KR101699201B1 - Phamaceutical composition for preventing or treating cancer comprising 2,6-Dibenzyl-4-chlorophenol compound - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating a cancer comprising 2,6-dibenzyl-4-chlorophenol compound. The 2,6-dibenzyl-4-chlorophenol compound suppresses proliferation of the cancer cells in a concentration-dependent manner at A549 cells, DU145 cells, Hela cells and HepG2 cells. The 2,6-dibenzyl-4-chlorophenol compound of the present invention can be utilized as the composition for preventing or treating the cancer.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for preventing and treating cancer, which comprises 2,6-Dibenzyl-4-chlorophenol compound as an active ingredient,

The present invention relates to a composition for preventing and treating cancer, which comprises 2,6-Dibenzyl-4-chlorophenol compound synthesized by a novel method as an active ingredient.

With the development of modern science and technology, along with the development of medical technology, biotechnology and genetic engineering, various researches and methods for treating cancer have been developed, but cancer is recognized as a serious disease that can not be cured yet. Many methods such as incision, chemotherapy, radiotherapy, and incision have been developed to treat these cancers, but radiation therapy or incision is mainly effective only when the initial diagnosis of cancer is made, It is of no use and can only be treated with chemotherapy. Since the 1940s, the chemotherapy introduced into cancer therapy has been attracting much attention because of its advantage that it can be applied relatively easily regardless of the timing of cancer, and various chemotherapeutic agents have been developed.

However, when the above-mentioned anticancer drugs are administered repeatedly for a long time or when the cancer recurs, the cancer cells lose the therapeutic effect by acquiring resistance to the anticancer drug. In addition, most anticancer drugs have the effect of inhibiting the synthesis of nucleic acid in the cell or directly binding to nucleic acid to impair its function. These anticancer drugs do not only act selectively on cancer cells, but also on normal cells, Which causes various side effects such as a decrease in bone marrow function, gastrointestinal mucosal damage, hair loss, and the like.

Recently, alternative medical treatments such as Chinese traditional medicine have been rapidly increasing not only in the western countries but also in Asia. So far, many natural compounds derived from nature have been shown to exhibit anticancer effects induced by apoptosis, and such natural compounds can be used as new and effective natural compounds based on the discovery of new drugs for the treatment and prevention of cancer.

Apoptosis is known to be a programmed cell death, a genetic control mechanism that plays an important role in maintaining cell homeostasis. Apoptosis can generally be induced through an exogenous (death receptor) pathway or endogenous (in mitochondria) pathway. In the exogenous pathway, the ligation of the TNF / Fas-receptor by its ligand induces cleavage of the caspase-8 initiator and directly activates the effector caspase-3 or activates Bcl-2 (Mellier et al., 2010; Tan et al., 2009; Wu, 2009) induces the disruption of Bax in the mitochondrial membrane after inducing cleavage of the family member Bid. On the other hand, the endogenous pathway is mediated by mitochondria, and in response to aortotoxic stimuli, cytochrome c and apoptosis-inducing factor (AIF) are released from the mitochondria and these factors are caspase-dependent and independent It is involved in apoptosis. Cytochrome c binds to Apaf-1

To form a structure called apoptosome, to activate caspase-9, followed by activation of caspase-3, and finally to apoptotic cell death. AIF is a marker of caspase-independent apoptosis, which, after apoptotic stimulation, translocates into the nucleus and induces DNA fragmentation (Huerta et al, 2006; Millan

and Huerta, 2009).

The prior art of the present invention is known from Korean Patent No. 10-1465238, but it relates to a composition comprising 4-isopropyl-2,6-bis (1-phenylethyl) phenol as an active ingredient, The technology is known from Korean Patent No. 10-1483915, but it relates to a composition containing 4-isopropyl-2,6-bis (1-phenylethyl) aniline as an active ingredient. European Patent EP 186255 discloses a method of synthesizing 2,6-Dibenzyl-4-chlorophenol. However, since the reaction time is longer than 7 hours and the yield is low, the efficiency is low (18.9%) and the polymer material ), But it has not been disclosed or implied for cancer prevention and treatment.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, or a composition for preventing and / or improving cancer, which contains 2,6-Dibenzyl-4-chlorophenol compound prepared by the novel method as an active ingredient.

The above object of the present invention is achieved by providing a natural anticancer agent through identification of a 2,6-Dibenzyl-4-chlorophenol compound synthesized by a novel method having excellent anticancer properties.

The present invention has an effect of providing a composition containing 2,6-Dibenzyl-4-chlorophenol compound synthesized by the novel method as an active ingredient. The compound 2,6-Dibenzyl-4-chlorophenol inhibits the proliferation of cancer cells in various cancer cell lines, not only increases the activity of signal transduction proteins that induce apoptosis of cancer cells, but also inhibits tumorigenesis in vivo There is an excellent effect in cancer prevention and treatment or cancer prevention and improvement.

1 is a graph showing the results of ¹ H-NMR analysis of 2,6-Dibenzyl-4-chlorophenol which is newly synthesized according to the present invention.
2 is a graph showing the results of ¹³ C-NMR analysis of 2,6-Dibenzyl-4-chlorophenol, which is newly synthesized according to the present invention.
FIG. 3 is a graph showing that 2,6-Dibenzyl-4-chlorophenol newly synthesized according to the present invention is not toxic to the normal cell line NIH-3T3.
FIG. 4 is a graph showing the killing effect of the newly synthesized 2,6-Dibenzyl-4-chlorophenol according to the present invention on various cancer cells (A549 cell, DU145 cell, Hela cell and HepG2 cell).
FIG. 5 is a photograph showing the cell death of A549 cells when A549 cells were treated with 2,6-Dibenzyl-4-chlorophenol at different concentrations.
FIG. 6 is a western blot result showing the change in the amount of pro-Caspase-3 by 2,6-Dibenzyl-4-chlorophenol in A549 cell.
FIG. 7 is a western blot result showing the change of Bcl-2 and Bax by 2,6-Dibenzyl-4-chlorophenol in A549 cell.
8 is a graph showing a change in the ratio of Bcl-2 to Bax by 2,6-Dibenzyl-4-chlorophenol in A549 cell.
9 is a western blot result obtained by confirming the change of the activity of p53 protein by 2,6-Dibenzyl-4-chlorophenol in A549 cell.

The present invention provides a composition for prevention and treatment of cancer, which comprises 2,6-Dibenzyl-4-chlorophenol compound as an active ingredient synthesized by a novel method.

The cancer may be selected from the group consisting of liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, colon cancer, lung cancer, ovarian cancer, rectum cancer, esophageal cancer, small intestine cancer, fallopian tube carcinoma, endometrial carcinoma, And is preferably selected from the group consisting of lung, prostate, cervical, and liver cancer, and is preferably selected from the group consisting of lung cancer, prostate cancer, vulvar cancer, , But it is not limited thereto.

The 2,6-Dibenzyl-4-chlorophenol compound reduces the amount of pro-caspase-3 in cancer cells, decreases the ratio of Bax and Bcl-2, which are regulators of cell death, and increases the activity of p53 protein to induce apoptosis But is not limited thereto.

When the composition of the present invention is used as a medicine, the pharmaceutical composition containing 2,6-Dibenzyl-4-chlorophenol compound as an active ingredient may be formulated into oral or parenteral dosage forms described below, It is not.

 Examples of the formulations for oral administration include tablets, pills, light / soft capsules, liquids, suspensions, emulsions, syrups, granules and elixirs. These formulations may contain, in addition to the active ingredient, a diluent (e.g., lactose, dextrose, (E.g., silica, talc, stearic acid and magnesium or calcium salts thereof and / or polyethylene glycols), such as, for example, water, rosin, sucrose, mannitol, sorbitol, cellulose and / or glycine. The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine and may optionally contain additives such as starch, agar, alginic acid or its sodium salt A disintegrating or boiling mixture and / or an absorbent, a colorant, a flavoring agent, and a sweetening agent.

The pharmaceutical composition containing 2,6-Dibenzyl-4-chlorophenol as an active ingredient can be administered parenterally, and parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. At this time, 2,6-Dibenzyl-4-chlorophenol may be prepared as a solution or suspension by mixing with water or a stabilizer or a buffer in order to formulate the preparation for parenteral administration, and it may be prepared into an ampule or vial unit dosage form. The composition may be sterilized or may contain other therapeutically useful substances such as preservatives, stabilizers, wettable or emulsifying accelerators, salts for controlling osmotic pressure and / or buffers, and other therapeutically useful substances, It can be formulated according to the coating method.

The present invention will be described in more detail with reference to the following specific examples and experimental examples. However, the following examples and experimental examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention.

Example 1. Synthesis by a novel method of 2,6-Dibenzyl-4-chlorophenol compound.

The 2,6-Dibenzyl-4-chlorophenol compound was newly synthesized as shown in Structural Formula 1 and Structural Formula 2 below.

… [구조식1]

... [Structural formula 1]

or,

… [구조식2]

... [Structural formula 2]

6.17 g of p- chlorophenol and 0.365 g of TsOH were placed in a 100 mL round bottom flask equipped with a magnetic bar. 1 mL of benzyl alcohol was added dropwise over 1 hour while stirring at 120 ° C. After addition, the reaction was further continued for 30 minutes and then cooled at room temperature. Ether (60 mL) was added and transferred to a separating funnel, washed three times with 30 mL of water, and washed seven times with 20 mL of 1 M NaOH solution. After washing twice with 30 mL of water, water was removed with MgSO ₄ and the ether was removed by rotary distillation. After drying in vacuo, the silica gel column was separated and purified with MeOH: CHCl ₃ = 1: 80 ratio. The ¹ H-NMR spectrum and ¹³ C-NMR spectrum of the final synthesized compound, 2,6-Dibenzyl-4-chlorophenol were confirmed in FIG. 1 and FIG. 2, respectively, and the three-dimensional structural formula of the compound was as follows.

… [2,6-Dibenzyl-4-chlorophenol의 3차원 구조식]

... [3-Dimensional Structure of 2,6-Dibenzyl-4-chlorophenol]

Yield of 2,6-Dibenzyl-4-chlorophenol: 1.37 g (25%);

¹ H-NMR (?): 3.92 s, 4H, CH ₂ ; 4.60 s, 1H, OH; 6.99 s, 2H, 3,5-H; 7.18 d, 4H, 2 ', 6 ' -H, J = 8.41 Hz; 7.22 t, 2H, 4 ' -H, J = 7.43 Hz; 7.30 t, 4H, 3 ', 5 ' -H, J = 7.57 Hz;

¹³ C-NMR (ppm): 36.90 CH ₂ , 125.72 2-C, 127.17 3-C, 129.06 3'-C, 129.25 4'-C, 129.40 4-C, 139.20 1'-C, 151.18.

Experimental Example 1 It was confirmed that 2,6-Dibenzyl-4-chlorophenol was not toxic.

NIH-3T3 cells (fibroblasts) were used to confirm the toxicity of 2,6-Dibenzyl-4-chlorophenol, which was synthesized through Example 1, and confirmed by MTT assay. The MTT assay is an experimental method used to measure the viability of cells by colorimetric assay using the light absorbance method.

To evaluate this, NIH-3T3 cells were plated on a 96-well plate at 1 × 10 ⁴ cells / well and treated with 12.5-50 μM of 2,6-Dibenzyl-4-chlorophenol compound by concentration. After incubation at 37 ° C in a 5% CO ₂ incubator for 24 hours, MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphinytetrazolium bromide) was added at a concentration of 1 mg / Lt; / RTI > After completion of the reaction, 100uL of dimethyl sulfoxide was added to each well to dissolve formazan crystal. The cell viability was measured by measuring the absorbance at 570 nm of MTT reduced to formazan, and the OD value was obtained using the group without the addition of 2,6-Dibenzyl-4-chlorophenol as a control.

As shown in FIG. 3, the 2,6-Dibenzyl-4-chlorophenol compound showed almost no toxicity in the normal cell line NIH-3T3 as compared with the control group.

Experimental Example 2 Effect of 2,6-Dibenzyl-4-chlorophenol compound on cancer cell death.

A549 cell, DU145 cell (prostatic cancer cell), Hela cell (cervical cancer cell) and HepG2 cell were used to confirm the cancer cell killing effect of the new 2,6-Dibenzyl-4-chlorophenol compound synthesized in Example 2 above. cell (liver cancer cell) was used and confirmed by MTT assay.

To evaluate this, NIH-3T3 cells were plated on a 96-well plate at 1 × 10 ⁴ cells / well and treated with 12.5-50 μM of 2,6-Dibenzyl-4-chlorophenol compound by concentration. After incubation at 37 ° C in a 5% CO ₂ incubator for 24 hours, MTT was added at a concentration of 1 mg / mL and further reaction was induced for 2 hours. After completion of the reaction, 100uL of dimethyl sulfoxide was added to each well to dissolve formazan crystal. The cell viability was measured by measuring the absorbance at 570 nm of MTT reduced to formazan, and the OD value was obtained using the group without the addition of the 2,6-Dibenzyl-4-chlorophenol compound as a control. The killing effect on cancer cells was indicated by IC50.

As shown in FIG. 4, the cell death of the cancer cells against the 2,6-Dibenzyl-4-chlorophenol compound was inhibited in a concentration-dependent manner. IC50 was measured as 32uM for A549 cell, 29uM for DU145 cell, 31uM for Hela cell and 28uM for HepG2 cell.

Experimental Example 3. Morphological killing effect of 2,6-Dibenzyl-4-chlorophenol compound on lung cancer cell line A549 cells.

The morphological cell killing effect of the novel 2,6-Dibenzyl-4-chlorophenol compound synthesized in Example 2 on A549 cells, a lung cancer cell line, was confirmed by optical microscope.

To evaluate this, A549 cells were cultured on a 6-well plate at a concentration of 1 × 10 ⁴ cells / mL, treated with 30 to 60 μM of 2,6-Dibenzyl-4-chlorophenol compound by concentration, Type.

As a result, as shown in FIG. 5, A549 cell death was observed at 24 hours after treatment with 30 μM IC50, and the cancer cell death rate increased with increasing concentration.

Experimental Example 4. Identification of the induction of cell death of 2,6-Dibenzyl-4-chlorophenol compound on A549 cells, a lung cancer cell line.

The morphological apoptosis-inducing effect of the novel 2,6-Dibenzyl-4-chlorophenol compound synthesized in Example 2 on A549 cells, a lung cancer cell line, was confirmed by western blot.

To evaluate this, the amount of 2,6-Dibenzyl-4-chlorophenol compound was treated with 0 to 30 uM concentration in A549 cell to determine the amount of pro-capsase-3 before induction of apoptosis , And the ratio of Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic), which are regulators of cell death, was checked for 0-6 hours using western blot. In addition, changes in the activity of p53 protein, which regulates the activity of Bcl-2 and Bax, the regulators of apoptosis, were confirmed.

As a result, the amount of pro-caspase-3 decreased with increasing concentration of 2,6-Dibenzyl-4-chlorophenol compound (Fig. 6), and the ratio of Bcl-2 and Bax decreased with time And Fig. 8). In addition, it was confirmed that the activity of p53 protein increased with time (FIG. 9).

Thus, the 2,6-Dibenzyl-4-chlorophenol compound of the present invention was not toxic to the normal cell line NIH-3T3. However, as the concentrations of lung cancer cells, prostate cancer cells, It was observed that proliferation was suppressed (killed). In particular, when the 2,6-Dibenzyl-4-chlorophenol compound was treated with 30 μM of the IC50 in the lung cancer cell line A549 cells, the death of the cancer cells was confirmed when the 2,6-Dibenzyl-4-chlorophenol compound was treated for 24 hours . The action of 2,6-Dibenzyl-4-chlorophenol on the death of pancreatic cells increases the activity of p53 protein when 2,6-Dibenzyl-4-chlorophenol compound is administered to lung cancer cells, And activates Bax and inhibits pro-Caspase-3. In addition, Caspase-3 was activated and promoted the death of lung cancer cells.

As described above, the 2,6-Dibenzyl-4-chlorophenol compound newly synthesized according to the present invention has excellent anticancer properties and can induce cancer cell death. Therefore, the pharmaceutical composition containing the 2,6- Can be usefully used as a natural anticancer agent for prevention and treatment, and therefore, it is a very useful invention in medicine and health functional food industry.

Claims (8)

Next MgSO ₄ gave into the ρ-chlorophenol as TsOH in a flask with a magnetic bar is provided with stirring at 120 ° to wash the reaction the mixture was added dropwise benzyl alcohol was added to the cooled ether, washed with water and then transferred to a separating funnel and then NaOH solution and with water again by removal of water and removal of the ether by rotary evaporated and then vacuum dried, and then _{MeOH: CHCl 3 = 1: 2,6} -Dibenzyl- ratio of 80 to separation and purification by silica gel column chromatographic our having the following structural formula (ⅰ) characterized 4-chlorophenol compound.

(I)

A pharmaceutical composition for preventing and treating cancer, which comprises 2,6-Dibenzyl-4-chlorophenol compound prepared by the method of claim 1 as an active ingredient.

A composition for preventing and / or improving function of cancer, comprising 2,6-Dibenzyl-4-chlorophenol compound prepared by the method of claim 1 as an active ingredient.

The composition according to claim 2 or 3, wherein the cancer is any one selected from the group consisting of lung cancer, prostate cancer, cervical cancer and liver cancer.

The food composition according to claim 3, wherein the composition is any one of oral dosage forms selected from the group consisting of tablets, pills, capsules, liquids, suspensions, emulsions, syrups, granules and elixirs.

6. The food composition according to claim 5, wherein the formulation for oral administration contains at least one carrier selected from a diluent, a lubricant, a disintegrant, an absorbent, a colorant, a flavor and a sweetener.


3. The pharmaceutical composition according to claim 2, wherein the cancer is any one selected from the group consisting of lung cancer, prostate cancer, cervical cancer and liver cancer, and is injected by a parenteral administration method.

The pharmaceutical composition according to claim 7, wherein the formulation of the composition injected by the parenteral administration method is an ampule or vial prepared from a solution or suspension.

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