Reagent composition for inhibiting MMP11 expression comprising a compound with MMP11 inhibitory activity as an active ingredient

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of cardiovascular diseases comprising a compound exhibiting MMP11 (Matrix metalloproteinase 11) inhibitory activity as an active ingredient. By identifying a compound that exhibits MMP11 inhibitory activity and inhibits cellular aging of vascular endothelial cells, it can be usefully utilized as a composition for the prevention, treatment, or improvement of cardiovascular diseases.

Description

Technology Field

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of cardiovascular diseases comprising a compound exhibiting MMP11 (Matrix metalloproteinase 11) inhibitory activity as an active ingredient. Background Technology

[0002] Arteriosclerosis is a disease in which fatty substances containing cholesterol, phospholipids, and calcium accumulate on the inner lining (wall) of blood vessels over a long period, causing the vessel's inner lining to thicken. As these fatty substances accumulate, the arteries harden, lose elasticity, and narrow, which impedes blood supply or increases arterial pressure; in severe cases, this can lead to arterial rupture or dissection. Specifically, the endothelial cells composing the inner surface of the blood vessel are damaged first due to fatty substances and high pressure. Gradually, the accumulation of fatty substances within the inner lining leads to cell infiltration and inflammatory responses, resulting in the thickening of the inner lining and hindering smooth blood flow.

[0003] It is one of the adult diseases whose incidence is increasing with the development of modern society due to changes in dietary habits, lack of exercise, and internal and external stress. Major causes include hyperlipidemia, hypercholesterolemia, hypertension, and smoking. Furthermore, treatment and management are crucial as it can lead to various complications such as myocardial infarction and stroke.

[0004] Currently, various statins, which are HMG-CoA reductase inhibitors, are being developed as treatments for arteriosclerosis, and the development of more effective treatments is actively underway. Prior art literature

[0005] 1. Chinese Published Patent CN 113979851 (Published Jan. 28, 2022) The problem to be solved

[0006] The objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cardiovascular diseases comprising a compound exhibiting MMP11 (Matrix metalloproteinase 11) inhibitory activity as an active ingredient.

[0007] Another objective of the present invention is to provide a health functional food composition for preventing or improving cardiovascular diseases, comprising the above-mentioned compound as an active ingredient.

[0008] Another objective of the present invention is to provide a reagent composition for inhibiting MMP11 (Matrix metalloproteinase 11) expression comprising the above compound as an active ingredient. means of solving the problem

[0009] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of cardiovascular disease comprising, as an active ingredient, one or more selected from the group consisting of a compound represented by the following chemical formula 1, a pharmaceutically acceptable salt thereof, a solvate thereof, and a stereoisomer thereof.

[0010] [Chemical Formula 1]

[0011]

[0012] In the above chemical formula 1,

[0013] R 1 is a halogen, and R 2 is hydrogen or (C1-C5)alkyl.

[0014] In addition, the present invention provides a health functional food composition for preventing or improving cardiovascular disease, comprising as an active ingredient one or more selected from the group consisting of the above compound, a food-grade acceptable salt thereof, a solvate thereof, and a stereoisomer thereof.

[0015] In addition, the present invention provides a reagent composition for inhibiting MMP11 (Matrix metalloproteinase 11) expression, comprising one or more active ingredients selected from the group consisting of the above compound, its pharmaceutically acceptable salt, its solvate, and its stereoisomer. Effects of the invention

[0016] According to the present invention, by identifying a compound that exhibits MMP11 (Matrix metalloproteinase 11) inhibitory activity and inhibits cellular aging of vascular endothelial cells, it can be usefully utilized as a composition for preventing, treating, or improving cardiovascular diseases. Brief explanation of the drawing

[0017] FIG. 1 shows (E)-3-(4-((3-aminobenzyl)oxy)-3-iodo-5-methoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one [(E)-3-(4-((3-aminobenzyl)oxy)-3-iodo-5-methoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one] (hereinafter referred to as P300:103) and (E)-3-(3-chloro-5-methoxy-4-((3-(methylamino)benzyl)oxy)phenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one [(E)-3-(3-chloro-5-methoxy-4-((3-(methylamino)benzyl)oxy)phenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one] (hereinafter referred to as P300:104). This is the result of analyzing the inhibitory activity of MMP11 (Matrix metalloproteinase 11). Figure 2 shows the results of the analysis of the cytotoxicity of P300:103 and P300:104 on vascular endothelial cells. #; not significant, *; p<0.05, **; p<0.01, ***; p<0.001. Figure 3 shows the results of analyzing beta-galactosidase activity (A) and angiogenic ability (B) to confirm the inhibitory activity of P300:103 and P300:104 on vascular endothelial cell aging. #; not significant, *; p<0.05, **; p<0.01, ***; p<0.001. Figure 4 shows the results of analyzing BrdU incorporation (A) and aging marker (p53 and p21) expression to confirm the inhibitory activity of P300:103 and P300:104 on vascular endothelial cell aging. #; not significant, *; p<0.05, **; p<0.01, ***; p<0.001. Specific details for implementing the invention

[0018] The present invention will be described in more detail below.

[0020] The present invention provides a pharmaceutical composition for the prevention or treatment of cardiovascular diseases comprising, as an active ingredient, one or more selected from the group consisting of a compound represented by the following chemical formula 1, a pharmaceutically acceptable salt thereof, a solvate thereof, and a stereoisomer thereof.

[0021] [Chemical Formula 1]

[0022]

[0023] In the above chemical formula 1,

[0024] R 1 is a halogen, and R 2 is hydrogen or (C1-C5)alkyl.

[0025] In the above chemical formula 1, R 1 It is chlorine or iodine, and R 2 It can be hydrogen or methyl.

[0026] The above compound may be (E)-3-(4-((3-aminobenzyl)oxy)-3-iodo-5-methoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one [(E)-3-(4-((3-aminobenzyl)oxy)-3-iodo-5-methoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one [(E)-3-(3-chloro-5-methoxy-4-((3-(methylamino)benzyl)oxy)phenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one [(E)-3-(3-chloro-5-methoxy-4-((3-(methylamino)benzyl)oxy)phenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one].

[0027] The above cardiovascular diseases may be one or more selected from the group consisting of arteriosclerosis, hypertension, dyslipidemia, atherosclerosis, acute myocardial infarction, venous occlusion, peripheral arterial occlusion, and heart failure, but are not limited thereto.

[0028] In addition, the above cardiovascular disease may be caused by aging of vascular endothelial cells.

[0029] The above pharmaceutical composition can inhibit MMP11 (Matrix metalloproteinase 11).

[0030] The pharmaceutical composition of the present invention may be manufactured in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier according to a method that can be easily carried out by a person skilled in the art to which the invention belongs.

[0031] The above-mentioned pharmaceutically acceptable carriers are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0032] In the present invention, the content of the additive included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

[0033] The above pharmaceutical composition may be formulated in the form of one or more external skin preparations selected from the group consisting of injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, warning agents, lotions, liniments, pastes, and cataplasms, but is not limited thereto.

[0034] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include, but are not limited to, excipients such as starch, sugars, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinylpyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbate, cetyl alcohol, and glycerol. The pharmaceutically acceptable carriers and diluents may be biologically and physiologically affinities for the target. Examples of diluents include, but are not limited to, saline solution, aqueous buffer solution, solvent, and / or dispersion media.

[0035] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. In the case of oral administration, it may be formulated into tablets, troches, lozenges, water-soluble suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, elixirs, etc. In the case of parenteral administration, it may be formulated into injectable solutions, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for topical application, oils, creams, etc.

[0036] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition, weight, age, gender, health status, dietary constitutional specificity, properties of the formulation, degree of disease, time of administration of the composition, method of administration, duration or interval of administration, excretion rate, and form of the drug, and may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg / kg, but is not limited thereto, and may be administered once or several times a day.

[0037] The above pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. The pharmaceutical effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, method of administration, time of administration, route of administration, etc., and a person skilled in the art can easily determine and prescribe a dosage effective for the intended treatment. The pharmaceutical composition of the present invention may be administered once a day or divided into several doses.

[0039] In addition, the present invention provides a health functional food composition for preventing or improving cardiovascular disease comprising one or more active ingredients selected from the group consisting of a compound represented by the following chemical formula 1, a food-grade acceptable salt thereof, a solvate thereof, and a stereoisomer thereof.

[0040] [Chemical Formula 1]

[0041]

[0042] In the above chemical formula 1,

[0043] R 1 is a halogen, and R 2 is hydrogen or (C1-C5)alkyl.

[0044] The present invention can be generally used as a commonly used food.

[0045] The food composition of the present invention may be used as a health functional food. The term “health functional food” refers to a food manufactured and processed using raw materials or ingredients having functional properties useful to the human body in accordance with the Health Functional Foods Act, and the term “functional properties” refers to consuming the food for the purpose of obtaining beneficial effects for health purposes, such as regulating nutrients or physiological actions on the structure and function of the human body.

[0046] The above-mentioned health functional food composition may include ordinary food additives, and unless otherwise specified, suitability as a “food additive” shall be determined in accordance with the specifications and standards for the relevant item, based on the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety.

[0047] Examples of items listed in the above “Food Additives Codex” include chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid; natural additives such as persimmon dye, licorice extract, crystalline cellulose, sorghum dye, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkaline noodle additives, preservative preparations, and tar dye preparations.

[0048] The food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. For example, among health functional foods in capsule form, hard capsules can be manufactured by mixing and filling a conventional hard capsule with the composition according to the present invention and additives such as excipients, and soft capsules can be manufactured by mixing the composition according to the present invention with additives such as excipients and filling it into a capsule base such as gelatin. The soft capsule may contain plasticizers such as glycerin or sorbitol, coloring agents, preservatives, etc., as needed.

[0049] The definitions of terms regarding the above excipients, binders, disintegrants, lubricants, synergists, flavoring agents, etc., are those described in literature known in the art and include those with identical or similar functions. There are no special restrictions on the types of food mentioned above, and they include all health functional foods in the conventional sense.

[0050] In the present invention, the term “prevention” refers to any act of suppressing or delaying cardiovascular disease through the administration of a composition according to the present invention.

[0051] In this invention, the term “treatment” refers to any act of improving or beneficially altering the symptoms of a cardiovascular disease through the administration of a composition according to this invention.

[0052] In this invention, the term “improvement” refers to any act of administering or ingesting the composition of this invention to an individual to improve a poor condition of cardiovascular disease.

[0054] In addition, the present invention provides a reagent composition for inhibiting MMP11 (Matrix metalloproteinase 11) expression, comprising one or more active ingredients selected from the group consisting of a compound represented by the following chemical formula 1, a pharmaceutically acceptable salt thereof, a solvate thereof, and a stereoisomer thereof.

[0055] [Chemical Formula 1]

[0056]

[0057] In the above chemical formula 1,

[0058] R 1 is a halogen, and R 2 is hydrogen or (C1-C5)alkyl.

[0060] Hereinafter, to aid in understanding the present invention, examples and the like will be described in detail. However, the following examples and the like are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples and the like. The examples and the like of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0062] [ Experimental Example 1] Experiment Preparation

[0063] 1-1. Cells

[0064] Vascular endothelial cells were purchased from LONZA (Walkersville, MD, USA). Vascular endothelial cells were cultured in LONZA’s EBM-2 medium with the addition of FBS (fetal bovine serum), hFGF-B (human basic fibroblast growth factor), VEGF (vascular endothelial growth factor), R3 IGF-1 (R3 insulin-like growth factor-1), hEGF (human epidermal growth factor), hydrocortisone, ascorbic acid, heparin, gentamicin, and amphotericin B.

[0066] 1-2. P300:103 compound

[0067] The compound P300:103((E)-3-(4-((3-aminobenzyl)oxy)-3-iodo-5-methoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one) was obtained from the laboratory of Professor Na Young-hwa of the Department of Pharmacy, CHA University. The preparation method is as follows.

[0068] 3-iodo-5-methoxy-4-((3-nitrobenzyl)oxy)benzaldehyde [3-iodo-5-methoxy-4-((3-nitrobenzyl)oxy)benzaldehyde, 250.0 mg, 0.61 mmol] and 1-(3-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)ethan-1-one [1-(3-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)ethan-1-one, 173.0 mg, 0.79 mmol] were dissolved in a mixed solution of dichloromethane (2 mL), methanol (4 mL), and ethanol (10 mL) in a 100 mL round-bottom flask, then sodium hydroxide (NaOH, 40.0 mg, 1.82 mmol) was added, and the mixture was stirred at 40°C for 14 hours. After the reaction was complete, 4M-HCl was added dropwise to adjust the reaction solution to a pH of 1–2. After stirring for 1 hour, the reaction mixture was cooled to room temperature (root temperature; RT) and extracted with water and ethyl acetate. The organic layer was collected, washed with saturated sodium chloride (NaCl), anhydrous magnesium sulfate (MgSO4) was added, and filtered. The solvent was then distilled under reduced pressure and dried under vacuum. A 100 mL round-bottom flask containing the remaining yellow liquid was nitrogen-extruded, and 11 mL of anhydrous ethanol (EtOH) was added to dissolve the reaction product. Tin chloride (SnCl2, 289 mg, 1.52 mmol) was added, and the reaction mixture was refluxed and stirred at 70°C for 16 hours. After the reaction was finished, water was added while stirring at 0°C, the reaction mixture was adjusted to a pH of 7–8 with saturated sodium bicarbonate (NaHCO3), and extracted with ethyl acetate. The organic layer was collected, washed with saturated sodium chloride (NaCl), and the moisture in the organic layer was removed with anhydrous magnesium sulfate (MgSO4), after which the solvent was removed under reduced pressure. The remaining residue was separated and purified by silica gel column chromatography (ethyl acetate : n-hexane = 1:1 -> 3:1) to obtain the P300-103 compound in the form of a yellow solid (60.0mg, 19.8%)을 얻었다.

[0069] m.p. 77.8 - 80.3℃; R f 0.40 (Ethyl acetate : n-hexane = 1:1); 1 H-NMR(DMSO,400MHz)δ3.94(s,3H),4.84(s,2H),5.09(brs,2H),6.53(ddd, J = 8.0, 2.0, 1.2 Hz, 1H), 6.66 (d, J = 7.6 Hz, 1H), 6.74 (dd, J = 2.0, 1.6 Hz, 1H), 7.02 (dd, J = 8.0, 7.6 Hz, 1H), 7.07 (ddd, J = 8.0, 2.4, 0.8 Hz, 1H), 7.38 (dd, J = 8.0, 7.6 Hz, 1H), 7.48 (dd, J = 2.0, 2.0 Hz, 1H), 7.62 (d, J = 2.0 Hz, 1H), 7.63 (d, J = 15.2 Hz, 1H), 7.67 (d, J = 1.6 Hz, 1H), 7.86 (d, J = 15.2 Hz, 1H), 7.92 (d, J = 1.6 Hz, 1H), 9.79 (s, 1H); 13 C-NMR(DMSO,100MHz)56.3,74.3,93.6,113.3,113.6,113.8,114.7,115.8,119.7,120.3,122.4,128.6,129.8,131.0,133.0,137.3,139.0,142.3,148.6,148.9,152.5,157.7,188.9ppm.

[0071] 1-3. P300:104 compound

[0072] The compound P300:104((E)-3-(3-chloro-5-methoxy-4-((3-(methylamino)benzyl)oxy)phenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one) was obtained from the laboratory of Professor Na Young-hwa of the Department of Pharmacy, CHA University and used. The preparation method is as follows.

[0073] (E)-1-(3-(benzyloxy)phenyl)-3-(3-chloro-5-methoxy-4-((3-(methylamino)benzyl)oxy)phenyl)prop-2-en-1-one [(E)-1-(3-(benzyloxy)phenyl)-3-(3-chloro-5-methoxy-4-((3-(methylamino)benzyl)oxy)phenyl)prop-2-en-1-one, 60.0 mg, 0.12 mmol] was dissolved in anhydrous dichloromethane (dichloromethane, 12 mL) in a 50 mL round-bottom flask, boron trichloride (BCl3) (1.0 M in dichloromethane, 0.35 mL, 0.35 mmol) was added at 0 °C and stirred for 1 hour, after which water was added to terminate the reaction. This mixture was extracted with dichloromethane and saturated sodium bicarbonate (NaHCO3), the organic layer was collected, moisture was removed from the organic layer with anhydrous magnesium sulfate (MgSO4), and the solvent was removed under reduced pressure. The remaining residue was separated and purified by silica gel column chromatography (ethyl acetate : n-hexane = 1:2) to obtain the yellow liquid form of compound P300-104 (12.0 mg, 23.6%).

[0074] R f 0.50 (Ethyl acetate : n-hexane = 1:1); HPLC: 1 H-NMR (DMSO, 400MHz) δ2.68(s,3H),3.95(s,3H),4.96(s,2H),5.75(brs,1H),6.50(dd, J = 8.0, 1.6 Hz, 1H), 6.64 (d, J = 7.6 Hz, 1H), 6.90 (dd, J= 2.0, 2.0 Hz, 1H), 7.06 (dd, J = 8.0, 1.6 Hz, 1H), 7.07 (dd, J = 8.0, 7.6 Hz, 1H), 7.38 (dd, J = 8.0, 7.6 Hz, 1H), 7.48 (dd, J = 2.4, 2.0 Hz, 1H), 7.57 (d, J = 2.0 Hz, 1H), 7.64 (d, J = 2.0 Hz, 1H), 7.65 (d, J = 15.6 Hz, 1H), 7.88 (d, J = 15.6 Hz, 1H), 9.80 (s, 1H); 13 C-NMR(DMSO,100MHz)29.8,56.5,74.8,111.5,111.6,112.0,114.7,115.6,119.7,120.3,122.4,122.6,127.6,128.7,129.8,131.4,137.3,138.9,142.4,145.2,149.7,153.8,157.7,189.0ppm.

[0076] [ Experimental Example 2] MMP11 (Matrix metalloproteinase 11) Analysis of inhibitory activity

[0077] 2-1. Kumashi Brilliant Blue dyeing

[0078] To confirm the MMP1 inhibitory activity of P300:103 and P300:104, Coomassie Brilliant Blue (hereinafter referred to as CBB) staining was performed. After the electrophoresis was completed, the gel was fixed with a fixation solution (containing 50% methyl alcohol, 10% acetic acid, and H2O) for 1 hour, stained with a staining solution (containing 0.1% CBB, 50% methyl alcohol, 10% acetic acid, and H2O) for 1 hour, and then washed with a destaining solution (containing 40% methyl alcohol, 10% acetic acid, and H2O) until the bands were clearly visible.

[0080] 2-2. Western Blot

[0081] Western blot was performed to confirm the MMP1 inhibitory activity of P300:103 and P300:104. Cultured vascular endothelial cells were washed with PBS (phosphate-buffered saline) and harvested using a scraper. RIPA buffer [containing 5M NaCl, 10% NP-40, deoxycholic acid, 10% SDS (sodium dodecyl sulfate), 1.5M Tris-HCl (pH 8.0), 0.5M EDTA (ethylene-diamine-tetraacetic acid), and H2O] was added to the harvested cells, and the cells were crypted to extract proteins. The extracted proteins were quantified using a BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) and separated using an SDS-PAGE gel. The isolated proteins were transferred to a nitrocellulose membrane, and primary and secondary antibodies specific to each protein were attached in sequence. After washing with TBST, protein expression was confirmed using ECL reagents (Thermo Scientific).

[0083] [ Experimental Example 3] Cytotoxicity Analysis

[0084] To confirm the cytotoxicity of P300:103 and P300:104 on vascular endothelial cells, cultured vascular endothelial cells were washed with PBS. After removing the PBS, cells were detached by treatment with 1x Trypsin-EDTA. The cell suspension was transferred to a 1.5 ml tube and collected using a centrifuge. The collected cells were placed in 100 μl of PBS and resuspended, after which the cell suspension (10 μl) and Trypan-blue (10 μl) were mixed. Of this mixture, 10 μl was used to measure the number of cells under a microscope using a hemocytometer.

[0086] [ Experimental Example 4] Analysis of Inhibitory Activity on Vascular Endothelial Cell Aging

[0087] 4-1. Aging-associated beta-galactosidase staining method (SA-β-Gal assay)

[0088] To confirm the inhibitory activity of P300:103 and P300:104 on vascular endothelial cell senescence, senescence-associated beta-galactosidase (hereinafter referred to as SA-β-Gal) staining was performed. The degree of cellular senescence in human vascular endothelial cells was measured using the Enzolifescience Cellular Senescence Live Cell Analysis Assay Kit. Cells were cultured in 60 mm culture dishes, and after removing the culture medium, 2 ml of 1X cell pretreatment solution was added and incubated at 37°C for 2 hours. Subsequently, 10 μl of 200X SA-β-Gal substrate solution was added, and the cells were incubated at 37°C for 4 hours. After washing three times with 3 ml of PBS, aged cells stained green were imaged using a fluorescence microscope.

[0090] 4-2. Blood Vessels Formative ability analyze

[0091] To confirm the inhibitory activity of P300:103 and P300:104 on vascular endothelial cell senescence, angiogenesis was measured. 200 μL of Matrigel (BD Bioscience) was added to 48-well plates and polymerized in a 37°C incubator for 30 minutes. Vascular endothelial cells (4×10⁶) detached by trypsin were used. 4 (a) was placed in 200 μl of culture medium and seeded onto hardened Matrigel. After that, the tubes were incubated in a 37°C incubator for 16 hours, and the formed tubes were photographed with an optical microscope, and the tube area was measured using Fuji Multi Gauge V2.3 software (Fuji).

[0093] 4-3. BrdU incorporation assay

[0094] To confirm the inhibitory activity of P300:103 and P300:104 on vascular endothelial cell senescence, a BrdU incorporation assay was performed. Cell division of vascular endothelial cells seeded in 96-well plates was performed using the Roche BrdU (5-bromo-20-deoxy-uridine) Cell Proliferation Assay Kit (Cat# 11 647 229 001), and the experiment was conducted according to the product protocol. The absorbance of BrdU incorporation was measured at 450 nm.

[0096] 4-4. Aging Marker Expression analysis

[0097] To confirm the vascular endothelial cell aging inhibitory activity of P300:103 and P300:104, Western blot was performed in the manner described in Experimental Example 2-2 above to measure the expression of aging markers (p53 and p21).

[0099] [ Examples 1] MMP11 Inhibitory activity analysis

[0100] According to Experimental Example 2 above, the MMP11 inhibitory activity of P300:103 and P300:104 (100 μM) was analyzed. As shown in Figure 1, it was confirmed that α1-antitrypsin is cleaved by MMP11 when α1-antitrypsin recombinant protein, known as a representative substrate of MMP11, is mixed with MMP11 recombinant protein, and it was confirmed that P300:103 and P300:104 reduce α1-antitrypsin cleavage. From the above results, it was confirmed that P300:103 and P300:104 exhibit MMP11 inhibitory activity.

[0102] [ Examples 2] Cytotoxicity Analysis

[0103] According to Experimental Example 3 above, vascular endothelial cells were treated with P300:103 and P300:104 at different concentrations (5 and 10 μM), and after 1 day, the number of dead cells / total number of cells × 100 (%) was measured by trypan-blue staining. As shown in Figure 2, it was confirmed that no cytotoxicity occurred up to a concentration of 10 μM.

[0105] [ Examples 3] Analysis of Inhibitory Activity on Vascular Endothelial Cell Aging

[0106] According to Experimental Example 4 above, cellular senescence was induced in vascular endothelial cells by irradiating them with 6 Gy of radiation. After 1 day, P300:103 and P300:104 (10 μM) were treated, respectively, and after 2 days, SA-β-Gal activity, angiogenesis, BrdU incorporation, and the expression of senescence markers (p53 and p21) were measured. As a result, as shown in Figure 3, cellular senescence was induced in the irradiated group compared to the control group (non-irradiated group), resulting in increased SA-β-Gal activity and decreased angiogenesis. On the other hand, it was confirmed that in the P300:103 and P300:104 treated groups, SA-β-Gal activity decreased and angiogenesis increased compared to the irradiated group. Additionally, as shown in Figure 4, cellular senescence was induced in the irradiated group compared to the control group (non-irradiated group), resulting in decreased BrdU incorporation and increased expression of senescence markers p53 and p21. In contrast, BrdU incorporation increased and p53 and p21 expression decreased in the P300:103 and P300:104 treatment groups compared to the irradiated group. From the above results, it was confirmed that P300:103 and P300:104 exhibit inhibitory activity on vascular endothelial cell aging.

[0108] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 delete Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 A reagent for inhibiting MMP11 (Matrix metalloproteinase 11) expression comprising, as an active ingredient, a compound selected from (E)-3-(4-((3-aminobenzyl)oxy)-3-iodo-5-methoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one [(E)-3-(4-((3-aminobenzyl)oxy)-3-iodo-5-methoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one [(E)-3-(3-chloro-5-methoxy-4-((3-(methylamino)benzyl)oxy)phenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one [(E)-3-(3-chloro-5-methoxy-4-((3-(methylamino)benzyl)oxy)phenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one] Composition.

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