KR101204483B1 - Decursinol derivatives, method of the same and pharmaceutical composition comprising same - Google Patents

Abstract

The present invention relates to a novel decursinol derivative, a preparation method thereof and a pharmaceutical composition comprising the same as an active ingredient. Since the new decosinol derivatives not only protect neurons and inhibit glial activity but also inhibit NF-κB activity, the pharmaceutical composition comprising the same as an active ingredient is a composition for treating or preventing ischemic cerebrovascular disease. Can be used.

Description

DECURSINOL DERIVATIVES, METHOD OF THE SAME AND PHARMACEUTICAL COMPOSITION COMPRISING SAME}

The present invention relates to a novel decosinol derivative, a preparation method thereof and a pharmaceutical composition comprising the same as an active ingredient.

Of the total population released in 2005, the National Statistical Office recently made up 9.1% of the population aged 65 and over, and the proportion of young people aged 14 and younger is decreasing. The proportion of the population aged 65 and over is 7.2% in 2000. It is estimated that our society has already entered an aging society. In addition, as of 2003, the average life span of Korean people is 77.5 years old, and the elderly population is rapidly increasing. Meanwhile, Korea's GNI per capita announced by the National Statistical Office was $ 14,162 in 2004, and it is increasing by more than 10% every year. Due to such changes in social conditions, the number of deaths from infectious diseases, which are the major causes of death, is rapidly decreasing, while the number of deaths from degenerative diseases such as cancer, cerebrovascular disease, and diabetes is steadily increasing.

Cerebrovascular disease, which has a 12.31% incidence of a single disease, is the second most common cause of death in Korea. It is also called a stroke, and nerves that appear due to blockage or bursting of blood vessels that supply blood to the brain are damaged. One of the symptoms is divided into two types: ischemic stroke and hemorrhagic stroke caused by cerebral hemorrhage.

The ischemic cerebrovascular disease is a disease with a high incidence of 33.9% of cerebrovascular diseases, which is caused by the deposition of cholesterol on the cerebrovascular wall by the progression of arteriosclerosis or by the clot generated in the cardiovascular system to block the cerebrovascular vessel. Other wastes are caused by blocking blood vessels in the brain. When the cerebrovascular is blocked, blood flow of the brain is reduced and oxygen and glucose are not supplied, causing delayed neuronal death in the hippocampal CA1 region, resulting in the invention of ischemic cerebrovascular disease. When the cerebral ischemia occurs, the effect remains permanently, followed by deterioration or degradation of motor, sensory and cognitive functions according to the corresponding site. In addition, when the cerebrovascular is blocked, the supply of oxygen and glucose to the brain, in particular the hippocampal region is blocked, thereby reducing the ATP in neurons, resulting in edema.

There are two main causes of delayed neuronal death known to date: excessive inflow of calcium into cells (Hara et al. al ., Brain Res. Bull., 29, pp 659-665 (1992); Nedergaard, Acta Neurol. Scand., 77, pp81-101 (1988)), resulting in excitatory neuronal cell death or oxidation due to damage to DNA and membrane components due to free radical increase due to sudden oxygen supply during ischemia-reperfusion. Toxic neuronal death has been reported.

In relation to the excessive cellular influx of calcium, the reversal of active transport caused by the reduction of Adenosine Triphosphate (ATP) accelerates the accumulation of extracellular glutamic acid, and the accumulation of extracellular glutamic acid is NMDA (N-methyl-D Induces continuous activity of -aspartic acid), AMPA (α-amino-3-hydroxy-5-methyl-4 -isoxazole-propionic acid) and metabotropic glutamate receptors, leading to intracellular calcium excess Cause influx (Olney et al ., Science, 244, pp1360-1362, 1989).

Intracellular influx of calcium triggers the activity of calcium dependent proteases, lipases and modulators, in particular, resulting in cytotoxic molecules comprising free radicals. A-ketoglutarate dehydrogenase complex (KGDHC), a substrate enzyme present in the inner matrix of mitochondria, forms enzymes associated with the complex I and TCA cycles associated with mitochondrial respiratory changes to produce reactive oxygen species (Maas and Bisswanger, FEBS Lett, 270, pp 189-190, 1990; Sumegi and Srere, Biol Chem, 259. pp 15040-15045, 1984; Lyubarev and Kurgnov, Biosystems, 22, pp91-102, 1989). KGDHC which is activated by the low concentration of Ca ^{2 +} and the substrate ADP is only occur live nerve cell activity falls that endothelial cells (endothelial cell), astrocytes, glial cells, micro-projections is not affected.

Therefore, reactive oxygen species are known to cause neuronal death by destroying cellular structures such as DNA and cell membranes, and the reduction of KGDHC has become a marker of neurodegenerative diseases (Mastrogiacomo et. al ., Neurodegeneration 5, pp 27-33 (1996); Gibson et al ., Ann Neurol, 44, pp 676-681 (1998).

Based on these mechanisms, many researches have been conducted on the substances that effectively inhibit neuronal cell death induced by cerebral ischemia or the mechanisms of the substances, but no effective therapeutics have been developed.

Tissue plasminogen activator (Tissue plasminogen activator) is currently the only FDA-approved drug for treating cerebral ischemia, and it is a substance that induces rapid supply of oxygen and glucose by melting blood clots that cause cerebral ischemia. Therefore, it is not necessary to directly protect nerve cells, so it is necessary to use it quickly. Due to the characteristics of thrombolysis, excessive use or frequent use may cause thinning of the blood vessel wall and eventually cause hemorrhagic cerebrovascular disease.

In addition, MK-801, a calcium channel blocker (calcium channel blocker) for effectively inhibiting the initial calcium influx, has been clinically tested, but its side effects have prevented the use of drugs.

On the other hand, in Korea, many natural substances are marketed as health foods effective in preventing stroke, but most of them are not scientifically verified, and are often social problems because they cause health food abuse. Considering the characteristics of the cerebrovascular disease, cerebrovascular disease tends to emphasize prevention rather than treatment, and development of a material capable of effectively protecting brain neurons is required.

In order to meet the above demands, the present invention is excellent in neuronal cell protective ability, excellent in inhibiting glial activity, and effectively inhibiting the activation of NF-κB, which is useful for the prevention or treatment of ischemic cerebrovascular disease and has no side effects. It is an object to provide a small compound, a method for preparing the compound and a therapeutic composition containing the same.

In order to achieve the above object, the present invention provides a novel decursinol derivative (decursinol derivative) compound, a preparation method, a composition for treating or preventing ischemic cerebrovascular disease containing the same as an active ingredient.

The present inventors focus on the fact that a substance that is excellent in neuroprotective activity, excellent in inhibiting glial activity, and inhibiting NF-κB activation may be effective in treating or preventing ischemic cerebrovascular disease. While researching on a potent substance, a new decursinol derivative was derived and a preparation method thereof was established. The decusinol derivative is excellent in neuronal cell protection and glial activity, It was confirmed through experiments using animal models that the inhibition of NF-κB activation, the present invention was completed.

Hereinafter, the present invention will be described in more detail.

In one aspect of the invention, the invention provides a novel decursinol derivative compound. The decosinol derivative compound may be a compound having a structure of Formula 1 below, and may be a pharmaceutically acceptable salt thereof.

In Formula 1,

N is any integer from 0 to 4, and each R may be independently selected from the group consisting of alkyl, aryl, halogen, hydroxy, alkoxy, amino and thiol.

The alkyl may be alkyl or cyclo alkyl, preferably may be alkyl having 1 (C1) to 7 carbon atoms (C7) or carbon atoms 1 to 5 carbon atoms, the aryl is 6 to 30 carbon atoms or 6 to 12 carbon atoms. Phosphorus may be aryl, and the alkoxy may be alkoxy having 1 to 10 carbon atoms. The alkyl, aryl and alkoxy may be substituted or unsubstituted.

The decosinol derivative compound of the present invention may preferably be a compound having a structure of Formula 2 below.

In another aspect of the present invention, the present invention provides a method for preparing a compound having the structure of Formula 1.

The compounds of the present invention may be prepared by the method shown in Scheme 1 below, but are not limited to these examples. Scheme 1 below shows the preparation method of the representative compounds of the present invention in stages of preparation, and other compounds may be prepared by appropriate changes in reagents and starting materials known to those skilled in the art.

The method for preparing a compound having the structure of Formula 1 may be as described in Scheme 1 below.

[Reaction Scheme 1]

More specifically, the preparation method of Scheme 1,

(a) reacting decursinol, which is a compound having the structure of Formula 3, with a compound having the structure of Formula 4 to prepare a compound having the structure of Formula 6; And

(b) acetylating the compound having the structure of Formula 6 to prepare a compound having the structure of Formula 1

It may be a method for producing a decosinol derivative comprising a.

In Chemical Formulas 4 and 6, n is an integer of 0 to 4, and each R may be independently selected from the group consisting of alkyl, aryl, halogen, hydroxy, alkoxy, amino and thiol.

The alkyl may be alkyl or cyclo alkyl, preferably may be alkyl having 1 to 7 carbon atoms or 1 to 5 carbon atoms, the aryl may be aryl having 6 to 30 carbon atoms or 6 to 12 carbon atoms, The alkoxy may be alkoxy having 1 to 10 carbon atoms. The alkyl, aryl and alkoxy may be substituted or unsubstituted.

The method for producing the decosinol derivative of the present invention may preferably be as described in Scheme 2 below.

Scheme 2

More specifically, the manufacturing method of Scheme 2,

(a) reacting decursinol, which is a compound having the structure of Formula 3, with salicylic acid having the structure of Formula 5 to prepare a compound having the structure of Formula 7; And

(b) acetylating the compound having the structure of Formula 7 to prepare a compound having the structure of Formula 2

It may be a method for producing a decosinol derivative comprising a.

In the step (a), the compound having the structure of Formula 3 is dissolved in a methylene chloride solvent, and then reacted with salicylic acid having the structure of Formula 5 to prepare a compound having the structure of Formula 7. to be.

More specifically, step (a) is based on the salicylic acid having the structure of Formula 5, 0.8 equivalent to 1.2 equivalents of dicyclohexylcarboiimide (DCC) to 1 equivalent of salicylic acid (Dicyclohexylcarboiimide, DCC) and dimethylaminopyridine (Dimethylaminopyridine, DMAP) adding 0.05 equivalents to 0.2 equivalents together with stirring in anhydrous methylene chloride solvent for 20 to 40 minutes at room temperature, for example 10 to 25 ℃ or 15 to 20 ℃ and after the reaction the structure of formula 3 The process may include adding 0.4 to 0.6 equivalent of Decusinol having a DCC-couplilng reaction while refluxing at 50 to 70 ° C. for 36 to 60 hours or 40 to 56 hours.

After reacting the salicylic acid, the dicyclohexylcarbodiimide and the dimethylaminopyridine together in anhydrous methylene chloride solvent, a decosinol having the structure of Formula 3 is added and further reacted to perform a DCC-coupling reaction. Thus, a compound having the structure of Chemical Formula 7 was prepared.

The compound having the structure of Chemical Formula 7 was named HC-11, and the chemical formula was 7 R- (2-hydroxybenzoate) -8,8-dimethyl-8-oxo-7,8-dihydro-6 H -pyrano It was [3,2-g] chromen-2-one, and the compound having the structure of Chemical Formula 5 by the step (a) may be prepared in a yield of 93%.

The step (b) may be performed by a method of obtaining a compound having the structure of Chemical Formula 2 by acetylation by reacting the compound having the structure of Chemical Formula 5 with acetic anhydride.

More specifically, step (b) is based on the compound having the structure of Formula 7, 0.1 equivalent to 0.4 equivalent of dimethylaminopyridine (DMAP) and triethylamine to 1 equivalent of the compound having the structure of Formula 7 Adding 4 to 5 equivalents together and reacting with anhydrous methylene chloride solvent for 2 to 4 hours at room temperature, for example, 10-25 ° C or 15-20 ° C, to carry out an acetylation reaction. can do.

The compound having the structure of Chemical Formula 2 was prepared by reacting the compound having the structure of Chemical Formula 7, the dimethylaminopyridine, and the triethylamine together in anhydrous methylene chloride solvent to perform an acetylation reaction.

The compound having the structure of Chemical Formula 2 was named HC-12, and the chemical formula was 7 R- (2-acetoxybenzoate) -8,8-dimethyl-8-oxo-7,8-dihydro-6 H -pyrano It was [3,2-g] chromen-2-one, and the compound having the structure of Chemical Formula 2 by the step (b) may be prepared in a yield of 83%.

In another aspect of the present invention, the present invention also provides a pharmaceutical composition as an active ingredient a compound having the structure of Formula 1 or a pharmaceutically acceptable salt thereof.

More specifically, the present invention provides a composition for treating or preventing ischemic cerebrovascular disease comprising the compound having the structure of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. The compound having the structure of Formula 1 may preferably be a compound having the structure of Formula 2.

In the present invention, the cerebrovascular disease refers to a term that refers to various diseases caused by abnormalities of blood vessels of the brain, and cerebrovascular diseases are largely classified into hemorrhagic cerebrovascular disease and ischemic cerebrovascular disease.

The ischemic cerebrovascular disease is a disease caused by delayed neuronal death in neurons of the CA1 region of the hippocampus known to be sensitive to cerebral ischemia because brain blood flow is decreased and oxygen and glucose are not normally supplied. The ischemic cerebrovascular disease may include cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, leukemia, Alzheimer's disease, dementia or Huntington's disease.

The compound having the structure of Formula 1 of the present invention anesthetizes the whole neck artery by anesthesia to induce cerebral ischemia, and significantly inhibits neuronal cell death caused by cerebral ischemia in all experiments measuring the efficacy by staining neurons. It showed a cell protective effect.

The pharmaceutical composition of the present invention, specifically the composition for preventing or improving cerebral ischemic disease, 0.001 to 99.99% by weight of the compound having the structure of Formula 1 or a pharmaceutically acceptable salt thereof, preferably based on the total weight of the composition It may comprise 0.1 to 99% by weight.

The composition may contain a compound having the structure of Formula 1 or a pharmaceutically acceptable salt thereof, as well as nutrients, vitamins, electrolytes, flavors, coloring agents, neutralizing agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloids. Thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The composition may be added independently or in combination of the above components. The content of the additional component is preferably added in the range of 0.1 to 20 parts by weight per 100 parts by weight of the compound having the structure of Formula 1 or a pharmaceutically acceptable salt thereof.

The pharmaceutical composition may be directly applied to an animal, including a human, using the compound having the structure of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. The animal is a biological group corresponding to plants, and mainly refers to a nutrient ingested of organic matter, digestion, excretion and respiratory organs are differentiated, preferably mammals, and more preferably humans.

The compound having the structure of Formula 1 may be used alone in the pharmaceutical composition, specifically, the composition for preventing or improving the cerebral ischemic disease, and further includes other pharmacologically acceptable carriers, excipients, diluents or accessory ingredients. can do. More specifically, when a composition comprising a compound having the structure of Formula 1 is used as a medicament, or used for medicament or pharmaceutical use, a decosinol derivative having the structure of Formula 1 or a pharmaceutically acceptable thereof The salt to be used may be used in admixture with a pharmaceutically acceptable carrier or excipient or by dilution with a diluent according to conventional methods.

In this case, the content of the compound having the structure of Formula 1 in the composition may be 0.001% by weight to 99.9% by weight, 0.1% to 99% by weight or 1% to 20% by weight, but is not limited thereto. Depending on the mode of use and the method of use, the content of the compound may be appropriately adjusted to a desired content.

Examples of the pharmaceutically acceptable carrier, excipient or diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, propylhydroxybenzoate, talc, magnesium stearate and mineral oil , Dextrin, calcium carbonate, propylene glycol, liquid paraffin, and physiological saline. However, the present invention is not limited thereto, and any conventional carrier, excipient or diluent may be used. In addition, the pharmaceutical composition may further comprise at least one selected from the group consisting of conventional fillers, extenders, binders, disintegrants, anticoagulants, lubricants, humectants, pH adjusters, nutrients, vitamins, electrolytes, alginic acid and its salts, pectic acid and its salts, , Fragrances, emulsifiers, preservatives, and the like. The components may be added independently or in combination with the compound having the structure of Formula 1 or a pharmaceutically acceptable salt thereof as the active ingredient.

In addition, the pharmaceutical composition of the present invention may further include a known substance known to have the intended pharmacological effect or used for the pharmacological effect.

More specifically, the composition for preventing or improving cerebral ischemic disease may further include a substance known or used to have an effect for preventing or treating ischemic cerebrovascular disease in addition to the active ingredient.

When the composition is used as a medicine, it can be administered orally or parenterally. In one example, it can be administered through various routes including oral, transdermal, subcutaneous, intravenous or muscular.

In addition, the formulations of the compositions may vary depending on the method of use and may be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal . In general, solid dosage forms for oral administration include tablets, pills, soft or hard capsules, pills, powders and granules, which may contain one or more Excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Oral liquid preparations include suspending agents (SUSTESIONS), liquid solutions, emulsions (EMULSIONS) and syrups (SYRUPS) .Such as simple diluents such as water and liquid paraffin, various excipients such as wetting agents, Sweeteners, fragrances, preservatives and the like can be included.

Forms for parenteral administration are CREAM, LOTIONS, ONITMENTS, PLASTERS, LIQUIDS AND SOULTIONS, AEROSOLS, FRUIDEXTRACTS, Elixir (ELIXIR), INFUSIONS, SACHET, PATCH or INJECTIONS.

Furthermore, the compositions of the present invention may be formulated using suitable methods known in the art or using methods disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). Can be.

The dosage of the composition may be determined in consideration of the method of administration, age, sex, severity, condition of the patient, absorption of the active ingredient in the body, inactivation rate and the drug used in combination, for example, based on the daily effective ingredient 0.01 mg / kg (body weight) to 100 mg / kg (body weight), 0.1 mg / kg (body weight) to 50 mg / kg (body weight) or 0.5 mg / kg (body weight) to 10 mg / kg body weight It can be administered, and can be administered once or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The novel decosinol derivative having the structure of Formula 1, which is an active ingredient of the present invention, induces cerebral ischemia by anesthetizing an experimental animal, specifically a male Mongolian gerbil (Harlan, USA), which induced ischemia, and ligation of the whole neck artery. After staining, the cells have a superior neuronal cell protective effect that significantly inhibits neuronal cell death in experiments measuring the efficacy of staining and expressing NF-κB expression associated with inhibition of inflammation by cerebral ischemia. As a result, it was confirmed that the expression of the NF-κB can be effectively suppressed, and in association with the ischemic cerebrovascular disease caused by ischemia, it was confirmed that it can effectively block neuronal cell death.

The novel decosinol derivative compounds of the present invention can effectively prevent damage to nerve cells caused by cerebral ischemia and can inhibit neuronal cell death caused by cerebral ischemia, which can be useful for the prevention, treatment or improvement of ischemic cerebrovascular disease. It has been confirmed that the composition comprising the decosinol derivative compounds and pharmaceutically acceptable salts thereof and the decosinol derivative compounds and pharmaceutically acceptable salts thereof as an active ingredient can prevent, treat or prevent ischemic cerebrovascular disease. It can be used for various applications related to improvement.

1 is a stain of the hippocampal tissue of experimental animals 4 days after cerebral ischemia is induced with cresyl violet in order to determine the effect of HC-12 according to an embodiment of the present invention to prevent neuronal cell death by cerebral ischemia. In one picture, A, C, E and G represent the entire hippocampal region, B, D, F and H represent the CA1 region, Sham means a normal group that does not cause cerebral ischemia, Vehicle is administered only distilled water One control group, 20mg / kg and 40mg / kg refers to the experimental group administered 20mg or 40mg of HC-12 per kg of body weight of the experimental animals, respectively.
Figure 2 is a graph showing the relative value (%) based on the normal group (Sham) that did not cause cerebral ischemia after counting the viable cells in the CA1 region of FIG. 1 according to an embodiment of the present invention.
Figure 3 uses the NeuN antibody as a marker of neurons in the hippocampus tissue of experimental animals 4 days after cerebral ischemia induction in order to confirm the effect of HC-12 neuronal cell death by cerebral ischemia according to an embodiment of the present invention After performing immunohistochemical staining, the photographs confirmed, A, C, E and G represent the entire hippocampal region, B, D, F and H represent the CA1 region, Sham is normal without causing cerebral ischemia Vehicle means a control group administered only distilled water, 20mg / kg and 40mg / kg means an experimental group administered 20mg or 40mg of HC-12 per 1kg body weight of the test animals, respectively.
Figure 4 shows the relative value (%) based on the normal group (Sham) that did not cause cerebral ischemia after counting immune response cells in response to the NeuN antibody of the CA1 region of Figure 3 according to an embodiment of the present invention It is a graph.
5 is a GFAP marker of astrocytes in the CA1 region of experimental animals 4 days after the induction of cerebral ischemia in order to confirm the effect of inhibiting neuronal cell death by the activation of neuroglial cells of HC-12 according to an embodiment of the present invention. Immunohistochemical staining was performed using Iba-1, which is a marker of microprotuberant glial cells, and the photographs observed showed A, C, E and G immunohistochemical staining using GFAP. B, D, F and H show the results of immunohistochemical staining using Iba-1, Sham means a normal group that did not cause cerebral ischemia, Vehicle means a control group administered only distilled water, 20mg / kg And 40 mg / kg means an experimental group administered with 20 mg or 40 mg of HC-12, respectively, per kg of body weight of the experimental animal.
FIG. 6 is a graph showing the immunoreactivity of GFAP and Iba-1 of FIG. 5 measured by an optical density (OD) according to an embodiment of the present invention. FIG. 6A shows the immunoreactivity of GFAP as OD. Afterwards, the measurement result is a graph showing a relative value (%) based on the normal group (Sham) that did not cause cerebral ischemia, and FIG. 6B shows the result of measuring the immune response against Iba-1 after OD. Is a graph showing relative values (%) based on the normal group (Sham) that did not cause cerebral ischemia.
Figure 7 is immunohistochemistry using an antibody against NF-kB known as a very important inflammatory response factor in stroke in order to confirm the neuronal protective effect of the anti-inflammatory effect of HC-12 according to an embodiment of the present invention Sham (A) refers to the normal group that did not cause cerebral ischemia, and Vehicle (B) refers to the control group administered only distilled water, and 20 mg / kg (C) and 40 mg / kg ( D) refers to an experimental group in which 20 mg or 40 mg of HC-12 was administered per kg of body weight, respectively.
FIG. 8 is a graph showing the immunoreactivity measured by optical density (OD) using the antibody against NF-kB of FIG. 7 according to an embodiment of the present invention. After the measurement, the measurement result is a graph showing a relative value (%) based on the normal group (Sham) that did not cause cerebral ischemia.
9 is an immunohistochemical staining using an antibody against BDNF known as a factor that inhibits neuronal cell death by ischemia in order to confirm the neuronal protective effect of HC-12 according to an embodiment of the present invention The photographs observed afterwards, Sham (A) refers to the normal group that did not cause cerebral ischemia, Vehicle (B) refers to the control group administered only distilled water, 20mg / kg (C) and 40 mg / kg (D) Each experimental group to which 20 mg or 40 mg of HC-12 is administered per kg of body weight of each experimental animal, respectively.
FIG. 10 is a graph showing measured immunoreactivity using an antibody against BDNF of FIG. 9 according to an embodiment of the present invention in terms of optical density (OD). After measuring immunoreactivity against BDNF in OD, FIG. It is a graph showing the measurement results in relative values (%) based on the normal group (Sham) that did not cause cerebral ischemia.

Hereinafter, preferred embodiments of the present invention are described. However, the following examples are only preferred examples of the present invention, and are not limited to the following examples of the present invention.

Example 1 Preparation of HC-11 from Decosinol.

Salicylic acid (Salicylic acid, 0.033g, 0.243mmol), DCC (0.053g, 0.255mmol) and DMAP (0.003g, 0.024mmol) was dissolved in CH ₂ Cl ₂ (5mL), and then stirred under nitrogen for 30 minutes at room temperature Afterwards, (+)-decursinol (0.03 g, 0.121 mmol) was added. The mixture is refluxed under nitrogen stream at 60 ° C. for 48 hours and then cooled to room temperature. The cooled reaction solution was filtered through a Celite filter, and the filtrate was distilled under reduced pressure and diluted with CH ₂ Cl ₂ . The organic layer was washed with brine, dried over MgSO _4, and distilled under reduced pressure. The remaining material was separated by column chromatography (EtOAc: Hexane = 1: 1) to give a white solid, HC-11 (7 R- (2-hydroxybenzoate). 0.041 g of -8,8-dimethyl-8-oxo-7,8-dihydro-6 H -pyrano [3,2-g] chromen-2-one) was obtained. The yield of HC-11 was 93%.

R _f 0.67 (EtOAc: Hexane = 1: 1);

m.p. 121-124 ° C .;

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.43 (3H, s), 1.48 (3H, s), 3.01 (1H, dd, J = 4.8, 17.1 Hz), 3.31 (1H, dd, J = 4.8, 17.1 Hz), 5.31 (1H, t, J = 5.4 Hz), 6.23 (1H, d, J = 9.3 Hz), 6.80-6.85 (2H, m), 6.97 (1H, d, J = 7.8 Hz), 7.17 ( 1H, s), 7.44 (1H, brt, J = 7.8 Hz), 7.57 (1H, d, J = 9.3 Hz), 7.68 (1H, dd, J = 1.8, 7.8 Hz), 10.56 (1H, s) .

¹³ C NMR (75 MHz, CDCl ₃ ) δ 23.8 (Me), 25.3 (Me), 28.2, 71.5, 76.9, 105.1, 112.1, 113.3, 113.8, 115.4, 117.9, 119.6, 128.9, 130.1, 136.4, 143.2, 154.4 , 156.3, 161.3, 161.9, 169.4 (C = O).

Example 2 Preparation of HC-11 to HC-12.

Dissolve HC-11 (0.033 g, 0.090 mmol) and DMAP (0.002 g, 0.018 mmol) in CH ₂ Cl ₂ (5 mL), add Et ₃ N (0.06 mL, 0.450 mmol), and mix the mixture for 10 minutes. Stir at room temperature. After the stirring, acetic anhydride (0.04 mL, 0.360 mmol) was added thereto and reacted with stirring at room temperature for 3 hours. After the reaction, ethanol was added to the reaction, treated with water, and extracted with CH ₂ Cl ₂ . The extract was washed with brine, dried over MgSO ₄ and distilled under reduced pressure. The remaining material was separated by column chromatography (EtOAc: Hexane = 1: 1) to give a white solid of 7 R- (2-acetoxybenzoate) -8,8. 0.03 g of -dimethyl-8-oxo-7,8-dihydro-6 H -pyrano [3,2-g] chromen-2-one was obtained. The yield of HC-12 was 83%.

R _f 0.41 (EtOAc: Hexane = 1: 1);

m.p. 78-81 ° C .;

¹ H NMR (300 MHz, CDCl ₃ ) δ 1.40 (3H, s), 1.46 (3H, s), 2.30 (3H, s), 2.97 (1H, dd, J = 4.5, 17.1 Hz), 3.27 (1H, dd, J = 4.5, 17.1 Hz), 5.26 (1H, t, J = 4.5 Hz), 6.22 (1H, d, J = 9.6 Hz), 6.82 (1H, s), 7.07 (1H, d, J = 8.1 Hz), 7.16 (1H, s), 7.28 (1H, d, J = 7.8 Hz), 7.54-7.58 (2H, m), 7.89 (1H, dd, J = 1.8 8.1 Hz).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 21.4, 24.0 (Me), 25.3 (Me), 28.1, 71.1, 76.9, 105.0, 113.2, 113.6, 115.7, 122.7, 124.1, 126.3, 129.0, 131.9, 134.6, 143.3 , 151.0, 154.3, 156.4, 161.4, 163.8, 169.8 (C = O).

Example 3 Treatment Effect of Ischemic Brain Disease by HC-12

As described below for the biological efficacy of HC-12, the target compound obtained from the preparation method, specifically for the treatment of ischemic brain disease, neuroprotective effect, glial activity inhibitory activity, and inhibitory effect of NF-κB were confirmed.

Example 3-1. Breeding of Experimental Animals

40 male Mongolian gerbil, Meriones unguiculatus (Halan, USA), weighing between 65 and 75 grams, with constant contrast cycle conditions of light from 7 am to 7 pm, temperature conditions from 21 ° C. to 25 ° C., and 45% to It was bred at 65% relative humidity. For the feed of the experimental animals, general pellet dry feed (Korean experimental animals, Korea) was used, and the feed and water were always available.

Example 3-2. Measurement of Neuronal Protective Effects of HC-12 on Cerebral Ischemia (1)

In order to confirm the neuronal protective effect of HC-12 obtained in Example 2, the experimental animals bred in Example 3-1 were anesthetized by exposing the common carotid artery and ligation to induce cerebral ischemia. Then, the experiment of the method of staining the hippocampal tissue of the animal was observed. The specific method is as follows.

First, after adjusting the content of the HC-12 to 20mg or 40mg per kg body weight dissolved in 500ul of distilled water, it was intraperitoneally administered for 3 days before surgery. On the day of surgery, HC-12 was administered at the same dose and method 30 minutes prior to surgery. General anesthesia of the experimental animals was performed using a gas mixed with 3% isoflurane (isoflurane, Baxtor, USA) in a gas mixture of nitrogen and oxygen 7: 3.

Surgery was performed on the experimental animals while maintaining the anesthesia state of the experimental animals using the gas of 3% isoflurane mixed with the nitrogen and oxygen mixed gas. Surgery for the experimental animals was performed in the following manner.

First, the hair on the neck was cut and disinfected, and then the disinfected area was cut to expose both warm arteries. The exposed carotid artery was ligated for 5 minutes using an aneurysm clip (aneurysm clip, Staelting, USA) to cause cerebral ischemia, and then the clip was removed and reperfused. At this time, each experimental group confirmed the complete occlusion of the entire artery by observing the blood circulation of the central artery of retina using an ophthalmoscope. The body temperature was measured by inserting a rectal thermometer during the induction of the cerebral ischemia, and the body temperature was constantly maintained at a normal body temperature of 36.5 ° C. to 37.5 ° C. using a thermal pad automatically adjusted according to the temperature of the experimental animal.

Experiments on the control group were performed in the same manner as above except that only distilled water used to dissolve HC-12 was used. The normal group was not subjected to ischemia-reperfusion surgery, No substance was administered.

The experimental animals were anesthetized by intraperitoneal injection of thiopental sodium at 30 mg / kg body weight 4 days after induction of cerebral ischemia, and then containing 1,000 IU of heparin per 1,000 ml. Physiological saline at 4 ° C. was injected into the left ventricle and washed perfusion. Perfusion fixation was performed using 4% paraformaldehyde (in 0.1 M phosphate buffer (PB), pH 7.4) at 4 ° C. on the animals after the perfusion wash was completed.

The head bone space of the perfusion-fixed experimental animal was opened using a bone cutter, and the brain was extracted. The brains of the extracted experimental animals were post-fixed for 4 hours using 4% 4% paraformaldehyde (in 0.1 M phosphate buffer (PB), pH 7.4) at 4 ° C. The post-fixed brain is settled in a 30% sucrose solution (in 0.1M phosphate buffer (PB), pH 7.4) until it sinks to the bottom, and then the tissue of the settled brain is slide microtome (sliding microtome, Reichert-Jung, Germany) was cut into 30㎛ thickness to make a tissue section. The tissue sections were stored in 6-well plates containing a storage solution and stored at 4 ° C. until staining was performed.

Among the tissue sections, tissues with well-formed hippocampal formation were selected, and washed three times with 0.01 M PBS for 10 minutes to remove the preservatives from the tissue sections. The washed tissue sections were plated on gelatinized slides and dried sufficiently at 37 ° C. After sufficiently drying the tissue sections in distilled water, the tissue sections were stained for 1 minute in 2% cresyl violet acetate (Sigma, USA) solution. The stained tissue sections were sufficiently washed with running water to remove excess dye from the slides. The tissue sections from which the excess dye was removed were briefly immersed in distilled water and then treated with 50%, 70%, 80%, 90%, 95% and 100% ethanol solution to perform dehydration and excess cresyl violet washing. After confirming that the tissue section was visible in the Nissle body (Nissle body), it was immersed in xylene (Junsei, Japan) and made transparent, and then encapsulated in Canada Balsam (Kanto, Japan).

The encapsulated tissue sections were observed with an AxioM1 microscope (AxioM1 microscope, Carl Zeiss, Germany) attached with a digital camera (Axiocam, Cal Zeiss, Germany), and the observations taken through the camera attached to the microscope were observed. It is shown in Figure 1 divided into the normal group, the control group and the experimental group administered 20mg / kg or 40mg / kg HC-12. The photographing was performed by enlarging the tissue section by 25 times in the entire hippocampal region and 200 times in the CA1 region.

1, A, C, E and G represent the entire hippocampal region, B, D, F and H represent the CA1 region. In addition, A and B of Figure 1 is a normal group, C and D is a control group, E and F is a 20 mg / kg HC-12 administration group, G and H represents the total hippocampal region of 40 mg / kg HC-12 and CA1 region of the hippocampus In order to verify the significance, Figure 1 was taken to pick the most common part of each group.

As can be seen in Figure 1, the normal group (Sham) was able to observe the dark staining cells in the cresyl violet throughout the area of the hippocampus (Fig. 1 A and B). On the other hand, after administration of distilled water only, the control group (vehicle) that caused cerebral ischemia, delayed neuronal death occurred in the course of 4 days after the cerebral ischemia, it was observed that most cells in the CA1 region was not stained (Figures 1 C and D).

In addition, the experimental group administered HC-12, the compound of the present invention, specifically in the experimental group administered HC-12 in the content of 20 mg / kg (Fig. 1 E and F) and 40 mg / kg (Fig. 1 G and H) hippocampus It was confirmed that the cells were stained deeply in the whole area of the HC-12 was effective to protect the cell death caused by cerebral ischemia.

To quantitatively analyze the evaluation of the efficacy, neurons of the tissue sections stained in purple were counted using an image analyzer (Optimas 6.5, USA) program. One-way ANOVA test was performed to verify the significance of each group, and the average value of each data, specifically, the results of counting neurons in tissue sections, is shown in FIG. 2.

As shown in FIG. 2, the control group observed about 12.5% of CA1 region cells in the hippocampus compared to the normal group, but in the administration group administered with HC-12 at 20 mg / kg, about 75% of the neurons counted. In the administration group administered with 40 mg / kg, about 80% of neurons were counted in the normal group, and when the HC-12 was administered, high neuronal cell survival rate was confirmed even when ischemia was induced.

Example  3-3. HC -12 Cerebral ischemia  Nerve cell protection efficacy measurement (2)

In addition, in order to further confirm the protection of neurons by cerebral ischemia after HC-12 administration, immunohistochemistry staining was performed using an antibody against a neuronal specific nuclear protein (NEUN), a marker of neurons.

Were used in the experiments by selecting the above-described embodiment 3-2 tissue sections appear well-hippocampus complex of tissue prepared from the selected tissue section is 0.3% in order to remove endogenous peroxidase (peroxidase) present in the tissue H ₂ It was reacted with O ₂ (in 0.001 M PBS) for 30 minutes. To prevent nonspecific immune responses, the reacted tissue sections were reacted with 3% normal goat serum for 30 minutes. Tissue sections reacted with 3% normal goat serum and mouse anti-NeuN (Chemicon, USA), a primary antibody diluted 1: 800, were reacted at 4 ° C. for 48 hours. After the reaction, the tissue sections were reacted with anti-mouse IgG (Vector), a secondary antibody diluted 1: 200, at room temperature for 2 hours, and then, with a ABC solution (Vector), a third antibody diluted 1: 200. The reaction was carried out for 3 hours, and 3,3'-DAB (diaminobezidine) was developed as a substrate. In performing each step of the reaction with the antibody, the tissue sections were washed three times for 10 minutes using 0.01M PBS for each step.

After the tertiary reaction, the tissue sections were plated on a gelatin-coated slide glass, and dried at room temperature for 12 hours, and the dried tissue sections were enclosed through a conventional dehydration and transparency process. The encapsulated tissue sections were observed using an electron microscope, and photographs of the observation results are shown in FIG. 3.

As shown in FIG. 3, in the normal group, a large number of cells showing immune response to NeuN were observed in the whole hippocampus including CA1, but in the hippocampus of the control group that induced ischemia after administration of a solvent (distilled water), anti- It was confirmed that the cells that showed an immune response to the NeuN antibody, that is, neurons, were significantly reduced.

In the experimental group in which ischemia was induced after administration of the compound of the present invention, HC-12 at 20 mg / kg and 40 mg / kg, a number of NeuN immune-responsive neurons similar to those of the normal group were observed. It was confirmed again that neuroprotective effect by 12-valent cerebral ischemia.

In order to confirm neuronal survival, the nerve cells of the tissue sections stained by immunohistochemical staining using anti-NeuN antibody using an image analyzer (Optimas 6.5, USA) program in the same manner as in Example 3-2 Was counted, and the counted result was found as a relative value.

As shown in FIG. 4, the control group had only about 8.5% of the number of neurons stained by immunohistochemical staining using anti-NeuN antibody in CA1 region cells of the hippocampus compared to the normal group, but HC-12 In the experimental group administered at 20 mg / kg or 40 mg / kg, about 65% and 68% of neurons were counted, respectively, and the HC-12 was found to have a remarkably excellent effect of preventing neuronal death by ischemia. .

Example  3-4. HC -12 Inhibition of Glucoblastic Activity

In order to confirm the effect of HC-12 on the neuronal cell death by ischemia using the experimental animals, the activity change of the glial cells (microglia, astroglia) was confirmed through immunohistochemistry staining.

Tissue sections showing hippocampal complexes among the tissues prepared in Example 3-2 were selected and used in the experiment. The selected tissue sections were subjected to immunohistochemical staining by applying the method described in Example 3-3.

In the immunohistochemical staining process, the primary antibody used for the primary antibody reaction is rabbit anti-GFAP (Glial fibrillary acidic protein) diluted at 1: 1000 as an antibody for confirming glial change. USA), and rabbit anti-Iba-1 (Wako, USA) diluted 1: 1000 was used as an antibody to confirm the change of microprogenitor cells. After performing an immunohistochemical staining process using the primary antibody, the encapsulated tissue sections were observed using an electron microscope, and the photographs of the observation results are shown in FIG. 5.

As shown in FIG. 5, in the experimental group to which HC-12 was administered, the expression of astrocytes was remarkably reduced compared to the control group to which the solvent was administered (FIG. 5E and G), in particular, a marker of microglia. When the expression of Iba-1 was confirmed, Iba-1 expression was clearly observed in the control group (Vehicle) in which the solvent was administered, whereas Iba-1 was hardly observed in the experimental group in which HC-12 was administered. (Sham) was confirmed to a similar degree (FIGS. 5F and 5H). From the above results, it was confirmed that HC-12 not only has a very excellent effect on neuronal cell protection, but also inhibits the expression of microglia and astroglia.

In addition, Fig. 6 shows the result of a graph obtained by calculating the result counted by the method of Example 3-3 as a relative value.

As shown in FIG. 6, in the case of GFAP, which is a marker of astrocytic cells, a value of about 250% of the experimental group was confirmed in the control group (FIG. 6A), and in the case of Iba-1, which is a marker of microglial cells, about 1,700 in the control group. While the value of% was confirmed, the experimental group administered HC-12 showed almost the same value as the normal group (FIG. 6B), and the HC-12 showed activity of glial cells (microglia, astroglia) by ischemia. It was confirmed that the change can be effectively suppressed.

Example  3-5. HC -12 NF - kappaB  Expression inhibition effect measurement

In order to confirm the effect of HC-12 on the neuro-inflammation using the experimental animals, immunohistochemistry staining was performed on the change of NF-κB which is a very important factor in neuro-inflammation induced by neuronal cell death by cerebral ischemia. It was confirmed through.

Tissue sections showing hippocampal complexes among the tissues prepared in Example 3-2 were selected and used in the experiment. The selected tissue sections were subjected to immunohistochemical staining by applying the method described in Example 3-3.

In the immunohistochemical staining, the primary antibody used for the primary antibody reaction was rabbit anti-NF-κB (Vector, CA, USA) diluted 1: 200. After performing an immunohistochemical staining process using the primary antibody, the encapsulated tissue sections were observed using an electron microscope, and the photographs of the observation results are shown in FIG. 7. In addition, the result counted by the method of Example 3-3 was calculated | required as the relative value (relative value when the normal group is set to 100%) with respect to a normal group, and the graph which showed the result is shown in FIG.

As shown in FIG. 7, in the normal group, there was almost no expression of NF-κB (FIGS. 7A and 8), but in the control group, the expression of NF-κB increased rapidly, indicating that neuritis was induced (FIG. 7B). And FIG. 8). In addition, in the experimental group to which the HC-12 of the present invention was administered, the expression level of NF-κB was measured to be almost similar to that of the normal group, and it was confirmed that there was no further expression of NF-κB due to ischemia. The decrease in the expression of NF-κB was measured, and it was confirmed that the HC-12 can suppress neuroinflammation caused by cerebral ischemia, and can effectively prevent the occurrence and further deepening of the disease caused by the neuroinflammation (FIG. 7C, D and FIG. 8).

Example  3-6. HC -12 brain - derived neurotrophic factor ( BDNF ) Expression promotion effect measurement

In order to confirm the neuronal cell death inhibitory effect of HC-12 using the experimental animals, changes in the amount of expression of BDNF known as neuronal cell death protective factor by cerebral ischemia were confirmed by immunohistochemistry staining.

Tissue sections showing hippocampal complexes among the tissues prepared in Example 3-2 were selected and used in the experiment. The selected tissue sections were subjected to immunohistochemical staining by applying the method described in Example 3-3.

In the immunohistochemical staining, the primary antibody used for the primary antibody reaction was rabbit anti-BDNF (Chemicon International, USA) diluted 1: 1,00. After performing an immunohistochemical staining process using the primary antibody, the encapsulated tissue sections were observed using an electron microscope, and the photographs of the observation results are shown in FIG. 9. In addition, a result obtained by counting by the method of Example 3-3 was calculated as a relative value with respect to the normal group, and a graph showing the result is shown in FIG.

As shown in FIG. 9, in the normal group, expression of BDNF was confirmed in the pyramid layer of the hippocampus CA1 region (FIGS. 9A and 10), but in the control group, BDNF immunoreactivity could not be confirmed, and neuronal cell death caused by cerebral ischemia was observed. It was confirmed that the expression level of BDNF was sharply reduced (FIGS. 9B and 10). In addition, in the experimental group administered HC-12 of the present invention, although slightly lower than the normal group, the expression level of BDNF is significantly increased compared to the control group, the HC-12 induces the expression of BDNF in neurons It was confirmed that neuronal cell death by cerebral ischemia can be suppressed (FIGS. 9C, D and 10).

Claims (8)

A composition for treating or preventing ischemic cerebrovascular disease comprising a decursinol derivative having a structure of Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient.
(2)

The method of claim 1,
The ischemic cerebrovascular disease is a composition for treating or preventing ischemic cerebrovascular disease is any one selected from the group consisting of cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage and leukemia. (a) dissolving a compound having a structure of Formula 3 in a methylene chloride solvent and then reacting with a compound having a structure of Formula 5 to prepare a compound having a structure of Formula 7; And
(b) acetylating the compound having the structure of Chemical Formula 7 by acetic anhydride to prepare a compound having the structure of Chemical Formula 2 according to claim 1;
Method for producing a decosinol derivative having a therapeutic or prophylactic effect of ischemic cerebrovascular disease comprising a.
(3)

[Chemical Formula 5]

(7)

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