Hz) ¹³ C-NMR spectrum (125 MHz, DMSO-
d ₆ ) δ ppm: 168.49 (s), 14
4.53 (d), 143.72 (d), 133.
88 (s), 130.16 (d), 129.9
7 (d), 128.19 (d), 126.45
(D), 122.86 (s), 79.42
(D), 76.10 (d), 71.72
(D), 71.37 (d), 70.01
(D), 69.02 (d), 56.34
(D), 55.66 (q), 54.77 (d),
40.29 (d), 39.00 (d), 38.
06 (t), 37.82 (t), 37.19
(D), 35.40 (t), 34.39
(T), 34.11 (d), 16.41 (q),
16.15 (q), 15.88 (q), 14.
93 (q), 13.22 (q), 9.35
(Q), 8.53 (q) Solubility; easily soluble in organic solvents such as methanol and dimethylsulfoxide, but poorly soluble in water. Distinction between acidic, neutral and basic substances; Neutral substance Color reaction; Sulfuric acid reaction positive Phosphomolybdic acid reaction positive Rf value; 0.6 [Merck Kieselgel 60F ₂₅₄ used, developing solvent: chloroform / methanol (10 1)] Biological activity of BE-45653 (antitumor effect) In order to determine the growth inhibitory effect of the antitumor substance BE-45653 on mouse experimental tumor cells, an in vitro test was conducted. The antitumor effect test on mouse leukemia cells P388 was carried out by dissolving BE-45653 in dimethyl sulfoxide and then serially diluting it with dimethyl sulfoxide, and then measuring 10% fetal bovine serum-containing RPMI 1640 medium (2
(Including 0 mM 2-mercaptoethanol). Cell culture medium containing 1 × 10 ³ tumor cells (RPMI 1640 medium containing 10% fetal bovine serum, 2
50 μl containing 0 mM 2-mercaptoethanol)
Was dispensed into a 96-well microplate, incubated at 37 ° C. for 24 hours under 5% CO ₂ , and then 50 μl of the above test solution was added.
After culturing at 37 ° C. for 72 hours under 5% CO ₂ ,
It was compared with the control group by the TT measurement method. The antitumor test against mouse colon cancer cell colon 26 was BE-45.
After dissolving 653 in dimethylsulfoxide and serially diluting it with dimethylsulfoxide, it was added to RPMI 1640 medium containing 10% fetal bovine serum to give a test solution. 1x10 ³
100 μl of cell culture medium containing 10 tumor cells (RPMI 1640 medium containing 10% fetal bovine serum) was dispensed into a 96-well microplate, cultured at 37 ° C. for 24 hours under 5% CO ₂ , and then the above test solution was added. After 100 μl was added, the mixture was incubated at 37 ° C. for 72 hours under 5% CO ₂ , fixed with 50% trichloroacetic acid, and stained with 0.4% sulforhodamine B.
The dye was extracted from the cells using 0 mM Tris solution. 45
The absorbance at 550 nm was measured using 0 nm as a control wavelength and compared with the control group. As a result, BE-45653
Shows strong growth inhibitory activity against both tumor cells,
The growth inhibitory concentration (IC ₅₀ ) was as shown in Table 1. Furthermore, the antitumor activity of BE-45653 against human tumor cells was tested in vitro. The cells are human colon cancer cells D
LD-1, human lung cancer cells PC-13 and human gastric cancer cells MKN-45 were used, and the cell culture medium was RPMI 1640 medium containing 10% fetal bovine serum for all tumor cells. colon 26
It measured using the method similar to. As a result, BE-45
653 also showed strong growth inhibitory activity against human tumor cells, and its 50% growth inhibitory concentration (IC ₅₀ ) was as shown in Table 1.

[0007]

[Table 1] As described above, BE-45653 shows 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. Next, a method for manufacturing BE-45653 will be described. The microorganism used in the production of the antitumor substance BE-45653 of the present invention or a mutant strain thereof is the antitumor substance B.
Any one may be used as long as it produces E-45653, but examples thereof include microorganisms having the following mycological properties. 1. Morphology A 45653 strain forms aerial hyphae and basal hyphae that are well elongated and branched, and no division of the hyphae and hyphae is observed. A long chain of spores (50 or more) is formed on the aerial mycelium, and its morphology is a spiral type. The surface of the spores is a hump and has an elliptical shape with a size of about 2.2 to 1.6 × 1.0 to 0.8 μm, and no special organs such as sporangia, flagella spores and sclerotia are observed. 2. Cultural properties on various agar plate media (28 ° C, 14
Days culture)

[0008]

[Table 2] 3. Growth temperature (yeast / malt agar medium, cultured for 14 days) 7 ℃; No growth 10 ℃; Good growth 13 ℃; Good growth, few aerial mycelia formation 18 ℃; Very good growth, Good aerial mycelia formation 21 ℃; Growth And aerial hyphae formation very good 25 ° C; growth and aerial hyphae formation very good 29 ° C; growth and aerial hyphae formation very good 34 ° C; growth and aerial hyphae formation 36 ° C; good growth and aerial hyphae formation very low 42 ° C Not grown 4. Physiological properties (1) Negative liquefaction of gelatin (glucose / peptone / gelatin medium) (2) Positive hydrolysis of starch (starch / inorganic salt agar medium) (3) Negative coagulation of skim milk powder (skimmed milk medium) (4) Peptonization of skim milk powder is positive (skimmed milk medium) (5) Melanin-like pigment formation is negative (ISP-1, ISP-6, ISP-7) (6) Salt tolerance Grows with a sodium content of 7% or less (yeast malt agar) Medium) 5. Utilization of carbon source Using Pridham Godleybe agar as a basal medium, the following sugars were added and cultured at 28 ° C for 14 days.

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-mentioned mycological properties, the A45653 strain is considered to belong to the genus Streptomyces of actinomycete. Therefore, the A 45653 strain was designated as Streptomyces sp.
sp. A 45653). Also,
Vergie's Manual of Determinative
Bacteriology (Bergey's Manual)
of Determinative Bacterio
), 8th Edition, 1974 and Applied
Microbiology (Applied Microb)
As a result of literature searches such as Iology), Vol. 22, 1971, Streptomyces torulosus (Strepto
myces torulosus) was estimated to be a closely related species. The strain has been deposited at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry, and the deposit number is FERM.
P-14937. The mutant strain of the microorganism that produces the antitumor substance BE-45653 used in the present invention is, for example, a radiation treatment such as X-ray or ultraviolet ray, for example, nitrogen mustard, azaserine, nitrous acid, 2-aminopurine or N-methyl-. Treatment with a mutagenesis agent such as N'-nitro-N-nitrosoguanidine (NTG),
It is a microorganism obtained by mutating a BE-45653-producing bacterium by a commonly used bacterial species conversion treatment method such as phage contact, transformation, transduction or conjugation. BE-456 of the present invention
In producing 53, a BE-45653-containing culture can be obtained by inoculating a nutrient source-containing medium with a BE-45653-producing strain to aerobically grow. As the nutrient source, those known as the 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. 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 that can be used by the producing bacterium and are useful for the production of BE-45653, 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 10 to 36 ° C, preferably 21 to 29 ° C. The preferred pH of the medium is 4-8.
In the range, the culture time is 48 hours to 168 hours, preferably 72 hours to 120 hours. In order to collect the desired BE-45653 from the culture, a separation means usually used for collecting from the metabolite produced by the microorganism can be appropriately used. BE-45653 is present in the bacterial cells, and therefore purified from the bacterial cells by an ordinary 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 alone or in combination. it can. 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 was partitioned with water-ethyl acetate, and the residue obtained after distilling off the ethyl acetate layer was subjected to silica gel chromatography (eluted with hexane / ethyl acetate) to give BE-45653. Can be obtained as a powder or a solid. The compound BE-45653 of the present invention inhibits the growth of tumor cells and exerts an antitumor effect, but various forms can be selected as an administration form when the compound of the present invention is used as an antitumor agent, for example, tablets, Examples include oral preparations such as capsules, powders, granules or solutions, or sterilized liquid parenteral preparations such as solutions or 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, 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. , Other additives commonly used such as water, gelatin, talc, vegetable oil or gum arabic And the like. The solid preparations such as tablets, capsules, granules and powders are generally 0.1-10.
It contains 0% by weight, preferably 5-100% by weight, of the active ingredient. Liquid formulations include water, alcohols or soybean oil, for example.
Using suitable additives usually used in liquid formulations such as plant-derived oils such as peanut oil or sesame oil,
It is manufactured as a suspension, syrup or injection. 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, glucose aqueous solution, ethanol, Liquid for intravenous injection (eg aqueous solution of citric acid and sodium citrate) or electrolyte solution (for intravenous drip and intravenous injection)
Or a mixed solution of these. These injections may be pre-dissolved, or may be in the form of powder or dissolved with a suitable additive before use. These injections are usually 0.1 to 10% by weight,
It preferably contains 1 to 5% by weight of active ingredient. In addition, liquid preparations such as suspensions or syrups for oral administration are 0.5 to 1
Contains 0% by weight of 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. For example, the dose for adults per day is
10 to 500 mg, and 10 to 100 mg per day for parenteral administration, preferably for intravenous injection. The frequency of administration depends on the administration method and symptoms, but
Times 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.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION

[0012]

【Example】

Example 1: Method for producing BE-45653 Actinomycete A 45653 strain inoculated on a slant soft agar medium was glucose 0.1%, dextrin 2.0%, fish meal 0.5.
%, Gluten meal 1.0%, yeast extract 0.1%, sodium chloride 0.1%, magnesium sulfate 0.05%,
Zinc sulfate 0.00008%, calcium chloride 0.05
%, Ferrous sulfate 0.0002%, cuprous chloride 0.000
04%, manganese chloride 0.00004%, cobalt chloride 0.00004%, sodium borate 0.00008
%, Ammonium molybdate 0.00024%, 3-
Inoculate four Erlenmeyer flasks of 500 ml volume containing 100 ml of a medium (pH 6.8) consisting of 0.5% of (N-morpholino) propanesulfonic acid, and incubate at 28 ° C. for 72 hours on a rotary shaker (180 rpm). It was cultured in. 2 this culture
100 ml of a 500 ml Erlenmeyer flask containing 110 ml of the above medium was inoculated and cultured at 28 ° C. for 96 hours on a rotary shaker (180 rpm). The cells were separated from the thus obtained culture solution (about 10 L) by filtration, 8 L of methanol was added to the cells, and after stirring for several hours, the cells were filtered off to obtain a methanol extract. The methanol of the methanol extract was distilled off under reduced pressure, 600 ml of deionized water was added to the residue, and 3 L of ethyl acetate was added for extraction. The obtained ethyl acetate extract was concentrated under reduced pressure, the residue was applied to a silica gel chromatography column (2.4 × 28 cm), and eluted with a mixed solvent of hexane / ethyl acetate (1: 1). The eluted active fraction was concentrated to dryness under reduced pressure, and BE-
448 mg of a mixture containing 45653 was obtained. Next, Sephadex LH-20 (Pharmacia, 1.5x
It was applied to a chromatography column (80 cm) and eluted with methanol. The fraction of interest was concentrated under reduced pressure, and the ODS column (Chromato)
rex DU0005MT, Fuji Silysia Chemical, 2.0
x25 cm) and eluted with 75% methanol to give a fraction purely containing BE-45653, which was concentrated to dryness under reduced pressure,
371 mg of white powder of BE-45653 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-45653) 10 parts Heavy magnesium oxide 15 parts Lactose 75 parts are uniformly mixed to obtain a powdery or fine-granular powder of 350 μm or less. This powder was placed in a capsule container to obtain a capsule. Formulation example 2 This substance (BE-45653) 45 parts Starch 15 parts Lactose 16 parts Crystalline cellulose 21 parts Polyvinyl alcohol 3 parts Distilled water 30 parts After mixing uniformly, it is crushed and granulated, dried and then sieved to a diameter. Granules having a size of 1410 to 177 μm were prepared. 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.

[0013]

The BE-45653 described in the present invention is
Since it shows 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.

Front Page Continuation (72) Inventor Katsuhisa Kojiri 3 Okubo, Tsukuba City, Ibaraki Prefecture, Mana Pharmaceutical Co., Ltd. Tsukuba Research Institute (72) Inventor Hiroyuki Suda 3 Okubo, Tsukuba City, Ibaraki Manwa Pharmaceutical Co., Ltd. Tsukuba Research In-house

Claims (6)

[Claims] [Claim 1] The formula: The compound represented by. 2. Streptomyces
es) culturing a microorganism or a mutant strain thereof belonging to the genus and having the ability to produce the compound represented by the formula [1] according to claim 1, and collecting the compound represented by the formula [1]. A method for producing the compound represented by the formula [1], which is characterized. 3. The method according to claim 2, wherein the microorganism or its mutant strain is Streptomyces sp. 4. An antitumor agent comprising the compound represented by the formula [1] according to claim 1 as an active ingredient. 5. Streptomyces having an ability to produce the compound represented by the formula [1] according to claim 1.
A microorganism belonging to the genus ptomies) or a mutant strain thereof. 6. The microorganism is Streptomyces sp.
A45653 (Streptomyces sp. A)
45653), The microorganism according to claim 5 or a mutant strain thereof.