20(S)-Ginsenoside Rg2 amino acid ester derivative, its preparation method and uses

Abstract

This invention provides a 20(S)-ginsenoside Rg2 amino acid ester derivative, its preparation method, and its uses, belonging to the pharmaceutical field. This invention relates to its application in drugs for treating shock diseases. In vitro cell experiments show that the compound of this invention can not only improve the survival rate of hypoxic-damaged cardiomyocytes, inhibit the increase of LDH, MDA, TNF-α, and IL-6 levels in hypoxic-damaged cells, and increase SOD activity, but also promote the expression of p-PI3K and p-Akt in hypoxic-damaged cells, exhibiting a strong protective effect against hypoxic cardiomyocytes. This invention can also increase blood pressure in shock model rats, inhibit the increase of heart rate, reduce lactate content to improve tissue perfusion, and reduce PCO2 content while increasing HCO3 content. ‑ It reduced the levels of TCO2 and BE, corrected the acid-base balance in the body, significantly reduced the content of MDA, LDH, TNF-α and IL-6, increased the activity of SOD, inhibited oxidative stress and inflammatory response, and showed good anti-shock biological activity.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to a method for preparing and using a 20(S)-ginsenoside Rg2 amino acid ester derivative. Background Technology

[0002] Hemorrhagic shock (HS) is a type of shock caused by a sharp decrease in effective circulating blood volume due to the loss of whole blood or plasma. It is the most common type of shock. With a mortality rate as high as 40%, hemorrhagic shock is a serious threat to human life. When hemorrhagic shock occurs, the rapid decrease in effective circulating blood volume alters hemodynamics, leading to microcirculatory disturbances. This results in decreased blood perfusion and hypoxia in various tissues and organs, increasing anaerobic metabolism and lactic acid levels, leading to acidosis. Furthermore, the balance between oxidation and antioxidation is disrupted, causing the accumulation of oxygen free radicals. These free radicals can react with various cellular components, causing acute or chronic damage to tissues and cells. In addition, the ischemic and hypoxic state of various tissues and organs triggers immune dysfunction, releasing large amounts of inflammatory mediators and cytokines, thus initiating systemic inflammatory response syndrome (SIRS) and exacerbating damage to various tissues and organs, potentially leading to multiple organ dysfunction syndrome (MODS). It is evident that the pathophysiological mechanism of hemorrhagic shock is extremely complex, involving multiple systemic responses such as microcirculatory disturbances, oxidative stress, inflammatory responses, and apoptosis.

[0003] There are currently no specific drugs for treating shock in clinical practice. Instead, appropriate drug treatment is required based on the type and stage of shock, and the timing and dosage of medication must be carefully controlled to avoid worsening the condition. Currently, drugs commonly used in clinical practice to treat shock can be classified into several types according to their mechanisms of action, including volume expanders, vasoconstrictors, vasodilators, myocardial contractility enhancers, and cytoprotective agents. However, each type of drug has certain limitations. Volume expanders can quickly replenish effective circulating blood volume to normal levels, but they are prone to causing tissue edema and are often accompanied by renal dysfunction and coagulation abnormalities. Vasoconstrictors can increase blood pressure to increase blood flow to vital organs, but they increase peripheral vascular resistance and cardiac afterload, exacerbating microcirculatory disturbances. Vasodilates can reduce peripheral vascular resistance, thereby improving microcirculation and tissue perfusion, but they can cause a drop in blood pressure. Myocardial contractility enhancers can improve cardiac output and stroke volume while increasing myocardial contractility, but they also increase myocardial oxygen consumption, exacerbating myocardial damage. Cytoprotective agents have the effects of scavenging free radicals, reducing the release of inflammatory factors, and inhibiting cell necrosis, mainly targeting secondary cellular metabolic disorders following shock. All types of shock treatments have certain limitations; therefore, it is essential to research an anti-shock drug with broad pharmacological effects and few side effects.

[0004] 20(S)-Ginsenoside Rg2 (20(S)-Ginsenoside Rg2, 20(S)-Rg2) is a triol-type ginsenoside. Modern pharmacological studies have shown that 20(S)-Rg2 not only has strong effects in improving cardiovascular function and antioxidation, but also reduces myocardial oxygen consumption index, oxygen uptake rate, and peripheral resistance, making it a potential compound for treating shock. However, 20(S)-Rg2 has poor solubility and does not easily cross absorption barriers and phospholipid bilayer structures, leading to difficulty in oral absorption and limited direct clinical application. Therefore, designing drug structures with suitable solubility and dissociation is crucial for improving the drug-likeness of ginsenosides. Amino acids contain amino (-NH2), carboxyl (-COOH), and side-chain functional groups, giving them amphiphilic properties. Studies have shown that introducing amino acid groups into the structure modification of natural products can not only increase the permeability and solubility of the modified products, but also improve the efficacy, selectivity, and reduce toxicity of the modified products due to the diversity of amino acid side-chain functional groups. Summary of the Invention

[0005] This invention provides a 20(S)-ginsenoside Rg2 amino acid ester derivative, its preparation method, and its uses. By selecting Fmoc-amino acids to modify the structure of 20(S)-Rg2 and synthesizing the 20(S)-Rg2 amino acid ester derivative, the anti-shock activity of 20(S)-Rg2 is improved, providing a candidate compound library for further development of anti-shock drugs.

[0006] The technical solution adopted in this invention is:

[0007] 20(S)-Ginsenoside Rg2 amino acid ester derivatives and their pharmaceutically acceptable salts or solvates have the following structures:

[0008]

[0009] Wherein: R is selected from Fmoc-aminoacyloxy or aminoacyloxy;

[0010] Furthermore, Fmoc is a fluorenemethyloxycarbonyl group;

[0011] Furthermore, the Fmoc-aminoacyloxy groups mentioned above include the following eight types: Fmoc-glycyloxy, Fmoc-leucyloxy, Fmoc-isoleucyloxy, Fmoc-valineyloxy, Fmoc-methionyloxy, Fmoc-tryptophanyloxy, Fmoc-threonyloxy, and Fmoc-seryloxy.

[0012] Furthermore, the amino acyloxy groups mentioned above include the following eight types: glycyloxy, leucyloxy, isoleucyloxy, valine acyloxy, methionyloxy, tryptophan acyloxy, threonyloxy, and serine acyloxy.

[0013] The compounds described in this invention include the following compounds: 6-O-[6'-O-fluorenylmethoxycarbonylglycyl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglycosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol;

[0014] 6-O-[6'-O-fluorenylmethoxycarbonylleucyl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol;

[0015] 6-O-[6'-O-fluorenylmethoxycarbonylisoleucyl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucopyranosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol;

[0016] 6-O-[6'-O-fluorenylmethoxycarbonylvaline-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglycosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol;

[0017] 6-O-[6'-O-fluorenylmethoxycarbonylmethylthioyl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglycosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol;

[0018] 6-O-[6'-O-fluorenylmethoxycarbonyltryptamine-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol;

[0019] 6-O-[6'-O-fluorenylmethoxycarbonylthreonyl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol;

[0020] 6-O-[6'-O-fluorenylmethoxycarbonylserine-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucopyranosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol;

[0021] 6-O-[6'-O-glycyl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucopyranosyl]-damar-24-en-3β,6α,12β,20S-tetraol; 6-O-[6'-O-leucyl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucopyranosyl]-damar-24-en-3β,6α,12β,20S-tetraol; 6-O-[6'-O-isoleucyl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucopyranosyl]-damar-24-en-3β,6α,12β,20S-tetraol;

[0022] 6-O-[6'-O-valine-α-L-pyrano-rhamnosyl-(1,2)-β-D-pyrano-glucopyranosyl]-damar-24-ene-3β,6α,12β,20S-tetraol; 6-O-[6'-O-methionyl-α-L-pyrano-rhamnosyl-(1,2)-β-D-pyrano-glucopyranosyl]-damar-24-ene-3β,6α,12β,20S-tetraol;

[0023] 6-O-[6'-O-tryptophanyl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucopyranosyl]-damar-24-en-3β,6α,12β,20S-tetraol; 6-O-[6'-O-threonyl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucopyranosyl]-damar-24-en-3β,6α,12β,20S-tetraol; 6-O-[6'-O-seryl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucopyranosyl]-damar-24-en-3β,6α,12β,20S-tetraol;

[0024] And its pharmaceutically acceptable salts or solvates.

[0025] The preparation method of the 20(S)-ginsenoside Rg2 amino acid ester derivative of the present invention is as follows:

[0026] 20(S)-Rg2, Fmoc amino acids, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N,N-diisopropylethylamine (DIPEA), and 4-dimethylaminopyridine (DMAP) were added sequentially to anhydrous tetrahydrofuran in a ratio of 1:2:5:2:0.8. The mixture was shaken to dissolve the compounds, and the reaction was carried out in an ice-water bath for 8 hours. The solvent was then removed from the reaction solution under reduced pressure and dried. The residue was dissolved in dichloromethane, and then washed thoroughly with water and saturated NaCl solution, followed by drying with Na2SO4. After filtration, the crude product was concentrated and purified by column chromatography to obtain 20(S)-ginsenosides. The Fmoc-amino acid ester derivative of Rg2 was obtained using a dichloromethane:methanol:ethyl acetate:water ratio of 4:1.3:4:1 as the eluent. The 20(S)-Rg2-Fmoc-amino acid ester derivative was dissolved in a dichloromethane solution containing 10% piperidine (v / v). The reaction was carried out at room temperature for 1 hour. After the reaction was completed, the solvent was removed from the reaction solution under reduced pressure and dried to obtain the crude product. The crude product was purified by column chromatography to obtain the amino acid ester derivative of 20(S)-ginsenoside Rg2, using a dichloromethane:methanol:ethyl acetate:water ratio of 4:2:4:1 or a dichloromethane:methanol:ethyl acetate:water ratio of 2:2:7:0.2. The reaction formula is as follows:

[0027]

[0028] This invention provides a method for synthesizing amino acid ester derivatives using 20(S)-ginsenoside Rg2 as a lead compound and Fmoc-amino acid as a modifying group, and an evaluation of its anti-shock activity.

[0029] A pharmaceutical composition comprising at least 20(S)-ginsenoside Rg2 amino acid ester derivative or its pharmaceutically acceptable salt or hydrate.

[0030] A pharmaceutical preparation comprising an active ingredient and pharmaceutically acceptable excipients and / or carriers, said active ingredient comprising at least a 20(S)-ginsenoside Rg2 amino acid ester derivative or a pharmaceutically acceptable salt or hydrate thereof.

[0031] The application of the 20(S)-ginsenoside Rg2 amino acid ester derivative described in this invention in the preparation of a drug for treating cellular hypoxic injury.

[0032] The application of the 20(S)-ginsenoside Rg2 amino acid ester derivative described in this invention in the preparation of a drug for treating shock.

[0033] The use of the pharmaceutical composition described in this invention in the preparation of a medicament for treating cellular hypoxic injury.

[0034] The use of the pharmaceutical composition described in this invention in the preparation of a medicament for treating shock.

[0035] The application of the pharmaceutical preparation described in this invention in the preparation of a drug for treating cellular hypoxic injury.

[0036] The application of the pharmaceutical preparation described in this invention in the preparation of a drug for treating shock.

[0037] The beneficial effects of this invention are as follows:

[0038] 1. This invention provides a 20(S)-ginsenoside Rg2 amino acid ester derivative or a pharmaceutically acceptable salt thereof. The 20(S)-ginsenoside Rg2 amino acid ester derivative of this invention has a novel structure, a simple synthesis method, and high product purity, and has good application prospects.

[0039] 2. In vitro experiments on the protection against hypoxic injury to cardiomyocytes showed that the 20(S)-ginsenoside Rg2 amino acid ester derivative provided by this invention exhibited a significant protective effect against hypoxic-injured cardiomyocytes. Specifically, the 20(S)-ginsenoside Rg2 glycine ester derivative showed significantly stronger anti-hypoxic activity than the lead compound 20(S)-ginsenoside Rg2, and its effect was close to that of the positive control drug. Further studies on the anti-hypoxic activity of the 20(S)-ginsenoside Rg2 glycine ester derivative showed that it possesses multiple pharmacological effects, including inhibiting the increase of LNH and MDA, increasing SOD activity to reduce oxidative stress damage, inhibiting the increase of TNF-α and IL-6 to alleviate inflammatory damage, and promoting the expression of p-PI3K and p-Akt to activate the PI3K / Akt signaling pathway.

[0040] 3. In vivo anti-shock activity experiments showed that the 20(S)-ginsenoside Rg2 glycine ester derivative provided by this invention has a significant protective effect on shock model rats. It can increase the blood pressure of rats after shock, inhibit the abnormal increase of heart rate, reduce the content of lactic acid in the blood, correct disordered blood gas indicators, and also inhibit oxidative stress and inflammatory response, showing good anti-shock biological activity. Attached Figure Description

[0041] Figure 1 This is a graph showing the effect of compound 1 on changes in LDH content in hypoxic-damaged cardiomyocytes;

[0042] Figure 2 This is a graph showing the effect of compound 1 on changes in MDA content in hypoxic-injured cardiomyocytes;

[0043] Figure 3 This is a graph showing the effect of compound 1 on the changes in SOD content in hypoxic-damaged cardiomyocytes;

[0044] Figure 4 This is a graph showing the effect of compound 1 on the changes in TNF-α content in hypoxic-injured cardiomyocytes;

[0045] Figure 5This is a graph showing the effect of compound 1 on changes in IL-6 levels in hypoxic-injured cardiomyocytes;

[0046] Figure 6 This is a graph showing the effect of compound 1 on the changes in p-PI3K content in hypoxic-injured cardiomyocytes;

[0047] Figure 7 This is a graph showing the effect of compound 1 on the changes in p-Akt content in hypoxic-injured cardiomyocytes;

[0048] Figure 8 This is a graph showing the effect of compound 1 on changes in blood lactate levels in shock model rats;

[0049] Figure 9 This is a graph showing the effect of compound 1 on changes in blood pH in a shock model rat.

[0050] Figure 10 This is a graph showing the effect of compound 1 on changes in PCO2 in the blood of shock model rats;

[0051] Figure 11 Compound 1 affects the level of HCO3- in the blood of shock model rats. - Impact diagram of changes;

[0052] Figure 12 This is a graph showing the effect of compound 1 on changes in TCO2 in the blood of shock model rats;

[0053] Figure 13 This is a graph showing the effect of compound 1 on changes in BE levels in the blood of shock model rats;

[0054] Figure 14 This is a graph showing the effect of compound 1 on changes in PO2 in the blood of shock model rats;

[0055] Figure 15 This is a graph showing the effect of compound 1 on changes in SO2 levels in the blood of shock model rats;

[0056] Figure 16 This is a graph showing the effect of compound 1 on changes in LDH levels in the plasma of rats with shock.

[0057] Figure 17 This is a graph showing the effect of compound 1 on changes in MDA content in the plasma of shock model rats;

[0058] Figure 18 This is a graph showing the effect of compound 1 on the changes in SOD content in the plasma of shock model rats;

[0059] Figure 19 This is a graph showing the effect of compound 1 on changes in plasma TNF-α levels in shock model rats;

[0060] Figure 20This is a graph showing the effect of compound 1 on changes in IL-6 levels in the plasma of shock model rats. Detailed Implementation

[0061] The present invention will be further illustrated below with specific examples, but the present invention is not limited to these embodiments.

[0062] Example 1: Synthesis of 6-O-[6'-O-fluorenylmethoxycarbonylglycyl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglycosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (Intermediate 1)

[0063]

[0064] 20(S)-Rg2, Fmoc-glycine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N,N-diisopropylethylamine (DIPEA), and 4-dimethylaminopyridine (DMAP) were dissolved in anhydrous tetrahydrofuran in a ratio of 1:2:5:2:0.8. The mixture was shaken to dissolve and reacted in an ice-water bath for 8 hours. The solvent was recovered under reduced pressure and dried. The residue was dissolved in dichloromethane and then washed thoroughly with water and saturated NaCl solution. Na2SO4 was added for drying. The mixture was filtered and concentrated to obtain a crude product. The crude product was purified by column chromatography to obtain the Fmoc-glycine ester derivative of 20(S)-ginsenoside Rg2. The eluent used was dichloromethane:methanol:ethyl acetate:water = 4:1.3:4:1.

[0065] MS(m / z): [M+Na] + =1086.5699.

[0066] 1 H-NMR (400MHz, C5D5N): δ8.87(1H,t), 7.59(2H,d), 7.46(2H,d), 7.16(2H,t), 7.04(2H,t), 1.86(3 H, s), 1.56 (3H, d), 1.37 (6H, s), 1.15 (3H, s), 1.09 (3H, s), 1.04 (3H, s), 0.80 (3H, s), 0.77 (3H, s).

[0067] 13CNMR (100MHz, C5D5N): δ171.29, 157.82, 144.75, 141.82, 130.83, 128.20, 127.64, 126.48, 125.83, 120. 54, 102.22, 101.60, 79.15, 78.54, 78.40, 75.48, 74.28, 73.96, 73.11, 72.54, 72.41, 72.29, 71.16, 69.5 6, 67.06, 65.83, 60.83, 54.97, 51.82, 49.89, 48.43, 47.84, 46.28, 43.34, 41.37, 40.05, 39.77, 39.50, 35.75, 32.25, 32.14, 30.10, 27.87, 27.11, 26.92, 25.90, 23.07, 18.85, 17.8, 17.72, 17.54, 17.31, 17.17.

[0068] Example 2: Synthesis of 6-O-[6'-O-fluorenemethoxycarbonylleucyl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (Intermediate 2)

[0069]

[0070] Using 20(S)-Rg2 and Fmoc-leucine as raw materials, and with other operations the same as in Example 1, a Fmoc-leucine ester derivative of 20(S)-ginsenoside Rg2 was obtained.

[0071] MS(m / z): [M+Na] + =1142.6331.

[0072] 1 H-NMR (400MHz, C5D5N): δ9.01 (1H, d), 7.69 (2H, d), 7.62 (2H, d), 7.32 (2H, t), 7.20 (2H, t), 2.03 (3H, s), 1.73 (3 H, d), 1.58 (3H, s), 1.55 (3H, s), 1.34 (3H, s), 1.28 (6H, s), 1.03 (3H, s), 0.95 (3H, s), 0.92 (3H, d), 0.86 (3H, d).

[0073] 13CNMR (100MHz, C5D5N): δ174.04, 157.24, 144.63, 144.28, 141.53, 130.51, 127.87, 127.33, 126.17, 125.51, 120 .22, 101.88, 101.24, 78.87, 78.15, 75.59, 73.99, 73.29, 72.81, 72.22, 72.08, 71.96, 70.89, 69.20, 66.41, 65.6 6, 60.58, 54.60, 53.39, 51.55, 49.57, 48.16, 47.66, 46.26, 41.08, 40.93, 39.77, 39.49, 39.21, 35.58, 32.02, 31.96, 31.3, 27.58, 26.85, 26.72, 25.66, 24.93, 23.20, 22.78, 21.29, 18.55, 17.72, 17.52, 17.45, 17.38, 16.86.

[0074] Example 3: Synthesis of 6-O-[6'-O-fluorenemethoxycarbonylisoleucyl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (Intermediate 3)

[0075]

[0076] Using 20(S)-Rg2 and Fmoc-isoleucine as raw materials, and with other operations the same as in Example 1, a Fmoc-isoleucine ester derivative of 20(S)-ginsenoside Rg2 was obtained.

[0077] MS(m / z): [M+Na] + =1142.4325.

[0078] 1 H-NMR (400MHz, C5D5N): δ8.91 (1H, d), 7.76 (1H, d), 7.74 (1H, d), 7.61 (1 H, d), 7.60 (1H, d), 7.33 (1H, t), 7.31 (1H, t), 7.22 (1H, t), 7.21 (1H, t), 2.02(3H,s), 1.73(3H,d), 1.71(3H,s), 1.57(3H,s), 1.33(3H,s), 1.27( 3H, s), 1.06 (3H, s), 1.04 (3H, s), 1.00 (3H, d), 0.96 (3H, d), 0.83 (3H, s).

[0079] 13CNMR (100MHz, C5D5N): δ172.78, 157.25, 144.6, 144.28, 141.52, 130.53, 127.87, 127.33, 126.13, 125.53, 125.49, 1 20.22, 120.20, 101.84, 101.31, 78.92, 78.20, 78.14, 75.45, 73.98, 73.56, 72.82, 72.23, 72.08, 71.84, 70.90, 69.2 5, 66.47, 65.19, 60.60, 59.72, 54.58, 51.57, 49.59, 48.15, 47.67, 46.04, 41.06, 39.77, 39.48, 39.20, 37.68, 35.63, 32.01, 31.94, 31.31, 27.58, 26.86, 26.76, 25.63, 25.48, 22.79, 18.58, 17.51, 17.44, 17.19, 16.88, 16.11, 11.40.

[0080] Example 4: Synthesis of 6-O-[6'-O-fluorenylmethoxycarbonylvaline-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (intermediate 4)

[0081]

[0082] Using 20(S)-Rg2 and Fmoc-valine as raw materials, and with other operations the same as in Example 1, a Fmoc-valine ester derivative of 20(S)-ginsenoside Rg2 was obtained.

[0083] MS(m / z): [M+Na] + =1128.6172.

[0084] 1 H-NMR (400MHz, C5D5N): δ8.94(1H,d), 7.76(1H,d), 7.75(1H,d), 7.61(1H,d), 7.60(1H,d), 7.33(1H,t), 7.31(1H,t), 7.20(2H,t), 2.02(3 H, s), 1.73 (3H, d), 1.56 (3H, s), 1.53 (3H, s), 1.33 (3H, s), 1.28 (3H, s), 1.25 (3H, s), 1.06 (3H, d), 1.04 (3H, d), 1.00 (3H, s), 0.89 (3H, s).

[0085] 13CNMR (100MHz, C5D5N): δ172.75, 157.38, 144.58, 144.31, 141.52, 130.52, 127.88, 127.33, 126.14, 125.54, 125.4 9. 120.23, 101.85, 101.20, 78.95, 78.25, 78.16, 75.39, 73.99, 73.48, 72.82, 72.25, 72.11, 71.81, 70.90, 69.27, 66.51, 65.34, 60.61, 60.47, 54.59, 51.57, 49.59, 48.16, 47.67, 45.95, 41.05, 39.77, 39.48, 39.19, 35.64, 31.98, 31.92, 31.30, 29.8, 27.58, 26.87, 26.74, 25.62, 22.79, 19.54, 18.60, 18.15, 17.50, 17.44, 17.41, 17.15, 16.87.

[0086] Example 5: Synthesis of 6-O-[6'-O-fluorenylmethoxycarbonylmethylthioyl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (intermediate 5)

[0087]

[0088] Using 20(S)-Rg2 and Fmoc-methionine as raw materials, and with other operations the same as in Example 1, a Fmoc-methionine ester derivative of 20(S)-ginsenoside Rg2 was obtained.

[0089] MS(m / z): [M+Na] + =1160.5933.

[0090] 1 H-NMR (400MHz, C5D5N): δ9.10 (1H, d), 7.75 (2H, d), 7.61 (2H, d), 7.32 (2H, t), 7.21 (2H, t), 2.02 (3H, s), 1.99 (3 H, s), 1.72 (3H, d), 1.58 (3H, s), 1.56 (3H, s), 1.33 (3H, s), 1.28 (3H, s), 1.25 (3H, s), 1.01 (3H, s), 0.95 (3H, s).

[0091] 13CNMR (100MHz, C5D5N): δ173.02, 157.2, 144.57, 144.27, 141.53, 130.54, 127.89, 127.34, 126.20, 125.50, 120. 22, 101.87, 101.2, 78.84, 78.26, 78.15, 75.41, 73.98, 73.31, 72.83, 72.22, 72.09, 71.88, 70.89, 69.22, 66.45 65.65, 60.58, 54.66, 54.09, 51.54, 49.58, 48.15, 47.64, 46.19, 41.07, 39.76, 39.47, 39.20, 35.52, 31.97, 31.91, 31.24, 30.67, 29.81, 27.58, 26.82, 26.71, 25.65, 22.79, 18.55, 17.61, 17.53, 17.44, 17.20, 16.89, 15.12.

[0092] Example 6: Synthesis of 6-O-[6'-O-fluorenylmethoxycarbonyltryptophanyl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (intermediate 6)

[0093]

[0094] Using 20(S)-Rg2 and Fmoc-tryptophan as raw materials, and with other operations the same as in Example 1, a Fmoc-tryptophan ester derivative of 20(S)-ginsenoside Rg2 was obtained.

[0095] MS(m / z): [M+K] + =1231.0535.

[0096] 1 H-NMR (400MHz, C5D5N): δ11.81 (1H, s), 8.98 (1H, m), 7.93 (1H, d), 7.91 (1H, d), 7.74(1H,d), 7.73(1H,d), 7.56(1H,m), 7.54(2H,m), 7.47(1H,m), 7.46(1H,m), 7.43(1H,m), 7.30(1H,t), 7.28(1H,t), 7.23(1H,m), 2.03(3H,s), 1.73(3H,d), 1.53(6H,s), 1.30(3H,s), 1.27(3H,s), 1.12(3H,s), 0.98(3H,s), 0.95(3H,s).

[0097] 13 CNMR (100MHz, C5D5N): δ173.03, 156.96, 144.41, 141.46, 137.42, 130.48, 128.40, 127.84, 127.32, 126.19, 125.55, 124. 21, 121.71, 120.17, 119.30, 119.02, 111.91, 110.65, 101.87, 101.2, 78.92, 78.41, 78.15, 75.38, 74.01, 73.46, 72.82, 7 2.26, 72.11, 71.89, 70.91, 69.27, 66.62, 65.51, 60.60, 56.04, 54.66, 51.56, 49.61, 48.16, 47.55, 46.08, 41.06, 39.79, 39.45, 39.16, 35.51, 32.00, 31.24, 29.82, 28.53, 27.57, 26.83, 26.70, 25.61, 22.80, 18.59, 17.52, 17.42, 17.14, 16.90.

[0098] Example 7: Synthesis of 6-O-[6'-O-fluorenemethoxycarbonylthreonyl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (intermediate 7)

[0099]

[0100] Using 20(S)-Rg2 and Fmoc-threonine as raw materials, and with other operations the same as in Example 1, a Fmoc-threonine ester derivative of 20(S)-ginsenoside Rg2 was obtained.

[0101] MS(m / z): [M+K] + =1146.2833.

[0102] 1 H-NMR (400MHz, C5D5N): δ8.68 (1H, s), 7.77 (1H, t), 7.76 (2H, s), 7.65 (1H, t), 7.33 (1H, d), 7.32 (1H, d), 7.21 (1H, t), 7.20 (1H, t), 3.95 (1H, m), 2.02 (3H, s), 1.72 (3H, d), 1.54 (6H, s), 1.45 (3H, d), 1.32 (3H, s), 1.28 (3H, s), 1.27 (3H, s), 1.02 (3H, s), 0.96 (3H, s).

[0103] 13 CNMR (100MHz, C5D5N): δ171.93, 157.65, 144.62, 144.32, 141.52, 130.5, 127.89, 127.37, 126.16, 125.61, 125 .58, 120.24, 101.86, 101.26, 78.84, 78.30, 78.17, 75.35, 73.99, 73.62, 72.79, 72.24, 72.12, 70.90, 69.27, 6 7.72, 66.79, 65.48, 61.21, 60.63, 54.62, 51.57, 49.61, 48.15, 47.64, 45.96, 41.06, 39.77, 39.50, 39.22, 35.55, 31.97, 31.91, 31.25, 27.58, 26.85, 26.73, 25.61, 22.77, 20.80, 18.59, 17.51, 17.48, 17.40, 17.12, 16.88.

[0104] Example 8: Synthesis of 6-O-[6'-O-fluorenylmethoxycarbonylserine-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (Intermediate 8)

[0105]

[0106] Using 20(S)-Rg2 and Fmoc-serine as raw materials, and with other operations the same as in Example 1, a Fmoc-serine ester derivative of 20(S)-ginsenoside Rg2 was obtained.

[0107] MS(m / z): [M+K] + =1132.2539.

[0108] 1 H-NMR (400MHz, C5D5N): δ8.98 (1H, d), 7.76 (2H, s), 7.63 (2H, d), 7.51 (2H, s), 7.33 (2H, d), 3.56 (1H, m), 3.54 (1H, m), 1.95(3H,s), 1.72(3H,s), 1.65(3H,s), 1.56(3H,s), 1.30(3H,s), 1.19(3H,s), 1.11(3H,s), 0.85(3H,s), 0.79(3H,s).

[0109] 13CNMR (100MHz, C5D5N): δ171.87, 157.21, 144.63, 144.35, 141.49, 129.81, 127.89, 127.36, 126.18, 125.62, 120.22, 106.13, 104.72, 78.42, 78.15, 77.92, 76.49, 75.02, 73.97, 72.81, 72.78, 72.23, 72.10, 71.23, 70.8 6, 67.66, 66.95, 62.94, 62.64, 58.10, 54.64, 51.53, 50.22, 48.38, 48.19, 47.51, 39.80, 39.66, 38.96, 35.72, 31.90, 30.60, 28.98, 27.91, 26.92, 25.65, 24.03, 23.01, 22.83, 18.58, 17.52, 16.82, 16.37, 16.22, 15.62.

[0110] Example 9: Synthesis of 6-O-[6'-O-glycyl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (Compound 1)

[0111]

[0112] The 20(S)-Rg2-Fmoc-glycine ester derivative (intermediate 1) was dissolved in a 10% piperidine / dichloromethane solution and reacted at room temperature for 1 h. After the reaction was stopped, the solvent was recovered under reduced pressure to dryness to obtain the crude product. The crude product was purified by column chromatography (dichloromethane: methanol: ethyl acetate: water) to obtain the glycine ester derivative of 20(S)-ginsenoside Rg2. The eluent used was dichloromethane: methanol: ethyl acetate: water = 4:2:4:1.

[0113] MS(m / z):[M+H] + =842.5266.

[0114] 1 H-NMR (400MHz, C5D5N): δ3.85 (1H, m), 3.54 (2H, t), 3.14 (1H, m), 2.00 (3H, s), 1.70 (3H, d), 1.58(3H,s), 1.56(3H,s), 1.32(3H,s), 1.23(3H,s), 1.11(3H,s), 0.89(3H,s), 0.87(3H,s).

[0115] 13CNMR (100MHz, C5D5N): δ170.81, 130.60, 126.17, 101.89, 101.34, 78.77, 78.24, 78.10 ,75.08,73.94,73.70,72.79,72.20,72.11,71.68,70.84,69.29,65.63,60.51,54.59 51.49, 49.55, 48.07, 45.90, 41.52, 41.00, 39.72, 39.44, 39.17, 35.49, 31.90, 31.77, 29.80, 27.52, 26.79, 26.56, 25.64, 22.77, 18.57, 17.51, 17.41, 16.88, 16.82, 16.74.

[0116] Example 10: Synthesis of 6-O-[6'-O-leucyl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (Compound 2)

[0117]

[0118] Using intermediate 2 as raw material, the eluent used was dichloromethane:methanol:ethyl acetate:water = 2:2:7:0.2, and other operations were the same as in Example 9, to obtain a leucine ester derivative of 20(S)-ginsenoside Rg2.

[0119] MS(m / z):[M+H] + =898.7789.

[0120] 1 H-NMR (400MHz, C5D5N): δ4.55 (2H, d), 3.63 (1H, m), 2.03 (3H, s), 1.76 (1H, m), 1.72 (3H, d), 1.71 (3H, s), 1.60 (2H , m), 1.59(3H,s), 1.34(3H,s), 1.24(3H,s), 1.22(3H,s), 0.97(3H,s), 0.93(3H,2d), 0.89(3H,2d), 0.88(3H,s).

[0121] 13CNMR (100MHz, C5D5N): δ174.52, 130.56, 126.12, 101.86, 101.38, 78.78, 78.09, 75.48, 73. 97, 73.36, 72.78, 72.20, 72.06, 71.52, 70.84, 69.21, 65.33, 60.51, 54.56, 52.90, 51.53, 49 .53, 48.13, 46.23, 42.64, 41.05, 39.75, 39.48, 39.16, 35.60, 32.00, 31.89, 31.26, 27.55, 26.84, 26.64, 25.65, 24.71, 23.00, 22.78, 21.73, 18.57, 17.62, 17.51, 17.48, 17.21, 16.85.

[0122] Example 11: Synthesis of 6-O-[6'-O-isoleucyl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (compound 3)

[0123]

[0124] Using intermediate 3 as the raw material, and following the same procedures as in Example 9, an isoleucine ester derivative of 20(S)-ginsenoside Rg2 was obtained. MS (m / z): [M+H] + =898.5826.

[0125] 1 H-NMR (400MHz, C5D5N): δ4.69 (2H, m), 3.89 (1H, m), 2.06 (1H, m), 2.02 (3H, s), 1.71 (3H, d), 1.59 (3H, s), 1.55 (3 H, s), 1.52 (2H, m), 1.34 (3H, s), 1.25 (3H, s), 1.24 (3H, s), 1.02 (3H, s), 1.01 (3H, t), 0.96 (3H, s), 0.95 (3H, d).

[0126] 13CNMR (100MHz, C5D5N): δ175.02, 130.58, 126.10, 101.86, 101.24, 78.89, 78.14, 78.12, 75. 59, 73.98, 73.33, 72.81, 72.22, 72.10, 71.73, 70.86, 69.22, 64.67, 60.55, 59.62, 54.56, 51 .57, 49.55, 48.13, 46.02, 41.05, 39.76, 39.48, 39.16, 38.93, 35.65, 31.97, 31.89, 31.30, 27.56, 26.86, 26.69, 25.64, 24.89, 22.79, 18.57, 17.47, 17.44, 17.16, 16.85, 16.08, 11.54.

[0127] Example 12: Synthesis of 6-O-[6'-O-valine-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglycosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (compound 4)

[0128]

[0129] Using intermediate 4 as the raw material, and following the same procedures as in Example 10, a valine ester derivative of 20(S)-ginsenoside Rg2 was obtained. MS (m / z): [M+H] + =884.7642.

[0130] 1 H-NMR (400MHz, C5D5N): δ4.87 (2H, m), 3.64 (1H, m), 2.28 (1H, m), 2.00 (3H, s), 1.71 (3H, d), 1.58 (3H, s), 1.54(3H,s), 1.33(3H,s), 1.24(3H,s), 1.20(3H,s), 1.04(3H,t), 0.97(3H,d), 0.94(3H,s), 0.93(3H,s).

[0131] 13CNMR (100MHz, C5D5N): δ174.57, 130.58, 126.09, 101.83, 101.19, 78.85, 78.18, 78.12, 75 .48, 73.94, 73.33, 72.82, 72.20, 72.08, 71.62, 70.85, 69.23, 64.87, 60.55, 60.24, 54.53 51.55, 49.53, 48.11, 45.96, 41.02, 39.73, 39.46, 39.15, 35.65, 32.04, 31.93, 31.85, 31.25, 27.53, 26.84, 26.66, 25.64, 22.79, 19.55, 18.57, 17.57, 17.51, 17.39, 17.09, 16.83.

[0132] Example 13: Synthesis of 6-O-[6'-O-methionyl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (compound 5)

[0133]

[0134] Using intermediate 5 as a raw material, and following the same procedures as in Example 9, a methionine ester derivative of 20(S)-ginsenoside Rg2 was obtained. MS (m / z): [M+H] + =916.4614.

[0135] 1 H-NMR (400MHz, C5D5N): δ4.72 (1H, m), 4.69 (1H, m), 3.61 (1H, m), 2.86 (2H, t), 2.06 (2H, m), 2.02 (6H, s), 1.71(3H,d), 1.59(3H,s), 1.56(3H,s), 1.33(3H,s), 1.24(3H,s), 1.21(3H,s), 0.96(3H,s), 0.93(3H,s).

[0136] 13CNMR (100MHz, C5D5N): δ174.00, 130.58, 126.17, 101.86, 101.32, 78.80, 78.20, 78.10, 75 .36, 73.96, 73.38, 72.80, 72.20, 72.08, 71.59, 70.85, 69.24, 65.40, 60.52, 54.62, 53.42 51.54, 49.55, 48.12, 46.18, 41.06, 39.74, 39.47, 39.17, 35.55, 33.12, 31.95, 31.87, 31.21, 30.51, 27.55, 26.82, 26.65, 25.65, 22.79, 18.57, 17.52, 17.46, 17.14, 16.85, 15.02.

[0137] Example 14: Synthesis of 6-O-[6'-O-tryptophanyl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (compound 6)

[0138]

[0139] Using intermediate 6 as a raw material, and following the same procedures as in Example 9, a tryptophan ester derivative of 20(S)-ginsenoside Rg2 was obtained. MS (m / z): [M+H] + =971.4802.

[0140] 1 H-NMR (400MHz, C5D5N): δ11.83 (1H, s), 7.90 (1H, d), 7.46 (2H, s), 7.22 (2H, s), 4.59 (1H, d), 4.57 ( 1H, d), 2.03 (3H, s), 1.72 (3H, d), 1.54 (6H, s), 1.31 (3H, s), 1.27 (3H, s), 1.26 (3H, s), 0.95 (6H, s).

[0141] 13CNMR (100MHz, C5D5N): δ175.39, 137.45, 130.52, 128.49, 126.14, 124.46, 121.70, 119.20, 111.91, 1 10.92, 101.91, 101.10, 78.92, 78.38, 78.14, 75.56, 74.00, 73.26, 72.81, 72.26, 72.12, 71.85, 70.88 69.26, 64.93, 60.57, 55.98, 54.61, 51.57, 49.58, 48.14, 46.02, 41.06, 39.76, 39.46, 39.15, 35.55, 31.93, 31.86, 31.22, 31.18, 27.56, 26.83, 26.63, 25.61, 22.80, 18.58, 17.48, 17.41, 17.17, 16.87.

[0142] Example 15: Synthesis of 6-O-[6'-O-threonyl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (compound 7)

[0143]

[0144] Using intermediate 7 as the raw material, and following the same procedures as in Example 9, a threonine ester derivative of 20(S)-ginsenoside Rg2 was obtained. MS (m / z): [M+H] + =886.4875.

[0145] 1 H-NMR (400MHz, C5D5N): δ4.86 (1H, m), 4.69 (1H, m), 4.44 (1H, m), 2.01 (3H, s), 1.71 (3H, d), 1.57 (3 H, s), 1.55 (3H, s), 1.32 (3H, s), 1.25 (3H, s), 1.21 (3H, s), 0.96 (3H, s), 0.93 (3H, s), 0.89 (3H, s).

[0146] 13CNMR (100MHz, C5D5N): δ174.32, 130.58, 126.12, 101.87, 101.28, 78.85, 78.23, 78.12, 75.47, 73.98, 73.48, 72.79, 72.22, 72.09, 71.81, 70.85, 69.24, 68.37, 64.95, 61.15, 60 .56, 54.57, 51.55, 49.56, 48.11, 46.00, 41.03, 39.75, 39.47, 39.17, 35.58, 31.95, 31.87, 31.21, 27.55, 26.84, 26.66, 25.63, 22.78, 20.95, 18.57, 17.51, 17.41, 17.08, 16.84.

[0147] Example 16: Synthesis of 6-O-[6'-O-seryl-α-L-pyranorhamnosyl-(1,2)-β-D-pyranoglucosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (compound 8)

[0148]

[0149] Using intermediate 8 as the raw material, and following the same procedures as in Example 9, a serine ester derivative of 20(S)-ginsenoside Rg2 was obtained. MS (m / z): [M+H] + =872.4565.

[0150] 1 H-NMR (400MHz, C5D5N): δ5.12 (1H, m), 4.87 (1H, m), 4.01 (1H, m), 3.87 (1H, m), 2.02 (3H, s), 1.73(3H,d), 1.56(6H,s), 1.32(3H,s), 1.25(3H,s), 1.19(3H,s), 0.95(3H,s), 0.92(3H,s).

[0151] 13CNMR (100MHz, C5D5N): δ173.26, 130.57, 126.17, 101.90, 101.29, 78.84, 78.29, 78.11, 75.34, 74.00, 73.49, 72.78, 72.23, 72.11, 71.66, 70.86, 69.27, 65.34, 63.21, 60.55, 56 .94, 54.62, 51.90, 51.53, 49.56, 48.12, 41.02, 39.75, 39.45, 39.18, 35.53, 31.94, 29.81, 29.42, 27.57, 27.33, 26.83, 25.63, 22.80, 18.59, 17.51, 17.41, 17.04, 16.99, 16.84.

[0152] Example 17 Study on the protective effect of the newly synthesized 20(S)-ginsenoside Rg2 amino acid ester derivative of the present invention on a hypoxia model of cardiomyocytes

[0153] (I) Effects of compounds 1-8 on the survival rate of hypoxic-injured cardiomyocytes

[0154] The amino acid ester derivatives (compounds 1-8) of 20(S)-ginsenoside Rg2 obtained in this invention were tested for their anti-cardiomyocyte hypoxia activity using the CCK-8 assay. Trimetazidine was selected as the positive control group, and 20(S)-ginsenoside Rg2 was selected as the unmodified control group.

[0155] The specific method is as follows:

[0156] Accurately weigh appropriate amounts of compound 1-compound 8, 20(S)-ginsenoside Rg2, and trimetazidine, add a small amount of DMSO to aid dissolution, and then add DMEM complete culture medium to prepare stock solutions of each test compound with concentrations of 40, 80, and 120 μM.

[0157] Accurately weigh an appropriate amount of cobalt chloride hexahydrate (CoCl2), dissolve it in deionized water, and then add serum-free low-glucose DMEM medium to prepare an anaerobic medium with a concentration of 600 μM.

[0158] H9c2 cells in logarithmic growth phase were harvested, digested with trypsin, and their density was adjusted to 8 × 10⁶ cells / year using fresh DMEM medium. 4 / mL, prepare cell suspension, seed 100μL of cell suspension in each well of a 96-well plate, and incubate in a 37℃ CO2 cell culture incubator for 24h.

[0159] After 24 hours, the culture medium was discarded, and the cells were divided into three groups: blank group, hypoxia model group, 20(S)-Rg2 group (containing three dose groups of 40, 80, and 120 μM), compound 1-compound 8 group (containing three dose groups of 40, 80, and 120 μM), and trimetazidine group (containing three dose groups of 40, 80, and 120 μM). The blank group and hypoxia model group were added to DMEM culture medium, while the 20(S)-Rg2 group, compound 1-compound 8 group, and trimetazidine group were added to the culture medium of the respective concentration of compounds, and cultured for another 24 hours.

[0160] After 24 hours, the culture medium was discarded. DMEM medium was added to the blank group, while hypoxia model group, 20(S)-Rg2 group, compound 1-compound 8 group and trimetazidine group were added to hypoxia medium and cultured for 18 hours to replicate the cell hypoxia model.

[0161] After modeling, 10 μL of LCK-8 solution was added to each well and incubated for 2 h. Then, the OD value of each well was measured at a wavelength of 450 nm, and the cell viability was calculated. The results are shown in Table 1.

[0162] Table 1. Effects of compounds 1-8 on the survival rate (%) of hypoxic H9c2 cells.

[0163]

[0164] Compared with the model group, *p<0.05, **p<0.01; compared with the 20(S)-Rg2 group, ▲ p<0.05.

[0165] Experimental results showed that, compared with the blank group (102.03±2.88), cell viability decreased to 50.51±1.38 after 18 h of treatment with 600 μM CoCl2 (P<0.001), indicating a significant difference. This demonstrates the successful establishment of the CoCl2-induced hypoxic injury model in H9c2 cells. Compared with the model group, trimetazidine significantly improved cell viability at all concentrations (P<0.01), showing a good protective effect against hypoxic cardiomyocytes. Compared with the model group, pretreatment with different concentrations of 20(S)-Rg2 and compounds 1-8 showed varying degrees of protective effect against hypoxic injury. Compound 5 showed good protective effects against H9c2 hypoxic cells at a concentration of 80 μM (P<0.05 compared with the model group); at a concentration of 120 μM, all compounds showed good protective effects against H9c2 hypoxic cells (P<0.05, P<0.01 compared with the model group), among which compound 1 showed significantly better protective effects than lead compound 20(S)-Rg2 (P<0.05 compared with the 20(S)-Rg2 group), and was closest to the protective effect of trimetazidine, showing the best anti-hypoxia activity.

[0166] (II) Effects of Compound 1 on Oxidative Stress Indicators in Hypoxic-Injured Cells

[0167] The effects of compound 1 on the changes in SOD, LDH, and MDA levels in hypoxic cells were investigated using a superoxide dismutase (SOD) activity assay kit, a lactate dehydrogenase (LDH) activity assay kit, and a malondialdehyde (MDA) content assay kit.

[0168] The specific method is as follows:

[0169] H9c2 cells in the logarithmic growth phase were harvested and treated with 1×10⁻⁶ cells. 6 Cells were seeded at a density of 1 cell per well in 6-well plates and cultured in a 37°C CO2 incubator for 24 hours.

[0170] After 24 hours, the culture medium was discarded, and the cells were divided into a blank group, a hypoxia model group, a 20(S)-Rg2 120μM group, a compound 1 group (containing three dose groups of 40, 80, and 120μM), and a trimetazidine 120μM group. The blank group and the hypoxia model group were added to DMEM culture medium, while the 20(S)-Rg2 group, compound 1 group, and trimetazidine group were added to the culture medium of the respective concentrations of the compound, and cultured for another 24 hours.

[0171] After 24 hours, the culture medium was discarded. DMEM medium was added to the blank group, while hypoxia model group, 20(S)-Rg2 group, compound 1 group and trimetazidine group were added to hypoxia medium and cultured for 18 hours to replicate the cell hypoxia model.

[0172] After modeling was completed, cells and cell culture medium from each group were collected. The LDH, SOD, and MDA reagent kits were then administered according to their operating procedures. The OD values ​​of each well were measured using a microplate reader at the specified wavelengths specified in the kits. Finally, the contents of LDH, SOD, and MDA in each group were calculated according to the instructions based on the measurement results. The kit results are shown below. Figure 1-3 As shown.

[0173] Depend on Figure 1-3 It was found that, compared with the normal group, the levels of LDH and MDA in the model group were significantly increased, while the activity of SOD was significantly decreased, indicating oxidative stress damage in hypoxic cells. After intervention with 40μM, 80μM, and 120μM of compound 1, the levels of LDH and MDA and the activity of SOD all significantly recovered, and the intervention effect of 120μM compound 1 was better than that of 20(S)-Rg2 at the same dose, and similar to that of trimetazidine, indicating that compound 1 has the effect of improving oxidative stress damage in hypoxic cells.

[0174] (III) Effects of Compound 1 on Inflammatory Markers in Hypoxic-Injured Cells

[0175] The effect of compound 1 on the changes in TNF-α and IL-6 levels in hypoxic cells was detected using an enzyme-linked immunosorbent assay kit for rat tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6).

[0176] The specific method is the same as that used in the experiment to detect the effect of compound 1 on the changes in SOD, LDH, and MDA content in hypoxic cells using the SOD, LDH, and MDA kit.

[0177] After modeling was completed, cells and cell culture medium from each group were collected. The procedures for the TNF-α and IL-6 kits were followed, and the OD values ​​of each well were measured using a microplate reader at the specified wavelengths. Finally, the levels of TNF-α and IL-6 in each group were calculated according to the instructions based on the measurement results. The kit results are shown below. Figure 4-5 As shown.

[0178] Depend on Figure 4-5 It was found that, compared with the normal group, the levels of TNF-α and IL-6 in the model group were significantly increased, indicating that inflammatory damage occurred in hypoxic cells. After intervention with compound 1, the levels of TNF-α and IL-6 were reduced in a dose-dependent manner, and the reducing effect of 120 μM compound 1 was superior to that of 20(S)-Rg2 at the same dose, and the effect was similar to that of trimetazidine, indicating that compound 1 has the effect of improving intracellular inflammatory damage in hypoxic cells.

[0179] (iv) Effects of compound 1 on the expression of PI3K / Akt pathway proteins in hypoxic-injured cells

[0180] The effect of compound 1 on the expression of p-PI3K and p-AKT in hypoxic cells was detected using an enzyme-linked immunosorbent assay kit for phosphorylated inositol 3-kinase (PI3K) and phosphorylated AKT protein (AKT) in rats.

[0181] The specific method is the same as that used in the experiment to detect the effect of compound 1 on the changes in SOD, LDH, and MDA content in hypoxic cells using the SOD, LDH, and MDA kit.

[0182] After modeling, cells and cell culture medium from each group were collected. The p-PI3K and p-AKT kits were then operated according to their instructions. The OD values ​​of each well were measured using a microplate reader at the specified wavelengths. Finally, based on the measurement results, the changes in p-PI3K and p-AKT protein expression in each group were calculated according to the instructions. The kit results are shown below. Figure 6-7 As shown.

[0183] Depend on Figure 6-7It was found that, compared with the normal group, the expression levels of p-PI3K and p-Akt in the model group were significantly reduced, indicating that the PI3K / Akt pathway was inhibited in hypoxic cells. However, intervention with compound 1 significantly promoted the expression levels of p-PI3K and p-Akt in a dose-dependent manner. The promoting effect of compound 1 was significantly better than that of 20(S)-Rg2 and trimetazidine, indicating that compound 1 has the effect of activating the PI3K / Akt pathway. It is speculated that its effect in alleviating hypoxic cell damage may be related to the activation of the PI3K / Akt pathway.

[0184] Conclusion: Compounds 1-8 all showed protective effects against hypoxic-damaged cardiomyocytes, indicating that the introduction of amino acid groups into the 20(S)-Rg2 structure helps to enhance anti-hypoxia activity. Among them, compound 1 showed the best protective effect, superior to the anti-hypoxia protective effect of the lead compound 20(S)-Rg2. Further studies revealed that compound 1 dose-dependently inhibited the increase of LDH, MDA, TNF-α, and IL-6 levels, while increasing SOD activity and promoting the expression of p-PI3K and p-Akt. This suggests that compound 1 can inhibit oxidative stress and inflammatory responses in damaged cells and activate the PI3K / Akt signaling pathway to protect hypoxic-damaged cells.

[0185] Example 18: Study on the protective effect of compound 1 of the present invention against shock model rats.

[0186] (I) Effect of Compound 1 on Blood Gas Indicators in Shock Model Rats The anti-shock activity of Compound 1 obtained in this invention was tested using a blood gas analyzer, and Shenfu injection was selected as a positive control group.

[0187] The specific method is as follows:

[0188] A rat model of hemorrhagic shock was established using the constant-volume blood loss method. First, the total blood volume (TBV / mL) of the rat was calculated as: rat body weight (g) × 0.0612 (mL / g). Then, the blood loss at shock was calculated for each rat at 45% of its TBV. For 12 hours prior to the experiment, the rats were fasted but allowed water. After weighing, the rats were anesthetized with anhydrous ether, and their tails were connected to an electronic blood pressure monitor (model BP-2010) to monitor and record the rats' noninvasive mean blood pressure (MBP) and heart rate (HR). The anesthetized rats were fixed flat, and blood was collected from the retinal venous plexus using a blood collection tube moistened with physiological saline. The rate of blood loss was initially rapid and then slowed down, which better reflects the actual situation during shock and resulted in better model replication. The rat model of hemorrhagic shock was successfully established when the blood loss reached 45% of the total blood volume within 10 minutes.

[0189] Experimental rats were randomly divided into 5 groups of 6 rats each: Blank group, Shock model group, Positive drug group, Compound 1 group, and Solvent control group. The Shock model group, Positive drug group, Compound 1 group, and Solvent control group were used to replicate the shock model, while the Blank group underwent no other procedures.

[0190] After modeling was completed, the positive drug group was injected intraperitoneally with Shenfu injection at a dose of 0.84 mL / Kg; the compound 1 administration group was injected intraperitoneally with compound 1 solution at a dose of 10 mg / Kg; the solvent control group was injected intraperitoneally with 25% 1,2-propanediol solution at a volume of 10 mL / Kg; the blank group and the shock model group were not administered any drugs.

[0191] Physiological parameters such as mean blood pressure (MBP) and heart rate (HR) were recorded for rats in each group at baseline, 0.5 h post-drug administration (AD 0.5 h), 1.5 h post-drug administration (AD 1.5 h), 3.5 h post-drug administration (AD 3.5 h), and 5.5 h post-drug administration (AD 5.5 h). Blood samples were collected from rats at baseline, 0.5 h post-drug administration (AD 0.5 h), and 1.5 h post-drug administration (AD 1.5 h) for lactate and blood gas analysis. Remaining blood samples were allowed to stand at room temperature for 30 min, then centrifuged at 3000 rpm for 10 min. Plasma was collected in EP tubes and stored at -20°C for later use.

[0192] The effects of compound 1 on blood pressure and heart rate in rats before and after shock are shown in Table 2.

[0193] Table 2. Effects of compound 1 on blood pressure and heart rate in rats before and after shock (x±SD, n=6)

[0194]

[0195] Compared with the blank group, ### p<0.001; compared with the model group, *p<0.05, **p<0.01, ***p<0.001.

[0196] Table 2 shows that the blood pressure and heart rate of the shock model group rats were significantly different from those of the blank group rats (P<0.001), indicating that the shock model was successfully replicated. Compared with the model group, compound 1 significantly increased the blood pressure reduction caused by shock, with a greater effect than the solvent control group, and the increase in blood pressure by compound 1 was greater than that of the positive control group at 3.5 h after administration; at the same time, compound 1 also significantly inhibited the abnormal increase in heart rate of shock rats, with an inhibitory effect superior to that of the solvent control group and the positive control group.

[0197] The changes in whole blood lactate levels in rats in each group after drug administration are as follows: Figure 8 As shown.

[0198] Depend on Figure 8 It was found that at 0.5 h after administration, the lactate content in the model group was significantly increased compared with the blank group (P<0.001), while compound 1 showed a trend of inhibiting the increase of lactate content compared with the model group (P<0.05), and the inhibitory effect was stronger than that of the solvent control group and the positive drug group; at 1.5 h after administration, the lactate content in each group was significantly restored, and there was no significant difference in lactate content between the groups (P>0.05).

[0199] After administration, the pH, partial pressure of carbon dioxide (PCO2), and bicarbonate (HCO3) levels in whole blood of rats in each group were measured. - The changes in indicators such as total carbon dioxide (TCO2) and alkali residue (BE) are as follows: Figure 9-13 As shown.

[0200] Depend on Figure 9-13 It can be seen that, compared with the blank group, the model group rats had lower pH and HCO3 levels at 0.5 h. - The levels of TCO2 and BE were significantly decreased (P<0.05, P<0.01), while PCO2 significantly increased at 1.5 h (P<0.01). These significant changes indicated metabolic acidosis in the model rats. After drug intervention, all drug-treated groups showed a trend towards pH recovery, but the difference was not statistically significant compared with the model group (P>0.05). At 0.5 h of drug administration, compared with the model group, compound 1 inhibited BE and HCO3. - The reduction in content was significant (P<0.05, P<0.01), significantly better than the solvent control group and the positive drug group. Furthermore, compound 1 also significantly increased TCO2 (P<0.01), with a stronger effect than the solvent control group and the positive drug group. At 1.5 h after administration, compared to the model group, compound 1 showed a significant trend of inhibiting the increase of PCO2 (P<0.01), with a stronger inhibitory effect than the solvent control group and the positive drug group.

[0201] The changes in partial pressure of oxygen (PO2) and oxygen saturation (SO2) in whole blood of rats in each group after drug administration are as follows: Figure 14-15 As shown. By Figure 14-15 It can be seen that, compared with the blank group, the PO2 and SO2 of the rats in the model group were significantly reduced (P<0.01), while compared with the model group, the compound 1 group showed a certain trend of increasing PO2 and SO2, but the difference was not statistically significant compared with the model group (P>0.05).

[0202] (II) Effects of Compound 1 on Oxidative Stress Indicators in Shock Model Rats

[0203] The effects of compound 1 on the changes in SOD, LDH, and MDA levels in the plasma of shocked rats were investigated using a superoxide dismutase (SOD) activity assay kit, a lactate dehydrogenase (LDH) activity assay kit, and a malondialdehyde (MDA) content assay kit.

[0204] The specific method is as follows:

[0205] Plasma was collected from each group separately, and the LDH, SOD, and MDA reagent kits were operated according to their instructions. The OD values ​​of each well were then measured using a microplate reader at the specified wavelengths. Finally, the contents of LDH, SOD, and MDA in each group were calculated according to the instructions based on the measurement results. The kit results are shown below. Figure 16-18 As shown.

[0206] Depend on Figure 16-18 It was found that, compared with the blank group, the levels of LDH and MDA in the model group were significantly increased (P<0.05, P<0.01), and the activity of SOD was significantly decreased (P<0.01, P<0.001), indicating that oxidative stress damage occurred in the rats in the model group. Compared with the model group, compound 1 showed a trend of inhibiting the increase of LDH and MDA levels. Its inhibitory effect on MDA was significant at 0.5 h after administration (P<0.05), and its inhibitory effect on LDH was significant at 1.5 h after administration (P<0.05), and the inhibitory effect was stronger than that of the solvent control group and the positive drug group. In addition, compound 1 showed a significant effect of increasing SOD activity at 1.5 h after administration (P<0.01), and the effect of increasing activity was stronger than that of the solvent control group but weaker than that of the positive drug group.

[0207] (III) Effects of Compound 1 on Inflammatory Markers in Shock Model Rats

[0208] The effect of compound 1 on the changes in TNF-α and IL-6 levels in the plasma of shocked rats was detected using an enzyme-linked immunosorbent assay kit for rat tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6).

[0209] The specific method is as follows:

[0210] Plasma was collected from each group separately, and the TNF-α and IL-6 kits were operated according to the specified procedures. The OD values ​​of each well were then measured using a microplate reader at the wavelengths specified in the kits. Finally, the levels of TNF-α and IL-6 in each group were calculated according to the instructions based on the measurement results. The kit results are shown below. Figures 19-20 As shown.

[0211] Depend on Figures 19-20 It was found that, compared with the blank group, the levels of TNF-α and IL-6 in the model group were significantly increased (P<0.001), indicating that there was inflammatory damage in the model rats; while compared with the model group, compound 1 group showed a better effect in reducing the expression of TNF-α and IL-6 (P<0.01), with better effect than the solvent control group and slightly stronger than the positive drug group.

[0212] Conclusion: During hemorrhagic shock, the effective circulating blood volume decreases sharply, leading to a rapid drop in blood pressure. This reduction in circulating blood volume also results in a decrease in stroke volume, while the heart rate compensates by increasing. However, all tissues remain in a state of insufficient blood perfusion and hypoxia, resulting in metabolic acidosis accompanied by oxidative stress and inflammation. After intervention with compound 1, the decreasing trend of blood pressure and increasing trend of heart rate in shock rats were significantly inhibited, and the levels of lactic acid, PCO2, and HCO3- were reduced. - Blood gas parameters such as TCO2 and BE were significantly improved. At the same time, the levels of LDH, MDA, TNF-α and IL-6 in plasma decreased significantly, and the activity of SOD increased significantly. This indicates that compound 1 can exert a protective effect on shock rats through multiple pharmacological effects, such as improving cardiac function, enhancing tissue perfusion, correcting metabolic acidosis, and inhibiting oxidative stress and inflammatory response.

[0213] The above embodiments are merely exemplary embodiments of the present invention and are not intended to limit the present invention. The scope of protection of the present invention is defined by the claims. Those skilled in the art can make various modifications or equivalent substitutions to the present invention within its spirit and scope of protection, and such modifications or equivalent substitutions should also be considered to fall within the scope of protection of the present invention.

Claims

1. A 20(S)-ginsenoside Rg2 amino acid ester derivative, characterized in that... Selected from the following structure: (1) ； (2) ； (3) ； (4) ； (5) ； (6) ； (7) ； (8) ； Or its pharmaceutically acceptable salt.

2. A 20(S)-ginsenoside Rg2 amino acid ester derivative, characterized in that... The molecular structure is as follows: 。 3. The method for preparing a 20(S)-ginsenoside Rg2 amino acid ester derivative as described in claim 1, characterized in that: 20(S)-Rg2, Fmoc amino acids, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N-diisopropylethylamine, and 4-dimethylaminopyridine were added sequentially to anhydrous tetrahydrofuran in a ratio of 1:2:5:2:0.

8. The mixture was shaken to dissolve the compounds, and the reaction was carried out in an ice-water bath for 8 h. The solvent was then removed from the reaction solution under reduced pressure and dried. The residue was dissolved in dichloromethane, and then washed thoroughly with water and saturated NaCl solution, dried with Na2SO4, filtered, and concentrated to obtain the crude product. The crude product was purified by column chromatography to obtain the Fmoc-amino acid ester derivative of 20(S)-ginsenoside Rg2. The eluent used was dichloromethane:methanol:ethyl acetate:water. The 20(S)-Rg2-Fmoc-amino acid ester derivative was dissolved in 10% piperidine / dichloromethane solution and reacted at room temperature for 1 h. h, the reaction was stopped, the solvent was recovered from the reaction solution under reduced pressure and dried to obtain crude product, which was purified by column chromatography to obtain amino acid ester derivative of 20(S)-ginsenoside Rg2, with dichloromethane: methanol: ethyl acetate: water as the eluent.

4. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises at least the compound as described in claim 1 or 2, or a pharmaceutically acceptable salt thereof.

5. A pharmaceutical formulation comprising an active ingredient and pharmaceutically acceptable excipients and / or a carrier, said active ingredient comprising at least the compound as described in claim 1 or 2 or a pharmaceutically acceptable salt thereof.

6. Use of the compound of claim 1 or 2 in the preparation of a medicament for treating cellular hypoxic injury.

7. Use of the compound of claim 1 or 2 in the preparation of a medicament for treating shock diseases.

8. Use of the pharmaceutical composition of claim 4 in the preparation of a medicament for treating cellular hypoxic injury.

9. Use of the pharmaceutical composition of claim 4 in the preparation of a medicament for treating shock.

10. The use of the pharmaceutical formulation of claim 5 in the preparation of a medicament for treating cellular hypoxic injury.

11. Use of the pharmaceutical preparation of claim 5 in the preparation of a medicament for treating shock diseases.

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