KR102303899B1 - Pharmaceutical composition comprising oleracone as an active ingredient for preventing or treating brain disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating brain disease comprising oleracone as an active component. Oleracone provided in the present invention has an inhibitory effect on VCAM1 expression, an effect on inhibiting leukocyte adhesion to brain microvascular endothelial cells, and an effect of promoting cognitive functions, so that it is possible to inhibit pathological brain inflammation, and the compounds can be used for preventing or treating brain diseases.

Description

A pharmaceutical composition for preventing or treating brain diseases comprising oleracone as an active ingredient {Pharmaceutical composition comprising oleracone as an active ingredient for preventing or treating brain disease}

The present invention relates to a pharmaceutical composition for preventing or treating brain disease comprising oleracone as an active ingredient.

Alzheimer's disease (Alzheimer's disease) is a disease that shows symptoms such as memory impairment and decreased cognitive function due to selective damage to the brain regions related to cognitive function, particularly the neocortex and hippocampus neurons. It shows pathological features such as neuronal cell death and synaptic loss of the circuit. However, until now, the mechanisms of neuronal cell death, synapse loss, and the formation of neurofibrillary tangles have not been clearly identified, making it difficult to prevent and treat Alzheimer's dementia.

The cytotoxicity of cholinergic neurons due to aggregation of β-amyloid beta (Aβ) is known to be the main cause of Alzheimer's dementia, thus inhibiting the production of Aβ (inhibiting β-secretase and γ-secretase), generating reactive oxygen species due to Aβ, or A variety of approaches to treat Alzheimer's disease are being developed, such as inhibiting inflammation or removing Aβ in the brain using an Aβ antibody. However, since the above strategy is not expected to be significantly effective when the accumulation of Aβ has progressed significantly, the discovery of a new target is required.

Recent systemic inflammation causes oligaemia due to damage to the brain blood barrier or atherosclerosis, leading to increased Aβ production, inhibition of Aβ clearance, and accumulation of harmful substances, leading to Alzheimer’s It has been reported to cause Alzheimer's disease (Zlokovic BV., 2011, Nat Rev Neurosci), and control of the inflammatory response is attracting attention as a new target for Alzheimer's dementia treatment.

Vascular Cell Adhesion Molecule 1 (VCAM1) is a representative cell adhesion molecule that increases due to an inflammatory response. The fact that the concentration of VCAM1 in the plasma of Alzheimer's dementia patients is 1.3 times higher than that of normal people (Zuliani et al., 2008) in relation to Alzheimer's dementia This has been reported In addition, VCAM1 present on the vascular endothelial cell surface is cleaved by activated Matrix Metalloproteinase 9 (MMP9), causing damage to the blood brain barrier (BBB), an entry barrier that primarily protects the brain from invasion of bacteria or lesions ( Kong DH et al., 2018), reported that induced expression of VCAM1 contributes to vascular inflammation (Libby et al., 2002).

In addition, when tumor necrosis factor alpha (TNFα), an inflammation-inducing factor, is treated in human brain microvecular endothelia cells (HBMVEC), VCAM1 expression is increased, resulting in leukocyte adhesion and blood cell leakage. It has been reported that by promoting transmigraion, it promotes the migration of leukocytes into the brain (Choi et al., 2017). Therefore, suppression of VCAM1 expression is expected to be an important target for the development of therapeutic agents for brain diseases including Alzheimer's disease.

Therefore, while the present inventors were trying to find a new compound that inhibits VCAM1 expression in order to prevent or treat brain diseases including Alzheimer's dementia, oleracone inhibits VCAM1 expression, and inhibits leukocyte microvascular endothelial cell adhesion. And the present invention was completed by confirming that there is an effect of enhancing cognitive function.

It is an object of the present invention to provide a cell adhesion inhibitor capable of improving memory and improving cognitive function and preventing or treating brain diseases including Alzheimer's dementia, and providing a pharmaceutical composition for preventing or treating brain diseases comprising the same as an active ingredient will do

In order to achieve the above object,

The present invention provides a pharmaceutical composition for preventing or treating encephalopathy comprising a compound represented by the following Chemical Formula 1, an optical isomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

(In Formula 1,

R ¹ is hydrogen, a hydroxy group or a C _1-5 alkoxy group;

R ² is hydrogen, a hydroxy group or a C _1-5 alkoxy group;

R ³ is hydrogen, a hydroxy group, or a C _1-5 alkoxy group, and R ⁴ is hydrogen, or R ³ and R ⁴ are linked together to form a hexagonal ring, wherein the ring is a carbocyclic ring or at least one oxygen a heterocyclic ring containing atoms, and the bond of the ring consists of a saturated single bond or contains at least one unsaturated double bond; and

R ⁵ is hydrogen, a hydroxy group or a C _1-5 alkoxy group).

Oleracone provided in the present invention can suppress pathological brain inflammation through the effect of inhibiting VCAM1 expression, the effect of inhibiting the adhesion of leukocytes to the brain microvascular endothelial cells, and the effect of enhancing cognitive function. can

1 is a diagram showing TNFα-induced VCAM1 expression levels according to the concentrations of oleracone D, E and F in human brain microvascular endothelial cells (n=3).
1a is a diagram showing the TNFα-induced VCAM1 expression level according to the concentrations of oleacon D, E and F in human brain microvascular endothelial cells through western blotting.
Figure 1b is a graph showing the level of TNFα-induced VCAM1 expression according to the concentration of oleracone D in human brain microvascular endothelial cells.
1c is a graph showing the TNFα-induced VCAM1 expression level according to the concentration of oleracone E in human brain microvascular endothelial cells.
1d is a graph showing the TNFα-induced VCAM1 expression level according to the concentration of oleracone F in human brain microvascular endothelial cells.
2 is a view confirming the degree of HL-60 adherence after culturing oleracone D, E and F with TNFα in human brain microvascular endothelial cells.
3 is a graph showing the adhesion level of HL-60 according to the concentration of oleracone D, E and F cultured with TNFα in human brain microvascular endothelial cells.
3A is a graph showing the level of HL-60 adhesion according to the concentration of oleracone D cultured with TNFα in human brain microvascular endothelial cells.
3B is a graph showing the level of HL-60 adhesion according to the concentration of oleracone E incubated with TNFα in human brain microvascular endothelial cells.
3c is a graph showing the level of HL-60 adhesion according to the concentration of oleracone F cultured with TNFα in human brain microvascular endothelial cells.
4 is 2X AD Tg mice divided into four groups of vehicle (10% cremopher in saline)/wild, vehicle/Tg, oleracone F/wild and oleracone F/Tg, showing whether cognitive function is recovered after treatment with oleracone F It is also

Hereinafter, the present invention will be described in detail.

One aspect of the present invention provides a pharmaceutical composition for preventing or treating brain disease containing a compound represented by the following Chemical Formula 1, an optical isomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient to provide.

[Formula 1]

(In Formula 1,

R ¹ is hydrogen, a hydroxy group or a C _1-5 alkoxy group;

R ² is hydrogen, a hydroxy group or a C _1-5 alkoxy group;

R ³ is hydrogen, a hydroxy group, or a C _1-5 alkoxy group, and R ⁴ is hydrogen, or R ³ and R ⁴ are linked together to form a hexagonal ring, wherein the ring is a carbocyclic ring or at least one oxygen a heterocyclic ring containing atoms, and the bond of the ring consists of a saturated single bond or contains at least one unsaturated double bond; and

R ⁵ is hydrogen, a hydroxy group or a C _1-5 alkoxy group).

On the other hand,

The compound represented by the formula (1) is a compound represented by the following formula.

(In Formula 1,

R ¹ is hydrogen, a hydroxy group or a methoxy group;

R ² is hydrogen, a hydroxy group or a methoxy group;

이고, 여기서

는 단일결합 또는 이중결합을 나타내고; 및R ³ is hydrogen, a hydroxy group or a methoxy group and R ⁴ is hydrogen, or R ³ and R ⁴ are taken together

and where

represents a single bond or a double bond; and

R ⁵ is hydrogen, a hydroxy group or a methoxy group.)

In another aspect,

The compound represented by Formula 1 is preferably any one selected from the following compound group.

(1) 5-hydroxy-3-(2-hydroxybenzyl)-7-methoxychroman-4-one;

(2) 1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(2-hydroxyphenyl)propa-1-one; and

(3) 3-(2-hydroxybenzyl)-5,7-dimethoxy-4H-chromen-4-one.

delete

(1) 5-hydroxy-3-(2-hydroxybenzyl)-7-methoxychroman-4-one

(2) 1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(2-hydroxyphenyl)propa-1-one

(3) 3-(2-hydroxybenzyl)-5,7-dimethoxy-4H-chromen-4-one

The compound represented by Formula 1 of the present invention binds to the promoter region of VCAM1, demethylates the promoter region of VCAM1 to regulate the expression of VCAM1, thereby reducing the expression of vascular adhesion proteins such as VCAM1 induced by TNFα, and , thereby reducing the adhesion level of HL-60, a white blood cell, and reducing vascular inflammation. As a result, the compound inhibits the functional decline or dysfunction of brain microvasculature and improves cognitive function to prevent or treat brain diseases.

The compound represented by Formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, etc., aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. Non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids, etc., organic acids such as trifluoroacetic acid, acetate, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, etc. get it from Examples of such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and Odide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, sube Late, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and the like.

The acid addition salt according to the present invention can be prepared by a conventional method, for example, a precipitate formed by dissolving the derivative of Formula 1 in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc. and adding an organic or inorganic acid It can be prepared by filtration and drying, or by distilling the solvent and excess acid under reduced pressure, followed by drying, and crystallization in an organic solvent.

In addition, bases can be used to prepare pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. In this case, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt. The corresponding salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg silver nitrate).

Furthermore, the present invention includes not only the compound represented by Formula 1 and pharmaceutically acceptable salts thereof, but also solvates, optical isomers, hydrates, and the like, which can be prepared therefrom.

The term “hydrate” refers to a compound of the present invention comprising a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolec μLar forces. or salts thereof. The hydrate of the compound represented by Formula 1 of the present invention may include a stoichiometric or non-stoichiometric amount of water that is bound by non-covalent intermolecular forces. The hydrate may contain 1 equivalent or more, preferably, 1 to 5 equivalents of water. Such a hydrate may be prepared by crystallizing the compound represented by Formula 1 of the present invention, an isomer thereof, or a pharmaceutically acceptable salt thereof from water or a solvent containing water.

The term “solvate” means a compound of the invention or a salt thereof which contains either a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Preferred solvents therefor include solvents that are volatile, non-toxic, and/or suitable for administration to humans.

The term “isomer” refers to a compound of the present invention or a salt thereof that has the same chemical formula or molecular formula but differs structurally or sterically. Such isomers include structural isomers such as tautomers, stereoisomers such as R or S isomers having an asymmetric carbon center, geometric isomers (trans, cis), and optical isomers (enantiomers). All these isomers and mixtures thereof are also included within the scope of the present invention.

The brain disease is stroke, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, alcoholic cranial nerve disease, spinal cord injury ) and alcoholic dementia and Wernicke-Korsakoff's syndrome (Wernicke-Korsakoff's syndrome) is preferably any one selected from the group consisting of.

The compound represented by Formula 1 or a pharmaceutically acceptable salt thereof may be administered in various oral and parenteral formulations during clinical administration. In the case of formulation, it is prepared using diluents or excipients, such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in one or more compounds, for example, starch, calcium carbonate, sucrose or lactose ( lactose), gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, and emulsions. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

A pharmaceutical composition comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient may be administered parenterally, and parenteral administration is administered by subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. depending on how

At this time, in order to formulate a formulation for parenteral administration, the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof is mixed with water together with a stabilizer or buffer to prepare a solution or suspension, which is an ampoule or vial unit dosage form. can be manufactured with The composition may be sterilized and/or contain adjuvants such as preservatives, stabilizing agents, wetting or emulsifying agents, salts and/or buffers for regulating osmotic pressure, and other therapeutically useful substances, and mixing, granulation, in a conventional manner. It can be formulated according to the method of formulation or coating.

Formulations for oral administration include, for example, tablets, pills, hard/soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, troches, and the like. , dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine), lubricants (eg silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol). Tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine and the like, and optionally boron such as starch, agar, alginic acid or its sodium salt. It may contain releasing or boiling mixtures and/or absorbents, colorants, flavoring agents, and sweetening agents.

The dosage of the pharmaceutical composition of the present invention varies depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate and severity of disease, and any method is within the scope of the present invention. is not limited to Individual dosages specifically contain the amount in which the active drug is administered at one time.

In a specific embodiment of the present invention, when oleracone D, E and F are treated, the compounds demethylate the promoter region of VCAM1 to regulate the expression of VCAM1, so that the expression level of VCAM is reduced in a concentration-dependent manner (Fig. 1). See), the degree of adhesion of HL-60 induced by TNFα is decreased in a concentration-dependent manner when treated with oleracone D, E and F (see FIGS. 2 and 3), and lowered in Alzheimer's dementia model mice administered with vehicle. By confirming that memory is improved upon administration of oleracone F (see FIG. 4 ), oleracone of the present invention can be usefully used as a pharmaceutical composition for the prevention or treatment of brain diseases.

Hereinafter, the present invention will be described in detail by way of Examples and Experimental Examples.

However, the following Examples and Experimental Examples are only for illustrating the present invention, and the present invention is not limited by the following Examples and Experimental Examples.

<Preparation Example 1> Preparation of oleracone D, E and F

1-1. Overview of Manufacturing Reaction

Unless otherwise noted, all reactions were performed in dry solvent under anhydrous conditions and an argon atmosphere. Reaction flasks were flamedried prior to use, and all solvents for extraction and chromatography were reagent grade. All reagents were purchased from suppliers and used without further purification. Thin-layer chromatography (TLC) was performed to monitor the reaction progress on 0.25 mm silica gel plates (Merck. Kenilworth, NJ, USA), and silica gel 60 (230-400 mesh, Merck, Kenilworth, NJ, USA) was used for flash column chromatography with the indicated solvents. 1H and 13C spectra were recorded with a Brucker Analytik ADVANCE digital 500 (500 MHz) (Billerica, MA, USA) or BRUKER AVANCE-800 (800 MHz) (Billerica, MA, USA). 1H-NMR data are chemical shift, multiplicity (singlet, s; doublet, d; triplet, t; quartet, q; broad b; and/or multiple resonances), coupling constants in Hertz (Hz) and The number of protons is indicated. Chemical shifts (unit: ppm) are expressed downfield from tetramethylsilane (TMS) and refer to deuterated solvents (CDCl3 and DMSO-d6). IR (infrared) spectra were recorded with an FT-IR-4200 (JASCO, Tokyo, Japan) spectrometer. In addition, low-resolution and high-resolution mass spectra were recorded with a JMS-700 (JEOL, Tokyo, Japan).

[Scheme 1]

1-2. Synthesis of starting materials

Starting material (E)-3-(2-(benzyloxy)phenyl)-1-(2-hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one ((E)- To synthesize 3-(2-(Benzyloxy)phenyl)-1-(2-hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one), ethyl alcohol (11 mL) and KOH (400 mg, 7.14) in a solution of 4,6-dimethoxy-2-hydroxybenzophenone 4 (700 mg, 3.57 mmol) and 2-(benzyloxy)benzaldehyde 5(2-( benzyloxy)benzaldehyde 5) (1100 mg, 5.35 mmol) was added at room temperature. Then, after stirring at the same temperature for 48 hours, the reaction mixture was concentrated in vacuo. The reaction mixture was quenched with 1N aqueous hydrochloric acid solution and extracted with EtOAc (ethyl acetate). The combined organic layers were washed with saturated sodium bicarbonate solution, dried over anhydrous magnesium sulfate, and concentrated in vacuo. Purification of the concentrated residue by flash column chromatography (EtOAc/n-hexane = 1:40 to 1:10) gave 1500 mg (75%) of chalcone 6 as a yellow powder.

m.p.: 114-115 °C;

Rf = 0.25 (EtOAc: n-Hexane = 1: 10);

¹ H NMR (800 MHz, CDCl3) δ 14.37 (s, 1H), 8.17 (d, 1H, J = 15.8 Hz), 8.02 (d, 1H, J = 15.7 Hz), 7.62 (dd, 1H, J = 1.5) , 7.7 Hz), 7.46 (d, 2H, J =7.6 Hz), 7.39-7.37 (m, 2H), 7.33-7.30 (m, 2H), 7.00 (t, 1H, J = 7.5 Hz), 6.87 (d , 1H, J = 8.2 Hz), 6.10 (d, 1H, J = 2.3 Hz), 5.91 (d, 1H, J = 2.3 Hz), 5.20 (s, 2H), 3.83 (s, 3H), 3.72 (s) , 3H);

¹³ C NMR (200 MHz, CDCl3) δ 193.3, 168.4, 166.2, 162.6, 157.9, 138.1, 136.9, 131.4, 129.5, 128.8, 128.5, 128.1, 127.3, 125.1, 121.2, 112.9, 106.6, 93.8, 93.8, 91.3, 70.5, 55.7, 55.7;

IR (thin film, neat) v _max 1620, 1595, 1553, 1452, 1344, 1213, 1107, 984, 816, 760 cm ^-1 ;

LR-MS (Low-Resolution Mass Spectroscopy) (FAB+) m/z 391 (M+H+);

HR-MS (High-Resolution Mass Spectroscopy) (FAB+) C ₂₄ H ₂₃ O ₅ (M + H+) The calculated value is 391.1545; The observed figure is 391.1541.

<Example 1> Preparation of oleracone E

Oleracone E

To a solution of ethyl alcohol (10 mL) and the above chalcone 6 (500 mg, 1.28 mmol) was added a catalytic amount of 5% activated palladium on carbon at room temperature. After stirring under H ₂ atmosphere for 48 h, the reaction mixture was filtered through a pad of celite and concentrated in vacuo. Purification of the concentrated residue by flash column chromatography (EtOAc : n-hexane = 1 : 10 to 1 : 3) gave 340 mg (88 %) of oleracone E as a yellow powder.

m.p.: 137-138 ° C; Rf = 0.20 (EtOAc/n-Hexane = 1:5);

¹ H NMR (500 MHz, DMSO-d6) δ 13.63 (brs, 1H), 9.30 (brs, 1H), 7.06 (dd, 1H, J = 1.5, 7.4 Hz), 6.99 (brtd, 1H, J = 1.6, 7.7 Hz), 6.77 (dd, 1H, J = 1.0, 8.0 Hz), 6.70 (td, 1H, J = 1.0, 7.4 Hz), 6.11 (d, 1H, J = 2.4 Hz), 6.09 (d, 1H, J = 2.4 Hz), 3.82 (s, 3H), 3.80 (s, 3H), 3.20 (brt, 2H, J = 7.7 Hz), 2.81 (brt, 2H, J = 7.7 Hz);

¹³ C NMR (200 MHz, DMSO-d6) δ 205.4, 166.1, 166.0, 162.9, 155.5, 130.3, 127.7, 127.5, 119.5, 115.3, 106.0, 94.2, 91.2, 56.4, 56.1, 43.9, 25.5;

IR (thin film, neat) v _max 1618, 1578, 1489, 1462, 1418, 1362, 1204, 1109, 999, 829, 758 cm ^-1 ;

LR-MS (FAB+) m/z 303 (M + H ⁺ );

HR-MS (FAB+) C ₁₇ H ₁₉ O ₅ (M + H ⁺ ) calculated 303.1232; The observed figure is 1226.

<Example 2> Preparation of oleracone F

Oleracone F

N,N-Dimethylformamide dimethyl acetal (0.053 mL, 0.40 mmol) was added to a solution of the above oleracone E (100 mg, 0.33 mmol) in toluene (5.5 mL). The reaction mixture was refluxed for 2 h, then cooled to room temperature and the reaction mixture was concentrated in vacuo. Purification of the concentrated residue by flash column chromatography (EtOAc : n-hexane = 1 : 2) gave 93 mg (90 %) of oleracone F as a yellow powder.

m.p.: 224-225 °C; Rf = 0.23 (EtOAc/n-Hexane = 1:2);

¹ H NMR (500 MHz, DMSO-d6) δ 9.46 (s, 1H), 7.88 (s, 1H), 7.07 (dd, 1H, J = 1.5, 7.5 Hz), 7.01 (brtd, 1H, J = 1.6, 7.7 Hz), 6.78 (dd, 1H, J = 1.0, 8.0 Hz), 6.70 (brtd, 1H, J = 1.1, 7.4 Hz), 6.61 (d, 1H, J = 2.3 Hz), 6.48 (d, 1H, J = 2.3 Hz), 3.86 (s, 3H), 3.81 (s, 3H), 3.52 (s, 2H);

¹³ C NMR (200 MHz, DMSO-d6) δ 175.8, 164.1, 160.9, 160.0, 155.4, 151.9, 130.5, 127.9, 125.7, 123.8, 119.6, 115.6, 108.7, 96.4, 93.3, 56.5, 56.3, 25.6;

IR (thin film, neat) v _max 1643, 1595, 1566, 1454, 1373, 1207, 1152, 1078, 824, 752 cm ^-1 ;

LR-MS (FAB+) m/z 313 (M + H ⁺ );

HR-MS (FAB+) C ₁₈ H ₁₇ O ₅ (M + H ⁺ ) calculated 313.1076; The observed figure is 313.1078.

<Example 3> Preparation of oleracone D (olerecone D)

Oleracone D

To a solution of the above oleracone F (150 mg, 0.48 mmol) in methylene chloride (5 mL), 1.92 mL of BCl ₃ (1 M CH ₂ Cl ₂ solution, 1.92 mmol) was added slowly at 0 °C. After stirring at the same temperature for 1 h, the reaction mixture was _{quenched with H 2} O and diluted with methylene chloride. After stirring for an additional 1 hour, extraction was performed with methylene chloride. The combined organic layers were washed with brine, dried over anhydrous magnesium sulfate and concentrated in vacuo. Purification of the concentrated residue by flash column chromatography (EtOAc: n-hexane = 1: 5) gave 114 mg (80 %) of oleracone D as a yellow powder.

m.p.: 154-155 °C;

Rf = 0.31 (EtOAc: n-Hexane = 1: 5);

¹ H NMR (500 MHz, DMSO-d6) δ 12.76 (s, 1H), 9.47 (brs, 1H), 8.06 (s, 1H), 7.08 (dd, 1H, J = 1.4, 7.5 Hz), 7.03 (brtd) , 1H, J = 1.6, 7.7 Hz), 6.81 (dd, 1H, J = 0.8, 8.0 Hz), 6.71 (brtd, 1H, J = 1.4, 7.4 Hz), 6.61 (d, 1H, J = 2.3 Hz) , 6.38 (d, 1H, J = 2.3 Hz), 3.84 (s, 3H), 3.61 (s, 2H);

¹³ C NMR (200 MHz, DMSO-d6) δ 181.6, 165.6, 161.6, 158.2, 155.4, 155.3, 130.4, 128.0, 125.0, 121.4, 119.5, 115.4, 105.5, 98.4, 92.8, 56.4, 25.0;

IR (thin film, neat) v _max 1647, 1585, 1510, 1435, 1368, 1240, 1163, 1051, 822, 754 cm ^-1 ;

LR-MS (FAB+) m/z 299 (M + H ⁺ );

HR-MS (FAB+) C ₁₇ H ₁₅ O ₅ (M + H ⁺ ) calculated value is 299.0919; The observed figure is 299.0915.

Chemical structural formulas of the examples are shown in the table below.

Example constitutional formula One

2

3

<Experimental Example 1> Analysis of the effect of oleracone D, E and F on TNFα-induced adhesion protein (VCAM1) expression in HBMVEC

Increased VCAM1 expression by inflammation in vascular endothelial cells is an early pathological phenomenon of Alzheimer's dementia, leading to Aβ accumulation and encephalitis. Therefore, in the present invention, a compound that modulates the expression of VCAM1 is searched for and used for the prevention and treatment of Alzheimer's dementia.

To search for compounds that modulate VCAM1 expression in brain microvascular endothelial cells, human brain microvascular endothelial cells (HBMVEC, Cell Systems (Kirkland, WA)) were treated with 20% fetal bovine serum (Fetal bovine). M199 medium containing serum; FBS), penicillin (100 U/ml), streptomycin (100 μg/ml), 3 ng/ml basal fibroblast growth factor (bFGF) and 5 U/ml heparin incubated in

When the cell density reached 100% (100% confluency) after dispensing the cells into the medium, oleracone D, E and F (0, 0.2, 1, 5, 25 and 50 μM) were added to TNFα (10 ng /ml; sigma T6674) was treated 1 hour before treatment, and further cultured for 8 hours after treatment with TNFα. Then, the cells were lysed and the protein was extracted to analyze the VCAM1 protein level (FIG. 1).

Specifically, the VCAM1 protein level was measured by western blotting. First, TNFα-treated cells were washed twice with 1x ice-cold PBS, and then 100 μl Lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate) , SDS) and 1 mM ethylenediamine tetraacetic acid (EDTA) were added, the cells were collected with a scrapper, placed in a 1.5 ml tube, and reacted on ice for 30 minutes. After centrifugation at 4° C. at 13,000 rpm for 15 minutes, the supernatant was collected and protein was quantified using the BCA protein assay (Pierce) method.

After that, 20 μg of the separated protein was electrophoresed on 8% SDS-PAGE, and then washed at 100 V for 90 minutes using a transfer kit (Invitrogen) to transfer the protein to PVDF membrane (Millipore Cat# IPVH 00010). After loading, the membrane to which the protein has been transferred is treated with blocking buffer (5% skim milk in Tris-buffered saline-tween20; TBS-T; 25 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.1% (v/v) Tween 20). ) and reacted for 1 hour. Then, the blocking buffer was removed, and the membrane was reacted at 4° C. for 16 hours in blocking buffer to which the primary antibody anti-β-actin (Abcam ab6276) was added. After reacting for 1 hour at room temperature in blocking buffer to which HRP-conjugated secondary antibody was added, non-specific binding was removed with TBS-T, and protein expression was observed by ECL (enhanced chemiluminescence, Amersham Life Science).

As a result of the experiment, it was confirmed that the expression level of VCAM1 protein induced by TNFα in cells treated with oleacon D, E and F was decreased in a concentration-dependent manner ( FIGS. 1b , 1c and 1d ). These results indicate that oleracone D, E and F inhibit the expression of VCAM1 induced by TNFα.

<Experimental Example 2> Analysis of the effect of oleracone D, E and F on the adhesion of HL-60 in HBMVEC

Adhesion assay was performed to determine whether oleacon D, E and F inhibited TNFα-induced adhesion of HL-60 to brain microvascular endothelial cells.

HL-60, a human leukemia cell, was obtained from the Korean cell line bank (Seoul, Korea) and used, and 10% Fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 μg/ml) was cultured in RPMI-1640 (Hyclone).

After dispensing brain microvascular endothelial cells in a 12-well plate, they were cultured in an incubator until the endothelial cell density reached 100% (100% confluency). Thereafter, oleracone D, E and F (0, 0.2, 1, 5 and 25 μM) were treated with TNFα (10 ng/ml; sigma T6674) 1 hour before treatment, and further cultured for 8 hours after TNFα treatment. Then, the growth medium was removed, and leukocytes HL-60 (1×10 ⁵ cells/500 μl growth media) were put into each well, and then cultured at _{37° C./CO 2 in an incubator for 1 hour.} HL-60 cells not attached to endothelial cells were removed by suction and washed with endothelial cell proliferation medium (M199 culture medium).

As a result of the experiment, it was confirmed that the adhesion of HL-60 induced by TNFα by oleacon D, E and F was decreased in a drug concentration-dependent manner ( FIGS. 2 and 3 ). These results indicate that oleracone D, E and F have the effect of inhibiting HL-60 from adhering to brain microvascular endothelial cells.

<Experimental Example 3> Cognitive behavioral experiment using Alzheimer's dementia model mice

In order to confirm whether the compound of the present invention has an effect of improving cognitive function, a memory behavioral experiment with oleracone F among the compounds was performed.

In this experiment, a 12-month-old swedish mutant APP/PS Δ9 deletion double transgenic mouse (2X AD Tg mouse) was used. AD Tg mice were divided into four groups: vehicle (10% cremopher in saline)/wild, vehicle/Tg, oleracone F/wild, and oleracone F/Tg, and each drug (200 μl) was administered by intraperitoneal injection for 28 days. did it After that, the passive avoidance task test was conducted to evaluate whether it helps improve memory ability.

Specifically, as an experiment to evaluate the long-term memory ability of mice, the experiment was conducted over a total of 2 days using a box divided into a dark field and a light field. Before entering this experiment, they were acclimatized for 5 minutes in a light field. First, put the mouse in a bright area and wait 30 seconds, then open the door to the dark field, measure the time it takes to enter, wait up to 120 seconds, and close the door when the mouse enters the dark room. After 5 seconds of entering the dark room, 0.3 mA of electrical stimulation was applied for 3 seconds, and after waiting 1 minute, the mice were taken out. After 24 hours, the memory test was performed, and the time it took for the mouse to open and enter the dark field was measured after putting the mouse into the light field and waiting 30 seconds without any adaptation process for 5 minutes. After that, when the mouse enters, it waits 300 seconds for the door to close.

As a result of the experiment, it was confirmed that the reduced memory of the vehicle-administered Alzheimer's dementia model mice was improved upon administration of oleracone F (FIG. 4).

Through the above results, oleracone D, E and F inhibit the dysfunction of the blood-brain barrier by blocking the migration of leukocytes to the brain caused by the increase in the expression of VCAM1 and the increase in the expression of the vascular adhesion protein, and prevent pathological brain inflammation. By suppressing it, it was confirmed that the cognitive decline seen in Alzheimer's dementia could be suppressed. Therefore, the compound can be usefully used as a composition for preventing or treating brain diseases.

Claims (8)

delete delete 3-(2-hydroxybenzyl)-5,7-dimethoxy-4H-chromen-4-one as an active ingredient,
Characterized in suppressing brain inflammation by reducing the expression of VCAM1 and inhibiting the adhesion of leukocytes to brain microvascular endothelial cells,
A pharmaceutical composition for preventing or treating Parkinson's disease, Alzheimer's disease, Huntington's disease, or alcoholic dementia.
delete delete According to claim 3, wherein the white blood cells (leukocyte) is neutrophil (neutrophil), eosinophil (eosinophil), characterized in that any one or more of basophils (basophil), the pharmaceutical composition.
The pharmaceutical composition according to claim 3, wherein the composition enhances cognitive function.

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