Anticancer composition using flavone derivative

Abstract

Disclosed is an anticancer composition using a flavone derivative. In the present invention, the flavone derivative exhibits cytotoxicity against: H827, H1299, H522, and A549, which are human non-small cell lung cancer cell lines; MCF-7, which is a human breast cancer cell line; and PANC-1, which is a human pancreatic cancer cell line, in a time-dependent manner and in a treatment concentration-dependent manner, and not only exhibits a synergistic effect when used in combination with other conventional anticancer agents, such as osimertinib, which is an anticancer agent targeting for lung cancer, tamoxifen, which is a hormone agent against breast cancer, and gemcitabine, which is a cytotoxic anticancer agent against pancreatic cancer, but also exhibits the effect of synergistically inhibiting the expression of HIF-1α when used alone or in combination with an existing anticancer agent.

Description

Anticancer composition using flavone derivatives

[0001] The present invention relates to an anticancer composition using a flavone derivative.

[0002] Cancer is a disease caused by a series of mutations that lead to uncontrolled, uncontrolled cell proliferation. It is one of the greatest threats to human health. Causes of cancer include external factors such as chemicals, viruses, bacteria, and ionizing radiation, as well as internal factors such as congenital genetic mutations.

[0003] Despite remarkable progress over the past several decades in the search for new targets, including regulation of the cell cycle and apoptosis and oncogenes and tumor suppressor genes, cancer still accounts for approximately 25% of deaths.

[0004] Currently, surgery, radiation therapy, and chemotherapy are used to treat cancer. Surgery is effective in removing cancer in its early stages, but in some cases, it has side effects such as having to remove organs, and it cannot prevent metastasis. In the case of radiation therapy, while it has the advantage of being highly effective in treating cancer in specific areas, it has the problem of causing new carcinogenesis due to radiation exposure, cannot prevent metastasis, and causes pain to the patient during treatment. Chemotherapy is a method that uses cytotoxic anticancer drugs, targeted anticancer drugs, immuno-oncology drugs, and other hormonal drugs to treat malignant tumors. Cytotoxic anticancer drugs interfere with the metabolic pathways or signal transmission of cancer cells, blocking the replication, transcription, and translation of DNA, or interfering with the synthesis of nucleic acid precursors, thereby inhibiting cell division, causing toxicity in cancer cells that divide faster than normal cells. Targeted anticancer drugs inhibit the growth or proliferation of cancer cells by blocking the signal transmission necessary for cancer cell growth and proliferation. Immunotherapy drugs exert their anticancer effects by preventing cancer cells from evading the human immune system or by inducing the human immune system to attack cancer cells, while hormonal drugs suppress the production of hormones that control the growth of cancer cells or by inhibiting their action.

[0005] However, cytotoxic anticancer agents lack specific selectivity for cancer cells and can also affect rapidly dividing normal cells (such as bone marrow hematopoietic cells, gastrointestinal epithelial cells, and hair root epithelial cells), causing side effects. Furthermore, targeted anticancer agents can only be used in patients with identified targets, and long-term use can lead to resistance. Furthermore, immunotherapy agents can cause side effects such as autoimmune diseases, thyroid disease, hepatitis, and pneumonia, while hormonal agents can cause systemic fatigue and depression. These side effects can ultimately lead to the discontinuation of anticancer treatment.

[0006] Therefore, it can be said that the development of a substance that can minimize the side effects of anticancer drugs and increase the effectiveness of anticancer treatment is required.

[0007] Meanwhile, solid tumors are surrounded by a tumor microenvironment with a hypoxic environment. Cancer cells in the tumor microenvironment adapt to the hypoxic environment by overexpressing factors such as HIF-1α (Hypoxia Inducible Factor-1α). HIF-1α not only plays a crucial role in the survival, growth, and proliferation of cancer cells in this hypoxic environment, but also plays a crucial role in cancer cell resistance to cytotoxic anticancer drugs. Therefore, research targeting this factor has been actively conducted recently (Future Med Chem. 2013 Apr; 5(5); Cancers (Basel). 2022 Dec; 14(24): 6054).

[0008] The purpose of the present invention is to provide an anticancer composition using a flavone derivative that has anticancer activity on its own and can increase the therapeutic effect of another anticancer agent when used in combination with the other anticancer agent.

[0009] Other or specific purposes of the present invention will be presented below.

[0010] The present invention was completed by confirming that, as confirmed in the following examples and experimental examples, the novel flavone derivative of the following chemical formula 1 exhibits cytotoxicity in a time-dependent and treatment concentration-dependent manner against human non-small cell lung cancer cell lines H827, H1299, H522, and A549, human breast cancer cell line MCF-7, and human pancreatic cancer cell line PANC-1, and not only exhibits a synergistic effect when used in combination with other existing anticancer agents, such as osimertinib, a targeted anticancer agent for lung cancer, tamoxifen, a hormonal agent for breast cancer, and gemcitabine, a cytotoxic anticancer agent for pancreatic cancer, but also synergistically suppresses the expression of HIF-1α when used alone or in combination with existing anticancer agents.

[0011] <Chemical Formula 1>

[0012]

[0013] The present invention is provided based on these experimental results, and in one aspect, the present invention can be understood as an anticancer composition comprising a flavone derivative of <Chemical Formula 1> or a hydrate thereof or a solvate thereof as an active ingredient, and in another aspect, the present invention can be understood as a composition for enhancing the anticancer effect of an anticancer agent comprising a flavone derivative of <Chemical Formula 1> or a hydrate thereof or a solvate thereof as an active ingredient.

[0014] The anticancer composition of the present invention or the composition for enhancing the anticancer effect of an anticancer agent may be a composition for human non-small cell lung cancer, breast cancer, or pancreatic cancer.

[0015] The anticancer composition of the present invention may additionally contain a known anticancer agent in addition to the flavone derivative of <Chemical Formula 1> or a hydrate or solvate thereof as an effective ingredient.

[0016] In this case, the anticancer composition of the present invention can increase the therapeutic effect of a conventionally known anticancer agent, thereby producing an anticancer effect even with a small amount, and consequently, can minimize the side effects of the anticancer agent.

[0017] Any known anticancer agent that may be additionally included in the composition of the present invention is an anticancer agent.

[0018] These anticancer drugs include cytotoxic anticancer drugs, targeted anticancer drugs, immunotherapy drugs, and other hormonal drugs.

[0019] Cytotoxic anticancer agents can be broadly classified into antimetabolites, microtubulin inhibitors, alkylating agents, antimitotic agents, DNA cleavage agents, DNA cross-linker agents, DNA intercalator agents, and DNA topoisomerase inhibitors. As for antimetabolites, folic acid derivatives such as methotrexate, purine analogs such as cladribine, pyrimidine analogs such as gemcitabine and azacitidine, doxifluridine, fluorouracil, etc. are known in the art, and as for microtubule targeting agents, Drugs of the auristatin series, such as monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), and dolastatin, and maytansines, are known in the art. As alkylating agents, alkyl sulfonate preparations, such as busulfan and treosulfan, nitrogen mustard derivatives, such as bendamustine, and platinum preparations, such as cisplatin and heptaplatin, are known in the art.Also known in the art as antimitotic agents are taxanes such as docetaxel and paclitaxel, vinca alkalis such as vinflunine, and podophyllotoxin derivatives such as etoposide. Calicheamicins are known as DNA cleavage agents, and PBD duplexes are known as DNA cross-linker agents. Also known in the art as DNA intercalation agents are doxorubicin, and known in the art as DNA topoisomerase inhibitors are SN-28, etc.

[0020] Known targeted anticancer drugs include monoclonal antibodies such as cetuximab, trastuzumab, and bevacizumab, as well as signal transduction inhibitors such as erlotinib, gefitinib, vandetanib, afatinib, and osimertinib.

[0021] Known immunotherapy drugs include immune checkpoint inhibitors such as ipilimumab, pembrolizumab, nivolumab, and atezolizumab, and immune cell enhancers such as blinatumomab.

[0022] Hormonal agents known include androgen inhibitors such as bicalutamide and enzalutamide, and female hormone inhibitors such as tamoxifen, anastrozole, and letrozole.

[0023] In the composition for enhancing the anticancer effect of the anticancer agent of the present invention, the anticancer agent may be a cytotoxic anticancer agent, a targeted anticancer agent, an immune anticancer agent, a hormonal agent, etc. as described above.

[0024] In this specification, “hydrate” means a compound to which water is bound, and includes an inclusion compound in which there is no chemical bond between water and the compound.

[0025] Also, in this specification, “solvate” means a compound formed between molecules or ions of a solute and molecules or ions of a solvent.

[0026] Also, in this specification, “active ingredient” means an ingredient that exhibits the desired activity alone or can exhibit the activity together with a carrier that is inactive in itself.

[0027] In the composition of the present invention, the active ingredient may be included in any amount (effective amount) depending on the specific use, formulation, etc., as long as it can exhibit an anticancer effect, an anticancer effect enhancing effect of an anticancer agent, etc., and a typical effective amount will be determined within the range of 0.001 wt % to 99 wt % based on the total weight of the composition. Here, the "effective amount" refers to the amount of the active ingredient included in the composition of the present invention that can exhibit the intended medical and pharmacological effects, such as an anticancer effect, an anticancer effect enhancing effect of an anticancer agent, etc., when the composition of the present invention is administered to a mammal, preferably a human, which is the subject of application, for an administration period as recommended by a medical professional, etc. Such an effective amount can be experimentally determined within the normal ability of a person skilled in the art.

[0028] The composition of the present invention can be understood as a food composition in a specific aspect.

[0029] The food composition of the present invention can be manufactured in any form, for example, beverages such as tea, juice, carbonated beverages, and sports beverages; processed dairy products such as milk and yogurt; foods such as gums, rice cakes, Korean traditional sweets, bread, confectionery, and noodles; and health functional food preparations such as tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, and bars. In addition, the food composition of the present invention can have any product classification as long as it complies with the laws and regulations in effect at the time of manufacturing and distribution in terms of legal and functional classification. For example, it can be a health functional food according to the Korean "Health Functional Food Act", or confectionery, beans, tea, beverages, special-purpose foods, etc. according to each food type according to the Food Code of the Korean "Food Sanitation Act" (the "Standards and Specifications of Foods" announced by the Ministry of Food and Drug Safety).

[0030] The food composition of the present invention may contain food additives in addition to its effective ingredients. Food additives can generally be understood as substances added to, mixed with, or infiltrated into food during the manufacturing, processing, or preservation of food. Since they are consumed daily and over a long period of time with food, their safety must be guaranteed. The Food Additive Codex, which is based on the laws of each country that regulate the manufacturing and distribution of food (in Korea, the "Food Sanitation Act"), provides limited regulations on food additives with guaranteed safety in terms of ingredients or functions. The Korean Food Additive Codex (Ministry of Food and Drug Safety Notice "Food Additive Standards and Specifications") classifies food additives into chemically synthesized products, natural additives, and mixed preparations in terms of ingredients. These food additives are classified into sweeteners, flavoring agents, preservatives, emulsifiers, acidulants, and thickeners in terms of functions.

[0031] Sweeteners are used to impart an appropriate sweetness to foods, and both natural and synthetic sweeteners can be used in the food composition of the present invention. Preferably, a natural sweetener is used. Examples of natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

[0032] Flavoring agents are used to enhance taste or aroma, and both natural and synthetic flavors can be used. Natural flavoring agents are preferred. When using natural flavoring agents, they can also serve the purpose of enhancing nutrition in addition to flavor. Natural flavoring agents can be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or from green tea leaves, Polygonum multiflorum, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. Also, flavoring agents obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo biloba, etc. can be used. Natural flavoring agents can be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents can be used, such as esters, alcohols, aldehydes, and terpenes.

[0033] Preservatives that can be used include calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc.; emulsifiers that can be used include acacia gum, carboxymethylcellulose, xanthan gum, pectin, etc.; and acidulants that can be used include citric acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, etc. In addition to the purpose of enhancing taste, acidulants can be added to ensure that the food composition has an appropriate acidity for the purpose of inhibiting the growth of microorganisms. Thickeners that can be used include suspending agents, sedimentation agents, gel-forming agents, and puffing agents.

[0034] In addition to the food additives described above, the food composition of the present invention may include physiologically active substances or minerals known in the art and guaranteed to be safe as food additives for the purpose of supplementing and reinforcing functionality and nutrition.

[0035] Examples of such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, and dibenzoylthiamine, and examples of minerals include calcium preparations such as calcium citrate, magnesium preparations such as magnesium stearate, iron preparations such as ferrous citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, and zinc.

[0036] The food composition of the present invention may include the aforementioned food additives in an appropriate amount that can achieve the purpose of addition depending on the product type.

[0037] With regard to other food additives that may be included in the food composition of the present invention, reference may be made to the food code or food additive code according to the laws of each country.

[0038] The composition of the present invention may be considered as a pharmaceutical composition in other specific embodiments.

[0039] The pharmaceutical composition of the present invention may be prepared as an oral or parenteral formulation, depending on the route of administration, by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. "Pharmaceutically acceptable" herein means that the composition does not inhibit the activity of the active ingredient and does not exhibit toxicity exceeding that tolerated by the intended subject.

[0040] When the pharmaceutical composition of the present invention is prepared as an oral dosage form, it can be prepared in the form of powder, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers, etc., using a suitable carrier and a method known in the art. At this time, examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, xylitol, etc.; starches such as corn starch, potato starch, wheat starch, etc.; celluloses such as cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyols, and vegetable oils. In case of formulation, the formulation may include diluents and / or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants, as needed.

[0041] When the pharmaceutical composition of the present invention is prepared as a parenteral dosage form, it can be formulated in the form of eye drops, injections, transdermal administration agents, inhalation agents (agents for delivering drugs directly into the nasal cavity, oral cavity, respiratory tract, bronchial tubes, etc. using a nebulizer, etc.), suppositories, etc., with a suitable carrier according to a method known in the art. When formulated as eye drops, suitable carriers include sterile water, saline, isotonic solutions such as 5% dextrose, etc., and, if necessary, benzalkonium chloride, mephylparaben, ethylparaben, etc. can be added for preservative purposes. When formulated as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, and preferably, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, isotonic solutions such as 5% dextrose, etc. When formulated as a transdermal agent, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, pastes, liniments, aerosols, etc. In the case of inhalation, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc. When formulated as a suppository, the base can be witepsol, tween 61, polyethylene glycols, cacao butter, laurin butter, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, etc.

[0042] Specific formulations of pharmaceutical compositions are known in the art and can be found, for example, in Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.

[0043] The preferred dosage of the pharmaceutical composition of the present invention may range from 0.001 mg / kg to 10 g / kg per day, preferably from 0.001 mg / kg to 1 g / kg, depending on the patient's condition, weight, sex, age, severity of the condition, and route of administration. Administration may be administered once daily or divided into several doses. Such dosage should not be construed as limiting the scope of the present invention in any way.

[0044] As described above, according to the present invention, an anticancer composition using a flavone derivative can be provided, and a composition for enhancing the anticancer effect of an anticancer agent can also be provided.

[0045] The composition of the present invention can be commercialized as a food product such as a health functional food or a drug product such as a medicine.

[0046] Figures 1 and 2 are 1H-NMR and 13C-NMR data of a novel flavone derivative, respectively.

[0047] Figure 3 is an LC-MS spectrum of a novel flavone derivative.

[0048] Figures 4 to 6 show the results of evaluating the effect of novel flavone derivatives on the survival rate of cancer cells.

[0049] Figures 7 to 9 show the results of evaluating the effects of existing anticancer drugs on the survival rate of cancer cells.

[0050] Figures 10 to 14 show the results of evaluating cell viability by combined treatment of a novel flavone derivative with an existing anticancer drug, and the Chou-Talalay combination index (CI) calculated to confirm the synergistic effect according to the combined treatment.

[0051] Figure 15 shows the results of confirming the level of HIF-1α protein expression according to single treatment with a novel flavone derivative and combined treatment with osimertinib, an existing anticancer drug.

[0052] <Example> Preparation of a novel flavone derivative and its anticancer activity test

[0053] <Example 1> Preparation and identification of novel flavone derivatives

[0054] 1. Preparation of novel flavone derivatives

[0055] Step 1

[0056]

[0057] Potassium carbonate (K2CO3, 32.86 g, 238.10 mmol), benzylbrimide (BnBr, 22.39 g, 130.95 mmol) was added and stirred at room temperature for 5 hours. The solvent was removed, the residue was diluted with 100 mL of water, extracted with ethyl acetate (100 mL x 2), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain methyl 3,4-bis(benzyloxy)benzoate (2) (19 g, yield: 91.73%) as a yellow solid.

[0058] MS (ESI) m / z 349.2 [M+1] +

[0059] Step 2

[0060]

[0061] To a solution of methyl 3,4-bis(benzyloxy)benzoate (2) (19 g, 54.60 mmol) in THF(tetrahydrofuran) / MeOH (50 mL / 50 mL) was added 1 M NaOH (aq) (60 mL, 60.0 mmol), and the mixture was stirred at 20°C for 12 h. The mixture was concentrated, diluted with water, adjusted to pH 5 with 1 M HCl (aq), and extracted with DCM (dichloromethane). The organic solvent was dried over Na2SO4, filtered, and concentrated to obtain 3,4-bis(benzyloxy)benzoic acid (3) (13 g, crude) as a white solid.

[0062] MS (ESI) m / z 335.2 [M+1] +

[0063] Step 3

[0064]

[0065] To a solution of 3,4-bis(benzyloxy)benzoic acid (3) (7 g, 20.96 mmol) in DCM (50 mL) at 0°C were added thionyl chloride (4.99 g, 41.92 mmol) and DMF (15.33 mg, 0.21 mmol), and the mixture was stirred at 0°C for 4 hours. The resulting mixture was concentrated to obtain 3,4-bis(benzyloxy)benzoyl chloride (4) (8 g, crude) as a yellow solid, which was then immediately used for the next step 4.

[0066] Step 4

[0067]

[0068] To a DCM (50 mL) solution (0°C) of 3,4-bis(benzyloxy)benzoyl chloride(4)(8 g, 20.96 mmol) was added TEA (triethylamine, 2.12 g, 20.96 mmol) and 1H-benzo[d][1,2,3]triazole(5)(2.49 g, 20.96 mmol), and the mixture was stirred at 0°C for 4 h. Aqueous ammonium chloride solution was added to the obtained product, extracted three times with ethyl acetate, and the organic layer was washed with 3 M aqueous sodium hydroxide solution. Next, the residue was dried over Na2SO4, concentrated under reduced pressure, and recrystallized from n-hexane-DCM to obtain (1H-benzo[d][1,2,3]triazol-1-yl)(3,4-bis(benzyloxy)phenyl)methanone(6)(8 g, yield: 87.74%) as a white solid.

[0069] MS (ESI) m / z 436.2 [M+1] +

[0070] Step 5

[0071]

[0072] To a THF (50 mL) solution (-70°C) of 1-(2-((tert-butyldiphenylsilyl)oxy)-4,5-dimethoxyphenyl)ethan-1-one (8 g, 18.43 mmol) (14, prepared in Step 11 below) was added LiHMDS (Lithium bis(trimethylshily)amide, CAS No. 4939-32-1) (55 mL, 55 mmol, 1 M solution in THF) and stirred at -70°C for 1 h. To the resulting mixture was added a THF (30 mL) solution (-70°C) of (1H-benzo[d][1,2,3]triazol-1-yl)(3,4-bis(benzyloxy)phenyl)methanone(6)(8 g, 18.43 mmol) and stirred at 0°C for 2 h. A saturated aqueous solution of ammonium chloride was added to the resultant, extraction was performed with ethyl acetate, the organic solvent was dried over Na2SO4, and the mixture was concentrated under reduced pressure to obtain 1-(3,4-bis(benzyloxy)phenyl)-3-(2-((tert-butyldiphenylsilyl)oxy)-4,5-dimethoxyphenyl)propane-1,3-dione(7)(19 g, crude) as a yellow oil, which was then immediately proceeded to the next step 6.

[0073] Step 6

[0074]

[0075] TBAF (Tetrabutylammonium fluoride, 5.23 g, 20.00 mmol) was added to a THF (50 mL) solution of 1-(3,4-bis(benzyloxy)phenyl)-3-(2-((tert-butyldiphenylsilyl)oxy)-4,5-dimethoxyphenyl)propane-1,3-dione(7)(19 g, crude) and stirred at 20°C for 3 hours. Saturated aqueous ammonium chloride solution was added to the resultant, extracted three times with ethyl acetate, dried the organic solvent over Na2SO4, and concentrated under reduced pressure. The resulting product was purified by silica column chromatography (petroleum ether: ethyl acetate = 4:1) to obtain 1-(3,4-bis(benzyloxy)phenyl)-3-(2-hydroxy-4,5-dimethoxyphenyl)propane-1,3-dione (8) (5.3 g, yield: 56.17%) as a yellow solid.

[0076] MS (ESI) m / z 513.3[M+1] +

[0077] Step 7

[0078]

[0079] To a solution of 1-(3,4-bis(benzyloxy)phenyl)-3-(2-hydroxy-4,5-dimethoxyphenyl)propane-1,3-dione (8) (5.3 g, 10.35 mmol) in MeOH / THF=1 / 5 (30 mL) at 0°C was added TFA (7.92 mL, 103.50 mmol), which was stirred at 60°C for 5 h. The solvent was removed from the resulting mixture, and recrystallization from n-hexane-DCM gave 2-(3,4-bis(benzyloxy)phenyl)-6,7-dimethoxy-4H-chromen-4-one (9) (3 g, yield: 58.68%) as a white solid.

[0080] MS (ESI) m / z 495.2 [M+1] +

[0081] Step 8

[0082]

[0083] To a solution of PhI(OAc)2 (2.93 g, 9.11 mmol) in DCM (30 mL) was added TBAB (Tetrabutylammonium bromide, 2.93 g, 9.11 mmol), and the mixture was stirred at 20°C for 1 h under a hydrogen atmosphere. To the resulting mixture was added anhydrous DCM (20 mL) solution of 2-(3,4-bis(benzyloxy)phenyl)-6,7-dimethoxy-4H-chromen-4-one(9) (3 g, 6.07 mmol), and the mixture was stirred at 20°C for 8 h. Saturated aqueous ammonium hydroxide solution was added to the resulting mixture, and the mixture was extracted three times with DCM. The organic solvent was removed with Na2SO4, and the mixture was concentrated under reduced pressure. The resulting product was purified by silica column chromatography (petroleum ether: ethyl acetate = 2:1) to obtain 2-(3,4-bis(benzyloxy)phenyl)-3-bromo-6,7-dimethoxy-4H-chromen-4-one (10) (3.2 g, yield: 92.16%) as a yellow solid.

[0084] MS (ESI) m / z 575.2 [M+1] +

[0085] Step 9

[0086]

[0087] To a THF / EtOH = 1 / 1 (30 mL) solution of 2-(3,4-bis(benzyloxy)phenyl)-3-bromo-6,7-dimethoxy-4H-chromen-4-one (10) (3.2 g, 5.59 mmol) was added TBAB (1.80 g, 5.59 mmol), K2CO3 (1.54 g, 11.18 mmol), and Pd(PPh3)4 (347 mg, 0.30 mmol), and stirred at 80°C in a hydrogen atmosphere for 5 h. The solvent was removed, and the resultant product was purified by silica column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain 2-(3,4-bis(benzyloxy)phenyl)-6,7-dimethoxy-3-(4-methoxyphenyl)-4H-chromen-4-one (11) (2.3 g, yield: 68.57%) as a yellow solid.

[0088] MS (ESI) m / z 601.3 [M+1] +

[0089] Step 10

[0090]

[0091] To a solution of 2-(3,4-bis(benzyloxy)phenyl)-6,7-dimethoxy-3-(4-methoxyphenyl)-4H-chromen-4-one (11) (1.6 g, 2.67 mmol) in THF / EtOH = 1 / 1 (30 mL), Pd / C (500 mg) was added and stirred overnight at room temperature under H2 conditions. The resultant was filtered through celite, concentrated, and recrystallized from Et2O (diethylether) to obtain the final product, 2-(3,4-dihydroxyphenyl)-6,7-dimethoxy-3-(4-methoxyphenyl)-4H-chromen-4-one (UC-224425) (750 mg, yield: 66.88%) as a green solid of chemical formula 1.

[0092] MS (ESI) m / z 421.1[M+1] +

[0093] Step 11

[0094]

[0095] 1-(2-hydroxy-4,5-dimethoxyphenyl)ethan-1-one (12) (1 g, 5.1 mmol) was added to DMF (5 mL) (0°C), imidazole (624 mg, 9.18 mmol) and tert-butylchlorodiphenylsilane (13) (3.0 g, 11.2 mmol) and stirred at 100°C for 10 hours. Saturated aqueous ammonium chloride solution was added, extracted three times with DCM, and the organic layer was dried over sodium sulfate (Na2SO4), concentrated under reduced pressure, and then recrystallized from n-hexane-EA to obtain 1-(2-((tert-butyldiphenylsilyl)oxy)-4,5-dimethoxyphenyl)ethan-1-one (14) (800 mg, yield: 87.74%) as a white solid. The obtained product was used as a reactant in the fifth step.

[0096] 2. Nuclear magnetic resonance spectroscopy experiments on novel flavone derivatives

[0097] 1H-NMR and 13C-NMR were performed on the final compound obtained above.

[0098] Samples for nuclear magnetic resonance (NMR) spectroscopy experiments were dissolved in deuterium-substituted dimethyl sulfoxide to a final concentration of 100 mM and transferred to a 5-mm NMR tube. NMR experiments were measured at room temperature using a JEOL 500 spectrometer system (11.7 T; JEOL Ltd. JAPAN). Chemical mobilities were obtained using tetramethylsilane as a reference. The relaxation delay, 90° pulse, spectral width, number of data points, and digital resolution for hydrogen NMR spectroscopy were 1 s, 11.6 μs, 5,500 Hz, 32 K, and 0.34 Hz / point, respectively. The parameters for carbon NMR spectroscopy experiments were 3 s, 15.0 μs, 21,000 Hz, 64 K, and 0.64 Hz / point.

[0099] The 1H-NMR results are shown in Figure 1, and the 13C-NMR results are shown in Figure 2.

[0100] 3. High-resolution mass spectrometry experiments on novel flavone derivatives

[0101] High-resolution mass spectrometry experiments were performed using ultraperformance liquid chromatography-hybrid quadrupole-time-of-flight mass spectrometry (QTOF / MS) using a Waters Acquity UPLC system (Waters Corp., Milford, MA). The results are shown in Fig. 3.

[0102] MS (ESI) m / z 353.3 [M+1] +

[0103] <Example 2> Anticancer activity test of novel flavone derivatives

[0104] 1. Experimental method

[0105] 1.1 Reagents and compounds

[0106] Roswell Park Memorial Institute (RPMI) 1640 and Dulbecco's Modified Eagle Medium (DMEM) media, 10,000 units / mL penicillin, and 10,000 μg / mL streptomycin were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Fetal bovine serum (FBS) was purchased from Merck & Co, Inc. (Darmstadt, Land Hessen, Germany). 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), tamoxifen, and gemcitabine were purchased from Sigma Aldrich Co. (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) was purchased from Biosesang Co. (Youngin, Gyeonggi, Korea). Osimertinib was purchased from MedChemExpress Co. (Yongin, Gyeonggi, Korea). (Monmouth Junction, NJ, USA). Antibody to HIF-1α (NB100-105) was purchased from Novus Biologicals (Littleton, CO, USA).

[0107] 1.2 Cell culture

[0108] Human non-small cell lung cancer cell lines H827, H1299, H522, and A549 were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 units / mL penicillin, and 100 μg / mL streptomycin. Human breast cancer cell line MCF-7 and human pancreatic cancer cell line PANC-1 were cultured in DMEM medium supplemented with 10% FBS, 100 units / mL penicillin, and 100 μg / mL streptomycin. All cell lines were cultured under 5% CO2 at 37°C.

[0109] 1.3 Cell viability analysis

[0110] The cell viability of human non-small cell lung cancer cell lines, human breast cancer cell lines, and human pancreatic cancer cell lines was measured using the MTT assay. All cell lines were seeded (2.5 × 10 4 (dog / mL) and cultured for 24 hours. Then, the material was treated and cultured for additional 24, 48, and 72 hours. After culture, the culture medium was removed and treated with MTT solution (5 mg / mL) for 3 hours, then dissolved in 150 μL of DMSO and the absorbance was measured at 570 nm using a spectrophotometer. Cell viability was calculated as a percentage compared to the untreated group (control group), and all results were repeated three times.

[0111] 1.4 Western blot

[0112] Human non-small cell lung cancer cell lines were seeded in 100 mm tissue culture dishes (4 × 10 5(dog / plate) and cultured for 24 hours. Then, the material was treated and cultured for 24 and 48 hours. After culture, the cells were collected, lysed by adding lysis buffer, and the lysate was centrifuged to remove cell membrane components. The obtained proteins were quantified using bovine serum albumin (BSA). The quantified proteins were electrophoresed using sodium dodecylsulfate-polyacrylamide gel (SDS-PAGE), and the separated proteins were transferred to a polyvinyl difluoride (PVDF) membrane. The membrane was blocked with 5% skim milk powder dissolved in tris-buffered saline containing 0.1% Tween-20 (TBST) for 12 hours at 4°C, and the primary antibody was attached for 12 hours at 4°C. The membrane was then washed with TBST, treated with secondary antibody, and treated with enhanced chemiluminescent (ECL) substrate before being visualized with ChemiDoc. TM Protein bands were visualized using an Imaging System (Bio-Rad, Inc., Hercules, CA, USA).

[0113] 2. Experimental Results

[0114] 2.1 Check cell viability

[0115] (1) Treatment with novel flavone derivatives alone

[0116] The cell viability of human non-small cell lung cancer cell lines H827, H1299, H522, and A549, human breast cancer cell line MCF-7, and human pancreatic cancer cell line PANC-1 was examined by treating with novel flavone derivatives (Achem I, ACH, or A) alone at various concentrations. As a result, as confirmed in Figures 4 to 6, cell viability decreased in a concentration-dependent and time-dependent manner in all cases.

[0117] IC 50As a result of comparing the antiproliferative effects of novel flavone derivatives by cell line through values, the lowest IC was found in H1299 cells, as shown in Table 1. 50 H1299 cells were found to be the most sensitive cell line to the novel flavone derivatives.

[0118]

[0119] (2) Combined treatment with existing cytotoxic anticancer drugs

[0120] The synergistic effect was confirmed through the cell viability of each cancer cell line by combining treatment with osimertinib (OSI or O), an anticancer drug for lung cancer, tamoxifen (TAM), an anticancer drug for breast cancer, and gemcitabine (GEM), an anticancer drug for pancreatic cancer, of the novel flavone derivative. The synergistic effect was confirmed through the Chou-Talalay combination index (CI) using the CompuSyn (Informer Technologies, Inc., Los Angeles, CA, USA) program. In order to more clearly confirm the synergistic effect, the treatment concentrations of the novel flavone derivative and each anticancer drug were used in the range where the cell viability was 50% or higher. The determination of this range of concentrations was made based on the results of the cell viability according to the treatment concentration of each anticancer drug, and the cell viability results are shown in Figures 7 to 9.

[0121] The results of cell viability and the synergistic effect confirmed through the CI index by combined treatment of the novel flavone derivative with existing anticancer drugs are shown in Figures 10 to 14. It can be seen that the novel flavone derivative, when combined treatment with existing cytotoxic anticancer drugs, reduces cell viability in a concentration-dependent and time-dependent manner and also shows a synergistic effect. For reference, the Chou-Talalay combination index (CI) means that when 1 (CI=1) there is an additive effect, when it is greater than 1 (CI>1) there is an offset effect, and when it is less than 1 (CI<1) there is a synergistic effect (ancer Res. 2010 Jan 15;70(2):440-6).

[0122] 2.2 Confirmation of HIF-1α protein expression through Western blot

[0123] The effects of treatment with a novel flavone derivative (A) alone (6.25 μM), osimertinib (O) alone (0.01 μM), and combined treatment with the novel flavone derivative and osimertinib (A+O) on HIF-1α protein expression in H827 cells were evaluated by Western blot, and the results are shown in Fig. 15. It can be confirmed that combined treatment with the novel flavone derivative and osimertinib (A+O) further reduces HIF-1α expression compared to treatment with each substance alone.

[0124] The present invention can be used as a functional food, pharmaceutical, etc.

Claims

1. An anticancer composition comprising a flavone derivative of <chemical formula 1> or a hydrate or solvate thereof as an effective ingredient. <Chemical Formula 1> 2. In paragraph 1, A composition characterized in that the above anticancer agent is anticancer against human non-small cell lung cancer, breast cancer or pancreatic cancer.

3. In paragraph 1, The above anticancer composition is characterized in that it additionally contains one or more anticancer agents selected from the group consisting of a cytotoxic anticancer agent, a targeted anticancer agent, an immunological anticancer agent, and a hormonal agent.

4. In paragraph 1, The above anticancer composition is characterized in that it comprises at least one of osimertinib, tamoxifen, a hormonal agent for breast cancer, and gemcitabine, a cytotoxic anticancer agent for pancreatic cancer.

5. A composition for enhancing the anticancer effect of an anticancer agent, comprising a flavone derivative of <chemical formula 1> or a hydrate or solvate thereof as an effective ingredient. <Chemical Formula 1> 6. In paragraph 5, A composition characterized in that the anticancer agent is at least one of a cytotoxic anticancer agent, a targeted anticancer agent, an immune anticancer agent, and a hormonal agent.

7. In paragraph 1, A composition characterized in that the anticancer agent is osimertinib, the hormonal agent tamoxifen for breast cancer, and the cytotoxic anticancer agent gemcitabine for pancreatic cancer.

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