Preparation and application of a protac degrader

Abstract

The application provides preparation and application of a PROTAC degrading agent, and relates to the field of medicines.The ganoderic acid A-PROTAC compound with degrading activity provided by the application takes ganoderic acid A as raw material, retains the structural skeleton of ganoderic acid A, and is connected with E3 ubiquitin ligase Cereblon (CRBN) or VHL ligand by using different lengths of fatty chains or polyethylene glycol (PEG) chains to obtain ganoderic acid A derivatives GAA C1-C10 and V1-V10.The ganoderic acid A derivative with novel antitumor effect and the like is synthesized by a simple synthesis method.The ganoderic acid A derivative has the structure shown in the following general formula I.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to the preparation and application of a PROTAC degrading agent based on Ganoderma lucidum triterpenes. Background Technology

[0002] Reishi mushroom is the dried fruiting body of the fungus *Ganoderma lucidum* Karst or *Ganoderma sinese*, belonging to the family Polyporaceae of the class Basidiomycetes. It is believed to have the effects of tonifying qi, calming the mind, relieving cough, and alleviating asthma. As a traditional edible and medicinal fungus, reishi has a long history of use, primarily treating restlessness, insomnia, palpitations, and consumptive cough. Modern research and clinical practice have demonstrated that reishi has preventative and therapeutic effects on various diseases, including enhancing immunity, anti-tumor activity, anti-cardiovascular disease activity, anti-aging, and liver protection. Reishi is rich in triterpenes, polysaccharides, heteroterpenes, steroids, alkaloids, and nucleosides, with triterpenes being the main component. Currently, over 300 triterpenoid compounds have been extracted from reishi medicinal materials, exhibiting a wide range of pharmacological effects, such as anti-tumor, antioxidant, anti-inflammatory, hypoglycemic, and liver-protective activities.

[0003] Ganoderic acid A (GAA) is a triterpenoid active ingredient isolated from Ganoderma lucidum (reishi mushroom). It has anti-tumor, sedative, analgesic, hepatoprotective, and immunomodulatory effects. Studies have confirmed that GAA exerts its anti-tumor effects through mechanisms such as arresting the cell cycle, inhibiting tumor cell proliferation, and inducing tumor cell apoptosis. It has been widely used as an adjuvant therapy for various tumors, including lung cancer, liver cancer, cervical cancer, prostate cancer, breast cancer, and colon cancer.

[0004] Patent CN 102219822 B discloses a ganoderic acid T amide derivative TLTO-A in the field of biomedicine, its synthesis, and its application. Using ganoderic acid T as a lead compound, its structure was modified to obtain the amide derivative TLTO-A with better antitumor activity. The compound of this invention significantly inhibits the growth and proliferation of human cervical cancer cell line HeLa, human highly metastatic lung cancer 95D, and human liver cancer cell line HepG2, and can induce apoptosis in tumor cells. The structural formula is shown below:

[0005]

[0006] Patent CN 111171025 A relates to the preparation of a degrading agent targeting eEF2K and its application in anti-breast cancer technology, belonging to the field of anti-tumor pharmaceutical technology. The technical problem solved by this invention is to provide a compound as an eEF2K degrading agent. The compound of this invention, or its pharmaceutically acceptable salt, has the activity of degrading eEF2K protein, can act as an eEF2K degrading agent, and can induce apoptosis in tumor cells, thereby exerting an anti-breast cancer effect. The structural formula is shown below:

[0007]

[0008] In recent years, a novel targeted protein degradation technology—proteolysis-targeting chimeras (PROTACs)—has attracted widespread attention from researchers due to its potential to degrade druggable targets, achieve stronger cellular activity than corresponding inhibitors in inhibiting target protein function, and overcome drug resistance. Drug development targeting PROTAC technology has become a new hot topic. PROTAC technology involves linking a target protein ligand with an E3 ubiquitin-ligase ligase (E3) ligand through a suitable linker chain, forming a targeted-induced protein degradation chimera. In vivo, PROTACs can simultaneously recruit the target protein and specific E3 to achieve ubiquitination and degradation of the target protein, representing a promising disease treatment strategy utilizing the ubiquitin-proteasome pathway to degrade target proteins.

[0009] Traditional Chinese medicine (TCM) active ingredients are an important source of lead compounds due to their abundant resources, complex structures, diverse skeletons, good drug-like properties, and significant advantages and characteristics in anti-tumor activity. Therefore, leveraging the advantages of PROTAC, the preparation of GAA-based PROTAC degrading agents and their application in the targeted degradation of disease-related proteins is of great significance for tumor treatment. Summary of the Invention

[0010] To address the aforementioned problems, this invention provides a method for preparing and applying a PROTAC degrading agent. Using ganoderic acid A as a raw material, the structural framework of ganoderic acid A is retained. By using fatty acid chains or polyethylene glycol (PEG) chains of different lengths to link it with E3 ubiquitin ligase Cereblon or VHL ligands, ganoderic acid A derivatives GAA C1-C10 and V1-V10 are obtained. The synthesis method is simple and suitable for widespread application.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] On one hand, the present invention provides a PROTAC degrading agent having the structure shown in general formula I:

[0013]

[0014] E3 ligase is a protein ligand in the E3 ubiquitin ligase complex, and is at least one of Cereblon protein ligand or VHL protein ligand; linker is a linking group between ganoderic acid A and the protein ligand in the E3 ubiquitin ligase complex.

[0015] Preferably, the linker can be at least one of aliphatic chain or polyethylene glycol (PEG) chain.

[0016] Preferably, the specific protein ligand includes, but is not limited to, at least one of Cereblon protein ligand and VHL protein ligand.

[0017] Preferably, the Cereblon protein ligand includes, but is not limited to, pomalidomide or its derivatives.

[0018] Preferably, the general formula of the Cereblon protein ligand is as follows:

[0019]

[0020] Preferably, the general formula of the VHL protein ligand is as follows:

[0021]

[0022] Preferably, the linking group includes, but is not limited to, any one of the following structures:

[0023]

[0024] Preferably, the compound may have the following structure:

[0025]

[0026]

[0027] On the other hand, the present invention provides a method for preparing a PROTAC degrading agent, comprising the following steps:

[0028] 1) Using 3-fluorophthalic anhydride and 3-aminopiperidine-2,6-dione hydrochloride as raw materials, a solvent and sodium acetate were added, and the mixture was heated under reflux to obtain compound S1;

[0029] 2) Using compound S1 and 3-azidopropylamine as raw materials, a solvent was added and N,N-diisopropylethylamine was reacted to obtain compound S2;

[0030] 3) Compound S3, ganoderic acid A, was added to a solvent, and O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate, N,N-diisopropylethylamine and propargylamine were added respectively. The reaction was carried out to obtain compound S4.

[0031] 4) Using products S2 and S4 as raw materials, add solvent and cuprous thiophene-2-carboxylic acid to react and obtain the final product.

[0032] Preferably, the solvent in step 1) includes, but is not limited to, acetic acid.

[0033] Preferably, the solvent in step 2) can be DMF.

[0034] Preferably, the reaction temperature in step 2) can be 85-95°C, and more preferably, the reaction temperature is 90°C.

[0035] Preferably, the solvent in step 3) includes, but is not limited to, dichloromethane.

[0036] Preferably, the solvent in step 4) can be dichloromethane.

[0037] Preferably, the above preparation method further includes purification: the crude product obtained after the reaction in step 4) is concentrated and purified by column chromatography with dichloromethane / methanol = 20 / 1 to obtain the product.

[0038] In another aspect, the present invention provides the application of the above-mentioned degrading agent or the degrading agent prepared by the preparation method described above in the preparation of antitumor drugs and antitumor-related targeted drugs.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] 1. Using ganoderic acid A as a raw material, while retaining its structural framework, ganoderic acid A was linked to E3 ubiquitin ligase Cereblon or VHL ligand using different linking strands to obtain ganoderic acid A derivatives GAA C1-C10 and V1-V10. Novel ganoderic acid A derivatives with antitumor and other effects were synthesized using a simple synthetic method.

[0041] 2. Ganoderic acid A, as the most active and abundant compound among Ganoderma lucidum triterpenes, has enhanced antitumor activity and other biological effects after structural modification. Modifying ganoderic acid A saves natural plant resources and is conducive to sustainable development. It also exhibits better antitumor activity than the original. Attached Figure Description

[0042] Figure 1 The antiproliferative activity of compounds C1-C10 and V1-V10 against SJSA-1 cells;

[0043] Figure 2The antiproliferative activity of compounds C1-C10 and V1-V10 against MCF-7 cells;

[0044] Figure 3 The antiproliferative activity of compounds V1-V10 against MDA-MB-231 cells;

[0045] Figure 4 To detect the levels of NR2E1, MDM2, p53, p21 and β-actin proteins in cells treated with V9 (75, 85, 95, 105, 115 μM) for 48 h using Western blotting;

[0046] Figure 5A Cells were treated with V9 (100 μM) for 48 h and then with the proteasome inhibitor MG-132 (25 μM) for 6 h. The expression levels of NR2E1, MDM2 and β-actin proteins were detected by Western blotting.

[0047] Figure 5B To detect the levels of NR2E1, MDM2, and β-actin proteins in cells after 48 h of treatment with GAA (100 μM), VHL ligand (100 μM), a mixture of GAA and VHL ligands (100 μM), and V9 (100 μM) by immunoblotting. Detailed Implementation

[0048] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the invention is further illustrated below with specific embodiments. However, these embodiments are merely preferred embodiments and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the scope of protection of this invention. It is worth noting that the raw materials used in this invention are all common commercially available products, and their sources are not specifically limited. The technical and scientific terms used in the embodiments have the meanings commonly understood by those skilled in the art to which this invention pertains.

[0049] All reagents used in the examples were commercially available analytical grade.

[0050] Example 1

[0051] GAA-C1 Synthesis Route:

[0052]

[0053] Dissolve S0 (300 mg, 1.81 mmol) and 3-aminopiperidine-2,6-dione hydrochloride (298 mg, 1.81 mmol) in acetic acid, add sodium acetate (223 mg, 2.72 mmol), heat to reflux, and stir until the reactants have reacted completely. After concentration, perform dichloromethane / methanol = 50 / 1 column chromatography (ball mill flash column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain white solid product S1 (482 mg, 96%).

[0054] S1 (50 mg, 0.18 mmol) and 3-azidopropylamine (18 mg, 0.18 mmol) were dissolved in DMF, and DIPEA (7 mg, 0.05 mmol) was added. The mixture was stirred at 90 °C until the reactants were completely reacted. After concentration, the mixture was subjected to dichloromethane / methanol = 50 / 1 column chromatography (ball mill flash column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain yellow solid S2 (55 mg, 85%).

[0055] S3 (500 mg, 0.97 mmol) and propargylamine (59 mg, 1.06 mmol) were dissolved in dichloromethane. TBTU (342 mg, 1.06 mmol) and DIPEA (138 mg, 1.06 mmol) were added, and the mixture was stirred until the reactants were completely reacted. After concentration, dichloromethane / methanol = 30 / 1 column chromatography (ball mill flash chromatography column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) yielded a white solid S4 (550 mg, 95%).

[0056] S4: 1 H NMR (600MHz, CDCl3): δ6.24 (t, J = 4.8Hz, 1H, NH), 4.76-4.73 (m, 1H), 4.61-4.58

[0057] (m,1H),4.03-3.94(m,2H),2.87-2.72(m,4H),2.50-2.37(m,5H),2.22(t,J=2.4 Hz,1H),2.21(dd,J=16.2,9.6Hz,1H),2.03-2.00(m,1H),1.99-1.96(m,1H),1.8 0-1.77(m,3H),1.69-1.65(m,2H),1.48-1.43(m,1H),1.24(d,J=7.8Hz,6H,),1. 14(d,J=7.2Hz,3H),1.08(d,J=14.4Hz,6H),0.96(s,3H),0.85(d,J=6.6Hz,3H);

[0058] 13 C-APT (150MHz, CDCl3): δ217.6,209.8,199.8,175.5,159.5,140.2,79.5,72.4,71.8,68.9,54.0,51.8,49.9,48.7 ,48.1,47.2,46.8,46.7,38.0,36.3,35.7,35.6,34.4,32.8,29.4,29.0,27.5,20.8,19.8,19.6,19.5,17.8,17.3.

[0059] Dissolve the above S2 (50 mg, 0.14 mmol) and S4 (84 mg, 0.14 mmol) in dichloromethane, add an appropriate amount of cuprous thiophene-2-carboxylate, stir until the reactants react completely, concentrate, and then perform dichloromethane / methanol = 20 / 1 column chromatography (ball mill flash chromatography column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain yellow-green solid product S5 (56 mg, 44%).

[0060] C1: 1H NMR(600MHz, CDCl3):~50:50mixture of rotamers*indicates minorrotamer δ7.62(s,1H),7.47(t,J=6Hz,1H),7.07(d,J=6.8Hz,1H),6.88-6.87(m,1H),6.81(d,J=8.4Hz),6.25(m,1H,NH), 4.69-4.68(m,1H),4.57-4.51(m,3H),4.43-4.41(m,2H),4.27-4.22(m,1H,OH),4.03(bs,1H,OH),3.31-3.30(m,2H),2.88-2.6 9(m,8H),2.49-2.39(m,4H),2.23-2.21(m,1H),2.10-2.07(m,4H),2.01-1.99(m,1H),1.75-1.63(m,5H),1.47-1.41(m,1H),1. 22-1.20(m,6H,2×CH3),1.10-1.08(m,6H,2×CH3),1.07-1.06(m,3H,1×CH3),0.87(s,3H,1×CH3),0.80(d,J=6.2Hz,3H,1×CH3).

[0061] 13 C-APT (150MHz, CDCl3): δ217.5,210.0,*210.0,199.8,176.2,*172.0,171.9,169.5,169.4,167 .7,159.7,146.4,*145.5,145.4,140.1,136.5,132.5,122.8,116.7,112.0,110.4,72.3,68.8,5 4.0,51.8,49.8,49.0,48.8,48.1,47.9,47.2,*46.8,46.7,39.7,38.0,36.1,35.9,35.6,35.1,34.4,32.8,*32.7,31.5,29.8,29.5,28.9,27.5,22.9,20.8,*19.8,19.8,19.6,19.5,18.0,17.2.

[0062] The preparation methods for GAA C2-10 and V1-10 are the same as those for GAA-1, and the specific steps are as follows:

[0063] Synthetic route of GAA-C2:

[0064]

[0065] S1 (50 mg, 0.18 mmol) and azido-PEG1-amine (23 mg, 0.18 mmol) were dissolved in DMF, and DIPEA (7 mg, 0.05 mmol) was added. The mixture was stirred at 90 °C until the reactants were completely reacted. After concentration, the mixture was subjected to dichloromethane / methanol = 50 / 1 column chromatography (ball mill flash column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain a yellow solid product S6 (52 mg, 74%).

[0066] Dissolve the above S6 (50 mg, 0.13 mmol) and S4 (77 mg, 0.13 mmol) in dichloromethane, add an appropriate amount of cuprous thiophene-2-carboxylate, stir until the reactants react completely, concentrate, and then perform dichloromethane / methanol = 20 / 1 column chromatography (ball mill flash chromatography column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain yellow-green solid product S7 (46 mg, 38%).

[0067] C2: 1H NMR(600MHz,CDCl3):~50:50mixture of rotamers*indicates minorrotamerδ7.80(s,1H),7.53-7.49(m,1H),7.12(t,J=6.6Hz,1H),6.88-6.86(m,1H),6.74-6.73(m,1H),6.45(q,J=5.4Hz,1H),5.07-5.01(m,1H),4.74-4.69(m,1H),4.60-4.50(m,4H),4.48-4.42(m,1H),3.87-3.84(m,2H),3.70(t,J=4.8Hz,2H),3.47-3.43(m,2H),2.85-2.68(m,7H),2.49-2.45(m,2H),2.40-2.35(m,2H),2.15-2.12(m,1H),2.01-1.97(m,1H),1.88(m,4H),1.79-1.74(m,2H),1.70-1.64(m,2H),1.47-1.41(m,1H),1.25(s,3H,1×CH3),1.23(s,3H,1×CH3),1.19(s,3H,1×CH3),1.10(s,3H,1×CH3),1.08(s,3H,1×CH3),0.93(d,J=6.6Hz,3H,1×CH3),0.87-0.84(m,3H,1×CH3).

[0068] 13C-APT (150MHz, CDCl3): δ217.6,*217.5,210.1,*209.9,199.9,176.0,*172.2,172.0,*169.7,169.7,169.6,*169.4,167.7,*167.7,*159.8 ,159.8,146.6,*146.6,144.5,*144.4,140.0,136.4,132.6,*123.6,1 23.6,116.9,*116.8,*112.1,112.1,*110.6,110.6,*72.4,72.4,69.6 ,*69.4,69.4,68.8,*68.8,*54.0,53.9,51.8,50.6,*49.8,49.8,49.0,48.9,*48.8,*47.9,47.9,*47.1,47.0,46.8,*46.8,*42.2,42.2,38.0,36.1,*35.9,35.8,35.6,35.2,34.4,32.7,31.5,29.8,28.8,27.5,*23.1,23.1,20.8,*19.9,19.8,19.6,*19.5,19.4,*18.0,17.9,17.3.

[0069] Synthetic route of GAA-C3:

[0070]

[0071] S1 (50 mg, 0.18 mmol) and 2-[2-(2-azidoethoxy)ethoxy]ethylamine (31 mg, 0.18 mmol) were dissolved in DMF, and DIPEA (7 mg, 0.05 mmol) was added. The mixture was stirred at 90 °C until the reactants were completely reacted. After concentration, the mixture was subjected to dichloromethane / methanol = 50 / 1 column chromatography (ball mill flash column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain a yellow solid product S8 (64 mg, 82%).

[0072] Dissolve the above S8 (50 mg, 0.12 mmol) and S4 (69 mg, 0.12 mmol) in dichloromethane, add an appropriate amount of cuprous thiophene-2-carboxylate, stir until the reactants react completely, concentrate, and then perform dichloromethane / methanol = 20 / 1 column chromatography (ball mill flash chromatography column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain yellow-green solid product S9 (57 mg, 50%).

[0073] C3: 1 H NMR(600MHz, CDCl3):~50:50mixture of rotamers*indicates minorrotamerδ7.69(s,1H),7.50(t,J=7.8Hz,1H),7.11(d,J=7.2Hz,1H),6.91 (dd,J=6.6,1.8Hz,1H),6.69-6.66(m,1H),6.55(t,J=5.4Hz,1H),4.94-4.89(m, 1H),4.73(t,J=7.8Hz,1H),4.61(s,1H),4.52-4.50(m,3H),4.36-4.31(m,1H),4 .28(s,1H,OH),3.89-3.86(m,2H),3.72-3.71(m,2H),3.64(s,4H),3.48-3.45(m ,2H),2.87-2.71(m,7H),2.50-2.47(m,2H),2.39-2.34(m,2H),2.26-2.21(m,1H ),2.12-2.11(m,1H),2.03-2.00(m,1H),1.92-1.89(m,1H),1.72(bs,4H),1.69- 1.65(m,2H),1.48-1.42(m,1H),1.26(s,3H,1×CH3),1.24(s,6H,2×CH3),1.11(s ,3H,1×CH3),1.09(s,3H,1×CH3),0.94(s,3H,1×CH3),0.86-0.85(m,3H,1×CH3).

[0074] 13C-APT (150MHz, CDCl3): δ217.4,*210.1,210.0,199.8,*175.9,175.8,*171.8,171.7,169.5,*169.5,*169.2,169.1,167.8,159.7,146.8, 144.5,*140.1,140.0,136.3,132.6,*132.6,123.6,*117.0,116.9,* 111.9,111.8,110.4,72.5,70.7,70.5,69.6,69.3,*69.2,68.8,54.0 ,51.8,50.3,49.9,*49.8,*49.1,49.1,48.9,*48.0,47.9,47.2,*47.1,46.8,*46.8,42.3,38.1,36.2,*35.9,35.8,35.7,35.1,*35.1,34.4,*32.7,32.7,*31.6,31.6,29.8,28.9,*28.8,27.5,*27.5,22.9,*22.9,20.8,20.0,*19.9,19.7,*19.6,19.5,17.9,*17.9,17.3,*17.3.

[0075] Synthetic route of GAA-C4:

[0076]

[0077] S1 (50 mg, 0.18 mmol) and 11-azido-3,6,9-trioxaundecan-1-amine (39 mg, 0.18 mmol) were dissolved in DMF, and DIPEA (7 mg, 0.05 mmol) was added. The mixture was stirred at 90 °C until the reactants were completely reacted. After concentration, the mixture was subjected to dichloromethane / methanol = 50 / 1 column chromatography (ball mill flash column: Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; silica gel for column chromatography: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain a yellow solid product S10 (60 mg, 70%).

[0078] Dissolve the above S10 (50 mg, 0.11 mmol) and S4 (63 mg, 0.11 mmol) in dichloromethane, add an appropriate amount of cuprous thiophene-2-carboxylate, stir until the reactants react completely, concentrate, and then perform dichloromethane / methanol = 20 / 1 column chromatography (ball mill flash chromatography column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain yellow-green solid product S11 (55 mg, 51%).

[0079] C4: 1 H NMR(600MHz, CDCl3):~50:50mixture of rotamers*indicates minorrotamer δ7.68(s,1H),7.48-7.46(m,1H),7.06(dd,J=7.2,3Hz,1H),6.90(d,J=8. 4Hz,1H),6.86(t,J=5.4Hz,1H),6.49(q,J=4.8Hz,1H),4.95-4.89(m,1H),4.72-4.68(m, 2H),4.59(s,1H),4.47-4.44(m,2H),4.40-4.34(m,1H),4.15-4.12(m,1H,OH),3.82(q,J =4.8Hz,2H),3.70(t,J=5.4Hz,2H),3.64-3.56(m,8H),3.45(q,J=5.4Hz,2H),2.88-2.69 (m,7H),2.48-2.42(m,3H),2.39-2.34(m,2H),2.28(bs,1H),2.19(dd,J=16.8,9Hz,1H) ,2.10-2.08(m,1H),2.01-1.98(m,1H),1.93-1.90(m,1H),1.83-1.64(m,4H),1.46-1.41 (m,1H),1.23(s,3H,1×CH3),1.21(d,J=1.8Hz,3H,1×CH3),1.09(s,3H,1×CH3),1.08(s,3 H,1×CH3),1.06(s,3H,1×CH3),0.92(d,J=1.2Hz,3H,1×CH3),0.81(d,J=6Hz,3H,1×CH3).

[0080] 13C-APT (150MHz, CDCl3): δ217.6,210.0,*210.0,199.9,175.9,*172.1,172.0,169.4,*169.2,169.2,*167.9,167.8,159.9 ,146.8,144.5,140.0,136.2,132.5,123.6,116.9,111.7,*111.7,110.2,72.3,70.8,*70.7,70.6,70.5,*70.5,70.5,69.5 ,69.4,68.8,54.0,51.8,*50.3,50.3,*49.9,49.8,48.9,48.8,48.1,*47.1,47.0,*46.8,46.7,42.4,38.0,36.1,35.8,35.6,*35.0,35.0,34.4,32.7,*32.7,31.6,29.8,28.8,27.5,*22.8,22.8,20.8,19.8,*19.8,19.6,19.5,*18.0,18.0,17.3.

[0081] Synthetic route of GAA-C5:

[0082]

[0083] S1 (50 mg, 0.18 mmol) and azido-PEG4-amine (47 mg, 0.18 mmol) were dissolved in DMF, and DIPEA (7 mg, 0.05 mmol) was added. The mixture was stirred at 90 °C until the reactants were completely reacted. After concentration, the mixture was subjected to dichloromethane / methanol = 50 / 1 column chromatography (ball mill flash column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain a yellow solid product S12 (53 mg, 56%).

[0084] Dissolve the above S12 (50 mg, 0.10 mmol) and S4 (58 mg, 0.10 mmol) in dichloromethane, add an appropriate amount of cuprous thiophene-2-carboxylate, stir until the reactants react completely, concentrate, and then perform dichloromethane / methanol = 20 / 1 column chromatography (ball mill flash chromatography column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain yellow-green solid product S13 (56 mg, 54%).

[0085] C5: 1 H NMR(600MHz, CDCl3):~50:50mixture of rotamers*indicates minorrotamer δ7.71(s,1H),7.48(t,J=7.8Hz,1H),7.08(d,J=7.2Hz,1H),6.90(d,J=8.4 Hz,1H),6.78-6.77(m,1H),6.50(t,J=5.4Hz,1H),4.94-4.90(m,1H),4.74-4.71(m,1H), 4.60-4.58(m,1H),4.52-4.47(m,3H),4.43-4.37(m,1H),4.02(bs,1H,OH),3.84-3.82(m ,2H),3.71(t,J=4.8Hz,2H),3.66-3.63(m,6H),3.59-3.57(m,6H),3.45(q,J=5.4Hz,2H) ,2.86-2.80(m,3H),2.77-2.71(m,4H),2.49-2.44(m,2H),2.41-2.35(m,2H),2.21(dd,J =16.2,8.4Hz,1H),2.11-2.00(m,4H),1.95-1.92(m,1H),1.86-1.82(m,1H),1.76-1.72( m,1H),1.70-1.64(m,2H),1.47-1.42(m,1H),1.25(s,3H,1×CH3),1.23(s,3H,1×CH3),1. 10(s,6H,2×CH3),1.08(s,3H,1×CH3),0.93(s,3H,1×CH3),0.84(d,J=6.6Hz,3H,1×CH3).

[0086] 13C-APT (150MHz, CDCl3): δ217.5,209.9,199.8,175.8,171.9,169.4,*169.1,169.1,167.9,159.8,146.9,*144.5 ,144.5,140.0,136.2,132.6,*123.7,123.6,116.9,111.7,110.3,72.3,70.8,70.7,70.6,70.6,70.6,70.4,69. 4,69.4,68.8,54.0,51.8,50.3,*49.9,49.9,49.0,48.8,48.1,*47.1,47.0,*46.8,46.7,42.4,38.0,36.2,35.9,35.6,*35.1,35.0,34.4,*32.7,32.7,31.6,29.8,28.8,27.5,22.9,20.8,*19.9,19.9,19.6,19.5,18.0,17.3.

[0087] Synthetic route of GAA-C6:

[0088]

[0089] S1 (300 mg, 1.08 mmol) and propargylamine (338 mg, 1.08 mmol) were dissolved in DMF, and DIPEA (42 mg, 0.32 mmol) was added. The mixture was stirred at 90 °C until the reactants were completely reacted. After concentration, the mixture was subjected to dichloromethane / methanol = 50 / 1 column chromatography (ball mill flash column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain a yellow solid product S14 (310 mg, 92%).

[0090] S3 (100 mg, 0.19 mmol) and 3-azidopropylamine (21 mg, 0.21 mmol) were dissolved in dichloromethane. TBTU (68 mg, 0.21 mmol) and DIPEA (28 mg, 0.21 mmol) were added, and the mixture was stirred until the reactants were completely reacted. After concentration, dichloromethane / methanol = 30 / 1 column chromatography (ball mill flash column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) yielded a white solid S15 (103 mg, 89%).

[0091] S15: 1 H NMR (600MHz, CDCl3): δ6.07(s,1H,NH),4.76-4.74(m,1H),4.61-4.57(m,1H),3.66-3.60(m,14H),3.53-3.51( m,2H),3.40-3.37(m,4H),2.85-2.71(m,5H),2.48-3.36(m,5H),2.20(dd,J=16.2,9.6Hz,1H),2.01-1.96(m,2H ),1.79-1.77(m,3H),1.69-1.64(m,2H),1.47-1.42(m,1H),1.24(s,3H,1×CH3),1.22(s,3H,1×CH3),1.12(d,J =7.2Hz,3H,1×CH3),1.09(s,3H,1×CH3),1.06(s,3H,1×CH3),0.96(s,3H,1×CH3),0.84(d,J=6.0Hz,3H,1×CH3).

[0092] 13 C-APT (150MHz, CDCl3): δ217.5,209.9,199.8,176.1,159.5,140.2,72.4,68.9,54.0,51.8,49.9,49.2,48.7,48.1 ,47.3,46.8,46.7,38.0,37.1,36.3,36.1,35.6,34.4,32.9,29.0,28.8,27.5,20.8,19.7,19.6,19.5,18.1,17.4.

[0093] Dissolve the above S14 (26 mg, 0.08 mmol) and S15 (50 mg, 0.08 mmol) in dichloromethane, add an appropriate amount of cuprous thiophene-2-carboxylate, stir until the reactants react completely, concentrate, and then perform dichloromethane / methanol = 20 / 1 column chromatography (ball mill flash chromatography column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain yellow-green solid product S16 (30 mg, 39%).

[0094] C6: 1 H NMR (600MHz, CDCl3): ～ 50:50mixture of rotamers*indicates minorrotamer δ7.69(s,1H),7.46(t,J=6Hz,1H),7.07(d,J=7.0Hz,1H),6.99(d,J=8.5Hz,1H),6.68-6.65(m,1H),6.43(bs,1H) ,4.96-4.93(m,1H),4.71(s,1H),4.56(s,3H),4.36(d,J=5.0Hz,2H),4.00(bs,1H,OH),3.31-3.29(m,1H),3.07-3.06(m,1H),2. 89-2.63(m,7H),2.53-2.34(m,5H),2.10-2.06(m,3H),2.00-1.99(m,2H),1.91(s,1H),1.77-1.63(m,5H),1.46-1.41(m,1H),1. 23(d,J=5.6Hz,3H,1×CH3),1.21(d,J=8.5Hz,3H,1×CH3),1.08(s,6H,2×CH3),0.88(d,J=5.9Hz,3H,1×CH3),0.79(s,3H,1×CH3).

[0095] 13C-APT (150MHz, CDCl3): δ217.6,210.4,199.9,*176.6,176.5,172.1,169.4,169.4,167.7,159.8,146.2 ,145.0,*144.9,140.1,136.4,132.4,122.9,*122.8,117.3,112.3,110.6,*110.6,72.3,68.8,54.0,51 .8,49.8,*49.8,49.0,48.7,48.2,*48.1,*47.7,47.7,47.3,*46.7,46.7,38.5,38.0,*36.3,36.2,36.1,36.1,35.6,34.4,32.9,31.5,30.3,29.8,28.9,27.5,22.8,20.8,19.7,19.6,19.5,18.1,17.2,*17.2.

[0096] Synthetic route of GAA-C7:

[0097]

[0098] S3 (100 mg, 0.19 mmol) and azido-PEG1-amine (28 mg, 0.21 mmol) were dissolved in dichloromethane. TBTU (68 mg, 0.21 mmol) and DIPEA (28 mg, 0.21 mmol) were added, and the mixture was stirred until the reactants were completely reacted. After concentration, dichloromethane / methanol = 30 / 1 column chromatography (ball mill flash column: Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; silica gel for column chromatography: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) yielded a white solid S17 (105 mg, 86%).

[0099] S17: 1 H NMR (600MHz, CDCl3): δ6.19 (t, J = 5.4Hz, 1H, NH), 4.76-4.74 (m, 1H), 4.60-

[0100] 4.58(m,1H),3.69-3.64(m,2H),3.54-3.52(m,2H),3.43-3.36(m,4H),2.86-2.79(m, 2H),2.74-2.71(m,2H),2.48-2.36(m,5H),2.20(dd,J=16.2,7.8Hz,1H),2.03-1.96( m,2H),1.79-1.78(m,3H),1.69-1.64(m,2H),1.47-1.42(m,1H),1.24(d,J=9.6Hz,6H ),1.13(d,J=6.6Hz,3H),1.08(d,J=13.8Hz,6H),0.96(s,3H),0.85(d,J=6.6Hz,3H);

[0101] 13 C-APT (150MHz, CDCl3): δ217.4,209.4,199.7,175.9,159.4,140.1,72.3,70.2,69.8,68.8,54.0,51.7,50.6,49.8,4 8.7,48.1,47.0,46.8,46.6,39.2,37.9,36.3,36.0,35.5,34.3,32.7,28.9,27.4,20.7,19.7,19.5,19.4,18.0,17.3.

[0102] Dissolve the above S17 (50 mg, 0.08 mmol) and S14 (26 mg, 0.08 mmol) in dichloromethane, add an appropriate amount of cuprous thiophene-2-carboxylate, stir until the reactants react completely, concentrate, and then perform dichloromethane / methanol = 20 / 1 column chromatography (ball mill flash column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain yellow-green solid product S18 (30 mg, 40%).

[0103] C7: 1 H NMR (600MHz, CDCl3): ～50:50mixture of rotamers*indicates minorrotamerδ7.75(s,1H),7.50(t,J=7.8Hz,1H),7.13(dd,J=7.2,2.4Hz,1H),7.04(d,J=9Hz,1H),6.68-6.66(m,1H),6.18(t,J=6Hz,1H),4.94-4.90(m,1H),4.72-4.68(m,1H),4.63(t,J=5.4Hz,2H),4.60-4.56(m,1H),4.54-4.53(m,2H),3.82-3.79(m,2H),3.49-3.45(m,2H),3.40-3.28(m,2H),2.90-2.65(m,7H),2.49-2.46(m,2H),2.43-2.35(m,2H),2.22-2.17(m,1H),2.14-2.10(m,1H),2.04-2.00(m,1H),1.98-1.95(m,1H),1.77(bs,4H),1.70-1.64(m,2H),1.47-1.42(m,1H),1.26-1.24(m,6H,2×CH3),1.21(d,J=3Hz,3H,1×CH3),1.10-1.08(m,6H,2×CH3),0.91(d,J=3.6Hz,3H,1×CH3),0.82(t,J=6Hz,3H,1×CH3).

[0104] 13C-APT (150MHz, CDCl3): δ217.4,*210.5,210.4,*199.8,199.8,176.0,*176.0,*171.8,171.7,*169.5,169.5,*169.2,169.2,167.7,*159.6,159 .5,146.3,*146.3,145.3,*145.2,*140.2,140.2,136.4,132.5,*123.3, 123.2,117.4,112.4,*110.7,110.7,72.3,*69.9,69.8,*69.1,69.0,*68 .9,68.9,54.0,*54.0,51.8,50.4,*50.0,50.0,49.0,*48.8,48.8,*48.3,48.3,*47.5,47.4,46.8,*46.7,39.2,38.7,*38.0,38.0,*36.2,36.1,35.9,*35.9,35.6,34.4,32.9,*32.8,31.5,29.8,*29.0,28.9,27.5,22.9,*22.9,20.8,19.9,*19.8,19.6,19.5,*19.5,*18.1,18.1,17.3,*17.2.

[0105] Synthetic route of GAA-C8:

[0106]

[0107] S3 (100 mg, 0.19 mmol) and 2-[2-(2-azidoethoxy)ethoxy]ethylamine (37 mg, 0.21 mmol) were dissolved in dichloromethane. TBTU (68 mg, 0.21 mmol) and DIPEA (28 mg, 0.21 mmol) were added, and the mixture was stirred until the reactants were completely reacted. After concentration, dichloromethane / methanol = 30 / 1 column chromatography (ball mill flash chromatography column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) yielded a white solid S19 (110 mg, 84%).

[0108] S19: 1 H NMR (600MHz, CDCl3): δ6.29 (t, J = 5.3Hz, 1H, NH), 4.76-4.74 (m, 1H), 4.60-

[0109] 4.58(m,1H),3.70-3.39(m,12H),2.86-2.72(m,4H),2.49-2.36(m,5H),2.21(dd,J=1 6.2,9.6Hz,1H),2.03-1.96(m,2H),1.80-1.78(m,3H),1.71-1.66(m,2H),1.47-1.41 (m,1H),1.25(s,3H,1×CH3),1.23(s,3H,1×CH3),1.13(d,J=7.0Hz,3H,1×CH3),1.10( s,3H,1×CH3),1.07(s,3H,1×CH3),0.97(s,3H,1×CH3),0.85(d,J=6.4Hz,3H,1×CH3);

[0110] 13 C-APT (150MHz, CDCl3): δ217.5,209.5,199.8,175.9,159.5,140.2,72.4,70.6,70.3,70.2,69.9,68.8,54.0,51.8,50.7,5 0.0,48.8,48.2,47.0,46.8,46.7,39.3,38.0,36.4,36.1,35.6,34.4,32.8,29.0,27.5,20.8,19.7,19.6,19.5,18.2,17.4.

[0111] Dissolve the above-mentioned S19 (50 mg, 0.08 mmol) and S14 (26 mg, 0.08 mmol) in dichloromethane, add an appropriate amount of cuprous thiophene-2-carboxylate, stir until the reactants react completely, concentrate, and then perform dichloromethane / methanol = 20 / 1 column chromatography (ball mill flash chromatography column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain yellow-green solid product S20 (44 mg, 54%).

[0112] C8: 1 H NMR (600MHz, CDCl3): ～50:50mixture of rotamers*indicates minorrotamerδ7.76(s,1H),7.51-7.48(m,1H),7.14-7.12(m,1H),7.03(d,J=8.4Hz,1H),6.69(bs,1H),6.30-6.27(m,1H),4.94-4.91(m,1H),4.73(bs,1H),4.65-4.60(m,3H),4.55-4.54(m,2H),3.87-3.86(m,2H),3.56-3.55(m,2H),3.51-3.50(m,2H),3.47-3.42(m,2H),3.40-3.34(m,2H),2.91-2.71(m,7H),2.50-2.45(m,2H),2.41-2.36(m,2H),2.23-2.17(m,1H),2.15-2.11(m,1H),2.04(s,1H),2.03-2.02(m,1H),1.96-1.95(m,1H),1.77(bs,3H),1.71-1.65(m,2H),1.48-1.42(m,1H),1.26(d,J=2.4Hz,3H,1×CH3),1.25(s,3H,1×CH3),1.23(d,J=3.6Hz,3H,1×CH3),1.11(s,3H,1×CH3),1.09(s,3H,1×CH3),0.93(s,3H,1×CH3),0.84(dd,J=6.6,2.4Hz,3H,1×CH3).

[0113] 13C-APT (150MHz, CDCl3): δ216.3,*209.1,209.0,198.7,174.8,170.6,*170.5,168.3,168.0,*167.9,166.6,158.5 ,145.1,143.9,*143.9,139.0,135.2,131.3,122.1,116.2,111.2,*109.6,109.6,71.2,69.4,*68.9,68.9,68.7,6 8.3,67.7,52.9,50.6,49.4,48.9,47.9,47.6,47.1,46.1,*45.7,45.6,*38.1,38.1,*37.6,37.6,36.9,*35.1,35.1,*34.9,34.9,34.5,33.3,*31.7,31.7,30.4,28.7,27.8,26.3,21.7,19.7,18.7,18.5,18.3,*17.0,17.0,16.1.

[0114] Synthetic route of GAA-C9:

[0115]

[0116] S3 (100 mg, 0.19 mmol) and 11-azido-3,6,9-trioxaundecan-1-amine (46 mg, 0.21 mmol) were dissolved in dichloromethane. TBTU (68 mg, 0.21 mmol) and DIPEA (28 mg, 0.21 mmol) were added, and the mixture was stirred until the reactants were completely reacted. After concentration, dichloromethane / methanol = 30 / 1 column chromatography (ball mill flash chromatography column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) yielded a white solid S21 (116 mg, 84%).

[0117] S21: 1 H NMR (600MHz, CDCl3): δ6.43 (t, J = 5.3Hz, 1H, NH), 4.76-4.74 (m, 1H), 4.59-

[0118] 4.58(m,1H),4.22(s,1H),3.68-3.65(m,8H),3.63-3.61(m,2H),3.54-3.52(m,2H),3.41-3.39(m,4H),2. 87-2.71(m,4H),2.49-2.37(m,5H),2.21(dd,J=16.2,9.6Hz,1H),2.14(s,1H),2.03-1.95(m,2H),1.81-1 .77(m,3H),1.71-1.64(m,2H),1.47-1.42(m,1H),1.25(s,3H,1×CH3),1.23(s,3H,1×CH3),1.13(d,J=7.0 Hz,3H,1×CH3),1.10(s,3H,1×CH3),1.07(s,3H,1×CH3),0.97(s,3H,1×CH3),0.85(d,J=6.0Hz,3H,1×CH3).

[0119] 13 C-APT (150MHz, CDCl3): δ217.5,209.5,199.8,175.9,159.5,140.2,72.4,70.8,70.7,70.6,70.3,70.1,69.9,68.8,54.0,51.8,5 0.7,50.0,48.8,48.2,47.0,46.8,46.7,39.4,38.0,36.4,36.1,35.6,34.4,32.8,29.0,27.5,20.8,19.8,19.6,19.5,18.2,17.4.

[0120] Dissolve the above-mentioned S21 (50 mg, 0.08 mmol) and S14 (26 mg, 0.08 mmol) in dichloromethane, add an appropriate amount of cuprous thiophene-2-carboxylate, stir until the reactants react completely, concentrate, and then perform dichloromethane / methanol = 20 / 1 column chromatography (ball mill flash chromatography column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain yellow-green solid product S22 (40 mg, 47%).

[0121] C9: 1 H NMR (600MHz, CDCl3): ～50:50mixture of rotamers*indicates minorrotamer δ7.75(s,1H),7.50-7.47(m,1H),7.13-7.11(m,1H),7.02-7.00(m,1H),6.69-6.68(m,1H),6.58-6.57(m,1H),4.95-4.91( m,1H),4.71(bs,1H),4.62(bs,2H),4.59-4.58(m,1H),4.52(bs,2H),3.85(bs,2H),3.57-3.55(m,8H),3.50(bs,2H),3.38(bs,2H),2.86- 2.70(m,7H),2.49-2.44(m,2H),2.39-2.35(m,2H),2.21-2.17(m,1H),2.12-1.94(m,5H),1.77-1.74(m,2H),1.72-1.64(m,2H),1.47-1.4 1(m,1H),1.25-1.24(m,6H,2×CH3),1.22(s,3H,1×CH3),1.10(s,3H,1×CH3),1.08(s,3H,1×CH3),0.91(s,3H,1×CH3),0.82(s,3H,1×CH3).

[0122] 13 C-APT (150MHz, CDCl3): δ217.5,210.1,*210.1,199.8,176.0,*171.8,171.7,*169.5,169.5,*169.0,169.0,167 .7,159.7,146.2,144.9,140.1,136.4,132.5,123.4,117.3,112.4,110.7,72.3,70.6,70.4,70.4,70.2,*69.9,6 9.9,69.4,68.9,54.0,51.8,50.5,50.1,49.0,48.8,48.2,47.2,*46.8,46.7,39.3,*39.2,38.7,38.0,36.2,*36.0,36.0,35.6,34.4,*32.8,32.7,31.5,29.8,28.9,27.5,22.9,20.8,*19.9,19.9,19.6,19.5,*18.2,18.2,17.3.

[0123] Synthetic route of GAA-C10:

[0124]

[0125] S3 (100 mg, 0.19 mmol) and azido-PEG4-amine (56 mg, 0.21 mmol) were dissolved in dichloromethane. TBTU (68 mg, 0.21 mmol) and DIPEA (28 mg, 0.21 mmol) were added, and the mixture was stirred until the reactants were completely reacted. After concentration, dichloromethane / methanol = 30 / 1 column chromatography (ball mill flash column: Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; silica gel for column chromatography: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) yielded a white solid S23 (118 mg, 79%).

[0126] S23: 1 H NMR (600MHz, CDCl3): δ6.68 (t, J = 4.8Hz, 1H, NH), 4.75-4.73 (m, 1H), 4.59-

[0127] 4.57(m,1H),3.66-3.60(m,14H),3.53-3.51(m,2H),3.40-3.37(m,4H),2.85-2.71(m,5H),2.4 8-3.36(m,5H),2.20(dd,J=16.2,9.6Hz,1H),2.01-1.96(m,2H),1.79-1.77(m,3H),1.69-1.64 (m,2H),1.47-1.42(m,1H),1.24(s,3H,1×CH3),1.22(s,3H,1×CH3),1.12(d,J=7.2Hz,3H,1×CH 3),1.09(s,3H,1×CH3),1.06(s,3H,1×CH3),0.96(s,3H,1×CH3),0.84(d,J=6.0Hz,3H,1×CH3).

[0128] 13 C-APT (150MHz, CDCl3): δ217.5,209.5,199.8,175.9,159.6,140.1,72.3,70.7,70.6,70.6,70.5,70.5,70.2,70.1,69.9,68.8,54.0,5 1.8,50.7,50.1,48.7,48.2,46.9,46.8,46.7,39.4,38.0,36.3,36.0,35.6,34.4,32.8,28.9,27.5,20.8,19.7,19.6,19.5,18.2,17.4.

[0129] Dissolve the above-mentioned S23 (50 mg, 0.08 mmol) and S14 (26 mg, 0.08 mmol) in dichloromethane, add an appropriate amount of cuprous thiophene-2-carboxylate, stir until the reactants react completely, concentrate, and then perform dichloromethane / methanol = 20 / 1 column chromatography (ball mill flash chromatography column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain yellow-green solid product S24 (32 mg, 36%).

[0130] C10: 1 H NMR (600MHz, CDCl3): ～ 50:50mixture of rotamers*indicates minorrotamerδ7.78(s,1H),7.51-7.48(m,1H),7.12(d,J=6.6Hz,1H),7.03(d,J=9Hz,1 H),6.77-6.74(m,1H),6.68(m,1H),4.94-4.91(m,1H),4.74-4.72(m,1H),4.62(d,J=4. 8Hz,1H),4.53(t,J=4.8Hz,2H),4.43(bs,1H,OH),3.85(t,J=5.4Hz,2H),3.61-3.55(m, 12H),3.51(t,J=4.8Hz,2H),3.40-3.36(m,2H),2.87-2.70(m,7H),2.49-2.44(m,2H),2 .38-2.37(m,1H),2.35-2.34(m,1H),2.19(dd,J=16.2,9Hz,1H),2.12-2.11(m,1H),2.0 4-2.00(m,3H),1.96-1.95(m,1H),1.78-1.73(m,2H),1.71-1.64(m,2H),1.47-1.42(m, 1H),1.25(d,J=3.6Hz,3H,1×CH3),1.24(s,3H,1×CH3),1.22(s,3H,1×CH3),1.10(s,3H, 1×CH3), 1.08 (s, 3H, 1×CH3), 0.92 (d, J=2.4Hz, 3H, 1×CH3), 0.83 (d, J=6.6Hz, 3H, 1×CH3).

[0131] 13C-APT (150MHz, CDCl3): δ217.5,210.0,199.8,176.0,*171.8,171.7,169.4,*169.0,168.9,167.7,159. 7,146.3,144.9,140.1,136.4,132.5,123.4,117.3,112.4,110.7,72.3,70.6,70.5,70.5,70.5,70.4,70 .2,70.0,69.5,68.8,54.0,51.8,50.5,50.1,49.0,48.8,48.3,47.1,*46.8,46.7,39.3,38.7,38.0,36.2,*36.0,36.0,35.6,34.4,32.7,31.6,29.8,28.9,27.5,22.9,20.8,*19.9,19.9,19.6,19.5,18.2,17.3.

[0132] Synthetic route of GAA-V1:

[0133]

[0134] S25 (300 mg, 0.67 mmol) and 5-hexyneic acid (83 mg, 0.74 mmol) were dissolved in dichloromethane. TBTU (238 mg, 0.74 mmol) and DIPEA (96 mg, 0.74 mmol) were added, and the mixture was stirred until the reactants were completely reacted. After concentration, dichloromethane / methanol = 30 / 1 column chromatography (ball mill flash column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) yielded a white solid product S26 (300 mg, 83%).

[0135] Dissolve the above-mentioned S26 (50 mg, 0.09 mmol) and S15 (48 mg, 0.09 mmol) in dichloromethane, add an appropriate amount of cuprous thiophene-2-carboxylate, stir until the reactants react completely, concentrate, and then perform dichloromethane / methanol = 20 / 1 column chromatography (ball mill flash chromatography column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain white solid product S27 (51 mg, 48%).

[0136] V1:1 1H NMR (600 MHz, CDCl3): δ 8.67 (s, 1H), 7.61 (d, J = 7.7 Hz, 1H), 7.47 (s, 1H), 7.37

[0137] (dd, J = 8.6, 11.9 Hz, 4H), 5.08 (m, 1H), 4.73 - 4.31 (m, 7H), 4.07 (d, J = 11.3 Hz, 1H), 3.62 - 3.59 (m, 1H), 3.33 - 3.27 (m, 1H), 3.08 - 3.03 (m, 1H), 2.94 (dd, J = 9.9, 18.0 Hz, 1H), 2.83 - 2.78 (m, 1H), 2.73 - 2.67 (m, 4H), 2.51 (s, 3H), 2.49 - 2.33 (m, 10H), 2.30 - 2.26 (m, 1H), 2.19 - 2.11 (m, 4H), 2.02 - 1.99 (m, 2H), 1.93 - 1.90 (m, 2H), 1.83 - 1.59 (m, 6H), 1.46 (d, J = 7.0 Hz, 3H, 1×CH3), 1.25 (s, 3H, 1×CH3), 1.22 (s, 3H, 1×CH3), 1.12 (d, J = 7.0 Hz, 3H, 1×CH3), 1.09 (s, 3H, 1×CH3), 1.07 (s, 3H, 1×CH3), 1.03 (s, 9H, 3×CH3), 0.92 (s, 3H, 1×CH3), 0.84 (d, J = 6.3 Hz, 3H, 1×CH3).

[0138] 13 C - APT (150 MHz, CDCl3): δ 217.5, 210.6, 199.9, 176.4, 173.8, 172. In0, 170.2, 160.0, 150.6, 148.5, 146.9, 143.4, 140.0, 131.8, 130.9, 129.6, 129.6, 126.6, 126.6, 122.4, 72.1, 70.0, 68.8, 58.8, 58.0, 57.1, 54.1, 51.8, 50.2, 48.9, 48.8, 48.3, 47.2, 46.9, 46.8, 46.7, 38.1, 36.3, 36.2, 36.0, 35.6, 35.4, 35.2, 34.4, 33.2, 30.2, 29.8, 28.8, 27.5, 26.7, 26.7, 26.7, 25.6, 24.6, 22.4, 20.8, 19.8, 19.7, 19.6, 18.1, 17.4, 16.2.

[0139] Synthesis route of GAA - V2:

[0140]

[0141] Dissolve the above S17 (50 mg, 0.08 mmol) and S26 (43 mg, 0.08 mmol) in dichloromethane, add an appropriate amount of cuprous thiophene-2-carboxylate, stir until the reactants react completely, concentrate, and then perform dichloromethane / methanol = 20 / 1 column chromatography (ball mill flash chromatography column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain white solid product S28 (23 mg, 25%).

[0142] V2: 1 H NMR (600MHz, CDCl3): δ8.67(s,1H),7.83(s,1H),7.45(d,J=8.2Hz,2H),7.38

[0143] (d,J=8.2Hz,2H),6.39(t,J=4.7Hz,1H),5.12-5.07(m,1H),4.77(t,J=8. 4Hz,1H),4.57-4.49(m,6H),3.86-3.77(m,3H),3.67-3.65(m,1H),3.58-3 .48(m,3H),3.46(s,2H),3.34-3.30(m,1H),2.96-2.95(m,1H),2.88(dd,J =10.2,18.2Hz,1H),2.76-2.74(m,3H),2.71-2.67(m,1H),2.52(s,3H),2. 50-2.44(m,3H),2.38(d,J=15.2Hz,1H),2.24-1.94(m,9H),1.79-1.64(m ,4H),1.59(d,J=13.3Hz,1H),1.48(d,J=7.0Hz,3H,1×CH3),1.39(s,3H,1× CH3),1.22(s,3H,1×CH3),1.12(d,J=7.0Hz,3H,1×CH3),1.09(s,3H,1×CH3),1.07(s,12H,4×CH3),0.83(s,3H,1×CH3),0.80(d,J=6.2Hz,3H,1×CH3).

[0144] 13C-APT (150MHz, CDCl3): δ217.0,210.3,201.6,176.1,173.8,171.4,170.3,160.0,150.4,148.4,147.3,14 4.0,140.3,131.9,130.6,129.6,129.6,126.5,126.5,122.7,72.1,70.1,69.9,69.3,68.8,59.4,58.2,57 .8,54.1,51.9,50.2,49.5,49.0,48.9,48.2,47.4,47.2,46.8,39.6,37.9,36.4,36.3,36.2,36.0,35.8,35.5,34.4,32.4,29.8,29.2,27.2,27.0,27.0,27.0,24.8,22.7,21.0,20.0,19.4,19.3,18.2,17.3,16.2.

[0145] GAA-V3 synthesis route:

[0146]

[0147] Dissolve the above-mentioned S19 (50 mg, 0.07 mmol) and S26 (40 mg, 0.07 mmol) in dichloromethane, add an appropriate amount of cuprous thiophene-2-carboxylate, stir until the reactants react completely, concentrate, and then perform dichloromethane / methanol = 20 / 1 column chromatography (ball mill flash chromatography column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain white solid product S29 (43 mg, 48%).

[0148] V3: 1 H NMR (600MHz, CDCl3): δ8.66 (s, 1H), 7.59 (s, 1H), 7.39 (dd, J = 8.4Hz, 4H), 6.60

[0149] (t,J=5.2Hz,1H),5.09-5.04(m,1H),4.77(t,J=8.2Hz,1H),4.62-4.47(m,7H),3.93(d,J=11.2Hz,1H),3.84(t,J=4.9Hz ,2H),3.64-3.31(m,9H),2.85(dd,J=9.6,17.6Hz,1H),2.80-2.67(m,5H),2.51(s,3H),2.47(t,J=7.6Hz,2H),2.42-2.37 (m,5H),2.28-1.62(m,14H),1.46(d,J=7.0Hz,3H,1×CH3),1.33(s,3H,1×CH3),1.22(s,3H,1×CH3),1.12(d,J=7.0Hz,3H ,1×CH3),1.09(s,3H,1×CH3),1.07(s,3H,1×CH3),1.06(s,9H,3×CH3),0.87(s,3H,1×CH3),0.80(d,J=6.2Hz,3H,1×CH3).

[0150] 13 C-APT (150MHz, CDCl3): δ217.3,210.2,200.9,176.2,173.7,171.5,170.3,160.2,150.4,148.4,146.9,143. 8,140.1,131.9,130.6,129.6,129.6,126.5,126.5,122.7,72.0,70.5,70.2,70.1,69.9,69.5,68.8,59.0,58 .1,57.4,54.1,51.8,50.3,50.0,48.9,48.9,48.1,47.2,47.0,46.7,39.2,38.0,36.3,36.1,36.1,36.1,36.1,35.7,35.5,34.4,32.6,29.8,29.0,27.4,26.9,26.9,26.9,24.6,22.5,20.9,19.9,19.5,19.4,18.2,17.1,16.2.

[0151] Synthetic route for GAA-V4:

[0152]

[0153] Dissolve the above-mentioned S21 (50 mg, 0.07 mmol) and S26 (38 mg, 0.07 mmol) in dichloromethane, add an appropriate amount of cuprous thiophene-2-carboxylate, stir until the reactants react completely, concentrate, and then perform dichloromethane / methanol = 20 / 1 column chromatography (ball mill flash chromatography column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain white solid product S30 (32 mg, 37%).

[0154] V4: 1 H NMR (600MHz, CDCl3): δ8.67 (s, 1H), 7.65 (d, J = 7.4Hz, 1H), 7.55 (s, 1H), 7.38

[0155] (dd,J=12.1,8.6Hz,4H),7.19(d,J=7.8Hz,1H),6.63(t,J=5.2Hz,1H),5.10 -5.05(m,1H),4.73(t,J=8.1Hz,1H),4.69-4.48(m,7H),4.05(d,J=11.3Hz, 1H),3.86(t,J=5.0Hz,2H),3.61-3.59(m,8H),3.52-3.50(m,2H),3.39-3.3 6(m,2H),2.87(dd,J=17.8,8.9Hz,1H),2.83-2.67(m,5H),2.51(s,3H),2.49 -2.35(m,6H),2.32-2.16(m,7H),2.12-2.08(m,1H),2.04-1.92(m,4H),1.7 9-1.65(m,5H),1.46(d,J=6.9Hz,3H,1×CH3),1.27(s,3H,1×CH3),1.23(s,3 H,1×CH3),1.11(d,J=7.1Hz,3H,1×CH3),1.10(s,3H,1×CH3),1.07(s,3H,1× CH3),1.05(s,9H,3×CH3),0.90(s,3H,1×CH3),0.83(d,J=6.4Hz,3H,1×CH3).

[0156] 13C-APT (150MHz, CDCl3): δ217.5,210.0,200.0,175.9,173.6,171.9,170.1,160.0,150.5,148.5,146.9,143.4,1 40.1,131.8,130.8,129.6,129.6,126.6,126.6,122.6,72.1,70.6,70.5,70.4,70.2,70.0,69.9,69.5,68.8,58 .7,58.0,57.1,54.1,51.8,50.3,50.2,48.9,48.8,48.4,47.2,46.8,46.7,39.3,38.1,36.2,36.0,36.0,35.6,35.4,35.3,34.4,32.7,28.9,27.5,26.7,26.7,26.7,25.9,24.4,22.4,20.8,20.0,19.6,19.5,18.2,17.3,16.2.

[0157] GAA-V5 synthesis route:

[0158]

[0159] Dissolve the above S23 (50 mg, 0.06 mmol) and S26 (35 mg, 0.06 mmol) in dichloromethane, add an appropriate amount of cuprous thiophene-2-carboxylate, stir until the reactants react completely, concentrate, and then perform dichloromethane / methanol = 20 / 1 column chromatography (ball mill flash chromatography column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain white solid product S31 (45 mg, 54%).

[0160] V5: 1 H NMR (600MHz, CDCl3): δ8.67 (s, 1H), 7.59 (d, J = 7.9Hz, 1H), 7.57 (s, 1H), 7.38

[0161] (dd,J=8.3,14.8Hz,4H),7.11(d,J=8.4Hz,1H),6.89(t,J=5.4Hz,1H),5.10-5 .05(m,1H),4.74-4.70(m,2H),4.62-4.59(m,1H),4.56(d,J=8.6Hz,1H),4.52- 4.48(m,3H),4.07(d,J=11.4Hz,1H),3.85(t,J=5.0Hz,2H),3.63-3.59(m,10H ),3.52-3.50(m,2H),3.40-3.37(m,2H),2.88-2.68(m,6H),2.52(s,3H),2.49- 2.16(m,15H),2.12-2.09(m,1H),2.01(dd,J=6.8,11.8Hz,1H),1.97-1.94(m, 3H),1.79-1.65(m,5H),1.47(d,J=7.0Hz,3H,1×CH3),1.26(s,3H,1×CH3),1.24 (s,3H,1×CH3),1.11(d,J=6.8Hz,3H,1×CH3),1.10(s,3H,1×CH3),1.08(s,3H, 1×CH3),1.05(s,9H,3×CH3),0.91(s,3H,1×CH3),0.83(d,J=6.4Hz,3H,1×CH3).

[0162] 13 C-APT (150MHz, CDCl3): δ217.5,209.9,200.0,175.9,173.6,172.0,170.1,159.9,150.5,148.5,146.9,143.4,140 .0,131.8,130.9,129.6,129.6,126.6,126.6,122.7,72.1,70.6,70.6,70.5,70.5,70.4,70.2,70.1,70.0,69.6,68 .8,58.7,58.0,57.1,54.0,51.8,50.2,48.9,48.8,48.4,47.1,46.8,46.7,39.3,38.1,36.3,36.0,35.9,35.6,35.3,35.2,34.4,32.6,29.8,28.8,27.5,26.7,26.7,26.7,25.8,24.4,22.4,20.8,20.0,19.6,19.5,18.3,17.3,16.2.

[0163] GAA-V6 synthesis route:

[0164]

[0165] S25 (300 mg, 0.67 mmol) and 6-heptenynic acid (94 mg, 0.74 mmol) were dissolved in dichloromethane. TBTU (238 mg, 0.74 mmol) and DIPEA (96 mg, 0.74 mmol) were added, and the mixture was stirred until the reactants were completely reacted. After concentration, dichloromethane / methanol = 20 / 1 column chromatography (ball mill flash column: Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; silica gel for column chromatography: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) yielded a white solid product S32 (320 mg, 86%).

[0166] Dissolve the above-mentioned S32 (50 mg, 0.09 mmol) and S15 (54 mg, 0.09 mmol) in dichloromethane, add an appropriate amount of cuprous thiophene-2-carboxylate, stir until the reactants react completely, concentrate, and then perform dichloromethane / methanol = 20 / 1 column chromatography (ball mill flash chromatography column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain white solid product S33 (29 mg, 28%).

[0167] V6: 1 H NMR (600MHz, CDCl3): δ8.67(s,1H),7.62(s,1H),7.44(s,1H),7.37(dd,J=8.5,

[0168] 12.2Hz,4H),6.78-6.76(m,1H),6.55-6.54(m,1H),5.10-5.06(m,1H),4.73-4.58(m,5H),4.48(s,1H),4.38-4.30(m,2H),4.01(d,J= 11.2Hz,1H),3.31-3.30(m,1H),3.08-3.05(m,1H),2.89(dd,J=9.7,17.9Hz,1H),2.83-2.78(m,1H),2.73-2.65(m,4H),2.50(s,3H),2 .49-2.37(m,8H),1.28-1.92(m,8H),1.81-1.62(m,9H),1.46(d,J=7.0Hz,3H,1×CH3),1.25(s,3H,1×CH3),1.22(s,3H,1×CH3),1.11(d ,J=7.1Hz,3H,1×CH3),1.09(s,3H,1×CH3),1.07(s,3H,1×CH3),1.02(s,9H,3×CH3),0.90(s,3H,1×CH3),0.83(d,J=6.4Hz,3H,1×CH3).

[0169] 13 C-APT (150MHz, CDCl3): δ217.5,210.5,199.9,176.4,173.8,171.8,170.2,160.0,150.6,148.5,147.6,14 3.4,140.1,131.8,130.8,129.6,129.6,126.5,126.5,121.9,72.1,69.9,68.8,58.9,57.6,57.2,54.1,51 .8,50.1,48.9,48.8,48.3,47.5,47.2,46.8,46.7,38.0,36.3,36.2,36.2,36.2,35.9,35.6,35.5,34.4,33.1,30.2,28.8,28.5,27.5,26.6,26.6,26.6,25.1,25.0,22.4,20.8,19.9,19.7,19.5,18.1,17.3,16.2.

[0170] GAA-V7 synthesis route:

[0171]

[0172] Dissolve the above-mentioned S17 (50 mg, 0.08 mmol) and S32 (44 mg, 0.08 mmol) in dichloromethane, add an appropriate amount of cuprous thiophene-2-carboxylate, stir until the reactants react completely, concentrate, and then perform dichloromethane / methanol = 20 / 1 column chromatography (ball mill flash chromatography column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain white solid product S34 (37 mg, 39%).

[0173] V7: 1 H NMR (600MHz, CDCl3): δ8.66 (s, 1H), 7.56 (s, 1H), 7.37 (dd, J = 8.4, 14.3Hz, 4H),

[0174] 5.10-5.06(m,1H),4.70-4.48(m,7H),3.97(d,J=11.3Hz,1H),3.80(t,J=5.1Hz,2H),3.62-3.59(m, 1H),3.48-3.28(m,4H),2.86-2.63(m,6H),2.50(s,3H),2.48-2.35(m,6H),2.26-2.11(m,4H),2.01- 1.95(m,2H),1.80-1.60(m,10H),1.47(d,J=7.0Hz,3H,1×CH3),1.25(s,3H,1×CH3),1.23(s,3H,1×CH 3),1.09-1.06(m,9H,3×CH3),1.01(s,9H,3×CH3),0.90(s,3H,1×CH3),0.82(d,J=6.4Hz,3H,1×CH3).

[0175] 13C-APT (150MHz, CDCl3): δ217.4,210.0,199.9,176.0,173.7,171.8,170.1,159.9,150.5,148.5,147.5,143 .3,140.0,131.7,130.8,129.6,129.6,126.5,126.5,122.4,72.0,69.9,69.8,69.2,68.8,58.9,57.7,57.3 ,54.0,51.7,50.2,50.1,48.9,48.8,48.4,47.4,46.8,46.7,39.2,38.0,36.3,36.1,35.9,35.9,35.6,34.4,32.7,29.8,28.8,28.6,27.4,26.7,26.7,26.7,25.2,25.1,22.4,20.8,20.0,19.6,19.5,18.1,17.3,16.2.

[0176] GAA-V8 synthesis route:

[0177]

[0178] Dissolve the above-mentioned S19 (50 mg, 0.07 mmol) and S32 (41 mg, 0.07 mmol) in dichloromethane, add an appropriate amount of cuprous thiophene-2-carboxylate, stir until the reactants react completely, concentrate, and then perform dichloromethane / methanol = 20 / 1 column chromatography (ball mill flash chromatography column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain white solid product S35 (46 mg, 51%).

[0179] V8: 1 H NMR (600MHz, CDCl3): δ8.66 (s, 1H), 7.55 (d, J = 7.8Hz, 1H), 7.51 (s, 1H), 7.37

[0180] (dd,J=8.4,15.4Hz,4H),6.77(d,J=8.9Hz,1H),6.52(t,J=5.4Hz,1H),5.10-5.05(m,1H),4.70-4.49(m,7H),4.01(d,J=11.2Hz,1H ),3.86(t,J=5.0Hz,2H),3.62-3.31(m,9H),2.88(dd,J=9.2,17.8Hz,1H),2.83-2.66(m,5H),2.51(s,3H),2.48-2.35(m,7H),2.28- 2.11(m,4H),2.01-1.94(m,2H),1.79-1.64(m,9H),1.47(d,J=7.0Hz,3H,1×CH3),1.25(s,3H,1×CH3),1.23(s,3H,1×CH3),1.10(d,J =7.0Hz,3H,1×CH3).1.09(s,3H,1×CH3),1.07(s,3H,1×CH3),1.02(s,9H,3×CH3),0.91(s,3H,1×CH3),0.83(d,J=6.4Hz,3H,1×CH3).

[0181] 13 C-APT (150MHz, CDCl3): δ217.5,210.0,199.8,175.9,173.7,171.9,170.1,159.9,150.5,148.5,147.4,143.3 ,140.0,131.7,130.9,129.6,129.6,126.5,126.5,122.4,72.0,70.5,70.1,69.9,69.8,69.5,68.8,58.8,57.6 ,57.2,54.0,51.8,50.2,50.1,48.9,48.8,48.3,47.2,46.8,46.7,39.3,38.0,36.3,36.0,36.0,36.0,35.9,35.6,35.5,34.4,32.8,28.8,28.6,27.5,26.6,26.6,26.6,25.2,25.1,22.3,20.8,19.9,19.6,19.5,18.1,17.3,16.2.

[0182] GAA-V9 synthesis route:

[0183]

[0184] Dissolve the above-mentioned S21 (50 mg, 0.07 mmol) and S32 (39 mg, 0.07 mmol) in dichloromethane, add an appropriate amount of cuprous thiophene-2-carboxylate, stir until the reactants react completely, concentrate, and then perform dichloromethane / methanol = 20 / 1 column chromatography (ball mill flash chromatography column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain white solid product S36 (59 mg, 67%).

[0185] V9: 1 H NMR (600MHz, CDCl3): δ8.67 (s, 1H), 7.52-7.51 (m, 2H), 7.37 (dd, J = 8.4, 15.4

[0186] Hz,4H),6.67-6.64(m,2H),5.10-5.05(m,1H),4.70-4.48(m,7H),4.02(d,J=11.4Hz,1H),3.85(t,J=5.1Hz,2H),3.60(s,4H),3.58 (s,4H),3.52-3.49(m,2H),3.39-3.36(m,2H),2.87(dd,J=8.9,17.8Hz,1H),2.83-2.79(m,1H),2.74-2.66(m,4H),2.51(s,3H),2. 49-2.34(m,8H),2.30-2.10(m,4H),2.02-1.94(m,2H),1.79-1.63(m,9H),1.47(d,J=7.0Hz,3H,1×CH3),1.26(s,3H,1×CH3),1.23( s,3H,1×CH3),1.11-1.10(m,6H,2×CH3),1.07(s,3H,1×CH3),1.02(s,9H,3×CH3),0.90(s,3H,1×CH3),0.83(d,J=6.4Hz,3H,1×CH3).

[0187] 13C-APT (150MHz, CDCl3): δ217.5,210.0,199.8,175.9,173.6,171.9,170.0,159.9,150.5,148.5,147.4,143.3,14 0.0,131.7,130.9,129.6,129.6,126.5,126.5,122.4,72.1,70.5,70.5,70.4,70.2,69.9,69.9,69.5,68.8,58.8 ,57.6,57.1,54.0,51.8,50.2,50.1,48.9,48.8,48.3,47.2,46.8,46.7,39.3,38.0,36.2,35.9,35.9,35.9,35.6,35.5,34.4,32.8,28.8,28.6,27.5,26.6,26.6,26.6,25.2,25.0,22.3,20.8,19.9,19.6,19.5,18.2,17.3,16.2.

[0188] Synthesis route of GAA-V10:

[0189]

[0190] Dissolve the above-mentioned S23 (50 mg, 0.06 mmol) and S32 (36 mg, 0.06 mmol) in dichloromethane, add an appropriate amount of cuprous thiophene-2-carboxylate, stir until the reactants react completely, concentrate, and then perform dichloromethane / methanol = 20 / 1 column chromatography (ball mill flash chromatography column: brand Synthware, catalog number C195173, outer diameter 17 mm, inner diameter 13.4 mm, wall thickness 1.8 mm, effective length 305 mm, ground glass joint 35 / 20, gate 2 mm; column chromatography silica gel: Qingdao Marine Chemical Plant Branch, batch number 0220195, particle size 200-300 mesh) to obtain white solid product S37 (46 mg, 54%).

[0191] V10: 1 H NMR (600MHz, CDCl3): δ8.67 (s, 1H), 7.52 (s, 1H), 7.46 (d, J = 7.9Hz, 1H), 7.37

[0192] (dd,J=8.2,19.4Hz,4H),6.81(t,J=5.2Hz,1H),6.56(d,J=9.0Hz,1H),5.10-5.05(m,1H),4.71-4.47(m,7H),4.02(d,J=11.3H z,1H),3.84(t,J=5.2Hz,2H),3.63-3.59(m,12H),3.52-3.50(m,2H),3.40-3.37(m,2H),2.88-2.66(m,6H),2.52(s,3H),2.49- 2.44(m,4H),2.37-2.11(m,8H),2.02-1.94(m,2H),1.79-1.62(m,10H),1.47(d,J=7.0Hz,3H,1×CH3),1.26(s,3H,1×CH3),1.23 (s,6H,2×CH3),1.10(s,3H,1×CH3),1.08(s,3H,1×CH3),1.02(s,9H,3×CH3),0.90(s,3H,1×CH3),0.83(d,J=6.4Hz,3H,1×CH3).

[0193] 13 C-APT (150MHz, CDCl3): δ217.5,209.8,199.9,175.9,173.6,172.0,170.0,159.9,150.5,148.5,147.4,143.3,140. 0,131.7,130.9,129.7,129.7,126.6,126.6,122.4,72.1,70.6,70.6,70.5,70.5,70.4,70.2,70.1,69.9,69.6,68.8 ,58.7,57.6,57.2,54.0,51.8,50.2,50.2,48.9,48.8,48.4,47.2,46.8,46.7,39.3,38.1,36.3,36.0,36.0,35.8,35.6,35.5,34.4,32.7,28.8,28.6,27.5,26.6,26.6,26.6,25.2,25.1,22.3,20.8,20.0,19.6,19.5,18.2,17.3,16.2.

[0194] Example 2

[0195] The molecular weights of the protein degradation chimeras of all embodiments of the present invention (GAA C1-C10 and V1-V10) are shown in Tables 1 and 2 below.

[0196] Table 1. Molecular weight of compound GAA C1-10

[0197]

[0198]

[0199] Table 2. Molecular weight of compound GAA V1-10

[0200]

[0201]

[0202] Example 3

[0203] In vitro antitumor activity of compounds in all embodiments of this invention (GAA C1-C10 and V1-V10):

[0204] The antiproliferative activity of the protein degradation chimera in this invention against human osteosarcoma cells SJSA-1, human breast cancer cells MCF-7, and human breast cancer cells MDA-MB-231 was tested using an MTT assay kit. 100 μL of SJSA-1 and MCF-7 cell suspensions were prepared in 96-well plates, and the plates were pre-cultured at 37 °C in a 5% CO2 incubator for 24 hours. The next day, the original culture medium was discarded, and 100 μL of serum-free medium containing 50 μM or 100 μM of the protein degradation chimera and 5 μM doxorubicin hydrochloride (DOX) was added to each well. The plates were incubated for 48 or 72 hours. After drug administration, 10 μL of MTT solution was added to each well. After incubating the culture plate in an incubator for 4 hours, add 100 μL of dissolving solution, and then incubate the culture plate in an incubator for another 4 hours. Measure the absorbance at 570 nm in each well using an ELISA reader.

[0205] The effect of the drug on the proliferation of SJSA-1, MCF-7, and MDA-MB-231 cells was calculated using the following formula:

[0206] Cell viability = [(As-Ab)] / [(Ac-Ab)]*100%

[0207] As: Experimental wells (containing cell culture medium, MTT, and the drug to be tested),

[0208] Ac: Control wells (containing cell culture medium, MTT, and no test drug)

[0209] Ab: Blank wells (culture medium and MTT containing no cells or test drug).

[0210] The in vitro antitumor experimental results of compounds GAA C1-C10 or V1-V10 of the present invention are shown in the figure. Figure 1-3 .

[0211] Example 4

[0212] Proteomics analysis of the ganoderic acid A-PROTACs compounds of the present invention

[0213] Six MCF-7 cell samples were used, divided into two groups: the V9 group (PROTAC-treated group) and the control group, with three biological replicates for each group. Data were compared between the treated group and the control group. The data were functionally annotated using Blast2GO version 5, differentially expressed proteins were enriched using GOATOOLS, and pathway analysis was performed using KOBAS.

[0214] Experimental Results: Based on TMT proteomics, quantitative analysis of proteins was performed, identifying a total of 6614 protein groups and 51244 peptides. Statistical methods were used to screen differentially expressed proteins, with the screening criteria being: one-way ANOVAtest p-value < 0.05 and FOLD CHANGE > 1.5. The summary data of all differentially expressed proteins are shown in Table 3. According to the screening criteria, a total of 140 differentially expressed proteins were identified in the drug-treated group and the control group, including 11 upregulated proteins and 129 downregulated proteins. Since the mechanism of action of PROTACs (small molecules) is to directly degrade proteins, we focused on the downregulated differentially expressed proteins among the total differentially expressed proteins. (Based on the screening principles) we screened the differentially expressed proteins between the two groups and ranked them according to the degree of difference, identifying the three proteins with the most significant differences: DOLK, PRAG1, and NR2E1 (TLX). Studies have shown that dysregulation of the NR2E1 (TLX) transcription factor plays a crucial role in the pathogenesis of human neurological diseases and brain tumors. A series of gene expression profiling studies have demonstrated that NR2E1 (TLX) is overexpressed in various types of human brain tumors, and knockout of NR2E1 (TLX) significantly inhibits the proliferation of human glioma cells and improves the survival rate of mice transplanted with glioblastoma stem cells. As a transcription factor, NR2E1 (TLX) can also affect the Wnt / β-catenin signaling pathway, activating the expression of its target genes and regulating the expression of cyclin-dependent kinase inhibitors, such as p21, p57, and p53, and several downstream genes involved in brain tumor development. Combined with differential proteomics results, NR2E1 (TLX) is speculated to be a potential anti-tumor target of ganoderic acid A.

[0215] Table 3. Results of differentially expressed protein screening

[0216]

[0217]

[0218] Example 5

[0219] Western blotting analysis of the ganoderic acid A-PROTACs compounds of the present invention

[0220] We further investigated the effects of compound V9 on the downregulation of the protein NR2E1 and the levels of MDM2, a potential target of GAA identified in previous studies, as well as related genes p53 and p21, using Western blotting experiments. MCF-7 cells were seeded in 6-well plates. After cell attachment, cells were treated with the appropriate concentration of the drug. RIPA lysis buffer was added after the appropriate treatment time, and the entire process was performed on ice. Protein was extracted, and protein concentration was determined using the BCA protein quantification method. The plates were then boiled after adding bromophenol blue. 20 μg of protein was loaded into each well, and electrophoresis was stopped when the bromophenol blue dye reached the bottom of the gel. Wet transfer was performed, followed by washing the membrane three times with TBST for 10 min each time, and blocking with 5% skim milk for 2 h. The membrane was then incubated overnight at 4°C with primary antibodies against NR2E1 (1:1000), MDM2 (1:1000), and β-actin (1:1000). The following day, wash the membrane three times with TBST for 10 minutes each time, add the appropriate secondary antibody (1:10000), incubate at room temperature for 1 hour, wash the membrane three times with TBST for 10 minutes each time, and develop the color using ECL chemiluminescence solution. Figure 3 It is known that compound V9 not only effectively degrades NR2E1 and increases the expression of the downstream target gene p21, but also affects the MDM2-p53 negative feedback regulatory pathway, activating p53 while degrading MDM2. MG-132, a proteasome inhibitor, has been reported to completely inhibit the action of PROTAC. Figure 5A / Figure 5B It can be seen that 25 μM MG-132 effectively blocked V9-induced consumption of NR2E1 and MDM2, indicating that the consumption of NR2E1 and MDM2 by compound V9 is a proteasome-dependent degradation. Figure 5A Furthermore, GAA, VHL ligands, and mixtures thereof had limited effects on NR2E1 and MDM2 protein levels, while the same concentration of V9 led to significant depletion of NR2E1 and MDM2. Figure 5B ).

[0221] The Western blotting results of compound V9 of this invention are shown below. Figures 4-5A / Figure 5B .

[0222] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A PROTAC degrading agent, characterized in that, Selected from the following structures: 。 2. A method for preparing a PROTAC degrading agent, characterized in that, Includes the following steps: 1) Using 3-fluorophthalic anhydride and 3-aminopiperidine-2,6-dione hydrochloride as raw materials, a solvent and sodium acetate were added, and the mixture was heated under reflux to obtain compound S1; 2) Using compound S1 and 3-azidopropylamine as raw materials, a solvent was added and N,N-diisopropylethylamine was reacted to obtain compound S2; 3) Compound S3, ganoderic acid A, was added to a solvent, and then O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate, N,N-diisopropylethylamine and propargylamine were added respectively. The reaction was carried out to obtain compound S4. 4) Using products S2 and S4 as raw materials, a solvent was added, and cuprous thiophene-2-carboxylic acid was added to react and the final product was obtained.

3. The preparation method according to claim 2, characterized in that, It also includes purification: after the reaction in step 4), the crude product is concentrated and purified by column chromatography with dichloromethane / methanol = 20 / 1 to obtain the product.

4. The use of the degrading agent according to claim 1 or the compound prepared by the preparation method according to any one of claims 2-3 in the preparation of antitumor drugs and antitumor-related targeted drugs.

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