JPH08198861A - Antitumor antibiotic be-39907b - Google Patents

Abstract

PURPOSE: To obtain a new antitumor antibiotic useful as a curing agent for cancers, etc., and having antitumor activities by inhibiting proliferation of tumor cells in mice and humans. CONSTITUTION: This new antitumor antibiotic is a compound (BE-39907B) expressed by the formula or its pharmaceutically allowable salt. This antitumor antibiotic BE-39907B has the following characteristics. Appearance: orange red solid; molecular formula: C14 H11 N3 O3 ; mass analysis [high resolution FAB-MS] as (M+H)<+> : 270.0906 (found value), 270.0879 (calculated value); solubility: easily soluble in DMSO, slightly soluble in MeOH, acetone, EtOAc and insoluble in water, hexane, CHCl3 ; color reaction: positive for phosphomolybdic acid reaction; basic material; etc. The compound of the formula can be obtained by culturing microorganisms of Saccharopolyspora-sp. A39907 (FERM-P-14071) belonging to the genus Saccharopolyspora or its variant and harvesting from the culture medium and the mycelia.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention is useful in the field of medicine, and more specifically, a novel compound having an antitumor activity by inhibiting the growth of tumor cells, a method for producing the same, a use thereof and a production of the compound. It relates to microorganisms that do.

[0002]

In the field of cancer chemotherapy, many compounds have already been put to practical use as pharmaceuticals. However, its effect is not always sufficient for various types of tumors, and its clinical applicability is becoming complicated as the phenomenon of tumor cell resistance to these drugs is revealed clinically [47th Japanese Cancer Society General Assembly Article, 12
Pp.-15 (1988)].

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

[0004]

The object of the present invention is to provide a novel antitumor substance which can meet the above-mentioned needs. That is, it is a problem to be solved by the present invention to provide a compound that exhibits an antitumor effect against various tumors for which existing antitumor substances cannot sufficiently exert the effect.

[0005]

Means for Solving the Problems In order to solve the above problems, the present inventors extensively screened secondary microbial metabolites for substances having antitumor activity, and as a result, are represented by the following formula [1]. The present invention has been completed by finding that the compound has an excellent antitumor effect.

That is, the present invention has a novel formula [1]

[0007]

[Chemical 4] Or a pharmaceutically acceptable salt thereof, a process for producing the same, and an ability to produce the compound of formula [1].
Lyspora) genus.

The physicochemical properties of the novel antitumor substance BE-39907B according to the present invention are shown below.

The meanings of 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: physicochemical properties of Hertz BE-39907B : red-orange solid molecular formula: C ₁₄ H ₁₁ N ₃ O ₃ mass spectrometry: [High-resolution FAB-MS] (M + H) ⁺ : Actual value 270.0906 Calculated value 270.0879 Ultraviolet and visible absorption spectrum: λmax (MeO
H, nm) 248, 262, 417, 431 Red external absorption spectrum: (KBr, cm ^-1 ) 3385,
2924, 1670, 1624, 1593, 1508,
1410 ¹ H-NMR spectrum: (400 MHz, DMSO-
d ₆ ) δppm: 2.85 (3H, d, J = 5.4H
z), 6.10 (1H, s), 6.40 (1H, s),
7.29 (1H, q, J = 5.4 Hz), 7.48 (1
H, br. s), 7.54 (1H, d, J = 8.5H
z), 7.92 (1H, dd, J = 8.5, 2.2H
z), 8.13 (1H, br.s), 8.20 (1H,
d, J = 2.2 Hz) ¹³ C-NMR spectrum (100 MHz, DMSO-d
₆ ) δppm: 29.0, 95.0, 103.8, 11
5.8, 127.0, 127.6, 131.1, 13
3.3, 143.7, 147.1, 148.5, 14
9.2, 166.6, 180.0 Solubility: Easily soluble in dimethylsulfoxide, sparingly soluble in methanol, acetone and ethyl acetate, insoluble in water, hexane and chloroform.

Distinction between acidic, neutral and basic substances: Basic substance Rf value: 0.34 [Kieselgel 60F ₂₅₄ manufactured by Merck & Co., Inc.
Used / developing solvent: chloroform / methanol (10:
1)] Color reaction: phosphomolybdic acid reaction Positive The compound of the present invention can exist in the form of a salt, and examples of such a salt include inorganic acid salts such as hydrochloride and sulfate, citrate and lactic acid. An organic acid salt such as a salt may be used.

Biological activity of BE-39907B (antitumor
To determine the growth inhibitory action against 瘍作for) antitumor experimental mouse tumor cells of a substance BE-39907B, the test was conducted in vitro. The antitumor effect test against mouse leukemia cells P388 was carried out by dissolving BE-39907B in dimethylsulfoxide and then serially diluting it with dimethylsulfoxide, and then adding 10% fetal bovine serum-containing RPMI1640 medium (2
0 mM 2-mercaptoethanol was included) to give a test solution. Cell culture medium containing 1 × 10 ³ tumor cells (RPMI1640 medium containing 10% fetal bovine serum, 20 m
M-containing 2-mercaptoethanol) 50 μl 9
Dispense into a 6-well microplate and incubate at 37 ° C for 24 hours, 5
After culturing under% CO ₂ , 50 μl of the above test solution was added, and after culturing at 37 ° C. for 72 hours under 5% CO ₂ , MTT
The measurement method was used to compare with the control group.

In the antitumor test against mouse colon cancer cell colon 26, BE-39907B was dissolved in dimethylsulfoxide and then serially diluted with dimethylsulfoxide, and then added to RPMI1640 medium containing 10% fetal bovine serum to prepare a test solution. Cell culture medium containing 1 × 10 ³ tumor cells (RPMI1640 medium containing 10% fetal bovine serum)
Dispense 100 μl into a 96-well microplate, 37 ℃
After culturing in 5% CO ₂ for 24 hours, the above test solution 1
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 10 mM Tris solution. . 5 with 450nm as reference wavelength
The absorbance at 50 nm was measured and compared with the control group.
As a result, BE-39907B showed a strong growth inhibitory activity against both tumor cells and had a 50% growth inhibitory concentration (IC ₅₀ ).
Was as shown in Table 1.

Furthermore, the antitumor activity of BE-39907B against human tumor cells was tested in vitro. Human colon cancer cells DLD-1, human lung cancer cells PC-13 and human gastric cancer cells MKN-45 were used as cells, and all tumor cells were used as a cell culture medium in RPMI containing 10% fetal bovine serum.
Using the 1640 medium, the above mouse colon cancer cell colo
It measured using the method similar to n26 cell. As a result, BE-39907B showed a strong growth inhibitory activity against human tumor cells, and its 50% growth inhibitory concentration (I
C ₅₀ ) was as shown in Table 1.

[0015]

[Table 1] As described above, BE-39907B 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-39907B will be described.

Antitumor substance BE-39907B of the present invention
The microorganism used for the production of the above or a mutant strain thereof may be any as long as it produces the antitumor substance BE-39907B, and examples thereof include microorganisms having the following mycological properties.

Bacteriological Properties of A39907 Strain 1. Morphology A39907 strain is a spore (1.
3 to 1.0 × 0.7 to 0.6 μm) are connected to form long straight and curved long spore chains, and the surface of the spore sheath is hair-like. Rupture of basal hyphae is observed, but special organs such as sporangia, flagella spores, and sclerotium are not observed. 2. Culture properties in various agar plates (28 ° C 14
Day culture)

[0019]

[Table 2] 3. Growth temperature (starch / inorganic salt agar culture for 14 days) Optimal growth conditions 12 ° C: No growth 16 ° C: Growth is trace level 19 ° C: Good growth, no aerial mycelium formation 23 ° C: Very good growth, aerial mycelia Good 28 ° C: Very good growth, good formation of aerial mycelium 32 ° C: Very good growth, good formation of aerial mycelium 37 ° C: Good growth, no formation of aerial mycelium 43 ° C: No growth 4. Physiological properties (1) Gelatin liquefaction positive (glucose / peptone / gelatin medium) (2) Starch hydrolysis positive (starch / inorganic salt agar medium) (3) Skim milk powder coagulation negative (skimmed milk medium) (4) 4. Peptonization of skim milk powder positive (skim milk medium) (5) Formation of melanin-like pigment negative (6) Salt tolerance Growing at 13% or less salt content (yeast / malt agar medium) 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.

[0020]

[Table 3] 6. Cell wall composition meso-diaminopimelic acid, arabinose and galactose were detected in the cell wall, and the cell wall type was I.
It is considered to be V-shaped. The whole cell sugar components characteristic of classification are arabinose and galactose, and the sugar pattern is A
It was a mold. Further, mycolic acid is not detected, the main menaquinone was MK-9 (H ₄₎ and MK-10 (H _4).

From the above mycological characteristics, the A39907 strain is considered to belong to the actinomycete Saccharopolispora.
Therefore, the A39907 strain was replaced with Saccharopolyspora sp. A39907 (Saccharopolyspor).
a sp. A 39907).

The bacterial strain has been deposited at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry, and the deposit number of the Institute of Microindustry is Microindustry Research Institute No. 14071 (FERM P-1).
4071).

The antitumor substance BE-39 used in the present invention
The mutant strain of the 907B-producing microorganism is, for example, irradiated with X-rays or ultraviolet rays, for example, nitrogen mustard, azaserine, nitrous acid, 2-aminopurine or N-methyl-N′-nitro-N-nitrosoguanidine (NTG). ) Etc. treatment with a mutagen, phage contact,
It is a microorganism obtained by mutating a BE-39907B-producing bacterium by a commonly used bacterial species conversion treatment method such as transformation, transduction or conjugation.

In producing BE-39907B of the present invention, BE-39907B-producing strain is inoculated into a nutrient-source-containing medium to aerobically grow to obtain BE-3.
A culture containing 9907B is obtained. As a nutrient source,
Known sources of actinomycetes can be used. For example, as the carbon source, commercially available glucose, maltose, starch, saccharose, molasses, dextrin, etc. are used alone or as a mixture. As a nitrogen source, commercially available soybean flour, corn stay liquor, meat extract,
Yeast extract, dry yeast, cottonseed flour, peptone, wheat germ,
Fish meal, meat meal, defatted rice bran, defatted meat and bone meal, inorganic ammonium salts, sodium nitrate, etc. may be 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, a trace amount of a heavy metal salt such as iron, manganese, zinc, cobalt, molybdic acid can be added. When the foaming is severe, a vegetable oil such as soybean oil or linseed oil, higher alcohols such as octadecanol, and various silicon compounds may be appropriately added as an antifoaming agent. In addition to these, BE-
Anything useful for the production of 39907B, 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 solid culture or liquid culture. In the case of liquid culture, static culture, stirring culture,
Either shaking culture or aeration culture may be carried out, but shaking culture or deep aeration stirring culture is particularly desirable. The culture temperature is appropriately 19 to 37 ° C., but preferably 23
~ 32 ° C. The preferable pH of the medium is in the range of 4 to 8, and the culture time is 72 hours to 288 hours, preferably 14 hours.
It is 4 hours to 240 hours. Targeted BE from culture
To collect -39907B, a separation means usually used for collecting from metabolites produced by microorganisms is appropriately used.

BE-39907B is present in the culture filtrate and in the cells, mainly in the filtrate, and therefore, it is usually separated from the culture filtrate and the cells by the solvent extraction method, ion exchange resin method or adsorption or partition chromatography. The purification can be carried out by a single method or a combination of the methods such as a graphic method and a gel filtration method.

The following method is mentioned as an example of preferable separation and purification. First, the culture solution is filtered to obtain a filtrate. The filtrate was extracted with ethyl acetate, the extract was concentrated under reduced pressure, and then silica gel column chromatography (chloroform / methanol).
Then, a fraction containing BE-39907B is obtained. A solid of BE-39907B can be obtained by purifying this by gel filtration chromatography.

The compound BE-39907B 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 agents such as tablets, capsules, powders, granules or solutions, or sterile liquid parenteral agents such as solutions or suspensions.

The solid preparation can be produced as it is in the form of tablets, capsules, granules or powders, or can be produced using appropriate additives. Examples of such additives include 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, for example. Synthesis of synthetic polymers such as polyvinylpyrrolidone or polyalkylene glycol, fatty acid salts such as calcium stearate or magnesium stearate, alcohols such as stearyl alcohol or benzyl alcohol, such as methyl cellulose, carboxymethyl cellulose, ethyl cellulose or hydroxypropyl methyl cellulose Cellulose derivatives or commonly used additives such as water, gelatin, talc, vegetable oil, gum arabic Thing, and the like.

The solid preparations such as tablets, capsules, granules and powders are generally 0.1 to 100% by weight,
It preferably contains from 5 to 100% by weight of active ingredient.

The liquid preparation uses suspensions, syrups or injections which are prepared by using appropriate additives usually used in liquid preparations such as water, alcohols or plant-derived oils such as soybean oil, peanut oil or sesame oil. It is manufactured as a form such as.

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, a liquid for intravenous injection (for example, an aqueous solution of citric acid and sodium citrate) or an electrolyte solution (for intravenous drip and intravenous injection), or a mixed solution thereof.

These injections may be pre-dissolved, or may be in the form of powder or dissolved with a suitable additive before use. These injection solutions usually contain 0.1-10% by weight, preferably 1-5% by weight of the active ingredient.

Further, the liquid preparation such as suspension or syrup for oral administration 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 depending on the type of compound used, the type of composition formulated, the frequency of application and the particular site to be treated, host and tumor. Is. For example, the daily dose for adults is 10 to 50 in the case of oral administration.
0 mg, preferably 10 to 100 mg per day for parenteral administration, preferably for intravenous injection. The administration frequency is 1 to 5 times, though it varies depending on the administration method and symptoms.

[0036]

The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

Example 1: Method for producing BE-39907B Actinomycete A39907 strain inoculated on a slope agar medium was glucose 0.2%, dextrin 2.0%, meat meal 0.5%, defatted rice rice bran 0.5%. , Defatted meat and bone meal 0.2%,
Dry yeast 0.1%, magnesium sulfate 0.05%, sodium bromide 0.05%, sodium chloride 0.5%, dipotassium hydrogen phosphate 0.1%, ferrous sulfate 0.0002
%, Cupric chloride 0.00004%, manganese chloride 0.0
0004%, cobalt chloride 0.00004%, zinc sulfate 0.00008%, sodium borate 0.00008
%, And 500 ml Erlenmeyer flasks containing 100 ml of a medium (pH 7.2) consisting of 0.00024% ammonium molybdate were inoculated and cultured at 28 ° C. for 96 hours on a rotary shaker (180 rpm). . Add this culture to 3
100 ml of a 500 ml Erlenmeyer flask containing 100 ml of the above medium was inoculated and cultured at 28 ° C. for 240 hours on a rotary shaker (180 rpm).

The culture broth thus obtained (about 10
10 L of ethyl acetate was added to L), and the mixture was stirred at room temperature for 30 minutes. Celite was added thereto, and the cells were separated by a filtration method to obtain an ethyl acetate layer. The ethyl acetate layer was dehydrated with anhydrous sodium sulfate and then concentrated to dryness under reduced pressure. The residue was dissolved in 100 ml of chloroform and the silica gel column (100
g, adsorbed on Kieselgel 60 manufactured by Merck & Co., Inc., and eluted successively with chloroform, chloroform / methanol (20: 1), chloroform / methanol (10: 1), chloroform / methanol (5: 1). BE-39907B eluted with chloroform / methanol (5: 1)
The fractions containing were collected, reapplied to a 40 g silica gel column, and eluted with chloroform / methanol (20: 1) and chloroform / methanol (5: 1). BE-399
The fractions containing 07B were collected, dissolved in 8 ml of chloroform / methanol (1: 1), applied to a Sephadex LH-20 column (1.7 × 89 cm, manufactured by Pharmacia) twice 4 ml each, and chloroform / methanol (1: 1). 1)
The development was carried out with to obtain 4.9 mg of a reddish orange solid of BE-39907B.

Formulation examples of the compound of the present invention are shown below.
The formulation of the compound of the present invention is not limited to this formulation example.

Formulation Example 1 10 parts of this substance (BE-39907B), 15 parts of heavy magnesium oxide, and 75 parts of lactose are uniformly mixed to obtain a powdery or finely divided powder having a particle size of 350 μm or less. This powder was put into a capsule container to give a capsule.

Formulation Example 2 This substance (BE-39907B) 45 parts Starch 15 parts Lactose 16 parts Crystalline cellulose 21 parts Polyvinyl alcohol 3 parts Distilled water 30 parts Evenly mix, then crush granulate and dry, then It was sieved to obtain granules having a diameter of 1410 to 177 μm.

Formulation Example 3 A granule was prepared in the same manner as in Formulation Example 2, and then 96 parts of this granule was added with 3 parts of calcium stearate and compression-molded 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 give tablets having a diameter of 8 mm, and then syrup was added thereto. Sugar-coated tablets were prepared by adding a mixed suspension of gelatin and precipitated calcium carbonate.

[0044]

INDUSTRIAL APPLICABILITY BE-39907B described in the present invention
Shows 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.

[0045]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display area // (C12N 1/20 C12R 1:01) (C12P 17/14 C12R 1:01) (72) Inventor Hajime Suzuki, 3 Okubo, Tsukuba City, Ibaraki Prefecture, within the Tsukuba Research Center, Banyu Pharmaceutical Co., Ltd. (72) Inventor, Hiroyuki Suda, 3 Okubo, Tsukuba City, Ibaraki Prefecture, within the Tsukuba Research Center, Banyu Pharmaceutical Co., Ltd.

Claims (6)

[Claims] 1. A formula [1]: The compound represented by or a pharmaceutically acceptable salt thereof. 2. Saccharo
belongs to the genus polyspora) and has the formula [1] A microorganism or a mutant strain thereof having the ability to produce a compound represented by the formula (1) is cultured, the compound represented by the formula [1] is collected from the culture solution and cells, and if necessary, a pharmaceutically acceptable salt. A method for producing a compound represented by the formula [1] or a pharmaceutically acceptable salt thereof: 3. A microorganism or its mutant strain is Saccharopolyspora sp. A39907 (Saccharopo).
lyspora sp. A39907).
The manufacturing method described. 4. A formula [1]: An antitumor agent comprising a compound represented by: or a pharmaceutically acceptable salt thereof as an active ingredient. 5. A microorganism belonging to the genus Saccharopolispora or a mutant strain thereof having the ability to produce the compound represented by the formula [1]. 6. The microorganism is Saccharopolyspora sp. A39907 (Saccharopolyspor).
a sp. A39907), The microorganism or a mutant strain thereof according to claim 5.