KR101283004B1 - Anticancer composition containig the benzochalcone - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of cancer diseases.
According to the present invention as described above, benzochalcone having a growth inhibitory and killing effect of cancer cells through poly ADP-ribose polymerase-1 (PARP-1) cleavage by a caspase activity, a cell death regulating protein By providing a pharmaceutical composition for the prevention and treatment of cancer diseases, including), there is an effect that can be usefully used to prevent and treat cancer diseases.

Description

Anticancer composition comprising benzochalcone as an active ingredient {ANTICANCER COMPOSITION CONTAINIG THE BENZOCHALCONE}

The present invention relates to a pharmaceutical composition for the prevention and treatment of cancer diseases, and more particularly, poly ADP-ribose polymerase-1 (PARP-1, poly (ADP) by caspase (caspase) enzyme activity, apoptosis control protein. The present invention relates to a novel benzochalcone compound represented by the following Chemical Formula 1 having a growth inhibitory and killing effect of cancer cells through cleavage, and a pharmaceutical composition for preventing and treating cancer diseases.

[Formula 1]

Colorectal cancer is one of the advanced national diseases, and Korea is entering advanced countries, and its eating habits are also becoming western. Colorectal cancer is the number one mortality rate among Korean women and third among males. Like other cancers, surgery, radiation therapy, chemotherapy, etc. are used to treat the postoperative cure for cancer, and many anticancer drugs have not been developed yet. Therefore, the development of new anticancer drugs is necessary. In addition, anticancer drugs can be used, but because they are far from cured, there is still a need to develop new colorectal cancer treatments. In addition, since colon cancer has no early symptoms, it is often found after significant progress, and thus development of a preventive agent is also necessary.

In order to develop anticancer drugs, screening methods for measuring anticancer effects are required. Among the various methods, poly ADP-ribose polymerase-1 (PARP-1) is a nicotinamide adenine dinucleotide. An enzyme that catalyzes poly ADP-ribosyl action, a kind of post-translational modification, to various proteins by converting it into nicotinamide using a substrate as a substrate. to be. When the DNA is severely damaged, the enzyme increases its expression, activating proteins poly ADP-ribosyl. DNA lyase (Dnase) also is one of the protein, calcium ²⁺ / Mg ²⁺ through the process - to inhibit the operation of the dependent DNA lyase ^{Ⅰ (Ca 2+ / Mg 2+ -dependentDnaseⅠ} ). In other words, when PARP-1 is increased, the activity of DNAase I is inhibited. In contrast, when PARP-1 is cleaved and loses its activity, it induces the activation of DNAase I, which is involved in DNA degradation. This leads to cleavage of chromosomal DNA, which induces apoptosis (Genes to Cell, 9, 1103, 2004; Cell Death Differentiation, 14, 992, 2007). Apoptosis occurs differently between normal cells and cancer cells, so it is more lethal to cancer cells, so the substance causing apoptosis can be used as an anticancer agent.

As a result of the cell apoptosis, while searching for a substance showing an anticancer effect, it was found that a substance of benzochalcone compound is effective for cell apoptosis. Calcon is a type of flavonoid that has a skeleton composed of two rings. The two rings at both ends are connected to each other with pentenone, and have been found during the production of the secondary metabolism of plants or as one of the secondary metabolites. However, since the known chalcon derivatives have difficulty in passing through cell membranes composed of hydrophobic lipid structures because the hydroxy group is mainly substituted on two rings, it is expected that the substitution of such hydroxy groups through the methyl group will more easily pass through the cell membrane. have. The present inventors designed a derivative having three methoxy groups by adding one more ring to two rings, and found that the derivative is a novel substance and shows an effective anticancer effect and named DK74. To produce the compound.

Poly ADP-ribose polymerase-1 (PARP-1) cleavage of this compound was performed to inhibit cancer cell proliferation and induce apoptosis. As a result, the present invention was completed by effectively killing cancer cell lines. .

Conventional techniques using a chalcone or a derivative thereof as a pharmaceutical composition have been disclosed in Korean Patent No. 10-0567125 (A matrix composition for inhibiting matrix metalloprotease activity containing a chalcone or a derivative thereof) by inhibiting MMP activity. Patent No. 10-0751899 (new chalcone derivatives, pharmaceutically acceptable salts thereof, preparation method thereof and glycosidase containing them as an active ingredient) which has the effect of preventing and treating diseases caused by glycosidase. Composition for the prevention and treatment of diseases caused by).

An object of the present invention is benzochalcone having growth inhibition and killing activity of cancer cells through poly ADP-ribose polymerase-1 (PARP-1) cleavage by caspase enzyme activity, an apoptosis control protein. It provides a compound and a pharmaceutical composition for the prevention and treatment of cancer diseases comprising the same.

In addition, another object of the present invention to provide a health functional food for the prevention and improvement of cancer diseases comprising a benzochalcone (benzochalcone) compound.

Finally, the present invention provides (E) -1- (2-hydroxynaphthalen-1-yl) -3- (2,4,6-trimeth, which is a benzochalcone compound having the activity of cleaving PARP-1. Methoxyphenyl) prop-2-en-1-one ((E) -1- (2-hydroxynaphthalen-1-yl) -3- (2,4,6-trimethoxyphenyl) prop-2-en-1-one ) Compound (DK74) and its use as an anticancer agent.

In order to achieve the above object, the present invention provides a novel benzochalcone compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Formula 1]

In addition, the present invention is a method for producing a benzochalcone compound represented by the formula (1), (1) 2-hydroxy-1-acetonaphthone (2-hydroxy-1-acetonaphthone) and 2,4, Dissolving 6-trimethoxybenzaldehyde (2,4,6-trimethoxybens-aldehyde) in ethanol; and (2) adding a 50% KOH aqueous solution to the mixed solution of step (1); and (3 A) stirring the mixed solution prepared by step (2) at room temperature and then cooling; And (4) neutralizing by adding 6N HCl solution to the mixed solution cooled by step (3); And (5) subjecting the benzochalcon compound solid produced in the neutralization process of step (4) to filtration and washing with ethanol; (6) recrystallizing the benzochalcon compound solid washed by the step (5) in ethanol; provides a method for producing a benzochalcone compound comprising a.

In addition, the present invention is a caspase, apoptosis control protein for 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 Pharmaceutically acceptable with the benzochalcone compound represented by the formula (1) having the inhibition and killing effect of cancer cells through poly ADP-ribose polymerase-1 (PARP-1) cleavage by the enzyme activity (caspase) It provides a pharmaceutical composition for the prevention and treatment of cancer diseases comprising salts.

The pharmaceutical composition is characterized in that it comprises one or more pharmaceutically acceptable carriers, diluents or excipients.

The pharmaceutical composition is characterized in that it comprises a second anticancer agent or anticancer adjuvant.

The second anticancer agent is interferon, interleukin-2, paclitaxel, vincristine, vinblastin, doxorrubicin, etoposide, etoposide, Irinotecan hydrochloride, cisplatin, amsacrine, cytosine arabinoside, fluorouracil and taxol It is done.

In addition, the present invention provides a health functional food for preventing and improving cancer diseases, including a benzochalcone compound represented by the formula (1) and a food supplement acceptable food supplement.

The health functional food is characterized in that the tablet, capsule, pills or liquid form.

According to the present invention as described above, a novel benzochalcon having growth inhibition and killing action of cancer cells through poly ADP-ribose polymerase-1 (PARP-1) cleavage by caspase enzyme activity, apoptosis control protein (benzochalcone) compound 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, can be usefully used to prevent and treat cancer diseases, There is an effect that can be usefully used in dietary supplements for prevention and improvement.

1 is a hydrogen nuclear magnetic resonance spectra of the benzochalcon compound (using a 400 MHz Bruker nuclear magnetic resonance spectrometer).
2 is a carbon nuclear magnetic resonance spectroscopy spectrum of a benzochalcone compound (using a 100 MHz Bruker nuclear magnetic resonance spectrometer).
3 is a Western blot investigation of the effect of PARP protein cleavage of the benzochalcon compound in SW620 cells.
Figure 4 shows the SW620 colon cancer cell proliferation inhibitory ability of the benzochalcon compound.
FIG. 5 is a flow cytometer of the apoptosis effect of the benzochalcon compound in SW620 cells (2N, diploid DNA; 4N, tetraploid DNA, M1, sub-G1; M2, G1; M3, S; M4, G2 / M).

Hereinafter, the present invention will be described in detail.

The present invention provides a novel benzochalcone compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[Formula 1]

The present invention is a method for producing a benzochalcone compound represented by the formula (1), (1) 2-hydroxy-1-acetonaphthone (2-hydroxy-1-acetonaphthone) and 2,4,6- Dissolving trimethoxybenzaldehyde (2,4,6-trimethoxybens-aldehyde) in ethanol; and (2) adding 50% KOH aqueous solution to the mixed solution according to step (1); and (3) Stirring the mixed solution prepared by step (2) at room temperature and then cooling; and (4) adding 6N HCl solution to the mixed solution cooled by step (3) to neutralize; and (5 A) filtration of the benzochalcon compound solid produced in the neutralization process of step (4) and washing with ethanol; And (6) recrystallizing the benzochalcon compound solid washed by the step (5) in ethanol. It provides a method for producing a benzochalcone compound comprising a.

The present invention is a caspase (caspase) which is an apoptosis regulating protein for 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, and blood cancer. A benzochalcone compound represented by Formula 1 and a pharmaceutically acceptable salt having growth inhibition and death of cancer cells through cleavage of poly ADP-ribose polymerase-1 (PARP-1) by enzyme activity It provides a pharmaceutical composition for the prevention and treatment of cancer diseases comprising.

The pharmaceutical composition may comprise 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 may include a second anticancer agent or an anticancer adjuvant. The second anticancer agent or anticancer agent includes interferon, interleukin-2, paclitaxel, vincristine, vinblastin, doxorrubicin, etoposide (etoposide), irinotecan hydrochloride, cisplatin, amsacrine, cytosine arabinoside, fluoro uracil and taxol It is not limited.

Pharmaceutical compositions containing the benzohydroxymethoxychalcone compounds of the present invention may be oral formulations, external preparations, suppositories, or sterile injections of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. according to conventional methods, respectively. It can be formulated and used in the form of a solution. Specifically, when formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like 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 benzohydroxymethoxychalcone compound. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. 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 sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

Preferred dosages of the benzohydroxymethoxychalcone compounds of the present invention vary depending on the condition and weight of the patient, the severity 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. However, for the desired effect, the benzohydroxymethoxychalcone compound of the present invention or a pharmaceutically acceptable salt thereof is 0.0001 to 100 mg / kg, preferably in an amount of 0.001 to 100 mg / kg once a day It can be administered in divided doses. The benzohydroxymethoxychalcone compound or pharmaceutically acceptable salt thereof of the present invention in the entire pharmaceutical composition 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. .

In addition, the present invention provides a health functional food for preventing and improving cancer diseases, including a benzochalcone compound represented by the formula (1) and a food supplement acceptable food supplement. For example, 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 benzohydroxymethoxychalcone compound or a pharmaceutically acceptable salt thereof to a vitamin complex and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example 1 Synthesis of Benzochalcone Compound (DK74)

In the present invention, the benzochalcone (DK-74) compound represented by Chemical Formula 1 was synthesized using the method shown in Scheme 1 below.

[Reaction Scheme 1]

Dissolve 2-hydroxy-1-acetonaphtone (1.86 g, 10 mmol) and 2,4,6-trimethoxybenzaldehyde (1.96 g, 10 mmol) in 40 ml ethanol with stirring and lower the temperature by about 3 ^° C. Then 3 ml of 50% KOH solution was slowly added for 3 minutes. Then, after stirring for 20 hours at room temperature, the reaction mixture was added to iced water and neutralized with 6N hydrochloric acid (HCl). The solid formed during the neutralization was filtered under reduced pressure and washed with cold ethanol to obtain a benzochalcone compound as a target compound. This benzochalcon compound was recrystallized in ethanol to give pure benzochalcon (DK-74) in 62% yield (226 mg). Mp; 110-111 ^o C.

 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. Hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectra were shown in FIGS. 1 and 2, respectively, and chemical mobility was as follows.

1 H-NMR (400 MHz) δ (multiplicity, filling constant / Hz): 2.57 (dd, 16.8, 3.2), 3.92 (dd, 16.8, 14.5), 3.78 (s), 3.83 (s), 6.05 (dd, 14.4, 3.2), 6.31 (s), 7.19 (d, 9.0), 7.46 (m), 7.65 (ddd, 8.5, 6.9, 1.3), 7.91 (d, 7.6), 8.09 (d, 9.0), 9.37 (d , 8.5)

13C-NMR (100 MHz) δ: 42.0, 55.4, 56.0, 71.5, 91.3, 105.4, 111.5, 119.2, 124.6, 125.0, 128.7, 128.7, 129.4, 131.1, 137.2, 159.9, 161.9, 164.4, 194.1

The benzochalcone compound (DK-74) is a novel substance whose molecular formula is C22H20O5 and has a molecular weight of 364.4.

Example 2. PARP (poly (ADP-ribose) polymerase) cleavage effect of benzochalcone compound (DK74)

In the present invention, in order to confirm the cancer suppressive activity effect of the benzochalcon compound (DK74), SW620 cells (human colon cancer cell line) were distributed in the ATCC (American Type Culture Collection, USA), the benzochalcon compound (DK74) according to the present invention ), And then the degree of fragment fragmentation of poly (ADP-ribose) polymerase (PARP) was investigated using Western blot analysis.

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 μM concentration of benzochalcone compound (DK74) compound, and cells were harvested after 0, 6, 12, 24 and 48 hours. 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 μg / 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. Primary antibody that binds to caspase-.3 protein (purchased from Cell Signaling Technology) and primary antibody that recognizes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) that does not change protein expression as a control (purchased from Santa Cruz technology) After reacting for 5 hours, the secondary antibody (purchased by Cell Signaling Technology, Inc.), which prints the primary antibody, was reacted for 1 hour. The change of expression of each protein on the X-ray film was analyzed using a chemistry fluorescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ).

As a result, as shown in FIG. 3, it was observed that when the benzochalcone compound (DK74) compound was treated to SW620 colorectal cancer cells, PARP fragment fragments of 89-kDa size were generated from 24 hours after treatment. PARP protein cleavage is induced by caspase-3 or caspase-7 enzyme activity, a cysteine protease. Caspase-3 and caspase-7 enzymes are usually present as inactive precursors and are cleaved and activated when apoptosis stimulation signals are delivered. In order to determine whether PARP cleavage by the benzochalcone compound (DK74) was induced by caspase enzyme activity, the production of active caspase-3 and caspase-7 fragments was investigated using the Western blot method. . When SW620 colorectal cancer cells were treated with DK74, the amount of GAPDH, the control protein, did not change, but the cleaved caspase-3 and caspase-7 proteins were increased after 24 hours of treatment. These results indicate that the benzochalcone compound (DK74) of the present invention induces cleavage of PARP protein through caspase-3 and caspase-7 enzyme activity.

Example 3. Growth Inhibition of Colon Cancer Cell Line by Benzocalcon Compound (DK74)

Growth inhibition of SW620 colorectal cancer cells by the benzochalcone compound (DK74) compound of the present invention was measured using a CCK-8 kit (Cell Counting Kit-8; Dojindo, Japan). 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 μM benzochalcone compound (DK74) compounds and CCK-8 solution after 24 and 48 hours, respectively. 10 μl was added. After 2 hours, the absorbance of the cell culture was measured at 450 nm to analyze cell viability.

As a result, as shown in FIG. 4, the cell growth capacity increased with time in the control cell group which was not treated, but in the cell group treated with the benzocalcon compound (DK74) compound, the cells were treated in a time-dependent manner with the concentration of the benzocalcon compound (DK74). Growth was reduced. These results indicate that apoptosis of SW620 colon cancer cells is induced by the benzochalcone compound (DK74) compound, thereby inhibiting cancer cell proliferation.

Example 4 Induction of Apoptosis of Colon Cancer Cell Lines by Benzochalcon Compound (DK74)

The main process of apoptosis by drug treatment can be confirmed from the contraction of the cells and the accompanying chromatin condensation and DNA fragmentation of DNA. This phenomenon occurs when the DNA content is measured in the cell cycle (Cell cycle) is less than the 2N DNA content of G1 phase cells (sub-G1) appears. Therefore, cells having a sub-G1 DNA content are generally considered to be cells in which apoptosis is in progress. In the present invention, in order to determine the effect of apoptosis induced by the benzochalcone compound (DK74) compound in colorectal cancer cells was measured intracellular DNA content using a flow cytometer (BD Science, USA).

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.

As a result, as shown in Figure 5, when the benzochalcon compound (DK74) of the present invention was treated at 5 and 10μM concentrations, the sub-G1 DNA content increased from 1.2% to 6.41% and 18.54%, respectively. Therefore, it was confirmed that the benzochalcone compound (DK74) compound according to the present invention induces apoptosis of SW620 colon cancer cells.

Having described specific portions of the present invention in detail, it will be apparent to those skilled in the art that this specific description is only a preferred embodiment and that the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

delete delete delete delete delete A method for inducing the activity of caspase-3 and -7 enzymes in vitro by treating benzochalcone compounds represented by Formula 1 to colon cancer cells in vitro.
[Formula 1]

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