KR101690665B1 - Novel 2-hydroxy curcuminoid derivatives, a method for preparing the same and pharmaceutical compositions for anticancer property comprising the same - Google Patents

Abstract

More particularly, the present invention relates to a novel 2-hydroxycercuminoid derivative having anticancer activity that inhibits the growth of cancer cells by inducing active oxygen and apoptosis, A method for producing the same, and an anticancer composition comprising the same.

Description

[0001] The present invention relates to a novel 2-hydroxycocuminoid derivative, a process for preparing the same, and a novel anticancer composition comprising the same,

More particularly, the present invention relates to a novel 2-hydroxycercuminoid derivative having anticancer activity that inhibits the growth of cancer cells by inducing active oxygen and apoptosis, A method for producing the same, and an anticancer composition comprising the same.

Active oxygen occurs mainly in mitochondria, a small organ within the cell. It refers to various types of oxygen that are more unstable than normal oxygen in the body and thus have increased reactivity. Oxygen is usually a stable molecular state and active oxygen has more electrons attached to it.

It is inevitable that active oxygen occurs in the body. However, it acts as a deadly poison when it is over-produced in the body, but an appropriate amount is essential for life maintenance. Thus, active oxygen is a double-edged sword in the body.

It is known that 90% of modern diseases are related to active oxygen. Just as oxygen is delivered to all parts of our body, active oxygen can occur in any organ of our body. Since active oxygen damages cells and prevents regeneration, it is especially related to degenerative diseases such as cardiovascular disease, dementia, arthritis, and cataracts. Therefore, active oxygen is the source of panic and the main cause of aging.

When bacteria or viruses invade the body, white blood cells start attacking, and at this time, they make active oxygen and attack. Active oxygen acts as an essential defense mechanism to dissolve invading germs and foreign matter, and it informs other cells of this situation that our body becomes defensive. When an anticancer drug is added, active oxygen is generated to attack cancer cells. Thus, active oxygen is essential for immune function. However, if the amount of active oxygen is excessively increased, it will attack normal tissues and cause various diseases.

The proliferation and differentiation of the cells that make up the body are fundamental to the maintenance of life phenomena. Cell proliferation and growth for normal function is controlled by a sophisticated intracellular signaling pathway, which is a process in which a cell receives a signal from the outside of a cell, such as a PLC, PKC, Shc, Grb2, Raf, MAPK, MEK, etc.) and molecular mediators (GTP, cAMP, etc.). If any part of this process occurs, it will be balanced by its own regulation mechanism, but in many cases it will develop into a disease.

Among the genes involved in cell growth, anticancer drugs have been developed targeting genes that are specifically activated or inactivated by cancer cells, and many multinational pharmaceutical companies are developing anticancer drugs targeting these targets. However, cancer cells have the ability to induce tolerance to substances that regulate specific targets in an effort to survive, thereby neutralizing the efficacy of these drugs. Areas receiving attention from drug development strategy that emerged in order to overcome this problem is to target a specific biochemical function of cancer cells (Frantz, S. Drug approval triggers debate on future direction for cancer treatments. Nature Rev. Drug Discov., 5 , 91, 2006). A representative example is the rapid metabolism of cancer cells and the high concentration of active oxygen in cancer cells. As mentioned earlier, small amounts of free radicals are necessary for cell growth and differentiation, but excessive amounts of free radicals induce oxidation of nuclei and lipids in cells, leading to death. Therefore, homeostasis of free radicals is very important for cell growth and survival. To maintain intracellular homeostasis of these active oxygen species, various antioxidants exist (superoxide dismutases, glutathione peroxidase, peroxiredoxins, glutaredoxin, thioredoxin, catalase, etc.).

Many researchers have demonstrated that cancer cells have more active oxygen than normal cells (Szatrowski, TP & Nathan, CF Production of large amounts of hydrogen peroxide by human tumor cells. Cancer Res. 51 , 794, 1991 ). If a substance that can increase the amount of active oxygen from the outside is administered to the cancer cells, the amount of active oxygen of the cancer cell will be increased, and the cancer cells may be seriously affected. Thus, drugs that increase the amount of reactive oxygen species in cancer cells may selectively induce the death of cancer cells (Nature Review Drug Discovery 8, 579, 2009).

The cell cycle is divided into four distinct phases, as the clock we use on a daily basis is divided into second, minute, and hour. That is, Gap1 (G1), DNA synthesis (S phase), Gap2 (G2), and mitosis phase. In addition, when the cells are present at high density or left in the environment of a low concentration of growth factors for a long period of time, the cells are folded into a growth stop (Go) state called the dormant phase. This series of nuclear reactions is called the cell line.

Inside the watch, the clock is further vibrated by electrical or physical force, and accordingly the second, minute, and hour hands move sequentially to give us the correct time. To this end, a certain amount of force must be applied to the watch, and various accessories that control it constitute the watch. Likewise, the cell cycle also has a complex network called a checkpoint, which leads the cell clock to proceed exactly in the order of G1-S-G2-M. After confirming that the conditions for progressing from the checkpoint to the next stage of the cell cycle are met, the S or M phase proceeds if all conditions are met. The lack of this checkpoint control mechanism increases the instability of the genetic material, resulting in uncontrolled cell growth, and sometimes even cancer-like cell growth.

If signal transmission and nutritional status from outside the nucleus is good, the cell size of the G1 phase becomes larger and allows it to enter the cell cycle. Cell cycle activation occurs at the G1 checkpoint called START in yeast, and restriction point in mammalian cells. After this point, if there is no special braking, the cell will automatically clone and divide its genome through a four-step cell cycle. These steps in mammalian cells are detailed below. The G1 phase, where checkpoints are present, is a preparation phase for the production of new cells. At this time, if the growth factors and sufficient nutrients are not supplied, the cell cycle is stopped and the growth state stops. However, when sufficient nutrients are supplied and various growth factors are prepared, the cell cycle proceeds to S phase. At this time, the cell not only replicates its own genome, but also replicates several factors in the cytoplasm to divide into two cells. At the end of the S phase, the cells enter the G2 stage, known as the second checkpoint. The main mechanism during this G2 regulates DNA replication and completion and prepares for entry into the splitter. Therefore, various factors necessary for cell organization are produced at this time. After sufficient factors are established to divide into two cells, the process proceeds to the M-group, the cleavage site where substantial cell division occurs, and the M-group is the shortest and most dramatic stage in time. This is because the cloned genome is separated into the cell's poles and two daughter cells are created. This series of processes is a process that all cells undergo in order for a cell to grow and divide into two cells. Therefore, the study of the cell cycle and the development of a substance that regulates them are essential for the study of cell growth mechanism and for the development of a preventive and therapeutic agent for cancer caused by abnormal cell cycle (Nature Review Cancer 2001, 1, 222-231).

Under these circumstances, the present inventors have proposed a novel 2-hydroxycercuminoid derivative which inhibits abnormal growth and induces the death of cancer cells by inducing reactive oxygen species in cancer cells to stop the growth of cancer cells in the cell cycle G2 / M phase Thereby completing the present invention.

It is an object of the present invention to provide a novel 2-hydroxycercuminoid derivative or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a process for preparing the novel 2-hydroxycercuminoid derivative or a pharmaceutically acceptable salt thereof.

Still another object of the present invention is to provide an anticancer composition comprising the novel 2-hydroxycecuminoid derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

In one aspect, the present invention provides a 2-hydroxycercuminoid derivative represented by the following formula (I): or a pharmaceutically acceptable salt thereof.

In this formula,

R is hydrogen, a C ₆ -C ₁₀ aryl group or a C ₆ -C ₁₀ arylcarbonyl.

Preferably, in Formula 1,

R is hydrogen or benzoyl.

In a more preferred embodiment, the present invention provides a compound selected from the group consisting of the following Chemical Formulas 2 to 3, or a pharmaceutically acceptable salt thereof.

The term " aryl " as used herein means a mono- or poly-cyclic aromatic ring having 6 to 10 carbon atoms and includes, for example, phenyl, naphthyl and the like. A phenyl group is particularly preferred.

The compounds of formula (I) of the present invention may be prepared by pharmaceutically acceptable salts and solvates according to methods conventional in the art.

Salts are useful as acid addition salts formed by pharmaceutically acceptable free acids. The acid addition salt is prepared in a conventional manner, for example, by dissolving the compound in an excess amount of an aqueous acid solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The same molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be subjected to suction filtration.

As the free acid, organic acids and inorganic acids can be used. As the inorganic acids, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used. Examples of the organic acids include methanesulfonic acid, p- toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycollic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid and hydroiodic acid may be used. It is not limited.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble compound salt, and evaporating and drying the filtrate. At this time, as the metal salt, it is preferable to produce sodium, potassium or calcium salt particularly, but not limited thereto. The corresponding silver salt can also be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

The pharmaceutically acceptable salts of the compounds of formula (1) include, unless otherwise indicated, salts of acidic or basic groups which may be present in the compounds of formula (1). For example, pharmaceutically acceptable salts may include sodium, calcium and potassium salts of hydroxy groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, (Mesylate) and p -toluenesulfonate (tosylate) salt, and the like, and a method for producing a salt known in the art ≪ / RTI >

In another aspect, the present invention provides a process for producing a 2-hydroxycercuminoid derivative represented by the general formula (1).

Compounds of formula (I) wherein R is hydrogen may be prepared as in Scheme 1 below, comprising the steps of:

Dissolving the hydroxybenzaldehyde in an organic solvent and reacting with acetylacetone in the presence of boroxide and tributylborate.

[Reaction Scheme 1]

In the above Reaction Scheme 1, dimethylformamide or the like can be used as an organic solvent.

Compounds of formula (I) wherein R is benzoyl may be prepared as in scheme 2, comprising the steps of:

1) dissolving hydroxybenzaldehyde in an organic solvent and reacting with acetylacetone in the presence of boroxide and tributylborate; And

2) reacting the compound prepared in the above step with benzoyl chloride in the presence of a carbonate compound.

[Reaction Scheme 2]

In the above Reaction Scheme 2, as the organic solvent in the first step, dimethylformamide or the like may be used. In addition, the second-step reaction of the above-mentioned Reaction Scheme 2 is carried out in the presence of a carbonate compound for at least 10 hours and for 11 to 13 hours. At this time, as the carbonate compound, potassium carbonate and the like can be used.

In order to analyze the structure of the compound prepared in the present invention, first, the molecular weight and the molecular formula of the pure purified compound are determined by performing a UV absorbance analysis, an infrared (IR) absorbance analysis and a high-resolution mass spectrometry analysis. Specifically, the UV absorbance was measured with a Shimadzu UV-265 spectrophotometer (Shimadzu UV-265 spectrophotometer), and the IR absorbance was measured with a Bio-Rad Digilab Division FTS-80 spectrophotometer from Bio- 80 spectrophotometer, and the molecular weight and the molecular formula are determined by measuring a high resolution MS using a VG70-SEQ mass spectrometry (MS). Furthermore, ¹ H, ¹³ C-NMR spectra can be obtained using a nuclear magnetic resonance machine (Varian 300 MHz, 500 MHz NMR), and these spectra can be analyzed synthetically to finally determine the structure.

In another aspect, the present invention provides an anticancer composition comprising a 2-hydroxycercuminoid derivative represented by the formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.

In the present invention, anti-cancer includes both prevention and therapeutic effects of cancer.

Wherein the cancer is selected from the group consisting of colon cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, head cancer, head cancer, cervical cancer, skin melanoma, intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, Endometrioid cancer, thyroid cancer, pituitary cancer, soft tissue sarcoma, soft tissue sarcoma, urethral cancer, penile cancer, endometrial carcinoma, endometrial carcinoma, endometrial carcinoma, endometrial cancer, uterine cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, Cancer of the prostate, chronic leukemia, acute leukemia, lymphocytic lymphoma, bladder cancer, renal cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, and central nervous system (CNS) tumors.

The compounds according to the present invention are excellent in the effect of inhibiting the abnormal growth of cancer cells and thus can be usefully used for the treatment and prevention of cancer diseases.

In one embodiment, treating the compounds of formula (1) with 10 microM of the human colon cancer cell line SW620 inhibits cell growth by stopping the cell cycle G2 + M period. Specifically, the cell growth inhibitory effect experiment can be carried out as follows. First, the compound dissolved in DMSO was treated with the culture solution of the human colorectal cancer cell line SW620, and 20 hours later, the cell DNA was stained. The stained cells were stained with a Becton-Dickinson FACS calibur (Becton-Dickinson, The cell cycle was measured using a Becton-Dickinson Modifit cell cycle analysis program and the amount of cells in the G1, S, G2 + M phase of the cell cycle Is calculated as a percentage.

The pharmaceutical composition comprising the compound of formula (I) according to the present invention may further comprise an appropriate carrier, excipient or diluent according to a conventional method. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, oral preparations, suppositories or sterilized injection solutions according to a conventional method.

In detail, when formulating, it can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, Can be mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of liquid formulations for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the non-aqueous solution and suspension include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the compound according to the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention can be administered in an amount of 1 to 50 mg, preferably 5 to 50 mg per kg of body weight per day, once or several times.

The pharmaceutical dosage forms of the compounds according to the invention may also be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in suitable aggregates.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The 2-hydroxycercuminoid derivative of formula (I) or a pharmaceutically acceptable salt thereof according to the present invention is effective for inducing apoptosis by specifically inducing reactive oxygen species in cancer cells and for inhibiting the cell cycle G2 + M group To inhibit abnormal growth, thereby exhibiting an excellent anti-cancer effect.

Figure 1 shows the NMR spectrum of the compound of formula (2) of the present invention.
Fig. 2 shows the results of comparing the effect of generating active oxygen on various structures of cucuminoid compounds.
FIG. 3 shows the results of measurement of active oxygen generation effect over time for the compound of formula (2) of the present invention.
FIG. 4 is a graph illustrating the results of measuring the effect of the compound of formula (2) according to the present invention on the production of active oxygen.
FIG. 5 shows the results of analysis of the effect of the compound of formula (2) of the present invention on the cell cycle.
FIG. 6 shows the results of measurement of inhibition of human cancer cell growth by the compound of formula (2) of the present invention using WST-1.
FIG. 7 shows the results of measuring the inhibition rate of cancer cell proliferation according to the concentration of the control (compound untreated) concentration in the colorectal cancer cell line SW620 for the compound of formula 2 of the present invention.
Fig. 8 shows the results of investigating the effect of the compound of formula (2) of the present invention on apoptosis.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Preparation of compound of formula (2)

(0.7 g, 10 mmole) and acetylacetone (1 g, 10 mmole) were stirred at 70 ° C for 30 minutes, then tributylborate (1 g, 10 mmole) and salicylic aldehyde (1.2 g, 10 mmole) And the mixture was stirred at 70 캜 for one hour. Butylamine (0.7 g, 10 mmole) diluted in ethyl acetate was then slowly added dropwise over 15 minutes and further stirred at 80 ° C for 3 hours. 10 mL of 1N hydrochloric acid was added, followed by stirring for 30 minutes to complete the reaction. The reaction product was separated and purified, and then reacted with sodium hydride (0.3 g, 12 mmole) and bromo (methoxy) methane (1.2 g, 10 mmole) using tetrahydrofuran as a solvent. As a result of the reaction, the alcohol functional group was protected with methoxymethyl ether, and the synthesized intermediate was prepared by dissolving iodomethane (3.1 g, 22 mmole) with acetone (20 mL) in which potassium carbonate (2 g) Lt; / RTI > After separation and purification of the reaction substance, 10 ml of 1N hydrochloric acid was added to 25 ml of methanol as a solvent to remove the protecting group, and the mixture was reacted at 70 ° C for 3 hours. The organic solvent layer containing the active material was collected and concentrated under reduced pressure. The concentrated extract was dissolved in methylene chloride and the active fraction was separated and purified by column chromatography on silica gel (Merck, Art No. 9385) at a ratio of ethyl acetate to hexane of 20:80 to obtain 1.7 g of a pale yellow compound (2) (Yield: 50%). The physicochemical properties of the compound of formula (2) are shown in Table 1, and the hydrogen NMR spectrum is shown in FIG.

_{^{1 H-NMR (CDCl 3)}} : 8.01 (2H, d, J = 15.9), 7.45 (2H, d, J = 8.1), 7.22 (2H, t, J = 7.2), 6.93 (4H, m), 6.79 (2H, d, J = 8.1), 1.48 (6H, s)

Appearance Light yellow Molecular formula C ₂₁ H ₂₀ O ₄ Molecular Weight 336.38 Melting point (캜) 110 Solubility Availability Methanol, ethylacetate, acetone Insoluble water

Example 2: Preparation of compound of formula (3)

The compound of Formula 2 (302 mg, 0.9 mmole) obtained in Example 1 was dissolved in acetone to which potassium carbonate (2 g) was added, benzoyl chloride (200 mg, 1.8 mmole) was added, and the mixture was reacted at room temperature for 2 hours. After the reaction was completed, the organic solvent layer containing the active material was collected and concentrated under reduced pressure. The concentrated extract was dissolved in methylene chloride, and the active fraction was separated by column chromatography on silica gel (Merck, Art. No. 9385) at a ratio of ethyl acetate to hexane of 10: 90 to obtain 390 mg of a pale yellow compound (3) Yield: 80%). The physicochemical properties of the compounds of formula (3) are shown in Table 2 below.

¹ H-NMR (CDCl ₃ ): 8.19 (4H, d, 7), 7.79 (2H, d, J = 15.5), 7.63 , < / RTI > m), 7.23 (4H, m), 1.30 (6H, s)

Appearance Light yellow Molecular formula C ₃₅ H ₂₈ O ₆ Molecular Weight 544.59 Melting point (캜) 127 Solubility Availability Methanol, ethylacetate, acetone Insoluble water

Experimental Example 1: Comparison of active oxygen induction of various structures of cucuminoid compounds

The effects of active oxygen generation on the various structures of the following cucuminoid compounds were compared.

A specific experimental method was performed as follows.

First, to measure the total amount of active oxygen in human colorectal cancer cells (SW 620), the cells were cultured in RPMI 1640 medium containing 10% FBS at 37 ° C in the presence of 5% carbon dioxide. The cultured cells were suspended in 0.05% trypsin Cells were detached using EDTA (trypsin-EDTA) and cells were seeded at a concentration of 5000 cells / well in a 96-well black plate. After 24-hour incubation, the cells were treated with 10 μM DCF-DA (Sigma-aldrich), a fluorescent reactive substance sensitive to reactive oxygen species, in each well for 10 minutes. After washing of DCF-DA with PBS, fluorescence was measured using a Wallac 1420 VICTOR2 multilabel counter. The fluorescence intensities of the cells treated with the control and each sample were measured with an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

The measurement results are shown in Fig.

2, it was found that compounds having a 2-hydroxy group produced a relatively large amount of active oxygen. In particular, it was found that the compounds of the present invention, compound No. 7 (compound of formula 3) It was found that it has the highest active oxygen generating effect among the cucuminoid compounds.

On the other hand, the activity of generating active oxygen over time was measured for the 7th compound (compound of formula 2).

As a specific experimental method, the same method as described above was used except that the incubated cells were treated with time in the treatment of the samples.

The results are shown in Fig.

3, it was confirmed that the effect of the 7th compound (compound of formula 2) was increased with time.

In addition, the active oxygen generation effect of the compound of Chemical Formula 2 of Example 1 according to the concentration was measured.

As a specific experimental method, the same method as described above was used except that the incubated cells were treated with the concentration in treating the sample.

The results are shown in Fig.

FIG. 4 shows that when the compound of Chemical Formula 2 is treated, the effect of producing active oxygen increases with increasing concentration.

Experimental Example 2: Cell cycle analysis

For cell cycle analysis, the human colon cancer cell line SW620 was divided into T25 flasks (7.5 ml of culture) using RPMI 1640 and DMEM medium containing 10% FBS, and cultured in a 37 ° C incubator for 12 hours. After 12 hours of culture, DMSO was added to a final concentration of 0.1% (7.5 μl) in a cell culture used as a control, and 7.5 μl of a sample dissolved in DMSO at various concentrations was added to the cell culture used as a treatment group . Cell lines treated with DMSO or samples were separated and used for cell analysis at 48 hours after each treatment.

For cell cycle analysis, the cultured cells were removed from the flask and then centrifuged (300 g, 5 minutes) using trypsin in a culture flask. Separated cells were washed twice with phosphate buffer to remove media components. The cells thus prepared were treated with 3 ml of 70% ethanol and allowed to stand at -20 ° C for 12 hours to fix the cells. Cells immobilized with ethanol were centrifuged (300 g, 3 minutes), and the cells were washed twice with cold PBS to remove residual ethanol. After adding 50 μl of 100 μg / ml RNase A, the cells were incubated at 37 ° C for 30 minutes and treated with 10 μl of 1 mg / ml propidium iodide (in PBS) DNA was stained. The stained cells were counted in 20,000 cells using a Becton-Dickinson FACS calibur (Becton-Dickinson, San Jose, Calif., USA), and the cell cycle of Becton-Dickinson Modifit) cell cycle analysis program was used to calculate the percentage of cells in the G1, S, G2 + M phase of the cell cycle.

The results of analyzing the effect of the compound of formula (2) on the cell cycle are shown in Table 3 and FIG.

Cell Cycle Inhibitory Activity in Cancer Cells Cell line compound density % of Cells G0-G1 S G2-M SW620 Control group 10 μM 42 21 20 Compound of formula 2 37 14 32

As shown in Table 3 and FIG. 5, the compound of Formula 2 of the present invention showed an excellent effect of inhibiting cell division by stopping G2 / M-phase cell growth, which is an important factor in the cell cycle. Thus, it was confirmed that the compound of formula 2 of the present invention is useful for treating or preventing cancer by inhibiting abnormal growth of cancer cells.

Experimental Example 3: Anticancer activity in cell line

The inhibition of human cancer cell growth by the compound of formula (2) of the present invention was measured using WST-1. Each human cancer cell line was cultured in a medium containing 10% fetal bovine serum (FBS) at 37 ° C with 5% carbon dioxide, and cells were detached using 0.05% trypsin-EDTA. Each well of a 96-well plate was dosed with 4,000 cells each calculated with a hematocytometer. The cells were cultured in a medium containing 10% FBS in a 37 ° C. incubator containing 5% carbon dioxide, and after 24 hours, the cells were cultured in the control (0.1% DMSO) or 5, 10, 15, 20, 25, 30 μg / Was replaced with fresh medium containing the concentration-dependent (compound dissolved in DMSO diluted with the medium). After treating the drug for 48 hours, 10 ㎕ of WST-1 (manufactured by Roche) was added to each well and incubated for 2 hours. Then, the absorbance at 450 nm was measured using an ELISA reader (Bio-Rad) Were measured.

The results are shown in Fig.

6, DLD-1, HCT116, and SW620 showed a 50% growth inhibitory effect (GI ₅₀ ) at a concentration of 10 μM or less after 48 hours, indicating that the growth of colon cancer tissue was inhibited. In addition, SK-MEL-28, MCF-7, MDA-MB-468 and MDA-MB-231 exhibited a 50% growth inhibitory effect (GI ₅₀ ) at a concentration of 20 μM below the inhibition of skin cancer and breast cancer tissue growth MiaPaCa2 and A549 showed a 50% growth inhibitory effect (GI ₅₀ ) at a concentration of about 30 μM, and the growth of pancreatic cancer and lung cancer tissues was also inhibited.

On the other hand, anti-cancer effect was investigated by measuring inhibition rate of cancer cell proliferation according to the concentration of control (compound untreated) in colorectal cancer cell line SW620 against the compound of formula (2).

The results are shown in Fig.

7, it was confirmed that the growth of the SW620 cell line was further suppressed as the concentration of the compound of Chemical Formula 2 was increased. In particular, the compound of formula (2) exhibited a 50% growth inhibitory effect (GI ₅₀ ) at a concentration of 10 μM in SW620.

Experimental Example 4: Influence on cell death

The effect of the compound of formula (2) of the present invention on apoptosis was examined.

Poly (ADP-Ribose) polymerase (hereinafter referred to as PARP) is a marker for confirming the induction of apoptosis, and is a typical intracellular repair system that enables the maintenance of gene stability against external stimuli. When cell death occurs, PARP is cleaved and cleavage of such PARP is considered to be a typical signal of apoptosis.

Therefore, in order to confirm the cell death-inducing activity of the compound of formula (2) of the present invention, the following experiment was conducted.

SW620 cells were inoculated into a 100 mm cell culture dish at 10 x 10 ⁵ cells. After 18 hours, the compound of formula 2 was treated at a concentration of 10 uM and incubated for 48 hours. To analyze the cleavage of PARP by the compound of formula 2, the treated cells were dissolved in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, 0.5% sodium deoxycholate, 0.05% sodium deoxy sulfate) . The cell lysate was centrifuged at 1080 rpm to recover the supernatant, and the protein concentration of the recovered cell lysate was measured using a Bradford reagent (Bio-Rad protein assay, USA).

Proteins were separated by 30% by mass of SDS-polyacrylamide gel electrophoresis (7.5% SDS-PAGE), electrophoresed on PVDF membrane (Millipore, USA) and incubated with TBST (50 mM Tris- , 150 mM NaCl, 0.1% tween 20). The cells were reacted with PARP antibody (Cell signaling, USA) and Actin antibody (Sigma, USA) for 1 hour and reacted with HRP conjugated secondary antibody (Jackson Immunolab, USA) for 1 hour. Then, chemiluminescence POD reagent ). ≪ / RTI >

The results are shown in Fig.

8, it was confirmed that the cells treated with the compound of Chemical Formula 2 had a reduced amount of normal PARP protein and an increased amount of cleaved form of PARP protein as compared with the cells treated with DMSO. Therefore, it was found that the compound of formula (2) induces the death of cancer cells. On the other hand, when the active oxygen inhibitor GSH or NAC was treated simultaneously, the cleaved form of PARP protein could not be observed. Through this, it was confirmed that apoptosis by the compound of formula (2) is related to active oxygen.

Claims (10)

A 2-hydroxycercuminoid derivative represented by the following formula (1): < EMI ID =
[Chemical Formula 1]

In this formula,
And R is C ₆ -C ₁₀ arylcarbonyl. The compound according to claim 1, wherein in Formula 1,
Wherein R is benzoyl, or a pharmaceutically acceptable salt thereof. The 2-hydroxycercuminoid derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein the 2-hydroxycercuminoid derivative is a compound represented by the following formula (3).
(3)

delete delete A process for preparing a 2-hydroxycercuminoid compound represented by the following formula (1), wherein R is benzoyl, which comprises the steps of:
1) dissolving hydroxybenzaldehyde in an organic solvent and reacting with acetylacetone in the presence of boroxide and tributylborate; And
2) reacting the compound prepared in the above step with benzoyl chloride in the presence of a carbonate compound.
[Chemical Formula 1]

The process for preparing a 2-hydroxycercuminoid compound according to claim 6, wherein the organic solvent in the first step is dimethylformamide. 7. The process for producing a 2-hydroxycercuminoid compound according to claim 6, wherein the carbonate compound of the second step is potassium carbonate. An anticancer composition comprising the compound of any one of claims 1 to 3 as an active ingredient. 10. The method of claim 9, wherein the cancer is selected from the group consisting of colon cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, cervical cancer, skin melanoma, intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, The present invention relates to a method for treating cancer in a patient suffering from cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophagus cancer, small bowel cancer, endocrine cancer, (CNS) tumors characterized in that they are cancer of the urethra, cancer of the urethra, penile cancer, prostate cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureter cancer, kidney cell carcinoma, renal pelvic carcinoma or central nervous system Composition.

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