KR20100001837A - Composition comprising 3',4'-dimethyl flavanone compound showing over-expression effect on p21 for the prevention and treatment of cancer disease - Google Patents

Abstract

PURPOSE: A 3',4'-dimethyl flavanone compound activating p21 expression is provided to suppress cell growth and induce apoptosis. CONSTITUTION: A 3',4'-dimethyl flavanone compound of chemical formula 1 suppresses cancer cell proliferation and induces apoptosis by activating p21 expression. The 3',4'-dimethyl flavanone compound is isolated or synthesized from a plant. A pharmaceutical composition for preventing and treating cancer contains 0.01~50 weight% of 3',4'-dimethyl flavanone compound or its pharmaceutically acceptable salt. The pharmaceutical composition is administered by injection to vein, muscle or through subcutaneous or intracerebroventricular injection for mammal including rat, mouse, domestic animals, and human.

Description

Composition comprising 3 ′, 4′-dimethyl flavanone compound showing over-expression effect on p21 for the prevention and treatment of cancer disease}

The present invention relates to a pharmaceutical composition for the prevention and treatment of cancer diseases containing the 3 ', 4'-dimethoxyflavanone compound of formula 1 to activate the expression of p21, more specifically by activating p21 expression It relates to a pharmaceutical composition for the prevention and treatment of cancer diseases comprising a 3 ', 4'-dimethoxyflavanone compound of formula 1 or a pharmaceutically acceptable salt thereof that inhibits the proliferation of cancer cells and induces apoptosis .

Normal cells maintain a balance between their growth / differentiation and self-killing functions, but cancer cells break this balance and the number of cells grows exponentially and grows rapidly.

The cell cycle process for cell growth and differentiation is largely divided into interphase and mitotic phase. The former is further divided into three processes: G (1) -phase, where cells synthesize synthesis of proteins necessary for cell division, S-phase, where DNA synthesis occurs, and mitosis-phase, a cell differentiation process. .

Cyclin proteins required for cell division form a cyclin / CDK complex with a phosphorylation enzyme called Cyclin Dependent Kinase (CDK) to regulate the phase of the cell cycle.

One of the checkpoints to eliminate fatal mistakes in cell division is the DNA synthesis process, D1 / S-phase, where p53, a protein that checks for DNA damage, repairs DNA damage. Until the cell cycle progresses. p53 is known to induce the synthesis of a CDK inhibitory protein called p21, which delays the cell cycle.

Selective inhibitors of CDK, such as p21, have attracted attention as new anticancer agents, as it has been found that the regulation of these proteins involved in the cell cycle in many cancer tissues is poor.

Cyclin kinase inhibitors (CKIs) are attached to the G1-phase cyclin / CDK complex, which prevents the complex from activating and inhibits cell cycle progression, which in turn inhibits cell growth. Thus, in cells lacking p21, DNA damage does not inhibit cell cycle progression.

In addition, various oncogenes inhibit p21 to promote cell growth and form tumors. As such, p21, which inhibits proliferation of cells, also functions as a tumor suppressor. As a result, it is possible to increase the expression of p21 and more effectively inhibit the proliferation of cancer cells.

Currently used anticancer drugs are expensive and have serious side effects, so it is necessary to develop new anticancer drugs that are less toxic and effective.

Flavonoids are secondary metabolites of plants and have a variety of biological activities. Flavanone, one of them, is particularly included in citrus fruits, and flavanones present in nature are less toxic than conventional anticancer drugs, and have a basic skeleton structure different from those of conventional anticancer agents, and thus can be used for the development of new leading substances. Can be.

Accordingly, the present inventors have made an effort to find a compound having an effect of inhibiting cancer cells by overexpressing p21, a cell cycle progression inhibitor, and inhibiting cell inhibition. As a result, 3 ', 4'-dimethoxyflavanone activates p21 expression. The present invention was completed by confirming that the test was performed.

After all, the present invention has a main object to provide a pharmaceutical composition for the prevention and treatment of cancer diseases, including 3 ', 4'-dimethoxyflavanone compound or a pharmaceutically acceptable salt thereof that activates p21 expression. .

In order to achieve the above object, the present invention provides a 3 ', 4'-dimethoxyflavanone compound of the formula (1) to inhibit the proliferation of cancer cells and induce cell death by activating p21 expression.

[Formula 1]

The present invention also provides a pharmaceutical composition for the prevention and treatment of cancer diseases, including the 3 ', 4'-dimethoxyflavanone compound or a pharmaceutically acceptable salt thereof.

The cancer disease includes general cancer diseases, preferably gastric cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or ocular melanoma, uterine cancer, ovarian cancer, rectal cancer , Anal leiomyoma, fallopian tube dysfunction, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma Cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, One or more diseases selected from brain stem glioma, pituitary adenoma, and the like.

Since the 3 ', 4'-dimethoxyflavanone of the present invention has an effect of inhibiting cell growth by activating p21 expression, a cell cycle progression inhibitor, cancer prevention and treatment by inhibiting the proliferation of cancer cells and inducing cell death It can be usefully used as a pharmaceutical composition for.

Hereinafter, the present invention will be described in more detail.

The present invention provides a 3 ', 4'-dimethoxyflavanone compound represented by the following Chemical Formula 1 to inhibit the proliferation of cancer cells and induce cell death by activating p21 expression.

[Formula 1]

In the present invention, the 3 ', 4'-dimethoxyflavanone compound can be extracted and separated from plants or prepared by synthetic methods well known in the art.

The present invention also provides a pharmaceutical composition for the prevention and treatment of cancer diseases, including the 3 ', 4'-dimethoxyflavanone compound or a pharmaceutically acceptable salt thereof.

In the above, the 3 ', 4'-dimethoxyflavanone compound of the present invention can also be synthesized through the synthesis and fractionation of conventional substituents. (Herbert O. House, Modern Synthetic Reactions, 2nd Ed., The Benjamin / Cummings Publishing Co., 1972).

In addition, the compounds of the present invention can be prepared with pharmaceutically acceptable salts and solvates according to methods conventional in the art.

As the pharmaceutically acceptable salt, acid addition salts formed by free acid are useful. Acid addition salts are prepared by conventional methods, for example by dissolving a compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equimolar amounts of the compound and acid or alcohol (eg, glycol motomethylether) in water can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered.

In this case, organic acids and inorganic acids may be used as the free acid, and hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, and the like may be used as the inorganic acid, and methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, Citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid ( gluconic acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanic acid, hydroiodic acid, and the like.

Bases can also be used to make pharmaceutically acceptable metal salts. An alkali metal or alkaline earth metal salt is obtained by, for example, dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate. In this case, as the metal salt, it is particularly suitable to prepare sodium, potassium or calcium salt, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Pharmaceutically acceptable salts of the chemicals of the invention include salts of acidic or basic groups which may be present in the compounds of the invention, unless otherwise noted. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, Hydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts. It can be prepared through the process.

The compound of the present invention obtained by the above method was tested to determine the effect of inhibiting the proliferation of cancer cells and inducing apoptosis, can confirm the expression activity of p21.

The pharmaceutical composition for preventing and treating cancer diseases of the present invention contains the chemical at 0.01 to 50% by weight based on the total weight of the composition.

Pharmaceutical compositions comprising a compound of formula 1 according to the invention may further comprise a suitable carrier, excipient or diluent according to conventional methods. Carriers, excipients and diluents that may be included in the compositions of the present invention 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 compositions according to the invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories or sterile injectable solutions according to conventional methods.

Specifically, when formulated, it may be formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like that are commonly used. Formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in the compound, such as starch, calcium carbonate, sucrose. ), Lactose, gelatin and the like can be mixed. In addition to the simple excipients, lubricants such as magnesium stearate, talc and the like may also be used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups.In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the compounds according to the invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention may be administered in an amount of 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg, divided once or several times a day. In the composition of the present invention, the compound of formula 1 should be present in an amount of 0.001 to 50% by weight, preferably 0.01 to 50% by weight based on the total weight of the total composition.

Pharmaceutical dosage forms of the compounds according to the invention can also be used in the form of their pharmaceutically acceptable salts, or can be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes, and all modes of administration can be expected, for example, oral, rectal or intravenous, muscle, subcutaneous, It can be administered by intrauterine dural or intracerebroventricular injection.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Experimental Example 1. Confirmation of increased expression of p21 Ⅰ

In the present invention, in order to observe whether the 3 ', 4'-dimethoxyflavanone is activated for p21 expression, it is expressed through a luciferase reporter assay of how much p21 works in the intracellular transcriptional stage. The amount was investigated.

Plasmid DNA was prepared by transfecting 0.5 μg of 21 promoter into U-87MG cells and inserting a pRL-null plasmid containing luciferase.

After incubating the cells transformed with the prepared plasmid DNA for 12 hours, 20 µg / ml of 3 ', 4'-dimethoxyflavanone of the present invention was administered and reacted for 12 hours, followed by luminescence by luciferase. 50 μl of luciferin substrate was administered, and the degree of luciferase expressed was measured using a Dual-Glo Luciferase Assay System.

The expression level of luciferase expressed by the p21 promoter, that is, the expression level of p21 is as shown in FIG. 2, and the 3 ', 4'-dimethoxyflavanone of the present invention was treated as compared to the control group which did not process anything. One experimental group confirmed that the expression level of p21 increased by about 6 times (see FIG. 2).

Experimental Example 2. Confirmation of Increased Expression of p21 Ⅱ

In the present invention, in order to confirm whether the expression of 3 ', 4'-dimethoxyflavanone is increased to p21 at the cellular stage, Western blot for measuring the expression level of a specific protein using the antibody of the protein at the cellular stage ( Western blot assay) was performed.

Cell proteins were dissolved in 20 mM HEPES buffer + 1% Triton X-100 + 10% glycerol + 150 mM NaCl + 10 ug / ml leupeptin + 1 mM PMSF buffer and subjected to electrophoresis.

Proteins separated by molecular weight were transferred to a nitrocellulose filter and the expression level of p21 was measured using an antibody against p21.

As a result, as shown in Figure 3, the experimental group treated with 3 ', 4'-dimethoxyflavanone of the present invention increased the expression level of p21 protein compared to the control group treated with nothing, 24 hours rather than 12 hours It was confirmed that the expression level after the further increase.

Examples of the pharmaceutical compositions containing the compounds of the present invention will be described, but the present invention is not intended to be limited thereto, but is intended to be described in detail.

Formulation Example 1 Preparation of Powder

20 mg of the compound of the present invention

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2 Preparation of Tablet

10 mg of compound of the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation Example 3 Preparation of Capsule

10 mg of compound of the present invention

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

10 mg of compound of the present invention

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄ 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

20 mg of the compound of the present invention

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve it, and lemon flavor is added, the above components are mixed, and then, purified water is added to adjust the total amount to 100 ml, and then sterilized by filling into a brown bottle. do.

Figure 1 shows the effect of the expression activity of p21 according to 3 ', 4'-dimethoxyflavanone of the present invention.

Figure 2 is a Western blot confirming the effect of the expression activity of p21 according to the 3 ', 4'-dimethoxyflavanone of the present invention.

Claims (3)

3 ', 4'-dimethoxyflavanone compound of formula 1 to inhibit the proliferation of cancer cells and induce cell death by activating p21 expression. [Formula 1]

A pharmaceutical composition for the prevention and treatment of cancer diseases comprising the 3 ', 4'-dimethoxyflavanone compound of claim 1 or a pharmaceutically acceptable salt thereof for activating the expression of p21. The method of claim 2, The cancer diseases include gastric cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal muscle cancer, fallopian tube, and uterus. Endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic Or acute leukemia, lymphocytic lymphoma, bladder cancer, renal or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma, or pituitary adenoma Pharmaceutical composition characterized by.

Images