Protac compound for targeted degradation of CDK2, preparation method therefor and use thereof

Abstract

The present invention relates to a PROTAC compound for targeted degradation of CDK2, a preparation method therefor and a use thereof, and provides a PROTAC compound that is capable of targeted degradation of CDK2. Compared with existing CDK2 inhibitors, the small-molecule compound prepared by the present invention can effectively induce the degradation of CDK2 protein in MCF-7 cells, exhibits good ability to inhibit the proliferation of MCF-7 cells, demonstrates favorable therapeutic effects against breast cancer, and has the potential to become an effective treatment modality for malignant tumors.

Description

A PROTAC compound for targeted degradation of CDK2, its preparation method and application Technical Field

[0001] This invention belongs to the field of drug synthesis technology, and in particular relates to a PROTAC compound that targets and degrades CDK2, its preparation method, and its application. Background Technology

[0002] Targeted protein degradation (TPD) is an emerging therapeutic approach. PROTACs generally consist of three parts: a target protein ligand, an E3 protein ligase ligand, and two ligands linked by a linker to form a ternary complex. PROTACs hijack the ubiquitin-proteasome system by simultaneously binding to the target protein and the E3 ubiquitin ligase through bifunctional small molecules, inducing ubiquitination of the target protein and its degradation by the proteasome. This directly eliminates disease-related proteins using the cell's own destructive mechanisms, whereas traditional small-molecule targeted therapies for these pathogenic proteins have been extremely challenging. In the 20 years since the concept of PROTAC molecules utilizing the ubiquitin-proteasome system to degrade target proteins was reported, TPD has moved from academia to industry. Many companies have disclosed preclinical and early-stage clinical development plans, and this field is poised to conquer targets previously considered "undrugable."

[0003] CDK2 is a key regulatory site in multiple oncogenic signaling pathways, and its activity is crucial for uncontrolled tumor proliferation. CDK2 inhibitors have the potential to induce growth arrest and apoptosis in cancer cells. Studies have shown that CDK2 gene knockout mice can still survive without significant abnormal responses, suggesting that CDK2 inhibitors may preferentially target cancer cells while preserving normal tissue. Furthermore, research indicates that overexpression of the CDK2 regulatory subunits Cyclin A / E is a key oncogenic process in some cancers. Cells with low Cyclin E expression can resist oncogenic transformation, while overexpression of Cyclin E may bypass CDK4 / 6 by activating CDK2, leading to resistance to CDK4 / 6 inhibitors and thus promoting tumor proliferation, such as in triple-negative breast cancer. Tumors often exhibit either a lack of Rb protein expression or high Cyclin E expression, both of which lead to resistance to CDK4 / 6 inhibitor treatment. Since these cyclins are neither enzymes nor receptors, developing small molecules that directly target cyclins is undoubtedly a significant challenge. Therefore, considering the relative specificity of Cyclin E for CDK2 and its deregulation in certain types of cancer, CDK2 is a very attractive target for treating tumors of specific gene types. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a PROTAC compound for targeted degradation of CDK2, its preparation method, and its applications.

[0005] The technical solution adopted in this invention is: a PROTAC compound with the structure shown in Formula 1;

[0006] R1 is an alkyl group containing 1-3 carbon atoms or a cycloalkane containing 3-6 carbon atoms;

[0007] X1 is a carbonyl group or a sulfonic acid group;

[0008] X2 is -CH2- or carbonyl;

[0009] Linker is a connecting group, which is a 4-6 member nitrogen-containing monocyclic or spirocyclic structure.

[0010] Preferably, the linker structure is as shown in any one of Equations 2a, 2b, 2c, 2d, 2e and 2f;

[0011] Where p is 1 or 2, q is 1 or 2, r is 1 or 2, and s is 1 or 2;

[0012] Where p is 1 or 2, q is 1 or 2, r is 1 or 2, and s is 1 or 2;

[0013] Where m is an integer between 0 and 2, n is 1 or 2, and o is 1 or 2;

[0014] Where p is 1 or 2, q is 1 or 2, r is 1 or 2, and s is 1 or 2;

[0015] Where p is 1 or 2, q is 1 or 2, r is 1 or 2, and s is 1 or 2.

[0016] Preferably, the structure is as shown in formulas b1-b14, and any one of c1 and c2;

[0017] One method for preparing PROTAC compounds involves synthesizing a target protein ligand POI molecule, linking it to a linker, and then linking it to an E3 ligase ligand; or linking the E3 ligase ligand to a linker and then linking it to a POI molecule.

[0018] The molecular structure of the target protein ligand POI is shown in formula a1, a2 or a3;

[0019] The structure of the E3 ligase is shown in formula b1-4;

[0020] Application of PROTAC compounds in the preparation of antitumor drugs.

[0021] Preferably, it targets and degrades intracellular CDK2.

[0022] Preferably, the tumor is a tumor that highly expresses CDK2, or a tumor resistant to CDK2 / 4 / 6 inhibitors.

[0023] Preferably, the tumor is one or more of the following: breast cancer, leukemia, lung cancer, liver cancer, esophageal cancer, pancreatic cancer, colorectal cancer, stomach cancer, cervical cancer, brain cancer, and prostate cancer.

[0024] A pharmaceutical composition comprising a PROTAC compound.

[0025] Preferably, it includes a PROTAC compound or a pharmacologically or physiologically acceptable salt thereof, and also includes one or more pharmaceutically acceptable carriers, excipients, diluents, adjuvants and mediators.

[0026] The advantages and positive effects of this invention are: a new PROTAC compound is synthesized that can effectively induce the selective degradation of CDK2 protein in cancer cells in a dose-dependent manner, and can show good therapeutic effects on multiple types of cancer cells. Attached Figure Description

[0027] Figure 1 shows compound b8 of the present invention. 1 H-NMR spectrum;

[0028] Figure 2 shows compound b8 of the present invention. 13 C-NMR spectrum;

[0029] Figure 3 shows compound b10 of the present invention. 1 H-NMR spectrum;

[0030] Figure 4 shows compound b10 of the present invention. 13 C-NMR spectrum;

[0031] Figure 5 shows the screening effect of the compounds of the present invention on the degradation of CDK2 and CDK1 in MCF-7 cells. Detailed Implementation

[0032] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0033] This invention relates to a PROTAC compound for targeted degradation of CDK2, its preparation method, and its application. Firstly, a PROTAC compound is provided, the structure of which is shown in Formula 1;

[0034] Wherein, R1 is an alkyl group containing 1-3 carbon atoms or a cycloalkane containing 3-6 carbon atoms; X1 is a carbonyl group or a sulfonic acid group; X2 is a carbonyl group; and Linker is a linking group, preferably a 4-6 member nitrogen-containing monocyclic or spirocyclic structure.

[0035] The linker structure is shown in any one of Equations 2a, 2b, 2c, 2d, 2e, and 2f;

[0036] Where p is 1 or 2, q is 1 or 2, r is 1 or 2, and s is 1 or 2;

[0037] Where p is 1 or 2, q is 1 or 2, r is 1 or 2, and s is 1 or 2;

[0038] Where m is an integer between 0 and 2, n is 1 or 2, and o is 1 or 2;

[0039] Where p is 1 or 2, q is 1 or 2, r is 1 or 2, and s is 1 or 2;

[0040] Where p is 1 or 2, q is 1 or 2, r is 1 or 2, and s is 1 or 2.

[0041] The preparation method of the PROTAC compound shown in Formula 1 is as follows: There are two methods for preparing the PROTAC molecule targeting CDK2 protein. The first method is to first synthesize the target protein ligand POI molecule, link it to the linker, and then link it to the E3 ligase ligand. The second method is to link the E3 ligase ligand to the linker and then link the POI molecule.

[0042] The molecular structure of the target protein ligand POI is shown in formula a1, a2 or a3;

[0043] The structure of the E3 ligase is shown in formula b1-4;

[0044] PROTACs are heterogeneous bifunctional small molecules composed of two ligands: one ligand recruits and binds to the protein of interest (POI), while the other recruits and binds to the E3 ubiquitin ligase. These two ligands are linked by linkers of different lengths. PROTAC simultaneously binds to both the POI and the E3 ubiquitin ligase, inducing the ubiquitination of the POI, which is then subsequently degraded by the ubiquitin-proteasome system (UPS). This process repeats, allowing a single PROTAC molecule to cyclically catalyze the degradation of numerous POI proteins.

[0045] CDK2 is expressed at low levels in most normal tissues: except for a few tissues with sustained proliferative function, it is in a quiescent state in most normal cells. CDK2 exhibits high activity in the testes, thus it is believed to play a unique role in meiotic cell division; CDK2-deficient mice are infertile due to their inability to undergo meiosis in gametes. Simultaneously, CDK2 is a key regulatory site in multiple oncogenic signaling pathways, and its enhanced activity is crucial for uncontrolled tumor proliferation. CDK2 inhibitors have the potential to induce growth arrest and apoptosis in cancer cells. Consistent with this, mice with the CDK2 gene knocked out still survive without significant abnormal responses. These basic studies suggest that CDK2 inhibitors, as anticancer drugs, may offer a good therapeutic window by balancing safety and efficacy.

[0046] The CDK family exhibits high structural sequence similarity; for CDK2, which we are interested in, it shares 65% similarity with CDK1. CDK1 is central to the regulation of many biological processes, including cell cycle regulation, DNA replication, and DNA damage repair, tightly linking these processes to the cell cycle progression. CDK1 interacts with Cyclin B to activate cells into the M phase and maintain it to ensure normal mitosis, preventing premature entry into the G1 phase. CDK1 is also an essential isoform in the cell cycle, and literature has shown that CDK1 deficiency can damage normal cells. Therefore, designing inhibitors selective between CDK1 and CDK2 is a key challenge and a major research focus, representing the primary issue in the development of selective CDK2 inhibitors. The early clinical trials of CDK2 inhibitors were terminated due to high toxicity, possibly as a result of simultaneous CDK1 inhibition.

[0047] Meanwhile, due to the high similarity of ATP binding sites among other CDKs, especially CDK1, the development of selective small molecule inhibitors for CDK2 has always been a research challenge, and currently, there are no commercially available small molecule inhibitors targeting CDK2. In recent years, the rise of PROTAC compounds has attracted keen attention from researchers. Utilizing the ability of PROTAC molecules to increase selectivity between targets, applying PROTACs to the study of CDK2 targets will become a key technology to overcome the current difficulties faced by small molecule inhibitors.

[0048] The prepared PROTAC compounds can form protein hydrolysis-targeting chimeras, enabling targeted degradation of CDK2. PROTAC compounds or their pharmacologically or physiologically acceptable salts can be added to drugs for the treatment or prevention of CDK2-related diseases. PROTAC compounds or their pharmacologically or physiologically acceptable salts can be added to antitumor drugs for tumors such as leukemia, lung cancer, liver cancer, esophageal cancer, pancreatic cancer, colorectal cancer, gastric cancer, cervical cancer, brain cancer, or prostate cancer; they are particularly suitable for tumors with high CDK2 expression or tumors resistant to CDK2 / 4 / 6 inhibitors, such as in drugs for the treatment of breast cancer. Antitumor drugs also include pharmaceutically acceptable carriers, excipients, diluents, adjuvants, mediators, or combinations thereof. The synthesized PROTAC compounds can effectively induce selective degradation of CDK2 protein in cancer cells in a dose-dependent manner, showing good therapeutic effects.

[0049] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in terms of operation steps are performed in accordance with the corresponding product manuals. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can be purchased from commercial companies.

[0050] Example 1: Synthesis of target protein ligand a1

[0051] The synthetic route for compound a1 is shown below:

[0052] The specific preparation steps of intermediate a1-3 in the technical route are as follows: Compound a1-1 (1 g, 3.9 mmol), CuI (75 mg, 0.39 mg, 0.10 equiv.), Pd(PPh3)2Cl2 (273 mg, 0.39 mmol, 0.10 equiv.), and compound a2-2 (820 mg, 3.9 mmol) were weighed and transferred to a 100 mL round-bottom flask. Oxygen and residual moisture were then removed under vacuum. Under nitrogen protection, 30.0 mL of anhydrous DMF and TEA (1.1 mL, 7.8 mmol, 2.0 equiv.) were added sequentially, and the reaction was carried out at 100 °C under nitrogen protection. The reaction mixture was stirred at room temperature for 12 h. TLC monitoring showed product a1-3 (PE:EA = 1:1, Rf = 0.4). Most of the DMF and TEA were removed by rotary evaporation under reduced pressure, followed by the addition of 100 mL of LDM and washing with 100 mL of saturated sodium chloride solution. The DCM solution was dried over anhydrous sodium sulfate, concentrated, and then purified by silica gel column chromatography (PE:EA = 3:1) to give a yellow oily substance a1-3920 mg, with a yield of 68%.

[0053] The intermediate a1-3 was tested, and the test data are as follows: 1 HNMR(400MHz, CDCl3)δ7.89(d,J=3.9Hz,1H),6.71(d,J=5.5Hz,1H),5.29(s,1H),3.78–3.59(m,2H),3 .33(d,J=21.0Hz,2H),2.94(dt,J=6.8,3.8Hz,1H),1.96–1.83(m,2H),1.78–1.67(m,2H),1.46(s,9H). 13 CNMR(101MHz, CDCl3)δ158.74,153.70,145.85,144.88,113.40,102.50,78.60,73.60,41.04,35.02,30.29,27.43,26.93.

[0054] The specific preparation steps of intermediate a1-6 in the technical route are as follows: weigh compound a1-4 (200 mg, 0.59 mmol), compound a1-5 (226 mg, 0.72 mmol, 1.2 equiv.), Cs2CO3 (392 mg, 1.2 mmol, 2.0 equiv.), and Xphos2 nd(48 mg, 0.06 mmol, 0.10 equiv.) were transferred to a 10 mL double-necked flask. Oxygen and residual moisture were removed under vacuum. A mixed solution of 3.0 mL 1,4-Dioxane and 0.6 mL H₂O was added. The reaction was carried out at 100 °C for 6 h under nitrogen protection. TLC monitoring showed product a1-6 (PE:EA = 1:3, Rf = 0.3). Most of the 1,4-Dioxane / H₂O and TEA were removed by rotary evaporation under reduced pressure. Then, 10 mL of DCM was added, and the mixture was washed with 10 mL of saturated sodium chloride solution. The DCM solution was dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (PE:EA = 1:2) to give 235 mg of white solid a1-6, yield 80%. The intermediate a1-6 was analyzed, and the results were as follows: 1 HNMR(400MHz, CDCl3)δ12.24(s,1H),7.62(s,1H),7.34(d,J=10.8Hz,1H),7.20(s,1H),6.46(s,1H),4.82–4.66(m,1H),4.40–4.23(m,2H),3.07( t,J=11.0Hz,1H),2.95(t,J=11.7Hz,2H),2.69(s,3H),2.13(d,J=12.6Hz ,2H),1.86(dd,J=23.2,12.1Hz,2H),1.70(d,J=6.6Hz,6H),1.50(s,9H). 13 CNMR(101MHz,CD Cl3)δ154.79,152.30,149.50,145.27,141.41,141.36,141.16,141.13,136.91,136.82,132. 05,131.88,108.01,107.82,107.28,95.32,79.67,48.47,36.35,31.74,28.51,21.46,15.04.

[0055] The specific preparation steps of intermediate a1-7 in the technical route are as follows: Weigh compound a1-6 (200 mg, 0.31 mmol), transfer to a 10 mL round-bottom flask, and add sequentially 3.0 mL of anhydrous DCM and 0.6 mL of LTFA. The compound is reacted at room temperature for 2 h. TLC monitoring: Product a1-7 (DCM:MeOH = 5:1, R f =0.1). Add saturated sodium carbonate aqueous solution to a pH of around 9, then add 10 mL of EA. The EA solution is dried over anhydrous sodium sulfate and concentrated to give compound a1-7 140 mg, with a yield of 96%.

[0056] The specific preparation steps of compound a1 in the technical route are as follows: Compound a1-7 (60 mg, 0.153 mmol) and TEA (0.056 mL, 0.41 mmol, 2.7 equiv.) were weighed and transferred to a 5 mL round-bottom flask. Oxygen and residual moisture were removed under vacuum. 1.5 mL of anhydrous DMF and Ms2O (28 mg, 0.24 mmol) were added sequentially, and the reaction was carried out at room temperature for 2 h. TLC monitoring showed that product a1 (PE:EA = 1:2, R...) f =0.5). The reaction compound was removed by rotary evaporation under reduced pressure to remove most of the DMF and TEA. Then, 5 mL of DCM was added, and the mixture was washed with 5 mL of saturated sodium chloride solution. The DCM solution was dried over anhydrous sodium sulfate and concentrated, then purified by silica gel column chromatography (PE:EA = 1:1) to give 16.2 mg of a white solid a1, with a yield of 16.9%. The detection data for compound a1 are as follows: 1 H NMR (400MHz, DMSO) δ11.77(s,1H),8.21(s,1H),7.76(s,1H),7.32(d,J=11. 7Hz,1H),7.22(d,J=5.0Hz,1H),6.39(d,J=1.6Hz,1H),4.90–4.78(m,1H),3 .66(d,J=11.9Hz,2H),2.90(s,3H),2.85(dd,J=11.9,2.1Hz,2H),2.63(s,3 H),2.20–2.05(m,2H),1.78(qd,J=12.6,3.9Hz,2H),1.61(d,J=6.9Hz,6H). 13 C NMR(101MHz,DMSO)δ153.50,149.66,145.35,142.32,139.71,118.42,115.0 9,108.20,107.73,94.94,48.53,45.94,34.81,34.59,30.92,21.36,14.99.

[0057] Example 2: Synthesis of target protein ligand a2

[0058] The synthetic route for compound a2 is shown below:

[0059] The specific preparation steps of compound a2 in the technical route are as follows: the synthesis steps are the same as those of a1, and the crude product is purified by silica gel column chromatography to obtain 36 mg of compound a2 (mobile phase: PE:EA = 1:1), with a yield of 64%.

[0060] The detection data for compound a2 are as follows: 1H NMR (400MHz, CDCl3) δ12.32(s,1H),8.26(d,J=5.1Hz,1H),7.61(s,1H),7.33(d,J=1 1.0Hz,1H),7.23(d,J=5.2Hz,1H),6.46(s,1H),4.77–4.61(m,1H),3.98(d,J=13.6H z,1H),3.45–3.23(m,2H),3.16(dd,J=15.8,7.6Hz,1H),2.80(t,J=12.8Hz,1H),2.7 0(s,3H),2.26–2.18(m,2H),2.16(s,3H),1.94–1.76(m,2H),1.70(d,J=6.9Hz,6H). 13 C NMR (101MHz, CDCl3) δ169.05,152.56,148.41,145.23,140.01,115.10,108.06,107.87,107 .38,95.63,48.53,46.54,41.65,36.18,32.25,31.27,21.58,21.50,15.07.HRMS(ESI)calcd for[M+H] + C 25 H 29 FN5O + 434.2356, found 434.2278.

[0061] Example 3: Synthesis of target protein ligand a3

[0062] The synthetic route for compound a3 is shown below:

[0063] The specific preparation steps of intermediate a3-2 in the technical route are as follows: Weigh a3-1 (4.12 g, 72.3 mmol), transfer it to a 250 mL single-necked flask, and remove oxygen and residual moisture under vacuum. Under nitrogen protection, sequentially add 70.0 mL of anhydrous DCM, TEA (10.0 mL, 86.8 mmol, 1.2 equiv.), and dropwise add acetic anhydride (7.5 mL, 79.5 mmol, 1.1 equiv.) in an ice bath. After adding the mixture, react at room temperature for 12 h. TLC monitoring: Product a4-2 (EA:MeOH = 50:1, R...) f=0.2). The reaction compound was removed by rotary evaporation under reduced pressure to remove most of the acetic anhydride and TEA. Then, 70 mL of anhydrous diethyl ether was added to dissolve it. 6 g of anhydrous K₂CO₃ was added and stirred for 12 h. A large amount of white precipitate formed, which was then filtered. The filtrate was concentrated under reduced pressure to obtain a yellow oily substance. The crude product was purified by silica gel column chromatography (EA) to obtain 2.7 g of a white solid (hygroscopic) a₃-2, with a yield of 38%. The intermediate a₃-2 was analyzed, and the results were as follows: 1 H NMR (400MHz, CDCl3) δ2.74–2.64(m,1H),2.14(s,1H),1.94(s,3H),0.75(q,J=6.8Hz,2H),0.54–0.40(m,2H). 13 C NMR (101MHz, CDCl3) δ171.81, 23.07, 22.73, 8.21, 6.50.

[0064] The specific preparation steps of intermediate a3-4 in the technical route are as follows: Compounds a3-2 (2.3 g, 23 mmol, 2.0 equiv.) and a3-3 (2.4 g, 11.5 mmol) were weighed and transferred to a 250 mL double-necked flask. Oxygen and residual moisture were removed under vacuum. Under nitrogen protection, 100.0 mL of anhydrous toluene and TEA (2.5 mL, 17.25 mmol, 1.5 equiv.) were added sequentially. The mixture was stirred at room temperature for 30 min. POCl3 (2.1 mL, 23 mmol, 2.0 equiv.) was added, and the mixture was heated to 100 °C and reacted for 3 h. The solution changed from colorless to yellow initially. TLC monitoring showed that product a4-4 (PE:EA = 5:1, R...)... f =0.5). The reactants were removed by rotary evaporation under reduced pressure to remove most of the toluene and TEA. Then, 200 mL of DCM was added to dissolve the compound, followed by washing with 200 mL of saturated sodium chloride solution. The DCM solution was dried over anhydrous sodium sulfate. The filtrate was concentrated under reduced pressure to obtain a yellow, oily crude product, which was purified by column chromatography (PE:EA = 7:1) to give 2.8 g of a white solid a3-4, with a yield of 84.2%. The intermediate a3-4 was analyzed, and the results were as follows: 1 H NMR (400MHz, DMSO) δ7.41 (s, 1H), 7.31 (d, J = 6.3Hz, 2H), 2.80–2.63 (m, 1H), 1.63 (s, 3H), 0.70–0.54 (m, 2H), 0.50–0.34 (m, 2H). 13 C NMR (101MHz, DMSO) δ160.03,156.62,154.36,115.68,115.39,24.19,6.56.

[0065] The specific preparation steps of intermediate a3-5 in the technical route are as follows: a3-4 (2.8 g, 10 mmol) and t-BuOK (2.2 g, 20 mmol, 2.0 equiv.) were weighed into a 250 mL double-necked flask. Oxygen and residual moisture were removed under vacuum. 100.0 mL of anhydrous THF was added under nitrogen protection to dissolve the compound. The mixture was heated to 80 °C, and the solution changed from anhydrous to yellow. The reaction time was 4 h. TLC monitoring: Product a4-5 (PE:EA = 3:1, R...) f =0.4), cooled to room temperature and filtered. The filter cake was washed with dichloromethane. The filtrate was concentrated under reduced pressure to remove most of the THF, dissolved in 100 mL of DCM, washed with 100 mL of saturated sodium chloride, and dried over anhydrous sodium sulfate. The concentrated solution yielded a yellow crude solid, which was purified by column chromatography (PE:EA = 4:1) to give 2.5 g of white solid a3-5, with a yield of 95%. The intermediate a3-5 was analyzed, and the results were as follows: 1 H NMR (400MHz, CDCl3) δ7.42(d,J=1.5Hz,1H),7.06(dd,J=9.6,1.6Hz,1H),3.25–3.13(m,1H),2.66(s,3H),1.35–1.18(m,2H),1.13–0.93(m,2H). 13 C NMR (101MHz, CDCl3) δ154.70,154.06,151.51,139.67,139.57,129.89,129.72 ,114.47,114.42,111.78,111.57,109.68,109.64,28.66,25.03,14.79,7.02.

[0066] The specific preparation steps of intermediate a3-6 in the technical route are as follows: Weigh compound a3-5 (2.5 g, 9.3 mmol), bis(pinacol)diboron (3.5 g, 13.9 mmol, 1.5 equiv.), (C6H5)3P (400 mg, 1.39 mmol, 1.5 equiv.), Pd(OAC)2 (200 mg, 0.93 mmol, 0.10 equiv.), and AcOK (3.1 g, 32.5 mmol, 5.0 euqiv.). Transfer them to a 250 mL double-necked flask, purge with nitrogen three times, add 100.0 mL of 1,4-Dioxane, the solution turns slightly black, and the reaction is heated to 100 °C under reflux. After 10 h, the solution turns black. After 8 h, monitor the reaction; TLC shows PE:EA = 3:1, indicating the reaction is complete. The solution was filtered through diatomaceous earth, concentrated under reduced pressure, and then redissolved in 100 mL of DCM. The solution was washed three times with 100 mL of saturated saline solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a yellow crude solid. 1.5 g of a white solid, a3-6, was purified by column chromatography (PE:EA = 3:2), with a yield of 51%. The intermediate a3-6 was analyzed, and the results are as follows: 1 H NMR (400MHz, CDCl3) δ7.72(s,1H),7.35(d,J=10.6Hz,1H),3.28–3.17(m,1H),2.71(s,3H),1.36(s,12H),1.29(q,J=6.7Hz,2H),1.09(t,J=7.8Hz,2H). 13 C NMR (101MHz, CDCl3) δ155.07,113.28,113.12,112.89,84.03,25.23,24.90,24.61,7.28.

[0067] The specific preparation steps of intermediate a3-7 in the technical route are as follows: the compound synthesis steps are the same as those for a1-6. The crude product is purified by silica gel column chromatography (PE:EA = 1:3) to obtain 235 mg of compound a3-7, with a yield of 80%. The intermediate a3-7 was analyzed, and the detection data are as follows: 1H NMR (400MHz, CDCl3) δ12.24(s,1H),7.62(s,1H),7.34(d,J=10.8Hz,1H),7.20(s,1H),6.46(s,1H),4.82–4.66(m,1H),4.40–4.23(m,2H),3.07(t ,J=11.0Hz,1H),2.95(t,J=11.7Hz,2H),2.69(s,3H),2.13(d,J=12.6Hz,2H),1.86(dd,J=23.2,12.1Hz,2H),1.70(d,J=6.6Hz,6H),1.50(s,9H). 13 C NMR(101MHz, CDCl3)δ154.79,152.30,145.27,108.01,107.82,107.28,95.32,79.67,48.47,36.35,31.74,28.51,21.46,15.04.HRMS(ESI)calcd for[M+H] + C 28 H 33 FN5O2 + 490.2614, found 490.2540.

[0068] The specific preparation steps of intermediate a3-8 in the technical route are as follows: the compound synthesis steps are the same as those for a1-7. The crude product is purified by silica gel column chromatography (DCM:MeOH = 5:1) to obtain 140 mg of compound a3-8, with a yield of 96%. Intermediate a3-8 was analyzed, and the detection data are as follows: HRMS (ESI) calcd for [M+H] + C 23 H 25 FN5 + 390.2090, found 390.2016.

[0069] The specific preparation steps of compound a3 in the technical route are as follows: the synthesis steps of compound a3 are the same as those of a1. The crude product is purified by silica gel column chromatography (PE:EA = 1:1) to obtain 14 mg of compound a3, with a yield of 45%. The detection data of compound a3 are as follows: 1H NMR (400MHz, DMSO) δ11.77(s,1H),8.21(s,1H),7.76(s,1H),7.32(d,J=11. 7Hz,1H),7.22(d,J=5.0Hz,1H),6.39(d,J=1.6Hz,1H),4.90–4.78(m,1H),3 .66(d,J=11.9Hz,2H),2.90(s,3H),2.85(dd,J=11.9,2.1Hz,2H),2.63(s,3 H),2.20–2.05(m,2H),1.78(qd,J=12.6,3.9Hz,2H),1.61(d,J=6.9Hz,6H). 13 C NMR (101MHz, DMSO) δ153.50,149.66,145.35,142.32,139.71,118.42,115.09,108.2 0,107.73,94.94,48.53,45.94,34.81,34.59,30.92,21.36,14.99.HRMS(ESI)calcd for[M+H] + C 24 H 27 FN5O2S + 468.1759, found 468.1719.

[0070] Example 4: Synthesis of compound b1

[0071] The synthetic route for compound b1 is shown below;

[0072] The specific preparation steps of intermediate b1-1 in the technical route are as follows: Weigh a1-7 (0.20 g, 0.39 mmol) and N-Boc piperazine acetic acid (0.18 mg, 0.78 mmol, 2.0 equiv.), and transfer them to a 10 mL flask; then, remove oxygen and residual moisture under vacuum. Under nitrogen protection, add 1.0 mL of anhydrous DMF, TEA (0.11 mL, 0.78 mmol, 2.0 equiv.), and HATU (0.18 mg, 0.78 mmol, 2.0 equiv.) in sequence in a DMF solution (1.0 mL). The reaction mixture is stirred at room temperature for 10 h. TLC monitoring: Product b1-1 (MeOH:DCM = 5:95, R f=0.5). Most of the DMF and TEA were removed by rotary evaporation under reduced pressure. Then, 10 mL of DCM was added, followed by washing with 10 mL of saturated NaHCO3 and 10 mL of saturated sodium chloride solution. The DCM solution was dried over anhydrous sodium sulfate, concentrated, and then purified by silica gel column chromatography (DCM:MeOH = 20:1) to obtain a pale yellow solid powder, b1-1190 mg, in 67% yield.

[0073] The intermediate b1-1 was tested, and the test data are as follows: 1 HNMR (400MHz, CDCl3) δ12.02 (s, 1H), 8.26 (d, J = 4.6Hz, 1H), 7.61 (s, 1H), 7.32 (d, 3 J HF =11.0Hz,1H),7.20(d,J=4.8Hz,1H),6.44(s,1H),4.82-4.66(m,2H),4.23(d,J=12.8Hz,1H),3.44(s,4H),3.39-3.10(m,4H),2.84(t ,J=12.5Hz,1H),2.69(s,3H),2.54(s,4H),2.23(d,J=11.7Hz,2H),1.83(dd,J=22.7,11.1Hz,2H),1.70(d,J=6.9Hz,6H),1.42(s,9H). 13 CNMR(101MHz,CDCl3)δ167.42,154.81(d, 1 J CF =22? Hz),154.65,152.43,149.24,144.69,141.77,140.99,136.94,136.85,133.26,133.19,131.93,119.70,115.17,108.01,107 .82,107.31,95.45,79.83,61.03,52.94,48.49,45.71,42.01,32.48,31.26,29.69,28.38,21.48.HRMS(ESI)calcdfor[M+H] + C 34 H 45 FN7O3 + 618.3596, found618.3490.

[0074] The specific preparation steps of intermediate b1-2 in the technical route are as follows: Weigh b1-1 (200 mg, 0.41 mmol), transfer it to a 25 mL flask, add 4.0 mL of DCM to dissolve it, the system is a pale yellow solution, and the addition of 1.0 mL of LTFA solution changes the fluorescence from green to yellow. After 1 h, TLC monitoring of the reaction shows: DCM:MeOH = 10:1, the reaction is complete. Adjust the pH of the system to ≈9 with saturated Na2CO3, add 10 mL of DCM to dilute, and extract to obtain the organic phase. Dry the organic phase with anhydrous sodium sulfate, filter and concentrate to finally obtain b1-2 as a yellow solid of 190 mg, with a yield of 93%. The product does not need to be purified and can be directly used for the next reaction. The detection data of intermediate b1-1 are as follows: HRMS (ESI) calcd for [M+H] + C 29 H 37 FN7O + 518.3039, found518.2965.

[0075] The specific preparation steps of intermediate b1-3 in the technical route are as follows: Weigh b1-2 (200 mg, 0.39 mmol) and 4-N-Boc piperidinecarboxaldehyde (175 mg, 0.78 mmol, 2.0 equiv.) into a 10 mL flask, add 3.0 mL of anhydrous DCE, and react at 70 °C for 3 h under nitrogen protection. At this time, the reaction system is a yellow solution. Stop heating and let the reaction cool to room temperature. Then add sodium triacetylborohydride (496 mg, 2.34 mmol, 6.0 equiv.). After the addition, the system becomes turbid. React overnight at room temperature. After 10 h, monitor the reaction by TLC: DCM:MeOH = 10:1. The product shows blue fluorescence under TLC. f =0.5. After the reaction was complete, 3 mL of water was added to quench the reaction. The organic phase was extracted and collected by layering. The organic phase was washed three times with 3 mL of saturated brine and backwashed twice with 3 mL of DCM. The organic phases were combined. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a yellow oily crude product. Column chromatography (DCM:MeOH = 30:1-20:1) yielded 190 mg of yellow solid powder b1-3, with a yield of 69%. The intermediate b1-3 was analyzed, and the results were as follows: 1HNMR (400MHz, CDCl3) δ11.94(s,1H),8.23(d,J=5.1Hz,1H),7.60(s,1H),7.32(d,J=11.0 Hz,1H),7.19(d,J=5.1Hz,1H),6.43(s,1H),4.80–4.67(m,2H),4.20(d,J=13.1Hz,1H),4 .06(s,2H),3.32(d,J=13.9Hz,1H),3.26–3.07(m,3H),2.88–2.39(m,14H),2.30–2.15(m ,4H),1.88–1.76(m,2H),1.75–1.57(m,9H),1.43(s,9H),1.06(dd,J=20.7,11.3Hz,2H). 13 CNMR(101MHz, CDCl3)δ154.85,152.43,149.35,144.59,141.59,141.08,119.60,115.13,107.99,107.80,107.27,95.36,79.33 ,64.17,53.38,48.48,45.60,41.97,36.06,33.15,32.44,31.26,30.74,29.71,28.46,21.50,15.06.HRMS(ESI)calcdfor[M+H] + C 40 H 56 FN8O3 + 715.4367, found 715.4381.

[0076] The specific preparation steps of compound b1 in the technical route are as follows: Weigh b1-3 (50 mg, 0.1 mmol) into a 5 mL round-bottom flask, add 1.0 mL of HCl / MeOH solution (2 mol / L), and react at room temperature. After 30 min, TLC monitoring indicates the reaction is complete. Remove the solvent under reduced pressure to obtain a white solid. Add the obtained white solid, b1-4 (46 mg, 0.15 mmol), and DIPEA (113 μL, 0.75 mmol, 5.0 equiv.) to a 5 mL round-bottom flask, add 1.0 mL of anhydrous DMF to dissolve, and heat to 90 °C for 8 h under nitrogen protection. The system gradually turns dark green. After 8 h, TLC monitoring shows DCM:MeOH = 10:1, and product R... f=0.45. After the reaction was complete, heating was stopped, and the reaction solution was cooled to room temperature. Most of the DMF and DIPEA were removed under reduced pressure. The resulting product was redissolved in 3 mL of DCM and washed three times with 3 mL of saturated brine. The product was backwashed twice with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Column chromatography yielded b1 as a fluorescent green solid (30 mg), with a yield of 49%. The detection data for compound b1 were as follows: 1 H NMR (400MHz, CDCl3) δ11.28(s,1H),9.92(s,1H),8.27(d,1H),7.64(d,J=8.3Hz,1H),7.59(s,1H),7.32(d,J=10. 9Hz,1H),7.23(s,1H),7.17(s,1H),7.01(d,J=8.2Hz,1H),6.42(s,1H),4.94(dd,J=10.9,4.6Hz,1H),4.71(dd,J= 13.3,7.3Hz,2H),4.16(d,J=9.3Hz,1H),3.90(d,J=12.0Hz,2H),3.34(d,J=13.6Hz,1H),3.28–3.06(m,3H),3.00– 2.74(m,8H),2.75–2.46(m,11H),2.43–2.26(m,2H),2.22–2.09(m,3H),1.98–1.73(m,5H),1.69(d,J=6.6Hz,6H). 13 C NMR (101MHz, CDCl3) δ170.92,168.04,167.08,166.67,166.28,154.30,151.37,148.28,143. 30,140.49,140.40,135.83,133.37,132.37,124.41,118.28,117.59,116.80,114.17,107.4 8,106.96,106.78,106.25,94.44,62.76,52.25,48.12,47.45,46.85,44.57,41.00,34.94,3 0.91,30.54,30.21,29.09,28.68,28.34,21.85,21.67,20.46,14.03,13.10.HRMS(ESI)calcd for[M+H] + C 48 H 56 FN 10 O5 + 871.4409, found 817.4341.

[0077] Example 5: Synthesis of compound b2

[0078] The synthetic route for compound b2 is as follows:

[0079] The specific preparation steps of intermediate b2-1 in the technical route are as follows: the synthesis steps are the same as those for compound b1-3. The crude product was purified by column chromatography to obtain 190 mg of yellow solid powder b2-1, with a yield of 67% (DCM:MeOH = 30:1-20:1). The detection data for intermediate b2-1 are as follows: 1 HNMR (400MHz, CDCl3) δ11.03(s,1H),8.27(d,J=4.6Hz,1H),7.60(s,1H),7.33(d,J=11.0Hz,1H),7. 20(d,J=5.0Hz,1H),6.44(s,1H),4.80–4.67(m,2H),4.25–4.15(m,1H),4.00(t,J=8.4Hz,2H),3.57 (t,2H),3.35-3.07(m,4H),2.89-2.75(m,2H),2.69(s,1H),2.58(d,J=42.9Hz,8H),2.19(d,J=11.9 Hz,2H),1.81(dd,J=19.3,9.4Hz,2H),1.70(d,J=6.9Hz,6H),1.42(s,9H).HRMS(ESI)calcdfor[M+H] + C 38 H 52 FN8O3 + 687.4141, found687.4068.

[0080] The specific preparation steps for compound b2 in the technical route are as follows: Following the same steps as compound b1, the crude product was purified by column chromatography to obtain 26 mg of fluorescent green solid b2, with a yield of 53%. The detection data for compound b2 are as follows: 1H NMR (400MHz, DMSO) δ11.78(s,1H),11.09(s,1H),8.21(d,J=5.0Hz,1H),7.75(s,1H),7.62(d,J=8.3Hz,1H),7.32(d,J=11.6Hz,1H), 7.22(d,J=5.0Hz,1H),6.75(s,1H),6.61(d,J=10.2Hz,1H),6.34(s,1H),5.12–4.98(m,1H),4.88–4.76(m,1H),4.44(d,J=11.9Hz,2H ),4.10(dd,J=18.1,10.1Hz,3H),3.70–3.52(m,4H),3.45(s,2H),3.18–2.99(m,4H),2.96–2.78(m,2H),2.77–2.65(m,2H),2.61(s,3 H),2.60–2.55(m,2H),2.46–2.35(m,3H),2.09–1.93(m,4H),1.71(dd,J=11.4Hz,1H),1.60(d,J=6.9Hz,6H),1.52(d,J=14.0Hz,1H). 13 C NMR(101MHz,DMSO)δ173.29,170.59,167.64,155.50,154.30,153.47,149.89,145.75,142.50, 139.54,137.36,134.27,133.18,131.64,129.24,125.32,122.25,120.36,118.30,117.14,115 .01,114.43,108.00,107.20,104.71,94.54,77.75,77.01,76.38,70.14,67.86,66.15,63.21, 62.52,56.08,49.16,48.33,45.66,35.50,31.47,29.50,22.67,21.41,15.10.HRMS(ESI)calcd for[M+H] + C 46 H 52 FN 10 O5 + 843.4103, found 843.4208.

[0081] Example 6: Synthesis of compound b3

[0082] The synthetic route for compound b3 is as follows:

[0083] The specific preparation steps of compound b3 in the technical route are as follows: weigh b1-2 (50 mg, 0.1 mmol), b1-4 (46 mg, 0.15 mmol), and DIPEA (0.09 mL, 0.5 mmol) into a 5 mL round-bottom flask, add 1 mL of anhydrous DMF to dissolve, heat to 80 °C under nitrogen protection and react for 8 h, the system gradually turns dark green.

[0084] After 8 hours, the reaction was monitored by TLC. The ratio of DCM to MeOH was 10:1, and the product R was... f =0.45. After the reaction was complete, heating was stopped, and the reaction solution was cooled to room temperature before removing DMF under reduced pressure. The resulting product was redissolved in 3 mL of DCM and washed three times with 3 mL of saturated brine. The product was backwashed twice with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Column chromatography yielded 30 mg of b3 as a fluorescent green solid, with a yield of 57%. The detection data for compound b3 are as follows: 1 H NMR (400MHz, CDCl3) δ11.54(s,1H),10.26(s,1H),8.28(d,J=5.1Hz,1H),7.64(d,J=8.5Hz,1H),7.58(s ,1H),7.31(d,J=11.0Hz,1H),7.23(s,1H),7.18(d,J=5.1Hz,1H),6.99(d,J=7.5Hz,1H),6.43(s,1H),4. 95(dd,J=12.0,5.2Hz,1H),4.80–4.64(m,2H),4.18(d,J=12.2Hz,1H),3.39(s,5H),3.30–3.07(m,3H),2 .93–2.69(m,8H),2.67(s,3H),2.25–2.07(m,3H),1.80(dd,J=21.8,10.1Hz,2H),1.67(d,J=6.9Hz,6H). 13C NMR (101MHz, CDCl3) δ172.19,169.24,167.91,167.28,155.36,154.75,152.47,152.24,149.1 1,144.42,141.65,141.12,136.97,136.85,134.22,133.27,133.19,132.04,131.86,125.29,1 19.74,119.50,118.04,115.15,108.66,107.98,107.80,107.27,95.58,60.73,52.57,49.24, 48.47,47.37,45.72,42.04,35.98,32.50,31.60,31.18,29.70,22.80,21.47.HRMS(ESI)calcd for[M+H] + C 42 H 45 FN9O5 + 774.3523, found 774.3449.

[0085] Example 7: Synthesis of compound b4

[0086] The synthetic route for compound b4 is as follows:

[0087] The specific preparation steps of intermediate b4-1 in the technical route are as follows: The synthesis steps are the same as for compound b1-3. The crude product was purified by column chromatography to obtain 178 mg of yellow solid powder of b4-1, with a yield of 63% (mobile phase: DCM:MeOH = 30:1-20:1). The detection data for intermediate b4-1 are as follows: 1 H NMR (400MHz, CDCl3) δ12.04(s,1H),8.26(s,1H),7.61(s,1H),7.33(d,J=10.7Hz,1H),7.20(s,1H) ,6.44(s,1H),4.75(d,J=12.8Hz,2H),4.21(d,J=12.2Hz,1H),4.13–3.95(m,2H),3.23(ddd,J=26.5 ,21.3,14.6Hz,5H),2.90–2.76(m,2H),2.69(s,5H),2.65(d,J=6.1Hz,12H),2.21(d,J=12.2Hz,2H ),1.82(dd,J=22.9,11.2Hz,2H),1.69(d,J=6.4Hz,6H),1.63(dd,J=21.2,9.3Hz,3H),1.43(s,9H).13 C NMR (101MHz, CDCl3) δ167.65,154.83,152.39,149.58,144.46,141.45,136.96,136. 87,133.36,133.29,132.07,131.90,119.47,115.19,107.97,107.79,107.27,107.2 5,95.43,79.29,55.77,52.81,48.47,45.54,41.98,36.12,34.35,32.51,32.06,31. 92,31.28,29.70,29.66,29.36,28.46,22.69,21.49,15.07,14.12.HRMS(ESI)calcd for[M+H] + C 41 H 58 FN8O3 + 729.4576, found 729.4538.

[0088] The specific preparation steps for compound b4 in the technical route are as follows: The synthesis steps are the same as for compound b1. Crude product column chromatography yielded 23 mg of b4 as a fluorescent green solid, with a yield of 54%. The detection data for compound b4 are as follows: 1 H NMR (400MHz, CDCl3) δ11.46(s,1H),10.19(s,1H),8.28(s,1H),7.63(d,J=8.3Hz,1H),7.59(s,1H),7.23( s,1H),7.17(d,J=4.1Hz,1H),7.00(d,J=8.5Hz,1H),6.42(s,1H),5.00–4.88(m,1H),4.72(dt,J=13.1,6. 6Hz,2H),4.19(d,J=10.6Hz,1H),3.89(d,J=12.2Hz,2H),3.36–3.05(m,5H),2.99–2.71(m,8H),2.68(s,3 H), 2.52 (d, J = 64.7Hz, 10H), 2.15 (dd, J = 22.1, 13.4Hz, 4H), 1.77 (d, J = 12.2Hz, 5H), 1.68 (d, J = 6.6Hz, 6H). 13C NMR (101MHz, CDCl3) δ172.09,169.11,168.13,167.91,167.35,155.34,154.77,152.38,152.26,149.38,144.41 ,141.46,141.37,136.94,136.85,134.42,133.42,133.36,132.00,131.83,125.39,119.36,118.45,117.69,115 .16,108.49,107.98,107.79,107.24,95.37,61.10,55.79,53.45,53.03,52.83,49.16,48.47,48.10,45.66,41. 98,36.00,34.15,32.97,32.42,31.59,31.53,31.28,31.25,29.70,22.84,22.68,21.48,15.05.HRMS(ESI)calcd for[M+H] + C 49 H 58 FN 10 O5 + 885.4568, found 885.4497.

[0089] Example 8: Synthesis of compound b5

[0090] The synthetic route for compound b5 is as follows:

[0091] The specific preparation steps of intermediate b5-1 in the technical route are as follows: The synthesis steps are the same as those for compound b1-3. The crude product was purified by column chromatography to obtain 168 mg of yellow solid powder of b5-1, with a yield of 64% (eluent: DCM:MeOH = 30:1-20:1). The detection data for intermediate b5-1 are as follows: 1H NMR (400MHz, CDCl3) δ12.05(s,1H),8.26(s,1H),7.60(s,1H),7.33(d,J=10.9Hz,1H),7.20 (s,1H),6.45(s,1H),4.86–4.60(m,2H),4.23(d,J=12.2Hz,1H),3.87(d,J=6.7Hz,2H),3.76 (s,2H),3.38–3.10(m,4H),3.03(s,1H),2.83(t,J=12.0Hz,1H),2.69(s,3H),2.60(s,4H), 2.38(s,4H),2.22(d,J=12.6Hz,2H),1.89–1.75(m,2H),1.70(d,J=6.4Hz,6H),1.40(s,9H). 13 C NMR (101MHz, CDCl3) δ167.73,156.27,154.81,152.41,152.30,149.59,144.5 6,141.47,136.97,136.87,133.37,133.30,132.08,131.91,119.52,115.19,1 07.99,107.80,107.26,107.23,95.41,79.44,61.14,53.75,52.74,49.49,48 .47,45.70,41.98,36.14,32.59,31.28,29.70,28.37,21.49.HRMS(ESI)calcd for[M+H] + C 37 H 50 FN8O3 + 673.3987, found 673.3912.

[0092] The specific preparation steps of compound b5 in the technical route are as follows: the synthesis steps are the same as those for compound b1. Crude product b5 was obtained by column chromatography as a fluorescent green solid (30 mg), with a yield of 57%. The detection data for compound b5 are as follows: 1H NMR (400MHz, CDCl3) δ11.06(s,1H),9.46(d,J=7.4Hz,1H),8.27(d,J=5.1Hz,1H),7.62(d,J=8.3Hz,1H),7.59(s,1H),7.32(d, J=11.0Hz,1H),7.20(d,J=5.1Hz,1H),6.76(d,J=1.7Hz,1H),6.50(dd,J=8.3,1.7Hz,1H),6.43(s,1H),4.93(dd,J=12.1,5.3Hz ,1H),4.81–4.65(m,2H),4.16(d,J=12.3Hz,1H),4.06(dd,J=13.6,6.8Hz,2H),3.88(s,2H),3.40(d,J=13.0Hz,2H),3.33–3.05 (m,3H),2.93–2.71(m,7H),2.71–2.59(m,5H),2.60–2.39(m,3H),2.26–2.04(m,3H),1.89–1.72(m,2H),1.69(d,J=6.9Hz,6H). 13 C NMR (101MHz, CDCl3) δ171.70,168.93,167.90,167.50,154.88,152.52,152.26,13 4.24,125.26,119.59,118.10,115.21,114.23,108.00,107.82,107.31,107.28,10 5.07,95.68,55.09,54.61,52.52,49.35,49.11,48.50,45.65,41.99,35.86,31.9 4,31.54,29.72,29.67,29.38,22.83,22.71,21.50,15.08,14.15.HRMS(ESI)calcd for[M+H] + C 45 H 50 FN 10 O5 + 829.3942, found829.3871.

[0093] Example 9: Synthesis of compound b6

[0094] The synthetic route for compound b6 is as follows:

[0095] The specific preparation steps of intermediate b6-1 in the technical route are as follows: The synthesis steps are the same as for compound b1-3. Crude product column chromatography yielded 195 mg of yellow solid powder b6-1, with a yield of 71% (eluent: DCM:MeOH = 30:1-20:1). The detection data for intermediate b6-1 are as follows: 1 H NMR (400MHz, CDCl3) δ12.36(s,1H),8.26(s,1H),7.61(s,1H),7.33(d,J=10.7Hz,1H),7.20( s,1H),6.44(s,1H),4.74(t,J=9.9Hz,2H),4.22(d,J=13.0Hz,1H),4.08(s,2H),3.34–3.11( m,4H),2.82(t,J=11.9Hz,1H),2.68(s,3H),2.66–2.33(m,10H),2.21(d,J=12.3Hz,2H),1.7 9(dd,J=15.8,15.4Hz,4H),1.69(d,J=6.5Hz,6H),1.42(s,9H),1.33(d,J=21.4,9.0Hz,3H). 13 C NMR (101MHz, CDCl3) δ167.85,154.80,154.62,152.39,152.29,149.70,144.71 ,141.39,136.96,136.87,133.40,133.33,132.05,131.88,107.97,107.78,10 7.24,95.34,79.51,62.00,60.99,53.24,48.95,48.47,45.64,41.98,37.09,3 6.22,32.64,31.92,31.25,29.69,29.35,28.42,22.68,21.49.HRMS(ESI)calcd for[M+H] + C 39 H 54 FN8O3 + 701.4935, found 701.4225.

[0096] The specific preparation steps of compound b6 in the technical route are as follows: The synthesis steps are the same as for compounds b1-3. Crude product column chromatography yielded 30 mg of b6 as a fluorescent green solid, with a yield of 57% (eluent: DCM:MeOH = 30:1-20:1). The detection data for compound b6 are as follows: 1H NMR(400MHz,CDCl3)δ11.47(s,1H),10.18(s,1H),8.28(d,J=5.1Hz,1H),7.64(d,J=8.5Hz,1H),7.59(s,1H),7.32(d,J=11.0Hz,1H),7.24(d,J=2.0Hz,1H),7.18(d,J=5.1Hz,1H),7.01(dd,J=8.7,1.9Hz,1H),6.42(s,1H),4.94(dd,J=12.2,5.4Hz,1H),4.72(dt,J=13.7,6.8Hz,2H),4.19(d,J=12.4Hz,1H),3.93(d,J=12.6Hz,2H),3.31(d,J=13.8Hz,1H),3.26–3.07(m,3H),2.99–2.71(m,7H),2.68(s,3H),2.66–2.44(m,8H),2.22–2.07(m,3H),1.94(d,J=10.9Hz,2H),1.81(dd,J=23.9,11.4Hz,2H),1.68(d,J=6.9Hz,6H),1.57(dd,J=21.2,10.3Hz,2H). 13 C NMR(101MHz,CDCl3)δ172.18,169.15,168.03,167.85,167.32,155.05,154.78,152.42,152.24,149.46,144.46,141.51,141.32,136.97,136.87,134.38,133.42,133.36,131.99,131.83,125.38,119.36,118.91,117.91,115.16,108.59,107.97,107.79,107.27,107.24,95.40,61.38,60.94,53.17,49.19,48.96,48.48,47.24,45.64,42.02,36.04,32.48,31.92,31.60,31.30,29.70,29.66,29.36,27.57,27.46,22.85,22.69,21.48,15.05,14.13.HRMS(ESI)calcd for[M+H] + C 47 H 54 FN 10 O5 + 857.4257,found 857.4184。

[0097] Example 10: Synthesis of compound b7

[0098] Synthetic route of compound b7:

[0099] The specific preparation steps of intermediate b7-1 in the technical route are as follows: The synthesis steps are the same as for compound b1-3. Crude product column chromatography yielded 184 mg of yellow solid powder b7-1, with a yield of 66% (eluent: DCM:MeOH = 30:1-20:1). The detection data for intermediate b7-1 are as follows: 1 H NMR (400MHz, CDCl3) δ11.92 (s, 1H), 8.25 (d, J = 5.0Hz, 1H), 7.60 (s, 1H), 7.31 (d, J=11.0Hz,1H),7.19(d,J=5.0Hz,1H),6.43(s,1H),4.79–4.67(m,2H),4.05(d,J= 9.8Hz,1H),3.39–3.11(m,9H),3.09–2.75(m,7H),2.68(s,3H),2.21(d,J=12.3H z,2H),1.95–1.73(m,6H),1.69(d,J=6.9Hz,6H),1.49–1.44(m,2H),1.43(s,9H). 13 C NMR (101MHz, CDCl3) δ166.21,153.92,153.78,151.41,151.27,148.66,143.43,140.47, 140.36,135.96,135.87,132.36,132.28,131.03,118.43,114.16,106.92,106.74,106. 27,94.38,78.34,47.94,47.47,44.30,41.00,38.48,35.08,33.34,31.41,30.91,30.22 ,29.58,28.67,28.64,28.34,27.44,21.67,21.39,20.49,14.05,13.10.HRMS(ESI)calcd for[M+H] + C 40 H 54 FN8O3 + 713.4302, found 713.4225.

[0100] The specific preparation steps for compound b7 in the technical route are as follows: The synthesis steps are the same as for compounds b1-3. Crude compound b7 was obtained by column chromatography as a fluorescent yellow-green solid (30 mg), with a yield of 57% (eluent: DCM:MeOH = 30:1-20:1). The detection data for compound b7 are as follows: 1 H NMR (400MHz, DMSO) δ11.78(s,1H),11.08(s,1H),8.21(d,J=5.0Hz,1H),7.75(s,1H),7.63(d,J=8.3Hz,1H),7.32(d,J=11.7Hz,1H),7. 23(d,J=5.0Hz,1H),6.75(s,1H),6.61(d,J=10.1Hz,1H),6.34(s,1H),5.05(dd,J=12.8,5.3Hz,1H),4.89–4.73(m,1H),4.45(d,J=11.7 Hz,1H),4.04(s,2H),3.95(s,2H),3.20–2.98(m,3H),2.96–2.77(m,2H),2.75–2.65(m,2H),2.62(s,3H),2.59(d,J=2.8Hz,1H),2.54( d,J=8.9Hz,2H),2.48–2.38(m,6H),2.38–2.26(m,5H),2.16–1.92(m,4H),1.77–1.65(m,1H),1.60(d,J=6.9Hz,6H),1.55–1.42(m,1H). 13 C NMR (101MHz, CDCl3) δ172.23,169.22,168.15,167.78,167.38,155.28,154.76,152.39,152.25,149. 42,144.45,141.33,136.84,134.36,133.41,131.99,125.35,118.31,117.25,108.08,107.95,107.77 ,107.28,95.35,77.39,77.07,76.76,49.15,48.47,45.55,43.63,43.54,41.99,38.93,36.00,34.72, 32.38,31.60,31.20,30.86,30.52,29.69,29.35,23.38,22.86,21.48,15.05,14.13.HRMS(ESI)calcd for[M+H] + C 48 H 54 FN 10 O5+ 869.4263, found 869.4184.

[0101] Example 11: Synthesis of compound b8

[0102] The synthetic route for compound b8 is as follows:

[0103] The specific preparation steps of intermediate b8-1 in the technical route are as follows: The synthesis steps are the same as for compound b1-3. Crude product column chromatography yielded 152 mg of yellow solid powder of b8-1, with a yield of 51% (eluent: DCM:MeOH = 30:1-20:1). The detection data for intermediate b8-1 are as follows: 1 H NMR (400MHz, CDCl3) δ11.86(s,1H),8.19(d,J=5.0Hz,1H),7.54(s,1H),7.25(d,J=11.0Hz,1H),7.1 3(d,J=5.0Hz,1H),6.37(s,1H),4.82–4.63(m,2H),3.99(d,J=6.3Hz,1H),3.36–3.20(m,6H),3.22– 3.05(m,3H),3.05–2.69(m,9H),2.63(s,3H),2.58–2.41(m,1H),2.15(d,J=12.3Hz,2H),1.92–1.67 (m,7H),1.63(d,J=6.9Hz,6H),1.61–1.48(m,2H),1.40(t,2H),1.37(s,9H),1.03(t,J=12.3Hz,2H). 13 C NMR (101MHz, CDCl3) δ166.21,153.92,153.78,151.41,151.27,148.66,143.43,140.47,140 .36,135.96,135.87,132.36,132.28,131.03,130.86,118.43,114.16,106.92,106.74,106. 27,94.38,78.34,47.94,47.47,44.30,41.00,38.48,35.08,33.34,31.41,30.91,30.22,29 .97,29.58,28.67,28.64,28.34,27.44,21.67,21.39,20.49,14.05,13.10.HRMS(ESI)calcd for[M+H] + C 44 H 62FN8O3 + 769.4927, found 769.4851.

[0104] The specific preparation steps for compound b8 in the technical route are as follows: The synthesis steps are the same as for compound b1. Crude compound b8 was obtained by column chromatography as a fluorescent yellow-green solid (30 mg), with a yield of 57%. The detection data for compound b8 are as follows: 1 H NMR (400MHz, CDCl3) δ11.61(s,1H),10.38(s,1H),8.27(s,1H),7.70–7.57(m,2H),7.31(d,J=1 0.8Hz,1H),7.22(s,1H),7.18(s,1H),6.97(d,J=7.9Hz,1H),6.42(s,1H),5.06–4.86(m,1H),4. 76–4.63(m,2H),4.15(d,J=12.0Hz,1H),3.34(d,J=12.6Hz,5H),3.22–3.02(m,3H),2.96–2.45( m,16H),2.43–2.05(m,5H),1.86–1.71(m,6H),1.68(d,J=6.5Hz,6H),1.61(s,2H),1.44(s,4H). 13 C NMR (101MHz, CDCl3) δ172.23,169.22,168.15,167.78,167.38,155.28,154.76,152.39,152.25,149.42,144.45,141.42 ,141.33,136.93,136.84,134.36,133.41,133.34,131.99,131.82,125.35,119.36,118.31,117.25,115.14,108.08,10 7.95,107.77,107.28,107.25,95.35,64.06,49.15,49.11,48.47,45.55,43.63,43.54,41.99,38.93,36.00,34.79,34. 72,32.38,31.92,31.60,31.20,30.86,30.52,29.69,29.35,23.38,22.86,22.68,21.48,15.05,14.13.HRMS(ESI)calcd for[M+H] + C 52 H 62 FN 10 O5 +925.4886, found 925.4801.

[0105] Compound B8 1 The H-NMR spectrum is shown in Figure 1. 13 The C-NMR spectrum is shown in Figure 2.

[0106] Example 12: Synthesis of compound b9

[0107] The synthetic route for compound b9 is as follows:

[0108] The specific preparation steps of intermediate b9-1 in the technical route are as follows: The synthesis steps are the same as for compound b1-3. Crude product column chromatography yielded 210 mg of b9-1 as a pale yellow solid powder, with a yield of 72% (eluent: DCM:MeOH = 30:1-20:1). The detection data for intermediate b9-1 are as follows: 1 H NMR (400MHz, CDCl3) δ11.73(s,1H),8.26(d,J=4.8Hz,1H),7.61(d,J=1.1Hz,1H),7.33(dd,J=11.1,0.9Hz,1H ),7.20(d,J=5.0Hz,1H),6.45(s,1H),4.92–4.62(m,2H),4.23–3.97(m,1H),3.49–3.31(m,3H),3.30–3.20(m, 3H),3.22–3.07(m,1H),2.87(ddd,J=35.4,25.7,13.6Hz,6H),2.70(s,3H),2.68–2.36(m,2H),2.31–2.12(m, 2H),2.11–1.92(m,3H),1.81(dd,J=23.7,11.6Hz,3H),1.71(d,J=6.9Hz,6H),1.65–1.48(m,6H),1.44(s,9H). 13 C NMR (101MHz, CDCl3) δ154.91,152.40,149.51,141.48,136.94,136.88,133.35,133.28,132.09,131.91,107.98,107.26,95.49,79.34,55.52,4 9.57,48.48,45.48,41.98,36.09,32.49,31.93,31.55,31.27,30.04,29.70,29.66,29.37,28.45,22.69,21.50,15.06,14.12.HRMS(ESI)calcd for[M+H]+ C 42 H 58 FN8O3 + 741.4615, found 741.4538.

[0109] The specific preparation steps of compound b9 in the technical route are as follows: the synthesis steps are the same as those for compound b1. Crude product column chromatography yielded 34 mg of b9 as a fluorescent yellow-green solid, with a yield of 47% (eluent: DCM:MeOH = 30:1-20:1). The detection data for compound b9 are as follows: 1H... NMR (400MHz, CDCl3) δ11.26(s,1H),10.06(s,1H),8.27(d,J=5.1Hz,1H),7.64(d,J=8.5Hz,1H),7.59(d,J=0.7Hz,1H),7.32(d,J=11.1H z,1H),7.23(d,J=1.7Hz,1H),7.18(d,J=5.1Hz,1H),7.01(dd,J=8.7,1.9Hz,1H),6.42(s,1H),4.94(dd,J=12.1,5.5Hz,1H),4.72(dt,J =14.0,7.0Hz,2H),4.13(d,J=14.1Hz,1H),3.34(dd,J=22.3,6.9Hz,5H),3.25–3.06(m,3H),2.94–2.72(m,7H),2.71–2.51(m,7H),2.14 (dd,J=19.0,10.1Hz,4H),2.09–2.00(m,2H),1.83–1.73(m,2H),1.69(d,J=6.9Hz,9H),1.26(d,J=9.6Hz,2H),1.22(d,J=7.0Hz,2H).13C NMR (101MHz, CDCl3) δ171.04,168.07,167.07,166.28,154.25,151.37,151.22,148.3 2,143.22,140.56,133.37,124.41,118.29,116.74,107.45,106.95,106.26,94.45,7 6.34,76.22,76.02,75.70,57.38,48.46,48.11,47.45,44.13,43.93,34.94,30.90,3 0.55,30.42,28.68,28.34,21.83,21.67,20.46,17.43,14.04,13.11.HRMS(ESI)calcd for[M+H]+C 50 H 58 FN10 O5 + 897.4573, found 897.4997.

[0110] Example 13: Synthesis of compound b10

[0111] The synthetic route for compound b10 is as follows:

[0112] The specific preparation steps of intermediate b10-1 in the technical route are as follows: The synthesis steps are the same as for compound b1-3. Crude product column chromatography yielded 170 mg of b10-1 as a pale yellow solid powder, with a yield of 59% (eluent: DCM:MeOH = 30:1-20:1). The detection data for intermediate b10-1 are as follows: 1 H NMR (400MHz, CDCl3) δ11.61(s,1H),8.26(d,J=3.7Hz,1H),7.60(s,1H),7.32(d,J=11 .0Hz,1H),7.20(d,J=4.8Hz,1H),6.44(s,1H),4.80–4.66(m,2H),4.10(d,J=14.0Hz, 1H),3.56(d,J=14.6Hz,4H),3.46–2.62(m,17H),2.61–2.38(m,2H),2.21(d,J=11.5H z,3H),1.97(d,J=11.3Hz,4H),1.87–1.74(m,3H),1.70(d,J=6.9Hz,6H),1.42(s,9H). 13 C NMR (101MHz, CDCl3) δ156.46,154.79,152.41,149.52,144.32,144.28,14 1.57,141.42,107.97,107.79,107.29,107.26,95.49,79.42,48.90,48.8 6,48.49,45.39,41.97,36.05,34.48,34.41,32.42,32.41,31.94,31.24, 29.71,29.67,29.38,28.41,22.71,21.50,15.08,14.14.HRMS(ESI)calcd for[M+H] + C 42 H 58 FN8O3 + 741.4611, found 741.4538.

[0113] The specific preparation steps of compound b10 in the technical route are as follows: The synthesis steps are the same as for compound b1. Crude compound b10 was obtained by column chromatography as a fluorescent yellow-green solid, 16 mg, with a yield of 34% (eluent: DCM:MeOH = 30:1-20:1). The detection data for compound b10 are as follows: 1 H NMR (400MHz, CDCl3) δ11.49(s,1H),10.24(s,1H),8.27(d,J=5.1Hz,1H),7.66–7.57(m,1H),7.32(d,J=11.0Hz,1H),7.18(d,J=5 .1Hz,1H),6.72(d,J=1.7Hz,1H),6.46(dd,J=8.4,1.8Hz,1H),6.42(s,1H),5.07–4.86(m,1H),4.81–4.62(m,1H),4.13(d,J=8.6 Hz,1H),3.65(d,J=17.2Hz,1H),3.33(d,J=13.9Hz,4H),3.26–3.05(m,3H),2.96–2.70(m,8H),2.71–2.54(m,5H),2.49–2.25(m, 1H),2.22–2.08(m,3H),2.06–1.86(m,4H),1.89–1.72(m,2H),1.68(d,J=6.9Hz,6H),1.52(t,J=12.2Hz,3H),1.42–1.29(m,3H). 13 C NMR (101MHz, CDCl3) δ172.23,169.27,168.01,167.72,167.58,155.06,154.75,152.41,152.25,149.42,144.3 9,141.52,141.33,136.94,136.85,134.31,133.41,133.34,131.99,131.82,125.25,119.33,117.52,115.17,1 13.87,107.97,107.79,107.30,107.27,104.72,95.40,61.60,60.77,49.10,48.97,48.49,45.53,42.04,35.9 7,34.83,32.34,31.94,31.59,31.22,29.71,29.67,29.37,22.89,22.71,21.49,15.07,14.15.HRMS(ESI)calcd for[M+H] + C 50 H 58 FN 10O5 + 897.4556, found 897.4497.

[0114] Compound B10 1 The H-NMR spectrum is shown in Figure 3. 13 The C-NMR spectrum is shown in Figure 4.

[0115] Example 14: Synthesis of compound b11

[0116] The synthetic route for compound b11 is as follows:

[0117] The specific preparation steps of intermediate b11-1 in the technical route are as follows: The synthesis steps are the same as for compound b1-3. Crude product column chromatography yielded 175 mg of white solid powder b11-1, with a yield of 62% (eluent: DCM:MeOH = 30:1-20:1). The detection data for intermediate b11-1 are as follows: 1 H NMR (400MHz, CDCl3) δ12.03(s,1H),8.27(d,J=5.0Hz,1H),7.62(s,1H),7.34(d,J=11.0Hz,1H ),7.21(d,J=5.1Hz,1H),6.45(s,1H),4.82–4.66(m,2H),4.19(d,J=14.3Hz,1H),3.52–3.40( m,2H),3.37–3.08(m,6H),2.90–2.76(m,2H),2.78–2.48(m,12H),2.23(d,J=11.7Hz,2H),2.1 4(d,J=4.4Hz,2H),1.89–1.75(m,3H),1.71(d,J=6.9Hz,6H),1.44(s,9H),1.35–1.28(m,2H). 13 C NMR (101MHz, CDCl3) δ154.50,152.40,149.59,144.52,141.41,136.95,136. 85,133.38,133.30,132.04,131.87,119.48,115.18,107.97,107.79,107.26 ,95.38,79.15,68.18,52.02,48.48,45.56,41.99,36.12,32.45,31.94,31. 32,29.71,29.67,29.37,28.51,22.70,21.50,15.08,14.14.HRMS(ESI)calcd for[M+H] + C41 H 56 FN8O3 + 727.4455, found 727.4381.

[0118] The specific preparation steps of compound b11 in the technical route are as follows: The synthesis steps are the same as for compound b1. Crude compound b11 was obtained by column chromatography as a fluorescent yellow-green solid (30 mg), with a yield of 57% (eluent: DCM:MeOH = 30:1-20:1). The detection data for compound b11 are as follows: 1 H NMR (400MHz, CDCl3) δ11.68(s,1H),10.59(d,J=15.9Hz,1H),8.28(d,J=4.4Hz,1H),7.60(d,J=8.4Hz,2H),7.32(d,J=11.0Hz,1H) ,7.17(d,J=4.9Hz,1H),6.93(s,1H),6.64(d,J=8.2Hz,1H),6.41(s,1H),4.93(dd,J=11.8,5.4Hz,1H),4.72(dt,J=13.6,6.7Hz,2H ),4.18(d,J=10.8Hz,1H),3.59–3.46(m,2H),3.40–3.27(m,2H),3.27–3.03(m,4H),2.80(m,J=20.9,13.5Hz,8H),2.68(s,3H),2.6 6–2.42(m,6H),2.17(d,J=9.9Hz,4H),2.13–2.06(m,1H),1.79(dd,J=27.1,13.9Hz,2H),1.68(d,J=6.9Hz,6H),1.58–1.38(m,3H). 13 C NMR (101MHz, CDCl3) δ172.37,169.43,168.22,167.83,167.68,152.41,152.34,149.43,144 .51,141.39,141.33,134.35,125.29,119.36,116.85,115.73,107.97,107.79,107.31,106. 78,95.30,68.12,54.44,54.36,52.83,52.15,49.07,48.49,45.64,41.98,40.93,40.87,36 .01,32.36,31.94,31.63,29.71,29.38,22.90,22.71,21.49,15.08,14.16.HRMS(ESI)calcd for[M+H] + C49 H 56 FN 10 O5 + 883.4418, found 883.4341.

[0119] Example 15: Synthesis of compound b12

[0120] The synthetic route for compound b12 is as follows:

[0121] The specific preparation steps of intermediate b12-2 in the technical route are as follows: Take b12-1 (250 mg, 1.1 mmol) and b1-4 (458 mg, 1.65 mmol) in a 50 mL round-bottom flask, add 10 mL of anhydrous DMF to dissolve them, b12-2 dissolves, thalidomide is slightly soluble, add DIPEA (0.95 mL, 5.5 mmol), heat to 80 °C for 8 h under nitrogen protection, the system gradually dissolves and turns dark green. After 8 h, TLC monitoring shows that PE:EA = 1:1, and product R... f =0.25. After the reaction was complete, heating was stopped, and the reaction solution was cooled to room temperature before removing DMF under reduced pressure. The resulting product was redissolved in 20 mL of DCM and washed three times with 20 mL of saturated brine. The product was backwashed twice with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Column chromatography yielded 300 mg of b12-2 as a fluorescent bright yellow solid, with a yield of 57% (eluent: PE:EA = 2:1). The intermediate b12-2 was analyzed, and the results were as follows: 1 H NMR (400MHz, CDCl3) δ8.24(s,1H),7.67(d,J=8.5Hz,1H),7.03(dd,J=8.6,2.2Hz,1H),3.48–3. 36(m,8H),2.93–2.66(m,3H),2.18–2.02(m,1H),1.64(t,4H),1.54–1.49(m,4H),1.46(s,9H). 13 C NMR (101MHz, CDCl3) δ171.11,168.37,168.05,167.29,155.28,154.95,134.37,125.45,118. 63,117.46,108.31,79.56,49.11,43.41,34.57,31.46,29.81,28.47,22.76.HRMS(ESI)calcd for[M+H] + C 27 H 35 FN4O6 +511.2552, found 511.2478.

[0122] The specific preparation steps of intermediate b12-3 in the technical route are as follows: Take 200 mg (0.41 mmol) of b12-2 in a 25 mL round-bottom flask, add 4 mL of DCM to dissolve it, resulting in a fluorescent green solution. Adding 1 mL of TFA solution changes the fluorescent green color to brownish-yellow. After 1 hour, TLC monitoring shows that the ratio of DCM:MeOH = 10:1, indicating the disappearance of the starting material and the completion of the reaction. Remove DCM and TFA under reduced pressure, and remove TFA several times with water, finally obtaining 190 mg of b12-3 as a bright yellow solid, with a yield of 93%. The product does not require purification and can be directly used in the next reaction. The detection data for intermediate b12-3 are as follows: HRMS (ESI) calcd for [M+H] + C 22 H 27 FN4O4 + 411.2030, found 411.1954.

[0123] The specific preparation steps of intermediate B12-4 in the technical route are as follows: Take B12-3 (140 mg, 0.28 mmol) in a 10 mL single-necked flask, add 3 mL of DCM to dissolve it; most of the starting material is insoluble. Add DIPEA (0.97 mL, 0.56 mmol). During the addition, white fumes are emitted, and the starting material dissolves into a fluorescent green solution. Add tert-butyl bromoacetate (0.61 mL, 0.42 mmol), and react under nitrogen for 6 hours. After 6 hours, TLC monitoring shows that PE:EA = 1:1, and product R... f =0.1. After the reaction was complete, 5 mL of DCM was added, and the mixture was washed three times with 10 mL of saturated saline solution. The mixture was backwashed twice with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Column chromatography yielded 130 mg of a fluorescent bright yellow solid (b12-4), with a yield of 95% (eluent PE:EA = 1:1). The intermediate b12-4 was analyzed, and the results were as follows: 1 H NMR (400MHz, CDCl3) δ8.32(s,1H),7.66(d,J=8.6Hz,1H),7.02(dd,J=8.6,2.1Hz,1H),4.93(dd,J=12.3,5.3Hz,1H),3. 44–3.35(m,4H),3.32(s,2H),2.95–2.66(m,7H),2.17–2.06(m,1H),1.78–1.68(m,4H),1.68–1.59(m,4H),1.47(s,9H). 13C NMR (101MHz, CDCl3) δ171.15,168.41,168.04,167.28,155.28,134.35,125.44,118.61,1 17.46,108.31,49.11,47.98,43.44,34.48,31.46,28.89,28.14,22.76.HRMS(ESI)calcd for[M+H] + C 28 H 37 FN4O6 + 525.2713, found 525.2635.

[0124] The specific preparation steps of compound b12 in the technical route are as follows: Weigh b12-4 (200 mg, 0.39 mmol) and HATU (175 mg, 0.78 mmol) into a 10 mL flask, dissolve them in 3 mL of anhydrous DMF, add DIPAE (200 mg, 0.39 mmol), and react under nitrogen protection for 20 min. At this point, the reaction system is a yellow solution. After 20 min, add a1-7 (200 mg, 0.39 mmol) and react overnight at room temperature. After 10 h, monitor the reaction by TLC: DCM:MeOH = 10:1. The product shows blue fluorescence under TLC. f =0.5. After the reaction was complete, DMF was removed under reduced pressure, and the organic phase was extracted and collected. The organic phase was washed three times with 10 mL of saturated brine and backwashed twice with 10 mL of DCM. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a yellow oily crude product. Column chromatography purified the product to obtain 190 mg of fluorescein b12 solid powder, with a yield of 67% (eluent DCM:MeOH = 30:1-20:1). The detection data for compound b12 are as follows: 1H NMR (400MHz, DMSO) δ11.81(s,1H),11.10(s,1H),8.22(d,J=5.0Hz,1H),7.75(s,1H),7.67(d,J=8.5Hz,1H),7.33(dd ,J=6.8,4.3Hz,3H),7.24(d,J=5.0Hz,2H),6.35(s,1H),5.07(dd,J=12.9,5.5Hz,1H),4.91–4.77(m,1H),4.46(d,J= 9.0Hz,1H),3.65–3.56(m,1H),3.53–3.37(m,5H),3.17(d,J=5.2Hz,2H),3.14–2.98(m,2H),2.94–2.79(m,2H),2.63 (s,3H),2.61–2.53(m,2H),2.17–2.04(m,2H),2.00(dd,J=16.8,8.8Hz,2H),1.81–1.56(m,12H),1.55–1.39(m,6H). 13 C NMR (101MHz, DMSO) δ173.31,170.60,168.16,167.43,155.39,153.54,149.83,142 .59,139.64,134.46,125.45,118.31,118.01,117.90,115.09,108.14,108.01,107 .93,107.28,107.16,94.61,53.94,49.19,49.06,48.96,48.34,43.18,31.64,31.4 4,29.17,28.91,22.56,22.42,21.40,18.41,17.10,15.13,14.41.HRMS(ESI)calcd for[M+H] + C 47 H 53 FN9O5 + 842.4174, found 842.4075.

[0125] Example 16: Synthesis of compound b13

[0126] The synthetic route for compound b13 is as follows:

[0127] The specific preparation steps of intermediate b13-2 in the technical route are as follows: The synthesis steps are the same as those for compound b12-2. Crude product b13-2 was obtained by column chromatography as a fluorescent bright yellow solid (300 mg), with a yield of 57% (eluent: PE:EA = 2:1). The detection data for intermediate b13-2 are as follows: 1 H NMR (400MHz, CDCl3) δ8.08(s,1H),7.64(d,J=8.3Hz,1H),6.79(d,J=1.8Hz,1H),6.52(dd,J=8.3,1.9Hz,1H),4.93( dd,J=12.2,5.2Hz,1H),3.77(s,4H),3.42(t,4H),2.95–2.64(m,3H),2.20–2.09(m,1H),1.80(t,4H),1.47(s,9H). 13 C NMR (101MHz, CDCl3) δ171.01,168.31,167.86,167.46,155.05,154.77,134.34,125.31,117.99, 114.01,104.94,79.85,61.04,49.11,35.42,35.01,31.44,29.69,28.44,22.78.HRMS(ESI)calcd for[M+H] + C 25 H 31 FN4O6 + 483.2204, found 483.2165.

[0128] The specific preparation steps of intermediate b13-3 in the technical route are as follows: The synthesis steps are the same as for compound b12-3. Crude product b13-3 was obtained by column chromatography as a bright yellow solid (190 mg), with a yield of 93%. The product does not require purification and can be directly proceeded to the next reaction. The detection data for intermediate b13-3 are as follows: HRMS (ESI) calcd for [M+H] + C 20 H 23 FN4O4 + 383.1719, found 383.1641.

[0129] The specific preparation steps of intermediate b13-4 in the technical route are as follows: The synthesis steps are the same as for compound b12-4. Crude product b13-4 was obtained by column chromatography as a fluorescent bright yellow solid, 130 mg, with a yield of 95% (eluent: PE:EA = 1:1). The detection data for intermediate b13-4 are as follows: 1H NMR (400MHz, CDCl3) δ8.33(s,1H),7.63(d,J=8.3Hz,1H),6.77(d,J=1.6Hz,1H),6.50(d,J=8.3Hz,1H),4.92(dd,J=12.1,5. 2Hz,1H),3.73(s,4H),3.15(s,2H),2.94–2.68(m,3H),2.59(s,4H),2.17–2.04(m,1H),1.90(t,J=5.1Hz,4H),1.47(s,9H). 1 H NMR (400MHz, CDCl3) 13 C NMR (101MHz, CDCl3) δ171.18,169.48,168.44,167.91,167.51,155.10,134.34,125.29,117.68,113.87,10 4.85,81.30,61.22,59.84,49.92,49.09,35.49,34.39,31.92,31.46,29.65,29.35,28.17.HRMS(ESI)calcd for[M+H] + C 26 H 33 FN4O6 + 497.2400, found 497.2322.

[0130] The specific preparation steps of compound b13 in the technical route are as follows: The synthesis steps are the same as for compound b12. Crude product column chromatography yielded 190 mg of fluorescein b13 solid powder, with a yield of 67% (eluent: DCM:MeOH = 30:1-20:1). The detection data for compound b13 are as follows: 1H NMR (400MHz, CDCl3) δ11.49(s,1H),10.10(d,J=7.5Hz,1H),8.28(d,J=4.9Hz,1H),7.60(d,J=8.1Hz,2H),7.32(d ,J=11.0Hz,1H),7.18(d,J=5.0Hz,1H),6.73(s,1H),6.46(d,J=8.2Hz,1H),6.41(s,1H),5.01–4.84(m,1H),4.81 –4.61(m,2H),4.20(d,J=12.0Hz,1H),3.69(s,4H),3.35(d,J=13.5Hz,1H),3.27–3.03(m,3H),2.93–2.71(m,4H) ,2.68(s,3H),2.55(s,4H),2.18(d,J=11.2Hz,2H),2.15–2.07(m,1H),1.92–1.72(m,6H),1.68(d,J=6.8Hz,6H). 13 C NMR (101MHz, CDCl3) δ172.11,169.23,167.96,167.66,167.57,155.00,154.75,152.43,152.24,149.3 7,144.35,141.45,141.43,136.95,136.86,134.30,133.40,133.32,132.00,131.83,125.25,119.38, 117.68,115.16,113.90,107.98,107.80,107.28,107.25,104.80,95.40,61.11,50.42,49.13,48.47, 45.70,41.99,35.93,35.46,34.33,32.43,31.58,31.21,29.70,22.85,21.48,15.05.HRMS(ESI)calcd for[M+H] + C 45 H 49 FN9O5 + 814.3834, found 814.3762.

[0131] Example 17: Synthesis of compound b14

[0132] The synthetic route for compound b14 is as follows:

[0133] The specific preparation steps of intermediate b14-2 in the technical route are as follows: The synthesis steps are the same as for compound b12-2. Crude product b14-2 was obtained by column chromatography as a fluorescent bright yellow solid (300 mg), with a yield of 57% (eluent: PE:EA = 2:1). The detection data for intermediate b14-2 are as follows: 1 H NMR (400MHz, CDCl3) δ8.23(s,1H),7.68(d,J=8.4Hz,1H),7.31(d,J=1.5Hz,1H),7.12(d,J=7.1Hz,1H),4.9 7–4.87(m,1H),3.70(s,1H),3.39(t,1H),2.92–2.67(m,1H),2.18–2.06(m,1H),1.89(t,1H),1.45(s,1H). 13 C NMR (101MHz, CDCl3) δ171.05,168.30,167.83,167.12,156.40,154.96,134.39,125.48,119.69,118 .49,109.13,79.67,58.75,49.19,45.51,34.53,33.45,31.44,29.69,28.41,22.74.HRMS(ESI)calcd for[M+H] + C 25 H 31 FN4O6 + 482.2217, found 482.2165.

[0134] The specific preparation steps of intermediate b14-3 in the technical route are as follows: The synthesis steps are the same as for compound b12-3. Crude product b14-3 was obtained by column chromatography as a bright yellow solid (190 mg), with a yield of 93%. The product does not require purification and can be directly proceeded to the next reaction. The detection data for intermediate b14-3 are as follows: HRMS (ESI) calcd for [M+H] + C 25 H 31 FN4O6 + 383.1715, found 383.1641.

[0135] The specific preparation steps of intermediate b14-4 in the technical route are as follows: The synthesis steps are the same as for compound b12-4. Crude product column chromatography yielded 130 mg of b14-4 as a fluorescent bright yellow solid, with a yield of 95% (eluent: PE:EA = 1:1). The detection data for intermediate b14-4 are as follows: 1H NMR (400MHz, CDCl3) δ7.65(d,J=8.5Hz,1H),7.25(s,1H),7.03(dd,J=8.6,2.1Hz,1H),5.01–4.81( m,1H),3.37(t,4H),3.27(s,6H),2.94–2.62(m,3H),2.15–2.03(m,1H),1.89(t,4H),1.45(s,9H). 13 C NMR (101MHz, CDCl3) δ171.16,168.39,167.98,167.24,155.21,134.39,125.47,118.85,117.93,108.71,81.47,63 .92,59.77,49.12,45.34,35.06,34.98,31.94,31.47,29.73,29.69,29.39,28.13,22.76,14.15.HRMS(ESI)calcd for[M+H] + C 26 H 33 FN4O6 + 497.2400, found 497.2322.

[0136] The specific preparation steps of compound b14 in the technical route are as follows: The synthesis steps are the same as for compound b12. Crude product column chromatography yielded 190 mg of b14 fluorescein solid powder, with a yield of 67% (eluent: DCM:MeOH = 30:1-20:1). The detection data for compound b14 are as follows: 1 H NMR (400MHz, DMSO) δ11.84(s,1H),11.10(s,1H),8.46(s,2H),8.31(d,J=8.4Hz,1H),8.22(d,J=5.0Hz,1H),7.74(d, J=8.0Hz,1H),7.65(d,J=8.5Hz,1H),7.31(s,1H),7.23(d,J=5.0Hz,1H),6.35(d,J=1.5Hz,1H),5.10–5.01(m,1H),4 .87–4.78(m,1H),4.46–4.25(m,3H),3.86(s,4H),3.79–3.69(m,2H),3.25–3.13(m,2H),3.13–3.01(m,1H),2.94–2. 72(m,3H),2.62(s,4H),2.58–2.53(m,1H),2.18–1.98(m,4H),1.90(s,4H),1.79–1.68(m,2H),1.60(d,J=6.9Hz,6H).13 C NMR (101MHz, DMSO) δ173.31,170.59,168.08,167.42,155.15,153.51,151.87,149.90,149.26 ,145.37,142.58,139.95,139.63,137.40,134.97,134.49,133.19,128.27,125.48,120.01,1 18.30,118.23,115.08,108.51,107.99,107.40,107.22,94.63,63.52,62.48,49.22,48.35,4 4.63,41.63,35.17,35.09,33.79,31.45,31.07,25.95,22.65,21.40,15.12.HRMS(ESI)calcd for[M+H] + C 47 H 57 FN9O5 + 846.4415, found 846.4388.

[0137] Example 18: Synthesis of compound c1

[0138] The synthetic route for compound c1 is as follows:

[0139] The specific preparation steps of intermediate c1-2 in the technical route are as follows: Compound c1-1 (5 g, 15.4 mmol) was placed in a 250 mL double-necked round-bottom flask, and the flask was evacuated three times to purge with nitrogen. Under nitrogen protection, 75 mL of anhydrous DMF was added, and compound c1-1 dissolved into a colorless solution. Subsequently, potassium thioacetate (2.1 g, 18.5 mmol, 1.2 equiv.) was added to the reaction flask, and the temperature was raised to 95 °C. The system gradually changed from a colorless solution to a yellow solution. After 4 hours, a sample was taken for TLC analysis to monitor the reaction progress: PE:EA = 8:1, and product R... f =0.5, the starting material spot disappears, and the reaction is complete. Stop the reaction, and after cooling to room temperature, distill off DMF under reduced pressure. Dissolve the compound again in 200 mL of DCM, wash three times with 100 mL of saturated brine, backwash twice, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the crude product by column chromatography to obtain a pale yellow oily compound c1-2 3.9 g, yield 93% (mobile phase: PE:EA = 20:1). The intermediate c1-2 was analyzed, and the results are as follows: 1HNMR(400MHz, CDCl3) δ4.09(d,J=13.3Hz,2H),2.82(d,J=6.8Hz,2H),2.65(td,J=13.3,2.5Hz,2H),2. 33(s,3H),1.72(d,J=13.2Hz,2H),1.62–1.51(m,1H),1.44(s,9H),1.13(qd,J=24.6,12.5,4.4Hz,2H). 13 C NMR (101MHz, CDCl3) δ195.66,154.76,79.39,43.66,36.50,34.96,31.32,30.66,28.45.

[0140] The specific preparation steps of intermediate c1-3 in the technical route are as follows: Compound c1-2 (3.2 g, 11.7 mmol) was placed in a 250 mL double-necked round-bottom flask, and the flask was evacuated three times to purge with nitrogen. AcOH / H2O (200 mL / 20 mL) was added under ice bath conditions, and compound c1-2 dissolved into a colorless solution. Then, N-chlorosuccinimide (4.6 g, 35.1 mmol, 3.0 equiv.) was added to the reaction flask, and the system gradually changed from a pale yellow solution to a colorless solution. After 1 hour, a sample was taken for TLC analysis to monitor the reaction progress: PE:EA = 8:1, the starting material spot disappeared, and the reaction was complete. The reaction was stopped, and AcOH was distilled off under reduced pressure. The crude product was purified by column chromatography to obtain a white solid compound c1-3 (2.9 g), with a yield of 85% (mobile phase: PE:EA = 5:1). The detection data for intermediate c1-3 are as follows: 1 H NMR (400MHz, CDCl3) δ4.14(d,J=13.6Hz,1H),3.66(d,J=6.4Hz,1H),2.83–2.70(m,1H),2. 41–2.25(m,1H),1.95(d,J=13.2Hz,1H),1.45(s,4H),1.34(qd,J=24.9,12.3,4.2Hz,1H). 13 C NMR (101MHz, CDCl3) δ154.63,79.89,71.33,43.23,33.14,31.23,28.41.

[0141] The specific preparation steps of intermediate c1-4 in the technical route are as follows: Compound c1-3 (1.1 g, 3.7 mmol) was weighed into a 100 mL double-necked round-bottom flask, and residual oxygen and moisture were removed by vacuum. 15.0 mL of anhydrous DMF was added under ice bath conditions, and compound c1-3 dissolved into a colorless solution. Subsequently, 15.0 mL of a1-7 (1.6 g, 4.1 mmol, 1.1 equiv.) DMF solution and DIPEA (0.71 mL, 4.1 mmol, 1.1 equiv.) were added sequentially to the reaction flask, and the system changed from a colorless solution to a yellow solution. After 2 hours, a sample was taken for TLC analysis of the reaction. The product c1-4:(DCM:MeOH = 20:1, R f =0.6). Most of the DMF and DIPEA were removed under reduced pressure. The crude product was purified by column chromatography (DCM:MeOH = 40:1-30:1) to give 1.7 g of a white solid compound c1-4, yield 72%. Intermediate c1-4 was analyzed, and the results were as follows: 1 H NMR (400MHz, CDCl3) δ12.28(s,1H),8.33(d,J=5.1Hz,1H),7.61(s,1H),7.33(d,J=11.0Hz,1H),7.23(d,J= 5.1Hz,1H),6.46(s,1H),4.81-4.66(m,J=13.9,6.9Hz,1H),4.19–4.03(m,2H),3.96(d,J=12.1Hz,2H),3.10 -3.00(m,1H),2.99-2.90(m,2H),2.83(d,J=6.4Hz,2H),2.80–2.71(m,2H),2.70(s,3H),2.29-2.21(m,2H), 2.21–2.12(m,1H),2.11–2.00(m,2H),2.00–1.90(m,2H),1.70(d,J=6.9Hz,6H),1.45(s,9H),1.29(qt,2H). 13 C NMR (101MHz, CDCl3) δ154.74,152.45,149.13,144.21,141.02,119.73,115.23,108.03,107.85,107.32 ,95.71,79.60,54.39,48.49,45.88,35.53,31.98,31.79,31.46,28.44,21.50,15.06.HRMS(ESI)calcd for[M+H] + C 34 H 46 FN6O4S +653.3281, found 653.3207.

[0142] The specific preparation steps of intermediate C1-5 in the technical route are as follows: Take 1.5 g (2.3 mmol) of C1-4 in a 100 mL round-bottom flask, add 20 mL of DCM to dissolve it, resulting in a pale yellow solution. Adding 4.0 mL of LTF solution changes the color from pale yellow to yellow. After 1 hour, TLC monitoring shows that DCM:MeOH = 10:1, indicating the disappearance of the starting material and the completion of the reaction. Adjust the reaction pH to approximately 9 with saturated sodium carbonate solution. After adjustment, add 20 mL of DCM for dilution and extract to obtain an organic phase (a white viscous substance). Add 10 mL of MeOH to the organic phase to completely dissolve it. Dry the organic phase with anhydrous sodium sulfate, filter, and concentrate to obtain 1.1 g of C1-5 as a yellow solid, with a yield of 86%. The product does not require purification and can be directly used for the next reaction. The detection data for intermediate C1-5 are as follows: HRMS (ESI) calcd for [M+H] + C 29 H 38 FN6O2S + 553.2761, found 553.2683.

[0143] The specific preparation steps of intermediate C1-6 in the technical route are as follows: Weigh C1-5 (200 mg, 0.39 mmol), tert-butyl 7-oxo-2-azaspiro[3.5]non-2-carboxylic acid (175 mg, 0.78 mmol, 2 equiv.) into a 10 mL flask, then remove residual water and air by vacuum, and sequentially add CH3COOH (2 μL, 0.039 mmol, 0.1 equiv.) and 3.0 mL of anhydrous MeOH. React at 70 °C for 3 h under nitrogen protection. At this time, the reaction system is a yellow solution. Stop heating and wait for the reaction to cool to room temperature. Then add sodium cyanoborohydride (49 mg, 0.78 mmol, 2 equiv.). After the addition, the system becomes slightly turbid. React overnight at room temperature. After 10 h, monitor the reaction by TLC: DCM:MeOH = 10:1. The product shows blue fluorescence under TLC. f =0.5. After the reaction was complete, most of the MeOH and CH3COOH were removed under reduced pressure. The mixture was dissolved in 6 mL of DCM, washed three times with 3 mL of saturated brine, and backwashed twice with 6 mL of DCM. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a yellow oily crude product. Column chromatography (DCM:MeOH = 30:1-20:1) yielded 190 mg of c1-6 yellow solid powder, with a yield of 67%. The intermediate c1-6 was analyzed, and the results were as follows: 1H NMR (400MHz, MeOD) δ8.18(d,J=5.1Hz,1H),7.77(s,1H),7.33(d,J=11.4Hz,1H),7.21(d,J= 5.1Hz,1H),6.42(s,1H),3.84(d,J=11.9Hz,2H),3.74–3.42(m,6H),3.34(s,2H),3.24–2.92 (m,8H),2.68(s,3H),2.28(d,J=11.7Hz,2H),2.18(d,J=11.1Hz,2H),2.05(d,J=10.6Hz,4H) ,1.83(qd,J=15.7,12.7,3.7Hz,2H),1.69(d,J=6.9Hz,6H),1.65–1.45(m,5H),1.42(s,9H). 13 C NMR(101MHz,MeOD)δ156.95,154.07,153.28,148.77,145.03,141.36,140.89,136.44,133.81,133.74,119.15,114.63,107.78,107.7 5,107.67,107.49,94.73,79.65,48.98,48.76,48.47,45.51,34.92,33.78,33.43,31.23,27.24,23.07,20.08,13.12.HRMS(ESI)calcd for[M+H] + C 42 H 59 FN7O4S + 776.4331, found 776.4255.

[0144] The specific preparation steps for compound c1 in the technical route are as follows: The synthesis steps are the same as for compound b1. Crude product column chromatography yielded 15 mg of fluorescein solid powder c1, with a yield of 34%. The detection data for compound c1 are as follows: 1H NMR (400MHz, DMSO) δ11.80(s,1H),11.07(s,1H),8.21(d,J=5.0Hz,1H),7.76(s,1H),7.65(d,J=8.5Hz,1H),7.32( d,J=6.8Hz,2H),7.23(d,J=5.1Hz,2H),6.38(s,1H),5.10–5.03(m,1H),4.88–4.79(m,1H),3.70(d,J=11.5Hz,2H), 3.60–3.53(m,2H),3.49(s,2H),3.48–3.39(m,6H),3.04(d,J=4.7Hz,4H),2.97–2.89(m,3H),2.63(s,3H),2.21–2. 09(m,3H),2.08–1.91(m,7H),1.87–1.79(m,3H),1.76(d,J=10.1Hz,3H),1.61(d,J=6.9Hz,6H),1.50–1.35(m,4H). 13 C NMR(101MHz,DMSO)δ173.11,170.80,169.03,168.24,167.19,153.58,149.93,1 45.37,145.12,142.58,139.64,134.56,131.47,130.16,118.12,117.59,108.10 ,77.74,76.50,70.25,65.95,63.23,48.21,45.64,40.62,40.41,40.20,39.99, 39.79,39.58,39.37,29.19,28.93,22.52,21.42,15.03,14.53.HRMS(ESI)calcd for[M+H] + C 50 H 59 FN9O6S + 932.4263, found 932.4215.

[0145] Example 19: Synthesis of compound c2

[0146] The synthetic route for compound C12 is as follows:

[0147] The specific preparation steps of intermediate c2-1 in the technical route are as follows: The synthesis steps are the same as for compound c1-4. The crude compound was purified by column chromatography (EA) to obtain 1.7 g of a white solid, c2-1, with a yield of 72%. The detection data for intermediate c2-1 are as follows: 1HNMR (400MHz, CDCl3) δ12.23(s,1H),8.36(d,J=5.0Hz,1H),7.63(s,1H),7.35(d,J=10.8Hz,1H),7.25(s ,1H),6.49(s,1H),4.11(s,2H),3.98(d,J=11.9Hz,2H),3.38-3.27(m,1H),3.05(t,J=11.3Hz,1H),2.96( t,J=11.2Hz,2H),2.83(d,J=6.4Hz,3H),2.74(s,3H),2.26(d,J=12.4Hz,2H),2.21–2.12(m,1H),2.13-2. 03(m,2H),1.96(d,J=12.2Hz,2H),1.45(s,9H),1.35–1.28(m,3H),1.28-1.22(m,2H),1.17-1.06(m,2H). 13 CNMR(101MHz, CDCl3)δ155.21,154.74,144.24,142.02,140.96,119.61,115.29,108.43,108.25,106.40,95 .89,79.61,54.38,45.90,35.56,31.98,31.79,31.53,28.44,25.06,15.03,7.14.HRMS(ESI)calcdfor[M+H] + C 34 H 44 FN6O4S + 651.3129, found651.3051.

[0148] The specific preparation steps for intermediate c2-2 in the technical route are as follows: the synthesis steps are the same as for compound c1-5, and product c2-2 does not require purification and is directly used in the next step. Intermediate c2-2 was analyzed, and its detection data are: HRMS (ESI) calcd for [M+H] + C 34 H 44 FN6O4S + 551.2574, found 551.2526.

[0149] The specific preparation steps of intermediate C2-3 in the technical route are as follows: The synthesis steps are the same as for compound C1-6. C2-3 was purified by column chromatography to obtain 110 mg of a pale yellow solid, with a yield of 63%. The detection data for intermediate C2-3 are as follows: 1HNMR(400MHz,MeOD)δ8.17(d,J=5.1Hz,1H),7.72(s,1H),7.31(d,J=11.2Hz,1H),7.18(d,J=5.1Hz,1H), 6.45(s,1H),3.85(d,J=11.8Hz,2H),3.68(s,2H),3.62–3.49(m,4H),3.37(s,3H),3.25–3.10(m,2H),3.1 1–2.91(m,5H),2.72(s,2H),2.30(d,J=11.8Hz,3H),2.17(d,J=12.1Hz,2H),2.07(d,J=10.3Hz,4H),1.8 2(qd,J=12.9,3.8Hz,2H),1.75–1.63(m,2H),1.59(d,J=11.8Hz,2H),1.53(d,J=11.7Hz,2H),1.44(s,9H) ,1.31(dd,J=12.6,11.0Hz,2H),1.17–1.09(m,2H).13CNMR(101MHz,MeOD)δ156.88,155.95,153.77,151 .28,148.66,144.99,141.18,140.75,139.16,139.07,134.00,133.93,131.98,129.51,129.34,119.08, 119.00,114.52,107.93,107.75,106.76,94.89,79.63,64.54,52.41,48.96,48.47,45.50,34.88,33.77 ,33.44,31.20,29.65,29.36,29.01,27.25,24.84,23.05,13.23,12.10,6.16.HRMS(ESI)calcdfor[M+H] + C 50 H 57 FN9O6S + 774.4173, found774.1099.

[0150] The specific preparation steps for compound c2 in the technical route are as follows: The synthesis steps are the same as for compound b1. Crude product column chromatography yielded 23 mg of fluorescein solid powder c2, with a yield of 43%. The detection data for compound c2 are as follows: 1HNMR (400MHz, DMSO) δ11.83(s,1H),11.10(s,1H),8.22(d,J=4.9Hz,1H),7.70(s,1H),7.65(d,J=8.5Hz,1H),7.39–7.27(m,2H), 7.21(d,J=4.9Hz,2H),6.44(s,1H),5.12–5.00(m,1H),3.70(d,J=10.4Hz,2H),3.47(d,J=22.5Hz,7H),3.30–3.13(m,2H),3.05( d,J=5.8Hz,2H),3.01–2.89(m,4H),2.90–2.81(m,1H),2.64(s,3H),2.62–2.53(m,1H),2.14(d,J=12.5Hz,3H),2.10–1.98(m,3H ),1.98–1.88(m,2H),1.87–1.73(m,6H),1.65(d,J=6.3Hz,2H),1.59(s,2H),1.43–1.34(m,3H),1.19–1.13(m,2H),1.12(s,2H). 13 CNMR(101MHz,DMSO)δ173.33,170.63,168.14,167.46,155.94,155.40,151.69,149.88,145 .26,142.59,139.51,134.44,133.47,133.40,130.65,130.11,125.43,118.24,117.76,108 .02,107.67,107.48,107.08,94.96,49.17,48.56,45.72,43.45,43.39,34.76,34.03,31.4 5,31.17,30.55,29.00,25.36,22.65,22.57,21.55,15.09,6.91.HRMS(ESI)calcdfor[M+H] + C50H 57 FN9O6S + 930.4103, found930.4058.

[0151] Example 20: Analysis of the anti-tumor cell proliferation activity of PROTACs

[0152] Using MCF-7 cells as a model, the antitumor effects of compounds prepared from the b-series were examined. The antiproliferative activity of each compound in MCF-7 cells was detected using the MTT assay, and the results are shown in Table 1. Analysis revealed that all synthesized compounds exhibited antitumor cell proliferation activity, with compounds b8, b9, and b10 showing strong activity and low IC50 values. This indicates that changes in the linker can affect the antitumor effect.

[0153] Table 1. Effects of 13 PROTACs on MCF-7 cell proliferation

[0154] Subsequently, the ability of PROTAC molecules to degrade CDK2 and CDK1 was evaluated using Western blotting. The inventors performed Western blotting analysis in MCF-7 cells, and the results are shown in Figure 5. Through structural modification and optimization of PROTAC, compounds capable of effectively degrading CDK2 protein at low nanomolar concentrations without degrading CDK1 protein were screened. The results showed that compounds b8 and b10 exhibited better selectivity and targeting, significantly degrading CDK2 in cells in a concentration-dependent manner without affecting CDK1. Therefore, compounds b8 and b10 possess both degradative activity and selectivity, demonstrating promising development and application prospects.

[0155] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A PROTAC compound, characterized in that: The structure is shown in Equation 1; Wherein, R1 is an alkyl group containing 1-3 carbon atoms, or a cycloalkane containing 3-6 carbon atoms; X1 is a carbonyl group or a sulfonic acid group; X2 is a carbonyl group; Linker is a connecting group, including 3-6 member nitrogen-containing monocyclic and / or spirocyclic structures.

2. The PROTAC compound according to claim 1, characterized in that: The linker structure is shown in any one of Equations 2a, 2b, 2c, 2d, 2e, and 2f; Where p is 1 or 2, q is 1 or 2, r is 1 or 2, and s is 1 or 2; Where p is 1 or 2, q is 1 or 2, r is 1 or 2, and s is 1 or 2; Where m is an integer between 0 and 2, n is 1 or 2, and o is 1 or 2; Where p is 1 or 2, q is 1 or 2, r is 1 or 2, and s is 1 or 2; Where p is 1 or 2, q is 1 or 2, r is 1 or 2, and s is 1 or 2.

3. The PROTAC compound according to claim 1, characterized in that: The structure is shown in equations b1-b14, and any one of c1 and c2; 4. A method for preparing the PROTAC compound according to claim 1 or 2, characterized in that: The target protein ligand POI molecule is synthesized, linked to the linker, and then linked to the E3 ligase ligand. Alternatively, the E3 ligase ligand can be linked to the linker, and then the POI molecule can be linked. The molecular structure of the target protein ligand POI is shown in formula a1, a2 or a3. The structure of the E3 ligase is shown in formula b1-4; 5. The use of any of the PROTAC compounds according to claims 1-3 in the preparation of antitumor drugs.

6. The application according to claim 5, characterized in that: Targeted degradation of intracellular CDK2.

7. The application according to claim 5, characterized in that: The tumor is a CDK2-overexpressing tumor or a CDK2 / 4 / 6 inhibitor-resistant tumor.

8. The application according to claim 5, characterized in that: The tumor is one or more of the following: breast cancer, leukemia, lung cancer, liver cancer, esophageal cancer, pancreatic cancer, colorectal cancer, stomach cancer, cervical cancer, brain cancer, and prostate cancer.

9. A pharmaceutical composition, characterized in that: Includes the PROTAC compound described in any one of claims 1-3.

10. The pharmaceutical composition according to claim 9, characterized in that: This includes PROTAC compounds or their pharmacologically or physiologically acceptable salts, as well as one or more pharmaceutically acceptable carriers, excipients, diluents, adjuvants, and mediators.

Images