Use of flavonoids in aortic valve disease or cardiovascular aging

Abstract

The application belongs to the technical field of medicine, and particularly relates to a use of a flavonoid compound in aortic valve disease or cardiovascular aging. The flavonoid compound shown in formula I is found to be capable of inhibiting the osteogenic differentiation of valve interstitial cells for the first time. In animal implementation, the flavonoid compound can significantly reduce the peak flow rate and peak pressure difference of the aortic valve of a calcified aortic valve mouse, and has a significant effect of relieving aortic valve calcification. Therefore, the flavonoid compound has a good application prospect in the preparation of a drug for relieving aortic valve disease, relieving cardiovascular aging and other related cardiovascular diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of a flavonoid compound in the preparation of drugs to alleviate aortic valve disease or cardiovascular aging. Background Technology

[0002] Cardiovascular aging is one of the most critical pathophysiological events in the aging process, characterized by structural remodeling and functional decline of the heart and blood vessels. With age, cardiomyocytes gradually undergo hypertrophy, apoptosis, and fibrosis; vascular endothelial function is impaired; arterial wall elasticity decreases and stiffness increases, ultimately leading to decreased cardiac output, elevated blood pressure, and insufficient tissue perfusion. Recent studies have shown that cardiovascular aging is not simply a degenerative change, but rather an active regulatory process driven by multiple factors, including oxidative stress, chronic inflammation, cellular senescence, and metabolic disorders. Among these, vascular calcification, as an important pathological marker of cardiovascular aging, significantly increases the risk of myocardial infarction, heart failure, and stroke, becoming a major public health problem threatening the health of the elderly. Currently, interventions for cardiovascular aging mainly focus on risk factor control (such as lowering blood pressure, lipids, and blood sugar) and lifestyle modifications, but there is a lack of effective drugs that can directly delay or reverse the cardiovascular aging process. Therefore, developing novel treatment strategies has significant clinical implications.

[0003] Calcified aortic valve disease (CAVD) is one of the most typical valvular diseases in the cardiovascular aging process. Its essence is no longer simply "age-related degeneration," but rather an active pathological process similar to atherosclerosis, driven by multiple risk factors. With the increasing aging of the global population, CAVD has become an increasingly heavy public health burden. Epidemiological data shows that its prevalence in people over 75 years of age is as high as 12%, and it is a significant cause of heart failure and sudden cardiac death. Once clinical symptoms such as angina, syncope, or heart failure appear, the average survival time of patients will plummet to 2-3 years. The one-year mortality rate for untreated patients with severe symptoms can exceed 50%, making it as dangerous as malignant tumors.

[0004] Currently, the clinical management of CAVD faces a dilemma at both ends. In the early stages of the disease, namely the asymptomatic calcification progression phase, there are no proven drug treatments that can slow down or reverse the valvular calcification process; only passive observation and risk factor control are possible. Once the disease progresses to the symptomatic, severe stenosis stage, the treatment window narrows drastically to mechanical valve replacement (surgical SAVR or transcatheter aortic valve implantation (TAVI)). While these are effective life-saving methods, they are all "replacement therapies" for end-stage disease and cannot change the fundamental course of the disease, and they come with surgical risks, complications, and high medical costs. Currently, there are no marketed drugs for the treatment of calcific aortic valve disease. Therefore, there is an urgent need to develop drugs specifically for calcific aortic valve disease.

[0005] Flavonoids are a class of polyphenolic secondary metabolites widely found in plants, possessing various biological activities such as antioxidant, anti-inflammatory, lipid metabolism regulation, and improvement of vascular endothelial function, showing promising application prospects in the prevention and treatment of cardiovascular diseases. Existing research has confirmed that flavonoids exert cardiovascular protective effects in disease models such as hypertension, atherosclerosis, and myocardial ischemia-reperfusion injury. For example, representative flavonoids such as quercetin, catechins, and puerarin have been reported to have pharmacological activities such as antihypertensive, anti-atherosclerotic, and anti-myocardial fibrosis effects. However, research reports on the therapeutic effects of flavonoids in alleviating cardiovascular aging, particularly targeting aortic valve calcification, a key pathological link in cardiovascular aging, are currently lacking. Therefore, developing new uses for flavonoids in the preparation of drugs to alleviate cardiovascular aging and aortic valve calcification is of significant scientific and practical value for expanding the clinical applications of flavonoids. Summary of the Invention

[0006] In view of the problems of the prior art, the present invention provides the use of flavonoids in aortic valve disease or cardiovascular aging.

[0007] The use of the compound shown in Formula I, or a pharmaceutically acceptable salt thereof, or a deuterated derivative thereof, or a tritated derivative thereof, or a stereoisomer thereof, or a prodrug thereof, or a crystal form thereof, in the preparation of a medicament for the treatment and / or prevention of aortic valve disease and / or cardiovascular aging, wherein the compound has the following structural formula: in: R1, R2, R3, and R4 are each independently selected from H, deuterium, tritium, halogens, -OH, -NH2, -NO2, -CF3, -COOH, C1-C8 alkoxy groups substituted or unsubstituted with 1-3 Rb groups, C1-C8 ester groups, and -NHR. h1 C1-C8 alkyl groups substituted with or unsubstituted with 1-3 Ra atoms; R5, R6, R7, R8, and R9 are each independently selected from H, deuterium, tritium, halogen, -OH, -NH2, -NO2, -CF3, -COOH, C1~C8 alkoxy groups, -OCF3, C1~C8 ester groups, and -NHR. h1 C1-C8 alkyl groups substituted with or unsubstituted with 1-3 Ra atoms; R 10 Selected from H, deuterium, tritium, halogens, -OH, -NH2, -NO2, -CF3, -COOH, C1-C8 alkoxy groups substituted or unsubstituted with 1-3 Rb, C1-C8 alkyl groups substituted or unsubstituted with 1-3 Ra, C1-C8 ester groups substituted or unsubstituted with 1-3 Rc, -NHR h1 ; Ra is independently selected from deuterium, tritium, halogen, hydroxyl group, and 5-10 membered heterocyclic alkyl groups; Rb is independently selected from deuterium, tritium, halogen, and C6-C group. 10 Aryl, 5-10 membered heterocyclic alkyl; Rc is independently selected from C6~C 10 Aryl; R h1 The heterocycle is selected from hydroxyl, C1-C5 alkyl, phenyl-substituted C1-C8 alkyl, substituted 6-12 aryl, and substituted 5-10 heterocycles, wherein the heteroatom of the heterocycle is selected from at least one of N, O, or S, and the number of heteroatoms is 1-4. The substituents in the substituted phenyl, substituted 6-12 aryl, or substituted 5-10 heterocycles are selected from deuterium, tritium, halogen, -OH, methoxy, C1-C8 alkyl, C1-C8 alkoxy, phenyl, etc. -CF3, -OCF3, C1~C8 alkenyl, carboxyl.

[0008] Preferably, R1, R2, R3, and R4 are independently selected from H, deuterium, tritium, halogen, -OH, -NH2, -NO2, -CF3, -COOH, C1-C4 alkoxy groups substituted or unsubstituted with 1-3 Rb groups, C1-C4 ester groups, and -NHR. h1 C1-C8 alkyl groups substituted with or unsubstituted with 1-3 Ra atoms; R5, R6, R7, R8, and R9 are each independently selected from H, deuterium, tritium, halogen, -OH, -NH2, -NO2, -CF3, -COOH, C1~C4 alkoxy group, -OCF3, C1~C4 ester group, and -NHR. h1 C1-C8 alkyl groups substituted with or unsubstituted with 1-3 Ra atoms; R 10Selected from H, deuterium, tritium, halogens, -OH, -NH2, -NO2, -CF3, -COOH, C1-C4 alkoxy groups substituted or unsubstituted with 1-3 Rb, C1-C4 alkyl groups substituted or unsubstituted with 1-3 Ra, C1-C4 ester groups substituted or unsubstituted with 1-3 Rc, -NHR h1 .

[0009] Preferably, R1, R2, R3, and R4 are each independently selected from H, deuterium, tritium, halogen, -OH, -NH2, -NO2, -CF3, -COOH, C1~C4 alkoxy, and C1~C4 alkyl groups substituted with 1~3 Ra; Ra is each independently selected from deuterium, tritium, halogen, and hydroxyl. R5, R6, R7, R8, and R9 are each independently selected from H, deuterium, tritium, halogen, -OH, -NH2, -NO2, -CF3, -COOH, C1~C8 alkyl, and C1~C4 alkoxy groups; R 10 Selected from H, deuterium, tritium, halogen, -OH, -NH2, -NO2, -CF3, -COOH, and C1~C4 ester groups.

[0010] Preferably, R1 is selected from H, deuterium, -OH, and methoxy groups; R2 is selected from H, Cl, -OH, -NH2, -COOH, C1~C2 alkyl, and methoxy. R3 is selected from H, deuterium, -OH, methoxy, and phenyl-substituted methoxy groups; R4 is selected from H, deuterium, -OH, and methoxy groups; R5 and R9 are independently selected from H, deuterium, -OH, and methoxy groups, respectively; R6 and R8 are independently selected from H, deuterium, -OH, -NO2, -CF3, methoxy, and deuterium-substituted methoxy groups, respectively; R7 is selected from H, F, Cl, -OH, -CF3, -COOH, C1~C4 alkoxy, deuterated methoxy, fluorinated methoxy, methyl, deuterated methyl, and 6-membered nitrogen-containing heterocyclic alkyl-substituted ethyl. R 10 Selected from H, deuterium, -OH, methoxy, deuterium-substituted methoxy, phenyl-substituted C1 ester group, ethyl ester group, and ethoxy with a 6-membered nitrogen-containing heterocyclic alkyl group.

[0011] Preferably, R1 is selected from H or -OH; R2 is selected from H, Cl, -OH, -NH2, and -COOH; R3 is selected from H, -OH, and methoxy groups; R4 is selected from H or -OH; R5 and R9 are independently selected from H, deuterium, and -OH, respectively; R6 and R8 are independently selected from H, deuterium, -OH, -CF3, and methoxy, respectively; R7 is selected from H, -OH, methoxy, deuterated methoxy, methyl, and deuterated methyl. R 10 Selected from H, deuterium, -OH, methoxy, and deuterium-substituted methoxy groups.

[0012] Preferably, the compound has the structural formula shown in Formula II: in: R1 is selected from H or -OH; R2 is selected from H or -OH; R3 is selected from -OH and methoxy groups; R5 and R9 are independently selected from H and -OH, respectively; R7 is selected from H and -OH; R8 is selected from H, -OH, and methoxy groups; R 10 Selected from H, -OH, and methoxy groups.

[0013] Preferably, the compound has the following structural formula: .

[0014] Preferably, the drug is used to delay or inhibit aortic valve calcification.

[0015] Preferably, the drug is used to achieve at least one of the following functions: reducing the pressure gradient across the valve, reducing the maximum peak blood flow velocity, and improving aortic valve function.

[0016] Preferably, the drug is used to inhibit osteogenic differentiation of valvular interstitial cells.

[0017] Preferably, the drug is a formulation made by using the compound, or a pharmaceutically acceptable salt thereof, or a deuterated derivative thereof, or a tritated derivative thereof, or a stereoisomer thereof, or a prodrug thereof, or a crystal form thereof as the active ingredient, plus pharmaceutically acceptable excipients.

[0018] Preferably, the dosage form of the preparation is selected from tablets, capsules, oral liquids, granules, pills, powders, injections, or powder injections.

[0019] The compounds and derivatives provided in this invention can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature systems.

[0020] Regarding the definition of terms used in this invention: Unless otherwise stated, the initial definitions provided for groups or terms herein apply to the groups or terms used throughout this specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and context.

[0021] "Substitution" refers to the replacement of hydrogen atoms in a molecule by other different atoms or molecules.

[0022] The minimum and maximum carbon atom content in hydrocarbon groups are indicated by a prefix, for example, the prefix C. a - C b Alkyl indicates any alkyl group containing "a" to "b" carbon atoms. Therefore, for example, "C1-C6 alkyl" refers to alkyl groups containing 1 to 6 carbon atoms.

[0023] "Alkyl" refers to a saturated hydrocarbon chain having a specified number of member atoms. For example, C1-C6 alkyl refers to an alkyl group having 1 to 6 member atoms, such as 1 to 4 member atoms. Alkyl groups can be straight-chain or branched. Representative branched alkyl groups have one, two, or three branches. Alkyl groups may optionally be substituted by one or more substituents as defined herein. Alkyl groups include methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, and tert-butyl), pentyl (n-pentyl, isopentyl, and neopentyl), and hexyl. Alkyl groups may also be part of other groups, such as C1-C6 alkoxy groups.

[0024] "Halogen" refers to fluorine, chlorine, bromine, or iodine.

[0025] "Heterocyclic" and "heterocyclic alkyl" refer to saturated rings or non-aromatic unsaturated rings containing at least one heteroatom and having a single ring or multiple rings (including fused, bridged, and spirocyclic systems); where heteroatoms refer to nitrogen atoms, oxygen atoms, and sulfur atoms; "Alkoxy" refers to a group in which an alkyl group is attached to a linker site via an oxygen atom. For example, methoxy is -OCH3.

[0026] "Ester group" refers to a group that is connected to a carbon chain via a linking site, and the carbon chain contains at least one ester bond (i.e., -COO-).

[0027] A "carboxyl group" is a group that is connected to a linking site through a carbon chain, and the carbon chain contains at least one -COOH group.

[0028] The term "pharmaceutically acceptable" means that a carrier, delivery substance, diluent, excipient, and / or the salt formed therefrom is generally chemically or physically compatible with other components constituting a drug dosage form and physiologically compatible with receptors.

[0029] The terms "salt" and "pharmaceutical salt" refer to acidic and / or basic salts formed by the above-described compounds or their stereoisomers with inorganic and / or organic acids and bases, including zwitterionic salts (internal salts) and quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly during the final separation and purification of the compounds. Alternatively, they can be obtained by mixing the above-described compounds or their stereoisomers with an appropriate amount (e.g., equimolar amounts) of an acid or base. These salts may be obtained by precipitating in solution and collecting by filtration, by recovery after solvent evaporation, or by freeze-drying after reaction in an aqueous medium. The salts described in this invention can be hydrochlorides, sulfates, citrates, benzenesulfonates, hydrobromides, hydrofluoric acids, phosphates, acetates, propionates, succinates, oxalates, malates, succinates, fumarates, maleates, tartrates, or trifluoroacetates of the compounds.

[0030] In some embodiments, one or more compounds of the present invention may be used in combination with each other. Alternatively, the compounds of the present invention may be used in combination with any other active agent to prepare a medicament or pharmaceutical composition for regulating cell function or treating disease. If a group of compounds is used, these compounds may be administered to the test subject simultaneously, separately, or sequentially.

[0031] This invention is the first to discover a class of flavonoids that can inhibit osteogenic differentiation of valvular interstitial cells. In animal studies, these flavonoids significantly reduced peak aortic flow velocity and peak pressure gradient in mice with calcified aortic valves, demonstrating a significant effect in alleviating valve calcification. They show potential application in the development of drugs to alleviate aortic valve disease and improve cardiovascular aging and other related cardiovascular diseases. Therefore, the flavonoids provided by this invention have promising applications in the development of drugs to improve cardiovascular aging and alleviate aortic valve disease.

[0032] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0033] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0034] Figure 1 The figure shows the experimental results of compounds 1-18 in Example 1 inhibiting osteogenic differentiation of valvular interstitial cells. In this figure, A is a graph of ALP staining results of different compounds, and B is a statistical graph of ALP staining results of different compounds. Figure 2 The figure shows the experimental results of compounds 19-36 in Example 1 inhibiting osteogenic differentiation of valvular interstitial cells. In this figure, A is a graph of ALP staining results of different compounds, and B is a statistical graph of ALP staining results of different compounds. Figure 3 The figure shows the experimental results of compounds 37-54 in Example 1 inhibiting osteogenic differentiation of valvular interstitial cells. In this figure, A is a graph of ALP staining results of different compounds, and B is a statistical graph of ALP staining results of different compounds. Figure 4 The figure shows the experimental results of compounds 55-72 inhibiting osteogenic differentiation of valvular interstitial cells in Example 1. In this figure, A is a graph of ALP staining results of different compounds, and B is a statistical graph of ALP staining results of different compounds. Figure 5 The figure shows the experimental results of compounds 73-90 in Example 1 inhibiting osteogenic differentiation of valvular interstitial cells. In this figure, A is a graph of ALP staining results of different compounds, and B is a statistical graph of ALP staining results of different compounds. Figure 6 The figure shows the experimental results of compounds 91-102 inhibiting osteogenic differentiation of valvular interstitial cells in Example 1. In this figure, A is a graph of ALP staining results of different compounds, and B is a statistical graph of ALP staining results of different compounds. Figure 7 The figures shown are experimental results of the therapeutic effects of each compound in Example 2 on alleviating aortic valve calcification. In the figure, A is an ultrasound result of the mouse heart, B is the result of the peak pressure gradient of the aortic valve, and C is the result of the peak flow velocity of the aortic valve.

[0035] Figure 8 This is for the immunoblotting assay used in Experiment Example 3.

[0036] Figure 9 The figure shows the experimental results of compound 69 in Experiment 4, which effectively alleviated the valve structural abnormalities and calcification in an aged mouse CAVD model. In the figure, A is a mouse cardiac ultrasound image, B is a mouse H&E result image, C is a crossvalve peak jet velocity statistical graph, and D is a aortic valve leaflet thickness statistical graph. Detailed Implementation

[0037] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0038] The commercial information for the compounds used in the following experiments is as follows: Example 1: Cellular experiments revealed the inhibitory effect of flavonoids on osteogenic differentiation of valvular interstitial cells. I. Experimental Methods 1. Isolation of primary aortic valve interstitial cells This experiment was approved by the ethics committee (Approval No. 93, 2017), and informed consent was obtained from the patients. Aortic valve tissue was obtained from 10 patients who underwent cardiac surgery (mean age 52.8 ± 10.6 years). The primary valve interstitial cell isolation procedure is as follows: After endothelial removal and wiping of the valve surface, the tissue was washed with PBS. Enzymatic digestion: The tissue was transferred to a centrifuge tube containing 20-25 mL of type II collagenase and digested with shaking at 37°C. The digestion solution was filtered through a 70 μm filter, centrifuged at 1100 rpm for 4 minutes, and resuspended in complete culture medium. Cells were seeded in 100 mm culture dishes and cultured at 37°C in a 5% CO2 incubator until confluence >90%, then passaged. Experiments were performed or the cells were passaged again after cell confluence reached the target (>90% in culture dishes, 70-80% in 12-well plates).

[0039] 2. Osteogenic induction differentiation Cells were induced using osteogenic differentiation medium consisting of basal medium supplemented with 10 mM sodium β-glycerophosphate, 50 μM vitamin C, and 100 nM dexamethasone. Induction began when cell confluence reached 70–80%, with compound 1-102 (final concentration 5 μM) added to each experimental group simultaneously, and induction continued for 3 days.

[0040] In addition, a control group was set up: CM: Cultured on normal culture medium without the addition of osteogenic differentiation-inducing reagents. OM: Cultured in normal culture medium, with the addition of relevant reagents for osteogenic induction differentiation, without drug treatment.

[0041] 3. Alkaline phosphatase (ALP) staining Alkaline phosphatase (ALP) is a key early marker of osteoblast differentiation. The BCIP / NBT method was used to assess the degree of calcification in valvular interstitial cells: Cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 10 minutes. After washing with PBS, freshly prepared BCIP / NBT staining working solution was added to each well. Incubation at room temperature was performed until the desired color depth was reached, followed by washing with distilled water to terminate the staining. Cells were then air-dried and observed and recorded under a microscope.

[0042] II. Experimental Results Experimental results are as follows Figure 1-6 As shown, the results indicate that compounds 5, 7, 27, 28, 37, 39, 41, 43, 52, 53, 54, 55, 59, 60, 63, 64, 66, 69, 77, 78, 81, 82, 83, 84, and 87 all significantly inhibit osteogenic differentiation of valvular interstitial cells (reducing ALP activity). Subsequently, we selected compounds 27, 28, 41, 43, 66, and 69 for further animal experiments to evaluate their effects.

[0043] The above experimental results indicate that these flavonoids have the effect of inhibiting osteogenic differentiation of valvular interstitial cells and are potential active ingredients for improving cardiovascular aging and alleviating aortic valve disease.

[0044] Example 2: Evaluation in vivo in animal experiments—In vivo validation of activity in guidewire injury-induced calcified aortic valve disease I. Experimental Methods 1. Establishment of a model of calcific aortic valve disease induced by guidewire injury A mouse model of calcified aortic valve disease was established using the guidewire injury method. The specific procedure was as follows: Mice were anesthetized in an isoflurane induction device. After shaving and thorough disinfection of the neck, the skin on the right side of the neck was incised, and the neck muscles were separated to expose the right common carotid artery. A guidewire was then inserted at approximately 30° to the predetermined depth, and the process was repeated approximately 50 times with each rotation, inducing mechanical damage to the aortic valve through friction, thereby inducing calcified aortic valve lesions. After the animal model was established, mice were administered the corresponding drugs (compounds 27, 28, 41, 43, 66, and 69) daily via gavage for 60 consecutive days.

[0045] Experimental Groups: The experiment was divided into four groups: Blank group, Sham group, Model group, and Experimental group. The Blank group received no surgery, normal diet, and no medication. The Sham group had only the right common carotid artery exposed, without guidewire injury induction, normal diet, and no medication. The Experimental groups administered compounds 27, 28, 41, 43, 66, and 69 of the examples to the model mice, respectively, at a daily dose of 50 mg / kg via gavage. The Model group received an equal volume of physiological saline.

[0046] 2. Assessment of cardiac structure and aortic valve function in mice Two months after guidewire injury modeling, mice underwent cardiac color Doppler echocardiography to assess aortic valve hemodynamics and cardiac function. Mice were placed in a transparent anesthesia box and induced anesthesia with 3% isoflurane, maintaining a stable level of anesthesia throughout the examination. The mice were then fixed supine on a small animal ultrasound platform, and a high-frequency ultrasound probe was used to locate cardiac structures and acquire relevant ultrasound parameters, including: transaortic peak velocity, left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), and left ventricular ejection fraction (LVEF). After the cardiac ultrasound assessment, blood and heart specimens were finally obtained from the mice.

[0047] II. Experimental Results Experimental results are as follows Figure 7 As shown in the figure. The results indicate that compounds 27, 28, 41, 43, 66, and 69 can significantly reduce the peak aortic valve velocity and peak pressure gradient, and all can significantly alleviate aortic valve calcification.

[0048] Example 3: Inhibitory effect of flavonoids on osteogenic differentiation and senescence of valvular interstitial cells I. Experimental Methods 1. Osteogenic induction differentiation Primary aortic valve interstitial cells were induced using an osteogenic differentiation medium consisting of basal medium supplemented with 10 mM sodium β-glycerophosphate, 50 μM vitamin C, and 100 nM dexamethasone. Induction began when cell confluence reached 70–80%. The experimental group received compound 69 (final concentration 5 μM) and underwent induction for 3 days. Control groups were also established: CM: basal medium without osteogenic differentiation-inducing agents (sodium β-glycerophosphate, vitamin C, dexamethasone); OM: normal medium with osteogenic differentiation-inducing agents, without treatment.

[0049] The dosage concentration of compound 69 was 5 μM. Samples were collected 3 days after drug treatment.

[0050] 2. Western blot assay to detect differences in the expression of different proteins. Prepare the separating and stacking gels for SDS-PAGE according to the instructions. Prepare electrophoresis buffer and transfer buffer. Electrophoresis separation: Pour the electrophoresis buffer into the electrophoresis tank. Place the gel in the electrophoresis tank and connect it to the power supply. Add protein markers (5-10 μL) and samples (5-20 μL) to each well. First, run the concentration at a low voltage (80V); then run the separation at a higher voltage (120V), adjusting the run time as needed. Transfer: Cut the polyvinylidene fluoride (PDVF) membrane to the same width as the desired protein band. Moisten the sponge and thick filter paper in transfer buffer and activate the PDVF membrane with methanol. Separate the glass plate and remove the gel. Prepare a transfer "sandwich" by combining the gel, filter paper, and PVDF membrane in the following order: (black side) sponge → 3 sheets of filter paper → gel → PVDF membrane → 3 sheets of filter paper → sponge (red side). Transfer the "sandwich" to the transfer apparatus, which should be placed on ice. The transfer time depends on the gel thickness and the molecular weight of the protein. Blocking. Transfer the PVDF membrane to 5% skim milk prepared with TBS / T and block on a shaker at room temperature for 1 hour. Primary antibody incubation: Dilute the antibody with the primary antibody diluent according to the antibody instructions, place the corresponding PVDF membrane in the antibody incubation box, and incubate overnight at 4°C. The next day, wash the bands three times with TBST for 5 minutes each time. Secondary antibody incubation: Dilute the secondary antibody with 5% skim milk prepared with TBST and incubate at 37°C for 1 hour. Wash the membrane three times with TBS / T for 5 minutes each time. Chemiluminescent development: Prepare ECL developer by mixing ECL A and B solutions according to the instructions, preparing fresh each time. Incubate the membrane uniformly for 1-2 minutes. Develop using a chemiluminescence exposure apparatus. The antibodies used were: RUNX2 antibody Abcam ab192256, P21 antibody Abcam ab188224, P53 antibody Proteintech 60283-2-Ig, osteopontin (OPN) antibody Abcam ab214050, Osteocalcin (OCN) antibody Abclonal A20800, and β-actin antibody CST 4970.

[0051] II. Experimental Results like Figure 8As shown, the effects of compound 69 on the aging and osteogenic differentiation of VICs were investigated. After stimulation with osteogenic induction medium, the expression of aging-related proteins P21 and P53, as well as osteogenic marker proteins OCN, RUNX2, and OPN, were significantly upregulated in VICs, indicating that cells undergo significant aging and osteogenic differentiation under calcification-induced conditions. Further comparison of the effects of different drug interventions revealed that the compound 69 treatment group significantly reduced the expression levels of P21 and P53, while significantly inhibiting the upregulation trend of RUNX2, OCN, and OPN, indicating that AAI can effectively delay the aging process of VICs and block their osteogenic phenotypic transformation. The comprehensive analysis results suggest that compound 69 exhibits significant anti-aging and anti-calcification effects.

[0052] Example 4: The allergic effect of compound 69 on aged mice with calcified aortic valve. I. Experimental Methods A mouse model of calcified aortic valve disease was established using the guidewire injury method. The experimental groups were: ① young sham-operated group; ② elderly sham-operated group; ③ elderly model group; ④ elderly model drug-treated group. Mice in the elderly model drug-treated group were administered compound 69 (50 mg / kg) orally by gavage once daily for two months after surgery. The other groups received an equal volume of the solvent as a control.

[0053] Two months after guidewire injury modeling, mice underwent cardiac color Doppler echocardiography to assess aortic valve hemodynamics and cardiac function. Mice were placed in a transparent anesthesia box and induced anesthesia with 3% isoflurane, maintaining a stable level of anesthesia throughout the examination. The mice were then fixed supine on a small animal ultrasound platform, and a high-frequency ultrasound probe was used to locate cardiac structures and acquire relevant ultrasound parameters, including: transaortic peak velocity, left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), and left ventricular ejection fraction (LVEF). After the cardiac ultrasound assessment, blood and heart specimens were finally obtained from the mice.

[0054] After tissue sampling, the tissue was rinsed with pre-cooled sterile PBS to remove blood, blotted dry, and fixed in 4% paraformaldehyde at room temperature for 24 hours. It was then rinsed slowly with tap water overnight. Subsequently, it was dehydrated by sequentially applying 70%, 80%, 95%, and 100% ethanol for 60 minutes each, and then soaked in molten paraffin at 60°C three times (1 hour each time) for infiltration. During embedding, the tissue was placed in a preheated mold and its orientation was adjusted. It was then rapidly solidified on a cooling plate at 4°C. Sections with a thickness of 5 μm were cut using a rotary microtome, spread in warm water at 45°C, transferred to glass slides, and dried at 60°C for 2 hours. For H&E staining, the sections were first dried at 60°C for 1 hour, then dewaxed with xylene I and II for 5 minutes each, then rehydrated with 100%, 95%, 80%, and 70% ethanol for 2 minutes each, rinsed with distilled water, stained with hematoxylin for 5 minutes, rinsed with tap water for 1 minute, differentiated with 1% hydrochloric acid ethanol for a few seconds, rinsed with running water, and blued with 0.1% ammonia for 1 minute; rinsed with distilled water for 1 minute and stained with eosin for 1 minute; finally, dehydrated with 70%, 80%, 95%, and 100% ethanol for 1 minute each, cleared with xylene I and II for 5 minutes each, mounted with neutral resin, covered with a coverslip, and allowed to air dry before microscopic examination and photography.

[0055] II. Experimental Results The peak velocity of transvalvular jet flow in the aortic valve of aged mice in the sham-operated group was significantly higher than that in the younger group, suggesting that aging itself can lead to valvular functional degeneration and mild calcification. The transvalvular jet velocity further increased in the aged model group, indicating that the stress response induced by mechanical injury significantly aggravated valvular dysfunction. However, after intervention with compound 69, this velocity significantly decreased, suggesting that compound 69 can improve valvular hemodynamic abnormalities. Figure 9 A). Histological examination further validated the ultrasound results. H&E staining showed that the aortic valve leaflets in the elderly sham-operated group were significantly thicker than those in the younger group. After the model was created and the operation was performed, the leaflets became even thicker. Treatment with compound 69 significantly reduced the increase in leaflet thickness. Figure 9 B). In summary, aging can cause mild structural degeneration and calcification of the aortic valve, and guidewire injury exacerbates this pathological process. However, the intervention of compound 69 can effectively alleviate the valve structural abnormalities and calcification in an aged mouse CAVD model, indicating that it has a significant in vivo protective effect in delaying the valvular calcification process.

[0056] As can be seen from the above embodiments, this invention, through in vivo and in vitro experiments, demonstrates that a class of flavonoid compounds can significantly inhibit osteogenic differentiation of valvular interstitial cells, significantly reduce peak aortic valve flow velocity and peak pressure gradient in mice with calcified aortic valves, and have a significant effect in alleviating aortic valve calcification. It can inhibit significant senescence and osteogenic differentiation of valvular interstitial cells under calcification-induced conditions, and alleviate valvular structural abnormalities and calcification in aged mouse CAVD models. Therefore, the compounds provided by this invention have good application prospects in the preparation of drugs to alleviate aortic valve disease and other related cardiovascular diseases such as cardiovascular aging.

Claims

1. The use of the compound of Formula I, or a pharmaceutically acceptable salt thereof, or a deuterated derivative thereof, or a tritated derivative thereof, or a stereoisomer thereof, or a prodrug thereof, or a crystal form thereof, in the preparation of a medicament for the treatment and / or prevention of aortic valve disease and / or cardiovascular aging, characterized in that, The structural formula of the compound is: in: R1, R2, R3, and R4 are each independently selected from H, deuterium, tritium, halogens, -OH, -NH2, -NO2, -CF3, -COOH, C1-C8 alkoxy groups substituted or unsubstituted with 1-3 Rb groups, C1-C8 ester groups, and -NHR. h1 C1-C8 alkyl groups substituted with or unsubstituted with 1-3 Ra atoms; R5, R6, R7, R8, and R9 are each independently selected from H, deuterium, tritium, halogen, -OH, -NH2, -NO2, -CF3, -COOH, C1~C8 alkoxy groups, -OCF3, C1~C8 ester groups, and -NHR. h1 C1-C8 alkyl groups substituted with or unsubstituted with 1-3 Ra atoms; R 10 Selected from H, deuterium, tritium, halogens, -OH, -NH2, -NO2, -CF3, -COOH, C1-C8 alkoxy groups substituted or unsubstituted with 1-3 Rb, C1-C8 alkyl groups substituted or unsubstituted with 1-3 Ra, C1-C8 ester groups substituted or unsubstituted with 1-3 Rc, -NHR h1 ; Ra is independently selected from deuterium, tritium, halogen, hydroxyl group, and 5-10 membered heterocyclic alkyl groups; Rb is independently selected from deuterium, tritium, halogen, and C6-C group. 10 Aryl, 5-10 membered heterocyclic alkyl; Rc is independently selected from C6~C 10 Aryl; R h1 The heterocycle is selected from hydroxyl, C1-C5 alkyl, phenyl-substituted C1-C8 alkyl, substituted 6-12 aryl, and substituted 5-10 heterocycles, wherein the heteroatom of the heterocycle is selected from at least one of N, O, or S, and the number of heteroatoms is 1-4. The substituents in the substituted phenyl, substituted 6-12 aryl, or substituted 5-10 heterocycles are selected from deuterium, tritium, halogen, -OH, methoxy, C1-C8 alkyl, C1-C8 alkoxy, phenyl, etc. -CF3, -OCF3, C1~C8 alkenyl, carboxyl.

2. The use according to claim 1, characterized in that: R1, R2, R3, and R4 are each independently selected from H, deuterium, tritium, halogens, -OH, -NH2, -NO2, -CF3, -COOH, C1-C4 alkoxy groups substituted or unsubstituted with 1-3 Rb groups, C1-C4 ester groups, and -NHR. h1 C1-C8 alkyl groups substituted with or unsubstituted with 1-3 Ra atoms; R5, R6, R7, R8, and R9 are each independently selected from H, deuterium, tritium, halogen, -OH, -NH2, -NO2, -CF3, -COOH, C1~C4 alkoxy group, -OCF3, C1~C4 ester group, and -NHR. h1 C1-C8 alkyl groups substituted with or unsubstituted with 1-3 Ra atoms; R 10 Selected from H, deuterium, tritium, halogens, -OH, -NH2, -NO2, -CF3, -COOH, C1-C4 alkoxy groups substituted or unsubstituted with 1-3 Rb, C1-C4 alkyl groups substituted or unsubstituted with 1-3 Ra, C1-C4 ester groups substituted or unsubstituted with 1-3 Rc, -NHR h1 .

3. The use according to claim 2, characterized in that: R1, R2, R3, and R4 are each independently selected from H, deuterium, tritium, halogen, -OH, -NH2, -NO2, -CF3, -COOH, C1~C4 alkoxy, and C1~C4 alkyl groups substituted with 1~3 Ra; Ra is independently selected from deuterium, tritium, halogen, and hydroxyl. R5, R6, R7, R8, and R9 are each independently selected from H, deuterium, tritium, halogen, -OH, -NH2, -NO2, -CF3, -COOH, C1~C8 alkyl, and C1~C4 alkoxy groups; R 10 Selected from H, deuterium, tritium, halogen, -OH, -NH2, -NO2, -CF3, -COOH, and C1~C4 ester groups.

4. The use according to claim 1, characterized in that: R1 is selected from H, deuterium, -OH, and methoxy groups; R2 is selected from H, Cl, -OH, -NH2, -COOH, C1~C2 alkyl, and methoxy. R3 is selected from H, deuterium, -OH, methoxy, and phenyl-substituted methoxy groups; R4 is selected from H, deuterium, -OH, and methoxy groups; R5 and R9 are independently selected from H, deuterium, -OH, and methoxy groups, respectively; R6 and R8 are independently selected from H, deuterium, -OH, -NO2, -CF3, methoxy, and deuterium-substituted methoxy groups, respectively; R7 is selected from H, F, Cl, -OH, -CF3, -COOH, C1~C4 alkoxy, deuterated methoxy, fluorinated methoxy, methyl, deuterated methyl, and 6-membered nitrogen-containing heterocyclic alkyl-substituted ethyl. R 10 Selected from H, deuterium, -OH, methoxy, deuterium-substituted methoxy, phenyl-substituted C1 ester group, ethyl ester group, and ethoxy with a 6-membered nitrogen-containing heterocyclic alkyl group.

5. The use according to claim 4, characterized in that: R1 is selected from H or -OH; R2 is selected from H, Cl, -OH, -NH2, and -COOH; R3 is selected from H, -OH, and methoxy groups; R4 is selected from H or -OH; R5 and R9 are independently selected from H, deuterium, and -OH, respectively; R6 and R8 are independently selected from H, deuterium, -OH, -CF3, and methoxy, respectively; R7 is selected from H, -OH, methoxy, deuterated methoxy, methyl, and deuterated methyl. R 10 Selected from H, deuterium, -OH, methoxy, and deuterium-substituted methoxy groups.

6. The use according to claim 5, characterized in that: The structural formula of the compound is shown in Formula II: in: R1 is selected from H or -OH; R2 is selected from H or -OH; R3 is selected from -OH and methoxy groups; R5 and R9 are independently selected from H and -OH, respectively; R7 is selected from H and -OH; R8 is selected from H, -OH, and methoxy groups; R 10 Selected from H, -OH, and methoxy groups.

7. The use according to claim 1, characterized in that: The structural formula of the compound is: 。 8. The use according to claim 1, characterized in that: The drug is used to delay or inhibit aortic valve calcification.

9. The use according to claim 8, characterized in that: The drug is used to achieve at least one of the following functions: reducing the pressure gradient across the valve, reducing the peak blood flow velocity, and improving aortic valve function.

10. The use according to claim 8, characterized in that: The drug is used to inhibit osteogenic differentiation of valvular interstitial cells.

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