JP5718450B2 - Anti-neoplastic agent and its production method and use - Google Patents

Description

  The present invention relates to a novel 3787-5-1 substance useful for the prevention and treatment of diseases caused by abnormal cell proliferation such as malignant tumors, and a production method and use thereof.

  The cells that make up the human body repeat division, proliferation, and apoptosis, and a control mechanism that keeps the number of cells almost constant under normal conditions. However, if control is lost due to gene mutation or the like, the cells grow indefinitely and tumors are formed.

  There are benign and malignant tumors. Tumors that do not invade or metastasize to other parts of the body and do not appear enlarged are called benign tumors, while tumors that invade or metastasize to other parts are called malignant tumors. Particularly problematic are malignant tumors.

  It is thought that important factors involved in the development of malignant tumors are constitutive activation of cells due to mutations in genes called proto-oncogenes such as src, ras, and myc, and p53, Rb, BRCA1 It is a dysfunction of cells due to mutations in a group of genes called tumor suppressor genes (Non-patent Document 1). The functions of tumor suppressor proteins are diverse, including cell cycle checkpoint control, transcription factor control, transcription, and DNA repair.

  For the treatment of malignant tumors, 1) alkylating agents represented by nitrogen mustards, nitrosoureas, etc., 2) antimetabolites represented by purine analogues, pyrimidine analogues, folate antimetabolites, etc. 3) Antitumor antibiotics represented by anthracyclines, anthracenediones, bleomycins, mitomycins, etc. 4) Plant alkaloids represented by vinca alkaloids, taxoids, DNA topoisomerase inhibitors, etc. 5) Platinum compounds 6) Hormonal drugs such as antiestrogens, aromatase inhibitors, LH-RH stimulants, luteinizing hormones, antiandrogens, glucocorticoids, estrogens, androgens, 7) molecular targeting drugs, 8 ) Various drugs such as cytokines have been developed and used in clinical settings.

  However, it is generally known that most antineoplastic drugs are more toxic than other therapeutic agents and have problems with side effects (Non-patent Document 2). Therefore, development of a new type of antineoplastic agent that suppresses the growth of cancer cells is desired.

One of the new drugs is an anti-malignant tumor activity enhancer that can enhance the pharmacological activity of an anti-malignant tumor agent in a specific manner for malignant tumor cells.
Normal cells have a mechanism to repair DNA damage in the G1 and G2 phases (cell cycle checkpoints), and DNA damage is repaired mainly at checkpoints in the G1 phase (G1 checkpoint). Is called. However, in many malignant tumor cells with mutated tumor suppressor genes, this G1 repair mechanism (G1 checkpoint) does not function and DNA damage is repaired by the G2 repair mechanism (G2 checkpoint). (Non-Patent Document 3).

  Therefore, when a G2 checkpoint inhibitor is administered in combination with administration of a minute amount of DNA damaging agent and / or treatment such as slight irradiation, normal cells can repair DNA damage with G1 checkpoint, Since malignant tumor cells cannot repair DNA damage by either G1 checkpoint or G2 checkpoint, it is considered that DNA damage accumulates in malignant cells and cell proliferation becomes impossible. That is, a drug that inhibits G2 checkpoint is expected to become a new type of antitumor agent that can enhance the effect of an antineoplastic agent specifically on malignant tumor cells (Patent Document 1 and Non-Patent Document 4). ).

WO 2001/021771

Hanahan et al., Cell, Vol. 100, pp. 57-70, 2000 Edited by Yasuhiko Yamada, Pharmacotherapeutics of Cancer for Pharmacists and Pharmacists, Chemistry Dojinsha, pp. 43-57, 2009 Zhou et al., Cancer Biol. Ther., Vol. 4, Suppl. 1, pp. S16-S22, 2003 Zabludoff et al., Mol. Cancer Ther., Vol. 7, pp. 2955-2966, 2008.

  The present invention relates to a novel compound that is a safe and effective pharmaceutical agent capable of suppressing the onset and progression of a malignant tumor when administered alone or in combination with other antineoplastic agents, a method for producing the same, and a use thereof. The purpose is to provide.

  As a result of searching for a metabolite produced by a microorganism and having an anti-neoplastic tumor activity alone and further enhancing the activity of another anti-neoplastic agent, the present inventors have found that Ishigaki, Okinawa, Japan. Substance with the desired activity in the culture solution of actinomycete Phytohabitans sp. 3787-5 (patent microorganism deposit center accession number NITE BP-1137) newly isolated from the soil of the island Was found to be produced. Subsequently, as a result of separating and purifying an anti-malignant tumor activity enhancing substance from the culture, it was found that this substance was a novel compound having the chemical structure described below, and this substance was newly named 3787-5-1.

  The present invention has been completed based on such findings, and relates to a 3787-5-1 substance and a salt thereof which are compounds represented by the following formula [I].

  The present invention also includes a microorganism belonging to the genus Phytohabitans and capable of producing the above 3787-5-1 substance, which is cultured in a medium to accumulate the 3787-5-1 substance in the culture, and from the culture, The present invention relates to a method for producing a 3787-5-1 substance, characterized by collecting the 3787-5-1 substance.

In the above production method, the microorganism is preferably Phytohabitans sp. 3787-5 strain (NITE BP-1137).
Furthermore, the present invention relates to an antineoplastic agent or an antineoplastic activity enhancer comprising the above 3787-5-1 substance or a pharmaceutically acceptable salt thereof, or the above 3787-5-1 substance or a pharmaceutically acceptable salt thereof. It also relates to the use of an acceptable salt as an anti-neoplastic agent or an anti-neoplastic activity enhancer.

  The present invention also comprises the above 3787-5-1 substance or a pharmaceutically acceptable salt thereof as an active ingredient, particularly for the prevention or treatment of diseases caused by abnormal cell growth, typically malignant tumors. Diseases resulting from abnormal cell proliferation in mammals including humans, comprising administering to a patient in need of a pharmaceutical composition, and an effective amount of the above 3787-5-1 substance or a pharmaceutically acceptable salt thereof In particular, it also relates to a method for preventing or treating malignant tumors. The present invention also relates to a method for enhancing the activity of an antineoplastic agent comprising administering an effective amount of the above 3787-5-1 substance or a pharmaceutically acceptable salt thereof to mammals including humans. Related.

  According to the present invention, there is provided a novel substance 3787-5-1 which exhibits an anti-malignant tumor activity alone and has an anti-malignant tumor activity enhancing action when administered in combination with other antineoplastic agents. A pharmaceutical composition comprising this substance as an active ingredient can be used for the prevention or treatment of malignant tumors.

  Furthermore, according to the present invention, there are provided a microorganism that produces the novel substance 3787-5-1 and a method for producing the novel substance 3787-5-1 using the microorganism.

It is a figure which shows the ultraviolet absorption spectrum (in methanol solution) of 3787-5-1 substance based on this invention. It is a figure which shows the infrared region absorption spectrum (potassium bromide method) of 3787-5-1 substance based on this invention. It is a figure which shows the proton nuclear magnetic resonance spectrum (in deuterated chloroform) of 3787-5-1 substance based on this invention. It is a figure which shows the carbon nuclear magnetic resonance spectrum (in deuterochloroform) of 3787-5-1 substance based on this invention.

  The 3787-5-1 substance, which is a novel compound according to the present invention, belongs to the genus Phytohabitans, and a microorganism having the ability to produce the 3787-5-1 substance is cultured in a medium, and 3787-5- It can be produced by accumulating one substance and collecting 3787-5-1 substance from the culture.

  As an example of a strain used for producing the 3787-5-1 substance of the present invention, Phytohabitans sp. 3787-5 newly isolated from soil by the present inventors (Phytohabitans sp. 3787-5) ) (Deposited with the Patent Microorganisms Center, National Institute of Technology and Evaluation, August 26, 2011, deposit number NITE BP-1137, deposit date September 28, 2011). The mycological properties of this strain are as follows.

  This strain is grown at 27 ° C. at 2 ° C. for growth on a general liquid medium for actinomycetes such as yeast extract / dextrose medium or starch / glucose medium (starch, glucose, peptone, meat extract, yeast extract, calcium carbonate). It takes 3 weeks and the cells are orange. On starch / inorganic salt agar (ISP 4) and yeast / malt extract agar (ISP 2), a slight ivory colony is formed by culturing at 27 ° C. for 2 weeks, and white short aerial hyphae are formed. No soluble pigment is produced.

  The sequence of 1281 bases in the 16S rRNA gene of this strain was determined, and the sequence was searched from the DNA database of approved actinomycetes using EzTaxon server 2.1 (http://147.47.212.35:8080/) As a result, this strain showed 100% homology with Phytohabitans suffuscus and was determined to be one species belonging to the genus Phytohabitans.

  In order to produce the 3787-5-1 substance of the present invention, it is preferable to use the Phytohabitans sp 3787-5 strain, but not limited thereto, artificial mutants and natural mutants of the strains are also available. Any microorganism that belongs to the genus Phytohabitans and has the ability to produce the 3787-5-1 substance can be used.

  As the medium for culturing the microorganism, a nutrient medium containing a carbon source that can be assimilated by the microorganism into a nutrient source, a nitrogen source that can be digested, and an inorganic salt, a vitamin, or the like as required. As carbon sources that can be assimilated, sugars such as glucose, fructose, maltose, lactose, galactose, dextrin, starch, and vegetable oils such as soybean oil are used alone or in combination. Digestible nitrogen sources include peptone, yeast extract, meat extract, soy flour, cottonseed flour, corn steep liquor, malt extract, casein, amino acids, urea, ammonium salts, nitrates alone or in combination It is done. Other salts such as phosphate, magnesium salt, calcium salt, sodium salt, potassium salt, heavy metal salts such as iron salt, manganese salt, copper salt, cobalt salt, zinc salt, vitamins, etc. Ingredients suitable for production of -1 substances may be added as appropriate.

  During the cultivation, when foaming is severe, an antifoaming agent such as liquid paraffin, animal oil, vegetable oil, silicone, surfactant or the like may be added as necessary. The medium used for the culture may be liquid or solid as long as the nutrient source is contained, but it is usually preferable to use a liquid medium for culture. When the target substance is industrially produced in large quantities, it is preferable to culture with aeration and stirring. When culturing in a large tank, in order to prevent the growth delay of the bacteria in the production process, first inoculate and inoculate the produced bacteria in a relatively small amount of medium, and then transfer the culture to a large tank. Production culture is preferred. In this case, the composition of the medium used for the preculture and the medium used for the production culture may be the same or different. When the culture is performed under aerated stirring conditions, known methods such as stirring with a propeller or other machine, rotation or shaking of a fermenter, pumping, air blowing, etc. are appropriately used. Use sterilized air for ventilation.

  The culture temperature can be appropriately changed within the range in which the microorganism producing this 3787-5-1 substance produces this substance, but is usually 20 to 30 ° C., preferably around 27 ° C. Culturing can be performed by appropriately combining one or both of shaking culture and stationary culture. For the production of the 3787-5-1 substance, it is preferable to use shaking culture, and the culture time varies depending on the culture conditions, but is usually about 10 to 50 days in the case of shaking culture.

  In order to collect the novel substance of the present invention accumulated in the culture, a method usually used for collecting a metabolite from a microorganism culture can be used. For example, the target substance is separated and purified by one or more methods selected from extraction with organic solvents, concentration, drying, adsorption, filtration, centrifugation, chromatography and the like.

  An example of a method for collecting the 3787-5-1 substance of the present invention from the culture of Phytohabitans sp. Strain 3787-5 is as follows. The cells are extracted from the culture with a water-miscible organic solvent such as ethanol, the extract is distilled under reduced pressure to distill off the organic solvent, and the resulting residue is extracted with a water-immiscible organic solvent such as ethyl acetate. To do. For the substances collected in this way, known chromatographic treatments used for the purification of fat-soluble substances, such as adsorption chromatography, gel filtration chromatography, thin layer chromatography, centrifugal countercurrent distribution chromatography, high performance liquid chromatography, etc. The 3787-5-1 substance can be purified by appropriately combining or repeating methods selected from

The physicochemical properties of the 3787-5-1 substance of the present invention thus obtained will be described below.
(1) Property: brown powder;
(2) Molecular formula: C ₂₆ H ₃₈ O
HREI-MS (m / z) [M] ⁺ calculated value 366.2923, actual value 366.2929;
(3) Molecular weight: 366
Observe [M] ⁺ 366 with EI-MS (m / z);
(4) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in a methanol solution is as shown in FIG. 1, and λmax (MeOH, ε): 221 (8052), 234 (3294), 284 (3587) nm Indicates absorption;
(5) Infrared absorption spectrum: The infrared absorption spectrum measured by the potassium bromide tablet method is as shown in FIG. 2, and νmax 3509, 3428, 2929, 2851, 1711, 1614, 1461, 1380, 1338, 1264, Shows characteristic absorption maximum at 1185 cm ^-1 etc .;
(6) Specific rotation: [α] D22 -41.60 ° (c = 0.1, methanol);
(7) Solubility in solvents: Soluble in methanol, ethanol, acetonitrile, ethyl acetate, dimethyl sulfoxide. Easily soluble in chloroform;
(8) Proton and carbon nuclear magnetic resonance spectra: The hydrogen chemical shift (ppm) and carbon chemical shift (ppm) measured in a deuterated chloroform with a Varian 600 MHz nuclear magnetic resonance spectrometer are as shown below. is there;
δH: 0.33 (3H), 0.73 (3H), 0.78 (3H), 0.82 (1H), 0.84 (1H) 0.85 (3H, 1H), 1.02 (1H), 1.06 (3H), 1.15 (1H, 1H), 1.26 (1H), 1.36 (1H), 1.40 (1H), 1.43 (1H), 1.46 (1H), 1.54 (1H), 1.56 (1H), 1.72 (1H, 1H), 1.82 (1H), 2.25 (1H ), 2.58 (1H), 2.94 (1H), 4.68 (1H), 6.54 (1H), 6.55 (1H), 6.96 (1H) ppm;
δC: 16.4, 16.4, 18.2, 18.4, 18.6, 21.5, 31.0, 33.3, 33.4, 33.5, 35.0, 37.4, 37.4, 40.1, 42.2, 43.0, 46.2, 56.8, 58.0, 63.1, 107.8, 112.6, 124.6, 135.9, 153.8, 154.2 ppm.

The proton nuclear magnetic resonance spectrum in deuterated chloroform is shown in FIG. 3, and the carbon nuclear magnetic resonance spectrum is shown in FIG.
As a result of detailed examination of various physicochemical properties and spectral data of the above 3787-5-1 substance, it was determined that the 3787-5-1 substance has a chemical structure represented by the following formula [I].

  The present invention also includes salts of the 3787-5-1 substance represented by the above formula [I], particularly pharmaceutically acceptable salts. Examples of the salt include alkali metal salts such as sodium salts and ammonium salts. These salts can be produced according to a conventional method. For example, the 3787-5-1 substance is dissolved in a water-miscible organic solvent such as ethanol, and the resulting solution is reacted with an alkali metal hydroxide solution dissolved in the same solvent, or ammonia gas is added to this solution. By passing through, an alkali metal salt or an ammonium salt can be produced.

  The 3787-5-1 substance of the present invention exhibits an anti-malignant tumor action alone as shown in the test examples described later, and further has an anti-malignant tumor activity enhancing action of other anti-neoplastic agents used in combination. . Therefore, this substance can suppress the onset and progression of malignant tumors alone or in combination with other antineoplastic agents, resulting from malignant tumors and diseases resulting from them, more broadly, abnormal cell proliferation. It is useful as a preventive or therapeutic agent for diseases.

  Examples of diseases for which compounds that exhibit antineoplastic activity alone or in combination are effective include gastric malignancy, interstitial pneumonia, rheumatoid arthritis, intrahepatic bile duct malignancy, liver malignancy, pneumothorax, Acute bronchitis, acute bronchiolitis, acute leukemia, chest wall tumor, pleural tumor, retroperitoneal disease, melanoma, myeloproliferative disease, bone soft tissue malignant tumor, cervical malignant tumor, uterine body malignant tumor, Uterine appendage malignant tumor, autoimmune disease, small intestine malignant tumor, esophageal malignant tumor, mediastinal malignant tumor, mediastinal / pleural malignant tumor, renal pelvis malignant tumor, renal tumor, pancreatic tumor, testicular tumor, prostate Malignant tumor, colon malignant tumor, multiple myeloma, extrabiliary bile duct malignant tumor, cholangitis, bile duct stone, gall bladder malignant tumor, intestinal obstruction, rectal anal malignant tumor, head and neck malignant tumor, breast malignant tumor, Malignant tumor of ureter, cerebral infarction, encephaloma , Pneumonia, lung malignant tumor, spleen tumor, non-Hodgkin lymphoma, bladder tumor, Hodgkin disease, chronic hepatitis C, chronic leukemia, immune system malignant neoplasm, ovarian malignant tumor, glaucoma, etc. It is not limited.

  When the 3787-5-1 substance of the present invention is used alone or in combination with other antineoplastic agents as a prophylactic or therapeutic agent for malignant tumors and related diseases, the formulation may be according to conventional methods. For example, the substance of the present invention is used as an active ingredient, and conventional carriers and excipients, if necessary, binders, disintegrants, lubricants, buffers, suspending agents, stabilizers, pH regulators, colorants A flavoring agent, a fragrance | flavor, etc. can be added and it can formulate in a solution, suspension, a tablet, a granule, a powder, a capsule.

  When the 3787-5-1 substance of the present invention is used as a drug for enhancing the antineoplastic activity of other antineoplastic agents, the anti-malignant tumor activity is enhanced by the 3787-5-1 substance of the present invention. Examples of the antineoplastic agent include antitumor antibiotics such as bleomycin and adriamycin (doxorubicin). Although many antineoplastic agents have problems with side effects, by increasing the activity of the antineoplastic agent by co-administering the 3787-5-1 substance of the present invention, the dosage of the antineoplastic agent can be increased. Reduction and / or shortening of the administration period can be achieved, and side effects of the drug can be reduced.

The other antineoplastic agents and the 3787-5-1 substance of the present invention can be administered simultaneously or sequentially.
The pharmaceutical composition containing the 3787-5-1 substance of the present invention or a pharmaceutically acceptable salt thereof can be formulated according to a conventional method with a carrier or the like, if necessary, according to the administration form. Examples of the preparation for oral administration include tablets, pills, granules, capsules, powders, solutions, suspensions, syrups, sublinguals and the like. Examples of the preparation for parenteral administration include injections, transdermal absorption agents, inhalants, suppositories and the like. In formulating, pharmaceutical additives such as surfactants, excipients, stabilizers, wetting agents, disintegrants, solubilizers, isotonic agents, buffers, coloring agents, and flavoring agents are appropriately used. When used in combination with other antineoplastic agents, the dosage form and administration route of the 3787-5-1 substance are not necessarily the same as those of other antineoplastic agents. For example, one can be administered orally and the other parenterally.

  As the carrier, pharmaceutically acceptable ones can be used, and the type and composition can be appropriately determined depending on the administration route and administration method. For example, water, alcohol, soybean oil, sesame oil or the like can be used as the liquid carrier. As the solid carrier, sugars such as maltose and sucrose, amino acids such as lysine, cellulose derivatives such as hydroxypropyl cellulose, polysaccharides such as cyclodextrin, organic acid salts such as magnesium stearate, and the like can be used. When formulated as an injection, the liquid carrier can be generally a physiological saline, various buffers, or a sugar solution such as glucose.

  When the 3787-5-1 substance of the present invention or a pharmaceutically acceptable salt thereof is administered in the form of a pharmaceutical composition, the dosage depends on the patient's age, weight, type and degree of disease, administration route, etc. It depends on factors such as the presence or absence of these drugs, and can be determined within a range where the total dose does not exceed a certain amount in consideration of various situations such as the results of animal tests. Specifically, in the case of oral administration to humans, it can be administered within the range of 1-1000 mg / kg per day for each adult, and in the case of intravenous administration, it is similarly 0.1-100 mg / kg. The specific dose is an amount effective for the prevention or treatment of malignant tumors and varies from patient to patient, and is determined by the attending physician.

  When the pharmaceutical composition further contains other antineoplastic agents in addition to the 3787-5-1 substance, it can be formulated in the same manner as described above. In that case, the dosage of other antineoplastic agents is the amount prescribed for the antineoplastic agent. The dose of the 3787-5-1 substance is an amount effective for enhancing the antineoplastic activity of the antineoplastic agent to be combined. Preferably, the enhancement activity ratio obtained by the method shown in Test Example 2 described later is 2 or more. More preferably, the amount is 5 or more.

  Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

3787-5-1 Substance Production Method Agar Slope Medium (Starch 1.0% (Kanto Chemical), N-Zet Amine 0.3% (Wako Pure Chemical Industries), Yeast Extract 0.1% (Oriental Yeast), Meat Extract 0.1% (Kyokuto Pharmaceutical) Industrial), calcium carbonate 0.3% (Kanto Chemical), agar 1.2% (Shimizu Foods) adjusted to pH 7.0), Phytohabitans sp 3787-5 strain, seed medium (glucose 2% (Wako Pure Chemical Industries), 0.5% of polypeptone (Wako pure _{Chemical), MgSO 4 · 7H 2 O} 0.05% ( Wako pure Chemical), 0.2% yeast extract (Oriental yeast), KH ₂ PO ₄ 0.1% (Kanto Chemical), 0.1% agar (Shimizu foods) (Adjusted to pH 6.0) A large test tube into which 10 ml was dispensed was inoculated with one platinum loop, and cultured at 27 ° C. for 3 days on a rotary shaker (210 rpm). The obtained seed culture solution was added to a production medium (soluble starch 1.0% (Becton Dickinson), glucose 1.0% (Kanto Chemical), yeast extract 0.2% (Oriental yeast), soy flour 2.0% (Becton Dickinson), metal salt solution 0.1 % (FeSO ₄ · 7H ₂ O 0.1% (Kanto Chemical), MnCl ₂ · 4H ₂ O 0.1% (Kanto Chemical), ZnSO ₄ · 7H ₂ O 0.1% (Kanto Chemical), CuSO ₄ · 5H ₂ O 0.1% ( Kanto Chemical), CoCl ₂ · 6H ₂ O 0.1% (Wako Pure Chemical Industries, Ltd., pH 7.0), inoculate 1% each into a 500 ml Erlenmeyer flask containing 100 ml of 100 l, and shake at 27 ° C for 30 days Culture was performed.

  After completion of the culture, the combined culture solution (4.5 l) is centrifuged at 3,000 rpm for 10 minutes, ethanol (2.0 l) is added to the precipitated cells, and after sufficient stirring, sonicated for 1 hour. An extract was obtained. Ethanol was distilled off from the extract under reduced pressure to obtain a 0.8 l concentrate. The active ingredient was extracted from this concentrated solution with ethyl acetate (0.8 l), and the ethyl acetate layer was concentrated to dryness to obtain a brown active crude substance (2.34 g). This crude material was roughly purified by chromatography using an ODS (octadecylsilyl) column (PEGASIL, manufactured by Senshu Kagaku, 125 g). In chromatography, 800 ml of 60% acetonitrile aqueous solution, 1.2 l of 100% acetonitrile, and 1.2 l of 100% acetone were used as developing solvents, and 200 ml each of the eluate was fractionated with any developing solvent. By concentrating fractions containing the 3787-5-1 substance (first and second 100% acetone fractions), 81.4 mg of a brown substance was obtained. This material was dissolved in a small amount of methanol and subjected to final purification by preparative HPLC (column: PEGASIL C4, 20φ × 250 mm, Senshu Kagaku Co., Ltd.). Acetonitrile aqueous solution containing 0.05% phosphoric acid was used as a mobile phase, and elution was performed with a linear concentration gradient from 85% to 90% acetonitrile in 45 minutes. Absorption at UV 210 nm was monitored at a flow rate of 8 ml / min. A peak showing activity was observed at a retention time of 27 minutes. This peak was collected, and the fraction was concentrated under reduced pressure to isolate a brown powder of 3787-5-1 substance in a yield of 16.41 mg. The identification data is as described above.

(Test Example 1)
Anticancer activity of 3787-5-1 substance of the present invention by MTT evaluation method using thiazolyl blue tetrazolyl bromide (Mosmann et al., J Immunol. Methods, Vol. 65, pp. 55-63, 1983) Examined.

10 μl of a solution of 3787-5-1 substance adjusted to a concentration of 0.001, 0.01, 0.1, 1 mg / ml with methanol was added to each 96-well plate and air-dried in a clean bench.
Next, Jurkat cells, a human T-cell leukemia cell line, were prepared at 5.0 × 10 ⁵ cells in RPMI-1640 medium (Invitrogen) containing 5% fetal calf serum (Hyclone) and penicillin / streptomycin (Invitrogen). After preparing 100 ml in each well, it was cultured overnight at 37 ° C. in a 5% carbon dioxide incubator.

Next, 10 μl of thiazolyl blue tetrazolyl bromide aqueous solution dissolved in 5.5 mg / ml was added to each well and further cultured for 3 hours.
90 μl of a solution (40% N, N-dimethylformamide, 2% acetic acid, 20% sodium dodecyl sulfate, 0.03N hydrochloric acid) was added to each well and shaken for 3 hours.

The absorbance at 570 nm of each hole was measured with a microplate reader (Elx808, manufactured by BIO-TEK Instruments), and the survival rate was measured by the following formula.
Survival rate (%) = 100 × [1-((absorbance when test compound is added) − (background)) / ((absorbance of control) − (background))]
As a result of obtaining a concentration (IC ₅₀ ) that inhibits the survival rate by 50% as an index of antineoplastic activity, the IC ₅₀ value of the 3787-5-1 substance of the present invention was calculated to be 142 μM. It became clear that malignant tumor activity was shown.

(Test Example 2)
Jurkat cells are known to fail G1 checkpoint due to mutations in p53 and to repair DNA damage depending on G2 checkpoint (Suganuma et al., Cancer Res., Vol. 59, pp. 5887-5891, 1999). That is, the anti-malignant tumor activity of DNA damaging agents in Jurkat cells is thought to reflect G2 checkpoint inhibitory activity (Arai et al., Biochem. Biophys. Res. Commun., Vol. 317, pp. 817-822 , 2004).

The MTT evaluation method was used to examine the effect of enhancing the antineoplastic activity of the 3787-5-1 substance of the present invention against bleomycin.
9.5 μl of 3787-5-1 substance adjusted to 0.01 mg / ml with methanol was added to a 96-well plate and air-dried in a clean bench.

Next, Jurkat cells were mixed with RPMI-1640 medium (Invitrogen) containing 5% fetal calf serum (Hyclone), penicillin / streptomycin (Invitrogen) and 30 μg / ml bleomycin hydrochloride (Nippon Kayaku Co., Ltd.). After preparing 10 × 10 ⁵ cells / ml, 100 μl was poured into each well and cultured overnight at 37 ° C. in a 5% carbon dioxide incubator.

Next, 10 μl of thiazolyl blue tetrazolyl bromide aqueous solution dissolved in 5.5 mg / ml was added to each well and further cultured for 3 hours.
90 μl of a solution (40% N, N-dimethylformamide, 2% acetic acid, 20% sodium dodecyl sulfate, 0.03N hydrochloric acid) was added to each well and shaken for 3 hours.

The absorbance at 570 nm of each hole was measured with a microplate reader (Elx808, manufactured by BIO-TEK Instruments), and the IC ₅₀ value was measured in the same manner as in Test Example 1.
Furthermore, based on the antineoplastic activity of bleomycin, the activity enhancement ratio of the antineoplastic activity by 3787-5-1 substance was calculated by the following formula.

Activity enhancement ratio = (bleomycin IC ₅₀ ) / (bleomycin IC ₅₀ in the presence of 3787-5-1 substance)
The obtained results are shown in Table 1 below for the IC ₅₀ value and the enhancement activity ratio.

When only bleomycin was allowed to act on Jurkat cells, the IC ₅₀ value of bleomycin was calculated to be 236 μg / ml. When the 3787-5-1 substance was reacted with bleomycin at 0.78 μg / ml, the IC ₅₀ value of bleomycin was calculated to be 23.9 μg / ml, and the antineoplastic activity of bleomycin was enhanced by 9.9 times.

(Test Example 3)
Measure the cell cycle using a flow cytometer, and refer to the method of Suganuma et al. (Suganuma et al., Cancer Res., Vol. 59, pp. 5887-5891, 1999). The checkpoint inhibitory activity was analyzed.

1 μl each of 3787-5-1 substance adjusted to 0.1, 1, 10 mg / ml with methanol was added to a 96-well plate and air-dried in a clean bench.
Next, Jurkat cells were cultured in RPMI-1640 medium containing 5% fetal calf serum (Hyclone), penicillin / streptomycin (Invitrogen) and 30 μg / ml bleomycin hydrochloride (Nippon Kayaku Co., Ltd.) Invitrogen) was prepared to 5.0 × 10 ⁵ cells / ml, and 100 μl was poured into each well and cultured overnight at 37 ° C. in a 5% carbon dioxide incubator.

  100 μl of staining solution (0.2% sodium citrate (SIGMA), 100 mg / ml propidium iodide (SIGMA), 40 mg / ml ribonuclease A (SIGMA), 0.6% IGEPAL CA-630 (SIGMA))) in each hole Add each and shake for 10 minutes.

The cell cycle was measured with a flow cytometer (FACSCalibur, manufactured by Becton Dickinson), and the proportion of cells distributed in each phase was calculated with Cell Quest.
Furthermore, 3787-5-1 reduces the proportion of cells in G2 / M phase to 50% based on the case where 3787-5-1 substance is not allowed to act in both the absence and presence of 30 μg / ml bleomycin hydrochloride. calculating the concentration of -5-1 material was the EC _50.

The results obtained for EC ₅₀ are shown in Table 2 below.

The EC ₅₀ when only 3787-5-1 substance was allowed to act on Jurkat cells was calculated to be 273 μM or more. When the 3787-5-1 substance was allowed to act together with bleomycin, the EC ₅₀ of the 3787-5-1 substance was calculated to be 3.55 μM. That is, it became clear that the 3787-5-1 substance inhibits G2 checkpoint (G2 phase repair mechanism) caused by bleomycin.

  The compound according to the present invention exhibits G2 checkpoint inhibitory activity, and further exhibits the activity of enhancing the anti-malignant tumor activity of bleomycin. Therefore, as an anti-neoplastic agent and an anti-malignant tumor activity enhancer, a malignant tumor caused by abnormal cell proliferation It is expected to be useful for the treatment of diseases such as these.

  NITE BP-1137

Claims (8)

A 3787-5-1 substance or a salt thereof which is a compound represented by the following formula [I].

Antineoplastic agents containing Claim 1 substance or a pharmaceutically acceptable salt thereof. An anti-malignant tumor activity enhancer comprising the substance according to claim 1 or a pharmaceutically acceptable salt thereof .   A pharmaceutical composition for preventing or treating a disease caused by abnormal cell proliferation, comprising the substance according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.   The pharmaceutical composition according to claim 4, further comprising another antineoplastic agent. Other antineoplastic agent is an antitumor antibiotic substances, the pharmaceutical composition according to claim 5. A pharmaceutical composition for inhibiting G2 phase repair mechanism , comprising the substance according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. The method for producing 3787-5-1 substance according to claim 1, wherein Phytohabitans sp 3787-5 strain (accession number NITE BP-1137) having the ability to produce 3 787-5-1 substance or A method comprising culturing a microorganism which is the mutant strain in a medium, accumulating 3787-5-1 substance in the culture, and collecting 3787-5-1 substance from the culture.

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