Asiatic acid PROTACs as well as preparation method and application thereof

Abstract

The invention discloses asiatic acid PROTACs and a preparation method and application thereof, the structural formula of the asiatic acid PROTACs is # imgabs0, the asiatic acid PROTACs can degrade target protein in tumor cells so as to exert anti-tumor activity, the anti-tumor activity of the asiatic acid PROTACs is superior to that of asiatic acid, and the asiatic acid PROTACs have good potential application prospects in the aspects of anti-cancer drugs and anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic synthesis, and specifically relates to asiatic acid PROTACs and a preparation method and application thereof. Background Art

[0002] Protein degradation targeting chimeras (PROTACs) are a new type of drug with broad prospects. Its structure is similar to a dumbbell and consists of three parts: an E3 ligase ligand (E3 ligand), a target protein ligand (POI ligand), and a linker connecting the two. One end of the PROTACs molecule binds to the target protein, and the other end binds to the E3 ubiquitin ligase. The E3 ubiquitin ligase "sticks" a small protein called ubiquitin to the target protein, marking the target protein as a defective or damaged protein, and then uses the 26S proteasome in the cell to specifically recognize and degrade the marked target protein. The E3 ligand is responsible for recruiting the E3 ligase, while the POI ligand recognizes and binds to the target protein. Therefore, inside the cell, the PROTACs molecule forms a ternary complex of POI:PROTAC:E3 ligase. In the patient's body, the POI ligand of PROTACs binds to the target protein, and the E3 ligand binds to the substrate binding region of the E3 ubiquitin ligase in the cell. The target protein is "pulled" to the side of the E3 ubiquitin ligase through the Linker, and the target protein is degraded by the ubiquitin-proteasome (UPS) system. PROTACs molecules do not need to be highly chimeric with the highly active region of the target. They can achieve degradation of the target molecule through weak binding interactions with low bond energy such as some special intermolecular forces. Because PROTACs-mediated protein degradation has a unique driving model and huge therapeutic potential, this technology is currently mainly used in drug development, and some PROTACs have entered the clinical trial stage.

[0003] PROTAC drugs have many advantages over traditional small molecule drugs, as follows:

[0004] 1. The design of PROTAC drugs is not limited by Lipinski's five-fold rule.

[0005] 2. The pharmacological effects of traditional small molecule drugs rely on occupancy of key sites on the target protein (occupancy-driven), that is, they work by binding to the active sites of enzymes or receptors. This requires the drug to maintain a certain concentration in the body and has high requirements for small molecule binding sites. PROTACs can achieve degradation at any position of the target protein. Its pharmacological mechanism is to activate potential targets to achieve pharmacodynamic effects. Studies have shown that very high drug concentrations are not required during the pharmacodynamic process, the requirements for binding sites are relatively low, and the efficacy is good.

[0006] 3. Approximately 50% of proteins in the human body are associated with human diseases, such as cancer. However, only about 20% of proteins can be bound by traditional small molecules. In theory, PROTACs can bind to any part of a protein.

[0007] 4. Compared with traditional small molecules, PROTACs have advantages in terms of drug resistance. In theory, PROTACs can better tolerate mutations in target proteins.

[0008] 5. PROTACs are more durable and have stronger inhibitory power than traditional drug molecules.

[0009] Furthermore, asiatic acid, a major component of the triterpene extract of the natural plant Centella asiatica, possesses an ursane-type skeleton structure. Since its discovery in 1979 as a treatment for skin wounds, studies have shown that asiatic acid possesses a variety of benefits, including anti-irritation properties, skin repair, collagen synthesis inhibition, keratinization improvement, and skin cell stimulation. Summary of the Invention

[0010] The present invention aims to provide asiatic acid PROTACs.

[0011] Another object of the present invention is to provide a method for preparing the above-mentioned Centella asiatica PROTACs.

[0012] Another object of the present invention is to provide the application of the above-mentioned Centella asiatica PROTACs.

[0013] The technical solutions of the present invention are as follows:

[0014] A Centella asiatica acid PROTACs, the structural formula of which is in,

[0015] E3 Ligase is selected from lenalidomide and pomalidomide,

[0016] Linker is the first fatty chain, the second fatty chain or the PEG chain,

[0017] The first fatty chain is -NH-(CH2) n1 -NH-(CH2)3-CO-, n1 is 1, 2, 3, 4, 5 or 6,

[0018] The second fatty chain is -NH-(CH2) n2 -CH2-NH-, n2 is 1, 2, 3, 4, 5 or 7,

[0019] The PEG chain is -NH-(CH2-CH2-O) n3 -CH2-CH2-NH-, n3 is 1, 2 or 3.

[0020] In a preferred embodiment of the present invention, its structural formula is selected from at least one of the following:

[0021]

[0022]

[0023]

[0024]

[0025] The preparation method of the above-mentioned Centella asiatica PROTACs has the following reaction scheme:

[0026]

[0027]

[0028] Use of the above-mentioned Centella asiatica PROTACs or pharmacologically or physiologically acceptable salts thereof in preparing a composition for treating tumor diseases.

[0029] In a preferred embodiment of the present invention, the tumor diseases include brain cancer, breast cancer, cervical cancer, stomach cancer, liver cancer and lung cancer.

[0030] A composition for treating tumor diseases, the active ingredient of which includes the above-mentioned Centella asiatica PROTACs or a pharmacologically or physiologically acceptable salt thereof.

[0031] Use of the above-mentioned Centella asiatica PROTACs or pharmacologically or physiologically acceptable salts thereof in the preparation of an anti-inflammatory composition.

[0032] An anti-inflammatory composition, the active ingredient of which includes the above-mentioned Centella asiatica PROTACs or a pharmacologically or physiologically acceptable salt thereof.

[0033] The beneficial effects of the present invention are: the present invention uses Centella asiatica as the POI ligand, uses lenalidomide and pomalidomide as the E3 ligand, and selects PEG chains and fatty chains as linkers. Its anti-cancer activity is better than that of Centella asiatica, and it has good potential application prospects in anti-cancer drugs and the treatment of inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a diagram of the experimental results of Example 8 of the present invention.

[0035] Figure 2 This is a diagram showing the experimental results of Example 9 of the present invention.

[0036] Figure 3 This is a diagram showing the experimental results of Example 10 of the present invention. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further illustrated and described below through specific implementation methods in conjunction with the accompanying drawings.

[0038] The following list of Centella asiatica PROTACs prepared in Examples 1 to 6 is as follows:

[0039]

[0040]

[0041]

[0042]

[0043]

[0044] Example 1: Preparation of Intermediates S1-S6 (Preparation of E3 Ligand)

[0045] A. The structural formula of intermediate BS1 (n1=1) / BS2 (n1=2) / BS3 (n1=3) / BS4 (n1=4) / BS5 (n1=5) / BS6 (n1=6) is:

[0046] The specific synthesis method is as follows:

[0047] Boc-3-aminopropionic acid (1 g, 4.9 mmol) and DIPEA (1.71 mL, 9.8 mmol) were dissolved in 1 mL of dry DMF and stirred at room temperature for 5 min. HATU (1.49 g, 3.92 mmol) was added and the reaction was monitored by TLC for approximately 0.5 h. Lenalidomide (826 mg, 3.2 mmol) was added and stirred at room temperature. TLC followed the reaction, indicating completion in approximately 12 h. Crushed ice was added to the reaction solution to quench the reaction. The mixture was extracted three times with ethyl acetate. The organic layers were combined, washed sequentially with saturated ammonium chloride solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The reaction was then analyzed by PTLC (dichloromethane:methanol = 20:1) to afford intermediate BS1 (white solid, 1.28 g, 90%).

[0048] Boc-4-aminobutyric acid (1 g, 5.2 mmol) and DIPEA (1.81 mL, 10.4 mmol) were dissolved in 1 mL of dry DMF and stirred at room temperature for 5 min. HATU (1.6 g, 4.16 mmol) was added and the reaction was monitored by TLC for approximately 0.5 h. Lenalidomide (876 mg, 3.38 mmol) was added and stirred at room temperature. TLC followed the reaction, indicating completion in approximately 12 h. Crushed ice was added to the reaction solution to quench the reaction. The mixture was extracted three times with ethyl acetate. The organic layers were combined, washed sequentially with saturated ammonium chloride solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The reaction was analyzed by PTLC (dichloromethane:methanol = 20:1) to afford intermediate BS2 (white solid, 1.3 g, 90%).

[0049] Boc-5-aminovaleric acid (1 g, 4.6 mmol) and DIPEA (1.6 mL, 9.2 mmol) were dissolved in 1 mL of dry DMF and stirred at room temperature for 5 min. HATU (1.4 g, 3.68 mmol) was added and the reaction was monitored by TLC for approximately 0.5 h. Lenalidomide (775 mg, 2.99 mmol) was added and stirred at room temperature. TLC followed the reaction, indicating completion in approximately 12 h. Crushed ice was added to the reaction solution to quench the reaction. The mixture was extracted three times with ethyl acetate. The organic layers were combined, washed sequentially with saturated ammonium chloride solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The reaction was analyzed by PTLC (dichloromethane:methanol = 20:1) to afford intermediate BS3 (white solid, 1.25 g, 91%).

[0050] Boc-6-aminohexanoic acid (1 g, 4.3 mmol) and DIPEA (1.5 mL, 8.6 mmol) were dissolved sequentially in 1 mL of dry DMF and stirred at room temperature for 5 min. HATU (1.31 g, 3.44 mmol) was added and the reaction was monitored by TLC for approximately 0.5 h. Lenalidomide (725 mg, 2.8 mmol) was then added and stirred at room temperature. TLC followed the reaction, indicating completion in approximately 12 h. Crushed ice was added to the reaction solution to quench the reaction. The mixture was extracted three times with ethyl acetate. The organic layers were combined, washed sequentially with saturated ammonium chloride solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The reaction was then analyzed by PTLC (dichloromethane:methanol = 20:1) to afford intermediate BS4 (white solid, 1.2 g, 92%).

[0051] Boc-7-aminoheptanoic acid (1 g, 4.1 mmol) and DIPEA (1.43 mL, 8.2 mmol) were dissolved in 1 mL of dry DMF and stirred at room temperature for 5 min. HATU (1.25 g, 3.28 mmol) was added and the reaction was monitored by TLC for approximately 0.5 h. Lenalidomide (691 mg, 3.38 mmol) was added and stirred at room temperature. TLC followed the reaction, indicating completion in approximately 12 h. Crushed ice was added to the reaction solution to quench the reaction. The mixture was extracted three times with ethyl acetate. The organic layers were combined, washed sequentially with saturated ammonium chloride solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The reaction was analyzed by PTLC (dichloromethane:methanol = 20:1) to afford intermediate BS5 (white solid, 1.53 g, 93%).

[0052] Boc-8-aminooctanoic acid (1 g, 3.8 mmol) and DIPEA (1.3 mL, 7.6 mmol) were dissolved in 1 mL of dry DMF and stirred at room temperature for 5 min. HATU (1.16 g, 3.04 mmol) was added and the reaction was monitored by TLC for approximately 0.5 h. Lenalidomide (640 mg, 2.47 mmol) was added and stirred at room temperature. TLC followed the reaction, indicating completion in approximately 12 h. Crushed ice was added to the reaction solution to quench the reaction. The mixture was extracted three times with ethyl acetate. The organic layers were combined, washed sequentially with saturated ammonium chloride solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The reaction was analyzed by PTLC (dichloromethane:methanol = 20:1) to afford intermediate BS6 (white solid, 1.11 g, 90%).

[0053] B. The structural formula of intermediate S1(n=1) / S2(n=2) / S3(n=3) / S4(n=4) / S5(n=5) / S6(n=6) is

[0054] The specific synthesis method is as follows:

[0055] Intermediate BS1 (1 g, 2.25 mmol) was dissolved in a DCM / MeOH (v:v = 3:1) mixture (10 mL). TFA (3 mL) was added dropwise under an ice bath. After addition, the mixture was slowly brought to room temperature. The reaction was monitored by TLC and completed in approximately 12 hours. After cessation of the reaction, the solvent was removed by concentration under reduced pressure to obtain intermediate S1 (white solid, 713 mg, 96.0%), which was used directly in the next step without purification.

[0056] Intermediate BS2 (1 g, 2.25 mmol) was dissolved in a DCM / MeOH (v:v = 3:1) mixture (10 mL). TFA (3 mL) was added dropwise under an ice bath. After addition, the mixture was slowly brought to room temperature. The reaction was monitored by TLC and completed in approximately 12 hours. After cessation of the reaction, the solvent was removed by concentration under reduced pressure to afford intermediate S2 (white solid, 675 mg, 90%), which was used directly in the next step without purification.

[0057] Intermediate BS3 (1 g, 2.25 mmol) was dissolved in a DCM / MeOH (v:v = 3:1) mixture (10 mL). TFA (3 mL) was added dropwise under an ice bath. After addition, the mixture was slowly brought to room temperature. The reaction was monitored by TLC and completed in approximately 12 hours. After the reaction ceased, the solvent was removed by concentration under reduced pressure to obtain intermediate S3 (white solid, 705 mg, 93.0%), which was used directly in the next step without purification.

[0058] Intermediate BS4 (1 g, 2.25 mmol) was dissolved in a DCM / MeOH (v:v = 3:1) mixture (10 mL). TFA (3 mL) was added dropwise under an ice bath. After addition, the mixture was slowly brought to room temperature. The reaction was monitored by TLC and completed in approximately 12 hours. After cessation of the reaction, the solvent was removed by concentration under reduced pressure to obtain intermediate S4 (white solid, 690 mg, 92.0%), which was used directly in the next reaction without purification.

[0059] Intermediate BS5 (1 g, 2.25 mmol) was dissolved in a DCM / MeOH (v:v = 3:1) mixture (10 mL). TFA (3 mL) was added dropwise under an ice bath. After addition, the mixture was slowly brought to room temperature. The reaction was monitored by TLC and completed in approximately 12 hours. After the reaction ceased, the solvent was removed by concentration under reduced pressure to obtain intermediate S5 (white solid, 668 mg, 93.0%), which was used directly in the next step without purification.

[0060] Intermediate BS6 (1 g, 2.25 mmol) was dissolved in a DCM / MeOH (v:v = 3:1) mixture (10 mL). TFA (3 mL) was added dropwise under an ice bath. After addition, the mixture was slowly brought to room temperature. The reaction was monitored by TLC and completed in approximately 12 hours. After cessation of the reaction, the solvent was removed by concentration under reduced pressure to obtain intermediate S6 (white solid, 683 mg, 91.0%), which was used directly in the next step without purification.

[0061] Example 2: Preparation of final products A1 to A6

[0062] A 25 mL round-bottom flask was charged with asiatic acid (100 mg, 0.26 mmol, 1 eq), linker and E3 ligase ligands S1 / S2 / S3 / S4 / S5 / S6 (0.51 mol, 2 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) (0.51 mol, 2 eq), 4-dimethylaminopyridine (DMAP) (0.26 mol, 1 eq), and DMF (5 mL). The reaction was stirred at room temperature for 5–8 h. Upon completion, ice water was added to quench the reaction. The mixture was extracted with dichloromethane (DCM), and the organic phase was washed with 5% sodium bicarbonate solution and saturated sodium chloride, then dried over anhydrous Na₂SO₄. The solid was removed by filtration, and the solvent was evaporated under reduced pressure to obtain the crude product, which was then purified by column chromatography (PE-EA = 10:1 to 1:1, v / v).

[0063] Final product A1 (white solid, yield 33.5%)

[0064] Final product A2 (white solid, yield 39.6%)

[0065] Final product A3 (white solid, yield 38.8%)

[0066] Final product A4 (white solid, yield 39.9%)

[0067] Final product A5 (white solid, yield 40.2%)

[0068] Final product A6 (white solid, yield 38.9%)

[0069] Example 3: Preparation of intermediates P1-P6

[0070] A. The structural formula of the intermediate BP1 (n2 = 1) BP2 (n2 = 2) / BP3 (n2 = 3) / BP4 (n2 = 4) / BP5 (n2 = 5) / BP6 (n2 = 7) is

[0071] The specific synthesis method is as follows:

[0072] Fluorothalidamide (2 g, 7.241 mmol) was dissolved in 20 mL of DMF with stirring. DIEA (1.872 mL, 14.481 mmol) and N-Boc-1,2-ethylenediamine (2 mL, 8.689 mmol) were then added and the mixture was heated to 90°C and refluxed for 12 h. After completion, the reaction was quenched with ice water and extracted with ethyl acetate. The combined organic phases were washed with saturated NaHCO₃ and then saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to obtain BP1 (yellow-green powder, 41.2% yield).

[0073] Fluorothalidamide (2 g, 7.241 mmol, 1 eq) was dissolved in 20 mL of DMF with stirring. DIEA (1.872 mL, 14.481 mmol) and N-Boc-1,3-propylenediamine (2 eq) were then added and the mixture was heated to 90°C and refluxed for 12 h. After completion, the reaction was quenched with ice water and extracted with ethyl acetate. The combined organic phases were washed sequentially with saturated NaHCO₃ and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to afford BP2 (yellow-green powder, 55.4% yield).

[0074] Fluorothalidamide (2 g, 7.241 mmol, 1 eq) was dissolved in 20 mL of DMF with stirring. DIEA (1.872 mL, 14.481 mmol) and N-Boc-1,4-butanediamine (2 eq) were then added and the mixture was heated to 90°C and refluxed for 12 h. After completion, the reaction was quenched with ice water and extracted with ethyl acetate. The combined organic phases were washed with saturated NaHCO₃ and then saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to afford BP3 (yellow-green powder, 48.3% yield).

[0075] Fluorothalidamide (2 g, 7.241 mmol, 1 eq) was dissolved in 20 mL of DMF with stirring. DIEA (1.872 mL, 14.481 mmol) and N-Boc-1,5-pentanediamine (2 eq) were then added and the mixture was heated to 90°C and refluxed for 12 h. After completion, the reaction was quenched with ice water and extracted with ethyl acetate. The combined organic phases were washed sequentially with saturated NaHCO₃ and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to yield BP3 (yellow-green powder, 48.3% yield).

[0076] Fluorothalidamide (2 g, 7.241 mmol, 1 eq) was dissolved in 20 mL of DMF with stirring. DIEA (1.872 mL, 14.481 mmol) and N-Boc-1,6-hexanediamine (2 eq) were then added and the mixture was heated to 90°C and refluxed for 12 h. After completion, the reaction was quenched with ice water and extracted with ethyl acetate. The combined organic phases were washed with saturated NaHCO₃ and then saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to afford BP3 (yellow-green powder, 46.5% yield).

[0077] Fluorothalidamide (2 g, 7.241 mmol, 1 eq) was dissolved in 20 mL of DMF with stirring. DIEA (1.872 mL, 14.481 mmol) and N-Boc-1,8-octanediamine (2 eq) were then added and the mixture was heated to 90°C and refluxed for 12 h. After completion, the reaction was quenched with ice water and extracted with ethyl acetate. The combined organic phases were washed with saturated NaHCO₃ and then saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to afford BP3 (yellow-green powder, 39.1% yield).

[0078] B. The structural formula of intermediate = P1(n2=1)P2(n2=2) / P3(n2=3) / P4(n2=4) / P5(n2=5) / P6(n2=7) is

[0079] The specific synthesis method is as follows:

[0080] Dissolve 1 g of intermediate BP1 in 10 ml of dichloromethane and place in an ice bath. Slowly add 2.5 ml of trifluoroacetic acid (TFA) dropwise in an ice bath. After the addition is complete, slowly bring the reaction mixture to room temperature and continue the reaction. Monitor the reaction progress using thin-layer chromatography (TLC), and the reaction reaches completion after approximately 12 hours. After completion of the reaction, remove the solvent by concentrating under reduced pressure to obtain intermediate P1 (yellow-green powder) in a 97% yield. No purification is required and the product can be used directly in the next reaction.

[0081] 1 g of intermediate BP2 was dissolved in 10 ml of dichloromethane and placed in an ice bath. 2.5 ml of trifluoroacetic acid (TFA) was slowly added dropwise under ice bath conditions. After the addition was complete, the reaction mixture was slowly brought to room temperature and the reaction continued. The reaction progress was monitored by thin layer chromatography (TLC), and the reaction reached completion after approximately 12 hours. After completion of the reaction, the solvent was removed by concentrating under reduced pressure to obtain intermediate P2 (yellow-green powder) in a yield of 97%. No purification was required and the product could be used directly in the next step.

[0082] Dissolve 1 g of intermediate BP3 in 10 ml of dichloromethane and place in an ice bath. Slowly add 2.5 ml of trifluoroacetic acid (TFA) dropwise in an ice bath. After the addition is complete, slowly bring the reaction mixture to room temperature and continue the reaction. Monitor the reaction progress using thin-layer chromatography (TLC), and the reaction reaches completion after approximately 12 hours. After completion of the reaction, remove the solvent by concentrating under reduced pressure to obtain intermediate P3 (yellow-green powder) in a yield of 98%. No purification is required and the product can be used directly in the next reaction.

[0083] Dissolve 1 g of intermediate BP4 in 10 ml of dichloromethane and place in an ice bath. Slowly add 2.5 ml of trifluoroacetic acid (TFA) dropwise in an ice bath. After the addition is complete, slowly bring the reaction mixture to room temperature and continue the reaction. Monitor the reaction progress using thin-layer chromatography (TLC), and the reaction reaches completion after approximately 12 hours. After completion of the reaction, remove the solvent by concentrating under reduced pressure to obtain intermediate P4 (yellow-green powder) in a 97% yield. No purification is required and the product can be used directly in the next reaction.

[0084] Dissolve 1 g of intermediate BP5 in 10 ml of dichloromethane and place in an ice bath. Slowly add 2.5 ml of trifluoroacetic acid (TFA) dropwise in an ice bath. After the addition is complete, slowly bring the reaction mixture to room temperature and continue the reaction. Monitor the reaction progress using thin-layer chromatography (TLC), and the reaction reaches completion after approximately 12 hours. After completion of the reaction, remove the solvent by concentrating under reduced pressure to obtain intermediate P5 (yellow-green powder) in a 98% yield. No purification is required and the product can be used directly in the next reaction.

[0085] Dissolve 1 g of intermediate BP6 in 10 ml of dichloromethane and place in an ice bath. Slowly add 2.5 ml of trifluoroacetic acid (TFA) dropwise in an ice bath. After the addition is complete, slowly bring the reaction mixture to room temperature and continue the reaction. Monitor the reaction progress using thin-layer chromatography (TLC), and the reaction reaches completion after approximately 12 hours. After completion of the reaction, remove the solvent by concentrating under reduced pressure to obtain intermediate P6 (yellow-green powder) in a yield of 98%. No purification is required and the product can be used directly in the next reaction.

[0086] Example 4: Preparation of final products A7-A12

[0087] Centella asiatica (1 eq) and DIPEA (2 eq) were dissolved in a dry DMF solution in sequence. Stir at room temperature for 5 minutes, then add HATU (1.5 eq) and monitor by TLC. After about 30 minutes, add P1 (1.5 eq) and continue stirring at room temperature. The reaction was complete after 12 hours. The reaction was quenched with ice water, extracted with ethyl acetate three times, and the organic layers were combined. Subsequently, the mixture was washed with saturated ammonium chloride solution and saturated brine in sequence. After drying over anhydrous sodium sulfate, the mixture was filtered, concentrated under reduced pressure, and purified by PTLC (dichloromethane: methanol = 20:1) to obtain the final product A7. Its NMR data are as follows: A7 (yellow-green powder, 36.3%): ESI-MS m / z 809.5 [M+Na] + . 1H NMR(500MHz,Chloroform-d)δ9.53(d,J=123.4Hz,1H),7.59–7.48(m,1H),7.13(d,J=7.1Hz,1H),6.98(dd,J=22.0,8.6Hz,1H),6.36(d,J=8.2Hz,1H),5.39–5.13(m,1H),5.05–4.93(m,1H),4.42(s,2H),3.78–3.63(m,2H),3.63–3.49(m,2H),3.45–3.38(m,2H),3.35–3.21(m,2H),2.87(q,J=13.2,9.9Hz,1H),2.82–2.72(m,2H),2.23–2.10(m,2H),2.00(q,J=6.3Hz,1H),1.95–1.88(m,2H),1.86(s,1H),1.79–1.69(m,2H),1.55(d,J=12.1Hz,2H),1.47(t,J=9.6Hz,2H),1.40(d,J=13.8Hz,2H),1.37(s,1H),1.33–1.28(m,2H),1.28–1.26(m,2H),1.25(s,1H),1.24(s,2H),1.23–1.16(m,2H),1.04(s,3H),0.92(s,3H),0.89–0.83(m,3H),0.80(dd,J=12.9,6.0Hz,3H),0.74(d,J=6.8Hz,3H),0.62(d,J=36.3Hz,3H). 13 C NMR(126MHz,CDCl3)δ179.25,172.24,169.55,168.85,167.63,147.82,139.72,136.76,132.65,127.00,117.53,113.20,110.74,81.39,71.13,68.76,54.06,49.92,48.27,48.02,47.10,43.24,42.62,40.35,39.63,39.22,38.65,37.99,37.08,36.07,29.83,29.05,27.91,27.35,25.90,23.47,22.43,21.79,18.26,17.41,17.09,17.00,16.90,14.47,13.64.

[0088] Centella asiatica (1 eq) and DIPEA (2 eq) were dissolved in a dry DMF solution. Stir at room temperature for 5 minutes, then add HATU (1.5 eq). Monitor by TLC. After approximately 30 minutes, add P2 (1.5 eq) and continue stirring at room temperature. The reaction is complete after 12 hours. The reaction is quenched with ice water and extracted with ethyl acetate three times. The organic layers are then combined. The mixture is then washed with saturated ammonium chloride solution and saturated brine. After drying over anhydrous sodium sulfate, the mixture is filtered, concentrated under reduced pressure, and purified by PTLC (dichloromethane:methanol = 20:1) to obtain the final product A8. Its NMR data are as follows: A8 (yellow-green powder, 38.2%): ESI-MS m / z 823.5 [M+Na] + . 1 H NMR(500MHz,Chloroform-d)δ8.75(d,J=11.4Hz,1H),7.49(q,J=7.5,7.1Hz,1H),7.10(d ,J=7.1Hz,1H),6.88(d,J=8.6Hz,1H),6.29(s,1H),6.09(s,1H),5.27(d,J=4.3Hz,1H),5. 01-4.83(m,1H),3.75(s,1H),3.63(d,J=14.3Hz,1H),3.51-3.44(m,1H),3.39(s,1H),3.2 9(t,J=6.1Hz,2H),3.11(d,J=11.8Hz,1H),2.86(t,J=15.8Hz,1H),2.76(q,J=12.0,11.5H z,2H),2.19-2.04(m,2H),1.95(s,1H),1.87(s,1H),1.87-1.84(m,1H),1.82(d,J=10.0Hz ,2H),1.72(d,J=12.8Hz,1H),1.61(s,1H),1.61(s,1H),1.50-1.45(m,2H),1.41(t,J=3.3 Hz,2H),1.37(d,J=7.3Hz,2H),1.34(d,J=15.1Hz,2H),1.28(s,2H),1.27(s,2H),1.07(s, 3H),0.97(d,J=4.2Hz,3H),0.94(d,J=4.1Hz,3H),0.84(s,3H),0.83(s,3H),0.74(s,3H). 13C NMR (126MHz, DMSO) δ176.32,172.81,170.04,168.14,165.97,146.29,138.61,135.90,132 .25,124.49,117.83,110.46,109.18,75.96,67.33,64.46,51.91,48.55,47.43,46.88,46. 60,45.97,42.49,41.69,38.79,38.45,37.21,37.15,36.25,32.19,31.39,30.44,28.56,27.04,26.04,23.50,23.35,22.98,21.16,17.39,17.17,16.86,16.77,16.42,13.75,12.44.

[0089] Centella asiatica (1 eq) and DIPEA (2 eq) were dissolved in a dry DMF solution in sequence. Stir at room temperature for 5 minutes, then add HATU (1.5 eq). Monitor by TLC. After approximately 30 minutes, add P3 (1.5 eq) and continue stirring at room temperature. The reaction is complete after 12 hours. The reaction is quenched with ice water and extracted with ethyl acetate three times. The organic layers are then combined. The mixture is then washed with saturated ammonium chloride solution and saturated brine in sequence. After drying over anhydrous sodium sulfate, the mixture is filtered, concentrated under reduced pressure, and purified by PTLC (dichloromethane:methanol = 20:1) to obtain the final product A9. Its NMR data are as follows: A9 (yellow-green powder, 40.1%): ESI-MS m / z 837.5 [M+Na] + . 1H NMR(500MHz,Chloroform-d)δ9.28(d,J=118.1Hz,1H),7.50(p,J=8.5,8.1Hz,1H),7.10(dd,J=7.1,4.5Hz,1H),6.95-6.84(m,1H),6.21(dt,J=10.3,5.6Hz,1H),5.99(s,1H),5.40-5.29(m,1H),5.00-4.87(m,1H),3.76-3.67(m,1H),3.66-3.58(m,1H),3.41(s,1H),3.36-3.33(m,1H),3.27(q,J=6.8,6.3Hz,2H),3.11(d,J=13.0Hz,1H),2.96(s,1H),2.87(d,J=12.8Hz,1H),2.80-2.68(m,2H),2.27(dt,J=54.2,7.6Hz,2H),2.12(d,J=8.9Hz,1H),1.99-1.93(m,2H),1.90(d,J=15.1Hz,2H),1.81(d,J=10.4Hz,1H),1.74(d,J=13.4Hz,1H),1.66(dd,J=13.8,7.0Hz,2H),1.61(d,J=6.5Hz,2H),1.58(d,J=5.4Hz,1H),1.49-1.44(m,2H),1.40(q,J=5.1,4.4Hz,2H),1.31(d,J=11.9Hz,2H),1.28(s,2H),1.25(s,2H),1.14(d,J=4.1Hz,1H),1.08(d,J=4.9Hz,3H),0.93(d,J=3.1Hz,3H),0.89(d,J=12.9Hz,3H),0.83(d,J=6.2Hz,3H),0.78(d,J=6.3Hz,3H),0.73(d,J=10.5Hz,3H). 13C NMR (126MHz, DMSO) δ177.53,172.81,170.05,168.97,166.61,146.44,138.59,136.24,132.18,117.6 0,111.60,108.99,75.56,67.37,63.89,51.88,51.66,51.26,48.56,46.98,46.87,46.56,45.98,42. Asiatic acid (1 eq) and DIPEA (2 eq) were dissolved sequentially in dry DMF. The mixture was stirred at room temperature for 5 min, followed by the addition of HATU (1.5 eq). The mixture was monitored by TLC. After approximately 30 min, P4 (1.5 eq) was added and stirring continued at room temperature. The reaction was complete after 12 hours. The reaction was quenched with ice water and extracted three times with ethyl acetate. The organic layers were then combined and washed sequentially with saturated ammonium chloride and saturated brine. After drying over anhydrous sodium sulfate, the product was filtered, concentrated under reduced pressure, and purified by PTLC (dichloromethane:methanol = 20:1) to obtain the final product A10. Its NMR data are as follows: A10 (yellow-green powder, 42.3%): ESI-MS m / z 851.5 [M+Na] + . 1H NMR(500MHz,Chloroform-d)δ8.64(d,J=21.8Hz,1H),7.50(dd,J=8.5,7.1Hz,1H),7.10(d,J=7.1Hz,1H),6.88(d,J=8.6Hz,1H),6.21(td,J=5.6,2.1Hz,1H),5.94(t,J=5.5Hz,1H),5.28(d,J=3.7Hz,1H),4.91(dd,J=12.1,5.4Hz,1H),3.73(dt,J=10.4,5.2Hz,1H),3.63(d,J=11.0Hz,1H),3.39(t,J=10.4Hz,2H),3.33(dq,J=10.8,3.9,3.2Hz,1H),3.27(q,J=6.6Hz,2H),3.09-3.02(m,1H),2.90-2.86(m,1H),2.80-2.72(m,2H),2.15-2.10(m,1H),2.00(d,J=13.8Hz,1H),1.95(dd,J=13.7,8.5Hz,2H),1.91(dd,J=8.8,3.9Hz,1H),1.89-1.82(m,2H),1.73(s,1H),1.70(d,J=6.8Hz,2H),1.66(dd,J=13.0,5.7Hz,2H),1.60(dd,J=11.3,6.0Hz,2H),1.56-1.50(m,2H),1.49(s,1H),1.48-1.44(m,2H),1.42(d,J=4.8Hz,2H),1.39(t,J=4.2Hz,1H),1.38-1.33(m,2H),1.28(s,2H),1.25(d,J=3.1Hz,3H),1.14-1.06(m,3H),1.06-1.01(m,2H),0.96(d,J=6.2Hz,3H),0.94(s,3H),0.91-0.87(m,1H),0.86-0.84(m,2H),0.83(d,J=2.9Hz,3H),0.75(s,3H). 13C NMR (126MHz, CDCl3) δ177.96,171.31,169.65,168.71,167.80,149.63,141.17,137.41,133.65,125.85,117.75,112.83,110.59,83.05,72.2 8,69.18,54.12,49.02,48.93,47.85,47.24,46.55,46.42,43.17,42.7 3,42.59,39.89,39.65,39.37,39.22,38.11,37.34,32.64,31.57,31.0 0,29.19,27.92,25.32,24.74,23.56,23.44,22.95,21.37,18.67,17.36,17.30,17.20,12.93.

[0090] Centella asiatica (1 eq) and DIPEA (2 eq) were dissolved in a dry DMF solution in sequence. Stir at room temperature for 5 minutes, then add HATU (1.5 eq). Monitor by TLC. After approximately 30 minutes, add P5 (1.5 eq) and continue stirring at room temperature. The reaction is complete after 12 hours. The reaction is quenched with ice water and extracted with ethyl acetate three times. The organic layers are then combined. The mixture is then washed with saturated ammonium chloride solution and saturated brine in sequence. After drying over anhydrous sodium sulfate, the mixture is filtered, concentrated under reduced pressure, and purified by PTLC (dichloromethane:methanol = 20:1) to obtain the final product A11. Its NMR data are as follows: A11 (yellow-green powder, 45.6%): ESI-MS m / z 865.5 [M+Na] + . 1H NMR(500MHz,Chloroform-d)δ9.11(d,J=154.1Hz,1H),7.54-7.45(m,1H),7.09(d,J=7.1Hz,1H),6.87(d,J=8.5Hz,1H),6.18(s,1H),6.01-5.78(m,1H),5.30(s,1H),4.96-4.86(m,1H),3.71(dd,J=13.3,6.3Hz,1H),3.64(d,J=8.2Hz,1H),3.43(d,J=53.0Hz,2H),3.25(q,J=7.5Hz,2H),3.08(dt,J=13.0,6.3Hz,1H),2.96(s,1H),2.86(t,J=11.0Hz,1H),2.75(q,J=16.0,14.8Hz,2H),2.42(s,1H),2.38-2.21(m,2H),2.21-2.02(m,2H),1.97(d,J=23.4Hz,2H),1.90(d,J=19.8Hz,2H),1.87-1.76(m,2H),1.74(d,J=13.2Hz,1H),1.71-1.65(m,2H),1.60(d,J=13.4Hz,2H),1.48(d,J=7.4Hz,2H),1.42(d,J=7.7Hz,2H),1.37(d,J=13.9Hz,2H),1.28(s,2H),1.26(s,2H),1.25(s,2H),1.08(s,3H),1.01(d,J=26.4Hz,3H),0.94(s,3H),0.89(d,J=6.4Hz,1H),0.85(d,J=6.6Hz,3H),0.79(d,J=8.7Hz,3H),0.76(d,J=3.0Hz,3H). 13C NMR (126MHz, DMSO) δ176.74,172.84,171.39,170.09,167.32,146.22,138.68,136.28,132.21 ,126.28,110.82,109.03,75.56,67.38,64.66,51.90,51.25,49.14,47.00,46.90,46.56,46. 01,43.07,41.87,41.72,38.42,37.25,37.10,31.89,31.00,30.56,30.46,28.95,28.76,27.33,26.37,26.19,24.18,23.53,23.34,23.01,22.20,21.16,17.77,17.15,16.96,16.80,14.56.

[0091] Centella asiatica (1 eq) and DIPEA (2 eq) were dissolved in a dry DMF solution in sequence. Stir at room temperature for 5 minutes, then add HATU (1.5 eq) and monitor by TLC. After approximately 30 minutes, add P6 (1.5 eq) and continue stirring at room temperature. The reaction was complete after 12 hours. The reaction was quenched with ice water and extracted with ethyl acetate three times. The organic layers were combined. The mixture was then washed with saturated ammonium chloride solution and saturated brine in sequence. After drying over anhydrous sodium sulfate, the mixture was filtered, concentrated under reduced pressure, and purified by PTLC (dichloromethane:methanol = 20:1) to obtain the final product A12. Its NMR data are as follows: A12 (yellow-green powder, 38.2%): ESI-MS m / z 879.5 [M+Na] + . 1 H NMR(500MHz,Chloroform-d)δ8.92(d,J=84.5Hz,1H),7.54-7.44(m,1H),7.11- 7.04(m,1H),6.89(dd,J=12.5,8.6Hz,1H),6.19(t,J=5.7Hz,1H),5.89(s,1H),5 .34(t,J=4.9Hz,1H),5.29(d,J=3.7Hz,1H),4.91(dt,J=13.1,3.5Hz,1H),3.80 -3.68(m,2H),3.67-3.52(m,2H),3.49-3.31(m,2H),3.24(q,J=6.5Hz,2H),3.15

[0092] -3.06(m,1H),2.97(d,J=11.2Hz,1H),2.87(d,J=13.6Hz,1H),2.75(td,J=12.4,6.3Hz,2H),2.33(t,J=7.6Hz,1H),2.21(t,J=7 .7Hz,1H),2.10(d,J=24.5Hz,2H),2.05-1.99(m,2H),1.99-1.94(m,2H),1.94-1.88(m,2H),1.85(dd,J=19.2,10.5Hz,2H),1.7 4(d,J=13.5Hz,1H),1.66(q,J=7.1Hz,2H),1.64-1.61(m,2H),1.60(s,1H),1.47-1.44(m,2H),1.40(d,J=6.5Hz,2H),1.32(s,2 H),1.29(s,2H),1.25(s,2H),1.08(s,3H),1.01(s,3H),0.94(s,3H),0.87(d,J=6.9Hz,3H),0.84(d,J=5.8Hz,3H),0.77(s,3H). 13 C NMR (126MHz, CDCl3) δ179.62,172.54,169.63,168.88,167.83,147.09,140.23,136.3 4,131.91,126.64,119.84,111.71,109.00,79.87,69.50,68.63,54.12,49.46,48.23 ,47.85,47.09,45.01,41.87,39.88,39.65,39.37,39.22,37.74,37.33,32.45,31.56,30.99,29.15,27.91,24.96,24.40,23.56,23.45,22.94,20.57,18.30,17.19,13.00.

[0093] Example 5: Preparation of intermediates O1 to O3

[0094] A. Intermediate BO1 (n3 = 1) / BO2 (n3 = 2) / BO3 (n3 = 3)

[0095] The specific synthesis method is as follows:

[0096] Fluorothalidamide (2 g, 7.241 mmol, 1 eq) was dissolved in 20 ml of DMF and stirred to dissolve. DIEA (1.872 ml, 14.481 mmol, 2 eq) and tert-butyl (2-(2-aminoethoxy)ethyl)carbamate (1.2 eq) were then added and the mixture was heated to 90°C and refluxed for 12 h. After completion, the reaction was quenched with ice water and extracted with ethyl acetate. The combined organic phases were washed sequentially with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to yield intermediate O1 (yellow-green powder, 32.6%).

[0097] Fluorothalidamide (2 g, 7.241 mmol, 1 eq) was dissolved in 20 ml of DMF with stirring. DIEA (1.872 ml, 14.481 mmol, 2 eq) and N-Boc-3,6-dioxa-1,8-octanediamine (1.2 eq) were then added and the mixture was heated to 90°C and refluxed for 12 h. After completion, the reaction was quenched with ice water and extracted with ethyl acetate. The combined organic phases were washed with saturated sodium bicarbonate solution and then saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to yield intermediate O2 (yellow-green powder, 29.3%).

[0098] Fluorothalidamide (2 g, 7.241 mmol, 1 eq) was dissolved in 20 ml of DMF and stirred to dissolve. DIEA (1.872 ml, 14.481 mmol, 2 eq) and N-Boc-1,11-diamino-3,6,9-trioxaundecane (1.2 eq) were then added and the mixture was heated to 90°C and refluxed for 12 h. After completion, the reaction was quenched with ice water and extracted with ethyl acetate. The combined organic phases were washed sequentially with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to obtain intermediate O3 (yellow-green powder, 34.1%).

[0099] B. Intermediate O1(n3=1) / O2(n3=2) / O3(n3=3)

[0100] The specific synthesis method is as follows:

[0101] Intermediate BO1 was dissolved in 10 ml of dichloromethane and the solution was placed in an ice bath. In the ice bath, 3 ml of trifluoroacetic acid (TFA) was slowly added dropwise. After the addition was complete, the reaction mixture was slowly brought to room temperature and the reaction continued. The reaction progress was monitored by thin-layer chromatography (TLC), and completion was achieved after approximately 12 hours. After completion of the reaction, the solvent was removed by concentration under reduced pressure to yield BP8 as a yellow-green powder in a yield of 32.6%.

[0102] Intermediate BO2 was dissolved in 10 ml of dichloromethane and the solution was placed in an ice bath. In the ice bath, 3 ml of trifluoroacetic acid (TFA) was slowly added dropwise. After the addition was complete, the reaction mixture was slowly brought to room temperature and the reaction continued. The reaction progress was monitored by thin-layer chromatography (TLC), and completion was achieved after approximately 12 hours. After completion of the reaction, the solvent was removed by concentration under reduced pressure to yield BP8 as a yellow-green powder in a yield of 29.3%.

[0103] Intermediate BO3 was dissolved in 10 ml of dichloromethane and the solution was placed in an ice bath. In the ice bath, 3 ml of trifluoroacetic acid (TFA) was slowly added dropwise. After the addition was complete, the reaction mixture was slowly brought to room temperature and the reaction continued. The reaction progress was monitored by thin-layer chromatography (TLC), and completion was achieved after approximately 12 hours. After completion of the reaction, the solvent was removed by concentration under reduced pressure to yield BP8 as a yellow-green powder in a yield of 34.1%.

[0104] Example 6: Preparation of final products A13 to A15

[0105] Centella asiatica (1 eq) and DIPEA (2 eq) were dissolved in a dry DMF solution in sequence. Stir at room temperature for 5 minutes, then add HATU (1.5 eq) and monitor by TLC. After about 30 minutes, add O1 (1.5 eq) and continue stirring at room temperature. The reaction was complete after 12 hours. The reaction was quenched with ice water, extracted with ethyl acetate three times, and the organic layers were combined. Subsequently, the mixture was washed with saturated ammonium chloride solution and saturated brine in sequence. After drying over anhydrous sodium sulfate, the mixture was filtered, concentrated under reduced pressure, and purified by PTLC (dichloromethane: methanol = 20:1) to obtain the final product A13. Its NMR data are as follows: A13 (yellow-green powder, 34.8%): ESI-MS m / z 853.5 [M+Na] +.1H NMR(500MHz,Chloroform-d)δ9.35(s,1H),7.52(td,J=7.9,3.9Hz,1H),7.13(d,J=7.0Hz,1H),6.95(dd,J =8.6,3.9Hz,1H),6.47(q,J=6.2Hz,1H),6.36(d,J=7.6Hz,1H),5.20(dd,J=22.1,3.9Hz,1H),4.93(td,J=1 1.3,5.2Hz,1H),3.70(d,J=5.2Hz,1H),3.68(d,J=5.3Hz,2H),3.58-3.53(m,2H),3.51(d,J=12.2Hz,2H),3 .47(s,2H),3.38(dd,J=9.6,4.8Hz,2H),3.31(td,J=10.9,5.1Hz,2H),2.86(d,J=12.7Hz,1H),2.76(t,J=1 1.8Hz,2H),2.09(dd,J=10.6,4.7Hz,2H),2.00(q,J=6.5Hz,1H),1.98-1.89(m,2H),1.84(d,J=6.8Hz,2H), 1.80(d,J=6.6Hz,2H),1.68(d,J=12.4Hz,1H),1.61-1.52(m,2H),1.48(d,J=6.0Hz,1H),1.46-1.42(m,2H) ,1.42-1.36(m,2H),1.36-1.31(m,2H),1.31-1.28(m,1H),1.25(d,J=4.9Hz,3H),1.18(d,J=14.7Hz,2H),1 .04(s,3H),0.91(s,3H),0.87(d,J=6.0Hz,3H),0.81-0.78(m,2H),0.74(d,J=5.1Hz,3H),0.73(s,3H).13C NMR (126MHz, CDCl3) δ178.96,173.69,169.46,168.96,167.04,146.94,139.71,136.46,132.99,127.35,120.78,111.88,108.56,81.60,70.04,69.96,69.83,68.64,53.27,50.24,49.06,47.86,4 7.41,43.40,42.79,42.56,39.64,39.14,37.95,37.30,32.01,31.58,31.00,29.82,29.45,27.66,27.18,25.11,23.48,22.81,22.62,20.68,18.72,17.32,17.12,13.06.

[0106] Centella asiatica (1 eq) and DIPEA (2 eq) were dissolved in a dry DMF solution. Stir at room temperature for 5 minutes, then add HATU (1.5 eq). Monitor by TLC. After approximately 30 minutes, add O2 (1.5 eq) and continue stirring at room temperature. The reaction is complete after 12 hours. The reaction is quenched with ice water and extracted with ethyl acetate three times. The organic layers are combined. The mixture is then washed with saturated ammonium chloride solution and saturated brine. After drying over anhydrous sodium sulfate, the mixture is filtered, concentrated under reduced pressure, and purified by PTLC (dichloromethane:methanol = 20:1) to obtain the final product A14. Its NMR data are as follows: A14 (yellow-green powder, 31.2%): ESI-MS m / z 897.5 [M+Na] + . 1H NMR(500MHz,Chloroform-d)δ8.87(d,J=22.9Hz,1H),7.53-7.46(m,1H),7.11(d,J=7.2Hz,1H),6.91(dd,J=8.5,2.3Hz,1H),6.48(t,J=5.7Hz,1H),6.37(d,J=5.6Hz,1H),5.31(d,J=9.5Hz,1H),4.98-4.88(m,1H),3.74-3.71(m,2H),3.67-3.65(m,2H),3.65-3.60(m,2H),3.60(d,J=4.9Hz,1H),3.51(q,J=4.8Hz,2H),3.47(q,J=5.6,4.2Hz,2H),3.41(td,J=7.8,7.2,3.9Hz,2H),3.28(dt,J=10.4,4.7Hz,1H),2.87(d,J=13.6Hz,1H),2.74(qd,J=14.9,13.3,4.0Hz,2H),2.46-2.31(m,1H),2.14-2.09(m,1H),1.98(q,J=5.9,5.2Hz,2H),1.90-1.81(m,2H),1.74(s,2H),1.71(s,2H),1.67(s,2H),1.63(s,2H),1.60(s,1H),1.50-1.46(m,2H),1.42(dd,J=13.5,4.1Hz,2H),1.37(s,2H),1.28(s,2H),1.22(d,J=7.3Hz,2H),1.08(s,3H),1.01(s,3H),0.93(s,3H),0.86(d,J=3.6Hz,3H),0.84(d,J=3.2Hz,3H),0.77(s,3H). 13C NMR (126MHz, CDCl3) δ178.57,173.15,172.16,169.44,167.80,146.80,139.51,136.21,132.67, 125.76,117.87,113.46,110.32,80.94,70.83,70.24,69.66,69.45,68.81,68.46,54.29,51.95 ,50.36,48.98,47.84,47.41,46.30,42.76,42.56,39.79,39.68,39.37,39.17,38.40,36.76,32.77,31.50,30.97,27.88,24.90,24.37,23.56,23.41,20.98,18.23,17.36,17.29,17.02,13.09.

[0107] Asiatic acid (1 eq) and DIPEA (2 eq) were dissolved sequentially in a dry DMF solution. Stir at room temperature for 5 minutes, then add HATU (1.5 eq). Monitor by TLC. After approximately 30 minutes, add O3 (1.5 eq) and continue stirring at room temperature. The reaction was complete after 12 hours. The reaction was quenched with ice water and extracted three times with ethyl acetate. The organic layers were combined. The mixture was then washed sequentially with saturated ammonium chloride solution and saturated brine. After drying over anhydrous sodium sulfate, the mixture was filtered, concentrated under reduced pressure, and purified by PTLC (dichloromethane:methanol = 20:1) to obtain the final product A15. Its NMR data are as follows: A15 (yellow-green powder, 36.7%): ESI-MS m / z 941.5 [M+Na] + . 1H NMR(500MHz,Chloroform-d)δ9.26(d,J=18.3Hz,1H),7.48(dd,J=8.5,7.1Hz,1H),7.09(d,J=7.2Hz,1H),6.91(dd,J=8.5,2.5Hz,1H),6.47(t,J=5.6Hz,1H),6.40(q,J=5.0Hz,1H),5.34-5.24(m,1H),4.92(ddt,J=9.0,5.4,3.3Hz,1H),3.72(t,J=5.4Hz,2H),3.68(s,2H),3.67(s,2H),3.66-3.62(m,2H),3.60(d,J=4.3Hz,2H),3.58-3.53(m,1H),3.52-3.49(m,2H),3.47(t,J=2.9Hz,2H),3.40(dd,J=9.6,2.0Hz,1H),3.35(d,J=10.7Hz,1H),3.27(td,J=8.9,4.4Hz,1H),2.87-2.82(m,1H),2.75(dtt,J=8.7,6.1,3.7Hz,2H),2.10(dd,J=7.6,3.9Hz,1H),2.00(d,J=5.8Hz,1H),1.98-1.94(m,2H),1.92(d,J=12.1Hz,2H),1.88-1.81(m,2H),1.71(d,J=13.2Hz,1H),1.60(dd,J=10.5,6.5Hz,2H),1.49-1.44(m,2H),1.44-1.38(m,2H),1.37-1.32(m,2H),1.32(d,J=2.5Hz,1H),1.29-1.26(m,2H),1.26(s,1H),1.24(d,J=3.5Hz,2H),1.23(s,2H),1.06(s,3H),1.01(d,J=3.7Hz,1H),0.98(s,3H),0.92(s,3H),0.87(d,J=6.6Hz,1H),0.83(dd,J=6.4,3.3Hz,3H),0.78(d,J=3.1Hz,3H),0.75(s,3H). 13C NMR (126MHz, CDCl3) δ178.50,171.93,169.46,169.12,167.81,145.65,139.56,136.74,133.29,132. 66,125.77,116.92,112.33,110.44,82.37,70.86,70.80,70.77,70.69,70.42,69.73,69.67,68.64,5 3.80,49.00,48.64,47.88,47.47,46.28,43.65,42.59,40.32,39.69,39.30,39.18,38.11,37.23,33.54,31.57,31.00,29.82,29.05,27.92,27.34,25.67,24.57,23.45,22.81,21.35,17.39,16.70,13.0

[0108] Example 7: In vitro anti-tumor activity test (IC50) of the asiatic acid PROTACs compound of the present invention

[0109] The asiatic acid PROTACs compounds of the present invention were tested for their ability to inhibit tumor cell proliferation using the conventional CCK-8 assay. When tumor cells (B16 cells (mouse skin melanoma cells)) grew to 80%-90% of the culture dish, the cells were digested from the culture dish, centrifuged, and resuspended in fresh DMEM / 1640 complete medium. The cells were counted under a microscope, and the cell suspension was diluted to 50 cells / μL. A circle of sterile PBS was placed on the outermost perimeter of a 96-well plate, 100 μL per well, and 100 μL of the diluted cell suspension was placed in each well of the remaining wells. The plate was then incubated at 37°C in a 5% CO2 incubator. After 24 hours, the medium in the 96-well plate was aspirated, and the drug was diluted with fresh complete medium in a certain concentration gradient and then added to the 96-well plate in sequence. After 48 hours, the medium containing the drug was removed, and CCK-8 liquid diluted with culture medium (100 μL culture medium + 10 μL CCK-8) was added. The cells were incubated in a 37°C, 5% CO2 incubator for 30-40 minutes. The absorbance was measured at OD450 on a microplate reader, and the inhibition rate was calculated based on the absorbance at OD450. The data were used to calculate the half-maximal inhibitory concentration (IC50) using SPSS software.

[0110] The calculation formula is as follows:

[0111] Inhibition rate = [(Ac-As) / (Ac-Ab)] × 100%

[0112] As: absorbance of experimental wells (containing cells, culture medium, CCK-8 solution, and drug solution);

[0113] Ac: absorbance of control well (containing cells, culture medium, and CCK-8 solution, but no drug);

[0114] Ab: absorbance of blank wells (containing culture medium and CCK-8 solution, but not cells or drugs).

[0115] Experimental Results: To further evaluate the in vitro antitumor activity of the target compound, B16 cells (mouse skin melanoma), MCF-7 cells (human breast cancer), A549 cells (lung cancer), and HELA cells (cervical cancer) were used as test tumor lines, with Asiatic acid A serving as the control group. The test results are shown in Tables 1 and 2. Overall, the Asiatic acid PROTAC compound exhibited superior antitumor activity compared to the Asiatic acid derivative itself. Based on the IC50 values and compound structure, A2 was selected as the dosing group for further western blotting studies.

[0116] Table 1 In vitro antitumor activity (IC50) of asiatic acid and A1-A15 against B16 cells

[0117]

[0118]

[0119] Table 2: In vitro antitumor activity (IC50) of asiatic acid and A1-A15

[0120]

[0121] Example 8: Degradation efficiency test of the asiatic acid PROTACs compound of the present invention on the target protein

[0122] The protein degradation efficiency of the asiatic acid PROTAC compounds of the present invention was tested using a conventional Western blotting method. Based on the existing in vitro anti-tumor activity test data (IC50), the degradation efficiency of the asiatic acid PROTAC compounds against the target protein STAT3 was further tested. MCF-7 cells were treated with PROTAC compounds with better activity and different structures to observe the degradation efficiency of the target protein STAT3.

[0123] Experimental results: After the initial screening at the protein level ( Figure 1 ), A2 showed good STAT3 degradation efficiency.

[0124] Example 9: Degradation efficiency test of target protein by different concentrations of A2 in the asiatic acid PROTACs compound of the present invention

[0125] Through the previous testing of the asiatic acid PROTACs compound on the target protein degradation efficiency, A2 showed good STAT3 degradation efficiency. Therefore, different concentrations of A2 were used to treat MCF-7 cells to study the target protein degradation efficiency at different concentrations and explore whether the degradation of STAT3 is dependent on the treatment concentration of A2. The test method used was conventional Western blotting.

[0126] Experimental results: The degradation of STAT3 is concentration-dependent on A2 ( Figure 2 ).

[0127] Example 10: Degradation efficiency test of target protein by A2 at different action times in the Asiatic acid PROTACs compound of the present invention

[0128] Because PROTACs are time-dependent in their degradation of target proteins, MCF-7 cells were treated with A2 for varying durations. Conventional Western blotting was used for the assay.

[0129] Experimental results: A2 can increase the degradation efficiency of target protein STAT3 by prolonging the time ( Figure 3 ).

[0130] The above description is merely a preferred embodiment of the present invention and therefore cannot be used to limit the scope of the present invention. In other words, equivalent changes and modifications made within the scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. An asiatic acid PROTACs, characterized by: Its structural formula is in, E3 Ligase is selected from lenalidomide and pomalidomide, Linker is the first fatty chain, the second fatty chain or the PEG chain, The first fatty chain is -NH-(CH2) n1 -NH-(CH2)3-CO-, n1 is 1, 2, 3, 4, 5 or 6, The second fatty chain is -NH-(CH2) n2 -CH2-NH-, n2 is 1, 2, 3, 4, 5 or 7, The PEG chain is -NH-(CH2-CH2-O) n3 -CH2-CH2-NH-, n3 is 1, 2 or 3.

2. The asiatic acid PROTACs according to claim 1, characterized in that: Its structural formula is selected from at least one of the following:

3. The method for preparing asiatic acid PROTACs according to claim 1 or 2, characterized in that: The reaction route is:

4. Use of the asiatic acid PROTACs or pharmacologically or physiologically acceptable salts thereof according to claim 1 or 2 in preparing a composition for treating tumor diseases.

5. The use according to claim 4, characterized in that: The tumor diseases include brain cancer, breast cancer, cervical cancer, stomach cancer, liver cancer and lung cancer.

6. A composition for treating tumor diseases, characterized in that: The active ingredient comprises the asiatic acid PROTACs or a pharmacologically or physiologically acceptable salt thereof according to claim 1 or 2.

7. Use of the asiatic acid PROTACs or pharmacologically or physiologically acceptable salts thereof according to claim 1 or 2 in the preparation of an anti-inflammatory composition.

8. An anti-inflammatory composition, characterized in that: The active ingredient comprises the asiatic acid PROTACs or a pharmacologically or physiologically acceptable salt thereof according to claim 1 or 2.

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