KR20050071832A - Novel pyrazoline derivatives, a process for the preparation thereof and composition comprising the same for prevention and treatment of cardiac circuit disease - Google Patents

Abstract

The present invention relates to a novel pyrazoline derivative, a method for preparing the same, and a composition for preventing and treating a cardiovascular disease containing the same.

The pyrazoline derivatives of the present invention not only have excellent antioxidant activity against low density lipid proteins, but also effectively inhibit activity against ACAT.

Therefore, the composition of the present invention can be usefully used for the prevention and treatment of cardiovascular diseases such as hyperlipidemia and arteriosclerosis caused by the synthesis and accumulation of cholesteryl esters.

Description

Novel pyrazoline derivatives, a process for the preparation conjugate and composition comprising the same for prevention and treatment of cardiac circuit disease}

The present invention relates to a novel pyrazoline derivative, a method for preparing the same, and a composition for preventing and treating a cardiovascular disease containing the same.

In recent years, coronary cardiovascular diseases such as atherosclerosis and hypercholesterolemia have become a major cause of death.

Atherosclerosis, a representative disease of the cardiac circulatory system, is one of the major diseases that occur with aging, as the artery thickens and hardens, loses elasticity, and becomes weak. Atherosclerosis is more likely to occur in the cerebral artery or coronary arteries. In the case of cerebral arteriosclerosis, headache, dizziness, and mental disorders are indicated and cause encephalopathy. It is known to become. In addition, this causes high blood pressure, heart disease, cerebral hemorrhage, and diseases caused by arteriosclerosis are emerging as the leading cause of death in modern society, especially among men in their 50s and 60s.

High blood cholesterol levels are likely to cause coronary cardiovascular disease. Therefore, to reduce blood cholesterol levels, it is necessary to reduce the absorption of cholesterol by administering a diet that reduces cholesterol and fat intake or by inhibiting enzymes related to lipid metabolism.

Thus, attempts have been made to reduce plasma LDL levels by inhibiting cholesterol absorption and inhibiting biosynthesis in order to prevent such diseases (Principles in Biochemistry, lipid biosynthesis, 770-817, 3rd Edition, 2000 Worth Publishers). , New York; Steinberg, N. Engl. J. Med., 1989, 320, 915-924).

Recently, the production of LDL oxide in the blood as a factor of atherosclerosis has been the main concern (Circulation, 1995, 91, 2488-2496; Arterioscler. Thromb. Vasc. Biol., 1997, 17, 3338- 3346), in particular, the production of LDL peroxides as the formation of foam cells following the influx of HM-LDL (highly modified LDL), which is produced through overoxidation and structural modification, into macrophage Studies on factors and elimination are being actively conducted (Curr. Atheroscler. Res., 2000, 2, 363-372).

Plaque formation and rupture in the vessel wall are the main factors in the development of myocardial infarction, and atherosclerosis is a chronic inflammatory process for damage of the vessel wall and is suggested as a defense mechanism rather than an injury mechanism (Circ. Res. 2001, 89, 298-304).

Acyl-CoA: cholesterol acyltransferase (ACAT) is an enzyme that generally esterifies cholesterol, and its mechanism of action occurs largely in three parts of the body (intestinal, liver, and vascular wall cells).

First, in the intestine, ACAT converts ingested cholesterol into the form of esters to facilitate its absorption into the intestine. Second, cholesterol absorbed from the outside or biosynthesized in the body is accumulated in a carrier called VLDL (very low density lipoprotein) in the liver, and then supplied to each organ through the blood vessels, whereby ACAT converts cholesterol into cholesteryl ester form. This enables the accumulation of cholesterol in the carrier. Third, in the cells that make up the arterial vessel wall, ACAT converts cholesterol into its ester form to promote the accumulation of cholesterol in the cell, which is a direct cause of atherosclerosis.

In addition, since foam cells contain a large amount of cholesteryl ester derived from cholesterol by ACAT activity, the formation of foam cells derived from macrophages and smooth muscle cells is very important from an experimental and clinical aspect. Since the proliferation of foam cells in the vessel wall is directly related to the increase in ACAT activity, the development of ACAT inhibitors as a potent anti-arterial agent is desirable.

Therefore, the drug that inhibits the activity of ACAT may first reduce the amount of cholesterol that enters the body by inhibiting the absorption of intestinal cholesterol, and secondly, by inhibiting the release of cholesterol into the blood vessels in the liver to reduce blood cholesterol levels. Third, it is expected to prevent arteriosclerosis by preventing cholesterol from accumulating in blood vessel wall cells.

ACAT activity inhibitors reported to date are activity inhibitors for rat hepatic microsomal ACAT or rat hepatic macrophage (J774) ACAT.

Human ACAT includes human ACAT-1 and human ACAT-2. Human ACAT-1 (50 kDa) acts mainly on the liver, adrenal glands, macrophages and kidneys of adults. Human ACAT-2 (46 kDa) is a small intestine. (Rudel, LL et al., Curr. Opin. Lipidol 12, 121-127, 2001). Substances that inhibit human ACAT activity are targets for the prevention and treatment of hypercholesterolemia, cholesterol stones or atherosclerosis through mechanisms that inhibit the absorption of cholesterol from food and the accumulation of cholesteryl esters in the blood vessel walls. (Buhman, KK et al., Nature Medicine 6, 1341-1347, 2000).

Probucol, N, N'-diphenylenediamine (N, N'-diphenylenediamine), and phenolic synthetic antioxidants BHA (butylatedhydroxyanisol) and BHT (butylated hydroxy toluene), which are currently used as a treatment for hyperlipidemia, use LDL cholesterol. It is reduced, weakens the degree of oxidation and reduces the formation of lesions, the antioxidant power is excellent, but many side effects are limited use.

Therefore, there is a growing interest in the concurrent administration of lipid-lowering agents with LDL antioxidants in atherosclerosis patients.

Therefore, the present inventors designed and synthesized a new pyrazoline derivative in order to develop a therapeutic agent superior to existing hyperlipidemia and arteriosclerosis, and the antioxidant activity against low-density lipid protein and the inhibitory activity against the ACAT enzyme in these pyrazoline derivatives It was confirmed that the present invention was completed.

The present invention seeks to provide novel pyrazoline derivatives.

In addition, the present invention is to provide a method for preparing the pyrazoline derivatives.

In addition, the present invention is to provide a composition for the prevention and treatment of cardiovascular diseases containing the pyrazoline derivatives.

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

(R ¹ is hydrogen, C ₁ -C ₄ alkyl, R ² is hydrogen, C ₁ -C ₄ alkyl, hydroxy, halogen, phenyl, nitro group, R ³ is hydrogen, C ₁ -C ₄ Alkyl, methoxy, halogen, phenyl)

The pyrazoline derivatives of the present invention can be used in the form of pharmaceutically acceptable salts and include all salts, hydrates and solvates prepared by conventional methods.

As the pharmaceutically acceptable salt, acid addition salts formed by pharmaceutically acceptable free acid are useful. The inorganic acid and organic acid may be used as the free acid, and hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, lactic acid, maleic acid, umarin acid, gluconic acid, methanesulfonic acid, Glyconic acid, succinic acid, 4-toluenesulfonic acid, trifluoroacetic acid, galluxuronic acid, embonic acid, glutamic acid, or aspartic acid can be used.

Preferred compounds among the pyrazoline derivatives of the present invention are specifically as follows.

1) 3,5-bis- (3,5-di-t-butyl-4-hydroxy) phenyl-4,5-dihydro-1H-pyrazole

2) 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (3,5-di-isopropyl-4-hydroxy) phenyl-4,5-dihydro-1H- Pyrazole

3) 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (3,5-dimethyl-4-hydroxy) phenyl-4,5-dihydro-1H-pyrazole

4) 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (2,3,5-trimethyl-4-hydroxy) phenyl-4,5-dihydro-1H-pyra Sol

5) 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (3,5-di-fluoro-4-hydroxy) phenyl-4,5-dihydro-1H- Pyrazole

6) 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (5-methoxy-4-hydroxy) phenyl-4,5-dihydro-1H-pyrazole

7) 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (3,4-di-hydroxy) phenyl-4,5-dihydro-1H-pyrazole

8) 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (3,5-di-phenyl-4-hydroxy) phenyl-4,5-dihydro-1H-pyra Sol

9) 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (3-nitro-4-hydroxy) phenyl-4,5-dihydro-1H-pyrazole

10) 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (4-hydroxy) phenyl-4,5-dihydro-1H-pyrazole

In addition, the present invention provides a method for preparing a pyrazoline derivative, represented by the following Scheme 1.

(R ¹ is hydrogen, C ₁ -C ₄ alkyl, R ² is hydrogen, C ₁ -C ₄ alkyl, hydroxy, halogen, phenyl, nitro group, R ³ is hydrogen, C ₁ -C ₄ Alkyl, methoxy, halogen, phenyl)

The preparation method of the pyrazoline derivatives of the present invention

1) hydrogen, α-β-unsaturated propenone substituted with a phenyl substituted with a substituent selected from the group consisting of alkyl, hydroxy, methoxy, halogen, phenyl or nitro group of C ₁ to C ₄ at 1,3 position; Reacting hydrazine to obtain a pyrazoline derivative; And

2) purifying the pyrazoline derivative obtained in step 1) by silica gel column chromatography.

Specifically, the first step is hydrogen, phenyl substituted with a substituent selected from the group consisting of alkyl, hydroxy, methoxy, halogen, phenyl or nitro group of C ₁ ~ C ₄ at position 1,3 The prepared α, β-unsaturated propenone is dissolved in ethanol or methanol solvent, and 1 to 5 molar equivalents of hydrazine are added thereto. After the reaction mixture is stirred or refluxed for 24 hours, the reaction is cooled to room temperature. After dilution with excess water and extraction with an organic solvent (ethyl acetate, dichloromethane), the organic layer is washed with saturated aqueous sodium hydrogen carbonate solution and brine, then dried, filtered and concentrated to obtain a pyrazoline derivative.

In the second step, the pyrazoline derivatives obtained in step 1 are purified by silica gel column chromatography to obtain pure pyrazoline derivatives.

Another method for preparing the pyrazoline derivatives of the present invention is to cool the reaction to room temperature, remove the ethanol solvent and add excess primary distilled water. Ethyl acetate was added thereto, and n-hexane was added and filtered. The filtered solid is again dissolved in a methanol solvent, and then distilled water is slowly added, the precipitated solid is filtered and dried in air to obtain a pure pyrazoline compound.

In addition, the present invention provides a composition for the prevention and treatment of cardiovascular diseases containing the pyrazoline derivatives.

The pyrazoline derivatives of the present invention have a low IC ₅₀ value and show excellent antioxidant activity against low-density lipid proteins as measured by the TVA method.

In addition, the pyrazoline derivatives of the present invention effectively inhibit activity against ACAT in human ACAT-1.

Therefore, the composition of the present invention can be usefully used for the prevention and treatment of cardiovascular diseases such as hyperlipidemia and arteriosclerosis caused by the synthesis and accumulation of cholesteryl esters.

The composition of the present invention may contain one or more active ingredients exhibiting the same or similar functions in addition to the pyrazoline derivatives.

The composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as antioxidants, buffers And other conventional additives such as bacteriostatic agents can be added. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

The compositions of the present invention may be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method, and the dosage may be based on the weight, age, sex, health status, The range varies depending on diet, administration time, administration method, excretion rate and severity of disease. The daily dosage is about 0.1 to 100 mg / kg of pyrazoline derivatives, preferably 0.5 to 10 mg / kg, and more preferably administered once to several times a day.

As a result of oral administration of the pyrazoline derivatives of the present invention to mice, 50% lethal dose (LD ₅₀ ) by oral toxicity test was determined to be a safe substance of at least 1 g / kg or more.

The composition of the present invention can be used alone or in combination with methods using surgery, hormonal therapy, drug therapy and biological response modifiers for the prevention and treatment of cardiovascular diseases.

The composition of the present invention can be added to health foods for the purpose of improving cardiovascular disease. When the pyrazoline derivative of the present invention is used as a food additive, the pyrazoline derivative may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, in the preparation of food or beverages, the pyrazoline derivatives of the present invention are added in an amount of 1 to 20% by weight, preferably 5 to 10% by weight based on the raw materials. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. .

There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, and the like and include all of the health foods in the conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonic acid. Carbonating agents and the like used in beverages. In addition, the composition of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, preferred examples and experimental examples are presented to help understand the present invention. However, the following Examples and Experimental Examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited by the Examples.

Example 1  Preparation of 3,5-bis- (3,5-di-t-butyl-4-hydroxy) phenyl-4,5-dihydro-1H-pyrazole

1) 1,3-bis- (3,5-di -t- butyl-4-hydroxyphenyl) propenone [1,3-Bis- (3,5-di- tert -butyl-4-hydroxyphenyl) propenone After dissolving 19 g (0.04 mmol) in 300 ml of ethanol, 11.2 ml (0.12 mmol) of hydrazine (55% in water) was added to a dropping funnel and slowly added thereto. The reaction solution was refluxed for 9 hours, cooled to room temperature, ethanol solvent was removed, and 300 ml of water was added and stirred for 30 minutes. Extracted twice with 200 ml of ethyl acetate, dried over magnesium sulfate (MgSO ₄ ), filtered and concentrated.

The obtained compound was purified by silica gel chromatography to obtain 10.5 g (55%) of pure pyrazoline compound.

2) Alternatively, the reaction was cooled to room temperature, the ethanol solvent was removed and excess primary distilled water was added. 100 ml of ethyl acetate was added thereto and then 500 ml of n-hexane was added and filtered. The filtered solid was dissolved in methanol solvent again, and primary distilled water was added slowly, and the precipitated solid was filtered and dried in air to obtain 8.0 g (42%) of pure pyrazoline derivatives.

The analysis results are as follows.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.45 (s, 18H), 1.46 (s, 18H), 3.0 (dd, J = 10.5, 16.2 Hz, 1H), 3.43 (dd, J = 10.2, 15.9 Hz, 1H), 4.83 (t like, J = 10.5 ㎐, 1H), 5.20 (s, 1H, -OH), 5.37 (s, 1H, -OH), 5.84 (br, 1H, -NH), 7.23 (s, 2H), 7.53 (s, 2H).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 30.23, 30.27, 34.38, 34.41, 42.2, 64.9, 123.1, 123.3, 124.4, 133.1, 136.0, 136.1, 152.6, 153.3, 154.7.

Example 2  : 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (3,5-di-isopropyl-4-hydroxy) phenyl-4,5-dihydro-1H-pyra Preparation of the sol

1- (3,5-di-t-butyl-4-hydroxyphenyl) -3 instead of 1,3-bis- (3,5-di-t-butyl-4-hydroxyphenyl) propenone as starting material The title compound was obtained in the same manner as in Example 1, except that-(3,5-di-isopropyl-4-hydroxyphenyl) propenone was added.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.25 (d, J = 6.6 Hz, 3H), 1.27 (d, J = 6.6 Hz, 3H), 1.46 (s, 18H), 2.99 (dd, J = 10.2, 16.5 ㎐, 1H), 3.14 (m, 1H), 3.44 (dd, J = 10.8, 16.5 ㎐, 1H), 4.83 (t like, J = 10.5 ㎐, 1H), 5.36 (s, 1H, -OH), 7.10 (s, 2 H), 7.52 (s, 2 H).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 22.6, 22.7, 27.4, 30.2, 34.4, 64.6, 121.8, 123.1, 124.4, 134.0, 134.6, 136.0, 149.5, 152.7, 154.7.

Example 3  Preparation of 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (3,5-dimethyl-4-hydroxy) phenyl-4,5-dihydro-1H-pyrazole

1- (3,5-di-t-butyl-4-hydroxyphenyl) -3 instead of 1,3-bis- (3,5-di-t-butyl-4-hydroxyphenyl) propenone as starting material The title compound was obtained in the same manner as in Example 1, except that-(3,5-dimethyl-4-hydroxyphenyl) propenone was added.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.46 (s, 18H), 2.23 (s, 6H), 3.02 (dd, J = 8.7, 16.8 ㎐, 1H), 3.42 (dd, J = 10.8, 16.2 ㎐ 1H ), 4.77 (dd, J = 8.4, 10.2 μs, 1H), 5.29 (s, 1H, -OH), 5.38 (s, 1H, -OH), 5.38 (br, 1H, -NH), 6.98 (s, 2H), 7.53 (s, 2H).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 15.9, 30.2, 34.4, 41.9, 63.5, 123.2, 123.4, 124.3, 126.5, 134.8, 135.9, 151.7, 152.4, 154.7.

Example 4  3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (2,3,5-trimethyl-4-hydroxy) phenyl-4,5-dihydro-1H-pyrazole Manufacture

1- (3,5-di-t-butyl-4-hydroxyphenyl) -3 instead of 1,3-bis- (3,5-di-t-butyl-4-hydroxyphenyl) propenone as starting material The title compound was obtained in the same manner as in Example 1, except that-(2,3,5-trimethyl-4-hydroxyphenyl) propenone was added.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.45 (s, 18H), 2.21 (s, 6H), 2.26 (s, 3H), 2.88 (dd, J = 8.4, 15.6 Hz, 1H), 3.45 (dd, J = 10.2, 15.9 ㎐, 1H), 5.10 (dd, J = 10.2, 10.8 ㎐, 1H), 5.36 (s, 1H), 5.78 (br, 1H, -NH), 7.13 (s, 1H), 7.51 ( s, 2H).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 12.2, 15.3, 15.9, 30.2, 34.4, 40.9, 60.6, 120.3, 122.5, 123.1, 124.4, 125.0, 132.3, 132.7, 135.9, 151.1, 152.0, 154.7.

Example 5  : 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (3,5-di-fluoro-4-hydroxy) phenyl-4,5-dihydro-1H-pyra Preparation of the sol

1- (3,5-di-t-butyl-4-hydroxyphenyl) -3 instead of 1,3-bis- (3,5-di-t-butyl-4-hydroxyphenyl) propenone as starting material The title compound was obtained in the same manner as in Example 1, except that-(3,5-di-fluoro-4-hydroxyphenyl) propenone was added.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.45 (s, 18H), 2.98 (dd, J = 8.7, 16.5 μs, 1H), 3.48 (dd, J = 10.2, 16.2 μs, 1H), 4.80 (t like , J = 9.6 kPa, 1H), 5.42 (s, 1H, -OH), 6.93 (s, 1H), 6.96 (s, 1H), 7.51 (s, 2H).

Example 6 Preparation of 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (5-methoxy-4-hydroxy) phenyl-4,5-dihydro-1H-pyrazole

1- (3,5-di-t-butyl-4-hydroxyphenyl) -3 instead of 1,3-bis- (3,5-di-t-butyl-4-hydroxyphenyl) propenone as starting material The title compound was obtained in the same manner as in Example 1, except that-(5-methoxy-4-hydroxyphenyl) propenone was added.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.46 (s, 18H), 3.18 (dd, J = 8.1, 15.9 Hz, 1H), 3.43 (dd, J = 11.1, 16.5 Hz 1H), 3.87 (s, 3H ), 4.79 (dd, J = 10.2, 10.5 Hz, 1H), 5.37 (br, 1H, -OH), 6.78-6.95 (m, 3H), 7.51 (s, 2H).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 30.2, 34.4, 41.7, 56.0, 63.5, 110.7, 112.5, 117.7, 123.2, 124.2, 136.0, 136.6, 145.9, 146.1, 152.3, 154.8.

Example 7  Preparation of 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (3,4-dihydroxy) phenyl-4,5-dihydro-1H-pyrazole

1- (3,5-di-t-butyl-4-hydroxyphenyl) -3 instead of 1,3-bis- (3,5-di-t-butyl-4-hydroxyphenyl) propenone as starting material The title compound was obtained in the same manner as in Example 1, except that-(3,4-dihydroxyphenyl) propenone was added.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.39 (s, 18H), 2.74 (dd, J = 9.9, 15.9 Hz, 1H), 3.31 (dd, J = 10.5, 16.2 Hz, 1H), 4.59 (t like , J = 10.2 ㎐, 1H), 6.59 (dd, J = 1.8, 8.1 ㎐, 1H), 6.66 (d, J = 8.1 ㎐, 1H), 6.76 (d, J = 1.8 ㎐, 1H), 7.08 (br , 1H, -NH), 7.11 (s, 1H, -OH), 7.38 (s, 2H), 7.39 (s, 2H), 8.74 (s, 1H, -OH), 8.85 (s, 1H, -OH) .

¹³ C NMR (75 MHz, CDCl ₃ ) δ 30.2, 34.5, 41.0, 63.2, 113.7, 115.3, 117.4, 122.0, 124.9, 134.3, 139.0, 144.3, 145.2, 149.5, 154.3.

Example 8  Of 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (3,5-diphenyl-4-hydroxy) phenyl-4,5-dihydro-1H-pyrazole Produce

1- (3,5-di-t-butyl-4-hydroxyphenyl) -3 instead of 1,3-bis- (3,5-di-t-butyl-4-hydroxyphenyl) propenone as starting material The title compound was obtained in the same manner as in Example 1, except that-(3,5-diphenyl-4-hydroxyphenyl) propenone was added.

¹ H NMR (300 MHz, CDCl ₃ ) δ 4.46 (s, 18H), 3.11 (dd, J = 9.0, 15.9 Hz, 1H), 3.50 (dd, J = 10.2, 16.5 Hz, 1H), 4.93 (t like , J = 9.9 kPa, 1H), 5.37 (s, 1H, -OH), 5.40 (br, 1H, -NH), 7.32-7.57 (m, 14H).

Example 9 Preparation of 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (3-nitro-4-hydroxy) phenyl-4,5-dihydro-1H-pyrazole

1- (3,5-di-t-butyl-4-hydroxyphenyl) -3 instead of 1,3-bis- (3,5-di-t-butyl-4-hydroxyphenyl) propenone as starting material The title compound was obtained in the same manner as in Example 1, except that-(3-nitro-4-hydroxyphenyl) propenone was added.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.46 (s, 18H), 2.96 (dd, J = 9.9, 15.9 Hz, 1H), 3.52 (dd, J = 10.5, 15.9 Hz, 1H), 4.91 (t like , J = 9.6 μs, 1H), 5.40 (s, 1H, -OH), 7.16 (d, J = 8.7 μs, 1H), 7.51 (s, 2H), 7.69 (d, J = 8.4 μs, 2H), 8.13 (d, J = 1.2 Hz, 1H), 10.56 (br, 1H, -OH).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 30.2, 34.4, 41.9, 62.9, 120.6, 122.9, 123.2, 123.7, 135.4, 135.9, 136.1, 152.3, 154.5, 155.0.

Example 10 Preparation of 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (4-hydroxy) phenyl-4,5-dihydro-1H-pyrazole

1- (3,5-di-t-butyl-4-hydroxyphenyl) -3 instead of 1,3-bis- (3,5-di-t-butyl-4-hydroxyphenyl) propenone as starting material The title compound was obtained in the same manner as in Example 1, except that-(4-hydroxyphenyl) propenone was added.

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.39 (s, 18H), 3.04 (dd, J = 7.8, 15.9 Hz, 1H), 3.45 (dd, J = 10.2, 16.5 Hz, 1H), 4.82 (dd, J = 10.5, 10.2 kPa, 1H), 5.39 (br, 1H, -OH), 6.79 (d, J = 8.4 kPa, 2H), 7.21 (d, J = 8.7 kPa, 2H), 7.52 (s, 2H) .

¹³ C NMR (75 MHz, CDCl ₃ ) δ 30.2, 34.4, 41.7, 63.4, 115.7, 123.3, 123.9, 127.6, 135.0, 136.0, 152.9, 154.9, 155.5.

Experimental Example 1  Antioxidant Activity of Pyrazoline Derivatives by TBARS Method

In order to determine the antioxidant activity of the pyrazoline derivatives of the present invention on the low density lipid protein, the following experiment was performed.

Cu ²⁺ is known to induce oxidation of low density lipid proteins (Cu ²⁺ mediated LDL-oxidation). Therefore, in the present invention, dialdehyde (dialdehyde), which is an oxidation product of the unsaturated fatty acid produced at this time, was measured by TBA (thiobarbituric acid) method to investigate the antioxidant activity of pyrazoline derivatives (Packer, L. Ed. (1994) Methods in Enzymology Vol. 234, Oxygen radicals in biological systems Part D. Academic press, San Diego).

300 ml of human plasma was centrifuged at 100,000 xg for 24 hours using an ultracentrifuge to remove the high-density lipoprotein (VLDL) / chylomicron layer suspended in the upper layer and the specific solution to 1.063 g / ml. After centrifugation at 100,000 xg for 24 hours, 25 ml (1.5-2.5 mg protein / ml) of low density lipid protein suspended in the upper layer was separated again.

20 μl (protein concentration, 50-100 μg / ml) of the separated low-density lipid protein was mixed with 210 μl of 10 mM phosphate-buffered saline (PBS) and pyrazoline prepared in Examples 1 to 10. 10 μl of each of the derivatives was added.

Pyrazoline derivatives were dissolved in DMSO (dimethylsulfoxide) and diluted to various concentrations before use in experiments. As the negative control, only the solvent was added, and as the positive control, the probucol was added.

10 μl of 0.25 mM CuSO ₄ was added to the solution for 4 hours at 37 ° C., and 1 ml of 20% trichloroacetic acid (TCA) solution was added to stop the reaction. 1 ml of a 0.67% TBA solution dissolved in 0.05N NaOH solution was added thereto, stirred for 10 seconds, and heated at 95 ° C. for 5 minutes to cause a color reaction. The solution was cooled with ice water. The solution was centrifuged at 3000 rpm for 5 minutes to separate the supernatant, and the absorbance at 540 nm was measured by UV-vis spectrophotometry to determine the amount of malondialdehyde (MDA) produced by the color reaction. .

Meanwhile, using a stock solution of tetramethoxypropane malonaldehyde bis (dimethylacetal), 250 μl of PBS standard solution containing 0-10 nmol malondialdehyde was prepared.

The standard solution was developed in the same manner as described above, the absorbance at 540 nm was measured, and the standard curve of malondialdehyde was obtained.

The amount of malondialdehyde in this experiment with pyrazoline derivatives was quantified using this standard curve.

The results are shown in Table 1.

Antioxidant Activity of Pyrazoline Derivatives on Low Density Lipid Proteins compound IC ₅₀ (μM) R ¹ = hydrogen, R ² = R ³ = t-butyl 0.1 R ¹ = hydrogen, R ² = R ³ = isopropyl 0.7 R ¹ = hydrogen, R ² = R ³ = methyl 0.2 R ¹ = R ² = R ³ = methyl 0.6 R ¹ = hydrogen, R ² = R ³ = fluoro 0.2 R ¹ = R ² = hydrogen, R ³ = methoxy 0.1 R ¹ = R ³ = hydrogen, R ² = hydroxy 0.3 R ¹ = hydrogen, R ² = R ³ = phenyl 0.6 R ¹ = R ³ = hydrogen, R ² = nitro 0.3 R ¹ = R ² = R ³ = Hydrogen 0.2 Positive control group Probucol 0.6

As shown in Table 1, the IC ₅₀ value of the pyrazoline derivatives of the present invention is low, it can be seen that the antioxidant activity against low-density lipid protein.

Therefore, the pyrazoline derivatives according to the present invention can be usefully used for the prevention or treatment of cardiovascular diseases such as hyperlipidemia and arteriosclerosis, which are known to be caused by oxidation of low-density lipid proteins.

Experimental Example 2  : Effect on ACAT Activity of Pyrazoline Derivatives of the Present Invention

In order to determine the effect on the ACAT activity of the pyrazoline derivatives of the present invention, the following experiment was performed.

1. Preparation of ACAT Enzyme Source

1) Rat liver ACAT

Liver of white rat (male Sprague-Dawley 150-200g) was extracted as ACAT enzyme source. 1 g of liver was homogenized in 5 ml of homogenization solution (0.1 M KH ₂ PO ₄ , pH 7.4, 0.1 mM EDTA and 10 mM β-mercaptoethanol). The homogenate was centrifuged at 3,000 × g for 10 minutes at 4 ° C., and the obtained supernatant was centrifuged at 15,000 × g for 15 minutes at 4 ° C. to obtain a supernatant. The supernatant was placed in an ultracentrifuge tube (Beckman) and centrifuged at 100,000 xg for 1 hour at 4 ° C. to obtain microsomal pellets, which were then suspended in 3 ml of homogenization solution at 100,000 ° C. at 4 ° C. for 1 hour. Centrifuged. The obtained pellet was suspended in 1 ml of homogenization solution. The protein concentration in the obtained suspension was measured by the method of Lowry et al., And the protein concentration was adjusted to 4-8 mg / ml. The suspension obtained was stored in a deep freezer (Forma Scientific Inc.).

2) Human ACAT-1 and Human ACAT-2

Human ACAT-1 and ACAT-2 proteins were obtained using baculovirus expression systems to investigate the effects on human ACAT-1 and ACAT-2 activity.

The cDNAs of hACAT-1 and hACAT-2 obtained through human cDNA library screening were inserted into baculovirus delivery vectors and introduced into insect cells, sf9 cells, to prepare viruses. Subsequently, the recombinant viruses of hACAT-1 and hACAT-2 were isolated by plaque purification, and the titer of the viral stock was increased through three amplification processes. Recombinant virus was infected to Hi5 insect cells having good protein expression efficiency to 1 (Mutiplicity of Infection), and then cultured for one day at 27 ° C.

The cultured hACAT-1 and hACAT-2 were collected by centrifugation at 500xg for 15 minutes to separate microsomal fractions from overexpressed Hi5 cells, and then quenched by rapid freeze thawing in hypotonic buffer. After breaking the ultracentrifugation for 100,000 hours at 100,000xg.

The obtained microsome fractions were suspended in storage buffer so that the protein concentration was 8 mg / ml and stored in a cold freezer until use.

2. ACAT activity measurement

6.67 μl of a cholesterol solution dissolved in acetone at a concentration of 1 mg / ml was mixed with 6 μl of 10% triton WR-1339 (Sigma Co.) in acetone, and acetone was removed by evaporation using nitrogen gas. It was. Distilled water was added to the obtained mixture to adjust the concentration of cholesterol to 30 mg / ml.

10 μl of aqueous solution of cholesterol, 10 μl of 1M KH ₂ PO ₄ (pH 7.4), 5 μl of 0.6 mM bovine serum albumin (BSA), 10 μl of microsome solution obtained in 1, 10 μl A sample (pyrazoline derivative) and 45 μl of distilled water were added (90 μl total). The mixture was pre-reacted for 30 minutes in a 37 ° C. water bath.

10 μl of (1- ¹⁴ C) oleyl-CoA solution (0.05 μCi, final concentration: 10 μM) was added to the pre-reacted mixture and the resulting mixture was reacted for 30 minutes in a 37 ° C. water bath. To this was added 500 μl isopropanol: heptane mixture (4: 1 (v / v)), 300 μl heptane and 200 μl 0.1 M KH ₂ PO ₄ (pH 7.4) and the mixture was vigorously vortexed. After mixing, the mixture was left at room temperature for 2 minutes.

The resulting 200 µl supernatant was placed in a scintillation bottle, and 4 ml of scintillation liquid (Lumac) was added. The radiation dose of this mixture was measured with a 1450 Microbeta liquid scintillation counter, Wallacoy, Finland.

ACAT activity was calculated from cholesteryl oleate picolol (picomol / min / mg protein) synthesized per mg of protein for 1 minute by calculating the radiation dose per hour from the measured radiation dose.

The results are shown in Table 2.

Inhibitory Activity of Pyrazoline Derivatives on ACAT compound IC ₅₀ (μM) Rat liver ACAT hACAT-1 hACAT-2 R ¹ = hydrogen, R ² = R ³ = t-butyl 87.1 12.8 14.7 R ¹ = hydrogen, R ² = R ³ = isopropyl 3.7 20.5 11.1 R ¹ = hydrogen, R ² = R ³ = methyl 23.9 12.8 19.9 R ¹ = R ² = R ³ = methyl 53.2 27.0 31.5 R ¹ = hydrogen, R ² = R ³ = fluoro 121.4 35.6 164.3 R ¹ = R ² = hydrogen, R ³ = methoxy 92.5 32.1 64.5 R ¹ = R ³ = hydrogen, R ² = hydroxy 100.7 112.5 170.2 R ¹ = hydrogen, R ² = R ³ = phenyl 84.0 23.3 127.4 R ¹ = R ³ = hydrogen, R ² = nitro 169.9 65.4 125.8 R ¹ = R ² = R ³ = Hydrogen 66.2 32.4 68.5

As shown in Table 2, it can be seen that the pyrazoline derivatives of the present invention have an excellent inhibitory activity against acyl-CoA: cholesterol transferase in human ACAT-1.

Therefore, the pyrazoline derivatives according to the present invention can be usefully used for the prevention or treatment of cardiovascular diseases such as hyperlipidemia and arteriosclerosis caused by the synthesis and accumulation of cholesteryl esters.

Experimental Example 3  : Acute Toxicity in Oral Administration in Mice

In order to determine the acute toxicity of the pyrazoline derivatives according to the present invention, the following experiment was performed.

12 females and 12 males (3 males and 3 females each) were used as specific pathogens free ICR mice at 4 weeks of age in an animal room of temperature 22 ± 3 ° C, humidity 55 ± 10%, and illumination 12L / 12D. Breeding in. Mice were allowed to acclimate for about a week before being used in the experiment. Feed for experimental animals (JeilJedang Co., Ltd., mice and rats) and negative water were supplied after sterilization and free ingestion.

3,5-bis- (3,5-di-t-butyl-4-hydroxy) phenyl-4,5-dihydro-1H-pyrazole prepared in Example 1 was prepared using 0.5% Tween 80 as a solvent. After the preparation at 50mg / ㎖ concentration, 0.04ml (100mg / kg), 0.2ml (500mg / kg), 0.4ml (1000mg / kg) was administered orally per 20g body weight of the mouse. Samples were administered orally once and observed for side effects or lethality for 7 days after administration. That is, on the day of administration, changes in general symptoms and the presence or absence of dead animals were observed at least once in the morning, at least once every afternoon from 1 hour, 4 hours, 8 hours, 12 hours, and the next day after administration.

In addition, on the 7th day of administration, animals were killed and dissected, and the internal organs were visually examined. Changes in body weight were measured at daily intervals from the day of administration to observe the weight loss phenomenon of animals caused by pyrazoline derivatives.

As a result, all mice treated with test substance showed no clinical symptoms and no dead mice, and no toxicity change was observed in weight change, blood test, blood biochemistry test and autopsy findings.

Thus, the 3,5-bis- (3,5-di-t-butyl-4-hydroxy) phenyl-4,5-dihydro-1H-pyrazole according to the present invention is up to 1,000 mg / kg in all mice. There was no change in toxicity, and oral administration minimum dose (LD ₅₀ ) was judged to be safe substance with more than 1,000mg / kg.

Examples of preparations for the compositions of the present invention are illustrated below.

Formulation Example 1  : Preparation of Pharmaceutical Formulations

1. Preparation of powder

2 g of pyrazoline derivatives

1g lactose

The above ingredients were mixed and filled in airtight cloth to prepare a powder.

2. Preparation of Tablets

Pyrazoline Derivatives 100mg

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

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

3. Preparation of Capsule

Pyrazoline Derivatives 100mg

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

4. Preparation of Injection Solution

10 μg / ml pyrazoline derivative

Dilute hydrochloric acid BP until pH 3.5

Injectable sodium chloride BP up to 1 ml

The pyrazoline derivative was dissolved in an appropriate volume of sodium chloride BP for injection, the pH of the resulting solution was adjusted to pH 3.5 with dilute hydrochloric acid BP, and the volume was adjusted with sodium chloride BP for injection and thoroughly mixed. The solution was filled into a 5 ml Type I ampoule of clear glass, dissolved in glass and enclosed under an upper grid of air, sterilized by autoclaving at 120 ° C. for at least 15 minutes to prepare an injection solution.

Formulation Example 2  : Manufacture of food

Foods containing pyrazoline derivatives were prepared as follows.

1. Preparation of Cooking Seasonings

Pyrazoline derivatives 0.2 to 10% by weight to prepare a cooking seasoning for health promotion.

2. Preparation of Tomato Ketchup and Sauce

Health promotion tomato ketchup or sauce was prepared by adding 0.2-1.0 wt% pyrazoline derivative to tomato ketchup or sauce.

3. Manufacturing of Flour Foods

0.1 to 5.0% by weight of pyrazoline derivatives were added to the flour, and bread, cake, cookies, crackers and noodles were prepared using the mixture to prepare foods for health promotion.

4. Preparation of soups and gravy

0.1 to 1.0% by weight of pyrazoline derivatives were added to soups and broths to prepare health products, noodle soups and broths.

5. Preparation of Ground Beef

10% by weight of pyrazoline derivative was added to ground beef to prepare ground beef for health promotion.

6. Manufacture of Dairy Products

0.1 to 1.0% by weight of pyrazoline derivatives were added to milk and various milk products were prepared, such as butter and ice cream.

7. Manufacture of wire

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh using a grinder.

Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.

The pyrazoline derivatives were decompressed and concentrated in a vacuum concentrator, and the dried product obtained by drying with a spray and a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

The dry powders of the grains, seeds and benzylidine thiazolidinedione derivative compounds prepared above were prepared by blending in the following ratios.

Cereals (30% by weight brown rice, 15% by weight radish, 20% by weight barley),

Seeds (7% by weight perilla, 8% by weight black beans, 7% by weight black sesame),

Dry powder of pyrazoline derivative (1% by weight),

Ganoderma lucidum (0.5% by weight),

Foxglove (0.5 wt%)

Formulation Example 3  : Preparation of Beverages

1. Preparation of Carbonated Drinks

5-10% of sugar, 0.05-0.3% citric acid, 0.005-0.02% caramel, 0.1-1% of vitamin C are mixed, and 79-94% purified water is mixed to make syrup, and the syrup is 85-98 Sterilizing at 20 ℃ for 180 seconds, mixed with cooling water in a ratio of 1: 4, and then injected with carbon dioxide gas 0.5 ~ 0.82% to prepare a carbonated beverage containing pyrazoline derivatives.

2. Manufacture of health drinks

Instant sterilization by homogeneous mixing of subsidiary ingredients such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%), water (75%) and pyrazoline derivatives , Packed in a small packaging container such as plastic bottles to prepare a healthy beverage.

3. Preparation of Vegetable Juice

0.5 g of pyrazoline derivatives were added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.

4. Preparation of Fruit Juice

0.1 g of pyrazoline derivative was added to 1,000 ml of apple or grape juice to prepare fruit juice for health promotion.

The pyrazoline derivatives of the present invention not only have excellent antioxidant activity against low density lipid proteins, but also effectively inhibit activity against ACAT.

Therefore, the composition of the present invention can be usefully used for the prevention and treatment of cardiovascular diseases such as hyperlipidemia and arteriosclerosis caused by the synthesis and accumulation of cholesteryl esters.

Claims (8)

A pyrazoline derivative having antioxidant activity and ACAT inhibitory activity on a low density lipid protein represented by the following formula (1). <Formula 1> (R ¹ is hydrogen, C ₁ -C ₄ alkyl, R ² is hydrogen, C ₁ -C ₄ alkyl, hydroxy, halogen, phenyl, nitro group, R ³ is hydrogen, C ₁ -C ₄ Alkyl, methoxy, halogen, phenyl) The pyrazoline derivative according to claim 1, wherein R ¹ is hydrogen and R ² , R ³ are methyl, isopropyl, t-butyl. The pyrazoline derivative of claim 1 is 1) 3,5-bis- (3,5-di-t-butyl-4-hydroxy) phenyl-4,5-dihydro-1H-pyrazole 2) 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (3,5-di-isopropyl-4-hydroxy) phenyl-4,5-dihydro-1H- Pyrazole 3) 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (3,5-dimethyl-4-hydroxy) phenyl-4,5-dihydro-1H-pyrazole 4) 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (2,3,5-trimethyl-4-hydroxy) phenyl-4,5-dihydro-1H-pyra Sol 5) 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (3,5-di-fluoro-4-hydroxy) phenyl-4,5-dihydro-1H- Pyrazole 6) 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (5-methoxy-4-hydroxy) phenyl-4,5-dihydro-1H-pyrazole 7) 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (3,4-di-hydroxy) phenyl-4,5-dihydro-1H-pyrazole 8) 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (3,5-di-phenyl-4-hydroxy) phenyl-4,5-dihydro-1H-pyra Sol 9) 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (3-nitro-4-hydroxy) phenyl-4,5-dihydro-1H-pyrazole 10) pyrazoline derivatives characterized in that 3- (3,5-di-t-butyl-4-hydroxy) phenyl-5- (4-hydroxy) phenyl-4,5-dihydro-1H-pyrazole . 1) hydrogen, α-β-unsaturated propenone substituted with a phenyl substituted with a substituent selected from the group consisting of alkyl, hydroxy, methoxy, halogen, phenyl or nitro group of C ₁ to C ₄ at 1,3 position; Reacting hydrazine to obtain a pyrazoline derivative; And 2) purifying the pyrazoline derivative obtained in step 1) by silica gel column chromatography. A method for producing the pyrazoline derivative of claim 1 represented by Scheme 1 below. <Scheme 1> (R ¹ is hydrogen, C ₁ -C ₄ alkyl, R ² is hydrogen, C ₁ -C ₄ alkyl, hydroxy, halogen, phenyl, nitro group, R ³ is hydrogen, C ₁ -C ₄ Alkyl, methoxy, halogen, phenyl) A pharmaceutical composition for preventing and treating cardiovascular diseases containing the pyrazoline derivative of claim 1. The pharmaceutical composition for preventing and treating cardiovascular diseases of claim 5, wherein the cardiovascular disease is hyperlipidemia or arteriosclerosis. Health food composition for the prevention and treatment of cardiovascular diseases containing the pyrazoline derivative of claim 1. The method of claim 7, wherein the cardiovascular disease is a hyperlipidemia or atherosclerosis health food composition for the prevention and treatment of cardiovascular disease, characterized in that.