Degradable agent based on benzimidazole fused covalent warhead and preparation method and application thereof

Abstract

The application discloses a degrader based on benzimidazole fusion covalent warhead and a preparation method and application thereof, and relates to the technical field of drug development. A structural formula of the degrader based on benzimidazole fusion covalent warhead is shown as follows: wherein, is a phenyl group or a substituted phenyl group; R is a methyl group or a tert-butyl group; is selected from one of the following structures:,,,,,,,,,,,, and ; and indicates a connecting site. The application utilizes a modular design of a benzimidazole directing group and an acrylate covalent warhead, and then connects the BRD4 protein inhibitor JQ1 through a linker to obtain a series of degraders based on benzimidazole fusion covalent warhead. The degrader based on benzimidazole fusion covalent warhead can target the BRD4 protein and efficiently degrade the BRD4 protein. Therefore, the application realizes the targeted degradation of the BRD4 protein while enriching a library of protein degradation targeting chimeric molecules.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug development, and particularly relates to a degradation agent based on a benzimidazole fusion covalent warhead and a preparation method and application thereof. BACKGROUND

[0002] In the past few decades, the drug discovery model has changed from phenotype research to mechanism-based target selective development, i.e. the targeted drug strategy. In recent years, a new targeted drug strategy is emerging, i.e. the targeted protein degradation (TPD) strategy. This strategy utilizes the main endogenous pathways for protein and organelle degradation in eukaryotic cells: the lysosomal pathway and the ubiquitin-proteasome system (UPS) pathway. The emergence of the TPD strategy enriches the action targets of targeted drugs, has the characteristics of high efficiency and high selectivity, and provides a solution to the problem of drug-resistant proteins and traditional drugs.

[0003] Among them, the protein degradation targeting chimera (PROTAC) based on the UPS pathway is the main research object in the TPD strategy, such as Figure 1 as shown in formula (I) (Ub represents ubiquitin), which is composed of three parts, i.e. a ligand for recruiting an E3 ubiquitin ligase (i.e. an E3 ubiquitin ligase ligand), a ligand for specifically binding a target protein (i.e. a target protein ligand), and a linker connecting the two. The protein degradation targeting chimera forms a ternary complex by connecting a specific E3 ubiquitin ligase and a target protein, promotes the transfer of a ubiquitin tag activated by three ubiquitin enzymes from the E3 ubiquitin ligase to the target protein, and the multiple ubiquitinated target proteins are ultimately degraded after being recognized by the proteasome, while the protein degradation targeting chimera is released in this process. Therefore, the protein degradation targeting chimera has the characteristics of event-driven action, i.e. even if it is weakly bound or bound to a non-active site, the protein degradation targeting chimera can achieve degradation in a catalytic amount.

[0004] At present, the field of protein degradation targeting chimeras is constantly producing results, and can degrade multiple types of proteins such as endosomes, protein kinases and transcriptional regulators, and even proteins that are considered to be undruggable. In the face of challenges such as drug resistance, protein degradation targeting chimeras also provide new solutions and have broad prospects. There are still many potentials of protein degradation targeting chimeras to be developed, which brings both opportunities and new challenges. The problem that needs to be solved at present is the limited available E3 ubiquitin ligase ligands. It has been found through research that there are more than 600 E3 ubiquitin ligases in the human body, but only 3% of the E3 ubiquitin ligases are currently used in protein degradation targeting chimeras, more than 90% of which rely on E3 ubiquitin ligases CRBN or VHL, but protein degradation targeting chimeras based on CRBN or VHL have drug resistance.

[0005] Bromodomain-containing protein 4 (BRD4) is a member of the bromodomain and extraterminal domain (BET) protein family, which can recognize acetylated histones and locate to the promoter or enhancer region of target genes, initiating and maintaining the expression of tumor-related genes. BRD4 protein is closely related to the regulation of various transcription factors and chromatin modification, and is involved in DNA damage repair and maintenance of telomere function, thereby maintaining the survival of tumor cells.

[0006] Therefore, developing new E3 ubiquitin ligase ligands to overcome the above challenges, tapping the potential of protein degradation targeting chimera technology, constantly enriching the protein degradation targeting chimera molecule library, and achieving the degradation of target proteins such as BRD4 protein are the research focus in this field.

[0007] Therefore, the prior art still needs to be improved and developed. SUMMARY

[0008] Based on the deficiencies of the prior art described above, the purpose of the present application is to provide a degradation agent based on a benzimidazole fusion covalent warhead and a preparation method and application thereof, aiming to enrich the protein degradation targeting chimera molecule library and simultaneously achieve the targeted degradation of BRD4 protein.

[0009] The technical scheme of the present application is as follows:

[0010] In a first aspect of the present application, a degradation agent based on a benzimidazole fusion covalent warhead is provided, wherein the structural formula of the degradation agent based on a benzimidazole fusion covalent warhead is:

[0011] ; wherein, is a phenyl group or a substituted phenyl group; R is a methyl group or a tert-butyl group; the linker is selected from one of the following structures:

[0012] , , , , , , , , , ,

[0013] , , , , , and ;

[0014] indicates a connection site (if no special instructions are given, the connection site involved in the structural formula in the following text is All those marked as connection sites will not be elaborated again when they appear later. (The meaning will not be repeated when it appears again later).

[0015] Based on the E3 ubiquitin ligase DCAF16, the present invention utilizes the modular design of a benzimidazole-directed group and an acrylate covalent warhead, and then couples it with the BRD4 protein inhibitor JQ1 ( ) through an adjustable linker to obtain a series of E3 ubiquitin ligase DCAF16 activity-dependent covalent protein degrader molecules, namely degraders based on benzimidazole-fused covalent warheads. The degraders based on benzimidazole-fused covalent warheads provided by the present invention can recruit the E3 ubiquitin ligase DCAF16 to form a ternary complex with the target protein (i.e., BRD4 protein), induce ubiquitination of the BRD4 protein, and the ubiquitinated BRD4 protein is finally degraded after being recognized by the proteasome. In addition, the degraders based on benzimidazole-fused covalent warheads exhibit efficient BRD4 protein degradation characteristics in breast cancer, colon cancer, and glioma cells, indicating their potential as cancer therapeutic drugs.

[0016] Therefore, the degraders based on benzimidazole-fused covalent warheads provided by the present invention can target the BRD4 protein and degrade the BRD4 protein, enriching the molecular library of proteolysis-targeting chimeras while achieving targeted degradation of the BRD4 protein.

[0017] Optionally, the substituted phenyl is a phenyl substituted with an alkoxy group (the alkoxy group can be mono-substituted or di-substituted), a phenyl substituted with fluorine (fluorine can be mono-substituted or di-substituted), a phenyl substituted with chlorine (chlorine can be mono-substituted or di-substituted), a phenyl substituted with bromine (bromine can be mono-substituted or di-substituted), or a phenyl substituted with a nitro group (the nitro group can be mono-substituted or di-substituted).

[0018] Optionally, is selected from one of the following structures:

[0019] , , , , , , , and .

[0020] In the second aspect of the present invention, a preparation method of the degrader based on benzimidazole-fused covalent warhead as described above in the present invention is provided, wherein the preparation method of the degrader based on benzimidazole-fused covalent warhead comprises the following steps:

[0021] Dissolve (wherein and , the same as and in the structural formula of the degradation agent based on the benzimidazole fusion covalent warhead above) and (R is methyl or tert-butyl) to obtain the degradation agent based on the benzimidazole fusion covalent warhead; Boc represents tert-butyloxycarbonyl.

[0022] Optionally, The preparation method of the degradation agent based on the benzimidazole fusion covalent warhead comprises the following steps:

[0023] The reaction of with (wherein and , the same as and in the structural formula of the degradation agent based on the benzimidazole fusion covalent warhead above) to obtain .

[0024] In a third aspect of the present application, the degradation agent based on the benzimidazole fusion covalent warhead is used for preparing a medicament for treating breast cancer.

[0025] Optionally, the breast cancer is triple-negative breast cancer.

[0026] In a fourth aspect of the present application, the degradation agent based on the benzimidazole fusion covalent warhead is used for preparing a medicament for treating colon cancer.

[0027] In a fifth aspect of the present application, the degradation agent based on the benzimidazole fusion covalent warhead is used for preparing a medicament for treating glioma.

[0028] Optionally, the medicament comprises the degradation agent based on the benzimidazole fusion covalent warhead, and the medicament further comprises a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable additive.

[0029] Specifically, the pharmaceutically acceptable carrier comprises at least one of a glidant, a diluent, a wetting agent, a suspending agent, a solvent and an emulsifying agent; and the pharmaceutically acceptable additive comprises at least one of a preservative, a coloring agent, a flavoring agent, a stabilizer and an isotonic agent.

[0030] Beneficial effects: the application utilizes the modular design of benzimidazole guiding group and acrylic ester covalent warhead, and then connects with BRD4 protein inhibitor JQ1 through a linker, to obtain a series of benzimidazole-based degradation agent fused with covalent warhead. The benzimidazole-based degradation agent fused with covalent warhead can target BRD4 protein and efficiently degrade BRD4 protein. Therefore, the application enriches the library of protein degradation targeting chimera molecules while realizing the targeted degradation of BRD4 protein. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 It is a schematic diagram of the mechanism of the protein degradation targeting chimera in the prior art to degrade target proteins.

[0032] Figure 2 It is a graph of the activity test results of each compound in Example 2 and Example 4.

[0033] Figure 3 It is a graph of the degradation activity results of compound LGF327 on BET family proteins, wherein (a) is a graph of the degradation effect of compound LGF327 on BET family proteins, (b) is a semi-quantitative result graph of the degradation of compound LGF327 on BET family proteins, and (c) is a DC 50 Result graph.

[0034] Figure 4 It is a graph of the degradation results of compound LGF327 on BRD4 protein in different cancer cells, wherein (a) is a graph of the degradation effect of compound LGF327 on BRD4 protein in MDA-MB-231 cells at different times, (b) is a graph of the degradation effect of compound LGF327 on BRD4 protein after 12h elution and culture in drug-free medium for different times, (c) is a graph of the degradation effect of compound HL345 on BRD4 protein in different cancer cells, and (d) is a graph of the degradation effect of compound LGF327 on BRD4 protein in different cancer cells.

[0035] Figure 5 It is a graph of the degradation mechanism verification results of compound LGF327, wherein (a) is a graph of the degradation mechanism results of compound LGF327 under high-content analysis microscope, and (b) is a semi-quantitative result graph of the degradation mechanism verification of LGF327 under high-content analysis microscope.

[0036] Figure 6 It is a graph of the lysosome pathway inhibition experiment results of compound LGF327, wherein (a) is a graph of the lysosome pathway inhibition experiment results of compound LGF327, and (b) is a semi-quantitative result graph of the lysosome pathway inhibition experiment of compound LGF327.

[0037] Figure 7 Figure 2 shows the results of proteasome pathway inhibition experiments for compound LGF327, wherein (a) is a graph of the results of proteasome pathway inhibition experiments for compound LGF327, and (b) is a semi-quantitative graph of the results of proteasome pathway inhibition experiments for compound LGF327. DETAILED DESCRIPTION

[0038] The present application provides degraders based on benzimidazole fused covalent warheads and preparation methods and applications thereof. In order to make the purpose, technical solutions and effects of the present application clearer and more explicit, the present application is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application.

[0039] 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 application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0040] The present application is further described below through specific examples.

[0041] In the following examples and synthetic routes, the meanings of some symbols are as follows:

[0042] HBTU: benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate;

[0043] DCM: dichloromethane;

[0044] DIPEA: N,N-diisopropylethylamine;

[0045] DBU: 1,8-diazabicyclo(5.4.0)undec-7-ene;

[0046] AcOH: acetic acid;

[0047] HOBt: 1-hydroxybenzotriazole;

[0048] DMAP: 4-dimethylaminopyridine;

[0049] DABCO: 1,4-diazabicyclo[2.2.2]octane;

[0050] equiv.: equivalent;

[0051] TFA: trifluoroacetic acid;

[0052] r.t.: room temperature;

[0053] 5% aqueous citric acid solution: the mass percentage of citric acid in the aqueous citric acid solution is 5%;

[0054] Fmoc: fluorenylmethyloxycarbonyl;

[0055] tBu: tert-butyl.

[0056] Example 1

[0057] This example first synthesizes compounds 3a, 3b, 3c and 3d, whose structural formulas are specifically shown in Table 1.

[0058] Table 1, structural formulas of compounds 3a to 3d and mScarlet / EGFP ratio

[0059]

[0060] It is found (see Table 1) by high-content cell fluorescence imaging analysis that compound 3a cannot mediate the targeted degradation of BRD4 protein, but when it is connected with a covalent warhead MBH ester (such as compounds 3b and 3c), the fluorescence signal intensity ratio related to BRD4 protein degradation shows a significant downward trend, indicating that compounds 3b and 3c have the function of degrading BRD4 protein. The experimental results show that the strategy of molecular chimerization of benzimidazole as a guide group and MBH ester covalent warhead may effectively improve the directional recruitment ability of protein degradation targeting chimera molecules by enhancing the synergistic recognition of target proteins and E3 ubiquitin ligase.

[0061] The change in the fluorescence intensity ratio of mScarlet (red fluorescent protein, as a target signal) and EGFP (green fluorescent protein, as an internal reference signal) can be quantitatively analyzed by a high-content imaging system, which can objectively characterize the efficiency of the degradation agent based on benzimidazole fusion covalent warhead in inducing target protein degradation. The smaller the mScarlet / EGFP ratio, the stronger the ability of the degradation agent based on benzimidazole fusion covalent warhead to degrade BRD4 protein (principle see below).

[0062] Next, based on the skeletal structure of lead compound 3c, a series of degradation agents based on benzimidazole fusion covalent warhead (see below) are designed and synthesized by introducing substituents (single substituent or double substituent) at different positions of the benzene ring, and a preparation method of compound 3b is provided.

[0063] In the following examples, the structural formulas of compounds 3c, 3ca to 3ch are: ; wherein, The specific structures of are shown in Table 2.

[0064] Table 2, specific structures of in the structural formulas of compounds 3c, 3ca to 3ch

[0065] ​

[0066] The structural formulas of compounds 4c to 19c are as follows:

[0067] ;in, The specific structure is shown in Table 3.

[0068] Table 3 Specific structure

[0069]

[0070] Example 2 Synthesis of compounds 3c, 3ca to 3ch

[0071] The synthetic route is as follows:

[0072] .

[0073] 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.

[0074] 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.

[0075] Synthesis of compound 3c:

[0076] (1) Into a 50 mL round-bottom flask, compound b (1.0 mmol, 1 equiv.) was added, followed by HBTU (1.25 mmol, 1.25 equiv.) and DCM (30 mL). DIPEA (2.0 mmol, 2 equiv.) was slowly added under ice bath (0 °C) condition. After stirring for 10 min, compound a (1.05 mmol, 1.05 equiv.) was slowly added into the reaction system. After maintaining the ice bath condition for 30 min, the temperature was gradually increased to room temperature and the stirring was continued for 16 h. After the reaction was completed, the mixture was extracted with DCM (30 mL x 3). The combined organic phase was washed with 5% citric acid aqueous solution (30 mL), saturated sodium bicarbonate solution (30 mL) and saturated sodium chloride solution (40 mL x 2) successively. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was separated by silica gel column chromatography (petroleum ether / ethyl acetate gradient elution) to obtain compound c (0.39 g, yield 98%).

[0077] (2) Compound c (0.39 g) prepared in the previous step was dissolved in 20 mL of acetic acid and stirred at 80 °C for 10 h. After the reaction was completed, the acetic acid was removed by evaporation under reduced pressure. The residue was added to 30 mL of water and extracted with DCM (20 mL x 3). The combined organic phase was dried over anhydrous sodium sulfate. After being concentrated under reduced pressure, compound d (0.36 g, yield 95%) was obtained by purification through silica gel column chromatography (petroleum ether / ethyl acetate gradient elution).

[0078] (3) Into a 25 mL round-bottom flask, compound d (0.5 mmol, 1 equiv.) and DCM (10 mL) were added. DBU (0.5 mmol, 1 equiv.) was slowly added by syringe. The reaction was stirred at room temperature for 10 min. After the reaction was completed, the solvent was concentrated under reduced pressure. The residue was purified by flash column chromatography to obtain compound e (0.077 g, yield 95%).

[0079] (4) Into a 25 mL round-bottom flask, 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. DIPEA (0.2 mmol, 2 equiv.) was added by syringe. After stirring for 10 min, compound e (0.12 mmol, 1.2 equiv.) was added. The reaction was stirred overnight (i.e. 12 h). After the reaction was completed, the mixture was extracted with DCM and washed with 5% citric acid aqueous solution (30 mL), saturated sodium bicarbonate solution (30 mL) and saturated brine (20 mL x 2) successively. The combined organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Compound 1 (0.05 g, yield 95%) was obtained by purification through flash column chromatography.

[0080] (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%).

[0081] (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%).

[0082] 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: 13C NMR (101 MHz, CDC13) 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 is HRMS (ESI) m / z: [M+H] + calcd for C 33 H 33 O3N7ClS + : 642.2049; found: 642.2028; Purity: 95% (calcd for represents theoretical calculation result; found represents actual analysis result, purity represents purity, hereinafter, calcd for, found and purity refer to the same meaning as here).

[0083] Synthesis of compound 3ca:

[0084] According to the synthetic route of this example, compound 3ca (24.1 mg, yield 35.8%) was obtained by referring to the synthesis method of compound 3c. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (500 MHz, CDC13) δ 7.40-7.32 (m, 1H), 7.27-7.22 (m, 2H), 7.15 (d, J = 2.6 Hz, 2H), 7.14-7.13 (m, 1H), 7.13-7.09 (m, 1H), 6.79-6.74 (m, 1H), 6.15 (d, J = 1.6 Hz, 1H), 5.00-4.95 (m, 1H), 4.83-4.78 (m, 2H), 4.53-4.49 (m, 1H), 3.73 (d, J = 7.2 Hz, 3H), 3.69 (d, J = 8.8 Hz, 3H), 3.40-3.27 (m, 2H), 2.95 (s, 2H), 2.89 (q, J = 6.6 Hz, 2H), 2.51 (d, J = 2.6 Hz, 3H), 2.28 (s, 3H), 1.57-1.51 (m, 3H). Its nuclear magnetic resonance carbon spectrum data is 13C NMR (126 MHz, CDC13) 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 spectrum data is HRMS (ESI) m / z: [M+H] + calcd for C 34 H 35 O4N7ClS + : 672.2141; found: 672.2154; Purity: 95.3%.

[0085] Synthesis of compound 3cb:

[0086] Compound 3cb (48.4 mg, 73.3%) was obtained according to the synthetic route of this example, referring to the synthetic method of compound 3c. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (500 MHz, CDC13) δ 7.36 (d, J = 6.3 Hz, 1H), 7.26 (d, J = 2.0 Hz, 1H), 7.25 (d, J = 1.9 Hz, 1H), 7.17 (d, J = 2.6 Hz, 1H), 7.16 (d, J = 2.6 Hz, 1H), 7.14 (s, 1H), 6.90-6.85 (m, 1H), 6.20-6.16 (m, 1H), 5.05 (d, J = 5.5 Hz, 1H), 4.83 (d, J = 15.6 Hz, 2H), 4.51-4.46 (m, 1H), 3.85-3.71 (m, 2H), 3.68 (d, J = 2.5 Hz, 3H), 3.38-3.20 (m, 2H), 2.93 (d, J = 6.2 Hz, 2H), 2.50 (d, J = 2.5 Hz, 3H), 2.28 (s, 3H), 1.55 (s, 3H). Its nuclear magnetic resonance carbon spectrum data is 13CNMR (126 MHz, CDC13) 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 spectrum data is HRMS (ESI) m / z: [M+H] + calcd for C 33 H 31 O3N7Cl3S + : 660.1923; found: 660.1954; Purity: 99%.

[0087] Synthesis of compound 3cc:

[0088] According to the synthetic route of this example, compound 3c was synthesized, compound 3cc (45.1 mg, yield 66.77%) was obtained. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (400 MHz, CDC13) 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 = 1.9 Hz, 1H), 5.00 - 4.90 (m, 2H), 4.60 (q, J = 6.8 Hz, 1H), 3.99 - 3.82 (m, 2H), 3.80 (d, J = 5.3 Hz, 3H), 3.56 - 3.31 (m, 2H), 3.05 (q, J = 4.7, 3.1 Hz, 2H), 2.61 (d, J = 3.7 Hz, 3H), 2.40 (s, 3H), 1.66 (s, 3H). Its nuclear magnetic resonance carbon spectrum data is 13C NMR (126 MHz, CDC13) 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 spectral data is HRMS (ESI) m / z: [M+H] + calcd for C 33 H 32 O3N7Cl2S + : 676.1659; found: 676.1643; Purity: 96%.

[0089] Synthesis of compound 3cd:

[0090] Compound 3cd (45.3 mg, yield 62.88%) was obtained according to the synthetic route of this example, referring to the synthetic method of compound 3c. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (400 MHz, CDC13) δ 7.73 - 7.61 (m, 1H), 7.51 (d, J = 8.5 Hz, 1H), 7.39 (d, J = 2.9 Hz, 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 = 7.0 Hz, 1H), 3.98 - 3.82 (m, 2H), 3.80 (d, J = 6.3 Hz, 3H), 3.54 - 3.31 (m, 2H), 3.04 (q, J = 6.0 Hz, 2H), 2.61 (d, J = 4.8 Hz, 3H), 2.40 (s, 3H), 1.66 (s, 3H). Its nuclear magnetic resonance carbon spectrum data is 13C10 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%.

[0091] Synthesis of compound 3ce:

[0092] 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%.

[0093] Synthesis of compound 3cf:

[0094] Compound 3cf (71.8 mg, yield 90%) was obtained following the synthetic route of this example, referring to the synthetic method of compound 3c. Its1H NMR data was 1 H NMR (400 MHz, CDC13) δ 7.77 (s, 1H), 7.65 (t, J = 6.1 Hz, 1H), 7.49 (s, 1H), 7.38 (d, J = 8.5 Hz, 2H), 7.31 (d, J = 8.7 Hz, 2H), 6.38 - 6.20 (m, 1H), 5.17 (d, J = 1.8 Hz, 1H), 5.02 - 4.84 (m, 2H), 4.53 (t, J = 6.6 Hz, 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). Its13C NMR data was 13 C NMR (101 MHz, CDC13) 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 spectrum data was HRMS (ESI) m / z: [M+H] + calcd for C 33 H 31 O3N7Br2ClS + : 798.0259; found: 798.0228; Purity: 97%.

[0095] Synthesis of compound 3cg:

[0096] Compound 3cg (50 mg, yield 73.73%) was obtained according to the synthetic route of this example, referring to the synthetic method of compound 3c. Its proton nuclear magnetic resonance spectrum data was 1 H NMR (400 MHz, CDCI3) δ 7.58 (d, J = 5.8 Hz, 1 H), 7.40 (d, J = 7.1 Hz, 1 H), 7.38 (q, J = 2.7 Hz, 2 H), 7.30 (d, J = 8.6 Hz, 2 H), 7.06 - 6.99 (m, 1 H), 6.30 (d, J = 1.6 Hz, 1 H), 5.19 (d, J = 1.8 Hz, 1 H), 4.92 (s, 2 H), 4.59 (d, J = 6.7 Hz, 1 H), 3.98 - 3.81 (m, 2 H), 3.80 (s, 3 H), 3.53 - 3.30 (m, 2 H), 3.04 (q, J = 6.8 Hz, 2 H), 2.62 (s, 3 H), 2.40 (s, 3 H), 1.67 (s, 3 H). Its carbon nuclear magnetic resonance spectrum data was 13 C NMR (126 MHz, CDCI3) 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 spectrum data was HRMS (ESI) m / z: [M+H] + calcd for C 33 H 31 O3N7ClF2S + : 678.1860; found: 678.1847; Purity: 95.5%.

[0097] Synthesis of compound 3ch:

[0098] Compound 3ch (24 mg, yield 34.99%) was obtained according to the synthetic route of this example, referring to the synthetic method of compound 3c. Its proton nuclear magnetic resonance spectrum data was 1 H NMR (600 MHz, CDCI3) δ 8.02 - 7.97 (m, 1 H), 7.84 - 7.79 (m, 1 H), 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 = 5.3Hz, 3H), 2.40 (s, 3H), 2.29 - 2.23 (m, 2H), 1.67 (s, 3H). Its carbon nuclear magnetic resonance spectrum data is 13 C NMR (126 MHz, CDC13) 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 spectrum data is HRMS (ESI) m / z: [M+H] + calcd for C 33 H 32 O5N8ClS + : 687.1899; found: 687.1885; Purity: 99%.

[0099] Example 3 Synthesis of compound 3b

[0100] Referring to the synthesis method of compound 3c in Example 2, compound g therein is replaced by (R is tBu), after the reaction, compound 3b (52 mg, yield is 76%) is obtained. Its hydrogen nuclear magnetic resonance spectrum data is 1 H NMR (400 MHz, CDC13) 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: 13 C10 NMR (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%.

[0101] Example 4: Synthesis of compounds 4c to 8c and 14c

[0102] Synthesis of compound 4c:

[0103] 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 = 6.9Hz, 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 nuclear magnetic resonance spectrum data is 13 CNMR (101 MHz, CDC13) 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 spectrum data is HRMS (ESI) m / z: [M+H] + calcd for C 32 H 31 O3N7ClS + : 628.1892; found: 628.1880; Purity: 99.7%.

[0104] Synthesis of compound 5c:

[0105] Referring to the synthesis steps (1) to synthesis step (6) of compound 3c in Example 2, the compound b therein is replaced by , after reaction, compound 5c (45.5 mg, yield 69.3%) is obtained. Its hydrogen nuclear magnetic resonance spectrum data is 1 H NMR (400 MHz, CDC13) 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= 6.9Hz, 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 nuclear magnetic resonance spectrum data is 13 C NMR (126 MHz, CDC13) 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 spectrum data is HRMS (ESI) m / z: [M+H] + calcd for C 34 H 35 O3N7ClS + : 656.2205; found: 656.2205; Purity: 97%.

[0106] Synthesis of compound 6c:

[0107] Referring to the synthesis steps (1) to (6) of compound 3c in Example 2, the compound b therein is replaced by After reaction, compound 6c (32.5 mg, yield 48.5%) is obtained. Its hydrogen nuclear magnetic resonance spectrum data is 1 H NMR (500 MHz, CDC13) δ 7.71 - 7.67 (m, 1H), 7.33 (d, J = 2.0 Hz, 1H), 7.32 (d, J = 2.0 Hz, 1H), 7.24 (s, 1H), 7.22 (d, J = 1.5 Hz, 1H), 7.19 (s, 2H), 7.18 - 7.16 (m, 1H), 7.16 (d, J = 1.8 Hz, 1H), 6.93 (t, J = 5.3 Hz, 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 = 7.6Hz, 2H), 1.66-1.61 (m, 2H), 1.59 (s, 3H). Its carbon nuclear magnetic resonance spectrum data is 13 C NMR (126 MHz, CDC13) 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 spectrum data is HRMS (ESI) m / z: [M+H] + calcd for C 35 H 37 O3N7ClS + : 670.2360; found: 670.2362; Purity: 96%.

[0108] Synthesis of compound 7c:

[0109] Referring to the synthesis steps (1) to synthesis step (6) of compound 3c in Example 2, the compound b therein is replaced by , after reaction, compound 7c (14.7 mg, yield 21.5%) is obtained. Its hydrogen nuclear magnetic resonance spectrum data is 1 H NMR (500 MHz, CDC13) 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: 13 C10 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%.

[0110] Synthesis of compound 8c:

[0111] 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 H NMR (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 =7.1Hz, 2H), 1.26 (s, 2H). Its carbon nuclear magnetic resonance spectrum data is 13 C NMR (151 MHz, CDC13) 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 spectrum data is HRMS (ESI) m / z: [M+H] + calcd for C 37 H 41 O3N7ClS + : 698.2667; found: 698.2675; Purity: 96.5%.

[0112] Synthesis of compound 14c:

[0113] Referring to the synthesis steps (4) to (6) of compound 3c in Example 2, the compound e therein is replaced by (R 1 = , which is directly purchased), after the reaction, compound 14c (64.1 mg, yield 94%) is obtained. Its hydrogen nuclear magnetic resonance spectrum data is 1 H NMR (600 MHz, CDC13) 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 C NMR (151 MHz, CDC13) 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 spectrum data is HRMS (ESI) m / z: [M+H] 13 C NMR (151 MHz, CDC13) 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 spectrum data is HRMS (ESI) m / z: [M+H] + C NMR (151 MHz, CDC13) 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 spectrum data is HRMS (ESI) m / z: [M+H] 36 H 37 O3N7ClS + : 682.2362; found: 682.2347; Purity: 99.9%.

[0114] Synthesis of compound 9c to compound 13c, compound 15c to compound 19c

[0115] Synthesis route 1:

[0116] .

[0117] Synthesis of compound g1 to compound g8:

[0118] 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.

[0119] Refer to the synthesis method of compound g1 mentioned above:

[0120] Replace “connector precursor A1” with “connector precursor A2”. ", to obtain compound g2;

[0121] Replace “connector precursor A1” with “connector precursor A3”. ", to obtain compound g3;

[0122] Replace “connector precursor A1” with “connector precursor A4”. ", to obtain compound g4;

[0123] Replace “connector precursor A1” with “connector precursor A5”. ", to obtain compound g5;

[0124] Replace “connector precursor A1” with “connector precursor A6”. ", to obtain compound g6;

[0125] Replace “connector precursor A1” with “connector precursor A7”. ", to obtain compound g7;

[0126] Replace “connector precursor A1” with “connector precursor A8”. ”, thus obtaining compound g8.

[0127] Synthesis Route 2:

[0128] .

[0129] In the process of preparing compounds 9c to 13c, 15c to 19c, the specific structure of in the structural formula of compound 1a in the above synthesis route is respectively the same as the specific structure of in the structural formula of the corresponding prepared compounds 9c to 13c, 15c to 19c. For example, if in the structural formula of compound 9c is , then in the structural formula of compound 1a used to prepare compound 9c is also . The rest of the compound preparation is similar.

[0130] Synthesis of compound 9c:

[0131] According to the above synthesis route, 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 with a syringe, and after stirring for 10 minutes, compound j1 (0.12 mmol, 1.2 equiv.) was added and stirred overnight. After the reaction was completed, the mixture was extracted with DCM, and then washed successively with 5% aqueous citric acid solution (30 mL), saturated sodium bicarbonate solution (30 mL) and saturated brine (20 mL x 2). The combined organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by flash column chromatography to obtain compound 1a.

[0132] A dry 10 mL glass test tube was taken, and compound 2 (0.15 mmol, 1.5 equiv.), compound 1a (0.1 mmol, 1 equiv.) and DABCO (0.02 mmol, 20 mol%) were added successively, and then DCM (1 mL) was injected, and stirred at room temperature for 1 hour. The reaction progress was monitored by TLC, and after completion, the solvent was removed under reduced pressure, and the residue was purified by flash column chromatography to obtain compound 9c (40.1 mg, yield 57.31%). The nuclear magnetic resonance hydrogen spectrum data thereof is 1 H NMR (400 MHz, CDCl3) δ 8.21 (d, J = 6.3 Hz, 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 = 6.4Hz, 2H), 1.68 (s, 3H). Its carbon nuclear magnetic resonance spectrum data is 13 CNMR (151 MHz, CDC13) 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 spectrum data is HRMS (ESI) m / z: [M+H] + calcd for C 35 H 36 O4N8ClS + : 699.2263; found: 699.2252; Purity: 99%.

[0133] Synthesis of compound 10c:

[0134] According to synthetic route 2 above, taking compound j2 and compound g1 as raw materials, referring to the synthetic method of compound 9c, compound 10c (19 mg, yield was 26.69%) was obtained. Its hydrogen nuclear magnetic resonance spectrum data is 1 H NMR (600 MHz, CDC13) δ 8.76 (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 nuclear magnetic resonance spectrum data is 13 C NMR (151 MHz, CDC13) 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 spectrum data is HRMS (ESI) m / z: [M+H] + calcd for C 36 H 38 O4N8ClS + : 713.2420; found: 713.2404; Purity: 95%.

[0135] Synthesis of compound 11c:

[0136] According to synthetic route 2 above, taking compound j3 and compound g2 as raw materials, referring to the synthetic method of compound 9c, compound 11c (54.7 mg, yield 84.8%) was obtained. Its hydrogen nuclear magnetic resonance spectrum data is 1 H NMR (600 MHz, CDC13) δ 7.93-7.85 (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 nuclear magnetic resonance spectrum data is 13 C NMR (151 MHz, CDC13) 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 spectrum data is HRMS (ESI) m / z: [M+H] + calcd for C 37 H 40 O4N8ClS + : 727.2576 : found: 727.2557; Purity: 99.7%.

[0137] Synthesis of compound 12c:

[0138] According to synthetic route 2 above, taking compound j3 and compound g5 as raw materials, referring to the synthetic method of compound 9c, compound 12c (61.4 mg, yield 72%) was obtained. Its hydrogen nuclear magnetic resonance spectrum data is 1 H NMR (600 MHz, CDC13) δ 7.68-7.62 (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 = 12.7Hz, 1H), 1.63 (s, 4H), 1.57 (s, 4H). Its nuclear magnetic resonance carbon spectrum data is 13 C NMR (151 MHz, CDC13) δ 173.1, 171.1, 170.0, 165.5, 163.8, 155.7, 153.3, 149.8, 142.3, 139.2, 136.7, 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 nuclear magnetic high resolution mass spectrum data is HRMS (ESI) m / z: [M+H] + calcd for C 44 H 51 O5N9ClS + : 852.3417; found: 852.3391; purity: 99.9%.

[0139] Synthesis of compound 13c:

[0140] According to synthetic route 2 above, with compound j3 and compound g4 as raw materials, referring to the synthesis method of compound 9c, compound 13c (80.0 mg, yield 92.3%) was obtained. Its nuclear magnetic resonance hydrogen spectrum data is 1H NMR (600 MHz, CDC13) δ 7.68 - 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 - 3.11 (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 = 7.5 Hz, 2H), 1.99 - 1.77 (m, 2H), 1.64 - 1.52 (m, 7H), 1.30 - 1.23 (m, 2H). Its carbon nuclear magnetic resonance spectrum data is 13 C NMR (151 MHz, CDC13) δ 173.2, 171.3, 170.0, 165.5, 163.8, 155.7, 153.3, 149.8, 142.4, 138.1, 136.7, 136.6, 136.6, 134.9, 134.7, 132.1, 130.9, 130.9, 130.5, 129.8, 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 spectrum data is HRMS (ESI) m / z: [M+H] + calcd for C 45 H 53 O5N9ClS + : 866.3573; found: 866.3546; purity: 97.8%.

[0141] Synthesis of compound 15c:

[0142] According to synthetic route 2 above, taking compound j3 and compound g3 as raw materials, referring to the synthesis method of compound 9c, compound 15c (64 mg, yield 79%) was obtained. Its hydrogen nuclear magnetic resonance spectrum data is 1 H NMR (400 MHz, CDC13) 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 = 5.4Hz, 3H). Its carbon nuclear magnetic resonance spectrum data is 13 C NMR (101 MHz, CDC13) 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 spectrum data is HRMS (ESI) m / z: [M+H] + calcd for C 41 H 45 O5N9ClS + : 810.2947; found: 810.2922; Purity: 98%.

[0143] Synthesis of compound 16c:

[0144] Compound 16c (73.3 mg, yield 87.4%) was obtained according to synthetic route 2 above by using compound j3 and compound g6 as raw materials, and referring to the synthetic method of compound 9c. Its proton nuclear magnetic resonance spectral data were 1 H NMR (600 MHz, CDC13) δ 7.73 (d, J = 7.5 Hz, 1H), 7.43 (d, J = 8.2 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 7.31-7.22 (m, 4H), 6.30 (s, 1H), 5.14 (s, 1H), 5.01 (s, 2H), 4.82 (d, J = 5.1 Hz, 1H), 4.03-3.72 (m, 10H), 3.64-3.42 (m, 5H), 3.06 (t, J = 6.1 Hz, 2H), 2.68 (s, 3H), 2.43 (s, 3H), 2.35 (d, J = 9.4 Hz, 2H), 2.25 (s, 2H), 1.69 (d, J = 17.8 Hz, 7H). Its carbon nuclear magnetic resonance spectral data were 13 C NMR (151 MHz, CDC13) 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 spectral data were HRMS (ESI) m / z: [M+H] + calcd for C 43 H 49 O5N9ClS + : 838.3260; found: 838.3235; purity: 99.5%.

[0145] Synthesis of compound 17c:

[0146] Compound 17c (70 mg, yield 82.1%) was obtained according to synthetic route 2 above by using compound j3 and compound g7 as raw materials, referring to the synthetic method of compound 9c. Its proton nuclear magnetic resonance spectral data were 1 H NMR (600 MHz, CDC13) δ 7.76-7.71 (m, 1H), 7.40 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 8.2 Hz, 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 nuclear magnetic resonance spectral data were 13 C NMR (126 MHz, CDC13) δ 173.2, 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 spectral data were HRMS (ESI) m / z: [M+H] + calcd for C 44 H 51 O5N9ClS + : 852.3417; found: 852.3393; purity: 99.8%.

[0147] Synthesis of compound 18c:

[0148] Compound 18c (79.1 mg, yield 90%) was obtained according to synthetic route 2 above by using compound j4 and compound g6 as raw materials, referring to the synthetic method of compound 9c. Its proton nuclear magnetic resonance spectral data were 1 H NMR (600 MHz, CDC13) δ 7.61 - 7.53 (m, 1H), 7.27 (d, J = 8.1 Hz, 2H), 7.17 (d, J = 8.2 Hz, 2H), 7.10 - 7.05 (m, 3H), 6.12 (s, 1H), 4.90 (s, 3H), 4.65 (t, J = 6.7 Hz, 1H), 4.61 - 4.51 (m, 1H), 3.89 (d, J = 13.5 Hz, 1H), 3.70 - 3.32 (m, 15H), 3.06 (t, J = 12.8 Hz, 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 nuclear magnetic resonance spectral data were 13 C NMR (151 MHz, CDC13) 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, 122.6, 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 spectral data were HRMS (ESI) m / z: [M+H] + calcd for C 46 H 53 O5N9ClS + : 878.3573; found: 878.3546; purity: 99%.

[0149] Synthesis of compound 19c:

[0150] Following Synthetic Route 2, compound 19c was obtained (44.6 mg, in 44.54% yield) using compound j3 and compound g8 as the starting materials, by referring to the synthetic method of compound 9c. Its1H NMR data was 1 H NMR (500 MHz, CDC13) δ 7.71 (d, J = 7.1 Hz, 1H), 7.41 (d, J = 8.2 Hz, 2H), 7.32 (d, J = 8.2 Hz, 3H), 7.24 (s, 3H), 6.27 (s, 1H), 5.08 (s, 1H), 4.99 (s, 2H), 4.79 (t, J = 6.6 Hz, 1H), 3.84 (s, 3H), 3.82 (s, 3H), 3.74 (d, J = 8.9 Hz, 1H), 3.72-3.67 (m, 3H), 3.67-3.59 (m, 12H), 3.55 (d, J = 5.1 Hz, 4H), 3.52-3.45 (m, 5H), 3.04 (t, J = 6.2 Hz, 2H), 2.69 (s, 1H), 2.66 (s, 3H), 2.61 (t, J = 6.3 Hz, 2H), 2.54 (d, J = 12.8 Hz, 1H), 2.48 (s, 1H), 2.46 (t, J = 5.7 Hz, 2H), 2.39 (s, 3H), 1.67 (s, 3H). Its13C NMR data was 13 C NMR (126 MHz, CDC13) 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 spectrum data was HRMS (ESI) m / z: [M+2H] 2+ calcd for C 50 H 66 O9N9ClS 2+ : 501.7179; found: 501.7191; Purity: 97%.

[0151] Test:

[0152] (1) High content analysis screening

[0153] a、Construction of a dual fluorescence screening model containing BD1 domain, BD2 domain and BD1-BD2 double domain

[0154] In the expression vector, the FLAG-tagged BRD4 protein domain (BD1 domain, BD2 domain and BD1-BD2 double domain, respectively) was fused with the mScarlet protein (red fluorescent protein) and coupled with the EGFP protein (green fluorescent protein) through the P2A self-cleavage peptide. Flag-BD1-mScarlet-P2A-EGFP recombinant plasmid, Flag-BD2-mScarlet-P2A-EGFP recombinant plasmid and Flag-BD1-BD2-mScarlet-P2A-EGFP recombinant plasmid were constructed, respectively. Then, the recombinant plasmids were transfected into HEK-293T cells, respectively. After successful transfection, the cells expressed Flag-BD1-mScarlet-P2A-EGFP dual fluorescent protein fusion, Flag-BD2-mScarlet-P2A-EGFP dual fluorescent protein fusion and Flag-BD1-BD2-mScarlet-P2A-EGFP dual fluorescent protein fusion, respectively.

[0155] Based on the ribosome skipping effect, the P2A peptide is broken at the glycine-proline site during translation, so that a single transcription sample can express two independent products, i.e. BRD4 domain (BD1 domain, BD2 domain and BD1-BD2 double domain, respectively)-mScarlet fusion protein and free EGFP protein.

[0156] The compounds (i.e. degradation agents based on benzimidazole fusion covalent warhead) prepared in the above examples 2, 4 and 5 were gradient diluted with complete medium to prepare compound solutions with different concentration gradients.

[0157] The required cells were transfected with the above recombinant plasmids, and after digestion and counting of the transfected cells, 10,000 cells per well were inoculated in a 96-well plate and cultured overnight. When the cells were fully adherent and the density was appropriate, fresh complete medium was replaced, compound solutions were added, 3 replicates per concentration, and an equal amount of PBS (phosphate buffered saline) was added to the edge of the well. After incubation in the incubator for 12 h, the cells were placed in a high-content analysis instrument for detection.

[0158] The transfected cells, when the benzimidazole-based fusion covalent warhead-based degrader is added, effectively degrade the target domain (BD1 domain, BD2 domain or BD1-BD2 double domain) of the BRD4 protein, resulting in a synchronous reduction of the mScarlet fluorescence covalently bound thereto, while the independently expressed EGFP remains stable. Thus, by quantitatively analyzing the fluorescence intensity ratio of mScarlet (as a target signal) to EGFP (as an internal reference signal) through a high-content imaging system, the efficiency 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 BRD4 protein.

[0159] (2) Western blotting experiment

[0160] The cell plate is rinsed with PBS, RIPA lysis buffer (containing protease inhibitors) is added, and the cells are scraped and transferred to an EP tube for crushing (20% power for 8 times, 2 seconds each time). Centrifuge at 12000 rpm for 20 min at 4°C, transfer the supernatant to a new EP tube. Take the above supernatant (fixed volume), add 5xLoading Buffer (loading buffer), mix well, heat at 95°C for 8 min, use or store at -80°C. Perform protein quantification, and prepare protein electrophoresis gel of different concentrations as needed.

[0161] Install the electrophoresis device, add electrophoresis liquid to the top, pull out the comb to leak the loading hole, add Maker (molecular weight standard protein) and protein sample in turn, set the electrophoresis conditions (70V, 30min), run the gel, and when it is flat, separate it under the voltage condition (120V, 60min), and stop when the double-color Loading Buffer reaches the bottom. Soak the appropriate size PVDF (polyvinylidene fluoride) membrane in methanol for 1 min, rinse with transfer solution, cover it on the gel block after electrophoresis, cut off the excess glue, transfer to the electrotransfer clamp to remove bubbles, clamp tightly and place in the electrotransfer tank, and transfer under the condition of 230mA constant current in ice bath, set the electrotransfer time according to the protein molecular weight.

[0162] After the end of the electrotransfer, the membranes were placed in 5% skimmed milk at room temperature for 1 h of slow shaking. After removal, they were rinsed 3 times for 20 min each time with 1x TBST buffer (Tris-Buffered Saline-Tween), the membrane was cut in the area where the protein of interest was located and marked, incubated in the primary antibody, rotating at 4°C overnight, after removal it was rinsed 3 times for 20 min each time with 1x TBST buffer, the membrane was incubated at room temperature in the secondary antibody for 1 h, after removal it was rinsed 3 times for 20 min each time with 1x TBST buffer, in a light-proof box the developing solution was prepared by mixing equal volumes of A and B from the developing kit, the membrane was immersed and incubated for 3 min, after removal it was developed and analyzed.

[0163] (3) Cell viability assay

[0164] After cell digestion and counting, 3000-5000 cells per well were inoculated in a 96-well plate, and the incubator was used overnight. The above prepared compound was prepared into a gradient concentration with complete culture medium by gradient dilution, and then the culture medium was replaced and added to the 96-well plate, 3 replicates for each concentration, PBS was added to the edge wells of the plate, and the plate was incubated in the incubator for 48 h. 10 μL of CCK-8 reagent was added to the 96-well plate, shaken and mixed, and incubated in the cell incubator for 2 h. After removal, the absorbance OD value at 450 nm was detected by a microplate reader, and the cell survival rate was calculated.

[0165] (4) Elution experiment (for evaluating the sustained effect of the compound on degrading BRD4 protein)

[0166] 6-well plates were used to culture MDA-MB-231 cells to 80% confluence, and 100 nM of the compound was added to the complete culture medium, and 0.1% DMSO (methyl sulfoxide) was used as a control. Incubate at 37°C, 5% CO2 for 12 h. After 12 h, wash with 37°C PBS twice, and replace the drug-free medium. Collect cells at 0 h, 6 h, 12 h, 24 h, 36 h, and 48 h after elution, and perform Western blotting test.

[0167] The results are as follows:

[0168] (1) The activity test results of each compound in the above Example 2, Example 4 and Example 5 are shown in Table 1. Figure 2 The con represents the control group, row min represents mScarlet / EGFP is 0.6, and row max represents mScarlet / EGFP is 1.0. Figure 2The degradation effects of each compound on the BD1 domain, BD2 domain and BD1-BD2 double domain in HEK293T cells stably expressing a high-content screening model are shown. It can be seen that compound 4c (i.e. compound LGF327) exhibits the best BRD4 degradation activity at concentrations of 100 nM and 1 μM, and its selective degradation ability is significantly better than that of other compounds.

[0169] The high-content screening results based on the double-domain fluorescence reporter system show that the series of compounds generally exhibit BRD4 degradation activity. In the comparison experiment between the BD1-BD2 double-domain reporter model and the single-domain (BD1 domain and BD2 domain) reporter model, except for compound 3ch, the degradation efficiency of other compounds in the double-domain system is significantly better than that in the single-domain system. This indicates that such compounds can target the BD1 domain and the BD2 domain synergistically, and more efficiently trigger the ubiquitin-proteasome-dependent degradation pathway.

[0170] Among the compounds singly substituted on the benzene ring, the degradation activity of the fluorine-substituted compound (such as 3cb) is relatively weak compared to other halogen- or alkyl-substituted compounds (3ca, 3cc and 3cd). It is speculated that the strong lipophilic fluorine atom can cause the electron cloud distribution of the molecule to deviate or affect the overall solubility of the molecule at the target binding interface. The activity of all double-substituted compounds (compounds ce to cg) is lower than that of the corresponding single-substituted compounds (compounds 3cb to 3cd), which may be due to the formation of steric hindrance effect by the double substituent group near the JQ1 binding pocket, which weakens the binding stability of the compound to the target protein. Therefore, it is speculated that the degradation efficiency of the above compounds on BRD4 protein is related to the synergistic targeting ability of the compounds to the BD1-BD2 double domain, and the benzene ring substituent affects the degradation activity through the dual mechanisms of electronic effect and steric effect.

[0171] 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 cause insufficient target binding adaptability due to fixed spatial conformation, resulting in reduced or even lost degradation efficiency (at low concentration).

[0172] In addition, for the flexible linker series, the present application further finds that the increase in linker length is slightly negatively correlated with the degradation effect, and the degradation activity of the compound 4c containing a single carbon chain is the best, with a fluorescence signal intensity of 62%, while the fluorescence signal intensity is 88% when the chain length increases to 5 or 6 carbons. This may be due to the fact that the too long alkane chain not only affects the efficiency of the compound penetrating the cell membrane, but also hinders the formation of a stable ternary complex between the BRD4 protein and the E3 ubiquitin ligase.

[0173] (2) The degradation activity of compound 4c (i.e. compound LGF327) on BET family proteins is shown in Figure 3 . It can be seen that compound LGF327 exhibits extremely high degradation potency (DC 50 = 5.3 nM, DC 50 represents the concentration of the compound required to degrade 50% of the target protein; D max > 90%, D max represents the maximum degradation rate) on BRD4 protein, and has no degradation effect on BRDT protein (testis-specific bromodomain-containing protein). In addition, compound LGF327 has certain degradation activity on BRD2 protein (bromodomain-containing protein 2) and BRD3 protein (bromodomain-containing protein 3), but the potency is significantly lower than that on BRD4 protein (about 3-5 times lower). For BRD4 protein, BRD4 protein degradation can be detected 1 hour after administration, 50% clearance rate is reached 3 hours later, and complete degradation is basically achieved 6 hours later.

[0174] (3) The degradation of BRD4 protein by compound LGF327 in different cancer cells is shown in Figure 4 . It can be seen that, compared with HL435 , compound LGF327 exhibits high-efficiency degradation of BRD4 protein only in specific cell types. Compound LGF327 can mediate significant degradation of BRD4 protein at a concentration of 100 nM in triple-negative breast cancer and colon cancer cell lines, and at a concentration of 1 μM in glioma cell U251, and no obvious degradation effect is observed in other tested cancer cell lines. In addition, as shown in (b) in Figure 4 , compound LGF327 has good sustained effect on degradation of BRD4 protein.

[0175] (3) In order to verify the degradation pathway of the compound LGF327, the MDA-MB-231 cells were treated with the compound LGF327 at a concentration of 100 nM and the autophagy inhibitors CQ (chloroquine) and BafA1 (bafilomycin), the 26S proteasome inhibitors MG132 (N-[(benzyloxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide) and PS341 (bortezomib), the E1 ubiquitin-activating enzyme inhibitor PYR41 (4-[4-[(5-nitro-2-furanyl)methylene]-3,5-dioxo-1-pyrazolidinyl]benzoic acid ethyl ester), and the NEDD8 activating enzyme inhibitor MLN4924 (aminosulfonic acid [(1S,2S,4R)-4-[4-[[(1S)-2,3-dihydro-1H-inden-1-yl]amino]-7H-pyrrolo[2,3-D]pyrimidin-7-yl]-2-hydroxycyclopentyl]methyl ester) at the same time, and then it was analyzed whether the degradation of the target protein was competitively inhibited by high content analysis, and the results are shown in Figures Figure 5 , Figure 6 and Figure 7 wherein ns represents no significant difference, p<0.05, p<0.01, p<0.001, p<0.0001.

[0176] Through high content screening and cell level experiments, the present application uses two types of specific inhibitors for verification: the first group of inhibitors inhibits the lysosome pathway, and the autophagy inhibitors CQ and BafA1 are used; the second group of inhibitors inhibits the ubiquitin-proteasome pathway, and the 26S proteasome inhibitors MG132 and PS341, the E1 ubiquitin-activating enzyme inhibitor PYR-41, and the NEDD8 activating enzyme inhibitor MLN4924 are used. The experimental results show that in the lysosome pathway inhibition group, whether chloroquine is used to inhibit autophagosome acidification or bafilomycin is used to interfere with lysosome maturation, the degradation activity of the compound LGF327 cannot be effectively hindered. However, the four ubiquitin-proteasome pathway inhibitors can significantly inhibit the degradation effect of the compound LGF327 on BRD4 protein. Therefore, it can be illustrated that the compound LGF327 mediates the degradation of BRD4 protein through the classical ubiquitin-proteasome pathway.

[0177] It should be understood that the application of the present application is not limited to the above examples, and those of ordinary skill in the art can make improvements or changes according to the above description, and all these improvements and changes shall belong to the protection scope of the appended claims of the present application.

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; The substituted phenyl group is Polylene substituted with fluorine, phenylene substituted with chlorine, phenylene substituted with bromine, or phenylene substituted with nitro; 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, Choose from one of the following structures: , , , , , , , and .

3. A method for preparing a degrading agent based on a benzimidazole fused covalent warhead as described in any one of claims 1-2, 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.

4. The preparation method according to claim 3, characterized in that, The preparation method includes the following steps: Will and After the reaction, we get .

5. The use of a benzimidazole-based degrading agent according to any one of claims 1-2 in the preparation of a medicament for treating breast cancer.

6. The application according to claim 5, characterized in that, The breast cancer mentioned is triple-negative breast cancer.

7. The use of a benzimidazole-based degrading agent according to any one of claims 1-2 in the preparation of a medicament for treating colon cancer.

8. The use of a benzimidazole-based degrading agent according to any one of claims 1-2 in the preparation of a medicament for treating glioma.

9. The application according to any one of claims 5-8, 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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