Anti-cancer composition that induces cell senescence and cell death

Abstract

The present invention relates to an anti-cancer composition containing a compound that induces cell senescence and cell death as an active ingredient. The compound of the present invention is characterized in that it can distinguish between cancer cells and normal cells and simultaneously induce cell senescence and cell death specifically in cancer cells, does not require the attachment of a cancer cell-specific target substance, can minimize frequent side effects caused by anti-cancer drugs attacking normal cells, and can exert a strong anti-cancer effect based on the above-mentioned dual action.

Description

[Technical field]

[0001] The present invention relates to an anti-cancer composition containing, as an active ingredient, a compound that induces cellular senescence and cell death, and more specifically, to an anti-tumor or anti-cancer composition containing, as an active ingredient, a compound represented by Chemical Formula 1 or a salt thereof, which specifically induces cellular senescence and cell death in tumor or cancer cells and exhibits an anti-tumor or anti-cancer effect. [Background technology]

[0002] A tumor is a mass that grows abnormally due to the autonomous overgrowth of bodily tissues, and can be divided into benign and malignant tumors. Benign tumors grow relatively slowly and do not metastasize (moving far away from where the tumor originally originated), whereas malignant tumors infiltrate surrounding tissues, grow rapidly, and spread or metastasize to various parts of the body, threatening life. Therefore, malignant tumors can be considered to have the same meaning as cancer.

[0003] Cells, the smallest units that make up the body, usually divide and grow through their own regulatory functions, and when they reach the end of their life or become damaged, they die (disappear) to maintain the overall balance of their numbers. However, when problems arise in the regulatory functions of these cells due to various causes, abnormal cells that would normally die grow excessively, and in some cases invade surrounding tissues and organs, forming tumors and destroying or deforming existing structures. This condition can be defined as cancer.

[0004] Small molecules that target the cell cycle have been considered to be cancer-inducing due to unfavorable medical risks such as systemic inhibition of stem cell differentiation or deterioration of homeostatic immunity. However, natural drug scaffolds such as chromone-scaffolds, which are mainly found in flavones and isoflavones, have the advantage of being developed for medical use because they have been proven to be mostly safe as natural products. In fact, one of the chromone scaffold derivatives, SB203580, was reported to have anticancer activity (Yan W et al., Toxicol Lett 2016;259:28-34. DOI:10.1016 / j.toxlet.2016.07.591).

[0005] Flavones are members of the polyphenol family, a group of over 10,000 compounds found exclusively in plant systems. Generally, these phytochemicals function to protect plants from radiation damage. Flavones have long been used to treat inflammatory diseases such as arthritis and asthma due to their antioxidant or anti-inflammatory potential, and chromone (1,4-benzopyrone-4-one) is the central chemical backbone that makes up flavones and isoflavones.

[0006] The present inventors have recently confirmed that eupatilin, a chromone scaffold-containing compound from Artemisia species, and the compound of the present invention (Korean Patent Registration No. 10-1871166) inhibit fibrosis by degrading actin polymers, thereby inhibiting epithelial-mesenchymal transition (EMT) (Kim HS et al., bioRxiv 2020;770404.DOI:10.1101 / 770404).

[0007] In this regard, the present inventors are conducting further studies and have found that the compounds of the present invention inhibit replicative senescence (RS) and oncogene-induced senescence (OAS) in various aggressive cancer cell lines. The present invention has been completed by confirming that the compound is a potent inducer of OIS, which inhibits unlimited proliferation of cancer cells through such a cellular senescence mechanism, induces escape from tumorigenesis by reversing the NAD / NADH ratio through NAD biosynthesis and upregulating ROS, and can function as a potent therapeutic agent for cancer treatment through a series of actions that induce cell death. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention aims to provide antitumor and / or anticancer applications of chromone scaffold derivatives that simultaneously induce cell senescence and cell death. [Means for solving the problem]

[0009] In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating tumors or cancer diseases, comprising a compound represented by the following chemical formula 1 or a salt thereof as an active ingredient:

[0010] [Chemical formula 1] [ka]

[0011] In the above chemical formula 1, R 1 is C 1-5 Alkyl, C 5-6 cyclic alkyl, C containing one or more heteroatoms of O or N 5-6 Cyclic alkyl, C 6-12 or C containing one or more heteroatoms of O or N 5-6 is a heteroaryl of the formula R 2 is hydrogen, ethyl, acetyl, acetoxy, carboxy, benzoyloxy or 3,4,5-trihydroxybenzoyloxy, R 3is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 4 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 5 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy.

[0012] The present invention also provides a quasi-drug composition for preventing or ameliorating tumors or cancer diseases, comprising a compound represented by the following chemical formula 1 or a salt thereof as an active ingredient:

[0013] [Chemical formula 1] [ka]

[0014] In the above chemical formula 1, R 1 is C 1-5 Alkyl, C 5-6 cyclic alkyl, C containing one or more heteroatoms of O or N 5-6 Cyclic alkyl, C 6-12 or C containing one or more heteroatoms of O or N 5-6 is a heteroaryl of the formula R 2 is hydrogen, ethyl, acetyl, acetoxy, carboxy, benzoyloxy or 3,4,5-trihydroxybenzoyloxy, R 3 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 4 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 5 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy.

[0015] The present invention also provides a food composition for preventing or ameliorating tumors or cancer diseases, comprising as an active ingredient a compound represented by the following chemical formula 1 or a salt thereof:

[0016] [Chemical formula 1] [ka]

[0017] In the above chemical formula 1, R 1 is C 1-5 Alkyl, C 5-6 cyclic alkyl, C containing one or more heteroatoms of O or N 5-6 Cyclic alkyl, C 6-12 or C containing one or more heteroatoms of O or N 5-6 is a heteroaryl of the formula R 2 is hydrogen, ethyl, acetyl, acetoxy, carboxy, benzoyloxy or 3,4,5-trihydroxybenzoyloxy, R 3 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 4 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 5 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy.

[0018] The present invention also provides a method for preventing or treating a tumor or cancer disease, comprising administering to an individual in need thereof a compound represented by the following Chemical Formula 1 or a salt thereof:

[0019] [Chemical formula 1] [ka]

[0020] In the above chemical formula 1, R 1 is C 1-5 Alkyl, C 5-6 cyclic alkyl, C containing one or more heteroatoms of O or N 5-6 Cyclic alkyl, C 6-12 or C containing one or more heteroatoms of O or N 5-6 is a heteroaryl of the formula R 2 is hydrogen, ethyl, acetyl, acetoxy, carboxy, benzoyloxy or 3,4,5-trihydroxybenzoyloxy, R 3 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 4 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 5 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy.

[0021] The present invention also provides a use of a compound represented by the following chemical formula 1 or a salt thereof for preventing or treating a tumor or a cancer disease:

[0022] [Chemical formula 1] [ka]

[0023] In the above chemical formula 1, R 1 is C 1-5 Alkyl, C 5-6 cyclic alkyl, C containing one or more heteroatoms of O or N 5-6 Cyclic alkyl, C 6-12 or C containing one or more heteroatoms of O or N 5-6 is a heteroaryl of the formula R 2is hydrogen, ethyl, acetyl, acetoxy, carboxy, benzoyloxy or 3,4,5-trihydroxybenzoyloxy, R 3 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 4 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 5 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy.

[0024] The present invention also provides a use of a compound represented by the following chemical formula 1 or a salt thereof for the manufacture of a medicament for the prevention or treatment of a tumor or cancer disease:

[0025] [Chemical formula 1] [ka]

[0026] In the above chemical formula 1, R 1 is C 1-5 Alkyl, C 5-6 cyclic alkyl, C containing one or more heteroatoms of O or N 5-6 Cyclic alkyl, C 6-12 or C containing one or more heteroatoms of O or N 5-6 is a heteroaryl of the formula R 2 is hydrogen, ethyl, acetyl, acetoxy, carboxy, benzoyloxy or 3,4,5-trihydroxybenzoyloxy, R 3 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 4 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 5is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy.

[0027] In the present invention, R 1 is methyl, ethyl, cyclopentyl, cyclohexyl or phenyl.

[0028] In the present invention, R 1 is methyl, R 2 is hydrogen, R 3 is hydrogen, hydroxy or methoxy; R 4 is hydroxy or methoxy, R 5 is hydrogen, hydroxy or methoxy.

[0029] In the present invention, the compound is characterized by being represented by any one of the following chemical formulas 2 to 5:

[0030] [Chemical formula 2] [ka]

[0031] [Chemical formula 3] [ka]

[0032] [Chemical formula 4] [ka]

[0033] [Chemical formula 5] [ka] .

[0034] In the present invention, the cancer is characterized in that it is a blood cancer or a solid cancer.

[0035] In the present invention, the blood cancer is characterized by being leukemia, malignant lymphoma, multiple myeloma, or aplastic anemia.

[0036] In the present invention, the solid cancer is characterized in that it is brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, craniopharyngioma, ependymoma, brain stem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cavity cancer, nasopharyngeal cancer, salivary gland tumor, hypopharyngeal cancer, thyroid cancer, oral cancer, breast tumor, small cell lung cancer, non-small cell lung cancer, thymic cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, gastric cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, colon cancer, anal cancer, bladder cancer, kidney cancer, male reproductive organ tumor, penile cancer, prostate cancer, female reproductive organ tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external genital organ tumor, female urethral cancer, or skin cancer.

[0037] In the present invention, The compound is (i) Suppression of tumor- or cancer-cell-specific proliferation by induction of replicative senescence (RS) and / or oncogene-induced senescence (OIS); (ii) reversal of the tumor or cancer cell NAD / NADH ratio; (iii) upregulation of tumor or cancer cell ROS; and (iv) induction of tumor or cancer cell-specific cell death; It is characterized by exhibiting antitumor or anticancer activity due to the action of Effect of the Invention

[0038] The compound of the present invention is characterized in that it can distinguish between cancer cells and normal cells, and can simultaneously induce cell senescence and cell death specifically in cancer cells, does not require the attachment of a cancer cell-specific targeting substance, can minimize frequent side effects caused by anticancer drugs attacking normal cells, and can exert a strong anticancer effect based on the above-mentioned dual action. [Brief description of the drawings]

[0039] [Figure 1] FIG. 1 shows the results of comparing the effects of inducing cellular senescence in A549 cells by treatment with the compound of Chemical Formula 2 (ONG41008) according to an embodiment of the present invention, eupatilin (ONG21002), and other chromone scaffold-containing derivatives. [Figure 2a] FIG. 2a shows the results of observing the expression level and intracellular location of p53 in A549 cells after 24 hours of treatment with the compound of Chemical Formula 2 (ONG41008) at different concentrations according to an embodiment of the present invention. [Figure 2b] FIG. 2b shows the results of observing the expression level and intracellular location of p21 in A549 cells after 24 hours of treatment with the compound of Chemical Formula 2 (ONG41008) at different concentrations according to an embodiment of the present invention. [Figure 2c] FIG. 2c shows the results of observing the expression level and intracellular location of p16 in A549 cells after 24 hours of treatment with the compound of Chemical Formula 2 (ONG41008) at different concentrations according to an embodiment of the present invention. [Diagram 3] FIG. 3 shows the results of observing the expression level and intracellular location of p16 in A549 cells after treatment with the compound of Chemical Formula 2 (ONG41008) (10 μM) according to an embodiment of the present invention for 72 hours. [Figure 4a] FIG. 4a shows the results of Western blot analysis of the expression levels of p21, p16, p53, and p-p53 after 24 hours of treatment with the compound of Chemical Formula 2 (ONG41008) at different concentrations according to an embodiment of the present invention (the positions of the protein bands are indicated in yellow). [Figure 4b]FIG. 4b shows the results of observing changes in cells caused by treatment with the compound of Chemical Formula 2 (ONG41008) according to an embodiment of the present invention after inducing A549 cell proliferation with TGFβ. [Figure 5a] FIG. 5a shows the results of examining the degree of cellular senescence and multinucleation of A549 cells over time ((a) 24 hours) after treatment with the compound of Chemical Formula 2 (ONG41008) according to an embodiment of the present invention. [Figure 5b] FIG. 5b shows the results of examining the degree of cell senescence and multinucleation of A549 cells over time ((b) 48 hours) after treatment with the compound of Chemical Formula 2 (ONG41008) according to an embodiment of the present invention. [Figure 5c] FIG. 5c shows the results of examining the degree of cell senescence and multinucleation of A549 cells over time ((c) 72 hours) after treatment with the compound of Chemical Formula 2 (ONG41008) according to an embodiment of the present invention. [Figure 6] FIG. 6 shows the results of observing the mitochondrial membrane potential and confirming the cell proliferation inhibitory effect by treating the compound of Chemical Formula 2 (ONG41008) or SAHA according to an embodiment of the present invention at different concentrations. [Figure 7a] FIG. 7a shows the effect of the compound of Chemical Formula 2 (ONG41008) according to an embodiment of the present invention on the cell cycle of A549 cells as determined by Western blotting. [Figure 7b] FIG. 7b shows the results of analyzing the effect of the compound of Chemical Formula 2 (ONG41008) according to an embodiment of the present invention on the cell cycle of A549 cells treated at different concentrations using PI staining. [Figure 7c] FIG. 7c shows the effect of the compound of Chemical Formula 2 (ONG41008) or SAHA on the NAD / NADH ratio of A549 cells at different concentrations. [Figure 7d] FIG. 7d shows the effect of the compound of Chemical Formula 2 (ONG41008) according to an embodiment of the present invention on ROS production in A549 cells. [Figure 8] FIG. 8 shows the results of confirming cell death of A549 cells by treatment with the compound of Chemical Formula 2 (ONG41008) according to an embodiment of the present invention. [Figure 9] FIG. 9 shows the results of CCK-8 assay to confirm the cell proliferation inhibitory effect of H358 cells treated with the compound of Chemical Formula 2 (ONG41008) according to an embodiment of the present invention or paclitaxel. [Figure 10] FIG. 10 is an electron micrograph showing mitotic disruption of H358 cells by treatment with the compound of Chemical Formula 2 (ONG41008) according to an embodiment of the present invention. [Figure 11] FIG. 11 shows the results of confirming the multinucleation of H1299 cells by treatment with the compound of Chemical Formula 2 (ONG41008) according to one embodiment of the present invention at different concentrations. [Figure 12] FIG. 12 shows the results of CCK-8 assay to confirm the cell proliferation inhibitory effect of the compound of Chemical Formula 2 (ONG41008) according to an embodiment of the present invention or paclitaxel on PANC1 cell proliferation. [Figure 13] FIG. 13 shows the results of confirming the multinucleation of PANC1 cells by treatment with the compound of Chemical Formula 2 (ONG41008) according to an embodiment of the present invention. [Figure 14] FIG. 14 shows the results of CCK-8 assay to confirm the cell proliferation inhibitory effect of MCF7 cells treated with the compound of Chemical Formula 2 (ONG41008) or SAHA according to an embodiment of the present invention. [Figure 15] FIG. 15 shows the results of confirming the multinucleation of MCF7 cells by treatment with the compound of Chemical Formula 2 (ONG41008) according to an embodiment of the present invention. [Figure 16] FIG. 16 shows the results of CCK-8 assay to confirm the cell proliferation inhibitory effect of the compound of Chemical Formula 2 (ONG41008) or SAHA according to an embodiment of the present invention on PC3 cells. [Figure 17] FIG. 17 shows the results of Caspase 3 / 7 assay to confirm cell death of PC3 cells treated with the compound of Chemical Formula 2 (ONG41008) or SAHA according to an embodiment of the present invention. [Figure 18] FIG. 18 shows the results of confirming the multinucleation of PC3 cells by treatment with the compound of Chemical Formula 2 (ONG41008) according to an embodiment of the present invention. [Figure 19]FIG. 19 shows the results of comparing the cell proliferation inhibitory effects of treating human normal lung fibroblasts with the compound of Chemical Formula 2 according to an embodiment of the present invention (ONG41008), nintedanib, or SAHA using CCK-8 assay. [Figure 20] FIG. 20 shows the results of treating normal human lung fibroblasts with the compound of Chemical Formula 2 according to an embodiment of the present invention (ONG41008), nintedanib, or SAHA to compare whether cell death is induced. [Figure 21] FIG. 21 is a diagram summarizing the mechanism of action of the compound of Chemical Formula 2 (ONG41008) and eupatilin (ONG21002) according to an embodiment of the present invention for inducing an anti-cancer (or anti-tumor) effect in comparison with other chromone scaffold derivatives. [Figure 22] FIG. 22 is a diagram showing that the effect induced by the combined administration of a conventional anticancer drug and a chromone scaffold derivative can be exerted by the single administration of the compound of the present invention. BEST MODE FOR CARRYING OUT THEINVENTION

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein is one that is well known and commonly used in the art.

[0041] In vivo, the cell cycle is exquisitely regulated to coordinate proliferation, differentiation, and apoptosis. However, if such a fine regulatory function is impaired, it can cause serious illnesses, and in particular, unlimited cell proliferation can cause extremely fatal and intractable diseases such as cancer. Therefore, in order to treat cancer, it is necessary to inhibit cell proliferation, and anticancer drugs that exhibit cell proliferation inhibition function also cause many side effects.

[0042] In the present invention, it was confirmed that the compound of the present invention, which is one of the chromone scaffold derivatives, can eliminate the unlimited proliferation state of various cancer cell lines by cellular senescence (i.e., replicative senescence and senescence due to oncogene induction). Interestingly, it was confirmed that the compound of the present invention does not induce such cellular senescence in normal cells and does not inhibit normal cell proliferation, and has the advantage that it does not require the attachment of a substance that targets cancer cells, as in the case of general anticancer drugs. In addition, it was further verified that no pathological abnormalities or toxicity were observed when the compound of the present invention was orally administered to mice or rats (preclinical study number: U-18156, dosage 1g), and it was found that the compound of the present invention can safely exert an anticancer effect without side effects.

[0043] Therefore, in a first aspect, the present invention relates to a pharmaceutical composition for preventing or treating a tumor or a cancer disease, comprising, as an active ingredient, a compound represented by the following chemical formula 1 or a salt thereof:

[0044] [Chemical formula 1] [ka]

[0045] In the above chemical formula 1, R 1 is C 1-5 Alkyl, C 5-6 cyclic alkyl, C containing one or more heteroatoms of O or N 5-6 Cyclic alkyl, C 6-12 or C containing one or more heteroatoms of O or N 5-6 is a heteroaryl of the formula R 2 is hydrogen, ethyl, acetyl, acetoxy, carboxy, benzoyloxy or 3,4,5-trihydroxybenzoyloxy, R 3 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 4is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 5 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy.

[0046] In a second aspect, the present invention relates to a quasi-drug composition for preventing or ameliorating a tumor or cancer disease, comprising a compound represented by the following chemical formula 1 or a salt thereof as an active ingredient:

[0047] [Chemical formula 1] [ka]

[0048] In the above chemical formula 1, R 1 is C 1-5 Alkyl, C 5-6 cyclic alkyl, C containing one or more heteroatoms of O or N 5-6 Cyclic alkyl, C 6-12 or C containing one or more heteroatoms of O or N 5-6 is a heteroaryl of the formula R 2 is hydrogen, ethyl, acetyl, acetoxy, carboxy, benzoyloxy or 3,4,5-trihydroxybenzoyloxy, R 3 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 4 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 5 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy.

[0049] In addition, in a third aspect, the present invention provides a food composition for preventing or ameliorating tumors or cancer diseases, comprising a compound represented by the following chemical formula 1 or a salt thereof as an active ingredient:

[0050] [Chemical formula 1] [ka]

[0051] In the above chemical formula 1, R 1 is C 1-5 Alkyl, C 5-6 cyclic alkyl, C containing one or more heteroatoms of O or N 5-6 Cyclic alkyl, C 6-12 or C containing one or more heteroatoms of O or N 5-6 is a heteroaryl of the formula R 2 is hydrogen, ethyl, acetyl, acetoxy, carboxy, benzoyloxy or 3,4,5-trihydroxybenzoyloxy, R 3 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 4 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 5 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy.

[0052] In addition, in a fourth aspect, the present invention relates to a method for preventing or treating a tumor or a cancer disease, comprising administering a compound represented by the following chemical formula 1 or a salt thereof to an individual in need thereof:

[0053] [Chemical formula 1] [ka]

[0054] In the above chemical formula 1, R 1 is C 1-5 Alkyl, C 5-6 cyclic alkyl, C containing one or more heteroatoms of O or N 5-6 Cyclic alkyl, C 6-12 or C containing one or more heteroatoms of O or N 5-6 is a heteroaryl of the formula R 2 is hydrogen, ethyl, acetyl, acetoxy, carboxy, benzoyloxy or 3,4,5-trihydroxybenzoyloxy, R 3 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 4 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 5 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy.

[0055] In a fifth aspect, the present invention relates to use of a compound represented by the following chemical formula 1 or a salt thereof for preventing or treating a tumor or a cancer disease:

[0056] [Chemical formula 1] [ka]

[0057] In the above chemical formula 1, R 1 is C 1-5 Alkyl, C 5-6 cyclic alkyl, C containing one or more heteroatoms of O or N 5-6 Cyclic alkyl, C 6-12 or C containing one or more heteroatoms of O or N 5-6 is a heteroaryl of the formula R 2is hydrogen, ethyl, acetyl, acetoxy, carboxy, benzoyloxy or 3,4,5-trihydroxybenzoyloxy, R 3 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 4 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzo yloxy, R 5 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy.

[0058] In a sixth aspect, the present invention relates to a use of a compound represented by the following chemical formula 1 or a salt thereof for the manufacture of a medicament for preventing or treating a tumor or a cancer disease:

[0059] [Chemical formula 1] [ka]

[0060] In the above chemical formula 1, R 1 is C 1-5 Alkyl, C 5-6 cyclic alkyl, C containing one or more heteroatoms of O or N 5-6 Cyclic alkyl, C 6-12 or C containing one or more heteroatoms of O or N 5-6 is a heteroaryl of the formula R 2 is hydrogen, ethyl, acetyl, acetoxy, carboxy, benzoyloxy or 3,4,5-trihydroxybenzoyloxy, R 3 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 4 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy, R 5 is hydrogen, hydroxy, methyl, methoxy, acetoxy, carboxy or benzoyloxy.

[0061] In any one of the above aspects of the present invention, R 1 is methyl, ethyl, cyclopentyl, cyclohexyl or phenyl.

[0062] In any one of the above embodiments of the present invention, R 1 is methyl, R 2 is hydrogen, R 3 is hydrogen, hydroxy or methoxy; R 4 is hydroxy or methoxy, R 5 is hydrogen, hydroxy or methoxy.

[0063] In any one of the above-mentioned aspects of the present invention, the compound is characterized by being represented by any one of the following chemical formulas 2 to 5:

[0064] [Chemical formula 2] [ka]

[0065] [Chemical formula 3] [ka]

[0066] [Chemical formula 4] [ka]

[0067] [Chemical formula 5] [ka] .

[0068] In any one of the above aspects of the present invention, the cancer is characterized in that it is a blood cancer or a solid cancer.

[0069] In any one of the above aspects of the present invention, the blood cancer is characterized by being, but not limited to, leukemia, malignant lymphoma, multiple myeloma, or aplastic anemia.

[0070] In any one of the above-mentioned aspects of the present invention, the solid cancer is selected from the group consisting of brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, craniopharyngioma, ependymoma, brain stem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cavity cancer, nasopharyngeal cancer, salivary gland tumor, hypopharyngeal cancer, thyroid cancer, oral cancer, breast tumor, small cell lung cancer, non-small cell lung cancer, thymic cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, colon cancer, anal cancer, bladder cancer, and kidney cancer. , male reproductive tract tumors, penile cancer, prostate cancer, female reproductive tract tumors, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external genital tract tumors, female urethral cancer, or skin cancer.

[0071] In any one of the above embodiments of the present invention, The compound is (i) Suppression of tumor- or cancer-cell-specific proliferation by induction of replicative senescence (RS) and / or oncogene-induced senescence (OIS); (ii) reversal of the tumor or cancer cell NAD / NADH ratio; (iii) upregulation of tumor or cancer cell ROS; and (iv) induction of tumor or cancer cell-specific cell death; The antitumor or anticancer activity is characterized by, but not limited to, the action of the above.

[0072] The composition of the present invention may be the compound of the present invention or a salt thereof by itself, or a composition in which the compound is mixed with a pharma- ceutically or food-acceptable carrier.

[0073] The composition of the present invention may contain the compound of the present invention or a salt thereof in an amount of 0.0001 to 100% by weight based on the weight of the total composition.

[0074] The composition of the present invention can be administered orally or parenterally in clinical administration, and when administered parenterally, it can be administered by intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, intracerebrovascular injection or intrathoracic injection, and can be used in the form of a general pharmaceutical formulation.

[0075] The compositions of the present invention may be used alone or in combination with surgery, radiation therapy, hormonal therapy, chemotherapy and other methods using biological response modifiers.

[0076] The daily dose of the composition of the present invention may be about 0.0001 to 100 mg, or 0.001 to 10 mg, per kg of body weight based on the compound of the present invention or a salt thereof contained in the composition, and may be administered once or several times a day. The range varies depending on the body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, severity of disease, etc. of the subject.

[0077] When administered clinically, the composition can be formulated into various oral or parenteral dosage forms, and in this case, the composition can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0078] The pharmaceutical composition of the present invention can contain, in addition to the compound of the present invention or a pharma- ceutically acceptable salt thereof, one or more other active ingredients exhibiting the same or similar function.

[0079] The quasi-drug composition of the present invention means an article used for the purpose of diagnosing, curing, ameliorating, mitigating, treating or preventing a disease in humans or animals, which has a milder effect than a pharmaceutical drug. For example, according to the Pharmaceutical Affairs Law, quasi-drugs exclude articles used for pharmaceutical purposes, and include products used for the treatment or prevention of diseases in humans or animals, and products that have a mild effect on the human body or that do not have a direct effect.

[0080] The quasi-drug composition of the present invention may be in the form of a transdermal administration formulation such as a lotion, ointment, gel, cream, patch or spray. In each formulation, the quasi-drug composition may contain other ingredients. The amount of the active ingredient can be selected and mixed according to the dosage form or intended use of the quasi-drug. The amount of the active ingredient mixed can be appropriately determined according to the intended use (suppression or alleviation). For example, it may contain usual auxiliary agents such as thickeners, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers. In addition, in the quasi-drug composition of each dosage form, other ingredients than the above-mentioned essential ingredients can be selected and mixed appropriately by a person skilled in the art according to the dosage form or intended use without difficulty.

[0081] The food composition of the present invention may be a composition in which the compound of the present invention or a pharma- ceutically acceptable salt thereof is mixed with a food-acceptable carrier, or the compound of the present invention or a pharma-ceutically acceptable salt thereof is mixed with the food-acceptable carrier, and the content of the compound of the present invention or a pharma-ceutically acceptable salt thereof may be appropriately adjusted by a conventional method based on the content and dosage in the pharmaceutical composition.

[0082] The most preferred embodiment of the food composition of the present invention may be a functional health food composition, but is not limited thereto, and other embodiments may include common food forms such as processed meat products, fish products, tofu, porridge, noodles such as ramen and buckwheat, seasoned foods such as soy sauce, miso, gochujang, and mixed sauces, sauces, confectionery, dairy products such as fermented milk and cheese, pickled foods such as kimchi and pickles, fruits, vegetables, soy milk, and drinking water such as fermented drinks. In another embodiment, the food composition of the present invention may be a food additive.

[0083] In addition, the above-mentioned pharma- ceutical acceptable carriers can also be used as the food-based acceptable carriers.

[0084] Working Example The present invention will be described in more detail with reference to the following examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention.

[0085] Example 1. Anticancer effect on A549 cell line 1-1. Cell culture and reagents A549 cell line was purchased from the Korea Cell Line Bank and cultured in RPMI1640 (10% FBS + 1% P / S). Chemically synthesized ONG41008 (chemical formula 2) and ONG21001 (eupatilin) ​​were obtained from Syngene International Ltd. (Bangalore, India), dissolved in DMSO at a stock concentration of 50 mM, and stored in aliquots at -20°C. DMSO at the appropriate concentration was used as a control. Hispidulin was purchased from Sigma (SML0582), Jaceosidin from Selleckchem (S0931), and Apigenin from Sigma (10798).

[0086] The ONG41008 used in one embodiment of the present invention is a compound represented by the following formula 2.

[0087] [Chemical formula 2] [ka]

[0088] 1-2. Effect of the compounds of the present invention on induction of cellular senescence in A549 cells A549 cells, a non-small cell lung cancer cell line, were inoculated into the medium and cultured for 24 hours. The changes in gene expression levels due to the presence or absence of treatment with the compound of the present invention (ONG41008) (20 μM) were confirmed, and up- or down-regulated genes were analyzed.

[0089] For this, the cultured cells were washed twice with cold PBS and harvested using a TaKaRa MiniBEST Universal RNA extraction kit (Takara, Japan). RNA was purified using the same kit according to the manufacturer's protocol. RNA was reverse transcribed using a cDNA synthesis kit (PCRBio Systems, London, UK). The synthesized cDNA was purified using StepOne Plus (Applied The mRNA levels were amplified using PCR with qPCRBio Probe Mix Hi-ROX (PCRBio). Comparisons between mRNA levels were performed using the ΔΔCt method, and GAPDH was used as an internal control.

[0090] Processed reads were mapped to the Homo sapiens reference genome using Tophat and Cufflink along with base parameters. Differential analysis was performed using Cuffdiff with base parameters. Cuffdiff FPKM values ​​were also normalized and quantified using RTCC (comparison of package tag counts) to determine statistical significance (e.g. P-value) and differential expression (e.g. fold change). Gene expression values ​​were plotted in Volcano plots with fold change values ​​using in-house developed R scripts.

[0091] As a result, it was confirmed that the expression patterns of genes related to cellular senescence were significantly changed depending on whether or not the compound of the present invention was treated (data not shown).

[0092] Considering that the compound according to the present invention is a chromone scaffold-containing derivative, immunocytochemistry was performed to confirm whether the effect of inducing cellular senescence is also exerted by other chromone scaffold-containing derivatives.

[0093] A549 cell line was treated with 20 μM each of the compound of formula 2 of the present invention (ONG41008), eupatilin (ONG21001), or other chromone scaffold-containing derivatives, hispidulin, jaceosidin, and apigenin, for 72 hours, after which the cells were fixed with 4% paraformaldehyde, permeabilized with 0.4% TritonX100, and blocked with 1% BSA. Rhodamine Phalloidin (Thermo After washing, cells were incubated with Alexa Fluor 488 (Abcam, Cambridge, UK)-conjugated secondary antibodies. Images were analyzed using an EVOS M7000 (Invitrogen, CA, USA). Nuclei were counterstained with DAPI.

[0094] As a result, cell flatness, which is a morphological change of cell senescence, was observed specifically in cells treated with the compound of Formula 2 of the present invention (ONG41008) and eupatiline (ONG21001), as shown in Figure 1. That is, flat cell morphology was observed in A549 cells treated with the compound of Formula 2 of the present invention (ONG41008) and eupatiline (ONG21001), but such morphological characteristics of cell senescence were not observed in experimental groups treated with other chromone scaffold-containing derivatives, indicating that cell senescence is a specific effect of the compound of the present invention among various chromone scaffold-containing derivatives.

[0095] 1-3. Replicative senescence of A549 cells induced by the compounds of the present invention It is known that various tumor suppressor proteins are involved in the interruption of cell cycle progression. TP53 inhibits cell cycle by phosphorylating and binding mainly with p21 or p16. To confirm whether the compound treatment according to the present invention induces replicative senescence centered on TP53-p21-p16 in A549 cells, immunocytochemistry was performed.

[0096] A549 cell line was treated with the compound of formula 2 (ONG41008) of the present invention at 1, 5, 10, and 20 μM for 24 or 72 hours, and the cells were fixed with 4% paraformaldehyde, permeabilized with 0.4% TritonX100, and blocked with 1% BSA. The cells were incubated with Anti-p53 (Cell signaling technology, Beverly, MA), Anti-p21 (Abcam, Cambridge, UK), and Anti-p16-INK4A (Proteintech, IL, USA) for 4 hours at room temperature. After washing, the cells were cultured with Alexa Fluor 488 (Abcam, Cambridge, UK)-conjugated secondary antibodies. Images were analyzed using EVOS M7000 (Invitrogen, CA, USA). Nuclei were counterstained with DAPI.

[0097] As a result, as shown in Figures 2a to 2c, TP53 protein expression level did not change when treated with 1 μM, but nuclear localization was observed, and p21 was upregulated and localized to the nucleus. p16 was upregulated in proportion to the concentration of the compound of Formula 2 of the present invention (ONG41008) and was simultaneously localized in the nucleus and perinuclear area.

[0098] The cell morphology was confirmed to be more uniform at 72 hours after compound treatment than at 24 hours after compound treatment, reflecting the elimination of multinucleated A549 cells. Maximum translocation of p16 to the nucleus was completed at 72 hours, and thus A549 cells saturated with p16 are predicted to undergo cell death (Figure 3).

[0099] Since phosphorylation of TP53 is known to be essential for regulating cell cycle arrest, Western blot analysis was performed using a phospho-specific TP53 antibody.

[0100] For this, A549 cells were cultured in a 100 mm cell culture dish at 1x10 6Cells / well were dispensed and incubated overnight, then treated with various concentrations of the compound of formula 2 (ONG41008). After 24 hours, the cell lysates were clarified by centrifugation at 14,000xg for 10 minutes, and the supernatants were collected. Protein concentrations were quantified by Bradford assay (Thermo Fisher, Massachusetts, USA). Then, 25 μg of cell protein was loaded onto a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. After blocking with 5% BSA, the membrane was incubated with anti-p53 (Cell signaling technology, Beverly, MA), anti-phosphor-p53 (Cell signaling technology, Beverly, MA), anti-p21 (Abcam, Cambridge, UK), anti-p16-INK4A (Proteintech, IL, USA), and anti-GAPDH (Abcam, Cambridge, UK) overnight at 4°C. After thorough washing, the membrane was incubated with HRP-conjugated secondary antibodies. Protein bands were visualized using ECL reagent (Abfrontier, Korea) and Uvitec HD9 (UVITEC, UK).

[0101] As a result, the TP53 was phosphorylated (Fig. 4a), while the total amount of TP53 remained the same. In addition, both p21 and p16 were induced in a concentration-dependent manner, consistent with the results in Fig. 2a-c.

[0102] In addition, in order to further proliferate the A549 cells, the A549 cells were stimulated by treatment with TGFβ, and the changes in the cells were observed with or without treatment with the compound of the present invention (ONG41008) (10 μM).

[0103] As a result, it was observed that a significant portion of A549 cells treated with the compound of formula 2 of the present invention (ONG41008) began to die from day 6 and complete cell death was induced by day 15, whereas the control A549 cells remained viable. Blue and red circles indicate the control A549 cells and the A549 cells treated with the compound of formula 2 of the present invention, respectively. All cells exposed to 10 μM of the compound of formula 2 of the present invention (ONG41008) were observed to receive cell death signals for 72 hours (FIG. 4b).

[0104] 1-4. Cell senescence-mediated cell death of A549 cells by the compounds of the present invention We predicted that the compound of the present invention would rapidly convert A549 cells into replicative senescence and induce cell death, and observed cell death upon treatment with the compound of the present invention. One of the characteristics of cell death is multinucleation (MNC), and it is known that multinucleation of cancer cells in particular is induced by oncogene-induced senescence (OIS).

[0105] A549 cells were treated with 10 μM of the compound of formula 2 (ONG41008) for 24, 48 or 72 hours to establish cellular senescence, and immunocytochemistry was performed to observe morphological changes as well as multinucleation. ZEB1, a positive regulator of epithelial-mesenchymal transition (EMT), was used as an expression control.

[0106] As a result, 24 hours after treatment with the compound of Chemical Formula 2 (ONG41008), cell flattening was clearly observed in A549 cells, and multinucleation also began to be observed. The highest number of multinucleated cells was observed 48 hours after treatment, and the number actually decreased by 72 hours after treatment, which is expected to be the result of cell death (FIGS. 5a to 5c).

[0107] 1-5. Senescence of A549 cells by induction of oncogenes by the compounds of the present invention The mechanism of action (MOA) of the compound of the present invention that removes cancer cells is expected to be senescence caused by induction of oncogenes. Generally, anticancer drugs target cell cycle regulation, DNA replication or chromatin remodeling, but in A549 cells treated with the compound of the present invention, changes in the expression level of HIST1H4K (H4 Clustered Histone 12), which is involved in DNA replication and DNA repair, have been observed (data not shown). Based on this, the effects of SAHA (suberoylanilide hydroxamic acid), a reversible pan-histone deacetylase (HDAC) inhibitor, and the compound of the present invention on cell cycle regulation were compared.

[0108] For this purpose, A549 cells were plated in 96-well plates for 24 hours and then exposed to different concentrations of the compound of formula 2 (ONG41008) or SAHA. The cells were then co-incubated with TMRE (abcam, ab113852) for 30 minutes in the dark at 37°C, and the mitochondrial membrane potential was examined according to the manufacturer's protocol. 20 μM FCCP-treated cells were used as a positive control.

[0109] As a result, cell viability was rapidly decreased 24 hours after SAHA treatment, and it was confirmed by mitochondrial membrane potential analysis that the substantial inhibition of cell viability was maximized at 72 hours. Meanwhile, the inhibitory effect of the compound of Formula 2 of the present invention (ONG41008) started at 48 hours and continued for all The increase continued up to 4 days until the cells were eliminated. This was in the same context as the immunocytochemistry data, which confirmed that oncogene-induced senescence began 48 hours after treatment with the compound of formula 2 of the present invention (ONG41008) and continued for up to 72 hours (Figures 5a to 5c). The IC of SAHA and the compound ONG41008 50 showed a difference of approximately 20-fold (Figure 6).

[0110] In summary, SAHA induces rapid cell death through its broad-spectrum HDAC inhibitory effect on the chromatin remodeling process, but also induces various undesirable effects during this process, whereas the compound of the present invention is interpreted as inducing cell death through senescence due to induction of oncogenes, and it is believed that due to these differences, the compound of the present invention has superior cell death-inducing effect and duration.

[0111] 1-6. Changes in G2 / M cell cycle arrest and NAD / NADH ratio in A549 cells by the compounds of the present invention The association of the compound of the present invention with TP53, p16, and p21 means that the compound of the present invention plays an important role in controlling the cell cycle. In order to confirm how the compound of the present invention acts on the cell cycle, A549 cells were treated with the compound of the present invention, and Western blotting was performed 24 hours after the treatment, and PI staining was performed 24 and 48 hours after the treatment to perform cell cycle analysis.

[0112] For Western blot, A549 cells were plated at 1 x 10 in a 100 mm cell culture dish. 6 After overnight incubation, the cells were treated with 50 μM of the compound of formula 2 of the present invention (ONG41008) or 50 μM of Fisetin (Glantham, GL7384-100MG). Fisetin is a cellular senescence inducer that induces arrest in the G0 / G1 phase. After 24 hours, the cell lysate was clarified by centrifugation at 14,000×g for 10 minutes, and the supernatant was collected. Protein concentration was measured using a Bradford ELISA. The protein content was quantified by a ELISA assay (Thermo Fisher, Massachusetts, USA). Then, 25 μg of cellular protein was loaded onto a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. After blocking with 5% BSA, the membrane was incubated with anti-CDK2 (Cell signaling Technology, MA), anti-CDK4 (Cell signaling Technology, MA), and anti-GAPDH (Abcam, Cambridge, UK) antibodies overnight at 4°C. After thorough washing, the membrane was incubated with HRP-conjugated secondary antibodies. Protein bands were visualized using ECL reagent (Abfrontier, Korea) and Uvitec HD9 (UVITEC, UK).

[0113] For PI staining, A549 cells were plated in a 100 mm cell culture dish at 2 × 10 5 After 24 hours, the cells were harvested, washed with PBS, and then added with 10 μM, 20 μM, or 50 μM of the compound of formula 2 of the present invention (ONG41008) or a control medium. After 24 hours, the cells were harvested, fixed in chilled 70% ethanol, and stained with propidium iodide containing 200 μg / ml RNase (Abcam, 139418) in a dark room at 37°C for 30 minutes. Each stage of the cell cycle was confirmed using Accuri C6 Plus (BD Bioscience).

[0114] As a result, it was confirmed that the compound of formula 2 of the present invention (ONG41008) inhibited CDK2 and CDK6 expression at 50 μM and induced arrest of the G1-S-G2 phase (FIG. 7a, FIG. 7b). Therefore, the compound of the present invention induces mitotic collapse through oncogene-induced senescence (OIS), resulting in cell death. I found out.

[0115] Metabolic regulation has been regarded as an important factor controlling cell cycle regulation or cell death. It is well known that the NAD / NADH ratio plays a central role in regulating energy metabolism, including glycolysis and the TCA cycle, and has a significant effect on mitochondrial function, such as disease onset or aging. Therefore, we attempted to confirm whether the compounds of the present invention affect the NAD / NADH ratio in A549 cells.

[0116] For this purpose, total NAD was extracted and quantified from A549 cell lysates using the NAD+ / NADH Colorimetric Assay Kit (Abcam, ab65348) according to the manufacturer's protocol. 6 Cells were lysed in 400 μl NAD / NADH extraction buffer, filtered through a 10 kD spin column (ab93349), and measured in pure water or diluted 1 in 5. Briefly, the amount of total NAD was calculated by dividing the standard curve (pmol) by the sample volume (μL) added to the reaction well and multiplying by the dilution factor.

[0117] As a result, it was confirmed that when A549 cells were stimulated with the compound of formula 2 of the present invention (ONG41008), NAD biosynthesis increased and the NAD / NADH ratio increased significantly (Figure 7c). On the other hand, SAHA did not induce such effects at all. The NAD / NADH ratio of the A549 cells themselves showed a negative value, indicating that A549 cells can exclusively use the aerobic glycolysis process. Since the compound of the present invention can increase the intracellular concentration of NAD, Nampt (nicotinamide adenosyltransferase), which is the rate-limiting enzyme in the above pathway, can be the main target of the compound of the present invention.

[0118] The compound of the present invention can normalize the NAD / NADH ratio in the range of 60 to 100, and it was confirmed whether such a normalized NAD / NADH ratio affects ROS generation in cancer cells treated with the compound of the present invention.

[0119] For this, 1x10 4A549 cells at 1000 cells / ml were dispensed into a 96-well plate and incubated for 24 hours in a dark room at 37°C, after which the cells were treated with 10 μM H2DCFDA (Invitrogen, CA). Then, the cells were treated with various concentrations of the compound of formula 2 of the present invention (ONG41008) or TBHP (Abcam, UK), an ROS inducer, as a positive control, for 24 hours. Intracellular ROS was examined using a fluorescence microscope (Thermo, MA) and quantified using a fluorescence spectrometer (Perkin Elmer, MA).

[0120] As a result, it was shown that treatment with the compound of formula 2 (ONG41008) of the present invention increased ROS in a concentration-dependent manner (FIG. 7d), which is interpreted as an increase in the NAD / NADH ratio affecting ROS production in cancer cells and causing senescence through the induction of oncogenes.

[0121] As can be seen from the comparative examples, the compounds of the present invention distinguish between cancer cells and normal cells and act specifically on cancer cells, because the chromone scaffold of the compounds of the present invention recognizes some components of the intracellular microenvironment, including NAD, NADH, pH, or ATP, as energy sensors.

[0122] 1-7. Kinetic analysis of cell death induced by the compound of the present invention After treatment with the compound of the present invention, TUNEL assay and 7AAD assay were carried out to observe cell death of A549 cells over time.

[0123] TUNEL analysis was performed using a TUNEL assay kit (Abca) according to the manufacturer's instructions. The A549 cells were cultured in a 100 mm cell culture dish at 2 × 10 5After 24 hours, the cells were harvested, washed with PBS, and then the compound of formula 2 of the present invention (ONG41008) was added at 10 μM, 20 μM, or 50 μM or the control medium was added. After 24 hours, the cells were harvested, fixed in chilled 70% ethanol, and added with 50 μL of DNA labeling solution (10 μL of TdT reaction buffer, 0.75 μL of TdT enzyme, 8 μL of Br-dUTP, ddH 2 After incubation at 37°C for 1 h, the cells were washed twice with 300 μL rinse buffer, and an antibody solution (5 μL Anti-BrdU-Red antibody, 95 μL rinse buffer) was prepared and added to the washed cells and incubated for 30 min in the dark at room temperature. Finally, 300 μL of 7-AAD / RNase solution was added. Each sample was analyzed using Accuri C6 Plus (BD Bioscience).

[0124] As a result, cell death began to be observed 48 hours after treatment with the compound of the present invention (ONG41008), and cell death was clearly observed in a compound concentration-dependent manner 72 hours after treatment with the compound of the present invention (ONG41008) (FIG. 8).

[0125] Example 2. Anticancer effect on H358 and H1299 cells 2-1. Cell culture and reagents Non-small cell lung cancer primary cells, H358 and H1299, were purchased from the Korean Cell Line Bank and cultured in RPMI1640 (10% FBS + 1% P / S). Chemically synthesized ONG41008 was obtained from Syngene International Ltd. (Bangalore, India), dissolved in DMSO at a stock concentration of 50 mM, and stored in aliquots at -20°C.

[0126] 2-2. Inhibition of H358 cell proliferation by the compounds of the present invention The inhibitory effect of the compounds of the present invention on the proliferation of H358 cells was confirmed by CCK-8 assay and compared with that of paclitaxel, an anti-cancer therapeutic agent approved for use in the treatment of ovarian, breast, lung or gastric cancer.

[0127] H358 cells were cultured at 3x10 for 24 h. 4 The cells were dispensed into a 96-well plate so that the cells were divided into cells / well. The cell viability after 72 hours of treatment with the compound of the present invention (ONG41008) or paclitaxel (Abcam, ab120143) at 0.1, 1, 10, 20, 30, and 50 μM was determined by cell counting according to the manufacturer's protocol. The measurement was performed using kit-8 (Dojindo, CK04). After 72 hours of treatment with the compound of the present invention (ONG41008), H358 cells were observed under an optical microscope (EVOS M7000).

[0128] The experimental results showed that both the compound of formula 2 (ONG41008) and paclitaxel inhibited the proliferation of non-small cell lung cancer cells at similar levels at treatments of 30 μM or more, but the IC 50 showed a difference of about 14-fold (FIG. 9). After 72 hours of treatment with 10 μM of the compound of the present invention (ONG41008), mitotic collapse of H358 cells was observed (FIG. 10).

[0129] 2-3. Multinucleation of H1299 cells by the compounds of the present invention The compound of Formula 2 of the present invention (ONG41008) was treated at different concentrations, and immunocytochemistry was carried out in the same manner as in Example 1 to observe the changes in cells.

[0130] As a result of the experiment, as shown in FIG. 11, it was confirmed that multinucleation, which is an indicator of aging due to induction of oncogenes, was observed by treatment with the compound of Formula 2 of the present invention (ONG41008).

[0131] Example 3. Anticancer effect on PANC1 cells 3-1. Cell culture and reagents PANC1 cells, a human pancreatic cancer cell line, were purchased from the Korea Cell Line Bank and cultured in RPMI1640 (10% FBS + 1% P / S).

[0132] Chemically synthesized ONG41008 was obtained from Syngene International Ltd. (Bangalore, India), dissolved in DMSO to a stock concentration of 50 mM, and stored in aliquots at -20°C.

[0133] 3-2. Inhibition of proliferation of PANC1 cells by the compounds of the present invention The inhibitory effect of the compound of formula 2 (ONG41008) on the proliferation of PANC1 cells was confirmed by CCK-8 assay and compared with the effect of paclitaxel.

[0134] The experimental method was the same as that described in Example 2, except for the cells used.

[0135] As a result of the experiment, the compound of formula 2 of the present invention (ONG41008) exhibited a more excellent inhibitory effect on the proliferation of pancreatic cancer cells than paclitaxel at a concentration of 10 μM or more, and the IC 50 is 11.8 μM, IC of paclitaxel against PANC1 50 was confirmed to be 84.7 μM (FIG. 12).

[0136] 3-3. Multinucleation of PANC1 cells by the compounds of the present invention The compound of Formula 2 of the present invention (ONG41008) was treated at different concentrations, and immunocytochemistry was carried out in the same manner as in Example 1 to observe the changes in cells.

[0137] As a result, as shown in FIG. 13, multinucleation, which is an indicator of aging due to induction of oncogenes, was observed by treatment with the compound of Formula 2 of the present invention (ONG41008).

[0138] Example 4. Anticancer effect on MCF7 cells 4-1. Cell culture and reagents MCF7 cells, a human breast cancer cell line, were purchased from the Korea Cell Line Bank and cultured in RPMI1640 (10% FBS + 1% P / S).

[0139] Chemically synthesized ONG41008 was obtained from Syngene International Ltd. (Bangalore, India), dissolved in DMSO to a stock concentration of 50 mM, and stored in aliquots at -20°C.

[0140] 4-2. Inhibition of MCF7 cell proliferation by the compounds of the present invention The inhibitory effect of the compound of formula 2 (ONG41008) on the proliferation of MCF7 cells was confirmed by CCK-8 assay and compared with the effect of SAHA.

[0141] The experimental method was the same as that described in Example 2, except for the cells and the positive control reagent used.

[0142] As a result of the experiment, the compound of formula 2 of the present invention (ONG41008) exhibited a cell proliferation inhibitory effect very similar to that of SAHA, and the IC 50 IC of SAHA against MCF7 was 15.38 μM. 50 was confirmed to be 11.18 μM (FIG. 14).

[0143] 4-3. Multinucleation of MCF7 cells by the compounds of the present invention The compound of Formula 2 of the present invention (ONG41008) was treated at different concentrations, and immunocytochemistry was carried out in the same manner as in Example 1 to observe the changes in cells.

[0144] As a result of the experiment, as shown in FIG. 15, multinucleation, which is an indicator of aging due to induction of oncogenes, was observed by treatment with the compound of Formula 2 of the present invention (ONG41008).

[0145] Example 5. Anticancer effect on PC3 cells 5-1. Cell culture and reagents PC3 cells, a human prostate cancer cell line, were purchased from the Korean Cell Line Bank and cultured in RPMI1640 (10% FBS + 1% P / S).

[0146] Chemically synthesized ONG41008 was obtained from Syngene International Ltd. (Bangalore, India), dissolved in DMSO to a stock concentration of 50 mM, and stored in aliquots at -20°C.

[0147] 5-2. Inhibition of proliferation of PC3 cells by the compounds of the present invention The inhibitory effect of the compound of Formula 2 of the present invention (ONG41008) on the proliferation of PC3 cells was confirmed by CCK-8 assay and compared with the effect of SAHA.

[0148] The experimental method was the same as that described in Example 2, except for the cells and the positive control reagent used.

[0149] As a result of the experiment, the cell proliferation inhibitory effect of the compound of formula 2 of the present invention (ONG41008) was confirmed. Specifically, the IC 50 was confirmed to be 32.05 μM, and the IC 50 The IC value for PC3 was confirmed to be 3.84 μM (Figure 16). 50 It was confirmed that SAHA had an even lower level of death in PC3 cells, but over time, the compound of Formula 2 (ONG41008) of the present invention also induced PC3 cell death at a level similar to that of SAHA.

[0150] 5-3. Induction of cell death in PC3 cells by the compounds of the present invention The effect of the compound of Formula 2 (ONG41008) on inducing cell death in PC3 cells was examined by measuring caspase-3 / 7 activity, and compared with the effect of SAHA.

[0151] Caspase-3 / 7 activity was measured using a Caspase-3 / 7 assay kit (Promega, G8091). Cells treated with various concentrations of the compound of formula 2 (ONG41008) or SAHA were harvested and lysed on ice. Protein was then measured with BCA (Thermo, 23227) and adjusted to 50 μg protein per 50 μL of cell lysis buffer. Caspase-3 / 7 activity was then analyzed according to the manufacturer's protocol.

[0152] As a result, it was confirmed that both the compound of formula 2 of the present invention (ONG41008) and SAHA induced PC3 cell death in a concentration-dependent manner, as shown in Figure 17. In this case, the EC 50 EC of SAHA is 20.37 μM. 50 was confirmed to be 13.51 μM.

[0153] 5-4. Multinucleation of PC3 cells by the compounds of the present invention The compound of Formula 2 of the present invention (ONG41008) was treated at different concentrations, and immunocytochemistry was carried out in the same manner as in Example 1 to observe the changes in cells.

[0154] As a result, as shown in FIG. 18, the compound of the present invention (ONG41008) was treated. Multinucleation, an indicator of senescence due to oncogene induction, was observed.

[0155] Comparative Example: Effects of the Compound of the Present Invention on Normal Cells 1-1. Cell culture and reagents Normal human lung fibroblasts (NHLF) were purchased from Lonza (CC-2512) and cultured in Fibroblast Growth medium (0.1% insulin, 0.1% hFGF-B, 0.1% GA-1000, 2% FBS).

[0156] Chemically synthesized ONG41008 was obtained from Syngene International Ltd. (Bangalore, India), dissolved in DMSO to a stock concentration of 50 mM, and stored in aliquots at -20°C.

[0157] 1-2. Confirmation of cell proliferation inhibition by the compound of the present invention The compound of formula 2 of the present invention (ONG41008) was used to confirm whether it inhibits cell proliferation of normal NHLF cells by CCK-8 analysis. In this case, the effects of SAHA and nintedanib, an anticancer drug, on normal cells were compared.

[0158] The experimental method was the same as that described in Example 2, except for the cells and the positive control reagent used.

[0159] As a result of the experiment, as shown in FIG. 19, both SAHA and nintedanib inhibited cell proliferation in normal human lung fibroblastoma (NHLF) and showed very strong toxicity, whereas the compound of Chemical Formula 2 of the present invention (ONG41008) did not inhibit cell proliferation.

[0160] 1-3. Confirmation of cell death caused by the compound of the present invention To confirm whether the compound of Formula 2 (ONG41008) of the present invention induces cell death in NHLF cells, three assays were performed: (i) mitochondrial membrane potential assay, (ii) Caspase-3 activity assay, and (iii) LDH cytotoxicity assay.

[0161] (i) Mitochondrial membrane potential analysis was carried out in the same manner as described in Example 1.

[0162] (ii) Caspase-3 activity assay was performed using a Caspase-3 assay kit (Abcam, ab37401). Briefly, cells treated with various concentrations of the compound of formula 2 (ONG41008) or nintedanib were harvested and lysed on ice. Protein was then measured by BCA (Thermo, 23227) and adjusted to 50 μg protein per 50 μL of cell lysis buffer. Caspase-3 activity was then assayed according to the manufacturer's protocol.

[0163] (iii) LDH cytotoxicity assay was performed by detecting LDH release using an LDH assay kit (abcam, ab56393). Briefly, cell culture plates treated with various concentrations of the compound of formula 2 (ONG41008) or nintedanib or SAHA were centrifuged at 480×g for 10 minutes, and the supernatant (10 μL / well) was extracted into another 96-well plate, after which 100 μL of LDG reaction mixture was added to each well and incubated at room temperature for 30 minutes. The absorbance value was measured at 450 nm with a microplate reader.

[0164] As a result of the experiment, as shown in Figure 20, when measured by MTMP, Caspase 3 and LDH, SAHA and nintedanib induced strong cell death in normal cells ([SAHA]M TMP:IC 50 22.36μM, LDH:EC 50 115.0μM / [nintedanib]MTMP:IC 50 41.02μM, Caspase3:EC 50 128.2μM, LDH:EC 50 218.2 μM), the compound of formula 2 of the present invention (ONG41008) did not induce clear cell death in normal cells.

[0165] Further, in the caspase 3 / 7 analysis, it was confirmed again that the compound of formula 2 of the present invention (ONG41008) does not induce caspase 3 / 7 activity, unlike the cancer cell line (PC3), and does not induce cell death in normal cells (data not shown).

[0166] The compounds according to the present invention are potent inducers of tumor or cancer cell-specific cellular senescence (replicative senescence (RS) and oncogene-induced senescence (OIS)), releasing these cells from an unlimited proliferation loop in the form of cellular senescence while at the same time inducing cancer cell-specific cell death, thereby exhibiting excellent antitumor or anticancer properties.

[0167] These properties can be said to be unique properties exhibited only by the compound of the present invention among various chromone scaffold derivatives. Other chromone scaffold derivatives, such as histidulin and jaceocidin, which have oxygen at the C6 position, exhibit anti-fibrotic effects but do not induce cancer cell-specific cellular senescence or remove senescent cells, apigenin does not induce any of the effects of anti-fibrosis, cancer cell-specific cellular senescence, or removing senescent cells, and fisetin and quercetin induce cell death but do not remove senescent cells or induce anti-fibrotic effects. However, the compound of the present invention can effectively remove senescent cells (senolytic) as well as induce cancer cell-specific cellular senescence (senogenic), and is therefore interpreted as exhibiting excellent anti-tumor or anti-cancer properties as confirmed in the present invention (see FIG. 21).

[0168] Recently, there have been reports of co-administration of Dasatinib and Quercetin, or of co-administration of Doxorubicin (Adriamycin (registered trademark)) and Fisetin, in order to enhance the therapeutic effect of anti-cancer drugs. This is because the senolytic effect induced by anti-cancer drugs such as Dasatinib and Doxorubicin and the synolytic effect induced by chromone scaffold derivatives such as Quercetin and Fisetin are simultaneously exerted on cancer cells, thereby exerting a synergistic effect on anti-cancer. The compound according to the present invention exerts senolytic and senolytic effects simultaneously, and the effects of the above-mentioned combined administration system, which has been actively researched recently, can be exerted by the single use of one compound (see Figure 22).

[0169] Although the specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0170] CS: Chromone Scaffold CSD: Chromone Scaffold-containing derivatives Derivatives NHLF: Normal Human Lung Fibroblasts RS: Replicative senescence OIS: Oncogene-Induced Senescence MNC: Multinucleation MTMP: Mitochondrial Membrane Potential NAD: Nicotinamide adenine dinucleotide NAMPT: Nicotinamide phosphoribosyltransferase [Industrial Applicability]

[0171] The compound of the present invention can distinguish between cancer cells and normal cells and simultaneously induce cell senescence and cell death specifically in cancer cells, and can exert a strong anti-cancer effect based on the above-mentioned dual action, without the need to attach a cancer cell-specific target substance, and can minimize frequent side effects caused by anti-cancer drugs attacking normal cells.

Claims

1. A composition for preventing, treating or ameliorating a tumor or cancer disease, comprising as an active ingredient a compound represented by the following chemical formula 2 or a salt thereof: [Chemical formula 2] 【Chemistry 1】 。

2. The composition of claim 1, wherein the cancer is a blood cancer or a solid cancer.

3. The composition according to claim 2, wherein the blood cancer is leukemia, malignant lymphoma, multiple myeloma or aplastic anemia.

4. 3. The composition of claim 2, wherein the solid cancer is brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, craniopharyngioma, ependymoma, brain stem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cavity cancer, nasopharyngeal cancer, salivary gland tumor, hypopharyngeal cancer, thyroid cancer, oral cavity cancer, breast tumor, small cell lung cancer, non-small cell lung cancer, thymic cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, gastric cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, colon cancer, anal cancer, bladder cancer, kidney cancer, male reproductive organ tumor, penile cancer, prostate cancer, female reproductive organ tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external genital organ tumor, female urethral cancer, or skin cancer.

5. The compound is (i) replicative senescence (RS) and / or or suppression of tumor or cancer cell-specific proliferation by induction of oncogene-induced senescence (OIS); (ii) reversal of the tumor or cancer cell NAD / NADH ratio; (iii) upregulation of tumor or cancer cell ROS; and (iv) tumor or cancer cell-specific cell death induction; The composition according to claim 1, characterized in that it exhibits antitumor or anticancer activity by the action of

6. The composition according to any one of claims 1 to 5, which is a pharmaceutical agent.

7. The composition according to any one of claims 1 to 5, which is a quasi-drug.

8. The composition according to any one of claims 1 to 5, which is a food product.

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