WO2013065922A1 - Pharmaceutical composition including a diphenylpropenone compound as an active ingredient for preventing and treating an inflammatory disorder in the nervous system - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating an inflammatory disorder in the nervous system. According to the above-described present invention, a novel diphenylepropenone compound ((E)-3-(3,5-dimethoxyphenyl)-1-(2-hydroxy-5-methoxyphenyl)prop-2-en-1-one) having an anti-inflammatory effect by inhibiting the activity of the nuclear factor-kappa B (NF-kB) of a microglia cell inducing the inflammatory reaction in the nervous system, and a pharmaceutical composition for preventing and treating an inflammatory disorder in the nervous system including same, are provided. The pharmaceutical composition may be effectively used in preventing and treating inflammatory disorders in the nervous system.

Description

Pharmaceutical composition for the prevention and treatment of inflammatory diseases of the nervous system, including diphenylpropenone compound as an active ingredient

The present invention relates to a pharmaceutical composition for the prevention and treatment of inflammatory diseases of the nervous system, and more particularly, to inhibit inflammation through inhibiting NF-kB (nuclear factor-kappa B) activity of microglia cells that cause neurological inflammatory responses. Novel diphenylpropenone compound (E-3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-ene represented by the following general formula (1) having the effect -1-one, (E) -3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one) and prevention of neurological inflammatory diseases including the same The present invention relates to a therapeutic pharmaceutical composition.

[Formula 1]

The chalcone compound is one of the compounds produced in the plant's secondary metabolite biosynthesis process and has been reported to have various structures, and several derivatives thereof are known to exhibit anti-inflammatory effects (Bioorg Med Chem Lett. 2011 21 (15) 4480-4).

In the present invention, in order to further improve the anti-inflammatory effects of known chalcone compounds and to find new chalcone compounds having novelty, a new compound modified by adding a hydroxyl group and a methoxy group to an existing chalcone compound through a structure-activity relationship study It was designed and produced a compound named DK139.

A number of autoimmune diseases, including atopy, allergies, lupus and arthritis, are 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).

NF-kB (Nuclear Factor-kappa B) protein is a transcription factor protein that regulates various signaling synthesis related to inflammatory response, immune function, aging and tumor. NF-kB consists of Rel homodimers or heterodimers such as p50 (NFκB1), p52 (NFκB2), p65 (Rel-A), c-Rel and Rel-B, and inhibitory It is present in the cytoplasm in an inactivated state by binding to an inhibitory molecule called κB (IκB). When the inflammatory signal is transmitted intracellularly, IκB is phosphorylated by IKB (IκB kinase) and proteolyses through a series of degradation processes, whereby NFkB becomes active and moves to the nucleus, causing transcription of the target gene ( Annu Rev). Immunol , 1998, 16, 225). Factors involved in NFkB activity include TNF-α, lymphotoxin and IL-1β, mitogens, and lipopolysaccharide (LPS) ( Int Rev Cytol , 1993, 143: 1).

One of the cells that drives the inflammatory response in the nervous system is microglia cells (J Neuroinflammation 2004, 1:14). Microglia cells are immune cells of brain tissue that have a function similar to macrophage, a cell for phagocytosis, and induce NFkB activity by tissue damage or invasion of external pathogens to secrete various inflammatory mediators. It enters the site of encephalitis and activates astroglia to maintain brain homeostasis (J Neurosci Res 2005, 81: 302). However, abnormally excessive activation of NF-kB in microglia cells leads to chronic inflammatory conditions and abnormally increased production of inflammatory mediators such as inflammatory cytokines, chemokines, prostaglandins, eicosanoids, and nitric oxide. (Life Science 2011, 89: 141). It excessively activates the brain's immune response, causing a variety of degenerative diseases, including neuronal damage and degeneration ( Neurisci. Lett , 1997, 22:61). In degenerative brain diseases such as Alzheimer's disease, inflammatory reactions are also considered as a major cause ( Brain Res , 1993, 629: 245) (Glia 2002, 40: 232). Denatured amyloid protein, characteristic of Alzheimer's, induces NF-kB activity in microglia cells and secretes a variety of inflammatory mediators, ultimately leading to degeneration and death of neuronal cells such as neurons. Schizophrenia is also associated with encephalitis responses (Medical Hypotheses, 1995, 45: 135), and reports have shown that suppressing encephalitis may help treat depression (Biol Psychiatry 2009, 65: 732). Indeed, anti-depressant drugs such as Imipramine, trazodone and Fluoxetine have been shown to have anti-inflammatory effects (Hum Psychopharmacol Clin Exp 2001, 16:95) (Pharmacol Res 2004, 49: 119).

More than 60 NF-kB target genes have been reported to date, and are known to play a pivotal role in various inflammatory reactions. Therefore, if NF-kB activity can be properly controlled, new initiatives in the prevention and treatment of degenerative brain diseases such as dementia, depression and Parkinson's disease, as well as various chronic inflammatory diseases such as chronic arthritis, degenerative arthritis, gastritis and hepatitis Can be developed as a substance.

In the present invention, NF-kB activity by LPS that mediates inflammation by bacterial infection was used as an inflammation model signal. The DK139 compound of the present invention has completed the present invention by obtaining the results of inhibiting NF-kB activity by LPS in microglia cells leading to inflammatory response in the nervous system.

Conventional techniques using a chalcone or a derivative thereof as a pharmaceutical composition have been disclosed in Korean Patent No. 10-0567125 (A matrix composition for inhibiting matrix metalloprotease activity containing a chalcone or a derivative thereof) by inhibiting MMP activity. Patent No. 10-0751899 (new chalcone derivatives, pharmaceutically acceptable salts thereof, preparation method thereof and glycosidase containing them as an active ingredient) which has the effect of preventing and treating diseases caused by glycosidase. Composition for the prevention and treatment of diseases caused by).

An object of the present invention is to provide a novel diphenylpropenone compound (E-3- (E-3- (E-)) which has an inhibitory effect through the inhibition of NF-kB (nuclear factor-kappa B) activity of microglia cells that cause nervous system inflammation. 3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one, (E) -3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one) and a pharmaceutical composition for preventing and treating neurological inflammatory diseases including the same.

Another object of the present invention is to provide a novel diphenylpropenone compound (E-3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-ene- For the prevention and treatment of inflammatory diseases of the nervous system, including 1-one, (E) -3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one) To provide a pharmaceutical composition.

In order to achieve the above object, the present invention is a diphenyl propenone compound represented by the following formula (E-3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) Prop-2-en-1-one, (E) -3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one) or a pharmaceutical thereof To provide acceptable salts.

[Formula 1]

In addition, the present invention is a diphenyl propenone compound represented by the formula (1) (E-3- (3, 5-dimethoxyphenyl) -1- (2-hydroxy-5- methoxyphenyl) prop-2- In the method for producing en-1-one, (E) -3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one), (1) Dissolving 2-hydroxy-6-methoxy-acetophenone and 3,5-dimethoxybenzaldehyde in ethanol; and (2 A) adding 50% KOH aqueous solution to the mixed solution of step (1); and (3) stirring the mixed solution prepared by step (2) at room temperature and then cooling to 3 to 5 ° C; And (4) neutralizing by adding 6N HCl solution to the mixed solution cooled by step (3) and then extracting twice with dichloromethane; and (5) each organic extracted by step (4). Combining the solvents and then adding magnesium sulfate to remove water; and (6) the Diphenylpropenone compound (E-3- (3,5-dimethoxyphenyl) -1) comprising the step of removing the water in step (5) and filtration and drying the remaining mixture under reduced pressure, and then separating and purifying by column chromatography. -(2-hydroxy-5-methoxyphenyl) prop-2-en-1-one, (E) -3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop -2-en-1-one) is provided.

In addition, the present invention is a diphenylpropenone compound represented by the formula (1) of claim 1 having an inhibitory effect through the inhibition of NF-kB (nuclear factor-kappa B) activity of the microglia (microglia) cells causing nervous system inflammation (E-3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one, (E) -3- (3,5 It provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases of the nervous system comprising -dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one) and a pharmaceutically acceptable salt.

The pharmaceutical composition is characterized in that it comprises one or more pharmaceutically acceptable carriers, diluents or excipients.

In addition, the present invention is a diphenyl propenone compound represented by the formula (1) (E-3- (3, 5-dimethoxyphenyl) -1- (2-hydroxy-5- methoxyphenyl) prop-2- En-1-one, (E) -3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one) and food acceptable additives It provides a health functional food for the prevention and improvement of inflammatory diseases including nervous system.

The health functional food is characterized in that the tablet, capsule, pills or liquid form.

Hereinafter, the present invention will be described in detail.

The present invention provides a diphenylpropenone compound represented by the following formula (E-3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-ene- 1-one, (E) -3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one) or a pharmaceutically acceptable salt thereof .

[Formula 1]

The present invention provides a diphenylpropenone compound represented by the formula (E-3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-ene- 1-one, (E) -3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one), wherein (1) 2- Dissolving hydroxy-6-methoxy-acetophenone and 2-hydroxy-5-methoxy-1-acetophenone in 3,5-dimethoxybenzaldehyde (3,5-dimethoxybenzaldehyde); and (2) Adding 50% KOH aqueous solution to the mixed solution of step (1); and (3) stirring the mixed solution prepared by step (2) at room temperature and then cooling to 3 to 5 ° C; and ( 4) neutralizing by adding 6N HCl solution to the mixed solution cooled by step (3), and then extracting twice with dichloromethane; and (5) each organic solvent extracted by step (4). Combining to remove water by adding magnesium sulfate; (6) diphenylpropenone compound (E-3- (3,5-dimethic), comprising the step of removing the water and removing the remaining mixture in step (5) under reduced pressure filtration and drying, followed by column chromatography. Methoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one, (E) -3- (3,5-dimethoxyphenyl) -1- (2-hydroxy- It provides a method for preparing 5-methoxyphenyl) prop-2-en-1-one).

The present invention has a diphenylpropenone compound represented by Formula 1 of claim 1 having an inhibitory effect through the inhibition of NF-kB (nuclear factor-kappa B) activity of microglia cells that cause nervous system inflammation (E -3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one, (E) -3- (3,5-dimethoxyphenyl It provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases of the nervous system, comprising) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one) and a pharmaceutically acceptable salt.

Such salts 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 pharmaceutical composition may comprise one or more pharmaceutically acceptable carriers, diluents or excipients. The carrier, diluent or excipient may 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 pharmaceutical composition of the present invention may be formulated in various forms, such as powders, granules, tablets, capsules, suspensions, emulsions, oral formulations such as syrups, aerosols, and sterile injectable solutions according to conventional methods for the purpose of each use. It may be used orally, or may be administered through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may include 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. Preparations for parenteral administration include sterile aqueous solutions, non-aquatic solutions, 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, “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. , Drug sensitivity, time of administration, route of administration and rate of release, time of treatment, factors including concurrent use of drugs, 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 achieve the maximum effect with a minimum amount without side effects, which can be easily nominated by one 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 ~ 150mg, preferably 0.01 ~ 100mg per kg 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.

In addition, the present invention is a diphenyl propenone compound represented by the formula (1) (E-3- (3, 5-dimethoxyphenyl) -1- (2-hydroxy-5- methoxyphenyl) prop-2- En-1-one, (E) -3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one) and food acceptable additives It provides a health functional food for the prevention and improvement of inflammatory diseases including nervous system. For example, dietary supplements include meat, sausages, breads, chocolates, candy, snacks, confectionery, pizzas, ramen noodle, dairy products including gums, ice creams, various soups, beverages, teas, drinks, alcoholic beverages, It can be prepared by adding a benzohydroxymethoxychalcone compound or a pharmaceutically acceptable salt thereof to a vitamin complex and the like.

According to the present invention as described above, a novel diphenylpropenone compound (E-3) having an inhibitory effect through the inhibition of NF-kB (nuclear factor-kappa B) activity of microglia cells that cause nervous system inflammation (E-3) -(3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one, (E) -3- (3,5-dimethoxyphenyl)- 1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one) and pharmaceutical compositions for the prevention and treatment of neurological inflammatory diseases, including the same, and health functional foods for the prevention and improvement of inflammatory diseases, In addition to being useful for preventing and treating neurological inflammatory diseases, there is an effect that can be usefully used in health food for the prevention and improvement of inflammatory diseases.

1 is a hydrogen nuclear magnetic resonance spectra of the diphenylpropenone compound (DK139) (using a 400MHz Bruker nuclear magnetic resonance spectrometer).

Figure 2 is a carbon nuclear magnetic resonance spectra of the diphenylpropenone compound (DK139) (using a 100MHz Bruker nuclear magnetic resonance spectrometer).

Figure 3 shows the effect of inhibiting NF-kB phosphorylation of diphenylpropenone compound (DK139) using the immunoblot method.

Figure 4 shows the inhibitory effect of NF-kB transcriptional activity (discriptional activity) of the diphenylpropenone compound (DK139) using a luciferase reporter experiment.

Figure 5 shows the effect of inhibiting NF-kB activity of the diphenylpropenone compound (DK139) using a fluorescence microscope,

6 shows the effect of inhibiting NF-kB target gene mRNA expression of diphenylpropenone compound (DK139) using reverse transcriptase-polymerase chain reaction.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1 Synthesis of Diphenylpropenone Compound (DK139)

In the present invention, the diphenylpropenone compound represented by Chemical Formula 1 (DK139) (E-3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2 -En-1-one, (E) -3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one) is shown in Scheme 1 below. And synthesized as follows.

Scheme 1

2-hydroxy-6-methoxy-acetophenone (116 mg, 1 mmol) and 3.5-dimethoxybenzaldehyde (3,5-dimethoxybenzaldehyde, 116 mg, 1 mmol) ) Was dissolved in 15 ml of ethanol, the temperature was lowered to about 4 ° C, and 1 ml of 50% KOH aqueous solution was added slowly. Then, after stirring for 48 hours at room temperature, the temperature was cooled to about 4 ° C, neutralized by adding 4 ml of 6N HCl solution, and the aqueous solution was extracted twice with 30 ml of dichloromethane. Each of the extracted organic solvents was combined and magnesium sulfate was added to remove water, and the remaining mixture was filtered under reduced pressure. The filtered mixture was filtered under reduced pressure using a rotary evaporator to remove the solvent from the filtrate, and the remaining mixture was purified by column chromatography to obtain the target compound. Diphenylpropenone compound (DK139) (E-3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one, (E 217 mg (69% yield) of) -3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one) was obtained. M. P.; 80-82 ℃

In order to confirm the production of the diphenylpropenone compound (DK139) was confirmed through the hydrogen nuclear magnetic resonance spectra (Bruker 400MHz) and carbon nuclear magnetic resonance spectrum (Bruker 100MHz). The device used was a Bruker 400 MHz device. Hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectra are shown in FIGS. 1 and 2, respectively, and chemical mobility is as follows.

1 H-NMR (400 MHz) δ (multiple, coupling constant / Hz): 3.81 (s), 3.81 (s), 6.62 (t, 2.2), 6.96 (d, 9.0), 7.09 (d, 2.2), 7.22 (dd, 9.0, 3.1), 7.66 (d, 3.1), 7.75 (d, 15.5), 7.98 (15.5), 11.97 (bs)

13C-NMR (400 MHz) δ: 55.4, 55.9, 103.0, 107.1 113.9, 118.6, 120.8, 122.6, 123.6, 136.4, 144.9, 151.7, 155.9, 160.7, 193.3

Example 2 Inhibitory Effect of Diphenylpropenone Compound (DK139) on NF- kB (nuclear factor-kappa B) Activity by Immunoblotting

BV2 microglia cells were purchased from the American Type Culture Collection (ATTC) and incubated with DMEM / F12 (Invitrogen Life Technologies) cultures containing 10% Fetal Bovine Serum (Invitrogen Life Technologies) and Antibiotic-Antimycotic solution (Invitrogen Life Technologies) Once in a 100-mm cell culture dish was incubated in a 37%, 5% CO ₂ incubator with a seed density of 1 x 10 ⁶ .

To analyze the inhibitory effect of NF-kB activity by diphenylpropenone compound (DK139), treatment with LPS (lipopolysaccharide; purchased from sigma) at a concentration of 0.5 µg / ml, which induces inflammation in BV2 microglia cells, or LPS treatment Thirty minutes before treatment with diphenylpropenone compound (DK139) at 20 μM concentration. Cells were harvested 10 min after LPS treatment, 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 1% Triton X-100, 10% glycerol, 150 mM NaCl, 10 μg / μL Leupeptin, Cells were lysed with a buffer solution containing 1 mM PMSF (phenylmethylsulfonyl fluoride), followed by high-speed centrifugation to harvest only cell lysates. Samples prepared to contain the same amount of protein were subjected to SDS-polyacrylamide gel electrophoresis to separate proteins present in the cells. After electrophoresis, the proteins were transferred to a polystyrene membrane, followed by primary antibodies (purchased from Cell Signaling Technology, Inc.) that recognize only NF-kB and IkB in phosphorylated form and Glyceraldehyde 3-phosphate with unchanged protein expression as a control. After reacting dehydrogenase (GAPDH) -recognized primary antibody (purchased from Santa Cruz technology company) for 5 hours, the secondary antibody recognizing primary antibody (purchased from Cell Signaling Technology company) was reacted for 1 hour. Protein changes were analyzed using a chemiluminescence system (purchased from Amersham Pharmacia Biotechnology).

As shown in FIG. 3, the LPS-treated BV2 microglia cells did not change the amount of GAPDH, the control protein, but the phosphorylation was increased at the serine 32 residues of IkB and the serine 468 residues of the p65 / NFkB protein. These results indicate that LPS phosphorylates IkB, an NF-kB inhibitory protein, via IKK (IκB kinase) activity to proteolyze IkB, thereby increasing the phosphorylated form of NF-kB active forms. At this time, when the diphenylpropenone compound (DK139) of the present invention was treated in parallel with LPS, phosphorylation of IkB and NF-kB was not increased by LPS. These results confirm that the diphenylpropenone compound (DK139) of the present invention inhibits NF-kB activated by the inflammatory response.

Example 3 Inhibitory Effect of Diphenylpropenone Compound (DK139) on NF-kB Transcriptional Activity Using Reporter Experiment

In order to investigate whether the NF-kB phosphorylation inhibitory effect of the diphenylpropenone compound (DK139) of the present invention directly inhibits NF-kB activity, luciferase gene expression is reduced when the transcription factor NF-kB is activated. Luciferase reporter assay was used in which luciferase enzyme activity was increased by increasing. Injecting 0.5 μg of a reporter plasmid (pNFkB-Luc; purchased from Staratagene) containing NF-kB binding sequence into the luciferase gene expression control region into BV2 microglia cells using a Fugene 6 (purchased from Roche) reagent After 48 hours, LPS alone at 0.05 μg / ml concentration or LPS and 20 μM diphenylpropenone compound (DK139) were treated in parallel. The drug-treated cells were further cultured for 12 hours, harvested and lysed in the same manner as in the immunoblot method, and then luciferin fluorescence was measured by adding 50 μl of luciferin as a substrate to the cell solution protein. Luciferin fluorescence measurements were measured on a Centro LB960 luminometer (Berthold Technologies) using a Dual-Glo Luciferase Assay System purchased from Promega.

As a result, as shown in Figure 4 LPS increased about 18 times the luciferase enzyme activity. These results indicate that LPS induces NF-kB activity and activates the NF-kB-regulatory element present in the gene promoter, thereby increasing the expression of the luciferase enzyme gene, a target gene, resulting in luciferase. It means that the enzyme activity is increased. At this time, it was observed that in the experimental group treated with the diphenylpropenone compound (DK139) of the present invention in parallel with LPS, the luciferase enzyme activity increased by LPS was suppressed depending on the diphenylpropenone compound (DK139) treatment concentration. . Therefore, it was confirmed that the diphenylpropenone compound (DK139) of the present invention acts on microglial cells to inhibit NF-kB activity activated by the inflammation inducer LPS. These results indicate that the diphenylpropenone compound (DK139) of the present invention can be used as a prophylactic and therapeutic agent for inflammatory diseases by inhibiting NF-kB activity.

Example 4 Inhibitory Effect of Diphenylpropenone Compound (DK139) on NF-kB Activity by Fluorescence Microscopy

To investigate the effect of inhibiting NF-kB activity by the diphenylpropenone compound (DK139) at the cellular level, the presence of activated and phosphorylated NF-kB protein in the cells was investigated using fluorescence microscopy. The BV2 microglia cells were cultured in cover glass, and then treated with LPS at 0.5 μg / ml alone, or treated with 20 μM diphenylpropenone compound (DK139) 30 minutes before LPS treatment. After 10 minutes of LPS treatment, 4% formaldehyde was added to fix the cells. A 0.1% Triton X-100 solution containing 2% bovine serum albumin was added to create holes for the antibody to pass through the cell membrane. After reacting the ionized calcium binding adapter molecule 1 (Iba1), a microglia cell marker protein, with a primary antibody that binds to the phosphorylated serine 468 residue of the p65 / NFkB protein for 2 hours, Alexa-Fluor is a green phosphor that recognizes the Iba1 antibody. A mixture of a secondary antibody (purchased from Invitrogen), which binds to 488, and a secondary antibody (purchased by Invitrogen), bound to the red phosphor Alexa-Fluor 555, which recognizes the phosphorylated p65 / NFkB protein, was reacted for 30 minutes. The cover glass was washed with PBS buffer solution for 10 minutes, and the reagent Hoechst 33258 reagent (purchased from Invitrogen) was added for 10 minutes to selectively stain DNA with blue fluorescence. Then, using an EVOSf1 fluorescence microscope (purchased from Advanced Microscopy Group) Observed. As a result, as shown in FIG. 5, the LPS-treated cells showed no change in the green fluorescence indicating Iba1 in the cytoplasm, but the red fluorescence indicating the phosphorylated p65 / NFkB protein was strongly increased in the nucleus. At this time, when the diphenylpropenone compound (DK139) of the present invention is present, red fluorescence induced by LPS did not appear. These results indicate that DK139 compound inhibits NF-kB activity activated by LPS.

Example 5 Inhibitory Effect of Diphenylpropenone Compound (DK139) on NF-kB Target Gene Expression by Reverse Transcription-Polymerase Chanin Reaction (RT-PCR)

  To determine whether NF-kB target gene expression is inhibited by inhibition of NF-kB activity by the diphenylpropenone compound (DK139), mRNA of the NF-kB target gene using reverse transcriptase-polymerase chain reaction (RT-PCR) The amount of expression was measured. LPS at a concentration of 0.5 μg / ml, which induces inflammation in BV2 microglia cells, was treated alone or treated with diphenylpropenone compounds (DK139) at 5, 10, and 20 μM concentrations 30 minutes before LPS treatment. Twelve hours after LPS treatment, cells were harvested and total RNA was isolated using Trizol RNA extraction kit (purchased from QIAGEN). 0.5 μg of total RNA was synthesized by the first strand cDNA by reverse transcription and double helix cDNA was synthesized using an iScript cDNA Synthesis Kit (purchased from Bio-RAD). INOS, COX-2, IL-1β, and IL-6 mRNA were amplified by PCR with 0.0125 µg of double helix cDNA. The PCR reaction was initially denatured by heating the first cDNA at 94 ° C. for 5 minutes, and then amplifying the reaction 30 times at 94 ° C. 30 seconds, 55 ° C. 30 seconds, and 72 ° C. for 1 minute. Primer sequences used for PCR reactions were prepared as follows based on known nucleotide sequences. IL-1β (+ 141 / + 319; forward, 5′-GTTGACGGACCCCAAAAGAT-3 ′; reverse, 5′-AAGGTCCACGGGAAAGACAC-3 ′), COX2 (+ 1323 / + 1692; forward, 5′-TTGTTGAGTCATTCACCAGACAGAT-3 ′; reverse , 5′- CAGTATTGAGGAGAACAGATGGGATT-3 ′), iNOS (+ 606 / + 1412; forward, 5′-CAACCAGTATTATGGCTCCT-3 ′; reverse, 5′-GTGACAGCCCGGTCTTTCCA-3 ′), IL-6 (+ 66 / + 468; forward , 5′-TTGCCTTCTTGGGACTGATGCT-3 ′; reverse, 5′-GTATCTCTCTGAAGGACTCTGG-3 ′), and β-actin (+ 885 / + 1233; forward, 5′-TGGAATCCTGTGGCATCCATGAAAC-3 ′; reverse, 5′-TAAAACGCAGCTCAGTAACAGTCCG-3 ′ ). PCR products were electrophoresed on a 1% agar gel (agarose gel) and DNA stained with ethidium bromide. As a result, as shown in FIG. 6, mRNA expression levels of iNOS, COX-2, IL-1β, and IL-6, which are NF-kB target genes induced by LPS, were dependent on the concentration of diphenylpropenone compound (DK139). Decreased.

These results suggest that the diphenylpropenone compound (DK139) of the present invention can be used as a prophylactic and therapeutic agent for various neurological inflammatory diseases by inhibiting NF-kB activated during an inflammatory response.

As described above, specific portions of the present disclosure have been described in detail, and for those skilled in the art, these specific techniques are merely preferred embodiments, and the scope of the present disclosure is not limited thereto. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (6)

Diphenylpropenone compound represented by the following formula (E-3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one , (E) -3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one) and pharmaceutically acceptable salts thereof. [Formula 1]

Diphenylpropenone compound (E-3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-ene- represented by Formula 1 of claim 1 1-one, (E) -3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one), a) dissolving 2-hydroxy-6-methoxy-acetophenone and 3,5-dimethoxybenzaldehyde (3,5-dimethoxybenzaldehyde) in a solvent; b) adding a base to the mixed solution of step a); c) cooling the mixed solution prepared by step b) at room temperature after stirring; d) neutralizing by adding acid to the mixed solution cooled by step c) and extracting with a solvent; e) removing water after combining the respective solvents extracted by step d); And f) distilling off the water and removing the remaining mixture in step e) under reduced pressure filtration and drying; diphenylpropenone compound comprising E-3- (3,5-dimethoxyphenyl) -1- ( 2-hydroxy-5-methoxyphenyl) prop-2-en-1-one, (E) -3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2 -en-1-one). Diphenylpropenone compound represented by Chemical Formula 1 of claim 1 having an inhibitory effect through NF-kB (nuclear factor-kappa B) activity inhibition of microglia cells that cause nervous system inflammation (E-3) -(3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one, (E) -3- (3,5-dimethoxyphenyl)- A pharmaceutical composition for preventing and treating inflammatory diseases of the nervous system, including 1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one) and a pharmaceutically acceptable salt. The method of claim 3, wherein The pharmaceutical composition is a pharmaceutical composition for the prevention and treatment of inflammatory diseases of the nervous system, characterized in that it comprises at least one pharmaceutically acceptable carrier, diluent or excipient. Diphenylpropenone compound (E-3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-ene- represented by Formula 1 of claim 1 1-one, (E) -3- (3,5-dimethoxyphenyl) -1- (2-hydroxy-5-methoxyphenyl) prop-2-en-1-one) and food acceptable food supplement additives Health functional foods for the prevention and improvement of inflammatory diseases. The method of claim 5, The health functional food is a health functional food for the prevention and improvement of inflammatory diseases of the nervous system, characterized in that the tablets, capsules, pills or liquid form.

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