Stat3 inhibitors, protac molecules, and methods of making and using the same

Abstract

The application relates to the technical field of pharmaceutical chemistry, in particular to a STAT3 inhibitor, a PROTAC molecule and a preparation method and application thereof. A novel STAT3 inhibitor is designed and synthesized as a ligand combined with a target protein, thalidomide is used as a ligand of an E3 ligase CRBN, a PROTAC molecule for targeted degradation of STAT3 protein is synthesized by connection of a connection chain Linker, the PROTAC molecule has a good degradation effect on STAT3 protein, and has good proliferation inhibition activity on various malignant tumor cells, for example, lung cancer cells, liver cancer cells and the like, and especially has a good industrial prospect in the application to the preparation of a drug for treating non-small cell lung cancer.

Description

Technical Field

[0001] This invention relates to the technical field of medicinal chemistry, and in particular to STAT3 inhibitors, PROTAC molecules, their preparation methods and applications. Background Technology

[0002] Signal transducer and activator of transcription (STAT) proteins belong to the cytoplasmic transcription factor family and are responsible for the transduction of extracellular cytokine and growth factor signals, as well as the activation of gene transcription. STATs have dual functions of signal transduction and transcription activation. When cytokines or growth factors activate their corresponding receptors, STAT proteins are activated, transducing extracellular signals into the cell nucleus, thereby regulating the transcription and expression of related genes (J.Med.Chem.2021,64,8884-8915.). There are seven STAT family members in mammalian cells, including STAT1, STAT2, STAT3, STAT4, STAT5α, STAT5β, and STAT6, which share 20-50% homology. Many genes related to cell cycle control, cell survival, and immune responses are regulated by STAT proteins. STAT3 controls cell cycle progression and anti-apoptosis; therefore, it is most commonly associated with the development and poor prognosis of various human cancers. Besides its involvement in cancer progression, STAT3 is also involved in the development of autoimmune and inflammatory diseases such as rheumatoid arthritis, Crohn's disease, atherosclerosis, and inflammatory bowel disease. Therefore, STAT3 has become an attractive therapeutic target for cancer, immune, and inflammatory diseases, and developing inhibitors for STAT3-related disease treatment has become an important direction in drug research. Over the past two decades, numerous compounds that directly inhibit STAT3 activity have been discovered using strategies such as virtual screening and high-throughput screening (Acta Pharmaceutica Sinica, 2018, 53(10):1598-1608). However, no drugs have yet been approved for clinical use.

[0003] In 2001, Sakamoto et al. first proposed the concept of protein hydrolysis target chimeras (PROTACs), demonstrating that the degradation of target proteins (POIs) could be artificially induced through PROTAC technology (Prc. Natl. Acad. Sci. USA 2001, 98(15), 8554-8559.). A PROTAC is a bifunctional molecule composed of three parts: a ligand for binding to the target protein, a ligand for the E3 ubiquitin ligase, and an intermediate chemical linker connecting the two functional ligands. PROTAC molecules can specifically bind to the target protein and induce POI degradation by catalyzing the binding to the E3 ubiquitin ligase through a given E3 ubiquitin ligand ligand. PROTAC technology can not only solve the problems of drug resistance and off-target effects commonly found in current antitumor drug research, but also, designing PROTAC degraders of STAT3 as a new strategy for effectively targeting STAT3 may provide a feasible approach for the treatment of STAT3-related diseases.

[0004] In 2019, Wang Shaomeng's team reported a STAT3 protein degrader designed based on SI-109 [J.Med.Chem.2019,62,24,11280–11300], SD-36PROTAC molecule can effectively degrade STAT3 protein in leukemia cells and inhibit Molm-16 cell proliferation; in 2021, Neamati's team reported a STAT3 protein degrader designed based on Napabucasin (J.Med.Chem.2021,64,3,1626–1648), XD2-149 PROTAC molecule can inhibit STAT3 signal transduction in pancreatic cancer cell lines without causing proteasome-dependent STAT3 degradation; in 2022, Qin Jiangjiang's team reported a STAT3 protein degrader designed based on S3I-201 (Front.Pharmacol.13:944455), SDL-1PROTAC molecule achieved in vitro degradation of STAT3 protein and has good anti-gastric cancer cell proliferation activity. Currently, there are very few applications of PROTAC technology on STAT3 protein. Therefore, it is necessary to develop a PROTAC molecule that can significantly target and degrade STAT3 protein in tumor cells, especially STAT3 protein in non-small cell lung cancer cells. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide STAT3 inhibitors, PROTAC molecules, their preparation methods and applications, and to solve the problem of how to provide a PROTAC molecule that can significantly target and degrade STAT3 protein in cells, especially STAT3 protein in non-small cell lung cancer cells.

[0006] The technical solution of the present invention is as follows:

[0007] This invention provides a STAT3 inhibitor, wherein the chemical structural formula of the STAT3 inhibitor is shown in Figure II:

[0008] Where n is selected from an integer between 0 and 3; R is selected from hydrogen, alkyl, halogen, hydroxyl or nitro.

[0009] Optionally, the chemical structural formula of the STAT3 inhibitor is shown below:

[0010]

[0011] This invention provides a method for preparing the STAT3 inhibitor, comprising the following steps:

[0012] S1. Dissolve tert-butyl sarcosine and N,N-diisopropylethylamine in acetonitrile, then add 2,3,4,5,6-pentafluorobenzene-1-sulfonyl chloride, and after the reaction, tert-butyl N-methyl-N-((pentafluorophenyl)sulfonyl)glycine salt is obtained.

[0013] S2. Dissolve the tert-butyl N-methyl-N-((pentafluorophenyl)sulfonyl)glycine salt in dichloromethane, add trifluoroacetic acid and mix, and after the reaction, N-methyl-N-((pentafluorophenyl)sulfonyl)glycine is obtained;

[0014] S3. Dissolve the N-methyl-N-((pentafluorophenyl)sulfonyl)glycine in thionyl chloride, reflux, and react to obtain N-methyl-N-((pentafluorophenyl)sulfonyl)glycyl chloride;

[0015] S4. Prepare a mixed solution of p-aminobenzoic acid, dichloromethane and triethylamine, and add N-methyl-N-((pentafluorophenyl)sulfonyl)glycyl chloride dropwise to the mixed solution. After the reaction, 4-(2-((2,3,4,5,6-pentafluoro-N-tolyl)sulfonylamino)acetamido)benzoic acid, i.e., the STAT3 inhibitor.

[0016] This invention provides a PROTAC molecule, the chemical structural formula of which is shown in Figure I:

[0017] Wherein, n is selected from an integer between 0 and 3; R is selected from hydrogen, alkyl, halogen, hydroxyl or nitro; and Linker is the linking chain between the STAT3 inhibitor and thalidomide.

[0018] Optionally, the connecting chain is selected from...

[0019]

[0020] Optionally, the chemical structural formula of the PROTAC molecule is shown below:

[0021] Where n is selected from integers between 1 and 10.

[0022] This invention provides a method for preparing the PROTAC molecule, comprising the following steps:

[0023] 4-(2-((2,3,4,5,6-pentafluoro-N-tolyl)sulfonamide)acetamido)benzoic acid and compound 11 were added to dichloromethane to obtain a mixed solution. N,N-diisopropylethylamine and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate were added to the mixed solution, and the PROTAC molecule was obtained after the reaction.

[0024] The chemical structural formula of compound 11 is shown below:

[0025] n is an integer between 1 and 10.

[0026] The method for preparing the PROTAC molecule, wherein the method for preparing compound 11 includes:

[0027] N,N-diisopropylethylamine and N,N-dimethylformamide were mixed, and 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione and compound 9 were added. The mixture was heated and stirred until the reaction was complete, yielding compound 10.

[0028] Compound 10 was dissolved in dichloromethane, and trifluoroacetic acid was added to react and give compound 11.

[0029] The chemical structural formula of compound 9 is shown below:

[0030] (The Boc group is a tert-butyloxycarbonyl group), and n is taken from an integer between 1 and 10;

[0031] The chemical structural formula of compound 10 is shown below:

[0032] n is an integer between 1 and 10.

[0033] This invention provides the use of the PROTAC molecule, its pharmaceutically acceptable salt, its stereoisomer, its geometric isomer, its tautomer, its ester, its prodrug, its solvate, its metabolite, its nitride, or its deuterated compound in the preparation of a medicament for treating tumors.

[0034] This invention provides the use of the PROTAC molecule, its pharmaceutically acceptable salt, its stereoisomer, its geometric isomer, its tautomer, its ester, its prodrug, its solvate, its metabolite, its nitride, or its deuterated compound in the preparation of a medicament for treating non-small cell lung cancer.

[0035] The beneficial effects of this invention are as follows:

[0036] This invention provides a STAT3 inhibitor, a PROTAC molecule, its preparation method, and its applications. The invention designs and synthesizes a novel STAT3 inhibitor as a ligand for binding to the target protein, using thalidomide as a ligand for the E3 ligase CRBN, and synthesizing a PROTAC molecule that targets and degrades STAT3 protein through a linker. The PROTAC molecule exhibits excellent degradation effects on STAT3 protein and demonstrates good proliferative inhibitory activity against various malignant tumor cells, such as lung cancer cells and liver cancer cells, especially non-small cell lung cancer, showing promising industrialization prospects. Attached Figure Description

[0037] Figure 1 The following is an example of the effect of PROTAC molecule S3D1 on the STAT3 protein level in H1299 cells. (a) shows the effect of different concentrations of S3D1 on the STAT3 protein level in H1299 cells, and (b) shows the change in the degradation of STAT3 protein in H1299 cells by a certain concentration of S3D1 over time.

[0038] Figure 2 The following is an example of the effect of PROTAC molecule S3D1 on the STAT3 protein level in HepG2 cells. (a) shows the effect of different concentrations of S3D1 on the STAT3 protein level in HepG2 cells; (b) shows the change in the degradation of STAT3 protein in HepG2 cells by a certain concentration of S3D1 over time. Detailed Implementation

[0039] This invention provides STAT3 inhibitors, PROTAC molecules, their preparation methods, and applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0040] The pentafluorophenylsulfonamide derivative BP-1-102 is an orally available STAT3-SH2 domain inhibitor and has the greatest potential for clinical application. BP-1-102 has a strong binding affinity for STAT3 (Kd = 504 nM), inhibits the DNA-binding activity of STAT3 (IC50 = 6.8 μM), and can selectively inhibit the growth, survival, migration, and invasion of STAT3-dependent tumor cells, while also inhibiting the growth of MDA-MB-231 and A549 xenograft tumors (PNAS. 109(24) 9623-9628). Currently, there are no reports on its use for the targeted degradation of STAT3 in cancer treatment.

[0041] The inventors conceived of designing and modifying the BP-1-102 molecule as a target protein binding ligand. Based on the predictions of the molecular docking model, they retained the phenylsulfonamide and pentafluorobenzene fragments, which bind well to the binding pocket, while removing the isopropylbenzene fragment. This yielded a novel STAT3 inhibitor as a ligand (POI) for binding to the target protein. Hypothesizing that its carboxyl moiety might be the solvent-exposed region of a linker of varying lengths, they used thalidomide as a ligand for the E3 ligase CRBN and designed and synthesized a series of PROTACs molecules that target and degrade STAT3 proteins via linker linkage.

[0042] Specifically, this invention provides a STAT3 inhibitor, wherein the chemical structural formula of the STAT3 inhibitor is shown in Figure II:

[0043] Where n is selected from an integer between 0 and 3; R is selected from hydrogen, alkyl, halogen, hydroxyl or nitro.

[0044] In one embodiment, the chemical structural formula of the STAT3 inhibitor is shown below:

[0045]

[0046] This invention provides a method for preparing the STAT3 inhibitor, comprising the following steps:

[0047] S1. Dissolve tert-butyl sarcosine and N,N-diisopropylethylamine in acetonitrile, then add 2,3,4,5,6-pentafluorobenzene-1-sulfonyl chloride, and after the reaction, tert-butyl N-methyl-N-((pentafluorophenyl)sulfonyl)glycine salt is obtained.

[0048] S2. Dissolve the tert-butyl N-methyl-N-((pentafluorophenyl)sulfonyl)glycine salt in dichloromethane, add trifluoroacetic acid, and stir to obtain N-methyl-N-((pentafluorophenyl)sulfonyl)glycine;

[0049] S3. Dissolve the N-methyl-N-((pentafluorophenyl)sulfonyl)glycine in thionyl chloride and reflux to obtain N-methyl-N-((pentafluorophenyl)sulfonyl)glycyl chloride;

[0050] S4. Prepare a mixed solution of p-aminobenzoic acid, dichloromethane and triethylamine, and add N-methyl-N-((pentafluorophenyl)sulfonyl)glycyl chloride dropwise to the mixed solution. After the reaction, 4-(2-((2,3,4,5,6-pentafluoro-N-tolyl)sulfonylamino)acetamido)benzoic acid is obtained, which is the STAT3 inhibitor.

[0051] This invention provides a PROTAC molecule, the chemical structural formula of which is shown in Figure I:

[0052] Wherein, n is selected from an integer between 0 and 3; R is selected from hydrogen, alkyl, halogen, hydroxyl or nitro; and Linker is the linking chain between the STAT3 inhibitor and thalidomide.

[0053] In some embodiments, the connecting chain is selected from...

[0054] In one embodiment, the chemical structural formula of the PROTAC molecule is shown below:

[0055] Where n is selected from integers between 1 and 10.

[0056] This invention provides a method for preparing the PROTAC molecule, comprising the following steps:

[0057] 4-(2-((2,3,4,5,6-pentafluoro-N-tolyl)sulfonamide)acetamido)benzoic acid and compound 11 were added to dichloromethane and stirred to obtain a mixed solution. N,N-diisopropylethylamine and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate were added to the mixed solution, and the reaction was carried out to obtain the PROTAC molecule.

[0058] The chemical structural formula of compound 11 is shown below:

[0059] n is an integer between 1 and 10.

[0060] In one embodiment, the method for preparing compound 11 includes:

[0061] N,N-diisopropylethylamine and N,N-dimethylformamide were mixed, and 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione and compound 9 were added. The mixture was heated and stirred until the reaction was complete, yielding compound 10.

[0062] Compound 10 was dissolved in dichloromethane, and trifluoroacetic acid was added to react and give compound 11.

[0063] The chemical structural formula of compound 9 is shown below:

[0064] n is an integer between 1 and 10;

[0065] The chemical structural formula of compound 10 is shown below:

[0066] n is an integer between 1 and 10.

[0067] This invention provides the use of the PROTAC molecule, its pharmaceutically acceptable salt, its stereoisomer, its geometric isomer, its tautomer, its ester, its prodrug, its solvate, its metabolite, its nitride, or its deuterated compound in the preparation of a medicament for treating tumors.

[0068] This invention provides the use of the PROTAC molecule, its pharmaceutically acceptable salt, its stereoisomer, its geometric isomer, its tautomer, its ester, its prodrug, its solvate, its metabolite, its nitride, or its deuterated compound in the preparation of a medicament for treating non-small cell lung cancer.

[0069] The present invention will be further described below through specific embodiments.

[0070] Example 1

[0071] The synthetic route for 4-(2-((2,3,4,5,6-pentafluoro-N-tolyl)sulfonamide)acetamido)benzoic acid (STAT3 inhibitor) is as follows:

[0072]

[0073] The specific steps are as follows: 20 mmol of tert-butyl sarcosinate (compound 1 in the synthetic route) and 3 eq of 2,3,4,5,6-pentafluorobenzene-1-sulfonyl chloride (DIPEA) were dissolved in anhydrous acetonitrile and cooled to 0°C. Then, 22 mmol of 2,3,4,5,6-pentafluorobenzene-1-sulfonyl chloride was added. The resulting solution was stirred overnight at room temperature. The reaction was quenched with water, and the aqueous layer was extracted 2 to 3 times with ethyl acetate. The organic layer was washed with saturated NaHCO3 aqueous solution and brine. The organic layer was dried with anhydrous Na2SO4. The crude product was purified by silica gel column chromatography in a mobile phase PE:EA (20:1) to obtain tert-butyl N-methyl-N-((pentafluorophenyl)sulfonyl)glycine salt (compound 3 in the synthetic route). The obtained tert-butyl N-methyl-N-((pentafluorophenyl)sulfonyl)glycine salt (16.3 mmol) was dissolved in 50 mL of DCM and stirred. 50 mL of TFA was added, and the mixture was stirred overnight at room temperature. The resulting mixture was concentrated under reduced pressure to give N-methyl-N-((pentafluorophenyl)sulfonyl)glycine (compound 4 in the synthetic route). The obtained N-methyl-N-((pentafluorophenyl)sulfonyl)glycine was directly dissolved in sulfoxide, refluxed for 3 hours, and then directly evaporated to dryness for the next step. Add 10 mmol of p-aminobenzoic acid (compound 6 in the synthetic route), 10 mL of dichloromethane, and 30 mmol of triethylamine to a 100 mL round-bottom flask. Add 15 mmol of N-methyl-N-((pentafluorophenyl)sulfonyl)glycyl chloride (compound 5 in the synthetic route) dropwise under ice bath conditions. After the addition is complete, remove the ice bath and let the mixture incubate overnight at room temperature. After the reaction is complete, acidify directly with 1 mol / L hydrochloric acid. A solid precipitates; filter and collect the precipitate. Recrystallization from tetrahydrofuran-hexane solution yields 4-(2-((2,3,4,5,6-pentafluoro-N-tolyl)sulfonylamino)acetamido)benzoic acid (compound 7 in the synthetic route), i.e., the STAT3 inhibitor.

[0074] Example 2

[0075] The synthetic route for N-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)ethoxy)ethyl)-4-(2-((2,3,4,5,6-pentafluoro-N-tolyl)sulfonylamino)acetamido)benzamide (S3D1) is as follows:

[0076]

[0077] Take DIPEA (0.7 mL), N,N-dimethylformamide (10 mL), add to a three-necked flask, then add 0.5 g of 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione (compound 8 in the synthetic route) (1.8 mmol), and compound 9a (2.0 mmol). Stir at 90°C for 12 h. Add 100 mL of water to the reaction mixture, extract with ethyl acetate 2 to 3 times, 200 mL each time, combine the ethyl acetate layers, evaporate to dryness under reduced pressure, and purify by silica gel column chromatography (methanol:dichloroethane = 1:20) to obtain compound 10a (yield 56%).

[0078]

[0079] Synthesis of intermediate 11a: 0.2 mmol of compound 10a was dissolved in 4 mL of dichloromethane, and 0.5 mL of trifluoroacetic acid was added to the reaction system. The reaction was carried out at room temperature for 2 h, and the mixture was concentrated under reduced pressure to obtain compound 11a.

[0080]

[0081] DIPEA (3.0 equivalents) was added to a solution of compound 7 (1.0 equivalents) and compound 11a (1.1 equivalents) in dichloromethane (15 mL) under stirring at room temperature. HATU (1.5 equivalents) was added to the mixture, and the reaction was allowed to proceed overnight at room temperature. The mixture was extracted with water and DCM, and the organic layer was dried over anhydrous Na2SO4. Excess solvent was removed by filtration under reduced pressure. The crude product was purified by gradient elution using column chromatography to obtain the final product S3D1. 1 H NMR(400MHz,Chloroform-d)δ9.79(s,1H),9.23(s,1H),7.90(d,J=8.3Hz,2H) ,7.79–7.65(m,3H),7.26(d,J=7.1Hz,2H),7.16(d,J=8.6Hz,1H),6.78(s,1H) ,5.21(dd,J=11.5,5.2Hz,1H),4.43(d,J=18.3Hz,2H),4.09–3.84(m,6H),3.7 2(s,2H),3.36(s,3H),3.00(dd,J=30.8,14.0Hz,3H),2.33(d,J=11.5Hz,1H). 13C NMR(101MHz,Chloroform-d)δ172.29,169.80,169.39,167.59,167.15,165.72,146.79,140.26,129.75, 127.98,119.20,116.99,111.75,109.92,69.35,69.22,53.47,48.83,42.12,39.81,36.01,31.30,22.64. 19 F NMR(376MHz,Chloroform-d)δ-134.11–-135.46(m,2F),-145.49(ddd,J=29.4,22.4,7.6Hz,1F),-158.25–-159.25(m,2F).HRMS(ESI-TOF):m / z calculated for C 33 H 29 F5N6O9S[M+Na] + :803.1637,found:803.1531.

[0082] Example 3

[0083] The synthetic route for N-(2-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)ethoxy)ethyl)-4-(2-((2,3,4,5,6-pentafluoro-N-tolyl)sulfonylamino)acetamido)benzamide (S3D2) is as follows:

[0084]

[0085] Synthesis of intermediate 10b: DIPEA (0.7 mL) and N,N-dimethylformamide (10 mL) were added to a three-necked flask, followed by 0.5 g of 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione (compound 8) (1.8 mmol) and compound 9b (2.0 mmol). The mixture was stirred at 90°C for 12 h. 100 mL of water was added to the reaction mixture, and the mixture was extracted 2 to 3 times with 200 mL of ethyl acetate each time. The combined ethyl acetate layers were evaporated to dryness under reduced pressure and purified by silica gel column chromatography (methanol:dichloroethane = 1:20) to obtain compound 10b (yield 48%).

[0086]

[0087] Synthesis of intermediate 11b: 0.2 mmol of compound 10b was dissolved in 4 mL of dichloromethane, and 0.5 mL of trifluoroacetic acid was added to the reaction system. The reaction was carried out at room temperature for 2 h, and the mixture was concentrated under reduced pressure to obtain compound 11b.

[0088]

[0089] DIPEA (3.0 equivalents) was added to a solution of compound 7 (1.0 equivalents) and compound 11b (1.1 equivalents) in dichloromethane (15 mL) under stirring at room temperature. HATU (1.5 equivalents) was added to the mixture, and the reaction was allowed to proceed overnight at room temperature. The mixture was extracted with water and DCM, and the organic layer was dried over anhydrous Na₂SO₄. The solution was then concentrated by vacuum filtration to remove excess solvent. The crude product was purified by gradient elution using column chromatography to obtain the final product S3D₂. 1 H NMR(400MHz,Chloroform-d)δ9.79(s,1H),8.93(s,1H),7.51–7.21(m,6H),6.97(d,J=7.0Hz,1H),6.52(s,1H),6.24(s, 1H),5.12–4.90(m,1H),4.38–3.91(m,2H),3.89–3.10(m,12H),3.07(s,3H),2.82(dd,J=36.5,15.2Hz,3H),2.15(s,1H). 13 C NMR(101MHz,Chloroform-d)δ172.10,170.39,169.58,167.56,166.51,165.33,146.46,140.19,136.27,131.76,127 .84,118.58,116.84,111.76,109.61,70.38,69.89,69.74,68.80,53.15,48.77,42.07,39.53,35.97,31.37,22.49. 19 F NMR(376MHz,Chloroform-d)δ-135.00(d,J=22.0Hz,2F),-145.96(d,J=22.0Hz,1F),-159.27(t,J=20.8Hz,2F).HRMS(ESI-TOF):m / z calculated for C 35 H 33 F5N6O 10 S[M+Na] + :847.1899,found:847.1793.

[0090] Example 4

[0091] Synthesis of N-(2-(2-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)ethoxy)ethyl)-4-(2-((2,3,4,5,6-pentafluoro-N-tolyl)sulfonylamino)acetamido)benzamide (S3D3):

[0092]

[0093] Synthesis of intermediate 10c: DIPEA (0.7 mL) and N,N-dimethylformamide (10 mL) were added to a three-necked flask, followed by 0.5 g of 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione (compound 8) (1.8 mmol) and compound 9c (2.0 mmol). The mixture was stirred at 90°C for 12 h. 100 mL of water was added to the reaction mixture, and the mixture was extracted 2 to 3 times with 200 mL of ethyl acetate each time. The combined ethyl acetate layers were evaporated to dryness under reduced pressure and purified by silica gel column chromatography (methanol:dichloroethane = 1:20) to obtain compound 10c (yield 43%).

[0094]

[0095] Synthesis of intermediate 11c: 0.2 mmol of compound 10c was dissolved in 4 mL of dichloromethane, and 0.5 mL of trifluoroacetic acid was added to the reaction system. The reaction was carried out at room temperature for 2 h, and the mixture was concentrated under reduced pressure to obtain compound 11c.

[0096]

[0097] DIPEA (3.0 equivalents) was added to a solution of compound 7 (1.0 equivalents) and compound 11c (1.1 equivalents) in dichloromethane (15 mL) under stirring at room temperature. HATU (1.5 equivalents) was added to the mixture, and the reaction was allowed to proceed overnight at room temperature. The mixture was extracted with water and DCM, and the organic layer was dried over anhydrous Na2SO4. Excess solvent was removed by filtration under reduced pressure. The crude product was purified by gradient elution using column chromatography to obtain the final product S3D3. 1H NMR (400MHz, Chloroform-d) δ9.71 (s, 1H), 9.30 (s, 1H), 7.92 (d, J = 8.2Hz, 2H ),7.76-7.66(m,3H),7.40(s,1H),7.29(d,J=7.0Hz,1H),7.05(d,J=8.6Hz,1H ),6.63(t,J=5.4Hz,1H),5.31-5.20(m,1H),4.46(s,2H),4.00-3.84(m,14H), 3.61(q,J=5.4Hz,2H),3.36(s,3H),3.13-2.99(m,3H),2.39(d,J=7.5Hz,1H). 13 C NMR(101MHz,Chloroform-d)δ172.00,169.78,169.23,167.68,166.95,165.54,146.55,140.32,136.04,132.10,129.65,127.9 9,119.02,116.83,111.51,109.81,70.50,70.38,70.08,69.70,69.28,53.45,53.21,48.81,42.13,39.79,35.87,31.32,22.62. 19 F NMR(376MHz,Chloroform-d)δ-134.25--135.72(m,2F),-145.87(td,J=21.8,21.2,10.3Hz,1F),-158.48--159.51(m,2F).HRMS(ESI-TOF):m / zcalculated for C 37 H 37 F5N6O 11 S[M+Na] + :891.2161,found:891.2054.

[0098] Example 5

[0099] Synthesis of N-(14-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)-3,6,9,12-tetraoxatetradecyl)-4-(2-((2,3,4,5,6-pentafluoro-N-tolyl)sulfonylamino)acetamido)benzamide (S3D4):

[0100]

[0101] Synthesis of intermediate 10d: DIPEA (0.7 mL) and N,N-dimethylformamide (10 mL) were added to a three-necked flask, followed by 0.5 g of 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione (compound 8) (1.8 mmol) and compound 9d (2.0 mmol). The mixture was stirred at 90°C for 12 h. 100 mL of water was added to the reaction mixture, and the mixture was extracted 2 to 3 times with 200 mL of ethyl acetate each time. The combined ethyl acetate layers were evaporated to dryness under reduced pressure and purified by silica gel column chromatography (methanol:dichloroethane = 1:20) to obtain compound 10d (yield 58%).

[0102]

[0103] Synthesis of intermediate 11d: 0.2 mmol of compound 10d was dissolved in 4 mL of dichloromethane, and 0.5 mL of trifluoroacetic acid was added to the reaction system. The reaction was carried out at room temperature for 2 h, and the mixture was concentrated under reduced pressure to obtain compound 11d.

[0104]

[0105] DIPEA (3.0 equivalents) was added to a solution of compound 7 (1.0 equivalents) and compound 11d (1.1 equivalents) in dichloromethane (15 mL) under stirring at room temperature. HATU (1.5 equivalents) was added to the mixture, and the reaction was allowed to proceed overnight at room temperature. The mixture was extracted with water and DCM, and the organic layer was dried over anhydrous Na2SO4. Excess solvent was removed by filtration under reduced pressure. The crude product was purified by gradient elution using column chromatography to obtain the final product S3D4. 1 H NMR(400MHz,Chloroform-d)δ9.40(s,1H),9.08(s,1H),7.67(d,J=8.3Hz,2H),7.40(dd,J=18.5,8.3Hz,4H),6.99(d,J=7.1Hz,1H),6.78(d,J=8.5Hz,1 H),6.34(t,J=5.4Hz,1H),4.99-4.84(m,1H),4.16(s,2H),3.61(s,18H),3. 34(q,J=5.5Hz,2H),3.05(s,3H),2.85-2.70(m,3H),2.09(q,J=10.0Hz,1H). 13C NMR(101MHz,Chloroform-d)δ171.78,169.66,169.22,167.66,166.94,165.43,146.64,140.21,136.08,132.18,129.92,128.10,119. 06,116.86,111.58,109.90,70.59,70.49,70.40,70.34,70.16,69.86,69.25,53.46,53.36,48.83,42.17,39.90,35.98,31.35,22.71. 19 F NMR(376MHz,Chloroform-d)δ-133.90--135.19(m,2F),-145.53--146.50(m,1F),-158.42--159.50(m,2F).HRMS(ESI-TOF):m / z calculated for C 39 H 41 F5N6O 12 S[M+Na] + :935.2423,found:935.2314.

[0106] Example 6

[0107] Synthesis of N-(17-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)-3,6,9,12,15-pentaheptadecyl)-4-(2-((2,3,4,5,6-pentafluoro-N-tolyl)sulfonylamino)acetamido)benzamide (S3D5)

[0108]

[0109] Synthesis of intermediate 10e: DIPEA (0.7 mL) and N,N-dimethylformamide (10 mL) were added to a three-necked flask, followed by 0.5 g of 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione (compound 8) (1.8 mmol) and compound 9e (2.0 mmol). The mixture was stirred at 90°C for 12 h. 100 mL of water was added to the reaction mixture, and the mixture was extracted 2 to 3 times with 200 mL of ethyl acetate each time. The combined ethyl acetate layers were evaporated to dryness under reduced pressure and purified by silica gel column chromatography (methanol:dichloroethane = 1:20) to obtain compound 10e (yield 46%).

[0110]

[0111] Synthesis of intermediate 11e: 0.2 mmol of compound 10e was dissolved in 4 mL of dichloromethane, and 0.5 mL of trifluoroacetic acid was added to the reaction system. The reaction was carried out at room temperature for 2 h, and the mixture was concentrated under reduced pressure to obtain compound 11e.

[0112]

[0113] DIPEA (3.0 equivalents) was added to a solution of compound 7 (1.0 equivalents) and compound 11e (1.1 equivalents) in dichloromethane (15 mL) under stirring at room temperature. HATU (1.5 equivalents) was added to the mixture, and the reaction was allowed to proceed overnight at room temperature. The mixture was extracted with water and DCM, and the organic layer was dried over anhydrous Na₂SO₄. The solution was then concentrated by vacuum filtration to remove excess solvent. The crude product was purified by gradient elution using column chromatography to obtain the final product S3D5. 1 H NMR(400MHz,Chloroform-d)δ9.33(s,1H),9.07(s,1H),7.70(d,J=8.2Hz,2H),7.45 (dd,J=22.4,8.1Hz,3H),7.26(s,1H),7.03(d,J=7.1Hz,1H),6.83(d,J=8.5Hz,1H), 6.41(t,J=5.5Hz,1H),4.94(dd,J=10.7,6.8Hz,1H),4.20(s,2H),3.70-3.56(m,22H ),3.38(q,J=5.4Hz,2H),3.08(s,3H),2.86-2.72(m,3H),2.12(q,J=8.6,7.7Hz,1H). 13 C NMR(101MHz,Chloroform-d)δ171.79,169.41,169.19,167.64,166.95,165.47,146.61,140.31,136.03,132.22,129.86,128.09,119.06,116 .80,111.51,109.94,70.51,70.48,70.37,70.34,70.28,70.24,69.99, 69.67,69.24,53.44,53.22,48.82,42.13,39.78,35.90,31.34,22.66. 19 F NMR(376MHz,Chloroform-d)δ-134.60--135.18(m,2F),-145.63--146.24(m,1F),-158.76--159.39(m,2F).HRMS(ESI-TOF):m / z calculated for C 41 H45 F5N6O 13 S[M+Na] + :979.2685,found:979.2572.

[0114] Example 7

[0115] Synthesis of N-(20-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)-3,6,9,12,15,18-hexaoxaecoalkyl)-4-(2-((2,3,4,5,6-pentafluoro-N-tolyl)sulfonylamino)acetamido)benzamide (S3D6)

[0116]

[0117] Synthesis of intermediate 10f: DIPEA (0.7 mL) and N,N-dimethylformamide (10 mL) were added to a three-necked flask, followed by 0.5 g of 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione (compound 8) (1.8 mmol) and compound 9f (2.0 mmol). The mixture was stirred at 90°C for 12 h. 100 mL of water was added to the reaction mixture, and the mixture was extracted 2 to 3 times with 200 mL of ethyl acetate each time. The combined ethyl acetate layers were evaporated to dryness under reduced pressure and purified by silica gel column chromatography (methanol:dichloroethane = 1:20) to obtain compound 10f (yield 61%).

[0118]

[0119] Synthesis of intermediate 11f: 0.2 mmol of compound 10f was dissolved in 4 mL of dichloromethane, and 0.5 mL of trifluoroacetic acid was added to the reaction system. The reaction was carried out at room temperature for 2 h, and the mixture was concentrated under reduced pressure to obtain compound 11f.

[0120]

[0121] DIPEA (3.0 equivalents) was added to a solution of compound 7 (1.0 equivalents) and compound 11f (1.1 equivalents) in dichloromethane (15 mL) under stirring at room temperature. HATU (1.5 equivalents) was added to the mixture, and the reaction was allowed to proceed overnight at room temperature. The mixture was extracted with water and DCM, and the organic layer was dried over anhydrous Na2SO4. Excess solvent was removed by filtration under reduced pressure. The crude product was purified by gradient elution using column chromatography to obtain the final product S3D6. 1H NMR(400MHz,Chloroform-d)δ9.63(s,1H),9.44(s,1H),8.01(d,J=8.2Hz,2H),7.80(d ,J=8.3Hz,2H),7.73(t,J=7.8Hz,1H),7.53(s,1H),7.33(d,J=7.0Hz,1H),7.12(d,J=8. 5Hz,1H),6.71(t,J=5.5Hz,1H),5.24(dd,J=10.5,6.7Hz,1H),4.52(s,2H),3.99-3.83 (m,26H),3.68(q,J=5.4Hz,2H),3.38(s,3H),3.19-3.04(m,3H),2.40(d,J=7.3Hz,1H). 13 C NMR(101MHz,Chloroform-d)δ171.76,169.27,169.19,167.62,166.93,165.48,146.57,140.36,136.00,132.21,129.80,128.06,119.02, 116.74,111.49,109.96,70.45,70.44,70.37,70.33,70.25,69.94,6 9.61,69.22,53.43,53.16,48.82,42.08,39.71,35.85,31.34,22.63. 19 F NMR(376MHz,Chloroform-d)δ-134.56--135.27(m,2F),-145.75--146.44(m,1F),-158.84--159.58(m,2F).HRMS(ESI-TOF):m / z calculated for C 43 H 49 F5N6O 14 S[M+Na] + :1023.2948,found:1023.2837.

[0122] Example 8

[0123] Synthesis of N-(23-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)-3,6,9,12,15,18,21-heptaoxatrialkyl)-4-(2-((2,3,4,5,6-pentafluoro-N-tolyl)sulfonylamino)acetamido)benzamide (S3D7)

[0124]

[0125] Synthesis of 10 g of intermediate: DIPEA (0.7 mL) and N,N-dimethylformamide (10 mL) were added to a three-necked flask, followed by 0.5 g of 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione (compound 8) (1.8 mmol) and 9 g of compound (2.0 mmol). The mixture was stirred at 90°C for 12 h. 100 mL of water was added to the reaction mixture, and the mixture was extracted 2 to 3 times with 200 mL of ethyl acetate each time. The combined ethyl acetate layers were evaporated to dryness under reduced pressure and purified by silica gel column chromatography (methanol:dichloroethane = 1:20) to obtain 10 g of compound (yield 43%).

[0126]

[0127] Synthesis of intermediate 11g: 0.2 mmol of compound 10g was dissolved in 4 mL of dichloromethane, 0.5 mL of trifluoroacetic acid was added to the reaction system, and the reaction was carried out at room temperature for 2 h. After concentration under reduced pressure, compound 11g was obtained.

[0128]

[0129] DIPEA (3.0 equivalents) was added to a solution of compound 7 (1.0 equivalents) and compound 11 g (1.1 equivalents) in dichloromethane (15 mL) under stirring at room temperature. HATU (1.5 equivalents) was added to the mixture, and the reaction was allowed to proceed overnight at room temperature. The mixture was extracted with water and DCM, and the organic layer was dried over anhydrous Na2SO4. Excess solvent was removed by filtration under reduced pressure. The crude product was purified by gradient elution using column chromatography to obtain the final product S3D7. 1 H NMR(400MHz,Chloroform-d)δ9.55(s,1H),9.45(s,1H),8.02(d,J=8.3Hz,2H),7.82(d,J =8.3Hz,2H),7.74(t,J=7.8Hz,1H),7.54(s,1H),7.34(d,J=7.1Hz,1H),7.12(d,J=8.5Hz ,1H),6.72(t,J=5.5Hz,1H),5.23(dd,J=11.8,5.5Hz,1H),4.54(s,2H),3.98-3.82(m,30 H),3.67(q,J=5.4Hz,2H),3.39(s,3H),3.12(dd,J=26.2,12.6Hz,3H),2.47-2.39(m,1H). 13C NMR(101MHz,Chloroform-d)δ171.71,169.19,169.16,167.63,166.94,165.53,146.56,140.43,135.99,132.23,129.77,128.08,118. 98,116.72,111.47,109.98,70.46,70.36,70.25,70.20,69.92,69.65,69.21,53.13,48.82,42.06,39.71,35.83,31.35,29.58,22.62. 19 F NMR(376MHz,Chloroform-d)δ-134.73--135.07(m,2F),-146.15(ddd,J=29.6,22.3,7.4Hz,1F),-159.08--159.45(m,2F).HRMS(ESI-TOF):m / z calculated for C 45 H 53 F5N6O 15 S[M+Na] + :1067.3210,found:1067.3100.

[0130] Example 9

[0131] Synthesis of N-(26-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)-3,6,9,12,15,18,21,24-octaoxahexadecyl)-4-(2-((2,3,4,5,6-pentafluoro-N-tolyl)sulfonylamino)acetamido)benzamide (S3D8)

[0132]

[0133] Synthesis of intermediate 10h: DIPEA (0.7 mL), N,N-dimethylformamide (10 mL), and compound 9h were added to a three-necked flask. Then, 0.5 g of 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione (compound 8) (1.8 mmol) and compound 9h (2.0 mmol) were added. The mixture was stirred at 90°C for 12 h. 100 mL of water was added to the reaction mixture, and the mixture was extracted 2 to 3 times with 200 mL of ethyl acetate each time. The combined ethyl acetate layers were evaporated to dryness under reduced pressure and purified by silica gel column chromatography (methanol:dichloroethane = 1:20) to obtain compound 10h (yield 62%).

[0134]

[0135] Synthesis of intermediate 11h: 0.2 mmol of compound 10h was dissolved in 4 mL of dichloromethane, and 0.5 mL of trifluoroacetic acid was added to the reaction system. The reaction was carried out at room temperature for 2 h, and the mixture was concentrated under reduced pressure to obtain compound 11h.

[0136]

[0137] DIPEA (3.0 equivalents) was added to a solution of compound 7 (1.0 equivalents) and compound 11h (1.1 equivalents) in dichloromethane (15 mL) under stirring at room temperature. HATU (1.5 equivalents) was added to the mixture, and the reaction was allowed to proceed overnight at room temperature. The mixture was extracted with water and DCM, and the organic layer was dried over anhydrous Na2SO4. Excess solvent was removed by filtration under reduced pressure. The crude product was purified by gradient elution using column chromatography to obtain the final product S3D8. 1 H NMR(400MHz,Chloroform-d)δ9.15(s,2H),7.74(d,J=8.2Hz,2H),7.54(d,J=8.3Hz,2 H),7.45(t,J=7.8Hz,1H),7.29(d,J=10.4Hz,1H),7.05(d,J=7.1Hz,1H),6.85(d,J=8 .5Hz,1H),6.43(t,J=5.6Hz,1H),4.93(dd,J=11.8,5.4Hz,1H),4.25(s,2H),3.66-3. 47(m,34H),3.39(q,J=5.5Hz,2H),3.10(s,3H),2.90-2.72(m,3H),2.19-2.09(m,1H). 13 C NMR(101MHz,Chloroform-d)δ171.66,169.18,169.04,167.63,167.04,165.58,146.62,140.48,135.98,132.28,129.86,128.14,119.00,116.74,11 1.47,110.01,70.48,70.45,70.40,70.35,70.30,70.28,70.22,70.19,70. 16,69.92,69.75,69.24,53.14,48.82,42.12,39.74,35.83,31.36,22.65. 19F NMR(376MHz,Chloroform-d)δ-134.69--135.07(m,2F),-146.19(ddd,J=29.5,22.3,7.5Hz,1F),-159.07--159.50(m,2F).HRMS(ESI-TOF):m / z calculated for C 47 H 57 F5N6O 16 S[M+Na] + :1111.3472,found:1111.3367.

[0138] Example 10

[0139] Synthesis of N-(29-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)-3,6,9,12,15,18,21,24,27-nonazo-nonazoalkyl)-4-(2-((2,3,4,5,6-pentafluoro-N-tolyl)sulfonylamino)acetamido)benzamide (S3D9)

[0140]

[0141] Synthesis of intermediate 10i: DIPEA (0.7 mL) and N,N-dimethylformamide (10 mL) were added to a three-necked flask, followed by 0.5 g of compound 8 (1.8 mmol) and compound 9i (2.0 mmol). The mixture was stirred at 90°C for 12 h. 100 mL of water was added to the reaction mixture, and the mixture was extracted 2 to 3 times with 200 mL of ethyl acetate each time. The combined ethyl acetate layers were evaporated to dryness under reduced pressure and purified by silica gel column chromatography (methanol:dichloroethane = 1:20) to obtain compound 10i (yield 42%).

[0142]

[0143] Synthesis of intermediate 11i: 0.2 mmol of compound 10i was dissolved in 4 mL of dichloromethane, and 0.5 mL of trifluoroacetic acid was added to the reaction system. The reaction was carried out at room temperature for 2 h, and the mixture was concentrated under reduced pressure to obtain compound 11i.

[0144]

[0145] DIPEA (3.0 equivalents) was added to a solution of compound 7 (1.0 equivalents) and compound 11i (1.1 equivalents) in dichloromethane (15 mL) under stirring at room temperature. HATU (1.5 equivalents) was added to the mixture, and the reaction was allowed to proceed overnight at room temperature. The mixture was extracted with water and DCM, and the organic layer was dried over anhydrous Na2SO4. Excess solvent was removed by filtration under reduced pressure. The crude product was purified by gradient elution using column chromatography to obtain the final product S3D9. 1 H NMR(400MHz,Chloroform-d)δ9.48(d,J=13.8Hz,2H),8.05(d,J=8.3Hz,2H),7.85(d,J =8.4Hz,2H),7.76(t,J=7.8Hz,1H),7.62-7.55(m,1H),7.35(d,J=7.1Hz,1H),7.17(d,J =8.6Hz,1H),6.74(t,J=5.6Hz,1H),5.29-5.18(m,1H),4.56(s,2H),3.99-3.84(m,38H) ,3.71(q,J=5.4Hz,2H),3.40(s,3H),3.10(dt,J=23.6,11.9Hz,3H),2.47-2.37(m,1H). 13 C NMR(101MHz,Chloroform-d)δ171.63,169.13,168.96,167.58,166.99,165. 60,146.60,140.51,135.93,132.25,129.78,128.09,118.95,116.71,111.4 1,109.98,70.45,70.39,70.35,70.33,70.31,70.28,70.26,70.22,70.19,69.91,69.71,69.23,53.08,48.77,42.12,39.70,35.77,31.32,29.52,22.61. 19 F NMR(376MHz,Chloroform-d)δ-134.74--135.03(m,2F),-146.30(dt,J=28.6,13.1Hz,1F),-159.17--159.54(m,2F).HRMS(ESI-TOF):m / z calculated for C 49 H 61 F5N6O 17 S[M+Na] + :1155.3734,found:1155.3629.

[0146] Example 11

[0147] Synthesis of N-(32-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)-3,6,9,12,15,18,21,24,27,30-decaoxatridodecyl)-4-(2-((2,3,4,5,6-pentafluoro-N-tolyl)sulfonylamino)acetamido)benzamide (S3D10)

[0148]

[0149] Synthesis of intermediate 10j: DIPEA (0.7 mL) and N,N-dimethylformamide (10 mL) were added to a three-necked flask, followed by 0.5 g of compound 8 (1.8 mmol) and compound 9j (2.0 mmol). The mixture was stirred at 90°C for 12 h. 100 mL of water was added to the reaction mixture, and the mixture was extracted 2 to 3 times with 200 mL of ethyl acetate each time. The combined ethyl acetate layers were evaporated to dryness under reduced pressure and purified by silica gel column chromatography (methanol:dichloroethane = 1:20) to obtain compound 10j (yield 40%).

[0150]

[0151] Synthesis of intermediate 11j: 0.2 mmol of compound 10j was dissolved in 4 mL of dichloromethane, and 0.5 mL of trifluoroacetic acid was added to the reaction system. The reaction was carried out at room temperature for 2 h, and the mixture was concentrated under reduced pressure to obtain compound 11j.

[0152]

[0153] DIPEA (3.0 equivalents) was added to a solution of compound 7 (1.0 equivalents) and compound 11j (1.1 equivalents) in dichloromethane (15 mL) under stirring at room temperature. HATU (1.5 equivalents) was added to the mixture, and the reaction was allowed to proceed overnight at room temperature. The mixture was extracted with water and DCM, and the organic layer was dried over anhydrous Na2SO4. Excess solvent was removed by filtration under reduced pressure. The crude product was purified by gradient elution using column chromatography to obtain the final product S3D10. 11H NMR (400 MHz, Chloroform-d) δ 9.50 (s, 1H), 9.39 (s, 1H), 8.05 (d, J = 8.4 Hz, 2H), 7.86 (d, J = 8.3 Hz, 2H), 7.76 (t, J = 7.8 Hz, 1H), 7.62 - 7.50 (m, 1H), 7.36 (d, J = 7.0 Hz, 1H), 7.18 (d, J = 8.6 Hz, 1H), 6.75 (t, J = 5.6 Hz, 1H), 5.22 (dd, J = 11.8, 5.3 Hz, 1H), 4.57 (s, 2H), 3.95 - 3.85 (m, 42H), 3.74 - 3.68 (m, 2H), 3.40 (d, J = 7.4 Hz, 4H), 3.19 - 3.04 (m, 3H), 2.47 - 2.35 (m, 1H). 13 13C NMR (101 MHz, Chloroform-d) δ 171.59, 169.11, 168.90, 167.56, 166.95, 165.61, 146.60, 140.54, 135.91, 132.24, 129.73, 128.06, 118.93, 116.69, 111.39, 109.98, 70.45, 70.38, 70.33, 70.29, 70.25, 70.19, 70.16, 69.91, 69.68, 69.23, 53.42, 53.05, 48.75, 42.12, 39.68, 35.75, 31.31, 29.53, 22.60. 19 19F NMR (376 MHz, Chloroform-d) δ -134.61--135.12 (m, 2F), -146.03--146.56 (m, 1F), -159.33 (dd, J = 38.7, 15.5 Hz, 2F). HRMS (ESI-TOF): m / z calculated for C 51 1 65 19F5N6O 18 1S [M+Na] + : 1199.3996, found: 1199.3886.

[0154] Example 12

[0155] Using lung cancer cells H1299 and liver cancer cells HepG2 as test cells, the in vitro antiproliferative activity of compounds against H1299 and HepG2 cells was determined using the CCK-8 assay. An appropriate amount of cells in the logarithmic growth phase were resuspended in culture medium, inverted 10 times to mix, and seeded at a density of 2000 cells / well in 96-well plates (100 μL of cell suspension per well). The plates were incubated overnight at 37°C with 5% CO2. The next day, 100 μL of different concentrations of the test compound prepared with culture medium were added to each well (with a blank control). After incubation for 96 hours at 37°C with 5% CO2, 10 μL of CCK-8 was added to each well, and incubation continued for 2 hours. The OD450 absorbance was read using a microplate reader, and the experiment was repeated three times. Data were analyzed using Graphpad Prism software to determine the IC50. 50 Table 1 shows the IC50 values ​​of the compounds disclosed in this invention for inhibiting the proliferation of H1299 and HepG2 cells. 50 .

[0156] Table 1

[0157]

[0158] The results in Table 1 show that the PROTAC molecules prepared in Examples 2-11 have different linker chains. PROTAC molecules with shorter linker chains exhibit better inhibitory effects on tumor cell proliferation than those with longer linker chains. It is speculated that this is because the different linker chain lengths result in different spatial structures for the PROTAC molecules. These different spatial structures can form a more suitable spatial relationship with the STAT3 protein, thus producing better adaptability and influencing the formation of the "target protein-PROTAC-E3 ubiquitin ligase" ternary complex, ultimately affecting the selectivity of the molecule for the target protein and ligand.

[0159] Taking the PROTAC molecule S3D1 prepared in Example 2 as an example, the degradation effect of the compound on STAT3 protein in H1299 and HepG2 cells was investigated by Western blot. Experimental method: H1299 and HepG2 cells in logarithmic growth phase were treated with different concentrations of the compound (DMSO as solvent control) and cultured at 37°C and 5% CO2 for 24 h; or H1299 and HepG2 cells in logarithmic growth phase were treated with 5 μM of the compound and cultured at 37°C and 5% CO2 for 0 h, 3 h, 6 h, 12 h, 24 h, 30 h, 36 h, and 48 h, respectively. Cells were lysed with RIPA lysis buffer, proteins were collected, and sonicated. The expression of STAT3 protein in the cells was detected by Western blot.

[0160] like Figure 1 and Figure 2As shown, the results indicate that the PROTAC molecule S3D1 prepared in the embodiments of the present invention can significantly degrade STAT3 protein in H1299 and HepG2 cells. Figure 1 (a) and Figure 2 The results in (a) indicate that, within a certain concentration range, the higher the concentration of PROTAC molecule S3D1, the more significant the degradation effect on STAT3 protein. Figure 1 As shown in (b), at this concentration, PROTAC molecule S3D1 significantly degraded STAT3 protein in H1299 cells within 3 hours. Figure 2 As shown in (b), the degradation effect of PROTAC molecule S3D1 on STAT3 protein in HepG2 cells gradually increased with time.

[0161] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A PROTAC molecule, characterized in that, The chemical structural formula of the PROTAC molecule is shown below: , where n can be 1, 3, 4 or 5.

2. A method for preparing the PROTAC molecule according to claim 1, characterized in that, Including the following steps: 4-(2-((2,3,4,5,6-pentafluoro-N-tolyl)sulfonamide)acetamido)benzoic acid and compound 11 were added to dichloromethane to obtain a mixed solution. N,N-diisopropylethylamine and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate were added to the mixed solution, and the PROTAC molecule was obtained after the reaction. The chemical structural formula of compound 11 is shown below: In compound 11, n can be 1, 3, 4 or 5.

3. The method for preparing PROTAC molecules according to claim 2, characterized in that, The preparation method of compound 11 includes: N,N-diisopropylethylamine and N,N-dimethylformamide were mixed, and 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione and compound 9 were added. The mixture was heated and stirred until the reaction was complete, yielding compound 10. Compound 10 was dissolved in dichloromethane, and trifluoroacetic acid was added to react and give compound 11. The chemical structural formula of compound 9 is shown below: In compound 9, n can be 1, 3, 4, or 5; The chemical structural formula of compound 10 is shown below: In compound 10, n can be 1, 3, 4 or 5.

4. The use of a PROTAC molecule as described in claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating lung cancer or liver cancer.

5. The use of a PROTAC molecule as described in claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating non-small cell lung cancer.

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