Silibinin derivative and use thereof

Abstract

Provided are a silibinin isopentenyl derivative and a use thereof, belonging to the technical field of medicine. Specifically disclosed are a silibinin isopentenyl derivative and a use thereof in the preparation of a drug for preventing and / or treating ischemic cardiovascular diseases. The silibinin isopentenyl derivative greatly improves bioavailability compared with silibinin, overcomes the drawback of the poor bioavailability of silibinin, and effectively protects H9c2 cells from oxygen-glucose deprivation injury and acute myocardial ischemia injury, thereby providing more approaches for the development of drugs for the prevention and treatment of ischemic cardiovascular diseases.

Description

A silymarin derivative and its uses Technical Field

[0001] This invention discloses a silybin derivative and its uses, specifically a silybin isopentenyl derivative and its use in the preparation of drugs for the prevention and / or treatment of ischemic heart disease. This invention belongs to the field of pharmaceutical technology. Background Technology

[0002] Cardiovascular disease is a leading cause of death worldwide, ranking first in mortality rates and continuing to rise annually, seriously endangering human health. Myocardial infarction (MI) is a serious coronary artery disease characterized by ischemic necrosis of the myocardium caused by transient or persistent occlusion of the distal coronary arteries. During a myocardial infarction, myocardial ischemia and hypoxia lead to infiltration of inflammatory cells such as macrophages and monocytes, inducing a local inflammatory response, which in turn causes myocardial cell death, resulting in cardiac dysfunction. Clinical symptoms often manifest as severe and persistent retrosternal pain, endangering the patient's life. This disease has a high incidence rate and is one of the leading causes of death and disability worldwide.

[0003] Thrombolysis and percutaneous coronary intervention are currently the main treatments for myocardial infarction. Although they have significantly reduced mortality, many patients still experience complications such as ischemia-reperfusion injury and coronary restenosis. Therefore, finding safer and more effective drugs for the prevention and treatment of myocardial infarction, especially those targeting myocardial cell damage caused by myocardial infarction, is of great significance.

[0004] Silymarin is a flavonoid lignan isolated from the silymarin plant (Silybum marianum) of the Asteraceae family. It is formed by the condensation of dihydroflavonols and phenylpropanoid derivatives, and mainly comprises four components: silybin, isosilybin, silybinine, and silybinine. Silymarin exhibits good inhibitory effects on prostate cancer, colon cancer, bladder cancer, and liver cancer. Furthermore, it possesses various pharmacological activities, including treating hyperlipidemia, anti-gastric ulcer activity, lipid-lowering effects, anti-platelet aggregation, and immunomodulatory effects, making it highly valuable for development.

[0005] Among them, silibinin has the highest content and possesses pharmacological activities such as scavenging free radicals, resisting lipid peroxidation, protecting hepatocyte membranes, promoting the synthesis of DNA and structural proteins in damaged hepatocytes, and anti-fibrosis. Recent studies have also shown that silibinin has cardioprotective effects. Cao et al. (Cao Xudan, Ren Chunmei, He Tiantian, et al. Protective effect and mechanism of silybin on ischemia-reperfusion injury in isolated heart[J]. Modern Journal of Integrated Traditional and Western Medicine, 2019, 28(26):2860-2864.) showed that silybin can improve myocardial function in rats after myocardial ischemia-reperfusion injury, inhibit cardiomyocyte apoptosis, inhibit oxidative stress and improve hemodynamics; Chen et al. (Chen Y, Lin H, Wang Q, et al. Protective Role of Silibinin Against Myocardial Ischemia / Reperfusion Injury-Induced Cardiac Dysfunction[J]. International Journal of Biological Sciences, 2020, 16(11):1972-1988.) showed that silybin can reduce endoplasmic reticulum stress in myocardial cells in mice after myocardial ischemia-reperfusion injury and counteract myocardial ischemia-reperfusion injury by regulating inflammatory response through inactivation of NF-κB signaling pathway.

[0006] However, due to the poor solubility of silymarin, which is almost insoluble in water and oils, its oral absorption is poor and its bioavailability is low, thus affecting its clinical efficacy. Therefore, there is an urgent need to develop a silymarin derivative with high bioavailability and good efficacy. Summary of the Invention

[0007] To solve the above problems, the present invention adopts the following technical solution:

[0008] This invention provides a silymarin derivative as shown in Formula I and its pharmaceutically acceptable meso, racemic, enantiomer, and diastereomer.

[0009] This invention modifies the structure of silybin to provide a silybin isopentenyl derivative (Sil-1), chemically named "3,5,7-trihydroxy-2-(3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-6-(3-methylbut-2-en-1-yl)benzodihydropyran-4-one", with a molecular weight of 550.18. Its specific structure is shown in Formula I.

[0010] The silybin isopentenyl derivative of the present invention is obtained by isopentenylation modification of silybin via in vitro enzymatic catalysis using dimethylpropene pyrophosphate (DMAPP) as the isopentenyl donor.

[0011] The present invention provides a pharmaceutical composition comprising a compound of Formula I or thereof, or a racemic, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, and pharmaceutical excipients.

[0012] The present invention also provides the use of the compound of Formula I or a racemic, racemic, enantiomer, diastereomer, or mixture of isomers thereof, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof, in the preparation of a medicament for the prevention and / or treatment of ischemic heart disease.

[0013] Furthermore, the ischemic heart disease is ischemic myocardial injury or myocardial infarction.

[0014] Furthermore, the myocardial infarction mentioned is an acute myocardial infarction.

[0015] Furthermore, the drug is a drug that reduces cardiomyocyte apoptosis.

[0016] Furthermore, the drug is a drug that reduces the area of ​​myocardial infarction.

[0017] Furthermore, the drug is a drug that lowers serum creatine kinase.

[0018] Furthermore, the drug is a drug for reducing cardiac inflammation caused by myocardial infarction.

[0019] Furthermore, the drug is a drug for preventing and treating ischemic myocardial injury.

[0020] Compounds of Formula I, when mixed with commonly used auxiliary additives acceptable in oral formulations such as disintegrants, excipients, lubricants, binders, and fillers, can be formulated into solid dosage forms such as tablets, pills, capsules, or various corresponding sustained-release and controlled-release agents using conventional methods. When mixed with commonly used solubilizers, emulsifiers, wetting agents, foaming or defoaming agents, surfactants, diluents, preservatives, stabilizers, flavoring agents, and thickeners, they can be formulated into liquid dosage forms such as aqueous solutions and syrups using appropriate methods. When combined with appropriate solvents and excipients commonly used in injections, they can also be formulated into corresponding intramuscular or intravenous injection dosage forms.

[0021] In the technical solution of the present invention, the drug is prepared into any drug dosage form by using Sil-1 or its pharmaceutically acceptable salt as the active pharmaceutical ingredient.

[0022] The dosage form is an oral preparation, an injectable preparation, or a topical preparation.

[0023] Furthermore, the dosage form is an injectable formulation.

[0024] The pharmaceutically acceptable salts are those commonly used in pharmaceuticals, including but not limited to acetates, hydrochlorides, hydrobroms, nitrates, sulfates, phosphates, benzoates, fumarates, maleates, succinates, tartrates, citrates, oxalates, glyoxylates, aspartate, tartrates, 2,5-dihydroxybenzoates, methanesulfonates, ethanesulfonates, benzenesulfonates, lauryl sulfonates, hydroquinone sulfonates, and p-toluenesulfonates.

[0025] In the uses described in this invention, the content of Sil-1 in each unit of the pharmaceutical preparation is 0.1-99 wt%.

[0026] Unless otherwise defined, the technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0027] The term "pharmaceutical acceptable" in this invention means that a compound, composition, or carrier is suitable for administration to an individual to achieve the use described herein without undue harmful side effects.

[0028] The term "pharmaceutically acceptable salt" in this invention refers to a salt that, within the bounds of reliable medical judgment, is suitable for use in humans and other mammals without causing excessive toxicity, irritation, allergic reactions, etc., and is commensurate with a reasonable benefit / risk ratio.

[0029] The terms "individual" and "patient" in this application include both humans and mammals. A mammal means any animal species of the mammal class. Examples of mammals include humans; non-human primates such as monkeys; laboratory animals such as rats, mice, and guinea pigs; livestock such as cats, dogs, rabbits, cattle, sheep, goats, horses, and pigs; and domesticated wild animals such as lions and tigers.

[0030] In this invention, the term "treatment" refers to the process of intervening in or changing a particular health condition, including eliminating the cause, symptomatic treatment, or supportive treatment.

[0031] In this invention, the term "prevention" refers to both preclinical prevention and clinical prevention. Preclinical prevention refers to preventing changes in the disease in its preclinical or early clinical stages through early detection and diagnosis, and appropriate treatment, enabling the disease to be detected and treated at an early stage, avoiding or reducing complications, sequelae, and disabilities, or shortening the time to disability. Clinical prevention refers to timely treatment using various clinical treatment methods to prevent the patient's disease from worsening, promote early recovery, reduce the adverse effects of the disease, and prevent complications and disabilities.

[0032] The term "pharmaceutical composition" in this invention refers to a composition containing at least one bioactive compound.

[0033] The "ischemic heart disease" in this invention refers to a class of heart diseases caused by insufficient blood supply to the coronary arteries, resulting in myocardial ischemia, hypoxia, or even necrosis.

[0034] The terms "myocardial injury," "myocardial infarction," and related regulations in this invention comply with the provisions of the "thygesen et al. 2018-fourth-universal-definition-of-myocardial-infarction(2018)" formulated by the European Society of Cardiology (ESC), the American College of Cardiology (ACC), the American Heart Association (AHA), and the World Heart Federation (WHF). Furthermore, the entire "thygesen et al. 2018-fourth-universal-definition-of-myocardial-infarction(2018)" is cited in the terminology explanation section of this application, and will not be repeated here due to space limitations. Specifically, myocardial infarction is caused by a sudden reduction or interruption of coronary blood flow, leading to persistent ischemia and hypoxia of myocardial cells, ultimately resulting in myocardial cell necrosis and deterioration of cardiac function. This includes, but is not limited to: acute ST-segment elevation myocardial infarction, acute non-ST-segment elevation myocardial infarction, ischemic cardiomyopathy, etc.

[0035] In this invention, the term "oral preparation" refers to a form of preparation that is administered orally and absorbed into the bloodstream through the gastrointestinal tract. This includes, but is not limited to, tablets, granules, capsules, pills, and oral solutions.

[0036] In this invention, the term "injectable formulation" refers to a pharmaceutical preparation injected into the body via a syringe, which can directly act on the blood system or specific tissues. This includes, but is not limited to, solutions, suspensions, or emulsions.

[0037] The term "topical preparation" in this invention refers to pharmaceutical preparations that are applied directly to the skin or specific areas of the skin. This includes, but is not limited to, creams, gels, lotions, and medicated sprays.

[0038] The structure or name of the compound used in this invention covers the compound and all possible isomers therein, such as its tautomers, meso compounds, racemates, enantiomers, and / or diastereomers and any mixtures thereof.

[0039] The present invention has the following beneficial effects:

[0040] The bioavailability of Sil-1 in this invention is significantly improved, at least three times higher than that of Silibinin, exhibiting superior biological activity and making it more suitable for clinical application.

[0041] The Sil-1 of the present invention has a clear protective effect on cardiomyocytes in both in vivo and in vitro experiments.

[0042] The Sil-1 of this invention significantly improves the cell viability and morphology of H9c2 cells under glucose-oxygen deprivation, and its effect is superior to that of Silibinin.

[0043] The Sil-1 of this invention reverses the increase in LDH release from H9c2 cells under glucose-oxygen deprivation conditions, significantly reduces the degree of glucose-oxygen deprivation damage in H9c2 cells, and has a better effect than Silibinin.

[0044] The Sil-1 of this invention inhibited the increase of ROS levels in H9c2 cells under glucose-oxygen deprivation conditions, and showed better antioxidant stress effects after the introduction of isopentenyl groups.

[0045] The Sil-1 of this invention significantly inhibits the release of inflammatory factors in H9c2 cells under glucose-oxygen deprivation, exhibiting definite anti-inflammatory activity, and its anti-inflammatory activity is higher than that of Silibinin.

[0046] The Sil-1 of this invention reduced the ST segment elevation trend in the electrocardiogram of a rat model of myocardial infarction, and Sil-1 was more significant than Silibinin in reducing T wave potential.

[0047] The Sil-1 of this invention reduced serum CK, CK-MB, and LDH levels in rats with a myocardial infarction model, alleviating the degree of myocardial cell damage and necrosis during acute myocardial infarction, and its effect was superior to silymarin. In particular, the medium and high dose groups showed extremely significant differences in reducing serum CK levels in rats (P<0.05).

[0048] The Sil-1 of this invention reduces the proportion of myocardial infarction area in rats with myocardial infarction, and is more effective than silymarin.

[0049] The Sil-1 of this invention reduces the serum inflammatory factor levels in rats with myocardial infarction, reduces the increase in intercellular space caused by myocardial ischemia, and reduces the degree of inflammatory factor infiltration in myocardial cells. Sil-1 can more effectively alleviate the inflammatory response of myocardial cells during acute myocardial infarction. Attached Figure Description

[0050] Figure 1 shows the effects of Sil-1 and Silibin on the morphology of H9c2 cells under glucose-oxygen deprivation.

[0051] Figure 2 shows the effects of Sil-1 and Silibin on the viability of H9c2 cells under glucose-oxygen deprivation.

[0052] Figure 3 shows the effects of Sil-1 and Silibinin on LDH release from H9c2 cells after glucose and oxygen deprivation.

[0053] Figure 4 shows the effects of Sil-1 and Silibin on ROS levels in H9c2 cells after glucose and oxygen deprivation (DCFH-DA, 100×). (A) is the observation under a fluorescence microscope, and (B) is the fluorescence intensity statistics.

[0054] Figure 5 shows the effects of Sil-1 and Silibinin on the excretion of inflammatory factors in H9c2 cells under glucose-oxygen deprivation treatment: A represents the effect on TNF-α excretion in H9c2 cells, B represents the effect on IL-β excretion in H9c2 cells, and C represents the effect on IL-6 excretion in H9c2 cells.

[0055] Figure 6(A) shows the electrocardiograms of rats with myocardial infarction after being treated with different doses of silbinin and sil-1; Figure 6(B) shows the comparison of ST segment elevation potentials of rats with myocardial infarction after being treated with different doses of silbinin and sil-1.

[0056] Figure 7 shows the serum myocardial enzyme levels after Silibinin and Sil-1 were administered at different doses to rats with myocardial infarction. A represents the serum creatine kinase (CK) level, B represents the serum creatine kinase isoenzyme (CK-MB), and C represents the serum lactate dehydrogenase (LDH) level.

[0057] Figure 8 shows the serum inflammatory factor levels in rats with myocardial infarction after treatment with different doses of silbinin and sil-1. A represents the serum TNF-α level, B represents the serum IL-1β level, and C represents the serum IL-6 level.

[0058] Figure 9(A) shows the TTC staining results of heart sections in rats after treatment with different doses of Silibinin and Sil-1 in a rat model of myocardial infarction. Figure 9(B) shows the comparison of the area of ​​the myocardial infarction region in rats after treatment with different doses of Silibinin and Sil-1 in a rat model of myocardial infarction.

[0059] Figure 10 shows HE staining of heart sections after different doses of Silibinin and Sil-1 were applied to rats with myocardial infarction.

[0060] Note: Compared with the normal group # P < 0.05 ## P < 0.01, ### P < 0.001; compared with the model group, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with the Silibin group, △P < 0.05, △△ P < 0.01, △Δ△ P < 0.001. Detailed implementation manners

[0061] Before further describing the specific implementation manners of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific implementation manners. The terms used in the embodiments of the present invention are for describing specific implementation manners, rather than limiting the protection scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out according to conventional methods or methods recommended by the manufacturer. Unless otherwise defined, the technologies and terms used in the present invention have the same meanings as those commonly understood by those skilled in the art.

[0062] Example 1 Pharmacokinetic experiment of Sil-1

[0063] 1. Sample preparation

[0064] Sil-1 suspension: Weigh an appropriate amount of Sil-1, grind it with 0.5% sodium carboxymethylcellulose (CMC-Na) to the required volume, and mix evenly to obtain;

[0065] Sil-1 injection: Weigh an appropriate amount of Sil-1, dissolve it by ultrasonic treatment with an aqueous solution containing 5% polyethylene glycol 15-hydroxystearate (HS-15), and then make up the volume to the required volume with 0.9% NaCl injection and mix evenly to obtain.

[0066] 2. Test method

[0067] Twelve SPF-grade male SD rats, with body weight (190 - 210) g, were provided by Jinan Pengyue Laboratory Animal Breeding Co., Ltd., production license number: SCXK (Lu) 20220006. All rats were housed in a standard room with 12h / 12h light-dark alternating lighting, at a constant temperature of 23 - 25°C, and a relative humidity of 40 - 60%. The rats were randomly divided into 2 groups according to body weight, with 6 rats in each group. Group 1 was given the compound Sil-1 suspension (concentration: 0.5 mg / mL) by gavage, and Group 2 was given the compound Sil-1 injection (concentration: 0.2 mg / mL) by intravenous injection. The gavage volume for Group 1 was 10 ml / kg, and the intravenous injection volume for Group 2 was 5 ml / kg. After administration, whole blood was collected at 0.083, 0.25, 0.5, 1,​​​​​​​​​脉注射给药 *Extravascular dosing dose). The calculated bioavailability F value of Sil-1 was 3.78%, while that of Silibinin was 0.93% (data from https: / / old.tcmsp-e.com / molecule.php?qn=7450). The bioavailability of Sil-1 was significantly improved, being 3 times higher than that of Silibinin, demonstrating a higher bioavailability.

[0070] Table 1 Pharmacokinetic test data

[0071] Example 2: Effects of Sil-1 on a glucose-oxygen deprivation model in H9c2 cells

[0072] 1. Experimental Materials and Methods

[0073] 1.1 Cell lines

[0074] H9c2 cells were purchased from the Chinese Academy of Sciences Type Culture Collection Committee.

[0075] 1.2 Materials and Reagents

[0076] Sil-1 (provided by Shandong New Era Pharmaceutical Co., Ltd., batch number: Y032230201-DG);

[0077] Silibinin (Sigma-Aldrich, S0417);

[0078] Sugar-free DMEM medium (Gibco, 11966025);

[0079] Cobalt chloride hexahydrate (CoCl2) (Shanghai Test, 10007216);

[0080] CCK-8 reagent kit (Beyotime, C0039);

[0081] Lactate dehydrogenase cytotoxicity assay kit (Beyotime, C0017);

[0082] Reactive oxygen species detection kit (Beyotime, S0033M);

[0083] DAPI staining solution (Beyotime, C1006);

[0084] Rat Tumor Necrosis Factor α (TNF-α) ELISA Detection Kit (Enzyme-Linked Biotechnology, ml002859);

[0085] Rat interleukin-1β (IL-1β) ELISA kit (Enzyme-Linked Biotechnology, ml003057);

[0086] Rat interleukin-6 (IL-6) ELISA kit (Enzyme-Linked Biotechnology, ml102828).

[0087] 1.3 Cultivation and Subculturing of H9c2

[0088] H9c2 cells were cultured in a high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C and 5% CO2. Cells were passaged using 0.25% trypsin containing EDTA and their state was observed using an optical microscope.

[0089] 1.4 Establishment of the Glucose-Oxygen Deprivation Model

[0090] Four groups were set up for cell experiments: control group, model group, silibin group, and sil-1 group. Before glucose-oxygen deprivation and drug administration, cells in the model group, silibin group, and sil-1 group were starved for 24 h using high-glucose DMEM containing 1% fetal bovine serum. Cells in these groups were then cultured for 18 h in glucose-free DMEM containing CoCl2 to establish glucose-oxygen deprivation conditions. The culture media for the silibin and sil-1 groups contained the corresponding concentrations of silibin and sil-1, respectively. To avoid affecting the viability of H9c2 cells, 10 μM was selected as the maximum drug treatment concentration for both drugs, and three concentration groups of 2.5 μM, 5 μM, and 10 μM were also set up.

[0091] 1.5 Detection Indicators

[0092] 1.5.1 Cell State

[0093] Cell status was observed using an optical microscope after glucose-oxygen deprivation and drug treatment.

[0094] 1.5.2 Cell viability assay

[0095] After glucose-oxygen deprivation and drug treatment, cell viability was measured according to the instructions of the CCK-8 kit (Beyotime).

[0096] 1.5.3 Cytotoxicity (LDH) Assay

[0097] After glucose and oxygen deprivation and drug treatment, 1000g of cell culture medium was collected, centrifuged for 5 minutes, and the supernatant was collected. Cytotoxicity was determined according to the instructions of the lactate dehydrogenase cytotoxicity assay kit (Beyotime).

[0098] 1.5.4 Determination of Reactive Oxygen Species (ROS) Levels

[0099] After glucose and oxygen deprivation and drug treatment, the cell culture medium was removed, and the cells were fixed with 4% paraformaldehyde for 30 min. The cells were washed three times with serum-free culture medium, and the ROS level of the adherent cells was measured according to the instructions of the reactive oxygen species detection kit (Beyotime) and DAPI staining solution (Beyotime).

[0100] 1.5.5 Measurement of inflammatory factors in cell culture medium

[0101] The levels of TNF-α, IL-1β, and IL-6 in the cell culture medium were measured according to the instructions provided by the ELISA kit manufacturer.

[0102] 2. Experimental Results and Analysis

[0103] 2.1 Observation of cell state

[0104] As shown in Figure 1, the model group exhibited a large number of suspended dead cells. Compared to the model group, H9c2 cells treated with 10 μM Sil-1 still showed significant cell death, while H9c2 cells treated with 10 μM Sil-1 showed fewer dead cells, a relatively higher cell density, and maintained a relatively healthy spindle shape. These experimental results indicate that Sil-1 is more effective than Silbinin in improving the cell morphology and survival rate of H9c2 cells under glucose-oxygen deprivation conditions.

[0105] 2.2 Cell viability assay

[0106] The results of CCK-8 cell viability detection are shown in Figure 2. The cell viability of the model group cells decreased to about 50% of that of the normal group (P<0.01). Compared with the model group, the cell viability of H9c2 cells was improved by all concentrations of Silibinin and Sil-1, especially the 2.5 μM Sil-1 group, which showed a significant difference compared with the 5 μM Silibinin group (P<0.01), indicating that the efficacy of lower concentrations of Sil-1 was significantly better than that of medium concentrations of Silibinin.

[0107] 2.3 LDH release

[0108] Figure 3 shows the results of LDH level detection in cell culture medium. The LDH release from the model group was significantly increased (P<0.01). Compared with the model group, all Sil-1 concentration groups, the 5 μM Silibinin group, and the 10 μM Silibinin group all reduced LDH release (P<0.01). Compared with 10 μM Silibinin, treatment with 10 μM Sil-1 significantly reduced LDH release (P<0.001). These results indicate that Sil-1 significantly reduced the degree of glucose-oxygen deprivation damage in H9c2 cells, and its effect was better than that of Silibinin.

[0109] 2.4 Intracellular ROS levels in H9c2 cells

[0110] ROS levels in H9c2 cells were detected using the DCFH-DA probe, and the results are shown in Figure 4(A). Under a fluorescence microscope, the model group of H9c2 cells showed significant green fluorescence, indicating a significant increase in intracellular ROS levels. The green fluorescence in H9c2 cells in the 10 μM Silibinin group and the 2.5 μM and 5 μM Sil-1 groups was reduced to some extent compared to the model group, with a significant reduction in the green fluorescence in the 10 μM Sil-1 group. As shown in Figure 4(B) of the relative fluorescence intensity statistics, compared to the model group, the ROS levels in H9c2 cells in the 10 μM Silibinin group and the 2.5 μM Sil-1 group were significantly lower (P<0.05), and the reduction in ROS levels in the 5 μM and 10 μM Sil-1 groups was even more significant (P<0.01), especially in the 10 μM Sil-1 group, which showed a significant difference compared to the 10 μM Silibinin group (P<0.01). The above experimental results show that silbinin and sil-1 can reverse the increase in ROS levels in H9c2 cells in the glucose-oxygen deprivation model and have antioxidant stress activation properties. The advantage of sil-1 in antioxidant stress is more obvious after the introduction of isopentenyl group.

[0111] 2.5 Levels of inflammatory factors in cell culture medium

[0112] The detection results of inflammatory factor levels in cell culture medium after oxygen-glucose deprivation are shown in Figure 5. Compared with the normal group, the level of inflammatory factor TNF-α in the cell culture medium of the model group was significantly increased (P<0.01); the TNF-α levels in the 10 μM Silibinin group and the 2.5 μM Sil-1 group decreased, but there was no significant difference compared with the model group; the TNF-α levels in the 5 μM Sil-1 group and the 10 μM Sil-1 group were significantly decreased compared with the model group, especially in the 5 μM Sil-1 group (P<0.01), and there was a significant difference compared with the Silibinin group (P<0.05). Compared with the normal group, the level of inflammatory factor IL-1β in the cell culture medium of the model group was significantly increased (P<0.05); the IL-1β levels in the 10 μM Silibinin group, the 5 μM Sil-1 group and the 10 μM Sil-1 group were all significantly decreased (P<0.01). Compared with the normal group, the level of inflammatory factor IL-6 in the cell culture medium of the model group was significantly increased (P<0.01); the IL-6 levels in the 10 μM Silibinin group and the 10 μM Sil-1 group were both significantly decreased (P<0.05), and the decrease amplitude in the 5 μM Sil-1 group was greater (P<0.01), and there was a significant difference compared with the Silibinin group (P<0.05). The above experimental results show that Sil-1 has anti-inflammatory activity, and its anti-inflammatory activity is higher than that of Silibinin.

[0113] Example 3 Pharmacodynamic study of Sil-1 on rats with acute myocardial infarction model induced by ligation of the left anterior descending coronary artery

[0114] 1. Experimental materials and methods

[0115] 1.1 Experimental animals

[0116] 72 SPF-grade male SD rats, weighing 190 - 210 g, were provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd., license number: SCXK(Shandong)20220006. The rats were housed in a SPF-grade animal breeding room with strictly controlled environmental conditions, temperature 20 - 26 °C, humidity 40 - 70%, light-dark cycle 12:12 h, fed with growth and reproduction feed, and allowed free access to food and water.

[0117] 1.2 Main reagents

[0118] Sil-1 (provided by Shandong New Times Pharmaceutical Co., Ltd., batch number: Y032230201-DG);

[0119] Silibinin (Sigma-Aldrich, S0417);

[0120] 2,3,5-Triphenyltetrazolium chloride (TTC) (Sigma-Aldrich, T8877);

[0121] Rat Tumor Necrosis Factor α (TNF-α) ELISA Detection Kit (Enzyme-Linked Biotechnology, ml002859);

[0122] Rat interleukin-1β (IL-1β) ELISA kit (Enzyme-Linked Biotechnology, ml003057);

[0123] Rat interleukin-6 (IL-6) ELISA kit (Enzyme-Linked Biotechnology, ml102828).

[0124] 1.3 Animal Model Making and Grouping

[0125] Seventy-two SD rats were randomly divided into six groups: normal group (C), model group (M), silymarin group (Y), low-dose Sil-1 group (L), medium-dose Sil-1 group (M), and high-dose Sil-1 group (H), with 12 rats in each group.

[0126] An acute myocardial infarction model in rats was established by ligation of the left anterior descending coronary artery (LAD). Preoperatively, rats were weighed, anesthetized, and fixed in a supine position. The skin of the left chest was disinfected and prepared, followed by endotracheal intubation and connection to a ventilator. An incision was made between the 3rd and 4th ribs on the left side of the sternum, and blunt dissection was performed layer by layer. The ribs were dissected along the intercostal spaces, with an incision length of approximately 1.0–2.0 cm. The pericardium was opened to expose the heart. A chest dilator was used to fully expose the heart. Using a 7-0 suture needle with sutures, the left anterior descending coronary artery was ligated 2 mm below the junction of the pulmonary conus and the left atrial appendage, with a needle depth of approximately 2 mm. Pneumothorax and effusion were manually expelled, the chest was closed, and the drainage tube was removed. In the normal group, only puncture was performed without ligation. Rats in group Y were injected with 3 mg / kg of silibin via the tail vein 30 minutes before surgery. Rats in groups L, M, and H were injected with 1 mg / kg, 3 mg / kg, and 10 mg / kg of sil-1 via the tail vein 30 minutes before surgery, respectively. Rats in group C underwent only thoracotomy and suture embedding without ligation (sham surgery). Both silibin and sil-1 were prepared by ultrasonic dissolution of 5% HS15 aqueous solution followed by addition of 0.9% sodium chloride injection solution, with an administration volume of 10 ml / kg. The normal group and the model group received the same dose of solvent via the tail vein.

[0127] 1.4 Statistical Processing

[0128] Experimental data are expressed as mean ± standard deviation. Statistical analysis was performed. One-way ANOVA was used for comparisons between groups, and multi-way ANOVA was used for repeated measures data. A p-value < 0.05 was considered statistically significant.

[0129] 1.5 Detection of Pharmacodynamic Indicators

[0130] 1.5.1 Electrocardiogram (ECG) testing

[0131] Twenty-four hours after the ligation of the left anterior descending coronary artery, the rats were anesthetized and fixed in a supine position. The subcutaneous tissue of both upper and lower limbs of the rats was punctured with needles, and electrocardiogram electrodes were connected to record standard lead II electrocardiograms to analyze the degree of ST segment elevation in the rats.

[0132] 1.5.2 Detection of myocardial enzyme levels

[0133] After completing the electrocardiogram test, the rats were dissected, and blood was collected from the abdominal venous duct. The serum was collected after centrifugation at 3000 r / min for 15 min. The levels of creatine kinase (CK), creatine kinase isoenzyme (CK-MB), and lactate dehydrogenase (LDH) in the rat serum were detected using a fully automated biochemical analyzer.

[0134] 1.5.3 Detection of inflammatory factor levels

[0135] Blood was collected from the abdominal vein, centrifuged, and serum was collected. The levels of TNF-α, IL-1β, and IL-6 in rat serum were detected according to the instructions provided by the ELISA kit manufacturer.

[0136] 1.5.4 Infarct Area Detection

[0137] After freezing rat hearts at -20°C for 24 hours, they were cut into 8-12 sections according to heart size. The sections were stained with 2% TTC solution at 37°C for 30 minutes and then photographed. The proportion of infarct area was analyzed using ImageJ.

[0138] 1.5.5 Observation of myocardial tissue pathological morphology

[0139] The remaining cardiac tissues fixed in paraformaldehyde and embedded in paraffin were cut into 4μm thick sections, dewaxed and hydrated, and stained with hematoxylin-eosin reagent. The structural changes of myocardial tissue were observed under a light microscope.

[0140] 2. Experimental Results

[0141] 2.1 Electrocardiogram (ECG) testing

[0142] As shown in Figure 6, the electrocardiogram (ECG) results indicated that 24 hours after ligation of the left anterior descending coronary artery, the model group rats exhibited tall T waves on their ECGs, suggesting acute myocardial infarction. The trend of tall T waves on the ECGs of rats in the low, medium, and high dose Sil-1 groups was reduced. Compared with the model group, the reduction in T wave potential in the medium and high dose Sil-1 groups was significantly different (P<0.01).

[0143] 2.2 Myocardial enzyme levels

[0144] Figure 7 shows the detection of myocardial enzyme levels in rats. Twenty-four hours after ligation of the left anterior descending coronary artery, serum CK levels in the model group were significantly higher than those in the normal group (P<0.01). Compared with the model group, serum CK levels in the Silibinin group and the low-dose Sil-1 group were significantly lower (P<0.05). The medium- and high-dose Sil-1 groups showed highly significant differences in reducing serum CK levels (P<0.01), with the medium-dose Sil-1 group showing the best effect and a significant difference compared to the Silibinin group (P<0.05). Serum CK-MB levels were also detected. Compared with the normal group, serum CK-MB levels in the model group were significantly higher (P<0.01). Compared with the model group, the Silibinin group and the low, medium, and high-dose Sil-1 groups showed highly significant differences in reducing serum CK-MB levels (P<0.01), especially the medium-dose Sil-1 group, which showed a significant difference compared to the Silibinin group (P<0.05). Serum LDH levels were detected, revealing a significant increase in the model group compared to the normal group (P<0.01). Compared to the model group, the silibin group and the low-dose Sil-1 group significantly reduced serum LDH levels (P<0.05), while the medium- and high-dose Sil-1 groups significantly reduced serum LDH levels (P<0.01). Furthermore, the medium-dose Sil-1 group showed a significant difference compared to the silibin group (P<0.05). These results indicate that Sil-1 can more effectively reduce the degree of cardiomyocyte damage and necrosis during acute myocardial infarction in rats, providing effective protection for cardiomyocytes.

[0145] 2.3 Inflammatory factor levels

[0146] The ELISA results are shown in Figure 8. Twenty-four hours after ligation of the left anterior descending coronary artery, the serum TNF-α level in the model group rats was significantly increased compared to the normal group (P<0.05). Compared to the model group, the silbinin group and the low-dose Sil-1 group significantly inhibited the increase in TNF-α levels (P<0.05). The medium- and high-dose Sil-1 groups showed highly significant differences in inhibiting the increase in TNF-α levels (P<0.01), especially the medium-dose Sil-1 group, which showed a significant difference compared to the silbinin group (P<0.05). By detecting the serum IL-1β level in rats, it was found that the serum IL-1β level in the model group was significantly increased compared with the normal group (P<0.01). Compared with the model group, the silbinin group and the low-dose sil-1 group significantly inhibited the increase of IL-1β level (P<0.05). The medium- and high-dose sil-1 groups showed extremely significant differences in inhibiting the increase of IL-1β level (P<0.01), especially the medium-dose sil-1 group, which showed a significant difference compared with the silbinin group (P<0.05). Serum IL-6 levels in rats were measured, revealing a significant increase in IL-6 levels compared to the normal group (P<0.01). Compared to the model group, the silibin group and the low- and high-dose Sil-1 groups significantly inhibited the rise in IL-6 levels (P<0.05). The medium-dose Sil-1 group showed a highly significant difference in inhibiting the rise in IL-6 levels (P<0.01), and a significant difference compared to the silibin group (P<0.01). These results indicate that Sil-1 can effectively alleviate the inflammatory response of cardiomyocytes during acute myocardial infarction in rats, and more effectively reverses the upward trend of inflammatory factors than silibin.

[0147] 2.4 Infarct area (TTC staining)

[0148] The TTC staining results are shown in Figure 9(A). Compared with the normal group, the model group showed obvious light-colored infarct areas in the cardiac slices; compared with the model group, the light-colored infarct areas in the cardiac slices of each treatment group were significantly reduced. The analysis results of the infarct area are shown in Figure 9(B). Compared with the model group, the proportion of infarct area in each treatment group was significantly reduced (P<0.01). Compared with the Silibinin group, the medium-dose Sil-1 group showed a significant difference in reducing the proportion of infarct area (P<0.01).

[0149] 2.5 Pathological morphology of myocardial tissue (HE staining)

[0150] The HE staining results are shown in Figure 10. Compared with the normal group, the intercellular spaces of cardiomyocytes in the model group were enlarged, and inflammatory factor infiltration was observed between cardiomyocytes. Low, medium, and high doses of Sil-1 significantly reduced the intercellular spaces of cardiomyocytes and decreased inflammatory factor infiltration compared with the Silibinin group.

[0151] In summary, at the cellular level, Sil-1 can improve the viability of cardiomyocytes under glucose-oxygen desquamation conditions, reduce LDH release levels, inhibit ROS elevation, and suppress the release of inflammatory factors, demonstrating definite anti-inflammatory and antioxidant activities. In animal models, Sil-1 can significantly reduce ST segment elevation on electrocardiograms after acute myocardial infarction in rats, reduce the infarct size, decrease serum levels of myocardial enzymes and inflammatory factors, improve myocardial tissue pathological morphology, and inhibit myocardial tissue inflammatory factor infiltration. Compared with silymarin, Sil-1 has significant advantages in the treatment of acute myocardial infarction.

[0152] The above embodiments are only used to further illustrate the technical solutions of the present invention in detail, and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention shall fall within the scope of protection of the present invention.

Claims

1. Compounds of Formula I and their pharmaceutically acceptable meso, racemic, enantiomers, and diastereomers:

2. A pharmaceutical composition comprising a compound of Formula I as claimed in claim 1, or a racemic, racemic, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutical excipient.

3. Use of the compound of Formula I as claimed in claim 1, or a racemic, racemic, enantiomer, diastereomer, or mixture of its isomers, or a pharmaceutically acceptable salt thereof, or a composition as claimed in claim 2, in the preparation of a medicament for the prevention and / or treatment of ischemic heart disease.

4. The use as described in claim 3, characterized in that, The ischemic heart disease refers to ischemic myocardial injury or myocardial infarction.

5. The use as described in claim 3, characterized in that, The ischemic heart disease is myocardial infarction, preferably acute myocardial infarction.

6. The composition as described in claim 2 and the use as described in claim 3, characterized in that, The drug in question is one that reduces cardiomyocyte apoptosis.

7. The composition as described in claim 2 and the use as described in claim 3, characterized in that, The drug in question is used to reduce the area of ​​myocardial infarction.

8. The composition as described in claim 2 and the use as described in claim 3, characterized in that, The drug in question is one that lowers serum creatine kinase.

9. The composition as described in claim 2 and the use as described in claim 3, characterized in that, The drug in question is used to reduce cardiac inflammation caused by myocardial infarction.

10. The composition as claimed in claim 2 and the use as claimed in claim 3, characterized in that, The drug is used to prevent and treat ischemic myocardial injury.

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