Anti-aging composition comprising oxazole derivative as active ingredient

Abstract

In the present invention, the KB2779 compound, which is most effective for cell proliferation, was screened for, and the target protein, mechanism of action, effect and the like of the drug were analyzed. The KB2779 compound possesses significant value as a potential compound for treating the described cellular senescence diseases and skin-aging diseases.

Description

Anti-aging composition containing an oxazole derivative as an active ingredient

[0001] The present invention relates to an anti-aging composition, and more particularly, to an anti-aging composition comprising an oxazole derivative represented by [chemical formula 1] as an active ingredient:

[0002] [Chemical Formula 1]

[0003] .

[0004] Research on aging has continued from the past to the present, as aging is considered a disease-causing factor. Cellular senescence is defined as a state in which cell division ceases permanently, and this is considered a fundamental mechanism of aging and a cause of age-related diseases such as cancer, arteriosclerosis, and degenerative arthritis. Early biologists hypothesized that tissue aging was caused by the suppression of the repair mechanisms of surrounding cells by cells that no longer divide. These cells, now called senescent cells (SNC), not only do not participate in the repair of damaged tissue, but their secreted substances are also known to induce inflammatory cell migration, restructuring of the extracellular matrix, unwanted cell death, and fibrosis. These senescent cells naturally accumulate with aging, accumulating damage to cellular tissue.

[0005] Current research technologies can selectively eliminate senescent cells, offering significant potential for developing methods to prevent and treat numerous age-related diseases. These treatments broadly include senolysis, immune-mediated senescent cell removal, and neutralization of the SASP (Severe Aging Proliferation Stimulation). Permanent senescent cell removal not only leads to a sustained decrease in SASP but also reduces the risk of cancer development if senescent cells survive. However, prior to applying these technologies, several considerations must be made. First, the method of inducing senescent cells from which cells and the disease models to be applied must be considered. Furthermore, when applying these therapies to mouse models, stress, surgery, or the use of aged mice can be used to induce senescent cells. The method of induction is also crucial. However, research on aging remains inconclusive, necessitating the development of novel substances and methods capable of suppressing aging.

[0006] Compounds that improve aging have diverse applications. Aging is associated with various health-related factors. Anti-aging drugs can be used in the health and anti-aging fields, and their efficacy can help prevent disease, enhance immunity, and improve metabolic function. Anti-aging drugs can also be used in the beauty and aging management industries. They can be used to address cosmetic and aging-related issues, such as improving skin health, reducing wrinkles, restoring elasticity, and regenerating hair. Anti-aging drugs have significant potential in the medical field. They can be used in research on longevity and the prevention and treatment of age-related diseases, as well as in studying the relationship between age-related diseases and aging. Anti-aging drugs can also be used in sports and performance enhancement. They can be used to improve physical strength, muscle strength, and recovery in athletes, and may also help prevent age-related decline in performance.

[0007] Aging is characterized by an increase in oxygen radicals (reactive oxygen species, ROS) generated in the mitochondria of cells. The same factors are also at play in skin aging. In the present invention, the oxazole derivative represented by [Chemical Formula 1] was found to significantly reduce ROS levels in aging fibroblasts, as well as enhance cell survival and inhibit apoptosis. Furthermore, the oxazole derivative's significant recovery of other aging markers was confirmed.

[0008] In summary, the present invention demonstrates that the senomorphic mechanism of an oxazole derivative represented by [chemical formula 1] works to reduce ROS and restore mitochondrial function in aging fibroblasts.

[0009] According to one embodiment of the present invention, an anti-aging composition is provided comprising an oxazole derivative represented by the following [chemical formula 1] as an active ingredient;

[0010] [Chemical Formula 1]

[0011] .

[0012] The above aging is characterized by being caused by mitochondrial dysfunction.

[0013] The above composition is characterized by reducing ROS (Reactive Oxygen Species) in aging fibroblasts.

[0014] The composition is characterized by improving intracellular mitochondrial respiration.

[0015] The composition is characterized by having a selective cell killing effect on senescent cells at a concentration of 4 μM or more.

[0016] The composition is characterized by restoring the autophagy system and increasing the metabolic activity of the corresponding function.

[0017] The above composition is characterized by reducing ROS in senescent cells.

[0018] The composition is characterized by reducing lipofuscin in senescent cells.

[0019] The composition is characterized by restoring telomeres of cells.

[0020] The composition is characterized by reducing β-galactosidase (SA-β-gal) accumulated inside cells.

[0021] The composition is characterized by decreasing the G1 / G0 phase ratio and increasing the S phase and G2 / M phase ratio in the cell cycle of the cell, and this characteristic activates the cell cycle.

[0022] According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating a disease related to cellular aging caused by a decline in mitochondrial function is provided, comprising an oxazole derivative represented by the following [chemical formula 1] or a pharmaceutically acceptable salt thereof;

[0023] [Chemical Formula 1]

[0024] .

[0025] Diseases related to cellular aging caused by the above mitochondrial dysfunction may be selected from the group consisting of Friedreich's Ataxia (FRDA), renal tubular acidosis, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, developmental pervasive disorder, hearing loss, deafness, diabetes, obesity, diabetic ulcer, insulin resistance, and hepatic steatosis (steatohepatitis), but are not limited thereto.

[0026] Provided is an anti-aging cosmetic composition comprising an oxazole derivative represented by the following [chemical formula 1] as an active ingredient;

[0027] [Chemical Formula 1]

[0028] .

[0029] In the case of the oxazole derivative represented by [Chemical Formula 1], which is an effective ingredient of the present invention, it reduces ROS (Reactive Oxygen Species) in senescent fibroblasts, improves intracellular mitochondrial respiration, has a selective cell death effect on senescent cells at a concentration of 4 μM or more, reduces intracellular ROS, lipofuscin, and β-galactosidase (SA-β-gal) in senescent cells, restores telomeres in cells, reduces the G1 / G0 phase ratio in the cell cycle, increases the S phase and G2 / M phase ratios, restores the autophagy system, and increases the metabolic activity of glycolysis, thereby inhibiting cellular senescence induced by mitochondria. Therefore, the oxazole derivative represented by [Chemical Formula 1] can be used as a composition for preventing or treating diseases related to cellular senescence induced by a decline in mitochondrial function.

[0030] Figures 1 and 2 are images showing the results of ROS identification of an oxazole library using high-throughput screening.

[0031] Figure 3 is a graph showing the results of KB2779 reducing cell proliferation in senescent fibroblasts.

[0032] Figure 4 is a graph showing the comparative results of KB2779 programmed cell death in young and aged fibroblasts.

[0033] Figure 5 is a graph showing the anti-aging phenotype improvement effect of KB2779.

[0034] Figure 6 is a graph showing the effect of KB2779 on cell cycle activation of senescent fibroblasts.

[0035] Figure 7 is a graph showing the effect of KB2779 on the recovery of autophagy function in senescent cells.

[0036] Figure 8 is a graph showing the metabolic regulation effect of KB2779 on senescent fibroblasts.

[0037] According to one embodiment of the present invention, an anti-aging composition is provided comprising an oxazole derivative represented by the following [chemical formula 1] as an active ingredient.

[0038] According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating a disease related to cellular aging caused by a decline in mitochondrial function is provided, comprising an oxazole derivative represented by the following [chemical formula 1] or a pharmaceutically acceptable salt thereof.

[0039] According to another embodiment of the present invention, an anti-aging cosmetic composition comprising an oxazole derivative represented by the following [chemical formula 1] as an active ingredient is provided.

[0040] The present inventors have confirmed that the oxazole derivative represented by [Chemical Formula 1], which is an effective ingredient of the present invention, reduces ROS (Reactive Oxygen Species) in senescent fibroblasts, improves intracellular mitochondrial respiration, has a selective cell death effect in senescent cells at a concentration of 4 μM or more, reduces intracellular ROS, lipofuscin, and β-galactosidase (SA-β-gal) in senescent cells, restores telomeres in cells, reduces the G1 / G0 phase ratio in the cell cycle, increases the S phase and G2 / M phase ratio, restores the autophagy system, and increases the metabolic activity of glycolysis, thereby inhibiting cellular aging induced by mitochondria, and therefore, the oxazole derivative represented by [Chemical Formula 1] can be used as a composition for preventing or treating a disease related to cellular aging induced by a decline in mitochondrial function or as a cosmetic composition for inhibiting skin aging.

[0041] Therefore, according to one embodiment of the present invention, an anti-aging composition is provided comprising an oxazole derivative represented by the following [chemical formula 1] as an active ingredient.

[0042] According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating a disease related to cellular aging is provided, comprising an oxazole derivative represented by the following [chemical formula 1] or a pharmaceutically acceptable salt thereof.

[0043] According to another embodiment of the present invention, a food composition for preventing or improving a disease related to cellular aging is provided, comprising an oxazole derivative represented by the following [chemical formula 1] or a pharmaceutically acceptable salt thereof.

[0044] According to another embodiment of the present invention, a cosmetic composition for inhibiting aging, specifically a cosmetic composition for inhibiting skin aging, is provided, which comprises an oxazole derivative represented by the following [chemical formula 1] as an active ingredient.

[0045] [Chemical Formula 1]

[0046] .

[0047] The above aging is characterized by being caused by mitochondrial dysfunction.

[0048] Diseases associated with cellular aging caused by mitochondrial dysfunction may be selected from the group consisting of, but are not limited to, Friedreich's Ataxia (FRDA), renal tubular acidosis, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, developmental pervasive disorder, hearing loss, deafness, diabetes, obesity, diabetic ulcers, insulin resistance, and hepatic steatosis (steatohepatitis).

[0049] The pharmaceutical composition for preventing or treating the above-mentioned cell aging-related disease can be prepared as a composition in a conventional formulation by selecting one or more pharmaceutically acceptable conventional carriers or one or more additives in an effective amount of an active ingredient.

[0050] The carrier may be selected from one or more of diluents, lubricants, binders, disintegrants, sweeteners, stabilizers, and preservatives, and the additive may be selected from one or more of flavorings, vitamins, and antioxidants.

[0051] In the present invention, any pharmaceutically acceptable carrier and additive may be used. Specifically, as a diluent, lactose monohydrate, trehalose, cornstarch, soybean oil, microcrystalline cellulose, or D-mannitol is preferable, as a lubricant, magnesium stearate or talc is preferable, and as a binder, polyvinyl pyrrolidone (PVP) or hydroxypropylcellulose (HPC) is preferable. In addition, it is preferable to select from among carboxymethylcellulose calcium (Ca-CMC), sodium starchglycolate, polacrylin potassium, or cross-linked polyvinylpyrrolidone as a disintegrant, white sugar, fructose, sorbitol, or aspartame as a sweetener, carboxymethylcellulosesodium (Na-CMC), β-cyclodextrin, white bee's wax, or xanthan gum as a stabilizer, and methyl p-hydroxy benzoate (methylparaben), propyl p-hydroxybenzoate (propylparaben), or It is preferable to choose from potassium sorbate, but it is not limited thereto.

[0052] The pharmaceutical composition of the present invention can be administered to a patient as a single dose, or can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. The term "pharmaceutically effective amount" as used herein refers to an amount that produces a greater response than a negative control group, and preferably refers to an amount sufficient to prevent or treat an inflammatory disease. In addition, the pharmaceutically effective amount may be appropriately varied depending on various factors, such as the disease and its severity, the patient's age, weight, health status, sex, route of administration, and treatment period.

[0053] The composition of the present invention can be formulated in various ways according to the route of administration using a method known in the art together with the pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable" refers to a non-toxic composition that is physiologically acceptable, does not inhibit the action of the active ingredient when administered to humans, and does not typically cause allergic reactions such as gastrointestinal disorders or dizziness or similar reactions. The composition of the present invention can be formulated in various ways according to the route of administration using a method known in the art together with the pharmaceutically acceptable carrier. The route of administration is not limited thereto, but may be administered orally or parenterally.

[0054] The above food composition may be a beverage (including tea or alcoholic beverage), fruit and its processed foods (e.g., canned fruit, bottled fruit, jam, marmalade, etc.), fish, meat and its processed foods (e.g., ham, sausage, corned beef, etc.), bread and noodles (e.g., udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g., butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, retort food, frozen food, various seasonings (e.g., soybean paste, soy sauce, sauce, etc.), etc.

[0055] In addition, the food composition may be a health functional food, and the health functional food may be formulated as a tablet, pill, powder, granule, powder, capsule, liquid, etc. These may be formulated by further including one or more of a carrier, a diluent, an excipient, and an additive.

[0056] Skin aging can be divided into i) intrinsic aging, which is naturally caused by the decline in the skin structure and physiological functions of the body with age regardless of environmental changes, and ii) extrinsic aging, which is caused by external factors such as ultraviolet rays, dry air, reactive oxygen species (ROS), and stress. Intrinsic aging shows mild clinical characteristics such as fine wrinkles, dry skin, and decreased elasticity, while extrinsic aging shows clinical characteristics such as the occurrence of many fine wrinkles along with thick and deep wrinkles. Therefore, such decreased skin elasticity or wrinkles can be said to be representative symptoms of skin aging as described above. Therefore, the above skin aging can be the occurrence of skin wrinkles or decreased skin elasticity.

[0057] That is, prevention or improvement of skin aging may include, but is not limited to, improvement of skin wrinkles, enhancement of skin elasticity, anti-oxidation, or anti-inflammation.

[0058] The cosmetic composition of the present invention may contain conventional auxiliary agents or carriers, such as fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering agents and chelating agents, preservatives, vitamins, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles, etc., in conventional amounts.

[0059] The cosmetic composition of the present invention can be manufactured into any formulation commonly manufactured in the art, such as emulsion, cream, toner, pack, foundation, lotion, beauty solution, hair cosmetic, etc. Specifically, the cosmetic composition can be manufactured into the formulation of skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, foundation, essence, nutrition essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, and body cleanser. If the formulation is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide can be used as a carrier component, and if the formulation is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder can be used as a carrier component, and in particular, in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether can be additionally included. If the formulation is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan. When the above formulation is a suspension, liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, etc. can be used as carrier components.When the above formulation is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linolenic derivative, or ethoxylated glycerol fatty acid ester may be used as a carrier component.

[0060] The present invention is described in more detail through the following examples. However, the present invention is not limited to these examples and may be embodied in any form. The examples presented are provided to aid understanding of the present invention.

[0061]

[0062] <Experimental Example>

[0063] 1. Synthesis of oxazole derivatives

[0064]

[0065] (1) Reagents and conditions

[0066] Reagents and conditions: (i) tert-butyl nitrite, copper(II) chloride, acetonitrile, 60 ℃ for 0.5 h, followed by 80 ℃ for 6hⅱ) 4-(trifluoromethyl)phenylboronic acid, Pd(PPh3)4, K2CO 3, 1,4-dioxane / H2O (5:1), 90 ℃ for 12 h; (ⅲ) 3-bromothioanisole, Cs2CO3, tri(o-tolyl)phosphine, Pd(OAc)2, Toluene, 90°C for 12 h; (iv) 1 N NaOH, EtOH, RT, for 6 h; (v) cyclopropylamine, HOBt, EDC-HCl, DMF, RT, for 12 h.

[0067]

[0068] Ethyl 2-chlorooxazole-4-carboxylate (1)

[0069] Ethyl 2-aminooxazole-4-carboxylate (2.0 g, 12.8 mmol) was added to a solution of tert-butyl nitrite (2.28 mL, 19.2 mmol, 1.5 equiv) and copper(II) chloride (2.60 g, 19.2 mmol, 1.5 equiv) in acetonitrile (60 mL) at 60 °C. The mixture was stirred under argon for 30 min and then at 80 °C for 6 h. The solution was cooled to room temperature and partitioned between dichloromethane, water, and concentrated hydrochloric acid. The aqueous layer was further extracted with dichloromethane, and the combined organic phase was washed with brine, dried over magnesium sulfate (MgSO4), and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (Hexane / Et2O = 11:1 to 9:1, v / v) to obtain compound 1 (702 mg, 62%) as a white solid. R f 0.41 (Hexane / Et2O = 3:1, v / v). 1 H NMR (600 MHz, CDCl3) δ 8.18 (s, 1H), 4.39 (q,J= 7.2 Hz, 2H), 1.38 (t,J= 7.2 Hz, 3H).

[0070]

[0071] Ethyl 2-(4-(trifluoromethyl)phenyl)oxazole-4-carboxylate(2)

[0072] Compound 1 (630 mg, 3.59 mmol), 4-(trifluoromethyl)phenylboronic acid (887 mg, 4.67 mmol, 1.3 equiv), tetrakis(triphenylphosphine)palladium (0) (208 mg, 0.18 mmol, 0.05 equiv) and potassium carbonate (1.49 g, 10.77 mmol, 3.0 equiv) were dissolved in a degassed 1,4-dioxane / water solution (5:1) (25 mL) under argon gas, and the reaction mixture was stirred at 90 °C for 12 h. After cooling to room temperature, the reaction mixture was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried over magnesium sulfate (MgSO4), and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (Hexane / Ethyl acetate = 9:1 to 5:1, v / v) to obtain compound 2 (586 mg, 57%) as a white solid. R f 0.48 (Hexane / Ethyl acetate = 4:1,v / v). 1 H NMR (600 MHz, CDCl3) δ8.32 (s, 1H), 8.25 (d,J= 8.3 Hz, 2H), 7.75 (d,J= 8.3 Hz, 2H), 4.45 (q,J= 7.1 Hz, 2H), 1.42 (t,J= 7.1 Hz, 3H).

[0073]

[0074] Ethyl 5-(3-(methylthio)phenyl)-2-(4-(trifluoromethyl)phenyl)oxazole-4-carboxylate(3)

[0075] A mixture of compound 2 (586 mg, 2.05 mmol), 3-bromothioanisole (330 μL, 2.46 mmol, 1.2 equiv), palladium acetate (92 mg, 0.41 mmol, 0.2 equiv), tri(o-tolyl)phosphine (125 mg, 0.41 mmol, 0.2 equiv), cesium carbonate (1.0 g, 4.10 mmol, 1.5 equiv), and toluene (20 mL) was stirred at 90 °C for 12 h under argon gas. The cooled mixture was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried over magnesium sulfate (MgSO4), and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (Hexane / Ethyl acetate = 9:1 to 5:1, v / v) to obtain compound 3 (471 mg, 56%) as a yellow solid. R f 0.53 (Hexane / Ethyl acetate = 4:1,v / v). 1 H NMR (600 MHz, CDCl3) δ 8.28 (d,J= 8.2 Hz, 2H), 8.04 (s, 1H), 7.86 (d,J= 7.7 Hz, 1H), 7.76 (d,J= 8.2 Hz, 2H), 7.43 (t,J= 7.8 Hz, 1H), 7.38 (d,J= 8.0 Hz, 1H), 4.47 (q,J= 7.1 Hz, 2H), 2.57 (s, 3H), 1.43 (t,J= 7.1 Hz, 3H).

[0076]

[0077] 5-(3-(methylthio)phenyl)-2-(4-(trifluoromethyl)phenyl)oxazole-4-carboxylic acid) (4)

[0078] To a solution of compound 3 (200 mg, 0.49 mmol) in ethanol (30 mL) was added 1 N sodium hydroxide solution (1.47 mL, 1.47 mmol, 3.0 equiv). The reaction mixture was stirred at room temperature for 6 h. The ethanol was evaporated, and a 3 N hydrogen chloride solution was added until no more precipitate was formed. The precipitate was filtered and dried to give compound 4 (181 mg, 98%) as a white solid, which was used in the next step without further purification. 1 H NMR (600 MHz, DMSO-d6) δ 8.32 (d,J= 8.2 Hz, 2H), 8.09 (s, 1H), 7.96 (d,J= 8.2 Hz, 2H), 7.89 (d,J= 7.7 Hz, 1H), 7.50 (t,J= 7.8 Hz, 1H), 7.43 (d,J= 8.0 Hz, 1H), 2.55 (s, 3H).

[0079]

[0080] N-cyclopropyl-5-(3-(methylthio)phenyl)-2-(4-(trifluoromethyl)phenyl)oxazole-4-carboxamide (5, KB2779)

[0081] Compound 4 (150 mg, 0.40 mmol), 1-hydroxybenzotriazole hydrate (HOBt) (85 mg, 0.63 mmol, 1.6 equiv), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (121 mg, 0.63 mmol, 1.6 equiv), and cyclopropylamine (33 μL, 0.47 mmol, 1.2 equiv) in dimethylformamide (5 mL) were added. The solution was stirred at room temperature for 12 h, and the solvent was removed. The residue was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried over magnesium sulfate (MgSO4), and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (Hexane / Ethyl acetate = 9:1 to 6:1, v / v) to obtain compound 5 (58 mg, 35%) as a white solid. R f 0.37 (Hexane / Ethyl acetate = 4:1,v / v). 1 H NMR (600 MHz, CDCl3) δ 8.33 (s, 1H), 8.24 - 8.19 (m, 3H), 7.77 (d,J= 8.2 Hz, 2H), 7.44 (s, 1H), 7.41 (t,J= 7.8 Hz, 1H), 7.34 (d,J= 7.9 Hz, 1H), 2.95 - 2.90 (m, 1H), 2.58 (s, 3H), 0.93 - 0.88 (m, 2H), 0.72 - 0.68 (m, 2H). HRMS m / z calculated for C21H17F3N2O2S [M+H] + : 419.1073; found: 419.0995.

[0082]

[0083] 2. Cell culture

[0084] Human dermal fibroblasts (HDF, PCS-201-010, ATCC, Manassas, VA, USA) were used. Cells were cultured in Dulbecco's modified Eagle's medium containing 25 mM glucose and supplemented with 10% fetal bovine serum (SH30919.03; Hyclone, Waltham, MA, USA), 100 U / ml penicillin, and 100 μg / ml streptomycin (SV30079.01; Hyclone). Senescent fibroblasts were defined as having a doubling time of >14 days. Cell viability and cell number were assessed using a Cedex HiRes analyzer (05650216001, Roche, Basel, Switzerland).

[0085]

[0086] 3. ROS screening

[0087] Senescent fibroblasts were cultured in 96-well plates at a density of 5,000 cells per well. Library components were diluted to a final concentration of 4 μM and added to the wells every 4 days. ROS screening was performed 12 days after drug treatment. For ROS screening, 100 μl of DHR123 (D23806; Thermo Fisher Scientific) was added per well to the 96-well plates and incubated for 30 minutes. After incubation, 200 μl of DPBS (20-030-CV; Corning, NY, USA) was added per well and washed twice. The washed 96-well plates were measured using a VICTOR Multilabel Plate Reader (485 nm / 535 nm 0.1 s; 2030-0050; PerkinElmer, Waltham, MA, USA).

[0088]

[0089] 4. Measurement of cell proliferation

[0090] Senescent fibroblasts were grown in 96-well plates at a density of 1,000 cells per well. Library components were diluted to a final concentration of 0–8 μM and added to the wells every 4 days. After 12 days of drug treatment, cells were washed twice with phosphate-buffered saline (PBS) and lysed in 50 μl of 0.2% SDS. The plates were incubated at 37°C for 1 h. GelGreen® nucleic acid gel stain (150 μl) (1:1,000 in DW; 41005; Biotium, Fremont, CA, USA) was added to the wells. Cell counts were determined by measuring fluorescence intensity using a VICTOR Multilabel Plate Reader (2030-0050; PerkinElmer, Waltham, MA, USA). The mean and standard deviation of 12 replicates were determined for each experimental group.

[0091]

[0092] 5. Flow cytometry (FACS)

[0093] FACS analysis was performed on a BD LSR II (LSR II, BD Bioscience, San Jose, CA, USA) at the Core Facility for Cell-to-In Vivo Imaging. The experimental methods using FACS are described in detail in a separate paragraph.

[0094]

[0095] 6. Measurement of mitochondrial membrane potential (MMP)

[0096] To measure mitochondrial membrane potential, cells were incubated with 0.6 μg / ml JC-10 (ab112134; abcam, Waltham, MA, USA) at 37°C for 30 min. After staining, mitochondrial membrane potential was measured by flow cytometry.

[0097]

[0098] 7. Measurement of ROS, mitochondrial mass, and lipofuscin

[0099] To quantify mitochondrial ROS, cells were incubated in medium containing 3 mM DHR123 (10056-1; Biotium) at 37°C for 30 min. To quantify mitochondrial mass, cells were incubated in medium containing 50 μM MitoTracker deep red (M46753, Thermofisher scientific) at 37°C for 30 min. Unstained cells were used to measure lipofuscin in HDFs, which was then measured by flow cytometry.

[0100]

[0101] 8. Autophagosome measurement

[0102] Autophagy flux was measured by flow cytometry using CYTO-ID® (ENZ-51031-0050, Enzo Lifescience, Farmingdale, NY, USA) autophagy detection according to the company protocol as previously described.

[0103]

[0104] 9. Age-associated β-galactosidase (SA-β-gal) staining

[0105] SA-β-gal staining was observed and analyzed after staining senescent fibroblasts according to the manufacturer's protocol.

[0106]

[0107] <Example>

[0108] 1. Exploration of compounds that improve ROS using high-throughput screening.

[0109] Cellular senescence is characterized by various aging phenotypes. Some phenotypes can be screened on a large scale through simple experiments. Traditionally, high-throughput screening (HTS) has been widely used to identify targets among compound libraries. In this invention, HTS methods were used to select a phenotype characterized by reduced ROS.

[0110] Aged fibroblasts (with a division time of 12 days or longer) were seeded in 96-well plates and treated with oxazole library compounds for 12 days (Fig. 1). After treatment, the medium was removed from the 96-well plates. 100 μl of cell culture medium diluted with DHR123 (1:1,000 dilution) was added to each well and incubated for 30 minutes (Fig. 1). After incubation, the 96-well plates were washed twice with PBS, and ROS levels were compared using 485 nm / 535 nm fluorescence (Fig. 1).

[0111] Among the oxazole library, KB2779 most significantly reduced ROS levels in senescent fibroblasts (Fig. 2). This suggests that the selected KB2779 has the potential to improve aging.

[0112]

[0113] 2. Senolytic effect of KB2779

[0114] KB2779, identified through ROS screening, can enhance additional senescent phenotypes. Senescent cells are generally known to cease proliferating. Cell proliferation can be an important indicator of senescent phenotypes. Therefore, the cell proliferation effect of KB2779 was observed at various concentrations. The cell proliferation effect was first observed in senescent fibroblasts (Fig. 2A). At relatively low concentrations (<2 μM), significant cell proliferation was observed over time (Fig. 3A). However, at relatively high concentrations (>4 μM), a significant decrease in the number of senescent fibroblasts was observed (Fig. 3A). These data suggest that KB2779 inhibits the proliferation of senescent fibroblasts at concentrations above 4 μM.

[0115] Senolytic therapy is known as an anti-aging treatment that selectively kills senescent cells and maintains a young cell pool. The inventors hypothesized that KB2779's inhibition of senescent fibroblasts is due to its senolytic effect. Therefore, the same experiment was performed on relatively young fibroblasts (with a division cycle of less than two days) (Figure 3B).

[0116] In young fibroblasts, significant cell proliferation was observed over time at all concentrations (Figure 2B). Significant cell proliferation was observed even at doses above 4 μM, indicating that no inhibitory effect occurred in young fibroblasts (Figure 3B). These data suggest that KB2779 at concentrations above 4 μM exhibits selective cell inhibition in senescent cells.

[0117] To reaffirm the selective inhibitory effect of KB2779, cell viability of fibroblasts was measured for 12 days (Fig. 3C). Young fibroblasts maintained greater than 95% viability for 12 days, regardless of KB2779 treatment (Fig. 3C). In contrast, senescent fibroblasts showed a significant decrease in viability from day 8 after KB2779 administration, and the viability decreased to 0% by day 12 (Fig. 3C). These data indicate that KB2779 selectively induces apoptosis in senescent fibroblasts.

[0118] Selective inhibition of senescent fibroblast proliferation may underlie the senolytic effect. The senolytic effect occurs through apoptosis in senescent cells. To confirm that the selective inhibition of senescent fibroblast proliferation by KB2779 is mediated by apoptosis, the rate of apoptosis was measured using flow cytometry (Fig. 4A). The apoptosis rate was 6% in young fibroblasts and 7.3% in young fibroblasts treated with KB2779 (Fig. 4B). However, a significant difference was observed: 19.5% in senescent fibroblasts and 23% in KB2779-treated senescent fibroblasts (Fig. 4B). The significant difference in KB2779-mediated apoptosis of senescent fibroblasts suggests selective inhibition via an apoptotic mechanism.

[0119]

[0120] 3. Improvement of aging phenotype

[0121] Cellular senescence and accelerated aging are characterized by excessive ROS generated from dysfunctional mitochondria. In this study, we focused on this issue and screened compounds that improve the ROS phenotype. As shown in Figure 1, KB2779 significantly increased ROS. To confirm the validity of the screening data, ROS was measured using flow cytometry. KB2779 significantly reduced ROS in senescent fibroblasts (Figure 5A). This data validated the ROS screening data in Figure 1.

[0122] Lipofuscin is a polymeric substance within lysosomes, primarily composed of cross-linked protein residues formed by iron-catalyzed oxidation. Because it cannot be degraded or removed through exocytosis, intracellular lipofuscin accumulation after mitosis is inevitable. Lipofuscin accumulates intracellularly during aging. Improvements in lipofuscin levels can be considered an indicator of improved aging. Therefore, in the present invention, lipofuscin in senescent fibroblasts was measured using flow cytometry. KB2779 significantly reduced the autofluorescence value of senescent fibroblasts (Figure 5B). The decrease in autofluorescence in senescent fibroblasts suggests a decrease in accumulated lipofuscin.

[0123] Telomeres are located at the ends of chromosomes and play a role in determining the lifespan of a cell. It is known that telomeres gradually fragment and shorten as cell division continues. Telomere length increased in senescent fibroblasts treated with the KB2779 reagent (Figure 5C). These data suggest that KB2779 restores telomeres in senescent fibroblasts.

[0124] β-galactosidase (SA-β), which is associated with senescence, accumulates inside cells. Traditionally, SA-β activity has been widely used as an indicator for improving aging. In the present invention, the proportion of SA-β-positive cells in senescent fibroblasts was significantly reduced after treatment with KB2779 (Fig. 5D). This indicates that KB2779 maintains a low amount of SA-β accumulated in the senescent fibroblast pool.

[0125] DNA plays a central role in life processes through replication and transcription. Typically, senescent cells experience semi-permanent DNA damage, accelerating aging. Damaged DNA exists in the nucleus in the form of small fragments. Therefore, to examine damaged DNA in senescent fibroblasts treated with KB2779, a comet assay was performed. Senescent fibroblasts exhibited significantly enlarged nuclei compared to KB2779-treated fibroblasts (Figure 5E). The shortened nuclei after KB2779 treatment indicate reduced DNA damage.

[0126] Senescent cells exhibit a decreased ability to divide over time. Senescent cells with reduced division capacity are characterized by cell cycle arrest. To determine the effect of KB2779 on the cell cycle of senescent cells, flow cytometry was performed (Fig. 6A). Senescent fibroblasts exhibited a G1 / G0 phase ratio of 63.8%, while KB2779-mediated senescent fibroblasts exhibited a significant decrease in the G1 / G0 phase ratio of 58.8% (Fig. 6B). A significant increase in the S phase was observed in senescent fibroblasts (18.6%) and in KB2779-mediated senescent fibroblasts (20%) (Fig. 6B). This resulted in a G1 / G0 phase ratio of 58.8% (Fig. 6B). A significant increase in the G2 / M phase ratio was observed in senescent fibroblasts (17.6%) and in KB2779-mediated senescent fibroblasts (20%) (Fig. 6B). In the cell cycle of senescent fibroblasts, KB2779 decreased the G1 / G0 phase ratio and increased the S phase and G2 / M phase ratios. These data suggest cell cycle activation after KB2779 treatment in senescent cells.

[0127]

[0128] 4. Improved autophagosome and mitochondrial function

[0129] Removing dysfunctional organelles is a crucial process within cells. Traditionally, cells remove dysfunctional organelles through the autophagy system. However, as cells age, problems arise in lysosomes and the autophagy system, disrupting normal processes. The accumulation of dysfunctional tubules accelerates cellular aging.

[0130] In this study, we conducted experiments to determine whether KB2779 improves the autophagy system. Flow cytometry analysis demonstrated a significant increase in autophagy flux in KB2779-mediated senescent fibroblasts (Figure 7A). Furthermore, a significant decrease in lysosomal mass was observed in KB2779-mediated senescent fibroblasts (Figure 7B). These data suggest that KB2779 mediates the improvement of the autophagy system in senescent fibroblasts. These findings suggest that KB2779-activated mitophagy restores the lysosomal / autophagy system in senescent fibroblasts.

[0131] Mitochondria are known to be cellular organelles closely associated with aging. As cells age, damage accumulates in mitochondria, leading to dysfunction. It is well known that dysfunctional mitochondria alter cellular metabolism. Considering enhanced autophagy, the inventors hypothesized that metabolic changes would also occur in aged fibroblasts. Therefore, in this study, we observed changes in cellular metabolism induced by KB2779 in fibroblasts. First, we measured KB2779-mediated mitochondrial respiration. A significant increase in oxygen consumption rate (OCR) was observed in KB2779-treated aged fibroblasts (Figure 8A). Significant increases in basal OCR and ATP production were also observed (Figures 8B and 8C). These data indicate that KB2779-mediated enhanced mitochondrial metabolism in aged fibroblasts.

[0132] After demonstrating the improvement of mitochondrial metabolism by KB2779, metabolic changes in glycolysis were also observed in KB2779-mediated senescent fibroblasts. A significant increase in extracellular acidification rate (ECAR) was observed in KB2779-mediated senescent fibroblasts (Figure 8D). Furthermore, significant increases in basal and compensatory glycolysis were observed (Figures 8E and 8F). These data suggest increased glycolysis production in senescent fibroblasts and potential KB2779-mediated restoration of glycolysis. In summary, KB2779-mediated treatment improved the autophagy system and actively altered cellular metabolic activity.

[0133]

[0134] Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims also fall within the scope of the present invention.

[0135]

[0136] <National Research and Development Project Supporting This Invention>

[0137] This work is the result of the University Key Research Institute Support Project, which was supported by the National Research Foundation of Korea (NRF) as an educational resource (Project No. 2020R1A6A1A0304195411).

[0138] This work is the result of the Innovation Growth Skin Health Technology Development Project (Project No. HP23C0024), which was supported by the Korea Health Industry Development Institute and funded by the Ministry of Health and Welfare.

[0139] This work is the result of a University Key Research Institute Support Project supported by the National Research Foundation of Korea and funded by the Ministry of Education (Project No. 2019R1A6A1A03031807).

Claims

1. An anti-aging composition comprising an oxazole derivative represented by the following [chemical formula 1] as an active ingredient; [Chemical Formula 1] .

2. In paragraph 1, An anti-aging composition characterized in that the above aging is caused by dysfunction of mitochondria.

3. In paragraph 1, The above composition is an anti-aging composition characterized in that it reduces ROS (Reactive Oxygen Species) in aging fibroblasts.

4. In paragraph 1, The above composition is an anti-aging composition characterized in that it improves intracellular mitochondrial respiration.

5. In paragraph 1, An anti-aging composition characterized in that the composition has a selective cell death effect on senescent cells at a concentration of 4 μM or more.

6. In paragraph 1, The composition is an anti-aging composition characterized in that it restores the autophagy system and increases the metabolic activity of the corresponding function.

7. A pharmaceutical composition for preventing or treating a disease related to cellular aging caused by a decline in mitochondrial function, comprising an oxazole derivative represented by the following [chemical formula 1] or a pharmaceutically acceptable salt thereof; [Chemical Formula 1] .

8. In paragraph 7, A pharmaceutical composition selected from the group consisting of diseases related to cellular aging caused by the above-mentioned decline in mitochondrial function, Friedreich's Ataxia (FRDA), renal tubular acidosis, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, developmental pervasive disorder, hearing loss, deafness, diabetes, obesity, diabetic ulcer, insulin resistance, and hepatic steatosis (steatohepatitis).

9. An anti-aging cosmetic composition comprising an oxazole derivative represented by the following [chemical formula 1] as an active ingredient; [Chemical Formula 1] .

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