WO2018021890A1 - Cromenone derivative and anticancer composition containing same - Google Patents

Cromenone derivative and anticancer composition containing same Download PDF

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WO2018021890A1
WO2018021890A1 PCT/KR2017/008204 KR2017008204W WO2018021890A1 WO 2018021890 A1 WO2018021890 A1 WO 2018021890A1 KR 2017008204 W KR2017008204 W KR 2017008204W WO 2018021890 A1 WO2018021890 A1 WO 2018021890A1
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cancer
compound
formula
present
hsp27
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French (fr)
Korean (ko)
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이윤실
나영화
권영주
이화정
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이화여자대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
    • C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
    • C07D407/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
    • C07D407/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the present invention relates to novel chromemenone derivatives, and more particularly to novel chromemenone derivatives having heat shock protein 27 inhibitory activity.
  • Heat shock protein is a chaperone protein and is known to regulate protein degeneration and cell death by expression by various physiological factors or environmental factors such as anticancer agents (Garrido, 2006). Activated heat shock proteins are chaperones that help fold up poorly folded proteins, prevent protein denaturation, and other important effects on various signaling pathways, interfering with cell death, immune regulation, and cell protection It has an effect of suppressing cell death by various functions such as effects (Khalil, 2011).
  • HSP27 Heat shock protein 27
  • ⁇ -crystallin domain and the N-terminal WDPF domain play an important role in the multimer production of HSP27 (Lambert, 1999). Cysteine residues in the ⁇ -Crystallin domain form disulfide bonds between monomers of HSP27 to form dimers of HSP27 (Mymrikov, 2010). The intracellular function of HSP27 is related to this degree of multimer production.
  • HSP27 prevents HSP27 from functioning as a chaperone by inhibiting the large multimeric form of HSP27, resistance to radiation or anticancer drugs will be overcome.
  • HSP90 is an ATP-dependent chaperone protein expressed in all eukaryotic cells.
  • HSP90 client proteins are involved in the survival of almost all cells, many of which are cell growth, cell division. And vital cell roles such as survival. Most of these processes are also involved in cancer growth. Therefore, targeting HSP90 simultaneously causes confusion of various carcinogenic signal transduction pathways, which is particularly effective in treating advanced cancers in which various carcinogenic signal transduction pathways occur.
  • HSP27 thermal shock protein 27
  • Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising the novel chromemenone derivative.
  • the present inventors have conducted intensive studies on the treatment of cancer, especially cancers resistant to radiation to anticancer agents, and have shown that the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof is modified to form a modified heat shock protein 27 (HSP27) dimer.
  • the present invention was completed by discovering the effect of inducing cancer cell death and reducing radiation and resistance to anticancer drugs.
  • the present invention provides a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof.
  • R 1 and R 2 are each independently hydrogen; Or heterocyclylC 1-4 alkyl composed of 1 or 2 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur and 1 to 6 carbon atoms,
  • R 3 is a heteroaryl group consisting of C 1- 10 alkyl, C 3- 10 cycloalkyl, C 6- 14 aryl, or oxygen, nitrogen and sulfur from the group consisting of one to four heteroatoms selected with 1 to 10 carbon atoms, Wherein at least one hydrogen of R 3 is each independently substituted or unsubstituted with R 4 ,
  • R 4 is F, Br, Cl, I, OH, OMe, OEt, NH 2 , NMe 2 , CN, COOH, COMe, COOMe, CONH 2 , or C 1-4 alkyl.
  • R 1 and R 2 are each independently hydrogen, or to be.
  • R 3 is a C 1- 10 alkyl, C 3- 10 cycloalkyl.
  • R 3 is methyl or phenyl.
  • the compound of Formula I may be selected from the group consisting of the compounds of Table 1 below.
  • pharmaceutically acceptable salts mean salts commonly used in the pharmaceutical industry, for example, inorganic ion salts, hydrochloric acid, nitric acid, phosphoric acid, bromic acid, prepared with calcium, potassium, sodium and magnesium, Inorganic acid salts made with iodic acid, perchloric acid, tartaric acid, sulfuric acid, etc.
  • Organic acid salts methanesulfonic acid, ethanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, which are made of cholic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanic acid, hydroiodic acid, etc.
  • amine salts made of nia, pyridine, picoline and the like the salts used in the present invention are not limited by these salts.
  • the compounds of the present invention induce the production of modified heat shock protein 27 (HSP27) dimers, thereby inhibiting the production of HSP27 macromers, thereby inhibiting the chaperone function of HSP27 and reducing cell protective function. It can also reduce resistance to anticancer agents and radiation treatments, including heat shock protein 90 (HSP90) inhibitors.
  • HSP27 modified heat shock protein 27
  • HSP90 heat shock protein 90
  • Preferred preparation method of the compound represented by the formula (I) of the present invention is the same as Scheme 1, and includes a preparation method modified to a level apparent to those skilled in the art.
  • reaction formula 1 composed of carbon atoms of the selected one or two hetero atoms and 1 to 6 from the group consisting of oxygen, nitrogen and sulfur in a compound of formula 1-1 heterocyclyl, C 1 - 4 alkyl
  • the compound represented by the formula (I) of the present invention can be synthesized by the same method as in Scheme 2, and includes a production method modified to a level apparent to those skilled in the art.
  • compositions comprising the novel chromemenone derivative compounds, uses thereof and methods of treatment using the same
  • the present invention also provides a pharmaceutical composition for the prevention or treatment of cancer, comprising a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the pharmaceutically acceptable salts are as described above in the pharmaceutically acceptable salts of the compounds represented by formula (I) of the present invention.
  • the pharmaceutical composition of the present invention has an effect on the prevention or treatment of cancer by inhibiting the activity of heat shock protein 27 (HSP27).
  • the pharmaceutical composition of the present invention has the effect of reducing resistance to treatment with other anticancer agents including heat shock protein 90 (HSP90), and radiation therapy.
  • the cancer is lung cancer, breast cancer, osteosarcoma, prostate cancer, cervical cancer, ovarian cancer, skin cancer, oral cancer, esophageal cancer, gastric cancer, pancreatic cancer, colon cancer, bladder cancer, ureter cancer, liver cancer, glioma, brain tumor, non-Hodgkin's disease Lymphomas, myelodysplastic syndromes, multiple myeloma, or plasmacytic tumors, including but not limited to, lung cancer, breast cancer or osteosarcoma.
  • the cancer may be a cancer particularly resistant to radiation to anticancer agents. Cancers resistant to the radiation to anticancer agents include, for example, lung cancer, breast cancer, etc., in which treatment by irradiation or anticancer agent administration was performed.
  • the pharmaceutical composition of the present invention may further comprise at least one other anticancer agent in addition to the compound represented by the formula (I).
  • the other anticancer agent may be, for example, an anticancer antibiotic, an alkylating agent, a tubulin depolymerization inhibitor, a platinum complex, or an HSP90 inhibitor, but is not limited thereto, and is preferably an HSP90 inhibitor.
  • Said other anticancer agents specifically include Taxol, Trastuzumab, Zephytinib, Cisplatin, Methotrexate, 6-mercaptopurine, 5-fluorouracil, gemcitabine, doxorubicin, dactinomycin, bleomycin, nitrourea, vincristine , Vinblastine, tamoxifen, or carboplatin, and the like, but are not limited thereto, and preferably Taxol, Trastuzumab, Zephytinib, or Cisplatin.
  • the HSP90 inhibitor may be a radicicol derivative, a geldanamycin derivative, a novobiocin derivative, or an artificially synthesized material based on purine, and specifically, ganetes.
  • Pep Ganetespib, STA-9090
  • Luminespib NBP-AUY922
  • Tanespimycin Tanespimycin, 17-AAG
  • Alvespimycin 17.-DMAG
  • Elesclomol STA- 4783
  • Onlaespib AT13387
  • BIIB021 (6-chloro-9-((4-methoxy-3,5-dimethylpyridin-2-yl) methyl) -9H-purin-2-amine
  • PU-H71 8-[(6-iodo-1,3-benzodioxol-5-yl) sulfanyl] -9- [3- (propan-2-ylamino) propyl] purin-6-amine
  • XL888 (2-
  • the pharmaceutical composition of the present invention may further include at least one pharmaceutically acceptable carrier in addition to the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof for administration.
  • Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as antioxidants, buffers And other conventional additives such as bacteriostatic agents can be added.
  • compositions of the present invention may be patches, solutions, pills, capsules, granules, tablets, suppositories, and the like.
  • formulations may be prepared by conventional methods used in the art for formulation or by methods disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA, and formulated into various formulations depending on the individual disease or component. Can be.
  • composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex and health of the patient.
  • parenterally eg, applied intravenously, subcutaneously, intraperitoneally or topically
  • the dosage is based on the weight, age, sex and health of the patient.
  • the time of administration, the method of administration, the rate of excretion and the severity of the disease the range varies, and may be administered once or several times a day.
  • the present invention also provides the use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of cancer.
  • the present invention also provides the use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for the prevention or treatment of cancer.
  • the present invention also provides a method of preventing or treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. To provide.
  • the "subject” includes mammals, especially humans.
  • therapeutically effective amount means an amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof that is effective for treating or preventing a subject disease.
  • HSP27 modified heat shock protein 27
  • the present invention can be effective in the prevention or treatment of cancer, and can also reduce the resistance of HSP90 and other anticancer agents, and can also reduce the resistance to radiation.
  • Figure 2 confirms the time dependent HSP27 dimer production capacity by Western blot.
  • Figure 3 is confirmed by Western blot HSP27 dimer generating ability in NCI-H460 cells.
  • Figure 4 confirms the concentration-dependent HSP27 dimer production ability in NCI-H460 cells by Western blot.
  • FIG. 5 shows Western blots of concentration dependent HSP70, HSP0 and HSF1 expression in NCI-H460 cells.
  • FIG. 6 shows modified HSP27 dimer, cleaved PARP and cleaved caspase-3 when NCI-H460 cells are administered with a compound of the present invention in combination with 17-AAG, taxol or cisplatin, respectively. -3) expression was confirmed by Western blot.
  • Fig. 7 shows the cell death rate and cell viability when NCI-H460 cells were co-administered with 17-AAG, Taxol or Cisplatin, respectively.
  • Figure 8 shows the tumor volume change and the number of cell death when the compound of the present invention in xenograft mice was administered alone or in combination with 17-AAG directly to tumors.
  • Figure 9 shows the tumor volume change and the number of cell death in the case of intraperitoneal administration of the compound of the present invention alone or in combination with Taxol.
  • Figure 10 shows the tumor volume change and the number of cell death when the compound of the present invention alone or in combination with Taxol to be administered directly to the tumor.
  • FIG. 11 is a compound of the present invention confirms the inhibitory effect of the signal transduction protein of the HER receptor and its substeps in a concentration-dependent manner by Western blot.
  • Figure 12 confirms the inhibitory effect of HER2 signal transduction when the compound of the present invention is treated in combination with trastuzumab.
  • Figure 13 confirms the effects of apoptosis-induced protein expression and cell death-inhibiting protein expression when the compound of the present invention in combination with trastuzumab by Western blot.
  • Fig. 14 shows the cell growth inhibition rate when the compound of the present invention is treated in combination with trastuzumab.
  • Figure 15 shows the tumor size, tumor volume change and survival rate when the compound of the present invention is treated in combination with trastuzumab.
  • Figure 16 confirms the effect of apoptosis inducing protein expression in the case of treatment with the compound of the present invention in combination with zephytinib by Western blot.
  • Fig. 17 shows the cell growth rate when the compound of the present invention is treated in combination with gefitinib.
  • Example 1 compound 1 (5-hydroxy-2- methyl -7- (Tyran-2- Ilmethoxy ) -4H- Chromen 4-one) and compound 2 (2-methyl-5,7-bis (thiran-2-ylmethoxy) -4H-chromen-4-one)
  • Example 2 compound 3 (5-hydroxy-7- ( Oxirane -2- Ilmethoxy ) -2-phenyl-4 H - Chromen -4-one) and compound 4 (5,7-bis (oxirane-2-ylmethoxy) -2-phenyl-4 H -Chromen-4-one)
  • Example 3 compound 5 (5-hydroxy-2-phenyl-7- (tyran-2- Ilmethoxy ) -4H- Chromen 4-one) and compound 6 (2-phenyl-5,7-bis (thiran-2-ylmethoxy) -4H-chromen-4-one)
  • HSP25 Wild type (HSP25WT) protein which is a rodent homolog of HSP27, was used.
  • HSP25WT was constructed using a plasmid expressing heat shock protein 25 (HSP25).
  • HSP25WT protein treated with Compound 1, Compound 4 and Compound 6 was subjected to Western blot using 1 ⁇ PBS (0.14 M sodium chloride (NaCl), 2.68 mM potassium chloride (KCl), 10 mM disodium phosphate (Na 2 HPO 4 ), After washing twice with 1.83 mM dipotassium phosphate (KH 2 PO 4 )), Radio Immunoprecipitation Polyacrylamide Assay buffer, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na 2 EDTA, 1% NP-40, 1% sodium dioxycholate, 1 mM ⁇ -glycerophosphate, 1 mM sodium orthovanadate (Na 3 VO 4 , 4 mM NaF)). The cell suspension was centrifuged at 13000 rpm for 30 minutes to extract only the supernatant.
  • the protein amount was measured by Bradford assay.
  • BSA Bovine Serum Albumin
  • Protein Dye protein dye
  • 2 ⁇ L of supernatant to 96-well 200 ⁇ L was aliquoted into the plate, and the absorbance was measured at a wavelength of 595 nm in an ELISA reader.
  • 6X sample buffer (0.35 M Tris (pH6.8), 3% glycerol, 1% Sodium Dodecyl Sulfate, 6 mM Dithiothreitol) was added to make a sample containing the same amount of protein. .
  • the samples were soaked in 100 ° C. water for 5 minutes and boiled and analyzed for protein using SDS-PAGE.
  • the primary antibodies for Western blot were Goat polyclonal anti-HSP27 (sc-1049) and ⁇ -actin ( ⁇ -actin, sc-47778).
  • the secondary antibodies were Donkey anti goat (sc-2020) and Goat anti rabbit ( sc-2004) and Goat anti mouse (sc-2005) were used.
  • Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, or Compound 6 of the present invention were each 10 ⁇ M in NCI-H460, a non-small cell lung cancer cell. The concentration was treated for 12 hours. The cells were then cultured with RPMI 1640 (RPMI, GIBCO-Invitrogen, 10% Fetal Bovine Serum (FBS)) and 1 ⁇ Antibiotic-Antimycotic (GIBCO-Invitrogen, Paisley, Scotland, UK). Paisley, Scotland, UK) using a medium at 37 ° C, 5% carbon dioxide (CO 2 ) Incubated for 12 hours in the incubator.
  • RPMI 1640 RPMI, GIBCO-Invitrogen, 10% Fetal Bovine Serum (FBS)
  • FBS Fetal Bovine Serum
  • GIBCO-Invitrogen 1 ⁇ Antibiotic-Antimycotic
  • Paisley, Scotland, UK Paisley, Scotland, UK
  • the cells treated with Compounds 1 to 6 were washed twice with 1 ⁇ PBS (0.14 M NaCl, 2.68 mM KCl, 10 mM Na 2 HPO 4 , 1.83 mM KH 2 PO 4 ), followed by Radio.
  • Immunoprecipitation Polyacrylamide Assay buffer (20 mM Tris-HCl, pH 7.5), 150 mM NaCl, 1 mM Na 2 EDTA, 1% NP-40, 1% Sodium Dioxycholate, 1 mM ⁇ -glycerophosphate, 1 mM Na 3 VO 4 , 4 mM NaF).
  • the cell suspension was centrifuged at 13000 rpm for 30 minutes to extract only the supernatant.
  • the protein amount was measured by Bradford assay. Add 8, 6, 4, 2, and 0 ⁇ L of 1% BSA solution as standard to 1 mL of Protein Dye, add 2 ⁇ L of supernatant, divide 200 ⁇ L into 96-well plate, and place in an ELISA reader at a wavelength of 595 nm. Absorbance was measured at.
  • 6X sample buffer (0.35 M Tris (pH6.8), 3% glycerol, 1% Sodium Dodecyl Sulfate, 6 mM Dithiothreitol) was added to make a sample containing the same amount of protein.
  • the samples were soaked in 100 ° C. water for 5 minutes and boiled and analyzed for protein using SDS-PAGE.
  • the primary antibodies for Western blot were Goat polyclonal anti-HSP27 (sc-1049) and ⁇ -actin (sc-47778).
  • the secondary antibodies were Donkey anti goat (sc-2020) and Goat anti rabbit (sc-2004). Goat anti mouse (sc-2005) was used.
  • Compound 1 was treated with NCI-H460, a non-small cell lung cancer cell, at a concentration of 5, 10, 20, or 40 ⁇ M, and cell culture and Western blot were performed in the same manner as in Experimental Example 2-1. Was performed.
  • HSF1 heat shock factor 1
  • Compound 1 of the present invention specifically acted on HSP27 to increase the HSP27 modified dimer in a concentration-dependent manner, and also reduced the expression of HSF1 in a concentration-dependent manner.
  • 17-AAG known as an inhibitor of HSP90
  • Taxol and Cisplatin anticancer agents used for anticancer treatment
  • NCI-H460 cells were treated with Compound 1 of the present invention at a concentration of 10 ⁇ M, treated with 0.01 ⁇ M Taxol, 2 ⁇ M Cisplatin, and 3 ⁇ M 17-AAG, and then incubated for 12 hours, in the same manner as in Example 2-1. Western blot was performed.
  • cleaved Parp Cleaved Parp
  • cleaved Caspase-3 Cleaved Caspase-3
  • apoptosis induction protein apoptosis induction protein
  • an MTT assay was performed.
  • NCI-H460 cells were treated with Compound 1 of the present invention at a concentration of 10 ⁇ M and co-treated with 0.01 ⁇ M Taxol, 2 ⁇ M Cisplatin, and 3 ⁇ M 17-AAG and incubated for 24 hours.
  • NCI-H460 cells Dispense the same amount of NCI-H460 cells in a 96 well plate, incubate until adhered well to the plate, and then incubate for 24 hours after treatment with the compound of the indicated concentration, remove the medium of the cell, wash with 1X PBS, 3- (4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT; Amersham Pharmacia Biotech, Little Chalfont, UK) reagent was diluted to 5 mg / ml in 1X PBS at 100 mg.
  • MTT 3- (4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide
  • the compound of the present invention can prevent Happ27 from forming a dimer and inhibiting multimer production, thereby preventing chaperone function, thereby increasing sensitivity to anticancer drugs.
  • Tumors were formed by injecting NCI-H460 cells 1 ⁇ 10 6 subcutaneously into the left leg of BALB / C nude mice, and when the formed tumors reached 100 to 300 mm 3 , the drug was divided into the following seven administration groups and control groups.
  • 17-AAG was intraperitoneally administered 100 ⁇ l three times a week at a concentration of 25 mg / kg
  • Taxol was intraperitoneally administered once a week at 100 ⁇ l at a concentration of 2 mg / kg
  • Compound 1 of the present invention was 6.8 mg / kg Intraperitoneal administration or injection directly into tumors three times a week at kg concentration.
  • the compound of the present invention has an excellent effect on increasing sensitivity and decreasing resistance of an anticancer agent such as Taxol.
  • Tumors of the xenograft mice of Experimental Example 6-1 were extracted, and cell death induced cell death was measured by immunohistochemical staining.
  • the compound of the present invention was able to clearly confirm the tendency to increase the cell death induced by 17-AAG.
  • Compound 1 of the present invention was able to confirm the tendency to increase apoptosis induced by Taxol. Therefore, it can be seen that the compound of the present invention is effective in increasing sensitivity and decreasing resistance of anticancer drugs including HSP90 inhibitor and taxol.
  • HER Human Epithelial Growth Factor Receptor, EGFR, human epidermal growth factor receptor
  • EGFR Human Epithelial Growth Factor Receptor
  • human epidermal growth factor receptor human epidermal growth factor receptor
  • Compound 1 of the present invention reduced the HER receptor and signal transduction proteins at lower levels thereof.
  • BT474 cell line that is highly reactive to trastuzumab, thus resistant cells (BT474-resistant) constructed by treatment with 3 mg / ml of trastuzumab for 16 weeks and patient-derived cell line resistant to trastuzumab (JIMT-1)
  • JIMT-1 patient-derived cell line resistant to trastuzumab
  • Each compound 1 of the present invention was treated with 10 ⁇ M, treated with 10 ⁇ g / ml trastuzumab, and then incubated for 12 hours.
  • Western blot was performed in the same manner as in Experimental Example 2-1 to confirm the expression of HER2, p-AKT, p-MAPK, HSP27 dimer, HSP27 dimer, HSP90 and GADPH.
  • the compounds of the present invention are effective in overcoming resistance to trastuzumab.
  • Cleaved Parp and Cleaved caspase-7 which are apoptosis inducing proteins
  • bcl-2 which is apoptosis inhibitory protein
  • BT474, BT474-resistant cell line and JIMT-1 cell line were treated with 10 ⁇ M of Compound 1 of the present invention and treated with 10 ⁇ g / ml trastuzumab, followed by incubation for 12 hours.
  • Western blot was carried out in the same manner as in Experimental Example 2-1, and c-PARP, pro-caspase 7, c-caspase 7, blc-2 and ⁇ - The expression of tubulin was confirmed.
  • the expression of apoptosis-inducing protein was increased in the group treated with the combination of trastuzumab and the compound 1 of the present invention alone in all cell lines, and the expression of the cell death suppressing protein. was found to decrease.
  • BT474, BT474-resistant cell line, and JIMT-1 cell line were each cultured at 1 ⁇ 10 4 per well of a 96-well plate for 24 hours. This was treated with Compound 1 of the present invention at a concentration of 10 ⁇ M and co-treated with 10 ⁇ g / ml trastuzumab, followed by further incubation for 24 hours. 10 ⁇ L of the WST reagent was treated per well and incubated for 1 hour at 37 ° C. and 5% CO 2. The absorbance was measured at 450 nm with an ELISA reader.
  • Tumors were formed by subcutaneous injection of BT474 cells 1 ⁇ 10 7 into the BALB / C nude mice under the right leg. When the formed tumors reached ⁇ 100 mm 3 , the drug was divided into three dose groups and a control group as follows.
  • Trastuzumab was intraperitoneally administered once a week at 100 ⁇ l at a concentration of 1 mg / kg, and Compound 1 of the present invention was intraperitoneally administered once every two days at a concentration of 20 mg / kg.
  • Formula 1 of the present invention was treated with HCC827 and PC9, which are sensitive to gefitinib, and NHCI-H1650, which is resistant to gefitinib, at 10 ⁇ M.
  • Zephytinib was treated with 0.05 ⁇ M and 0.01 ⁇ M for HCC827 cell line, 1 ⁇ M and 5 ⁇ M for PC9 cell line, and 5 ⁇ M and 10 ⁇ M for NCI-H1650 cell line, respectively, and then 24 hours.
  • Incubated for Western blot was performed in the same manner as in Experimental Example 2-1 to confirm the expression of EGFR, cleaved PARP, cleaved caspase 3 and ⁇ -actin.
  • MTT assay was performed.
  • HCC827 a cell line sensitive to gefitinib, and NCI-H150, a resistant cell line, were dispensed at 1 ⁇ 10 4 cells per well of a 96-well plate, respectively, and cultured for 24 hours. This was treated with Compound 1 of the present invention at a concentration of 10 ⁇ M and co-treatment with gefitinib at a concentration of 2 ⁇ M and further incubated for 24 hours. 100 ⁇ L of the MTT assay reagent was treated per well and incubated for 1 hour at 37 ° C. and 5% CO 2. The absorbance was measured at 540 nm using an ELISA reader.

Abstract

The present invention relates to a novel cromenone derivative compound having heat shock protein 27 (HSP27) inhibitory activity. The composition, a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the same, of the present invention, induces the formation of a modified HSP27 dimer so as to reduce the formation of a normal HSP27 multimer, thereby inhibiting the function of HSP27 as a chaperone. Therefore, the present invention can be effective in preventing or treating cancer, can reduce heat shock protein 90 (HSP90) and resistance to other anticancer drugs, and can reduce radioresistance.

Description

크로메논 유도체 및 이를 포함하는 항암용 조성물Chromenone derivative and anticancer composition comprising the same
본 발명은 신규한 크로메논 유도체, 보다 상세하게는 열충격 단백질(Heat shock protein) 27 억제 활성을 갖는 신규한 크로메논 유도체에 관한 것이다.The present invention relates to novel chromemenone derivatives, and more particularly to novel chromemenone derivatives having heat shock protein 27 inhibitory activity.
열충격 단백질(Heat Shock Protein, HSP)은 샤페론 단백질로 다양한 생리적인 요인이나 항암제와 같은 환경적인 요인에 의해 발현이 유도되어 단백질의 변성 및 세포사를 조절하는 것으로 알려져 있다(Garrido, 2006). 활성화된 열충격 단백질은 샤페론으로서 제대로 접히지 않은 단백질을 제대로 접히게 하고 단백질의 변성을 방지하는 역할을 하며 그 외에 다양한 신호 경로에 중요한 역할을 하는 효과기와 어울려 세포 사멸과정 간섭, 면역 조절에 관여, 세포 보호 효과 등의 다양한 기능으로 세포사를 억제하는 효과를 나타낸다(Khalil, 2011). Heat shock protein (HSP) is a chaperone protein and is known to regulate protein degeneration and cell death by expression by various physiological factors or environmental factors such as anticancer agents (Garrido, 2006). Activated heat shock proteins are chaperones that help fold up poorly folded proteins, prevent protein denaturation, and other important effects on various signaling pathways, interfering with cell death, immune regulation, and cell protection It has an effect of suppressing cell death by various functions such as effects (Khalil, 2011).
열충격 단백질 27 (HSP27)은 1980년대에 발견되었다. HeLa 세포를 배양할 때, 온도를 증가시키면 27 kDa의 알려지지 않은 단백질이 발견되었는데 이 단백질이 HSP27로 세포 내에 편재하며 스트레스에 의해 과다 발현이 유도된다. HSP27은 세포의 생존, 세포의 이동, 암세포의 침습성을 향상시키며 HSP27의 발현이 높은 암세포의 경우 전이가 잘되며 항암치료에 대한 내성이 증가하는 양상을 보이게 된다(Vargas-Roig, 1998). HSP27의 단백질 구조를 살펴보면 α-크리스탈린 도메인(α-crystallin domain)과 질소-말단(N-terminal)의 WDPF 도메인이 HSP27의 다량체 생성에 중요한 역할을 하게된다(Lambert, 1999). α-Crystallin domain내의 시스테인 잔기는 HSP27의 단량체와 단량체간의 이황화 결합을 형성하여 HSP27의 이량체를 형성하도록 한다(Mymrikov, 2010). HSP27의 세포내 기능은 이러한 다량체 생성 정도와 관련이 있다. Heat shock protein 27 (HSP27) was discovered in the 1980s. In culturing HeLa cells, increasing the temperature revealed an unknown protein of 27 kDa, which is ubiquitous in cells with HSP27 and induces overexpression by stress. HSP27 improves cell survival, cell migration, and cancer cell invasiveness. Cancer cells with high HSP27 expression are well metastasized and show increased resistance to chemotherapy (Vargas-Roig, 1998). Looking at the protein structure of HSP27, the α-crystallin domain and the N-terminal WDPF domain play an important role in the multimer production of HSP27 (Lambert, 1999). Cysteine residues in the α-Crystallin domain form disulfide bonds between monomers of HSP27 to form dimers of HSP27 (Mymrikov, 2010). The intracellular function of HSP27 is related to this degree of multimer production.
따라서 HSP27의 거대 다량체 형태를 저해함으로써 HSP27이 샤페론 기능을 수행하지 못하도록 한다면 방사선이나 항암제에 대한 내성이 극복될 것이다.Therefore, if the HSP27 prevents HSP27 from functioning as a chaperone by inhibiting the large multimeric form of HSP27, resistance to radiation or anticancer drugs will be overcome.
한편, 세포 내에는 HSP27외에도 HSP70, HSP90 등의 다양한 열충격 단백질들이 존재한다. HSP90은 모든 진핵세포에서 발현되는 ATP-의존성 샤페론 단백질(ATP-dependent chaperone protein)로 HSP90 클라이언트 단백질(HSP90 client protein)들은 거의 모든 세포의 생존 과정에 관여하며 클라이언트 단백질 중 다수는 세포 성장, 세포의 분열 및 생존 등의 중요한 세포의 역할에 연관이 있다. 이 중 대부분의 과정은 암의 성장에도 관련이 있다. 따라서 HSP90을 표적으로 하면 동시에 다양한 발암의 신호 전달 경로의 혼란을 야기하게 되므로 다양한 발암 신호 전달 경로의 변화가 일어나는 진행암 치료에 특별히 효과가 있다. Meanwhile, in addition to HSP27, various heat shock proteins such as HSP70 and HSP90 exist in the cell. HSP90 is an ATP-dependent chaperone protein expressed in all eukaryotic cells. HSP90 client proteins are involved in the survival of almost all cells, many of which are cell growth, cell division. And vital cell roles such as survival. Most of these processes are also involved in cancer growth. Therefore, targeting HSP90 simultaneously causes confusion of various carcinogenic signal transduction pathways, which is particularly effective in treating advanced cancers in which various carcinogenic signal transduction pathways occur.
그런데 특정 열충격 단백질을 억제하게 되면 그 외의 다른 열충격 단백질들의 발현이 더욱 증가하는 현상이 나타남이 이미 여러 논문 등에서 발표되었고, 현재 임상 시험 중인 HSP90 저해제의 가장 큰 부작용이 이러한 다른 열충격 단백질의 발현에 의한 항암효과 실패에 근거한다. 따라서 HSP90 저해제의 부작용을 억제할 수 있는 방안이 필요하다.However, it has been published in several papers that the inhibition of specific heat shock proteins further increases the expression of other heat shock proteins, and the biggest side effect of HSP90 inhibitors currently in clinical trials is anti-cancer caused by the expression of these heat shock proteins. Based on failure to effect. Therefore, there is a need for a method that can suppress the side effects of HSP90 inhibitors.
이러한 배경 하에, 방사선 치료 내지 항암제에 내성을 가진 암에 대한 신규 항암제 등에 대한 발굴이 절실한 실정이다. Under this background, there is an urgent need to discover new anticancer agents for cancer resistant to radiation therapy or anticancer agents.
본 발명의 목적은 열충격 단밸질 27 (HSP27) 억제 활성을 갖는 신규한 크로메논 유도체 또는 이의 약제학적으로 허용되는 염을 제공하는 것이다.It is an object of the present invention to provide novel chromenone derivatives or pharmaceutically acceptable salts thereof having thermal shock protein 27 (HSP27) inhibitory activity.
본 발명의 또 다른 목적은 상기 신규한 크로메논 유도체를 포함하는 암의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising the novel chromemenone derivative.
본 발명자들은 암, 특히 방사선 내지 항암제에 내성을 가진 암의 치료에 대해 예의 연구한 결과, 화학식 Ⅰ로 표시되는 화합물 또는 이의 약제학적으로 허용되는 염이 변형된 열충격 단백질 27 (HSP27) 이량체 형성에 효과를 보이며 암세포 사멸을 유도하고, 방사선 및 항암제의 내성 등을 감소시킴을 발견하여 본 발명을 완성하였다. The present inventors have conducted intensive studies on the treatment of cancer, especially cancers resistant to radiation to anticancer agents, and have shown that the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof is modified to form a modified heat shock protein 27 (HSP27) dimer. The present invention was completed by discovering the effect of inducing cancer cell death and reducing radiation and resistance to anticancer drugs.
신규한 크로메논 유도체 화합물Novel Chromone Derivative Compounds
상기 목적에 따라, 본 발명은 하기 화학식 Ⅰ로 표시되는 화합물 또는 이의 약제학적으로 허용 되는 염을 제공한다.In accordance with the above object, the present invention provides a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof.
[화학식 Ⅰ][Formula I]
Figure PCTKR2017008204-appb-I000001
Figure PCTKR2017008204-appb-I000001
상기 화학식 Ⅰ식에서, In Formula I,
R1 및 R2는 각각 독립적으로, 수소; 또는 산소, 질소 및 황으로 구성된 군에서 선택된 1 또는 2개의 헤테로원자와 1 내지 6의 탄소원자로 구성된 헤테로시클릴C1-4알킬이고, R 1 and R 2 are each independently hydrogen; Or heterocyclylC 1-4 alkyl composed of 1 or 2 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur and 1 to 6 carbon atoms,
R3는 C1- 10알킬, C3- 10시클로알킬, C6- 14아릴 또는 산소, 질소 및 황으로 구성된 군에서 선택된 1 내지 4개의 헤테로원자와 1 내지 10의 탄소원자로 구성된 헤테로아릴이고, 여기서 R3의 하나 이상의 수소는 각각 독립적으로 R4로 치환 또는 비치환되고,R 3 is a heteroaryl group consisting of C 1- 10 alkyl, C 3- 10 cycloalkyl, C 6- 14 aryl, or oxygen, nitrogen and sulfur from the group consisting of one to four heteroatoms selected with 1 to 10 carbon atoms, Wherein at least one hydrogen of R 3 is each independently substituted or unsubstituted with R 4 ,
R4는 F, Br, Cl, I, OH, OMe, OEt, NH2, NMe2, CN, COOH, COMe, COOMe, CONH2, 또는 C1-4알킬이다.R 4 is F, Br, Cl, I, OH, OMe, OEt, NH 2 , NMe 2 , CN, COOH, COMe, COOMe, CONH 2 , or C 1-4 alkyl.
본 발명의 바람직한 실시양태에 따르면, 상기 화학식 Ⅰ에서, R1 및 R2는 각각 독립적으로, 수소,
Figure PCTKR2017008204-appb-I000002
또는
Figure PCTKR2017008204-appb-I000003
이다.According to a preferred embodiment of the invention, in formula (I), R 1 and R 2 are each independently hydrogen,
Figure PCTKR2017008204-appb-I000002
or
Figure PCTKR2017008204-appb-I000003
to be.
본 발명의 바람직한 실시양태에 따르면, 상기 화학식 Ⅰ에서, R3는 C1- 10알킬 또는 C3- 10시클로알킬이다.According to a preferred embodiment of the invention, in Formula Ⅰ, R 3 is a C 1- 10 alkyl, C 3- 10 cycloalkyl.
본 발명의 보다 바람직한 실시양태에 따르면, 상기 화학식 Ⅰ에서, R3는 메틸 또는 페닐이다.According to a more preferred embodiment of the invention, in formula (I), R 3 is methyl or phenyl.
또한, 본 발명의 또 다른 바람직한 실시양태에 따르면, 상기 화학식 Ⅰ의 화합물은 하기 표 1의 화합물로 이루어진 군으로부터 선택된 것일 수 있다. In addition, according to another preferred embodiment of the present invention, the compound of Formula I may be selected from the group consisting of the compounds of Table 1 below.
[표 1]TABLE 1
Figure PCTKR2017008204-appb-I000004
Figure PCTKR2017008204-appb-I000004
본 발명에서, 약제학적으로 허용되는 염은 의약업계에서 통상적으로 사용되는 염을 의미하며, 예를 들어 칼슘, 칼륨, 나트륨 및 마그네슘 등으로 제조된 무기이온염, 염산, 질산, 인산, 브롬산, 요오드산, 과염소산, 주석산 및 황산 등으로 제조된 무기산염, 아세트산, 트리플루오로아세트산, 시트르산, 말레인산, 숙신산, 옥살산, 벤조산, 타르타르산, 푸마르산, 만데르산, 프로피온산, 구연산, 젖산, 글리콜산, 글루콘산, 갈락투론산, 글루탐산, 글루타르산, 글루쿠론산, 아스파르트산, 아스코르브산, 카본산, 바닐릭산, 하이드로 아이오딕산 등으로 제조된 유기산염, 메탄설폰산, 에탄설폰산, 벤젠설폰산, p-톨루엔설폰산 및 나프탈렌설폰산 등으로 제조된 설폰산염, 글리신, 아르기닌, 라이신 등으로 제조된 아미노산염 및 트리메틸아민, 트리에틸아민, 암모니아, 피리딘, 피콜린 등으로 제조된 아민염 등이 있으나, 열거된 이들 염에 의해 본 발명에서 의미하는 염의 종류가 한정되는 것은 아니다.In the present invention, pharmaceutically acceptable salts mean salts commonly used in the pharmaceutical industry, for example, inorganic ion salts, hydrochloric acid, nitric acid, phosphoric acid, bromic acid, prepared with calcium, potassium, sodium and magnesium, Inorganic acid salts made with iodic acid, perchloric acid, tartaric acid, sulfuric acid, etc. Organic acid salts, methanesulfonic acid, ethanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, which are made of cholic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanic acid, hydroiodic acid, etc. Amino acid salts made with sulfonates, glycine, arginine, lysine, etc., made of p-toluenesulfonic acid and naphthalenesulfonic acid, and trimethylamine, triethylamine, ammo Although there are amine salts made of nia, pyridine, picoline and the like, the salts used in the present invention are not limited by these salts.
본 발명의 화합물은 변형된 열충격 단백질 27 (HSP27) 이량체의 생성을 유도하여 HSP27 거대 다량체의 생성을 억제함으로써, HSP27의 샤페론 기능을 억제하며, 세포 보호 기능을 감소시키는 효과가 있다. 또한, 열충격 단백질 90 (HSP90) 억제제를 비롯한 항암제 및 방사선 치료에 대한 내성을 감소시킬 수 있다. The compounds of the present invention induce the production of modified heat shock protein 27 (HSP27) dimers, thereby inhibiting the production of HSP27 macromers, thereby inhibiting the chaperone function of HSP27 and reducing cell protective function. It can also reduce resistance to anticancer agents and radiation treatments, including heat shock protein 90 (HSP90) inhibitors.
본 발명의 화학식 Ⅰ로 표시되는 화합물의 바람직한 제조방법은 하기 반응식 1과 같으며, 통상의 기술자에게 자명한 수준으로 변형된 제조방법도 이에 포함된다.Preferred preparation method of the compound represented by the formula (I) of the present invention is the same as Scheme 1, and includes a preparation method modified to a level apparent to those skilled in the art.
[반응식 1] Scheme 1
Figure PCTKR2017008204-appb-I000005
Figure PCTKR2017008204-appb-I000005
상기 [반응식 1]에 도시된 바와 같이, 화학식 1-1의 화합물에 산소, 질소 및 황으로 구성된 군에서 선택된 1 또는 2개의 헤테로원자와 1 내지 6의 탄소원자로 구성된 헤테로시클릴C1 - 4알킬을 반응시켜 화학식 1-2 또는 화학식 1-3의 화합물을 합성할 수 있다.Wherein as shown in the reaction formula 1], composed of carbon atoms of the selected one or two hetero atoms and 1 to 6 from the group consisting of oxygen, nitrogen and sulfur in a compound of formula 1-1 heterocyclyl, C 1 - 4 alkyl By reacting the compound of Formula 1-2 or Formula 1-3 can be synthesized.
또한, 본 발명의 화학식 Ⅰ로 표시되는 화합물을 하기 반응식 2 와 같은 방법으로 합성 할 수 있으며, 통상의 기술자에게 자명한 수준으로 변형된 제조방법도 이에 포함된다.In addition, the compound represented by the formula (I) of the present invention can be synthesized by the same method as in Scheme 2, and includes a production method modified to a level apparent to those skilled in the art.
[반응식 2] Scheme 2
Figure PCTKR2017008204-appb-I000006
Figure PCTKR2017008204-appb-I000006
구체적으로, 상기 [반응식 2]에 도시된 바와 같이, 화학식 2-1의 화합물과 에피클로로히드린 또는 에피티오클로로히드린을 반응시켜 최종 화합물 1, 2, 3, 4, 5 및 6을 합성할 수 있다.Specifically, as shown in [Scheme 2], the compound of Formula 2-1 and epichlorohydrin or epithiochlorohydrin are reacted to synthesize final compounds 1, 2, 3, 4, 5, and 6. Can be.
신규한 크로메논 유도체 화합물을 포함하는 약학적 조성물, 이의 용도 및 이를 이용한 치료 방법Pharmaceutical compositions comprising the novel chromemenone derivative compounds, uses thereof and methods of treatment using the same
또한, 본 발명은 하기 화학식 Ⅰ로 표시되는 화합물 또는 이의 약제학적으로 허용되는 염을 유효성분으로 포함하는 암의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention also provides a pharmaceutical composition for the prevention or treatment of cancer, comprising a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.
[화학식 Ⅰ][Formula I]
Figure PCTKR2017008204-appb-I000007
Figure PCTKR2017008204-appb-I000007
상기 화학식 Ⅰ은 앞서 정의한 바와 같다.Formula I is as defined above.
상기 약제학적으로 허용가능한 염은 앞서 본 발명의 화학식 I 로 표시되는 화합물의 약제학적으로 허용되는 염에서 기술한 바와 같다.The pharmaceutically acceptable salts are as described above in the pharmaceutically acceptable salts of the compounds represented by formula (I) of the present invention.
본 발명의 약학적 조성물은 열충격 단백질 27 (HSP27)의 활성을 억제함으로써 암의 예방 또는 치료에 효과를 나타낸다. 또한, 본 발명의 약학적 조성물은 열충격 단백질 90 (HSP90)를 비롯한 다른 항암제 치료, 및 방사선 치료 등에 대한 내성을 감소시키는 효과가 있다.The pharmaceutical composition of the present invention has an effect on the prevention or treatment of cancer by inhibiting the activity of heat shock protein 27 (HSP27). In addition, the pharmaceutical composition of the present invention has the effect of reducing resistance to treatment with other anticancer agents including heat shock protein 90 (HSP90), and radiation therapy.
본 발명에 있어서, 암은 폐암, 유방암, 골육종, 전립선암, 자궁경부암, 난소암, 피부암, 구강암, 식도암, 위암, 췌장암, 대장암, 방광암, 요관암, 간암, 신경교종, 뇌종양, 비호지킨성림프종, 골수이형성증후군, 다발성골수종, 또는 형질세포성종양을 포함하나, 이에 한정되는 것은 아니며, 바람직하게는 폐암, 유방암 또는 골육종을 포함한다. 또한, 본 발명에 있어서, 암은 특히 방사선 내지 항암제에 내성을 가진 암일 수 있다. 상기 방사선 내지 항암제에 내성을 가진 암은 예를 들어, 방사선 조사 내지 항암제 투여에 의한 치료가 수행되었던 폐암, 유방암 등을 들 수 있다.In the present invention, the cancer is lung cancer, breast cancer, osteosarcoma, prostate cancer, cervical cancer, ovarian cancer, skin cancer, oral cancer, esophageal cancer, gastric cancer, pancreatic cancer, colon cancer, bladder cancer, ureter cancer, liver cancer, glioma, brain tumor, non-Hodgkin's disease Lymphomas, myelodysplastic syndromes, multiple myeloma, or plasmacytic tumors, including but not limited to, lung cancer, breast cancer or osteosarcoma. In addition, in the present invention, the cancer may be a cancer particularly resistant to radiation to anticancer agents. Cancers resistant to the radiation to anticancer agents include, for example, lung cancer, breast cancer, etc., in which treatment by irradiation or anticancer agent administration was performed.
본 발명의 바람직한 일 실시양태에서, 본 발명의 약학적 조성물은 화학식 Ⅰ로 표시되는 화합물 외에 다른 항암제를 1종 이상 더 포함할 수 있다.In one preferred embodiment of the present invention, the pharmaceutical composition of the present invention may further comprise at least one other anticancer agent in addition to the compound represented by the formula (I).
상기 다른 항암제는 예를 들어, 항암성 항생제, 알킬화제, 튜불린 탈중합 억제제, 백금 복합체, 또는 HSP90 억제제일 수 있으나, 이에 한정되는 것은 아니며, 바람직하게는 HSP90 억제제이다.The other anticancer agent may be, for example, an anticancer antibiotic, an alkylating agent, a tubulin depolymerization inhibitor, a platinum complex, or an HSP90 inhibitor, but is not limited thereto, and is preferably an HSP90 inhibitor.
상기 다른 항암제는 구체적으로, 탁솔, 트라스투주맙, 제피티닙, 시스플라틴, 메토트렉세이트, 6-머캅토퓨린, 5-플루오로우라실, 겜시타빈, 독소루비신, 닥티노마이신, 블레오마이신, 니트로유레아, 빈크리스틴, 빈블라스틴, 타목시펜, 또는 카보플라틴 등을 들 수 있으나, 이에 한정되는 것은 아니며, 바람직하게는 탁솔, 트라스투주맙, 제피티닙 또는 시스플라틴이다.Said other anticancer agents specifically include Taxol, Trastuzumab, Zephytinib, Cisplatin, Methotrexate, 6-mercaptopurine, 5-fluorouracil, gemcitabine, doxorubicin, dactinomycin, bleomycin, nitrourea, vincristine , Vinblastine, tamoxifen, or carboplatin, and the like, but are not limited thereto, and preferably Taxol, Trastuzumab, Zephytinib, or Cisplatin.
상기 HSP90 억제제는 라디시콜(Radicicol) 유도체, 겔다나마이신(Geldanamycin) 유도체, 노보비오신(Novobiocin) 유도체, 또는 퓨린(Purine)을 기본으로 한 인공적으로 합성된 물질 등일 수 있으며, 구체적으로 가네테스핍(Ganetespib, STA-9090), 루미네스핍(Luminespib, NVP-AUY922), 타네스피마이신(Tanespimycin, 17-AAG), 알베스피마이신(Alvespimycin, 17-DMAG), 엘레스클로몰(Elesclomol, STA-4783), 온날레스핍(Onlaespib, AT13387), BIIB021 (6-클로로-9-((4-메톡시-3,5-디메틸피리딘-2-일)메틸)-9H-퓨린-2-아민), PU-H71 (8-[(6-아이오도-1,3-벤조다이옥솔-5-일)설파닐]-9-[3-(프로판-2-일아미노)프로필]퓨린-6-아민), XL888 (2-(부탄-2-일아미노)-4-N-[(1R,5S)-8-[5-(사이클로프로판카보닐)피리딘-2-일]-8-아자바이사이클로[3.2.1]옥탄-3-일]-5-메틸벤젠-1,4-다이카복사마이드), KW-2478 (2-[2-에틸-3,5-다이하이드록시-6-[3-메톡시-4-(2-몰폴린-4-일에톡시)벤조일]페닐]-N,N-비스(2-메톡시에틸)아세타마이드), 또는 SNX-5422 ([4-[2-카바모일-5-[6,6-다이메틸-4-옥소-3-(트리플루오로메틸)-5,7-다이하이드로인다졸-1-일]아닐리노]사이클로헥실] 2-아미노아세테이트)일 수 있으며, 바람직하게는 가네테스핍(Ganetespib, STA-9090), 루미네스핍(Luminespib, NVP-AUY922), 타네스피마이신(Tanespimycin, 17-AAG) 또는 알베스피마이신(Alvespimycin, 17-DMAG)이다.The HSP90 inhibitor may be a radicicol derivative, a geldanamycin derivative, a novobiocin derivative, or an artificially synthesized material based on purine, and specifically, ganetes. Pep (Ganetespib, STA-9090), Luminespib (NVP-AUY922), Tanespimycin (Tanespimycin, 17-AAG), Alvespimycin (17-DMAG), Elesclomol, STA- 4783), Onlaespib (AT13387), BIIB021 (6-chloro-9-((4-methoxy-3,5-dimethylpyridin-2-yl) methyl) -9H-purin-2-amine), PU-H71 (8-[(6-iodo-1,3-benzodioxol-5-yl) sulfanyl] -9- [3- (propan-2-ylamino) propyl] purin-6-amine) , XL888 (2- (butan-2-ylamino) -4-N-[(1R, 5S) -8- [5- (cyclopropanecarbonyl) pyridin-2-yl] -8-azabicyclo [3.2 .1] octan-3-yl] -5-methylbenzene-1,4-dicarboxamide), KW-2478 (2- [2-ethyl-3,5-dihydroxy-6- [3-meth Methoxy-4- (2-morpholine-4- Ethoxy) benzoyl] phenyl] -N, N-bis (2-methoxyethyl) acetamide), or SNX-5422 ([4- [2-carbamoyl-5- [6,6-dimethyl- 4-oxo-3- (trifluoromethyl) -5,7-dihydroindazol-1-yl] anilino] cyclohexyl] 2-aminoacetate), preferably Ganetespib, STA-9090), Luminespib (NVP-AUY922), Tanespimycin (17-AAG) or Alvespimycin (Alvespimycin, 17-DMAG).
본 발명의 약제학적 조성물은 투여를 위해서 상기 화학식 I 로 표시되는 화합물 또는 이의 약제학적으로 허용되는 염 외에 추가로 약제학적으로 허용가능한 담체를 1 종 이상 더 포함할 수 있다. 약제학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다.The pharmaceutical composition of the present invention may further include at least one pharmaceutically acceptable carrier in addition to the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as antioxidants, buffers And other conventional additives such as bacteriostatic agents can be added.
또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 따라서, 본 발명의 조성물은 패치제, 액제, 환약, 캡슐, 과립, 정제, 좌제 등일 수 있다. 이들 제제는 당 분야에서 제제화에 사용되는 통상의 방법 또는 Remington's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA 에 개시되어 있는 방법으로 제조될 수 있으며 각 질환에 따라 또는 성분에 따라 다양한 제제로 제제화될 수 있다.Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Thus, the compositions of the present invention may be patches, solutions, pills, capsules, granules, tablets, suppositories, and the like. These formulations may be prepared by conventional methods used in the art for formulation or by methods disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA, and formulated into various formulations depending on the individual disease or component. Can be.
본 발명의 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여(예를 들어, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하고, 하루 일회 내지 수회에 나누어 투여할 수 있다.The composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex and health of the patient. Depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease, the range varies, and may be administered once or several times a day.
또한, 본 발명은 암의 예방 또는 치료에 있어서의 사용을 위한, 화학식 I로 표시되는 화합물 또는 이의 약제학적으로 허용 되는 염의 용도를 제공한다.The present invention also provides the use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of cancer.
또한, 본 발명은 암의 예방 또는 치료를 위한 약제의 제조에 있어서의 사용을 위한, 화학식 I로 표시되는 화합물 또는 이의 약제학적으로 허용 되는 염의 용도를 제공한다.The present invention also provides the use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for the prevention or treatment of cancer.
또한, 본 발명은 암의 예방 또는 치료를 필요로 하는 대상체에게 화학식 I로 표시되는 화합물 또는 이의 약제학적으로 허용 되는 염을 치료학적으로 유효한 양으로 투여하는 것을 포함하는, 암을 예방 또는 치료하는 방법을 제공한다.The present invention also provides a method of preventing or treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. To provide.
본 발명에 있어서, 상기 “대상체”는 포유류, 특히 인간을 포함한다.In the present invention, the "subject" includes mammals, especially humans.
본 발명에 있어서, “치료학적으로 유효한 양”은 대상 질환을 치료 또는 예방하는데 유효한 화학식 I의 화합물 또는 이의 약제학적으로 허용 되는 염의 양을 의미한다.In the present invention, "therapeutically effective amount" means an amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof that is effective for treating or preventing a subject disease.
본 발명의 화학식 Ⅰ로 표시되는 화합물, 이의 약제학적으로 허용되는 염, 또는 이들을 포함하는 약학적 조성물은 변형된 열충격 단백질 27 (HSP27) 이량체 형성을 유도하여 정상적인 HSP27 다량체의 형성을 감소시킴으로써, HSP27의 샤페론으로서의 기능을 억제한다. 따라서 본 발명은 암의 예방 또는 치료에 효과를 나타낼 수 있으며, 또한 HSP90 및 기타 항암제의 내성을 감소시킬 수 있고, 방사선에 의한 내성도 감소시킬 수 있다.Compounds represented by Formula I of the present invention, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising them induce the formation of modified heat shock protein 27 (HSP27) dimers, thereby reducing the formation of normal HSP27 multimers, It inhibits the function of HSP27 as chaperone. Therefore, the present invention can be effective in the prevention or treatment of cancer, and can also reduce the resistance of HSP90 and other anticancer agents, and can also reduce the resistance to radiation.
도 1은 셀 프리(cell-free) 시스템에서 재조합 단백질에서의 HSP27 이량체 생성능을 웨스턴 블랏으로 확인한 것이다.1 is a Western blot confirming the ability of HSP27 dimer production in recombinant proteins in a cell-free system.
도 2는 시간 의존적 HSP27 이량체 생성능을 웨스턴 블랏으로 확인한 것이다.Figure 2 confirms the time dependent HSP27 dimer production capacity by Western blot.
도 3은 NCI-H460 세포에서 HSP27 이량체 생성능을 웨스턴 블랏으로 확인한 것이다.Figure 3 is confirmed by Western blot HSP27 dimer generating ability in NCI-H460 cells.
도 4는 NCI-H460 세포에서 농도 의존적 HSP27 이량체 생성능을 웨스턴 블랏으로 확인한 것이다.Figure 4 confirms the concentration-dependent HSP27 dimer production ability in NCI-H460 cells by Western blot.
도 5는 NCI-H460 세포에서 농도 의존적 HSP70, HSP0 및 HSF1 발현을 웨스턴 블랏으로 확인한 것이다.FIG. 5 shows Western blots of concentration dependent HSP70, HSP0 and HSF1 expression in NCI-H460 cells.
도 6은 NCI-H460 세포에 본 발명의 화합물을 각각 17-AAG, 탁솔 또는 시스플라틴과 병용 투여했을 때의 변형된 HSP27 이량체, 절단된 PARP(Cleaved PARP) 및 절단된 카스파아제-3(Cleaved caspase-3)의 발현을 웨스턴 블랏으로 확인한 것이다.FIG. 6 shows modified HSP27 dimer, cleaved PARP and cleaved caspase-3 when NCI-H460 cells are administered with a compound of the present invention in combination with 17-AAG, taxol or cisplatin, respectively. -3) expression was confirmed by Western blot.
도 7은 NCI-H460 세포에 본 발명의 화합물을 각각 17-AAG, 탁솔 또는 시스플라틴과 병용 투여했을 때의 세포사멸률 및 세포생존율을 도식화한 것이다.Fig. 7 shows the cell death rate and cell viability when NCI-H460 cells were co-administered with 17-AAG, Taxol or Cisplatin, respectively.
도 8은 이종 이식 마우스에서 본 발명의 화합물을 단독 처리 또는 17-AAG와 병용 처리하여 종양에 직접 투여한 경우의 종양부피 변화 및 세포 사멸 수를 나타낸 것이다.Figure 8 shows the tumor volume change and the number of cell death when the compound of the present invention in xenograft mice was administered alone or in combination with 17-AAG directly to tumors.
도 9는 본 발명의 화합물을 단독 처리 또는 탁솔과 병용 처리하여 복강 투여한 경우의 종양부피 변화 및 세포 사멸 수를 나타낸 것이다.Figure 9 shows the tumor volume change and the number of cell death in the case of intraperitoneal administration of the compound of the present invention alone or in combination with Taxol.
도 10은 본 발명의 화합물을 단독 처리 또는 탁솔과 병용 처리하여 종양에 직접 투여한 경우의 종양부피 변화 및 세포 사멸 수를 나타낸 것이다.Figure 10 shows the tumor volume change and the number of cell death when the compound of the present invention alone or in combination with Taxol to be administered directly to the tumor.
도 11은 본 발명은 화합물이 농도 의존적으로 HER 수용체 및 그 하위 단계의 신호 전달 단백질의 저해 효과를 웨스턴 블랏으로 확인한 것이다.FIG. 11 is a compound of the present invention confirms the inhibitory effect of the signal transduction protein of the HER receptor and its substeps in a concentration-dependent manner by Western blot.
도 12는 본 발명의 화합물을 트라스투주맙과 병용 처리한 경우의 HER2 신호 전달 저해 효과를 웨스턴 블랏으로 확인한 것이다.Figure 12 confirms the inhibitory effect of HER2 signal transduction when the compound of the present invention is treated in combination with trastuzumab.
도 13은 본 발명의 화합물을 트라스투주맙과 병용 처리한 경우의 세포 사멸 유도 단백질 발현 증진 효과 및 세포 사멸 억제 단백질 발현 감소 효과를 웨스턴 블랏으로 확인한 것이다.Figure 13 confirms the effects of apoptosis-induced protein expression and cell death-inhibiting protein expression when the compound of the present invention in combination with trastuzumab by Western blot.
도 14는 본 발명의 화합물을 트라스투주맙과 병용 처리한 경우의 세포 성장 저해율을 도식화한 것이다.Fig. 14 shows the cell growth inhibition rate when the compound of the present invention is treated in combination with trastuzumab.
도 15는 본 발명의 화합물을 트라스투주맙과 병용 처리한 경우의 종양크기, 종양 부피 변화 및 생존율을 도식화한 것이다.Figure 15 shows the tumor size, tumor volume change and survival rate when the compound of the present invention is treated in combination with trastuzumab.
도 16은 본 발명의 화합물을 제피티닙과 병용 처리한 경우의 세포 사멸 유도 단백질 발현 증진 효과를 웨스턴 블랏으로 확인한 것이다.Figure 16 confirms the effect of apoptosis inducing protein expression in the case of treatment with the compound of the present invention in combination with zephytinib by Western blot.
도 17은 본 발명의 화합물을 제피티닙과 병용 처리한 경우의 세포 성장률을 도식화한 것이다.Fig. 17 shows the cell growth rate when the compound of the present invention is treated in combination with gefitinib.
이하, 실시예 및 실험예를 통하여 본 발명을 더욱 상세히 설명한다. 단, 이들 실시예 등은 본 발명의 예시일 뿐, 본 발명의 범위가 이들만으로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, these Examples etc. are only illustrations of this invention, The scope of the present invention is not limited only to these.
실시예Example 1: 화합물 1 (5-히드록시-2- 1: compound 1 (5-hydroxy-2- 메틸methyl -7-(티이란-2--7- (Tyran-2- 일메톡시Ilmethoxy )-4H-) -4H- 크로멘Chromen -4-온) 및 화합물 2 (2-메틸-5,7-비스(티이란-2-일메톡시)-4H-크로멘-4-온)의 합성4-one) and compound 2 (2-methyl-5,7-bis (thiran-2-ylmethoxy) -4H-chromen-4-one)
5,7-디히드록시-2-메틸-4H-크로멘-4-온 (0.50 g, 2.60 mmol)와 탄산 칼륨(K2CO3, 0.72g,5.20mmol)이 디메틸포름아마이드(Dimethylformaide, DMF)/아세톤(20 mL/10 mL) 용매에 들어있는 혼합물에 에피티오클로로히드린(1.13 g, 10.40 mmol)을 넣고 90℃ 에서 21 시간 교반한 후 실온이 될 때까지 정치하였다. 이 반응 혼합물에 물을 넣고 에틸 아세테이트로 추출하고 유기층을 모아 물과 브라인으로 세척하였다. 유기용매에 무수 황산 마그네슘(MgSO4)을 넣어 남은 수분을 제거하고, 용매는 감압하에서 증류 제거하였다 남은 잔사를 실리카 겔 컬럼 크로마토그래피(전개용매: 디클로로메탄(CH2Cl2)→에틸 아세테이트:n-헥세인=1:1)로 정제하여 화합물 1 (상아색 고체, 0.32 g, 46.8 %)과 화합물 2 (오렌지색 고체, 0.08 g, 8.7%)를 얻었다. 5,7-Dihydroxy-2-methyl-4H-chromen-4-one (0.50 g, 2.60 mmol) and potassium carbonate (K 2 CO 3 , 0.72 g, 5.20 mmol) are dimethylformamide (DMF) Epithiochlorohydrin (1.13 g, 10.40 mmol) was added to the mixture in the acetone (20 mL / 10 mL) solvent, and the mixture was stirred at 90 ° C. for 21 hours, and then allowed to stand at room temperature. Water was added to the reaction mixture, followed by extraction with ethyl acetate. The organic layers were collected and washed with water and brine. Anhydrous magnesium sulfate (MgSO 4 ) was added to the organic solvent to remove the remaining water, and the solvent was distilled off under reduced pressure. The remaining residue was subjected to silica gel column chromatography (developing solvent: dichloromethane (CH 2 Cl 2 ) → ethyl acetate: n Purification with -hexane = 1: 1) gave Compound 1 (ivory solid, 0.32 g, 46.8%) and Compound 2 (orange solid, 0.08 g, 8.7%).
화합물 1: Rf 0.36 (에틸 아세테이트:n-헥세인=1:3); 녹는점(melting point, mp): 152-153 ℃; 액체크로마토그래피(High Performance Liquid Chromatography, HPLC): RT 5.91 분(min) (순도(purity): 99.39%); 1H-NMR (CDCl3, 400 MHz) δ 2.33 (dd, J = 6.8, 1.2 Hz, 1H), 2.34 (s, 3H), 2.63 (dd, J = 6.4, 0.8 Hz, 1H), 3.24-3.30 (m, 1H), 3.97 (dd, J = 10.0, 2.4 Hz, 1H), 4.21 (dd, J = 10.4, 1.2 Hz, 1H), 6.03 (s, 1H), 6.33 (d, J = 2.4 Hz, 1H), 6.35 (d, J = 2.4 Hz, 1H), 12.70 (s, 1H); 13C-NMR (CDCl3, 100 MHz) 20.7, 24.0, 31.0, 73.1, 93.2, 98.6, 105.7, 109.1, 158.3, 162.5, 164.1, 167.1, 182.7 ppm Compound 1: R f 0.36 (ethyl acetate: n-hexane = 1: 3); Melting point (mp): 152-153 ° C .; High Performance Liquid Chromatography (HPLC): R T 5.91 min (purity: 99.39%); 1 H-NMR (CDCl 3 , 400 MHz) δ 2.33 (dd, J = 6.8, 1.2 Hz, 1H), 2.34 (s, 3H), 2.63 (dd, J = 6.4, 0.8 Hz, 1H), 3.24-3.30 (m, 1H), 3.97 (dd, J = 10.0, 2.4 Hz, 1H), 4.21 (dd, J = 10.4, 1.2 Hz, 1H), 6.03 (s, 1H), 6.33 (d, J = 2.4 Hz, 1H), 6.35 (d, J = 2.4 Hz, 1H), 12.70 (s, 1H); 13 C-NMR (CDCl 3 , 100 MHz) 20.7, 24.0, 31.0, 73.1, 93.2, 98.6, 105.7, 109.1, 158.3, 162.5, 164.1, 167.1, 182.7 ppm
화합물 2: Rf 0.14 (에틸 아세테이트:n-헥세인=1:1); mp: 95-96 ℃; HPLC: RT 4.77 min (purity: 99.0%); 1H-NMR (CDCl3, 400 MHz) δ 2.27 (s, 3H), 2.34 (dd, J = 5.6, 1.6 Hz, 1H), 2.48 (dd, J = 5.2, 0.8 Hz, 1H), 2.63-2.66 (m, 2H), 3.24-3.30 (m, 1H), 3.38-3.44 (m, 1H), 3.91 (dd, J = 10.0, 7.2 Hz, 1H), 3.99 (dd, J = 10.0, 6.8 Hz, 1H), 4.21 (dd, J = 10.0, 5.6 Hz, 1H), 4.39 (dd, J = 10.0, 4.8 Hz, 1H), 6.00 (d, J = 0.8 Hz, 1H), 6.37 (d, J = 2.4 Hz, 1H), 6.43 (d, J = 2.4 Hz, 1H); 13C-NMR (CDCl3, 100 MHz) 20.0, 23.9, 24.6, 30.9, 31.1, 73.1, 74.2, 94.5, 98.8, 109.9, 112.2, 159.9, 160.2, 162.4, 163.4, 177.4 ppm.Compound 2: R f 0.14 (ethyl acetate: n-hexane = 1: 1); mp: 95-96 ° C .; HPLC: R T 4.77 min (purity: 99.0%); 1 H-NMR (CDCl 3 , 400 MHz) δ 2.27 (s, 3H), 2.34 (dd, J = 5.6, 1.6 Hz, 1H), 2.48 (dd, J = 5.2, 0.8 Hz, 1H), 2.63-2.66 (m, 2H), 3.24-3.30 (m, 1H), 3.38-3.44 (m, 1H), 3.91 (dd, J = 10.0, 7.2 Hz, 1H), 3.99 (dd, J = 10.0, 6.8 Hz, 1H ), 4.21 (dd, J = 10.0, 5.6 Hz, 1H), 4.39 (dd, J = 10.0, 4.8 Hz, 1H), 6.00 (d, J = 0.8 Hz, 1H), 6.37 (d, J = 2.4 Hz , 1H), 6.43 (d, J = 2.4 Hz, 1H); 13 C-NMR (CDCl 3 , 100 MHz) 20.0, 23.9, 24.6, 30.9, 31.1, 73.1, 74.2, 94.5, 98.8, 109.9, 112.2, 159.9, 160.2, 162.4, 163.4, 177.4 ppm.
실시예Example 2: 화합물 3 (5-히드록시-7-( 2: compound 3 (5-hydroxy-7- ( 옥시란Oxirane -2--2- 일메톡시Ilmethoxy )-2-페닐-4) -2-phenyl-4 HH -- 크로멘Chromen -4-온) 및 화합물 4 (5,7-비스(옥시란-2-일메톡시)-2-페닐-4-4-one) and compound 4 (5,7-bis (oxirane-2-ylmethoxy) -2-phenyl-4 HH -크로멘-4-온)의 합성-Chromen-4-one)
5,7-디히드록시플라본 (1.00 g, 3.93 mmol)와 K2CO3(1.09 g, 7.86 mmol)이 DMF/아세톤 (12 mL/4 mL) 용매에 들어있는 혼합물에 에피클로로히드린 (1.82 g, 19.65 mmol)을 넣고 90 ℃ 에서 20 시간 교반한 후 실온이 될 때까지 정치하였다. 이 반응 혼합물에 물을 넣고 에틸 아세테이트로 추출하고 유기층을 모아 물과 브라인으로 세척하였다. 유기용매에 무수 MgSO4를 넣어 남은 수분을 제거하고, 용매는 감압하에서 증류 제거하였다 남은 잔사를 실리카 겔 컬럼 크로마토그래피 (전개용매: 메탄올(MeOH):클로로포름(CHCl3)=3:97→4:96)로 정제하여 화합물 3 (상아색 고체, 0.07 g, 5.7%)과 화합물 4 (상아색 고체, 0.30 g, 20.8%)를 얻었다. Epichlorohydrin (1.82) in a mixture containing 5,7-dihydroxyflavone (1.00 g, 3.93 mmol) and K 2 CO 3 (1.09 g, 7.86 mmol) in DMF / acetone (12 mL / 4 mL) solvent. g, 19.65 mmol), stirred at 90 ° C. for 20 hours, and allowed to stand until it reached room temperature. Water was added to the reaction mixture, followed by extraction with ethyl acetate. The organic layers were collected and washed with water and brine. Anhydrous MgSO 4 was added to the organic solvent to remove the remaining water, and the solvent was distilled off under reduced pressure. The remaining residue was subjected to silica gel column chromatography (developing solvent: methanol (MeOH): chloroform (CHCl 3 ) = 3: 97 → 4 :). 96) afforded Compound 3 (ivory solid, 0.07 g, 5.7%) and Compound 4 (ivory solid, 0.30 g, 20.8%).
화합물 3: Rf 0.24 (에틸 아세테이트:n-헥세인=1:3); HPLC: RT 5.67 min(purity: 100%); 1H-NMR (CDCl3, 400 MHz) δ2.79 (dd, J = 4.4, 2.4 Hz, 1H), 2.95 (dd, J = 4.4, 4.0 Hz, 1H), 3.37-3.41 (m, 1H), 4.00 (dd, J = 11.2, 6.0 Hz, 1H), 4.34 (dd, J = 11.2, 2.8 Hz, 1H), 6.39 (d, J = 2.4 Hz, 1H), 6.54 (d, J = 2.4 Hz, 1H), 6.68 (s, 1H), 7.51-7.56 (m, 3H), 7.87-7.90 (m, 2H), 12.73 (s, 1H); 13C-NMR (CDCl3, 100MHz) 44.6, 49.7, 69.2, 93.3, 98.6, 105.9, 106.0, 126.3, 129.1, 131.3, 131.9, 157.8, 162.3, 164.1, 164.3, 182.5 ppm; HRMS-ESI(m/z) [M-H]- C18H15O5 calcd 311.0914, found 311.0918. Compound 3: R f 0.24 (ethyl acetate: n -hexane = 1: 3); HPLC: R T 5.67 min (purity: 100%); 1 H-NMR (CDCl 3 , 400 MHz) δ 2.79 (dd, J = 4.4, 2.4 Hz, 1H), 2.95 (dd, J = 4.4, 4.0 Hz, 1H), 3.37-3.41 (m, 1H), 4.00 (dd, J = 11.2, 6.0 Hz, 1H), 4.34 (dd, J = 11.2, 2.8 Hz, 1H), 6.39 (d, J = 2.4 Hz, 1H), 6.54 (d, J = 2.4 Hz, 1H ), 6.68 (s, 1 H), 7.51-7.56 (m, 3H), 7.87-7.90 (m, 2H), 12.73 (s, 1H); 13 C-NMR (CDCl 3 , 100 MHz) 44.6, 49.7, 69.2, 93.3, 98.6, 105.9, 106.0, 126.3, 129.1, 131.3, 131.9, 157.8, 162.3, 164.1, 164.3, 182.5 ppm; HRMS-ESI (m / z) [M − H] − C 18 H 15 O 5 calcd 311.0914, found 311.0918.
화합물 4: Rf 0.39 (MeOH:CHCl3=1:24); HPLC: RT 7.72 min (purity: 98.89%); 1H-NMR (CDCl3, 400 MHz) δ 2.80 (dd, J = 4.8, 2.8 Hz, 1H), 2.95-2.97 (m, 2H), 3.14-3.16 (m, 1H), 3.38-3.43 (m, 1H), 3.44-3.48 (m, 1H), 6.50 (d, J = 2.4 Hz, 1H), 6.61 (d, J = 2.4 Hz, 1H), 6.64 (s, 1H), 7.48-7.52 (m, 3H), 7.85-7.88 (m, 2H); 13C-NMR (CDCl3, 100MHz) 44.6, 45.0, 49.8, 50.2, 69.2, 69.3, 94.5, 98.3, 109.1, 110.0, 126.0, 129.0, 131.3, 131.5, 159.7, 159.8, 160.8, 162.6, 177.2 ppm; HRMS-ESI(m/z) [M-H]- C21H19O6 calcd 367.1176, found 367.1183.Compound 4: R f 0.39 (MeOH: CHCl 3 = 1: 24); HPLC: R T 7.72 min (purity: 98.89%); 1 H-NMR (CDCl 3 , 400 MHz) δ 2.80 (dd, J = 4.8, 2.8 Hz, 1H), 2.95-2.97 (m, 2H), 3.14-3.16 (m, 1H), 3.38-3.43 (m, 1H), 3.44-3.48 (m, 1H), 6.50 (d, J = 2.4 Hz, 1H), 6.61 (d, J = 2.4 Hz, 1H), 6.64 (s, 1H), 7.48-7.52 (m, 3H ), 7.85-7.88 (m, 2H); 13 C-NMR (CDCl 3 , 100 MHz) 44.6, 45.0, 49.8, 50.2, 69.2, 69.3, 94.5, 98.3, 109.1, 110.0, 126.0, 129.0, 131.3, 131.5, 159.7, 159.8, 160.8, 162.6, 177.2 ppm; HRMS-ESI (m / z) [M − H] − C 21 H 19 O 6 calcd 367.1176, found 367.1183.
실시예Example 3: 화합물 5 (5-히드록시-2-페닐-7-(티이란-2- 3: compound 5 (5-hydroxy-2-phenyl-7- (tyran-2- 일메톡시Ilmethoxy )-4H-) -4H- 크로멘Chromen -4-온) 및 화합물 6 (2-페닐-5,7-비스(티이란-2-일메톡시)-4H-크로멘-4-온)의 합성4-one) and compound 6 (2-phenyl-5,7-bis (thiran-2-ylmethoxy) -4H-chromen-4-one)
5,7-디히드록시플라본 (0.50 g, 1.97 mmol)와 탄산 세슘(Cs2CO3 , 1.28g, 3.93mmol)이 DMF/아세톤 (5 mL/15 mL) 용매에 들어있는 혼합물에 에피클로로히드린 (0.64 g, 5.91 mmol)을 넣고 80℃ 에서 20 시간 교반한 후 실온이 될 때까지 정치하였다. 이 반응 혼합물에 물을 넣고 에틸 아세테이트로 추출하고 유기층을 모아 물과 브라인(brine)으로 세척하였다. 유기용매에 무수 MgSO4를 넣어 남은 수분을 제거하고, 용매는 감압하에서 증류 제거하였다 남은 잔사를 실리카 겔 컬럼 크로마토그래피 (전개용매: CH2Cl2→에틸 아세테이트:n-헥세인=2:1)로 정제하여 화합물 5 (노란색 고체, 0.05 g, 8.0%)와 화합물 6 (노란색 고체, 0.08 g, 8.9%)을 얻었다. Epichlorohib in a mixture of 5,7-dihydroxyflavone (0.50 g, 1.97 mmol) and cesium carbonate (Cs 2 CO 3 , 1.28 g, 3.93 mmol) in DMF / acetone (5 mL / 15 mL) solvent Drin (0.64 g, 5.91 mmol) was added thereto, stirred at 80 ° C. for 20 hours, and allowed to stand at room temperature. Water was added to the reaction mixture, followed by extraction with ethyl acetate. The organic layers were collected and washed with water and brine. Anhydrous MgSO 4 was added to the organic solvent to remove the remaining water, and the solvent was distilled off under reduced pressure. The remaining residue was purified by silica gel column chromatography (developing solvent: CH 2 Cl 2 → ethyl acetate: n -hexane = 2: 1). Purification was carried out to give compound 5 (yellow solid, 0.05 g, 8.0%) and compound 6 (yellow solid, 0.08 g, 8.9%).
화합물 5: Rf 0.47 (에틸 아세테이트:n-헥세인=1:3); HPLC: RT 8.30 min (purity: 98.56%); 1H-NMR (CDCl3, 400MHz) δ 2.36 (dd, J = 5.2, 1.2 Hz, 1H), 2.65 (dd, J = 6.4, 1.2 Hz, 1H), 3.27-3.33 (m, 1H), 4.00 (dd, J = 10.4, 6.8 Hz, 1H), 4.26 (dd, J = 10.0, 5.2 Hz, 1H), 6.38 (d, J = 2.0 Hz, 1H), 6.51 (d, J = 2.0 Hz, 1H), 6.68 (s, 1H), 7.50-7.56 (m, 3H), 7.87-7.90 (m, 2H), 12.73 (s, 1H); 13C-NMR (CDCl3, 100MHz) 23.8, 30.7, 73.0, 93.2, 98.7, 106.0, 126.3, 129.1, 131.3, 131.9, 157.8, 162.3, 164.1, 164.2, 182.5 ppm; 13C-NMR (CDCl3, 100 MHz) 44.9, 50.2, 69.3, 112.8, 113.2, 115.6, 121.7, 124.8, 129.6, 129.8, 130.8, 133.1, 145.0, 152.0, 157.2, 192.2 ppm; HRMS-ESI (m/z) [M-H]- C18H15O4S calcd 327.0686, found 327.0688. Compound 5: R f 0.47 (ethyl acetate: n -hexane = 1: 3); HPLC: R T 8.30 min (purity: 98.56%); 1 H-NMR (CDCl 3 , 400 MHz) δ 2.36 (dd, J = 5.2, 1.2 Hz, 1H), 2.65 (dd, J = 6.4, 1.2 Hz, 1H), 3.27-3.33 (m, 1H), 4.00 ( dd, J = 10.4, 6.8 Hz, 1H), 4.26 (dd, J = 10.0, 5.2 Hz, 1H), 6.38 (d, J = 2.0 Hz, 1H), 6.51 (d, J = 2.0 Hz, 1H), 6.68 (s, 1 H), 7.50-7.56 (m, 3 H), 7.87-7.90 (m, 2 H), 12.73 (s, 1 H); 13 C-NMR (CDCl 3 , 100 MHz) 23.8, 30.7, 73.0, 93.2, 98.7, 106.0, 126.3, 129.1, 131.3, 131.9, 157.8, 162.3, 164.1, 164.2, 182.5 ppm; 13 C-NMR (CDCl 3 , 100 MHz) 44.9, 50.2, 69.3, 112.8, 113.2, 115.6, 121.7, 124.8, 129.6, 129.8, 130.8, 133.1, 145.0, 152.0, 157.2, 192.2 ppm; HRMS-ESI (m / z) [M − H] − C 18 H 15 O 4 S calcd 327.0686, found 327.0688.
화합물 6: Rf 0.22 (에틸 아세테이트:n-헥세인=1:1); HPLC: RT 6.23 min (purity: 97.82%); 1H-NMR (CDCl3, 400 MHz)δ 2.36 (dd, J = 5.2, 1.2 Hz, 1H), 2.51 (dd, J = 5.2, 1.6 Hz, 1H), 2.64-2.69 (m, 2H), 3.27-3.33 (m, 1H), 3.41-3.47 (m, 1H), 3.95 (dd, J = 10.4, 3.2 Hz, 1H), 4.04 (dd, J = 10.4, 3.2 Hz, 1H), 4.25 (dd, J = 10.0, 5.6 Hz, 1H), 4.43 (dd, J = 10.4, 4.8 Hz, 1H), 6.42 (d, J = 2.4 Hz, 1H), 6.59 (d, J = 2.4 Hz, 1H), 6.64 (s, 1H), 7.48-7.52 (m, 3H), 7.84-7.87 (m, 2H); 13C-NMR (CDCl3, 100 MHz) 23.8, 30.7, 73.0, 93.2, 98.7, 106.0, 126.3, 129.1, 131.3, 131.9, 157.8, 162.3, 164.1, 164.2, 182.5 ppm; 13C-NMR (CDCl3, 100 MHz) 23.7, 24.4, 30.7, 31.0, 73.0, 74.0, 94.5, 98.8, 109.1, 110.1, 126.0, 129.0, 131.3, 131.5, 159.7, 160.8, 162.5, 177.2 ppm; HRMS-ESI (m/z) [M-H]- C21H19O4S2 calcd 399.0719, found 399.0723.Compound 6: R f 0.22 (ethyl acetate: n -hexane = 1: 1); HPLC: R T 6.23 min (purity: 97.82%); 1 H-NMR (CDCl 3 , 400 MHz) δ 2.36 (dd, J = 5.2, 1.2 Hz, 1H), 2.51 (dd, J = 5.2, 1.6 Hz, 1H), 2.64-2.69 (m, 2H), 3.27 -3.33 (m, 1H), 3.41-3.47 (m, 1H), 3.95 (dd, J = 10.4, 3.2 Hz, 1H), 4.04 (dd, J = 10.4, 3.2 Hz, 1H), 4.25 (dd, J = 10.0, 5.6 Hz, 1H), 4.43 (dd, J = 10.4, 4.8 Hz, 1H), 6.42 (d, J = 2.4 Hz, 1H), 6.59 (d, J = 2.4 Hz, 1H), 6.64 (s , 1H), 7.48-7.52 (m, 3H), 7.84-7.87 (m, 2H); 13 C-NMR (CDCl 3 , 100 MHz) 23.8, 30.7, 73.0, 93.2, 98.7, 106.0, 126.3, 129.1, 131.3, 131.9, 157.8, 162.3, 164.1, 164.2, 182.5 ppm; 13 C-NMR (CDCl 3 , 100 MHz) 23.7, 24.4, 30.7, 31.0, 73.0, 74.0, 94.5, 98.8, 109.1, 110.1, 126.0, 129.0, 131.3, 131.5, 159.7, 160.8, 162.5, 177.2 ppm; HRMS-ESI (m / z) [M − H] − C 21 H 19 O 4 S 2 calcd 399.0719, found 399.0723.
<실험예>Experimental Example
실험예 1: 재조합 단백질에서의 HSP27 변형 이량체 생성능 확인Experimental Example 1 Confirmation of HSP27 Modified Dimer Formation Capacity in Recombinant Protein
1-1. 화학식 Ⅰ의 화합물의 HSP27 변형 이량체 생성능 확인1-1. Confirmation of HSP27 Modified Dimer Formation Capability of Compounds of Formula (I)
본 발명의 화학식 Ⅰ의 화합물의 열충격 단백질 27 (HSP27) 변형 이량체 생성능을 확인하고자, HSP27의 설치류 호모로그인 HSP25 Wild type(HSP25WT) 단백질을 사용하였다. HSP25WT는 열충격 단백질 25 (HSP25)를 발현하는 플라스미드를 사용하여 제작하였다.In order to confirm the heat shock protein 27 (HSP27) modified dimer generating ability of the compound of formula (I) of the present invention, HSP25 Wild type (HSP25WT) protein, which is a rodent homolog of HSP27, was used. HSP25WT was constructed using a plasmid expressing heat shock protein 25 (HSP25).
화합물 1, 화합물 4 및 화합물 6을 HSP25WT 단백질에 각각 50 μM 농도로 3시간 동안 처리하고, 웨스턴 블랏으로 확인하였다. 50 μM each of compound 1, compound 4 and compound 6 in HSP25WT protein The concentration was treated for 3 hours and confirmed by Western blot.
웨스턴 블랏을 수행하기 위해 상기 화합물 1, 화합물 4 및 화합물 6을 처리한 HSP25WT 단백질을 1X PBS(0.14 M 염화나트륨(NaCl), 2.68 mM 염화칼륨(KCl), 10 mM 디소듐포스페이트(Na2HPO4), 1.83mM 디포타슘포스페이트(KH2PO4))로 2회 세척한 후 방사선 면역 촉진 분석 완충액(Radio Immunoprecipitation Polyacrylamide Assay buffer, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA,1% NP-40, 1% 소듐 디옥시콜레이트, 1mM β-글리세로포스페이트, 1 mM 오르토바나듐산 나트륨(Na3VO4, 4mM NaF))에 용해시켰다. 세포 현탁액을 13000 rpm으로 30분간 원심분리를 수행하여 상층액만을 취하였다. HSP25WT protein treated with Compound 1, Compound 4 and Compound 6 was subjected to Western blot using 1 × PBS (0.14 M sodium chloride (NaCl), 2.68 mM potassium chloride (KCl), 10 mM disodium phosphate (Na 2 HPO 4 ), After washing twice with 1.83 mM dipotassium phosphate (KH 2 PO 4 )), Radio Immunoprecipitation Polyacrylamide Assay buffer, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na 2 EDTA, 1% NP-40, 1% sodium dioxycholate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate (Na 3 VO 4 , 4 mM NaF)). The cell suspension was centrifuged at 13000 rpm for 30 minutes to extract only the supernatant.
같은 양의 단백질을 함유하는 샘플을 만들기 위하여, 브래드포드 분석(Bradford assay)으로 단백질 양을 측정하였다. 단백질 염료(Protein Dye) 1 mL에 기준(standard)으로 1% 소혈청알부민(Bovine Serum Albumin, BSA) 용액을 8, 6, 4, 2, 0 μL를 넣고, 상층액 2 μL를 넣어 96-웰 플레이트에 200 μL씩 분주하고, ELISA 리더기에 넣어 595 nm의 파장에서 흡광도를 측정하였다. To make a sample containing the same amount of protein, the protein amount was measured by Bradford assay. Add 1,8,6,4,2,0 μL of 1% Bovine Serum Albumin (BSA) solution to 1 mL of protein dye (Protein Dye), and add 2 μL of supernatant to 96-well 200 μL was aliquoted into the plate, and the absorbance was measured at a wavelength of 595 nm in an ELISA reader.
6X 시료 버퍼(0.35 M Tris(pH6.8), 3% 글리세롤, 1% 도데실황산나트륨(Sodium Dodecyl Sulfate), 6 mM 디티오크레이톨(Dithiothreitol))를 넣어 같은 양의 단백질을 함유하는 샘플을 만들었다. 샘플을 5분간 100 ℃ 물에 담가 끓이고 SDS-PAGE를 이용하여 단백질을 분석하였다. 웨스턴 블랏을 위한 일차 항체는 Goat polyclonal anti-HSP27(sc-1049) 및 β-액틴(β-actin, sc-47778)을 사용하였으며, 이차항체는 Donkey anti goat(sc-2020), Goat anti rabbit(sc-2004), Goat anti mouse(sc-2005)를 사용하였다.6X sample buffer (0.35 M Tris (pH6.8), 3% glycerol, 1% Sodium Dodecyl Sulfate, 6 mM Dithiothreitol) was added to make a sample containing the same amount of protein. . The samples were soaked in 100 ° C. water for 5 minutes and boiled and analyzed for protein using SDS-PAGE. The primary antibodies for Western blot were Goat polyclonal anti-HSP27 (sc-1049) and β-actin (β-actin, sc-47778). The secondary antibodies were Donkey anti goat (sc-2020) and Goat anti rabbit ( sc-2004) and Goat anti mouse (sc-2005) were used.
그 결과, 도 1에 나타낸 것과 같이, 본 발명의 화합물을 처리한 경우 HSP27 이량체의 생성이 증가함을 확인하였다. 따라서, 본 발명의 화합물이 HSP27의 활성을 억제할 수 있음을 알 수 있다.As a result, as shown in Figure 1, the treatment of the compound of the present invention was confirmed to increase the production of HSP27 dimer. Thus, it can be seen that the compounds of the present invention can inhibit the activity of HSP27.
1-2. 시간 의존적 HSP27 변형 이량체 생성능 확인1-2. Determination of Time-dependent HSP27 Strain Dimer Generation
화합물 1 50 μM을 0, 0.5, 1, 2, 3, 6, 12 또는 24시간 처리하였다. 상기 실험예 1-1과 동일한 방법으로 웨스턴 블랏을 수행하여 HSP27 변형 이량체의 생성 정도를 확인하였다. 50 μM of compound 1 was treated for 0, 0.5, 1, 2, 3, 6, 12 or 24 hours. Western blot was performed in the same manner as in Experimental Example 1-1 to confirm the production level of HSP27 modified dimer.
그 결과, 도 2에 나타낸 것과 같이, 본 발명의 화합물의 투여시간에 비례하여, HSP27 이량체의 생성이 증가함을 확인하였다.As a result, as shown in Figure 2, it was confirmed that the production of HSP27 dimer increases in proportion to the administration time of the compound of the present invention.
실험예 2: 세포에서의 HSP27 변형 이량체 생성능 확인Experimental Example 2 Confirmation of HSP27 Modified Dimer Formation Ability in Cells
2-1. NCI-H460 세포에서의 HSP27 변형 이량체 생성능 확인2-1. Confirmation of HSP27 Modified Dimer Formation Capacity in NCI-H460 Cells
세포에서의 HSP27의 변형 이량체 생성능 정도를 확인하기 위하여 본 발명의 화합물1, 화합물 2, 화합물 3, 화합물 4, 화합물 5 또는 화합물 6을 각각 비소세포폐암 세포인 NCI-H460에 10 μM 농도로 12시간 동안 처리하였다. 그 후, 세포를 10% 소태아혈청 (Fetal Bovine Serum, FBS)과 1X 항생제-항진균(Antibiotic-Antimycotic) (GIBCO-Invitrogen, Paisley, Scotland, UK)가 포함된 RPMI 1640 (RPMI, GIBCO-Invitrogen, Paisley, Scotland, UK)배지를 사용하여 37 ℃, 5 % 이산화탄소(CO2) 배양기에서 12시간 동안 배양하였다.In order to determine the degree of the ability of HSP27 to produce a modified dimer, Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, or Compound 6 of the present invention were each 10 μM in NCI-H460, a non-small cell lung cancer cell. The concentration was treated for 12 hours. The cells were then cultured with RPMI 1640 (RPMI, GIBCO-Invitrogen, 10% Fetal Bovine Serum (FBS)) and 1 × Antibiotic-Antimycotic (GIBCO-Invitrogen, Paisley, Scotland, UK). Paisley, Scotland, UK) using a medium at 37 ° C, 5% carbon dioxide (CO 2 ) Incubated for 12 hours in the incubator.
웨스턴 블랏을 수행하기 위해 상기 화합물 1 내지 화합물 6을 각각 처리한 세포를 1X PBS(0.14 M NaCl, 2.68 mM KCl, 10 mM Na2HPO4, 1.83mM KH2PO4)로 2회 세척한 후 Radio Immunoprecipitation Polyacrylamide Assay buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA,1% NP-40, 1% 소듐 디옥시콜레이트, 1mM β-글리세로포스페이트, 1 mM Na3VO4, 4mM NaF)에 용해시켰다. 세포 현탁액을 13000 rpm으로 30분간 원심분리를 수행하여 상층액만을 취하였다. In order to perform the Western blot, the cells treated with Compounds 1 to 6 were washed twice with 1 × PBS (0.14 M NaCl, 2.68 mM KCl, 10 mM Na 2 HPO 4 , 1.83 mM KH 2 PO 4 ), followed by Radio. Immunoprecipitation Polyacrylamide Assay buffer (20 mM Tris-HCl, pH 7.5), 150 mM NaCl, 1 mM Na 2 EDTA, 1% NP-40, 1% Sodium Dioxycholate, 1 mM β-glycerophosphate, 1 mM Na 3 VO 4 , 4 mM NaF). The cell suspension was centrifuged at 13000 rpm for 30 minutes to extract only the supernatant.
같은 양의 단백질을 함유하는 샘플을 만들기 위하여, Bradford assay로 단백질 양을 측정하였다. Protein Dye 1 mL에 standard로 1% BSA 용액을 8, 6, 4, 2, 0 μL를 넣고, 상층액 2 μL를 넣어 96-웰 플레이트에 200 μL씩 분주하고, ELISA 리더기에 넣어 595 nm의 파장에서 흡광도를 측정하였다. In order to make a sample containing the same amount of protein, the protein amount was measured by Bradford assay. Add 8, 6, 4, 2, and 0 μL of 1% BSA solution as standard to 1 mL of Protein Dye, add 2 μL of supernatant, divide 200 μL into 96-well plate, and place in an ELISA reader at a wavelength of 595 nm. Absorbance was measured at.
6X 시료 버퍼(0.35 M Tris(pH6.8), 3% 글리세롤, 1% Sodium Dodecyl Sulfate, 6 mM Dithiothreitol)를 넣어 같은 양의 단백질을 함유하는 샘플을 만들었다. 샘플을 5분간 100 ℃ 물에 담가 끓이고 SDS-PAGE를 이용하여 단백질을 분석하였다. 웨스턴 블랏을 위한 일차 항체는 Goat polyclonal anti-HSP27(sc-1049) 및 β-actin(sc-47778)를 사용하였으며, 이차항체는 Donkey anti goat(sc-2020), Goat anti rabbit(sc-2004), Goat anti mouse(sc-2005)를 사용하였다. 6X sample buffer (0.35 M Tris (pH6.8), 3% glycerol, 1% Sodium Dodecyl Sulfate, 6 mM Dithiothreitol) was added to make a sample containing the same amount of protein. The samples were soaked in 100 ° C. water for 5 minutes and boiled and analyzed for protein using SDS-PAGE. The primary antibodies for Western blot were Goat polyclonal anti-HSP27 (sc-1049) and β-actin (sc-47778). The secondary antibodies were Donkey anti goat (sc-2020) and Goat anti rabbit (sc-2004). Goat anti mouse (sc-2005) was used.
분석 결과, 도 3에 나타낸 것과 같이, 본 발명의 화합물을 처리한 경우 HSP27 이량체가 증가하고, HSP27 단량체가 감소하는 것을 확인하였다. 따라서, 본 발명의 화합물이 HSP27의 활성을 억제할 수 있음을 알 수 있다.As a result, as shown in Figure 3, when the compound of the present invention was treated, it was confirmed that the HSP27 dimer increases and the HSP27 monomer decreases. Thus, it can be seen that the compounds of the present invention can inhibit the activity of HSP27.
2-2. 농도 의존적 HSP27 변형 이량체 생성능 확인2-2. Concentration-dependent HSP27 modified dimer formation ability
농도 의존적 HSP27 변형 이량체 생성을 확인하고자 비소세포폐암 세포인 NCI-H460에 화합물 1을 5, 10, 20 또는 40 μM 농도로 처리하고, 상기 실험예 2-1과 동일한 방법으로 세포배양 및 웨스턴 블랏을 수행하였다. In order to confirm the production of concentration-dependent HSP27 modified dimers, Compound 1 was treated with NCI-H460, a non-small cell lung cancer cell, at a concentration of 5, 10, 20, or 40 μM, and cell culture and Western blot were performed in the same manner as in Experimental Example 2-1. Was performed.
그 결과, 도 4에 나타낸 것과 같이, 본 발명의 화합물이 투여량이 증가할수록 HSP27 이량체의 양이 증가하고, HSP27 단량체의 양이 감소함을 확인하였다.As a result, as shown in Figure 4, as the dosage of the compound of the present invention was confirmed that the amount of HSP27 dimer increases, the amount of HSP27 monomer decreases.
실험예 3: HSP70, HSP90 및 HSF1의 발현량에 미치는 영향 확인Experimental Example 3: Confirmation of the effect on the expression level of HSP70, HSP90 and HSF1
HSP70, HSP90 및 열충격 단백질의 발현을 유도시키는 열충격 인자 1 (HSF1) 발현량에 어떤 영향을 주는지 확인하기 위해, 본 발명의 화합물 1을 NCI-H460 세포에 0, 5, 10, 20, 40 μM가 되도록 12시간 처리하고, 상기 실험예 2와 동일한 방법으로 세포배양 및 웨스턴 블랏을 수행하였다.To determine the effect of heat shock factor 1 (HSF1) expression that induces the expression of HSP70, HSP90 and heat shock proteins, compound 1 of the present invention was added to NCI-H460 cells at 0, 5, 10, 20, 40 μM. 12 hours were treated, and cell culture and Western blot were performed in the same manner as in Experimental Example 2.
그 결과, 도 5에 나타낸 것과 같이, 본 발명의 화합물 1은 HSP27에 특이적으로 작용하여 HSP27 변형 이량체를 농도 의존적으로 증가시켰으며, 또한 HSF1의 발현을 농도 의존적으로 감소시켰다. As a result, as shown in FIG. 5, Compound 1 of the present invention specifically acted on HSP27 to increase the HSP27 modified dimer in a concentration-dependent manner, and also reduced the expression of HSF1 in a concentration-dependent manner.
실험예 4: 항암제와의 병용 처리시의 HSP27 변형 이량체 생성능 확인Experimental Example 4: Confirmation of HSP27 modified dimer formation ability in combination treatment with anticancer agent
HSP90의 억제제로 알려진 17-AAG와 항암 치료에 이용되는 항암제인 탁솔과 시스플라틴을 이용하여, 항암제 또는 HSP90 억제제의 투여시 촉진되는 HSP27의 활성을 본 발명의 화합물이 저해할 수 있는지 확인하였다. 17-AAG, known as an inhibitor of HSP90, and Taxol and Cisplatin, anticancer agents used for anticancer treatment, were used to determine whether the compounds of the present invention can inhibit the activity of HSP27 promoted when an anticancer agent or HSP90 inhibitor is administered.
NCI-H460 세포에 10 μM 농도로 본 발명의 화합물 1을 처리하고 0.01 μM 탁솔, 2 μM 시스플라틴과 3 μM 17-AAG를 처리한 후 12시간 동안 배양하고, 상기 실시예 2-1과 동일한 방법으로 웨스턴 블랏을 수행하였다.NCI-H460 cells were treated with Compound 1 of the present invention at a concentration of 10 μM, treated with 0.01 μM Taxol, 2 μM Cisplatin, and 3 μM 17-AAG, and then incubated for 12 hours, in the same manner as in Example 2-1. Western blot was performed.
그 결과, 도 6에 나타낸 바와 같이, 탁솔, 시스플라틴 또는 17-AAG과 본 발명의 화합물 1을 병용 처리한 경우 HSP27 이량체의 생성이 증가함을 확인할 수 있었다. 따라서, 본 발명 화합물의 투여가 항암제와 HSP90 저해제에 대한 내성 극복에 효과가 있음을 알 수 있다.As a result, as shown in Figure 6, when combined treatment with Taxol, cisplatin or 17-AAG and Compound 1 of the present invention it was confirmed that the production of HSP27 dimers increased. Therefore, it can be seen that administration of the compound of the present invention is effective in overcoming resistance to anticancer agents and HSP90 inhibitors.
실험예 5: 항암제와의 병용 처리시의 세포 사멸 유도 단백질 저해 확인Experimental Example 5: Confirmation of apoptosis-inducing protein inhibition in combination treatment with anticancer agent
세포 사멸 유도 단백질인 절단된 Parp(Cleaved Parp)와 절단된 카스파아제-3(Cleaved caspase-3)의 발현양을 측정하여 본 발명의 화합물과 항암제의 병용 투여가 세포 사멸에 미치는 영향을 확인하였다.Expression levels of cleaved Parp (Cleaved Parp) and cleaved Caspase-3 (Cleaved Caspase-3), apoptosis induction protein, were measured to determine the effects of the combination of the compound of the present invention and anticancer agents on cell death.
60mm 세포 배양 디쉬에 3 x 105개의 세포를 분주 후 배양하고, 실험예 4와 같은 각 화합물들을 24시간 병용 처리하여 Cleaved Parp와 Cleaved caspase-3의 발현을 웨스턴 블랏으로 확인하였다. After dispensing 3 x 10 5 cells in a 60 mm cell culture dish, and incubating each compound as in Experiment 4 for 24 hours to confirm the expression of Cleaved Parp and Cleaved caspase-3 by Western blot.
그 결과, 도 6에 나타낸 바와 같이, 탁솔, 시스플라틴 또는 17-AAG를 단독으로 처리했을 때보다 본 발명의 화합물 1과 병용으로 처리했을 때 세포 사멸 유도 단백질의 발현이 증가하는 것을 확인하였다. As a result, as shown in FIG. 6, it was confirmed that expression of apoptosis inducing protein was increased when treated in combination with Compound 1 of the present invention than when Taxol, Cisplatin or 17-AAG was treated alone.
실험예 6: 항암제와 병용 처리시 세포사멸 효과 확인Experimental Example 6: Confirmation of apoptosis effect when combined with anticancer agent
본 발명의 화합물의 세포사멸 효과 및 본 발명의 화합물과 다른 항암제와의 병용 투여시의 세포사멸 효과를 확인하기 위하여, MTT 검정을 수행하였다.In order to confirm the apoptosis effect of the compound of the present invention and the apoptosis effect when co-administration of the compound of the present invention with another anticancer agent, an MTT assay was performed.
NCI-H460 세포에 10 μM 농도로 본 발명의 화합물 1을 처리하고 0.01 μM 탁솔, 2 μM 시스플라틴과 3 μM 17-AAG를 병용처리한 후 24시간 배양하였다. NCI-H460 cells were treated with Compound 1 of the present invention at a concentration of 10 μM and co-treated with 0.01 μM Taxol, 2 μM Cisplatin, and 3 μM 17-AAG and incubated for 24 hours.
96 웰 플레이트에 NCI-H460 세포를 같은 양으로 분주하고 플레이트에 잘 붙을 때까지 배양한 후 제시한 농도의 화합물을 처리한 이후 24시간 동안 더 배양한 후 세포의 배지를 제거하고 1X PBS로 세척하고 3-(4,5-디메틸티아졸-2-일)2,5-디페닐테트라졸리움 브로마이드(MTT; Amersham Pharmacia Biotech, Little Chalfont, UK) 시약을 1X PBS에 5 mg/ml 농도로 희석하여 100 μL씩 칸마다 분주한 후 37 ℃, 5% CO2 배양기에서 4시간 정도 배양한 후 세포가 보라색으로 변한 것이 확인되면 상층액을 제거하고 디메틸설폭사이드(Dimethtl sulfoxide, DMSO)를 100 μL씩 넣어 생성된 포르마잔(Formazan) 결정을 잘 용해시켜주었다. 용해가 끝나나 후 ELISA 리더기로 540nm에서 흡광도를 측정하였다.Dispense the same amount of NCI-H460 cells in a 96 well plate, incubate until adhered well to the plate, and then incubate for 24 hours after treatment with the compound of the indicated concentration, remove the medium of the cell, wash with 1X PBS, 3- (4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT; Amersham Pharmacia Biotech, Little Chalfont, UK) reagent was diluted to 5 mg / ml in 1X PBS at 100 mg. After dispensing each μL each cell incubated for 4 hours at 37 ℃, 5% CO 2 incubator, if the cells turn purple, remove the supernatant and add 100 μL of dimethyl sulfoxide (DMSO) each Formazan crystals were dissolved well. After dissolution, the absorbance was measured at 540 nm with an ELISA reader.
그 결과, 도 7에 나타낸 바와 같이, 화합물 1을 단독 처리했을 때보다 다른 항암제와 병용처리 했을 때 세포사멸이 더 높게 나타남을 확인하였다. 상기 결과를 통해서, 본 발명의 화합물이 HSP27을 변형 이량체로 만들어 다량체 생성을 저해함으로써 샤페론 기능을 수행하지 못하게 하여, 항암제에 대한 민감성을 증가시킬 수 있음을 알 수 있다.As a result, as shown in Figure 7, it was confirmed that apoptosis was higher when compound 1 was used in combination with other anticancer agents than when treated alone. Through the above results, it can be seen that the compound of the present invention can prevent Happ27 from forming a dimer and inhibiting multimer production, thereby preventing chaperone function, thereby increasing sensitivity to anticancer drugs.
실험예 7: 항암제와 병용 처리시 이종이식 동물모델에서의 항암 효과 확인Experimental Example 7: Confirmation of anticancer effect in xenograft animal model when combined with anticancer agent
7-1. 종양 크기 확인7-1. Confirm tumor size
BALB/C 누드 마우스(BALB/C nude mouse)에 NCI-H460 세포 이종이식을 수행한 후에, in vivo에서 양상을 확인하였다. After performing NCI-H460 cell xenograft on BALB / C nude mouse, the pattern was confirmed in vivo .
BALB/C nude mouse에 NCI-H460 세포 1x106를 왼쪽 다리 피하에 주사하여 종양을 형성하였으며, 형성된 종양이 100~300 mm3에 도달했을 때 아래와 같은 7개 투여군 및 대조군으로 나누어 약물을 투여하였다.Tumors were formed by injecting NCI-H460 cells 1 × 10 6 subcutaneously into the left leg of BALB / C nude mice, and when the formed tumors reached 100 to 300 mm 3 , the drug was divided into the following seven administration groups and control groups.
[표 2]TABLE 2
Figure PCTKR2017008204-appb-I000008
Figure PCTKR2017008204-appb-I000008
17-AAG는 25 mg/kg의 농도로 100 μl 씩 일주일에 3번 복강 투여하였고, 탁솔은 2 mg/kg 농도로 100 μl씩 일주일에 1번 복강 투여하였고, 본 발명의 화합물 1은 6.8 mg/kg 농도로 일주일에 3회 복강투여 또는 종양에 직접 주사 하였다. 17-AAG was intraperitoneally administered 100 μl three times a week at a concentration of 25 mg / kg, Taxol was intraperitoneally administered once a week at 100 μl at a concentration of 2 mg / kg, and Compound 1 of the present invention was 6.8 mg / kg Intraperitoneal administration or injection directly into tumors three times a week at kg concentration.
그 결과, 도 8의 (A)에 나타낸 바와 같이, 화합물 1과 17-AAG를 병용 투여한 경우, 각각을 단독으로 투여한 경우보다 종양 크기가 더 많이 감소함을 확인할 수 있었다. 따라서 본 발명의 화합물이 효과적으로 HSP90 억제제의 민감성을 증가시키고, 내성을 감소시킴을 알 수 있다. As a result, as shown in (A) of FIG. 8, when compound 1 and 17-AAG were administered in combination, it was confirmed that tumor size was reduced more than when administered alone. Thus, it can be seen that the compounds of the present invention effectively increase the sensitivity of HSP90 inhibitors and reduce their resistance.
또한, 도 9 및 도 10의 (A)에 나타낸 바와 같이, 화합물 1과 탁솔을 병용 투여한 투여군의 경우, 화합물 1과 탁솔을 각각 단독으로 투여한 투여군보다 종양 크기가 크게 감소함을 확인할 수 있었다. 따라서 본 발명의 화합물이 탁솔과 같은 항암제의 민감성 증가 및 내성 감소에 뛰어난 효과가 있음을 알 수 있다. In addition, as shown in Figs. 9 and 10 (A), in the case of the administration of the compound 1 and Taxol in combination, it was confirmed that the tumor size is significantly reduced than the administration of the compound 1 and Taxol alone administration. . Therefore, it can be seen that the compound of the present invention has an excellent effect on increasing sensitivity and decreasing resistance of an anticancer agent such as Taxol.
7-2. 세포 사멸이 유도된 암 세포수 확인7-2. Identify the number of cancer cells that induced cell death
상기 실험예 6-1의 이종 이식 마우스의 종양을 적출하여 면역조직화학 염색법으로 세포 사멸이 유도된 세포수를 측정하였다.Tumors of the xenograft mice of Experimental Example 6-1 were extracted, and cell death induced cell death was measured by immunohistochemical staining.
그 결과, 도 8의 (B)에 나타난 것과 같이, 본 발명의 화합물은 17-AAG에 의해 유도되는 세포 사멸을 증가시키는 경향을 뚜렷하게 확인 할 수 있었다. 또한, 도 9 및 도 10의 (B)에 나타난 것과 같이, 본 발명의 화합물 1은 탁솔에 의해 유도되는 세포사멸을 증가시키는 경향을 확인할 수 있었다. 따라서 본 발명의 화합물이 HSP90 억제제, 탁솔을 비롯한 항암제의 민감성 증가 및 내성 감소에 효과가 있음을 알 수 있다.As a result, as shown in Figure 8 (B), the compound of the present invention was able to clearly confirm the tendency to increase the cell death induced by 17-AAG. In addition, as shown in Figure 9 and Figure 10 (B), Compound 1 of the present invention was able to confirm the tendency to increase apoptosis induced by Taxol. Therefore, it can be seen that the compound of the present invention is effective in increasing sensitivity and decreasing resistance of anticancer drugs including HSP90 inhibitor and taxol.
실험예 8: HER 수용체 신호전달 체계의 저해 효과 확인Experimental Example 8: Confirmation of Inhibitory Effect of HER Receptor Signaling System
본 발명의 화합물이 종양의 성장에 관여하는 HER(Human Epithelial Growth Factor Receptor, EGFR, 인간 표피성장인자 수용체) 및 그 하위 단계의 신호전달 단백질에 미치는 영향을 확인하기 위하여, 다음과 같은 실험을 수행하였다.In order to determine the effect of the compound of the present invention on HER (Human Epithelial Growth Factor Receptor, EGFR, human epidermal growth factor receptor) and its lower level signaling proteins involved in tumor growth, the following experiment was performed. .
6-웰 플레이트의 한 개 웰 당 3 x 105 개의 BT474 세포를 분주한 후, 10 % FBS와 0.1 % 페니실린-스트렙토마신(penicillin-streptomycin, Hyclone, USA)이 포함된 RPMI 배지 (hyclone, USA)를 사용하여 37 ℃, 5 % CO2 배양기에서 24시간 배양하고, 본 발명의 화합물 1을 각각 0, 5, 10, 20 μM로 16시간 처리하였다. 상기 실험예 2-1과 동일한 방법으로 웨스턴 블랏을 수행하여 HER, HER2, p-HER2, HER3, p-HER3, HER4, p-HER4, AKT, p-AKT, MAPK(미토겐활성화 단백질 키나아제), p-MAPK, HSP27 이량체, HSP27 단량체, HSP90 및 GADPH의 발현을 확인하였다.After dispensing 3 × 10 5 BT474 cells per well of a 6-well plate, RPMI medium containing 10% FBS and 0.1% penicillin-streptomycin (Hyclone, USA) (hyclone, USA) Was incubated for 24 hours in a 37 ℃, 5% CO 2 incubator, and compound 1 of the present invention was treated with 0, 5, 10, 20 μM for 16 hours, respectively. Western blot was performed in the same manner as in Experimental Example 2-1 to HER, HER2, p-HER2, HER3, p-HER3, HER4, p-HER4, AKT, p-AKT, MAPK (Mitogen Activation Protein Kinase), Expression of p-MAPK, HSP27 dimer, HSP27 monomer, HSP90 and GADPH was confirmed.
그 결과, 도 11에 나타낸 것과 같이, 본 발명의 화합물 1에 의해 농도 의존적으로 HSP27 이량체의 양이 증가함에 따라 HER 수용체 및 그 하위 단계의 신호 전달 단백질을 농도 의존적으로 감소시켰다.As a result, as shown in FIG. 11, as the amount of HSP27 dimer increased in a concentration-dependent manner, Compound 1 of the present invention reduced the HER receptor and signal transduction proteins at lower levels thereof.
실험예 9: 트라스투주맙과 병용 처리시 HER2 수용체 신호전달 체계 저해 효과 확인Experimental Example 9: Confirmation of Inhibitory Effect of HER2 Receptor Signaling System Upon Treatment with Trastuzumab
HER2 수용체의 세포외 도메인(extracellular domain, ECDs)을 표적으로 하는 재조합 인간화 단클론 항체인 트라스투주맙에 대한 반응성이 높은 세포와 저항성을 갖는 세포를 각각 이용할 경우, 본 발명의 화합물에 의해 트라스투주맙에 대한 내성이 극복될 수 있는지 확인하기 위하여, 다음과 같은 실험을 수행하였다.When using cells that are highly reactive and resistant to trastuzumab, a recombinant humanized monoclonal antibody that targets the extracellular domains (ECDs) of the HER2 receptor, the compounds of the present invention In order to confirm that the resistance to the test can be overcome, the following experiment was performed.
트라스투주맙에 대한 반응성이 높은 BT474 세포주, 이에 3 mg/ml의 트라스투주맙을 16주간 처리하여 구축한 내성 세포 (BT474-내성) 및 트라스투주맙에 대한 내성 환자 유래 세포주 (JIMT-1)를 각각 본 발명의 화합물 1을 10 μM로 처리하고, 10 μg/ml 트라스투주맙을 처리한 후, 12시간 동안 배양하였다. 상기 실험예 2-1과 동일한 방법으로 웨스턴 블랏을 수행하여, HER2, p-AKT, p-MAPK, HSP27 이량체, HSP27 딘량체, HSP90 및 GADPH의 발현을 확인하였다.BT474 cell line that is highly reactive to trastuzumab, thus resistant cells (BT474-resistant) constructed by treatment with 3 mg / ml of trastuzumab for 16 weeks and patient-derived cell line resistant to trastuzumab (JIMT-1) Each compound 1 of the present invention was treated with 10 μM, treated with 10 μg / ml trastuzumab, and then incubated for 12 hours. Western blot was performed in the same manner as in Experimental Example 2-1 to confirm the expression of HER2, p-AKT, p-MAPK, HSP27 dimer, HSP27 dimer, HSP90 and GADPH.
그 결과, 도 12에 나타낸 바와 같이, 트라스투주맙과 본 발명의 화합물 1을 병용 처리한 경우, 모든 세포주에서 트라스투주맙 및 본 발명의 화합물 1을 단독으로 처리했을 때 보다, HER2 수용체 및 그 하위단계 신호 전달 체계가 효과적으로 저해됨을 확인하였다. As a result, as shown in FIG. 12, when trastuzumab and Compound 1 of the present invention were treated in combination, HER2 receptor and its subunits were treated more than when trastuzumab and Compound 1 of the present invention were treated alone in all cell lines. It was confirmed that the step signaling system was effectively inhibited.
따라서, 본 발명의 화합물이 트라스투주맙에 대한 내성 극복에 효과가 있음을 알 수 있다.Thus, it can be seen that the compounds of the present invention are effective in overcoming resistance to trastuzumab.
실험예 10: 트라스투주맙과 병용 처리시 세포 사멸 유도 단백질 증진 효과 확인Experimental Example 10: Confirmation of apoptosis-inducing protein enhancement effect when combined with trastuzumab
세포 사멸 유도 단백질인 Cleaved Parp 및 Cleaved caspase-7과 세포 사멸 억제 단백질인 bcl-2의 발현양을 측정하여 본 발명의 화합물과 트라스투주맙의 병용 투여가 세포 사멸에 미치는 영향을 확인하였다.Expression of Cleaved Parp and Cleaved caspase-7, which are apoptosis inducing proteins, and bcl-2, which is apoptosis inhibitory protein, were measured to determine the effects of the combination of the present invention and trastuzumab on cell death.
BT474, BT474-내성 세포주 및 JIMT-1 세포주 각각에 본 발명의 화합물 1을 10 μM로 처리하고, 10 μg/ml 트라스투주맙을 처리한 후, 12시간 동안 배양하였다. 상기 실험예 2-1과 동일한 방법으로 웨스턴 블랏을 수행하여, c-PARP, 프로-카스파아제 7(pro-caspase 7), c-카스파아제 7(c-caspase 7), blc-2 및 α-튜블린의 발현을 확인하였다.BT474, BT474-resistant cell line and JIMT-1 cell line, respectively, were treated with 10 μM of Compound 1 of the present invention and treated with 10 μg / ml trastuzumab, followed by incubation for 12 hours. Western blot was carried out in the same manner as in Experimental Example 2-1, and c-PARP, pro-caspase 7, c-caspase 7, blc-2 and α- The expression of tubulin was confirmed.
그 결과, 도 13에 나타낸 바와 같이, 모든 세포주에서 트라스투주맙 및 본 발명의 화합물 1을 단독으로 처리한 군 보다 병용 처리한 군에서 세포 사멸 유도 단백질의 발현이 증가하였고, 세포 사멸 억제 단백질의 발현은 감소하는 것을 확인하였다.As a result, as shown in FIG. 13, the expression of apoptosis-inducing protein was increased in the group treated with the combination of trastuzumab and the compound 1 of the present invention alone in all cell lines, and the expression of the cell death suppressing protein. Was found to decrease.
실험예 11: 트라스투주맙과 병용 처리시 세포 성장 저해 효과 확인Experimental Example 11: Confirmation of cell growth inhibitory effect when combined with trastuzumab
본 발명의 화합물과 트라스투주맙의 병용 투여가 세포 성장에 미치는 영향을 확인하기 위하여, 다음과 같은 WST(Water Soluble Tetrazolium salt) 검정을 수행하였다.In order to confirm the effect of the combination of the present invention and trastuzumab on cell growth, the following Water Soluble Tetrazolium salt (WST) assay was performed.
BT474, BT474-내성 세포주, JIMT-1 세포주를 각각 96-웰 플레이트의 한 개 웰 당 1 x 104 개로 분주하여 24시간 배양하였다. 이에 10 μM 농도로 본 발명의 화합물 1을 처리하고 10 μg/ml 트라스투주맙을 병용 처리한 후, 24시간 더 배양하였다. WST 시약을 한 개 웰 당 10 μL처리하여 37 ℃, 5 % CO2 조건에서 1시간 정도 배양한 후 ELISA 리더기로 450 nm에서 흡광도를 측정하였다.BT474, BT474-resistant cell line, and JIMT-1 cell line were each cultured at 1 × 10 4 per well of a 96-well plate for 24 hours. This was treated with Compound 1 of the present invention at a concentration of 10 μM and co-treated with 10 μg / ml trastuzumab, followed by further incubation for 24 hours. 10 μL of the WST reagent was treated per well and incubated for 1 hour at 37 ° C. and 5% CO 2. The absorbance was measured at 450 nm with an ELISA reader.
그 결과, 도 14에 나타낸 바와 같이, 모든 세포주에서 트라스투주맙 및 본 발명의 화합물 1을 단독으로 처리한 군 보다 병용 처리한 군에서 세포 성장 저해율이 더욱 높게 나타나는 것을 확인하였다.As a result, as shown in FIG. 14, it was confirmed that the cell growth inhibition rate was higher in the group treated with the combination of trastuzumab and the compound 1 of the present invention alone in all the cell lines.
실험예 12: 트라스투주맙과 병용 처리시 이종이식 동물모델에서의 항암 효과 확인Experimental Example 12: Confirmation of anticancer effect in xenograft animal model when combined with trastuzumab
12-1. 종양 크기 확인12-1. Confirm tumor size
BALB/C 누드 마우스(BALB/C nude mouse)에 BT474 세포 이종이식을 수행한 후에, in vivo에서 양상을 확인하였다.After performing BT474 cell xenograft on BALB / C nude mouse, the pattern was confirmed in vivo .
BALB/C nude mouse에 BT474 세포 1x107을 오른쪽 다리 피하에 주사하여 종양을 형성하였으며, 형성된 종양이 ~100 mm3에 도달했을 때 아래와 같은 3개 투여군 및 대조군으로 나누어 약물을 투여하였다.Tumors were formed by subcutaneous injection of BT474 cells 1 × 10 7 into the BALB / C nude mice under the right leg. When the formed tumors reached ˜100 mm 3 , the drug was divided into three dose groups and a control group as follows.
[표 3]TABLE 3
Figure PCTKR2017008204-appb-I000009
Figure PCTKR2017008204-appb-I000009
트라스투주맙은 1 mg/kg의 농도로 100 μl 씩 일주일에 1번 복강 투여하였고, 본 발명의 화합물 1은 20 mg/kg 농도로 100 μl씩 이틀에 1번 복강 투여하였다. Trastuzumab was intraperitoneally administered once a week at 100 μl at a concentration of 1 mg / kg, and Compound 1 of the present invention was intraperitoneally administered once every two days at a concentration of 20 mg / kg.
그 결과, 도 15의 (A)에 나타낸 바와 같이, 화합물 1과 트라스투주맙을 병용 투여한 경우 트라스투주맙 및 본 발명의 화합물 1 각각을 단독으로 투여한 경우보다 종양 크기가 더 많이 감소함을 확인할 수 있었다. 따라서, 상기 결과를 통해 본 발명의 화합물이 트라스투주맙에 대한 반응성을 효과적으로 증진시킬 수 있음을 알 수 있다.As a result, as shown in (A) of FIG. 15, when the compound 1 and trastuzumab were administered in combination, the tumor size decreased more than when the trastuzumab and the compound 1 of the present invention were administered alone. I could confirm it. Thus, the results show that the compounds of the present invention can effectively enhance the responsiveness to trastuzumab.
* p < 0.05 vs 대조군, * p <0.05 vs control,
# p < 0.05 vs 화합물 1, # p <0.05 vs compound 1,
## p < 0.05 vs 트라스투주맙## p <0.05 vs trastuzumab
12-2. 종양 부피 변화 확인12-2. Identify tumor volume changes
도 15의 (B)에 나타낸 바와 같이, 화합물 1과 트라스투주맙을 병용 투여한 투여군의 경우, 화합물 1과 트라스투주맙을 각각 단독으로 투여한 투여군과 비교하여 종양 부피의 변화가 가장 적게 일어남을 확인할 수 있었다. 따라서, 본 발명의 화합물이 트라스투주맙에 대한 민감성을 증가시켜 약물 효능을 증진시키는데 효과가 있음을 알 수 있다.As shown in (B) of FIG. 15, in the case of the administration group in which Compound 1 and trastuzumab were administered in combination, the change in tumor volume occurred the least compared with the administration group in which Compound 1 and trastuzumab were administered alone. I could confirm it. Thus, it can be seen that the compounds of the present invention are effective in enhancing drug efficacy by increasing sensitivity to trastuzumab.
12-3. 생존율 확인12-3. Check survival rate
도 15의 (C)에 나타낸 바와 같이, 화합물 1과 트라스투주맙을 각각 단독으로 투여한 투여군이 대조군과 비교하여 생존률이 증가하는 것을 확인할 수 있었다. 또한, 화합물 1과 트라스투주맙을 병용 투여한 경우, 화합물 1과 트라스투주맙을 각각 단독으로 투여한 투여군보다 더 높은 생존률을 나타냄을 확인할 수 있었다. 따라서, 본 발명의 화합물이 트라스투주맙에 대한 치료 효능을 증진시켜 생존률의 개선을 유도하는데 탁월한 효과를 가짐을 알 수 있다.As shown in (C) of FIG. 15, it was confirmed that the administration group administered with Compound 1 and trastuzumab alone increased survival as compared to the control group. In addition, when Compound 1 and trastuzumab were administered in combination, it was confirmed that the survival rate was higher than that of Compound 1 and trastuzumab administered alone. Thus, it can be seen that the compounds of the present invention have an excellent effect in enhancing the therapeutic efficacy against trastuzumab and inducing an improvement in survival rate.
실험예 13: 제피티닙과 병용 처리시 세포 사멸 유도 단백질 증가 효과 확인Experimental Example 13: Confirmation of apoptosis-inducing protein increase effect when combined with zefitinib
세포 사멸 유도 단백질인 Cleaved Parp와 Cleaved caspase-3의 발현양을 측정하여 본 발명의 화합물과 EGFR(또는 HER1)의 타이로신 인산화 효소 도메인을 표적으로 하는 저해제인 제피티닙의 병용 투여가 세포 사멸에 미치는 영향을 확인하였다.Expression of Cleaved Parp and Cleaved caspase-3, the Apoptosis Inducing Proteins, and the Effect of Combination of Zephytinib, an Inhibitor Targeting the Tyrosine Phosphorylase Domain of EGFR (or HER1) The effect was confirmed.
제피티닙에 민감하게 반응하는 세포주인 HCC827과 PC9 그리고 제피티닙에 내성을 갖는 세포주인 NHCI-H1650 각각에 본 발명의 화학식 1을 10 μM로 처리하였다. 또한, 제피티닙을 HCC827 세포주에는 각각 0.05 μM 및 0.01 μM을 처리하고, PC9 세포주에는 각각 1 μM 및 5 μM을 처리하고, NCI-H1650 세포주에는 각각 5 μM 및 10 μM을 처리한 후, 24시간 동안 배양하였다. 상기 실험예 2-1과 동일한 방법으로 웨스턴 블랏을 수행하여, EGFR, 절단된 PARP, 절단된 카스파아제 3 및 β-액틴의 발현을 확인하였다. Formula 1 of the present invention was treated with HCC827 and PC9, which are sensitive to gefitinib, and NHCI-H1650, which is resistant to gefitinib, at 10 μM. Zephytinib was treated with 0.05 μM and 0.01 μM for HCC827 cell line, 1 μM and 5 μM for PC9 cell line, and 5 μM and 10 μM for NCI-H1650 cell line, respectively, and then 24 hours. Incubated for Western blot was performed in the same manner as in Experimental Example 2-1 to confirm the expression of EGFR, cleaved PARP, cleaved caspase 3 and β-actin.
그 결과, 도 16에 나타낸 바와 같이, 제피티닙에 민감한 세포주와 내성을 갖는 세포주 모두에서 제피티닙을 단독으로 처리했을 때보다 본 발명의 화합물 1과 병용으로 처리했을 때 세포 사멸 유도 단백질의 발현이 증가하는 것을 확인하였다.As a result, as shown in Fig. 16, expression of apoptosis inducing protein when treated in combination with Compound 1 of the present invention than when treated with zephytinib alone in both cell lines resistant to zephytinib and resistant cells It was confirmed that this increased.
실험예 14: 제피티닙과 병용 처리시 세포 성장 저해 효과 확인Experimental Example 14 Confirmation of Cell Growth Inhibition Effect in Combination Treatment with Gefitinib
본 발명의 화합물이 제피티닙과 병용 투여시 세포 성장이 저해되는지 확인하기 위하여, MTT 검정을 수행하였다.In order to confirm that the compound of the present invention is inhibited cell growth when administered in combination with gefitinib, MTT assay was performed.
제피티닙에 민감하게 반응하는 세포주인 HCC827과 내성을 갖는 세포주인 NCI-H150을 각각 96-웰 플레이트 한 웰 당 1 x 104 개로 분주하여 24 시간 배양하였다. 이에 10 μM 농도로 본 발명의 화합물 1을 처리하고 2 μM 농도로 제피티닙을 병용 처리한 후 24 시간 더 배양하였다. MTT 어세이 시약을 한 웰 당 100 μL 처리하여 37 ℃, 5 % CO2 조건에서 1시간 정도 배양한 후 ELISA 리더기로 540 nm에서 흡광도를 측정하였다. HCC827, a cell line sensitive to gefitinib, and NCI-H150, a resistant cell line, were dispensed at 1 × 10 4 cells per well of a 96-well plate, respectively, and cultured for 24 hours. This was treated with Compound 1 of the present invention at a concentration of 10 μM and co-treatment with gefitinib at a concentration of 2 μM and further incubated for 24 hours. 100 μL of the MTT assay reagent was treated per well and incubated for 1 hour at 37 ° C. and 5% CO 2. The absorbance was measured at 540 nm using an ELISA reader.
그 결과, 도 17에 나타낸 바와 같이, 제피티닙에 민감한 세포주와 내성을 갖는 세포주 모두에서 제피티닙 및 본 발명의 화합물 1을 단독으로 처리했을 때보다 본 발명의 화합물 1과 병용으로 처리했을 때 세포 성장이 더욱 많이 저해되는 것을 확인하였다.As a result, as shown in FIG. 17, when treated in combination with Compound 1 of the present invention, both Zephytinib and Compound 1 of the present invention were treated with both Zephytinib-sensitive and resistant cell lines alone. It was confirmed that cell growth was further inhibited.
본 명세서는 본 발명의 기술 분야에서 통상의 지식을 가진 자이면 충분히 인식하고 유추할 수 있는 내용은 그 상세한 기재를 생략하였으며, 본 명세서에 기재된 구체적인 예시들 이외에 본 발명의 기술적 사상이나 필수적 구성을 변경하지 않는 범위 내에서 보다 다양한 변형이 가능하다. 따라서 본 발명은 본 명세서에서 구체적으로 설명하고 예시한 것과 다른 방식으로도 실시될 수 있으며, 이는 본 발명의 기술 분야에 통상의 지식을 가진 자이면 이해할 수 있는 사항이다.In the present specification, those skilled in the art of the present invention can fully recognize and infer the details that have been omitted, and the technical spirit or essential configuration of the present invention in addition to the specific examples described in this specification are changed. Many more variations are possible without departing. Therefore, the present invention may be implemented in a manner different from that specifically described and illustrated herein, which can be understood by those skilled in the art.

Claims (15)

  1. 하기 화학식 Ⅰ로 표시되는 화합물 또는 이의 약제학적으로 허용되는 염:A compound represented by formula (I) or a pharmaceutically acceptable salt thereof:
    [화학식 Ⅰ][Formula I]
    Figure PCTKR2017008204-appb-I000010
    Figure PCTKR2017008204-appb-I000010
    상기 화학식 Ⅰ식에서, In Formula I,
    R1 및 R2는 각각 독립적으로, 수소; 또는 산소, 질소 및 황으로 구성된 군에서 선택된 1 또는 2개의 헤테로원자와 1 내지 6의 탄소원자로 구성된 헤테로시클릴C1-4알킬이고, R 1 and R 2 are each independently hydrogen; Or heterocyclylC 1-4 alkyl composed of 1 or 2 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur and 1 to 6 carbon atoms,
    R3는 C1-10알킬, C3-10시클로알킬, C6-14아릴 또는 산소, 질소 및 황으로 구성된 군에서 선택된 1 내지 4개의 헤테로원자와 1 내지 10의 탄소원자로 구성된 헤테로아릴이고, 여기서 R3의 하나 이상의 수소는 각각 독립적으로 R4로 치환 또는 비치환되고,R 3 is C 1-10 alkyl, C 3-10 cycloalkyl, C 6-14 aryl or heteroaryl consisting of 1 to 4 heteroatoms and 1 to 10 carbon atoms selected from the group consisting of oxygen, nitrogen and sulfur, Wherein at least one hydrogen of R 3 is each independently substituted or unsubstituted with R 4 ,
    R4는 F, Br, Cl, I, OH, OMe, OEt, NH2, NMe2, CN, COOH, COMe, COOMe, CONH2, 또는 C1- 4알킬이다.R 4 is F, Br, Cl, I, OH, OMe, OEt, NH 2, NMe 2, CN, a COOH, COMe, COOMe, CONH 2 , or C 1- 4 alkyl.
  2. 제1항에 있어서, R1 및 R2는 각각 독립적으로, 수소,
    Figure PCTKR2017008204-appb-I000011
    또는
    Figure PCTKR2017008204-appb-I000012
    인 화학식 Ⅰ로 표시되는 화합물 또는 이의 약제학적으로 허용되는 염.    The compound of claim 1, wherein R 1 and R 2 are each independently hydrogen,
    Figure PCTKR2017008204-appb-I000011
    or
    Figure PCTKR2017008204-appb-I000012
    A compound represented by formula (I) or a pharmaceutically acceptable salt thereof.
  3. 제1항에 있어서, R3는 C1- 10알킬 또는 C3- 10시클로알킬인 화학식 Ⅰ로 표시되는 화합물 또는 이의 약제학적으로 허용되는 염.The method of claim 1, wherein, R 3 is the compound or a pharmaceutically acceptable salt thereof, pharmaceutically represented by C 1- 10 alkyl, C 3- 10 cycloalkyl formula Ⅰ.
  4. 제1항에 있어서, R3는 메틸 또는 페닐인 화학식 Ⅰ의 화합물 또는 이의 약제학적으로 허용되는 염.The compound of formula I or a pharmaceutically acceptable salt thereof, according to claim 1, wherein R 3 is methyl or phenyl.
  5. 제1항에 있어서, 다음 화합물들로 이루어진 군으로부터 선택된 것인 화학식 Ⅰ의 화합물 또는 이의 약제학적으로 허용되는 염:The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, selected from the group consisting of:
    Figure PCTKR2017008204-appb-I000013
    Figure PCTKR2017008204-appb-I000013
  6. 제1항 내지 제5항 중 어느 한 항의 화학식 Ⅰ로 표시되는 화합물 또는 이의 약제학적으로 허용되는 염을 유효성분으로 포함하는 암의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for the prevention or treatment of cancer, comprising as an active ingredient a compound represented by the formula (I) of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof.
  7. 제6항에 있어서, 항암 활성이 있는 추가적인 유효성분을 더 포함하는 약학적 조성물.The pharmaceutical composition of claim 6, further comprising an additional active ingredient having anticancer activity.
  8. 제7항에 있어서, 상기 추가적인 유효성분은 열충격 단백질 90 (HSP90) 억제제인 약학적 조성물.The pharmaceutical composition of claim 7, wherein the additional active ingredient is a heat shock protein 90 (HSP90) inhibitor.
  9. 제8항에 있어서, 상기 열충격 단백질 90 (HSP90) 억제제는 17-알릴아미노-17-디메톡시겔다나마이신(17-AAG) 또는 17-디메틸아미노에틸아미노-17-디메톡시겔다나마이신(17-DMAG) 중 어느 하나 이상인 약학적 조성물.The method of claim 8, wherein the heat shock protein 90 (HSP90) inhibitor is 17-allylamino-17-dimethoxygeldanamycin (17-AAG) or 17-dimethylaminoethylamino-17-dimethoxygeldanamycin (17- Pharmaceutical composition of any one or more of DMAG).
  10. 제7항에 있어서, 상기 추가적인 유효성분은 탁솔, 트라스투주맙, 제피티닙 및 시스플라틴로 이루어진 군에서 선택되는 어느 하나 이상인 약학적 조성물.The pharmaceutical composition of claim 7, wherein the additional active ingredient is any one or more selected from the group consisting of Taxol, Trastuzumab, Gefitinib, and Cisplatin.
  11. 제6항에 있어서, 상기 암은 방사선 요법 내성 암 또는 항암제 내성 암인 약학적 조성물.The pharmaceutical composition of claim 6, wherein the cancer is radiation therapy resistant cancer or anticancer drug resistant cancer.
  12. 제6항에 있어서, 상기 암은 폐암, 유방암, 골육종, 전립선암, 자궁경부암, 난소암, 피부암, 구강암, 식도암, 위암, 췌장암, 대장암, 방광암, 요관암, 간암, 신경교종, 뇌종양, 비호지킨성림프종, 골수이형성증후군, 다발성골수종, 또는 형질세포성종양으로부터 선택되는 어느 하나 이상인 약학적 조성물.According to claim 6, The cancer is lung cancer, breast cancer, osteosarcoma, prostate cancer, cervical cancer, ovarian cancer, skin cancer, oral cancer, esophageal cancer, gastric cancer, pancreatic cancer, colon cancer, bladder cancer, ureter cancer, liver cancer, glioma, brain tumor, asylum A pharmaceutical composition, wherein the composition is any one or more selected from Zynkin's lymphoma, myelodysplastic syndrome, multiple myeloma, or plasmacytoma.
  13. 제6항에 있어서, 상기 암은 폐암, 유방암 또는 골육종으로부터 선택되는 어느 하나인 약학적 조성물.The pharmaceutical composition of claim 6, wherein the cancer is any one selected from lung cancer, breast cancer or osteosarcoma.
  14. 암의 예방 또는 치료를 필요로 하는 대상체에게 제1항 내지 제5항 중 어느 한 항에 따르는 화학식 I로 표시되는 화합물 또는 이의 약제학적으로 허용 되는 염의 치료학적으로 유효한 양으로 투여하는 것을 포함하는, 암의 예방 또는 치료 방법.Comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I) according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, How to prevent or treat cancer.
  15. 암의 치료를 위한 약제의 제조에 있어서, 제1항 내지 제5항 중 어느 한 항에 따르는 화학식 Ⅰ로 표시되는 화합물 또는 이의 약제학적으로 허용되는 염의 용도.Use of a compound represented by formula (I) according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of cancer.
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