KR20160025445A

KR20160025445A - New compounds having anti-inflammatory and anti oxidatant activity through TLR4 binding competition with LPS and medical use thereof

Info

Publication number: KR20160025445A
Application number: KR1020150101640A
Authority: KR
Inventors: 정해영; 문형룡; 문경미; 박윤정; 김남득; 박지영; 김혜림; 김철민; 정지원
Original assignee: 부산대학교 산학협력단
Priority date: 2014-08-25
Filing date: 2015-07-17
Publication date: 2016-03-08
Also published as: KR101686872B1

Abstract

The present invention relates to a novel compound having anti-oxidant and anti-inflammatory effects of inhibiting macrophage activation due to competitive bonding to TLR4 against LPS, and a medical use thereof. The compounds, according to the present invention, have anti-oxidant and anti-inflammatory activities, and thus can be helpfully used in preventing geriatric disease or treating inflammations. In addition, the compounds are competitively bonded to TLR4-MD2 against LPS, in particular, through the competitive bonding against LPS, thereby being able to be used as a pharmaceutical composition or a health food for preventing and treating sepsis, metabolic diseases, or cardiovascular diseases.

Description

TECHNICAL FIELD [0001] The present invention relates to a novel compound having antioxidant and anti-inflammatory activity through competitive binding to LPS in TLR4, and a medicinal use thereof for TLR4 binding competition with LPS and medical use thereof.

The present invention relates to novel compounds having antioxidant, antiinflammatory and anti-aging effects that inhibit the activity of macrophages through competitive binding to lipopolysaccharide (LPS) in TLR4 and their medical uses.

The inflammation reaction is promoted by oxidative stress in the body. Oxidative stress causes degeneration of the nucleic acid (DNA), the main component of the muscle, and fat, the main component of the cell membrane, Has a problem with the body function itself, which initiates or exacerbates an inflammatory reaction by increasing gene expression of certain cells that cause degenerative diseases as well as apoptosis.

Oxygen absorbed by normal respiration enters the mitochondria, the energy producing tissue in the cell, and produces energy (ATP) required for maintaining body temperature, cell activity, and physical activity. However, due to the fundamental defects of mitochondria, oxygen is not completely burned and by-products are produced, which is free radical, free radical. To cope with oxidative stress, organisms have a defense system that detoxifies, destroys, or neutralizes toxic oxygen through an evolutionary process. This is due to the fact that many resistance elements in the body that balance with oxidation / As it ages, it breaks down due to the destructive action of active oxygen and causes oxidative destruction to stress cells and tissues, resulting in a decline in body function and inflammation at the same time.

Macrophages are macrophages that are distributed in all tissues of animals and are the immune cells responsible for the innate immune response in the human body. They are white blood cells that play an important role in the human immune system. The macrophages activated by external stimuli induce an inflammatory reaction through the secretion of inflammatory mediates, thereby causing degenerative brain diseases such as asthma, bronchitis, arthritis, multiple sclerosis, arteriosclerosis, stroke, Alzheimer's disease or Parkinson's disease, And the like, and the disease is worsened.

Lipopolysaccharide (LPS), one of the endotoxins, is involved in pro-inflammatory processes such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin- inflammatory cytokines, and secretes inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2). In the inflammatory state, cyclooxygenase-2 (COX-2) and NO synthase (NOS) are induced and excessive amounts of PGE2 and NO are produced and various diseases and carcinogenesis are promoted. In particular, NOS, an enzyme that produces NO, and cyclooxygenase (COX), an enzyme that mediates the biosynthesis of various prostaglandins (PGs), are known to be important mediators of inflammatory response.

The Toll like receptor, a receptor for LPS, has been found to be closely related to oxidative stress. Among them, TLR2 / TLR4 mainly detects lipoprotein in the outer cell membrane / cell wall of bacteria and induces ROS (reactive oxygen species) causing oxidative stress. In the case of TLR4, even when macrophages are stimulated by stimulation of LPS, various inflammatory factors are generated due to NF-κB activation, leading to pathologies of various diseases and also involved in carcinogenesis. However, in vivo, it is known that there is no endogenous enzyme that can directly deactivate the produced active oxygen and the action of NO and ONOO, and studies on the development of antioxidants and anti-inflammation specifically suppressing and eliminating this are urgently required Has come. Studies on the development of antioxidants having ONOO scavenging activity have been carried out in order to protect cells from damage caused by active oxygen, NO, and ONOO, which are cytotoxic substances, and many natural products or synthetic eradicants have been developed.

Antioxidants are used not only to remove or absorb oxygen, but also to react with free radicals to minimize the loss of certain vitamins and essential amino acids, or to retard or prevent rancidity of preserved products. Synthetic antioxidants commonly used in foods or pharmaceuticals include butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG), tertiary butyl hydro- Quinone (TBHQ, Tertiary butyl hydroquinone). However, when they are administered to experimental animals at a high concentration, it is known that hepatic hypertrophy is induced or carcinogenic. In particular, butylated hydroxytoluene has been known to increase microsomal enzyme activity in the liver of experimental animals through various studies, and the safety of these phenolic synthetic antioxidants has been controversial. Usage is regulated by law. As a result, much research has been carried out in anticipation of developing a safe and economical natural antioxidant with a high antioxidant effect.

In recent years, studies on antioxidative substances have been actively carried out in researches on natural products. To date, natural antioxidants known to date include tocopherols, flavonoids, gossypol, sesamol, oryzanol, vitamin C, and the like. Among them, tocopherol and L-ascorbic acid are preferred as natural antioxidants. Of these, tocopherol has high safety, but has a disadvantage that it is low in oxidation inhibiting ability and expensive .

A TLR4 antagonist that binds directly to TLR4 has rarely been reported, but eritoran, which was created at the end of 2009 by Eisai in Japan, binds to MD2 of TLR4 and inhibits signaling mechanisms related to it. It is reported that the patient has entered clinical phase 3 with the purpose of treatment. However, the molecular weight is so large that it is difficult to mass-produce eritoran.

Korean Patent Laid-open No. 10-2011-0119279 (published on November 22, 2011)

It is an object of the present invention to provide novel compounds having antioxidant, anti-inflammatory and anti-aging effects that inhibit the activity of macrophages through competitive binding to lipopolysaccharide (LPS) in TLR4.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating an inflammatory disease containing the novel compound as an active ingredient.

Still another object of the present invention is to provide a health functional food for preventing or ameliorating inflammatory diseases containing the novel compound as an active ingredient, antioxidant or anti-aging health food.

In order to solve the above problems, there is provided a compound represented by the following general formula (1).

&Lt; Formula 1 >

In the above formula (1), R1 to R4 may be the same or different and are any one of H, OH, Br or C1 to C4 alkoxy, and X is O or S;

The present invention also provides a pharmaceutical composition for preventing or treating an inflammatory disease containing the above-mentioned compound as an active ingredient.

The present invention also provides a health functional food for preventing or ameliorating an inflammatory disease containing the above-mentioned compound as an active ingredient.

In addition, the present invention provides a health functional food for antioxidation containing the above-mentioned compound as an active ingredient.

The present invention also provides an anti-aging health functional food containing the above-mentioned compound as an active ingredient.

The present invention relates to a novel compound having antioxidant and antiinflammatory activity through competitive binding to LPS in TLR4 and its medical use. The compounds according to the present invention are useful as a small molecule that inhibits the activity of macrophages through competitive binding to LPS in TLR4 (ROS) and inflammatory mediators (NO, ONOO ^- ) and inflammatory genes (Cox - 2, iNOS) during the inflammatory reaction, inhibit the expression of inflammatory and activating oxygen species (ROS) In addition, it suppresses the activity of the transcription factor (NF-κB) that regulates inflammation, and thus has antioxidative and anti-inflammatory actions and exhibits a more effective anti-aging effect. In addition, this experiment was also validated in the liver of C57BL / 6 mice. Therefore, the novel compounds can be used in various fields such as pharmaceuticals or health foods that have less side effects and are safe in the prevention and treatment of sepsis, metabolic diseases or cardiovascular diseases.

1 is deulrosseo compound, ROS and ONOO which inhibit the TLR4 target ^a graph for the substance having an inhibitory effect with the screening (screening) a.
FIG. 2 shows that the compound according to the present invention binds to the MD2 portion of TLR4 and competitively binds to LPS through docking simulation.
FIG. 3 is a graph showing that compounds No. 42 and No. 50, which are novel compounds of the present invention, are stable to cytotoxicity at a concentration of 1 μM to 10 μM through a cell viability assay.
4A is a graph showing that oxidative stress (ROS, ONOO ^- , NO) increased by LPS in macrophages is reduced by compounds 42 and 50 of the present invention.
FIG. 4B is a graph showing that ROS and ONOO ^- increased by LPS in macrophages through fluorescent dyes are reduced by compounds 42 and 50 of the present invention.
FIG. 5 is a graph showing the inhibition of the expression of COX-2 and iNOS, which are inflammation-inducing proteins increased by LPS, by treating compounds No. 42 and No. 50.
FIG. 6 is a graph showing the expression and activity of NF-κB, a transcription factor of oxidative stress and inflammation-related protein, showing that the activity of NF-κB phosphorylation in ser536 among the phosphorylation sites of P65 was 42 and 50 The compound is inhibited by the compound.
FIG. 7 shows the activity of NF-kB in macrophages, showing inhibition of translocation of NF-kB by compounds No. 42 and No. 50.
FIG. 8 shows the effect of translocation into the nucleus using a ligation luciferase reporter vector with NF-κB binding site, wherein activation of NF-κB increased by LPS Is inhibited by the compounds 42 and 50 in a concentration-dependent manner.
FIG. 9 is a graph showing inhibitory effects of NF-κB translocation into the nucleus due to a decrease in IKKB activity and IκBα activity in a concentration-dependent manner in all of the compounds treated with the compounds No. 42 and No. 50.
FIG. 10 shows that the activity of AKT, which directly affects IKK in the NF-κB high signal transduction system, was inhibited by compounds 42 and 50 in a concentration-dependent manner, and the inhibitory effect of AKT on PTEN and NOX4 Is also inhibited by compounds 42 and 50 in a concentration-dependent manner.
FIG. 11 shows the results of ROS and ONOO ^- measurement of liver and blood of C57BL / 6 in order to confirm the inhibitory effect of oxidative stress on macrophages in animal models. ROS and ONOO ^- were increased in liver and blood in LPS group mice, and decreased in a dose dependent manner in compounds 42 and 50, respectively.
Figure 12 confirmed the phosphorylation and expression of NF-κB, an important transcription factor involved in inflammation in the liver of C57BL / 6. The increased phosphorylation of P65 and P65 in LPS was reduced in compounds 42 and 50 and also in COX-2, a pro-inflammatory gene, and iNOS also decreased COX -2 is the same trend.

The present invention relates to a method for inhibiting oxidative stress in Raw264.7 macrophage. The present invention relates to a method for inhibiting oxidative stress, comprising the steps of: (a) contacting TNF-α with TNF- Inhibits pro-inflammatory cytokines such as IL-6 and interleukin-1β (IL-1β) and inhibits inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2) (COX-2), NO synthase (NOS) and excessive amounts of PGE2, NO, etc., by blocking the AKT signaling mechanism by inhibiting transcription of NF-κB, a transcription factor that induces inflammation To prevent various diseases and cancer-related diseases (carcinogenesis) associated with sepsis.

The present invention provides a compound represented by the following formula (1).

&Lt; Formula 1 >

Preferably, the compound is selected from the group consisting of 5- (4-hydroxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) (1H, 3H, 5H) -triene, 5- (2,4-dihydroxybenzyl) pyrimidine- (1H, 3H, 5H) -triene, 5- (3-ethoxy-4-hydroxybenzyl) pyrimidine-2,4 , 6 (1H, 3H, 5H) -triene, 5- (3-hydroxy-4-methoxybenzyl) pyrimidine- (1H, 3H, 5H) -triene, 5- (3,4-dimethoxybenzyl) pyrimidine-2,4,6 3H, 5H) -thione, 5- (3,4,5-trimethoxybenzyl) pyrimidine (1H, 3H, 5H) -triene, 5- (4-hydroxy-3,5-dimethoxybenzyl) pyrimidine- (1H, 3H, 5H) -triene, 5- (3,5-dibromo-4-hydroxybenzyl) pyrimidine- Thioxo-dihydropyrimidine-4,6 (1H, 5H) - Dihydroxybenzyl) -2-thioxo dihydropyrimidine-4,6 (1H, 5H) -dione, 5- (2,4- (1H, 5H) -dione, 5- (4-hydroxy-3-methoxybenzyl) -2-thioxo dihydropyrimidine- -Dione, 5- (3-ethoxy-4-hydroxybenzyl) -2-thioxo dihydropyrimidine-4,6 (1H, 5H) -dione, 5- (4-methoxybenzyl) -2-thioxo dihydropyrimidine-4,6 -Dione, 5- (3,4-dimethoxybenzyl) -2-thioxo dihydropyrimidine-4,6 (1H, 5H) (1H, 5H) -dione, 5- (2-hydroxybenzyl) -2-thioxo- dihydropyrimidine-4,6 (1H, 5H) -dione and 5- (4-hydroxy-3,5-dimethoxybenzyl) -2-thioxo- dihydropyrimidine -Thioxo < / RTI > dihydropyrimidine-4,6 (1H, 5 H) -dione, 5- (3-bromo-4-hydroxybenzyl) -2-thioxodihydropyrimidine-4,6 -4-hydroxybenzyl) -2-thioxo dihydropyrimidine-4,6 (1H, 5H) -dione, but is not limited thereto.

More preferably, the compound is 5- (4-hydroxy-3-methoxybenzyl) -2-thioxo dihydropyrimidine-4,6 (1H, 5H) (Compound 42) or 5- (4-hydroxy-3,5-dimethoxybenzyl) -2-thioxo dihydropyrimidine-4,6 (lH, 5H) -dione (Compound 50) But is not limited thereto.

(2)

(3)

In addition, the present invention provides a composition for preventing or treating inflammatory diseases containing the above-mentioned compound as an active ingredient.

Preferably, the compound is capable of inhibiting the activity of macrophages through competitive binding to lipopolysaccharide (LPS) in TLR4.

Preferably, the inflammatory disease may be, but is not limited to, asthma, bronchitis, sepsis, arthritis, hepatitis, rheumatoid arthritis, osteoarthritis, ulcerative colitis, myocarditis, multiple sclerosis or viral infection.

In the case of the pharmaceutical composition of the present invention, the pharmaceutical composition may contain a pharmaceutically acceptable carrier in addition to the above-mentioned effective ingredient. Such a pharmaceutically acceptable carrier is usually used in the preparation of pharmaceuticals, But are not limited to, dextrose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, Tartrate, magnesium stearate, mineral oil, and the like, but the present invention is not limited thereto. In addition, the pharmaceutical composition may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. as an additive.

The dosage form of the pharmaceutical composition is determined depending on the severity of the symptoms, and local administration is usually preferred. The dosage of the active ingredient in the pharmaceutical composition may vary depending on the route of administration, the severity of the disease, the age, sex, and weight of the patient, and may be administered once to several times per day.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The pharmaceutical composition may be prepared in unit dose form by formulating it with a pharmaceutically acceptable carrier and / or excipient, or may be prepared by inserting it into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions, or may be in the form of elixirs, excipients, powders, granules, tablets, alerts, lotions, ointments and the like.

In the present invention, the term "health functional food" refers to a food having a biological control function such as prevention and treatment of diseases, bio-defense, immunity, recovery after disease and aging inhibition.

Meanwhile, the health functional food of the present invention can be prepared by various methods known in the field of pharmacy or pharmacy, and can be prepared by itself or in combination with any pharmaceutically acceptable carrier, excipient, diluent, Can also be produced. Preferably in the form of beverage, ring, granule, tablet or capsule.

The health functional food of the present invention may further comprise ingredients that are conventionally added at the time of food production and which are pharmaceutically acceptable.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example 1> Compound 1 to 12 Synthesis

The following table 1 shows the results of the synthesis of 5- (substituted benzylidene) pyrimidine-2,4,6 (1H, 3H, 5H) -trione analog [5- (substituted benzylidene) pyrimidine- 5H) -trione analog] To illustrate the substitution pattern of Compound 1-12.

&Lt; Formula 4 >

compound R ¹ R ² R ³ R ⁴ One H H OH H 2 H OH OH H 3 OH H OH H 4 H OMe OH H 5 H OEt OH H 6 H OH OMe H 7 H H OMe H 8 H OMe OMe H 9 OMe H OMe H 10 H OMe OMe OMe 11 H OMe OH OMe 12 H Br OH Br

OMe represents methoxy and OEt represents ethoxy.

Pyrimidine-2,4,6 (1H, 3H, 5H) -trione was used in place of 5- (substituted benzylidene) pyrimidine-2,4,6 analog] compounds 1 to 12 were synthesized as follows. That is, a suspension of substituted benzaldehyde (1.44-2.60 mmol) and barbituric acid (0.7-1.2 eq.) In EtOH (4 mL) and H ₂ O (4 mL) Lt; 0 > C. Before the reaction temperature reached 80 ° C, in most cases the reaction mixture became a clean solution. Meanwhile, during heating (1 to 18 hours), a precipitate was formed, and after cooling, the precipitate was filtered. In the case of the synthesis of Compound 12, the reaction was carried out at room temperature instead of 80 ° C, and the same operation was carried out at room temperature. In consideration of the characteristics of the remaining substituted benzaldehyde, the filter cake was washed with ethanol and / or methylene chloride and water to obtain the desired product (yield: 60.3 to 99.4%).

1. 5- (4- Hydroxybenzylidene (1H, 3H, 5H) -trione (Compound 1) Synthesis of pyrimidine-2,4,6 (1H,

Yellow solid; Reaction time, 6 hours; Yield, 82.6%; Melting point,> 300 ℃; ¹ H NMR (400 MHz, DMSO-d ₆ )? 11.23 (s, 1H), 11.10 (s, 1H), 10.79 (s, 1 H), 6.84 (d, 2H, J = 8.8 Hz); ¹³ C NMR (100 MHz, DMSO-d ₆ )? 164.8, 163.7, 163.0, 156.1, 150.9, 139.0, 124.4, 116.2, 114.9; LRMS (ESI) m / z 231 (MH) ^- .

2. 5- (3,4- Dihydroxybenzylidene (1H, 3H, 5H) -trione (Compound 2) was obtained in the same manner as in Example 1, synthesis

Orange solid; Reaction time, 8 hours; Yield, 99.3%; Melting point,> 300 ℃; ¹ H NMR (500 MHz, DMSO-d ₆ )? 11.14 (br s, 2 H), 9.76 (br s, 1H), 9.55 , 1H), 7.61 (d, 1H, J = 8.0 Hz), 6.83 (d, 1H, J = 7.5 Hz); ¹³ C NMR (100 MHz, DMSO-d ₆ )? 164.9, 162.9, 156.7, 153.0, 150.9, 145.5, 132.0, 124.9, 122.0, 116.0, 114.3; LRMS (ESI) m / z 247 (MH) ^- .

3. 5- (2,4- Dihydroxybenzylidene 2,4-dihydroxybenzylidene pyrimidine-2,4,6 (1H, 3H, 5H) -trione] (Compound 3) synthesis

Yellow solid; Reaction time, 10 hours; Yield, 60.3%; Melting point,> 300 ℃; ^{_{1 H NMR (500MHz, D 2}} O + NaOH) δ 8.07 (s, 1 H), 7.26 (d, 1 H, J = 8.5Hz), 6.44 (dd, 1H, J = 2.0, 9.0Hz), 6.24 ( s, 1H); ^{13 C NMR (100MHz, DMSO-} d 6) δ 168.5, 166.7, 162.2, 157.2, 156.8, 145.7, 134.0, 116.5, 113.1, 109.8, 103.3; LRMS (ESI) m / z 247 (MH) ^- .

4. 5- (4- Hydroxy -3- Methoxybenzylidene Pyridine-2,4,6 (1H, 3H, 5H) -trione] (Compound (5) 4) Synthesis

Thick yellow solid; Reaction time, 18 hours; Yield, 97%; Melting point, 288.6-290.7 DEG C; ¹ H NMR (400 MHz, DMSO-d ₆ )? 11.23 (s, 1H), 11.11 (s, 1H), 10.54 8.18 (s, 1H), 7.77 (dd, 1H, J = 2.0,8.4Hz), 6.86 (d, 1H, J = 8.4Hz), 3.79 (s, 3H); ¹³ C NMR (100 MHz, DMSO-d ₆ )? 164.8, 163.2, 156.6, 153.7, 150.9, 147.6, 133.2, 124.9, 118.6, 116.0, 114.6, 56.2; LRMS (ESI) m / z 261 (MH) ^- .

5. 5- (3- Ethoxy -4- Hydroxybenzylidene (1H, 3H, 5H) -trione, which is a compound of the formula 5) Synthesis

Orange solid; Reaction time, 15 hours; Yield, 77%; Melting point, 244.7-246.1 DEG C; ¹ H NMR (500 MHz, DMSO-d ₆ )? 11.24 (s, 1H), 11.11 (s, 1H), 10.46 2H), 7.74 (d, 1H, J = 8.5 Hz), 6.90 (d, 1H, J = 8.5 Hz), 4.08 ; ¹³ C NMR (100 MHz, DMSO-d ₆ )? 164.8, 163.1, 156.6, 154.0, 150.8, 146.8, 133.2, 124.9, 119.7, 116.1, 114.6, 64.5, 15.2; LRMS (ESI) m / z 275 (MH) ^- .

6. 5- (3- Hydroxy -4- Methoxybenzylidene Pyridine-2,4,6 (1H, 3H, 5H) -trione] (Compound (3) 6) Synthesis

Thick yellow solid; Reaction time, 17 hours; Yield, 93%; Melting point, 279.3-281.4 DEG C; ¹ H NMR (500 MHz, DMSO-d ₆ )? 11.26 (s, 1H), 11.13 (s, 1H), 9.42 H), 7.70 (d, 1H, J = 8.5 Hz), 7.03 (d, 1H, J = 9.0 Hz), 3.87 (s, 3H); ¹³ C NMR (100 MHz, DMSO-d ₆ )? 164.7, 162.8, 156.2, 153.6, 150.9, 146.4, 131.0, 126.1, 121.1, 115.7, 112.0, 56.4; LRMS (ESI) m / z 261 (MH) ^- .

7. 5- (4- Methoxybenzylidene (1H, 3H, 5H) -trione (Compound 7) Synthesis of pyrimidine-2,4,6 (1H,

Yellow solid; Reaction time, 13 hours; Yield, 93%; Melting point, 292.4-294.3 DEG C; ¹ H NMR (400 MHz, DMSO- d ₆ )? 11.27 (s, 1H), 11.14 (s, 1H), 8.33 (d, 2H, J = 9.2 Hz), 8.21 d, 2H, J = 8.8 Hz), 3.83 (s, 3H); ¹³ C NMR (100 MHz, DMSO-d ₆ )? 164.6, 164.1, 162.8, 155.6, 150.9, 138.1, 125.8, 116.2, 114.6, 56.4; LRMS (ESI) m / z 245 (MH) ^- .

8. 5- (3,4- Dimethoxybenzylidene Pyridine-2,4,6 (1H, 3H, 5H) -trione] (Compound 8) synthesis

Yellow solid; Reaction time, 9 hours; Yield, 96.6%; Melting point,> 300 ℃; ¹ H NMR (400 MHz, DMSO-d ₆ )? 11.27 (s, 1H), 11.15 (s, 1H), 8.37 (d, 1H, J = 2.0Hz) dd, 1H, J = 2.0, 8.4 Hz), 7.07 (d, 1H, J = 8.4 Hz), 3.84 (s, 3H), 3.77 (s, 3H); ¹³ C NMR (100 MHz, DMSO-d ₆ )? 164.7, 163.0, 156.1, 154.3, 150.9, 148.5, 132.4, 125.9, 117.4, 115.9, 111.8, 56.5, 56.1; LRMS (ESI) m / z 275 (MH) ^- .

9. 5- (2,4- Dimethoxybenzylidene Pyridine-2,4,6 (1H, 3H, 5H) -trione] (Compound 9) synthesis

Orange solid; Reaction time, 8 hours; Yield, 97%; Melting point, 291.1-291.7 DEG C; ^{1 H NMR (500MHz, DMSO-} d 6) δ 11.21 (s, 1 H), 11.06 (s, 1 H), 8.61 (s, 1 H), 8.53 (d, 1 H, J = 8.5Hz), 6.63 (d, 1H, J = 2.0 Hz), 6.61 (dd, 1H, J = 2.0, 9.0 Hz), 3.90 (s, 3H), 3.87 (s, 3H); ¹³ C NMR (100 MHz, DMSO-d ₆ )? 166.5, 164.8, 163.1, 162.8, 150.9, 149.7, 136.1, 115.3, 114.9, 106.5, 98.1, 56.9, 56.5; LRMS (ESI) m / z 275 (MH) ^- .

10. 5- (3,4,5- Trimethoxybenzylidene Pyrimidine-2,4,6 (1H, 3H, 5H) -trione] (Compound (5) 10) Synthesis

Yellow solid; Reaction time, 1 hour; Yield, 84.0%; Melting point, 274.8-275.4 DEG C; ¹ H NMR (400 MHz, DMSO- d ₆ )? 11.33 (s, 1H), 11.20 (s, 1H), 8.22 ), 3.75 (s, 3 H); ¹³ C NMR (100 MHz, DMSO-d ₆ )? 164.4, 162.8, 155.9, 152.6, 150.8, 142.6, 128.2, 117.9, 113.3, 61.0, 56.7; LRMS (ESI) m / z 305 (MH) ^- .

11. 5- (4- Hydroxy -3,5- Dimethoxybenzylidene ) Pyrimidine-2,4,6 (1H, 3H, 5H) - Tree [5- (4- Hydroxy -3,5- dimethoxybenzylidene ) 피리 미딘 -2,4,6 (1H, 3H, 5H) -trione] (Compound 11) Synthesis

Orange solid; Reaction time, 2 hours; Yield, 99.4%; Melting point,> 300 ℃; ¹ H NMR (500 MHz, DMSO-d ₆ )? 11.25 (s, 1H), 11.12 (s, 1H), 9.97 H), 3.82 (s, 6 H); ^{13 C NMR (100MHz, DMSO-} d 6) δ 164.8, 163.2, 157.0, 150.9, 147.8, 143.1, 123.5, 114.9, 114.6, 56.7; LRMS (ESI) m / z 291 (MH) ^- .

12. 5- (3,5- Dibromo -4- Hydroxybenzylidene ) Pyrimidine-2,4,6 (1H, 3H, 5H) -trione [5- (3,5- Dibromo -4- hydroxybenzylidene ) 피리 미딘 -2,4,6 (1H, 3H, 5H) - trione ] (Compound 12) Synthesis

Yellow solid; Reaction time, 10 hours; Yield 81.7%; ^{1 H NMR (500 MHz, DMSO-} d 6) d 11.33 (s, 1 H), 11.22 (s, 1 H), 8.54 (s, 2 H), 8.10 (s, 1 H); ¹³ C NMR (100 MHz, DMSO- d ₆ ) d 164.0, 162.7, 155.5, 152.4, 150.8, 139.0, 127.1, 118.2, 111.4.

< Example 2> Compound 13 to 26 Synthesis

The following Table 2 summarizes the results of the synthesis of 5- (substituted benzylidene) -2-thioxo dihydropyrimidine-4,6 (1H, 5H) -diyl analog [5- (substituted benzylidene) -2-thioxodihydropyrimidine- , 5H) -dione analog] To illustrate the substitution pattern of compounds 13-26.

&Lt; Formula 5 >

compound R ¹ R ² R ³ R ⁴ 13 H H OH H 14 H OH OH H 15 OH H OH H 16 H OMe OH H 17 H OEt OH H 18 H OH OMe H 19 H H OMe H 20 H OMe OMe H 21 OMe H OMe H 22 OH H H H 23 H OMe OMe OMe 24 H OMe OH OMe 25 H Br OH H 26 H Br OH Br

OMe represents methoxy and OEt represents ethoxy.

5- (substituted benzylidene) -2-thioxo dihydropyrimidine-4,6 (1H, 5H) -dione analog [5- (substituted benzylidene) -2-thioxodihydropyrimidine- dione analog] Compound 13-26 was synthesized as follows. That is, a solution of benzaldehyde (1.52-1.97 mmol) and thiobarbituric acid (0.9-1.1 eq.) Substituted in ethanol (4-8 mL) and H ₂ O (4-8 mL) The suspension was heated to 80 < 0 > C. Before the reaction temperature reached 80 캜, the reaction mixture became a clean solution in most cases. However, during the additional heating (5 minutes to 10.5 hours), a precipitate formed and after cooling the precipitate was filtered. In the case of the synthesis of compounds 25 and 26, the reaction was carried out at room temperature instead of 80 ° C, and the same operation as above was carried out at room temperature. In consideration of the characteristics of the remaining substituted benzaldehyde, the filter cake was washed with ethanol and / or methylene chloride and water to obtain Compound 13-26 (yield: 46.9-99.5%).

1. 5- (4- Hydroxybenzylidene )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (4- Hydroxybenzylidene )-2- thioxodihydropyrimidine -4,6 (1H, 5H) - dione ] (Compound 13) Synthesis

Orange solid; Reaction time, 3 hours; Yield, 96%; Melting point, 291.7-293.5 DEG C; ^{1 H NMR (400MHz, DMSO-} d 6) δ 12.30 (s, 1 H), 12.20 (s, 1 H), 10.93 (s, 1 H), 8.34 (d, 2 H, J = 8.8Hz), 8.19 (s, 1 H), 6.86 (d, 2H, J = 8.8 Hz); ¹³ C NMR (100 MHz, DMSO-d ₆ )? 178.8, 164.4, 163.1, 160.7, 157.2, 139.5, 124.6, 116.4, 114.9; LRMS (ESI) m / z 247 (MH) ^- .

2. 5- (3,4- Dihydroxybenzylidene )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (3,4- Dihydroxybenzylidene )-2- thioxodihydropyrimidine -4,6 (1H, 5H) -dione] (Compound 14) Synthesis

Orange solid; Reaction time, 3 hours; Yield, 99.5%; Melting point,> 300 ℃; ^{1 H NMR (400MHz, DMSO-} d 6) δ 12.28 (s, 1 H), 12.19 (s, 1 H), 10.55 (br s, 1 H), 9.56 (br s, 1 H), 8.25 (s, 1 H), 8.10 (s, 1H), 7.63 (d, 1H, J = 8.4 Hz), 6.83 (d, 1H, J = 8.4 Hz); ¹³ C NMR (100 MHz, DMSO-d ₆ )? 178.8, 163.2, 160.7, 157.7, 153.9, 145.7, 132.9, 125.2, 122.1, 116.2, 114.3; LRMS (ESI) m / z 263 (MH) ^- .

3. 5- (2,4- Dihydroxybenzylidene )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (2,4- Dihydroxybenzylidene )-2- thioxodihydropyrimidine -4,6 (1H, 5H) -dione] (Compound 15) Synthesis

Thick yellow solid; Reaction time, 5 min; Yield, 82.5%; Melting point,> 300 ℃; ¹ H NMR (400 MHz, DMSO-d ₆ )? 12.16 (s, 1H), 12.06 (s, 1H), 11.00 H, J = 8.8 Hz), 8.76 (s, 1H), 6.37 (d, 1H, J = 1.6 Hz), 6.31 (dd, 1H, J = 2.0, 9.2 Hz); ¹³ C NMR (100 MHz, DMSO-d ₆ )? 178.6, 167.3, 164.8, 163.6, 161.0, 151.2, 137.3, 113.5, 111.9, 109.1, 102.1; LRMS (ESI) m / z 263 (MH) ^- .

4. 5- (4- Hydroxy -3- Methoxybenzylidene )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (4- Hydroxy -3- methoxybenzylidene )-2- thioxodihydropyrimidine -4,6 (1H, 5H) -dione] (Compound 16) Synthesis

Red solid; Reaction time, 3 hours; Yield, 98.8%; Melting point, 260.9-263.6 캜; ¹ H NMR (400 MHz, DMSO-d ₆ )? 12.31 (s, 1H), 12.20 (s, 1H), 10.70 ), 7.83 (d, 1H, J = 8.8Hz), 6.88 (d, 1H, J = 8.4Hz), 3.80 (s, 3H); ¹³ C NMR (100 MHz, DMSO-d ₆ )? 178.7, 163.1, 160.9, 157.6, 154.5, 147.7, 133.9, 125.1, 118.9, 116.2, 114.7, 56.2; LRMS (ESI) m / z 277 (MH) ^- .

5. 5- (3- Ethoxy -4- Hydroxybenzylidene )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (3- Ethoxy -4- hydroxybenzylidene )-2- thioxodihydropyrimidine -4,6 (1H, 5H) -dione] (Compound 17) Synthesis

Light orange solid; Reaction time, 3 hours; Yield, 88.5%; Melting point, 285.0-287.3 캜; ¹ H NMR (400 MHz, DMSO- d ₆ )? 12.30 (s, 1H), 12.19 (s, 1H), 10.65 (br s, 1H), 8.47 1H, J = 8.4 Hz), 4.05 (q, 2H, J = 7.2 Hz), 1.33 (t, 3H , J = 7.2 Hz); ¹³ C NMR (100 MHz, DMSO-d ₆ )? 178.7, 163.1, 160.9, 157.7, 154.8, 146.9, 134.0, 125.1, 119.8, 116.2, 114.6, 64.5, 15.2; LRMS (ESI) m / z 291 (MH) ^- .

6. 5- (3- Hydroxy -4- Methoxybenzylidene )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (3- Hydroxy -4- methoxybenzylidene )-2- thioxodihydropyrimidine -4,6 (1H, 5H) -dione] (Compound 18) Synthesis

Orange solid; Reaction time, 8 hours; Yield, 97%; Melting point, 278.9-280.5 DEG C; ¹ H NMR (400 MHz, DMSO-d ₆ )? 12.32 (s, 1H), 12.23 (s, 1H), 9.58 8.13 (s, 1H), 7.73 (dd, 1H, J = 2.0 Hz, 8.4 Hz), 7.04 (d, 1H, J = 8.8 Hz), 3.86 (s, 3H); ^{13 C NMR (100 MHz, DMSO} -d 6) δ 178.9, 163.0, 160.6, 157.3, 154.2, 146.6, 131.8, 126.2, 121.2, 115.7, 112.1, 56.6; LRMS (ESI) m / z 277 (MH) ^- .

7. 5- (4- Methoxybenzylidene )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [Compound 5- (4-Methoxybenzylidene) -2-thioxodihydropyrimidine-4,6

Yellow solid; Reaction time, 4 hours; Yield, 77.9%; Melting point,> 300 ℃; ¹ H NMR (400 MHz, DMSO-d ₆ )? 12.34 (s, 1H), 12.25 (s, 1H), 8.38 (d, 2H, J = 8.8Hz) d, 2H, J = 9.2 Hz), 3.85 (s, 3H); ^{13 C NMR (100MHz, DMSO-} d 6) δ 179.0, 164.7, 162.9, 160.6, 156.6, 138.6, 126.0, 116.3, 114.8, 56.5; LRMS (ESI) m / z 261 (MH) ^- .

8. 5- (3,4- Dimethoxybenzylidene )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) - Dion [5- (3,4- Dimethoxybenzylidene )-2- thioxodihydropyrimidine -4,6 (1H, 5H) -dione] (Compound 20) Synthesis

Thick yellow solid; Reaction time, 4 hours; Yield, 91.9%; Melting point, 269.9-271.7 DEG C; ¹ H NMR (500 MHz, DMSO-d ₆ )? 12.37 (s, 1H), 12.26 (s, 1H), 8.41 , J = 8.5 Hz), 7.12 (d, 1H, J = 8.5 Hz), 3.88 (s, 3H), 3.80 (s, 3H); ¹³ C NMR (100 MHz, DMSO-d ₆ )? 178.8, 163.0, 160.8, 157.2, 154.9, 148.5, 133.0, 126.1, 117.6, 116.0, 111.9, 56.6, 56.1; LRMS (ESI) m / z 291 (MH) ^- .

9. 5- (2,4- Dimethoxybenzylidene )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) - Dion [5- (2,4- Dimethoxybenzylidene )-2- thioxodihydropyrimidine -4,6 (1H, 5H) -dione] (Compound 21) Synthesis

Orange solid; Reaction time, 4 hours; Yield, 98.4%; Melting point, 294.1-295.4 DEG C; ¹ H NMR (400 MHz, DMSO-d ₆ )? 12.27 (s, 1H), 12.16 (s, 1H), 8.62 (s, 1H), 6.60 (d, 1H, J = 8.0 Hz), 3.89 (s, 3H), 3.86 (s, 3H); ¹³ C NMR (100 MHz, DMSO-d ₆ )? 178.9, 167.3, 163.6, 163.1, 160.6, 150.5, 136.5, 115.1, 115.1, 106.9, 98.1, 57.1, 56.7; LRMS (ESI) m / z 291 (MH) ^- .

10. 5- (2- Hydroxybenzylidene )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (2- Hydroxybenzylidene )-2- thioxodihydropyrimidine -4,6 (1H, 5H) - dione ] (Compound 22) Synthesis

Light yellow solid; Reaction time, 4 hours; Yield, 65.9%; Melting point, 250.6-251.4 DEG C; ¹ H NMR (400 MHz, DMSO-d ₆ )? 11.40 (s, 1H), 9.75 (s, 1H), 9.60 J = 2.0, 8.0 Hz), 7.79 (td, 1H, J = 2.0, 8.0 Hz), 7.52 (d, 1H, J = 8.4 Hz), 7.44 (td, 1H, J = 0.8, 7.6 Hz); ¹³ C NMR (100 MHz, DMSO-d ₆ )? 181.2, 161.7, 161.1, 154.9, 150.7, 136.1, 131.5, 126.2, 119.0, 118.1, 117.1; LRMS (ESI) m / z 247 (MH) ^- .

11. 2- Thioxo -5- (3,4,5- Trimethoxybenzylidene ) Dihydropyrimidine -4,6 (1H, 5H) -dione [2- Thioxo -5- (3,4,5- trimethoxybenzylidene ) 다 히드 피르리 미딘 -4,6 (1H, 5H) -dione] (Compound 23) Synthesis

Orange solid; Reaction time, 1 hour; Yield, 65.5%; Melting point, 258.9-260.7 DEG C; ¹ H NMR (400 MHz, DMSO- d ₆ )? 12.41 (s, 1H), 12.30 (s, 1H), 8.24 ), 3.77 (s, 3 H); ¹³ C NMR (100 MHz, DMSO-d ₆ )? 179.0, 162.7, 160.6, 156.8, 152.6, 143.2, 128.3, 118.0, 113.6, 61.0, 56.7; LRMS (ESI) m / z 321 (MH) ^- .

12. 5- (4- Hydroxy -3,5- Dimethoxybenzylidene )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (4- Hydroxy -3,5- dimethoxybenzylidene ) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione] (Compound 24) Synthesis

Orange solid; Reaction time, 2 hours; Yield, 97.5%; Melting point,> 300 ℃; ¹ H NMR (500 MHz, DMSO-d ₆ )? 12.33 (s, 1H), 12.23 (s, 1H), 10.17 H), 3.83 (s, 6 H); ¹³ C NMR (100 MHz, DMSO-d ₆ )? 178.7, 163.2, 160.9, 158.0, 147.9, 144.0, 123.7, 115.0, 114.9, 56.7; LRMS (ESI) m / z 307 (MH) ^- .

13. 5- (3- Bromo -4- Hydroxybenzylidene )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (3- Bromo -4- hydroxybenzylidene )-2- thioxodihydropyrimidine -4,6 (1H, 5H) -dione] (Compound 25) Synthesis

Yellow solid; Reaction time, 10 hours; Yield, 46.9%; ^{1 H NMR (400 MHz, DMSO-} d 6) d 12.35 (s, 1 H), 12.26 (s, 1 H), 11.75 (br s, 1 H), 8.87 (d, 1 H, J = 2.0 Hz) , 8.13 (s, 1H), 8.07 (dd, 1H, J = 2.0,8.4 Hz), 7.00 (d, 1H, J = 8.4 Hz); ¹³ C NMR (100 MHz, DMSO- d ₆ ) d 178.9, 162.7, 160.7, 160.1, 155.3, 140.3, 138.5, 125.9, 116.5, 116.5, 110.2.

14. 5- (3,5- Dibromo -4- Hydroxybenzylidene )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (3,5- Dibromo -4- hydroxybenzylidene ) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione] (Compound 26) Synthesis

Yellow solid; Reaction time, 10.5 hours; Yield 55.9%; ^{1 H NMR (400 MHz, DMSO-} d 6) d 12.38 (s, 1 H), 12.28 (s, 1 H), 8.60 (s, 2 H), 8.09 (s, 1 H); ¹³ C NMR (100 MHz, DMSO- d ₆ ) d 179.0, 162.4, 160.6, 156.1, 153.3, 139.4, 127.1, 118.1, 111.6.

< Example 3> Compound 27 to 38 Synthesis

The following Table 3 shows the results of the synthesis of 5- (substituted benzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -trione analog [5- (substituted benzyl) pyrimidine- ) -trione analog] To illustrate the substitution pattern of compounds 27-38.

(6)

compound R ¹ R ² R ³ R ⁴ 27 H H OH H 28 H OH OH H 29 OH H OH H 30 H OMe OH H 31 H OEt OH H 32 H OH OMe H 33 H H OMe H 34 H OMe OMe H 35 OMe H OMe H 36 H OMe OMe OMe 37 H OMe OH OMe 38 H Br OH Br

5- (substituted-benzylidene) pyrimidin -2,4,6 (1 H, 3 H, 5 H) - one tree (corresponding to compounds 1-12, 30 mg) is suspended in ethanol (5 mL) Sodium borohydride (NaBH ₄ , 3 eq.) Was slowly added to the 0 ° C suspension and the mixture was stirred at room temperature for 0.5-4 hours. The solvent was evaporated under reduced pressure, water was added, and the pH was adjusted to 7 with 1N-hydrochloric acid (HCl). The water was volatilized under reduced pressure and then the resulting solid was filtered through ether / water (5-10: 1) or ethanol / water (5: 1, ) Or ethanol / water (5: 1, for compound 35) to give the desired product as a solid.

1. 5- (4- Hydroxybenzyl (1H, 3H, 5H) -trione] (Compound 27) Synthesis of thiophene-2-

Reaction time, 3 hours; Yield, 51.5%; ^{1 H NMR (500 MHz, DMSO-} d 6) d 11.10 (s, 2 H), 9.25 (s, 1 H), 6.84 (d, 2 H, J = 8.0 Hz), 6.61 (d, 2 H, J = 7.5 Hz), 3.73 (t, 1H, J = 4.5 Hz), 3.13 (d, 2H, J = 4.5 Hz); ¹³ C NMR (100 MHz, DMSO- d ₆ ) d 170.8, 156.8, 151.2, 130.6, 127.6, 115.7, 50.3, 33.9; LRMS (ESI) m / z 233 (MH) ^- .

2. 5- (3,4- Dihydroxybenzyl (3,4-dihydroxybenzyl) pyrimidine-2,4,6 (1 (1, 3H, 5H) H , 3 H , 5 H ) -trione] (Compound 28) Synthesis

Reaction time, 4 hours; Yield, 56.2%; ^{1 H NMR (400 MHz, D} 2 O) d 6.64 (br d, 1 H, J = 8.0 Hz), 6.61 (s, 1 H), 6.52 (d, 1 H, J = 8.0 Hz), 3.29 (s , 2 H); ¹³ C NMR (100 MHz, D ₂ O) d 166.7, 153.3, 143.9, 141.7, 135.3, 120.0, 116.3, 115.7, 89.5, 26.9; LRMS (ESI) m / z 249 (MH) ^- .

3. 5- (2,4- Dihydroxybenzyl (2,4-dihydroxybenzyl) pyrimidine-2,4,6 (1 (1, 3H, 5H) H , 3 H , 5 H ) -trione] (Compound 29) Synthesis

Reaction time, 3 hours; Yield, 83.3%; ^{1 H NMR (400 MHz, DMSO-} d 6) d 11.64 (s, 1 H), 10.94 (s, 2 H), 9.69 (s, 1 H), 7.02 (d, 1 H, J = 7.6 Hz), 6.53 (d, 1H, J = 7.6 Hz), 6.36 (s, 1H), 3.41 (s, 1H), 3.32 (s, 2H); ¹³ C NMR (100 MHz, DMSO- d ₆ ) d 164.8, 157.7, 154.4, 150.3, 131.1, 113.4, 110.3, 103.3, 84.8, 20.4; LRMS (ESI) m / z 249 (MH) ^- .

4. 5- (4- Hydroxy (4-hydroxy-3-methoxybenzyl) pyrimidine-2,4,6 (1 H , 3 H , 5 H ) -trione] (Compound 30) Synthesis

Reaction time, 2 hours; Yield, 26.8%; ^{1 H NMR (500 MHz, D} 2 O) d 6.79 (s, 1 H), 6.69 (d, 1 H, J = 8.0 Hz), 6.62 (d, 1 H, J = 8.0 Hz), 3.71 (s, 3 H), 3.40 (s, 2 H); ¹³ C NMR (100 MHz, D ₂ O) d 166.7, 153.4, 147.3, 142.6, 135.1, 120.4, 115.5, 112.3, 89.5, 56.0, 27.2; LRMS (ESI) m / z 263 (MH) ^- .

5. 5- (3- Ethoxy -4- Hydroxybenzyl 3-Ethoxy-4-hydroxybenzyl) pyrimidine-2,4,6 (1 (R) H , 3 H , 5 H ) -trione] (Compound 31) Synthesis

Reaction time, 2 hours; Yield, 30.8%; ^{1 H NMR (400 MHz, D} 2 O) d 6.73 (d, 1 H, J = 2.0 Hz), 6.65 (d, 1 H, J = 8.0 Hz), 6.58 (dd, 1 H, J = 2.0, 7.6 Hz), 3.93 (q, 2H, J = 6.8 Hz), 3.33 (s, 2H), 1.19 (t, 3H, J = 6.8 Hz); ¹³ C NMR (100 MHz, D ₂ O) d 166.7, 153.3, 146.2, 142.9, 135.1, 120.6, 115.5, 113.8, 89.5, 65.3, 27.1, 14.1; LRMS (ESI) m / z 277 (MH) ^- .

6. 5- (3- Hydroxy (3-hydroxy-4-methoxybenzyl) pyrimidine-2,4,6 (1 (R) H , 3 H , 5 H ) -trione] (Compound 32) Synthesis

Reaction time, 2 hours; Yield, 23.1%; ¹ H NMR (400 MHz, DMSO- d ₆ ) d 11.11 (s, 2H), 8.85 (s, 1H), 6.73 (d, 1H, J = 7.6 Hz), 6.48 6.40 (d, 1H, J = 7.2 Hz), 3.74 (br s, 1H), 3.66 (s, 3H), 3.08 (br s, 2H); ¹³ C NMR (100 MHz, DMSO- d ₆ ) d 170.7, 151.3, 147.1, 146.8, 130.1, 120.2, 117.0, 112.7, 56.2, 50.1, 33.7; LRMS (ESI) m / z 263 (MH) ^- .

7. Preparation of 5- (4-methoxybenzyl) pyrimidine-2,4,6 (1 (R) H , 3 H , 5 H ) -trione] (Compound 33) Synthesis

Reaction time, 3 hours; Yield, 67.9%; ^{1 H NMR (500 MHz, DMSO-} d 6) d 11.14 (s, 2 H), 6.98 (d, 2 H, J = 8.0 Hz), 6.80 (d, 2 H, J = 8.5 Hz), 3.80 (t , 1H, J = 3.5 Hz), 3.69 (s, 3 H), 3.19 (d, 2H, J = 3.5 Hz); ¹³ C NMR (100 MHz, DMSO- d ₆ ) d 170.7, 158.7, 151.2, 130.7, 129.6, 114.4, 55.6, 50.2, 33.5; LRMS (ESI) m / z 247 (MH) ^- .

8. 5- (3,4- Dimethoxybenzyl Pyrimidine-2,4,6 (1H, 3H, 5H) -triene [5- (3,4-Dimethoxybenzyl) pyrimidine-2,4,6 H , 3 H , 5 H ) -trione] (Compound 34) Synthesis

Reaction time, 30 minutes; Yield, 39.7%; ^{1 H NMR (500 MHz, D} 2 O) d 6.80 (s, 1 H), 6.79 (d, 1 H, J = 8.0 Hz), 6.70 (d, 1 H, J = 8.0 Hz), 3.69 (s, 3 H), 3.67 (s, 3 H), 3.40 (s, 2 H); ¹³ C NMR (100 MHz, D ₂ O) d 166.7, 153.3, 148.0, 146.1, 135.7, 120.1, 112.1, 111.8, 89.4, 55.9, 55.7, 27.2; LRMS (ESI) m / z 277 (MH) ^- .

9. 5- (2,4- Dimethoxybenzyl Pyrimidine-2,4,6 (1H, 3H, 5H) -triene [5- (2,4-Dimethoxybenzyl) pyrimidine-2,4,6 H , 3 H , 5 H ) -trione] (Compound 35) Synthesis

Reaction time, 2 hours; Yield, 87.7%; ^{1 H NMR (500 MHz, DMSO-} d 6) d 9.27 (s, 2 H), 6.77 (d, 1 H, J = 8.0 Hz), 6.40 (s, 1 H), 6.29 (d, 1 H, J = 8.0 Hz), 3.73 (s, 3H), 3.66 (s, 3H), 3.37 (s, 1H), 3.22 (s, 2H); ^{13 C NMR (100 MHz, DMSO-} d 6) d 165.6, 158.5, 158.4, 153.0, 128.9, 124.3, 104.3, 98.1, 82.6, 55.7, 55.7, 22.2; LRMS (ESI) m / z 277 (MH) ^- .

10. 5- (3,4,5- Trimethoxybenzyl Pyrimidine-2,4,6 (1H, 3H, 5H) -trione [5- (3,4,5-Trimethoxybenzyl) pyrimidine-2,4,6 H , 3 H , 5 H ) -trione] (Compound 36) Synthesis

Reaction time, 2 hours; Yield, 82.8%; ¹ H NMR (400 MHz, D ₂ O) d 6.45 (s, 2 H), 3.65 (s, 6 H), 3.56 (s, 3 H), 3.37 (s, 2 H); ¹³ C NMR (100 MHz, D ₂ O) d 166.7, 153.3, 152.3, 139.5, 134.6, 105.3, 89.0, 61.1, 56.1, 28.1; LRMS (ESI) m / z 307 (MH) ^- .

11. 5- (4- Hydroxy -3,5- Dimethoxybenzyl Pyrimidine-2,4,6 (1H, 3H, 5H) -tric acid [5- (4-Hydroxy-3,5-dimethoxybenzyl) H , 3 H , 5 H ) -trione] (Compound 37) Synthesis

Reaction time, 2 hours; Yield, 90%; ¹ H NMR (500 MHz, DMSO- d ₆ ) d 11.18 (br s, 1H), 9.31 (br s, 2H), 6.44 s, 1H), 3.27 (s, 2H); ¹³ C NMR (100 MHz, DMSO- d ₆ ) d 165.5, 152.4, 148.1, 135.5, 133.9, 106.7, 86.0, 56.5, 29.2; LRMS (ESI) m / z 293 (MH) ^- .

12. 5- (3,5- Dibromo -4- Hydroxybenzyl (3,5-Dibromo-4-hydroxybenzyl) pyrimidine-2,4,6 (1 H , 3 H , 5 H ) -trione] (Compound 38) Synthesis

Reaction time, 1 hour; Yield, 44.8%; ^{1 H NMR (500 MHz, CD} 3 OD) d 7.35 (s, 2 H), 3.48 (s, 2 H); ¹³ C NMR (100 MHz, CD ₃ OD) d 171.7, 150.8, 149.8, 137.9, 131.7, 110.8, 87.7, 26.8.

< Example 4> Compound 39 to 52 Synthesis

Table 4 5- (substituted benzyl) -2-thioxo-dihydro-pyrimidine -4,6 (1H, 5H) - dione analog of [5- (substituted benzyl) -2- thioxodihydropyrimidine-4,6 (1 H , 5 H) -dione analog] serve to explain the substitution pattern of the compounds 39-52.

&Lt; Formula 7 >

compound R ¹ R ² R ³ R ⁴ 39 H H OH H 40 H OH OH H 41 OH H OH H 42 H OMe OH H 43 H OEt OH H 44 H OH OMe H 45 H H OMe H 46 H OMe OMe H 47 OMe H OMe H 48 OH H H H 49 H OMe OMe OMe 50 H OMe OH OMe 51 H Br OH H 52 H Br OH Br

Dione are suspended in (for compounds 13 ~ 26, 30 mg) in ethanol (5 mL) - 5- (substituted-benzylidene) -2-thioxo-dihydro-pyrimidine -4,6 (1 H, 5 H) Sodium borohydride (NaBH ₄ , 3 eq.) Was slowly added to the stirred 0 ° C suspension and the mixture was stirred at room temperature for 10 min - 2 h. The solvent was evaporated under reduced pressure, water was added, and the pH was adjusted to 7 with 1N-hydrochloric acid (HCl). The water was volatilized under reduced pressure and the resulting solid was filtered and washed with ether / water (10-20: 1) or ethanol / water (10: 1 for compounds 41,46 and 47) ~ 20: 1) or ethanol / water (10: 1, for compounds 41, 46 and 47) to give the desired product as a solid.

1. 5- (4- Hydroxybenzyl )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (4-Hydroxybenzyl) -2-thioxodihydropyrimidine-4,6 H , 5 H ) -dione] (Compound 39) Synthesis

Reaction time, 10 min; Yield, 72.7%; ^{1 H NMR (500 MHz, DMSO-} d 6) d 11.71 (br s, 2 H), 9.03 (br s, 1 H), 6.94 (d, 2 H, J = 7.5 Hz), 6.59 (d, 2 H , J = 8.5 Hz), 3.44 (s, 1H), 3.43 (s, 2H); ¹³ C NMR (100 MHz, DMSO- d ₆ ) d 173.3, 161.6, 155.8, 131.9, 129.6, 115.4, 94.8, 26.9.

2. 5- (3,4- Dihydroxybenzyl )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (3,4- Dihydroxybenzyl )-2- thioxodihydropyrimidine -4,6 (1 H , 5 H ) - dione ] (Compound 40) Synthesis

Reaction time, 1 hour; Yield, 92.6%; ^{1 H NMR (400 MHz, D} 2 O) d 6.60 (dd, 1 H, J = 2.4, 8.0 Hz), 6.60 (d, 1 H, J = 2.4 Hz), 6.49 (d, 1 H, J = 8.4 Hz), 3.29 (s, 2H); ¹³ C NMR (100 MHz, D ₂ O) d 171.6, 165.1, 143.8, 141.8, 134.5, 120.1, 116.2, 115.8, 95.0, 26.9.

3. 5- (2,4- Dihydroxybenzyl )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (2,4- Dihydroxybenzyl )-2- thioxodihydropyrimidine -4,6 (1 H , 5 H ) - dione ] (Compound 41) Synthesis

Reaction time, 1 hour; Yield, 87.8%; ^{1 H NMR (500 MHz, D} 2 O) d 6.98 (d, 1 H, J = 9.0 Hz), 6.30-6.28 (m, 2 H), 3.34 (s, 2 H); ¹³ C NMR (100 MHz, D ₂ O) d 171.7, 165.2, 155.1, 154.8, 131.0, 120.4, 107.8, 103.8, 95.0, 22.4.

4. 5- (4- Hydroxy -3-methoxybenzyl) -2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (4- Hydroxy -3- methoxybenzyl )-2- thioxodihydropyrimidine -4,6 (1 H , 5 H ) -dione] (Compound 42) Synthesis

Reaction time, 2 hours; Yield, 36.8%; ^{1 H NMR (500 MHz, D} 2 O) d 6.76 (s, 1 H), 6.66 (dd, 1 H, J = 8.0 Hz), 6.59 (d, 1 H, J = 8.0 Hz), 3.68 (s, 3 H), 3.38 (s, 2 H); ¹³ C NMR (100 MHz, D ₂ O) d 171.7, 165.2, 147.3, 142.7, 134.4, 120.5, 115.5, 112.4, 94.9, 56.0, 27.2.

5. 5- (3- Ethoxy -4- Hydroxybenzyl )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (3- Ethoxy -4- hydroxybenzyl )-2- thioxodihydropyrimidine -4,6 (1 H , 5 H ) -dione] (Compound 43) Synthesis

Reaction time, 2 hours; Yield, 93.7%; ^{1 H NMR (500 MHz, D} 2 O) d 6.77 (s, 1 H), 6.68 (d, 1 H, J = 8.0 Hz), 6.62 (d, 1 H, J = 8.0 Hz), 3.95 (q, 2 H, J = 7.0 Hz), 3.41 (s, 2 H), 1.23 (t, 3 H, J = 7.0 Hz); ¹³ C NMR (100 MHz, D ₂ O) d 169.5, 163.1, 144.1, 140.9, 132.2, 118.5, 113.4, 111.7, 92.8, 63.1, 25.0, 12.0.

6. 5- (3- Hydroxy Methoxybenzyl) -2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (3- Hydroxy -4- methoxybenzyl )-2- thioxodihydropyrimidine -4,6 (1 H , 5 H ) -dione] (Compound 44) Synthesis

Reaction time, 1 hour; Yield, 96.9%; ^{1 H NMR (500 MHz, D} 2 O) d 6.78 (d, 1 H, J = 8.0 Hz), 6.67 (s, 1 H), 6.65 (d, 1 H, J = 8.0 Hz), 3.68 (s, 3 H), 3.37 (s, 2 H); ¹³ C NMR (100 MHz, D ₂ O) d 169.5, 163.1, 143.4, 142.6, 133.0, 117.8, 113.1, 110.8, 92.7, 54.2, 24.8.

7. 5- (4-Methoxybenzyl) -2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (4-Methoxybenzyl) -2-thioxodihydropyrimidine-4,6 H , 5 H ) -dione] (Compound 45) Synthesis

Reaction time, 30 minutes; Yield, 78.4%; ¹ H NMR (400 MHz, D ₂ O) d 7.02 (br s, 2 H), 6.71 (br s, 2 H), 3.61 (s, 3 H), 3.38 (s, 2 H); ¹³ C NMR (100 MHz, D ₂ O) d 171.6, 165.2, 157.0, 134.3, 129.1, 114.0, 95.0, 55.6, 26.9.

8. 5- (3,4- Dimethoxybenzyl )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (3,4-Dimethoxybenzyl) -2-thioxodihydropyrimidine-4,6 H , 5 H ) -dione] (Compound 46) Synthesis

Reaction time, 30 minutes; Yield, 91.4%; ^{1 H NMR (400 MHz, DMSO-} d 6) d 10.50 (s, 2 H), 6.81 (s, 1 H), 6.67 (d, 1 H, J = 8.4 Hz), 6.65 (d, 1 H, J = 8.4 Hz), 3.62 (s, 3H), 3.61 (s, 3H), 3.32 (s, 1H), 3.30 (s, 2H); ¹³ C NMR (100 MHz, DMSO- d ₆ ) d 172.6, 163.7, 148.8, 147.0, 137.4, 120.6, 113.3, 112.3, 90.8, 56.3, 56.0, 28.6.

9. 5- (2,4- Dimethoxybenzyl )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (2,4-Dimethoxybenzyl) -2-thioxodihydropyrimidine-4,6 H , 5 H ) -dione] (Compound 47) Synthesis

Reaction time, 30 minutes; Yield, 86.7%; ^{1 H NMR (400 MHz, DMSO-} d 6) d 10.55 (s, 2 H), 6.71 (d, 1 H, J = 8.0 Hz), 6.38 (s, 1 H), 6.27 (d, 1 H, J = 8.0 Hz), 3.71 (s, 3H), 3.64 (s, 3H), 3.38 (s, 1H), 3.22 (s, 2H); ¹³ C NMR (100 MHz, DMSO- d ₆ ) d 172.7, 164.0, 158.7, 158.4, 128.6, 123.2, 104.3, 98.2, 88.0, 55.8, 55.7, 22.1.

10. 5- (2- Hydroxybenzyl )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (2-Hydroxybenzyl) -2-thioxodihydropyrimidine-4,6 H , 5 H ) -dione] (Compound 48) Synthesis

Reaction time, 10 min; Yield, 72.7%; ^{1 H NMR (400 MHz, D} 2 O) d 7.06 (d, 1 H, J = 7.6 Hz), 6.97 (td, 1 H, J = 1.2, 7.2 Hz), 6.72 (t, 1 H, J = 6.8 Hz), 6.70 (d, 1H, J = 7.2 Hz), 3.36 (s, 2H); ¹³ C NMR (100 MHz, D ₂ O) d 171.8, 165.2, 153.7, 130.2, 128.2, 127.9, 121.0, 116.5, 94.5, 23.0.

11. 5- (3,4,5- Trimethoxybenzyl )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (3,4,5- Trimethoxybenzyl )-2- thioxodihydropyrimidine -4,6 (1 H , 5 H ) - dione ] (Compound 49) Synthesis

Reaction time, 1 hour; Yield, 100%; ¹ H NMR (500 MHz, D ₂ O) d 6.46 (s, 2 H), 3.67 (s, 6 H), 3.56 (s, 3 H), 3.41 (s, 2 H); ¹³ C NMR (100 MHz, D ₂ O) d 169.7, 163.0, 150.1, 136.6, 132.6, 103.2, 92.2, 58.9, 53.9, 25.9.

12. 5- (4- Hydroxy -3,5- Dimethoxybenzyl )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (4- Hydroxy -3,5- dimethoxybenzyl )-2- thioxodihydropyrimidine -4,6 (1 H , 5 H ) -dione] (Compound 50) Synthesis

Reaction time, 2 hours; Yield, 99.4%; ¹ H NMR (400 MHz, D ₂ O) d 6.44 (s, 2 H), 3.66 (s, 6 H), 3.37 (s, 2 H); ¹³ C NMR (100 MHz, D ₂ O) d 171.8, 165.2, 147.7, 133.8, 131.8, 105.4, 94.7, 56.4, 27.7.

13. 5- (3- Bromo -4- Hydroxybenzyl )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (3- Bromo -4- hydroxybenzyl )-2- thioxodihydropyrimidine -4,6 (1 H , 5 H ) -dione] (Compound 51) Synthesis

Reaction time, 1 hour; Yield, 46.7%; ^{1 H NMR (500 MHz, CD} 3 OD) d 7.32 (br d, 1 H, J = 1.5 Hz), 7.04 (dd, 1 H, J = 2.0, 8.5 Hz), 6.73 (d, 1 H, J = 8.5 Hz), 3.50 (s, 2H); ¹³ C NMR (100 MHz, CD ₃ OD) d 173.0, 164.6, 151.5, 135.3, 132.2, 128.3, 115.6, 109.0, 93.2, 26.7.

14. 5- (3,5- Dibromo -4- Hydroxybenzyl )-2- Thioxo dihydropyrimidine -4,6 (1H, 5H) -dione [5- (3,5- Dibromo -4- hydroxybenzyl )-2- thioxodihydropyrimidine -4,6 (1 H , 5 H ) -dione] (Compound 52) Synthesis

Reaction time, 1 hour; yield, 36.2%; ^{1 H NMR (400 MHz, CD} 3 OD) d 7.29 (s, 2 H), 3.52 (s, 2 H); ¹³ C NMR (100 MHz, CD ₃ OD) d 173.9, 162.2, 149.1, 134.7, 131.7, 110.8, 93.9, 25.8.

< Example 5> In vitro ROS ONOO ^- Scavenging activity analysis

1. Macrophages ( Raw264 .7) Preparation

Raw264.7 macrophagy cells (rat prostatic endothelial cell line) were obtained from ATCC (American Type Culture Collection, Manassas, Va., USA) and the cells were treated with 2 mM L-glutamine, 100 mg / ml streptomycin, 2.5 mg / L amphotericin B, and DMEM containing 5% inactivated fetal bovine serum (FBS) (Dulbecco's Modified Eagle Medium, Nissui, Tokyo, Japan). Cells were also maintained at 37 ° C in conditions such as humid atmospheres containing 5% CO ₂ and 95% air. And 5% FBS was used as serum-free medium (SFM). The cells were sub-cultured in a 100 mm plastic flask (Corning Co., New York, USA) once every two days to maintain the cell line.

2. In vitro ROS Measurement and ONOO ^- Measure

Was measured by the DCFDA (2 ', 7'-dichlorodihydrofluorescein diacetate) assay according to a known method (Chem Res Toxicol. 5: 227-231, 1992). That is, 12.5 mM DCFDA dissolved in 99.9% ethanol and 600 U / ml esterase dissolved in tertiary distilled water were stored at -20 ° C. in a stock solution, and DCFH (2 ') prepared by mixing 10 mM DCFDA and 6 U / , 7'-dichlorodihydrofluorescein) solution was incubated at 22 ° C for 20 minutes and stored frozen in a cow until use. Since the fat-soluble DCFDA is deacetylated with non-fluorescent DCFH by esterase or oxidative hydrolysis and DCFH is oxidized by active oxygen to become DCF (2 ', 7'-dichlorofluorescein) exhibiting strong fluorescence, the excitation wavelength is 485 nm And a fluorescence photometer (GENios, TECAN) at an emission wavelength of 530 nm. As a source of active oxygen, SOS-1 (3-morpholinosydnonimine hydrochloride) 50 μM is used to react with esterase to generate ROS arbitrarily.

As a result, compounds 28, 30, 31, 32, 37, 39, 42, 43, 44, 45, 48, and 48 as compounds having a large effect of eliminating ROS generated arbitrarily as shown in FIG. 1 as Trolox, 50, and 51, respectively.

The ONOO ^- elimination and the inhibitory activity on the 26 novel compounds were measured by ONOO ^- mediated by the method of Kooy et al., Free Radic. Biol. Med. 16: 149-156, Was measured and analyzed by fluorescence spectroscopy using the oxidation reaction of dihydrothodamine 123 (hereinafter referred to as "DHR"). Non-fluorescent DHR 123 is oxidized by ONOO ^- to form a fluorescent rhodamine 123. Using these properties, the compounds were reacted in DHR123 solution, respectively, and the ONOO ^{- -} scavenging activity of the new compounds was measured by measuring the change of fluorescence by ONOO ^- addition. The ONOO ^- SIN-1 (3-morpholinosydnonimine) was used to generate ONOO ^- (authentic peroxynitrite) as substrate and ONOO ^- as substrate for ONOO ^- production inhibition. As a result, as with 1 ONOO ^- erasing the screening of the compound 28, 29, 30, 31, 32, 37, 38, 41, 42, 43, 44, 45, 48, 50, 51 and 52 as large compound effect of Could.

< Example 6> TLR4 Bond intimacy measurement

In the present invention, the intimacy was confirmed by docking simulation in order to confirm whether the two new compounds selected in FIG. 1 have an inhibitory effect and an effect on the TLR4 receptor that directly affects the NF-KB activation pathway. 42 and 50 were found to bind directly to the MD2 portion of the TLR4 complex. As a result, the affinity of each of the compounds 42 and 50 was found to be 50 (methoxy) methoxy group In view of the fact that TLR4 has a higher affinity, Compound 50 has a more effective inhibitory effect on TLR4. That is, it was confirmed from the following experiment that the signal transduction pathway, oxidative stress, NF-κB activity and inflammatory reaction were both inhibited (FIG. 2).

< Example 7> Cytotoxicity experiment

For the toxicity test of this compound and the use of appropriate concentration, the cell viability assay was measured using the EZ-cy Tox kit. Compounds 42 and 50 were measured at concentrations of 1 μM to 50 μM, respectively, resulting in slight cell death at 50 μM (FIG. 3). As a result, the optimum concentration of 1,5, 10 μM was selected as the experimental concentration.

< Example 8> in macrophages ROS Measurement and ONOO ^- Measure

In order to induce oxidative stress and inflammation, 400 ng of LPS was used, and ROS measurement and fluorescence microscopy of macrophage RAW264.7 cells using DCFDA showed that ROS increased at 400 ng of LPS, And 50 times, respectively. In particular, it was confirmed that 10 μM of compounds 42 and 50 had better inhibitory effect than 10 μM of Trolox used as a positive control (FIG. 4A).

In addition, ROS can be confirmed by photographs, which means that the more fluorescent dye is expressed, the more amount of ROS is present. This fluorescence dye was found to be decreased by 10 μM compound 42, 5 μM, and 10 μM 50 compound 4b).

These results were similar to those of ONOO ^- measured with DHR123 fluorescent dye and NO measured with DAF2.

< Example 9> Test for the expression level of inflammatory cytokine

Western blotting was performed by boiling the sample lysed in the cell with loading buffer [0.125 M Tris-Hcl, pH 6.8, 4% SDS, 10% 2-mercaptoethanol and 0.2 bromophenol blue] for 1 minute . After dividing by protein size by SDS-PAGE with 10% acrylaminde, transfer to the PVDF membrane at 15V for 1 hour. After blocking with 5% skim milk for 1 hour, the first antibody is incubated overnight and the second antibody is developed after 1-3 hours.

Expression levels of INOS and COX-2, which are inflammatory cytokines induced by oxidative stress, by western blotting were also confirmed by the expression of inflammatory cytokines increased by LPS in Compound No. 42 and Compound No. 50 at 5 μM and 10 μM (Fig. 5).

< Example 10> Determination of expression level of inflammatory cytokine

NF-κB is a key factor involved in the regulation of the inflammatory response and regulation of the immune system. NF-κB is present in most cells and NF-κB is composed of p50, p52, RelA (p65), RelB, c-Rel and v-Rel. And have various names. Among them, the typical NF-κB activation pathway is a pathway essential for innate immunity, and the typical NF-κB activation pathway has been the subject of much research until now, mainly by the roles of P65 and P50. Phosphorylation of P65 has two sites, Ser536 and Ser276. The following results show different patterns in Ser536 and Ser276 (Fig. 6).

Compounds 42 and 50 were found to inhibit phosphorylation of P65 at Ser536 and the total amount of P65 expression was also decreased. However, the phosphorylated P65 phosphorylated at Ser276 did not show any difference in the groups treated with LPS, Compound 42 and Compound 50, respectively.

< Example 11> NF -KB < / RTI >

One. immunocytochemistry Through NF of -KB Into the nucleus Translocation confirmation

Based on the above results, we confirmed the translocation of NF-κB into the nucleus. We performed immunochemistry experiments using Alexa Fluor 488 Hoechst 33342. This data used a confocal microscope, Hoechst 33342 is blue as an dye staining nuclei, and also stains a portion of NF-κB in cells using NF-κB antibody and Alexa Fluor 488, Green.

These results suggest that NF-κB is cytoplasmic only in the untreated control group, because the middle part of the green part (nucleus part) is empty. In the LPS group as the negative control group, the green part is generally in the cell shape The presence of NF-κB translocates into the nucleus. Thus, it was confirmed that NF-κB translocated into the nucleus due to LPS decreased the translocation of NF-κB in a concentration-dependent manner by compounds 42 and 50 (FIG. 7).

2. Luciferase assay

Luciferase assays were performed to further validate the effect of compounds 42 and 50 on the translocation of NF-κB into the nucleus. The NF-κB binding site was transfected into a cell using a luciferase reporter vector, and then treated with LPS to activate NF-κB. Luciferase is expressed by activated NF-κB, and this luciferase reacts with luciferin to show phosphorescence, which is a marker of how active NF-κB is activated. As a result, it was confirmed that the activation of NF-κB by LPS was inhibited by the compounds 42 and 50 in a concentration-dependent manner (FIG. 8).

3. Conclusion

The NF-κB activation pathway, which is an essential signaling pathway for innate immunity, has been extensively studied. The NF-κB dimer is inactivated in the cytoplasm by binding to IkBa, which inhibits NF-κB activation, IκB is degraded by the activity of IKK, and NF-κB in the cytoplasm is transferred into the nucleus and activated. This process regulates the activity of IkB, which inhibits NF-κB in the cytoplasm, using the IKK complex. IKK is composed of IKKα, IKK, and IKKγ, among which the function of IKK is known to be strongest.

This result confirmed the inhibitory effect of the compounds 42 and 50 on the activity of NF-κB on the activity of NF-κB. First, the activity of IKBa and IKKb directly involved in translocation of NF-κB into the nucleus was confirmed by western blotting. In the case of treatment with compounds 42 and 50, it was confirmed that the translocation of NF-κB into the nucleus was inhibited by the concentration-dependent decrease in the activity of IKKb and the activity of IkBa (FIG. 9).

The upper signaling pathways regulating IKK in the NF-κB activation pathway (IKK-IKB-NF-KB) are mainly ERK, p38, MAPK and AKT. Induced the inhibition of compounds 42 and 50 in the signal transduction pathway of AKT / PI3K based on this. First, when AKT activity directly affecting IKK was measured, inhibition of AKT activity by compounds 42 and 50 was observed in a concentration-dependent manner. P-PTEN and NOX4, which regulate AKT activity, And 50, respectively (FIG. 10). That is, NOx4 is suppressed by the compounds 42 and 50, so that the subsequent oxidative stress is directly or indirectly suppressed. Thus, it was confirmed in the present invention that PTEN activation and AKT / NF-KB signaling pathway are both blocked, thereby reducing the expression of inflammatory cytokines.

< Example 12> in vivo test

The present inventors evaluated the efficacy thereof in vivo based on the previous in vitro experiments. C57BL / 6 mice were used as an animal model. In order to confirm the effect of inhibiting NF-κB transcription into the nucleus, compounds 42 and 50 were administered one hour later and 5 mg / kg LPS IP was dissected one hour later Respectively.

After dissection, ROS and ONOO ^- were measured in the blood and liver, indicating that oxidative stress increased by LPS was significantly inhibited by compounds 42 and 50 (FIG. 11). In addition, the expression of the target genes was confirmed by extracting the protein from the dissected liver. As a result, the activity of ser536 of P65 was markedly suppressed, and the inflammatory factors such as COX-2 and iNOS were also reduced to 42 and 50 (Fig. 12). &Lt; tb >< TABLE >

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that such detail is solved by the person skilled in the art without departing from the scope of the invention. will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims

A compound represented by the following formula (1).
&Lt; Formula 1 >

In Formula 1, R ₁ to R ₄ may be the same or different and are any one of H, OH, Br, or C1 to C4 alkoxy, and X is O or S;

The compound of claim 1, wherein the compound is selected from the group consisting of 5- (4-hydroxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) (1H, 3H, 5H) -triene, 5 (2,4-dihydroxybenzyl) pyrimidine-2,4,6 - (4-hydroxy-3-methoxybenzyl) pyrimidine-2,4,6 (1H, , 4,6 (1H, 3H, 5H) -triene, 5- (3-hydroxy-4-methoxybenzyl) pyrimidine- (1H, 3H, 5H) -triene, 5- (3,4-dimethoxybenzyl) pyrimidine-2,4,6 3H, 5H) -thione, 5- (3,4,5-trimethoxybenzyl) -2,3-dihydro- (1H, 3H, 5H) -triene, 5- (4-hydroxy-3,5-dimethoxybenzyl) pyrimidine- 4-hydroxybenzyl) -pyrimidine-2,4,6 (1H, 3H, 5H) -triene, 5- (3,5-dibromo-4- 2-thioxo-dihydropyrimidine-4,6 (1H, 5H) - Dihydroxybenzyl) -2-thioxo dihydropyrimidine-4,6 (1H, 5H) -dione, 5- (2,4-dihydroxybenzyl) -2- (1H, 5H) -dione, 5- (4-hydroxy-3-methoxybenzyl) -2-thioxo dihydropyrimidine- -Dione, 5- (3-ethoxy-4-hydroxybenzyl) -2-thioxo dihydropyrimidine-4,6 (1H, 5H) -dione, 5- (4-methoxybenzyl) -2-thioxo dihydropyrimidine-4,6 -Dione, 5- (3,4-dimethoxybenzyl) -2-thioxo dihydropyrimidine-4,6 (1H, 5H) (1H, 5H) -dione, 5- (2-hydroxybenzyl) -2-thioxo- dihydropyrimidine-4,6 (1H, 5H) -dione and 5- (4-hydroxy-3,5-dimethoxybenzyl) -2-thioxo- dihydropyrimidine -Thioxo < / RTI > dihydropyrimidine-4,6 (1H) , 5H) -dione, 5- (3-bromo-4-hydroxybenzyl) -2-thioxo dihydropyrimidine-4,6 4-hydroxybenzyl) -2-thioxo-dihydropyrimidine-4,6 (1H, 5H) -dione.

A pharmaceutical composition for preventing or treating an inflammatory disease containing the compound according to any one of claims 1 to 3 as an active ingredient.

A pharmaceutical composition for preventing or treating an inflammatory disease, comprising a compound represented by the following formula (2) or (3) as an active ingredient.
(2)

(3)

The pharmaceutical composition according to claim 3 or 4, wherein the compound inhibits the activity of macrophages through competitive binding to TLR4 with lipopolysaccharide (LPS).

The method according to claim 3 or 4, wherein the inflammatory disease is any one selected from asthma, bronchitis, sepsis, arthritis, hepatitis, rheumatoid arthritis, osteoarthritis, ulcerative colitis, myocarditis, multiple sclerosis and viral infection Wherein the pharmaceutical composition is for preventing or treating an inflammatory disease.

A health functional food for preventing or ameliorating an inflammatory disease containing the compound according to any one of claims 1 or 2 as an active ingredient.

An antioxidant health functional food containing the compound according to any one of claims 1 to 3 as an active ingredient.

A health functional food for anti-aging comprising the compound according to any one of claims 1 to 3 as an active ingredient.