KR101556861B1 - Pharmaceutical composition for preventing or treating of cancer comprising 4-isopropyl-2,6-bis(1-phenylethyl)aniline as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or treating a cancer including 4-isopropyl-2,6-bis(1-phenylethyl)aniline as an active ingredient. The 4-isopropyl-2,6-bis(1-phenylethyl)aniline according to the present invention controls propagation of cancer cell in various cancer cell hosts, and increases activity of signal transmitting protein inducing destruction of cancer cell, thereby being used as a pharmaceutical composition or a functional food composition for preventing, eliminating, or treating cancer.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for prevention or treatment of cancer comprising 4-isopropyl-2,6-bis (1-phenylethyl) aniline as an active ingredient. -phenylethyl) aniline as an active ingredient}

The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising 4-isopropyl-2,6-bis (1-phenylethyl) aniline as an active ingredient, or a health food composition comprising the same.

Cancer is one of the incurable diseases that the whole human race has to overcome, and it is one of the most common causes of mortality in Korea as well. In order to conquer cancer, development has been repeated by searching for new targets including the regulation of cell lineage or apoptosis and cancer genes or cancer suppressor genes, but the incidence of cancer is still increasing. At present, many methods such as incision, chemotherapy, and radiation therapy have been developed to treat cancer, but radiation therapy or incision is effective only when the initial diagnosis of cancer is made. However, There is no need to treat 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 repeatedly administered for a long period or when the cancer is recurred, 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.

Apoptosis, on the other hand, 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 extrinsic pathway, 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, activates caspase-9, then activates caspase-3, and eventually causes 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).

As described above, there is a growing interest in the effect of chemotherapy according to the induction of apoptosis, and the development of a cancer treatment agent that overcomes the disadvantages of currently used anticancer drugs is a major subject of study. No. 10-2010-0036145), but it is still not enough.

DISCLOSURE OF THE INVENTION The present invention has been made in order to solve the problems of the prior art as described above. The present inventors have made efforts to discover active compounds capable of preventing or treating cancer, and as a result, they have found that a derivative of a component derived from a yellow- It has been confirmed that isopropyl-2,6-bis (1-phenylethyl) aniline has an inhibitory effect on cancer cell proliferation and induces apoptosis, On this basis, the present invention has been completed.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising 4-isopropyl-2,6-bis (1-phenylethyl) aniline as an active ingredient, or a health food composition comprising the same .

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a process for preparing 4-isopropyl-2,6-bis (1-phenylethyl) aniline) A pharmaceutical composition for preventing or treating cancer comprising an acceptable salt as an effective ingredient, or a health food composition containing the same.

In one embodiment of the present invention the cancer is selected from the group consisting of bladder cancer, bone cancer, blood cancer, breast cancer, melanoma, thyroid cancer, pituitary cancer, bone cancer, rectal cancer, throat cancer, laryngeal cancer, lung cancer, esophageal cancer, pancreatic cancer, , Brain tumor, uterine cancer, head or neck cancer, gallbladder cancer, oral cancer, colon cancer, perianal cancer, glioma, liver cancer, and colon cancer.

In another embodiment of the present invention, the 4-isopropyl-2,6-bis (1-phenylethyl) aniline may increase the activity of caspase-3 protein in cancer cells and induce apoptosis.

The present invention relates to a method of administering 4-isopropyl-2,6-bis (1-phenylethyl) aniline or a pharmaceutically acceptable salt thereof to a subject The present invention provides a method for preventing or treating cancer.

The present invention relates to the use of 4-isopropyl-2,6-bis (1-phenylethyl) aniline or a pharmaceutically acceptable salt thereof for the prophylaxis or treatment of cancer Lt; / RTI >

The 4-isopropyl-2,6-bis (1-phenylethyl) aniline according to the present invention inhibits the proliferation of cancer cells in various cancer cell lines and increases the activity of signal transduction proteins that induce apoptosis of cancer cells It is expected that it can be usefully used as an active ingredient of a pharmaceutical composition or a functional food composition for preventing, ameliorating or treating cancer.

In addition, since no research has been conducted on the effect on 4-isopropyl-2,6-bis (1-phenylethyl) aniline to date, 4-isopropyl- Can be used not only for the purpose of attempting a new scientific study on cancer diseases but also for the development of medicines and quasi drugs.

1 is a result of confirming the inhibitory effect of 4-isopropyl-2,6-bis (1-phenylethyl) aniline (KTH-di2) on cancer cell proliferation in various cancer cell lines.
FIG. 2 shows the results of confirming the effect of marker generation of apoptosis by 4-isopropyl-2,6-bis (1-phenylethyl) aniline (KTH-di2).
FIG. 3 shows the activity of inducing the activity of caspase-3 protein of 4-isopropyl-2,6-bis (1-phenylethyl) aniline (KTH-di2).

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for preventing or treating cancer comprising 4-isopropyl-2,6-bis (1-phenylethyl) aniline as an active ingredient to provide.

As used herein, the term "prophylactic " means any act that inhibits cancer or delays the onset of cancer by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to any action that improves or alters the symptoms of cancer by administration of the pharmaceutical composition of the present invention.

"Cancer" as a disease to be improved, prevented or treated by the composition of the present invention is an aggressive characteristic that cells divide and grow by ignoring the normal growth limit, invasive characteristic penetrating into surrounding tissues And diseases caused by cells having metastatic characteristics spreading to other parts of the body. In the present specification, the cancer is also used in the same sense as a malignant tumor, including, but not limited to, solid tumors and blood born tumors. For example, in the present invention, the cancer may be selected from the group consisting of bladder cancer, bone cancer, blood cancer, breast cancer, melanoma, thyroid cancer, pituitary cancer, bone cancer, rectal cancer, , Head or neck cancer, gallbladder cancer, oral cancer, colon cancer, perineal cancer, glioma, liver cancer, and colorectal cancer.

4-isopropyl-2,6-bis (1-phenylethyl) aniline, which is an active ingredient of the pharmaceutical composition of the present invention, . ≪ / RTI >

[Chemical Formula 1]

The 4-isopropyl-2,6-bis (1-phenylethyl) aniline of the present invention can be used in the form of a pharmaceutically acceptable salt. The salt may be formed by a pharmaceutically acceptable free acid Acidophosphate is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Propyl sulphonate, naphthalene-1-yne, xylenesulfonate, phenylsulfate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, Sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention can be prepared by dissolving 4-isopropyl-2,6-bis (1-phenylethyl) aniline in an excess amount of an acid aqueous solution, For example, methanol, ethanol, acetone or acetonitrile.

By heating the same amount of 4-isopropyl-2,6-bis (1-phenylethyl) aniline and an acid or alcohol in water, followed by evaporating the mixture and drying or by suction filtration of the precipitated salt .

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 insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. In addition, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

The 4-isopropyl-2,6-bis (1-phenylethyl) aniline of the present invention can be used not only in the form of pharmaceutically acceptable salts, but also in all salts, hydrates and solvates .

The addition salt according to the present invention can be prepared by a conventional method, for example, by reacting 4-isopropyl-2,6-bis (1-phenylethyl) aniline with a water-miscible organic solvent such as acetone, methanol, ethanol , Or acetonitrile, etc., adding an excess amount of an organic acid, or adding an aqueous acid solution of an inorganic acid, followed by precipitation or crystallization. Subsequently, in this mixture, a solvent or an excess acid is evaporated and dried to obtain an additional salt, or the precipitated salt may be produced by suction filtration.

The 4-isopropyl-2,6-bis (1-phenylethyl) aniline of the present invention preferably induces apoptosis by increasing the activity of Caspase-3 protein in cancer cells, but is not limited thereto.

In a specific example of the present invention, when MDA-MB-231 cells and C6 cells are treated with 4-isopropyl-2,6-bis (1-phenylethyl) aniline, the cancer cell concentration- (See Example 2), blebbing, apoptotic bodies, which are markers of apoptosis in cancer cells, are generated, and caspase-3 is activated to induce apoptosis (Example 3 Respectively.

Therefore, the 4-isopropyl-2,6-bis (1-phenylethyl) aniline of the present invention effectively induces apoptosis of cancer cells in various cancer cell lines, and thus can be effectively used as an effective ingredient of a composition for preventing or treating cancer have.

The pharmaceutical composition of the present invention may be formulated and administered in a variety of oral or parenteral dosage forms at the time of clinical administration, but is not limited thereto.

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. 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 4-isopropyl-2,6-bis (1-phenylethyl) aniline as an active ingredient can be administered parenterally, and parenteral administration can be carried out by subcutaneous injection, intravenous injection, intramuscular injection, As shown in FIG.

At this time, 4-isopropyl-2,6-bis (1-phenylethyl) aniline or a pharmaceutically acceptable salt thereof is mixed with water together with a stabilizer or a buffer to formulate a formulation for parenteral administration, And can be prepared in an ampoule 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.

In addition, the pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the age, sex, condition, body weight, absorbency, inactivation rate, excretion rate, type of disease, May be administered in an amount of 0.001 to 150 mg, preferably 0.01 to 100 mg, per 1 kg of body weight per day, every other day, or one to three times per day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

In another aspect of the present invention, the present invention provides a pharmaceutical composition comprising 4-isopropyl-2,6-bis (1-phenylethyl) aniline or a pharmaceutically acceptable salt thereof, And a method of preventing or treating an inflammatory disease comprising the step of administering to a subject a possible salt.

The term "individual" as used herein refers to a subject in need of treatment for a disease, and more specifically refers to a human or non-human primate, mouse, rat, dog, cat, It means mammals.

In another aspect of the present invention, the present invention provides a pharmaceutical composition comprising 4-isopropyl-2,6-bis (1-phenylethyl) aniline or a pharmaceutically acceptable salt thereof, A health food composition for preventing or ameliorating an inflammatory disease comprising a salt as an effective ingredient.

In the functional food composition of the present invention, the active ingredient may be directly added to the food or may be used together with other food or food ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). In general, the composition of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, in the production of food or beverage. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range.

The functional food composition of the present invention has no particular limitation on the ingredients other than those containing the active ingredient as an essential ingredient in the indicated ratios and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary drinks . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above . The ratio of the above-mentioned natural carbohydrate can be appropriately determined by a person skilled in the art.

In addition to the above, the functional food composition of the present invention may contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, And salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. These components may be used independently or in combination. The ratios of these additives can also be appropriately selected by those skilled in the art.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Cell culture

Human breast cancer cell line MDA-MB 231 cells and Rat glioma cell line C6 were cultured in DMEM medium containing penicillin (100 IU / ml), streptomycin (100 μg / ml) and 10% culture dishes at a density of 70-80%.

Example 2 Confirmation of Cancer Cell Proliferation Inhibitory Effect of 4-isopropyl-2,6-bis (1-phenylethyl) aniline (KTH-13di2)

In order to examine the effect of 4-isopropyl-2,6-bis (1-phenylethyl) aniline (KTH-13di2) of the present invention on cancer cell proliferation in various cancer cell lines, KTH-13di2 Cell viability of each cancer cell line was confirmed.

More specifically, MDA-MB-231 cells (breast cancer cells) and C6 giloma cells (glioma cancer cells) were plated on a 96-well plate at 5 × 10 ⁵ cells / ml, respectively, and KTH-13di2 was treated by concentration. After incubation at 37 ° C for 24 hours and 48 hours in a CO ₂ incubator, MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphinyltetrazolium bromide) assay was performed. That is, 10 μl of MTT solution (stock concentration: 5 mg / ml) was added and additional reaction was induced for 3 hours. 100 μl of MTT stopping solution (10% sodium dodecyl sulfate in 0.01M HCl) was added to each well for reaction termination and formazan crystal dissolution. The cell viability was calculated from the OD value obtained by measuring the absorbance at 570 nm of the amount of MTT reduced to formazan, and the results are shown in FIG.

As shown in Fig. 1, when KTH-13di2 was treated, it was confirmed that the proliferation of each cancer cell was inhibited in dependence on the KTH-13di2 concentration.

Example 3. Confirmation of inducing effect of 4-isopropyl-2,6-bis (1-phenylethyl) aniline (KTH-13di2)

3-1. Confirmation of morphological changes of cells

MDA-MB 231 cells were cultured in a 24-well plate at a concentration of 5 × 10 ⁴ cells / ml. KTH-13di2 was treated at different concentrations and photographed by optical microscope at different time intervals. Respectively.

As shown in FIG. 2, when KTH-13di2 was treated, blebbing and apoptotic bodies, which are markers of apoptosis, were produced in cancer cells.

3-2. Confirming caspase activity induction effect

Since caspase plays an essential role in apoptosis, the proliferation inhibitory effect of 4-isopropyl-2,6-bis (1-phenylethyl) aniline (KTH-13di2) MDA-MB 231 cells were treated with KTH-13di2 to determine the activity of caspase.

More specifically, the Human breast cancer cell line in MDA-MB 231 cells, penicillin (100 IU / ml) and streptomycin 1X10 the (100 μg / ml) and 10% of a cell culture using a DMEM medium containing FBS ⁶ cells / ml in a 60 mm dish. Then, each fraction was treated with KTH-13di2 (100 μM) after a certain period of time. After 24 hours, the cells were collected and westernized by waking the cells using a lysis buffer and a sonicator. Protein concentration of each sample was measured with BSA as a standard. SDS-PAGE was performed with each sample amount based on the thus-obtained protein concentration, and the protein was blotted on a PVDF membrane using a wet blotting method. The membrane was blocked with 3% BSA, and the target protein antibody and the signal After washing, the secondary antibody solution was treated and washing was carried out using a solution of the transfer protein antibody (caspase 3, Bcl-2, β-actin). Then, the ECL solution (Amersham, England) was evenly distributed on the membrane in the dark room and sensitized with an X-ray film. The results are shown in FIG.

As shown in FIG. 3, it was confirmed that the amount of pro-Caspase 3, which is a step before KTH-13di2 induces apoptosis in MDA-MB 231 cells, is reduced and the amount of Bcl-2, an apoptosis regulator, is reduced. From the above results, it was found that the 4-isopropyl-2,6-bis (1-phenylethyl) aniline (KTH-13di2) of the present invention increases the activity of a signal transduction protein that induces apoptosis of cancer cells, And the effect of accelerating the death was found.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (6)

(1-phenylethyl) aniline, or a pharmaceutically acceptable salt thereof, as an active ingredient, and a method for treating breast cancer, including 4-isopropyl-2,6-bis Or a pharmaceutical composition for the prevention or treatment of glioma.
delete The composition according to claim 1, wherein the 4-isopropyl-2,6-bis (1-phenylethyl) aniline increases the activity of caspase-3 protein in cancer cells to induce apoptosis.
(1-phenylethyl) aniline, or a pharmaceutically acceptable salt thereof, as an active ingredient, and a method for treating breast cancer, including 4-isopropyl-2,6-bis Or a composition for preventing or ameliorating glioma.
delete 5. The composition according to claim 4, wherein the 4-isopropyl-2,6-bis (1-phenylethyl) aniline increases the activity of caspase-3 protein in cancer cells to induce apoptosis.

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