Alizarin A ring glycosylation derivative, synthesis method thereof and application of alizarin A ring glycosylation derivative in preparation of medicine for treating myocardial cell injury

Abstract

The invention discloses an alizarin A ring glycosylation derivative, a synthesis method thereof and application of the alizarin A ring glycosylation derivative in preparation of a medicine for treating myocardial cell injury. The structural formula of the alizarin A ring glycosylation derivative is # imgabs0 #. A series of alizarin A ring glycosylation derivatives are designed and synthesized through a computer-aided and chemical synthesis method; finally, the alizarin A ring glycosylation derivative, namely 1-amino-2-methyl-3-hydroxy anthraquinone, is found to be basically kept unchanged in antioxidant effect, increased in solubility in water, enhanced in binding capacity with 5 '-AMP activated protein kinase alpha2 protein, and enhanced in effect of treating and preventing myocardial cell dysfunction.

Description

Technical Field

[0001] The present invention belongs to the technical field of drugs for treating myocardial cell damage, and particularly relates to a glycosylated alizarin A ring derivative, a synthesis method thereof, and an application thereof in preparing drugs for treating myocardial cell damage. Background Art

[0002] Myocardial cell damage refers to the destruction of myocardial cell structural integrity or dysfunction due to ischemia, hypoxia, inflammation, toxic substances or mechanical stress, which manifests as pathological processes such as increased cell membrane permeability, organelle dysfunction (such as mitochondrial damage), energy metabolism disorders and decreased contractility. Cardiovascular disease is the main cause of myocardial cell damage. According to data from the World Health Organization (WHO), cardiovascular disease caused 17.9 million deaths in 2021, accounting for 32% of the global total death toll. In China, the "China Cardiovascular Health and Disease Report 2022" shows that the current number of people with cardiovascular disease has reached 330 million, and the annual incidence of myocardial infarction is about 1.5 million cases, and it is showing a trend of younger age (the proportion of people under 45 years old has risen to 15%).

[0003] The pathogenesis of cardiomyocyte injury involves multiple pathways, among which oxidative stress is a major mechanism of myocardial cell injury. Following myocardial cell injury, excessive production of reactive oxygen species (ROS) leads to lipid peroxidation, protein denaturation, and DNA damage, disrupting the mitochondrial electron transport chain (ETC) and exacerbating ATP synthesis disorders.

[0004] Current treatment strategies focus on etiological intervention and symptom management. Commonly used treatments and interventions include: ① Antioxidants such as coenzyme Q10 (ubiquinone) improve mitochondrial function by scavenging ROS, but their bioavailability is low; ② β-blockers (such as metoprolol) reduce myocardial oxygen consumption and inhibit sympathetic nerve overactivation; ③ RAAS inhibitors (such as enalapril) reduce cardiac afterload and delay ventricular remodeling; ④ Statins (such as atorvastatin) have anti-inflammatory and plaque-stabilizing effects in addition to lipid-lowering properties; ⑤ Interventional and surgical interventions, such as percutaneous coronary intervention (PCI) to restore blood flow and left ventricular assist devices (LVADs) to improve survival in patients with end-stage heart failure; and ⑥ Regenerative medicine, such as stem cell therapy (such as mesenchymal stem cells), promotes angiogenesis through paracrine effects but carries the risk of tumorigenesis. However, existing drugs often target a single pathological link, making it difficult to fully block the injury cascade. Furthermore, long-term use can easily lead to side effects such as hypotension and renal impairment.

[0005] Alizarin (1,2-dihydroxyanthraquinone) is a natural anthraquinone compound that has been recently shown to possess significant cardioprotective potential. Molecular docking revealed that the binding energy between alizarin and the AMPKα2 protein reached -7.41 kcal / mol, suggesting strong binding affinity between the two. Studies have shown that alizarin can activate AMPKα2 and potentially treat and prevent cardiomyocyte dysfunction. However, alizarin is virtually insoluble in water, which greatly hinders its practical application. Furthermore, traditional therapeutic agents for cardiomyocyte dysfunction primarily target beta-blockers, non-dihydropyridine calcium channel blockers, and sodium channel blockers. Summary of the Invention

[0006] The technical problem solved by the present invention is to provide a glycosylated derivative of alizarin A ring and a synthesis method thereof. The glycosylated derivative of alizarin A ring can be used as a targeted AMPKα2 agonist to resist myocardial cell damage, and further can be used to prepare a drug for treating myocardial cell damage.

[0007] The present invention adopts the following technical solution to solve the above technical problems, a glycosylated derivative of alizarin A ring, characterized in that the structural formula of the glycosylated derivative of alizarin A ring is:

[0008]

[0009] The method for synthesizing the glycosylated ring A derivative of alizarin of the present invention is characterized in that the specific synthesis steps are:

[0010] Step S1, adding alizarin, CH3CN and N,N-dimethylformamide (DMF) to a reaction vessel, and after the alizarin is completely dissolved, adding NaH solution at 0°C and stirring to mix evenly, then adding 3,5-di-O-(p-toluoyl)-2-deoxy-D-ribofuranose chloride, stirring at room temperature, adding acetic acid after the reaction is completed to quench the reaction, then adding water to the reaction system and extracting with ethyl acetate, combining the organic phases and drying with anhydrous magnesium sulfate, and removing the solvent by distillation under reduced pressure to obtain a crude product;

[0011] Step S2: Add CH3OH and DMF to the reaction vessel containing the crude product from step S1, stir and dissolve at room temperature, then add NaOMe and continue stirring at room temperature. After the reaction is completed, add acetic acid to quench the reaction, then add water to the reaction system and extract with ethyl acetate. Combine the organic phases and dry them over anhydrous magnesium sulfate. After removing the solvent by distillation under reduced pressure, purify on a silica gel column to obtain a yellow solid product, i.e., a glycosylated derivative of alizarin A ring.

[0012] Furthermore, the synthesis route of the alizarin A ring glycosylated derivative is:

[0013]

[0014] Furthermore, in step S1, the molar ratio of alizarin, NaH and 3,5-di-O-(p-toluoyl)-2-deoxy-D-ribofuranose chloride is 1:1.5-1:1.1-1.5.

[0015] Furthermore, the NaH solution in step S1 is a mineral oil solution containing 60 wt % NaH formed by NaH and mineral oil.

[0016] Furthermore, the molar ratio of NaOMe in step S2 to alizarin in step S1 is 5-10:1.

[0017] The invention relates to the use of the alizarin A ring glycosylated derivative in the preparation of a medicine for treating myocardial cell damage.

[0018] The application of the glycosylated alizarin A ring derivative of the present invention as an AMPKα2-targeted agonist reduces the apoptosis of myocardial cells by activating the ROS-NOX2 / 4-p-PI3K / p-AKT signaling pathway, thereby protecting myocardial cells.

[0019] The present invention has the following advantages and beneficial effects compared to existing technologies: A series of glycosylated alizarin A ring derivatives were designed and synthesized using computer-assisted and chemical synthesis methods. The final glycosylated alizarin A ring derivative, 1-amino-2-methyl-3-hydroxyanthraquinone, was selected and found to maintain substantially unchanged antioxidant activity, increase water solubility, enhance binding to 5'-AMP-activated protein kinase (AMPK) α2 protein (-9.06 kJ / mol), and enhance therapeutic and preventive effects on cardiomyocyte dysfunction. The glycosylated alizarin A ring derivatives prepared by the present invention have a significant advantage over alizarin and other drugs in that they can act as targeted drug molecules on AMPK α2, activating the ROS-NOX2 / 4-p-PI3K / p-AKT signaling pathway, reducing cardiomyocyte apoptosis, and protecting cardiomyocyte function. Therefore, the use of glycosylated alizarin A ring derivatives in the treatment of cardiomyocyte damage has the potential to become a first-line drug for the treatment of cardiovascular disease in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a molecular model diagram of the binding of alizarin A-ring glycosylated derivatives to AMPKα2.

[0021] Figure 2 The results of the cardiomyocyte proliferation experiment are shown in Figure 2. The data are expressed as mean ± standard deviation, n = 6. a P<0.05vs blank control group, b P<0.05vs myocardial cell injury group.

[0022] Figure 3 This is the immunofluorescence image of reactive oxygen species (ROS) in cardiomyocytes. Blue fluorescence represents the cell nucleus and red fluorescence represents ROS.

[0023] Figure 4 The immunofluorescence intensity analysis of myocardial reactive oxygen species (ROS) is shown in Figure 2. Data are expressed as mean ± standard deviation, n = 6. a P<0.05vs blank control group, b P<0.05vs myocardial cell injury group.

[0024] Figure 5 This is the immunoblotting of oxidative stress proteins NOX2 and NOX4.

[0025] Figure 6 The grayscale analysis of the oxidative stress proteins NOX2 and NOX4 by immunoblotting is shown in Figure 2. The data are expressed as mean ± standard deviation, n = 6. a P<0.05vs blank control group, b P<0.05vs myocardial cell injury group.

[0026] Figure 7 This is an immunofluorescence image of AMPKA2 in cardiomyocytes. The blue fluorescence represents the cell nucleus and the green fluorescence represents AMPKA2.

[0027] Figure 8 The immunofluorescence intensity analysis of AMPKα2 in cardiomyocytes is shown in Figure 5. The data are expressed as mean ± standard deviation, n = 6. a P<0.05vs blank control group, b P<0.05vs myocardial cell injury group.

[0028] Figure 9 Figure 3 is immunoblotting of AMPKα2 protein in cardiomyocytes.

[0029] Figure 10 Figure 2 is the grayscale analysis of AMPKα2 protein immunoblotting in cardiomyocytes. The data are expressed as mean ± standard deviation, n = 6. a P<0.05vs blank control group, b P<0.05vs myocardial cell injury group.

[0030] Figure 11 This is immunoblotting of P-AKT and P-PI3K proteins in cardiomyocytes.

[0031] Figure 12 Grayscale analysis of P-AKT and P-PI3K protein immunoblotting in cardiomyocytes. Data are expressed as mean ± standard deviation, n = 6. a P<0.05vs blank control group, bP<0.05vs myocardial cell injury group. DETAILED DESCRIPTION

[0032] The above contents of the present invention are further described in detail below through examples, but this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above contents of the present invention fall within the scope of the present invention.

[0033] Example

[0034] 1. Molecular Docking: Auto Dock Tools 1.5.6 is a molecular docking software based on network pharmacology. It consists of an automatic grid program (for energy calculation) and an automatic docking program (for conformational search and evaluation). It uses the Lamarckian genetic algorithm (LGA) to optimize the ligand-receptor binding sites. The 3D structure of the ligand small molecule was obtained from the PubChem database, while the receptor structure of the core target protein was obtained from the PDB database (https: / / www.rcsb.org / ). The small molecule serves as the ligand, and the target protein serves as the receptor. The ligand minimizes the structural energy in the ChemBioDraw 3D module. The receptor is modified (including dehydration and hydrogenation) using Autodock Tools 1.5.6 and exported to the pdbqt format. Results analysis and visualization are performed using PyMOL 2.3.0 and BIOVIA Discovery Studio 2016, which support intuitive display of binding modes and interaction forces.

[0035] 2. Drugs: Alizarin A ring glycosylated derivative (synthesized by the following method); Alizarin (purchased from Merck); CH3CN, NaH, NaOMe, 3,5-di-O-(p-toluoyl)-2-deoxy-D-ribofuranose chloride (Annaiji Chemical Co., Ltd.); Superoxide dismutase (SOD) (Sigma); ROS-antibody (A16669) (ABclonal), NOX2-antibody (PA5-76034) (Thermo Fisher Scientific), NOX4-antibody (MA5-32090) (Thermo Fisher Scientific), AMPKα2-antibody (ab105028) (abcam Trading Co., Ltd.); p-AKT (#AF8355), p-PI3K (#AF3242), GADPH (#AF7021), (Affinity Bioscience Trading Co., Ltd.).

[0036] The specific synthesis process of the glycosylated derivatives of alizarin A ring is as follows:

[0037] (1) Alizarin (1 mmol, 240 mg), CH3CN (1 mL) and DMF (3 mL) were added to a 25 mL round-bottom flask to completely dissolve the alizarin. NaH solution (1.5 mmol, 60 mg, 60 wt%, mineral oil solvent) was added at 0°C and the mixture was stirred for 10 minutes. 3,5-di-O-(p-toluoyl)-2-deoxy-D-ribofuranose chloride (1.1 mmol, 428 mg) was added and the mixture was stirred at room temperature for 1 hour. After the reaction, a few drops of acetic acid were added to quench the reaction. Water was added to the reaction system and the mixture was extracted with ethyl acetate (15 mL*3). The organic phases were combined and dried over anhydrous magnesium sulfate. The solvent was removed by distillation under reduced pressure. The crude product was directly used in the next reaction.

[0038] (2) CH3OH (2 mL) and DMF (10 mL) were added to a round-bottom flask containing the crude product and stirred at room temperature to dissolve. NaOMe (6 mmol, 254 mg) was then added and stirred at room temperature for 2 hours. After the reaction was completed, a few drops of acetic acid were added to quench the reaction. Water was then added to the reaction system and extracted with ethyl acetate (15 mL*3). The organic phases were combined and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure and then purified on a silica gel column (dichloromethane / ethyl acetate = 1 / 4, V / V) to obtain a yellow solid product (224 mg, total yield for two steps 63%).

[0039] The structural formula of the glycosylated derivative of alizarin A ring:

[0040]

[0041] 1-Hydroxy-2-(((2S,4R,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)oxy)anthracen e-9,10-dione:Eluent:dichloromethane:petroleum ether-ethylacetate(1:4);yellow solid;1HNMR(400MHz,DMSO-d6)δ:12.64(br s,1H),8.21-8.24(m,1H),8.16-8.20(m,1H),7.90-7.96(m,2H),7.20(d,J=8 .4Hz,1H),7.55(d,J=8.4Hz,1H),6.06(dd,J1=5.2Hz,J2=2.4Hz,1H),5.24(br s,1H),4.74(br s,1H),4.31-4.35(m,1H),3.87-3.91(m,1H),3.45-3.51(m,1H),3.34-3.37(m,1H),2. 43(ddd,J1=13.6Hz,J2=8.4Hz,J3=2.4Hz,1H),2.24(td,J1=13.6Hz,J2=8.4Hz,1H).13C NMR(150MHz,DMSO-d6)δ:189.0,181.3,152.7,151.5,135.7,134.8,133.9,13 3.4,127.3,127.1,126.1,121.7,120.7,116.7,103.2,88.9,70.8,63.0,41.6.

[0042] 3. Cells: H9c2 (rat myocardial) cell line (Cell Bank, Chinese Academy of Sciences), cultured in high-glucose DMEM medium (10% fetal bovine serum, 100 U / mL penicillin, and 100 mg / mL streptomycin) in a 37°C, 5% CO2 incubator, and passaged once every other day after digestion with 0.25 wt% trypsin.

[0043] 4. Experimental grouping: H9c2 cells were cultured according to the above conditions and treated in the exponential growth phase, and divided into 7 groups: (1) normal control group; (2) alizarin A ring glycosylated derivative control group: 100 μM alizarin A ring glycosylated derivative was added to the culture medium; (3) myocardial cell injury group: 10 μM H2O2 was added to the culture medium; (4) myocardial cell injury + SOD control group: 10 μM H2O2 and 200 U / L SOD were added to the culture medium at the same time; (5) myocardial cell injury + low-dose derivative group (10 μM H2O2 + 10 μM alizarin A ring glycosylated derivative); (6) myocardial cell injury + medium-dose derivative group (10 μM H2O2 + 50 μM alizarin A ring glycosylated derivative); (7) myocardial cell injury + high-dose derivative group (10 μM H2O2 + 100 μM alizarin A ring glycosylated derivative). The cells in all the above groups were in the exponential growth phase, and the culture conditions were as shown in Table 1. Figure 2 The incubation time was 24 h.

[0044] 5. Experimental content:

[0045] 5.1 Molecular docking

[0046] The structure of AMPKα2 was obtained from the PDB database (https: / / www.rcsb.org / ). A glycosylated derivative of the A ring of alizarin served as the ligand, and the target protein AMPKα2 served as the receptor. The glycosylated derivative of the A ring of alizarin was used to minimize the structure energy in the ChemBioDraw3D module. AMPKα2 was modified (including dehydration and hydrogenation) using AutodockTools 1.5.6 and exported to the pdbqt format. Results were analyzed and visualized using PyMOL 2.3.0 and BIOVIA Discovery Studio 2016, enabling intuitive visualization of binding modes and interaction forces.

[0047] 5.2 Cell proliferation assay

[0048] H9c2 cells were seeded in 96-well plates at 1×10 4 Each group was plated with six replicate wells. After cells adhered, different drugs were added according to group and the cells were incubated in a 37°C, 5% CO2 incubator for 24 hours. Fresh culture medium was replaced, and 10 μL of CCK8 was added to each well. After incubation in the incubator for 3 hours, the absorbance of the cells at 490 nm was measured using a microplate reader.

[0049] 5.3 Fluorescence detection of reactive oxygen species (ROS)

[0050] Follow the instructions in the ROS Detection Kit (chemiluminescence method). First, add the DCFH-DA fluorescent probe (1:2000, w / w) to the culture medium and incubate at 37°C for 1 hour. Collect the cells, wash twice with PBS, and centrifuge at 1000×g for 10 minutes to resuspend them. Measure cell fluorescence intensity at an excitation wavelength of 510 nm and an emission wavelength of 530 nm. The mean fluorescence intensity (MIF) represents the ROS level.

[0051] 5.4 Western Blot: PMSF (1:100, w / w) and cocktail (1:1000, w / w) were added to the cell homogenate. Tissue homogenate was added to the mixed tissue homogenate at a ratio of 15 μL / mg. The electric homogenizer was adjusted to 30 times / min, and then homogenized 50 times clockwise and 50 times counterclockwise. The tissue suspension was pipetted into a 5 mL centrifuge tube and 1 / 3 volume of buffer was added. The suspension was then mixed by vortexing. The tube was then placed in a test tube rack and boiled in a water bath for 10 minutes. The tube was then ultrasonically shaken 20 times at 20 kHz and centrifuged. After 5 minutes at 4°C / 12,000 g, the supernatant was collected and stored at -80°C. To prepare the BSA gradient solution, first prepare a 10 μg / μL BSA buffer solution. Then, sequentially dilute the solution with double-distilled water to a standard protein concentration of 0 μg / μL, 0.2 μg / μL, 0.4 μg / μL, 0.6 μg / μL, 0.8 μg / μL, and 1.0 μg / μL. Remove the protein supernatant from the -80°C freezer and place on ice. After slowly dissolving, pipette 2 μL of the protein sample and add 18 μL of double-distilled water. Mix thoroughly by vortexing. Set up three parallel wells on a 96-well plate. Then, add the protein sample and BSA gradient buffer solution at a volume of 5 μL / well. Mix 50 parts of Solution A and 1 part of Solution B from the kit, mix thoroughly, and prepare a working solution. Quickly add 95 μL to each well of the 96-well plate. Place the 96-well plate in an incubator at 37°C for 30 minutes. The spectrophotometer is set at 562nm light wave band, the absorbance value is measured and the standard protein curve is drawn to calculate the sample protein concentration value. Take 1 part bromophenol blue and 3 parts β-mercaptoethanol and mix and shake. Dilute the mixture 10 times with double distilled water, boil in a water bath for 10 minutes, and shake to mix. Add 10wt% separation gel to the gel plate and let it stand at room temperature. After the gel solidifies, pour out the water layer and dry it with filter paper. Then add 5wt% concentrated gel and let it stand at room temperature for solidification. Place the gel plate in the electrophoresis tank and fix it. Slowly add electrophoresis fluid along the electrophoresis tank, then mix the sample thoroughly, add the protein sample to the electrophoresis tank lane with a micropipette, use 80V constant voltage for electrophoresis for 30 minutes until the sample runs to the separation gel, and then use 120V constant voltage for electrophoresis separation. The electrophoresis time is determined according to the molecular weight of the target protein. Mark the nitrocellulose membrane with a marker and immerse it in pre-treated transfer solution. Peel off the separation gel and cut the gel horizontally according to the molecular weight range of the target protein. From negative to positive, stack the membrane in the following order: fiber sponge pad, three layers of filter paper, gel sheet, nitrocellulose membrane, three layers of filter paper, and fiber sponge pad. Stack these materials in a trapezoidal shape and place them in a transfer tank pre-incubated at 0°C. Transfer the membrane at a constant current of 252mA. The transfer time is determined by the molecular weight of the target protein and is generally 1-3 hours. After transfer, remove the nitrocellulose membrane from the transfer tank and rinse twice with PBS for 5 minutes each.Then, soak the sample in 100 μL of 2wt%-10wt% goat serum or 2wt%-5wt% bovine serum albumin at room temperature for 10-30 minutes, rinse with PBS three times, 5 minutes each time, and dilute the concentrate in an appropriate proportion to 1 cm. 2 Add 40 μL of primary antibody. Incubate at 37°C for 1-2 hours or at 4°C overnight. Wash three times with washing buffer, 10 minutes each time. The next day, remove the nitrocellulose membrane and recover the primary antibody. Add 0.02 wt% Tween 20 to TBS buffer and rinse three times, 5 minutes each time. Seal the bag and dilute the concentrate in an appropriate proportion to 1 cm 2 Add 40 μL of biotinylated secondary antibody and incubate at 37°C for 1-2 hours or overnight at 4°C. Rinse with PBS for 5 minutes and repeat three times. Remove the nitrocellulose membrane and recover the secondary antibody. Rinse three times with TBS buffer supplemented with 0.02 wt% Tween 20 for 5 minutes each. After rinsing, rinse three times with TBS buffer for 5 minutes each. Scan the nitrocellulose membrane at 700 nm.

[0052] 5.5 Cell immunofluorescence staining: In the culture plate, the slides with cells were soaked in PBS 3 times, each time for 3 minutes; fixed with 4wt% paraformaldehyde for 15 minutes, and soaked in PBS 3 times, each time for 3 minutes; permeabilized with 0.5wt% Triton X-100 (prepared in PBS) at room temperature for 20 minutes; soaked in PBS 3 times, each time for 3 minutes, blotted dry with PBS with absorbent paper, added normal goat serum on the slides, and blocked at room temperature for 30 minutes; absorbed the blocking solution with absorbent paper, added a sufficient amount of diluted primary antibody to each slide and placed in a humidified box, incubated at 4°C overnight; added fluorescent secondary antibody: soaked the slides in PBST 3 times, each time for 3 minutes, blotted dry with absorbent paper the excess liquid on the slides and added diluted fluorescent secondary antibody, incubated in a humidified box at 20-37°C for 1 hour, and soaked the sections in PBST 3 times, each time for 3 minutes; Note: from the addition of fluorescent secondary antibody, all subsequent operation steps should be performed in a darker place as much as possible. Re-stain the nucleus: add DAPI and incubate in the dark for 5 minutes to stain the nucleus of the specimen, then wash away the excess DAPI with PBST for 5 minutes x 4 times; dry the liquid on the slide with absorbent paper, seal the slide with a sealing solution containing an anti-fluorescence quencher, and then observe and collect images under a fluorescence microscope.

[0053] 6. Statistical methods: All data are expressed as mean ± standard deviation ( χ The differences among the groups were compared using ANOVA and Newman-Student multiple comparisons; t-test was performed using SPSS 13.0 statistical software. A two-sided P < 0.05 was considered significant.

[0054] 7. Results

[0055] 7.1 Analysis of molecular docking results:

[0056] In order to verify the binding effect of AMPKα2 as a core target with the glycosylated derivative of alizarin A ring (which may be a potential therapeutic drug), a molecular docking experiment was conducted. The results showed that the molecular docking binding energy between the glycosylated derivative of alizarin A ring and AMPKα2 was -9.06kcal / mol, which is less than -5.0kcal / mol. This indicates that the glycosylated derivative of alizarin A ring has a good affinity with the AMPKα2 target. These results provide a strong theoretical basis for further research and experiments. Figure 1 .

[0057] 7.2 Myocardial cell proliferation experiment: (1) Compared with the blank control group, the cell OD values of the low-, medium-, and high-dose treatment groups of the glycosylated derivatives of alizarin A were significantly lower (P < 0.01); (2) Compared with the myocardial cell injury model group, the cell OD values of the low-, medium-, and high-dose treatment groups of the glycosylated derivatives of alizarin A were significantly higher (P < 0.01). Figure 2 .

[0058] 7.3 Myocardial cell ROS immunofluorescence experiment: (1) The red fluorescence of ROS in myocardial cells of the injury group was stronger, and the content of ROS was significantly higher than that of the normal control group (P<0.01); (2) The red fluorescence of ROS in myocardial cells of the SOD treatment group was darker, and the content of ROS was significantly lower than that of the injury group (P<0.01); (3) The red fluorescence of ROS in myocardial cells of the low-, medium-, and high-dose treatment groups of the glycosylated derivative of alizarin A was darker, and the content of ROS was significantly lower than that of the injury group (P<0.01). Figure 3 and Figure 4 .

[0059] 7.4 Western Blot analysis of NOX2 and NOX4 expression: (1) The grayscale value of myocardial cells in the injury group was significantly increased, and the expression of NOX2 and NOX4 was significantly higher than that in the normal control group (P < 0.01); (2) The grayscale value of myocardial cells in the SOD treatment group was significantly decreased, and the expression of NOX2 and NOX4 was significantly lower than that in the injury group (P < 0.01); (3) The grayscale value of myocardial cells in the low-, medium-, and high-dose alizarin A ring glycosylated derivative treatment groups was significantly decreased, and the expression of NOX2 and NOX4 was significantly lower than that in the injury group (P < 0.01). Figure 5 and Figure 6 .

[0060] 7.5 Immunofluorescence and Western Blot analysis of AMPKα2 protein expression in myocardial cells: (1) The green fluorescence of myocardial cells in the injury group was stronger, and the gray value was significantly increased, and the AMPKα2 protein content was significantly higher than that in the normal control group (P<0.01); (2) The green fluorescence of myocardial cells in the SOD treatment group was weaker, and the gray value was significantly decreased, and the AMPKα2 protein content was significantly lower than that in the injury group (P<0.01); (3) The green fluorescence of myocardial cells in the low-, medium-, and high-dose treatment groups of the glycosylated derivative of alizarin A was darker, and the gray value was significantly decreased, and the AMPKα2 protein content was significantly lower than that in the injury group (P<0.01). Figure 7 、 Figure 8 、 Figure 9 and Figure 10 .

[0061] 7.6 Western Blot analysis of P-AKT and P-PI3K expression: (1) The grayscale value of myocardial cells in the injury group was significantly increased, and the expression of P-AKT and P-PI3K was significantly higher than that in the normal control group (P < 0.01); (2) The grayscale value of myocardial cells in the SOD treatment group was significantly decreased, and the expression of P-AKT and P-PI3K was significantly lower than that in the injury group (P < 0.01); (3) The grayscale value of myocardial cells in the low-, medium-, and high-dose alizarin A ring glycosylated derivative treatment groups was significantly decreased, and the expression of P-AKT and P-PI3K was significantly lower than that in the injury group (P < 0.01). Figure 11 and Figure 12 .

[0062] The basic principles, main features and advantages of the present invention are shown and described above. Without departing from the spirit and scope of the present invention, the present invention may also be subject to various changes and improvements, which fall within the scope of the present invention to be protected.

Claims

1. A glycosylated derivative of alizarin A ring, characterized in that The structural formula of the alizarin A ring glycosylated derivative is:

2. The method for synthesizing the glycosylated derivative of alizarin A ring according to claim 1, wherein The specific synthesis steps are: Step S1, adding alizarin, CH3CN and N,N-dimethylformamide to a reaction vessel, and after the alizarin is completely dissolved, adding NaH solution at 0°C and stirring to mix evenly, then adding 3,5-di-O-(p-toluoyl)-2-deoxy-D-ribofuranose chloride, stirring at room temperature, adding acetic acid after the reaction is completed to quench the reaction, then adding water to the reaction system and extracting with ethyl acetate, combining the organic phases and drying over anhydrous magnesium sulfate, and removing the solvent by distillation under reduced pressure to obtain a crude product; Step S2: Add CH3OH and DMF to the reaction vessel containing the crude product from step S1, stir and dissolve at room temperature, then add NaOMe and continue stirring at room temperature. After the reaction is completed, add acetic acid to quench the reaction, then add water to the reaction system and extract with ethyl acetate. Combine the organic phases and dry them over anhydrous magnesium sulfate. After removing the solvent by distillation under reduced pressure, purify on a silica gel column to obtain a yellow solid product, i.e., a glycosylated derivative of alizarin A ring.

3. The synthetic method of the glycosylated derivative of alizarin A ring according to claim 2, characterized in that The synthetic route of the alizarin A ring glycosylated derivative is:

4. The method for synthesizing the glycosylated ring A derivative of alizarin according to claim 2, wherein: The molar ratios of alizarin, NaH and 3,5-di-O-(p-toluoyl)-2-deoxy-D-ribofuranose chloride in step S1 are 1:1.5-1:1.1-1.5 respectively.

5. The method for synthesizing the glycosylated ring A derivative of alizarin according to claim 2, wherein: The NaH solution in step S1 is a mineral oil solution containing 60 wt % NaH formed from NaH and mineral oil.

6. The method for synthesizing the glycosylated ring A derivative of alizarin according to claim 2, wherein: The molar ratio of NaOMe in step S2 to alizarin in step S1 is 5-10:

1.

7. Use of the glycosylated ring A derivative of alizarin according to claim 1 in the preparation of a drug for treating myocardial cell damage.

8. Use of the glycosylated ring A derivative of alizarin according to claim 1 as an agonist targeting AMPKα2, which reduces cardiomyocyte apoptosis by activating the ROS-NOX2 / 4-p-PI3K / p-AKT signaling pathway, thereby protecting cardiomyocytes.

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