KR20120047513A - CALCONE DERIVATIVES HAVING INHIBITORY EFFECT ON NF-κB ACTIVATION - Google Patents

Abstract

PURPOSE: A 2',3',5'-trimethoxy chalcone compound is provided to retrain activation of NF-KB controlling infection reaction, to control initial path and process of an infection reaction system, thereby using as a pharmaceutical composition or a heath supplement food capable of preventing and treating various pathological diseases including cancel. CONSTITUTION: A 2',3',5'-trimethoxy chalcone compound is in chemical formula 1, and a manufacturing method thereof comprises: a step of dissolving 2-methoxyacetophenone and 3,5- dimethoxybenzaldehyde into ethanol; a step of adding 50% KOH aqueous solution into the solution; a step of stirring the mixture, and decreasing temperature to 3-5 °C; a step of adding 3 N HCl solution into the cooled solution; a step of extracting the aqueous solution by chloroform; a step of separating the extracted organic layer, and moisturizing by magnesium sulfate; a step of vacuum filtering the mixture in which water is removed, and conducting isolation and purification by using column chromatography.

Description

Calcon derivatives that inhibit the activity of NF-κＢ {CALCONE DERIVATIVES HAVING INHIBITORY EFFECT ON NF-κB ACTIVATION}

The present invention relates to a chalcone derivative that inhibits the activity of NF-κB, and more particularly, by inhibiting the activity of NF-κB induced by tumor necrosis factor alpha (TNF-α) Novel chalcone derivatives 2,3 ', 5'-trimethoxychalcone which can be used as compositions for the prophylaxis or treatment of related diseases, and methods for their preparation, pharmaceutical compositions and nutraceuticals containing the same.

[Formula 1]

Most human diseases are caused by abnormal components and substances produced by gene expression during the onset or progression of the disease. Generally, these genes are inactive or very insignificant in biological or physical processes. However, under certain conditions, such as exposure to environmental pollution, the expression of these genes increases exponentially due to preexisting genetic factors. These genetic factors are regulated by NF-κB (nuclear factor-κB), which is a cytokine, chemokine, growth factor, adhesion molecule, and acute stage protein. It activates the transcriptional phase of genes that express acute phase proteins.

NF-κB is a transcriptional protein that regulates various signaling pathways related to inflammatory responses, immune function, aging, and tumors, and has been identified in a wide range of organisms, from fruit flies to mammals. About 60 target genes for NF-κB have been studied to play a pivotal role in various reactions. Therefore, by regulating the activity of NF-κB, it is possible to selectively control the initial pathways and processes of various reactions and to use them as an indicator of disease treatment.

Especially in mammals, NF-κB consists of five proteins, p50 (NF-κB1), p52 (NF-κB2), p65 (Rel-A), c-Rel and Rel-B, and most of them are structurally similar proteins. The bond between groups consists of homodimers or heterodimers and exists in the cytoplasm in an inactivated state by an inhibitory molecule called IκB (inhibitoryκB). Through phosphorylation and a series of degradation processes of IκB, NF-κB is transported to the nucleus, leading to transcription of the target gene ( Annu Rev Immunol , 1998, 16,225). Factors involved in NF-κB activation include tumor necrosis factor-α (TNF-α), lymphotoxin and interleukin-1β (IL-1β), various cell growth factors (mitogens), and lipopolysaccharides. (lipopolysaccharide) and the like ( Int Rev Cytol , 1993, 143, 1).

In the present invention, the effect of inhibiting the activity of NF-κB induced by TNF-α, which acts as a ligand for inflammatory signaling activated by infection of external antigens, was investigated.

Proper coordination of activation and inactivation of NF-κB helps maintain body homeostasis, but abnormal production of inflammatory prostaglandins, eicosanoids and nitric oxide occurs when inflammatory reactions begin due to excessive activation of NF-κB. . It is associated with a number of pathological conditions that excessively sustain the body's immune response, cause aging, the onset, proliferation, and metastasis of tumors, including atherosclerosis and tissue transplantation host reactions ( Neurisci. Lett , 1997, 225, 61).

Diseases caused by excessive immune response include atopic dermatitis, allergies, lupus and arthritis. In addition, inflammatory responses induced by overexpression of NF-κB have been reported to be closely related to the aging process ( J. Invest Dermatol , 2000, 115, 177), ( Ann. NY Acad. Sci , 2001, 928, 327). , ( Proc. Natl. Acad. Sci. USA , 2001, 98, 10630). Various oxidative stresses in the aging process can continuously amplify the inflammatory response and damage various tissues. In particular, vascular endothelial cells and smooth muscle cells are prominent, suggesting that the inflammatory response is closely related to major vascular diseases ( Lab Invest , 1993, 68, 499). Atherosclerosis, a representative example of vascular aging, is a chronic inflammation caused by the continuous activation of NF-κB . It has been confirmed that NF-κB activity is increased even in atherosclerotic lesions ( Arterioscler. Thromb. Vasc. Biol , 19, 1623). Also in Alzheimer's, immune and inflammatory responses are emerging as new causes. Amyloid protein, characteristic of Alzheimer's, results in NF-κB activity. There is also a study on the induction of transcriptional activation of various genes that may be involved in neurodegeneration and response by NF-κB activity ( Brain Res , 1993, 629, 245).

Recently, studies showing that NF-κB activation is essential for cancer formation and metastasis have revealed that NF-κB plays an important role in carcinogenesis ( Oncogenic Ras. Science , 1997, 278, 1812). Aberrant expression and regulation of NF-κB is also found in breast cancer, non-small cell lung cancer, thyroid cancer, acute lymphocytic leukemia, and tumors caused by various viruses.

Therefore, by using compounds that can modulate the activity of NF-κB can be developed as an indicator for the development of new leading substances in the treatment as well as the treatment of the various diseases listed above.

Accordingly, the present inventors have the effect of preventing and treating various inflammatory diseases including cancer by blocking the early active pathways of various inflammatory response systems by regulating the activity of NF-κB, which plays an important role in the inflammatory response and cancer development process. I wanted to find. Thus, the present invention was completed by confirming that 2,3 ', 5'-trimethoxychalcone regulates the transcriptional activity of NF-κB induced by TNF-α, which is well known as a representative inflammatory cytokine.

The present invention has been made in view of the above problems and the need for the above, the technical problem to be achieved by the present invention is a novel compound that modulates the activity of NF-κB induced by TNF-α and containing it as an active ingredient It is to provide a pharmaceutical composition and dietary supplement.

In order to achieve the above object, the present invention provides a chalcone derivative 2,3 ', 5'-trimethoxychalcone represented by the following formula (1) or a pharmaceutically acceptable salt thereof and a method for preparing the same.

[Formula 1]

In the present invention, the preparation method comprises the steps of (1) dissolving 2-methoxyacetophenone and 3,5-dimethoxybenzaldehyde in ethanol; (2) adding 50% KOH aqueous solution to the solution at 3 to 5 ° C; (3) stirring the mixed solution for 15 to 30 hours and then lowering the temperature to 3 to 5 ° C; (4) neutralizing adding 3 N HCl solution to the cooled solution; (5) extracting the aqueous solution with chloroform; (6) separating the extracted organic layer with a separatory funnel and dehumidifying with magnesium sulfate; And (7) filtering the mixture from which the water was removed under reduced pressure, removing the solvent from the filtrate, and separating and purifying by using column chromatography.

In another aspect, the present invention provides a pharmaceutical composition and health functional food for the prevention or treatment of diseases containing 2,3 ', 5'-trimethoxychalcone represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. do.

In the present invention, the disease is characterized in that at least one selected from the group consisting of cancer, autoimmune diseases, hyperreactive inflammation, vascular diseases and Alzheimer's caused by the excessive activity of NF-κB, breast cancer, non-small cell lung cancer , Thyroid cancer, acute lymphocytic leukemia, tumors caused by various viruses, atopy, allergy, lupus, arthritis, arteriosclerosis or Alzheimer's is not limited thereto.

In addition, in the present invention, the 2,3 ', 5'-trimethoxychalcone or a pharmaceutically acceptable salt thereof prevents or treats the disease by inhibiting the activity of NF-κB, wherein the NF- κB is characterized by being activated by tumor necrosis factor alpha (TNF-α).

The present invention also provides a health functional food containing 2,3 ', 5'-trimethoxychalcone or a pharmaceutically acceptable salt thereof as an active ingredient.

The 2,3 ', 5'-trimethoxychalcone compound of the present invention inhibits the activity of NF-κB, an important transcription factor that modulates the inflammatory response, thereby regulating the early pathways and processes of the biological inflammatory response, including cancer. It can be usefully used as a pharmaceutical composition or health functional food that can prevent and treat pathological diseases.

1 is a hydrogen nuclear magnetic resonance spectrum ( ¹ H-NMR) of 2,3 ', 5'-trimethoxychalcone measured by Bruker 400 MHz nuclear magnetic resonance spectroscopy.
Fig. 2 is a carbon nuclear magnetic resonance spectrum ( ¹³ C-NMR) of 2,3 ', 5'-trimethoxychalcone measured by a Bruker 100 MHz nuclear magnetic resonance spectrometer.
Figure 3 is a graph showing the effect of TNF-α-induced NF-κB activity is regulated by treating the chalcone derivative, 2,3 ', 5'-trimethoxychalcone of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a chalcone derivative 2,3 ', 5'-trimethoxychalcone represented by Formula 1 below or a pharmaceutically acceptable salt thereof.

[Formula 1]

The present invention also provides a method for producing 2,3 ', 5'-trimethoxychalcone represented by Chemical Formula 1.

The preparation method of the present invention comprises the steps of (1) dissolving 2-methoxyacetophenone and 3,5-dimethoxybenzaldehyde in ethanol; (2) adding 50% KOH aqueous solution to the solution at 3 to 5 ° C; (3) stirring the mixed solution for 15 to 30 hours and then lowering the temperature to 3 to 5 ° C; (4) neutralizing adding 3 N HCl solution to the cooled solution; (5) extracting the aqueous solution with chloroform; (6) separating the extracted organic layer with a separatory funnel and dehumidifying with magnesium sulfate; And (7) filtering the mixture from which the water was removed under reduced pressure, removing the solvent from the filtrate, and separating and purifying by using column chromatography.

The novel chalcone derivative of the present invention, 2,3 ', 5'-trimethoxychalcone, has an effect of preventing or treating various diseases due to excessive inflammatory reactions and immune responses caused by the continuous increase of NF-κB activity. The disease is characterized in that at least one selected from the group consisting of cancer, autoimmune diseases and hyperreactive inflammation caused by excessive activity of NF-κB, breast cancer, non-small cell lung cancer, thyroid cancer, acute lymphocytic leukemia, various viruses Is preferably a tumor, atopy, allergy, lupus, arthritis, arteriosclerosis, or Alzheimer's disease.

In particular, it was confirmed that the 2,3 ', 5'-trimethoxychalcone of the present invention has an excellent effect of regulating the activity of NF-κB induced by tumor necrosis factor alpha (TNF-α).

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating a disease containing the 2,3 ', 5'-trimethoxychalcone or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the disease is cancer, autoimmune. It is preferably at least one disease selected from the group consisting of diseases and hyperreactive inflammation.

The composition comprising the compound of the present invention is preferably used for the prevention or treatment of colon cancer, stomach cancer, prostate cancer, breast cancer, kidney cancer, liver cancer, brain tumors, lung cancer, uterine cancer, colon cancer, bladder cancer, pancreatic cancer, blood cancer, etc. It is not limited to these.

Compounds of formula (1) included in the compositions of the present invention may also be used in the form of their salts, which include acid addition salts with various organic or inorganic acids that are pharmaceutically or physiologically acceptable. Suitable inorganic acids are, for example, halogen acids such as hydrochloric acid, sulfuric acid or phosphoric acid. Suitable organic acids include, for example, carboxylic acid, phosphonic acid, sulfonic acid, acetic acid, propionic acid, octanoic acid, decanoic acid, glycolic acid, lactic acid, fumaric acid, succinic acid, adipic acid, malic acid, tartaric acid, citric acid, glutamic acid, aspartic acid. , Maleic acid, benzoic acid, salicylic acid, phthalic acid, phenylacetic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, methyl sulfuric acid, ethyl sulfuric acid, dodecyl sulfate.

The composition of the present invention may further comprise a pharmacologically or physiologically acceptable carrier, excipient, diluent in addition to the compound of Formula 1 or a compound thereof.

Examples of suitable carriers, excipients and diluents that may be included in such compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Methyl cellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. When formulated, the composition may further include conventional fillers, extenders, binders, disintegrants, surfactants, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like.

The composition of the present invention may be formulated in various forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, sterile injectable solutions, etc. It may be used orally or may be administered through various routes including intravenous, intraperitoneal, subcutaneous, rectal, topical administration and the like.

Solid preparations 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, and the like. Mix and formulate. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to commonly used simple diluents such as water and liquid paraffin.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. Bases for injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

By "administration" in the present invention is meant to provide the patient with the desired material in any suitable way, the route of administration of the composition of the present invention being oral or parenteral via all common routes as long as the target tissue can be reached. May be administered. In addition, the composition can be administered by any device in which the active agent can migrate to the target cell.

In the present invention, "patient" means a human and an animal such as a monkey, dog, goat, pig, or rat having a disease that can improve symptoms by administering the composition of the present invention. The composition according to the invention can be applied not only for humans (treatment, inhibition or prevention) but also to other commercially useful animals.

In another aspect, the present invention provides a method for inhibiting, preventing and treating cancer and cancer metastasis by administering to a patient a composition comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof. The composition of the present invention can be administered in parallel with the conventional disease treatment.

The composition of 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 an effective dose level means the type, severity, and activity of the patient's disease. , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent drug use, and other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.

In addition, the dosage of the compound according to the present invention may be changed according to body absorption, weight, age, sex, health condition, diet, administration time, administration method, excretion rate, severity of disease, and the like. However, for the desired effect, the compound of the present invention is preferably administered at 0.001 to 150 mg, preferably 0.01 to 100 mg per kg of body weight per day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The compound of Formula 1 of the present invention can be synthesized using the synthesis method of Scheme 1 or various methods well known in the art .

The present invention also provides a health functional food containing 2,3 ', 5'-trimethoxychalcone or a pharmaceutically acceptable salt thereof as an active ingredient.

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

However, the following examples are only for illustrating the present invention, and the contents of the present invention are not limited to the following examples.

Example 1 Preparation of Novel Chalcone Derivatives, 2,3 ', 5'-trimethoxychalcone

In the present invention, the method of Scheme 1 below was used to synthesize a novel chalcone derivative, 2,3 ', 5'-trimethoxychalcone.

Scheme 1

2-methoxyacetophenone (2-methoxyacetophenone, 765 mg, 5 mmol) and 3,5-dimethoxybenzaldehyde (3,5-dimethoxybenzaldehyde, 840 mg, 5 mmol) were dissolved in 35 ml of ethanol and then at 4 ° C. Add 10 ml of 50% aqueous KOH solution slowly for about 10 minutes. After stirring the mixed solution at room temperature for 24 hours, the temperature is lowered to about 4 ℃. After neutralizing by adding 40 ml of 3 N HCl solution to the cooled solution, the aqueous solution was extracted twice with 50 ml of chloroform. Each of the extracted organic solvents was combined and magnesium sulfate was added to remove water and filtered under reduced pressure. Subsequently, after removing the solvent of the filtrate by using a rotary evaporator, the remaining mixture was purified by column chromatography to obtain 1072 mg (72%) of the target compound in a liquid state.

In order to confirm the formation of the target compound, the hydrogen nuclear magnetic resonance spectroscopy spectrum (Bruker 400 MHz) shown in FIG. 1 and the carbon nuclear magnetic resonance spectrum (Bruker 100 MHz) shown in FIG. , 5'-trimethoxychalcone (hereinafter referred to as "C23").

Example 2: Confirmation of the activity inhibitory effect of NF-κB by a novel chalcone derivative, 2,3 ', 5'-trimethoxychalcone (C23)

In the present invention, Cis- acting to observe the effect of inhibiting NF-κB activity by a novel chalcone derivative, C23 Luciferase reporter assay ( Cis- acting luciferase reporter assay) was used. In the luciferase reporter assay, when the transcription factor NF-κB is activated, it binds to the NF-κB binding DNA sequence present in the reporter plasmid (pNF-κB-Luc) to increase the expression of the luciferase gene. It uses the principles that appear. Thus, changes in luciferase enzyme activity refer to changes in NF-κB activity.

For use in this experiment, HCT116 human colon cancer cells were purchased from the American Type Culture Collection (USAC), and cultured in a 37 ° C cell incubator with DMEM culture containing 10% bovine fetal serum. . After dispensing 1 × 10 ⁵ cells in a 24-well cell culture dish and incubating for 24 hours, 0.5 μg of a reporter plasmid (pNF-κB-Luc) containing an NF-κB binding sequence was added to the fuGENE6 transfection reagent ( The cells were transfected with a transfection reagent. After 48 hours of plasmid injection, 10 ng / ml of TNF-α alone was treated as a control, and 10 ng / ml of TNF-α and 20 µg / ml of a chalcone derivative, C23, were treated as a control group.

The control and experimental cells were further incubated for 12 hours before harvesting to lyse the cells. 50 μl of luciferin, a substrate, was added to the cell lysate, and fluorescence was measured. Luciferin fluorescence was measured using a Dual-Glo Luciferase Assay System purchased from Promega.

As shown in FIG. 3, TNF-α increased luciferase enzyme activity by about 10-fold. These results indicate that NF-κB activity was increased by TNF-α. At this time, it was observed that the luciferase activity increased by TNF-α is inhibited by 50% or more in the experimental group treated with the novel chalcone derivative, C23, in parallel with TNF-α. From these results, it was confirmed that the novel chalcone derivative of the present invention, C23, inhibits the transcriptional activity of NF-κB induced by TNF-α.

Claims (9)

Chalcone derivative 2,3 ', 5'-trimethoxychalcone represented by the following formula (1) or a pharmaceutically acceptable salt thereof.
[Formula 1]

A pharmaceutical composition for preventing or treating a disease containing 2,3 ', 5'-trimethoxychalcone represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. The method of claim 2,
The 2,3 ', 5'-trimethoxychalcone or a pharmaceutically acceptable salt thereof is a pharmaceutical composition for preventing or treating a disease, characterized in that to prevent or treat the disease by inhibiting the activity of NF-κB. The method of claim 3, wherein
The NF-κB is a pharmaceutical composition for the prevention or treatment of diseases, characterized in that induced by tumor necrosis factor alpha (TNF-α). The method of claim 2,
The disease is a pharmaceutical composition for the prevention or treatment of a disease, characterized in that at least one selected from the group consisting of cancer, autoimmune diseases, hyperreactive inflammation, vascular diseases and Alzheimer's. 6. The method of claim 5,
The cancer is composed of breast cancer, colon cancer, stomach cancer, prostate cancer, kidney cancer, liver cancer, brain tumor, lung cancer, uterine cancer, colon cancer, bladder cancer, pancreatic cancer, blood cancer, non-small cell lung cancer, thyroid cancer, acute lymphocytic leukemia, and tumors caused by viruses. Pharmaceutical composition for the prevention or treatment of diseases, characterized in that at least one selected from the group. 6. The method of claim 5,
The autoimmune disease is a pharmaceutical composition for preventing or treating a disease, characterized in that at least one disease selected from the group consisting of atopy, allergy, lupus and arthritis. Health functional food containing 2,3 ', 5'-trimethoxychalcone represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. In the manufacturing method of 2,3 ', 5'-trimethoxychalcone represented by said Formula (1),
(1) dissolving 2-methoxyacetophenone and 3,5-dimethoxybenzaldehyde in ethanol;
(2) adding 50% KOH aqueous solution to the solution at 3 to 5 ° C;
(3) stirring the mixed solution for 15 to 30 hours and then lowering the temperature to 3 to 5 ° C;
(4) neutralizing adding 3 N HCl solution to the cooled solution;
(5) extracting the aqueous solution with chloroform;
(6) separating the extracted organic layer with a separatory funnel and dehumidifying with magnesium sulfate;
(7) filtering the mixture from which the water has been removed under reduced pressure, removing the solvent from the filtrate, and separating and purifying by using column chromatography;
Method for producing 2,3 ', 5'-trimethoxychalcone comprising a.

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