JP2803178B2 - Antitumor substance BE-16493 - Google Patents

Description

The present invention is useful in the field of medicine, and more specifically, a novel compound that inhibits the growth of tumor cells and exerts an anticancer effect, a method for producing the same, its use, and It relates to a novel microorganism Streptomyces sp.

Prior Art In the field of cancer chemotherapy, bleomycin (Bleo
Mycin) and many microbial metabolites such as Adriamycin have been clinically applied,
It is also used in clinical practice. However, its effect on various types of tumors is not always sufficient, and its clinical applicability is complicated as the phenomenon of tumor cell resistance to these drugs becomes clinically apparent. (The 47th Annual Meeting of the Japanese Cancer Society, 12 pages-
15 (1988)].

Problems to be Solved by the Invention Under such circumstances, there is a clinical demand for the development of new regulated cancer substances. In other words, there is a need for a substance that is effective in overcoming resistance to existing anticancer substances and that is effective against cancers of a type in which existing anticancer substances cannot sufficiently exert their effects.

Means for Solving the Problems In order to solve the above problems, the present inventors have screened a wide range of microbial metabolites for antitumor substances, and as a result, the compounds represented by the following general formula have excellent antitumor effects. The inventors have found what has been shown and completed the present invention.

That is, the present invention uses the formula Or a pharmaceutically acceptable salt thereof, a method for producing the same and a use thereof. In addition, the present invention relates to a novel microorganism belonging to the genus Streptomyces having the ability to produce BE-16493.

The physicochemical properties of the compound according to the present invention are shown below.

Physicochemical properties of BE-16493 Properties: orange amorphous solid or crystal Molecular formula: C ₃₈ H ₄₀ O ₁₅ Elemental analysis: theoretical 61.95% carbon, 5.47% hydrogen Found 61.81% carbon, 5.50% hydrogen Melting point: 180-188 ° C (decomposition point) Mass spectrum: FAB mass spectrum is shown in Fig. 1 (m /
z 738, M + 2H) UV spectrum: UV measured in methanol (30 μg / ml)
The spectrum is shown in FIG.

IR spectrum: The IR spectrum obtained by the KBr tablet method is shown in FIG. ¹ H-NMR spectrum: d ₆ - ¹ H-NMR was measured in acetone
The spectrum (300 MHz) is shown in FIG. ¹³ C-NMR spectrum: ¹³ C-NM measured in d ₆ -acetone
The R spectrum (75 MHz) is shown in FIG.

Solubility: Poorly soluble in benzene and n-hexene, soluble in alkaline water, and easily soluble in organic solvents such as methanol and acetone.

Acidic, neutral, basic substances distinguished: acidic substance Rf value: 0.36 (Merck, Kieselgel 60f ₂₅₄ used, developing solvent: chloroform / methanol / acetic acid (5: 1: 0.0
6)) Color reaction: Potassium permanganate reaction Positive sulfuric acid reaction Positive biological activity of BE-16493 To determine the inhibitory effect of the antitumor substance BE-16493 on experimental tumor cells in mice, an in vitro test was performed. went. The in vitro antitumor effect test on P388 tumor cells was performed by first dissolving the test compound in dimethyl sulfoxide, and then serially diluting it with a cell culture medium containing 20% dimethyl sulfoxide (20% DMSO-RPMI-1640 medium). 3 × 10
It was added 2μ to ⁴ for cell culture comprising tumor cell medium (calf serum 10% RPMI-1640 medium containing) 200 [mu]. 37
After culturing at 5 ° C. for 72 hours under 5% CO ₂ , the number of surviving cells was counted using a Coulter counter and compared with the control group. As a result, BE-16493, P388 tumor cells,
It exhibits a strong growth inhibitory effect, and the concentration (IC ₅₀ ) of BE-16493 at which the growth of the tumor cells is inhibited by 50% is 0. 0 against P388 / S cells.
9 μg / ml and 0.75 μg / ml for P388 / V cells.

Here, P388 / S is a kind of normal mouse leukemia cells, and P388 / V cells are P388 leukemia cells that have acquired resistance to the anticancer substance vincristine.

As described above, the compound of the present invention represented by the above formula (I) has a remarkable growth inhibitory effect on experimental mouse cancer cells. Therefore, the present invention is useful as a therapeutic agent for tumors in warm-blooded mammals.

The present invention also provides a pharmaceutical composition comprising an effective amount of the compound of the present invention alone or an effective amount of the compound of the present invention and an inert and pharmaceutically acceptable carrier when the compound of the present invention is used as an antitumor agent. Is done.

This pharmaceutical composition is manufactured by combining the compound of the present invention with an inert pharmaceutical carrier, and is orally administered in various formulations.
It is administered parenterally or topically. Examples of suitable compositions include solid compositions for oral administration such as tablets, capsules, pills, powders, granules and the like, liquid compositions for oral administration such as solutions, suspensions and emulsions. It can also be manufactured in the form of a sterile composition which can be dissolved in sterile water, physiological saline or another sterile injectable solvent immediately before use.

Typical examples of pharmaceutically acceptable salts of the compounds of the present invention include, for example, an equivalent of an alkali such as sodium hydroxide, potassium hydroxide or sodium hydrogen carbonate or triethylamine or 2-aminoethanol. It is an alkali addition salt formed by adding an organic amine.

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. The dose per adult per day is 10 to 500 mg for oral administration, and 10 to 500 mg per day for parenteral administration, preferably intravenous injection.
100 mg. The number of administrations varies depending on the administration method and symptoms, but is once or several times.

 Next, a method for producing BE-16493 will be described.

The microorganism or a mutant thereof used for producing the antitumor substance BE-16493 of the present invention may be any microorganism that produces the antitumor substance BE-16493, and for example, a microorganism having the following mycological properties: Can be mentioned.

1. Form BA16493 strain develops an almost straight chain of 20 or more spores from the tip of the aerial hyphae, which does not grow much under a microscope.

The spores are cylindrical with a size of about 0.5 × 1.0 μm, and no special organs such as sclerotium and spores are not observed. The spore surface is smooth.

2. Culture characteristics on various agar plate media Table 1 shows the results of culturing on various agar plate media at 28 ° C for 21 days.

3. Growth temperature (yeast / malt agar medium, cultivation for 21 days) 12 ° C: poor growth and no aerial mycelium formation 20 ° C: good growth and no aerial mycelium formation 28 ° C: good growth and aerial mycelium formation 37 ° C: good growth and no aerial mycelium formation 45 ° C: no growth 4. Physiological properties (1) Gelatin liquefaction 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) Peptone formation of skim milk powder Negative (Skim milk medium) (5) Melanin-like pigment formation Negative (6) Salt tolerance Salt content 2 5% or less (yeast / malt agar medium) 5. Utilization ability of carbon source Prideham / Godlive agar was used as a base medium, and the following various sugars were added thereto and cultured at 28 ° C. for 21 days. Table 2 shows the results.

6. Cell wall composition LL-diaminopimelic acid and glycine were detected.

From the above bacteriological properties, it was found that BA16493 strain is a radiobacterium belonging to the genus Streptomyces.

Therefore, the present inventors have established the BA16493 strain as Streptomyces sp. BA16493 (Streptomyces sp. BA1649).
3).

This strain has been deposited with the Research Institute of Microbial Industry and Technology of the Ministry of International Trade and Industry of Japan, and the deposit number of the microtechnical laboratory is No. 10750 (FERM P-10750).

The mutant strain of the microorganism producing the antitumor substance BE-16493 used in the present invention may be, for example, irradiated with X-rays or ultraviolet rays, for example, nitrogen mustard, azaserine, nitrite, 2-aminopurine or N-methyl. −
The antitumor substance BE-16493-producing bacterium can be obtained by a commonly used bacterial species conversion treatment method such as treatment with a mutagenic agent such as N'-nitro-N-nitrosoguanidine (NTG), phage contact, transformation, transduction or conjugation. Is a microorganism in which is mutated.

In producing the BE-16493 of the present invention, a culture containing the BE-16493 can be obtained by inoculating a medium containing a nutrient source containing the BA-16493 producing strain of the BE-16493 and allowing it to grow aerobically. As a nutrient source, a known nutrient source for radioactive bacteria can be used. For example, as a carbon source, commercially available glucose, glycerin, maltose, starch, sucrose, molasses, dextrin, or the like is used alone or as a mixture. As the nitrogen source, commercially available soybean flour, corn steep liquor, meat extract, yeast extract, cottonseed flour, peptone, wheat germ, fish meal, inorganic ammonium salt, sodium nitrate and the like are used alone or as a mixture. As the inorganic salt, commercially available calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate or various phosphates can be used. If necessary, a trace amount of a heavy metal salt such as iron, manganese or zinc can be added. When foaming is remarkable, 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, as long as the production bacterium utilizes and is useful for production of BE-16493,
Either can be used.

The culturing method may be the same as a general method for producing a metabolite of a microorganism, 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 preferable. Culture temperature is 20
C. to 37.degree. C. is suitable, but 25.degree. The preferred pH of the medium is in the range of 4 to 8, and the culture time is 24 hours to 19 hours.
2 hours, preferably 48 hours to 120 hours.

In order to collect the desired BE-16493 of the present invention from the culture, a separation means usually used for collecting from a culture of a microorganism is appropriately used. Since BE-16493 of the present invention is present in the culture filtrate and the cells, usual separation means from the culture filtrate or the cells, for example, a solvent extraction method, an ion exchange resin method or an adsorption or partition chromatography method and a gel filtration method Etc. can be purified alone or in combination. High performance liquid chromatography and thin layer chromatography can also be used for extraction and purification.

Preferred examples of separation-purification include the following methods. First, the culture filtrate is centrifuged to separate cells and culture supernatant. The cells are extracted with an organic solvent such as methanol or acetone, and the extract is concentrated under reduced pressure. PH of concentrated solution
Is acidified, extracted with an organic solvent such as ethyl acetate, and concentrated under reduced pressure to obtain a crude substance containing BE-16493.
If the obtained crude substance is purified using silica gel column chromatography or the like, the BE-16 of the present invention is obtained.
493 can be obtained as an orange crystalline powder.

Next, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the examples,
The modification means of the examples include, of course, all methods for producing, concentrating, extracting, and purifying BE-16493 using known means based on the properties of BE-16493 revealed by the present invention.

Example Radioactive bacteria BA16493 cultured on a slope agar medium were glycerin 2%, malt molasses 1%, meat extract 0.2%, cottonseed powder 0.5%, yeast extract 0.1%, wheat malt 0.5%, magnesium sulfate 0.2
110%, sodium chloride 0.2%, calcium carbonate 0.2%, monopotassium phosphate 0.1%, ammonium sulfate 0.1%, ferrous sulfate 0.001%, zinc sulfate 0.001% medium (pH 6.7) 110
inoculate two 500 ml Erlenmeyer flasks containing
The cells were cultured for 2 hours on a rotary shaker (180 rotations per minute). Each 1 ml of this culture solution was brought into contact with nine 500 ml Erlenmeyer flasks each containing 110 ml of the above medium, and cultured at 28 ° C. for 120 hours on a rotary shaker (180 rotations per minute). The culture solution (about 1) was filtered by a filtration method, and the obtained cells were washed with 500 ml of deionized water.
Stirred for hours. A methanol extract was obtained by a filtration method,
This methanol extract was concentrated to about 300 ml. The obtained concentrate was extracted with ethyl acetate 1 to obtain an ethyl acetate extract.

On the other hand, the pH of 900 ml of the culture filtrate obtained by the filtration method was adjusted to 2
The mixture was made up to about 2.5 with N hydrochloric acid, extracted with ethyl acetate 3, combined with the ethyl acetate extract from the previously extracted cells, added with anhydrous sodium sulfate, left overnight, and concentrated under reduced pressure.
The residue was washed with a small amount of n-hexane to obtain a crude material containing 2.6 g of BE-16493. The obtained crude substance was subjected to silica gel column chromatography (3 × 30 cm), developed with a mixed solution of chloroform-methanol (10: 1), and then developed with a mixed solvent of chloroform-methanol-acetic acid (10: 1: 0.1). Elution was performed, and the fraction containing BE-16493 was concentrated under reduced pressure to obtain 950 mg of a residue. Perform silica gel column chromatography (3 × 30 cm) again, eluting with a mixed solvent of chloroform-methanol-acetic acid (20: 1: 0.1),
By concentrating the fraction containing BE-16493 under reduced pressure, B
860 mg of E-16493 were obtained as an orange crystalline powder.

Effects of the Invention BE-16493 described in the present invention has a growth inhibitory effect on tumor cells having resistance to existing anticancer agents as well as cells that have not acquired resistance to the anticancer agents,
It is useful as a therapeutic agent for cancer in the field of medicine.

[Brief description of the drawings]

FIG. 1 is a diagram of the FAB-mass spectrum of BE-16493. FIG. 2 is a drawing of an absorption spectrum of ultraviolet and visible portions of BE-16493 in methanol. FIG.
It is a figure of the infrared absorption spectrum by the KBr tablet method of BE-16493. FIG. 4 shows the results of BE-16493 in d ₆ -acetone.
It is a figure of a 00MHz ¹ H-NMR spectrum. FIG. 5 shows BE-1
FIG. 6 is a diagram of a 75 MHz ¹³ C-NMR spectrum of 6493 in d ₆ -acetone.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1: 465) (C12P 17/16 C12R 1: 465) (72) Inventor Masanori Okanishi 2-9-1 Shimomeguro, Meguro-ku, Tokyo No. 3 Banyu Pharmaceutical Co., Ltd. Investigation Researcher Mayumi Saito (58) Field surveyed (Int. Cl. ⁶ , DB name) C12P 17/00-17/18 C07D 407/12 A61K 31/35 C12N 1/20 CA (STN) REGISTRY (STN) BIOSIS (DIALOG) WPI (DIALOG)

Claims (5)

(57) [Claims] (1) Expression Or a pharmaceutically acceptable salt thereof. (2) An antitumor agent comprising, as an active ingredient, an antitumor substance BE-16493 represented by the formula: or a pharmaceutically acceptable salt thereof. 3. The expression In producing the antitumor substance BE-16493 represented by or a pharmaceutically acceptable salt thereof, a microorganism producing the antitumor substance BE-16493 or a mutant thereof is cultured and the antitumor substance BE is produced.
-A production method characterized by accumulating and collecting 16493. 4. Streptomyces sp. BA16493 (Str.
The method according to claim 3, wherein the eptomyces sp. BA16493) strain or a mutant strain thereof is cultured. (5) A Streptomyces sp. BA16493 strain or a mutant thereof having the ability to produce the antitumor substance BE-16493 represented by