KR102024222B1 - Pharmaceutical composition for preventing or treating Prion disease containing 2-(((5,7-dimethylimidazo[1,2-a]pyrimidin-2-yl)methyl)thio)-N-(2-methylphenyl)benzo[d]oxazole-5-sulfonamide, its derivative ethyl 5-(2-((5-(N-(2-methoxyphenyl)sulfamoyl)benzo[d]oxazol-2-yl)thio)acetyl)-2,4-dimethyl-1H-pyrrole-3-carboxylate as an active ingredient - Google Patents

Abstract

The present invention relates to 2-(((5,7-dimethylimidazo [1,2-a] pyrimidin-2-yl) methyl) thio) -N- (2-methoxyphenyl) benzo [d] oxazole -5-sulfonamide [2-(((5,7-dimethylimidazo [1,2-a] pyrimidin-2-yl) methyl) thio) -N- (2-methylphenyl) benzo [d] oxazole-5-sulfonamide ] Compound or ethyl 5- (2-((5- (N- (2-methoxyphenyl) sulfamoyl) benzo [d] oxazol-2-yl) thio) acetyl) -2,4-dimethyl-1H- Pyrrole-3-carboxylate [ethyl 5- (2-((5- (N- (2-methoxyphenyl) sulfamoyl) benzo [d] oxazol-2-yl) thio) acetyl) -2,4-dimethyl-1H- A pyrrole-3-carboxylate] compound or a pharmaceutically acceptable salt of the compound as an active ingredient in a pharmaceutical composition for preventing or treating prion diseases, the compound inhibits the formation of needles of the prion protein and , Cytotoxicity, and inhibits the expression of the modified prion in a cell or animal model expressing the modified prion, so that the pharmaceutical composition for preventing or treating prion diseases It may be useful as Hyosung minutes.

Description

2-(((5,7-dimethylimidazo [1,2-a] pyrimidin-2-yl) methyl) thio) -N- (2-methoxyphenyl) benzo [d] oxazole-5- Sulfonamide or derivatives thereof ethyl 5- (2-((5- (N- (2-methoxyphenyl) sulfamoyl) benzo [d] oxazol-2-yl) thio) acetyl) -2,4-dimethyl Pharmaceutical composition for preventing or treating Prion disease containing 2-(((5,7-dimethylimidazo [1,2-a ] pyrimidin-2-yl) methyl) thio) -N- (2-methylphenyl) benzo [d] oxazole-5-sulfonamide, its derivative ethyl 5- (2-((5- (N- (2-methoxyphenyl) sulfamoyl ) benzo [d] oxazol-2-yl) thio) acetyl) -2,4-dimethyl-1H-pyrrole-3-carboxylate as an active ingredient}

The present invention relates to 2-(((5,7-dimethylimidazo [1,2-a] pyrimidin-2-yl) methyl) thio) -N- (2-methoxyphenyl) benzo [d] oxazole -5-sulfonamide or derivative thereof ethyl 5- (2-((5- (N- (2-methoxyphenyl) sulfamoyl) benzo [d] oxazol-2-yl) thio) acetyl) -2, It relates to a pharmaceutical composition for preventing or treating prion diseases, including 4-dimethyl-1H-pyrrole-3-carboxylate as an active ingredient.

Infectious spongiform encephalopathy is a fatal neurodegenerative disease caused by prion, a proteinaceous infectious particle. Examples include Bovine Spongiform Encephalopathy (BSE), Scrapie of sheep or goats, Creutzfeldt-Jacob Disease (CJD) in humans, Gerstmann-Straussler-Shinker syndrome (Gerstmann-Straussler-Scheinker Syndrome), Kuru, Mink's Transmissible Mink Encephalopathy, Chronic Wasting Disease of Deer, and Feline Spongiform Encephalopathies.

Infectious spongiform encephalopathy is the generation of a modified prion infectious protein causes, specifically, to develop as compass the formation of normal prion protein, protein folding (protein folding) a modified prion protein (Prp ^sc) blossomed over the (Prp ^c) . Modified prion protein, like normal prion protein in the body, does not have an immunogenicity and does not cause a host's immune response, structurally increased β-sheet, and partially resistant to proteinase K. Known.

To date, many researchers have been working on the mechanism of denaturation of normal prion proteins, but the exact cause of this is unknown. Therefore, the development of treatment based on the cause of the development is very insufficient. Aguzzi and A. of the University of Zurich have shown that the introduction of anti-prion genes in mice with prion removed prevents prion diseases by producing antibodies to denatured prions in the body (Science. 2001. 294: 178). -182), it was concluded that mice are difficult to use as therapeutic agents in that they have antibodies from birth. Stanley B. Prusiner and colleagues have reported that quinacrine, which is used to treat malaria, inhibits the conversion of prion proteins to pathogenic forms (PNAS. 2001. 98: 9836-9841), and Cashman N. The researchers found that antibodies that act directly on specific amino acid sequences of prion proteins selectively recognize only denatured prion proteins (Nature Medicine. 2003. 9: 819-820). In addition, in 2007, Kazuo Kuwata and colleagues at the Gifu University of Japan noted that the distance between the 159th aspartic acid (Asp) and the 196th glutamic acid (Glu) was 3 strains longer than normal prions, and it suppressed this shape change and contagious. We have designed and reported a compound called GN-8 that can inhibit the progression of cavernous encephalopathy (PNAS. 2007. 104: 11921-11926). The results suggest that the cause of degeneration can be used as a therapeutic agent for infectious spongiform encephalopathy by inhibiting degeneration of normal prions separately.

Prion disease, once onset, is very serious because it is very similar to dementia and has a 100% mortality rate. Until now, no appropriate prevention or treatment has been developed, and there is insufficient early diagnosis. Therefore, it is urgent to develop inhibitors to suppress prion diseases. Accordingly, the present inventors have tried to develop a novel therapeutic agent for prion disease that inhibits the formation of the prion protein, resulting in 2-(((5,7-dimethylimidazo [1,2-a] pyrimidin-2-yl. ) Methyl) thio) -N- (2-methoxyphenyl) benzo [d] oxazole-5-sulfonamide [2-(((5,7-dimethylimidazo [1,2-a] pyrimidin-2-yl) methyl) thio) -N- (2-methylphenyl) benzo [d] oxazole-5-sulfonamide] or a derivative thereof ethyl 5- (2-((5- (N- (2-methoxyphenyl) sulfamoyl) benzo [d] oxazol-2-yl) thio) acetyl) -2,4-dimethyl-1H-pyrrole-3-carboxylate [ethyl5- (2-((5- (N- (2-methoxyphenyl) sulfamoyl) benzo [d] oxazol-2-yl) thio) acetyl) -2,4-dimethyl-1H-pyrrole-3-carboxylate] inhibits the formation of acupuncture of prion protein and does not have cytotoxicity, so By confirming that it can be usefully used as an active ingredient, the present invention was completed.

An object of the present invention is to provide a pharmaceutical composition or health food for the prevention or treatment of prion disease comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

(In Formula 1,

또는

이다.), R ¹ is

or

to be.),

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating prion disease comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

(In Formula 1,

또는

이다.)R ¹ is

or

to be.)

In addition, the present invention provides a health food for preventing or improving a prion disease (Prion) comprising a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

2-(((5,7-dimethylimidazo [1,2-a] pyrimidin-2-yl) methyl) thio) -N- (2-methoxyphenyl) benzo [d] oxazole of the invention -5-sulfonamide [2-(((5,7-dimethylimidazo [1,2-a] pyrimidin-2-yl) methyl) thio) -N- (2-methylphenyl) benzo [d] oxazole-5-sulfonamide Or ethyl 5- (2-((5- (N- (2-methoxyphenyl) sulfamoyl) benzo [d] oxazol-2-yl) thio) acetyl) -2,4-dimethyl-1H-pyrrole 3-carboxylate [ethyl5- (2-((5- (N- (2-methoxyphenyl) sulfamoyl) benzo [d] oxazol-2-yl) thio) acetyl) -2,4-dimethyl-1H-pyrrole- 3-carboxylate] is useful as an active ingredient of the pharmaceutical composition for preventing or treating prion diseases because it inhibits the formation of the prion protein, has no cytotoxicity, and inhibits the expression of the modified prion in a cell or animal model expressing the modified prion. Can be used.

1 is a diagram showing a docked simulated binding mode of GN-8 known to bind to prion monomer stabilization sites.
2 is a diagram showing a hot spot region for stabilizing prion monomers.
Figure 3 is a diagram showing the docking simulated main binding site of the compound of the present invention (Formula 2) to bind to the prion monomer stabilization site.
Figure 4 is a diagram showing the docked simulated main binding site of the compound of the present invention (Formula 3) to bind to the prion monomer stabilization site.
5 is a diagram confirming the effect of inhibiting the formation of the needle on the prion protein according to the compound of the present invention.
Figure 6 is a diagram showing the toxicity test results of Na2 neurons according to the compound treatment of the present invention.
Figure 7 is a diagram showing the effect of inhibiting the expression of the modified prion by the compound treatment of the present invention in the cell line ScN2a cells expressing the modified prion.
Figure 8 is a diagram showing the effect of inhibiting the expression of the modified prion by the compound treatment of the present invention in the brain tissue of the animal model expressing the modified prion.
9 is a view showing the life extension effect of the animal model by the compound treatment of the present invention in an animal model expressing modified prion.

Hereinafter, the term of this invention is demonstrated.

As used herein, the term "prevention" means any action that inhibits or retards the onset of administration of the composition.

In the present invention, "improvement" or "treatment" means any action that improves or advantageously changes the condition of the disease by administration of the composition.

In the present invention, "administration" means providing the patient with the desired substance in any suitable way, and the route of administration of the composition of the present invention is oral or parenteral via all common routes as long as the target tissue can be reached. Oral administration. In addition, the composition can be administered by any device in which the active agent can migrate to the target cell.

In the present invention, "pharmacophore" is a three-dimensional arrangement of chemical groups (or features) that a drug (ligand) must have in order to be effective against a target disease or target protein. In particular, in order for a compound to bind well with a protein and increase or inhibit its activity, the definition of an active skeleton is very important. The active skeleton consists of a kind of features called hydrogen bond donor (HBA), hydrogen bond acceptor (HBD), and hydrophobic site (Lipo), and they are properly combined and expressed in a map form.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating prion disease, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

(In Formula 1,

또는

이다.)R ¹ is

or

to be.)

The compound represented by Formula 1 is 2-(((5,7-dimethylimidazo [1,2-a] pyrimidin-2-yl) methyl) thio) -N- (2-methoxyphenyl) benzo [d] oxazole-5-sulfonamide [2-(((5,7-dimethylimidazo [1,2-a] pyrimidin-2-yl) methyl) thio) -N- (2-methylphenyl) benzo [d] oxazole-5-sulfonamide] or ethyl 5- (2-((5- (N- (2-methoxyphenyl) sulfamoyl) benzo [d] oxazol-2-yl) thio) acetyl) -2,4- Dimethyl-1H-pyrrole-3-carboxylate [ethyl 5- (2-((5- (N- (2-methoxyphenyl) sulfamoyl) benzo [d] oxazol-2-yl) thio) acetyl) -2,4- dimethyl-1H-pyrrole-3-carboxylate].

The compound represented by Formula 1 may be to inhibit the prion protein sedimentation.

The prion disease is mad cow disease, mad cow disease, spongiform encephalophathy (BSE) or Creutzfeldt-Jakob disease (CJD), but is not limited thereto.

In a specific embodiment of the present invention, the present inventors have predicted that the 2-(((5,7-dimethylimidazo [1] is expected to be active in stabilizing prion monomers through protein structure-based virtual drug discovery. , 2-a] pyrimidin-2-yl) methyl) thio) -N- (2-methoxyphenyl) benzo [d] oxazole-5-sulfonamide [2-(((5,7-dimethylimidazo [1 , 2-a] pyrimidin-2-yl) methyl) thio) -N- (2-methylphenyl) benzo [d] oxazole-5-sulfonamide] and then tested for biologically denatured prion formation. (See FIGS. 2 and 3).

In addition, the present inventors described the above 2-(((5,7-dimethylimidazo [1,2-a] pyrimidin-2-yl) methyl) thio) -N- (2-methoxyphenyl) benzo [d ] Oxazole-5-sulfonamide [2-(((5,7-dimethylimidazo [1,2-a] pyrimidin-2-yl) methyl) thio) -N- (2-methylphenyl) benzo [d] oxazole- 5-sulfonamide] and a prion monomer were identified, and then ethyl 5- (2-((5- (N- (2-methoxyphenyl) sulfamoyl) benzo [d] oxazole 2-yl) thio) acetyl) -2,4-dimethyl-1H-pyrrole-3-carboxylate [ethyl 5- (2-((5- (N- (2-methoxyphenyl) sulfamoyl) benzo [d] oxazol- 2-yl) thio) acetyl) -2,4-dimethyl-1H-pyrrole-3-carboxylate] was tested in order to verify the inhibitory effect of denatured prion formation (see FIG. 4).

In addition, the present inventors confirmed the effect of inhibiting the needle formation of the prion protein of the compound of the present invention, it was confirmed that the compound of the present invention inhibits the needle formation of the prion protein (see Fig. 5).

In addition, the present inventors confirmed the cytotoxicity of the compound of the present invention, it was confirmed that the compound of the present invention does not show toxicity to N2a neurons (see Figure 6).

In addition, the present inventors confirmed the effect of inhibiting the expression of the modified prion of the compound of the present invention, it was confirmed that the compound of the present invention inhibits the expression of PrP ^res in the cell line ScN2a cells expressing the modified prion (see Fig. 7).

In addition, the present inventors confirmed the effect of inhibiting the expression of the modified prion of the compound of the present invention, it was confirmed that the compound of the present invention inhibits the expression of the modified prion in brain tissue in the animal model expressing the modified prion (see Fig. 8). ).

In addition, the present inventors confirmed the effect of extending the life of the compound of the present invention in a prion infected animal model, it was confirmed that the compound of the present invention extends the life of the animal model expressing modified prion (see Fig. 9).

Thus, Compound 2-(((5,7-dimethylimidazo [1,2-a] pyrimidin-2-yl) methyl) thio) -N- (2-methoxy represented by Formula 1 of the present invention Phenyl) benzo [d] oxazole-5-sulfonamide [2-(((5,7-dimethylimidazo [1,2-a] pyrimidin-2-yl) methyl) thio) -N- (2-methylphenyl) benzo [d] oxazole-5-sulfonamide] or ethyl 5- (2-((5- (N- (2-methoxyphenyl) sulfamoyl) benzo [d] oxazol-2-yl) thio) acetyl) -2 , 4-dimethyl-1H-pyrrole-3-carboxylate [ethyl5- (2-((5- (N- (2-methoxyphenyl) sulfamoyl) benzo [d] oxazol-2-yl) thio) acetyl) -2, 4-dimethyl-1H-pyrrole-3-carboxylate] inhibits the formation of needles of prion proteins, is not cytotoxic, and inhibits the expression of modified prions in cells or animal models expressing modified prions, thereby preventing or treating prion diseases. It can be usefully used as an active ingredient of a pharmaceutical composition.

The present invention includes all compounds represented by the formula (1) or pharmaceutically acceptable salts thereof, and possible solvates, hydrates, racemates, or stereoisomers which may be prepared therefrom.

It can be used in the form of a compound represented by the formula (1) of the present invention or a pharmaceutically acceptable salt thereof, acid salts formed by the pharmaceutically acceptable free acid is useful as the salt. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. Obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically nontoxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and iodide. Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suverate , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, meth Oxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesul Nate, Xylene Sulfonate, Phenyl Acetate, Phenylpropionate, Phenyl Butyrate, Citrate, Lactate, Hydroxybutyrate, Glycolate, Maleate, Tartrate, Methanesulfonate, Propanesulfonate, Naphthalene-1-sulfonate , Naphthalene-2-sulfonate or mandelate.

The acid addition salts according to the present invention can be dissolved in conventional methods, for example, by dissolving a compound represented by the formula (1) in an excess of an aqueous solution of an acid, which salt is a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. It can be prepared by precipitation using.

It is also possible to prepare an equivalent amount of the compound represented by the formula (1) and an aqueous acid solution or alcohol, followed by evaporation of the mixture to dryness or precipitation of the precipitated salt by suction filtration.

Bases can also be used to make pharmaceutically acceptable metal salts. An alkali metal or alkaline earth metal salt is obtained, for example, by dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. Corresponding silver salts are also obtained by reacting alkali or alkaline earth metal salts with a suitable negative salt (eg, silver nitrate).

When formulating the composition, it is prepared using commonly used diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants.

Solid preparations for oral administration include tablets, patients, powders, granules, capsules, troches, and the like, which solid preparations contain at least one excipient such as starch in at least one compound represented by Formula 1 of the present invention. , Calcium carbonate, sucrose or sucrose, lactose or gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, or syrups, and include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like.

As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used.

The composition according to the invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level means the type, severity, and activity of the patient's disease. , Drug sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.

Specifically, the effective amount of the compound according to the present invention may vary depending on the age, sex, and weight of the patient, and in general, 0.1 mg to 100 mg, preferably 0.5 mg to 10 mg per 1 kg of body weight is administered daily or every other day. Or divided into 1 to 3 times a day. However, the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.

The present invention provides a health food for preventing or improving a prion disease comprising a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

(In Formula 1,

또는

이다.)R ¹ is

or

to be.)

The compound represented by Formula 1 is 2-(((5,7-dimethylimidazo [1,2-a] pyrimidin-2-yl) methyl) thio) -N- (2-methoxyphenyl) benzo [d] oxazole-5-sulfonamide [2-(((5,7-dimethylimidazo [1,2-a] pyrimidin-2-yl) methyl) thio) -N- (2-methylphenyl) benzo [d] oxazole-5-sulfonamide] or ethyl 5- (2-((5- (N- (2-methoxyphenyl) sulfamoyl) benzo [d] oxazol-2-yl) thio) acetyl) -2,4- Dimethyl-1H-pyrrole-3-carboxylate [ethyl 5- (2-((5- (N- (2-methoxyphenyl) sulfamoyl) benzo [d] oxazol-2-yl) thio) acetyl) -2,4- dimethyl-1H-pyrrole-3-carboxylate].

The compound represented by Formula 1 may be to inhibit the prion protein sedimentation.

The prion disease is mad cow disease, mad cow disease, spongiform encephalophathy (BSE) or Creutzfeldt-Jakob disease (CJD), but is not limited thereto.

Compound 2-(((5,7-dimethylimidazo [1,2-a] pyrimidin-2-yl) methyl) thio) -N- (2-methoxyphenyl) represented by Formula 1 of the present invention Benzo [d] oxazole-5-sulfonamide [2-(((5,7-dimethylimidazo [1,2-a] pyrimidin-2-yl) methyl) thio) -N- (2-methylphenyl) benzo [d ] oxazole-5-sulfonamide] or ethyl 5- (2-((5- (N- (2-methoxyphenyl) sulfamoyl) benzo [d] oxazol-2-yl) thio) acetyl) -2,4 -Dimethyl-1H-pyrrole-3-carboxylate [ethyl5- (2-((5- (N- (2-methoxyphenyl) sulfamoyl) benzo [d] oxazol-2-yl) thio) acetyl) -2,4- dimethyl-1H-pyrrole-3-carboxylate] inhibits the formation of the prion protein, has no cytotoxicity and inhibits the expression of modified prion in cells or animal models expressing modified prion, thereby preventing or improving prion diseases. It can be usefully used as an active ingredient of.

There is no particular limitation on the kind of food to which the compound of the present invention is added. Examples of foods to which the above-mentioned substances may be added include dairy products, various soups, drinks, meat, sausages, breads, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, Beverages, alcoholic beverages and vitamin complexes, dairy products and dairy products, and the like, and include all health foods in the conventional sense.

The compound of the present invention may be added to a food as it is, or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient can be suitably determined according to the purpose of use (prevention or improvement). Generally, the amount of the compound in the health food can be added at 0.1 to 90 parts by weight of the total food weight. However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

When the health food composition according to the present invention is a beverage composition, there is no particular limitation on other ingredients except for containing the compound as essential ingredients in the indicated ratios, and various flavors or natural carbohydrates as additional ingredients are used as in general beverages. It may contain. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 10 g per 100 compositions of the present invention.

In addition, the health food composition according to the present invention is a flavor, such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like. Others may contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages.

These components can be used independently or in combination. The proportion of such additives is not limited, but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the compound of the present invention.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

< Example  1> Prion  Establishing an Active Skeletal Model of Proteins

Acupuncture formation by prion occurs by a mechanism in which normal prion monomers (monomers) form a denatured prion aggregate through a change in structure, and eventually cause neuronal cell death. Therefore, the stabilization of normal prion monomers through the conversion to denatured prion and the formation of aggregates to develop a material that inhibits the mad cow disease.

Specifically, literature studies were conducted on substances that inhibit the accumulation of denatured prions by binding to a hot spot region that induces stabilization of normal prion monomers. Major amino acids that interact with compounds at known key binding sites include asparagine (Asn) 159, glutamine (Gln) 160, lysine (Lys) 194 and glutamic acid (Glu) 196. In order to generate an active backbone suitable for the main binding site, the binding mode was first simulated through a representative known substance (GN-8) known to bind to the main active site (FIG. 1).

The coupling mode extracted by the docking simulation was compared with the binding mode of the data whose binding mode was determined through quantum calculation based on literature reviews. Analysis of the binding mode by the docking simulations revealed the interactions with key amino acids at key binding sites. The backbone carbonyl (C = O, carbonyl) group of glutamine 160 acts as a hydrogen bond acceptor, and the nitrogen of the backbone amide group acts as a hydrogen bond donor to hydrogen bond with GN-8. To achieve. Nitrogen in the side chain of lysine 194 acts as a hydrogen bond donor, confirming that there are two hydrogen bond interactions, and at the same time, hydrophobic interaction with the alkyl group of the side chain of lysine 194 is observed. It was.

Based on the interaction between the above-mentioned GN-8 and the key amino acids of the main binding site, an active skeleton was generated. The final selected active skeleton consists of the following elements: Hydrogen bond donor for backbone carbonyl group of asparagine 159, hydrogen bond acceptor for oxygen of amide group of side chain of glutamine 160, hydrogen bond donor for nitrogen of side chain of lysine 194 and main bond site Two hydrophobic seating elements in space across. A pharmacophore model was established based on the above interaction elements (FIG. 2).

< Example  2> Discovery of substances through virtual drug discovery based on protein structure

The library for virtual drug discovery was formed in the form of Accelrys' Discovery Studio / CatDB, and the compound could find multiple conformations of three-dimensional structure and include parameters to include all chemically possible conformal isomers. parameter was well controlled.

About 4 million kinds of compounds included in the virtual search compound library built using the Discovery Studio 'Search 3D Database' protocol were searched. Mapping the various isomers of each compound to the active skeleton established in Example 1 while evaluating whether the interaction elements set in the active skeleton and the atoms or substructures of the compound molecules are properly positioned Fitness) was measured. High conformity isomers are selected within one molecule, and the selected conformation isomers are evaluated for significant suitability. Through the above process, 1,110 compounds having the same characteristics as the active skeleton are finally selected from about 4 million compounds, and 1,110 compounds are selected using the FCFP (Functional Circular Fingerprint) method, which is one of molecular fingerprint descriptors. They were classified into structurally similar groups.

Prior to the selection of the final inducer, we have created a focused library that is expected to be active in stabilizing prion monomers and will be an important infrastructure for the discovery of denatured prion inhibitors. The final 37 compounds were selected by constructing the central database with a total of 682 compounds and then determining the similarity between molecules. Among these, 2-(((5,7-dimethylimidazo [1,2-a] pyrimidin-2-yl) methyl) thio) -N- (2-methoxyphenyl described by the following [Formula 2] ) Benzo [d] oxazole-5-sulfonamide [2-(((5,7-dimethylimidazo [1,2-a] pyrimidin-2-yl) methyl) thio) -N- (2-methylphenyl) benzo [ d] oxazole-5-sulfonamide] compound was selected to test the inhibitory effect of denatured prion formation.

[Formula 2]

< Example  3> Compound represented by Formula 2 and normal Prion  Docking simulation result of monomer

Docking simulation of the compound selected in Example 2 (Formula 2) and the normal prion monomer was carried out. Compounds selected in Example 2 are lysine (Lys) 192, glutamic acid (Glu) 196, asparagine (Asn) 159 and hydrogen among the hot spot region amino acids that induce the stabilization of the normal prion monomer It was found to bind prion protein through binding and hydrophobic interactions (FIG. 3).

< Example  4> Selection of derivative compounds

In order to improve the activity based on the binding structure of the compound (Formula 2) selected in Example 2 was to identify the derivative by replacing the structure. In view of the binding structure confirmed through the docking simulation of Example 3, it was predicted that the compound could be stably bound when the possibility of hydrogen bonding with the pocket formed with lysine (Lys) 194 and glutamic acid (Glu) 196 was increased. And ethyl 5- (2-((5- (N- (2-methoxyphenyl) sulfamoyl) benzo [d] oxazole) described by [Formula 3] including a carboxyl group among derivatives selected therefrom. 2-yl) thio) acetyl) -2,4-dimethyl-1H-pyrrole-3-carboxylate [ethyl 5- (2-((5- (N- (2-methoxyphenyl) sulfamoyl) benzo [d] oxazol- 2-yl) thio) acetyl) -2,4-dimethyl-1H-pyrrole-3-carboxylate] compound showed higher docking experiments through hydrogen bonding with Lys 194 and Glutamic Acid 196. It was confirmed to show the binding score (Fig. 4).

[Formula 3]

< Example  5> Prion  Protein Chimban  Formation inhibition effect verification

Normal prion protein consists of 42% α-helix and 3% β-sheet structure, but denatured prion protein has a secondary structure that increases β-sheet structure to 43%. It exhibits the property of aggregation while forming. The MDS (Multimer Detection System) developed using these characteristics is an experimental method for measuring only a multimer-type prion protein, and the effects of the compounds (Formula 2 and Formula 3) discovered using this were confirmed.

Specifically, the compounds selected in Examples 2 and 4 were solubilized at a concentration of 50 mM using DMSO (dimethylsulfoxide) and stored at -20 ° C until use. The compound was mixed with 80 ng of recombinant human prion protein (ajROBOSCREEN) at two concentrations of 5 μM and 20 μM, and reacted with stirring at 37 ° C. incubator for 3 hours. Then, the reaction was treated with two kinds of anti-prion antibodies (3E7-ajROBOSCREEN, T2-HRP-Japan NIAH Research Institute) required for capture and detection, stirred for 1 hour in a 37 ° C incubator, and the ECL substrate was reacted. After emitting light using the light emission value was measured by using a luminometer. In addition, a quinacrine compound was used as a positive control and a tris buffered saline-Tween 20 (TBS-T) was used as a negative control (only rHuPrP) instead of the compound.

As a result, as shown in Figure 5, compared to the negative control, the compound treatment group selected in Example 2 and Example 4 was confirmed that significantly reduced the production of multimers at 5 μM and 20 μM (Fig. 5 ).

< Example  6> Cytotoxicity Check

MTT (3- (4,5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide, invitrogen) analysis was performed to evaluate the toxicity of the compounds selected in Examples 2 and 4 to the cells. In living cells, MTT is reduced by reductase in the mitochondria to form a formazan crystal, which can be evaluated by the spectroscopic method of measuring the degree of death of the compound by toxicity. .

Specifically, MTT analysis was performed using N2a neurons (ATCC). Each cell was plated in a 96 well plate at 2 × 10 ⁵ cell counts / well and the compounds selected in Examples 2 and 4 were 0.5 μM, 2 μM, 10 μM, 50 μM, 200 μM and 1000 After treatment at a concentration of μM, the cells were incubated in a CO ₂ incubator at 37 ° C. for 24 hours. At this time, the negative control was treated with the same dose of PBS instead of the compound. Thereafter, 100 μl of MTT dissolved in PBS at a concentration of 5 mg / ml was added to each well and incubated for 4 hours at 37 ° C. in a CO ₂ incubator. Then, the MTT solution was removed and 100 μl of DMSO was added again. The reaction was carried out for a minute. Absorbance was measured at 550 nm using a spectrophotometer and analyzed by comparing the OD values of the inhibitors against the negative controls.

As a result, as shown in FIG. 6, it was confirmed that the compounds selected in Examples 2 and 4 for the cells did not show toxicity (FIG. 6).

< Example  7> Prion  Confirmation of inhibitory effect in infected cells

ScN2a (scrapie-infected neuroblastoma-2a) cells, which express the modified prion, were treated with compounds selected in Examples 2 and 4 at concentrations of 5 and 20 μM and then PrP ^res (protease K-resistant PrP ^Sc fragments). ) Inhibition of expression was confirmed by Western blotting. Western blot was carried out as follows. Each of the compounds (5 and 20 μM) selected in Example 2 and Example 4 were incubated with ScN2a cells, and the cells were lysed to extract proteins. The same amount of protein was digested with protease to precipitate only insoluble protein. This was separated by SDS-PAGE and transferred to PVDF membrane, and then treated with prion primary antibody (6H4, Prionics, PRN-01-011) diluted 1: 2,500 to react overnight. After washing the membrane, the secondary antibody (DAKO, P0260) to which the luminescent material was bound was treated and reacted for 1 hour. PrP ^res luminescence signal appeared on the membrane was induced by the light emitting reagent (ELpis Biotech, EBP-1073) and developed on the film. The same method was used to treat 1: 5,000 diluted β-actin antibody (Cell Signaling, 4970) to show that the samples used in this experiment were the same amount of total protein.

Quinacrine was treated as a positive control at 2 μM and DMSO as a negative control at 5 μM. As a result, it was confirmed that the inhibitory effect of PrP ^res was observed at 5 μM of the compound selected in Example 2 and 5 and 20 μM of the compound selected in Example 4 (FIG. 7).

< Example  8> Prion  Confirmation of inhibitory effect in infected mice

A mouse model expressing modified prions was prepared and treated with the compounds selected in Examples 2 and 4 at concentrations of 5 mg / kg and 10 mg / kg, respectively, to determine whether to suppress the expression of modified prions in the brain, spleen, and plasma. Investigate.

As an infectious agent, ME7 strain (Mouse adapted scrapie prion protein) was inoculated into the brain, and the compound selected in Example 2 and Example 4 was then intraperitoneally administered each week. Necropsy was performed at 100 dpi (daps post infection) and 170 dpi. Brain tissue and spleen were extracted, blood was collected, and plasma was separated. As a result of detecting the modified prion by Western blot, it was observed that the compounds selected in Examples 2 and 4 showed an inhibitory effect in brain tissue (FIG. 8). In the case of the compound selected in Example 2 (Formula 2) when administered 5 mg / kg than 20 mg / kg administration, the inhibitory effect was found to be superior, compared to the compound selected in Example 2 (Formula 2) Example It was observed that the compound selected in 4 (Formula 3) showed superior inhibitory effect in brain tissue. In addition, in the measurement of survival time of mice, the compounds selected in Examples 2 and 4 were observed to have a longer survival than the negative control (Prion only) (Fig. 9). The group treated with the compound selected in Example 4 (Formula 3) than the compound selected in Example 2 (Formula 2) showed a prolonged survival of about 1 month (FIG. 9).

Production Example 1 Preparation of Pharmaceuticals

1. Preparation of powder

500 ng of the compound of the present invention

1 g lactose

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

2. Preparation of Tablets

500 ng of the compound of the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

3. Preparation of Capsule

500 ng of the compound of the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

4. Preparation of Injectables

500 ng of the compound of the present invention

Mannitol 180 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

Distilled water 2974 mg

According to a conventional method for preparing an injectable preparation, an injectable preparation is prepared by containing the above components in the contents shown.

Preparation Example 2 Preparation of Health Food

1. Preparation of health food

500 ng of the compound of the present invention

Vitamin Mixture

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

0.13 mg of vitamin

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

Biotin 10 μg

Folic acid 50 mg

Mannitol 180 mg

Nicotinamide 1.7 mg

Calcium Pantothenate 0.5 mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

15 mg potassium monophosphate

Dicalcium Phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

2. Manufacture of health drinks

500 ng of the compound of the present invention

Citric acid 1000 mg

Plum concentrate 2 g

Calcium Pantothenate 0.5 mg

100 g oligosaccharides

Add 900 mL of purified water

After mixing the above components in accordance with the conventional method for preparing healthy beverages, and then stirring and heating at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2L container, sealed sterilization and refrigerated and then stored in a healthy beverage Used to prepare the composition.

Although the composition ratio is a composition that is relatively suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

Claims (6)

A pharmaceutical composition for preventing or treating prion diseases comprising a compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

(In Formula 1,
R ¹ is

or

to be.)
The method according to claim 1,
The compound is a pharmaceutical composition for preventing or treating prion disease, characterized in that it inhibits prion protein sedimentation.
The method according to claim 1,
The prion disease is mad cow disease (mad cow disease), spongiform encephalophathy (BSE) or Creutzfeldt-Jakob disease (CJD), characterized in that the pharmaceutical composition for preventing or treating prion disease.
A health food for preventing or improving a prion disease comprising a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

(In Formula 1,
R ¹ is

or

to be.)
The method according to claim 4,
The compound is a health food for preventing or improving prion disease, characterized in that to inhibit the prion protein sedimentation.
The method according to claim 4,
The prion disease is mad cow disease (mad cow disease), spongiform encephalophathy (BSE) or Creutzfeldt-Jakob disease (CJD), characterized in that the health food for preventing or improving prion disease.

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