JP2000007565A - Antitumor agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an antibiotic exhibiting an excellent antitumor action against solid tumor and multiple medicine-resistant tumor and capable of being used for cancer chemotherapy by compounding a specific compound (salt) as an active ingredient. SOLUTION: This antitumor agent contains a compound (salt) of the formula [R is CH3, CH(CH3)2 or CH(CH3)CH2CH3] as an active ingredient. The compound can be obtained by culturing Streptomyces sp. SNA 15896 strain to produce the compound in the culture product, and subsequently collecting the compound. The strain is deposited as FERM P-16667. The compound is obtained, for example, by a fermentation method using Streptomyces braegensis N617-29 strain.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an antitumor agent comprising a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient. The present invention is useful as an antitumor agent in cancer chemotherapy.

[0002]

2. Description of the Related Art In cancer chemotherapy, many compounds such as mitomycin C, fluorouracil and actinomycin D have already been put into practical use as pharmaceuticals. However, its effect on various tumors is not always sufficient, and the acquisition of resistance of tumor cells to these drugs is also a major obstacle, and the development of antitumor substances with new mechanisms of action has been required. I have.

[0003]

DISCLOSURE OF THE INVENTION In view of the above situation, the present inventors have sought to find a novel antitumor agent, and as a result, have found that a compound represented by the general formula (I) is a solid tumor or a multidrug resistant tumor. Have an excellent antitumor effect on Accordingly, an object of the present invention is to provide an antitumor agent comprising a compound represented by the general formula (I) or a pharmacologically acceptable salt thereof as an active ingredient.

[0004]

The present invention relates to an antitumor agent comprising a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient. The present invention is useful as an antitumor agent in cancer chemotherapy. The compound which is the active ingredient of the present invention includes UK-63,598 compounds (R = CH ₃ ), UK-65,662 compounds (R = CH (CH ₃ ) ₂ ) and UK-63,052 which are known as compounds having antibacterial activity. Compound (R = CH (C
H ₃ ) CH ₂ CH ₃ ) (Rance MJ et al., J. Antibiotics, Vo
l.42, No.2, 206 (1989)).

[0005]

Embedded image (Where R is CH ₃ , CH (CH ₃ ) ₂ , or CH (CH ₃ ) CH ₂ CH ₃
)

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The compound which is the active ingredient of the present invention was prepared by the present inventors using Streptomyces sp. Isolated from soil collected from Yuki City, Ibaraki Prefecture.
yces sp.) SNA15896 strain is cultured, the compound is produced in a culture, and the compound is collected. It should be noted that the strain used in the present invention was supplied to Life Science and Industrial Technology Research Institute, National Institute of Advanced Industrial Science and Technology by Life Science Bacteria No. 16667 (FERM P.
-16667). Further, the compound is known substances, for example, Streptomyces Burejenshisu (Streptomyces braegensis) fermentation method using N617-29 strain (Rance MJ et al., J. Antibiotics, Vol.4
2, No. 2, 206 (1989)). The compound of the present invention is produced in a culture of a nutrient-containing medium which can be used by ordinary microorganisms, by inoculating the microorganism with the production of the compound of the present invention and growing it. Any nutrient source may be used as long as it is used as a nutrient source for actinomycetes, and any of a synthetic medium, a semi-synthetic medium, and a natural medium can be used. For example, as a carbon source, glucose, glycerol, maltose, starch, sucrose, molasses, syrup, dextrin or the like is used alone or as a mixture. Nitrogen sources include soy flour, corn gluten meal, corn steep liquor, meat extract, yeast extract,
Organic nitrogen sources such as cottonseed meal, peptone, wheat germ, fish meal, and urea, and inorganic nitrogen sources such as ammonium sulfate and sodium nitrate are used alone or as a mixture. As the inorganic salt, calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate or various phosphates can be used.If necessary, heavy metal salts such as iron, copper, cobalt, molybdenum, manganese or zinc can be used. Can be added in a small amount. During culturing, when foaming is remarkable, various antifoaming agents known as antifoaming agents may be appropriately added to the medium. In addition, organic and inorganic substances which are used by the producing bacteria and are useful for producing the compound of the present invention can be appropriately used.

[0007] The method for culturing the strain 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, shaking culture or aeration culture may be performed, but when culturing Streptomyces sp.SNA 15896 strain, in particular, shaking culture or submerged culture is performed. Aeration stirring culture is preferred. Although it depends on the culture conditions, the preferred pH of the medium is in the range of 4 to 8, the culture temperature is 22 to 37 ° C,
Preferably, 25 to 30 ° C is appropriate. The culture time is 48 ~
168 hours, preferably 96-144 hours.

[0008] In order to isolate the desired compound which is the active ingredient of the present invention from the culture, a separation means usually used for isolating a metabolite produced by a microorganism may be appropriately used. Since the compound of the present invention produced by culturing is usually accumulated both inside and outside the cells in the culture, it is separated into a culture filtrate or cells by means such as centrifugation, filtration, etc. Normal separation means from the body, for example,
Separation / purification by dialysis, solvent extraction, method using difference in solubility with impurities, ion exchange resin method or adsorption or partition chromatography method and gel filtration method alone or in combination as appropriate, and in some cases repeated use can do.

When the preparation of the present invention is used as a medicine, the compound as an active ingredient of the present invention may be a pharmacologically acceptable salt. Salts with pharmacologically acceptable acids include inorganic acids with hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid And acid addition salts with organic acids such as succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, citric acid, tartaric acid, picric acid, methanesulfonic acid and glutamic acid. As a salt with a base, sodium, potassium, magnesium, calcium, an inorganic base such as aluminum, an organic base such as methylamine, ethylamine, ethanolamine, or lysine, arginine,
Examples thereof include salts with basic amino acids such as ornithine and ammonium salts. Further, the compound which is the active ingredient of the present invention may form a solvate with hydrate, ethanol or the like, or a crystalline polymorph. The compound or a pharmacologically acceptable salt thereof is safely orally and parenterally administered to humans and animals as a medicament. For parenteral administration, for example, intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, transdermal administration, pulmonary administration, nasal administration, enteral administration, buccal administration,
Transmucosal administration and the like are mentioned, and these preparations are administered.
For example, injections, suppositories, aerosols, transdermal absorption tapes and the like can be mentioned. Examples of oral administration preparations include tablets (including sugar-coated tablets, coated tablets, and buccal tablets), powders, capsules (including soft capsules), granules (coated substances, pills, troches, liquids, and preparations thereof. Sustained-release preparations that are chemically acceptable). Liquid preparations for oral administration include suspensions, emulsions, syrups (including dry syrups), elixirs and the like.

These preparations are administered as pharmaceutical compositions together with pharmacologically acceptable bases, carriers, excipients, disintegrants, lubricants, coloring agents, etc., according to known pharmaceutical preparation methods. Is done. As carriers and excipients used in these preparations, for example, lactose, glucose, sucrose, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, crystalline cellulose, licorice powder, gentian powder, etc. ,
For example, starch, tragacanth, gelatin, syrup, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, hydroxypropylcellulose, methylcellulose, ethylcellulose, carboxymethylcellulose, and the like, examples of disintegrants include starch, agar, gelatin powder, sodium carboxymethylcellulose, calcium carboxymethylcellulose, Crystalline cellulose, calcium carbonate, sodium bicarbonate, sodium alginate, etc., as lubricants, for example, magnesium stearate, talc, hydrogenated vegetable oil, macrogol, etc. , Respectively. tablet,
Granules include sucrose, gelatin, hydroxypropylcellulose, purified shellac, gelatin, glycerin,
Sorbitol, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, cellulose phthalate acetate,
It may be coated with hydroxypropyl methylcellulose phthalate, methyl methacrylate, methacrylic acid polymer, or the like, or may be coated with two or more layers. Further, capsules made of a substance such as ethyl cellulose or gelatin may be used. When preparing injections, a pH adjusting agent, a buffer, a stabilizing agent, a solubilizing agent, and the like are added to the main drug as needed, and each injection is prepared by a conventional method.

Examples of the form of the external preparation include solid, semi-solid, semi-solid, and liquid preparations for transdermal administration or transmucosal administration such as oral or nasal administration. Examples of the liquid preparation include pharmaceutically acceptable emulsions or emulsions such as lotions, tinctures for external use, and liquid preparations for transmucosal administration. This preparation contains, for example, ethanol, oils, emulsifiers and the like as commonly used diluents. Examples of semisolid preparations include ointments such as oily ointments and hydrophilic ointments. This preparation contains, for example, water, petrolatum, polyethylene glycol, oil, surfactant and the like as a commonly used base or carrier.
Examples of semi-solid or solid preparations include patches for transdermal administration such as plasters (rubber plaster, plaster, etc.), films, tapes, and cataplasms or for transmucosal (intraoral, nasal) administration. No. This formulation is usually used as a base or carrier, for example, natural rubber,
Rubber-based polymers such as butadiene rubber, synthetic rubber such as SBR and SIS, mud-forming agents such as gelatin, kaolin, and zinc oxide; hydrophilic polymers such as sodium carboxymethylcellulose and sodium polyacrylate; acrylic resins; liquid paraffin And tackifiers, water, other oils, and surfactants. These preparations may further use adjuvants such as stabilizers, solubilizers, and transdermal absorption enhancers, or additives such as fragrances and preservatives.

When the preparation of the present invention is administered to a patient, it depends on conditions such as the degree of symptoms, age, weight, and health of the patient, and is not particularly limited.
0 mg may be orally or parenterally administered once or more daily. When the pharmaceutical composition uses an external preparation, for example, an ointment containing 0.0001 to 1% of the active ingredient, it may be applied at least once a day.

[0013]

The present invention will be described in more detail with reference to the following examples, which are merely illustrative, and do not limit the present invention.

[0014]

Example 1 Production of compound as active ingredient of the present invention Streptomyces sp. SNA
A 1 cm square was cut out together with the mat from the slant medium (glucose / starch / asparagine agar medium) of strain 15896 (FERM P-16667), and 70 ml of a preculture medium (ammonium sulfate 0.1%) was cut out.
4%, potassium-phosphate 0.2%, calcium chloride 0.03
%, Magnesium sulfate 0.03%, urea 0.03%, polypeptone 0.5%, yeast extract 0.1%, soybean flour 3%, glucose 1%, soluble starch 0.5%, ferrous sulfate 0.0005
%, Manganese sulfate 0.00016%, zinc sulfate heptahydrate 0.000%
(15% 500 ml Erlenmeyer flask containing 14%, cobalt (II) chloride, 0.0002%, pH not adjusted) was inoculated and cultured on a rotary shaker at 27 ° C. for 6 days to obtain a precultured solution. One liter of this preculture medium was mixed with a main culture medium 120 having the same composition as the preculture medium.
Inoculate a 200 liter tank containing 27 liters
Aeration and agitation culture at 120 ° C (aeration rate 120 l / min, agitation 200
Rotation / min, internal pressure 0.1 kg / cm ² ). After 6 days of culture, 120 liters of the culture was centrifuged (17,000 rpm),
The supernatant and the cells were separated. The obtained cells were extracted with 36 liters of acetone, concentrated under reduced pressure to remove acetone, and extracted twice with an equal volume of ethyl acetate. The ethyl acetate extract was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude extract. This was dissolved in a small amount of a mixed solution of chloroform-methanol (98: 2), and applied to a 1-liter silica gel column equilibrated with the same mixed solution. After eluting impurities with 4 liters of the same mixture from this column,
The compound of the present invention was eluted with 1 liter of a mixed solution of chloroform-methanol (9: 1) and concentrated under reduced pressure to obtain a crude product. Further, this crude product was dissolved in a small amount of acetonitrile, and an HPLC column (TSK gel ODS-80TM _M Φ21.5 mm × 300 m) equilibrated with an acetonitrile-water (7: 3) mixed solution.
m, Tosoh Corporation) at a flow rate of 8 ml / min and eluted with the same solvent. The peaks of UK-63,598 (retention time: 26 minutes), UK-65,662 (retention time: 34 minutes), and UK-63,052 (retention time: 40 minutes), which are the active ingredients of the present invention, were collected by detection at 360 nm. And then concentrated under reduced pressure to give UK
-63,598 (R = CH ₃ ; 816 mg), UK-65,662 (R = CH (CH ₃ ) ₂ ;
253 mg), and UK-63,052 (R = CH (CH ₃ ) CH ₂ CH ₃ ; 85 mg)
Was obtained as a yellow powder. These were subjected to instrumental analysis and structural analysis, and confirmed to be compounds represented by the general formula (I), which are active ingredients of the present invention.

[0015]

Example 2 Cell growth inhibitory effect Mouse leukemia cell P388, vincristine multidrug resistant leukemia cell P388 / VCR, human ovarian cancer cell A278
0, A2780 multidrug resistant cells AD _10, human promyelocytic leukemia cells HL-60, using human nasopharyngeal cancer cells KB, and the murine colon cancer cell Colon 26, were subjected to drug susceptibility testing. Each cell was RPMI164 containing 10% fetal calf serum.
0 medium (P388 and P388 / VCR added with 20 μM mercaptoethanol), seeded in a 96-well microplate, and cultured at 37 ° C. in the presence of 5% carbon dioxide gas concentration. P388, P388 / VCR and HL-60 showed test compounds (UK-63,598, UK-65,66) after 4 hours and Colon 26, A2780, AD ₁₀ and KB after 24 hours.
2 or UK-63,052) at various concentrations,
Further, culturing was continued for 72 hours under the same conditions, and the MTT method (J.I.
mmun. Methods., 55 (1983)). 50% growth inhibitory activity (IC ₅₀ ) for each cell
Are shown in Table 1. From these results, it was confirmed that the compound as the active ingredient of the present invention was useful as an antitumor agent.

[0016]

[Table 1]

[0017]

Example 3 Antitumor activity against cultured human cancer cells The activity against cultured human cancer cells was tested according to Yamori's method (Cancer and Chemotherapy, 24 (2), 129 (1997)). That is, human cultured cancer cell line 38 (lung cancer line 7, gastric cancer line 6, colon cancer line 6,
Using ovarian cancer 5 lines, brain tumor 6 lines, breast cancer 5 lines, renal cancer 2 lines and melanoma 1 lines), each cell was seeded in a 96-well microplate, and cultured at 37 ° C. in the presence of 5% carbon dioxide gas concentration. After an hour, the active ingredient of the present invention obtained in Example 1, UK-63,
598 Compounds were added at various concentrations and
The culture is continued for a time and the protein-binding dye sulforhodamine B
(SRB) Colorimetric Method (J. Natl. Cancer Inst. 82:
1113, 1990), and the 50% growth inhibitory activity (IC ₅₀ ) was calculated. Table 2 shows the results. As a result,
It was confirmed that the compound as an active ingredient of the present invention was effective at a very low concentration on various human cultured cancer cells.

[0018]

[Table 2]

[0019]

EXAMPLE 4 Effect against P388 murine tumors (1) the CDF ₁ mice P388 leukemia cells were intraperitoneally transplanted at a rate of 10 ⁶ cells / mouse, once every day and 5 days after tumor implantation (intermittent Test compound (UK-63,598, UK-65,662) dissolved in a 10% saline solution of HCO-60 (polyoxyethylene hydrogenated castor oil, Nikko Chemicals).
, Or UK-63,052), respectively. The effect was evaluated by determining the survival rate from the average survival days of the drug administration group relative to the solvent administration group. Table 3 shows the results. As a result, the compound as the active ingredient of the present invention was effective against P388 mouse tumor at a low dose of 10 μg / kg.

[0020]

[Table 3]

[0021]

Example 5 P388 effect against mouse tumor (2) CDF ₁ mice P388 leukemia cells (10 ⁶ cells) were implanted intraperitoneally, once 9 days 1 day 1 day after tumor implantation sequential (continuous administration), Alternatively, once on the first and fifth days (intermittent administration), the UK-63,598 compound which is the active ingredient of the present invention obtained in Example 1 was treated with HCO-60 (polyoxyethylene hydrogenated castor oil, Nikko Chemicals Co., Ltd.). ) Was dissolved in a 10% saline solution and administered intraperitoneally. The effect was evaluated by determining the survival rate from the average survival days of the drug administration group relative to the solvent administration group. The results are shown in Table 4 (continuous administration) and Table 5 (intermittent administration). As a result, the compound as the active ingredient of the present invention was 2.5 μg / P each against the P388 mouse tumor.
It was confirmed that a very low dose of kg (continuous administration) or 5 μg / kg (intermittent administration) was effective.

[0022]

[Table 4]

[0023]

[Table 5]

[0024]

Example 6 Against Multidrug-Resistant P388 / VCR Mouse Tumors
Effect CDF ₁ mouse to P388 / VCR (vincristine)
The multidrug-resistant leukemia cells were intraperitoneally transplanted at a rate of 10 ⁶ cells / mouse, and once a day after the tumor transplantation, once a day for 9 consecutive days, the active ingredient of the present invention, UK, obtained in Example 1 was obtained in Example 1. -63,598 Compound was dissolved in a 10% saline solution of HCO-60 (polyoxyethylene hydrogenated castor oil, Nikko Chemicals) intraperitoneally, and the survival rate was calculated based on the average survival time of the drug administration group to the solvent administration group. I asked. Table 6 shows the results. As a result, the compound of the present invention shows that multi-drug resistant P388 /
Very low doses were found to be effective against VCR mouse tumors.

[0025]

[Table 6]

[0026]

Example 7 Preparation of Preparation (1) Preparation of Injection Compound (one of the compounds) which is an active ingredient of the present invention 1 mg Polyoxyethylene hydrogenated castor oil (HCO-60) 200 mg Absolute ethanol is added, and the total amount is 1 ml 1 mg The compound which is an active ingredient of the present invention (any one
Seed) and 200 mg of polyoxyethylene hydrogenated castor oil (HCO-60) were mixed with anhydrous ethanol to make a total volume of 1 ml, whereby an injection of the compound as the active ingredient of the present invention was obtained. This solution can be administered by diluting with an appropriate amount of physiological saline.

[0027]

According to the present invention, there is provided an antitumor agent comprising a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient. The present invention is useful as an antitumor agent in cancer chemotherapy.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Naohiro Washida 3-4-12 Hanaki, Ishibashi-cho, Shimotsuga-gun, Tochigi Prefecture (72) Inventor Kanji Higashio 1769-10 Yamada, Kawagoe-shi, Saitama F-term (reference) 4B064 AE50 AG37 CA04 DA05 4C086 AA01 AA02 CB31 MA01 MA04 NA14 ZB26 ZB35

Claims (1)

[Claims] 1. An antitumor agent comprising a compound represented by the following general formula (I) or a pharmacologically acceptable salt thereof as an active ingredient. Embedded image (Where R is CH ₃ , CH (CH ₃ ) ₂ , or CH (CH ₃ ) CH ₂ CH ₃
)