Degradation agent based on benzimidazole fused covalent warhead as well as preparation method and application of degradation agent

Abstract

The invention discloses a degradation agent based on benzimidazole fused covalent warheads as well as a preparation method and application of the degradation agent, and relates to the technical field of drug development. The structural formula of the degradation agent based on the benzimidazole fused covalent warhead is shown in the specification, wherein, is phenyl or substituted phenyl; r is methyl or tertiary butyl; the structure is selected from one of the following structures:,,,,,,,,, and; and a connection site is represented. According to the invention, a series of degradation agents based on benzimidazole fused covalent warheads are obtained by utilizing the modular design of a benzimidazole guiding group and an acrylate covalent warhead and then connecting with a BRD4 protein inhibitor JQ1 through a connexon. The degradation agent based on the benzimidazole fused covalent warhead can target the BRD4 protein and efficiently degrade the BRD4 protein. Therefore, targeted degradation of the BRD4 protein is realized while a protein degradation targeted chimera molecular library is enriched.

Description

Technical Field

[0001] This invention relates to the field of drug development technology, and in particular to a degrading agent based on a benzimidazole fusion covalent warhead, its preparation method, and its application. Background Technology

[0002] Over the past few decades, drug discovery has shifted from phenotypic research to mechanism-based target-selective development, i.e., targeted drug strategies. In recent years, a new targeted drug strategy has emerged: the protein degradation targeting (TPD) strategy. This strategy utilizes the main endogenous pathways of protein and organelle degradation in eukaryotic cells: the lysosomal pathway and the ubiquitin-proteasome system (UPS) pathway. The advent of the TPD strategy has enriched the range of targets for targeted drugs, offering high efficiency and selectivity, while also providing opportunities to address the problems of untreatable proteins and resistance to traditional drugs.

[0003] Protein degradation-targeting chimeras (PROTACs) based on the UPS pathway are a major research focus in TPD strategies, such as... Figure 1 As shown (Ub represents ubiquitin), it consists of three parts: a ligand that recruits E3 ubiquitin ligase (i.e., the E3 ubiquitin ligase ligand), a ligand that specifically binds to the target protein (i.e., the target protein ligand), and a linker connecting the two. The protein degradation-targeting chimera forms a ternary complex by linking a specific E3 ubiquitin ligase to the target protein, facilitating the transfer of the ubiquitin tag, activated and transferred by the three ubiquitinases, from the E3 ubiquitin ligase to the target protein. The multiubiquitinated target protein is then recognized by the proteasome and ultimately degraded, while the protein degradation-targeting chimera is released during this process. Therefore, the protein degradation-targeting chimera exhibits event-driven characteristics; even with weak binding or binding to inactive sites, it can achieve degradation at a catalytic level.

[0004] The field of protein degradation-targeting chimeras has seen continuous progress, now capable of degrading various types of proteins, including endosomes, protein kinases, and transcription factors, and even proteins previously considered undrug-resistant. Facing challenges such as drug resistance, protein degradation-targeting chimeras also offer new solutions and show great promise. However, many potential aspects of protein degradation-targeting chimeras remain to be explored, presenting both opportunities and new challenges. A pressing issue is the limited availability of E3 ubiquitin ligase ligands. Studies have found over 600 types of E3 ubiquitin ligases in the human body, but only 3% are currently used in protein degradation-targeting chimeras. Of these, over 90% rely on E3 ubiquitin ligases CRBN or VHL, but CRBN- or VHL-based protein degradation-targeting chimeras have developed drug resistance.

[0005] Bromodomain-containing protein 4 (BRD4) is a member of the bromodomain and extra-terminal domain (BET) protein family. It can recognize acetylated histones and localize to the promoter or enhancer regions of target genes, initiating and maintaining the expression of tumor-related genes. The BRD4 protein is closely related to the regulation of multiple transcription factors and chromatin modification, and is involved in DNA damage repair and maintenance of telomere function, thus maintaining the survival of tumor cells.

[0006] Therefore, developing new E3 ubiquitin ligase ligands to overcome the above challenges, deeply exploring the potential of proteolysis-targeting chimera technology, continuously enriching the proteolysis-targeting chimera molecular library, and achieving the degradation of target proteins such as BRD4 protein are the research focuses in this field.

[0007] Therefore, the existing technologies still need to be improved and developed. Summary of the Invention

[0008] Based on the deficiencies of the above-mentioned existing technologies, the purpose of the present invention is to provide a degrader based on benzimidazole-fused covalent warheads, its preparation method and application, aiming to enrich the proteolysis-targeting chimera molecular library and simultaneously achieve the targeted degradation of BRD4 protein.

[0009] The technical solution of the present invention is as follows: In the first aspect of the present invention, a degrader based on benzimidazole-fused covalent warheads is provided, wherein the structural formula of the degrader based on benzimidazole-fused covalent warheads is: ; wherein, is phenyl or substituted phenyl; R is methyl or tert-butyl; (linker) is selected from one of the following structures: , , , , , , , , , , , , , , , and ; [[ID=6 1]] represents the connection site (in the structural formulas involved hereinafter, represents the connection site. When appears again hereinafter, its meaning will not be repeated).

[0010] This invention is based on the E3 ubiquitin ligase DCAF16, utilizing a modular design of a benzimidazole directing group and an acrylate covalent warhead, and then combined with the BRD4 protein inhibitor JQ1 ( By coupling with tunable linkers, a series of E3 ubiquitin ligase DCAF16 activity-dependent covalent protein degraders were obtained, namely, degraders based on benzimidazole fusion covalent warheads. The benzimidazole fusion covalent warhead-based degraders provided by this invention can recruit E3 ubiquitin ligase DCAF16 to form a ternary complex with the target protein (i.e., BRD4 protein), inducing BRD4 protein ubiquitination. The ubiquitinated BRD4 protein is then recognized by the proteasome and ultimately degraded. Furthermore, the benzimidazole fusion covalent warhead-based degraders exhibited highly efficient BRD4 protein degradation characteristics in breast cancer, colon cancer, and glioma cells, indicating their potential as cancer therapeutic agents.

[0011] Therefore, the benzimidazole-based covalent warhead degrader provided by this invention can target and degrade the BRD4 protein. This invention enriches the protein degradation targeting chimeric molecule library while achieving targeted degradation of the BRD4 protein.

[0012] Optionally, the substituted phenyl group is an alkoxy-substituted phenyl group (the alkoxy group can be monosubstituted or disubstituted), a fluorine-substituted phenyl group (the fluorine group can be monosubstituted or disubstituted), a chlorine-substituted phenyl group (the chlorine group can be monosubstituted or disubstituted), a bromine-substituted phenyl group (the bromine group can be monosubstituted or disubstituted), or a nitro-substituted phenyl group (the nitro group can be monosubstituted or disubstituted).

[0013] Optionally, Choose from one of the following structures: , , , , , , , and .

[0014] A second aspect of the present invention provides a method for preparing a degrading agent based on a benzimidazole fusion covalent warhead as described above, wherein the method for preparing the degrading agent based on a benzimidazole fusion covalent warhead includes the following steps: Will (among them) and The structural formula of the degradation agent based on benzimidazole fused covalent warhead mentioned above is related to... and Same) and (R is methyl or tert-butyl) After the reaction, the degrading agent based on the benzimidazole fused covalent warhead is obtained; Boc represents tert-butyloxycarbonyl.

[0015] Optionally, The preparation method includes the following steps: Will and (among them) and The structural formula of the degradation agent based on benzimidazole fused covalent warhead mentioned above is related to... and After reacting with the same substance, we obtain... .

[0016] A third aspect of the present invention provides the use of the benzimidazole-based degrading agent described above in the preparation of a medicament for treating breast cancer.

[0017] Optionally, the breast cancer is triple-negative breast cancer.

[0018] In a fourth aspect, the present invention provides the use of the benzimidazole-based degrading agent described above in the preparation of a medicament for treating colon cancer.

[0019] A fifth aspect of the present invention provides the use of the benzimidazole-based degrading agent described above in the preparation of a medicament for treating glioma.

[0020] Optionally, the medicament includes a degrading agent based on a benzimidazole fusion covalent warhead, and the medicament further includes a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable additive.

[0021] Specifically, the pharmaceutically acceptable carrier includes at least one of glidants, diluents, wetting agents, suspending agents, solvents, and emulsifiers; the pharmaceutically acceptable adjuvants include at least one of preservatives, colorants, flavoring agents, stabilizers, and isotonic agents.

[0022] Beneficial Effects: This invention utilizes a modular design of a benzimidazole directing group and an acrylate covalent warhead, which is then linked to the BRD4 protein inhibitor JQ1 via a linker, resulting in a series of degradative agents based on benzimidazole fusion covalent warheads. These degradative agents can target and efficiently degrade the BRD4 protein. Therefore, this invention enriches the library of protein degradation-targeting chimeric molecules while achieving targeted degradation of the BRD4 protein. Attached Figure Description

[0023] Figure 1This is a schematic diagram illustrating the mechanism of target protein degradation by a protein degradation-targeting chimera in existing technologies.

[0024] Figure 2 The graph shows the activity test results of each compound in Examples 2 and 4.

[0025] Figure 3 The figures show the degradation activity of compound LGF327 on BET family proteins. (a) shows the degradation effect of compound LGF327 on BET family proteins, (b) shows the semi-quantitative results of compound LGF327's degradation of BET family proteins, and (c) shows the DC degradation of BRD4 protein by compound LGF327. 50 Result image.

[0026] Figure 4 The figures show the degradation results of compound LGF327 on BRD4 protein in different cancer cells. (a) shows the degradation effect of compound LGF327 on BRD4 protein in MDA-MB-231 cells at different time points. (b) shows the degradation effect of compound LGF327 on BRD4 protein in MDA-MB-231 cells after 12 h of culture and washing, followed by culture in drug-free medium for different time points. (c) shows the degradation effect of compound HL345 on BRD4 protein in different cancer cells. (d) shows the degradation effect of compound LGF327 on BRD4 protein in different cancer cells.

[0027] Figure 5 Figures show the results of the degradation mechanism verification of compound LGF327, where (a) is the degradation mechanism result of compound LGF327 under high content analysis microscopy, and (b) is the semi-quantitative result of the degradation mechanism verification of LGF327 under high content analysis microscopy.

[0028] Figure 6 The figures show the results of the lysosomal pathway inhibition experiment of compound LGF327, where (a) is the effect of the lysosomal pathway inhibition experiment of compound LGF327, and (b) is the semi-quantitative result of the lysosomal pathway inhibition experiment of compound LGF327.

[0029] Figure 7 The figures show the results of the proteasome pathway inhibition experiment of compound LGF327, where (a) is the effect of the proteasome pathway inhibition experiment of compound LGF327, and (b) is the semi-quantitative result of the proteasome pathway inhibition experiment of compound LGF327. Detailed Implementation

[0030] This invention provides a degrading agent based on a benzimidazole fused covalent warhead, its preparation method, and its application. 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 only for explaining the invention and are not intended to limit the invention.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0032] The present invention will be further described below through specific embodiments.

[0033] In the following embodiments and synthetic routes, the meanings of some symbols are as follows: HBTU: Benzotriazole-N,N,N',N'-Tetramethylurea hexafluorophosphate; DCM: Dichloromethane; DIPEA: N,N-diisopropylethylamine; DBU: 1,8-diazabicyclo(5.4.0)undec-7-ene; AcOH: Acetic acid; HOBt: 1-Hydroxybenzotriazole; DMAP: 4-Dimethylaminopyridine; DABCO: 1,4-diazabicyclo[2.2.2]octane; equiv.: equivalent; TFA: Trifluoroacetic acid; rt: room temperature; 5% citric acid aqueous solution: This means that the citric acid content in the citric acid aqueous solution is 5% by mass. Fmoc: fluorenemethyloxycarbonyl; tBu: tert-butyl.

[0034] Example 1 In this embodiment, compounds 3a, 3b, 3c and 3d were first synthesized, and their structural formulas are shown in Table 1.

[0035] Table 1. Structural formulas and mScarlet / EGFP ratios of compounds 3a to 3d

[0036] High-content cell fluorescence imaging analysis revealed (see Table 1) that compound 3a could not mediate the targeted degradation of BRD4 protein. However, when it was linked to the covalent warhead MBH ester (such as compounds 3b and 3c), the ratio of fluorescence signal intensity related to BRD4 protein degradation showed a significant decreasing trend, indicating that compounds 3b and 3c have the function of degrading BRD4 protein. The experimental results suggest that the strategy of using benzimidazole as a guiding group to perform molecular chimera with the MBH ester covalent warhead may effectively enhance the targeted recruitment ability of the protein degradation chimeric molecule by strengthening the synergistic recognition between the target protein and the E3 ubiquitin ligase.

[0037] By quantitatively analyzing the change in the fluorescence intensity ratio of mScarlet (a red fluorescent protein, serving as the target signal) to EGFP (a green fluorescent protein, serving as the internal control signal) using a high-content imaging system, the efficacy of the benzimidazole-based fusion covalent warhead-induced target protein degradation can be objectively characterized. The smaller the mScarlet to EGFP ratio, the stronger the ability of the benzimidazole-based fusion covalent warhead-induced degradation agent to degrade BRD4 protein (the principle is explained below).

[0038] Next, based on the skeletal structure of lead compound 3c, this invention designs and synthesizes a series of degrading agents based on benzimidazole fusion covalent warheads by introducing substituents (mono- or di-substituents) at different positions of the benzene ring (see below), and provides a method for preparing compound 3b.

[0039] In the following examples, the structural formulas of compounds 3c, 3ca to 3ch are as follows: ;in, The specific structure is shown in Table 2.

[0040] Table 2, Structural Formulas of Compounds 3c, 3ca to 3ch Specific structure

[0041] The structural formulas of compounds 4c to 19c are as follows: ;in, The specific structure is shown in Table 3.

[0042] Table 3 Specific structure

[0043] Example 2 Synthesis of compounds 3c, 3ca to 3ch The synthetic route is as follows: .

[0044] In this synthetic route, the structures of the final products 3c, 3ca, and 3ch are shown in the following diagrams. The specific structure is shown in Table 2.

[0045] In the preparation of compounds 3c, 3ca, and 3ch, the structures of compounds a, c, d, e, and 1 in the above synthetic route are... The specific structures are respectively related to the structural formulas of compounds 3c, 3ca to 3ch prepared accordingly. The specific structures are the same. For example, the 3C structure of compound... for Then, the corresponding compounds a, c, d, e, and 1 used to prepare compound 3c have the following structures: Also for The structural formula of compound 3ca for Then, the corresponding compounds a, c, d, e, and 1 used to prepare compound 3c have the following structures: Also for The preparation of the remaining compounds follows the same procedure.

[0046] Synthesis of compound 3c: (1) Compound b (1.0 mmol, 1 equiv.), HBTU (1.25 mmol, 1.25 equiv.), and DCM (30 mL) were added to a 50 mL round-bottom flask. Then, DIPEA (2.0 mmol, 2 equiv.) was slowly added under ice bath (0 °C). After stirring for 10 minutes, compound a (1.05 mmol, 1.05 equiv.) was slowly added to the reaction system. The reaction was maintained under ice bath conditions for 30 minutes, and then gradually raised to room temperature and stirred for 16 hours. After the reaction was completed, the mixture was extracted with DCM (30 mL × 3). The combined organic phases were washed successively with 5% citric acid aqueous solution (30 mL), saturated sodium bicarbonate solution (30 mL), and saturated sodium chloride solution (40 mL × 2). The organic layer was dehydrated with anhydrous sodium sulfate, concentrated by rotary evaporation under reduced pressure, and the residue was separated by silica gel column chromatography (petroleum ether / ethyl acetate gradient elution) to finally obtain compound c (0.39 g, yield 98%).

[0047] (2) The compound c (0.39 g) obtained in the previous step was dissolved in 20 mL of acetic acid and stirred at 80 °C for 10 hours. After the reaction was completed, the acetic acid was removed by vacuum distillation, and the residue was added to 30 mL of water and extracted with DCM (20 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the mixture was purified by silica gel column chromatography (petroleum ether / ethyl acetate gradient elution) to obtain compound d (0.36 g, yield 95%).

[0048] (3) Add compound d (0.5 mmol, 1 equiv.) and DCM (10 mL) to a 25 mL round-bottom flask, and then slowly add DBU (0.5 mmol, 1 equiv.) through a syringe. Stir the reaction at room temperature for 10 minutes. After the reaction is complete, concentrate the solvent under reduced pressure, and purify the residue by rapid column chromatography to obtain compound e (0.077 g, yield 95%).

[0049] (4) Compound f (0.1 mmol, 1 equiv.), HBTU (0.12 mmol, 1.2 equiv.), HOBt (0.12 mmol, 1.2 equiv.), and DCM (10 mL) were added to a 25 mL round-bottom flask. Then, DIPEA (0.2 mmol, 2 equiv.) was added using a syringe. After stirring for 10 minutes, compound e (0.12 mmol, 1.2 equiv.) was added, and the mixture was stirred overnight (12 h). After the reaction was complete, the mixture was extracted with DCM and then washed successively with 5% citric acid aqueous solution (30 mL), saturated sodium bicarbonate solution (30 mL), and saturated saline solution (20 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by rapid column chromatography to obtain compound 1 (0.05 g, yield 95%).

[0050] (5) Dissolve (5.0 mmol, 1 equiv.) in DCM (40 mL), then slowly add (5.25 mmol, 1.05 equiv.) was added, followed by a single addition of DMAP (0.25 mmol, 0.05 equiv.), and the mixture was stirred overnight (12 h) at room temperature. The reaction progress was monitored and confirmed by thin-layer chromatography (TLC). After the reaction was complete, the mixture was extracted with water and DCM, and the combined organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by rapid column chromatography to give a colorless oil, namely compound 2 (1.03 g, yield 95%).

[0051] (6) Take a dry 10 mL glass tube and add compound 2 (0.15 mmol, 1.5 equiv.), compound 1 (0.1 mmol, 1 equiv.), and DABCO (0.02 mmol, 0.2 equiv.) in sequence. Then inject DCM (1 mL) and stir at room temperature for 1 hour. The reaction process was monitored by TLC. After the reaction was completed, the solvent was removed under reduced pressure. The residue was purified by rapid column chromatography to obtain compound 3c (54.6 mg, yield 85%).

[0052] The proton NMR spectrum data of compound 3c are as follows: 1 H NMR (400MHz, CDCl3) δ 7.70 (dd, J = 7.4, 4.0Hz, 1H), 7.68–7.60 (m, 1H), 7.38–7.31 (m, 2H), 7.25 (s, 2H), 7.23 (s, 3H), 6.25 (s, 1H), 5.10 (s, 1H), 4.97 (s, 2H), 4.62 (t, J = 6.8Hz, 1H), 3.96–3.84 (m, 2H), 3.79 (s, 3H), 3.49 (dd, J = 14.8, 6.5Hz, 1H), 3.38 (dd, J = 14.8, 7.2Hz, 1H), 3.06 (t, J = 6.3 Hz, 2H), 2.61 (s, 3H), 2.39 (s, 3H), 1.65 (s, 3H). The carbon NMR data of compound 3c are: 13 C10 NMR (101 MHz, CDCl3) δ 170.7, 165.5, 155.6, 153.0, 136.7, 136.4, 134.8, 134.6, 132.0, 130.84, 130.75, 129.8, 128.6, 126.3, 122.7, 122.3, 119.3, 109.5, 54.2, 52.3, 43.9, 38.9, 36.7, 29.6, 27.0, 14.3, 13.0, 11.7. High-resolution mass spectrometry data for compound 3c are HRMS (ESI) m / z: [M+H] + calcd for C 33 H 33 O3N7ClS + : 642.2049; found: 642.2028; Purity: 95% (calcd for indicates theoretical calculation result; found indicates actual analysis result; purity indicates purity. The meanings of calcd for, found, and purity in the following text are the same as here).

[0053] Synthesis of compound 3ca: Following the synthetic route of this embodiment, and referring to the synthetic method of compound 3c, compound 3ca (24.1 mg, yield 35.8%) was obtained. Its 1H NMR spectral data are as follows: 1 H NMR (500MHz, CDCl3) δ 7.40–7.32 (m, 1H), 7.27–7.22 (m, 2H), 7.15 (d, J = 2.6Hz, 2H), 7.14–7.13 (m, 1H), 7.13–7.09 (m, 1H), 6.79–6.74 (m, 1H), 6.15 (d, J = 1.6Hz, 1H), 5.00–4.95 (m, 1H), 4.83–4.78 (m, 2H), 4.53–4.49 (m, 1H), 3.73 (d, J =7.2Hz, 3H), 3.69 (d, J = 8.8Hz, 3H), 3.40–3.27 (m, 2H), 2.95 (s, 2H), 2.89 (q, J = 6.6Hz, 2H), 2.51 (d, J = 2.6 Hz, 3H), 2.28 (s, 3H), 1.57–1.51 (m, 3H). Its carbon NMR data are as follows: 13 C10 NMR (126 MHz, CDCl3) δ 170.6, 156.6, 155.6, 152.0, 136.7, 132.1, 130.9, 130.7, 128.6, 119.9, 111.1, 109.9, 101.9, 93.5, 55.9, 55.8, 54.2, 52.31, 52.29, 45.2, 39.1, 28.3, 27.0, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+H] + calcd for C 34 H 35 O4N7ClS + :672.2141; found: 672.2154; Purity: 95.3%.

[0054] Synthesis of compound 3cb: Following the synthetic route of this embodiment, and referring to the synthetic method of compound 3c, compound 3cb (48.4 mg, 73.3%) was obtained. Its 1H NMR spectral data are as follows: 1 H NMR (500MHz, CDCl3) δ 7.36 (d, J= 6.3Hz, 1H), 7.26 (d, J =2.0Hz, 1H), 7.25 (d, J = 1.9Hz, 1H), 7.17 (d, J = 2.6Hz, 1H), 7.16 (d, J = 2.6Hz, 1H), 7.14 (s, 1H), 6.90–6.85 (m, 1H), 6.20–6.16 (m, 1H), 5.05 (d, J = 5.5Hz, 1H), 4.83 (d, J = 15.6Hz, 2H), 4.51–4.46 (m, 1H), 3.85–3.71 (m, 2H), 3.68 (d, J = 2.5Hz, 3H), 3.38–3.20 (m, 2H), 2.93 (d, J =6.2Hz, 2H), 2.50 (d, J = 2.5 Hz, 3H), 2.28 (s, 3H), 1.55 (s, 3H). Its carbon NMR data are: 13 CNMR (126 MHz, CDCl3) δ 170.7, 163.8, 155.6, 149.8, 136.8, 136.5, 134.5, 134.4, 132.1, 131.2, 130.9, 130.4, 129.8, 128.7, 126.7, 120.0, 96.7, 54.2, 52.4, 44.2, 39.1, 36.5, 27.1, 27.0, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+H] + calcd forC 33 H 31 O3N7Cl3S + :660.1923; found: 660.1954; Purity: 99%.

[0055] Synthesis of compound 3cc: Following the synthetic route of this embodiment and referring to the synthetic method of compound 3c, compound 3cc (45.1 mg, yield 66.77%) was obtained. Its 1H NMR data are as follows: 1H NMR (400MHz, CDCl3) δ 7.69–7.59 (m, 1H), 7.58–7.52 (m, 1H), 7.42–7.34 (m, 2H), 7.31–7.27 (m , 2H), 7.23–7.19 (m, 1H), 7.19–7.09 (m, 1H), 6.32–6.25 (m, 1H), 5.13 (t, J = 1.9Hz, 1H), 5.00–4.90 (m, 2H), 4.60 (q, J = 6.8Hz, 1H), 3.99–3.82 (m, 2H), 3.80 (d, J = 5.3Hz, 3H), 3.56–3.31 (m, 2H), 3.05 (q, J = 4.7, 3.1Hz, 2H), 2.61 (d, J = 3.7 Hz, 3H), 2.40 (s, 3H), 1.66 (s, 3H). Its carbon NMR data are: 13 C10 NMR (126 MHz, CDCl3) δ 170.7, 165.3, 163.8, 155.5, 154.0, 149.7, 136.7, 136.4, 135.4, 134.3, 130.81, 130.76, 129.8, 128.7, 128.6, 126.7, 123.0, 120.0, 119.0, 110.4, 109.8, 52.3, 44.1, 38.9, 36.6, 27.1, 27.0, 14.3, 13.0, 11.7. Its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+H] + calcd forC 33 H 32 O3N7Cl2S + :676.1659; found: 676.1643; Purity: 96%.

[0056] Synthesis of compound 3cd: Following the synthetic route of this embodiment, and referring to the synthetic method of compound 3c, compound 3cd (45.3 mg, yield 62.88%) was obtained. Its 1H NMR spectrum data is as follows: 1 H NMR (400MHz, CDCl3) δ 7.73–7.61 (m, 1H), 7.51 (d, J = 8.5Hz, 1H), 7.39 (d, J= 2.9Hz, 1H), 7.38–7.33 (m, 2H), 7.33–7.30 (m, 1H), 7.30–7.25 (m, 2H), 6.32–6.24 (m, 1H), 5.15–5.10 (m, 1H), 4.99–4.89 (m, 2H), 4.59 (q, J = 7.0Hz, 1H), 3.98–3.82 (m, 2H), 3.80 (d, J = 6.3Hz, 3H), 3.54–3.31 (m, 2H), 3.04 (q, J = 6.0Hz, 2H), 2.61 (d, J =4.8 Hz, 3H), 2.40 (s, 3H), 1.66 (s, 3H). Its carbon NMR data are: 13 C10 NMR (126 MHz, CDCl3) δ 155.6, 154.2, 153.8, 149.8, 136.8, 136.4, 135.9, 132.1, 130.9, 129.8, 128.7, 128.68, 126.7, 125.7, 122.0, 120.5, 115.9, 112.8, 110.9, 54.2, 52.4, 44.1, 39.0, 36.5, 27.0, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+H] + calcd for C 33 H 32 O3N7BrClS + :720.1154; found: 720.1139; Purity: 96%.

[0057] Synthesis of compound 3ce: Following the synthetic route of this embodiment, and referring to the synthetic method of compound 3c, compound 3ce (55.8 mg, yield 78.25%) was obtained. Its 1H NMR data (500 MHz, CDCl3) were δ 7.69–7.63 (m, 1H), 7.60 (s, 1H), 7.38 (d, 1H). J = 8.2Hz, 2H), 7.32 (d, J = 6.1Hz, 2H), 6.30 (s, 1H), 5.19 (s, 1H), 4.92 (s, 2H), 4.55 (t, J= 6.7Hz, 1H), 3.95–3.82 (m, 2H), 3.80 (s, 3H), 3.52–3.31 (m, 2H), 3.11–2.98 (m, 2H), 2.60 (s, 3H), 2.41 (s, 3H), 1.67 (s, 3H). Its carbon NMR data (126 MHz, CDCl3) are: δ 170.7, 165.2, 163.8, 155.5, 155.3, 141.6, 136.8, 136.4, 134.3, 134.1, 132.1, 130.8, 130.3, 129.8, 128.7, 126.8, 120.4, 111.0, 54.2, 52.4, 44.3, 38.9, 36.5, 29.6, 27.1, 14.3, 13.1, 11.7. Its high-resolution mass spectrometry data are: HRMS (ESI) m / z: [M+H] + calcd for C 33 H 31 O3N7Cl3S + :710.1269; found: 710.1255; Purity: 97%.

[0058] Synthesis of compound 3cf: Following the synthetic route of this embodiment, and referring to the synthetic method of compound 3c, compound 3cf (71.8 mg, yield 90%) was obtained. Its 1H NMR spectrum data is as follows: 1 H NMR (400MHz, CDCl3) δ 7.77 (s, 1H), 7.65 (t, J = 6.1Hz, 1H), 7.49 (s, 1H), 7.38 (d, J = 8.5Hz, 2H), 7.31 (d, J = 8.7Hz, 2H), 6.38–6.20 (m, 1H), 5.17 (d, J = 1.8Hz, 1H), 5.02–4.84 (m, 2H), 4.53 (t, J = 6.6Hz, 1H), 3.98–3.86 (m, 1H), 3.79 (s, 4H), 3.55–3.42 (m, 1H), 3.30 (dd, J = 14.7, 6.2 Hz, 1H), 3.14–2.89 (m, 2H), 2.58 (s, 3H), 2.40 (s, 3H), 1.66 (s, 3H). Its carbon NMR data are: 13C10 NMR (101 MHz, CDCl3) δ 170.8, 165.3, 163.8, 155.5, 155.3, 149.8, 142.6, 136.9, 136.4, 135.1, 134.3, 132.1, 130.9, 130.8, 130.3, 129.8, 128.7, 126.9, 123.7, 117.7, 117.3, 114.2, 54.2, 52.4, 44.3, 38.9, 36.4, 29.7, 27.1, 22.7, 14.4, 13.1, 11.7. Its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+H] + calcd forC 33 H 31 O3N7Br2ClS + :798.0259; found: 798.0228; Purity: 97%.

[0059] Synthesis of compound 3cg: Following the synthetic route of this embodiment, and referring to the synthetic method of compound 3c, compound 3cg (50 mg, yield 73.73%) was obtained. Its 1H NMR spectral data are as follows: 1 H NMR (400MHz, CDCl3) δ 7.58 (d, J = 5.8Hz, 1H), 7.40 (d, J = 7.1Hz, 1H), 7.38 (q, J = 2.7Hz, 2H), 7.30 (d, J = 8.6Hz, 2H), 7.06–6.99 (m, 1H), 6.30 (d, J = 1.6Hz, 1H), 5.19 (d, J = 1.8Hz, 1H), 4.92 (s, 2H), 4.59 (d, J = 6.7Hz, 1H), 3.98–3.81 (m, 2H), 3.80 (s, 3H), 3.53–3.30 (m, 2H), 3.04 (q, J = 6.8 Hz, 2H), 2.62 (s, 3H), 2.40 (s, 3H), 1.67 (s, 3H). Its carbon NMR data are: 13C10 NMR (126 MHz, CDCl3) δ 170.7, 165.3, 163.8, 155.5, 154.6, 149.8, 136.8, 136.4, 134.3, 132.1, 130.9, 130.4, 129.8, 128.7, 126.7, 106.8, 106.7, 97.9, 97.7, 54.2, 52.4, 44.3, 39.0, 36.6, 29.6, 27.2, 14.3, 13.0, 11.7. Its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+H] + calcd for C 33 H 31 O3N7ClF2S + :678.1860; found: 678.1847; Purity: 95.5%.

[0060] Synthesis of compound 3ch: Following the synthetic route of this embodiment, and referring to the synthetic method of compound 3c, compound 3ch (24 mg, yield 34.99%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (600MHz, CDCl3) δ 8.02–7.97 (m, 1H), 7.84–7.79 (m, 1H), 7.41 (d, J = 8.3Hz, 2H), 7.38 (s, 1H), 7.33 (d, J = 8.2Hz, 2H), 7.30 (s, 1H), 6.74 (d, J = 9.1Hz, 1H), 6.28 (d, J = 7.7Hz, 1H), 4.66–4.57 (m, 2H), 4.48–4.41 (m, 1H), 3.69 (d, J =10.2Hz, 3H), 3.57–3.50 (m, 2H), 3.32–3.20 (m, 2H), 2.69 (d, J = 5.3 Hz, 3H), 2.40 (s, 3H), 2.29–2.23 (m, 2H), 1.67 (s, 3H). Its carbon NMR data are: 13C10 NMR (126 MHz, CDCl3) δ 172.3, 170.0, 166.6, 155.5, 136.9, 134.8, 131.0, 130.9, 130.4, 129.8, 129.3, 128.7, 126.1, 124.2, 114.8, 54.3, 54.0, 53.4, 52.2, 38.4, 34.6, 34.2, 29.6, 22.6, 14.3, 14.14, 14.07, 13.1, 11.7. Its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+H] + calcd for C 33 H 32 O5N8ClS + :687.1899; found: 687.1885; Purity: 99%.

[0061] Example 3 Synthesis of compound 3b Refer to the synthesis method of compound 3c in Example 2, and replace compound g with... (R is tBu), after the reaction, compound 3b (52 mg, yield 76%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (400MHz, CDCl3) δ 7.67–7.59 (m, 1H), 7.46 (d, J = 5.3Hz, 1H), 7.31–7.25 (m, 2H), 7.18 (d, J = 2.0Hz, 1H), 7.18–7.16 (m, 3H), 7.15 (d, J = 2.3Hz, 1H), 6.05 (d, J = 1.4Hz, 1H), 4.84 (d, J = 1.9Hz, 3H), 4.59–4.52 (m, 1H), 3.91–3.76 (m, 2H), 3.44–3.38 (m, 1H), 3.36–3.29 (m, 1H), 2.96 (t, J =6.1 Hz, 2H), 2.54 (s, 3H), 2.31 (s, 3H), 1.58 (s, 3H), 1.43 (s, 9H). Its carbon NMR data are: 13CNMR (101 MHz, CDCl3) δ 170.7, 164.4, 163.8, 155.7, 153.0, 149.8, 142.3, 136.7, 136.2, 132.2, 130.9, 130.7, 129.8, 128.7, 124.8, 122.7, 122.3, 119.4, 109.6, 82.1, 54.2, 43.8, 39.1, 36.5, 29.7, 28.1, 27.0, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+H] + calcd for C 36 H 39 O3N7ClS + :684.2518; found: 684.2516; Purity: 95%.

[0062] Example 4: Synthesis of compounds 4c to 8c and compound 14c Synthesis of compound 4c: Referring to steps (4) to (6) of the synthesis of compound 3c in Example 2, replace compound e with (R) 1 = (obtained directly by purchase), after the reaction, compound 4c (55 mg, yield 87.5%) was obtained. Its 1H NMR spectrum data is as follows: 1 H NMR (600MHz, CDCl3) δ 7.73 (d, J = 7.3Hz, 1H), 7.60 (s, 1H), 7.37 (d, J =8.1Hz, 2H), 7.32–7.27 (m, 2H), 7.23 (d, J = 8.3Hz, 2H), 6.26 (s, 1H), 5.15 (d, J = 3.6Hz, 1H), 5.10 (d, J = 18.1Hz, 1H), 5.02 (d, J = 18.4Hz, 1H), 4.84–4.76 (m, 1H), 4.73–4.67 (m, 1H), 4.64 (t, J = 6.9 Hz, 1H), 3.78 (s, 3H), 3.72–3.63 (m, 1H), 3.59–3.53 (m, 1H), 3.53–3.47 (m, 1H), 2.65 (s, 3H), 2.40 (s, 3H), 1.66 (s, 3H). Its carbon NMR data are: 13C10 NMR (101 MHz, CDCl3) δ 170.8, 165.4, 164.1, 155.5, 151.2, 150.0, 141.7, 139.2, 136.7, 136.4, 135.0, 134.7, 132.2, 130.9, 130.8, 130.4, 129.9, 128.6, 126.5, 124.0, 123.3, 122.7, 119.5, 110.1, 54.3, 53.5, 52.3, 44.3, 38.7, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+H] + calcdfor C 32 H 31 O3N7ClS + :628.1892; found: 628.1880; Purity: 99.7%.

[0063] Synthesis of compound 5c: Referring to steps (1) to (6) of the synthesis of compound 3c in Example 2, replace compound b with After the reaction, compound 5c (45.5 mg, yield 69.3%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (400MHz, CDCl3) δ 7.72–7.64 (m, 1H), 7.33 (s, 1H), 7.30 (d, J = 8.4Hz, 2H), 7.22 (d, J = 8.4Hz, 2H), 7.20 (s, 1H), 7.17–7.14 (m, 2H), 6.21 (s, 1H), 5.06 (s, 1H), 4.92 (s, 2H), 4.57 (t, J = 6.9 Hz, 1H), 3.74 (s, 3H), 3.52–3.37 (m, 2H), 3.35–3.23 (m, 2H), 2.94–2.83 (m, 2H), 2.58 (s, 3H), 2.33 (s, 3H), 2.12–2.04 (m, 2H), 1.59 (s, 3H). Its carbon NMR data are as follows: 13CNMR (126 MHz, CDCl3) δ 170.6, 165.6, 163.9, 154.4, 149.9, 141.9, 136.7, 136.5, 134.7, 132.1, 130.9, 130.8, 130.4, 129.8, 128.7, 126.3, 122.6, 122.4, 119.1, 109.5, 54.4, 52.3, 43.9, 39.2, 38.9, 29.6, 26.7, 24.4, 14.3, 13.0, 11.8. Its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+H]. + calcd for C 34 H 35 O3N7ClS + :656.2205; found: 656.2205; Purity: 97%.

[0064] Synthesis of compound 6c: Referring to steps (1) to (6) of the synthesis of compound 3c in Example 2, replace compound b with After the reaction, compound 6c (32.5 mg, yield 48.5%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (500MHz, CDCl3) δ 7.71–7.67 (m, 1H), 7.33 (d, J = 2.0Hz, 1H), 7.32 (d, J = 2.0Hz, 1H), 7.24 (s, 1H), 7.22 (d, J = 1.5Hz, 1H), 7.19 (s, 2H), 7.18–7.16 (m, 1H), 7.16 (d, J =1.8Hz, 1H), 6.93 (t, J = 5.3Hz, 1H), 6.21 (s, 1H), 5.00 (s, 1H), 4.93 (d, J = 1.9Hz, 2H), 4.58–4.54 (m, 1H), 3.77 (s, 3H), 3.32–3.24 (m, 3H), 2.82–2.79 (m, 2H), 2.57 (s, 3H), 2.32 (s, 3H), 1.97 (d, J = 7.2Hz, 2H), 1.88 (d, J = 7.6 Hz, 2H), 1.66–1.61 (m, 2H), 1.59 (s, 3H). Its carbon NMR data are: 13C10 NMR (126 MHz, CDCl3) δ 170.5, 165.6, 154.7, 149.9, 136.8, 136.5, 134.6, 130.9, 130.8, 129.8, 128.7, 126.2, 122.7, 122.5, 119.1, 109.5, 54.5, 52.4, 43.9, 39.3, 39.1, 31.9, 29.7, 28.9, 26.5, 24.4, 22.7, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+H] + calcd for C 35 H 37 O3N7ClS + :670.2360; found: 670.2362; Purity: 96%.

[0065] Synthesis of compound 7c: Referring to steps (1) to (6) of the synthesis of compound 3c in Example 2, replace compound b with After the reaction, compound 7c (14.7 mg, yield 21.5%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (500MHz, CDCl3) δ 7.70–7.65 (m, 1H), 7.33 (d, J = 8.3Hz, 2H), 7.25 (d, J =8.6Hz, 2H), 7.19–7.16 (m, 1H), 7.16–7.14 (m, 1H), 7.14 (d, J = 2.0Hz, 1H), 6.74 (q, J =5.5Hz, 1H), 6.20 (d, J = 1.7Hz, 1H), 4.96 (d, J = 1.9Hz, 1H), 4.92 (d, J = 1.8Hz, 2H), 4.57–4.53 (m, 1H), 3.77 (s, 3H), 3.51–3.45 (m, 1H), 3.29–3.25 (m, 1H), 3.25–3.16 (m, 2H), 2.88 (s, 2H), 2.81 (s, 2H), 2.76 (t, J = 7.7 Hz, 2H), 2.59 (s, 3H), 2.33 (s, 3H), 1.88–1.80 (m, 2H), 1.60 (s, 3H). Its carbon NMR data are: 13C10 NMR (126 MHz, CDCl3) δ 170.4, 163.9, 162.6, 155.6, 155.0, 149.9, 142.2, 136.8, 136.6, 134.7, 132.1, 129.8, 128.7, 126.1, 122.5, 122.3, 119.2, 109.4, 54.5, 52.4, 43.8, 39.4, 36.5, 31.5, 29.7, 29.1, 27.0, 27.0, 26.7, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+H] + calcd for C 36 H 39 O3N7ClS + :684.2515; found: 684.2518; Purity: 98%.

[0066] Synthesis of compound 8c: Referring to steps (1) to (6) of the synthesis of compound 3c in Example 2, replace compound b with Compound 8c (6 mg, yield 8.6%) was obtained. Its 1H NMR spectrum data are as follows: 1 HNMR (500MHz, CDCl3) δ 7.78 (d, J = 7.0Hz, 1H), 7.33 (d, J = 8.2Hz, 2H), 7.25 (d, J = 8.3Hz, 2H), 7.22 (s, 1H), 7.20 (d, J = 4.3Hz, 2H), 6.77 (s, 1H), 6.26 (s, 1H), 5.11 (s, 1H), 4.99 (s, 2H), 4.57–4.53 (m, 1H), 3.77 (s, 3H), 3.55–3.34 (m, 2H), 3.21 (q, J = 7.2Hz, 2H), 2.93 (s, 2H), 2.57 (s, 3H), 2.32 (s, 3H), 1.84 (t, J = 7.5Hz, 2H), 1.60 (s, 3H), 1.49 (t, J = 7.1 Hz, 2H), 1.26 s, 2H. Its carbon NMR data are: 13C10 NMR (151 MHz, CDCl3) δ 170.4, 155.6, 155.1, 149.8, 136.8, 136.6, 134.6, 130.9, 130.8, 129.8, 128.7, 126.2, 122.8, 119.0, 109.5, 54.5, 53.4, 52.4, 43.9, 39.5, 39.4, 29.7, 29.6, 29.2, 28.8, 27.3, 26.3, 14.4, 13.1, 11.8; its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+H] + calcd for C 37 H 41 O3N7ClS + :698.2667; found: 698.2675; Purity: 96.5%.

[0067] Synthesis of compound 14c: Referring to steps (4) to (6) of the synthesis of compound 3c in Example 2, replace compound e with (R) 1 = (obtained directly by purchase), after the reaction, compound 14c (64.1 mg, yield 94%) was obtained. Its 1H NMR spectrum data is as follows: 1 H NMR (600MHz, CDCl3) δ 7.86–7.78 (m, 1H), 7.50 (d, J = 8.1Hz, 1H), 7.45 (d, J = 8.3Hz, 1H), 7.42–7.39 (m, 1H), 7.37 (d, J = 8.2Hz, 1H), 7.34–7.30 (m, 1H), 7.29–7.25 (m, 2H), 6.34 (s, 1H), 5.12–5.06 (m, 3H), 4.88–4.77 (m, 2H), 4.53–4.43 (m, 1H), 3.93–3.88 (m, 3H), 3.83–3.77 (m, 1H), 3.68–3 .61 (m, 1H), 3.46–3.36 (m, 1H), 3.17–3.09 (m, 1H), 2.94–2.85 (m, 1H), 2.75–2.68 (m, 3H), 2.44 (s, 3H), 2.31–2.22 (m, 1H), 2.14–2.06 (m, 2H), 2.05–1.98 (m, 1H), 1.73 (d, J = 10.3 Hz, 3H). Its carbon NMR data are: 13C10 NMR (151 MHz, CDCl3) δ 168.8, 165.6, 163.7, 156.9, 156.0, 149.8, 142.6, 139.3, 136.8, 136.6, 135.1, 134.7, 132.2, 131.0, 130.6, 130.0, 128.7, 126.0, 122.8, 122.4, 119.6, 116.7, 114.1, 109.6, 54.6, 54.3, 52.5, 45.7, 43.7, 41.7, 34.2, 31.3, 30.9, 14.4, 13.1, 11.9. Its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+H] + calcd for C 36 H 37 O3N7ClS + :682.2362; found: 682.2347; Purity: 99.9%.

[0068] Example 5: Synthesis of compounds 9c to 13c and compounds 15c to 19c Synthesis Route 1: .

[0069] Synthesis of compounds g1 to g8: Following synthetic route 1 above, compound f (0.1 mmol, 1 equiv.), HBTU (0.12 mmol, 1.2 equiv.), HOBt (0.12 mmol, 1.2 equiv.), and DCM (10 mL) were added to a 25 mL round-bottom flask. Then, DIPEA (0.2 mmol, 2 equiv.) was added using a syringe. After stirring for 10 minutes, the linker precursor A1, namely N-tert-butyloxycarbonyl-1,3-propanediamine, was added. 0.12 mmol (1.2 equiv.) was added and stirred overnight. After the reaction was complete, the mixture was extracted with DCM and washed successively with 5% citric acid aqueous solution (30 mL), saturated sodium bicarbonate solution (30 mL), and saturated saline solution (20 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by rapid column chromatography. The product was then dissolved in anhydrous DCM (5 mL), and an equal volume of TFA was slowly added dropwise to the reaction flask. The reaction was monitored by TLC. When the reaction was complete, the volatile solvent was removed by rotary evaporation under reduced pressure to obtain a white solid, namely compound g1.

[0070] Refer to the synthesis method of compound g1 mentioned above: Replace “connector precursor A1” with “connector precursor A2”. ", to obtain compound g2; Replace “connector precursor A1” with “connector precursor A3”. ", to obtain compound g3; Replace “connector precursor A1” with “connector precursor A4”. ", to obtain compound g4; Replace “connector precursor A1” with “connector precursor A5”. ", to obtain compound g5; Replace “connector precursor A1” with “connector precursor A6”. ", to obtain compound g6; Replace “connector precursor A1” with “connector precursor A7”. ", to obtain compound g7; Replace “connector precursor A1” with “connector precursor A8”. ”, thus obtaining compound g8.

[0071] Synthesis Route 2: .

[0072] In the preparation of compounds 9c to 13c and compounds 15c to 19c, the structure of compound 1a in the above synthetic route is shown in the figure. The specific structures are respectively related to the structures of compounds 9c to 13c and compounds 15c to 19c prepared. The specific structures are the same. For example, the structure of compound 9c is the same. for Then the corresponding structural formula of compound 1a used to prepare compound 9c is... Also for The preparation of the remaining compounds follows the same procedure.

[0073] Synthesis of compound 9c: Following the synthetic route described above, g1 (0.1 mmol, 1 equiv.), HBTU (0.12 mmol, 1.2 equiv.), HOBt (0.12 mmol, 1.2 equiv.), and DCM (10 mL) were added to a 25 mL round-bottom flask. Then, DIPEA (0.2 mmol, 2 equiv.) was added using a syringe. After stirring for 10 minutes, compound j1 (0.12 mmol, 1.2 equiv.) was added, and the mixture was stirred overnight. After the reaction was complete, the mixture was extracted with DCM and then washed successively with 5% citric acid aqueous solution (30 mL), saturated sodium bicarbonate solution (30 mL), and saturated saline solution (20 mL × 2). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by rapid column chromatography to give compound 1a.

[0074] Take a dry 10 mL glass tube and add compound 2 (0.15 mmol, 1.5 equiv.), compound 1a (0.1 mmol, 1 equiv.), and DABCO (0.02 mmol, 20 mol%) sequentially. Then inject DCM (1 mL) and stir at room temperature for 1 hour. The reaction progress is monitored by TLC. After completion, the solvent is removed under reduced pressure, and the residue is purified by rapid column chromatography to give compound 9c (40.1 mg, yield 57.31%). Its 1H NMR data are as follows: 1 H NMR (400MHz, CDCl3) δ 8.21 (d, J = 6.3Hz, 1H), 7.82–7.75 (m, 1H), 7.42 (s, 1H), 7.40 (s, 1H), 7.38–7.35 (m, 2H), 7.37–7.31 (m, 6H), 7.31 (d, J = 2.6Hz, 1H), 7.27 (s, 1H), 6.25 (s, 1H), 5.65 (d, J = 2.0Hz, 1H), 5.12–4.86 (m, 2H), 4.70–4.65 (m, 1H), 3.82 (s, 3H), 3.71–3.55 (m, 2H), 3.50 (q, J = 6.5Hz, 2H), 3.42–3.37 (m, 2H), 2.67 (s, 3H), 2.40 (s, 3H), 1.82 (d, J = 6.4 Hz, 2H), 1.68 (s, 3H). Its carbon NMR data are: 13CNMR (151 MHz, CDCl3) δ 170.9, 165.7, 159.1, 155.6, 149.9, 143.1, 136.8, 136.5, 135.3, 130.9, 130.5, 129.8, 128.7, 126.0, 125.1, 124.1, 120.5, 111.1, 54.4, 52.2, 45.4, 39.3, 36.8, 36.6, 31.4, 29.7, 29.4, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+H]. + calcd for C 35 H 36 O4N8ClS + :699.2263; found: 699.2252; Purity: 99%.

[0075] Synthesis of compound 10c: Following synthetic route 2 described above, using compounds j2 and g1 as starting materials, and referring to the synthetic method of compound 9c, compound 10c (19 mg, yield 26.69%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (600MHz, CDCl3) δ8.76 (d, J = 37.0Hz, 2H), 7.77–7.70 (m, 1H), 7.38 (d, J = 1.8Hz, 1H), 7.37 (s, 1H), 7.32 (d, J =1.4Hz, 1H), 7.31 (d, J = 1.4Hz, 1H), 7.30–7.29 (m, 1H), 7.28 (d, J = 3.5Hz, 1H), 7.27 (d, J =1.8Hz, 1H), 6.23 (d, J = 1.3Hz, 1H), 5.21 (d, J = 1.9Hz, 1H), 5.15–5.01 (m, 2H), 4.73 (dd, 1H), 4.15–4.08 (m, 1H), 3.80 (s, 3H), 3.78–3.72 (m, 2H), 3.72–3.64 (m, 2H), 3.21–3.14 (m, 2H), 3.13–3.07 (m, 1H), 2.68 (s, 3H), 2.42 (s, 3H), 1.85–1.75 (m, 2H), 1.68 (s, 3H). Its carbon NMR data are as follows: 13C10 NMR (151 MHz, CDCl3) δ 170.6, 167.3, 155.9, 150.7, 141.8, 136.8, 136.5, 134.6, 134.3, 130.8, 130.5, 129.8, 128.7, 126.9, 122.9, 122.7, 119.0, 110.0, 54.7, 53.4, 52.3, 44.1, 38.9, 38.6, 38.5, 35.3, 27.8, 14.3, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+H] + calcd for C 36 H 38 O4N8ClS + :713.2420; found: 713.2404; Purity: 95%.

[0076] Synthesis of compound 11c: Following synthetic route 2 described above, using compounds j3 and g2 as starting materials and referring to the synthetic method of compound 9c, compound 11c (54.7 mg, yield 84.8%) was obtained. Its 1H NMR spectrum data is as follows: 1 H NMR (600MHz, CDCl3) δ7.93–7.85 (m, 1H), 7.80 (t, J = 5.9Hz, 1H), 7.63 (d, J = 7.9Hz, 1H), 7.36 (d, J = 8.2Hz, 2H), 7.27 (s, 1H), 7.16–7.09 (m, 3H), 6.21 (s, 1H), 5.09 (s, 1H), 4.96 (d, J = 2.5Hz, 2H), 4.65–4.61 (m, 1H), 3.83–3.78 (m, 1H), 3.75 (s, 3H), 3.68–3.57 (m, 2H), 3.54–3.50 (m, 1H), 3.38–3.28 (m, 2H), 3.16–3.10 (m, 1H), 3.05–3.02 (m, 2H), 2.56 (s, 3H), 2.34 (s, 3H), 2.24–2.16 (m, 2H), 1.88–1.81 (m, 1H), 1.78–1.72 (m, 1H), 1.62 (s, 3H). Its carbon NMR data are as follows: 13C NMR (151MHz, CDCl3) δ 173.4, 170.7, 165.6, 163.9, 155.7, 153.3, 149.9, 142.2, 139.2, 136.7, 136.6, 134.8, 134.6, 132.0, 130.9, 130.8, 130.4, 129 .9, 128.7, 126.5, 122.6, 122.2, 118.9, 114.1, 109.8, 54.4, 53.7, 52.3, 44.0, 42.0, 38.5, 33.4, 27.2, 25.0, 14.4, 13.1, 11.7. Its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+H] + calcd for C 37 H 40 O4N8ClS + :727.2576: found: 727.2557; Purity: 99.7%.

[0077] Synthesis of compound 12c: Following synthetic route 2 described above, using compounds j3 and g5 as starting materials and referring to the synthetic method of compound 9c, compound 12c (61.4 mg, yield 72%) was obtained. Its 1H NMR spectrum data is as follows: 1 H NMR (600MHz, CDCl3) δ7.68–7.62 (m, 1H), 7.61–7.49 (m, 1H), 7.42–7.32 (m, 3H), 7.27 (d, J = 8.2Hz, 2H), 7.21–7.14 (m, 3H), 6.21 (s, 1H), 5.06 (s, 1H), 4.94 (s, 2H), 4.67–4.57 (m, 1H), 4.43–4.30 (m, 1H), 4.01–3.93 (m, 1H), 3.80–3.60 (m, 7H), 3.48–3.34 (m, 2H), 3.05–2.97 (m, 3H), 2.60 (s, 4H), 2.35 (s, 3H), 2.27–2.20 (m, 2H), 2.17 (t, J = 7.0Hz, 2H), 2.03–1.93 (m, 1H), 1.87 (d, J = 12.7 Hz, 1H), 1.63 (s, 4H), 1.57 (s, 4H). Its carbon NMR data are: 13C NMR (151MHz, CDCl3) δ 173.1, 171.1, 170.0, 165.5, 163.8, 155.7, 153.3, 149.8, 142.3, 139.2, 1 36.7, 136.6, 134.8, 134.7, 132.0, 130.9, 130.9, 130.5, 129.8, 128.7, 126 4, 122.6, 122.2, 119.1, 114.1, 109.7, 54.3, 53.7, 52.3, 46.5, 44.4, 43.9, 42.0, 40.6, 38.8, 36.6, 29.6, 29.1, 27.1, 25.2, 24.7, 14.4, 13.1, 11.8. Its high-resolution NMR mass spectrometry data are HRMS (ESI) m / z: [M+H] + calcd for C 44 H 51 O5N9ClS + :852.3417; found: 852.3391; purity: 99.9%.

[0078] Synthesis of compound 13c: Following synthetic route 2 described above, using compounds j3 and g4 as starting materials and referring to the synthetic method of compound 9c, compound 13c (80.0 mg, yield 92.3%) was obtained. Its 1H NMR spectral data are as follows: 1 H NMR (600MHz, CDCl3) δ7.68–7.63 (m, 1H), 7.57–7.41 (m, 1H), 7.39–7.33 (m, 2H), 7.29–7. 26 (m, 2H), 7.20–7.13 (m, 4H), 6.22 (s, 1H), 5.05 (s, 1H), 4.93 (s, 2H), 4.64–4.58 (m, 1H) , 4.45–4.34 (m, 1H), 4.03–3.94 (m, 1H), 3.85–3.59 (m, 7H), 3.49–3.32 (m, 2H), 3.23–3.1 1 (m, 1H), 3.07–2.96 (m, 3H), 2.76–2.57 (m, 4H), 2.36 (s, 3H), 2.25–2.17 (m, 2H), 2.13 (t, J = 7.5Hz, 2H), 1.99–1.77 (m, 2H), 1.64–1.52 (m, 7H), 1.30–1.23 (m, 2H). Its carbon NMR data are: 13C NMR (151MHz, CDCl3) δ173.2, 171.3, 170.0, 165.5, 163.8, 155.7, 153.3, 149.8, 1 42.4, 138.1, 136.7, 136.6, 136.6, 134.9, 134.7, 132.1, 130.9, 130.9, 130.5, 12 9.8, 128.7, 126.3, 122.6, 122.3, 119.1, 109.7, 54.3, 52.3, 51.9, 46.6, 44.5, 43.9, 40.6, 38.9, 36.5, 36.4, 32.9, 31.6, 28.9, 27.1, 25.3, 24.9, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+H]. + calcd forC 45 H 53 O5N9ClS + :866.3573; found: 866.3546; purity: 97.8%.

[0079] Synthesis of compound 15c: Following synthetic route 2 described above, using compounds j3 and g3 as starting materials and referring to the synthetic method of compound 9c, compound 15c (64 mg, yield 79%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (400MHz, CDCl3) δ 7.65 (d, J = 7.0Hz, 1H), 7.35–7.29 (m, 2H), 7.29–7.23 (m, 2H), 7.22–7.16 (m, 3H), 7.05–6.98 (m, 1H), 6.20 (s, 1H), 5.04 (d, J = 6.9Hz, 1H), 4.91 (t, J = 3.8Hz, 2H), 4.72 (d, J = 6.6Hz, 1H), 4.40 (s, 2H), 3.84–3.75 (m, 2H), 3.74 (d, J = 1.9Hz, 3H), 3.70–3.65 (m, 2H), 3.65–3.59 (m, 2H), 3.59–3.54 (m, 2H), 3.50 (d, J = 7.1Hz, 1H), 3.48–3.40 (m, 2H), 2.96 (d, J = 6.2Hz, 2H), 2.60 (d, J = 6.4Hz, 2H), 2.58 (d, J= 2.0Hz, 3H), 2.45 (d, J = 6.5Hz, 2H), 2.33 (d, J = 5.5Hz, 3H), 1.60 (d, J = 5.4 Hz, 3H). Its carbon NMR data are: 13 C10 NMR (101 MHz, CDCl3) δ 170.5, 165.5, 136.7, 134.8, 134.6, 132.1, 130.9, 130.4, 129.7, 128.7, 126.3, 122.7, 122.4, 119.1, 114.0, 109.6, 54.4, 54.3, 52.3, 43.9, 41.6, 36.4, 35.2, 31.1, 29.6, 29.3, 28.4, 26.9, 14.3, 13.0, 11.8. Its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+H] + calcd forC 41 H 45 O5N9ClS + :810.2947; found: 810.2922; Purity: 98%.

[0080] Synthesis of compound 16c: Following synthetic route 2 described above, using compounds j3 and g6 as starting materials and referring to the synthetic method of compound 9c, compound 16c (73.3 mg, yield 87.4%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (600MHz, CDCl3) δ7.73 (d, J = 7.5Hz, 1H), 7.43 (d, J = 8.2Hz, 2H), 7.35 (d, J = 8.0Hz, 2H), 7.31–7.22 (m, 4H), 6.30 (s, 1H), 5.14 (s, 1H), 5.01 (s, 2H), 4.82 (d, J = 5.1Hz, 1H), 4.03–3.72 (m, 10H), 3.64–3.42 (m, 5H), 3.06 (t, J = 6.1Hz, 2H), 2.68 (s, 3H), 2.43 (s, 3H), 2.35 (d, J = 9.4Hz, 2H), 2.25 (s, 2H), 1.69 (d, J = 17.8 Hz, 7H). Its carbon NMR data are: 13C NMR (151MHz, CDCl3) δ 172.9, 171.4, 169.3, 165.5, 163.9, 155.7, 153.3, 149.9, 142.3, 139.2, 136.8, 136.7, 134.9, 134.7, 132.2, 130.9, 130.7, 130.4, 129.8, 128.7, 126.4, 122.7, 122.3, 119.1, 114.1, 109.7, 54.5, 54.3, 52.3, 45.9, 45.5, 45.1, 43.9, 41.7, 41.4, 35.3, 33.8, 27.1, 25.1, 24.5, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+H] + calcd for C 43 H 49 O5N9ClS + :838.3260; found: 838.3235; purity: 99.5%.

[0081] Synthesis of compound 17c: Following synthetic route 2 described above, using compounds J3 and G7 as starting materials and referring to the synthetic method of compound 9C, compound 17C (70 mg, yield 82.1%) was obtained. Its 1H NMR spectrum data is as follows: 1 H NMR (600MHz, CDCl3) δ7.76–7.71 (m, 1H), 7.40 (d, J = 8.2Hz, 2H), 7.33 (d, J = 8.2Hz, 2H), 7.29–7.25 (m, 3H), 7.01–6.96 (m, 1H), 6.29 (s, 1H), 5.12 (s, 1H), 4.99 (s, 2H), 4.83–4.77 (m, 1H) ), 3.87–3.80 (m, 6H), 3.80–3.73 (m, 3H), 3.71–3.65 (m, 2H), 3.61–3.55 (m, 2H), 3.51–3.46 (m, 2H), 3.04 (t, J =5.9 Hz, 2H), 2.67 (s, 3H), 2.41 (s, 3H), 2.32–2.27 (m, 2H), 2.21–2.16 (m, 2H), 1.68 (s, 3H), 1.67–1.61 (m, 4H), 1.44–1.41 (m, 2H). Its carbon NMR data are as follows: 13C NMR (126MHz, CDCl3) δ173.2, 171.7, 169.4, 169.2, 165.5, 155.7, 153.3, 149.9, 142.1, 139.3, 136.7, 134.8, 134.7, 132.2, 130.9, 130.9, 130.9, 130.5, 129.8, 128.7, 126.4, 122.8, 122.5, 119.1, 114.1, 109.7, 54.5, 53.9, 52.4, 46.0, 45.6, 45.4, 45.2, 41.8, 41.3, 36.4, 35.4, 27.0, 25.3, 24.7, 22.7, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+H]. + calcd forC 44 H 51 O5N9ClS + :852.3417; found: 852.3393; purity: 99.8%.

[0082] Synthesis of compound 18c: Following synthetic route 2 described above, using compounds j4 and g6 as starting materials and referring to the synthetic method of compound 9c, compound 18c (79.1 mg, yield 90%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (600MHz, CDCl3) δ7.61–7.53 (m, 1H), 7.27 (d, J = 8.1Hz, 2H), 7.17 (d, J = 8.2Hz, 2H), 7.10–7.05 (m, 3H), 6.12 (s, 1H), 4.90 (s, 3H), 4.65 (t, J = 6.7Hz, 1H), 4.61–4.51 (m, 1H), 3.89 (d, J = 13.5Hz, 1H), 3.70–3.32 (m, 15H), 3.06 (t, J = 12.8Hz, 1H), 2.96–2.92 (m, 2H), 2.63–2.56 (m, 1H), 2.50 (s, 3H), 2.32–2.21 (m, 7H), 1.99–1.91 (m, 1H), 1.88–1.84 (m, 1H), 1.78 (d, J = 8.4 Hz, 2H), 1.57 (s, 4H). Its carbon NMR data are: 13C NMR (151MHz, CDCl3) δ 171.4, 170.8, 169.1, 165.4, 163.6, 156.8, 155.6, 149.7, 142.2, 139.0, 136. 6, 136.4, 135.0, 134.5, 132.0, 130.7, 130.7, 130.2, 129.7, 128.5, 126.0, 12 2.6, 122.2, 119.2, 114.0, 109.7, 54.2, 53.7, 52.3, 45.8, 45.3, 45.1, 43.7, 42.0, 41.3, 34.0, 33.6, 32.9, 31.7, 31.1, 29.5, 28.8, 24.9, 14.3, 13.0, 11.7. Its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+H]. + calcd for C 46 H 53 O5N9ClS + :878.3573; found: 878.3546; purity: 99%.

[0083] Synthesis of compound 19c: Following synthetic route 2 described above, using compounds J3 and G8 as starting materials and referring to the synthetic method of compound 9c, compound 19c (44.6 mg, yield 44.54%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (500MHz, CDCl3) δ7.71 (d, J = 7.1Hz, 1H), 7.41 (d, J = 8.2Hz, 2H), 7.32 (d, J = 8.2Hz, 3H), 7.24 (s, 3H), 6.27 (s, 1H), 5.08 (s, 1H), 4.99 (s, 2H), 4.79 (t, J = 6.6Hz, 1H), 3.84 (s, 3H), 3.82 (s, 3H), 3.74 (d, J = 8.9Hz, 1H), 3.72–3.67 (m, 3H), 3.67–3.59 (m, 12H), 3.55 (d, J = 5.1Hz, 4H), 3.52–3.45 (m, 5H), 3.04 (t, J = 6.2Hz, 2H), 2.69 (s, 1H), 2.66 (s, 3H), 2.61 (t, J = 6.3Hz, 2H), 2.54 (d, J= 12.8Hz, 1H), 2.48 (s, 1H), 2.46 (t, J = 5.7 Hz, 2H), 2.39 (s, 3H), 1.67 (s, 3H). Its carbon NMR data are: 13 C10 NMR (126 MHz, CDCl3) δ 168.8, 155.8, 136.7, 129.8, 128.6, 126.2, 122.6, 122.2, 119.2, 109.6, 70.5, 70.4, 70.3, 70.2, 67.1, 54.3, 53.5, 52.3, 43.8, 37.0, 36.5, 35.1, 29.6, 29.3, 27.1, 14.3, 13.0, 11.8. Its high-resolution mass spectrometry data are HRMS (ESI) m / z: [M+2H] 2+ calcd for C 50 H 66 O9N9ClS 2+ :501.7179; found: 501.7191; Purity: 97%.

[0084] test: (1) High-content analysis and screening a. Construction of dual fluorescence screening models containing BD1 domain, BD2 domain, and BD1-BD2 dual domain A tandem chimeric gene was designed in an expression vector, fusing the FLAG-tagged BRD4 protein domains (BD1, BD2, and BD1-BD2 dual domains, respectively) with mScarlet protein (red fluorescent protein), and then coupling it to EGFP protein (green fluorescent protein) via a P2A self-cleaving peptide. Recombinant plasmids Flag-BD1-mScarlet-P2A-EGFP, Flag-BD2-mScarlet-P2A-EGFP, and Flag-BD1-BD2-mScarlet-P2A-EGFP were constructed. These recombinant plasmids were then transfected into HEK-293T cells. After successful transfection, the cells will express the Flag-BD1-mScarlet-P2A-EGFP dual fluorescent protein fusion, the Flag-BD2-mScarlet-P2A-EGFP dual fluorescent protein fusion, and the Flag-BD1-BD2-mScarlet-P2A-EGFP dual fluorescent protein fusion, respectively.

[0085] Based on the ribosome jumping effect, the P2A peptide breaks at the glycine-proline site during translation, allowing a single transcription sample to simultaneously express two independent products: the BRD4 domain (BD1 domain, BD2 domain, and BD1-BD2 dual domain)-mScarlet fusion protein and the free EGFP protein.

[0086] The compounds prepared in Examples 2, 4 and 5 above (i.e., the degrading agents based on benzimidazole fused covalent warheads) were serially diluted with complete culture medium to prepare compound solutions of different concentration gradients.

[0087] The desired cells were transfected with the recombinant plasmid described above. After digestion and counting, the transfected cells were seeded at 10,000 cells per well in a 96-well plate and cultured overnight. When the cells were fully adhered and at an appropriate density, the medium was replaced with fresh complete medium, and the compound solution was added to three wells at each concentration. An equal volume of PBS (phosphate-buffered saline) was added to the edge of each well. The plates were incubated for 12 hours and then placed in a high-content analyzer for analysis.

[0088] In transfected cells, when a benzimidazole-based fusion covalent warhead-based degrader is added, the effective benzimidazole-based fusion covalent warhead-based degrader specifically degrades the target domains (BD1 domain, BD2 domain, or BD1-BD2 dual domain) of the BRD4 protein. This leads to a synchronous decrease in the fluorescence of the mScarlet covalently bound to the protein, while the independently expressed EGFP remains stable. Therefore, by quantitatively analyzing the change in the fluorescence intensity ratio of mScarlet (as the target signal) to EGFP (as the internal control signal) using a high-content imaging system, the efficacy of the benzimidazole-based fusion covalent warhead-based degrader in inducing target protein degradation can be objectively characterized. The smaller the mScarlet to EGFP ratio, the stronger the ability of the benzimidazole-based fusion covalent warhead-based degrader to degrade the BRD4 protein.

[0089] (2) Western blot experiment Wash the cell plate with PBS, add RIPA lysis buffer (containing protease inhibitors), scrape off cells and transfer to EP tubes for lysis (8 cycles at 20% power, 2 seconds each). Centrifuge at 12000 rpm for 20 min at 4°C, and transfer the supernatant to a new EP tube. Take a fixed volume of the supernatant, add 5× Loading Buffer, mix well, and heat at 95°C for 8 min before use or storage at -80°C. Perform protein quantification, preparing protein electrophoresis gels of different concentrations as needed.

[0090] Install the electrophoresis apparatus, add electrophoresis buffer until it is level with the top, remove the comb to expose the sample wells, and add the marker (molecular weight standard protein) and protein sample in sequence. Set the electrophoresis conditions (70V, 30min) and run the gel. Once the buffer is level, separate the gel under voltage conditions (120V, 60min). Stop the process when the two-color loading buffer reaches the bottom. Immerse a suitably sized PVDF (polyvinylidene fluoride) membrane in methanol for 1min, rinse with transfer buffer, and then cover the gel block after electrophoresis. Remove excess gel, transfer the membrane to an electrotransfer clamp to remove air bubbles, clamp it tightly, and place it in the electrotransfer tank. Transfer the membrane at a constant current of 230mA under ice bath conditions, setting the electrotransfer time according to the protein molecular weight.

[0091] After electroporation, the membrane was placed in 5% skim milk and slowly shaken at room temperature for 1 hour to block it. After removal, it was washed three times with 1×TBST buffer (tris(hydroxymethyl)aminomethane) buffer for 20 minutes each time. The membrane containing the target protein was excised and labeled, and incubated uniformly in primary antibody at 4°C overnight. After removal, it was washed three times with 1×TBST buffer for 20 minutes each time. The membrane was then incubated at room temperature in secondary antibody for 1 hour. After removal, it was washed three times with 1×TBST buffer for 20 minutes each time. In a light-proof container, equal volumes of solutions A and B from the development kit were mixed to prepare the developing solution. The membrane was soaked and incubated for 3 minutes. After development, it was removed and analyzed.

[0092] (3) Cell viability assay After cell digestion and counting, cells were seeded at a rate of 3000-5000 cells per well in 96-well plates and incubated overnight. The prepared compound was serially diluted with complete culture medium to create gradient concentrations, and the medium was replaced with each well in a 96-well plate, with three auxiliary wells for each concentration. PBS was added to the side wells of the plate, and the plates were incubated for 48 hours. 10 μL of CCK-8 reagent was added to each well, vortexed, and incubated for 2 hours. After incubation, the absorbance (OD) at 450 nm was measured using a microplate reader to calculate cell viability.

[0093] (4) Elution assay (used to evaluate the sustained effect of the compound on the degradation of BRD4 protein) MDA-MB-231 cells were cultured in 6-well plates to 80% confluence, and then incubated with complete medium containing 100 nM of the compound, with 0.1% DMSO (methyl sulfoxide) as a control. The cells were incubated at 37°C and 5% CO2 for 12 h. After 12 h, the cells were gently washed twice with 37°C PBS and then replaced with drug-free medium. Cells were harvested at six time points (0 h, 6 h, 12 h, 24 h, 36 h, and 48 h) for Western blotting analysis.

[0094] The results are as follows: (1) The activity test results of each compound in Examples 2, 4 and 5 above are as follows: Figure 2 As shown, con represents the control group, row min indicates mScarlet / EGFP is 0.6, and row max indicates mScarlet / EGFP is 1.0. Figure 2 The study demonstrated the degradation effects of various compounds on the BD1 domain, BD2 domain, and BD1-BD2 double domain in HEK293T cells with a stable high-content screening model. It was found that compound 4c (i.e., compound LGF327) exhibited the best BRD4 degradation activity at both 100 nM and 1 μM concentrations, with its selective degradation ability significantly superior to other compounds.

[0095] High-content screening results based on a dual-domain fluorescent reporter system showed that this series of compounds generally exhibited BRD4 degradation activity. In the comparison experiment between the BD1-BD2 dual-domain reporter model and the single-domain (BD1 and BD2 domains) reporter model, except for compound 3ch, the degradation efficiency of the other compounds in the dual-domain system was significantly better than that in the single-domain system. This suggests that these compounds may induce ubiquitin-proteasome-dependent degradation more efficiently by synergistically targeting the BD1 and BD2 domains.

[0096] Among compounds with monosubstituted benzene rings, fluorine-substituted compounds (such as 3cb) exhibit relatively weaker degradation activity than other halogen- or alkyl-substituted compounds (3ca, 3cc, and 3cd). It is speculated that the strong lipophilic fluorine atom may cause a shift in the electron cloud distribution at the molecule-target binding interface or affect the overall solubility of the molecule. Furthermore, all disubstituted compounds (compounds ce to cg) show lower activity than their corresponding monosubstituted compounds (compounds 3cb to 3cd). This may be attributed to the steric hindrance effect created by the disubstituted groups near the JQ1 binding pocket, weakening the binding stability of the compound to the target protein. Therefore, it is hypothesized that the degradation efficiency of the above compounds on the BRD4 protein is related to their synergistic targeting ability to the BD1-BD2 bidomain, with the benzene ring substituents influencing degradation activity through both electronic and steric effects.

[0097] Compounds containing flexible alkane chains (i.e., compounds 3c to 11c) generally exhibit significant degradation activity, while most rigid linker compounds (such as compounds 12c to 19c) may have insufficient target binding adaptability due to their fixed spatial configuration, resulting in reduced or even lost degradation efficiency (at low concentrations).

[0098] Furthermore, regarding the flexible linker series, this invention further discovered a slight negative correlation between the increase in linker length and the degradation effect. Compounds containing a single carbon chain (4c) exhibited the best degradation activity, with a fluorescence signal intensity of 62%. However, as the chain length increased to 5 or 6 carbons, the fluorescence signal intensity decreased to 88%. This may be because excessively long alkane chains not only affect the efficiency of the compound penetrating the cell membrane but also hinder the formation of a stable ternary complex between the BRD4 protein and the E3 ubiquitin ligase.

[0099] (2) The degradation activity of compound 4c (i.e., compound LGF327) against BET family proteins is as follows: Figure 3 As shown in the figure, compound LGF327 exhibits extremely high degradation efficacy (DC) against the BRD4 protein. 50 = 5.3nM, DC 50 This indicates the concentration of the compound required to degrade the target protein by 50%; D max >90%, D max The figure indicates the maximum degradation rate, while it has no degrading effect on BRDT protein (testis-specific bromine-containing domain protein). Furthermore, although compound LGF327 exhibits some degrading activity against BRD2 protein (bromine-containing domain protein 2) and BRD3 protein (bromine-containing domain protein 3), its potency is significantly lower than that against BRD4 protein (approximately 3-5 times lower). For BRD4 protein, degradation can be detected 1 hour after administration, reaching 50% clearance at 3 hours, and near-complete degradation by 6 hours.

[0100] (3) The degradation results of BRD4 protein by compound LGF327 in different cancer cells are as follows: Figure 4 As shown. It can be seen that compared to HL435 ( The compound LGF327 exhibited broad-spectrum degradation characteristics of BRD4 protein in various cancer cell lines (including breast cancer cells MDA-MB-231 and MCF-7, liver cancer cells HepG2, lung cancer cells A549, colon cancer cells HCT116, and glioma cells U251). However, LGF327 showed highly efficient degradation of BRD4 protein only in specific cell types. In triple-negative breast cancer and colon cancer cell lines, a concentration of 100 nM of LGF327 mediated significant degradation of BRD4 protein, while a concentration of 1 μM was required in glioma cells U251. No significant degradation was observed in other tested cancer cell lines. Furthermore, due to… Figure 4 As shown in (b), compound LGF327 has a good and sustained effect on degrading BRD4 protein.

[0101] (3) To verify the degradation pathway of compound LGF327, 100 nM of compound LGF327 was reacted with autophagy inhibitors CQ (chloroquine) and BafA1 (bafloxacin), 26S proteasome inhibitors MG132 (N-[(benzyloxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucine) and PS341 (bortezomib), and E1 ubiquitin activator inhibitor PYR41 (4-[4-[(5-nitro-2- [Furfural]methylene]-3,5-dioxo-1-pyrazolyl]ethyl benzoate, NEDD8 activator inhibitor MLN4924 (sulfamic acid [(1S,2S,4R)-4-[4-[[(1S)-2,3-dihydro-1H-indene-1-yl]amino]-7H-pyrrolo[2,3-D]pyrimidin-7-yl]-2-hydroxycyclopentyl]methyl ester), simultaneously treated MDA-MB-231 cells, and then high-content analysis was used to determine whether target protein degradation was competitively inhibited. The results are as follows: Figure 5 , Figure 6 and Figure 7 As shown (where ns indicates no significant difference), This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001. This indicates that p < 0.0001.

[0102] Through high-content screening and cellular-level experiments, this invention employed two classes of specific inhibitors for validation: the first group inhibited the lysosomal pathway using autophagy inhibitors CQ and BafA1; the second group inhibited the ubiquitin-proteasome pathway using 26S proteasome inhibitors MG132 and PS341, E1 ubiquitin activator inhibitor PYR-41, and NEDD8 activator inhibitor MLN4924. Experimental results showed that in the lysosomal pathway inhibition group, neither chloroquine inhibition of autophagosome acidification nor bafloxacin interference with lysosomal maturation effectively hindered the degradation activity of compound LGF327. However, all four ubiquitin-proteasome pathway inhibitors significantly inhibited the degradation effect of compound LGF327 on BRD4 protein. This demonstrates that compound LGF327 mediates BRD4 protein degradation through the classic ubiquitin-proteasome pathway.

[0103] 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 degrading agent based on benzimidazole fused covalent warhead, characterized in that, The structural formula of the degrading agent based on the benzimidazole fusion covalent warhead is: ； in, R is phenyl or substituted phenyl; R is methyl or tert-butyl; Choose from one of the following structures: 、 、 、 、 、 、 、 、 、 、 , , , , , and ; Indicates the connection site.

2. The degrading agent based on benzimidazole fused covalent warhead according to claim 1, characterized in that, The substituted phenyl group is an alkoxy-substituted phenyl group, a fluorine-substituted phenyl group, a chlorine-substituted phenyl group, a bromine-substituted phenyl group, or a nitro-substituted phenyl group.

3. The degrading agent based on benzimidazole fused covalent warhead according to claim 1, characterized in that, Choose from one of the following structures: , , , , , , , and .

4. A method for preparing a degrading agent based on a benzimidazole fused covalent warhead as described in any one of claims 1-3, characterized in that, The method for preparing the degrading agent based on benzimidazole fusion covalent warhead includes the following steps: Will and After the reaction, the degrading agent based on the benzimidazole fused covalent warhead is obtained; Boc represents tert-butyloxycarbonyl.

5. The preparation method according to claim 4, characterized in that, The preparation method includes the following steps: Will and After the reaction, we get .

6. The use of a benzimidazole-based degrading agent according to any one of claims 1-3 in the preparation of a medicament for treating breast cancer.

7. The application according to claim 6, characterized in that, The breast cancer mentioned is triple-negative breast cancer.

8. The use of a benzimidazole-based degrading agent according to any one of claims 1-3 in the preparation of a medicament for treating colon cancer.

9. The use of a benzimidazole-based degrading agent according to any one of claims 1-3 in the preparation of a medicament for treating glioma.

10. The application according to any one of claims 6-9, characterized in that, The drug includes a degrading agent based on a benzimidazole fusion covalent warhead, and the drug also includes a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable additive.

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