KR101705338B1 - A composition having anti-oxidant, anti-aging and autophagy activity and use thereof - Google Patents

Abstract

The present invention relates to a composition which has antioxidant, anti-aging, and autophagic activities and is also useful for preventing, ameliorating, or treating aging-related metabolic diseases including type 2 diabetes or neurodegenerative diseases. The present invention further relates to a use thereof.

Description

[0001] The present invention relates to compositions having anti-oxidative, anti-aging and autophagic activity,

The present invention relates to compositions useful for preventing, ameliorating or treating aging-related metabolic diseases including antioxidant, anti-aging and auto-phagocytic activities and thereby including neurodegenerative diseases or type 2 diabetes and uses thereof.

Due to the global aging phenomenon, it is predicted that people aged 65 and over will enter an aged society that accounts for more than 14% of the total population since 2018. The increase in the elderly population means that the percentage of the population suffering from a chronic disease or a decreased biomechanical function is increasing. Therefore, the aging of the aging industry such as medicines, functional foods, and cosmetics Is expected.

In addition, there is a growing demand for middle-aged people to improve their bio-indicators related to aging before they get older and to live in a healthier and younger state. In addition, since the proportion of women in economic and social activities is increasing, the demand for the development of anti-aging materials and products for women is very large. Among them, Demand is increasing.

In order to meet the demand for anti-aging, it is necessary to find out the main characteristics of aged tissues and cells and to find a way to improve them. As a main feature showing in aged tissues or cells constituting organs, , And / or a sharp decrease in autophagy activity. As the antioxidant activity and / or autophagic activity in the cells is decreased, the cell viability and activity are rapidly lowered and the morphological pattern of aging is shown. Thus, the cells develop into cell senescence, neurodegenerative diseases or metabolic diseases such as type 2 diabetes do.

Autophagy is a mechanism that removes energy regeneration and damaging substances by decomposing aging or damaged cellular materials and organs when the intracellular energy source is depleted or intracellular stressors are excessively generated. . Recently, various researches have reported that as the aging progresses or the aging accelerates, the intracellular autopoiesis activity decreases sharply. On the contrary, when the autopoiesis is suppressed, the aging mitochondria and the misfolded proteins accumulate excessively in the cells, The increase of free radicals and oxidative stress results in increased cell death and accelerated senescence.

In the mitochondria, the active oxygen group is formed as a byproduct while the energy generation process is activated. The external stress causes lipid peroxide, lipid peroxy radicals or peroxynitrite are generated and exert a wide range of oxidative stress. These oxidative stress products are very unstable and highly reactive with the surrounding substances, resulting in very strong binding with intracellular proteins, lipids and DNA, resulting in various denaturation and eventually a fatal problem.

Examples of substances that increase the antioxidant activity in cells include antioxidant substances such as vitamins, glutathione, coenzyme Q-10, and the like, or catalase, superoxide dismutase, glutathione- and antioxidant proteins such as glutathione-dependent peroxidase and peroxiredoxin. However, these conventional antioxidants have a problem in that the active oxygen scavenging ability is well exerted in-vitro and blood, but the side effects of increasing the formation of active oxygen rather than depending on the concentration in cells are problematic. In addition, it is a recycling mechanism that decomposes aging substances and organs in the cell. Through the activation of the predation, it rapidly removes proteins, lipids, and mitochondria transformed by the oxidative stress, and thereby allows the cells to survive in a healthier state Lt; / RTI >

Antioxidant activity and autophagic activation in cells are very important for eliminating such oxidative stress in cells and for restoring the health of individuals, tissues and cells. Therefore, in order to fundamentally solve the problems of conventional antioxidants and provide an excellent antioxidative effect, it is necessary to develop a substance having an ability to increase antioxidant activity by inducing intracellular antioxidant protein expression, .

Gan et al. Antioxidants & Redox Signaling, 2012, 17, 5, 719-932 Matsushima et al. Circulation, 2006, 113, 1779-1786 Zhao et al. Cell & Bioscience, 2012, 2, 22 Hung et al. Autophagy, 2009, 5, 4, 502-510 Qi et al. PLOS one, 2012, 7, 10, e46834 Xilouri et al. Brain, 2013, 136, 2130-2146 Kim et al. The Journal of Clinical Investigation, 2014, 124, 8, 3311-3324

Accordingly, the present inventors have made efforts to develop a material satisfying both the antioxidant activation and the activation of the self-sustaining mechanism. As a result, it has been found that the compound represented by the structural formula 1 of the present invention increases the expression of various antioxidant proteins in cells, The present invention has been completed by confirming the effect of increasing protein expression to activate autopoiesis and ultimately protecting cells from oxidative stress.

Accordingly, one object of the present invention is to provide a compound represented by the structural formula 1 of the present application, or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide an antioxidant and / or anti-aging composition comprising the compound represented by the structural formula 1 of the present invention or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a method for preventing and / or treating cell senescence-related diseases selected from the group consisting of neurodegenerative diseases and type 2 diabetes, comprising the compound represented by the structural formula 1 of the present application or a pharmaceutically acceptable salt thereof, Or a pharmaceutical composition for therapeutic use.

Another object of the present invention is to provide a method for preventing and / or treating cellular senescence-related diseases selected from the group consisting of neurodegenerative diseases and type 2 diabetes, comprising the compound represented by Structural Formula 1 of the present application or a pharmaceutically acceptable salt thereof, Or improving food composition.

Another object of the present invention is to provide an antioxidative and / or anti-aging method comprising the step of administering to a subject an effective amount of a compound represented by the structural formula 1 of the present invention or a pharmaceutically acceptable salt thereof.

It is another object of the present invention to provide a method of treating a neurodegenerative disease and a cell selected from the group consisting of type 2 diabetes, comprising administering to the subject an effective amount of a compound represented by formula 1 of the present application or a pharmaceutically acceptable salt thereof And to provide a method for treating an aging-related disease.

Antioxidant activity and autophagy activation in the cells are activated in tissues and cells of young people. However, as the aging progresses, the expression levels of antioxidant proteins and autopoietic related proteins in the cells are rapidly decreased, so that each individual cell is protected from oxidative stress I can not. Therefore, by protecting each cell from oxidative stress and activating autophagy, it is possible to inhibit the aging of each cell, tissue and individual and to treat various diseases caused by aging.

The compounds provided herein include Prx2 monomer, peroxiridoxin 3 (Prx3), Grp75, HSP90B1, epoxide dehydrogenase 2 (EPHX2), and stanniocalcin 2 (Fig. 5), and increased the expression of autopoiesis and detoxification-related proteins such as BiP, FoxO1 and sirtuin 5 (Fig. 5) 6). In addition, the ability to increase the antioxidant activity in-vitro and intracellularly (Figs. 3 and 4) and the ability to activate intracellular autophagy (Fig. 7 and Fig. 8) (Figs. 9 and 10).

Among various antioxidant proteins, peroxylidoxine is known to be a key protein that protects cells and intracellular organelles from oxidative stress in the cell, and its increased expression appears as an immediate cytoprotective effect. There has been no report on a substance that increases peroxiridoxin protein expression in the past, and only cases have been reported in which an effect of overexpressing a target protein by proving its effect by inserting into a cell in a plasmid form to produce genetically modified cells and individuals has been reported. It has been reported that overexpression of peroxylidoxine-2 can attain the effect of protecting nerve tissue from ischemic nerve injury (Gan et al. Antioxidants & Redox Signaling, 2012, 17, 5, 719-932) (Matsushima et al., Circulation, 2006, 113, 1779-1786) have shown that overexpression of peroxiridoxin-3 can attenuate tissue damage from cardiac dysfunction due to myocardial infarction. In addition, overexpression of peroxylidoxine-2 in pancreatic beta cells has been shown to protect beta cells from apoptosis induced by oxidative stress (Zhao et al., Cell & Bioscience, 2012, 2, 22) . Therefore, through these reports, it can be confirmed that accelerating the expression of peroxiridoxin in cells can treat various diseases caused by aging, for example, neurological diseases, myocardial diseases or diabetes.

In addition, the activation of autopoiesis enhances the survival rate of aged cells through the removal of harmful proteins and organs in the cell, and is also closely related to the lifespan of each individual.

According to previous reports, activation of autoregulation reduces the accumulation of beta-amyloid, a major cause of Alzheimer's disease, and thus protects cells from cytotoxicity resulting therefrom, thus minimizing damage to nerve cells (Hung et al. Autophagy , 2009, 5, 4, 502-510), and suggests the possibility of treatment and treatment of Huntington's disease by decomposing and eliminating the mutant huntingtin which is the main cause of Huntington's disease (Qi et al. , 10, < / RTI > e46834), inhibiting the accumulation of alpha-synuclein, a major cause of Parkinson's disease, and alleviating neurodegeneration by alpha-synuclein (Xilouri et al. Brain, 2013, 2146), the generation of type 2 diabetes is blocked by destroying the pancreatic beta cells to remove the toxic oligomer-forming form of the human pancreatic amyloid polypeptide causing type 2 diabetes, and the human pancreatic amyloidosis Suggests the possibility of symptom relief and treatment of type 2 diabetes resulting oligomer formed body toxicity of the peptide (Kim et al. The Journal of Clinical Investigation, 2014, 124, 8, 3311-3324). Thus, through these reports, it is possible to treat various diseases caused by aging, such as neurodegenerative diseases (Alzheimer's disease, Huntington's disease, Parkinson's disease, etc.) or type 2 diabetes, Can be confirmed.

As a result, the compounds provided herein increase the expression of antioxidant proteins, such as peroxylidoxine, and have the effect of activating autophagy, so they are useful for anti-aging applications, or for the treatment of neurodegenerative diseases (Alzheimer's disease, Huntington's disease, Parkinson's disease, Or for the prevention, amelioration or treatment of type 2 diabetes.

Accordingly, in one aspect, the present invention relates to a compound having excellent antioxidative and autophagic activating ability, or a pharmaceutically acceptable salt thereof.

More specifically, there is provided a compound of the following structural formula 1 or a pharmaceutically acceptable salt thereof.

[Structural formula 1]

This compound was synthesized from 2- [2,6-bis- (bis-carboxymethyl-amino) -hexanoyl-amino] -6- (bis- carboxymethyl-amino) -hexanoic acid {2- [ - (bis-carboxymethyl-amino) -hexanoyl-amino] -6- (bis-carboxymethyl-amino) -hexanoic acid}.

In another aspect, the present invention relates to an antioxidant and / or anti-aging composition comprising a compound represented by Structural Formula 1 or a pharmaceutically acceptable salt thereof:

In another aspect, the present invention provides a method for preventing and / or treating cellular senescence-related diseases selected from the group consisting of neurodegenerative diseases and type 2 diabetes, comprising the compound represented by the structural formula 1 or a pharmaceutically acceptable salt thereof, Or pharmaceutical compositions for therapeutic use.

In another aspect, the present invention provides a method for preventing and / or treating cellular senescence-related diseases selected from the group consisting of neurodegenerative diseases and type 2 diabetes, comprising the compound represented by the structural formula 1 or a pharmaceutically acceptable salt thereof, Or improving food composition.

In another aspect, the present invention relates to an antioxidant and / or anti-aging method comprising the step of administering to a subject an effective amount of a compound represented by the structural formula 1 or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides a method for treating a cell selected from the group consisting of neurodegenerative diseases and type 2 diabetes, comprising administering an effective amount of a compound represented by the structural formula 1 or a pharmaceutically acceptable salt thereof to an individual And a method for treating an aging-related disease.

The compounds of the invention, or pharmaceutically acceptable salts thereof, can be prepared using conventional techniques known in the art.

The term "pharmaceutically acceptable salt" as used herein includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Suitable acids include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, Sulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid and the like. Salts derived from suitable bases may include alkali metals such as sodium, alkaline earth metals such as magnesium, and ammonium.

Neurodegenerative diseases herein may include Alzheimer's disease, Huntington's disease or Parkinson's disease.

The composition of the present invention may be provided in various forms depending on the purpose such as a pharmaceutical composition, a food composition or a cosmetic composition.

When the composition of the present invention is provided as a cosmetic composition, the functional cosmetic composition for antioxidant or anti-aging can be administered extracutaneously, transdermally or subcutaneously, depending on the route of administration, May be a composition that can be administered.

The above-mentioned cosmetic composition refers to a composition which can be applied percutaneously to skin, scalp or hair and which can be used for the production of all cosmetic products such as foundation cosmetics, makeup cosmetics, body products, shaving products, hair products, And may be formulated in the form of a spray, a suspension, an emulsion, a cream, a gel or a foam, but there is no particular limitation on its form.

The content of the compound contained in the cosmetic composition can be appropriately adjusted according to the use, application form, purpose of use and desired effect of the composition. For example, the content of the compound may be 0.0001 to 1% by weight, Preferably 0.001 to 0.5% by weight, and most preferably 0.03 to 0.1% by weight. When the amount is less than the above range, substantial antioxidant protein expression increase and self-sustaining activation effect can not be obtained. If the amount is above the above range, the stability of the formulation may be deteriorated due to high hygroscopicity of the raw material.

The cosmetic composition may contain, in addition to the compound as an active ingredient, all kinds of components that can be used for ordinary commercialization or formulation such as a perfume, a pigment, a bactericide, an antioxidant, an antiseptic, a moisturizer, an agitator, an excipient, a diluent, Etc., and the kind and content thereof can be appropriately adjusted depending on the use of the final product and the purpose of use.

In addition, the cosmetic composition may contain a solvent ordinarily included in the application form, and examples thereof include ethanol, glycerin, butylene glycol, propylene glycol, polyethylene glycol, 1,2,4-butanetriol, sorbitol ester , 1,2,6-hexanetriol, benzyl alcohol, isopropanol, butanediol, diethylene glycol monoethyl ether, dimethylisosorbide, N-methyl-2-pyrrolidone, propylene carbonate, glycereth- At least one member selected from the group consisting of isosetyl myristate, isocetyl octanoate, octyldodecyl myristate, octyldodecanol, isostearyl diacetate, cetyl octanoate, neopentyl glycol dicaprate, etc. . ≪ / RTI > When the composition of the present invention is prepared using such a solvent, the solubility of the compound with respect to the solvent differs depending on the kind of the compound or the mixing ratio of the solvent. However, those skilled in the art will recognize that, Type and amount of usage can be appropriately selected and applied.

In addition, the cosmetic composition may contain various substances for enhancing transdermal permeation during transdermal administration. For example, there may be mentioned, for example, laurolactam derivatives and oleic acid, ester derivatives of monooleate derivatives, aderapalene, tritinoin, retinaldehyde, tazotrotin, salicylic acid, azelaic acid, glycolic acid, ethoxydiglycol, 80, lecithin olganogel, and the like. In order to impart additional functions, the cosmetic composition of the present invention may contain a cosurfactant, a surfactant, an anti-dandruff agent, an anti-dandruff agent, An antioxidant, a pH adjuster and purified water may be added to the composition according to the present invention. Depending on the application form, additives such as flavorings, pigments, preservatives, excipients and the like may be added .

When the composition of the present invention is provided as a pharmaceutical composition, the antioxidant protein expression can be enhanced and the effect of preventing or treating various aging-related diseases and aging-related diseases caused by aging of cells can be demonstrated by the action of autopoiesis activation.

For example, the pharmaceutical compositions provided herein may be used for the treatment of Alzheimer ' s disease caused by toxic oligomer-forming bodies of mutant alpha-amyloid, Huntingtin, alpha-synuclein and human pancreatic amyloid polypeptides by increased antioxidant protein expression and autophagy- , Huntington's disease, Parkinson's disease and type 2 diabetes.

The pharmaceutical composition of the present invention can be used mainly by oral, intravenous, intraperitoneal, muscular and subcutaneous administration methods. In addition, it may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppositories or sterilized injection solutions according to a conventional method, There are no special restrictions.

The compositions herein may further comprise pharmaceutically acceptable excipients customarily used in the manufacture of pharmaceutical compositions. Pharmaceutically acceptable excipient refers to a carrier or diluent that does not significantly stimulate the organism and does not interfere with the biological activity and properties of the administered compound. In addition, the additive can improve the preparation, compressibility, appearance and taste of the preparation, and can be used as a stabilizer, a surfactant, a lubricant, a solubilizer, a buffer, a sweetener, a base, an adsorbent, a mating agent, The present invention relates to an antioxidant and a thickening agent which can be used as a surface active agent, a curing agent, an antioxidant, a brightening agent, a flavoring agent, a flavoring agent, a pigment, a coating agent, a wetting agent, a wetting agent, A thickening agent, a foaming agent, a pH adjusting agent, an excipient, a dispersing agent, a disintegrant, a waterproofing agent, a preservative, a preservative, a solubilizing agent, a solvent and a fluidizing agent.

For example, formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient and / or lubricant. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. In addition, formulations for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

The content of the compound contained in the pharmaceutical composition may be appropriately adjusted according to the use of the composition, the application form, the purpose of use, and the desired effect, and may be, for example, from 0.0001 to 10% Preferably 0.001 to 5% by weight, and most preferably 0.03 to 1% by weight. When the amount is less than the above range, substantial antioxidant protein expression increase and self-sustaining activation effect can not be obtained. If the amount is above the above range, the stability of the formulation may be deteriorated due to high hygroscopicity of the raw material.

The preferred dosage of the pharmaceutical composition will vary depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. For a more preferable effect, the dose of the composition of the present invention is preferably 0.1 mg / kg to 100 mg / kg per day based on the active ingredient, but is not limited thereto. The administration may be carried out once a day or divided into several doses. The pharmaceutical dosage form of the composition of the present invention may be used in the form of a pharmaceutically acceptable salt of the active ingredient, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable combination.

When the composition of the present application is provided as a food composition, it may contain an acceptable food-aid additive and may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of functional foods.

In the present invention, the term " food " means a natural product or a processed product containing one or more nutrients, preferably a state of being able to be directly eaten through a certain processing step, , Health functional foods, beverages, food additives and beverage additives. Examples of the food include various foods, beverages, gums, tea, vitamin complex, and functional foods. In addition, in the present invention, the food may contain special nutritional foods (e.g., crude oil, spirits, infant food, etc.), meat products, fish products, tofu, jelly, noodles (Such as soy sauce, soybean paste, hot pepper paste, mixed sauce), sauces, confectionery (eg snacks), dairy products (eg fermented milk, cheese), other processed foods, kimchi, pickled foods But are not limited to, natural flavors (eg, ramen soup, etc.), vitamin complexes, alcoholic beverages, alcoholic beverages and other health supplement foods. The health functional food, beverage, food additive or beverage additive can be produced by a usual production method.

The amount of the compound contained in the food may be 0.00001% to 50% by weight of the total food. When the food is a beverage, the amount of the compound is preferably 0.001 g to 50 g based on 100 ml of the whole food, May be included in a ratio of 0.01 g to 10 g, but is not limited thereto.

Formulations comprising the compounds of the present invention can be used to increase the expression of antioxidant proteins and autopoiesis related proteins when applied to human derived cells to activate autophagy and protect cells from oxidative stress, Cells, tissues and individuals can be protected from various phenomena and diseases. Therefore, it is possible to anticipate the effects of treatment, improvement and deterioration of aging phenomenon and disease, and to apply them to various cosmetics, quasi-drugs, medicines and foods which are intended to provide effects on aging phenomenon and diseases .

Figure 1 shows the HPLC analysis results for compounds of formula 1.
Figure 2 shows the LC / MS analysis results for compounds of formula 1.
FIG. 3 shows the antioxidative activity of each of the compounds of formula 1, vitamin C and PCA (pyrrolidone carboxylic acid) using the ABTS radical scavenging assay.
FIG. 4 shows the result of confirming the intracellular antioxidant activity that inhibits the production of ROS (reactive oxygen species) by the compound of formula 1 using DCFH-DA staining and FACS scanning. The horizontal axis indicates the intensity of fluorescence sensed in each cell and the vertical axis indicates the number of cells. Red colorless control group, green color treated with H ₂ O ₂ , pink colored with H ₂ O ₂ and vitamin C treated group, blue colored with H ₂ O ₂ and compound of formula 1. Green is treated with H ₂ O ₂ only. It is red (untreated group) to green. Moving to the right means that ROS is generated in the cell. This means that my ROS has decreased.
FIG. 5 shows Western blotting results obtained by measuring changes in expression levels of antioxidation-related proteins when cells were treated with the compound of formula 1. FIG.
FIG. 6 shows Western blot results obtained by measuring changes in expression levels of autopoiesis related proteins when cells were treated with the compound of formula 1.
FIG. 7 shows the results of confirming activation of the self-predation through the formation of LC3 puncta when the compound of formula 1, rapamycin or vitamin C was treated or not treated.
FIG. 8A is a photograph of a human dermal fibroblast treated with a compound of formula 1 and cultured for 24 hours, followed by analysis of cell images through an electron microscope.
FIG. 8B shows the results of quantitative measurement of the number of intracellular phagophore, autophagosome and autophagolysosome in the electron micrograph. FIG.
FIG. 9 shows cell viability (%) when oxidative stress was induced by H ₂ O ₂ after pretreating cells with the compound of formula 1 or vitamin C for 24 hours.
FIG. 10 shows the cell viability (%) when oxidative stress was induced by H ₂ O ₂ after pretreating cells with the compound of formula 1 or vitamin C for 48 hours.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example 1. Synthesis of Compound of Structure 1

The synthesis of the compounds of formula 1 can be carried out by synthesis of the first step H-Lys-Lys-OH basic skeleton synthesis, the second step Alkylation reaction, the third step Hydrolysis reaction and finally the fourth step, Respectively. The concrete embodiment is as follows.

Example 1-1. H-Lys-Lys-OH (1c) Synthesis

One. Boc - Lys ( Boc ) - Lys ( Boc ) - OMe (1a) Synthesis

50 g of Boc-Lys (Boc) -OH (1 equivalent of 144.35 mmol) and 45.09 g of H-Lys (Boc) -OMe (1.2 equivalent) were added to a 2 L round flask and dissolved in 500 ml of dichloromethane. 1.76 g of DMAP (N, N-dimethylaminopyridine, 0.1 equivalent) was added and the temperature of the reaction was lowered to 0 ° C using an ice bath. 36 g of EDC (N- (3-Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride, 1.3 eq.) Was dissolved in 300 ml of MC and slowly added. When EDC was added, the temperature of the reaction was slowly raised to room temperature and allowed to react for 12 hours. The reaction was confirmed by TLC (Hexane: Ethyl acetate = 1: 1, Rf = 0.4). The solvent was removed using a concentrator. 500 ml of EA (Ethyl acetate) and 500 ml of 1N HCl solution were added, and the EA layer was extracted using separatory funnel. 500 ml of a 5% NaHCO ₃ aqueous solution was added thereto, and then an EA layer was extracted using a separatory funnel. After adding 20 ml of a saturated aqueous NaCl solution, the EA layer was extracted using a separatory funnel. Na ₂ SO ₄ was added to remove moisture, followed by concentration. 76.4 g was obtained in 90% yield.

2. Boc - Lys ( Boc ) - Lys ( Boc ) -OH (1b) Synthesis

50 g of 1a (84.98 mmol, 1 equivalent) was added to a 2 L round flask and dissolved by adding 500 mL of methanol. 170 ml (equivalent) of 1N NaOH aqueous solution was added and the mixture was reacted at room temperature for 12 hours. The reaction was confirmed by TLC (MC: MeOH = 10: 1, Rf = 0.4). Methanol was removed using a concentrator. 500 ml of water was added to dissolve it, and then the pH of the aqueous solution was adjusted to 5 by adding sulfuric acid. After adding 500 ml of MC, the MC layer was extracted using a separatory funnel. Na ₂ SO ₄ is added to remove moisture, and the mixture is concentrated. 46.4 g was obtained in 95% yield.

3. H- Lys - Lys Synthesis of -OH (1c)

40 g of 1b (69.64 mmol, 1 equivalent) was added to a 2 L round flask and dissolved in 200 mL of MC. Trifluoroacetic acid (TFA) (200 ml) was added and reacted at room temperature for 4 hours. The MC was removed using a concentrator, and 500 ml of diethyl ether was added to precipitate the product. The solid product was collected by filtration and washed with 300 ml of diethyl ether. The obtained solid product was dissolved in 300 ml of water and then lyophilized to obtain 31.5 g of a compound in the form of a TFA salt.

Examples 1-2. Alkylation reaction

One.( tert - butoxycarbonylmethyl ) ₂ - Lys ( tert - butoxycarbonylmethyl ) ₂ -Lys (tert-butoxycarbonylmethyl) ₂ -O (tert-butoxycarbonylmethyl) (1d) Synthesis

30 g of 1c (59.74 mmol, 1 equivalent) was added to a 2 L round flask and dissolved in 400 mL of water. 125 ml of DIPEA (N, N-diisopropylethylamime, 12 equivalents) was added and 400 ml of DMF (N, N-dimethylformamide) was added. Finally, 61.3 ml of tert-butylbromoacetate (7 equivalents) was added and reacted at room temperature for 3 days. The reaction was confirmed by TLC (Hexane: Ethyl acetate = 2: 1, Rf = 0.3). After adding 500 ml of EA, the EA layer was extracted using a separatory funnel. 500 ml of 1 N HCl aqueous solution was added, and the EA layer was extracted using a separatory funnel (repeated 6 times). After adding 500 ml of 5% aqueous NaHCO ₃ solution, the EA layer was extracted using a separatory funnel. After adding 20 ml of a saturated aqueous NaCl solution, the EA layer was extracted using a separatory funnel. Na ₂ SO ₄ was added to remove moisture, followed by concentration. 38.5 g in 60% yield were obtained.

Examples 1-3. Hydrolysis reaction

One.( tert - butoxycarbonylmethyl ) ₂ - Lys ( tert - butoxycarbonylmethyl ) ₂ -Lys (tert-butoxycarbonylmethyl) ₂ Synthesis of -OH (1e) Synthesis

30 g of 1a (27.95 mmol, 1 equivalent) was added to a 2 L round flask and dissolved by adding 600 mL of tetrahydrofuran (THF). After 216 ml of water was added, 84 ml (3 eq.) Of 1N NaOH aqueous solution was added and the mixture was reacted at room temperature for 12 hours. The reaction was confirmed by TLC (MC: MeOH = 10: 1, Rf = 0.4). THF was removed using a concentrator. 500 ml of water was added to dissolve it, and then the pH of the aqueous solution was adjusted to 5 by adding sulfuric acid. After adding 500 ml of MC, the MC layer was extracted using a separatory funnel. Na ₂ SO ₄ was added to remove moisture, followed by concentration. 25.5 g in 95% yield were obtained.

Example  1-4. Protecting group elimination reaction

1. Synthesis 1 Synthesis 1

20 g of 1e (20.85 mmol, 1 equivalent) was added to a 1 L round flask and dissolved in 200 ml of dioxane. 200 ml of a 35% HCl aqueous solution was added and reacted at room temperature for 12 hours. Reaction confirmations were performed with HPLC (Shimadzu LC-20AD, GraceSmart RP C18 5u 120A 4.6 * 250 mm, Rt = 3.668 min, various concentration gradient from 0% to 20% acetonitrile / water containing 0.01% TFA over 20 min) LC / MS (Waters SQD2, MS (ESI) m / e, [M + H] + = 623.03, [M + H] + / 2 = 312.11). Water and dioxane were removed using a concentrator. Dissolved in 300 ml of water, and then concentrated again (4 times). 300 ml of water was added to dissolve, and the mixture was frozen and frozen. 12.3 g of the compound of formula 1 was obtained in 95% yield. The obtained compound of formula 1 was subjected to HPLC analysis (FIG. 1) and LC / MS analysis (FIG. 2).

Example 2. Measurement of in-vitro antioxidative activity by the compound of formula 1

In order to measure the in vitro antioxidant activity of the compound of formula 1, the ABTS radical scavenging assay was performed. When ABTS [2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid)] reacts with potassium persulfate, the active cations, ABTS ^+, are produced. The ABTS ⁺ is removed by the force, and the unique index cyan color is discolored, and the antioxidant activity can be evaluated by measuring it with the absorbance value.

In order to activate the stabilized ABTS diammonium salt, 7 mM of ABTS diammonium salt and 2.45 mM of potassium persulfate were added to the distilled water and reacted at room temperature for 16 hours to produce ABTS radical. The mixture was diluted with PBS to prepare a reaction solution having an OD734 value of about 1.5. Then, vitamin C (VitC), PCA (Pyrrolidone carboxylic acid) or the compound of formula 1 described in the present invention were mixed to induce the reaction. The amount of activated ABTS radical was analyzed by measuring the absorbance at OD734.

As a result, as can be seen from Fig. 3, the compound of the formula 1 and the vitamin C showed the ability to abolish the ABTS radical activated in a concentration-dependent manner.

Example 3. Measurement of intracellular antioxidative activity by the compound of formula 1

In order to measure the intracellular antioxidant activity of the compound of formula 1, it was determined that the intracellular ROS (reactive oxygen species) using 6-carboxy-2 ', 7'-dichlorofluorescein diacetate staining and FACS (fluorescence activated cell sorter) ). When DCFH-DA is introduced into cells through the cell membrane, it is hydrolyzed to DCFH (6-carboxy-2 ', 7'-dichlorofluorescein) by the esterase present in the cytoplasm. When intracellular ROS is present, The band is oxidized to DCF (dichlorofluorescein) and exhibits a high fluorescence intensity of green at excitation 485 nm and emission 515 nm. Therefore, when DCFH-DA is treated with oxidative stress with hydrogen peroxide (H ₂ O ₂ ) after pretreatment of cells with the test substance, DCF fluorescence intensity is different according to the degree of antioxidant ability of the test substance. The intracellular antioxidant activity of the test substance can be measured.

As a specific experimental method, human epidermal keratinocyte cells (Gibco BRL) were divided into 3 × 10 ⁵ cells in a 60ø dish for culture, and cultured in epilife medium (Gibco BRL) containing human keratinocyte growth supplement (HKGS) And cultured in an incubator at 37, 5% CO ₂ for 24 hours. Subsequently, the test substances (H ₂ O ₂ , vitamin C, and the compound of formula 1) were each dissolved in water at a concentration of 10 mM to prepare a concentrate. The concentrate was diluted with the medium to a concentration of 100 μM, treated in a culture dish, and cultured for 48 hours After the completion of the culture, the medium was removed and washed, and the experiment was prepared. After incubation for 10 minutes with 10 uM hydrogen peroxide, the cells were incubated for 10 min. The cells were further incubated with 10 μM DCFH-DA for 60 min. After incubation with trypsin, whole cells were harvested and the fluorescence intensity of individual cells was measured using FACS calibur (Becton Dickinson) (Excitation 485 nm, emission 515 nm).

As a result, as shown in FIG. 4, when the vitamin C was pretreated for 48 hours, the amount of intracellular ROS produced by H ₂ O ₂ treatment did not decrease. However, when the compound of formula 1 was pretreated for 48 hours, H _It was confirmed that almost all the intracellular ROS produced by ₂ O ₂ treatment disappeared.

Example  4. Antioxidant protein and / Self-predation  Measurement of related protein expression level

In order to find the cause of the increase of intracellular antioxidant activity by the compound treatment of the structural formula 1, cDNA microarray was performed to confirm that the transcription of several important genes related to antioxidant and autophagy was increased, and the protein quantification of the selected gene was performed by western blot .

As a specific experimental method, human epidermal keratinocyte cells (Gibco BRL) were cultured in a 60ø dish for culture, and the compound of formula 1 was treated at a concentration of 50, 100 or 200 uM and cultured for 48 hours. RIPA lysis buffer containing inhibitor cocktail (2 ug / ml aprotinin, 5 ug / ml leupeptin, 1 ug / ml pepstatin A, 1 mM PMSF, 5 mM EDTA and 1 mM EGTA) was added to obtain a whole cell lysate. Total protein quantification was performed using a micro BCA assay (SMART micro BCA protein assay kit, Intron biotechnology), 10 ~ 20 ug of cell lysate was loaded on SDS-PAGE gel, each protein was separated by electrophoresis, (Anti-Prx2, anti-Prx3, anti-Grp75, anti-HSP90B1, anti-EPHX2, anti-Stanniocalcin2, anti-Bip, anti-Foxo1, anti- anti-Sirt5 and anti-β actin and HRP-conjugated secondary antibodies (anti-rabbit IgG HRP (sigma) and anti-mouse IgG HRP (sigma) Chemiluminescence (ECL) method was used to sensitize the film to the presence and quantification of each protein.

As shown in FIG. 5, when the compound of formula 1 was treated, the cells were treated with peroxiredoxin2 monomer (Prx2 monomer), peroxiredoxin3 (Prx3), Grp75, HSP90B1, epoxide dehydrogenase2 (EPHX2) and stanniocalcin2 As shown in FIG. 6, it was confirmed that expression of proteins important for autophagic and detoxification activities such as BiP, FoxO1 and sirtuin5 is increased.

Example  5. LC3 puncta  By analysis of the compound of formula 1 Self-predation  Activation measurement

The results of Example 4 confirmed the possibility of activation of intracellular autophagy by treatment with the compound of Formula 1, and LC3 puncta formation was confirmed in order to verify this.

As a specific experimental method, 200ul of poly-L-lysine (Sigma) was added to each well of 15ul slide glass (Ibidi) and incubated for 30 minutes at room temperature. Human epidermal keratinocyte cells (Gibco BRL) After incubation for 48 hours, the cells were fixed with formalin, blocked with anti-LC3 (Abfrontier), and then incubated with anti-LC3 (anti-LC3) And anti-rabbit IgG-FITC (sigma). The nuclei were stained with DAPI (4 ', 6'-diamidine-2'-phenylindole dihydrochloride) and confirmed by intracellular puncta formation using a confocal microscope.

As a result, as shown in Fig. 7, when treated with the compound of formula 1, as in the case of rapamycin, which is known to induce self-predation, a large amount of LC3 puncta (spot regarded as an autogranular body) In contrast, vitamin C treatment did not increase the production of LC3 puncta.

LC3 (light chain 3) is a protein similar to ubiquitin. LC3-I and phosphatidylethanolamine (PE) combine to form LC3-II. LC3-II is inserted into the membranes of autopagosomes (autophagosome) to form the structure of autogranular bodies. The incorporation of LC3-II into autopoetic somatosensory membranes is an important step that occurs consistently when autotrophs are formed and the amount of LC3 reflects the relative amount of autologous autologous cells in the cell. The LC3-II protein, when combined with an autophagous body, appears to be a puncta like a follicle when observed by cell immuno staining.

Thus, in this example, a large amount of LC3 puncta produced by the compound treatment of the structural formula 1 is an important basis for activating the intracellular self-predation.

Example  6. Electron microscopic analysis of the compound of formula 1 Self-predation  Activation measurement

The results of Example 5 confirmed the activation of intracellular autophagy by the treatment of the compound of Structural Formula 1. In order to more closely examine this, intracellular structural analysis on electron microscopy was carried out.

As a specific experimental method, a compound of the structural formula 1 was treated at a concentration of 50 uM and cultured for 24 hours while culturing human dermal fibroblast. After the incubation, the cells were fixed and a paraffin block was prepared. Was performed.

As a result, as can be seen from FIGS. 8A and 8B, it was confirmed that when the compound of formula 1 was treated, a large amount of phagophore, autophagosome and autophagolysosome were produced in the cells The numbers of phagophore, autophagosome and autophagolysosome in the cells were measured in a number of electron micrographs for quantitative analysis. As a result, the number of phagophore, (autophagosome) was significantly increased more than 2 times, and the number of phagophore and autophagolysosome was also significantly increased.

The self-trapping pathway begins with the formation of a cup-shaped discrete precursor membrane, called a phagophore. The pagophor forms a bilayer membrane known as the autophagosome through initiation, nucleation, elongation and closure processes. Autophagic bodies carry aggregated proteins, damaged intracellular organelles or pathogens, and carry them to lysosomes and fuse with lysosomes to form autophagolysosomes (autophagolysosomes). The autolysing solubles decompose the entrapped constituents in an acidic environment mediated by acid hydrolytic enzymes.

Therefore, the fact that the autopagosomes, pagophores and autopagoraisosomes were significantly increased by the compound treatment of the structural formula 1 in this example is an important basis for activating the intracellular autopatch.

Example 7. Measurement of cell protection effect by the compound of formula 1

The results of Examples 2 to 6 show that the expression of the antioxidant protein and the autopoiesis related protein in the cells was increased by the compound of the structural formula 1, thereby activating autopoiesis in the cells. Therefore, we carried out a follow-up study on the cytoprotective effect in order to confirm whether this autophagic activation protects cells from cytotoxicity caused by ROS.

As a specific experimental method, human epidermal keratinocyte cells (Gibco BRL) were cultured in a 96-well culture dish and treated with 0.1 mM or 1 mM of each test substance (vitamin C, compound of structural formula 1) for 24 or 48 hours and washed After addition of 1 mM H ₂ O ₂ , the cells were further cultured for 4 hours. Then, each well was washed, and 10 μl of 3- (4,5-dimethylthiazo1-2-yl) -2,5-diphenyltetrazolium bromide (MTT) dissolved in PBS at 5 mg / _{2 < / RTI} > conditions for 3 hours. After the supernatant was carefully removed, the MTT formazan precipitated in the cells was dissolved in 100 μl DMSO, and the absorbance at OD 570 was measured using an ELISA reader.

As a result, as can be seen from FIGS. 9 and 10, the concentration of 0.1 mM of vitamin C, which is known as an antioxidant, restored the cell survival rate to about 95% in the pretreatment for 24 hours and the recovery of the cell survival rate in the pretreatment for 48 hours In the case of 1 mM concentration, cell survival rate was rather lowered at 24 hours or 48 hours pretreatment. On the contrary, in the case of the pretreatment of the compound of the structural formula 1, it was confirmed that the cell survival rate was improved in the pretreatment for 24 hours or 48 hours.

Claims (9)

delete 1. A cosmetic composition having antioxidant and autopoietic activation function, comprising a compound of the following structural formula 1 or a cosmetically acceptable salt thereof as an active ingredient:
[Structural formula 1]

A food composition for antioxidation comprising a compound of the following structural formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Structural formula 1]

A pharmaceutical composition for preventing or treating a cell senescence-related disease selected from the group consisting of neurodegenerative diseases and type 2 diabetes, comprising a compound of the following structural formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Structural formula 1]

5. The pharmaceutical composition according to claim 4, wherein the composition increases the antioxidative activity and autophoric activity of the cells.
5. The pharmaceutical composition according to claim 4, wherein said neurodegenerative disease is Alzheimer's disease, Huntington's disease or Parkinson's disease.
A food composition for preventing or ameliorating a cell senescence-related disease selected from the group consisting of neurodegenerative diseases and type 2 diabetes, comprising a compound of the following structural formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Structural formula 1]

8. The food composition according to claim 7, wherein the composition increases the antioxidative activity and autophoric activity of the cells.
8. The food composition of claim 7, wherein said neurodegenerative disease is Alzheimer's disease, Huntington's disease or Parkinson's disease.

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