Scutellarin aglycone derivative as well as preparation method and application thereof

Abstract

The invention belongs to the field of medicines, and relates to a scutellarin aglycone derivative as well as a preparation method and application thereof. The scutellarin aglycone derivative has a structure as shown in a formula I. The preparation method comprises the following steps: reacting scutellarin (a compound II) with methanol to obtain a compound III, then reacting with bornyl chloroacetate (a compound IV) to obtain a compound V, and finally carrying out an alkaline hydrolysis reaction to obtain the compound I, the scutellarin aglycone derivative has the effect of preventing or treating cardiovascular and cerebrovascular related diseases, can improve the activity of superoxide dismutase (SOD), inhibit the production of lactic dehydrogenase (LDH) and reduce the malondialdehyde (MDA) level superior to that of scutellarin, has the inhibiting effect on inflammatory factors TNF-alpha, IL-6 and IL-1beta in cell supernatant superior to that of scutellarin, and can be used for preparing a medicine for treating cardiovascular and cerebrovascular related diseases, such as cardiovascular and cerebrovascular related diseases, cardiovascular and cerebrovascular related diseases, cardiovascular and cerebrovascular related diseases, cardiovascular and cerebrovascular related diseases, cardiovascular and cerebrovascular related diseases, cardiovascular and cerebrovascular related diseases and cardiovascular and cerebrovascular related diseases. The invention has the potential of preparing or preventing cardiovascular and cerebrovascular related diseases.

Description

Technical Field

[0001] This invention belongs to the field of chemical synthesis, specifically relating to a derivative of scutellaria baicalensis aglycone, its preparation method, and its application. Background Technology

[0002] As the cardiovascular and cerebrovascular diseases with the highest incidence and mortality rates, the treatment of stroke is particularly important. Traditional stroke treatments mainly consist of neuroprotective drugs and drugs that improve blood circulation. However, these drugs do not target the disease itself, so their clinical efficacy is not ideal. To date, there is no drug that is truly effective in treating stroke.

[0003] In recent years, traditional Chinese medicine has demonstrated significant effects in the prevention and treatment of cardiovascular and cerebrovascular diseases. Erigeron breviscapus extract is one such representative drug. However, the aglycone of erigeron breviscapus extract suffers from poor stability, low brain distribution, weak activity, and low water solubility and bioavailability (Liu Jingli, Li Na, Yan Hao, Liang Yanni. Study on the relationship between the structure and antioxidant activity of erigeron breviscapus extract and its aglycone. Natural Product Research and Development. 2018, 30: 1334-1339), which greatly restricts its clinical application. Currently, structural modifications of erigeron breviscapus extract are mostly performed semi-synthetically, primarily focusing on the etherification of the phenolic hydroxyl group, leaving many positions untouched. Based on these reasons, this application shifts its approach to modifying the 4” position to find a more suitable candidate compound. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to overcome the shortcomings of the existing technology and provide a derivative of *Erigeron breviscapus* glycoside, its preparation method, and its application.

[0005] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0006] A compound represented by Formula I:

[0007]

[0008] A compound represented by formula V:

[0009]

[0010] A method for preparing compound I according to claim 1, comprising:

[0011]

[0012] In the preparation of compound V from compound III, the molar ratio of compound III:compound IV:potassium iodide:potassium phosphate ranges from 1:1 to 1.1:0.3 to 0.5:2 to 2.5.

[0013] In the preparation of compound I from compound V, the molar ratio of compound V to base is in the range of 1:15 to 20, and the base is sodium borohydride or sodium hydroxide.

[0014] A pharmaceutical composition comprising the compound I or a stereoisomer thereof.

[0015] A pharmaceutical formulation comprising compound I or its stereoisomer and at least one pharmaceutically acceptable excipient, wherein the pharmaceutical composition or formulation uses a derivative of scutellarin aglycone represented by formula I of the present invention as the active ingredient, and the pharmaceutical formulation can be administered via the gastrointestinal tract or non-gastrointestinal route. The gastrointestinal dosage form includes powders, tablets, granules, capsules, solutions, emulsions, or suspensions, and the non-gastrointestinal dosage form includes injectable dosage forms, mucosal dosage forms, and cavity dosage forms. The excipients can serve functions such as filling, binding, disintegration, and lubrication. Commonly used excipients include starches (such as corn starch and potato starch), sugars (such as glucose and sucrose), celluloses (such as microcrystalline cellulose and carboxymethyl cellulose), and inorganic salts (such as inorganic calcium salts).

[0016] The use of compound I shown or its pharmaceutical composition or formulation in the preparation of medicaments for cardiovascular and cerebrovascular diseases.

[0017] The disease in question is an ischemic cardiovascular and cerebrovascular disease.

[0018] The ischemic cardiovascular and cerebrovascular diseases mentioned are sequelae caused by cerebral infarction, stroke, hyperviscosity and lipidemia, coronary heart disease, cerebral thrombosis, thromboangiitis obliterans, arteriosclerosis obliterans, venous thrombosis, angina pectoris, or cerebral thrombosis, cerebral ischemia, or cerebral hemorrhage.

[0019] Beneficial Effects: This invention provides a derivative of scutellarin aglycone, its preparation method, and its application. The preparation method includes: reacting compound II (baicalin) with methanol to obtain compound III, then reacting it with compound IV (borneol chloroacetate) to obtain compound V, and finally undergoing alkaline hydrolysis to obtain compound I. Experimental tests show that compound I of the scutellarin aglycone derivative can increase superoxide dismutase (SOD) activity, inhibit lactate dehydrogenase (LDH) production, and reduce malondialdehyde (MDA) levels better than scutellarin. It can effectively inhibit the production of reactive oxygen species (ROS) without significant cell damage, and its inhibitory effects on TNF-α, IL-6, and IL-1β in cell culture supernatant are also superior to those of scutellarin. It has the potential to prepare drugs for the treatment or prevention of cardiovascular and cerebrovascular diseases. Attached Figure Description

[0020] Figure 1 The photon spectrum of compound I in Example 1 is shown. Detailed Implementation

[0021] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are implemented under the premise of the technical solution of the present invention. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0022] Example 1

[0023] Synthesis of Compound III:

[0024]

[0025] Add 800 ml of methanol to a 1 L single-necked flask and cool to -5°C to 0°C. Then take 29 ml of thionyl chloride.

[0026] (0.388 mol, 10 eq) of compound II was slowly added dropwise to methanol under N2 protection. After the addition was complete, the mixture was stirred at room temperature for 1 h. Then, 18.48 g (0.0388 mol, 1.0 eq) of compound II was weighed and added, and the mixture was stirred at room temperature under N2 protection. After about 4 h, a large amount of yellow solid precipitated out. The solid was filtered, and the filter cake was washed with methanol. After drying the filter cake, approximately 16 g of yellow powdery solid compound III was obtained.

[0027] Synthesis of compound IV:

[0028]

[0029] Dissolve compound VI (0.065 mol, 1.0 eq) in 70 mL of dichloromethane by stirring. Then add 10 mL of triethylamine under N2 protection. At room temperature, add chloroacetyl chloride (0.097 mol, 1.5 eq) dropwise to the reaction system under N2 protection. After the addition is complete, stir overnight at room temperature.

[0030] After the TLC reaction was complete, the reaction solution was concentrated under reduced pressure. After concentration, 50 ml of dichloromethane and 50 ml of saturated saline were added for washing. The mixture was separated, and the organic phase was washed once more with 50 ml of water. The organic phase was separated again, dried with anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give 16.02 g of compound IV, a brown oily substance.

[0031] Synthesis of compound V:

[0032]

[0033] Compound III (0.03 mol, 1.0 eq) was added to a 250 mL three-necked flask, followed by 50 mL of N,N-dimethylformamide, and stirred until dissolved. Potassium phosphate (0.062 mol, 2.0 eq) and potassium iodide (0.009 mol, 0.3 eq) were then added. Compound IV (0.033 mol, 1.06 eq) was then added. 20 mL of acetonitrile was added, and the mixture was stirred under N2 protection. The mixture was heated to 50–55 °C and reacted for 3 h, then cooled to room temperature and allowed to stand for 36 h.

[0034] Add 300 ml of water to the reaction solution and stir; the solution becomes turbid. Then add 200 ml of ethyl acetate and extract three times. Combine the organic phases and wash once with 200 ml of water. Dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain approximately 15 g of a brown oily substance. Dry column chromatography yields 2.35 g of compound V as a yellow oily substance.

[0035] Synthesis of Compound I:

[0036]

[0037] Compound V (0.0035 mol, 1.0 eq) was dissolved in 60 mL of methanol and cooled in an ice bath at 0–5 °C. Sodium borohydride (0.07 mol, 20 eq) was then added in small, multiple batches. After the addition was complete, the mixture was stirred at room temperature for 5 h. The reaction was then analyzed by TLC after complete reaction.

[0038] Add 50 ml of 10% acetic acid aqueous solution to the reaction solution and stir for 0.5 h. Then add 250 ml of ethyl acetate and 150 ml of water for extraction. Separate the liquid and extract the aqueous phase twice with 100 ml of ethyl acetate. Combine the organic phases, dry to anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to give 2.44 g of orange-yellow solid. Dry column chromatography yields 1 g of the target product compound I, an off-white solid. NMR characterization is shown in the attached figure. Figure 1 .

[0039] Example 2

[0040] Synthesis of Compound III:

[0041]

[0042] Add 800 ml of methanol to a 1 L single-necked flask and cool to -5°C to 0°C. Then take 29 ml of thionyl chloride.

[0043] (0.388 mol, 10 eq) of compound II was slowly added dropwise to methanol under N2 protection. After the addition was complete, the mixture was stirred at room temperature for 1 h. Then, 18.48 g (0.0388 mol, 1.0 eq) of compound II was weighed and added, and the mixture was stirred at room temperature under N2 protection. After about 4 h, a large amount of yellow solid precipitated out. The solid was filtered, and the filter cake was washed with methanol. After drying the filter cake, approximately 16 g of yellow powdery solid compound III was obtained.

[0044] Synthesis of compound IV:

[0045]

[0046] Dissolve compound VI (0.065 mol, 1.0 eq) in 70 mL of dichloromethane by stirring. Then add 10 mL of triethylamine under N2 protection. At room temperature, add chloroacetyl chloride (0.097 mol, 1.5 eq) dropwise to the reaction system under N2 protection. After the addition is complete, stir overnight at room temperature.

[0047] After the TLC reaction was complete, the reaction solution was concentrated under reduced pressure. After concentration, 50 ml of dichloromethane and 50 ml of saturated saline were added for washing. The mixture was separated, and the organic phase was washed once more with 50 ml of water. The organic phase was separated again, dried with anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give 16.02 g of compound IV, a brown oily substance.

[0048] Synthesis of compound V:

[0049]

[0050] Compound III (0.03 mol, 1.0 eq) was added to a 250 mL three-necked flask, followed by 50 mL of N,N-dimethylformamide, and stirred until dissolved. Potassium phosphate (0.076 mol, 2.5 eq) and potassium iodide (0.015 mol, 0.5 eq) were then added. Compound IV (0.034 mol, 1.1 eq) was then added. 20 mL of acetonitrile was added, and the mixture was stirred under N2 protection. The mixture was heated to 50–55 °C and reacted for 3 h, then cooled to room temperature and allowed to stand for 36 h.

[0051] Add 300 ml of water to the reaction solution and stir; the reaction solution becomes turbid. Then add 200 ml of ethyl acetate and extract three times. Combine the organic phases and wash once with 200 ml of water. Dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain approximately 15 g of a brown oily substance. Dry column chromatography yields 2.36 g of compound V as a yellow oily substance.

[0052] Synthesis of Compound I:

[0053]

[0054] Compound V (0.0035 mol, 1.0 eq) was dissolved in 60 mL of methanol and cooled in an ice bath at 0–5 °C. Sodium hydroxide (0.53 mol, 15 eq) was then added in small, multiple batches. After the addition was complete, the mixture was stirred at room temperature for 5 h. The reaction was then analyzed by TLC after complete reaction.

[0055] Add 50 ml of 10% acetic acid aqueous solution to the reaction solution and stir for 0.5 h. Then add 250 ml of ethyl acetate and 150 ml of water for extraction. Separate the liquid and extract the aqueous phase twice with 100 ml of ethyl acetate. Combine the organic phases, dry to anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to give 2.43 g of orange-yellow solid. Dry column chromatography yields 1 g of the target product compound I, an off-white solid.

[0056] To illustrate the beneficial effects of the present invention, the following experimental examples are provided.

[0057] Experimental Example 1: CCK-8 assay for cell proliferation

[0058] The purpose of this experiment is to assess whether the compound causes damage to cells.

[0059] Using the drug scutellarin as a reference, cells were digested, counted, and prepared into a cell suspension with a concentration of 5 × 10⁴ cells / mL. 100 μL of the cell suspension was added to each well of a 96-well cell culture plate. The 96-well cell culture plates were incubated at 37°C in a 5% CO₂ incubator for 24 h until cell attachment. The drug was diluted with complete culture medium to the required concentrations (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625 μM), and 100 μL of the corresponding drug-containing culture medium was added to each well. A negative control group was also established. The 96-well cell culture plates were incubated at 37°C in a 5% CO₂ incubator for another 24 h. The 96-well plates were then stained with CCK-8 at λ = 450 nm, and the OD value was measured.

[0060] Add 10 μL of CCK-8 to each well and continue incubation in an incubator for 3 hours;

[0061] B. Shake gently for 10 minutes to mix thoroughly.

[0062] Cλ = 450nm, the OD value of each well is read by the microplate reader, and the inhibition rate is calculated;

[0063] The inhibition rate formula is as follows:

[0064]

[0065] The specific experimental procedures were performed according to Cell Counting Kit-8 (KGA9306-500, Jiangsu Kaiji Biotechnology Co., Ltd., China), and the results are shown in Table 1.

[0066] Table 1

[0067]

[0068] The CCK-8 results showed that scutellarin and its aglycone derivative compound I had no significant damaging effect on HT22 cells in the concentration range of 0-200 μM and even had a certain effect on promoting cell reproduction.

[0069] Experimental Example 2: CCK-8 assay using OGD / R model to detect cell proliferation.

[0070] The purpose of this experiment was to evaluate the effectiveness of the OGD / R model and to determine whether the compound caused damage to cells.

[0071] Using the drug scutellarin as a reference, cells were digested, counted, and prepared into a concentration of 5 × 10⁻⁶. 4 100 μL of cell suspension per well was added to each well of a 96-well cell culture plate. The 96-well cell culture plate was incubated at 37°C in a 5% CO2 incubator for 24 h until the cells adhered. The culture medium was then replaced with glucose- and serum-free DMEM (glucose-free basal medium) and incubated in a 5% CO2, 92% N2, and 3% O2 ​​environment for 4 h. Subsequently, the culture medium was replaced with complete medium containing the corresponding drug concentrations (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625 μM) and incubated in a 5% CO2, 95% air environment for 24 h. The 96-well plate was then stained with CCK-8 at λ = 450 nm, and the OD value was measured.

[0072] Add 10 μL of CCK-8 to each well and continue incubation in an incubator for 3 hours;

[0073] B. Shake gently for 10 minutes to mix thoroughly.

[0074] Cλ = 450nm, the OD value of each well is read by the microplate reader, and the inhibition rate is calculated;

[0075] The inhibition rate formula is as follows:

[0076]

[0077] The results are shown in Table 2.

[0078] Table 2

[0079]

[0080] The CCK-8 results showed that the OGD / R model was successfully established, and the model had a significant inhibition rate compared with the control group. In the model + drug group, both scutellarin and scutellarin aglycone derivative compound I showed different degrees of protective effects on HT22 cells in the concentration range of 0-200 μM, with the strongest protective effect at 50 μM. Furthermore, the protective effect increased with increasing concentration at three concentration gradients of 12.5 μM, 25 μM, and 50 μM. Therefore, 12.5 μM, 25 μM, and 50 μM were selected for subsequent experiments for both compounds.

[0081] Experimental Example 3: Observation of Calcein-AM / PI Cell Morphology

[0082] The purpose of this trial was to evaluate whether the addition of the drug could reduce the cell death / liveness ratio.

[0083] Using the drug scutellarin as a reference, cells were digested, counted, and prepared into a concentration of 2×10⁻⁶. 5 Cell suspension of 1 cell / mL was added to each well of a 6-well plate. The plates were incubated at 37°C in a 5% CO2 incubator for 24 hours until cell adhesion was achieved. The culture medium was then replaced with a glucose- and serum-free medium and incubated in a 5% CO2, 92% N2, 3% O2 ​​environment for 4 hours. Subsequently, the medium was replaced with complete medium containing the corresponding drug concentrations (50, 25, 12.5 μM) and incubated in a 5% CO2, 95% air environment for 24 hours. Live / Dead cell staining working solution was added, and staining was performed in the dark for 30 minutes. The cells were photographed using a fluorescence microscope (100× magnification). Each group was tested in triplicate, and the mean cell dead / live ratio was taken. Specific experimental procedures were performed according to the instructions of the cell viability (live / dead cell staining) assay kit (KGA9501-1000, Jiangsu Kaiji Biotechnology Co., Ltd., China). The results are shown in Table 3.

[0084] Table 3

[0085]

[0086] In fluorescence imaging, green represents live cells and red represents dead cells. The results of HT22 cell live / dead staining showed that the dead / live ratio of the OGD / R model group was significantly higher than that of the control group. The addition of scutellarin or scutellarin aglycone derivative compound I could reduce the cell dead / live ratio. Moreover, the cell dead / live ratio of scutellarin aglycone derivative compound I was lower than that of the control drug scutellarin at drug concentrations of 12.5-50 μM.

[0087] Experimental Example 4: Detection of Lactate Dehydrogenase (LDH) Content

[0088] The purpose of this experiment was to assess the intracellular lactate dehydrogenase (LDH) content.

[0089] The drug scutellarin was used as a reference and HT22 cell supernatant: HT22, HT22+OGD / R, HT22+OGD / R+scutellarin 12.5μM, HT22+OGD / R+scutellarin 25μM, HT22+OGD / R+scutellarin 50μM, HT22+OGD / R+compound I 12.5μM, HT22+OGD / R+compound I 25μM, and HT22+OGD / R+compound I 50μM. Reagent 1, Matrix buffer: 30ml*1 bottle; Reagent 2, Coenzyme I: powder; Coenzyme I solution preparation: dissolve each vial of powder in 1.3mL of double-distilled water; Reagent 3, 2,4-dinitrophenylhydrazine: 30mL*1 bottle; Reagent 4, 4mol / L NaOH: 30mL*1 bottle: dilute the 4mol / L NaOH solution 10 times with double-distilled water to prepare a 0.4mol / L NaOH solution, prepare as needed; Reagent 5, 2μmol / mL pyruvate standard solution: 1mL*1 bottle. The procedures are shown in Table 4 below.

[0090] Table 4

[0091]

[0092]

[0093] Mix well, incubate at room temperature for 3 minutes, and measure the absorbance of each well using a microplate reader at a wavelength of 440 nm. Calculate the LDH activity in the cell supernatant. Specific experimental procedures were performed according to the instructions of the lactate dehydrogenase (LDH) assay kit (KGA7403-24, Jiangsu Kaiji Biotechnology Co., Ltd., China). Each group was tested in triplicate, and the average LDH activity (U / L) was taken. The results are shown in Table 5. The formula is as follows:

[0094]

[0095] Table 5

[0096]

[0097] The results of LDH detection in HT22 cell supernatant showed that the LDH leakage rate of the OGD / R model group was increased compared with the control group. After adding scutellarin or scutellarin aglycone derivative compound I to the model, the LDH leakage rate decreased with the increase of drug concentration. Among them, the LDH leakage rate of compound I was lower than that of scutellarin in the range of 12.5-50 μM, and it could significantly inhibit the production of lactate dehydrogenase (LDH).

[0098] Experimental Example 5: Detection of Intracellular Reactive Oxygen Species (ROS) Content Using DCFH-DA Fluorescent Probe

[0099] The purpose of this experiment is to evaluate the levels of reactive oxygen species (ROS) in cells.

[0100] Using the drug scutellarin, cells were digested, counted, and formulated into a concentration of 2×10⁻⁶. 5 Cells were seeded at a concentration of 10 cells / mL, with 2 mL of the suspension injected into 6-well plates and incubated at 37°C in a 5% CO2 incubator for 24 h until cell adhesion was achieved. The culture medium was then replaced with glucose- and serum-free medium and incubated in a 5% CO2, 92% N2, 3% O2 ​​environment for 4 h. Subsequently, the medium was replaced with complete medium containing the corresponding drug concentrations (50, 25, 12.5 μM) and incubated in a 5% CO2, 95% air environment for 24 h. Cells were then collected by digestion with 0.25% trypsin, washed once with PBS (centrifuged at 1000 rpm for 5 min), and the cell concentration was adjusted to 1 × 10⁶ cells / mL. 6 / ml; Dilute DCFH-DA with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10μM. After cell collection, suspend the cells in the diluted DCFH-DA and incubate at 37℃ for 20 min. Invert and mix every 3-5 min to ensure full contact between the probe and cells; Wash cells three times with serum-free cell culture medium to remove any DCFH-DA that has not entered the cells; Detect intracellular reactive oxygen species (ROS) using flow cytometry (Ex = 488nm; Em = 525nm). Follow the instructions of the ROS reactive oxygen species detection kit (KGA7308-100, Jiangsu Kaiji Biotechnology Co., Ltd., China) for specific procedures. Each group was tested in triplicate, and the mean value was taken. The results are shown in Table 6.

[0101] Table 6

[0102]

[0103] Flow cytometry analysis of ROS changes in HT22 cells showed that the ROS level in the OGD / R model group was significantly increased compared with the control group. After adding scutellarin or scutellarin aglycone derivative compound I to the model, the ROS level in the cells decreased with increasing drug concentration. Among them, the ROS level in the cells of compound I was lower than that of scutellarin in the range of 12.5-50 μM, which can effectively inhibit the production of reactive oxygen species (ROS).

[0104] Experimental Example 6: Determination of MDA (Malondialdehyde) Content

[0105] The purpose of this experiment is to evaluate the intracellular MDA content.

[0106] Using the drug scutellarin as a reference and HT22 cells, HT22+OGD / R, HT22+OGD / R+scutellarin 12.5μM, HT22+OGD / R+scutellarin 25μM, HT22+OGD / R+scutellarin 50μM, HT22+OGD / R+compound I 12.5μM, HT22+OGD / R+compound I 25μM, and HT22+OGD / R+compound I 50μM were used. Cells were digested, centrifuged, and collected, and counted at 1×10⁻⁶ cells. 6 Centrifuge each tube with 1000 cells at 2000g for 10 minutes at 4℃, and discard the supernatant. Add 1 ml of pre-chilled cell lysis buffer, shake vigorously for 30 seconds every 5 minutes, lyse on ice for 20 minutes, centrifuge at 12000g for 5 minutes, and collect the supernatant for testing. Prepare reagents and plot the standard curve. Prepare cell samples: Take 0.2 mL of cell lysis buffer, add 1.5 mL of reagent C and 1.5 mL of reagent D, mix well, add 1 mL of double-distilled water to each tube, mix quickly, boil in a boiling water bath for 50 minutes, quickly cool in ice water, centrifuge at 3000 r / min and 4℃ for 15 minutes, collect the supernatant and read at 532 nm. The specific experimental operation is performed according to the instructions of the cell malondialdehyde (MDA) detection kit (KGA7101-100, Jiangsu Kaiji Biotechnology Co., Ltd., China). Each group is tested in parallel three times, and the mean value is taken. The results are shown in Table 7.

[0107] Table 7

[0108]

[0109]

[0110] The results of MDA changes in HT22 cells showed that the MDA content in the OGD / R model group was significantly increased compared with the control group. After adding scutellarin or scutellarin aglycone derivative compound I, the MDA content in the cells decreased with the increase of drug concentration. Moreover, the MDA content in the cells with compound I at concentrations of 12.5-50 μM was lower than that in the cells with scutellarin. It was better than scutellarin in reducing malondialdehyde (MDA) levels.

[0111] Experimental Example 7: Detection of Superoxide Dismutase (SOD)

[0112] The purpose of this experiment is to evaluate the detection of intracellular reactive oxygen species superoxide dismutase (SOD).

[0113] Using the drug scutellarin as a reference, and HT22 cells, HT22+OGD / R, HT22+OGD / R+scutellarin 12.5μM, HT22+OGD / R+scutellarin 25μM, HT22+OGD / R+scutellarin 50μM, HT22+OGD / R+compound I 12.5μM, HT22+OGD / R+compound I 25μM, HT22+OGD / R+compound I 50μM, reagent preparation is as follows: Reagent 1: 10ml stock solution; Reagent 2: 10ml liquid; Reagent 3: 10ml liquid; Reagent 4: 350ul stock solution; 10ml diluent; Preparation of reagent 4 working solution: stock solution: diluent = 1:4; Reagent 5: 1 vial of powder, add 75ml of double-distilled water before use; Reagent 6: 1 vial of powder, add 75ml of double-distilled water before use; Colorimetric reagent is prepared according to the volume ratio of reagent 5: reagent 6: glacial acetic acid = 3:3:2; Reagent 7: 30ml. The operation is shown in Table 8 below.

[0114] Table 8

[0115]

[0116] Mix well, incubate at room temperature for 10 minutes, and measure the absorbance using a microplate reader at a wavelength of 550 nm. The formula for calculating SOD activity in cell samples is as follows: SOD activity (U / mgprot) = (A 对照 -A 测定 ) / A 对照 *2*V 反总 / V 样 For the Cpr assay, the specific experimental procedures were performed according to the instructions of the Superoxide Dismutase (SOD) Assay Kit (KGA7301-96, Jiangsu Kaiji Biotechnology Co., Ltd., China). Each group was tested in triplicate, and the average value was taken. The results are shown in Table 9 below:

[0117] Table 9

[0118]

[0119] The results of detecting changes in SOD activity in HT22 cells showed that, compared with the control group, the SOD activity in the OGD / R model group cells was significantly decreased. After adding scutellarin or scutellarin aglycone derivative compound I to the model, the SOD activity in the cells increased with the increase of drug concentration. Moreover, the cell activity of scutellarin aglycone derivative compound I was higher than that of scutellarin in drug concentrations of 12.5-50 μM. Scutellarin aglycone derivative compound I can effectively increase superoxide dismutase (SOD) activity and is superior to scutellarin.

[0120] Experimental Example 8: Detection of Protein Expression by ELISA

[0121] The purpose of this study was to evaluate the detection of inflammatory factors TNF-α, IL-6, and IL-1β in the supernatant of HT22 cells.

[0122] Using the drug scutellarin as a reference, and HT22 cells, HT22+OGD / R, HT22+OGD / R+scutellarin 12.5μM, HT22+OGD / R+scutellarin 25μM, HT22+OGD / R+scutellarin 50μM, HT22+OGD / R+compound I 12.5μM, HT22+OGD / R+compound I 25μM, HT22+OGD / R+compound I 50μM were analyzed according to the mouse TNF-α ELISA kit (Jiangsu Kaiji Biotechnology Co., Ltd. KGC1211-48), mouse IL-6 ELISA kit (Jiangsu Kaiji Biotechnology Co., Ltd. KGC1204-48), and mouse IL-1β ELISA kits, the results were obtained. Following the instructions for the ELISA kit (KGC1201-48, Jiangsu Kaiji Biotechnology Co., Ltd., China), dilute the standard and add samples: Prepare blank wells (standard diluent), standard wells, and sample wells. Accurately add 100 μL of standard solution to each well and 100 μL of sample solution to each well on the ELISA plate. Add the sample to the bottom of the well, avoiding contact with the well walls, and gently shake to mix (do not dilute the sample). Incubation: Seal the plate with the sealing mold and incubate at 37°C for 90 minutes. Prepare the washing buffer: Dilute the 20-fold concentrated washing buffer 20 times with distilled water. Wash: Carefully remove the sealing mold, discard the liquid, and shake dry. Fill each well with washing buffer, let stand for 30 seconds, then discard. Repeat this process 5 times. Pat dry. Add antibody: Dilute biotinylated antibody 1:30 with antibody diluent, adding 100 μL to each well except the blank. Incubation: Seal the plate with the sealing mold and incubate at 37°C for 60 minutes. Repeat washing. Add enzyme: Add 100 μL of enzyme-labeled reagent to each well, except for the blank wells. Incubate: Seal the plate with the sealing mold and incubate at 37°C for 30 minutes. Repeat washing. Color development: Add 100 μL of color development solution to each well, gently shake to mix, and develop color at 37°C in the dark for 15 minutes. Termination: Add 100 μL of stop solution to each well to stop the reaction (at this time, the blue color will change to the diaphragm color). Measurement: Zero the plate with the blank wells and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution. The specific experimental procedures were performed according to the instructions for the mouse TNF-α ELISA kit (KGC1211-48, Jiangsu Kaiji Biotechnology Co., Ltd., China), the mouse IL-6 ELISA kit (KGC1204-48, Jiangsu Kaiji Biotechnology Co., Ltd., China), and the mouse IL-1β ELISA kit (KGC1201-48, Jiangsu Kaiji Biotechnology Co., Ltd., China). The TNF-α detection results are shown in Table 10 below, the IL-6 detection results are shown in Table 11 below, and the IL-1β detection results are shown in Table 12 below.

[0123] Table 10

[0124]

[0125] Table 11

[0126]

[0127] Table 12

[0128]

[0129] The results of detecting changes in inflammatory factors TNF-α, IL-6, and IL-1β in the supernatant of HT22 cells showed that, compared with the control group, the levels of TNF-α, IL-6, and IL-1β in the cell supernatant of the OGD / R model group were significantly increased. After adding scutellarin or compound I to the model, the levels of TNF-α, IL-6, and IL-1β in the cell supernatant decreased with increasing drug concentration. Moreover, in the drug concentration range of 12.5-50 μM, the levels of inflammatory factors TNF-α, IL-6, and IL-1β in the cell supernatant of compound I were significantly lower than those of the control drug scutellarin, which can effectively inhibit the production of inflammatory factors TNF-α, IL-6, and IL-1β in the cell supernatant.

[0130] The above specific embodiments further illustrate the purpose, technical solution and beneficial effects of this application. It should be understood that the above are only specific embodiments of this application and are not intended to limit the scope of protection of this application. Any modifications, equivalent substitutions, improvements, etc., made on the basis of the technical solution of this application should be included within the scope of protection of this application.

Claims

1. A compound represented by Formula I:

2. A compound of formula V:

3. A method for preparing compound I according to claim 1, comprising:

4. The preparation method according to claim 3, characterized in that: In the preparation of compound V from compound III, the molar ratio of compound III:compound IV:potassium iodide:potassium phosphate ranges from 1:1 to 1.1:0.3 to 0.5:2 to 2.

5.

5. The preparation method according to claim 3, characterized in that: In the preparation of compound I from compound V, the molar ratio of compound V to base is in the range of 1:15 to 20, and the base is sodium borohydride or sodium hydroxide.

6. A pharmaceutical composition comprising compound I as described in claim 1 or a stereoisomer thereof.

7. A pharmaceutical formulation comprising compound I as described in claim 1 or a stereoisomer thereof and at least one pharmaceutically acceptable excipient.

8. The use of compound I as described in claim 1, or a pharmaceutical composition or formulation thereof, in the preparation of a medicament for cardiovascular and cerebrovascular diseases.

9. The application according to claim 8, wherein the disease is ischemic cardiovascular and cerebrovascular disease.

10. The application according to claim 9, wherein the ischemic cardiovascular and cerebrovascular disease is a sequela of cerebral infarction, stroke, hyperlipidemia, coronary heart disease, cerebral thrombosis, thromboangiitis obliterans, arteriosclerosis obliterans, venous thrombosis, angina pectoris, or cerebral thrombosis, cerebral ischemia, or cerebral hemorrhage.

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