WO2016032127A2 - Novel compounds having antioxidant and anti-inflammatory activities due to competition with lps for binding to tlr4, and medical use thereof - Google Patents

Abstract

The present invention relates to novel compounds having antioxidant and anti-inflammatory effects of inhibiting the activity of macrophages due to competition with LPS for binding to TLR4, and a medical use thereof. The compounds, according to the present invention, have antioxidant and anti-inflammatory activities, and thus can be used for geriatric disease prevention or inflammation treatment. In addition, the compounds compete with LPS for binding to TLR4-MD2, in particular, thereby being used as a pharmaceutical composition useful in the prevention and treatment of sepsis, metabolic diseases, or cardiovascular diseases, or as a health food, by means of the competition with LPS for binding to TLR4-MD2.

Description

Novel compounds having antioxidant and anti-inflammatory activity through competitive binding with LPS to TLR4 and their medical uses

The present invention relates to a novel compound and its medical use having the effect of antioxidant, anti-inflammatory and anti-aging to inhibit the activity of macrophages through competitive binding of lipopolysaccharide (LPS) to TLR4.

Inflammatory reactions are stimulated by oxidative stress in the body, which in turn causes nucleic acid (DNA), the major protein in muscle, proteins, and fat, the major component of cell membrane, to deform or deactivate the cell. Is a problem in the body function itself, which increases the gene expression of specific cells causing not only cell death but also degenerative diseases, initiating or worsening the inflammatory response.

Oxygen, which is absorbed through normal respiration, enters the mitochondria, an energy-producing tissue within the cell, and burns, producing energy (ATP) necessary for maintaining body temperature, cell activity, and physical activity. However, the fundamental defect of mitochondria is that oxygen is not completely burned, producing by-products, which are free radicals, which are free radicals. To cope with oxidative stress, life has a defense system that detoxifies, destroys or neutralizes toxic oxygen throughout the evolutionary process, which is responsible for many resistances in the body that are balanced by a redox balance. As aging is broken by the force of destructive free radicals and oxidative destruction stresses cells and tissues, the body function is reduced and at the same time inflammation is generated.

Macrophage (macrophage) is an immune cell that is distributed in all tissues of the animal and is responsible for the innate immune response in the human body, and is a white blood cell that plays an important role in the human immune system. Macrophages activated by external stimuli trigger inflammatory responses through the secretion of inflammatory mediators, causing inflammation of asthma, bronchitis, arthritis, multiple sclerosis, arteriosclerosis, stroke, degenerative brain diseases such as Alzheimer's disease or Parkinson's disease It causes diseases and the like and makes the disease worse.

Lipopolysaccharide (LPS), one of the endotoxins, has been shown to contain pro- and lipases such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in Raw264.7 macrophage. It increases 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 to produce excess PGE2, NO, etc. and promote various diseases and carcinogenesis. In particular, NOS, an enzyme that produces NO, and cyclooxygenase (COX), an enzyme that mediates the biosynthesis of various prostaglandins (PGs), are known as important mediators of inflammatory responses.

Toll like receptor, LPS receptor, has been found to be closely related to oxidative stress. Among them, TLR2 / TLR4 mainly detects lipoproteins in bacteria's outer cell membrane / cell wall, and induces ROS (active oxygen species) causing oxidative stress. In the case of TLR4, even when macrophages are stimulated by LPS stimulation, several inflammatory factors are generated by NF-κB activation, which induces pathologies of various diseases and is also involved in carcinogenesis. However, it is known that there are no endogenous enzymes that can directly inactivate the generated active oxygen and NO, ONOO in vivo, and there is an urgent need for research on the development of antioxidants and anti-inflammatory agents that specifically inhibit and eliminate it. Has been. As such, research has been conducted on the development of antioxidants having ONOO scavenging activity in order to protect cells from damage caused by cytotoxic substances such as free radicals and NO, ONOO, and many natural-derived or synthetic scavengers have been developed.

Antioxidants are used for the purpose of minimizing the loss of certain vitamins and essential amino acids or by delaying or preventing rancidity of maintenance products by reacting with free radicals rather than removing or absorbing oxygen. Synthetic antioxidants commonly used in foods and pharmaceuticals include butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl galate (PG) and tertiary butyl hydro Quinone (TBHQ, Tertiary butyl hydroquinone), etc., but when administered to high concentrations in experimental animals are known to cause liver hypertrophy or carcinogenicity. In particular, butylated hydroxytoluene has been shown to increase microsomal enzyme activity in the liver of laboratory animals through several studies, and controversy has been raised about the safety of these phenolic synthetic antioxidants. Usage is legally regulated. Accordingly, many studies have been made in anticipation of the development of natural antioxidants of safe and economical plant origin with high antioxidant effects.

Recently, researches on antioxidants have been actively conducted. Among the known antioxidants, tocopherols, flavonoids, gossypol, sesamol (sesamol), oryzanol, vitamin C, and the like. Among them, tocopherol and L-ascorbic acid are preferred as natural antioxidants. Among them, tocopherol has high safety but low ability to stop oxidation reaction alone and is expensive. .

TLR4 antagonists that directly bind to TLR4 have been rarely reported. Among them, erytoran, made by Eisai of Japan in late 2009, binds to MD2 of TLR4 and suppresses its signaling mechanisms for sepsis. It is reported that it has entered the phase 3 clinical trial for the treatment of patients. However, due to its very high molecular weight, there is a difficulty in mass production of itoritoran.

An object of the present invention is to provide a novel compound having the effect of antioxidant, anti-inflammatory and anti-aging to inhibit the activity of macrophages through competitive binding of lipopolysaccharide (LPS) to TLR4.

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

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

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

<Formula 1>

In Formula 1, R1 to R4 may be the same or different, respectively, any one of H, OH, Br or C1 to C4 alkoxy, and X is O or S.

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

The present invention also provides a health functional food for preventing or improving inflammatory diseases containing the compound as an active ingredient.

The present invention also provides a dietary supplement for antioxidants containing the compound as an active ingredient.

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

The present invention relates to a novel compound having antioxidant and anti-inflammatory activity through competitive binding with LPS to TLR4 and its medical use, wherein the compounds according to the present invention are small molecules that inhibit the activity of macrophages through competitive binding with LPS to TLR4. as inflammation is secreted upon inflammation inducer and active oxygen species; generation and inflammatory mediators of a (reactive oxygen species ROS) (NO , ONOO -) inhibiting the expression of the proinflammatory gene (Cox-2, iNOS), and In addition, by inhibiting the activity of the transcription factor (NF-κB) that controls the inflammation, it has an antioxidant and anti-inflammatory action and shows a more effective anti-aging effect. In addition, this experiment was also verified in liver of C57BL / 6 mice. Therefore, the novel compounds can be used in various fields, such as drugs or health foods, which have little side effects and are safe in preventing and treating sepsis, metabolic diseases or cardiovascular diseases.

1 is a graph of compounds targeting TLR4 inhibition and having inhibitory effect through screening of ROS and ONOO ⁻ .

FIG. 2 shows that the compound according to the present invention binds to the MD2 portion of TLR4 and competes with LPS through docking simulation.

3 is a graph showing that compounds 42 and 50, which are novel compounds of the present invention, are stable to cytotoxicity at concentrations of 1 μM to 10 μM through cell viability assays.

Figure 4a is the increased oxidative stress in macrophages by ^LPS-a graph showing that (ROS, ONOO, NO) is present 42 times, 50 times reduced by the compound.

4B is a graph showing that ROS, ONOO ⁻ increased by LPS in macrophages through fluorescent dyes is reduced by compounds 42 and 50.

FIG. 5 is a diagram showing that expression of COX-2 and iNOS, which are inflammation-inducing proteins, increased by LPS by treating compounds 42 and 50, is inhibited.

6 is a diagram showing the expression and activity of NF-κB, a transcription factor of oxidative stress and inflammation-related proteins, wherein the activity of NF-κB phosphorylation at ser536 is 42 and 50 at the phosphorylation site of P65. It is a picture that is inhibited by burn compound.

Figure 7 shows the activity of NF-κB in macrophages, a picture of the effects of inhibiting the translocation of NF-κB by compounds 42, 50.

FIG. 8 shows the effect of translocation into the nucleus, using a luciferase reporter vector to which an NF-κB binding site is ligation, with activation of NF-κB increased by LPS. Is a concentration-dependent inhibition by compounds 42 and 50.

9 is a graph showing the effect of inhibiting translocation of NF-κB by decreasing the concentration of IKKB activity and IκBα activity in a substance treated with compounds 42 and 50.

10 is a concentration-dependent inhibition of AKT activity by the compounds 42 and 50 when the activity of AKT directly affects IKK in the NF-κB signaling system, PTEN and NOX4 In addition, the figure is inhibited by compounds 42 and 50 concentration-dependent.

Figure 11 is, C57BL / 6 and ONOO ROS in liver and blood in order to determine the inhibitory effect of oxidative stress in the macrophages in the animal model ^were measured. ROS and ONOO ^- were increased in both liver and blood in LPS group mice, and concentration-dependently decreased in compounds 42 and 50.

12 confirmed the phosphorylation and expression of NF-κB, an important transcription factor associated with inflammation in the liver of C57BL / 6. Phosphorylation of P65 and P65 increased in LPS decreased in compounds 42 and 50, and also decreased in COX-2, a pro-inflammatory gene. This figure shows the same trend as -2.

The present invention is a small molecular weight compound having scavenging ability of oxidative stress in Raw264.7 macrophage, which directly binds to TLR4 and increases tumor necrosis factor-alpha (TNF-α), interleukin-6 through competitive binding with LPS. It inhibits pro-inflammatory cytokines such as (IL-6) and interleukin-1β (IL-1β) and inhibits the secretion of inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2). In addition, by inhibiting the transcription of NF-κB, a transcription-inducing transcription factor, it blocks AKT signaling and induces cycloxygenase-2 (COX-2) and NO synthase (NOS) and excess PGE2 and NO. Inhibition of the production of a variety of diseases associated with sepsis and carcinogenesis (carcinogenesis) is to be prevented.

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

<Formula 1>

In Formula 1, R1 to R4 may be the same or different, respectively, any one of H, OH, Br or C1 to C4 alkoxy, and X is O or S.

Preferably, the compound is 5- (4-hydroxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -trione, 5- (3,4-dihydroxybenzyl) pyrimidine- 2,4,6 (1H, 3H, 5H) -trione, 5- (2,4-dihydroxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -trione, 5- ( 4-hydroxy-3-methoxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -trione, 5- (3-ethoxy-4-hydroxybenzyl) pyrimidine-2,4 , 6 (1H, 3H, 5H) -trione, 5- (3-hydroxy-4-methoxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -trione, 5- (4 -Methoxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -trione, 5- (3,4-dimethoxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -Trione, 5- (2,4-dimethoxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -trione, 5- (3,4,5-trimethoxybenzyl) pyrimidine -2,4,6 (1H, 3H, 5H) -trione, 5- (4-hydroxy-3,5-dimethoxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -tree On, 5- (3,5-dibromo-4-hydroxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -trione, 5- (4-hydroxybenzyl) -2- Thioxodihydropyrimidine-4,6 (1H, 5H)- On, 5- (3,4-dihydroxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione, 5- (2,4-dihydroxybenzyl) -2- Thioxodihydropyrimidine-4,6 (1H, 5H) -dione, 5- (4-hydroxy-3-methoxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -Dione, 5- (3-ethoxy-4-hydroxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione, 5- (3-hydroxy-4-methoxy Benzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione, 5- (4-methoxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -Dione, 5- (3,4-dimethoxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione, 5- (2,4-dimethoxybenzyl) -2-thi Oxodihydropyrimidine-4,6 (1H, 5H) -dione, 5- (2-hydroxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione, 5- ( 3,4,5-trimethoxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione, 5- (4-hydroxy-3,5-dimethoxybenzyl) -2 -Thioxodihydropyrimidine-4,6 (1H, 5 H) -dione, 5- (3-bromo-4-hydroxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione or 5- (3,5-dibromo -4-hydroxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione, but is not limited thereto.

More preferably, the compound is 5- (4-hydroxy-3-methoxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H)-represented by the following general formula (2) or (3): Dione (compound 42) or 5- (4-hydroxy-3,5-dimethoxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione (compound 50), It is not limited to this.

<Formula 2>

<Formula 3>

In another aspect, the present invention provides a composition for preventing or treating inflammatory diseases containing the compound as an active ingredient.

Preferably, the compound may inhibit the activity of macrophages through competitive binding to lipopolysaccharide (LPS) to TLR4.

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

In the case of the pharmaceutical composition of the present invention, the pharmaceutical composition may include a pharmaceutically acceptable carrier in addition to the active ingredient. Such pharmaceutically acceptable carriers are commonly used in pharmaceutical preparations, such as lactose, Dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzo Ate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but is not limited thereto. In addition, the pharmaceutical composition may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like as an additive.

The method of administration of the pharmaceutical composition is determined depending on the degree of symptoms, usually topical administration is preferred. In addition, the dosage of the active ingredient in the pharmaceutical composition may vary depending on the route of administration, the degree of the disease, the age, sex, and weight of the patient, and may be administered once to several times daily.

The pharmaceutical composition may be administered to various mammals such as rats, mice, livestock, humans, and the like. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The pharmaceutical compositions may be prepared in unit dose form or formulated using pharmaceutically acceptable carriers and / or excipients or may be prepared within a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion, or may be in the form of an exercicide, extract, powder, granule, tablet, warning, lotion, ointment, or the like.

The present invention also provides a health functional food for preventing or improving inflammatory diseases containing the compound as an active ingredient.

The present invention also provides a dietary supplement for antioxidants containing the compound as an active ingredient.

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

In the present invention, "health functional food" refers to a food having a bioregulatory function, such as prevention and treatment of diseases, biological defense, immunity, recovery of symptoms, aging inhibition, and should be harmless to the human body when taken in the long term.

On the other hand, the health functional food of the present invention can be prepared by a variety of methods known in the food science or pharmaceutical field, any food which can be ingested orally in combination with itself or a food-acceptable carrier, excipient, diluent, etc. It may also be prepared in form. Preferably in the form of beverages, pills, granules, tablets or capsules.

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

Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Example 1 Synthesis of Compounds 1 to 12

Table 1 shows 5- (substituted benzylidene) pyrimidine-2,4,6 (1H, 3H, 5H) -trione analogue [5- (substituted benzylidene) pyrimidine-2,4,6 (1H, 3H, 5H) -trione analog] For explaining the substitution pattern of compound 1-12.

<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 an ethoxy group.

5- (substituted benzylidene) pyrimidine-2,4,6 (1H, 3H, 5H) -trione analogue [5- (substituted benzylidene) pyrimidine-2,4,6 (1H, 3H, 5H) -trione analog] Compounds 1 to 12 were synthesized as follows. That is, a suspension of benzaldehyde (1.44-2.60 mmol) and barbituric acid (0.7-1.2 equivalents (eq.)) Substituted in EtOH (4 mL) and H ₂ O (4 mL) solvent was added. Heated to ° C. Before the reaction temperature reached 80 ° C., in most cases the reaction mixture became a clear solution. By the way, during further heating (1-18 hours), a precipitate formed and the precipitate was filtered after cooling. In the case of compound 12 synthesis, the reaction was performed at room temperature instead of 80 ° C., and the same operation was performed at room temperature. In view of the properties 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 Synthesis of pyrimidine-2,4,6 (1H, 3H, 5H) -trione (5- (4-Hydroxybenzylidene) pyrimidine-2,4,6 (1H, 3H, 5H) -trione)

Yellow solid; Reaction time, 6 hours; Yield, 82.6%; Melting point,> 300 ° C .; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.23 (s, 1 H), 11.10 (s, 1 H), 10.79 (s, 1 H), 8.29 (d, 2 H, J = 8.8 Hz), 8.17 (s, 1 H), 6.84 (d, 2H, J = 8.8 Hz); ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 164.8, 163.7, 163.0, 156.1, 150.9, 139.0, 124.4, 116.2, 114.9; LRMS (ESI) m / z 231 (M ⁻ H) ⁻ .

2. 5- (3,4- Dihydroxybenzylidene ) Pyrimidine-2,4,6 (1H, 3H, 5H) -trione [5- (3,4-Dihydroxybenzylidene) pyrimidine-2,4,6 (1H, 3H, 5H) -trione] (compound 2) synthesis

Orange solid; Reaction time, 8 hours; Yield, 99.3%; Melting point,> 300 ° C .; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 11.14 (br s, 2 H), 9.76 (br s, 1 H), 9.55 (br s, 1 H), 8.18 (s, 1 H), 8.10 (s , 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 ) δ 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 (M ⁻ H) ⁻ .

3. 5- (2,4- Dihydroxybenzylidene ) Pyrimidine-2,4,6 (1H, 3H, 5H) -trione [5- (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 ° C .; ¹ H NMR (500 MHz, D ₂ O + NaOH) δ 8.07 (s, 1 H), 7.26 (d, 1 H, J = 8.5 Hz), 6.44 (dd, 1H, J = 2.0, 9.0 Hz), 6.24 ( s, 1 H); ¹³ C NMR (100 MHz, 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 (M ⁻ H) ⁻ .

4. 5- (4- Hydroxy -3- Methoxybenzylidene Pyrimidine-2,4,6 (1H, 3H, 5H) -trione [5- (4-Hydroxy-3-methoxybenzylidene) pyrimidine-2,4,6 (1H, 3H, 5H) -trione] 4) synthetic

Dark yellow solid; Reaction time, 18 hours; Yield, 97%; Melting point, 288.6-290.7 ° C .; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.23 (s, 1 H), 11.11 (s, 1 H), 10.54 (s, 1 H), 8.44 (d, 1 H, J = 2.0 Hz), 8.18 (s, 1 H), 7.77 (dd, 1 H, J = 2.0, 8.4 Hz), 6.86 (d, 1H, J = 8.4 Hz), 3.79 (s, 3 H); ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 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 (M ⁻ H) ⁻ .

5. 5- (3- Ethoxy -4- Hydroxybenzylidene Pyrimidine-2,4,6 (1H, 3H, 5H) -trione [5- (3-Ethoxy-4-hydroxybenzylidene) pyrimidine-2,4,6 (1H, 3H, 5H) -trione] 5) synthetic

Orange solid; Reaction time, 15 hours; Yield 77%; Melting point, 244.7-246.1 ° C .; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 11.24 (s, 1 H), 11.11 (s, 1 H), 10.46 (s, 1 H), 8.48 (s, 1 H), 8.20 (s, 1 H ), 7.74 (d, 1H, J = 8.5 Hz), 6.90 (d, 1H, J = 8.5 Hz), 4.08 (q, 2H, J = 7.0 Hz), 1.36 (t, 3H, J = 7.0 Hz) ; ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 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 (M ⁻ H) ⁻ .

6. 5- (3- Hydroxy -4- Methoxybenzylidene Pyrimidine-2,4,6 (1H, 3H, 5H) -trione [5- (3-Hydroxy-4-methoxybenzylidene) pyrimidine-2,4,6 (1H, 3H, 5H) -trione] 6) synthetic

Dark yellow solid; Reaction time, 17 hours; Yield 93%; Melting point, 279.3-281.4 ° C .; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 11.26 (s, 1 H), 11.13 (s, 1 H), 9.42 (s, 1 H), 8.14 (s, 1 H), 8.10 (s, 1 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 ) δ 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 (M ⁻ H) ⁻ .

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

Yellow solid; Reaction time, 13 hours; Yield 93%; Melting point, 292.4-294.3 ° C .; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.27 (s, 1 H), 11.14 (s, 1 H), 8.33 (d, 2 H, J = 9.2 Hz), 8.21 (s, 1H), 7.02 ( d, 2H, J = 8.8 Hz), 3.83 (s, 3H); ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 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 (M ⁻ H) ⁻ .

8. 5- (3,4- Dimethoxybenzylidene ) Pyrimidine-2,4,6 (1H, 3H, 5H) -trione [5- (3,4-Dimethoxybenzylidene) pyrimidine-2,4,6 (1H, 3H, 5H) -trione] (compound 8) synthesis

Yellow solid; Reaction time, 9 hours; Yield, 96.6%; Melting point,> 300 ° C .; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.27 (s, 1 H), 11.15 (s, 1 H), 8.37 (d, 1 H, J = 2.0 Hz), 8.21 (s, 1H), 7.86 ( 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 ) δ 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 (M ⁻ H) ⁻ .

9. 5- (2,4- Dimethoxybenzylidene ) Pyrimidine-2,4,6 (1H, 3H, 5H) -trione [5- (2,4-Dimethoxybenzylidene) pyrimidine-2,4,6 (1H, 3H, 5H) -trione] (compound 9) synthesis

Orange solid; Reaction time, 8 hours; Yield, 97%; Melting point, 291.1-291.7 ° C .; ¹ H NMR (500 MHz, 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 = 8.5 Hz), 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 ) δ 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 (M ⁻ H) ⁻ .

10. 5- (3,4,5- Trimethoxybenzylidene Pyrimidine-2,4,6 (1H, 3H, 5H) -trione [5- (3,4,5-Trimethoxybenzylidene) pyrimidine-2,4,6 (1H, 3H, 5H) -trione] 10) synthetic

Yellow solid; Reaction time, 1 hour; Yield, 84.0%; Melting point, 274.8-275.4 ° C .; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.33 (s, 1 H), 11.20 (s, 1 H), 8.22 (s, 1 H), 7.80 (s, 2 H), 3.78 (s, 6 H ), 3.75 (s, 3H); ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 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 (M ⁻ H) ⁻ .

11. 5- (4-Hydroxy-3,5-dimethoxybenzylidene) pyrimidine-2,4,6 (1H, 3H, 5H) -trione [5- (4-Hydroxy-3,5-dimethoxybenzylidene Synthesis of pyrimidine-2,4,6 (1H, 3H, 5H) -trione]

Orange solid; Reaction time, 2 hours; Yield, 99.4%; Melting point,> 300 ° C .; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 11.25 (s, 1 H), 11.12 (s, 1 H), 9.97 (br s, 1 H), 8.24 (s, 1 H), 8.00 (s, 2 H), 3.82 (s, 6H); ¹³ C NMR (100 MHz, 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 (M ⁻ H) ⁻ .

12. 5- (3,5-Dibromo-4-hydroxybenzylidene) pyrimidine-2,4,6 (1H, 3H, 5H) -trione [5- (3,5-Dibromo-4- hydroxybenzylidene) pyrimidine-2,4,6 (1H, 3H, 5H) -trione] (Compound 12)

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> Synthesis of Compounds 13-26

Table 2 below shows 5- (substituted benzylidene) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione analogue [5- (substituted benzylidene) -2-thioxodihydropyrimidine-4,6 (1H). , 5H) -dione analog] To explain the substitution pattern of compound 13-26.

<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 an ethoxy group.

5- (substituted benzylidene) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione analogue [5- (substituted benzylidene) -2-thioxodihydropyrimidine-4,6 (1H, 5H)- dione analog] Compound 13-26 was synthesized as follows. That is, benzaldehyde (1.52-1.97 mmol) and thiobarbituric acid (0.9-1.1 equivalents (eq.)) Substituted in ethanol (4-8 mL) and H ₂ O (4-8 mL) solvents. The suspension was heated to 80 ° C. Before the reaction temperature reached 80 ° C., the reaction mixture became a clear solution in most cases. By the way, during additional heating (5 min-10.5 h), a precipitate formed and after cooling, the precipitate was filtered off. In the case of the synthesis of compounds 25 and 26, the reaction was performed at room temperature instead of 80 ° C., and the same operation was performed at room temperature. In view of the properties of the remaining substituted benzaldehyde, the filter cake was washed with ethanol and / or methylene chloride and water to give compound 13-26 (yield: 46.9-99.5%).

1.5- (4-Hydroxybenzylidene) -2-thioxodihydropyrimidine-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 ° C .; ¹ H NMR (400 MHz, 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 = 8.8 Hz), 8.19 (s, 1 H), 6.86 (d, 2H, J = 8.8 Hz); ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 178.8, 164.4, 163.1, 160.7, 157.2, 139.5, 124.6, 116.4, 114.9; LRMS (ESI) m / z 247 (M ⁻ H) ⁻ .

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

Orange solid; Reaction time, 3 hours; Yield 99.5%; Melting point,> 300 ° C .; ¹ H NMR (400 MHz, 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, 1 H), 7.63 (d, 1 H, J = 8.4 Hz), 6.83 (d, 1H, J = 8.4 Hz); ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 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 (M ⁻ H) ⁻ .

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

Dark yellow solid; Reaction time, 5 minutes; Yield, 82.5%; Melting point,> 300 ° C .; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.16 (s, 1 H), 12.06 (s, 1 H), 11.00 (s, 1 H), 10.89 (br s, 1 H), 8.77 (d, 1 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 ) δ 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 (M ⁻ H) ⁻ .

4. 5- (4-hydroxy-3-methoxybenzylidene) -2-thioxodihydropyrimidine-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 ° C .; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.31 (s, 1 H), 12.20 (s, 1 H), 10.70 (s, 1 H), 8.46 (s, 1 H), 8.20 (s, 1 H ), 7.83 (d, 1H, J = 8.8 Hz), 6.88 (d, 1H, J = 8.4 Hz), 3.80 (s, 3H); ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 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 (M ⁻ H) ⁻ .

5. 5- (3-Ethoxy-4-hydroxybenzylidene) -2-thioxodihydropyrimidine-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 ° C .; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.30 (s, 1 H), 12.19 (s, 1 H), 10.65 (br s, 1 H), 8.47 (d, 1 H, J = 2.0 Hz), 8.19 (s, 1H), 7.78 (dd, 1H, J = 2.0, 8.4 Hz), 6.89 (d, 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 ) δ 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 (M ⁻ H) ⁻ .

6. 5- (3-hydroxy-4-methoxybenzylidene) -2-thioxodihydropyrimidine-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 ° C .; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.32 (s, 1 H), 12.23 (s, 1 H), 9.58 (br s, 1 H), 8.16 (d, 1 H, J = 2.0 Hz), 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 (M ⁻ H) ⁻ .

7. 5- (4- Methoxybenzylidene )-2- Thioxodihydropyrimidine -4,6 (1H, 5H) -dione [5- (4-Methoxybenzylidene) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione] (Compound 19) Synthesis

Yellow solid; Reaction time, 4 hours; Yield, 77.9%; Melting point,> 300 ° C .; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.34 (s, 1 H), 12.25 (s, 1 H), 8.38 (d, 2 H, J = 8.8 Hz), 8.23 (s, 1H), 7.04 ( d, 2H, J = 9.2 Hz), 3.85 (s, 3H); ¹³ C NMR (100 MHz, 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 (M ⁻ H) ⁻ .

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

Dark yellow solid; Reaction time, 4 hours; Yield 91.9%; Melting point, 269.9-271.7 ° C .; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 12.37 (s, 1 H), 12.26 (s, 1 H), 8.41 (s, 1 H), 8.26 (s, 1 H), 7.95 (d, 1 H , 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 ) δ 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 (M ⁻ H) ⁻ .

9. 5- (2,4-Dimethoxybenzylidene) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione [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 ° C .; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.27 (s, 1 H), 12.16 (s, 1 H), 8.62 (s, 1 H), 8.61 (d, 1 H, J = 8.4 Hz), 6.61 (s, 1 H), 6.60 (d, 1 H, J = 8.0 Hz), 3.89 (s, 3H), 3.86 (s, 3H); ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 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 (M ⁻ H) ⁻ .

10. 5- (2-Hydroxybenzylidene) -2-thioxodihydropyrimidine-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 ° C .; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.40 (s, 1 H), 9.75 (s, 1 H), 9.60 (s, 1 H), 9.00 (s, 1 H), 8.01 (dd, 1 H , 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 ) δ 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 (M ⁻ H) ⁻ .

11. 2-Tioxo-5- (3,4,5-trimethoxybenzylidene) dihydropyrimidine-4,6 (1H, 5H) -dione [2-Thioxo-5- (3,4,5 -trimethoxybenzylidene) dihydropyrimidine-4,6 (1H, 5H) -dione] (Compound 23)

Orange solid; Reaction time, 1 hour; Yield 65.5%; Melting point, 258.9-260.7 ° C .; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.41 (s, 1 H), 12.30 (s, 1 H), 8.24 (s, 1 H), 7.85 (s, 2 H), 3.79 (s, 6 H ), 3.77 (s, 3H); ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 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 (M ⁻ H) ⁻ .

12. 5- (4-hydroxy-3,5-dimethoxybenzylidene) -2-thioxodihydropyrimidine-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 ° C .; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 12.33 (s, 1 H), 12.23 (s, 1 H), 10.17 (br s, 1 H), 8.27 (s, 1 H), 8.05 (s, 2 H), 3.83 (s, 6H); ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 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 (M ⁻ H) ⁻ .

13. 5- (3-Bromo-4-hydroxybenzylidene) -2-thioxodihydropyrimidine-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-thioxodihydropyrimidine-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 Synthesis of Compounds 27-38

Table 3 shows the 5- (substituted benzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -trione analogue [5- (substituted benzyl) pyrimidine-2,4,6 (1H, 3H, 5H). ) -trione analog] is for explaining the substitution pattern of compound 27-38.

<Formula 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) pyrimidine-2,4,6 (1 H , 3 H , 5 H ) -trione (corresponding to compound 1-12, 30 mg) suspended in ethanol (5 mL) Sodium borohydride (NaBH ₄ , 3 equiv) was slowly added into the 0 ° C. suspension and the mixed solution 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, then ether / water (5-10: 1) or ethanol / water (5: 1, for compound 35) was added and the resulting solid was filtered and ether / water (5-10: 1) ) Or ethanol / water (5: 1, for compound 35) to afford the desired product.

1. 5- (4- Hydroxybenzyl ) Synthesis of pyrimidine-2,4,6 (1H, 3H, 5H) -trione [5- (4-Hydroxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -trione]

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 (M ⁻ H) ⁻ .

2. 5- (3,4- Dihydroxybenzyl ) Pyrimidine-2,4,6 (1H, 3H, 5H) -trione [5- (3,4-Dihydroxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -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 (M ⁻ H) ⁻ .

3. 5- (2,4- Dihydroxybenzyl ) Pyrimidine-2,4,6 (1H, 3H, 5H) -trione [5- (2,4-Dihydroxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -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 (M ⁻ H) ⁻ .

4. 5- (4- Hydroxy 3-methoxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -trione [5- (4-Hydroxy-3-methoxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H ) -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, 2H); 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 (M ⁻ H) ⁻ .

5. 5- (3- Ethoxy -4- Hydroxybenzyl ) Pyrimidine-2,4,6 (1H, 3H, 5H) -trione [5- (3-Ethoxy-4-hydroxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -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 (M ⁻ H) ⁻ .

6. 5- (3- Hydroxy 4-methoxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -trione [5- (3-Hydroxy-4-methoxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H ) -trione] (compound 32) synthesis

Reaction time, 2 hours; Yield 23.1%; ¹ H NMR (400 MHz, DMSO- d ₆ ) d 11.11 (s, 2 H), 8.85 (s, 1 H), 6.73 (d, 1 H, J = 7.6 Hz), 6.48 (s, 1 H), 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 (M ⁻ H) ⁻ .

7. 5- (4-methoxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -trione [5- (4-Methoxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H ) -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 , 1 H, J = 3.5 Hz), 3.69 (s, 3 H), 3.19 (d, 2 H, 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 (M ⁻ H) ⁻ .

8. 5- (3,4- Dimethoxybenzyl ) Pyrimidine-2,4,6 (1H, 3H, 5H) -trione [5- (3,4-Dimethoxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -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, 3H), 3.40 (s, 2H); 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 (M ⁻ H) ⁻ .

9. 5- (2,4- Dimethoxybenzyl ) Pyrimidine-2,4,6 (1H, 3H, 5H) -trione [5- (2,4-Dimethoxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -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 (M ⁻ H) ⁻ .

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

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 (M ⁻ H) ⁻ .

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

Reaction time, 2 hours; Yield, 90%; ¹ H NMR (500 MHz, DMSO- d ₆ ) d 11.18 (br s, 1 H), 9.31 (br s, 2 H), 6.44 (br s, 2 H), 3.65 (s, 6 H), 3.61 ( s, 1 H), 3.27 (s, 2 H); ^{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 (M ⁻ H) ⁻ .

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

Reaction time, 1 hour; Yield 44.8%; ¹ H NMR (500 MHz, CD ₃ OD) d 7.35 (s, 2H), 3.48 (s, 2H); 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 Synthesis of Compounds 39-52

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

<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 equiv) was slowly added into the suspended solution at 0 ° C. and the mixed solution was stirred at room temperature for 10 minutes-2 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, then ether / water (10-20: 1) or ethanol / water (10: 1, for compounds 41, 46 and 47) was added and the resulting solid was filtered and ether / water (10 ˜20: 1) or washing with ethanol / water (10: 1, for compounds 41, 46 and 47) to afford the desired product.

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

Reaction time, 10 minutes; 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- Thioxodihydropyrimidine -4,6 (1H, 5H) -dione [5- (3,4-Dihydroxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -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- Thioxodihydropyrimidine -4,6 (1H, 5H) -dione [5- (2,4-Dihydroxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -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, 2H), 3.34 (s, 2H); 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-thioxodihydropyrimidine-4,6 (1H, 5H) -dione [5- (4-Hydroxy-3-methoxybenzyl) -2 -thioxodihydropyrimidine-4,6 (1H, 5H) -dione] (Compound 42)

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, 2H); 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-thioxodihydropyrimidine-4,6 (1H, 5H) -dione [5- (3-Ethoxy-4-hydroxybenzyl) -2 -thioxodihydropyrimidine-4,6 (1H, 5H) -dione] (Compound 43)

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, 2H), 1.23 (t, 3H, 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-4-methoxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione [5- (3-Hydroxy-4-methoxybenzyl) -2 -thioxodihydropyrimidine-4,6 (1H, 5H) -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, 2H); 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- Thioxodihydropyrimidine -4,6 (1H, 5H) -dione [5- (4-Methoxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -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- Thioxodihydropyrimidine -4,6 (1H, 5H) -dione [5- (3,4-Dimethoxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -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- Thioxodihydropyrimidine -4,6 (1H, 5H) -dione [5- (2,4-Dimethoxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -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- Thioxodihydropyrimidine -4,6 (1H, 5H) -dione [5- (2-Hydroxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione] (Compound 48) Synthesis

Reaction time, 10 minutes; 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-thioxodihydropyrimidine-4,6 (1H, 5H) -dione [5- (3,4,5-Trimethoxybenzyl) -2 -thioxodihydropyrimidine-4,6 (1H, 5H) -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-thioxodihydropyrimidine-4,6 (1H, 5H) -dione [5- (4-Hydroxy-3,5- dimethoxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -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-thioxodihydropyrimidine-4,6 (1H, 5H) -dione [5- (3-Bromo-4-hydroxybenzyl) -2 -thioxodihydropyrimidine-4,6 (1H, 5H) -dione] (Compound 51)

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-thioxodihydropyrimidine-4,6 (1H, 5H) -dione [5- (3,5-Dibromo-4 -hydroxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione] (Compound 52)

Reaction time, 1 hour; yield, 36.2%; ¹ H NMR (400 MHz, CD ₃ OD) d 7.29 (s, 2H), 3.52 (s, 2H); 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 Eradication activity analysis

1. Preparation of Macrophage (Raw264.7)

Raw 264.7 macrophagy cells (rat prostatic endothelial cell line) were obtained from American Type Culture Collection (ATCC), Manassas, VA, USA, which cells were 2 mM L-glutamine, 100 mg / ml streptomycin, 2.5 mg / L amphotericin. B, and cultured using DMEM (Dulbecco's Modified Eagle Medium, Nissui, Tokyo, Japan) containing 5% inactivated fetal bovine serum (FBS). The cells were also maintained at 37 ° C. under the same conditions as a humid atmosphere containing 5% CO ₂ and 95% air. And 5% FBS was not added was used as a serum-free medium (SFM, serum-free medium). Cell lines were maintained by subculture every two days in a 100 mm plastic flask (Corning Co., New York, USA).

2. In vitro ROS  Measure and ONOO- Measure

It was measured by 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 as stock solutions at -20 ° C. , 7'-dichlorodihydrofluorescein) solution was incubated at 22 ° C. for 20 minutes and then stored frozen in the dark until use. The fat-soluble DCFDA is deacetylated to non-fluorescent DCFH by esterase or oxidative hydrolysis, and DCFH is oxidized by free radicals to become DCF (2 ', 7'-dichlorofluorescein) which shows strong fluorescence, excitation wavelength 485nm And it was measured by a fluorescence photometer (GENios, TECAN) at an emission wavelength of 530nm. As a reactive oxygen generating source, ROS is optionally generated by reacting with esterase using 50 μM of 3-morpholinosydnonimine hydrochloride (SIN-1).

As a result, as shown in FIG. 1, compounds 28, 30, 31, 32, 37, 39, 42, 43, 44, 45, 48, which have a large effect of eliminating ROS randomly generated by the positive control Trolox 50, 51 could be selected.

ONOO ^- scavenging and production inhibitory activity of the 26 novel compounds was mediated by ONOO ^- according to the method of Kooy et al., Free Radic. Biol. Med. 16: 149-156,1994. It was measured and analyzed by fluorescence spectroscopy using an oxidation reaction of dihydroramine (dihidrorhodamine 123; hereinafter referred to as "DHR"). Non-fluorescent DHR 123 is oxidized by ONOO ⁻ to form fluorescent rhodamine 123. After using such a nature, each placed in DHR123 solution reaction of the compound of the 26 kinds of ONOO ^- by measuring the change in fluorescence of the added ONOO by the novel ^compounds was investigated scavenging activity, ONOO ^- measure the scavenging activity substrate with pure for ONOO ^- (authentic peroxynitrite) a, ONOO ^- a substrate to measure the inhibitory effect is produced ONOO ^- was used SIN-1 (3-morpholinosydnonimine) for generating. As a result, compounds 28, 29, 30, 31, 32, 37, 38, 41, 42, 43, 44, 45, 48, 50, 51, and 52 were screened as compounds having a high effect of ONOO ^- scavenging as shown in FIG. Could.

Example 6 Measurement of TLR4 Binding Intimacy

In the present invention, the affinity was confirmed through docking simulation in order to confirm whether the two novel compounds selected in FIG. 1 have an inhibitory effect and influence on the TLR4 receptor which directly affects the NF-κB activation pathway. No. 42 and No. 50 were found to bind directly to the MD2 portion of the TLR4 complex, and as a result of confirming the intimacy of each compound, Compound No. 42 had two more methoxy groups than Compound No. 42. Since the affinity was higher in the bun, compound 50 was more effective in inhibiting the effect on TLR4. In other words, it was confirmed from the following experiment that the signaling pathway, oxidative stress, NF-κB activity, and inflammatory response were all suppressed (FIG. 2).

Example 7 Cytotoxicity Experiment

Cell viability assay was measured using EZ-cy Tox kit for the toxicity test and proper concentration of this compound. Compounds 42 and 50 were measured at concentrations of 1 μM to 50 μM, respectively, and as a result, some cell deaths were observed at 50 μM (FIG. 3). As a result, the concentration of 1,5,10 μM, which is the most appropriate concentration, was selected as the experimental concentration.

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

400ng of LPS was used to induce oxidative stress and inflammatory reaction, and ROS measurement of macrophage RAW264.7 cell using fluorescence microscopy and fluorescence microscopy showed that ROS increased at 400ng of LPS. It was confirmed that the treatment by 50 times, decreased. In particular, it was confirmed that the inhibitory effect is better at 10μM of Compound 42 and 50 than 10μM Trolox used as a positive control (Fig. 4a).

In addition, ROS can be confirmed through a photograph, which means that the more fluorescence dye is expressed, the greater the amount of ROS, and it was confirmed that the fluorescence dye was significantly reduced in compounds 10μM 42 and 5μM and 10μM 50 (Fig. 4b).

These results showed similar trends for NO measured with DOO123 fluorescent dye and ONOO ^- and DAF2.

Example 9 Experiment of Confirming Expression of Inflammatory Cytokines

Western blotting boils the lysed sample in a cell with a loading buffer [0.125M Tris-Hcl, pH6.8, 4% SDS, 10% 2-mercaptoethanol and 0.2 bromophenol blue] for 1 to 5 minutes and loads onto the gel. . After SDS-PAGE using 10% acrylaminde by protein size, transfer to PVV membrane at 15V for 1 hour. After blocking for 1 hour with 5% skim milk, the primary antibody overnight and the secondary antibody are developed after 1-3 hours.

Expression of inflammatory cytokines INOS and COX-2 caused by oxidative stress was confirmed by western blotting.Also, the expression of inflammatory cytokines increased by LPS in compound 42 and compound 50 of 5μM and 10μM. The amount could be seen to decrease (FIG. 5).

Example 10 Confirmation of Expression of Inflammatory Cytokines

Next, NF-κB, a key factor involved in the regulation of inflammatory response and immune system, is present in most cells, and NF-κB consists of p50, p52, RelA (p65), RelB, c-Rel, and v-Rel. It has a variety of names. Among them, the typical NF-κB activation pathway constitutes a signaling system essential for innate immunity. The typical NF-κB activation pathway is a field in which many studies have been conducted until now, and the role of P65 and P50 is mainly played. Phosphorylation of P65 has two main sites, Ser536 and Ser276. Looking at the following results it can be seen that the different aspects in Ser536 and Ser276 (Fig. 6).

Compounds 42 and 50 inhibited the phosphorylation of P65 in Ser536, and it was confirmed that the total expression of P65 was also decreased. However, P65 phosphorylated at Ser276 showed no difference in the LPS group, Compound 42 and Compound 50, respectively.

Example 11 Transcription Inhibitory Activity of NF-κB

1. Confirmation of translocation of NF-κB through immunocytochemistry

The present inventors confirmed the translocation of NF-κB in the nucleus based on the above results, and performed an immunochemistry experiment using Alexa Fluor 488 Hoechst 33342. The data were taken using confocal microscopy, Hoechst 33342 is blue as a dye for nuclei staining, and also stains portions of NF-κB in cells using NF-κB antibody and Alexa Fluor 488. Shown in green.

In the present results, it can be seen that NF-κB is present only in the cytoplasm in the middle of the green part (nuclear part) in the untreated control group, and in the LPS group, the negative control group, the green part is generally cell-shaped. It can be seen that NF-κB translocation into the nucleus. Thus, NF-κB translocation into the nucleus due to LPS was found to decrease the concentration (translocation) of NF-κB by compounds 42 and 50 (Fig. 7).

2. Luciferase assay

Luciferase assay was performed once again to verify the effects of compounds 42 and 50 inhibiting NF-κB translocation into the nucleus. After transfection into cells using a luciferase reporter vector conjugated with an NF-κB binding site, LPS was treated to activate NF-κB. Luciferase is expressed by activated NF-κB, which luciferase reacts with luciferin to display phosphorescence, which is an indicator of how activated NF-κB is activated. As a result, it was confirmed that the activation of NF-κB increased by LPS was concentration-dependently inhibited by compounds 42 and 50 (FIG. 8).

3. Conclusion

NF-κB activation pathway, a pathway that forms an essential signaling system for innate immunity, has been studied in many fields. NF-κB dimers are inactivated in the cytoplasm by binding to IkBa that inhibits NF-κB activation. IKB is degraded by the activity of IKK, and NF-κB in the cytoplasm moves into the nucleus and is activated. This process modulates the activity of IkB, which inhibits NF-κB in the cytoplasm, using the IKK complex. IKK is composed of IKKα, IKK, and IKKγ.

In this study, compounds 42 and 50 were found to inhibit NF-κB activity in relation to NF-κB activity. First, the activity of IKBa and IKKb directly involved in translocation of NF-κB was confirmed by western blotting. When the compounds 42 and 50 were treated in both concentration-dependent manners, the activity of IKKb and IkBa decreased, indicating that translocation of NF-κB was inhibited (FIG. 9).

Higher signaling pathways that regulate IKK in the NF-κB activation pathway (IKK-IKB-NF-KB) include several higher signaling pathways such as ERK, p38, MAPK, and AKT. Inhibition of ser536 of NF-κB in a concentration-dependent manner, we confirmed the inhibitory effect of compounds 42 and 50 on the signaling pathway of AKT / PI3K. When measuring the activity of AKT directly affecting IKK, it was confirmed that compounds 42 and 50 inhibited AKT activity in a concentration-dependent manner, and that P-PTEN and NOX4, which regulates it, were also compound-dependent. And it was confirmed that it is suppressed by 50 (Fig. 10). In other words, NOx4 is inhibited by compounds 42 and 50, thereby directly and indirectly inhibiting oxidative stress generated. Accordingly, it was confirmed in the present invention that the expression of inflammatory cytokines is reduced by blocking both PTEN activation and AKT / NF-κB signaling pathways.

< Example  12> in vivo  test

The present inventors evaluated the efficacy against the same in vivo based on the experiment in vitro. As an animal model, C57BL / 6 mice were used, and one hour after feeding the compounds 42 and 50 to confirm that NF-κB was inhibited in the nucleus, 5 mg / kg LPS was IP dissected 1 hour later. It was.

After dissection, ROS and ONOO ⁻ were measured in blood and liver, and it was confirmed that the oxidative stress increased by LPS was significantly inhibited in compounds 42 and 50 (FIG. 11). In addition, as a result of extracting proteins from dissected liver and confirming the expression level of the preceding target genes, the activity of P65 ser536 was markedly inhibited similarly in vitro, and the inflammatory factors COX-2 and iNOS were also 42 and 50. It was confirmed that the expression was inhibited by the compound (Fig. 12).

Having described the specific parts of the present invention in detail, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

A compound represented by the following formula (1). <Formula 1>

In Formula 1, R _{One To} R _{4 It} may be the same or different, respectively, any one of H, OH, Br or C1 to C4 alkoxy, X is O or S. The compound of claim 1, wherein the compound is 5- (4-hydroxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -trione, 5- (3,4-dihydroxybenzyl) pyridine Midine-2,4,6 (1H, 3H, 5H) -trione, 5- (2,4-dihydroxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -trione, 5 -(4-hydroxy-3-methoxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -trione, 5- (3-ethoxy-4-hydroxybenzyl) pyrimidine-2 , 4,6 (1H, 3H, 5H) -trione, 5- (3-hydroxy-4-methoxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -trione, 5- (4-methoxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -trione, 5- (3,4-dimethoxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -trione, 5- (2,4-dimethoxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -trione, 5- (3,4,5-trimethoxybenzyl) Pyrimidine-2,4,6 (1H, 3H, 5H) -trione, 5- (4-hydroxy-3,5-dimethoxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -Trione, 5- (3,5-dibromo-4-hydroxybenzyl) pyrimidine-2,4,6 (1H, 3H, 5H) -trione, 5- (4-hydroxybenzyl)- 2-thioxodihydropyrimidine-4,6 (1H, 5H)- Dione, 5- (3,4-dihydroxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione, 5- (2,4-dihydroxybenzyl) -2- Thioxodihydropyrimidine-4,6 (1H, 5H) -dione, 5- (4-hydroxy-3-methoxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -Dione, 5- (3-ethoxy-4-hydroxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione, 5- (3-hydroxy-4-methoxy Benzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione, 5- (4-methoxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -Dione, 5- (3,4-dimethoxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione, 5- (2,4-dimethoxybenzyl) -2-thi Oxodihydropyrimidine-4,6 (1H, 5H) -dione, 5- (2-hydroxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione, 5- ( 3,4,5-trimethoxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione, 5- (4-hydroxy-3,5-dimethoxybenzyl) -2 -Tioxodihydropyrimidine-4,6 (1H , 5H) -dione, 5- (3-bromo-4-hydroxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione and 5- (3,5-dibro Mother-4-hydroxybenzyl) -2-thioxodihydropyrimidine-4,6 (1H, 5H) -dione. A pharmaceutical composition for preventing or treating inflammatory diseases, comprising the compound according to claim 1 or 2 as an active ingredient. A pharmaceutical composition for preventing or treating inflammatory diseases containing a compound represented by the following Chemical Formula 2 or Chemical Formula 3 as an active ingredient. <Formula 2>

<Formula 3>

[Claim 5] The pharmaceutical composition for preventing or treating inflammatory diseases according to claim 3 or 4, wherein the compound inhibits macrophage activity through competitive binding with lipopolysaccharide (LPS) to TLR4. The method of claim 3 or 4, wherein the inflammatory disease is any one selected from the group consisting of asthma, bronchitis, sepsis, arthritis, hepatitis, rheumatoid arthritis, osteoarthritis, ulcerative colitis, myocarditis, multiple sclerosis and viral infections. Pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that. A health functional food for preventing or improving an inflammatory disease containing the compound according to claim 1 or 2 as an active ingredient. A dietary supplement for antioxidants containing the compound according to claim 1 or 2 as an active ingredient. An anti-aging health functional food containing the compound according to claim 1 or 2 as an active ingredient.

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