KR101823768B1 - Phamaceutical composition for preventing or treating of vascular disease containing dihydroxyphenyl derivative - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of vascular diseases containing a dihydroxyphenyl derivative, wherein the dihydroxyphenyl derivative represented by the formula (1) according to the present invention inhibits the activity of arginase It is possible to improve the vascular endothelial dysfunction by increasing the production of NO and at the same time by inhibiting the production of active oxygen and to restore the reduced vascular relaxation ability in an animal model of arteriosclerosis, Composition can be usefully used as medicines, health functional foods or health foods for prevention, improvement and treatment of vascular diseases.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for preventing or treating vascular diseases containing dihydroxyphenyl derivatives,

The present invention relates to a pharmaceutical composition for preventing or treating vascular diseases containing a dihydroxyphenyl derivative.

In vivo, the blood is always balanced by a complex and elaborate regulatory system, so that the flow is not interrupted by bleeding or thrombosis under normal conditions. However, when the equilibrium state is broken due to various factors, the flow of blood vessels is not smooth and blood vessel diseases occur. The most common type of vascular disease is arteriosclerosis, which is one of the most dangerous diseases that cause myocardial infarction or cerebral infarction by causing ischemic conditions in important organs such as the heart and brain.

On the other hand, the heart and blood vessels supply oxygen and nutrients to the whole body, which is an essential part of human life. Therefore, if blood and oxygen supply to each tissue or organ is not smooth due to various causes of cardiovascular diseases, it may cause dangerous disease which threatens to life as well as local necrosis of tissue. Cardiovascular disease is becoming more frequent as a result of aging of the human body, especially as it gets older. For this reason, prevention and treatment of cardiovascular diseases is becoming very important.

The main factors that cause cardiovascular diseases are genetic factors, lifestyle, and diabetic complications. However, in modern medical terms, reactive oxygen species (ROS) and increased endothelial cell stress (ENOS) activity of endothelial-type Nitric Oxide Synthase (eNOS).

In particular, nitric oxide produced by endothelial nitric oxide synthase is a potent vasodilator and inhibits platelet aggregation, vascular myocyte proliferation, vascular deposition of mononuclear cells, and atherosclerosis-related protein expression, (Forstermann et al., Circulation, 113: 1708-1714, 2006). In addition, reactive oxygen species play a role in the regulation of adhesion molecule expression, the proliferation of vascular smooth muscle cells And lipoprotein regulation with mobility and oxidative potential, leading to cardiovascular disease.

To date, the ISMO of Schwarz has been introduced as a treatment for cardiovascular disease. However, this formulation has excellent effects and has side effects such as hypotension and palpitations. Therefore, hypotension, glaucoma, etc. (BETALOC) as a beta-blocker. This formulation is also effective, but patients with congestive heart failure should be used with caution due to rapid cardiac suppression.

On the other hand, normal production of endothelial nitric oxide is important for maintenance of vascular function. Increased release of nitric oxide in endothelial cells leads to decreased elasticity of smooth muscle cells and decreased vascular resistance. However, endothelial dysfunction under hypertensive and hyperlipidemic conditions causes a marked reduction in nitric oxide release, resulting in increased vasoconstriction and vasospasm, increased mononuclear cell and LDL infiltration, proliferation of vascular smooth muscle cells, Leading to increased oxidative stress and increased platelet aggregation. Therefore, endothelial function and substances restoring nitric oxide metabolism are effective in the treatment of vascular diseases including cardiac artery disease and cardiac dysfunction.

Most of the cells that synthesize nitric oxide also have an enzyme called arginase, which catalyzes the hydrolysis of L-arginine to produce L-ornithine and urea It is an enzyme group to generate. Arginase I is constitutively expressed in cells and arginase II is known to be induced by endotoxin. These enzymes play an important role in the production of nitric oxide under inflammatory conditions, but also play an important role in the synthesis of nitric oxide.

Arginase, on the other hand, competitively inhibits NOS (Nitric Oxide Synthase) through the use of a common substrate, L-arginine (Berkowitz et al., 2003; Holowatz et al. 2006; Morris et al., 1998; Peyton et al., 2009; Simon et al., 2003; Steppan et al., 2006). The expression and function of arginase I in macrophages, hepatocytes and vascular smooth muscle cells was assessed by the expression of lipopolysaccharide (LPS), IL-13, altered oxygen partial pressure and coronary arteries (Nelin et al., 2004), which can be stimulated by balloon dilatation (Chicoine et al., 2004; In addition, the activity and expression of arginase II was determined by a variety of methods including OxLDL, LPS, TNF-a, IFN-, 8-bromo-cGMP and hypoxia Can be induced by vascular factors.

On the other hand, recently reported data indicate that inhibition of arginase inhibits the production of nitric oxide actively and is associated with the development of normal cardiac function and atherogenesis, aging, erectile dysfunction and sickle cell disease, 2007; Morris et al., 2004; Steppan et al., 2004), and the results of this study are summarized as follows: (1) Bivalvia et al. , 2006; White et al., 2006; Xu et al., 2007).

Therefore, it is predicted that a substance that inhibits the activity of arginase may be useful for the improvement and treatment of vascular dysfunction. However, the conventionally developed arginase activity inhibitor has a side effect and its effect is poor Development of new arginase activity inhibitors is urgent.

Journal of Asian Natural Products Research, 2006, 8 (6), pp. 571-577

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of vascular diseases.

Another object of the present invention is to provide a health functional food composition for prevention or improvement of vascular diseases.

Another object of the present invention is to provide a health food composition for preventing or improving vascular diseases.

In order to achieve the above object,

The present invention provides a pharmaceutical composition for preventing or treating vascular diseases, which comprises a dihydroxyphenyl derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

The present invention also provides a health functional food composition for preventing or ameliorating a vascular disease, which comprises the dihydroxyphenyl derivative represented by the general formula (1) or a pharmaceutically acceptable salt thereof.

Further, the present invention provides a health food composition for preventing or ameliorating a vascular disease, comprising the dihydroxyphenyl derivative represented by the above formula (1) or a pharmaceutically acceptable salt thereof.

The dihydroxyphenyl derivative represented by formula (1) according to the present invention inhibits the activity of arginase and / or azine to increase production of nitrogen oxides (NO) and at the same time inhibit the production of active oxygen, And also has the effect of restoring reduced vascular relaxation ability in an animal model of atherosclerosis. Therefore, the composition containing it as an active ingredient is useful as a medicament for preventing, improving and treating vascular diseases, a health functional food or a health food Can be usefully used.

In FIG. 1, A shows the result of inhibiting the activity against arginase 2 when the dihydroxyphenyl derivative represented by the formula (1)
In FIG. 1, B shows the results of inhibiting the activity against arginase 1 when the dihydroxyphenyl derivative represented by formula (1) was treated by concentration in the liver lysate of mice,
In FIG. 1, C shows the results of inhibiting the activity against arginase when human diabetic endothelial cells (HUVEC) were treated with the dihydroxyphenyl derivative represented by the formula (1) by concentration.
In FIG. 2, A shows the result of confirming the effect of reducing the production of reactive oxygen species (ROS) when treating the mouse aortic endothelial cells with the dihydroxyphenyl derivative represented by the formula (1)
In FIG. 2, B is a result of confirming the effect of reducing NO production by treatment of the dihydroxyphenyl derivative represented by Chemical Formula 1 with mouse aortic endothelial cells.
In FIG. 3, when the dihydroxyphenyl derivative represented by formula (1) was treated, argininease 1, arginase 2, iNOS (inducible nitric oxide synthase and eNOS (endothelial nitric oxide synthase) Results,
In FIG. 3, B shows the results of western blotting of changes in the monomer and dimer form of eNOS when the dihydroxyphenyl derivative represented by formula (1) was treated.
In FIG. 3, C shows the result of Western blotting of the phosphorylation of Ser1177 and the amount of phosphorylation of Thr495 when the dihydroxyphenyl derivative represented by the formula (1) was treated.
A normal group (WT + ND) and atherosclerosis model ApoE A is a (normal diet) jeongsangsik to wild-type mice at 4 ^{- / -} cholesterol diet high in mice (high cholesterol diet, HCD) by atherosclerosis group (ApoE ^{- / -} + HCD) and the argininease activity when the arteriosclerosis group was treated with the dihydroxyphenyl derivative represented by the formula (1) according to the present invention,
In FIG. 4, B represents the amount of nitrogen monoxide produced in the normal group (WT + ND) and the arteriosclerosis group (ApoE ^{- / -} + HCD) and the dihydroxyphenyl derivative represented by the formula (1) The results of measurement of nitrogen monoxide production in the case of
In FIG. 4, C is a result of measuring changes in monomer and dimer form of eNOS when the dihydroxyphenyl derivative represented by Formula 1 according to the present invention is treated,
In FIG. 4, D represents an arterial hardening group (ApoE ^{- / -} + HCD + DHP) administered with a normal group (WT + ND), an arteriosclerosis group (ApoE ^{- / -} + HCD), a dihydroxyphenyl derivative according to the present invention, And the degree of blood vessel relaxation was evaluated.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating vascular diseases, which comprises a dihydroxyphenyl derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

The present invention also provides a health functional food composition for preventing or ameliorating a vascular disease, which comprises the dihydroxyphenyl derivative represented by the general formula (1) or a pharmaceutically acceptable salt thereof.

Further, the present invention provides a health food composition for preventing or ameliorating a vascular disease, comprising the dihydroxyphenyl derivative represented by the above formula (1) or a pharmaceutically acceptable salt thereof.

The vascular disease may be arteriosclerosis, hypertension, peripheral vascular disease, erectile dysfunction, angioma, vascular stenosis, cerebrovascular disease, stroke, cerebral hemorrhage, or ischemic disease, myocardial infarction, heart attack, angina pectoris, coronary artery disease, .

In the present invention, the dihydroxyphenyl derivative represented by the above formula (1) has an activity of inhibiting the activity of arginase for the first time. The inhibition of the activity of arginase is reported in recent reports Has been shown to have a beneficial effect on vascular dysfunction by increasing the production of nitric oxide.

Pharmaceutically acceptable salts

The derivatives of formula (1) of the present invention can be used in the form of pharmaceutically acceptable salts, and acid addition salts formed by pharmaceutically acceptable free acids are useful as salts. The term pharmaceutically acceptable salt means a concentration that is relatively non-toxic to a patient and has a beneficial effect that is harmless to the patient, such that the side effects caused by the salt are any organic or inorganic salt of the base compound of Formula 1 that does not impair the beneficial effects of the base compound of Formula An inorganic addition salt. Examples of the inorganic acid include hydrochloric acid, bromic acid, nitric acid, sulfuric acid, perchloric acid and phosphoric acid. Examples of the organic acid include citric acid, acetic acid, lactic acid, maleic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, maleic acid, tartaric acid, succinic acid, malonic acid, succinic acid, malonic acid, glutamic acid, aspartic acid, oxalic acid, P-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid or malonic acid. These salts also include alkali metal salts (sodium salts, potassium salts, etc.) and alkaline earth metal salts (calcium salts, magnesium salts, etc.). For example, the acid addition salt may be selected from the group consisting of acetate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camylate, citrate, eddylate, Hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, malate, glucoside, gluconate, gluconate, glucuronate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / Hydrogen phosphate, dihydrogen phosphate, dihydrogen phosphate, dihydrogen phosphate, dihydrogen phosphate, dihydrogen phosphate, dihydrogen phosphate, dihydroxyacetate, Lactate, stearate, succinate, tartrate, tosylate, trifluoroacetate Diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, zinc salts, and the like. Preferred is hydrochloride or trifluoroacetate.

The acid addition salt according to the present invention can be obtained by a conventional method, for example, by dissolving a derivative represented by the formula (1) in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile and the like, Followed by filtration and drying. Alternatively, the solvent and excess acid may be distilled off under reduced pressure, followed by drying or crystallization in an organic solvent.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Furthermore, the present invention encompasses derivatives of Formula 1 and pharmaceutically acceptable salts thereof, as well as possible solvates, hydrates, isomers, optical isomers and the like, which can be prepared therefrom.

Pharmaceutical composition

The derivatives of formula (I) of the present invention can be administered in various formulations for oral administration and parenteral administration at the time of clinical administration. In the case of formulation, diluents such as fillers, extenders, binders, wetting agents, disintegrants, Or an excipient.

Solid form preparations for oral administration include tablets, patients, powders, granules, capsules, troches and the like, which may contain one or more excipients such as starch, calcium carbonate, It is prepared by mixing sucrose, lactose or gelatin. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions or syrups. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like are included in addition to commonly used simple diluents such as water and liquid paraffin. .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol, gelatin and the like can be used.

The effective dose of the compound of the present invention on the human body may vary depending on the age, weight, sex, dosage form, health condition and disease severity of the patient, and is generally about 0.001-100 mg / kg / 0.0 > mg / kg / day. ≪ / RTI > It is generally from 0.07 to 7000 mg / day, preferably from 0.7 to 2500 mg / day, based on an adult patient weighing 70 kg, and may be administered once a day It may be divided into several doses.

Health functional food composition

The health functional food composition according to the present invention can be added to health functional food such as food, beverage, and the like.

There is no particular limitation on the kind of the food. Examples of the foods to which the above substances can be added include dairy products including dairy products, meat, sausage, bread, biscuits, rice cakes, chocolate, candies, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, Beverages, alcoholic beverages and vitamin complexes, dairy products, and dairy products, all of which include health functional foods in a conventional sense.

The health functional food composition containing the compound of the formula (1) or a pharmaceutically acceptable salt thereof according to the present invention as an active ingredient can be added directly to the food or can be used together with other food or food ingredients, Can be used. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). Generally, the amount of the composition in the health functional food may be 0.1 to 90 parts by weight of the total food. However, in the case of long-term intake for the purpose of health maintenance or health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health functional food composition of the present invention has no particular limitation on other components other than the above-mentioned compounds as essential components in the indicated ratios, and may contain various flavors or natural carbohydrates as additional components such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above . The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 health functional food composition of the present invention.

In addition to the above, the health functional food composition containing the compound of the formula (I) of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient may be in the form of a flavor such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors , A coloring agent and a thickening agent (cheese, chocolate, etc.), a pectic acid and its salt, an alginic acid and its salt, an organic acid, a protective colloid thickener, a pH adjusting agent, a stabilizer, a preservative, a glycerin, ≪ / RTI > In addition, the health functional food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.

These components may be used independently or in combination. Although the ratio of such additives is not so important, it is generally preferred that the ratio is 0.1 to about 20 parts by weight per 100 parts by weight of the health functional food composition containing the compound of the formula 1 of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient to be.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Materials and Methods

1. Experimental animals

An arginase lysate was prepared from liver and kidney of anesthetized mouse C57BL / 6.

2. HUVEC  cell

HUVECs cells were purchased from Cascade biologics and cultured and stored in media supplemented with Medium230 plus low-serum growth supplement (LSGS) under conditions of 5% CO 2 and 37 ° C according to supplier protocol.

3. Measurement of arginase activity

To measure the activity of arginase, the cells were first homogenized at 4 ° C and then centrifuged at 14,000 xg for 20 minutes and resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, (Protease inhibitors) to prepare lysates, and arginase activity assay used lysates in supernatants (Ryoo et al., 2006). The activity assay was also analyzed by measuring the activity of arginase in the presence of a known arginase inhibitor (ABH, 2 (S) -amino-6-boronohexanoic acid, 20 uM).

4. In isolated mouse aorta DAF -FM or DHE  Measurement of NO generation using

Mouse aortic rings were separated and cultured in Dulbeccos modified Eagles (R) supplemented with 2% FBS and antibiotic (1X) in the presence of picaetannol-3-O-beta-D-glucopyranoside (50 uMol / L) (DMEM) medium at 37 ° C and 5% CO 2 overnight (White et al., 2006). Thereafter, the aorta was cut in the longitudinal direction, and then the aorta was cut in the longitudinal direction and sealed with a seal-guard coating filled with HEPES buffer (NaCl 120 mM, KH 2 PO 4 2.6 mM, KCl 4 mM, CaCl 2 2 mM, MgCl 2 0.6 mM, HEPES 25 mM, glucose 14 mM, And fixed to the bottom of a sililged coated chamber (upper endothelial layer). The chamber was allowed to equilibrate with the heating stage for 30 minutes at 37 DEG C and the chamber maintained the static bath condition during fluorescence measurement. An Olympus 10x objective with an optimized excitation and emission wavelength (DAF-FM, 470/525 nm; or DHE, 470/580), an intensified camera (Luca 658M-TL) Fluorescence of the tissue background and DAF-FM (4-Amino-5-methylamino-2 ', 7'-difluorofluorescein) diacetate or DHE (dihydroethidine) was measured using an image acquisition program (Cell software, Olympus). Following the initial equilibrium, background fluorescence was recorded and the aorta was left at room temperature for 15 minutes. The aorta was then loaded into 5 uM DAF-FM or 5 uM DHE (molecular Probes) for 45 minutes in HEPES buffer, then washed with DAF-FM or DHE and maintained equilibrated at 37 ° C for 20 minutes . Fluorescence intensity was averaged from the entire view field (5 frames, 2x2 binning) and recorded with the acquisition program. After reprocessing L-NAME (mol / L) or MnTBAP (mol / L), it was washed with DAF-FM or DHE to establish a baseline change in intensity and recorded changes in DAF-FM or DHE fluorescence Respectively. The slope of fluorescence change over time was measured by linear regression in Origin (version 7.5, OrignLab Corp., Northampton, Mass.) And used for statistical comparisons.

5. Western Blot  analysis

Liver or kidneys of C57BL / 6 mice (10 weeks) were diluted in buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 1 ug / ml leupeptin, 1 ug / ml pepstatin, / ml aprotinin, 1 mM phenylmethylsulfonylflouride, 1 mM sodium orthovanadate and 1 mM NaF) and centrifuged at 14,000 xg for 30 minutes. Then, the amount of protein in the supernatant was measured by Bradford method, and the protein (100 ug) was electrophoresed on 10% SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad). Then, the membrane was incubated with monoclonal anti-arginase I (Santa Cruz), arginase II (Santa Cruz), anti-inducible nitric oxide synthase (iNOS, BD Bioscience), anti-endothelial nitric oxide synthase (BD Bioscience) or anti-beta tubulin (BD bioscience) antibody and reacted with a secondary antibody (Amersham). The signal analysis was then detected with X-ray film using a chemiluminescence detection reagent.

6. Statistical processing

All data were expressed as mean ± SD of at least four independent experiments. The unpaired Student's t-test between the two groups was used to evaluate the significance of the deviation. P <0.05 was considered significant.

7. Atherosclerotic animal models

Animals used in the experiment were C57BL / 6 mice aged 10-30 weeks. Apo-lipoprotein E knockout (ApoE ^{- / -} ) mouse was used as a model of atherosclerotic disease and 10-week-old males were fed high cholesterol diet for 4 weeks.

< Experimental Example  1> arginase ( arginase ) Inhibition of activity inhibition

In order to determine whether the dihydroxyphenyl derivative represented by formula (1) according to the present invention has an arginase inhibitory activity, the following experiment was conducted.

Specifically, lysates and HUVEC cells of the respective tissues were obtained from the liver and kidney of the mice in accordance with the above-mentioned method, and the dihydroxyphenyl derivatives represented by the formula (1) were treated with arginase I and arginase II were used to confirm the presence of an isomer of arginase through the above-described Western blotting method. The dihydroxyphenyl derivative represented by formula (1) according to the present invention was treated at each concentration The activity of arginase was measured according to the arginase activity assay described above. The results are shown in Fig.

In FIG. 1, A shows the result of inhibiting the activity against arginase 2 when the dihydroxyphenyl derivative represented by the formula (1)

In FIG. 1, B shows the results of inhibiting the activity against arginase 1 when the dihydroxyphenyl derivative represented by formula (1) was treated by concentration in the liver lysate of mice,

In FIG. 1, C shows the results of inhibiting the activity against arginase when human diabetic endothelial cells (HUVEC) were treated with the dihydroxyphenyl derivative represented by the formula (1) by concentration.

As shown in Fig. 1, the dihydroxyphenyl derivatives represented by the formula (1) according to the present invention exhibited enzyme activity in a concentration-dependent manner in both liver and kidney tissues of mice and HUVEC cells, arginase I and arginase II Respectively.

< Experimental Example  2> Increased production of nitrogen monoxide (NO) and active oxygen ROS ) Evaluation of generation reduction

In order to investigate whether the inhibition of arginase activity by the treatment of the dihydroxyphenyl derivative represented by the formula (1) according to the present invention has an effect of increasing the production of NO and simultaneously reducing the production of ROS, In the endothelial cells of the aorta treated with dihydroxyphenyl derivatives, O ₂ ^- generation was measured using O ₂ ^- - sensitive DHE, and NO production was measured using NO - sensitive sample DAF.

In FIG. 2, A shows the result of confirming the effect of reducing the production of reactive oxygen species (ROS) when treating the mouse aortic endothelial cells with the dihydroxyphenyl derivative represented by the formula (1)

In FIG. 2, B is a result of confirming the effect of reducing NO production by treatment of the dihydroxyphenyl derivative represented by Chemical Formula 1 with mouse aortic endothelial cells.

As shown in FIG. 2, the DHE fluorescence intensity was decreased by treatment with the dihydroxyphenyl derivative represented by the treated formula (1), and the DHE signal was completely inhibited by MnTBAP treatment. In addition, the results are consistent with a new concept that arginase is able to regulate NO production by NOS by limiting L-arginine substrate as a general mechanism for the regulation of NOS. Thus, inhibition of arginase can increase the amount of L-arginine and further inhibit the separation of eNOS from ROS production in the reduced endothelium.

< Experimental Example  3> Evaluation of expression amount of nitrogen monoxide (NO) -generating enzyme

In order to investigate the changes in the amount of NO production in HUVEC (human umbilical vein endothelial cell) cultured by treatment with the dihydroxyphenyl derivative represented by Chemical Formula 1 according to the present invention, Western blotting Respectively.

In FIG. 3, when the dihydroxyphenyl derivative represented by formula (1) was treated, argininease 1, arginase 2, iNOS (inducible nitric oxide synthase and eNOS (endothelial nitric oxide synthase) Results,

In FIG. 3, B shows the results of western blotting of changes in the monomer and dimer form of eNOS when the dihydroxyphenyl derivative represented by formula (1) was treated.

In FIG. 3, C shows the result of Western blotting of the phosphorylation of Ser1177 and the amount of phosphorylation of Thr495 when the dihydroxyphenyl derivative represented by the formula (1) was treated.

As shown in FIG. 3, it was found that the dihydroxyphenyl derivative according to the present invention did not induce the expression of iNOS without affecting the expression level of arginase 1, 2 and eNOS. In Fig. 3B, the dihydroxyphenyl derivatives according to the present invention were found to reduce the monomeric form of eNOS and increase the stability of the dimeric form. In Fig. 3C, the dihydroxyphenyl derivative according to the present invention showed an increase in the phosphorylation of Ser1177 of eNOS and a decrease in the phosphorylation of Thr495, which induce the activation of nitrogen monoxide (NO) synthase.

Accordingly, the dihydroxyphenyl derivative represented by formula (I) according to the present invention shows an increase in the expression amount of nitric oxide synthase, which is useful for treating vascular diseases.

< Experimental Example  4> Assessment of vascular function improvement in atherosclerotic mouse model

In order to examine the degree of enhancement of the vascular function of the dihydroxyphenyl derivative represented by the formula (1) according to the present invention, the following experiment was conducted.

Specifically, jeongsangsik the (normal diet) group (WT + ND) and atherosclerosis model ApoE in the wild type mice ^{- / -} A group of high-cholesterol diet (high cholesterol diet, HCD) in mice (ApoE ^{- / -} + HCD) Of arginase activity was measured (Fig. 4A). In addition, the amount of nitrogen monoxide produced in the WT + ND group and the ApoE ^{- / -} + HCD group was measured, and the negative control group was the sNOS inhibitor L-NAME (FIG. 4B). Furthermore, the amount of change in monomer and dimer form of eNOS was measured (Fig. 4C). In addition, the extent of vascular relaxation was measured (Fig. 4D).

A normal group (WT + ND) and atherosclerosis model ApoE A is a (normal diet) jeongsangsik to wild-type mice at 4 ^{- / -} cholesterol diet high in mice (high cholesterol diet, HCD) by atherosclerosis group (ApoE ^{- / -} + HCD) and the argininease activity when the arteriosclerosis group was treated with the dihydroxyphenyl derivative represented by the formula (1) according to the present invention,

In FIG. 4, B represents the amount of nitrogen monoxide produced in the normal group (WT + ND) and the arteriosclerosis group (ApoE ^{- / -} + HCD) and the dihydroxyphenyl derivative represented by the formula (1) The results of measurement of nitrogen monoxide production in the case of

In FIG. 4, C is a result of measuring changes in monomer and dimer form of eNOS when the dihydroxyphenyl derivative represented by Formula 1 according to the present invention is treated,

In FIG. 4, D represents an arterial hardening group (ApoE ^{- / -} + HCD + DHP) administered with a normal group (WT + ND), an arteriosclerosis group (ApoE ^{- / -} + HCD), a dihydroxyphenyl derivative according to the present invention, And the degree of blood vessel relaxation was evaluated.

As shown in FIG. 4, in the case of the arteriosclerosis group (ApoE ^{- / -} + HCD) in FIG. 4A, arginase activity and azeotropic activity were increased. However, when the dihydroxyphenyl derivative according to the present invention was administered, + ND), the activity of arginase was decreased. In FIG. 4B, the production of nitrogen monoxide is decreased in the case of the arteriosclerosis group (ApoE ^{- / -} + HCD) compared to the normal group (WT + ND), but when the dihydroxyphenyl derivative according to the present invention is administered, WT + ND), the amount of nitrogen monoxide was recovered. FIG. 4C shows that when the dihydroxyphenyl derivative according to the present invention is administered, the monomeric form of eNOS decreases and the dimeric form increases. In FIG. 4 D, the arterial hardening group (ApoE ^{- / -} + HCD + DHP) administered with the dihydroxyphenyl derivative according to the present invention showed restoration of blood vessel relaxation to the level of the normal group (WT + ND) .

Accordingly, the dihydroxyphenyl derivative represented by formula (I) according to the present invention appears to improve vascular function, and is useful for treating vascular diseases.

Example of preparation of pharmaceutical agent

The derivatives represented by the formula (1) according to the present invention can be formulated into various forms depending on the purpose. Hereinafter, some formulating methods in which the derivative represented by the formula (1) according to the present invention is contained as an active ingredient are exemplified, and the present invention is not limited thereto.

<Pharmaceutical preparation example 1> Manufacture of powder

2 g of the derivative of formula (1)

Lactose 1 g

After mixing the above components, the mixture was packed in an airtight container to prepare a powder.

&Lt; Pharmaceutical Preparation Example 2 > Preparation of tablet

100 mg of the derivative of formula (1)

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

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

&Lt; Pharmaceutical Preparation Example 3 > Preparation of capsules

100 mg of the derivative of formula (1)

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

&Lt; Pharmaceutical Preparation Example 4 > Preparation of injection

10 mu g / ml of the derivative of formula (1)

Until dilute hydrochloric acid BP pH 3.5

Sodium chloride BP injected up to 1 ml

The pH of the resulting solution was adjusted to pH 3.5 with dilute hydrochloric acid BP, and the volume was adjusted using sodium chloride BP for injection. . The solution was filled in a 5 ml type I ampoule made of transparent glass, sealed in an upper lattice of air by dissolving the glass, sterilized by autoclaving at 120 DEG C for 15 minutes or longer, and an injection solution was prepared.

Manufacturing Example of Health Functional Food

The derivatives represented by the formula (1) according to the present invention can be manufactured into various types of health functional foods according to purposes. Hereinafter, a method for preparing a health functional food containing the derivative represented by the formula 1 according to the present invention as an active ingredient is illustrated, but the present invention is not limited thereto.

&Lt; Health functional food production example 1 > Production of health functional food

100 mg of the derivative represented by the formula (1)

Vitamin mixture quantity

Vitamin A acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

Biotin 10 μg

Nicotinic acid amide 1.7 mg

Folic acid 50 μg

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a component suitable for a health functional food as a preferred embodiment, the compounding ratio may be arbitrarily changed, and the above components may be mixed , Granules may be prepared and used in the manufacture of health functional food compositions according to conventional methods.

&Lt; Health Functional Food Production Example 2 >

100 mg of the derivative represented by the formula (1)

Citric acid 100 mg

Oligosaccharide 100 mg

Plum concentrate 2 mg

Taurine 100 mg

Purified water was added to the whole 500 mL

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized container. The resulting solution was sealed and sterilized, &Lt; / RTI &gt; Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Of health food Manufacturing example

The derivative represented by the formula (1) according to the present invention can be manufactured into various types of health food according to the purpose. Hereinafter, the method of manufacturing some health foods containing the derivative represented by the formula 1 according to the present invention as an active ingredient is illustrated, but the present invention is not limited thereto.

<Health food Manufacturing example  1> Manufacture of dairy products

0.01-1 parts by weight of the derivative represented by the formula (1) of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

<Health food Manufacturing example  2> Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh. Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer. The derivative represented by the formula (1) of the present invention was concentrated under reduced pressure in a vacuum concentrator to obtain a dry powder. The above-prepared cereals, seeds and dry powders of the derivatives represented by the formula (1) were prepared in the following proportions.

Grain (34 parts by weight of brown rice, 19 parts by weight of yulmu, 20 parts by weight of barley)

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

(2 parts by weight) represented by the formula (1),

(1.5 parts by weight), and

Rhizome (1.5 parts by weight).

Claims (12)

A pharmaceutical composition comprising a dihydroxyphenyl derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof: atherosclerosis, hypertension, peripheral vascular disease, erectile dysfunction, hemangioma, vascular stenosis, cerebrovascular disease, ischemic disease, myocardial infarction, Seizures, angina pectoris, coronary artery disease, and aortic atherosclerotic plaques.
[Chemical Formula 1]

delete delete delete A pharmaceutical composition comprising a dihydroxyphenyl derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof: atherosclerosis, hypertension, peripheral vascular disease, erectile dysfunction, hemangioma, vascular stenosis, cerebrovascular disease, ischemic disease, myocardial infarction, Seizures, angina pectoris, coronary artery disease, and atherosclerosis atherosclerosis.
[Chemical Formula 1]

delete delete delete A pharmaceutical composition comprising a dihydroxyphenyl derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof: atherosclerosis, hypertension, peripheral vascular disease, erectile dysfunction, hemangioma, vascular stenosis, cerebrovascular disease, ischemic disease, myocardial infarction, Seizures, angina pectoris, coronary artery disease, and aortic atherosclerotic plaques.
[Chemical Formula 1]

delete delete delete

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