JP2002080446A - New compound f-15603 and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new compound having antitumor activity. SOLUTION: This compound is shown by the formula (I).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel compound having apoptosis-inducing activity, a method for producing the compound, an antitumor agent containing the compound as an active ingredient, and the like.

[0002]

2. Description of the Related Art The tumor suppressor gene p53 is one of the most important genes acting in the process of suppressing canceration, and p53 dysfunction caused by mutation of this gene is considered to be a cause of cell carcinogenesis. I have.

[0003] Mutations in the p53 gene have been found in most human cancer cells (Greenblatt, MS, et al., C
ancer Research, 54, 4855 (1994)), p53 gene knockout mice are 6-9 months old,
0% developed cancer (Donehower, LA, Nature, 356,
215 (1992)).

[0004] Taxol (Wahl, A. et al.) Is a compound that selectively induces apoptosis in cancer cells with p53 dysfunction.
F., Nature Medicine, 2, 72 (1996)), little is known, and new compounds have been awaited.

[0005]

DISCLOSURE OF THE INVENTION The present inventors have developed a cell line WI-38 VA-13 subline RA (hereinafter, referred to as "VA-13") dysfunctional tumor suppressor gene p53 (hereinafter, simply referred to as "p53"). As a result of searching for a substance having a growth inhibitory activity, a novel compound having a VA-13 cell growth inhibitory activity was found in a culture of a microorganism isolated from the natural world. The present invention was confirmed to have selective apoptosis-inducing ability and to exhibit growth inhibitory activity against various cancer cells, and thus completed the present invention.

[0006]

The present invention provides (1) the following formula (I)

[0007]

Embedded image (2) Compound having the following physicochemical properties: 1) Property: white powder, 2) Specific rotation: [α] _D ²⁰ −210 ° (c 0.2
5, methanol), 3) Solubility: soluble in dimethylsulfoxide, methanol, acetone, ethyl acetate, insoluble in water, 4) Molecular formula: C ₁₅ H ₂₁ O ₃ N (estimated from accurate mass measured by high-resolution mass spectrum) ), 5) Molecular weight: 263.3, 6) Ultraviolet absorption spectrum: λmax nm (ε), The ultraviolet absorption spectrum measured in a methanol solution is:
Shows a maximum absorption at 226 (29600) nm. 7) Infrared absorption spectrum (KBr): νmax cm
^The infrared absorption spectrum measured on the ^-1 KBr disc shows a maximum absorption at the following wave numbers: 3251, 2918, 26
35, 1943, 1664, 1623, 1538, 14
41, 1419, 1375, 1314, 1232, 11
89, 1116, 1068, 1026, 1012, 85
3,817,8) ¹ H-nuclear magnetic resonance spectrum; δ (ppm) The nuclear magnetic resonance spectrum (360 MHz) measured in a heavy methanol solution is as follows (internal standard is tetramethylsilane): 1.41 ( 1H, m), 1.71 (3H, br s),
1.76 (3H, br s), 2.00 (1H, m), 2.04 (1H, m), 2.15-
2.45 (4 overlapping signals), 4.09
(1H, dd, 12.4 Hz, 2.1 Hz), 4.12 (1H, dd, 12.4 Hz,
2.3 Hz), 5.58 (1H, m), 6.14 (1H, m), 6.49 (1H, br
s), 9) ¹³ C-nuclear magnetic resonance spectrum; δ (ppm) The nuclear magnetic resonance spectrum (90 MHz) measured in a heavy methanol solution is as follows (the internal standard is tetramethylsilane): 18.1 (q) ), 25.3 (t), 26.1 (q), 28.
6 (t), 32.3 (t), 34.2 (d), 63.1 (t), 94.7 (d), 11
0.6 (s), 121.2 (d), 132.0 (s), 134.1 (d), 134.9
(s), 169.3 (s), 204.8 (s), 10) Analysis by high performance liquid chromatography: Column: Symmetry C18 (4.6 mm in diameter × 150 mm in length, manufactured by Waters) Mobile phase: acetonitrile / 0.3% Triethylamine-phosphoric acid (pH 3.0) = 3/7 Flow rate: 1.0 mL / min Detection wavelength: 210 nm Retention time: 5.9 minutes (3) belongs to the genus Stereum, (1) or (2) A method for producing a compound according to (1) or (2), comprising culturing a bacterium producing the compound according to (1) and collecting the compound according to (1) or (2) from the culture.
(4) A bacterium that produces the compound according to (1) or (2), which belongs to the genus Stereum, is Stereum hirsutum SANK 16000 (FERM).
(BP-7200) strain, the production method according to (3),
(5) Stereum hirsutum S
ANK 16000 (FERM BP-7200) strain,
(6) a medicament containing the compound according to (1) or (2) as an active ingredient, (7) an apoptosis inducer containing the compound according to (1) or (2) as an active ingredient, and
(8) An antitumor agent comprising the compound according to (1) or (2) as an active ingredient.

In the present invention, the compound described in the above (1) or (2) is referred to as F-15603.

Since the compound represented by the formula (I) described in the above (1) has an asymmetric carbon atom and an allene bond, the compound has central chirality and axial chirality, and has various optical properties. There are isomers. Also, since the compound has some double bonds and hydroxamic acid, the compound has various geometric isomers. In the present invention, these isomers are all represented by a single formula.
The present invention also includes all of these isomers and mixtures of these isomers, for example, racemates and the like.

When a stereospecific synthesis method is used or when an optically active compound is used as a starting compound, individual isomers may be directly produced, or when produced as a mixture of isomers, individual isomers may be produced. The isomers may be purified and separated by a conventional method.

[0011] F-15603 can be converted into a salt using a known method. The present invention relates to such F-15603
And salts of

When F-15603 is used as a medicine, its salt is not particularly limited as long as it is used medically and is pharmacologically acceptable. For example, sodium salt, potassium salt, Alkali metal salts such as lithium salts: alkaline earth metal salts such as calcium salts and magnesium salts: metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, and cobalt salts: such as ammonium salts Inorganic salt: t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N , N '
Amine salts such as organic salts such as dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt and tris (hydroxymethyl) aminomethane salt: glycine Amino acid salts such as salts, lysine salts, arginine salts, ornithine salts, and asparagine salts: hydroxamic acid salts and the like, and more preferably sodium salts, potassium salts, lithium salts, calcium salts, and magnesium salts.

When F-15603 is used for purposes other than medicine, there is no limitation on its salt. Applications other than pharmaceuticals include, for example, applications as synthetic intermediates when producing F-15603 derivatives.

Further, F-15603 and a salt thereof may be a solvate. For example, when left in the air or recrystallized, it may absorb and adsorb moisture or become a hydrate. The present invention also includes such solvates.

Further, the present invention also includes a compound which is metabolized in the living body and converted into F-15603, a so-called prodrug.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION As an F-15603-producing bacterium,
Although not particularly limited, for example, filamentous fungi belonging to the genus Stereum (Stereum) and the like can be mentioned.
Preferably Stereum hirsutum
And more preferably, Sterium Hirstam SAN
K 16000 shares (hereinafter simply referred to as “SANK 16000
Stocks. ). 16,000 shares of SANK
It was isolated from basidiospores of filamentous fungal fruit bodies collected in July 997 in Kominato-cho, Tianjin, Japan.

[0017] In order to observe the mycological properties of the strain under culture, the strain was cultured on the following agar media.

Potato dextrose agar (Potato Dextr)
ose Agar: Hereafter referred to as “PDA”. ) Medium: Nissui potato dextrose agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.)
39 g was dissolved in 1 L of distilled water and sterilized at 121 ° C. for 15 minutes to prepare a plate.

WSH medium; Quaker Oatmeal 10
g, 1 g of magnesium sulfate heptahydrate, 1 g of potassium dihydrogen phosphate, 1 g of sodium nitrate and 20 g of agar were dissolved in 1 L of distilled water and sterilized at 121 ° C. for 15 minutes to prepare a plate.

The bacteriological properties of the SANK 16000 strain in culture are as follows:
The culture was 42 to 44 mm at 3 ° C for 1 week. The edge of the bacterium was slightly compressed and was the entire edge. The fungus was wool-like and white. The color of the medium on the back did not change. The growth on WSH was 26 ° C at 23 ° C for 1 week.
To 31 mm. The rim of the bacterium is crushed and the whole rim. Slightly wool-like to short fluffy, white. The color of the medium on the back did not change. Aerial hyphae are 1.3-6.5μ wide
m, from a thin wall to a slightly thick wall. There was no smell. No conidia were observed. There was little haze connection, and it was observed only in the hyphae formed in a ring shape and wide.

[0021] The identification of the SANK 16000 strain requires the acquisition of fruiting bodies, but the strain did not form fruiting bodies on the above-mentioned medium. Therefore, the bacteriological properties of the dried specimens of the fruiting bodies used for the separation were observed.

The bacteriological properties of the dried sample of the SANK 16000 strain are as follows: The fruiting bodies were dorsal to semi-dorsiform. The umbrella is 12 mm or less in width and 0.4 mm or less in thickness,
Shell-shaped or fan-shaped, side-by-side, firm. The surface was coarse to fluffy, ring-shaped and radially grooved, brown to light brown, and gray at the base. The edges were thin and sharp. The surface of the grain layer was smooth, slightly bumpy, and pale yellow. An underskin was present between the coat and the flesh. The underskin was thin and brown. Basidiospores are 4.9 to 6.7 μm × 1.7 to 2.
7 μm, oblong to cylindrical, smooth, colorless, amyloid. Basin device is 22 ~ 26μm × 4 ~ 5.8μ
m, elongate club-shaped, four petites, with no fascinating connections at the base. The skeletal cystidia had a width of 4 to 7 μm, a thick wall, and the tip had a circular or small protrusion. The walls were no more than 2 μm thick. The hyphae were of the two hypha type. The original hyphae had a width of 2 to 4 μm, a thin wall or a thick wall, and no partition was connected to the partition walls. The skeletal hyphae were 3-7 μm wide, thick walls, often with secondary septum. There were no Acanthophysis or pseudo-Acanthophysis.

The mycological properties of this strain are described in the Chamlis literature (Chamuris, GP, Mycotaxon, 22, 105).
(1985)) and the ratan literature (Rattan, SS, Bibl. Myc)
ol., 60, 156 (1977)). Therefore, SANK 16
000 strains were identified as S. hirsutum.

The SANK 16000 strain was
It was deposited on June 30, 2010 at the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science, Ministry of International Trade and Industry, 1-1-3 Higashi, Tsukuba, Ibaraki, Japan, and was assigned the accession number FERM BP-7200.

As is well known, basidiomycetes occur in nature.
Or artificial operation, for example, ultraviolet irradiation, radiation irradiation,
Mutation is easily caused by chemical treatment, etc.
The same applies to the ANK 16000 strain. SANK 160
Strain 00 includes all such mutants. These mutants also include those obtained by genetic methods, for example, recombination, transduction, transformation and the like. That is, SANK 16000 producing F-15603
Strains, their mutants and strains that are not clearly distinguished from them are all included in the SANK 16000 strain.

The culture of the F-15603-producing bacterium can be performed using a medium containing a carbon source, a nitrogen source, and an inorganic salt that can be assimilated by the microorganism.

As the carbon source, for example, glucose, fructose, maltose, sucrose, mannitol, glycerin, dextrin, oats, rye, corn starch, potato, corn flour, cottonseed oil, molasses, citric acid, tartaric acid, are selected from these. And the like. The upper limit of the content of the carbon source in the medium is 10 to 30% by weight, and the lower limit is 0.1 to 1% by weight.
0% by weight.

As the nitrogen source, proteins, substances containing hydrolysates thereof, and inorganic salts can be used. Such nitrogen sources include, for example, soybean flour, bran, peanut flour, cottonseed flour, casein hydrolyzate, pharmamine, fish meal, corn steep liquor, peptone, meat extract, yeast, yeast extract, malt extract, nitric acid Examples thereof include sodium, ammonium nitrate, ammonium sulfate, and a mixture of two or more selected from these.
The upper limit of the content of the nitrogen source in the medium is 10 to 30.
% By weight, with the lower limit being 0.01 to 0.2% by weight, with a preferred range being 0.2 to 10% by weight.

As nutrient inorganic salts, salts from which ions can be obtained can be used. Such nutrient inorganic salts include, for example, sodium, ammonium, calcium, phosphate, sulfate, chloride,
Carbonate and the like can be mentioned. Further, if necessary, a trace amount of metal such as potassium, calcium, cobalt, manganese, or magnesium may be contained in the medium.

In liquid culture, silicone oil, vegetable oil, surfactant and the like are used as antifoaming agents.

The upper limit of the pH of the medium is pH 7.0 to pH 7.0.
9.0, the lower limit is pH 4.0 to 5.0, and the preferred range is pH 5.0 to pH 7.0.

The culture temperature has an upper limit of 28 to 35 ° C. and a lower limit of 14 to 21 ° C., and a preferable range is 20 to 30 ° C.
C., a more preferred range is from 21 to 28.degree. C., optimally 23.degree.

The culture time depends on the culture temperature, aeration conditions, culture scale, etc., but the upper limit is 14 to 20 days.
The lower limit is 1 to 10 days, the preferred range is 2 to 15 days.
A more preferred range is 10 to 14 days.

F-15603 is produced by culturing the compound-producing bacterium under aerobic conditions. The aerobic culture method includes, for example, a solid culture method, a shaking culture method, an aeration stirring culture method, and the like.

The number of revolutions during shaking culture depends on the viscosity of the culture solution, the culture scale, etc., but the upper limit is 580 to 600 rpm, and the lower limit is 50 to 100 rpm.
A preferred range is 100 to 580 rpm, a more preferred range is 180 to 240 rpm, and optimally 210 rpm.

Culture of the F-15603-producing bacterium usually starts with a small-scale culture (hereinafter referred to as "seed culture").
If necessary, the culture is scaled up (hereinafter, a large-scale culture is referred to as "mass culture"). The culture can be scaled up by inoculating a part or all of the culture solution in which the producing bacterium has sufficiently grown into a larger amount of medium and allowing the bacterium to grow sufficiently. The scale-up from seed culture to large-scale culture is performed in one stage or divided into two or more stages.

For the seed culture, a Sakaguchi flask, an Erlenmeyer flask equipped with a water flow control wall, or an ordinary Erlenmeyer flask is used.

The incubator used for large-scale culture is selected depending on the purpose of the culture, and examples thereof include a Sakaguchi flask, an Erlenmeyer flask, a jar fermenter equipped with a stirrer and a ventilator, and a tank.

F-15603 present in the culture solution can be separated from the culture by filtration or centrifugation using diatomaceous earth or the like as a filter aid, and extracted from the filtrate or supernatant and from the cells. it can. In addition, by utilizing the physicochemical properties of F-15603, the compound can be purified and isolated from the extract.

When F-15603 is obtained from the filtrate or supernatant, the filtrate or supernatant can be directly subjected to purification. When the F-15603 compound is obtained from the cells, the cells are reduced by 50%.
After extraction with about 90% aqueous acetone or aqueous methanol, the organic solvent is removed by concentration under reduced pressure or the like, followed by purification.

The F-15603 compound present in the filtrate, supernatant or cell extract (hereinafter referred to as "crude fraction")
Adsorption chromatography, ion exchange chromatography, partition chromatography, high performance liquid chromatography (high pe
rformance liquid chromato
graph: Hereinafter, "HPLC". ) Can be isolated.

For the adsorption chromatography, activated carbon, Amberlite XAD-2, Amberlite XAD-4 (above, manufactured by Rohm and Haas), Diaion HP-10,
HP-20, HP-50, CHP20P (or more,
Adsorbents such as Mitsubishi Chemical Corporation, silica gel, and florisil can be used. When F-15603 is adsorbed on the adsorbent, the crude fraction is brought into contact with the adsorbent to remove the non-adsorbed fraction, and then F-15603 is eluted from the adsorbent. Examples of the organic solvent for eluting the F-15603 adsorbed on the adsorbent include, for example, a mixture of an organic solvent and water miscible with water, and preferably, aqueous methanol and aqueous acetone. When F-15603 does not adsorb to the adsorbent, the crude fraction is brought into contact with the adsorbent, and then F-15603 is recovered in the non-adsorbed fraction.

For ion exchange chromatography, ion exchange resins such as Dowex 50W × 4, 1 × 2, and SBR-P (both manufactured by Dow Chemical Company) can be used. When F-15603 adsorbs to the ion exchange resin, the crude fraction is brought into contact with the ion exchange resin to remove the non-adsorbed fraction.
Elute -15603. In order to elute F-15603 adsorbed on the ion exchange resin agent, the pH of the solvent phase is changed or the ionic strength is increased. Examples of the substance that changes the pH of the solvent phase include hydrochloric acid and aqueous ammonia.

Avicel (manufactured by Asahi Kasei Corporation), Sephadex LH-
20 (Pharmacia), Cosmosil 140C18
(Manufactured by Nacalai Tesque) can be used.

Further, HPLC using these chromatography supports can be used for the purification of F-15603.

The behavior of F-15603 in each purification step can be confirmed by the following methods 1) to 3). 1) Growth inhibition test of cancer suppressor gene p53 dysfunction cell A normal cell line and a p53 dysfunction mutant of the cell line are each cultured for a certain period of time in the presence of a test sample. Thereafter, the number of living cells is measured by a known method, and the influence of the test sample on each cell proliferation is compared. F-15603 selectively inhibits the growth of p53 dysfunctional cell lines. Examples of normal cell lines and p53 dysfunctional mutant combinations of the cell lines include WI-
38 and VA-13 are preferred. 2) Apoptosis induction test for p53 dysfunction cells After culturing a normal cell line and a p53 dysfunction cell line in the presence of a test sample for a certain period of time, respectively, whether or not apoptosis is induced is compared between the two lines. F-15603 selectively induces apoptosis in p53 dysfunctional mutants. As a method for confirming that apoptosis is induced, for example, measurement of chromatin fragmentation and the like can be mentioned. 3) HPLC analysis When the test sample was subjected to HPLC under the following conditions, F-
15603 is detected at a retention time of 5.9 minutes. Column: Symmetry C18 (4.6 mm in diameter × 150 mm in length: manufactured by Waters) Mobile phase: acetonitrile / 0.3% triethylamine-phosphoric acid (pH 3.0) = 3/7 Flow rate: 1.0 mL / min Temperature : 25 ° C Detection wavelength: 210 nm F-15603 has antitumor activity, and has gastric, esophageal, pancreatic, liver, colon, colon, rectal, cervical, cervical, ovarian cancer. It is effective for treating malignant tumors such as breast cancer, skin cancer, prostate cancer, testicular tumor, head and neck cancer, brain tumor, leukemia, osteosarcoma, malignant lymphoma, and bladder cancer.

When F-15603 is used as a medicine,
Various dosage forms can be taken. Examples of the administration form include oral administration, rectal administration, intravenous administration, intramuscular administration, intradermal administration, subcutaneous administration, intraperitoneal administration, transdermal administration and the like. The administration method is selected according to the patient's age, sex, site of the disease, degree of the disease, and the like.

The various dosage forms used in these administration methods include:
Formulated using ingredients commonly used in the pharmaceutical arts, such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspending agents, coating agents, etc. can do.

For oral administration, tablets, pills, solutions, suspensions, emulsions, granules, syrups, capsules and the like can be used.

For molding into tablets, carriers known in the art can be used. Examples of such carriers include excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, etc .: water, ethanol, propanol, simple syrup, glucose solution, Binders such as starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone: dry starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters , Disintegrators such as sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose: disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil: quaternary ammonium bases, absorption accelerators such as sodium lauryl sulfate: glycerin, Lees Humectants like; starch, lactose, kaolin, bentonite, adsorbent such as colloidal silicic acid: purified talc, stearates, boric acid powder, may be mentioned lubricants such as polyethylene glycol. In addition, a tablet can be coated as needed. As a tablet with a coating,
Examples include sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double tablets, multilayer tablets and the like.

For molding into a pill form, a carrier known in the art can be used. Such carriers include, for example, excipients such as glucose, lactose, cocoa butter, starch, hydrogenated vegetable oil, kaolin, and talc; binders such as gum arabic powder, tragacanth powder, gelatin, and ethanol; disintegration such as laminaran agar; Agents and the like.

For rectal administration, suppositories and the like can be used.

For shaping in the form of suppositories, carriers known in the art can be used. Such carriers include, for example, polyethylene glycol, cocoa butter, higher alcohols, higher alcohol esters,
Gelatin, semi-synthetic glyceride and the like can be mentioned.
Suppositories are suitable for rectal administration.

For intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal administration and the like, injections and the like can be used.

When prepared as an injection, desirably, it is sterile and isotonic with blood. Examples of the dosage form of an injection include a liquid preparation, an emulsion, a suspension and the like.
Known diluents in this field can be used. Examples of such a diluent include water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. Also, to make injections isotonic with blood,
An appropriate amount of salt, glucose, glycerin and the like may be contained. Further, the injection may contain a solubilizer, a buffer, a soothing agent and the like.

In these dosage forms, if necessary, a coloring agent,
Preservatives, flavors, flavors, sweeteners, nutrients, other medicines and the like may be included.

The upper limit of the content of F-15603 in these dosage forms is 30 to 70% by weight, the lower limit is 0.1 to 1% by weight, and the preferred range is 1 to 70% by weight. The range is 1 to 30% by weight.

The dose of F-15603 may vary depending on the condition of the patient,
Depending on the age, body weight, administration method, dosage form, etc., the daily dose usually administered to an adult has an upper limit of 1 g to 5 g.
g, the lower limit is 0.1 mg to 1 mg, and a preferred range is 1 to 1000 mg.

The frequency of administration of a drug containing F-15603 as an active ingredient depends on the patient's condition, age, body weight, administration method, dosage form, dose of F-15603, and the like. Once a day, or several times a day.

[0060]

The present invention will be described in more detail with reference to the following examples, test examples and preparation examples, but the present invention is not limited to these examples. Embodiment 1 FIG. Preparation of F-15603 [1] Culture of F-15603-producing bacterium
In 18 500mL Erlenmeyer flasks including 100mL,
One loopful of the loop was inoculated from the slant, and cultured at 210 rpm and 23 ° C. for 120 hours using a rotary shaking incubator to obtain a seed culture solution. Medium-2 15L having the following composition
Was placed in a 30 L jar, and sterilized by heating at 121 ° C. for 30 minutes. After cooling this to 23 ° C., the seed culture 900
mL was inoculated and cultured under aeration and stirring at 23 ° C. for 240 hours. The rotation speed of the rotary shaking incubator was controlled between 100 and 580 rpm so that the dissolved oxygen was 5 ppm at an internal pressure of 100 kPa and an aeration rate of 15 L / min. [Medium-1] Glucose 0.4% Malt extract (Difco) 1.0% Yeast extract (Difco) 0.4% Agar powder 0.3% Defoamer CB-442 0.01% pH 5.5 before sterilization Was adjusted. [Medium-2] Glucose 3.0% Glycerin 3.0% Soluble starch 2.0% Soybean flour 1.0% Gelatin 0.25% Yeast extract (Difco) 0.25% Ammonium nitrate 0.25% Defoamer CB -442 0.01% Adjusted to pH 6.5 before sterilization. [2] Isolation of F-15603 To 173 g of the culture solution obtained in [1], 850 g of Celite 545 (manufactured by Johns Manville Product Corporation) was added as a filter aid, followed by filtration. 0.8 kg was obtained.

The pH of the filtrate was adjusted to 3.4, and an equal volume of ethyl acetate was added for extraction. The obtained organic layer (14 L) was washed with saturated saline (10 L), dried over anhydrous sodium sulfate, and filtered. 12 L of the obtained filtrate was concentrated under reduced pressure,
200 ml of concentrated liquid was obtained.

On the other hand, 15 L of 80% acetone water was added to the above-described cell cake, and the mixture was stirred and extracted for 30 minutes, and then filtered to obtain 15 L of an acetone extract. The acetone extract was concentrated under reduced pressure to obtain 4.4 L of a concentrate. 3. Increase the pH of the concentrate to 4.
The mixture was adjusted to 5 and extracted twice with an equal volume of ethyl acetate. The obtained organic layer was washed with 6.5 L of saturated saline, dried over anhydrous sodium sulfate, and filtered. 6. Extract obtained
5 L was concentrated under reduced pressure to obtain 100 ml of a concentrated solution.

The filtrate and the respective concentrates obtained from the cells were mixed, and the solvent was distilled off under reduced pressure to obtain 9.7 g of an oil.

The oil was dissolved in 100 ml of methanol and subjected to column chromatography on Sephadex LH-20 (manufactured by Pharmacia). 3. Sephadex LH-20 column previously equilibrated with 70% aqueous methanol.
The above methanol solution was added to 8 L, developed using 70% methanol water, and 120 ml of eluate was collected. F-15603 was eluted in fractions 16 to 32. Fractions 16 to 24 and fractions 25 to 32 were separately mixed and concentrated to dryness under reduced pressure.
1.75 g of an oily substance was obtained from the fraction of the number 25, and 0.85 g of an oily substance was obtained from the fractions of the number 25 to 32.

Sephadex LH-20 (Pharmacia) column 1 was prepared by equilibrating 0.85 g of the oily substance obtained from fractions 25 to 32 with 70% aqueous methanol in advance.
L and developed using 70% aqueous methanol, and 20 ml of eluate was collected. F-15603 was eluted in the 57th to 62nd fractions. Mix these fractions,
The filtrate was concentrated to dryness under reduced pressure to obtain 255.9 mg of a crude powder.

255.9 mg of the powder was dissolved in 10 mL of 60% aqueous methanol to prepare a sample solution, which was subjected to HPLC.
HPLC column previously equilibrated with 0.05% trifluoroacetic acid-30% acetonitrile water: Symmetry C18
(Diameter 20 mm x length 100 mm: manufactured by Waters)
After injecting 0.7 ml of the sample solution into the column, the mixture was eluted with a mixed solvent used for equilibration at a flow rate of 6 mL / min. The eluate was monitored at a wavelength of 210 nm using a UV detector.
-15603 was eluted with a retention time of 10.2 to 11.4 minutes, and 7.2 ml of eluate was obtained. Similar preparative HPL
The procedure C was repeated 14 times to obtain a total amount of 100.8 ml of a fraction, which was concentrated and dried to obtain F-15603.
132.6 mg of a white powder were obtained.

A part of this white powder was collected and analyzed by HPLC under the following conditions. Column: Symmetry C18 (φ4.6 × 150m
Mobile phase: acetonitrile / 0.3% triethylamine-phosphoric acid (pH 3.0) = 3/7 Flow rate: 1.0 ml / min Temperature: 25 ° C. Detection wavelength: 210 nm Retention time: 5. 9 minutes Test example 1 Cytotoxicity test on p53 dysfunctional mutant and normal cell lines In this test example, p53 dysfunctional mutant VA-13 (American Type Culture Collection (hereinafter referred to as “ATCC”) CCL-) was used as a test cell. 75.
1) and a normal cell line WI-38 (AT
CC CCL-75) was used. 10% for culture of both strains
MEM (Gibco) containing fetal bovine serum (Hyclone) was used as a medium.

1560 VA-13 cells per well,
Alternatively, 4800 WI-38s per well, 96 wells
Well flat bottom plate 3598 (Corning Costar)
5% CO_TwoAnd cultured at 37 ° C for 2 days.
Was. 10% fetal bovine serum (high clone
F-15603 serially diluted 5-fold with MEM containing
The final volume of one well will be 200-220 μl
And 5% CO _TwoCulture at 37 ° C for 24 hours
Nourished. After removing the culture supernatant, add 200 μl of each well.
Was washed twice with MEM containing 10% fetal calf serum,
μl of MEM containing 10% fetal calf serum was added. 5%
CO_TwoAfter further culturing at 37 ° C. for 72 hours,
2,3-bis [2-methoxy-4-nitro-5-sulfo
Phenyl] -2H-tetrazolium-5-carboxani
Lido (manufactured by Sigma) was added to the medium at a concentration of 1 mg / ml.
Dissolved phenazine / methosulfate (SIG
(Manufactured by MA Co., Ltd.) dissolved to 25 mM
Below, it is called "XTT / PMS liquid". ) 50 μl of each well
And incubated at 37 ° C for 2-4 hours.
Plate reader (Spectra Max 250: More
(Manufactured by Cular Devices) to 450 nm absorption wavelength
The absorbance of the live cells was measured using the following formula.
The percentage (%) was calculated. Viable cell percentage (%) = (Suction of wells with sample / cell addition)
Luminous intensity-absorbance of sample-added / cell-unadded well) x 100
/ (Absorbance of well without sample added / cell added-sample added /
Absorbance of wells without cells) Percentage of live cells at each concentration of F-15603 was plotted
Create a semi-log graph to reduce live cell percentage by 50%
Concentration (hereinafter referred to as “ED₅₀Value ”). F-15
603 is 0.91 μM (0.24 μM) for VA-13.
g / ml) ED₅₀WI-38
ED against₅₀The value is about 37μM (10μg / ml) or more
There was a difference of 42 times or more. Test example 2. Cytotoxicity test for various cancer cell lines In this test example, HL-60 (Dainippon
Pharmaceutical: CCL-240), Saos-2 (ATCC H)
TB-85), VA-13 (ATCC CCL-75.
1), HeLa (ATCC CCL-2), MES-S
A (ATCC CRL-1976), MES-SA / Dx
5 (ATCC CRL-1977), U937 (ATC CRL-1977)
C CRL-1593), CaSki (ATCC CR)
L-1550) and DLD-1 (ATCC CCL-2).
21) was used. For culture of each strain, use the following media
10% fetal bovine serum (manufactured by Hyclone) was added.
Was used as a medium. RPMI1640 medium (made by Asahi Glass Co.): HL-60, U
937, CaSki, DLD-1 MEM medium (manufactured by Gibco): VA-13 McCoy5a medium (manufactured by Gibco): Saos-2, M
ES-SA, MES-SA / Dx5 Preliminarily diluted 3 to 4 times with a medium containing 10% fetal bovine serum.
F-15603, doxorubicin (Sigma)
Or bleomycin in a final volume of 50
to a 96-well flat bottom plate 3598 (co
3) for one drug concentration.
Each well was dispensed. Contains 10% fetal bovine serum
Each strain was grown at a density of 50,000 cells / ml using each medium described above.
100 μl per well of the solution suspended in
% CO_TwoAnd cultured at 37 ° C. for 4 days. End of culture
Thereafter, the ED was obtained by the method described in Test Example 1.₅₀Determine the values and see Table 1
Summarized in Table 1. Toxicity to various cancer cells ―――――――――――――――――――――――――――――――――― Cell line ED₅₀(ΜM) ―――――――――――――――――――――――――――― F-15603 Doxorubicin Bleomycin ―――――――――――― ―――――――――――――――――――――――― HL-60 0.27 0.072 16 U937 0.72 0.059 5.6 HeLa 0.65 1.8 1.5 CaSki 0.76 0.24 1.5 VA-13 0.38 1.7 0.76 Saos- 2 0.38 0.63 110 DLD-1 1.4 0.34 0.66 MES-SA 0.53 0.18 1.2 MES-SA / Dx5 0.72 4.5 0.42 ―――――――――――――――――――――――――― ―――――――――― As shown in Table 1, F-15603 is any of the tested
0.27 to 1.4 μM (0.072
ED of 0.36 μg / ml)₅₀The value was shown. Tested
DLD-1, MES-SA / Dx among 9 cancer cell lines
ED of F-15603 on 7 strains excluding 5₅₀The value is blurred
Smaller than omycin, the activity of the compound is generally bleoma
It was suggested to be more potent than Isin. MES-SA
/ Dx5 is a cancer cell line derived from MES-SA
Doxorubicin, by overexpressing P-glycoprotein,
Resistant to many drugs including taxol
Is known. F-15603 is MES-SA / Dx5
Again, it showed almost the same cytotoxicity as MES-SA,
Effective against multidrug-resistant cancer cells overexpressing glycoproteins
It was shown to be. Test Example 3 A against tumor deficient cells
Potostosis induction test Quantification of apoptosis using DNA fragmentation as an index
ISA kit (Cell Death Detection ELISA,
Roche Diagnostics)
Was. First, 96-well flat bottom plate 3598 (Corning
16,000 WI-38 or 4
000 VA-13 in MEM containing 10% fetal bovine serum
100 μl of the suspension suspended in each well and 5% C
O_TwoAt 37 ° C. for 24 hours. 10% cow
100 μl of the sample diluent prepared with MEM containing fetal serum
It was added to each well and cultured for another 24 hours. Culture supernatant
After removing the cell lysis buffer (included in the kit described above)
You. ) Add 200 μl to each well, plate mix
-(Micro Mixer E-36: manufactured by Taitec Co., Ltd.)
And stirred. Centrifuge plate at 200 xg for 10 minutes
After precipitating the high molecular weight DNA by separation,
Apply 20 μl of the well supernatant to a streptavidin-coated plate.
(Included in the kit described above). More bio
Tin-labeled anti-histone antibody and peroxidase-labeled anti-D
A mixture of NA antibodies (both included in the above kit)
Add 80 μl to each well and stir at room temperature for 2 hours.
Incubated for hours. After the reaction, remove the antibody solution and incubate
Incubation buffer (included in kit above) 250
Each well was washed three times with μl. Remove the water in the wells
After some removal, 2,2'-azinobis (3-ethyl
Benzthiazoline sulfonic acid substrate solution (included in the kit above)
included. ) Add 100 μl to each well and keep at room temperature.
Colored by heating, microplate reader
(Spectra Max 250: Molecular Debye
Absorbance at an absorption wavelength of 405 nm
The measurement was performed, and the measurement results are summarized in FIG.

As shown in FIG. 1, the F-
15603 induced a fragmentation of DNA, which is characteristic of apoptosis, on VA-13 in a concentration-dependent manner. on the other hand,
No remarkable DNA fragmentation was observed in WI-38. Therefore, it was suggested that F-15603 selectively induces apoptosis in mutants deficient in p53 function. After mixing the powder of the above formulation and passing through a 60 mesh sieve, the powder is placed in a gelatin capsule to make a capsule.

[0070]

The novel compound F found in the present invention
-15603 has p53-deficient cell-selective apoptosis-inducing activity and is useful as a medicine, especially as an antitumor agent. In addition, F-15603 has been shown to be effective against multidrug-resistant cancer cells, which has recently become a clinical problem, and is also effective in treating malignant tumors that are resistant to various anticancer agents.

[Brief description of the drawings]

FIG. 1 is a graph showing that F-15603 selectively induced apoptosis in p53 dysfunctional cells in a concentration-dependent manner.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) // (C12P 13/02 (C12P 13/02 C12R 1: 645) C12R 1: 645) (72) Inventor Hideyuki Shiozawa 1-2-58 Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. (72) Inventor Kazunori Tanaka 33 Miyukigaoka, Tsukuba, Ibaraki Prefecture Sankyo Co., Ltd. (72) Inventor Yoji Furukawa Shinagawa-ku, Tokyo 1-258 Hiromachi F-term in Sankyo Co., Ltd. (Reference) 4B064 AE02 BA03 BE09 BE12 BE14 BE19 BG01 BG09 BH01 BH02 BH04 BH05 BH06 CA07 DA05 4C206 AA01 AA02 GA08 MA01 MA04 NA14 ZB21 ZB26 4H006 AA01 AAAB

Claims (8)

[Claims] (1) The following formula (I): A compound represented by the formula: 2. A compound having the following physicochemical properties: 1) Property: white powder, 2) Specific rotation: [α] _D ²⁰ -210 ° (c 0.2
5, methanol), 3) Solubility: soluble in dimethylsulfoxide, methanol, acetone, ethyl acetate, insoluble in water, 4) Molecular formula: C ₁₅ H ₂₁ O ₃ N (estimated from accurate mass measured by high-resolution mass spectrum) ), 5) Molecular weight: 263.3, 6) Ultraviolet absorption spectrum: λmax nm (ε), The ultraviolet absorption spectrum measured in a methanol solution is:
Shows a maximum absorption at 226 (29600) nm. 7) Infrared absorption spectrum (KBr): νmax cm
^The infrared absorption spectrum measured on the ^-1 KBr disc shows a maximum absorption at the following wave numbers: 3251, 2918, 26
35, 1943, 1664, 1623, 1538, 14
41, 1419, 1375, 1314, 1232, 11
89, 1116, 1068, 1026, 1012, 85
3,817,8) ¹ H-nuclear magnetic resonance spectrum; δ (ppm) The nuclear magnetic resonance spectrum (360 MHz) measured in a heavy methanol solution is as follows (internal standard is tetramethylsilane): 1.41 ( 1H, m), 1.71 (3H, br s),
1.76 (3H, br s), 2.00 (1H, m), 2.04 (1H, m), 2.15-
2.45 (4 overlapping signals), 4.09
(1H, dd, 12.4 Hz, 2.1 Hz), 4.12 (1H, dd, 12.4 Hz,
2.3 Hz), 5.58 (1H, m), 6.14 (1H, m), 6.49 (1H, br
s), 9) ¹³ C-nuclear magnetic resonance spectrum; δ (ppm) The nuclear magnetic resonance spectrum (90 MHz) measured in a deuterated methanol solution is as follows (the internal standard is tetramethylsilane): 18.1 (q ), 25.3 (t), 26.1 (q), 28.
6 (t), 32.3 (t), 34.2 (d), 63.1 (t), 94.7 (d), 11
0.6 (s), 121.2 (d), 132.0 (s), 134.1 (d), 134.9
(s), 169.3 (s), 204.8 (s), 10) Analysis by high performance liquid chromatography: Column: Symmetry C18 (4.6 mm in diameter × 150 mm in length, manufactured by Waters) Mobile phase: acetonitrile / 0.3% Triethylamine-phosphoric acid (pH 3.0) = 3/7 Flow rate: 1.0 mL / min Detection wavelength: 210 nm Retention time: 5.9 minutes. 3. The method according to claim 1, wherein a bacterium producing the compound according to claim 1 or 2 belonging to the genus Stereum is cultured, and the compound according to claim 1 or 2 is collected from the culture. Item 10. The method for producing a compound according to Item 1 or 2. 4. The bacterium according to claim 1, which belongs to the genus Stereum, wherein the bacterium producing the compound according to claim 1 or 2 is Stereum hirsutum SANK 16000 (FE).
RM BP-7200) strain. (5) Stereum hirsut
um) SANK 16000 (FERM BP-720
0) strain. 6. A medicament comprising the compound according to claim 1 as an active ingredient. 7. An apoptosis-inducing agent comprising the compound according to claim 1 or 2 as an active ingredient. 8. An antitumor agent comprising the compound according to claim 1 as an active ingredient.