WO2005112951A1

WO2005112951A1 - Antitumor agent, method of producing antitumor agent, medicinal composition containing antitumor agent and microorganism producing antitumor agent

Info

Publication number: WO2005112951A1
Application number: PCT/JP2005/009185
Authority: WO
Inventors: Yoshikazu Takahashi; Ryuichi Sawa; Masaya Imoto; Yushi Futamura
Original assignee: Microbial Chemistry Research Foundation
Priority date: 2004-05-21
Filing date: 2005-05-19
Publication date: 2005-12-01
Also published as: JP4836783B2; JPWO2005112951A1

Abstract

It is intended to provide an antitumor agent, which has established a strengthened function selectivity of acting exclusively on caner cells, lessened side effects, mastery of drug tolerance and so on, and has an activity of inducing cell death in human lung small cell carcinoma Ms-1 cells and human lung small cell carcinoma Ms-1/Bcl-2 cells and Ms-1/Bcl-XL cells with the overexpression of an apoptosis-inhibitory protein Bcl-2 or Bcl-XL, and so on. Namely, an antitumor agent characterized by being a compound represented by the following general formula or its derivative. [Chemical formula 12] wherein R represents a hydrogen atom or an alkyl group.

Description

Specification

Antitumor agent, method for producing antitumor agent, pharmaceutical composition containing antitumor agent, and antitumor agent-producing bacterium

Technical field

[0001] The present invention relates to an antitumor agent having an activity of inducing cell death in tumor cells such as human small cell lung cancer Ms-1 cells, an efficient method for producing the antitumor agent, and a medicament containing the antitumor agent The present invention relates to a composition and a novel antitumor agent-producing bacterium that produces the antitumor agent.

Background art

[0002] In chemotherapy of tumors such as cancer, various anti-tumor drug S screenings have been conventionally performed (see, for example, Patent Documents 1 to 4), and among them, selective activity acting only on cancer cells. And those with few side effects have been used. By the way, in the above-described chemotherapy, tumor resistance such as cancer cells has acquired drug resistance, and the development of drug resistance has become a big problem because antitumor agents used so far become ineffective. Therefore, it is necessary to further search and screen for new antitumor agents every time the drug resistance is developed.

However, while it is not easy to search for and screen for new antitumor agents, there is a problem that if the development of the new antitumor agents is delayed, it will cause a serious hindrance to tumor chemotherapy such as cancer. .

Therefore, a novel antitumor agent that achieves enhancement of selective action acting only on the cancer cells, reduction of the side effects, overcoming the drug resistance, an efficient production method thereof, its producing bacteria, and the like At present, the development of pharmaceutical compositions is required.

[0003] Patent Document 1: Japanese Patent Laid-Open No. 8-143569

Patent Document 2: JP-A-8-239379

Patent Document 3: Japanese Patent Laid-Open No. 9-249647

Patent Document 4: Japanese Patent Laid-Open No. 11-21263

Disclosure of the invention

Problems to be solved by the invention [0004] An object of the present invention is to meet the above-mentioned demands, solve the conventional problems, and achieve the following objects. That is, the present invention achieves enhancement of selective action acting only on cancer cells, reduction of side effects, overcoming drug resistance, etc., and human small cell lung cancer Ms-1 cells and apoptosis inhibitory protein Bel-2 Or human small cell lung cancer that overexpresses Bel-XL Ms-lZBcl-2 cells, anti-tumor agent having activity to induce cell death in Ms-lZBcl-XL cells, efficient production method thereof, producing bacteria thereof, And a pharmaceutical composition containing the same.

Means for solving the problem

[0005] As a result of intensive studies by the present inventors in order to solve the above-mentioned problems, the following knowledge was obtained.

That is, in the process in which the cancer cells acquire drug resistance, apoptosis-inhibiting proteins such as Bel-2 and Bel-XL act and target these apoptosis-inhibiting proteins and are active against these apoptosis-inhibiting proteins. A compound having the above is effective as a novel antitumor agent. Then, the Streptomyces sp. ML6 94-90F3 strain isolated by the present inventors was cultured, and a specific compound isolated from the culture (named “ML694-90F3 substance”). Human small cell lung cancer Ms-1 cells and human small cell lung cancers that overexpress the apoptosis inhibitory protein Bel 2 or Bel-XL Ms—lZBcl—2 cells, Ms—1 ZBcl—XL cells It is a finding that it is effective as a tumor agent!

[0006] The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,

<1> An antitumor agent characterized by being a compound represented by the following general formula or a derivative thereof.

[Chemical 1]

R R

R

CH = CH_CH = CH_C = CH_CH = CH_C = CH_CH _CH

C -C- CH = CH-CH = CH-CH = C-CH = C- C -NH H / \ I I II

HO ROR 0 However, in said general formula, R represents a hydrogen atom or an alkyl group.

<2> An antitumor agent characterized by being either a compound represented by the following structural formula (1) or a derivative thereof.

[Chemical 2]

<3> The antitumor agent according to any one of <1> to <2>, wherein the derivative is any one of an inorganic acid salt and an organic acid salt.

<4> An antitumor agent characterized by being any one of a compound represented by the following structural formula (A) and a derivative thereof.

[Chemical 3]

R R

CH = CH-CH = CH-C = CH-CH = CH-C = CH-CH, -CH

• Structural formula (A)

HO One C One CH = CH-CH = CH-CH = C-CH = C- -NH

I / \ I I

HO R R OR

<5> The ability to produce the antitumor agent according to any one of <1> to <4> above, and a relatively long aerial hyphae is elongated from a well-branched basic hyphae, and the tip thereof is a key. , Looped or loosely wound spirals, oval-cylindrical spores are chained, and light yellow-brown to yellow-yellow aerial mycelia are grown on various mediums The soluble pigment is not observed or yellowish, is 2,6-diaminopimelate LL-type in the cell wall, and the partial base sequence of 16S rRNA gene is highly homologous to Streptomyces sp. T is an antitumor agent-producing bacterium characterized by having mycological properties.

<6> The antitumor agent-producing bacterium according to <5>, which belongs to the genus Streptomyces.

<7> The antitumor agent-producing bacterium according to <6>, which is Streptomyces sp. <8> Enter one of the above <5> to <7>, which has the accession number FERM P—19631 It is a listed antitumor agent-producing bacterium.

<9> The antitumor agent-producing bacterium according to any one of <5> to <8> is cultured, and the antitumor agent according to any one of <1> to <4> is separated from the culture. This is a method for producing an antitumor agent characterized by.

<10> The method for producing an antitumor agent according to <9>, wherein the culture is a liquid shaking culture.

<11> Centrifugation of the liquid culture of liquid shaking culture, concentrating the precipitate, purifying by centrifugal liquid distribution chromatography, and treating with acid to separate the antitumor agent from the culture The method for producing an antitumor agent according to <10>.

<12> A pharmaceutical composition comprising the antitumor agent according to any one of <1> to <4>.

The invention's effect

[0007] According to the present invention, the conventional problems can be solved, and enhancement of selective action acting only on cancer cells, reduction of side effects, overcoming drug resistance, etc. are achieved, and human small cell lung cancer Ms — Human small cell lung cancer that overexpresses 1 cell and apoptosis inhibitory protein Bel-2 or Bel-XL Ms— lZBcl— 2 cells, Ms— lZBcl— XL cells, an antitumor agent having an activity to induce cell death, An efficient production method thereof, its producing bacteria, and a pharmaceutical composition containing the same can be provided. Brief Description of Drawings

[0008] FIG. 1 is a chart of infrared absorption spectrum of ML694-90F3 substance measured by KBr tablet method.

[Figure 2] Figure 2 is a chart of the UV absorption spectrum of ML694-90F3 substance in methanol.

FIG. 3 is a chart of proton nuclear magnetic resonance spectrum at 500 MHz measured at room temperature in deuterated methanol of ML694-90F3 substance.

[FIG. 4] FIG. 4 is a chart of carbon-13 NMR ^ vector at 125 MHz measured in deuterated methanol of ML694-90F3 substance at room temperature.

BEST MODE FOR CARRYING OUT THE INVENTION [0009] (Antineoplastic agent)

The antitumor agent of the present invention is a compound represented by the following general formula or a derivative thereof.

[Chemical 4]

[0010] However, in the general formula, R represents a hydrogen atom or an alkyl group. Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, and a butyl group. Among these, a methyl group is particularly preferable. A plurality of R in the general formula may be the same or different from each other, but it is preferred that all Rs not bonded to a nitrogen atom are methyl groups. Of the two R bonded to the nitrogen atom, R bonded to the nitrogen atom bonded to the oxygen atom-containing hetero 6-membered ring bonded to the molecular end is preferably a methyl group or a hydrogen atom.

[0011] The compound represented by the following structural formula (A) constituting the general formula also exhibits antitumor activity, and therefore can be used as the antitumor agent.

[Chemical 5]

R R

R

CH = CH-CH = CH-C = CH-CH = CH-C = CH-CH, -CH

• Structural formula (A)

HO — C- CH = CH— CH = CH— CH = C— CH = C— C -NH

I / \ I I II

HO R O R 0

Among the antitumor agents represented by the general formula, compounds represented by the following structural formulas (1) to (4) and derivatives thereof are more preferred. Among these, the following structural formula (1) Particularly preferred are compounds represented by the formula and derivatives thereof. [0013] [Chemical 6]

CH

-CH -NH

Formula (1)

[0014] [I 匕 7]

[0015] [Chemical 8]

[0016] [Chemical 9]

)

[0017] The compound represented by the structural formula (1) is a novel compound separated by the present inventors, and may hereinafter be referred to as “ML694-90F3 substance”. The ML694-90F3 substance has two methylamino groups in the molecule.

[0018] The physicochemical properties of the compound represented by the structural formula (1), that is, the ML694-90F3 substance are as follows. That is, (1) Appearance is pale yellow powder.

(2) Melting point is 122-125 ° C.

(3) The molecular formula is represented by C H N O.

42 63 3 8

(4) The experimental value by high resolution mass spectrometry (HRESIMS: positive ion mode) is m / z 738. 4680 (M + H) ⁺ and the calculated value is mZz 738. 4693 (as CHNO)

42 64 3 8

(5) The specific rotation is [α] ²³ = —655 ° (cO.2, methanol).

D

(6) The infrared absorption spectrum is as shown in Fig. 1.

(7) The ultraviolet absorption spectrum is as shown in Fig.2.

(8) As a proton nuclear magnetic resonance spectrum, the proton NMR spectrum measured at room temperature in deuterated methanol at 500 MHz is as shown in FIG.

(9) As a carbon-13 nuclear magnetic resonance spectrum, the carbon-13 NMR spectrum measured at room temperature in deuterated methanol at 125 MHz is as shown in FIG.

(10) Silica gel 60F (Merck) thin layer chromatography as thin layer chromatography

254

In the graph, the Rf value is 0.26 when developed using chloroform: methanol: water (4: 1: 0.1, volume ratio) as the developing solvent.

[0019] Whether the compound has the structure represented by the general formula or the structural formula (1) can be confirmed by various appropriately selected analysis methods. For example, the proton nuclear magnetic resonance This can be confirmed by analyzing the spectrum, the carbon 13 nuclear magnetic resonance spectrum, the infrared absorption spectrum, the mass spectrum, and the like.

[0020] The derivative is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably pharmaceutically acceptable. For example, the general formula or the structural formula (1 Preferred examples include salts of compounds represented by The salt may be used alone or in combination of two or more. The salt is not particularly limited and may be appropriately selected depending on the purpose. For example, an inorganic acid salt And organic acid salts.

[0021] Examples of the inorganic salt include hydrochloride, sulfate, phosphate and the like.

Examples of the organic acid salt include acid addition salts of organic acids such as acetates and methanesulfonates. The acid addition salt such as the inorganic acid salt and the organic acid salt is represented by the general formula or the structural formula (1

It is obtained by adding an acid to the compound represented by

[0022] The antitumor agent of the present invention includes a compound represented by the general formula and the derivative thereof, preferably a compound represented by the structural formula (1), that is, an ML694-90F3 substance and the derivative thereof. These antitumor agents are human small cell lung cancer Ms-1 cells, human small cell lung cancer Ms-1ZBcl 2 cells, Ms-lZBcl-, which overexpress the apoptosis inhibitory protein Bel-2 or Bel-XL. It has the activity of inducing cell death in tumor cells such as XL cells. For this reason, the antitumor agent of this invention can be used conveniently for the pharmaceutical composition etc. of this invention mentioned later.

The antitumor agent of the present invention can be preferably produced by the method for producing an antitumor agent of the present invention using the antitumor agent-producing bacterium of the present invention described later.

[0023] (Antineoplastic agent-producing bacteria)

The antitumor agent-producing bacterium of the present invention can be appropriately selected without particular limitation, except that it has the ability to produce the antitumor agent of the present invention. Elongate aerial hyphae, the tip of which forms a hooked, looped or loosely wound helix, chained oval-cylindrical spores, and on various media, on the growth of light yellow to yellow-yellow Yellow-white to bright ash aerial mycelium is formed, soluble pigment is not observed or yellowish, is 2,6-diaminopimelate LL type in the cell wall, and the partial nucleotide sequence of 16S rRNA gene ^ Amongst the genus Streptomyces, which shows a high degree of homology with Streptomyces spp., And shows that it has a high degree of homology with the mycobacterial property, Streptomyces sp. From the favored soil of Ito Pass, Shizuoka Prefecture, The actinomycete ML694-90F3 strain isolated in September 2013 at the Institute of Microbial Chemistry (currently the Center for Microbial Chemistry) is particularly preferred.

[0024] The ML694-90F3 strain was filed with the National Institute of Advanced Industrial Science and Technology (1st, 1st, 1st, 1st, Tsukuba, Ibaraki, Japan 30 5-8566). On January 8, 2016, it was entrusted with the deposit number: FERM P—19631.

[0025] Here, the bacteriological properties of the ML694-90F3 strain will be described in detail as follows. That is, (1) Form: A relatively long aerial hyphae is elongated from a branched basic mycelium, and its tip forms a forceful, looped or loosely wound helix. The mature spore chain links 10-50 oval-cylindrical spores. The spore size is about 0.5 to 0.6 X O.8 to 0.9 m, and the spore surface is smooth. No roticular branches, mycelial bundles, spore capsules or motile spores are observed.

[0026] (2) Growth condition in various media: For the following color descriptions, the standard shown in parentheses is container ^ ~ · Corporation 'Ob' American color ^ ~ · Harmony ^ ~ · Many Was used.

1) Yeast malt agar medium (ISP—medium 2, 27 ° C culture)

On the growth of nibu-yellow to bright tea, yellow-white to light-yellow aerial hyphae are grown. No soluble pigment is observed.

2) Oatmeal agar medium (ISP—medium 3, 27 ° C culture)

On the growth of light yellow to yellow yellow, a bright ash aerial hyphae grows, and the soluble pigment is slightly yellowish.

3) Starch · Inorganic salt agar (ISP—medium 4, 27 ° C culture)

On the development of light yellow, yellowish white to bright ash aerial mycelium grows and no soluble pigment is observed.

4) Glycerin Asparagine Agar (ISP—medium 5, 27 ° C culture)

On the growth of nibu-yellow to bright tea, a slight yellowish white aerial hyphae is formed, and the soluble pigment is yellowish.

5) Sucrose · Nitrate agar medium (27 ° C culture)

On the growth of light yellow, bright aerial mycelia are steadily grown and no soluble pigment is observed.

[0027] (3) Physiological properties

1) Growth temperature range

East 'Starch agar medium (soluble starch 1.0 wt%, yeast extract 0.2 wt ^_0/0, the string agar 2.4 wt ^_0/0, pH 7. 0) using a 10. C, 20. C, 24. C, 27. C, 30. C, 37. As a result of testing at each temperature of C and 45 ° C, growth at 10 ° C, 37 ° C and 45 ° C was not observed. It grew at 0 ° C to 30 ° C. The optimal temperature for growth is around 27 ° C.

2) Starch hydrolysis (starch 'inorganic salt agar medium, ISP-medium 4, 27 ° C culture) Starch hydrolysis was observed slightly from around 18th day after cultivation, but its effect was weak.

[4] (4) Cell component: 2,6-Diaminopimelic acid in the cell wall is LL-type.

(5) 16S rRNA gene analysis: When the partial base sequence (458 bp) of 16S rRNA gene was determined and compared with the data of known strains registered in the DNA database, the ML

The base sequence of 694-90F3 strain is 16S r of Streptomyces genus actinomycetes.

High homology with RNA genes.

[0029] As described above, the ML694-90F3 strain belongs to the genus Streptomyces, and in the present invention, it is sometimes referred to as Streptomyces sp. ML694-90F3.

[0030] In the present invention, the antitumor agent-producing bacterium may be a strain itself such as the Streptomyces sp. ML694-90F3 strain, and irradiation, other mutation treatment, gene, etc. The ML694-90F3 substance-producing ability may be improved by performing a recombination treatment.

[0031] The antitumor agent-producing bacterium of the present invention can be suitably used for the production of the antitumor agent of the present invention, and can be preferably produced in the method for producing the antitumor agent of the present invention described later. it can.

[0032] (Method for producing antitumor agent)

In the method for producing an antitumor agent of the present invention, the method comprises culturing the antitumor agent-producing bacterium and separating the antitumor agent from the culture, and further selecting other treatments appropriately selected as necessary. including.

[0033] The culture can be performed using the antitumor agent-producing bacterium of the present invention (preferably the ML694-90F3 substance-producing bacterium, more preferably Streptomyces sp. ML694-90F3 strain). Conditions (eg, nutrient medium, temperature, inoculum, number of days, etc.), method, etc. can be appropriately selected according to the purpose without any particular limitation.

The antitumor agent-producing bacterium may be obtained from the National Institute of Advanced Industrial Science and Technology after receiving the distribution of the strain No. FERM P-19631, or from the natural world. You can obtain them by separating them by conventional methods.

[0034] The nutrient medium can be appropriately selected depending on the purpose without particular limitation, and examples thereof include a known actinomycete culture medium, bacteria (bacteria) culture medium, and the like. The nutrient source to be added to the nutrient medium can be appropriately selected from publicly known sources that are not particularly limited, and examples thereof include nitrogen sources, carbon sources, inorganic salts, trace metals, and the like. .

[0035] Examples of the nitrogen source include commercially available peptone, meat extract, yeast extract, corn steep liquor, ammonium sulfate, ammonium chloride, and corn 'Dalten' meal cotton. Seed, meal, toast sawer, etc. These may be used alone or in combination of two or more.

Examples of the carbon source include carbohydrates such as glycerol, starch, glucose, galactose, dextrin, corn starch, and maltose, fats, and the like. These may be used alone or in combination of two or more.

Examples of the inorganic salts include sodium chloride and calcium carbonate.

Examples of the trace metal include manganese.

[0036] The temperature can be appropriately selected depending on the purpose for which there is no particular limitation, and is usually about the optimum temperature for actinomycetes, preferably 25-30 ° C, particularly around 27 ° C. preferable. The inoculum can be appropriately selected according to the purpose for which there is no particular limitation, and preferred examples include slant cultures of the ML694-90F3 strain, liquid immersion cultures, and the like. The inoculation amount of the inoculum can be appropriately selected according to the purpose for which there is no particular limitation.

[0037] The number of days can be appropriately selected according to the purpose for which there is no particular limitation. Usually, the antitumor agent-producing bacteria grow to a sufficient amount in about 3 to 4 days, and the antitumor agent is A sufficient amount is produced. Whether a sufficient amount of the antitumor agent was produced is determined by measuring the time course of the titer of the antitumor agent (the L694-90F3 substance) in the culture medium of the antitumor agent-producing bacteria. Can be determined. The change in the titer of the antitumor agent (ML694-90F 3 substance) over time is, for example, the cytotoxicity given to human small cell lung cancer Ms-1 cells, Ms-lZBcl-2 cells, and Ms-lZBcl-XL cells. Measure by evaluating Can do.

[0038] As the method, for example, aerobic culture or anaerobic culture! /, Or may be a deviation! / Aerobic culture is preferred, and slant culture, flat plate (plate ) Solid (agar) culture such as culture or liquid culture may be used, but the liquid culture for which the liquid culture is preferred is any of shaking culture, stationary culture, and stirring culture. However, a rotary shake culture in which a shake culture is preferred is more preferable. In addition, when mass culture is performed, culture using a fermenter or the like.

[0039] Separation of the antitumor agent in the culture force can be performed by a method appropriately selected from known methods for separating metabolite products by microorganisms. For example, the antitumor agent produces the antitumor agent. When released outside the bacterial cells, after separating and removing the antitumor agent-producing bacteria from the culture solution of the antitumor agent-producing bacteria, the antitumor agent present in the culture solution is collected. When the antitumor agent is accumulated in the cells of the antitumor agent-producing bacterium, the antitumor agent-producing bacterium is isolated from the culture solution of the antitumor agent-producing bacterium. Thereafter, the anti-tumor agent-producing microbial cells can be crushed and then the anti-tumor agent can be collected. Among these, the liquid culture of the liquid shaking culture is centrifuged, and the precipitate is separated. After concentration, the product is purified by centrifuge liquid-liquid chromatography and treated with acid. The virtuous preferable.

[0040] The method for fractionating the antitumor agent can be appropriately selected according to the purpose without any particular limitation. For example, a solvent extraction method using a solvent, a difference in adsorption affinity to various adsorbents. Adsorptive separation methods using gels, gel filtration, and chromatographic methods using countercurrent distribution. These may be used alone or in combination of two or more. The centrifugal separation can be performed under appropriately selected centrifugal conditions using a known centrifugal separator. The acid can be appropriately selected according to the purpose for which there is no particular limitation, and examples thereof include inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid, and organic acids such as acetic acid. These may be used alone or in combination of two or more. When the treatment is performed using the acid, the pH of the added solution is preferably about 3 as a guide for stopping the addition of the acid.

[0041] The antitumor agent sorted as described above is usually the ML694-90F3 substance, Often it is a mixture containing several groups of compounds with very similar structures. In the present invention, the antitumor agent may be used as a mixture, or the ML694 90F3 substance may be further separated from the mixture and used.

In addition, as a method for identifying the strength of the antitumor agent as described above, it can be appropriately selected according to the purpose without any particular limitation. For example, high-performance liquid chromatography (HPLC), NMR, Analytical methods such as mass spectrometry (MS) can be used.

[0042] As a specific example of the separation of the antitumor agent from the culture, a culture (liquid) of the antitumor agent-producing bacterium (ML694-90F3 substance-producing bacterium) is overnight under basic conditions. After storage in an ice chamber, the supernatant and precipitate are separated by centrifugation. The precipitate was extracted with methanol and then concentrated to dryness. The resulting oily residue was subjected to centrifugal liquid-liquid partition chromatography (solvent system: black form: methanol: water = 5: 6: 4 (volume Ratio)) to obtain a primary refined oil containing the antitumor agent (ML694-90F3 substance). Next, hydrochloric acid is added to the antitumor agent (ML694-90F3 substance) in the primary refined oil to cause neutralization reaction, whereby the hydrochloride of the antitumor agent (ML694-90F3 substance) is obtained. The resulting reaction solution is directly subjected to centrifugal liquid-liquid partition chromatography (solvent system: black mouth form: methanol: water = 5: 6: 4 (volume ratio)) to obtain the desired antitumor agent (as described above). ML694-90F 3 substances) hydrochloride can be obtained as a pure product.

[0043] The antitumor agent of the present invention produced by the method for producing the antitumor agent of the present invention using the antitumor agent-producing bacterium of the present invention may be used as it is, or the present invention including the same. It may be used as a pharmaceutical composition.

[0044] (Pharmaceutical composition)

The pharmaceutical composition of the present invention contains other components that are appropriately selected as well as the known intermediate forces other than containing the antitumor agent.

Examples of the dosage form of the pharmaceutical composition include powder medicine, capsule medicine, tablet medicine, suppository, liquid medicine, and the like that are not particularly limited.

Suitable examples of the other components include pharmaceutically acceptable conventional solid or liquid carriers, such as ethanol, water, starch, and the like.

The pharmaceutical composition can be suitably used as an antitumor agent, and human small cell lung cancer Ms As an antitumor agent with the activity of inducing cell death in 1 cell and human small cell lung cancer that overexpresses apoptosis inhibitory protein Bel-2 or Bel-XL Ms-lZBcl-2 cells, Ms-lZBcl-XL cells It can be particularly preferably used.

Examples of the present invention will be described below, but the present invention is not limited to these examples.

[Example 1]

-Manufacture of antitumor agents (ML694-90F3 substances)

Streptomyces sp. ML694-90F3 strain (Accession No .: FERM P-19631) was used as the antitumor agent-producing bacterium, which was cultured on an agar slant medium and used as an inoculum.

Cotton 'seed' meal 1.0 wt ^_0/0, glucose 2.0 wt ^_0/0, glycerol 2.0 wt%, sodium chloride 0.5 wt ^_0/0, manganese chloride 0.0005 wt ^_0/0, carbonate Dispense 0.1 ml of liquid culture medium (pH 7.4) containing 0.3% by weight of calcium and 2% by weight of toast soaker into a triangular flask (500mL) at 120 ° C for 20 minutes. After sterilization, the inoculum was inoculated and cultured at 27 ° C for 5 days with rotary shaking.

[0047] The culture broth thus obtained was adjusted to pH 9 with sodium hydroxide and allowed to stand in an ice-cold room, and then separated into a supernatant and a precipitate by centrifugation. Then, with respect to the precipitate, methanol and (300 mL) was added, stirred, filtered, and concentrated to dryness to give an oily product quality crude (yield: 397. 3 mg) ₀ obtained The oily substance was purified by centrifugal liquid-liquid partition chromatography using a mixed solvent of chloroform: methanol: water = 5: 6: 4 (volume ratio), and the ML694-90F3 as the antitumor agent was purified. A primary purified product was obtained (yield: 96.6 mg).

[0048] After this primary purified product was dissolved in 1 ml of a mixed solvent of chloroform-form: methanol: water = 5: 6: 4 (volume ratio), this solution was added under ice-free until the pH reached 3. 1N HC1 was added and neutralized. As a result, hydrochloride of the ML694-90F3 substance as the antitumor agent was produced. The reaction mixture containing the ML694-90F3 substance hydrochloride was purified using a mixed solvent of chloroform: methanol: water = 5: 6: 4 (volume ratio), and the ML694-90F3 substance pure hydrochloride was purified. Product 5. 7 mg was obtained as a pale yellow powder. [0049] (Example 2)

-Manufacture of antitumor agents (ML694-90F3 substances)

Seed culture includes glucose 0.5 wt ^_0/0, glycerol 0.5 weight ^_0/0, glutamic acid sodium © beam 0.15 wt%, Ajiburon 0.5 wt%, and calcium carbonate 0.1% by weight Dispense lOmL of liquid culture medium (PH7.4) into Erlenmeyer flasks (500mL) at 120 ° C for 20 minutes in a conventional manner, and inoculate the inoculum above at 27 ° C. This was carried out by rotating and culturing for 3 days.

The obtained seed culture solution (2.5 L) was inoculated into a 200 L jar fermenter containing 100 L of the medium described above, and cultured with shaking at 27 ° C for 3 days.

[0050] The culture solution thus obtained was adjusted to pH 9 with sodium hydroxide and allowed to stand in an ice chamber for one week, followed by suction filtration. Then, a mixture of chloroform-form-methanol (1: 5) (40 L) is added to the obtained residue, stirred, filtered, concentrated, and dried to dryness. Mixed solvent of tanol: water = 5: 6: 4 (volume ratio) 2. Dissolved in 4 L and distributed to obtain 800 mL of organic layer containing active substance.

80mL of the obtained organic layer was concentrated and dried, and the resulting oily substance was mixed with centrifuge form: methanol: water = 5: 6: 4 (volume ratio) by centrifugal liquid-liquid partition chromatography. The product was purified using a solvent to obtain a primary purified product of the ML694-90F3 substance as the antitumor agent (yield: 1.47 g).

[0051] After this primary purified product was dissolved in the lower layer of a mixed solvent of black mouth form: methanol: water = 5: 6: 4 (volume ratio), it was subjected to centrifugal liquid-liquid partition chromatography to obtain a black mouth form. : Methanol: 5 mM aqueous hydrochloric acid = 5: 6: 4 (volume ratio) A mixed solvent was used for purification. To the fraction containing the ML694-90F 3 substance, add a lower layer of mixed solvent of chloroform mouth: methanol: water = 5: 6: 4 (volume ratio), adjust the pH to 8 with saturated aqueous sodium bicarbonate solution, and distribute. The lower layer containing the ML694-90F3 substance was concentrated and dried to obtain 30 lmg of a free form of the ML694-90F3 substance as the antitumor agent. The obtained free form was dissolved in 60 mL of methanol containing lOmM hydrochloric acid, concentrated, and dried to obtain the ML694-90F3 hydrochloride as the antitumor agent. Yield was 323 mg.

[0052] (Example 3)

-Evaluation of antitumor activity

In order to evaluate the activity of the hydrochloride of the ML694-90F3 substance obtained in Example 1 as an antitumor agent, the following experiment was conducted.

(1) Human small cell lung cancer Ms—1 cell, (2) Human small cell lung cancer constructed to overexpress Bel-2 or Bel-XL Ms—lZBcl—2 cells, (3) Ms—lZBcl— XL cells were seeded at 1 x 10 ⁴ cells in each well of a 96 well plate, and Roswell Park Memorial Institute (RPMI) 1640 medium (Nissui Pharmaceutical Co., Ltd.) was added to each well. company) to those high-pressure steam sterilization, © Shi fetal serum _{(FBS) (Bio S erum)} 5 wt%,據過sterilized 7 this Kanamycin (Sigma) lOOmgZ mL, Penicillin (Sigma) lOOunits ZmL, L (+) — Glutamine (Kantoi Gakaku Co., Ltd.) 30mgZmL and high-pressure steam sterilized NaHCO lOgZL in the medium, 37 ° C

3.Incubator with 5% CO concentration

2

Cultured in one. After 24 hours, various concentrations of the ML69 4-90F3 substance were added to the RPMI1640 medium, and the culture was continued at 37 ° C for 48 hours.

The cytotoxicity of the ML694-90F3 substance was evaluated by the cell viability using the trypan blue exclusion test method. As a result, (1) human small cell lung cancer Ms—1 cell, (2) human small cell lung cancer Ms—lZBcl 2 cell constructed to overexpress Bel-2 or Bel—XL, (3) Ms— In lZBcl-XL cells, the ML694-90F3 substance was found to cause 50% cell death at a concentration of lOOngZmL, respectively. From this result, it was confirmed that the ML694-90F3 substance has activity as a hydrochloride potent antitumor agent.

Industrial applicability

[0053] The antitumor agent of the present invention includes human small cell lung cancer Ms-1 cells and human small cell lung cancer Ms-lZBcl-2 cells, MslZBcl-, which overexpress the apoptosis-inhibiting protein Bel-2 or Bel-XL. Suitable for use as an antitumor agent with the activity of inducing cell death in XL cells And can be suitably used in the pharmaceutical composition of the present invention.

The antitumor agent-producing bacterium of the present invention can be suitably used for producing the antitumor agent of the present invention.

The pharmaceutical composition of the present invention can be suitably used as an antitumor agent.

Claims

The scope of the claims

[1] An antitumor agent, which is any one of a compound represented by the following general formula and a derivative thereof.

[Chemical 10]

However, in said general formula, R represents a hydrogen atom or an alkyl group.

[2] An antitumor agent characterized by being one of a compound represented by the following structural formula (1) and a derivative thereof.

[Chemical 11]

[3] The antitumor agent according to any one of claims 1 to 2, wherein the derivative is any one of an inorganic acid salt and an organic acid salt.

[4] It has the ability to produce the antitumor agent according to any one of claims 1 to 3, and extends a relatively long aerial hyphae from a well-branched basic hyphae, the tip of which is hooked, It forms a looped or loosely wound helix, links oval-cylindrical spores, and in various media, grows light yellow to yellow-yellow on a yellow-white to bright ash aerial mycelium, soluble Dye is not observed or yellowish, is 2,6-diaminopimelic acid LL-type in the cell wall, shows partial homology with 16S rRNA gene partial nucleotide sequence S Streptomyces sp. An antitumor agent-producing bacterium characterized by having gonococcal properties.

[5] The antitumor agent-producing bacterium according to claim 4, wherein the bacterium belongs to the genus Streptomyces.

[6] The antitumor agent-producing bacterium according to claim 5, which is Streptomyces sp.

[7] The antitumor agent-producing bacterium according to any one of claims 4 and 6, which has an accession number of FERM P-19631.

[8] The antitumor agent-producing bacterium according to any one of claims 4 to 7 is cultured, and the antitumor agent according to any one of claims 1 to 3 is isolated. A method for producing an antitumor agent.

[9] The method for producing an antitumor agent according to [8], wherein the culture is liquid shaking culture.

[10] The method according to claim 9, wherein the culture solution of liquid shaking culture is centrifuged, and the precipitate is concentrated and then purified by centrifugal liquid partition chromatography and treated with acid to separate the antitumor agent. The manufacturing method of antitumor agent as described.

[11] A pharmaceutical composition comprising the antitumor agent according to any one of claims 1 to 3.