JPH1067794A - BE-56980 antitumor substance and method for producing the same - Google Patents

Abstract

(57) [Summary] The present invention provides a novel compound represented by the following general formula [I]: Wherein R represents a hydrogen atom or a lower alkyl group. [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.].

[0003] Under such circumstances, in the field of cancer treatment, the development of new antitumor substances is always required.

[0004]

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.

[0005]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have screened a wide range of secondary metabolites of microorganisms for substances having antitumor activity, and as a result, they are represented by the following general 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 compound represented by the general formula [I]:

[0007]

Embedded image [Wherein, R represents a hydrogen atom or a lower alkyl group] or a pharmaceutically acceptable salt thereof, a production method thereof, a use thereof, and an ability to produce the compound. Actinoplanes
lanes).

Next, the terms used in this specification will be described.

The term "lower" means that the radical or compound to which it is attached has less than 6, preferably less than 4, carbon atoms.

The term "lower alkyl group" means a linear or branched alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec. -Butyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group,
Hexyl group and the like.

Hereinafter, R of the general formula [I] Is a hydrogen atom
The compound is designated BE-56980.

The novel antitumor substance BE according to the present invention
Physicochemical properties of -56980.

The meanings of the abbreviations in NMR measurement are shown below.
You. s: singlet d: doublet t: triplet q: quartet m: multiplet br: broad J: coupling constant Hz: hertzPhysicochemical properties of BE-56980 Properties: red-orange amorphous solid or crystal Molecular formula: C₄₆H₅₈O₂₀ Mass spectrometry: [High-resolution FAB-MS] (M)⁺As:
Observed value 930.3527; Calculated value 930.3522 Specific rotation: [α]²⁰ _D= −222 ° (c 0.10, C
HCl_Three) UV absorption spectrum: λ_max(MeOH, nm
(Ε)) 273 (28300), 312 (2220)
0), 479 (13400) Infrared external absorption spectrum: (KBr, cm^-1) 3446,
2929, 1707, 1680, 1647, 1622,
1554, 1458, 1396, 1306, 1263
1109, 1078, 608¹ H-NMR spectrum (500 MHz, CD Cl_Three) Δ
ppm: 14.7 (1H, s), 12.3 (1H,
s), 8.00 (1H, s), 7.46 (1H, s),
7.40 (1H, d, J = 1.8 Hz), 6.83 (1
H, d, J = 1.8 Hz), 5.79 (1H, br)
s), 5.62 (1H, brs), 5.25 (1H, b
rs), 4.25 (1H, brs), 4.07 (1H,
brs), 3.86 (1H, brs), 3.73-3.
63 (6H, m), 3.60 (6H, s), 3.56
(3H, s), 3.54 (3H, s), 3.52 (3
H, s), 3.51 (3H, s), 3.39 (1H, d
d, J = 9.0, 3.2 Hz), 3.29 (1H, t,
J = 9.5 Hz), 3.17 (2H, m), 3.10
(1H, t, J = 9.0 Hz), 1.70 (2H,
m), 1.30 (3H, d, J = 6.1 Hz), 1.2
7 (6H, d, J = 6.1 Hz), 1.02 (3H,
t, J = 7.3 Hz)¹³ C-NMR spectrum (125 MHz, CD Cl_Three)
δ ppm: 189.6 (s), 180.9 (s), 16
9.4 (s), 166.5 (s), 164.8 (s),
162.3 (s), 153.3 (s), 142.8
(S), 139.7 (s), 135.6 (s), 12
9.4 (s), 128.6 (s) 122.2 (d), 1
21.2 (s), 111.7 (s), 111.2
(D), 109.1 (d), 109.0 (d), 10
8.4 (s), 100.4 (d), 96.9 (d), 9
5.2 (d), 81.9 (d), 81.5 (d), 8
1.4 (d), 81.0 (d), 80.5 (d), 7
6.6 (d), 76.6 (d), 69.1 (d), 6
8.2 (d), 68.1 (d), 68.0 (d), 6
6.6 (d), 60.9 (q), 60.7 (q), 5
9.2 (q), 57.7 (q), 57.4 (q), 5
6.9 (q), 36.0 (t), 25.4 (t), 1
8.1 (q), 17.7 (q), 17.4 (q), 1
4.6 (q) Solubility: chloroform, methanol, dimethyl sulfoxide
It is easily soluble in organic solvents such as sides and hardly soluble in water.

Differentiation between acidic, neutral and basic substances: Acidic substance Color reaction: Sulfuric acid reaction Positive biological activity of BE-56980 (anti-tumor effect) Proliferation of anti-tumor substance BE-56980 on mouse experimental tumor cells The tests were performed in vitro to determine the inhibitory effect. In the antitumor test for mouse colon cancer cell colon 26, BE-56980 was dissolved in dimethylsulfoxide and then serially diluted with dimethylsulfoxide, and then added to RPMI 1640 medium containing 10% fetal bovine serum to prepare a test solution. Cell culture medium containing 1 × 10 ³ tumor cells (RPMI 1640 medium containing 10% fetal bovine serum)
Dispense 100 μl into a 96-well microplate,
The above test solution 1 after culturing for 24 hours in 5% CO ₂
After adding 00 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. . 55 with 450 nm as control wavelength
The absorbance at 0 nm was measured and compared with the control group. As a result, BE-56980 showed a strong growth inhibitory activity against tumor cells, and the 50% growth inhibitory concentrations were as shown in Table 1.

Furthermore, the antitumor activity of BE-56980 against human tumor cells was tested in vitro. Cells use human colon cancer cells DLD-1, human lung cancer cells PC-13 and human gastric cancer cells MKN-45, and the cell culture medium is
RPMI 1 containing 10% fetal calf serum in all tumor cells
The above mouse colon cancer cell colon was prepared using 640 medium.
It measured using the same method as 26. As a result, BE
-56980 also exhibited strong growth inhibitory activity against human tumor cells, and its 50% growth inhibitory concentration was as shown in Table 1.

[0016]

[Table 1] As described above, BE-56980 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-56980 will be described.

The microorganism used for producing the antitumor substance BE-56980 of the present invention is the antitumor substance BE-5698.
Any microorganism may be used as long as it produces 0, but for example, a microorganism having the following mycological properties collected from a grassland at the top of Mt. Hakodate, Hokkaido, ie, strain A56980, can be used. 1. Form A strain 56980 does not form aerial hyphae and forms spores one at a time at the tip of the spore stalk formed in the branched base hyphae. The shape of the spore sac is almost spherical and its diameter is 9
５5 μm. 0.9-1.1μ diameter for spores
Many m-shaped, substantially spherical spores are contained therein, and when mature spores are thrown into water, spores are released, and the spores have flagella and exhibit migratory properties.

[0019]

[Table 2]

[0020]

[Table 3]

[0021]

[Table 4]

[0022]

[Table 5] 6. Cell component: meso-diaminopimelic acid, 3-O
H-diaminopimelic acid and glycine were detected and the cell wall type is considered to be type II. In addition, all the cell sugar components characteristic of the classification were arabinose and xylose, and the sugar pattern was D-type. The main menaquinone is MK
It was -9 (H _4).

From the above mycological properties, the strain A56980 is considered to belong to the actinomycetes Actinoplanes genus. Therefore, the A56980 strain was transformed into Actinoplanes sp. A56980 (Actinoplanes sp.).
p. A 56980).

This strain has been deposited with the Institute of Biotechnology, Industrial Technology Institute of the Ministry of International Trade and Industry, and the deposit number is FE.
RM P-15716.

Actinoplanes SP A of the present invention
56980 (Actinoplanes sp. A
56980) can be obtained by irradiation with, for example, X-rays or ultraviolet rays, for example, nitrogen mustard, azaserine, nitrite, 2-aminopurine, or N-methyl-N′-nitro-N-nitrosoguanidine (NTG).
Treatment with a mutagen, etc., phage contact, transformation,
Actinoplanes SP A 56980 by a commonly used bacterial species conversion treatment method such as transduction or conjugation.
(Actinoplanes sp. A 5698
0) can be obtained by mutation.

[0026]

BEST MODE FOR CARRYING OUT THE INVENTION In producing BE-56980 of the present invention, a BE-56980-producing strain is inoculated on a nutrient-containing medium and allowed to grow aerobically.
A culture containing -56980 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 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-56980, 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 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 13 to 38 ° C. Preferred medium p
H ranges from 4 to 8, and the culture time is 168 hours to 268 hours, preferably 192 hours to 240 hours. In order to collect the desired BE-56980 from the culture, a separation means usually used for collecting from metabolites produced by microorganisms can be appropriately used.

Since BE-56980 is present in the cells and the filtrate, it can be separated from the cells and the filtrate by a conventional means such as a solvent extraction method, an ion exchange resin method, an adsorption or partition chromatography method and a gel filtration method. Can be purified alone or in combination.

Preferred examples of the separation and purification include the following methods. First, the culture solution is filtered to obtain a filtrate. The obtained filtrate was partitioned with a water-ethyl acetate system, and the residue obtained after distilling off ethyl acetate was subjected to Sephadex LH-2.
0 chromatography (ethyl acetate / acetonitrile /
(Eluted with water), etc. to give BE-56980
Can be obtained as a powder or a solid.

Among the compounds of the present invention, the compound represented by the formula [II] can be converted into a compound in which R of the general formula [I] is a lower alkyl group by a lower alkylation method well known in the field of chemistry. It can be easily converted.

For example, by reacting a compound represented by the formula [II] with a lower alkyl halide in the presence of a base, a compound of the formula [I] wherein R is a lower alkyl group can be obtained.

The compound of the present invention can exist in the form of a pharmaceutically acceptable salt. Examples of such a salt include a base addition salt at a carboxyl group.
For example, sodium salts, alkali metal salts such as potassium salts,
For example, aliphatic amine salts such as trimethylamine salt, triethylamine salt, dicyclohexylamine salt, ethanolamine salt, diethanolamine salt, triethanolamine salt, and procaine salt; aralkylamine salts such as N, N-dibenzylethylenediamine; Picoline salts, quinoline salts, heterocyclic aromatic amine salts such as isoquinoline salts, for example, tetramethylammonium salt, tetraethylammonium salt, benzyltrimethylammonium salt, benzyltriethylammonium salt,
Quaternary ammonium salts such as benzyltributylammonium salt, methyltrioctylammonium salt, and tetrabutylammonium salt, for example, basic amino acid salts such as arginine salt and lysine salt.

These salts and free carboxylic acids can be readily converted into each other by methods well known in the chemical arts.

The compound of the present invention inhibits the growth of tumor cells and exerts an antitumor effect. However, when the compound of the present invention is used as an antitumor agent, various administration forms can be selected. Examples include oral preparations such as capsules, powders, granules and liquids, and sterile liquid parenteral preparations such as solutions and suspensions.

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. Examples of such additives include 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, for example, polyvinyl, and the like. Synthetic polymers such as pyrrolidone or 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 , Other,
Examples of commonly used additives such as water, gelatin, talc, vegetable oil, and gum arabic are included.

These 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.

The liquid preparation is prepared by using an appropriate additive usually used in liquid preparations such as water, alcohols or plant-derived oils such as soybean oil, peanut oil or sesame oil, and is used as a suspension, syrup or injection. It is manufactured as such a form.

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, and 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), and the like, 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.

A liquid preparation such as a suspension or syrup for oral administration contains 0.5 to 10% by weight of an active ingredient.

It should be noted that the actually 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.

[0042]

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-56980 Production Method Actinomyces A56980 strain inoculated on a soft slope agar medium was prepared using 1.0% glucose, 2.0% dextrin, 0.2% soybean meal, 0.2% wheat germ, 0.6% polypeptone.
Medium (pH 7.0) 110 consisting of 5%, yeast extract 0.3%, humic acid 0.03%, and calcium carbonate 0.1%
inoculate three 500 ml Erlenmeyer flasks containing
The cells were cultured at 28 ° C. for 216 hours on a rotary shaker (180 rotations per minute).

The thus obtained culture solution (about 330
ml), the filtrate is separated by filtration, adjusted to pH 3 with 1N hydrochloric acid, adsorbed on HP-20, washed with water,
The active substance was eluted with methanol. The eluate was concentrated under reduced pressure, and ethyl acetate (150 ml × 2) was added. The obtained ethyl acetate extract was concentrated under reduced pressure, and the residue was purified using a column for chromatography (2.5 × 30 cm) of Sephadex LH-20 (manufactured by Pharmacia).
Elution with acetonitrile / water (10: 5: 1), BE-
The fraction containing 56980 was concentrated to dryness to give BE-
25.2 mg of 56980 was obtained.

The preparation 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 the substance (BE-56980), 15 parts of heavy magnesium oxide, and 75 parts of lactose were uniformly mixed to give 350 parts.
Powder or fine-grained powder having a particle size of μm or less. This powder was placed in a capsule container to obtain a capsule. Formulation Example 2 45 parts of the substance (BE-56980), 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 1410 to 177μ.
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. Formulation Example 5 0.6 part of this substance (BE-56980), 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.

[0045]

INDUSTRIAL APPLICABILITY 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.

[0046]

──────────────────────────────────────────────────続 き Continuing on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical indication location C12R 1: 045) (C12P 19/56 C12R 1: 045) (72) Inventor Katsuhisa Kojiri Ibaraki 3, Okubo, Tsukuba City, Tsukuba Research Laboratories of Banyu Pharmaceutical Co., Ltd. (72) Inventor Hiroyuki Suda, 3, Okubo Tsukuba City, Ibaraki Pref.

Claims (6)

[Claims] 1. A compound of the general formula [I] [Wherein R represents a hydrogen atom or a lower alkyl group] or a pharmaceutically acceptable salt thereof. 2. Actinoplanes
es) belongs to the genus and has the formula [II] A microorganism having the ability to produce the compound represented by the formula is cultured, and the compound represented by the formula [II] is collected from the culture solution and the cells, and if necessary, converted into a pharmaceutically acceptable salt. A method for producing a compound represented by the formula [II] or a pharmaceutically acceptable salt thereof. 3. A compound of the formula [II] A microorganism having the ability to produce the compound represented by the formula: Actinoplanes SP A 56980 (Ati
noplanes sp. A 56980) or a mutant thereof. 4. A compound of the general formula [I] [Wherein R represents a hydrogen atom or a lower alkyl group] or a pharmaceutically acceptable salt thereof as an active ingredient. 5. A compound of the formula [II] A microorganism belonging to the genus Actinoplanes, having the ability to produce the compound represented by the formula: 6. Actinoplan
es) is a microorganism belonging to the genus Actinoplanes sp A 56980 (Actinoplanes s).
p. A 56980) or a mutant thereof.
The microorganism according to the above.