KR101100624B1 - Farnesylacetone derivatives possessing depressive effects and pharmaceutical composite containing the same for antihypertensive drug - Google Patents

Abstract

The present invention relates to a farnesyl acetone derivative having a blood pressure-lowering effect and a pharmaceutical composition for an antihypertensive preparation containing the same, wherein the compound of the present invention selectively inhibits an increase in blood pressure by selectively inhibiting L-type calcium channels. By minimizing side effects, it can be used as a pharmaceutical composition very useful for treating hypertension.

Farnesyl Acetone Derivatives

Description

Farnesyllacetone derivatives possessing depressive effects and pharmaceutical composite containing the same for antihypertensive drug

The present invention relates to farnesyl acetone derivatives having an antihypertensive effect and a pharmaceutical composition for an antihypertensive preparation containing the same.

Blood pressure refers to the force that blood exerts on the vessel wall. In general, when blood pressure is expressed, the systolic blood pressure is applied to the vessel wall when blood is ejected during contraction of the ventricles in the cardiac cycle, and when it receives blood at the time of relaxation of the ventricles. Loss is divided by diastolic blood pressure.

Hypertension disorders often refer to patients with systolic blood pressure greater than 140 mg or diastolic blood pressure maintained above 90 mmHg in adults 18 years of age and older. The World Health and Health Organization (WHO) has a maximum blood pressure of 160 mmHg or more and a minimum blood pressure of 95 mmHg or more It is defined as hypertension, and the type of hypertension is classified into essential hypertension due to essential hypertension due to unclear causes, and more than 80% of these are known to belong to essential hypertension. Secondary hypertensive pressure includes kidney disease such as acute nephritis, chronic nephritis, pyelonephritis, vascular diseases such as aortic stenosis, peripheral vascular obstruction, endocrine diseases, neurological diseases such as encephalitis and brain tumors, and extreme mental anxiety and tension. It is known as a causative disease (Korean Society of Food and Nutrition, Food and Nutrition Dictionary, p.82, 1998).

In addition, risk factors for hypertension are known environmental and psychological factors such as family history, drinking, smoking, aging, lack of exercise, obesity, eating habits, and stress.

As reported recently, hypertension is a major threat to the life span of modern humans, as it frequently occurs as a major adult disease along with cardiovascular disease, diabetes, and cancer. Therefore, many researchers are actively researching new compounds or natural products, which are useful for treating hypertension.

On the other hand, the present inventors 311 and 312, the farnesyl acetone derivatives of novel compounds derived from prematurity hatban disclosed in the Republic of Korea Patent Publication No. 10-2009-0049171 in the Republic of Korea, as well as inhibiting the calcium channel as a new pharmacological use in addition to the prevention and treatment of cerebrovascular improvement By newly demonstrating that it has the effect of treating hypertension, the present invention has been completed.

In addition, the compounds disclosed as active ingredients in Publication No. 10-2009-0049171 have been reported extracted by Y Shizuri et al. (Phytochemistry, 1982, 21, 1808), anticholinesterase effect (Lee et al, Phytotherapy Res. 2007, 21 , 423; Arch. Pharm. Res. 2003, 26, 796), published in this paper, the use of the compounds in the treatment of diseases related to calcium channel inhibition is not known at all.

Accordingly, an object of the present invention is to provide a farnesyl acetone derivative having an effect of lowering blood pressure and a pharmaceutical composition for an antihypertensive preparation containing the same.

In order to achieve the above object, the present invention provides a farnesyl acetone derivative represented by the following formula (7).

[Formula 7]

In addition, to achieve another object, the present invention provides a pharmaceutical composition for an antihypertensive preparation comprising the farnesyl acetone derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, in order to achieve another object, the present invention provides a pharmaceutical composition for an antihypertensive preparation comprising a farnesyl acetone derivative represented by the following formula (311) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 311]

Furthermore, the present invention provides a pharmaceutical composition for an antihypertensive preparation comprising a farnesyl acetone derivative represented by the following Chemical Formula 312 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 312]

The pharmaceutical composition for an antihypertensive preparation according to the present invention is according to known methods such as Remington's Pharmaceutical Sciences Handbook, Mack Pub, Co., NY, USA, etc. Acceptable salts thereof may be mixed with pharmaceutically acceptable carriers to prepare into formulations such as tablets, capsules, solutions, syrups, injections and the like.

In addition, the formulation may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like to control the rapid, sustained or delayed release of the active ingredient after administration to a mammal.

In addition, pharmaceutically acceptable salts may be organic salts and inorganic salts, and preferably salts are water-soluble and non-toxic. Specifically, inorganic salts such as salts of alkali metals (such as potassium and sodium), salts of alkaline earth metals (such as calcium and magnesium) and ammonium salts, and tetramethylammonium, triethylamine, methylamine, dimethylamine, cyclopentylamine and benzyl Organic amine salts such as amine, phenethylamine, piperidine, monoethanolamine, diethanolamine, tris (hydroxymethyl) methylamine, lysine, arginine, N-methyl-D-glucamine, and the like. The invention is not limited to this.

On the other hand, the effective amount of the compound according to the present invention and a pharmaceutically acceptable salt thereof may be appropriately increased or decreased by those skilled in the art depending on the type of disease, route of administration, dosage form, purpose of use, sex, age, and the severity of patient symptoms. Can be. However, as a result of comparing effective amounts of other therapeutic agents from in vivo experiments, it is preferable to take 40 to 80 once a day on an adult basis.

Compounds 311, 312, and 7 of the present invention are obtained by separating and purifying from pretzel caps, as described in Korean Patent Publication No. 10-2009-0049171, or through organic synthesis, Prepare the salts that are allowed.

At this time, preferred methods of preparing the compounds of Formulas 311 and 7 and pharmaceutically acceptable salts thereof through organic synthesis are as follows.

1. 11-hydroxy-6,10-dimethylundeca-5,9-dien- 2 -one [ Formula 2 ]

   (11-hydroxy-6,10-dimethylundeca-5,9-dien-2-one)

2. 11-Bromo-6,10-dimethylundeca-5,9-dien-2-one [ Formula 3 ]

   (11-bromo-6,10-dimethylundeca-5,9-dien-2-one)

3. 3,7-Dimethyl-11-oxododeca-3,7-dienenitrile [ Formula 4 ]

   (3,7-dimethyl-11-oxododeca-3,7-dienenitrile)

4. 3,7-dimethyl-10- (2-methyl-1,3-dioxolan-2-yl) deca-3,7-dienenitrile [ Formula 5 ] (3,7-dimethyl-10- ( 2-methyl-1,3-dioxolan-2-yl) deca-3,7-dienenitrile)

5. 3,7-Dimethyl-10- (2-methyl-1,3-daxoran-2-yl) deca-2,7-dienenitrile [ Formula 6 ]

   (3,7-dimethyl-10- (2-methyl-1,3-dioxolan-2-yl) deca-2,7-dienenitrile)

6. 3,7-dimethyl-10- (2-methyl-1,3-daxoran-2-yl) deca-2,7-dieneal [ Formula 7 ]

   (3,7-dimethyl-10- (2-methyl-1,3-dioxolan-2-yl) deca-2,7-dienal)

7. 2,6,10-trimethyl-13- (2-methyl-1,3-daxoran-2-yl) trideca-5,10-dien-4-ol [ Formula 8 ] (2,6,10 -trimethyl-13- (2-methyl-1,3-dioxolan-2-yl) trideca-5,10-dien-4-ol)

8. 2,6,10-trimethyl-13- (2-methyl-1,3-daxoran-2-yl) trideca-5,10-dien-4-one [ Formula 9 ] (2,6,10 -trimethyl-13- (2-methyl-1,3-dioxolan-2-yl) trideca-5,10-dien-4-one)

9. 6,10,14-trimethylpentadeca-5,10-diene-2,12-dione [ Formula 311 ]

   (6,10,14-trimethylpentadeca-5,10-diene-2,12-dione)

The manufacturing method of Chemical Formulas 311 and 7 according to the present invention described above is capable of various substitutions, modifications, and changes within the scope of the technical spirit of the present invention to those skilled in the art to which the present invention pertains. Therefore, the present invention is not limited to the above method.

As described above, the present invention can provide a novel hypertension treatment similar to the existing verapamil or amlodipine as a selective L-type calcium channel blocker.

Hereinafter, the present invention will be described in more detail with reference to specific examples.

However, the present invention is not limited to the following examples, and it will be apparent to those skilled in the art that various changes or modifications can be made within the spirit and scope of the present invention.

[ Example ]

1.6,10,14- Trimethyl - Pentadeca -5,10 (E)- Dien -2,12- Dion [Formula 311] and 6,10,14-trimethyl- Pentadeca -5,10 (Z)- Dien -2,12- Dion [Formula 312]

1 kg of the collected undried pretzel hatch outpost was pulverized, placed in an extraction bag, immersed in 3 L of an 80% aqueous methanol solution, extracted at room temperature for 24 hours, and filtered through a filter paper. The extract is concentrated using a concentrator under reduced pressure until methanol is completely evaporated and extracted three times with 300 ml of ethyl acetate. Water in the ethyl acetate solution was removed with anhydrous magnesium sulfate, and then concentrated with a reduced pressure evaporator to obtain 13.5 g of a dark brown powder. 1 g of the obtained powder was taken and dissolved in a small amount of ethyl acetate, and then separated according to the separation conditions disclosed in Korean Patent Application Publication No. 10-2009-0049171 filed by the present inventors, and separated into single substances of Chemical Formulas 311 and 312.

These were named Farnesyl Acetone 311 and Farnesyl Acetone 312, respectively.

2. 3,7-dimethyl-10- (2- methyl -1,3- Daoxoran -2 days) Deca -2,7- Dienal [Formula 7]

end. 3,7-dimethyl-11- Oxododeca -3,7- Dienenitrile

Known 11-bromo-6,10-dimethylundec-5,9-dien-2-one (11-bromo-) in 120 ml of solvent in (DMF: CH ₃ CN = 1: 1) ratio 6,10-dimethylundeca-5,9-dien-2-one) was dissolved and stirred at room temperature. 4.3 g (87.84 mmol) of sodium cyanide was added to the reaction mixture, which was stirred for 4 hours at room temperature. When it was confirmed that the reaction was completed, 100 ml of water was added, extracted with CH ₂ Cl ₂ (100 ml × 3), and MgSO ₄ Water was removed and filtered. The solution was distilled off under reduced pressure, and the remaining filtrate was purified by flash column chromatography to give 6 g (93%) of light yellow oil as compound No. 4 [3,7-dimethyl-11-oxododeca-3,7-dienenitrile]. Was obtained (Chemical Formula: C ₁₄ H ₂₁ NO, Molecular Weight: 219.32).

¹ H-NMR (CDCl ₃ , 300 MHz) δ 5.44 (1H, m), 5.08 (1H, m), 3.02 (2H, s), 2.47 (2H, m), 2.28 (2H, m), 2.14 (3H, s), 2.12 (4H, m), 2.03 (2H, m), 1.72 (3H, s), 1.61 (3H, s) ppm

^l3 C-NMR (CDCl ₃ , 75 MHz) δ 209, 136, 129, 124, 123, 118, 44, 39, 31, 30, 27, 26, 22, 16 ppm

I. 3,7-dimethyl-10- (2- methyl -1,3- Dioxoran -2 days) Deca -3,7- Dienenitrile

Dissolve 4 in 100 ml of Benzene and stir at 100. 12.21 ml (218.88 mmol) of Ethylene glycol and 3.12 g (16.42 mmol) of p-Toluenesulfonic acid monohydrate were added to the reaction, followed by stirring for 5 hours at 100 ° C. When it is confirmed that the reaction is completed, 100 ml of water is added, extracted with CH ₂ Cl ₂ (100 ml × 3), and then water is removed with MgSO ₄ and filtered. The solution was distilled off under reduced pressure, and the remaining filtrate was purified by flash column chromatography to give the yellow oil form 4 g (56%) of compound 5 [3,7-dimethyl-10- (2-methyl-1,3-dioxolan). -2-yl) deca-3,7-dienenitrile].

Formula: C ₁₆ H ₂₅ NO ₂ , Molecular weight: 263.38)

¹ H-NMR (CDCl ₃ , 300 MHz) δ 5.48 (1H, m), 5.13 (1H, m), 4.00 (4H, m), 3.02 (2H, s), 2.08 (4H, m), 1.73 (3H, s), 1.68 (2H, m), 1.61 (3H, s), 1.32 (3H, s) ppm

^l3 C-NMR (CDCl ₃ , 75 MHz) δ 136, 134, 126, 124, 118, 110, 65 (2C), 39, 31, 27, 24, 23, 22, 16, 15 ppm

All. 3,7-dimethyl-10- (2- methyl -1,3- Daoxoran -2 days) Deca -2,7- Dienenitrile

Dissolve 5 in 100 ml of MeOH and stir at 80 ° C. 1.64 g (30.38 mmol) of sodium methoxide was added to the reaction and the mixture was stirred for 6 hours while maintaining the temperature of 80 ° C. When it is confirmed that the reaction is completed, add 100 ml of water, extract with Ethyl acetate (100 ml × 3), remove water with MgSO ₄ and filter. The solution was distilled off under reduced pressure, and the remaining filtrate was purified by flash column chromatography to give 1.9 g (87%) of a pale yellow oil. Compound 6 [ 3,7-dimethyl-10- (2-methyl-1,3- dioxolan-2-yl) deca-2,7-dienenitrile] (Formula: C ₁₆ H ₂₅ NO ₂ , Molecular Weight: 263.38).

la. 3,7-dimethyl-10- (2- methyl -1,3- Daoxoran -2 days) Deca -2,7- Dienal

Dissolve 6 in 20 ml of CH ₂ Cl ₂ and stir at -40 ° C for 30 minutes. Add 18.03 ml (18.03 mmol) of diisobutyl aluminum hydride and stir for 4 hours while maintaining at -40 ° C. When it was confirmed that the reaction was completed, CH ₂ Cl ₂ and H ₂ O were excessively added and passed through a Celite pad. Then, 50 ml of H ₂ O was added and extracted with CH ₂ Cl ₂ (50 ml × 3), followed by MgSO Remove the water with ₄ and filter. The solution was distilled off under reduced pressure, and the remaining filtrate was purified by flash column chromatography to give 1.12 g (68%) of a pale yellow oil. Compound 7 [3,7-dimethyl-10- (2-methyl-1,3- dioxolan-2-yl) deca-2,7-dienal]

(Formula: C ₁₆ H ₂₆ O _3, molecular weight: 266.38).

This was named Farnesyl Acetone 7 or YJ-7.

¹ H-NMR (CDCl ₃ , 300 MHz) δ 9.99 (1H, d, J = 9 Hz), 5.88 (1H, d, J = 9 Hz), 5.15 (1H, m), 3.95 (4H, m), 2.11 (2H , m), 2.06 (2H, m), 1.98 (2H, m), 1.67 (3H, s), 1.63 (2H, m), 1.62 (3H, s), 1.32 (3H, s) ppm

^l3 C-NMR (CDCl ₃ , 75 MHz) δ 191, 164, 134, 127, 126, 110, 65 (2C), 40, 39, 32, 25, 24, 23, 28, 16 ppm

[ Example  3] Preparation of Tablet

80 mg of farnesyl acetone 311, 120 mg of lactose, 40 mg of corn starch and 10 mg of magnesium stearate were compressed into 250 mg tablets according to a conventional tablet preparation method to prepare a pharmaceutical composition for tablet antihypertensive preparation.

[ Example  4] Preparation of capsule

40 mg of farnesyl acetone 312, lactose 32 mg, corn starch 26 mg, talc 1 mg and magnesium stearate are usually filled in 100 mg capsules according to the preparation method of the capsule preparation, and the pharmaceutical preparation for antihypertensive preparation of the capsule type The composition was prepared.

[ Example  5] Liquid  Produce

Farnesylacetone 7 100 mg, isomerized sugar 10 g, honey 500 mg, nicotinic acid amide (pharmaceutical) 20 mg, caffeine anhydrous (pharmaceutical) 30 mg and sodium benzoate 70 mg prepared according to a conventional liquid preparation method, 100 ml After filling into a brown bottle of dose, it was intimately pasteurized to prepare a pharmaceutical composition for antihypertensive preparation in liquid form.

[ Example  6] Preparation of Injection

 40 mg of farnesyl acetone 311 and an appropriate amount of sterile purified water were prepared according to a conventional injection method, and filled into 2 ml of ampoules, and then sealed and sterilized to prepare a pharmaceutical composition for treating hypertension in the form of an injection.

[ Experimental Example ]

1. Materials and Experimental Methods

end. Basal artery  Smooth muscle cell isolation

The basal artery used in this experiment was isolated by surgical method. First, a 2-2.5 kg white rabbit was inhaled anesthesia with an animal anesthetic, enflurane, and then the femoral artery was cut to remove blood, and the basal artery at the base of the brain was removed with the skull incised. It was. The extracted basal artery removed peripheral connective tissue and fat in Ca ²⁺ -free Tyrode solution saturated with 95% O ₂ and 5% CO ₂ . Ca ²⁺ -free Tyrode solution (mM) used for the separation of blood vessels was composed of 137 NaCl, 5.4 KCl, 1 MgCl ₂ , 23.8 NaHCO ₃ , 5.5 glucose, and the osmotic pressure was 300 mOsm / kg H ₂ O using sucrose. It was used according to.

Enzymatic dissociation was used to isolate single vascular smooth muscle cells. First, the extracted basal artery was soaked in cold Ca ²⁺ -free HEPES solution, gently shaken to remove residual blood, and then fixed in a 100 mm culture dish coated with Sylgard 184. The connective tissue surrounding the basal artery and carotid artery was removed, and the belly was split in the longitudinal direction, and then sliced into 1-2 mm pieces and equilibrated in Ca ²⁺ -free HEPES solution at 4 ° C. for 30 minutes. Ca ²⁺ papain the artery segment was removed -free HEPES solution (2 mg / mL), dithiothreitol (DTT; 1.5 mg / mL), bovine serum albumin (BSA; 1.5 mg / mL) Ca ²⁺ is included - Incubated for 15 minutes at 37 ℃ in free HEPES solution. Remove Papain solution and wash twice with Ca ²⁺ -free HEPES solution, then HEPES (200 μM Ca ² ) containing collagenase (2 mg / mL), DTT (1.5 mg / mL), BSA (1.5 mg / mL) ⁺ )) Was added to the solution and incubated for 15 minutes at 37 ℃. After the incubation, the supernatant was removed and washed three times. Fresh Ca ²⁺ -free HEPES solution (1 mL) was added, and the resultant was pulverized with blunt pasteur pipette until single smooth muscle cells fell off. Single smooth muscle cells were plated in 35 mm culture dish and used for patch clamp. The HEPES solution (mM) used to isolate single smooth muscle cells was composed of 135 NaCl, 5 KCl, 1 MgCl ₂ , 10 glucose, 10 HEPES, and 200 μM Ca ²⁺ was added to the collagenase treatment.

I. Ventricular muscle  Cell isolation and culture

Ventricular myocytes were isolated from enzymatic methods from around 200 g of Sprague-Dawley rats. Pentobarbital sodium (50 mg / Kg) was anesthetized by intraperitoneal injection, and the heart was quickly removed. The isolated heart was subjected to reverse perfusion of Ca ²⁺ -free Tyrode solution through the aorta for 5 minutes, followed by collagenase type A (1 mg / mL) and protease type XIV (0.1 mg / mL). Ca ²⁺ -free Tyrode solution was recycled for 15 minutes. The ventricular muscles were then dissected and dispersed, and then stored in a Tyrode solution containing 200 μM Ca ²⁺ for 2 hours at room temperature and consumed within 12 hours.

All. Heterologous Expression of Cell Culture and Calcium Channel Subtypes

HEK 293 cells derived from fetal kidney were distributed from Korea Cell Line Bank and 5% CO at 37 ° C. using DMEM medium containing 10% FBS, penicillin (100 units / mL) and streptomycin (100 mg / mL). _It was maintained and incubated in a humidified incubator saturated with ₂ and 95% air. HEK 293 cell lines stably expressing the T-type calcium channel subtypes a1G (GenBank code: AF027984), a1H (AF051946), and a1I (AF086827) were used by Dr. Perez-Reyes of Virginia State University. The medium was cultured with the addition of the selective antibiotic G418 (1 mg / mL). C2D7 cell lines heterologously expressing N-type calcium channels (a1B, a2δ, β1b) were used from Randall of Cambridge University. The a1C morphology, a molecular type of L-type calcium channel subtypes expressed in vascular smooth muscle cells and cardiomyocytes, was heterologously expressed in the manufacturer's protocol using CaPO ₄ transfection kit with auxiliary subunits of a1δ and β2a in HEK 293 cells. In brief, incubate a total of 2x10 ⁵ cells in a 35 mm culture dish (Corning) 24 hours prior to transfection of the desired cDNA vectors using CaPO ₄ . Replace with fresh medium 3 hours before transfection. CDNA for heterologous expression was used in the composition of a1C (3 μg), a2δ (1 μg), β1b (1 μg) and green fluorescence protein (GFP; 0.6 μg). 7.5 μL CaCl ₂ (2 M) and the desired cDNA combination were made in one eppendorf tube to a total volume of 60 μL, followed by gentle stirring. The other tube containing 60 μL 2X Hank's buffered saline (HBS) was stirred slowly using bubbling while slowly dropping cDNA for 1-2 minutes. When cDNA precipitate formed, it was left at room temperature for 30 minutes and slowly dropped into a 35 mm plate with cells plated. After shaking the dish gently so that the cDNA precipitate was mixed well, incubated for 24 hours in a wet incubator, and then exchanged with a fresh medium. 6-12 hours before the measurement of calcium current, the cells were detached from the culture dish and plated again in a 35 mm culture dish with single cells. Usually, 48 hours after transfection, calcium expression was measured after maximal expression.

la. Calcium Current Measurement Using Electrophysiological Method

Voltage-dependent calcium currents from basal artery smooth muscle cells, cardiac ventricular muscle cells, HEK 293 cells heterologously expressed in T- and L-type calcium channels and C2D7 cells heterologously expressed in N-type calcium channels The whole cell patch clamp method was used. Electrodes were fabricated using a P-97 Flaming-Brown micropipette puller from Corning 7052 borosilicate glass capillary. The fabricated electrode was coated with Sylgard 184 and heat treated with a microforge, and when the solution was filled in the electrode, the resistance was 2-3 MΩ. The culture dish containing the cells was placed on an inverted microscope and the extracellular perfusate was allowed to perfusion at a rate of about 1-2 mL / min by gravity. Capacitance and series resistance of the cell membranes were calibrated more than 80% with a patch clamp amplifier. Voltage stimulation protocol generation and calcium current recording were performed using pClamp 6.0 software on an IBM computer equipped with a digital converter Digidata 1200. Calcium current was stored after low pass filter at 2-5 kHz, and all experiments were performed at room temperature (20-25 ℃). The composition (mM) of the intracellular electrode solution used for calcium current measurement was 120 N-methyl-D-glucamine (NMG) -methanesulfonate (MS), 20 tetraethylammonium (TEA) -MS, 20 HCl, 11 EGTA, 10 HEPES, 1 CaCl ₂ , 4 MgATP, 0.3 Na ₂ GTP, 14creatine phosphate were used by titration with pH 7.2 and 290 mOsm / Kg H ₂ O. The composition of extracellular perfusate (mM) was titrated with pH 7.4 and 320 mOsm / Kg H ₂ O with 140 MS, 145 TEA-OH, 10 HEPES, 15 glucose, 10 CaCl ₂ , 0.0003 tetrodotoxin (TTX). In the case of L-type calcium current, the same concentration of BaCl ₂ was used instead of CaCl ₂ in the basic extracellular perfusate to suppress calcium-induced inactivation.

2. Basal artery  Experimental Effect of L-type Calcium Channel Inhibition on Smooth Muscle and Cardiomyocytes

In this experimental example, depolarization is induced when blood vessels are treated with a solution containing 50 mM K ⁺ , and the mechanism of contraction of blood vessels is caused by the introduction of calcium ions through L-type calcium channels from outside of smooth muscle cells, thus farnesylacetone 311. Vascular relaxation effect by and 312 was estimated to be induced by inhibition of L-type calcium channel.

Calcium currents were measured by electrophysiological methods from basal artery smooth muscle cells separated into single cells, and the results are shown in FIG. 1.

As shown in FIGS. 1A and D, the membrane voltage of the smooth muscle cells was fixed at -80 mV, and the concentration of farnesylacetone 311 and 312 was increased at 10 m intervals at 0 mV while increasing the concentration of farnesylacetone 311 and 312. It was confirmed that calcium current was suppressed. In addition, the treatment concentrations of Farnesylacetone 311 and 312 and the degree of inhibition of L-type calcium current were plotted, and fitting was performed using the Hill formula to confirm the concentration (IC ₅₀ ) of inhibiting L-type calcium current by 50%. , 1F and as shown in Table 1 below, IC ₅₀ of Farnesylacetone 311 and 312 was identified as 1.24 ± 0.14 (n = 6) and 3.01 ± 0.23 μM (n = 6), respectively, of the basal artery by farnesylacetone 311 and 312. It was confirmed that the relaxation mechanism is due to the efficient inhibition of L-type calcium channel. At this time, the Hill formula in Table 1 is fitted to {E = (1 + EC ₅₀ / [sargahydroquinoic acid] n) ^-1 } to calculate the IC ₅₀ value and the resulting value in the unit of μM is expressed as mean ± standard deviation, The number of experiments was six.

On the other hand, the calcium channel of ventricular myocytes of the heart, like the smooth muscle cells of the basal arteries, is reported to be L-type (a1C). Therefore, to determine whether farnesylacetone 311 and 312 show inhibition of L-type calcium currents of ventricular myocytes. In order to separate calcium current from current contamination by the action of Na ²⁺ / Ca ²⁺ exchanger of ventricular myocytes, the membrane voltage was fixed at -60 mV and depolarized stimulation was induced at 0 mV to induce calcium current. Since the capacitance is more than 100 pF, the measurement was performed by calibrating only about 60% together with the series resistance. As shown in FIGS. 1B and 1E, L-type calcium from ventricular muscle cells when administered with increasing concentrations of farnesylacetone 311 and 312. It was confirmed that the current was suppressed in a concentration-dependent manner as in the case of basal artery smooth muscle cells. In addition, IC ₅₀ of farnesylacetone 311 and 312 for L-type calcium currents of ventricular myocytes were found to be 1.51 ± 0.18 (n = 6) and 4.76 ± 0.28 μM (n = 6), respectively. It was similar to the sensitivity of farnesylacetone 311 and 312 to calcium currents (FIGS. 1B and 1E, Table 1).

3. Experimental Inhibitory Effect of L-type Calcium Channel

As shown in the results of Experiment 2, farnesylacetone 311 or 312 induced vascular relaxation by inhibiting L-type calcium channels, but if other calcium channel subtypes were inhibited with the same effect, they could affect the function of other cells or organs. In general, N-type calcium channels among the calcium channel subtypes account for about 60% of the calcium currents recorded in nerve cells, especially if farnesylacetone administered for the purpose of lowering blood pressure inhibits N-type calcium channels. It is thought that this may adversely affect the central function of neurons such as the transmission of sensory, pain, or motor signals by affecting the electrical properties of the cells. Therefore, in this experimental example, we tried to determine whether farnesylacetone 311 and 312 selectively sensitively inhibit heterologous L-type and N-type calcium channels. However, in general, selective sensitivity may be a difference of 10 times or more for two or more specific targets.

First, L-type calcium current was recorded by depolarizing stimulation at 0 mV while the membrane voltage of heterologously expressed HEK 293 cells was fixed at -80 mV. As a result, farnesylacetone 311 and 312 were increased as shown in FIGS. 2C and D. It was found that it inhibited L-type calcium current in a concentration-dependent manner. Similarly, N-type calcium channel stably heterologously expressed C2D7 cells at -80 mV and then depolarized at 0 mV when N-type calcium channels were fixed. As a result of recording the type calcium current, as shown in FIGS. 2A and 2B, it was confirmed that the concentration-dependent inhibition was achieved when farnesylacetone 311 and 312 were treated.

However, although the inhibition pattern of L-type and N-type calcium currents by Farnesylacetone 311 and 312 is similar to the concentration-dependent, the relationship between the treatment concentration and the degree of inhibition of farnesylacetone 311 and 312 is plotted and then fitted by Hill's equation. The value of IC ₅₀ was confirmed to show a difference of about 30 times as shown in Table 1 below. In other words, IC ₅₀ of L-type and N-type calcium currents by Farnesylacetone 311 showed 0.99 ± 0.51 and 27.8 ± 1.23 μM (n = 6), respectively, showing a 28-fold difference in sensitivity. IC ₅₀ for type and N-type calcium currents was 2.68 ± 0.24 and 41.2 ± 2.11 μM (n = 6), showing 15-fold difference in efficacy. At this time, the IC ₅₀ value is calculated by fitting the Hill formula in Table 1 below, and the results in units of μM are expressed as mean ± standard deviation, and the number of experiments is 6 times.

From these results, farnesylacetone 311 and 312 can be said to selectively selectively inhibit L-type calcium channels rather than N-type.

Therefore, administration of farnesylacetone 311 or 312 for the purpose of increasing cerebral blood flow or lowering blood pressure may affect the function of cells or organs caused by N-type calcium channel if treated at a critical concentration that does not inhibit N-type calcium channel. It is judged that the L-type can be selectively suppressed and its effect can be obtained without giving.

4. Effect on T-type Calcium Channel farnesylacetone  311 and 312 effects

In general, T-type calcium channel among calcium channel subtypes is expressed in excitable cells of human excitability such as autonomic nerve, cardiac nodule, brain nerve, spinal nerve, and is responsible for regulating cell electrical excitability. Inhibition of medication has the potential to pose a fatal hazard to humans. Therefore, in this experiment, the effect of farnesylacetone 311 and 312 selective on L-type calcium channel on T-type calcium channel was examined.

First, the membrane voltage of HEK 293 cells expressing T-type calcium channel subtypes was fixed at -100 mV and then stimulated with depolarization at -30 mV to induce T-type calcium current and increase the concentration of farnesylacetone 311. As shown in FIG. 3, it was confirmed that calcium currents by a1G (FIG. 3A), a1H (FIG. 3B), and a1I (FIG. 3C) were suppressed depending on the concentration of farnesylactone 311, and as shown in Table 1 above. After plotting the relationship between concentration and inhibition, IC50 was calculated to be 37.2 ± 2.23 (n = 6), 28.4 ± 1.98 (n = 6), and 33.9 ± 2.81 μM (n = 6), respectively. In addition, as shown in FIG. 4, as in farnesylaetone 311, the treatment concentration of 312 was increased, and the inhibition pattern for the T-type calcium channel subtypes a1G (FIG. 4A), a1H (FIG. 4B) and a1I (FIG. 4C) was confirmed. As a result, IC ₅₀ was found to be 40.6 ± 2.18 (n = 6), 32.2 ± 1.43 (n = 6), and 42.9 ± 1.75 μM (n = 6), respectively (see Table 1).

As a result, the inhibition pattern of L-type calcium channel and T-type calcium channel was similar, but compared with IC50, farnesylacetone 311 showed more than 30 times sensitivity difference, and farnesylacetone 312 also showed more than 15 times sensitivity difference. Seemed.

Therefore, as discussed in the sensitivity of L-type calcium channel to N-type calcium channel, it was confirmed that the sensitivity is also low for T-type calcium channel.

As a result of the experimental example described above, the sensitivity of the calcium channel subtype 311 showed HEK 293 (a1C)> basal artery smooth muscle L-type> ventricular muscle L-type> N-type (a1B)> T-type (a1H). )> T-type (a1I)> T-type (a1G), and for 312 HEK 293 (a1C)> basal artery smooth muscle L-type> ventricular muscle L-type> T-type (a1H)> It was identified as T-type (a1G)> N-type (a1B)> T-type (a1I). Due to this selective sensitivity, farnesylacetone 311 and 312, when treated in the human body for the purpose of increasing blood flow or lowering blood pressure, do not affect other calcium channel subtypes, thereby preventing side effects caused by inhibiting other calcium channel subtypes. It is thought that it is possible to increase the efficiency of lowering blood pressure without being shown.

5. Blood pressure drop effect experiment

The hypotensive effect was measured using a non-invasive blood pressure (NIBP) device. The experimental animals were 10 weeks old (SHR), a hypertension disease model, and the hypertension rats were placed in a fixed frame and stabilized for 15 minutes. Hypertension rats with blood pressure greater than 190 mHg were used for the experiments. Blood pressure was measured in the tail before the test, and drug administration was performed by injecting 0.2 ml of the tail vein to 3-10 μM and measuring blood pressure for 1 hour every 15 minutes. At this time, as a control group was administered dimopyridine-based nimodipine previously used as a blood pressure lowering agent.

Oral administration was compulsory diet using zonation at a concentration of 80 mg / kg body weight and blood pressure change was periodically observed for 24 hours.

As a result, as shown in Fig. 5, all of the penicyl acetone 311, 312 and 7 (YJ-7) of the present invention was excellent in blood pressure lowering effect, of which 311 is equivalent to the nimodipine treatment group control group at 10 μM It showed a blood pressure lowering effect and confirmed that the blood pressure lowering effect also lasted more than 8 hours (FIG. 6).

1 is a current record showing the L-type calcium current inhibitory effect of basal artery smooth muscle and extensor muscle cells by panesyl acetone 311 and 312 of the present invention, (A) is 311 (B) is 312, basal artery smooth muscle cells L-type calcium current records recorded by treatment of (B) and (C) are L-type calcium current records recorded by treating ventricular muscle cells at 311 and 312, respectively, and (C) and (F) are Hill formulas. Calculate the IC ₅₀ by fitting the test results. The results are plotted as mean ± standard deviation and the number of experiments (n) is 6 times.

Figure 2 is a current record showing the selective inhibitory effect of L-type calcium channel by panesyl acetone 311 and 312 of the present invention, (A) and (B) is the recording of N-type calcium current, (C) and ( D) is the L-type calcium current record and (E) and (F) are fitted by the Hill formula to calculate IC _{50. The} results are plotted as mean ± standard deviation and the number of experiments (n) is 6 circuits. It is.

Figure 3 is a current record showing the inhibitory effect of T-type calcium channel subtypes by Panesyl acetone 311 of the present invention, the (A) a1G, (B) a1H and (C) a1I of the subtypes of T-type calcium channel (D) is the L-type calcium current record and (E) and (F) are fitted by Hill's formula to calculate IC ₅₀ , and the results are plotted as mean ± standard deviation. (n) is 6 times.

Figure 4 is a current record showing the inhibitory effect of T-type calcium channel subtypes by Panesyl acetone 312 of the present invention, (A) a1G, (B) a1H and (C) a1I of the subtypes of T-type calcium channel (D) is the L-type calcium current record and (E) and (F) are fitted by Hill's formula to calculate IC ₅₀ , and the results are plotted as mean ± standard deviation. (n) is 6 times.

5 is administered to the high blood pressure pantyl acetone 311, 312 and 7 of the present invention and shows the blood pressure strengthening effect by periodically monitoring the blood pressure change for 1 hour.

FIG. 6 shows oral administration of panesyl acetone 311 of the present invention to a hypertension rat, and the change in blood pressure was periodically observed for 24 hours, thereby showing an effect of strengthening blood pressure.

Claims (4)

Farnesyl acetone derivative represented by the following formula (7). [Formula 7]

Pharmaceutical composition for an antihypertensive preparation comprising the derivative of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. delete delete

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