WO2013054998A1 - Nouveau dérivé de chalcone et composition anticancéreuse comprenant celui-ci comme principe actif - Google Patents

Nouveau dérivé de chalcone et composition anticancéreuse comprenant celui-ci comme principe actif Download PDF

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WO2013054998A1
WO2013054998A1 PCT/KR2012/004183 KR2012004183W WO2013054998A1 WO 2013054998 A1 WO2013054998 A1 WO 2013054998A1 KR 2012004183 W KR2012004183 W KR 2012004183W WO 2013054998 A1 WO2013054998 A1 WO 2013054998A1
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compound
cancer
cells
cell
caspase
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PCT/KR2012/004183
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Korean (ko)
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임융호
신순영
고동수
이영한
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건국대학교 산학협력단
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Priority claimed from KR1020110104580A external-priority patent/KR101333734B1/ko
Priority claimed from KR1020110105807A external-priority patent/KR101283004B1/ko
Priority claimed from KR1020110109128A external-priority patent/KR101290579B1/ko
Priority claimed from KR1020110109129A external-priority patent/KR101290578B1/ko
Priority claimed from KR1020110109130A external-priority patent/KR101333736B1/ko
Priority claimed from KR1020110109127A external-priority patent/KR101325783B1/ko
Application filed by 건국대학교 산학협력단 filed Critical 건국대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
    • C07C49/76—Ketones containing a keto group bound to a six-membered aromatic ring
    • C07C49/84—Ketones containing a keto group bound to a six-membered aromatic ring containing ether groups, groups, groups, or groups
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
    • C07C49/76—Ketones containing a keto group bound to a six-membered aromatic ring
    • C07C49/82—Ketones containing a keto group bound to a six-membered aromatic ring containing hydroxy groups

Definitions

  • the present invention relates to a novel chalcone derivative and an anticancer composition comprising the same as an active ingredient.
  • caspase-7 is one of apoptosis related cystein peptidases found in most mammals and humans. Caspase-7 is present as an inactive proprotein, which is then degraded by caspase-9. When a signal related to apoptosis is provided, caspase-7 is activated to induce apoptosis. Therefore, since caspase-7 cleavage is closely related to induction of apoptosis, the caspase-7 cleavage effect is recognized as a signal for apoptosis of cancer cells affected by apoptosis. It can be said that it has utility as.
  • Flavonoids have a structural skeleton of C 6 -C 3 -C 6, which is called a benzoflavonoid when the benzene ring is attached to another C 6 skeleton to have a naphthalene group.
  • Chalcone is also a flavonoid, so if one benzene ring is changed to a naphthalene group, it becomes benzochalcone. Since the hydrophobicity is increased in the case of benzochalcon than the known chalcone, permeation of the cell membrane becomes a little easier, and thus a change in biological activity can be expected.
  • the present inventors synthesized a compound having a new structure by adding three methoxy groups and one hydroxy group having a skeleton of benzochalcone and named it DK78. Since the DK78 compound, a kind of benzochalcone, has been reported to have a cleavage effect of caspase-7 as a new compound that has not been reported so far, further experiments related to cell cycle inhibition were performed to confirm whether the compound has an efficacy as an anticancer agent. The study was completed.
  • Chalcone (chalcone) compound is a kind of polyphenol-based compound that is found during the production of secondary metabolites of plants or known as secondary metabolites, and many kinds of chalcone compounds are found because it is possible to generate derivatives having various structures according to substituents. come. Some of these are known to have anticancer effects (Yadav VR, Prasad S, Sung B, Aggarwal BB. Int Immunopharmacol. 2011 Mar; 11 (3): 295-309).
  • the chalcone compound is a compound having a skeleton of C6-C3-C6 and has the same structure as the carbon skeleton of the flavonoid compound.
  • Increasing hydrophilicity has the disadvantage of facilitating affinity with water, which constitutes most of the living body, while making it difficult to penetrate the cell membranes, which constitute the cell. Therefore, adding a methoxy group and a benzo group as a substituent to the chalcone compound will have the advantage of improving the permeability of the cell membrane. Therefore, the present inventors attach a methoxy group and a benzo group as a substituent to the chalcone compound to have a little affinity with water constituting the living body. In order to test the anticancer effect was selected by selecting a benzohydroxy methoxychalcone compound having a hydroxyl group as a substituent.
  • the present invention has been made in view of the above necessity, and an object of the present invention is to provide a new anticancer drug candidate.
  • the present invention provides a chalcone (chalcone) derivative compound represented by one of the following Chemical Formulas 1 to 6 and pharmaceutically acceptable salts thereof.
  • the present invention is 1) 2-hydroxy-4-methoxy-acetophenone (2-hydroxy-4-methoxy-1-acetophenone) and 1-naphthaaldehyde (4-naphthaaldehyde); 2-hydroxy-1-acetonaphthone and 2,4,6-trimethoxybenzaldehyde; 2-hydroxy-1-aceto 2-hydroxy-1-acetonaphthone and 2-methoxybensaldehyde; 1-hydroxy-2-acetonaphthon and 3,5-di Methoxybenzaldehyde (3,5-dimethoxybenzaldehyde); or 2-hydroxy-6-methoxy-acetophenone and 2,4,6-trimethoxybenzaldehyde (2 , 4,6-trimethoxybenzaldehyde) in the organic solvent;
  • step (2) adding a base to the mixed solution of step (1);
  • (6) provides a method for preparing the chalcone derivative compound comprising the step of (5) the water is removed and the remaining mixture under reduced pressure filtration, drying and purification.
  • the present invention is to inhibit the growth and death of cancer cells against cancer cells selected from the group consisting of colon cancer, stomach cancer, prostate cancer, breast cancer, kidney cancer, liver cancer, brain tumor, lung cancer, uterine cancer, colon cancer, bladder cancer, pancreatic cancer, blood cancer It provides a pharmaceutical composition for the prevention and treatment of cancer diseases comprising a compound of the present invention having a pharmaceutically acceptable salt.
  • the pharmaceutical composition preferably comprises one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the carrier, diluent or excipient may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, Polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • the pharmaceutical composition preferably comprises a second anticancer agent or an anticancer adjuvant, wherein the second anticancer agent is an interferon, interleukin-2, paclitaxel ), Vincristine, vinblastin, doxorrubicin, etoposide, irinotecan hydrochloride, cisplatin, amsacrine, cytosine arabino At least one selected from the group consisting of seeds (cytosine arabinoside), fluorouracil, and taxol is not limited thereto.
  • the second anticancer agent is an interferon, interleukin-2, paclitaxel ), Vincristine, vinblastin, doxorrubicin, etoposide, irinotecan hydrochloride, cisplatin, amsacrine, cytosine arabino At least one selected from the group consisting of seeds (cytosine arabinoside), fluorouracil, and taxol is not limited thereto.
  • the pharmaceutical composition containing the chalcone compound of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and the like, oral preparations, suppositories, or sterile injectable solutions according to conventional methods. Can be used. Specifically, when formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used. Solid preparations for oral administration may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin and the like with the chalcone compound.
  • Liquid preparations for oral use include suspensions, solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories.
  • non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used.
  • utopsol macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • Preferred dosages of the chalcone compounds of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art.
  • the chalcone compound of the present invention or a pharmaceutically acceptable salt thereof may be administered in an amount of 0.0001 to 100 mg / kg, preferably in an amount of 0.001 to 100 mg / kg once or several times daily. have.
  • the total chalcone compound of the present invention or a pharmaceutically acceptable salt thereof should be included in 0.0001 to 10% by weight, preferably 0.001 to 1% by weight.
  • the pharmaceutical composition of the present invention can be administered to mammals such as stomach, mouse, livestock, human, etc. by various routes, and the mode of administration can be administered by injection in various ways such as oral, rectal, intravenous, intramuscular, subcutaneous. .
  • the present invention also provides a health functional food for preventing and improving cancer diseases including the chalcone compound and food chemically acceptable food supplement additives.
  • dietary supplements include meat, sausages, breads, chocolates, candy, snacks, confectionery, pizzas, ramen noodle, dairy products including gums, ice creams, various soups, beverages, teas, drinks, alcoholic beverages, It can be prepared by adding a chalcone compound or a food-acceptable salt thereof to a vitamin complex or the like.
  • the present invention provides a novel compound having a growth inhibitory and killing effect of cancer cells and a pharmaceutical composition for the prevention and treatment of cancer diseases and the health functional food for the prevention and improvement of cancer diseases, including the same
  • the results can be usefully used for preventing and treating cancer diseases, which is valuable as a candidate material that can be used for the prevention and treatment of cancer diseases.
  • Figure 1 shows the inhibitory effect of cancer cell colony formation by the benzohydroxymethoxychalcone compound (DK49) in Capan-1 human pancreatic cancer cells and HCT116 human colon cancer cells.
  • Figure 2 analyzes the cell cycle progression inhibition and apoptosis effect of benzohydroxymethoxychalcone compound (DK49) using a flow cytometry (fluorescent activating cell sorting, FACS).
  • Figure 3 is an analysis of the caspase (caspase) activity effect by the benzohydroxymethoxychalcone compound (DK49) by immunoblot method.
  • Figure 4 is observed by the fluorescence microscope the phenomenon that the caspase-7 (caspase-7) and the nuclear DNA is cut by the benzohydroxymethoxychalcone compound (DK49).
  • FIG 5 shows the tumor suppression effect of the benzohydroxymethoxychalcone compound (DK49)
  • Figure 6 shows the caspase activity effect of the present invention benzochalcone compound (DK78) in HCT116 cells by Western blot method.
  • FIG. 7 shows the inhibition of HCT116 colon cancer cell proliferation of the present invention benzochalcone compound (DK78).
  • Figure 8 is analyzed by the flow cytometer apoptosis effect of the present invention benzochalcon compound (DK78) in HCT116 cells (2N, dploid DNA; 4N, tetraploid DNA.M1, sub-G1; M2, G1; M3, S; M4, G2 / M)
  • Figure 9 was investigated by Western blot analysis of the PARP protein cleavage effect of the benzochalcon compound (DK74) in SW620 cells.
  • Figure 10 shows the ability of the benzochalcon compound (DK74) SW620 colon cancer cell proliferation inhibition.
  • Figure 11 shows the apoptosis effect of benzochalcon compound (DK74) in SW620 cells by flow cytometer (2N, diploid DNA; 4N, tetraploid DNA, M1, sub-G1; M2, G1; M3 , S; M4, G2 / M).
  • FIG. 12 is a diagram showing a process of synthesizing naphthalenylphenyldihydropyrazole compound (DK117).
  • Figure 13 shows the results of Western blot analysis of the inhibitory effect of cyclin D1 protein expression by the naphthalenylphenyldihydropyrazole compound of the present invention in HCT116 cells (DK117).
  • Figure 15 shows the proliferation inhibitory effect of HCT116 colorectal cancer cells by the present invention naphthalenylphenyldihydropyrazole compound (DK117).
  • Fig. 16 shows the results of Western blot analysis of the p21 protein expression inhibition effect by the hydroxymethoxybenzochalcon DK59 compound of the present invention.
  • FIG. 17 is a result of analyzing the cell cycle progression inhibiting effect by the hydroxymethoxy benzochalcon DK59 compound of the present invention by a fluorescent activating cell sorting (FACS).
  • FACS fluorescent activating cell sorting
  • Figure 18 shows the effect of inhibiting the proliferation of HCT116 colon cancer cells by the hydroxymethoxybenzochalcon DK59 compound of the present invention.
  • Figure 21 shows the results of the analysis of caspase enzyme activity and PARP protein cleavage effect by hydroxymethoxychalcon DK138 compound of the present invention by Western blot method.
  • Synthesis of the chalcone compound is 2-hydroxy-4-methoxy-acetophenone (2-hydroxy-4-methoxy-1-acetophenone, 830 mg, 5 mmol) and 1-naphthaldehyde (4-naphthaldehyde, 936 mg, 6 mmol) was dissolved in 30 mL of ethanol, and then 8 mL of 50% KOH aqueous solution was slowly added at about 5 minutes for about 5 minutes. After stirring the mixed solution at room temperature for 22 hours, the temperature was lowered to about 4 ° C. After neutralizing by adding 20 ml of 6 N HCl solution to the cooled solution, the aqueous solution was extracted twice with 70 ml of chloroform.
  • Nuclear magnetic resonance spectroscopy was performed to confirm the final product.
  • the device used was a Bruker 400 MHz device.
  • the compound was identified through hydrogen nuclear magnetic resonance spectroscopy and carbon nuclear magnetic resonance spectrum, the chemical mobility is as follows.
  • HCT116 colorectal cancer cells and Capan-1 pancreatic cancer cells were purchased from the American Type Culture Collection (ATTC) and DMEM (Invitrogen Life Technologies) including 10% FBS (Fetal Bovine Serum, Invitrogen Life Technologies), Antibiotic-Antimycotic solution (Invitrogen Life Technologies) Cultures were incubated in a 5% CO 2 incubator at 37 ° C., passaged once every two days at a seed density of 1 ⁇ 10 6 in a 100-mm cell culture dish. The effect of inhibiting cell proliferation by the benzohydroxymethoxychalcone compound (DK49) was measured by whether or not cell growth was inhibited through colony forming assay of cancer cells.
  • DK49 benzohydroxymethoxychalcone compound
  • HCT116 colorectal cancer cells and Capan-1 pancreatic cancer cells were dispensed at 6000 cells per well in a 24-well culture dish and treated with benzohydroxymethoxychalcone compounds (DK49) at concentrations of 0, 5, 10, and 20 ⁇ M, After 7 days, a mixture of 6% glutaraldehyde and 0.5% crystal violet solution in a 1: 1 ratio was added to the cells, and the remaining cells were stained by reacting for 15 minutes. As a result, as shown in FIG. 1, it was observed that colony forming ability of cancer cells was rapidly decreased when the benzohydroxymethoxychalcone compound (DK49) was treated at a concentration of 10 ⁇ M or more. From these facts, it was confirmed that the benzohydroxymethoxychalcone compound (DK49) has an effect of inhibiting the proliferation of colorectal cancer and pancreatic cancer cells.
  • DK49 benzohydroxymethoxychalcone compounds
  • FACS Fluorescent Activating Cell Sorting
  • Capan-1 pancreatic cancer cells had a G2 / M cycle but 69.3% after 1 day in the cell group treated with the benzohydroxymethoxychalcone compound (DK49). Increased. Since the G2 / M cycle cells gradually decreased, it was observed that the amount of sub-G0 / G1 cells increased.
  • Induction of apoptosis results in cells (sub-G0 / G1) containing a smaller amount of DNA than the 2N DNA content of G1 cycle cells, so that the DK49 compounds of the present invention are G2 / M cells in HCT116 colorectal cancer cells. Blocking the cycle progression was found to induce apoptosis.
  • Capan-1 pancreatic cancer cells were cultured in a 60 mm culture dish to 1.5 X 10 6 cells, and treated with benzohydroxymethoxychalcone compound (DK49) to a concentration of 20 ⁇ M, followed by 0, 6, 12, 24, 48 hours. The cells were harvested, lysed with cell lysis buffer, and then centrifuged at high speed to harvest only the cell lysate.
  • Samples prepared to contain the same amount of protein were subjected to SDS-polyacrylamide gel electrophoresis to separate proteins present in the cells.
  • the proteins separated by electrophoresis were transferred to a polystyrene membrane, and then cut into Caspase-9 and Caspase-7.
  • the cells expressing the active Caspase-7 were increased by treatment with the benzohydroxymethoxychalcone compound (DK49), and the characteristics of apoptosis were only observed in the cells in which the active Caspase-7 appeared.
  • Nuclear DNA fragmentation (indicated by arrows) was observed as a phenomenon.
  • FIG. 5B shows tumor size before autopsy at day 42.
  • FIG. 5C shows tumor removed by necropsy. It was confirmed that gross tumor size was significantly reduced in the benzohydroxymethoxychalcone compound (DK49) administration group than the control group (NT).
  • Example 2 anticancer composition comprising benzochalcone as an active ingredient
  • Benzochalcone compound (DK78) (E-1- (2-hydroxynaphthalen-1-yl) -3- (2,3,4-trimethoxyphenyl) prop-2-en-1-one, (E ) -1- (2-hydroxynaphthalen-1-yl) -3- (2,3,4-trimethoxyphenyl) prop-2-en-1-one) was synthesized as follows using the method shown in Scheme 2 below. .
  • the benzochalcone compound (DK78) (E-1- (2-hydroxynaphthalen-1-yl) -3- (2,3,4-trimethoxyphenyl) prop-2-en-1-one, ( E) -1- (2-hydroxynaphthalen-1-yl) -3- (2,3,4-trimethoxyphenyl) prop-2-en-1-one) is a new substance that has not been reported to date and has a molecular weight of 364.1.
  • Caspase-9 and caspase-7 enzymes which induce apoptosis, are normally inactive precursors, and are cleaved and activated when apoptosis stimulus signals are transmitted.
  • the caspase-7 and caspase-9 activating the cleaved caspase-7 and caspase-9 of the active form using Western blot analysis The degree of fragmentation of the substrate protein poly (ADP-ribose) polymerase (PARP) was investigated.
  • HCT116 cells human colorectal cancer cell line
  • ATCC American Type Culture Collection
  • Fetal Bovine Serum Fetal Bovine Serum
  • Antibiotic-Antimycotic solution Invitrogen Life Technologies Technologies Technologies
  • Cultures were added to a 60-mm cell culture dish every 2 days and incubated in a 37 ° C., 5% CO 2 incubator while maintaining a seed density of 1 ⁇ 10 6 .
  • the cultured cells were treated with a 20 ⁇ M concentration of benzochalcone compound (DK78), and cells were harvested after 0, 6, 12, and 24 hours.
  • DK78 benzochalcone compound
  • HCT116 colon cancer cells were grown using a CCK-8 kit (Cell Counting Kit-8; Dojindo, Japan). Dispense 1 x 10 4 HCT116 colorectal cancer cells into a 96-well cell culture plate and treat benzochalcone compounds (DK78) at 0, 5, 10, and 20 ⁇ M concentrations, and after 24 and 48 hours, respectively, use CCK-8 solution 10 ⁇ l was added. After 2 hours, the cell viability was measured at 450 nm to analyze cell viability.
  • CCK-8 kit Cell Counting Kit-8; Dojindo, Japan.
  • Dispense 1 x 10 4 HCT116 colorectal cancer cells into a 96-well cell culture plate and treat benzochalcone compounds (DK78) at 0, 5, 10, and 20 ⁇ M concentrations, and after 24 and 48 hours, respectively, use CCK-8 solution 10 ⁇ l was added. After 2 hours, the cell viability was measured at 450 nm to analyze cell viability.
  • HCT116 cells were treated with benzochalcone compound (DK78) and cells were harvested 24 hours later. The cells were fixed by adding 70% ethanol, and the DNA was stained by reacting PI (Propidium Iodine) for 30 minutes, and then the amount of sub-G1 DNA was analyzed by flow cytometry.
  • DK78 benzochalcone compound
  • Benzochalcon (DK-74) compounds were synthesized using the method shown in Scheme 3 below.
  • Hydrogen nuclear magnetic resonance spectroscopy (Bruker 400MHz) and carbon nuclear magnetic resonance spectra (Bruker 100MHz) were used to confirm the formation of benzochalcone compounds.
  • the device used was a Bruker 400 MHz device.
  • the compound was identified through hydrogen nuclear magnetic resonance spectroscopy and carbon nuclear magnetic resonance spectrum, the chemical mobility was as follows.
  • Benzochalcone compound (DK-74) is a novel substance with a molecular weight of 364.4.
  • PARP poly (ADP-ribose) polymerase
  • SW620 cells human colon cancer cell line
  • ATCC American Type Culture Collection
  • PARP poly (ADP-ribose) polymerase
  • SW620 cells were treated with 1 x 10 6 cells in a 60-mm cell culture dish every two days with 10% FBS (Fetal Bovine Serum, Invitrogen Life Technologies) and Antibiotic-Antimycotic solution (Invitrogen Life Technologies).
  • the cells were cultured in a 37 ° C., 5% CO 2 incubator with passage at a seed density of.
  • the cultured cells were treated with a 20 ⁇ M concentration of benzochalcone compound (DK74) compound, and cells were harvested after 0, 6, 12, 24 and 48 hours.
  • DK74 benzochalcone compound
  • the harvested cells were added with cell lysis buffer containing 20 mM HEPES (pH 7.2), 1% Triton X-100, 10% glycerol, 150 mM NaCl, 10 ⁇ g / ml leupeptin, 1 mM PMSF for 30 minutes. After the reaction, the cells were lysed, the cells were harvested by high-speed centrifugation, and the protein lysates prepared to contain the same amount of protein were subjected to SDS-polyacrylamide gel electrophoresis. Existing proteins were isolated. The electrophoretically separated protein was transferred to a polystyrene membrane, followed by PARP protein and cleaved caspase-7.
  • SW620 colon cancer cells were dispensed into 96-well cell culture plates in 1 ⁇ 10 4 cells and treated with 0, 5, 10, and 20 ⁇ M benzochalcone compound (DK74) compounds and CCK-8 solution after 24 and 48 hours, respectively. 10 ⁇ l was added. After 2 hours, the absorbance of the cell culture was measured at 450 nm to analyze cell viability.
  • SW620 colon cancer cells were cultured in the same manner as the Western blot method.
  • SW620 cells were treated with the benzochalcone compound (DK74) and harvested cells after 24 hours.
  • the cells were fixed by adding 70% ethanol, and the DNA was stained by reacting PI (Propidium Iodine) for 30 minutes, and then the amount of sub-G1 DNA was analyzed by flow cytometry.
  • PI Propidium Iodine
  • Example 3 Anticancer composition comprising naphthalenylphenyldihydropyrazole as an active ingredient
  • Naphthalenylphenyldihydropyrazole compound (DK117) was synthesized using the method shown in FIG. 12 as follows.
  • pyrazoline compound IV (2.81 g, 72%).
  • the pyrazoline compound ( IV , 319 mg, 1 mmol) and 3,4,5-trimethoxy isothiocyanate ( V , 225 mg, 1 mmol) were dissolved in 10 ml of ethanol and refluxed at about 90 ° C. for 8 hours.
  • naphthalenylphenyl dihydropyrazole compound (DK-117) was confirmed through the hydrogen nuclear magnetic resonance spectrum (Bruker 400MHz) and carbon nuclear magnetic resonance spectrum (Bruker 100MHz).
  • the device used was a Bruker 400 MHz device.
  • Compounds were identified through hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectra.
  • naphthalenylphenyldihydropyrazole compound DK117
  • HCT116 cells were treated with naphthalenylphenyldihydropyrazole compound (DK117), which is an important cell cycle regulator protein for cancer cell growth.
  • DK117 naphthalenylphenyldihydropyrazole compound
  • the expression level of cyclin D1 (cyclin D1) was analyzed by Western blot assay.
  • HCT116 colorectal cancer cells were purchased from the American Type Culture Collection (ATTC) and 1 x in DMEM (Invitrogen Life Technologies) cultures containing 10% Fetal Bovine Serum (Invitrogen Life Technologies) and Antibiotic-Antimycotic solution (Invitrogen Life Technologies). The cells were cultured in a 37 ° C., 5% CO 2 incubator with passage at a seed density of 10 6 .
  • the HCT116 cells were treated with a naphthalenylphenyldihydropyrazole compound (DK117) at a concentration of 20 ⁇ M, and the cells were harvested after 6, 12 and 24 hours. After harvesting, the cells were lysed by adding 20 mM HEPES buffer solution + 1% Triton X-100 + 10% glycerol + 150 mM NaCl + 10 ⁇ g / ml leupeptin + 1 mM PMSF and lysing the cells. Only cell lysate was harvested by centrifugation and cell lysates prepared to contain the same amount of protein were subjected to SDS-polyacrylamide gel electrophoresis to separate cell proteins by molecular weight.
  • DK117 naphthalenylphenyldihydropyrazole compound
  • the electrophoretically separated proteins were transferred to a nitrocellulose filter and GAPDH (purchased from Cell Signaling Technology, Inc.) as a control and a primary antibody against cyclin D1 protein. After reacting the primary antibody (purchased from Santa Cruz technology) for 5 hours to recognize the glyceraldehyde-3-phosphate dehydrogenase protein, the secondary antibody (purchased from Cell Signaling Technology) for 1 hour was reacted for 1 hour. . A change in the expression of each protein on the X-ray film was analyzed using a chemistry fluorescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ).
  • cell cycle progression is analyzed by measuring intracellular DNA content.
  • the G1 cycle cells contain 2N of DNA
  • the S phase cells contain an amount between 2N and 4N
  • the G2 and M cycle cells contain 4N DNA amount. Since cyclin D1 protein promotes the progression of the G1 cell cycle, the decrease of the Cyc D1 protein caused by naphthalenylphenyldihydropyrazole compound (DK117) may impair the cell cycle progression of cancer cells. Whether or not was examined using a flow cytometer (BD Science, USA).
  • the HCT116 cells were cultured in the same manner as in the Western blot analysis, followed by treatment with naphthalenylphenyldihydropyrazole compound (DK117), and after 0, 24 and 48 hours, trypsin-EDTA (1%) was added to remove the cells from the incubator. After removal, cells were fixed with 70% ethanol.
  • the HCT116 colon cancer cells growing normally had about 50.13% of the cells with the G1 cell cycle, but in the cell group treated with the naphthalenylphenyldihydropyrazole compound (DK117), 55.08 after 24 hours of treatment. It increased to% and to 60.18% after 48 hours of treatment.
  • the S phase of normal cells was 19.73%, but in the cells treated with naphthalenylphenyldihydropyrazole compound (DK117), S cycle cells gradually decreased with time to 14.28% and 8.04% after 24 and 48 hours, respectively. It was.
  • naphthalenylphenyldihydropyrazole compound (DK117) inhibits the growth of cancer cells by blocking G1 phase progression of colon cancer cells in HCT116 colon cancer cells.
  • DK117 naphthalenylphenyldihydropyrazole compound
  • the cell growth capacity of the control cell group that was not treated increased with time, but the naphthalenylphenyldihydropyrazole compound was treated in the cell group treated with the naphthalenylphenyldihydropyrazole compound (DK117).
  • DK117 Cell growth was reduced depending on treatment concentration and time.
  • Example 4 An anticancer composition comprising a hydroxymethoxy benzochalcone compound as an active ingredient
  • the reaction mixture was poured into iced water, neutralized by adding 12 N HCl, and extracted three times with 30 ml of ethyl acetate. The extracted organic layers were collected and collected to remove moisture with magnesium sulfate (MgSO 4 ). After filtration under reduced pressure, the filtrate was removed using a rotary evaporator and the residue was purified by column chromatography (Silica Gel, Merck, USA) to obtain the target compound DK59 in 59% yield (935 mg, mp). The target compound DK59 was analyzed by hydrogen nuclear magnetic resonance spectroscopy (Bruker 400 MHz) and carbon nuclear magnetic resonance spectroscopy (Bruker 100 MHz).
  • Hydroxymethoxybenzochalcone (DK59) prepared above was identified through hydrogen and carbon nuclear magnetic resonance spectra, and the chemical mobility is as follows.
  • the hydroxymethoxy benzochalcone (DK59) is a new substance that has not been reported to date, has a molecular formula of C 21 H 18 O 4 , molecular weight is 334.12, (E) -3- (3,5-dimethoxyphenyl)- It may be named 1- (1-hydroxynaphthalen-2-yl) prop-2-en-1-one.
  • the HCT116 colon cancer cells were purchased from the American Type Culture Collection (ATTC), and were cultured in DMEM (Invitrogen Life Technologies) cultures containing 10% Fetal Bovine Serum (Invitrogen Life Technologies) and Antibiotic-Antimycotic solution (Invitrogen Life Technologies). Incubation was carried out at 37 ° C., 5% CO 2 incubator, passaged at 1 ⁇ 10 6 seed density. HCT116 cells were treated with 20 ⁇ M concentration of DK59 compound and cells harvested after 6, 12, and 24 hours.
  • DMEM Invitrogen Life Technologies
  • Fetal Bovine Serum Invitrogen Life Technologies
  • Antibiotic-Antimycotic solution Invitrogen Life Technologies
  • Cell lysates prepared to contain the same amount of protein were subjected to SDS-polyacrylamide gel electrophoresis to separate cellular proteins by molecular weight.
  • the proteins separated by electrophoresis were nitrocellulose filter.
  • the primary antibody against p21 protein (Santa Cruz technology, USA) and the primary antibody that recognizes GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) protein that does not change protein expression as a control for 5 hours, respectively, A secondary antibody (Santa Cruz technology, USA) that recognizes the antibody was reacted for 1 hour.
  • cell cycle progression is analyzed by measuring intracellular DNA content.
  • cell cycle progression is analyzed by measuring intracellular DNA content.
  • p21 protein is increased by DK59, whether cell cycle progression of cancer cells is inhibited is flow cytometer; BD Science, USA).
  • HCT116 cells were cultured in the same manner as Western blot analysis, then treated with DK59, and after 0, 24, and 48 hours, trypsin-EDTA (1%) was removed from the incubator to fix the cells with 70% ethanol. . Intracellular DNA was stained by reacting Propidium Iodine (PI) for 30 minutes, and then the amount of DNA was measured.
  • PI Propidium Iodine
  • HCT116 colon cancer cells were dispensed into 1 ⁇ 10 4 cells in 96-well cell culture plates, and then 0, 5, 10, and 20 ⁇ M concentrations. DK59 compounds were treated and 10 ⁇ l of CCK-8 (Cell Counting Kit-8; Dojindo, Japan) solution was added after 24 and 48 hours, respectively. After 2 hours, the absorbance of the cell culture was measured at 450 nm to determine cell viability. Analyzed.
  • the cell growth ability increased with time in the control cell group which was not treated, whereas cell growth was decreased in time-dependent manner with the DK59 treatment concentration in the cell group treated with the DK59 compound of the present invention.
  • the DK59 compound of the present invention increases the amount of p21 protein, which is a cell cycle progression inhibiting protein, inhibits cell cycle progression and ultimately inhibits the growth capacity of colon cancer cells.
  • Example 5 An anticancer composition comprising a hydroxymethoxychalcone compound as an active ingredient
  • the target compound DK138 performs a nuclear magnetic resonance spectroscopy experiment using a Bruker (400 MHz) instrument for confirmation, and the compound is hydrogen nuclear magnetic resonance spectroscopy (Bruker 400 MHz) and carbon nuclear magnetic resonance spectroscopy ( Bruker 100 MHz) was confirmed, and the chemical mobility is as follows.
  • the hydroxymethoxychalcone (DK138) is a new substance that has not been reported to date, has a molecular formula of C 19 H 20 O 6 , molecular weight is 344.13, [E-1- (2-hydroxy-6-methoxy Phenyl) -3- (2,4,6-trimethoxyphenyl) prop-2-en-1-one ⁇ (E) -1- (2-hydroxy-6-methoxyphenyl) -3- (2,4 , 6-trimethoxyphenyl) prop-2-en-1-one ⁇ ].
  • HCT116 colorectal cancer cells are divided into 1 ⁇ 10 4 cells in a 96-well cell culture plate, DK138 compounds at concentrations of 0, 5, 10, and 20 ⁇ M were treated and 10 ⁇ l of CCK-8 (Cell Counting Kit-8; Dojindo, Japan) solution was added after 24 and 48 hours, respectively. The absorbance of was measured at 450 nm to analyze cell viability.
  • DK138 hydroxymethoxychalcone
  • the cell growth capacity increased with time in the control cell group which was not treated, but cell growth was decreased in time-dependent manner with the DK138 treatment concentration in the cell group treated with the DK138 compound of the present invention.
  • the DK138 compound of the present invention inhibits the growth ability of HCT116 colon cancer cells.
  • the G1 cycle cells were 60.67%, the S cycle cells were 15.04%, and the cells having the G2 / M cycle were about 22.43%.
  • G1 cells decreased to 3.06% after 24 hours of treatment, while G2 / M cycle cells increased to 88.43%, and after 48 hours of treatment, G2 / M stem cells gradually decreased to 28.76% and sub-G0 / G1 cells. The amount of increased to 65.53%.
  • the DK138 compound of the present invention is a G2 / M cell cycle in HCT116 colorectal cancer cells. Blocking the progression was found to gradually induce apoptosis.
  • HCT116 colon cancer cells were cultured to 1.5 ⁇ 10 6 cells in a 60 mm dish and treated with DK138 compounds to a concentration of 20 ⁇ M to harvest cells after 0, 6, 12, and 24 hours.
  • the harvested cells were lysed with cell lysis buffer, and then centrifuged at high speed to harvest only the cell solution.
  • Samples prepared to contain the same amount of protein were prepared using SDS-polyacrylamide gel. Electrophoresis was performed to separate proteins present in the cells. Electrophoretically isolated proteins were transferred to polystyrene membranes, followed by changes in protein expression as primary antibodies (Cell Signaling Technology, USA) and controls that cleaved caspase-9 and caspase-7 proteins and PAR.
  • the primary antibody (Santa Cruz technology, USA) that recognizes GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) is not reacted for 5 hours, and the secondary antibody (Cell Signaling Technology, USA) that recognizes the primary antibody was reacted for 1 hour.
  • the active form of each protein was analyzed using a chemiluminescence detection system (Chemiluminescence; Amersham Pharmacia Biotechnology, USA).
  • DK138 compound of the present invention exhibits anticancer effects by inducing apoptosis through caspase enzyme activity and PARP protein cleavage in colorectal cancer cells.

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Abstract

La présente invention concerne un nouveau dérivé de chalcone, et une composition anticancéreuse comprenant celui-ci comme principe actif.
PCT/KR2012/004183 2011-10-13 2012-05-25 Nouveau dérivé de chalcone et composition anticancéreuse comprenant celui-ci comme principe actif WO2013054998A1 (fr)

Applications Claiming Priority (12)

Application Number Priority Date Filing Date Title
KR1020110104580A KR101333734B1 (ko) 2011-10-13 2011-10-13 벤조히드록시메톡시칼콘을 유효성분으로 포함하는 항암제 조성물
KR10-2011-0104580 2011-10-13
KR1020110105807A KR101283004B1 (ko) 2011-10-17 2011-10-17 벤조칼콘을 유효성분으로 포함하는 항암제 조성물
KR10-2011-0105807 2011-10-17
KR10-2011-0109130 2011-10-25
KR10-2011-0109129 2011-10-25
KR1020110109128A KR101290579B1 (ko) 2011-10-25 2011-10-25 벤조칼콘을 유효성분으로 포함하는 항암제 조성물
KR1020110109129A KR101290578B1 (ko) 2011-10-25 2011-10-25 히드록시메톡시벤조칼콘 화합물을 유효성분으로 포함하는 항암용 조성물
KR1020110109130A KR101333736B1 (ko) 2011-10-25 2011-10-25 히드록시메톡시칼콘 화합물을 유효성분으로 포함하는 항암용 조성물
KR1020110109127A KR101325783B1 (ko) 2011-10-25 2011-10-25 나프탈레닐페닐디히드로피라졸을 유효성분으로 포함하는 항암제 조성물
KR10-2011-0109128 2011-10-25
KR10-2011-0109127 2011-10-25

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KR101566497B1 (ko) * 2013-08-29 2015-11-05 건국대학교 산학협력단 신규한 칼콘 유도체 및 그 유도체를 포함하는 항암 조성물
WO2015166040A3 (fr) * 2014-05-01 2016-01-14 De Montfort University Composé
CN105777520A (zh) * 2014-12-23 2016-07-20 浙江海洋学院 一种新型查尔酮化合物Chalcone-1203及其组合物、制备方法和应用
WO2019114125A1 (fr) * 2017-12-11 2019-06-20 Life-Garden Biotech Nouveau composé de quinochalcone et ses utilisations pour le traitement du cancer ou de l'inflammation
CN115260038A (zh) * 2022-07-18 2022-11-01 新乡医学院 一种治疗食管癌的新型查尔酮衍生物及其制备方法和医药用途

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KR101566497B1 (ko) * 2013-08-29 2015-11-05 건국대학교 산학협력단 신규한 칼콘 유도체 및 그 유도체를 포함하는 항암 조성물
WO2015166040A3 (fr) * 2014-05-01 2016-01-14 De Montfort University Composé
CN105777520A (zh) * 2014-12-23 2016-07-20 浙江海洋学院 一种新型查尔酮化合物Chalcone-1203及其组合物、制备方法和应用
WO2019114125A1 (fr) * 2017-12-11 2019-06-20 Life-Garden Biotech Nouveau composé de quinochalcone et ses utilisations pour le traitement du cancer ou de l'inflammation
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CN115260038A (zh) * 2022-07-18 2022-11-01 新乡医学院 一种治疗食管癌的新型查尔酮衍生物及其制备方法和医药用途
CN115260038B (zh) * 2022-07-18 2024-02-02 新乡医学院 一种治疗食管癌的新型查尔酮衍生物及其制备方法和医药用途

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