Anti-aging compounds, methods of preparation and use

Abstract

The application discloses an anti-aging compound, a preparation method and application, and experiment results in MEFs cell and MRC-5 cell anti-aging in vitro screening models show that the compound can significantly reduce the proportion of cells with positive staining of aging-related beta-galactosidase, down-regulate protein expression levels of aging markers p53, p21 and p16, and restore the proliferation ability of aging cells. In Caenorhabditis elegans, the compound prolongs the life span of the nematodes, improves the activity of the nematodes, and reduces lipofuscin accumulation caused by aging. Therefore, the compound can be applied to preparation of anti-aging drugs and health products. The application provides a new candidate compound and experimental basis for anti-aging new drug research and development and basic research, and has important practical significance.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an anti-aging compound, its preparation method, and its application. Background Technology

[0002] Faced with the increasing average life expectancy and the growing aging of human society, aging and anti-aging research has become a major scientific topic and research hotspot. Aging is a fundamental phenomenon and inevitable law of life. Aging mainly refers to the gradual degenerative changes that occur at multiple levels of the body, organs, tissues, cells, and biological macromolecules with increasing age, leading to impaired bodily functions and increased susceptibility to disease. At the same time, the aging of various bodily components is a significant contributing factor to aging and age-related diseases, which mainly include cardiovascular diseases, malignant tumors, diabetes, autoimmune diseases, and neurodegenerative diseases.

[0003] Although aging is an inevitable life process, it is possible to slow down aging, prevent and treat age-related diseases, and thus improve the quality of life for the elderly. In the research of drugs to delay aging, researchers have discovered some compounds that can slow down aging, but the variety and types are still insufficient, and the options available are limited. Therefore, developing novel anti-aging compounds is a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides an anti-aging compound, its preparation method, and its application, in order to develop another anti-aging compound.

[0005] One aspect of the present invention provides an anti-aging compound, the chemical formula of which is C. 18 H 14 N₂O₄, the structural formula of which is shown in the following formula W₂₅O₂: .

[0006] The compound achieves anti-aging by reducing the proportion of cells that are positive for aging-related β-galactosidase staining, downregulating the protein expression levels of aging markers p53, p21, and p16, and restoring the proliferative capacity of senescent cells.

[0007] Another aspect of the present invention provides a method for preparing the above-mentioned compound, which is achieved through the following preparation route: .

[0008] The preparation method includes the following steps: Raw materials SM-1 and SM-2 were placed in a three-necked round-bottom flask, and dry DMF was added and stirred to disperse them. Argon gas was purged three times, and the temperature was lowered to 0 °C. DIPEA and DMAP were added, followed by HATU. The mixture was stirred at 0 °C for 20 min, and the reaction was carried out at room temperature for 2 h. The reaction was then heated to 70 °C for 24 h. The reaction was detected by TLC to indicate that it was complete. The mixture was cooled to room temperature and added to water to precipitate a solid. The solid was washed with water, collected, dried, and then pulped with EA / PE. The solid was filtered and collected to obtain compound M1. Compound M1 was placed in a three-necked round-bottom flask, purged with argon three times, cooled to -78 °C, and BBr3 was added. The reaction was allowed to slowly return to room temperature and then allowed to proceed overnight. The reaction was detected as complete by LC-MS. The reaction was then cooled to -78 °C and quenched with MeOH. The reaction was evaporated to dryness, and the crude product was pulped with MeOH / EA. The solid was collected by filtration, and the solid was then mixed with purified water and ultrasonically pulped. The solid was then filtered, washed with water, and the solid was collected to obtain compound W2502.

[0009] This invention also provides the application of the above-mentioned compounds in the preparation of anti-aging drugs.

[0010] This invention also provides the application of the above-mentioned compounds in the preparation of anti-aging health products.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides novel anti-aging compounds. Experimental results in in vitro anti-aging screening models using MEF cells and MRC-5 cells show that the compounds significantly reduce the proportion of cells positive for aging-related β-galactosidase (SA-β-gal) staining, downregulate the protein expression levels of aging markers p53, p21, and p16, and restore the proliferative capacity of senescent cells. In *C. elegans*, the compound prolongs the lifespan of the nematodes, improves their motility, and reduces lipofuscin accumulation due to aging. Therefore, this compound can be used in the preparation of anti-aging drugs and health products. This invention provides new candidate compounds and experimental evidence for the development of new anti-aging drugs and basic research, and has significant practical implications. Attached Figure Description

[0012] Figure 1 The image shows the proton NMR spectrum of compound W2502. Figure 2 The image shows the carbon NMR spectrum of compound W2502. Figure 3 The mass spectrum of compound W2502 is shown below. Figure 4 This is a graph showing the results of viability testing of mouse embryonic fibroblast CCK8 cells treated with compound W2502 in Example 2; Figure 5 Microscopic images of mouse embryonic fibroblasts treated with compound W2502 in Example 2, showing SA-β-galactosidase staining. Figure 6 This is a bar chart showing the statistical results of the SA-β-galactosidase staining experiment in Example 2; Figure 7 This is a fluorescence micrograph of mouse embryonic fibroblast EdU cells treated with compound W2502 in Example 2, used to detect their proliferation capacity. Figure 8 This is a bar chart showing the statistical results of the fluorescence microscopy experiment for detecting the proliferation capacity of mouse embryonic fibroblasts (EdU cells) treated with compound W2502 in Example 2. Figure 9 The protein expression levels of P21 and P53, senescence-related proteins, in mouse embryonic fibroblasts treated with compound W2502 in Example 2 are shown. Figure 10 The image shows the results of viability testing of human fetal lung fibroblasts CCK8 cells treated with compound W2502 in Example 3. Figure 11 Microscopic image of human fetal lung fibroblasts treated with compound W2502 in Example 3, showing SA-β-galactosidase staining. Figure 12 This is a bar chart showing the statistical results of the SA-β-galactosidase staining experiment in Example 3; Figure 13 This is a fluorescence micrograph of human fetal lung fibroblast EdU cells treated with compound W2502 in Example 3, used to detect their proliferation capacity. Figure 14 This is a bar chart showing the statistical results of fluorescence microscopy of human fetal lung fibroblasts (EdU cells) treated with compound W2502 in Example 3 to detect their proliferation capacity. Figure 15 The protein expression levels of P21 and P16, senescence-related proteins, in human fetal lung fibroblasts treated with compound W2502 in Example 3 are shown. Figure 16 This is a line graph showing the lifespan statistics of wild-type *C. elegans* treated with compound W2502 in Example 4. Figure 17 This is a micrograph of wild-type *C. elegans* treated with compound W2502 in Example 4, stained with lipofuscin fluorescence. Figure 18 The bar chart shows the statistical results of lipofuscin fluorescence staining of wild-type *C. elegans* treated with compound W2502 in Example 4. Figure 19 This is a bar chart showing the statistical results of the number of body swings of wild-type nematodes treated with compound W2502 in Example 4 within 20 seconds on the seventh day. Figure 20 NL5901 treated with compound W2502 in Example 4 unc-54p::alphasynuclein::YFP Fluorescence micrograph of α-synuclein in the mutant strain on day 7; Figure 21 NL5901 treated with compound W2502 in Example 4 unc-54p::alphasynuclein::YFP A bar chart showing the fluorescence results of α-synuclein in the mutant strain on day 7. Detailed Implementation

[0013] To facilitate understanding of the present invention, a more complete description will be given below with reference to various embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0015] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the formulations involved in the following examples are all commercially available products that can be purchased from the market.

[0016] The present invention will be further described in detail below with reference to specific embodiments: Example 1: Synthesis of compound W2502 The synthetic route for compound W2502 is as follows: .

[0017] The specific preparation method of compound W2502 is as follows: In a 100 mL three-necked round-bottom flask, raw materials SM-1 (603.1 mg, 2.88 mmol, 1.0 eq.) and SM-2 (509.3 mg, 2.88 mmol, 1.0 eq.) were placed, followed by the addition of 30 mL of dry DMF. The mixture was stirred and dispersed, and argon gas was purged three times. The mixture was cooled to 0 °C, and DIPEA (1 mL, 5.76 mmol, 3.0 eq.) and DMAP (35.6 mg, 0.29 mmol, 0.1 eq.) were added, followed by HATU (1.69 g, 4.32 mmol, 1.5 eq.). The mixture was stirred at 0 °C for 20 min, and the reaction was maintained at room temperature for 2 h. The mixture was then heated to 70 °C for 24 h, and the reaction was monitored by TLC to confirm completion. The reaction was cooled to room temperature and added to water (200 mL), precipitating a solid. The solid was washed with water (3 × 30 mL), collected, dried, and pulped with EA / PE. The solid was then filtered and collected to give compound M1 (856.3 mg, purplish-gray solid, yield 81%).

[0018] Compound M1 (806.3 mg, 2.20 mmol, 1.0 eq.) was placed in a 50 mL three-necked round-bottom flask and purged with argon three times. The mixture was cooled to -78 °C and BBr3 (11 mL) (used as a solvent) was added. The reaction was allowed to slowly return to room temperature overnight, and the reaction was monitored by LC-MS to confirm completion. The reaction was then cooled to -78 °C and quenched with MeOH (30 mL). The reaction mixture was evaporated to dryness, and the crude product was slurried with MeOH / EA. The solid was collected by filtration, and the solid was then slurried again with 20 mL of purified water and ultrasonically slurried. The solid was then washed with water (3 × 3 mL) and collected to give compound W2502 (436.9 mg, off-white solid, yield 61%).

[0019] Please see Figure 1 The proton NMR spectrum of compound W2502 1 The H-NMR data are as follows: 1 H-NMR (500 MHz, DMSO- d 6), δ 10.00 (s, 1H), 9.87 (s, 1H), 9.49 (s,2H), 8.71 (dd, J = 4.2, 1.6 Hz, 1H), 8.08 (dd, J = 8.5, 1.6 Hz, 1H), 7.87 (d, J= 9.1 Hz, 1H), 7.56 - 7.32 (m, 3H), 6.89 (d, J = 15.8 Hz, 1H), 6.50 (d, J = 2.1Hz, 2H), 6.29 (t, J = 2.2 Hz, 1H).

[0020] Please see Figure 2 The carbon NMR spectrum of compound W2502 ( 13 C-NMR is: 13 C-NMR (126 MHz, DMSO- d 6), δ 165.07, 158.79, 150.33, 147.17, 142.88,140.61, 136.49, 131.03, 128.74, 126.27, 122.19, 121.28, 121.25, 116.51,105.83, 104.28.

[0021] Please see Figure 3 The mass spectrometry data for compound W2502 are as follows: HRMS (ESI-MS) m / z Calcd. For C 18 H 14 N2O4 + Exact mass = 322.0954, [MH] - =321.0880 (100%), [MH] - =322.0914(19.5%), [M+HCO2NH4-H] - =384.1201(100%), [M+HCO2NH4-H] - =385.1234(20.5%).

[0022] The above information confirms that the chemical formula of compound W2502 is C. 18 H 14 N2O4, structural formula: .

[0023] The anti-aging ability of compound W2502 will be tested below. Before the test, the cell model used in this application will be introduced.

[0024] Cellular senescence is a state of permanent proliferative arrest induced by telomere loss or stress damage. Senescent cells are not only morphologically enlarged and flattened, but also exhibit activation of the pro-inflammatory senescence-associated secretory phenotype (SASP) at the molecular level.

[0025] As a core model organism for aging biology research, *C. elegans* exhibits significant scientific advantages. Its small size, high productivity, ease of culture, and ability to support long-term cryopreservation, coupled with its short lifespan, constant cell count, and transparent body, allow researchers to directly track the dynamic evolution of tissues and cells during the aging process. Furthermore, as nematodes enter old age, they often exhibit typical phenotypic characteristics such as decreased motor function, behavioral disorders, reproductive cessation, and the accumulation of intracellular autofluorescent substances. These unique physiological attributes and clear indicators of aging establish its irreplaceable and ideal model status in the field of aging research.

[0026] Therefore, this application aims to explore the anti-aging potential of W2502 in different cell models and *C. elegans*.

[0027] 1. Selection of cell models: MRC-5 cell doxorubicin (Dox) induced senescence model and MEF cell replicative senescence model.

[0028] a. MRC-5 Cell Doxorubicin-Induced Senescence Model: The MRC-5 cell line, derived from human embryonic lung fibroblasts, is a physiological model for studying human cellular senescence due to its strict Hayflick limit, stable diploid karyotype, and high sensitivity to senescent stimuli. Using human MRC-5 cells enhances the clinical value of in vitro experimental results. Doxorubicin is one of the most commonly used drugs to induce stress-induced premature aging (SIPS) as a topoisomerase II inhibitor. Doxorubicin can intercalate into DNA base pairs, interfering with DNA replication and transcription, thereby generating severe DNA double-strand breaks (DSBs). Persistent DSBs activate the DNA damage response (DDR) sensor ATM / ATR kinase. ATM phosphorylates and activates the p53 protein, which then promotes the expression of the cyclin-dependent kinase inhibitor p21 (WAF1 / CIP1). p21 inhibits the Cyclin E / CDK2 complex, arresting cells in the G1 phase and ultimately driving the cells into a senescent state. This model can simulate damage-related aging caused by clinical chemotherapy.

[0029] b. MEF Cell Replicative Aging Model: Mouse embryonic fibroblasts (MEFs) are a typical representative of primary fibroblasts. Due to their rapid growth and ease of cross-species comparative studies, they are often used to verify the universality of anti-aging effects of drugs across different species. Replicative aging simulates the body's natural aging process. Its core principle is the successive shortening of telomeres caused by the "terminal replication problem." When telomere loss reaches a critical length, it triggers the p16INK4A-centered signaling pathway, maintaining long-term and irreversible growth arrest. This study established this model by passaged MEF cells to the P8 generation to evaluate the intervention effects of drugs on physiological aging.

[0030] 2. Scientific basis for detection indicators a. CCK-8 assay: This is a cellular metabolic activity assay based on WST-8. Before investigating the efficacy of anti-aging drugs, the safety of W2502 must be evaluated using CCK-8 to rule out its potential cytotoxicity and determine the non-toxic proliferation concentration range.

[0031] b. SA-β-gal staining assay: Senescence-associated β-galactosidase (SA-β-gal) is currently recognized as the most reliable biomarker for identifying senescent cells. The increased lysosomal content in senescent cells leads to a significant upregulation of β-galactosidase activity under pH 6.0 conditions, and the positive staining rate can directly reflect the proportion of senescent cells.

[0032] c. EdU Staining Assay: EdU (5-ethynyl-2'-deoxyuridine) is a thymidine nucleoside analogue. By labeling cells in the DNA synthesis phase using the Click reaction, cell proliferation activity can be precisely quantified. A decrease in EdU positivity is a key indicator for assessing cell entry into a state of proliferative arrest.

[0033] d. Western blot assay: p53-p21 and p16INK4A / pRB are two signaling axes mediating the core of cellular senescence. As a key node in tumor suppressor proteins and DNA damage response (DDR), p53 is activated and phosphorylated upon stress stimulation (such as Dox-induced DNA breaks), subsequently acting as a transcription factor to initiate the expression of downstream target gene p21 (WAF1 / CIP1). p21, as a knob protein, is mainly responsible for initiating early cell cycle arrest; while high expression of p16INK4A signifies the consolidation, deep locking, and irreversibility of the senescent state. By simultaneously detecting changes in the levels of these three proteins using Western blotting, we can systematically evaluate, from a molecular mechanism perspective, whether W2502 exerts its anti-aging effect by intervening in the early response pathway of "p53 / p21" or the late maintenance pathway of "p16", or both.

[0034] e. Lipofuscin Assay: Lipofuscin is an endogenous autofluorescent pigment and a biomarker of aging in *C. elegans*. As a widely recognized marker of aging, lipofuscin generally accumulates gradually with age and can be quantitatively measured using intestinal fluorescence. In *C. elegans*, lipofuscin is mostly concentrated in intestinal cells, and its fluorescence intensity is positively correlated with the physiological age of the worm. Furthermore, it is not affected by exogenous dye labeling. Therefore, lipofuscin has become an ideal endogenous indicator for assessing the aging process and the effectiveness of anti-aging interventions.

[0035] f. Motion Analysis Experiment: During the aging process, *C. elegans* exhibits many age-related physiological decline phenotypes, among which the gradual decrease in motor ability is the most direct and reliable indicator of aging. This is because the decline in motor ability is not only easy to observe and quantify, but also comprehensively reflects the functional integrity of the nervous, muscular, and metabolic systems. The movement trajectory of *C. elegans* is mainly propelled by the coordinated contraction of body wall muscles and the nervous system. The frequency of body swaying in *C. elegans* can sensitively reflect the integrity of the neuromuscular junction, as well as the contractile efficiency of muscle cells and the signal transmission efficiency of the nervous system. Therefore, body swaying frequency can serve as an indicator of the motor ability of *C. elegans*.

[0036] g. Parkinson's Disease Model Salvage Experiment: Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the accumulation and deposition of α-synuclein, neuronal loss, and the formation of α-synuclein aggregates in substantia nigra neurons. NL5901 pkIs 2386 [unc- 54p::alphasynuclein::YFP + unc-119(+)] It is a mutant strain of *C. elegans* associated with Parkinson's disease.

[0037] Example 2: Compound W2502 improves the cell phenotype of mouse embryonic fibroblasts (MEFs) during replicative aging. 1. Experimental reagents DMEM culture medium was purchased from Jiangsu Kaiji Biotechnology Co., Ltd., product number KGL1211-500; PBS buffer was purchased from Jiangsu Kaiji Biotechnology Co., Ltd., catalog number XB07; Trypsin digestion solution was purchased from Jiangsu Kaiji Biotechnology Co., Ltd., product number KGY001; Fetal bovine serum was purchased from CLARK BIOSCIENCE, catalog number FB15015 or FB25015; Tris-glycine SDS-PAGE electrophoresis buffer was purchased from Servicebio, catalog number G2018-15; The transfer buffer was purchased from Servicebio, catalog number G2017-15; SDS loading buffer was purchased from Beyotime, catalog number P0015; The primary antibodies P21, P16, P53 and GAPDH were all purchased from Wuhan Sanying Biotechnology Co., Ltd., with catalog numbers 10355-1-AP, 10883-1-AP, 21891-1-AP and 60004-1-Ig, respectively. Horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (HRP-conjugated Goat Anti-Rabbit IgG) was purchased from Santa Cruz Biotechnology, catalog number sc-2004; The β-galactosidase staining kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number C0602. EdU cell proliferation assay kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number C0071S; RNA extraction and separation kit was purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number DP424. The CCK-8 cell counting kit was purchased from TargetMol Chemicals Inc., catalog number C0005. The BCA protein concentration assay kit was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 20200ES; PAGE gel rapid preparation kit was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 20324ES; The ECL chemiluminescence detection kit was purchased from GlpBio Technology LLC, catalog number GK10008; The modified ammonium persulfate solution was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., product number 20325-F.

[0038] 2. Experimental Apparatus The cell culture incubator was purchased from ESCO (ESCO Micro Pte. Ltd., Singapore), model MCO-18AIC; the clean bench was purchased from ESCO (ESCO Micro Pte. Ltd., Singapore), model AC2-4S1; and the inverted fluorescence microscope was purchased from Zeiss (Carl Zeiss Microscopy GmbH, Germany), model Axio Observer 3. The microplate reader was purchased from BioTek (Agilent BioTek Instruments, USA), model Synergy H1. The real-time fluorescence quantitative PCR instrument was purchased from Yuanzan Life (Hangzhou Yuanzan Life Technology Co., Ltd., China), model YZ-96. The chemiluminescence imaging system (Western blot imaging system) was purchased from Sensitech (Shanghai Sensitech Technology Co., Ltd., China), model ChampChemi 610.

[0039] 3. Description of the testing method (1) CCK-8 detection After digesting and counting mouse embryonic fibroblasts, they were seeded evenly in 96-well plates at a rate of 4000 cells / well. After the cells were fully adhered, the corresponding concentration of W2502 was added to treat the cells for 48 h. After the intervention, fresh complete culture medium was mixed with CCK-8 reagent at a ratio of 10:1, the old culture medium was discarded, and fresh culture medium mixed with CCK-8 was added. After incubation for 50 min, the absorbance was measured at 450 nm using a microplate reader, and the cell viability was calculated.

[0040] (2) SA-β-gal staining Cells were pretreated with 100 μM MW2502 for 24 h, followed by treatment with 0.2 μM Dox for 24 h. After Dox treatment, cells were treated with 100 μM MW2502 for another 48 h. After intervention, cells were fixed with β-galactosidase fixative for 20 min, followed by washing three times with PBS for 3 min each time. After washing, 100 μL of SA-β-gal staining solution was added to each well. The plates were sealed with sealing film and incubated overnight at 37°C in a physiological and biochemical incubator. After incubation, images were taken under an inverted microscope. The degree of cell senescence was detected using a β-galactosidase kit; senescent cells were stained blue, and the more blue cells, the greater the degree of cell senescence.

[0041] (3) EdU staining Cells treated with 100 μM W2502 were inoculated into 12-well plates. After complete cell adhesion, an equal volume of pre-warmed (37°C) 2X EdU working solution (20 μM) was added to each well to bring the final EdU concentration to 1X. Incubation was performed for 4 h. After EdU labeling, the culture medium was removed, and 1 ml of fixative (4% paraformaldehyde) was added. The cells were fixed at room temperature for 30 min. The fixative was removed, and each well was washed three times with 1 ml of washing buffer for 3-5 minutes each time. The washing buffer was removed, and each well was incubated for 10-15 minutes at room temperature with 1 ml of permeabilization buffer (PBS containing 0.3% Triton X-100). The permeabilization buffer was removed, and each well was washed 1-2 times with 1 ml of washing buffer for 3-5 minutes each time. The Click additive solution was prepared. The Click reaction was prepared (to be used within 15 minutes). Add 300 μL of Click reaction solution to a 12-well plate and gently shake the plate to ensure the reaction mixture evenly covers the sample. Incubate at room temperature in the dark for 30 minutes. Aspirate the Click reaction solution and wash three times with washing buffer for 3-5 minutes each time. After aspirating the washing buffer, add 1 mL of 1X Hoechst 33342 solution to each well and incubate at room temperature in the dark for 15 minutes. Aspirate the 1X Hoechst 33342 solution. Wash three times with washing strip buffer for 3-5 minutes each time. Replace with PBS and take a picture. Count the number of EdU-positive cells.

[0042] The EdU cell proliferation assay kit was used to assess cell proliferation capacity. Cells with strong proliferation capacity exhibited very bright green fluorescence under a fluorescence microscope, while senescent cells that had lost their proliferative capacity did not show green fluorescence. After treatment with Hochest 33343 dye, all live cells showed blue fluorescence under a fluorescence microscope. The proportion of cells with proliferative capacity in the total number of cells was calculated, and the strength of cell proliferation capacity was finally assessed.

[0043] (4) Western blot assay Cells treated with 100 μM W2502 were inoculated into 6-well plates. Each well was washed once with 1 ml PBS, and the PBS was discarded. 60 μL of RIPA lysis buffer was added to each well and the plates were placed on ice for 5 min to lyse. Cells were then thoroughly pipetted until no obvious particles were observed. The supernatant was collected into 1.5 ml conical centrifuge tubes and centrifuged at 12000 rpm for 10 min at 4 ℃. After centrifugation, the supernatant was transferred to a new 1.5 ml centrifuge tube. A portion of the protein supernatant was used for BCA quantification (diluted 2-fold, then 5-fold). The remaining portion was added to the corresponding volume of 5x Loading buffer (15 μL) and boiled in a metal bath at 100 ℃ for 10 min. The plates were then stored at -20 ℃.

[0044] Take a clean 50 ml centrifuge tube and prepare four 12.5% ​​SDS-PAGE gels (add 10 ml of lower gel solution, 10 ml of lower gel buffer, and 250 μl of modified ammonium persulfate solution to the gel preparation glass plate). Then prepare the upper stacking gel (add 3 ml of upper gel solution, 3 ml of upper gel buffer, and 30 μl of modified ammonium persulfate solution to the gel preparation glass plate). Insert a 10-well comb and wait 30 min for the gel to solidify before loading the samples. For electrophoresis, run at 80 V for 30 min. After the proteins enter the lower separating gel, increase the pressure to 120 V. Once the bromophenol blue reaches the edge of the lower gel, begin the transfer process.

[0045] Add cooled transfer buffer to the iron pan, and assemble the membrane in the following order (transfer clamp negative electrode - sponge - gel - activated PVDF membrane - sponge - transfer clamp positive electrode) before transfer. After transfer, wash once with 1×TBST, block with rapid blocking buffer for 20 min, and incubate with primary antibody (4 ℃, overnight). After overnight incubation with primary antibody at 4 ℃, wash three times with 1×TBST, then incubate with secondary antibody at room temperature for 2 h. After incubation, wash three more times with 1×TBST, add developing solution, and develop in a Western blot instrument. Use the following antibodies for Western blotting: p21 (proteintech, 10355-1-AP), p53 (proteintech, 10442-1-AP), and GAPDH (proteintech, 60004-1-Ig).

[0046] 4. Experimental Procedure and Results Drug treatment and cell culture: Starting from passage 4, mouse embryonic fibroblasts (MEFs) were treated with either W2502 or the negative control solvent DMSO. The culture medium was replaced with fresh medium containing W2502 or the control solvent every other day. This treatment was repeated for three to six passages. The effect of W2502 on senescence-related phenotypes of MEFs was then examined.

[0047] Please see Figure 4 CCK-8 cell viability assay results showed that W2502 did not exhibit significant cytotoxicity against mouse embryonic fibroblasts (MEFs) within the concentration range of 1 μM to 200 μM.

[0048] Please see Figure 5 and Figure 6 , Figure 6 Statistical analysis was performed using SPSS 22.0 and GraphPad Prism 10 software. Comparisons between two groups were performed using the two-tailed unpaired Student's t-test; comparisons between three or more groups were performed using one-way ANOVA and Tukey's post-hoc multiple comparison test. All quantitative data are expressed as mean ± standard deviation (mean ± SD). This indicates a comparison with the model group. P<0.001. SA-β-galactosidase staining experiments showed that, compared with the model control group, mouse embryonic fibroblasts treated with W2502 100 μM for three generations (fourth to sixth generations) had a lower proportion of SA-β-gal positive cells, indicating that W2502 can effectively inhibit the replicative senescence process of MEFs.

[0049] Please see Figure 7 and Figure 8 , Figure 8 Statistical analysis was performed using SPSS 22.0 and GraphPad Prism 10 software. Comparisons between two groups were performed using the two-tailed unpaired Student's t-test; comparisons between three or more groups were performed using one-way ANOVA and Tukey's post-hoc multiple comparison test. All quantitative data are expressed as mean ± standard deviation (mean ± SD). This indicates a comparison with the model group. P<0.001. EdU staining experiments showed that W2502 100 μM treatment could promote cell proliferation, specifically characterized by a higher proportion of EdU-positive cells in mouse embryonic fibroblasts treated with W2502.

[0050] Please see Figure 9 Western blot analysis showed that W2502 treatment downregulated the protein expression levels of aging markers p21 and p53.

[0051] Example 3: Compound W2502 improves the cell phenotype of DOX-induced premature aging human fetal lung fibroblasts (MRC-5). 1. Experimental reagents Doxorubicin hydrochloride was purchased from Shanghai Lanmu Chemical Co., Ltd., product number S1208; DMEM culture medium was purchased from Jiangsu Kaiji Biotechnology Co., Ltd., product number KGL1211-500; PBS buffer was purchased from Jiangsu Kaiji Biotechnology Co., Ltd., catalog number XB07; Trypsin digestion solution was purchased from Jiangsu Kaiji Biotechnology Co., Ltd., product number KGY001; Fetal bovine serum was purchased from CLARK BIOSCIENCE, catalog number FB15015 or FB25015; Tris-glycine SDS-PAGE electrophoresis buffer was purchased from Servicebio, catalog number G2018-15; The transfer buffer was purchased from Servicebio, catalog number G2017-15; SDS loading buffer was purchased from Beyotime, catalog number P0015; The primary antibodies P21, P16, P53 and GAPDH were all purchased from Wuhan Sanying Biotechnology Co., Ltd., with catalog numbers 10355-1-AP, 10883-1-AP, 21891-1-AP and 60004-1-Ig, respectively. Horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (HRP-conjugated Goat Anti-Rabbit IgG) was purchased from Santa Cruz Biotechnology, catalog number sc-2004; The β-galactosidase staining kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number C0602. EdU cell proliferation assay kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number C0071S; RNA extraction and separation kit was purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number DP424. The CCK-8 cell counting kit was purchased from TargetMol Chemicals Inc., catalog number C0005. The BCA protein concentration assay kit was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 20200ES; PAGE gel rapid preparation kit was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 20324ES; The ECL chemiluminescence detection kit was purchased from GlpBio Technology LLC, catalog number GK10008.

[0052] 2. Experimental Apparatus The cell culture incubator was purchased from ESCO (ESCO Micro Pte. Ltd., Singapore), model MCO-18AIC; the clean bench was purchased from ESCO (ESCO Micro Pte. Ltd., Singapore), model AC2-4S1; and the inverted fluorescence microscope was purchased from Zeiss (Carl Zeiss Microscopy GmbH, Germany), model Axio Observer 3. The microplate reader was purchased from BioTek (Agilent BioTek Instruments, USA), model Synergy H1. The real-time fluorescence quantitative PCR instrument was purchased from Yuanzan Life (Hangzhou Yuanzan Life Technology Co., Ltd., China), model YZ-96. The chemiluminescence imaging system (Western blot imaging system) was purchased from Sensitech (Shanghai Sensitech Technology Co., Ltd., China), model ChampChemi 610.

[0053] 3. Description of the testing method In this embodiment, the specific procedures for CCK-8 detection, SA-β-gal staining, EdU staining, and Western blot experiment can be found in Example 2. Except for the incubation time of 6 h in EdU staining, the other steps are the same as in Example 2 and will not be repeated here.

[0054] 4. Experimental Procedure and Results Drug treatment and cell culture: 23rd generation human embryonic lung fibroblasts (MRC-5) were pretreated with 100 μM W2502 or negative control solvent DMSO for 24 h. Then, MRC-5 cells were treated with 0.2 μM Dox for 24 h to induce senescence. Finally, the cells were treated with 100 μM W2502 or negative control solvent DMSO for 48 h. The effect of W2502 on doxorubicin-induced senescence-related phenotypes in MRC-5 cells was then examined.

[0055] Please see Figure 10 CCK-8 cell viability assay results showed that W2502 did not exhibit significant cytotoxicity against human embryonic lung fibroblasts (MRC-5) within the concentration range of 1 μM to 200 μM.

[0056] Please see Figure 11 and Figure 12 , Figure 12 Statistical analysis was performed using SPSS 22.0 and GraphPad Prism 10 software. Comparisons between two groups were conducted using the two-tailed unpaired Student's t-test; comparisons between three or more groups were performed using one-way ANOVA and Tukey's post-hoc multiple comparison test. All quantitative data are expressed as mean ± standard deviation (mean ± SD). A # indicates that compared to the control group, ###P < 0.001. This indicates a comparison with the model group. P<0.01. SA-β-galactosidase staining experiments showed that the proportion of SA-β-gal positive cells in the W2502 treatment group was significantly reduced compared with the doxorubicin group (i.e., the model control group), indicating that W2502 can effectively inhibit the doxorubicin-induced senescence process in MRC-5 cells.

[0057] Please see Figure 13 and Figure 14 , Figure 14 Statistical analysis was performed using SPSS 22.0 and GraphPad Prism 10 software. Comparisons between two groups were conducted using the two-tailed unpaired Student's t-test; comparisons between three or more groups were performed using one-way ANOVA and Tukey's post-hoc multiple comparison test. All quantitative data are expressed as mean ± standard deviation (mean ± SD). A # indicates that compared to the control group, ###P < 0.001. This indicates a comparison with the model group. P<0.05. EdU staining experiments showed that treatment with 100 μM W2502 could promote cell proliferation, specifically characterized by a higher proportion of EdU-positive cells in human embryonic lung fibroblasts treated with W2502.

[0058] Please see Figure 15 Western blot analysis showed that W2502 treatment downregulated the protein expression levels of aging markers p16 and p21.

[0059] Example 4: Compound W2502 improves the aging phenotype of *C. elegans*. 1. Experimental reagents The main reagents used in the embodiments of this invention are as follows: Sodium chloride, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number C111533; Potassium dihydrogen phosphate, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number P104075; Disodium hydrogen phosphate, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., item number S112446; Disodium hydrogen phosphate dodecahydrate was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number S112619; Agar powder, purchased from Beijing Solarbio Technology Co., Ltd., product number A8190; Peptone, purchased from Beijing Solarbio Technology Co., Ltd., product number P8450; 5-Fluoro-2′-deoxyuridine, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number F110732; Isopropyl alcohol was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number I811925. Magnesium sulfate heptahydrate was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number M813597. Calcium chloride dihydrate, purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number C804986; Dipotassium hydrogen phosphate trihydrate, purchased from Shanghai Titan Technology Co., Ltd., General Reagent Brand, product number G28945C; Sodium hydroxide was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number S111518; Sodium hypochlorite was purchased from Shanghai Titan Technology Co., Ltd., under the general reagent brand, with product number DG82072C. Glycerin, purchased from Beijing Solarbio Technology Co., Ltd., product number G8190; Cholesterol, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number C104036.

[0060] 2. Experimental Apparatus The temperature and humidity chamber was purchased from Shanghai Yiheng Scientific Instruments Co., Ltd., model BPS-250CB. The clean bench was purchased from Shanghai Boxun Medical Bio-Instrument Co., Ltd., model BJ-1CD. The biosafety cabinet was purchased from Esco Lifesciences Group, model AC2-4E9. The benchtop high-speed micro-volume refrigerated centrifuge was purchased from Eppendorf AG, model Centrifuge 5425 R; The tabletop high-speed, large-capacity refrigerated centrifuge was purchased from Eppendorf AG, model Centrifuge 5810 R. The single-channel adjustable-range pipette was purchased from Eppendorf AG, model Research plus. The power supply for the electrophoresis apparatus was purchased from Beijing Junyi Oriental Electrophoresis Equipment Co., Ltd., model JY300E. The gel imaging system, model GenoSens 2000, was purchased from Shanghai Qinxiang Scientific Instruments Co., Ltd. A standard optical microscope, purchased from Carl Zeiss Microscopy GmbH, model Axio Lab.A1; The real-time PCR instrument was purchased from Bio-Rad Laboratories, Inc., model CFX96 Touch. The upright fluorescence microscope, purchased from Carl Zeiss Microscopy GmbH, is model Axio Imager A2.

[0061] 3. Description of the testing method Lifetime Experiment Before the experiment began, the identity of the *C. elegans* strain was determined through phenotypic or genotypic identification. After verification, the strain was placed under standard conditions for continuous subculturing for 3-5 generations to eliminate the potential impact of environmental stress on lifespan.

[0062] Synchronized L4 stage wild-type nematodes were plated onto control NGM plates containing OP50 and experimental NGM plates containing 200 μM W2502, respectively. All *C. elegans* were cultured uniformly in a constant temperature and humidity incubator. When repotting was required, pre-prepared 5-fluorouracil deoxyribonucleoside (FUDR, 5 mg / mL) was added to the culture medium. Approximately 30 *C. elegans* were transferred to each culture dish, with 3 plates per group, totaling approximately 70 worms. Every 3-4 days, the *C. elegans* in each culture dish were individually transferred to a fresh, corresponding culture dish. This procedure ensured the drug's effectiveness and provided sufficient food (except for *C. elegans* requiring special culture, all worms in this study were cultured using this method).

[0063] Lifespan statistics were started when *C. elegans* entered the natural death phase (the start time for lifespan statistics should not exceed 10 days). The specific method for determining whether *C. elegans* was dead was as follows: first, the platinum wire part of the nematode needle was sterilized by high temperature with the outer flame of a lighter. Then, one end of the platinum wire was gently touched to the head and tail of the *C. elegans*. If the *C. elegans* did not react and no pharyngeal pump activity was observed, it was determined that the *C. elegans* was dead. The number of dead *C. elegans* was counted daily. Conditions such as protrusion of the genital opening, leakage of intestinal contents, and hatching within the *C. elegans* were not included in the number of dead *C. elegans* (this was the method used to determine nematode death in this study).

[0064] Lipofuscin experiment The level of lipofuscin accumulation was detected on day 7 of adult nematodes. Wild-type nematodes synchronized to L4 were placed on control plates and experimental plates containing W2502 and cultured as described above. On day 7, the nematodes were anesthetized with a prepared 4% tetraimidazole hydrochloride solution and photographed using an upright fluorescence microscope under excitation wavelengths of 360-370 nm and emission wavelengths of 420-460 nm.

[0065] Motion analysis experiment The analysis of locomotor ability was performed on day 7 of the adult nematodes. The specific method was as follows: wild-type nematodes that had been synchronized and developed to the L4 stage were cultured according to the method described above. On day 7, M9 buffer was placed on a glass slide using a pipette, and the nematodes were transferred into the droplet. The number of body movements of the *C. elegans* within 20 seconds was recorded under a microscope, and the number of body movements was used to reflect changes in locomotor ability.

[0066] Parkinson's disease model rescue experiment NL5901 ( unc-54p::alphasynuclein::YFP The mutant strain reflects the accumulation level of α-synuclein in nematodes by detecting the fluorescence intensity of the body wall muscle. Synchronized nematodes cultured to L4 were placed on control plates and experimental plates containing W2502 and cultured as described above. On day 7 of adulthood, the nematodes were anesthetized with 4% tetraimidazole hydrochloride, placed on clean glass slides, and images were acquired using an upright fluorescence microscope (10x magnification).

[0067] 4. Experimental Procedure and Results Nematode elegans culture: Except for special cultures or specific experimental needs, Nematodes elegans are cultured uniformly in a constant temperature and humidity incubator at 20°C and 60% relative humidity. Each incubator needs to be equipped with an independent thermometer and hygrometer to monitor and calibrate the actual environmental parameters in real time to ensure the stability and reproducibility of the culture conditions.

[0068] Please see Figure 16 , Figure 16 Statistical analysis was performed using SPSS 22.0 and GraphPad Prism 10 software. Comparisons between two groups were performed using the two-tailed unpaired Student's t-test; comparisons between three or more groups were performed using one-way ANOVA and Tukey's post-hoc multiple comparison test. All quantitative data are expressed as mean ± standard deviation (mean ± SD). This indicates that compared with the control group, P<0.01, P < 0.001. Lifespan experiments on *C. elegans* showed that W2502 could prolong the lifespan of wild-type *C. elegans*, with the most significant effect observed at a concentration of 200 μM W2502, representing a 21.23% increase. Therefore, in subsequent *C. elegans* experiments, the concentration of compound W2502 was consistently 100 μM.

[0069] Please see Figure 17 and Figure 18 , Figure 18Statistical analysis was performed using SPSS 22.0 and GraphPad Prism 10 software. Two-tailed unpaired Student's t-test was used for comparisons between the two groups. All quantitative data are expressed as mean ± standard deviation (mean ± SD). This indicates that compared with the control group, P<0.001. The results of the lipofuscin experiment on *C. elegans* showed that W2502 significantly reduced the accumulation of lipofuscin in wild-type *C. elegans* on day 7.

[0070] Please see Figure 19 , Figure 19 Statistical analysis was performed using SPSS 22.0 and GraphPad Prism 10 software. Two-tailed unpaired Student's t-test was used for comparisons between the two groups. All quantitative data are expressed as mean ± standard deviation (mean ± SD). This indicates that compared with the control group, P<0.05, P<0.01. The results of the kinetic analysis experiment of *C. elegans* showed that W2502 prolonged the number of body swings of wild-type *C. elegans* on the seventh day.

[0071] Please see Figure 20 and Figure 21 , Figure 21 Statistical analysis was performed using SPSS 22.0 and GraphPad Prism 10 software. Two-tailed unpaired Student's t-test was used for comparisons between the two groups. All quantitative data are expressed as mean ± standard deviation (mean ± SD). This indicates that compared with the control group, P<0.001. The results of the Parkinson's disease model rescue experiment showed that W2502 significantly reduced the accumulation of α-synuclein in vivo on day 7 in the NL5901 (unc-54p::alphasynuclein::YFP) mutant strain.

[0072] In summary, this invention provides novel anti-aging compounds. Experimental results in in vitro anti-aging screening models using MEF cells and MRC-5 cells demonstrate that the compounds significantly reduce the proportion of cells positive for aging-related β-galactosidase (SA-β-gal) staining, downregulate the protein expression levels of aging markers p53, p21, and p16, and restore the proliferative capacity of senescent cells. In *C. elegans*, this compound prolongs the lifespan of the nematodes, improves their motility, and reduces lipofuscin accumulation due to aging. Therefore, this compound can be used in the preparation of anti-aging drugs and health products. This invention provides new candidate compounds and experimental evidence for the development of new anti-aging drugs and basic research, and has significant practical implications.

Claims

1. The application of an anti-aging compound in the preparation of anti-aging drugs, characterized in that, The chemical formula of the compound is C. 18 H 14 N₂O₄, the structural formula of which is shown in the following formula W₂₅O₂: ； The compound achieves anti-aging by reducing the proportion of cells that are positive for aging-related β-galactosidase staining, downregulating the protein expression levels of aging markers p53, p21, and p16, and restoring the proliferative capacity of senescent cells.

2. The use of the anti-aging compound according to claim 1 in the preparation of anti-aging drugs, characterized in that, The compound was obtained via the following preparation route: 。 3. The use of the anti-aging compound according to claim 2 in the preparation of anti-aging drugs, characterized in that, The method for preparing the compound includes the following steps: Raw materials SM-1 and SM-2 were placed in a three-necked round-bottom flask, and dry DMF was added and stirred to disperse them. Argon gas was purged three times, and the temperature was lowered to 0 °C. DIPEA and DMAP were added, followed by HATU. The mixture was stirred at 0 °C for 20 min, and the reaction was carried out at room temperature for 2 h. The reaction was then heated to 70 °C and carried out for 24 h. The reaction was detected by TLC to indicate that it was complete. The mixture was cooled to room temperature and added to water to precipitate a solid. The solid was washed with water, collected, dried, and then pulped with EA / PE. The solid was filtered and collected to obtain compound M1. Compound M1 was placed in a three-necked round-bottom flask, purged with argon three times, cooled to -78 °C, and BBr3 was added. The reaction was allowed to slowly return to room temperature and then allowed to proceed overnight. The reaction was detected as complete by LC-MS. The reaction was then cooled to -78 °C and quenched with MeOH. The reaction was evaporated to dryness, and the crude product was pulped with MeOH / EA. The solid was collected by filtration, and the solid was then mixed with purified water and ultrasonically pulped. The solid was then filtered, washed with water, and the solid was collected to obtain compound W2502.

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