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

Abstract

The invention discloses a degradation agent based on indole group fusion covalent warheads and a preparation method and application thereof, and relates to the technical field of drug development, the structural formula of the degradation agent is as follows: R is a single bond or-NH-; a single bond, a, a, a, or a; and is-NH-, or; or; r1 and R2 are respectively and independently-H,-F,-Cl,-Br,-CH3,-OCH3,-NO2,-CH2-O-Ph or-CN; ph is phenyl; and a connection site is represented. According to the invention, an E3 ubiquitin ligase ligand is constructed by using an indole skeleton and acrylate, and then the E3 ubiquitin ligase ligand is connected with a BRD4 protein inhibitor JQ1 through a linker, so that a series of degradation agents based on indole group fusion covalent warheads are obtained, and targeted degradation of BRD4 protein is realized while an E3 ubiquitin ligase ligand library and a protein degradation targeted chimera molecular library are enriched.

Description

Technical Field

[0001] This invention relates to the field of drug development technology, and in particular to degrading agents based on indole-based fused covalent warheads, their preparation methods, and applications. Background Technology

[0002] The development and progression of most diseases are related to the abnormal expression or aggregation of proteins. Traditional drug development approaches target these pathological mechanisms by developing various small molecule or protein inhibitors. These inhibitors aim to occupy and block the active sites of target proteins, thereby suppressing their functional activity. However, approximately 80% of the proteins in the human proteome lack druggable active sites, making them difficult to target using traditional small molecule inhibitors.

[0003] Targeted protein degradation (TPD) is a technology that has been developed in recent years. It utilizes the main endogenous pathways of protein and organelle degradation in eukaryotic cells: the lysosomal pathway and the ubiquitin-proteasome system (UPS) pathway. It achieves functional intervention by specifically inducing the degradation of target proteins. It has shown great potential in the treatment of various diseases, especially in the treatment of tumors, intervention of neurodegenerative diseases, and regulation of metabolic disorders. It provides a new solution for "undruggable" targets that are difficult to deal with by traditional small molecule inhibitors and gene interference technologies.

[0004] Protein degradation-targeting chimeras (PROTACs) based on UPS are an important research direction in the field of TPD, such as... Figure 1 As shown (Ub represents ubiquitin, ATP represents adenine triphosphate), its structure comprises three key components: an E3 ubiquitin ligase-binding ligand, a target protein (POI)-specific ligand (POI ligand for short), and a linker connecting the two. These molecules form a ternary complex by simultaneously binding to the E3 ubiquitin ligase and the target protein. Through the synergistic action of the E1 ubiquitin activator, E2 ubiquitin conjugate, and E3 ubiquitin ligase, the E3 ubiquitin ligase labels the ubiquitin molecule onto the target protein. The target protein, after multiple rounds of ubiquitination modification, is subsequently recognized and degraded by the proteasome, while the protein degradation-targeting chimeric molecule can be recycled in this process. Due to the catalytic properties of this mechanism, the protein degradation-targeting chimeric can effectively induce target protein degradation under substoichiometric conditions even with low affinity for the target protein or binding to an inactive site.

[0005] Significant progress has been made in the field of protein degradation-targeting chimeras in recent years, successfully achieving the degradation of a variety of proteins, including endosome proteins, protein kinases, transcription factors, and some proteins traditionally considered "undruggable." At least a dozen protein degradation-targeting chimeras have entered clinical trials. Despite the rapid development of protein degradation-targeting chimera technology, the limited variety and quantity of available E3 ubiquitin ligases and their ligands remain a key challenge. Although the human genome encodes hundreds of E3 ubiquitin ligases, most developed protein degradation-targeting chimera molecules to date rely primarily on CRBN or VHL. Recent drug resistance to CRBN or VHL-based protein degradation-targeting chimeras further underscores the urgent need to develop other E3 ubiquitin ligases and their ligands to fully utilize the capabilities of protein degradation-targeting chimeras. Furthermore, the specific expression of E3 ubiquitin ligases in cells and tissues provides a theoretical basis for developing protein degradation-targeting chimera molecules with higher targeting selectivity.

[0006] Bromo-containing domain protein 4 (BRD4) is a member of the bromo-containing domain and extra terminal domain (BET) protein family. It can recognize acetylated histones and locate in the promoter or enhancer regions 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, participating in DNA damage repair and maintaining telomere function, thereby maintaining the survival of tumor cells.

[0007] Therefore, exploring and developing novel E3 ubiquitin ligases and their ligands to overcome the limitations of existing technologies, continuously enrich the protein degradation targeting chimeric molecular library, and achieve the degradation of target proteins such as BRD4 protein to treat cancers related to the function of target proteins is of great significance.

[0008] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0009] In view of the shortcomings of the prior art, the purpose of this invention is to provide a degrading agent based on an indole group fused covalent warhead, its preparation method and application, aiming to enrich the protein degradation targeted chimeric molecular library and achieve targeted degradation of BRD4 protein.

[0010] The technical solution of the present invention is as follows: A first aspect of the present invention provides a degrading agent based on an indole-based fused covalent warhead, wherein the degrading agent based on the indole-based fused covalent warhead has the following structural formula: ; R is a single bond or -NH-; (i.e., linker 1) is a single bond, , , , , , , , , or ; (i.e., linker 2) is -NH-, , or ; is or ; R , ,

[0011] , , , , , Figure 2 ,

[0012] and R 2 are located at any connectable positions on the benzene ring or pyridine ring, R 1 and R 2 are each independently -H, -F, -Cl, -Br, -CH3, -OCH3, -NO2, -CH2-O-Ph or -CN; Ph is phenyl; represents the connection site (as in the structural formulas hereinafter, unless otherwise specified, involved all represent the connection site, and when it appears again hereinafter , its meaning will not be elaborated again).

[0011] Through research, the present invention discovers that by introducing an electrophilic group-containing covalent warhead into the backbone structure and using the covalent warhead to form stable covalent bonds with specific amino acid residues in the E3 ubiquitin ligase or its substrate receptor subunit, the binding ability and degradation efficiency of the E3 ubiquitin ligase ligand can be enhanced, and the E3 ubiquitin ligase lacking a traditional small molecule binding pocket can be targeted, thereby expanding the range of targetable E3 ubiquitin ligases. In addition, the present invention uses the BRD4 protein as a phenotypic research model. First, based on the characteristic that methyl acrylate can specifically covalently bind to cysteine (Cys) residues (HS, mercapto) in proteins (as shown in Figure 2 ), it is combined on the indole backbone to develop a novel E3 ubiquitin ligase ligand, and then the E3 ubiquitin ligase ligand (obtained by introducing the covalent warhead acrylate into the indole backbone) is conjugated with the well-targeted BRD4 protein inhibitor JQ1 to construct a degrader based on the BRD4 protein target.

[0012] The indole-based covalent warhead-based degradative agent provided by this invention can recruit E3 ubiquitin ligases (different from known E3 ubiquitin ligases such as CRBN, VHL, DCAF11, and DCAF16) to form a ternary complex with the target protein (BRD4 protein), inducing BRD4 protein ubiquitination. The ubiquitinated BRD4 protein is then recognized by the proteasome and ultimately degraded. Furthermore, the indole-based covalent warhead-based degradative agent can also recruit E3 ubiquitin ligases (different from known E3 ubiquitin ligases such as CRBN, VHL, DCAF11, and DCAF16) to form a ternary complex with BRD3 (bromine-containing domain protein 3), inducing BRD3 protein ubiquitination and ultimately degrading it. Moreover, the E3 ubiquitin ligase ligand in the indole-based covalent warhead-based degradative agent has a strong binding affinity to the E3 ubiquitin ligase, thereby improving the degradation efficiency of both BRD4 and BRD3 proteins. Furthermore, the indole-based fused covalent warhead degrader exhibited significant antiproliferative activity in various tumor cell lines and was able to downregulate downstream signaling pathway proteins of c-Myc (a homolog of the myeloma virus oncogene). The indole-based fused covalent warhead degrader also significantly inhibited the migration ability of MDA-MB-231 cells (triple-negative breast cancer cells), indicating its potential as a cancer therapeutic agent.

[0013] The degrading agent based on indole-like group fused covalent warhead provided by this invention can not only achieve targeted degradation of BRD4 protein, but also targeted degradation of BRD3 protein. It can recruit a class of previously unreported E3 ubiquitin ligases, enriching the E3 ubiquitin ligase ligand library and the protein degradation targeted chimeric molecule library.

[0014] Optionally, R is -NH-; for , , , , or ; It is -NH-.

[0015] Optionally, R is -NH-; for , , or ; for , or .

[0016] Optionally, R is a single bond. It is a single key. for , or .

[0017] Optionally, R in 1 For -H, R in 2 For -H, -F, -Cl, -Br, -CH3, -OCH3, -NO2, -CH2-O-Ph or -CN; R in 1 -H; R in 2 It can be -H, -F, -Cl, -Br, -CH3, -OCH3, -NO2, -CH2-O-Ph or -CN.

[0018] Optionally, for or R 1 and R 2 Each can be represented independently as -Cl or -OCH3.

[0019] A second aspect of the present invention provides a method for preparing a degrading agent based on an indole-like group fused covalent warhead as described above, comprising the following steps: After reacting compound A with compound B, the degrading agent based on the indole group-fused covalent warhead is obtained; The structural formula of compound A is: ; The structural formula of compound B is as follows: Boc represents tert-butyloxycarbonyl.

[0020] In this invention, the degradation agent based on the indole-based fused covalent warhead is obtained by N-allylation reaction of compound A and compound B.

[0021] Optionally, when R is -NH-, the preparation method of compound A includes the following steps: Will After reacting with compound C, compound A is obtained; The structural formula of compound C is: .

[0022] Specifically, the preparation method of compound C includes the following steps: Compound E was added to dichloromethane and trifluoroacetic acid, and after the reaction, compound C was obtained; The structural formula of compound E is: .

[0023] In the structural formulas of compounds A and C above, R, , , R 1 and R 2 The R in the degrading agent structure of the indole-based covalent warhead fused with the indole group mentioned above, respectively. , , R 1 and R 2 same.

[0024] A third aspect of the present invention provides the application of the indole-based fused covalent warhead degrader described above in the preparation of BRD4 protein degraders or BRD3 protein degraders.

[0025] Beneficial Effects: This invention utilizes an indole backbone and acrylate to construct an E3 ubiquitin ligase ligand, which is then linked to the BRD4 protein inhibitor JQ1 via a linker, yielding a series of degradative agents based on indole-based fused covalent warheads. These indole-based fused covalent warhead degradative agents can target and efficiently degrade both BRD3 and BRD4 proteins. Therefore, this invention enriches the E3 ubiquitin ligase ligand library and the protein degradation targeting chimeric molecule library, achieving not only targeted degradation of BRD4 protein but also degradation of BRD3 protein. Attached Figure Description

[0026] Figure 1 This is a schematic diagram illustrating the mechanism of target protein degradation by a protein degradation-targeting chimera in existing technologies.

[0027] Figure 2 This is a schematic diagram illustrating the mechanism by which the degrading agent based on the indole group-fused covalent warhead degrades the BRD4 protein in this invention.

[0028] Figure 3 The results of the construction and expression of the high-content screening platform are shown in the figure. (a) is the Western blot result of the dual-fluorescence screening model, and (b) is the fluorescence image of the dual-fluorescence screening model.

[0029] Figure 4 The graph shows the activity test results of each compound at different concentrations (100 nM, 1 μM) in the examples.

[0030] Figure 5 This figure shows the degradation effects of the four compounds with the highest degradation effects on endogenous BRD4 protein in the high-content analysis results.

[0031] Figure 6The graph shows the degradation activity results of compound D29, where (a) is the degradation effect of compound D29 on BET family proteins, (b) is the semi-quantitative result of compound D29 on BET family proteins, and (c) is the DC degradation of BRD3 and BRD4 proteins by compound D29. 50 The fitting results are shown in the following figures: (d) shows the time-dependent degradation of BRD4 protein by compound D29; (e) shows the semi-quantitative degradation of BRD4 protein by compound D29 at different times; (f) shows the elution effect of compound D29; and (g) shows the semi-quantitative elution result of compound D29.

[0032] Figure 7 Figures show the results of the degradation mechanism verification of compound D29, where (a) is the degradation mechanism result of compound D29 under a high content analysis microscope, and (b) is the semi-quantitative result of the degradation mechanism verification of compound D29 under a high content analysis microscope.

[0033] Figure 8 The figures show the results of the lysosomal pathway inhibition experiment and the proteasome pathway inhibition experiment of compound D29. Among them, (a) is the effect of the lysosomal pathway inhibition experiment of compound D29, (b) is the semi-quantitative result of the lysosomal pathway inhibition experiment of compound D29, (c) is the effect of the proteasome pathway inhibition experiment of compound D29, and (d) is the semi-quantitative result of the proteasome pathway inhibition experiment of compound D29.

[0034] Figure 9 The graph shows the test results for the E3 ubiquitin ligase recruited by compound D29. Detailed Implementation

[0035] This invention provides a degrading agent based on an indole-based 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 merely illustrative of the invention and are not intended to limit the invention.

[0036] 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.

[0037] If the embodiments of the present invention involve descriptions such as "first" or "second", such descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated.

[0038] The present invention will be further described below through specific embodiments.

[0039] The following examples and synthetic routes contain the meanings of some symbols: DIPEA: N,N-diisopropylethylamine; EDCl: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; HOBt: 1-Hydroxybenzotriazole; DMF: N,N-dimethylformamide; rt: room temperature; eq: equivalent; TFA: Trifluoroacetic acid; DCM: Dichloromethane; MeOH: Methanol; DABCO: 1,4-diazabicyclo[2.2.2]octane; EtOAc: Ethyl acetate; PE: Petroleum ether; JQ1: The structural formula is .

[0040] In the following examples, the structural formulas of compounds D1 to D16 are as follows: ,in, The specific structures of the (indole skeleton) are shown in Tables 1 and 2.

[0041] Table 1. Specific structures of the indole skeleton in the structural formulas of compounds D1 to D10

[0042] Table 2. Specific structures of the indole skeleton in the structural formulas of compounds D11 to D16

[0043] The structural formulas of compounds D17 to D21 are as follows: The specific structure of X is shown in Table 3.

[0044] Table 3. Specific structures of X in the structural formulas of compounds D17 to D21

[0045] The structural formulas of compounds D22 to D36 are as follows: ,in, and The specific structure is shown in Tables 4, 5 and 6.

[0046] Table 4. Structural formulas of compounds D22 to D26 and Specific structure

[0047] Table 5. Structural formulas of compounds D27 to D31 and Specific structure

[0048] Table 6. Structural formulas of compounds D32 to D36 and Specific structure

[0049] Example 1: Synthesis of compounds D1 to D16 The synthesis route is as follows: .

[0050] In this synthetic route, the structural formulas of the final products compounds D1 to D16 are as follows: The specific structure is shown in Table 1 and Table 2.

[0051] In the preparation of compounds D1 to D16, the structures of compounds 1, 3, 4, and 5 in the above synthetic route are as follows: The specific structures are respectively related to the structural formulas of compounds D1 to D16 prepared accordingly. The specific structures are the same. For example, the structure of compound D1 is the same. for (R) 2 = -H), then the corresponding structural formulas of compounds 1, 3, 4, and 5 used to prepare compound D1 are: Also for (R) 2 = -H). The preparation of other compounds follows the same procedure.

[0052] Synthesis of compound D1: Compound 1 (0.1612 g, 1 mmol), EDCI (0.29 g, 1.5 mmol), HOBt (0.27 g, 2 mmol), and DIPEA (0.52 mL, 3 mmol) were dissolved together in DMF (5 mL) and then activated in an ice bath for 30 min (to ensure the smooth progress of the reaction, the reaction system was cooled in an ice bath to lower the reaction temperature and control the reaction rate). After activation, compound 2 (0.21 g, 1.2 mmol) was added to the reaction system, and the reaction was allowed to proceed overnight at room temperature (i.e., 12 h to ensure the reaction proceeds fully). After the reaction was completed, the reaction solution was extracted with citric acid aqueous solution (citric acid mass percentage of 5%), saturated NaHCO3 aqueous solution and saturated saline solution in sequence with EtOAc at a volume ratio of 1:1 (i.e., a total of 3 extractions). The organic phase was dried and concentrated, and separated by column chromatography (using PE and EtOAc at a volume ratio of 4:1 as eluent) to obtain compound 3 (0.28 g, yield of 88.7%).

[0053] Compound 3 (0.28 g, 0.89 mmol) was mixed with DCM (1 mL) and TFA (1 mL) and stirred at room temperature for 0.5 h. After the reaction was completed, the mixture was extracted three times with DCM and H2O in a 1:1 volume ratio. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound 4 (0.18 g, yield 92.4%).

[0054] Compound 4 (43 mg, 0.2 mmol), EDCI (57.51 g, 0.3 mmol), HOBt (54 mg, 0.4 mmol), and DIPEA (104 μL, 0.6 mmol) were dissolved in DMF (1 mL). The reaction system was then activated in an ice bath for 30 min, followed by the addition of JQ1 (80 mg, 0.2 mmol), and the reaction was stirred at room temperature overnight (12 h). After the reaction was complete, the reaction solution was extracted with citric acid aqueous solution (5% by mass), saturated NaHCO3 aqueous solution, and saturated saline solution in a 1:1 volume ratio with EtOAc (i.e., a total of 3 extractions). The organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure to obtain the crude product. Subsequently, the crude product was purified by column chromatography (using DCM and MeOH in a 20:1 volume ratio as eluents) to obtain compound 5 (0.10 g, yield 84.7%).

[0055] Compound 5 (101 mg, 0.17 mmol) and compound B (44 mg, 1.2 mmol, see above for specific structure) were dissolved in DCM (1 mL). DABCO (4 mg, 0.034 mmol) was added to the reaction solution. The reaction system was stirred at room temperature for 0.5 h. The solvent was removed by rotary evaporation to obtain the crude product. Then, the crude product was separated by column chromatography (using DCM and MeOH in a volume ratio of 20:1) to obtain compound D1 (107 mg, yield 90.2%).

[0056] The proton NMR spectrum data of compound D1 are as follows: 1 H NMR (600MHz, CDCl3) δ 7.96–7.90 (m, 1H), 7.88–7.75 (m, 1H), 7.59 (d, J =8.0Hz, 1H), 7.42–7.38 (m, 2H), 7.32–7.29 (m, 2H), 7.28–7.24 (m, 2H), 7.13–7.09 (m, 2H), 6.15 (s, 1H), 5.51 (s, 2H), 4.86 (s, 1H), 4.79–4.73 (m, 1H), 3.81 (s, 3H), 3.61–3.50 (m, 4H), 3.45–3.37 (m, 2H), 2.66 (s, 3H), 2.40 (s, 3H), 1.81–1.75 (m, 2H), 1.63 (s, 3H).

[0057] The carbon NMR spectrum data of compound D1 are as follows: 13 C NMR (151MHz, CDCl3) δ 171.6, 166.2, 164.1, 162.3, 155.8, 150.0, 145.5, 142.6, 139.3, 138.3, 136.9, 136.4, 132.0, 131.9, 131.1, 131.0, 130.6, 1 29.8, 128.7, 126.4, 124.8, 124.1, 122.0, 120.6, 110.4, 104.9, 54.4, 54.3, 52.0, 44.8, 39.0, 36.3, 29.7, 14.4, 13.1, 11.8.

[0058] The high-resolution mass spectrometry data of compound D1 are as follows (HRMS-ESI, m / z): [M+H] + calcd for C 36 H 37 O4N7ClS +: 698.2311; found: 698.2309; purity: 99.32% (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).

[0059] Synthesis of compound D2: Following the synthetic method for compound D1, compound D2 (63.7 mg, yield 89.3%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (600MHz, CDCl3) δ 7.96–7.88 (m, 1H), 7.71–7.58 (m, 1H), 7.40–7.34 (m, 2H), 7.32–7.26 (m, 2H), 7.17–7.09 (m, 2H), 7.02 (d, J =8.3Hz, 1H), 6.78–6.70 (m, 1H), 6.14 (s, 1H), 5.51–5.41 (m, 2H), 4.87 (d, J =2.2Hz, 1H), 4.74–4.65 (m, 1H), 3.77 (d, J =4.1 Hz, 3H), 3.59–3.53 (m, 1H), 3.51–3.36 (m, 5H), 2.65 (s, 3H), 2.38 (s, 3H), 1.78–1.71 (m, 2H), 1.63 (s, 3H). Its carbon NMR data are as follows: 13 C10 NMR (151 MHz, CDCl3) δ 171.5, 166.1, 164.2, 161.8, 157.7, 156.0, 155.8, 150.1, 140.7, 140.6, 136.9, 136.6, 136.5, 132.2, 131.1, 131.0, 130.6, 129.8, 128.7, 125.0, 124.6, 124.5, 115.9, 115.8, 114.1, 105.2, 54.4, 54.3, 52.1, 45.3, 39.1, 36.3, 29.7, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd forC 36 H 36 O4N7ClFS + :716.2217; found: 716.2211; purity: 99.55%.

[0060] Synthesis of compound D3: Following the synthetic method for compound D1, compound D3 (38.4 mg, 52.5%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (400MHz, CDCl3) δ 7.97–7.87 (m, 1H), 7.69–7.57 (m, 1H), 7.36 (d, J =8.0Hz, 2H), 7.29–7.25 (m, 2H), 7.17–7.01 (m, 4H), 6.13 (s, 1H), 5.45 (s, 2H), 4.86 (s, 1H), 4.75–4.69 (m, 1H), 3.77 (d, J =2.2Hz, 3H), 3.56–3.36 (m, 6H), 2.63 (d, J =2.4Hz, 3H), 2.36 (d, J =2.3 Hz, 3H), 1.81–1.74 (m, 2H), 1.60 (s, 3H). Its carbon NMR data are: 13 C10 NMR (101 MHz, CDCl3) δ 171.4, 166.1, 164.2, 161.9, 155.7, 150.0, 138.8, 136.8, 136.6, 136.5, 132.5, 131.9, 131.1, 131.0, 130.6, 129.8, 128.7, 127.8, 127.0, 125.3, 125.1, 124.5, 120.3, 114.8, 109.2, 103.3, 54.3, 52.1, 45.2, 39.0, 36.5, 36.0, 29.7, 14.3, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 36 H 36 O4N7Cl2S + :732.1921; found: 732.1925; purity: 99.34%.

[0061] Synthesis of compound D4: Following the synthetic method for compound D1, compound D4 (65.3 mg, yield 84.3%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (400MHz, CDCl3) δ 8.09–7.95 (m, 1H), 7.80–7.66 (m, 1H), 7.39 (d, J =8.3Hz, 2H), 7.34–7.27 (m, 3H), 7.22 (d, J=8.3Hz, 1H), 7.13–7.07 (m, 2H), 6.16 (s, 1H), 5.48 (s, 2H), 4.89 (s, 1H), 4.81–4.73 (m, 1H), 3.80 (s, 3H), 3.60–3.41 (m, 6H), 2.66 (s, 3H), 2.39 (s, 3H), 1.86–1.77 (m, 2H), 1.62 (s, 3H). Its carbon NMR data are as follows: 13 C10 NMR (101 MHz, CDCl3) δ 171.3, 166.1, 164.2, 161.9, 155.7, 150.0, 139.3, 138.4, 136.8, 136.6, 136.5, 132.5, 131.9, 131.1, 131.0, 130.6, 129.8, 128.7, 127.1, 125.1, 124.8, 123.4, 115.8, 114.1, 109.7, 105.0, 54.3, 53.5, 52.1, 45.2, 38.9, 36.5, 29.7, 14.4, 13.1, 11.9. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 36 H 36 O4N7BrClS + :776.1416; found: 776.1417; purity: 99.77%.

[0062] Synthesis of compound D5: Following the synthetic method for compound D1, compound D5 (44 mg, yield 61.4%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (600MHz, CDCl3) δ 7.85–7.75 (m, 1H), 7.39 (d, J =7.8Hz, 3H), 7.30 (d, J =7.6Hz, 2H), 7.22–7.16 (m, 2H), 7.04 (s, 1H), 7.02–6.97 (m, 1H), 6.14 (s, 1H), 5.53–5.42 (m, 2H), 4.86 (s, 1H), 4.70–4.64 (m, 1H), 3.79 (s, 3H), 3.61–3.56 (m, 1H), 3.52–3.40 (m, 4H), 3.33 (s, 1H), 2.67 (s, 3H), 2.40 (s, 3H), 1.81–1.70 (m, 2H), 1.64 (s, 3H). Its carbon NMR data are as follows: 13CNMR (151 MHz, CDCl3) δ 171.6, 166.2, 164.4, 161.9, 157.4, 155.6, 150.1, 137.1, 136.8, 136.2, 134.9, 133.3, 132.0, 131.3, 131.0, 130.6, 129.9, 128.8, 126.5, 126.5, 125.0, 114.1, 113.0, 111.4, 106.3, 104.7, 54.4, 52.1, 45.0, 39.2, 36.3, 35.5, 29.7, 14.4, 13.2, 11.9. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 36 H 36 O4N7ClFS + :716.2217; found: 716.2213; purity: 99.65%.

[0063] Synthesis of compound D6: Following the synthetic method for compound D1, compound D6 (63.6 mg, yield 82.0%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (400MHz, CDCl3) δ 7.89–7.74 (m, 1H), 7.65–7.49 (m, 2H), 7.27 (d, J =8.3Hz, 2H), 7.22–7.16 (m, 3H), 7.03 (d, J =8.8 Hz, 1H), 6.88 (s, 1H), 6.03 (s, 1H), 5.34 (s, 2H), 4.75 (s, 1H), 4.66–4.56 (m, 1H), 3.68 (s, 3H), 3.50–3.23 (m, 6H), 2.53 (s, 3H), 2.29 (s, 3H), 1.72–1.62 (m, 2H), 1.52 (s, 3H). Its carbon NMR data are as follows: 13C10 NMR (101 MHz, CDCl3) δ 171.6, 166.1, 164.3, 161.9, 155.7, 150.1, 139.3, 137.0, 136.8, 136.6, 136.4, 133.0, 131.9, 131.2, 131.0, 130.6, 129.8, 128.8, 127.9, 126.9, 125.1, 124.3, 114.1, 113.7, 112.0, 104.2, 54.4, 52.1, 45.0, 39.0, 36.3, 35.7, 29.7, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 36 H 36 O4N7BrClS + :776.1416; found: 776.1416; purity: 99.91%.

[0064] Synthesis of compound D7: Following the synthetic method for compound D1, compound D7 (43.5 mg, yield 61.1%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (400MHz, CDCl3) δ 7.82–7.62 (m, 1H), 7.60–7.48 (m, 1H), 7.43–7.26 (m, 5H), 7.18–7.11 (m, 1H), 7.07 (d, J =8.5Hz, 1H), 7.01–6.93 (m, 1H), 6.11 (s, 1H), 5.44 (s, 2H), 4.83 (d, J =5.1Hz, 1H), 4.73–4.65 (m, 1H), 3.93–3.75 (m, 3H), 3.63–3.31 (m, 6H), 2.70–2.61 (m, 3H), 2.50–2.34 (m, 6H), 1.83–1.70 (m, 2H), 1.69–1.58 (m, 3H). Its carbon NMR data are as follows: 13CNMR (101MHz, CDCl3) δ 171.5, 166.3, 164.2, 162.4, 155.7, 150.0, 143.0, 142.6, 137.0, 136.8, 136.4, 132.0, 131.8, 131.1, 131.0, 130.6, 129.9, 129 .8, 128.8, 126.6, 126.0, 124.8, 121.4, 114.1, 110.1, 104.4, 54.4, 53.5, 52.0, 44.9, 39.1, 36.3, 29.7, 21.4, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 37 H 39 O4N7ClS + :712.2467; found: 712.2459; purity: 99.56%.

[0065] Synthesis of compound D8: Following the synthetic method for compound D1, compound D8 (53.9 mg, yield 74.1%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (600MHz, CDCl3) δ 7.71–7.63 (m, 1H), 7.60–7.49 (m, 1H), 7.39–7.34 (m, 2H), 7.30–7.27 (m, 2H), 7.13 (dd, J =9.1, 3.3 Hz, 1H), 7.01–6.96 (m, 2H), 6.92–6.88 (m, 1H), 6.15–6.08 (m, 1H), 5.42 (s, 2H), 4.82 (s, 1H), 4.70–4.61 (m, 1H), 3.78 (s, 3H), 3.76 (s, 3H), 3.56–3.51 (m, 1H), 3.48–3.37 (m, 4H), 3.34–3.29 (m, 1H), 2.62 (s, 3H), 2.37 (s, 3H), 1.77–1.67 (m, 2H), 1.61 (s, 3H). Its carbon NMR data are as follows: 13C NMR (151MHz, CDCl3) δ 171.7, 166.3, 164.3, 162.3, 155.6, 154.7, 150.2, 142.6, 138.3, 137.0, 136.4, 136.2, 133.7, 132.1, 131.9, 131.3, 131.0, 130 .6, 129.8, 128.8, 126.7, 124.9, 115.2, 111.3, 104.5, 102.7, 55.8, 55.7, 54.3, 52.0, 44.9, 38.9, 36.3, 29.2, 14.3, 13.1, 11.7. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 37 H 39 O5N7ClS + :728.2416; found: 728.2434; purity: 99.15%.

[0066] Synthesis of compound D9: Following the synthetic method for compound D1, compound D9 (44.9 mg, yield 60.5%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (600MHz, CDCl3) δ 8.56–8.48 (m, 1H), 8.21–8.08 (m, 2H), 7.48 (t, J =6.5Hz, 1H), 7.39–7.34 (m, 2H), 7.34–7.27 (m, 3H), 7.25 (s, 1H), 6.19 (s, 1H), 5.60–5.44 (m, 2H), 5.04–4.97 (m, 1H), 4.73–4.64 (m, 1H), 3.77 (s, 3H), 3.61–3.39 (m, 5H), 3.35–3.28 (m, 1H), 2.65 (s, 3H), 2.39 (s, 3H), 1.82–1.71 (m, 2H), 1.64 (s, 3H). Its carbon NMR data are as follows: 13C10 NMR (151 MHz, CDCl3) δ 171.7, 165.9, 164.4, 161.4, 155.7, 150.1, 142.3, 140.8, 139.3, 137.1, 136.3, 136.2, 135.4, 132.0, 131.3, 131.0, 130.5, 129.8, 128.8, 125.8, 125.6, 119.3, 119.2, 114.1, 110.7, 106.9, 54.4, 52.2, 45.4, 39.2, 36.4, 35.8, 29.7, 14.4, 13.2, 11.9. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 36 H 36 O6N8ClS + :743.2162; found: 743.2154; purity: 99.33%.

[0067] Synthesis of compound D10: Following the synthetic method for compound D1, compound D10 (60.5 mg, yield 75.2%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (400MHz, CDCl3) δ 7.79–7.65 (m, 1H), 7.64–7.50 (m, 1H), 7.39–7.27 (m, 6H), 7.24–7.18 (m, 3H), 7.10–7 .05 (m, 1H), 7.03–6.97 (m, 1H), 6.95–6.88 (m, 2H), 6.04 (s, 1H), 5.36 (s, 2H), 4.95 (d, J =3.7Hz, 2H), 4.76 (s, 1H), 4.67–4.57 (m, 1H), 3.75–3.66 (m, 3H), 3.52–3.25 (m, 6H), 2.55 (d, J =3.7Hz, 3H), 2.29 (d, J =3.8Hz, 3H), 1.71–1.60 (m, 2H), 1.54 (d, J =3.6Hz, 3H). Its carbon NMR data are: 13C NMR (101MHz, CDCl3) δ 171.5, 166.2, 164.2, 162.2, 155.7, 153.8, 150.0, 137.4, 137.0, 136.9, 1 36.4, 135.6, 133.8, 132.2, 132.0, 131.1, 131.0, 130.6, 129.8, 128.8, 128 0.5, 127.9, 127.5, 126.6, 124.8, 124.4, 118.1, 115.7, 114.1, 111.3, 104.5, 104.2, 70.7, 54.4, 52.0, 44.9, 39.0, 36.3, 35.5, 29.7, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 43 H 43 O5N7ClS + :804.2729; found: 804.2733; purity: 96.22%.

[0068] Synthesis of compound D11: Following the synthetic method for compound D1, compound D11 (63.5 mg, yield 88.8%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (600MHz, CDCl3) δ 7.78–7.70 (m, 1H), 7.51–7.48 (m, 1H), 7.43–7.35 (m, 3H), 7.30 (d, J =8.0Hz, 2H), 7.07 (s, 1H), 6.92 (dd, J =9.8, 2.2Hz, 1H), 6.89–6.84 (m, 1H), 6.15 (s, 1H), 5.42 (s, 2H), 4.88 (s, 1H), 4.67 (d, J =7.0Hz, 1H), 3.79 (s, 3H), 3.61–3.55 (m, 1H), 3.49–3.40 (m, 4H), 3.34–3.28 (m, 1H), 2.67 (s, 3H), 2.39 (s, 3H), 1.74 (s, 2H), 1.63 (s, 3H). Its carbon NMR data are as follows: 13C10 NMR (151 MHz, CDCl3) δ 171.6, 166.1, 164.3, 161.9, 160.3, 155.6, 150.1, 138.6, 138.6, 137.1, 136.5, 136.3, 132.6, 132.0, 131.3, 131.0, 130.6, 129.9, 128.8, 125.0, 123.1, 123.1, 122.8, 110.0, 105.1, 96.7, 59.5, 54.4, 52.1, 45.1, 39.2, 36.3, 29.7, 14.4, 13.2, 11.9. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd forC 36 H 36 O4N7ClFS + :716.2217; found: 716.2210; purity: 99.53%.

[0069] Synthesis of compound D12: Following the synthetic method for compound D1, compound D12 (72.2 mg, yield 93.0%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (400MHz, CDCl3) δ 7.93–7.83 (m, 1H), 7.74–7.63 (m, 1H), 7.41–7.34 (m, 4H), 7.27 (d, J =8.7Hz, 2H), 7.19–7.14 (m, 1H), 7.02 (s, 1H), 6.13 (s, 1H), 5.40 (s, 2H), 4.85–4.80 (m, 1H), 4.71–4.64 (m, 1H), 3.78 (s, 3H), 3.55–3.28 (m, 6H), 2.62 (s, 3H), 2.36 (s, 3H), 1.78–1.68 (m, 2H), 1.60 (s, 3H). Its carbon NMR data are as follows: 13C10 NMR (101 MHz, CDCl3) δ 171.6, 166.0, 164.2, 161.8, 155.7, 150.0, 139.3, 139.0, 136.9, 136.5, 136.3, 132.5, 131.9, 131.2, 130.9, 130.6, 129.8, 128.8, 125.2, 125.0, 124.1, 123.2, 117.8, 114.1, 113.3, 105.0, 54.3, 52.1, 45.0, 39.0, 36.2, 35.6, 29.7, 14.3, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 36 H 36 O4N7BrClS + :776.1416; found: 776.1420; purity: 98.78%.

[0070] Synthesis of compound D13: Following the synthetic method for compound D1, compound D13 (63.4 mg, yield 87.6%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (400MHz, CDCl3) δ 8.11–8.00 (m, 1H), 7.68–7.50 (m, 3H), 7.37 (d, J =8.2Hz, 2H), 7.33–7.27 (m, 3H), 7.12 (s, 1H), 6.18 (s, 1H), 5.59–5.41 (m, 2H), 4.96–4.91 (m, 1H), 4.68–4.63 (m, 1H), 3.78 (s, 3H), 3.60–3.26 (m, 6H), 2.64 (s, 3H), 2.39 (s, 3H), 1.79–1.68 (m, 2H), 1.62 (s, 3H). Its carbon NMR data are as follows: 13C NMR (101MHz, CDCl3) δ 171.7, 165.8, 164.3, 161.4, 155.6, 150.0, 146.8, 137.0, 136.9, 136.3, 136.3, 135.6, 132.0, 131.2, 130.9, 130.5, 129.8, 129. 5, 128.8, 125.5, 123.1, 122.9, 120.1, 118.8, 115.6, 106.4, 105.0, 54.4, 52.2, 45.1, 39.1, 36.3, 35.7, 29.1, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 37 H 36 O4N8ClS + :723.2263; found: 723.2260; purity: 97.10%.

[0071] Synthesis of compound D14: Following the synthetic method for compound D1, compound D14 (41.9 mg, yield 54.7%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (600MHz, CDCl3) δ 7.98–7.88 (m, 1H), 7.47 (d, J =8.1Hz, 2H), 7.40 (d, J =8.1 Hz, 2H), 7.35 (s, 2H), 7.23–7.15 (m, 2H), 6.27 (s, 1H), 5.58–5.46 (m, 2H), 5.01 (s, 1H), 4.81–4.74 (m, 1H), 3.89 (s, 3H), 3.69–3.44 (m, 6H), 2.77 (s, 3H), 2.49 (s, 3H), 1.91–1.81 (m, 2H), 1.73 (s, 3H). Its carbon NMR data are as follows: 13C10 NMR (151 MHz, CDCl3) δ 171.5, 165.9, 164.3, 161.5, 155.6, 150.1, 148.5, 142.7, 138.7, 137.0, 136.4, 136.3, 133.2, 131.9, 131.3, 131.0, 130.6, 129.9, 129.8, 128.8, 127.7, 125.4, 124.1, 121.0, 109.3, 103.4, 56.1, 54.3, 52.1, 45.4, 39.2, 36.0, 29.3, 14.4, 13.2, 11.8. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 36 H 35 O4N7Cl3S + :766.1531; found: 766.1528; purity: 99.89%.

[0072] Synthesis of compound D15: Following the synthetic method for compound D1, compound D15 (71.2 mg, yield 93.9%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (600MHz, CDCl3) δ 7.66–7.57 (m, 1H), 7.55–7.49 (m, 1H), 7.39–7.33 (m, 2H), 7.31–7.27 (m, 2H), 7.00 (d, J =2.8Hz, 1H), 6.98 (s, 1H), 6.65 (s, 1H), 6.12 (d, J =2.0Hz, 1H), 5.44 (d, J =2.2Hz, 2H), 4.83 (d, J =2.1Hz, 1H), 4.71–4.66 (m, 1H), 3.89 (s, 3H), 3.85 (s, 3H), 3.79 (s, 3H), 3.60– 3.55 (m, 1H), 3.51–3.43 (m, 3H), 3.42–3.37 (m, 1H), 3.34–3.27 (m, 1H), 2.65 (d, J =2.6 Hz, 3H), 2.37 (s, 3H), 1.77–1.68 (m, 2H), 1.61 (s, 3H). Its carbon NMR data are as follows: 13C NMR (151MHz, CDCl3) δ 171.6, 166.4, 164.2, 162.2, 155.7, 150.0, 149.1, 145.9, 142.6, 137.0, 137.0, 136.9, 136.4, 133.4, 132.0, 131.1, 131.0, 130.6, 130.0, 129.8, 128.8, 128.8, 124.9, 119.0, 105.0, 102.6, 92.6, 56.2, 56.2, 54.4, 52.0, 44.9, 39.2, 36.3, 29.4, 14.4, 13.1, 11.9. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd forC 38 H 41 O6N7ClS + :758.2522; found: 758.2521; purity: 99.57%.

[0073] Synthesis of compound D16: Following the synthetic method for compound D1, compound D16 (37.5 mg, yield 53.6%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (600MHz, CDCl3) δ 8.47 (d, J =5.4Hz, 1H), 8.19 (d, J =7.6Hz, 1H), 7.69 (d, J =7.3Hz, 1H), 7.47–7.36 (m, 2H), 7.35–7.29 (m, 2H), 7.25 (s, 1H), 7.23–7.10 (m, 2H), 6.20 (d, J =5.7Hz, 1H), 5.59–5.41 (m, 2H), 5.10 (d, J =5.5Hz, 1H), 4.69–4.62 (m, 1H), 3.76 (s, 3H), 3.61–3.50 (m, 2H), 3.50–3.43 (m, 1H), 3.42–3.20 (m, 3H), 2.67 (s, 3H), 2.39 (s, 3H), 1.84–1.72 (m, 2H), 1.65 (s, 3H). Its carbon NMR data are as follows: 13C10 NMR (151 MHz, CDCl3) δ 171.4, 166.0, 164.3, 161.7, 155.6, 150.1, 143.7, 143.4, 136.9, 136.6, 136.4, 132.1, 131.8, 131.1, 131.0, 130.5, 129.9, 128.8, 126.1, 124.4, 123.9, 120.2, 119.2, 118.4, 104.8, 56.5, 54.5, 52.2, 45.0, 39.4, 36.7, 29.7, 14.4, 13.2, 11.9. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 35 H 36 O4N8ClS + :699.2263; found: 699.2258; purity: 99.91%.

[0074] Example 2: Synthesis of compounds D17 to D21, D22, D27 and D32 The synthetic route is as follows: ; In the preparation of compounds D17 to D21, the specific structures of X in the structural formulas of compounds 7, 8, 9, and 10 in the above synthetic route are identical to the specific structures of X in the corresponding prepared structural formulas of compounds D17 to D21. For example, X in the structural formula of compound D17 is... Then, the X in the structural formulas of compounds 7, 8, 9, and 10 used to prepare compound D17 is also... The preparation of the remaining compounds follows the same procedure.

[0075] Synthesis of compound D17: Following the synthetic route of this embodiment and referring to the synthetic method of compound D1, compound D17 (61.2 mg, yield 85.7%) was obtained. Its 1H NMR spectral data are as follows: 1 H NMR (600MHz, CDCl3) δ 7.65 (m, 1H), 7.52 (t, J =6.0Hz, 1H), 7.34 (dd, J =8.6, 2.0Hz, 2H), 7.27 (s, 1H), 7.25 (d, J =2.5Hz, 1H), 7.14 (d, J =9.0Hz, 1H), 6.97 (t, J=3.0Hz, 1H), 6.92 (m, 2H), 6.13 (s, 1H), 5.49–5.37 (m, 2H), 4.86 (s, 1H), 4.67 (dd, J =8.2, 6.1Hz, 1H), 3.91–3.70 (m, 6H), 3.65–3.53 (m, 3H), 3.52–3.46 (m, 1H), 3.41 (m, 2H), 2.57 (d, J =5.8 Hz, 3H), 2.33 (s, 3H), 1.53 (s, 3H). Its carbon NMR data are: 13 C10 NMR (151 MHz, CDCl3) δ 172.2, 166.3, 164.3, 162.5, 155.6, 154.7, 150.0, 142.3, 139.5, 136.9, 136.9, 136.4, 133.7, 132.1, 131.8, 131.0, 130.9, 130.4, 129.8, 128.8, 126.7, 125.0, 115.2, 111.3, 104.6, 102.7, 55.7, 54.5, 52.0, 45.0, 40.7, 39.3, 39.2, 14.2, 13.0, 11.8. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 36 H 37 O5N7ClS + :714.2260; found: 714.2247; purity: 99.55%.

[0076] Synthesis of compound D18: Following the synthetic route of this embodiment and referring to the synthetic method of compound D1, compound D18 (46 mg, yield 60.8%) was obtained. Its 1H NMR spectral data are as follows: 1 H NMR (600MHz, CDCl3) δ 7.39 (d, J =8.2Hz, 2H), 7.35–7.30 (m, 2H), 7.15 (d, J =9.0Hz, 1H), 6.97 (d, J =2.5Hz, 1H), 6.96–6.93 (m, 2H), 6.91 (dd, J =9.0, 2.5Hz, 1H), 6.71 (s, 1H), 6.15 (s, 1H), 5.49–5.39 (m, 2H), 4.86 (s, 1H), 4.62 (m, 1H), 3.79 (d, J=1.5Hz, 6H), 3.58 (m, 1H), 3.48–3.42 (m, 1H), 3.36–3.24 (m, 4H), 2.62 (s, 3H), 2.37 (s, 3H), 1.86 (s, 2H), 1.64 (s, 3H), 1.59–1.53 (m, 2H), 1.43 (m, 2H). Its carbon NMR data are as follows: 13 C NMR (151MHz, CDCl3) δ 170.7, 166.2, 164.1, 162.3, 155.6, 154.7, 149.9, 142.5, 140.5, 137.0, 136.9, 136.6, 133.7, 132.2, 132.1, 130.9, 130.9, 130.4, 12 9.9, 128.8, 126.6, 125.0, 115.2, 111.4, 104.3, 102.5, 55.7, 54.7, 52.0, 45.0, 39.7, 39.4, 39.2, 29.1, 29.0, 24.1, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd forC 39 H 43 O5N7ClS + :756.2729; found: 756.2709; purity: 99.78%.

[0077] Synthesis of compound D19: Following the synthetic route of this embodiment and referring to the synthetic method of compound D1, compound D19 (66.3 mg, yield 83.0%) was obtained. Its 1H NMR spectrum data is as follows: 1 H NMR (600MHz, CDCl3) δ 7.39 (d, J =8.2Hz, 2H), 7.32 (d, J =8.7Hz, 2H), 7.17 (d, J =9.0Hz, 1H), 7.03 (d, J =2.4Hz, 1H), 6.93 (dd, J =9.0, 2.4Hz, 1H), 6.82–6.80 (m, 1H), 6.53–6.47 (m, 1H), 6.39 (t, J =5.8Hz, 1H), 6.17–6.14 (m, 1H), 5.43 (t, J=1.8Hz, 2H), 4.87 (s, 1H), 4.63–4.59 (m, 1H), 3.83 (s, 3H), 3.79 (s, 3H), 3.58–3.53 (m , 1H), 3.43–3.37 (m, 2H), 3.34–3.21 (m, 3H), 2.65 (s, 3H), 2.40–2.37 (m, 3H), 1.65 (d, J =1.1Hz, 3H), 1.61–1.56 (m, 2H), 1.55–1.49 (m, 2H), 1.38–1.33 (m, 2H), 1.30 (d, J =4.0Hz, 6H). Its carbon NMR data are: 13 C NMR (151MHz, CDCl3) δ 170.4, 166.2, 163.9, 162.1, 155.7, 154.8, 149.9, 136.9, 136.8, 136.6, 133.7, 132.2, 132.1, 130.9, 130.9, 130.4, 129.8, 128.7, 126. 5, 125.0, 115.3, 111.4, 103.8, 102.4, 55.7, 54.6, 52.0, 45.0, 39.6, 39.6, 39.6, 29.6, 29.4, 29.1, 29.1, 26.8, 26.7, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 42 H 49 O5N7ClS + :798.3199; found: 798.3166; purity: 96.72%.

[0078] Synthesis of compound D20: Following the synthetic route of this embodiment, and referring to the synthetic method of compound D1, compound D20 (58 mg, yield 72.3%) was obtained. Its 1H NMR spectral data are as follows: 1 H NMR (600MHz, CDCl3) δ 7.39 (d, J =8.1Hz, 2H), 7.33–7.29 (m, 2H), 7.14 (d, J =9.0Hz, 1H), 7.02 (m, 1H), 6.90 (dd, J=8.9, 2.4Hz, 1H), 6.88–6.82 (m, 1H), 6.26–6.12 (m, 2H), 5.76 (s, 1H), 5.16 (s, 2H), 5.08 (m, 1H), 4.64 (m, 1H), 3.82 (s, 2H), 3.80 (d, J =2.8Hz, 2H), 3.78 (s, 3H), 3.76 (s, 2H), 3.73–3.70 (m, 1H), 3.68 (d, J =1.8Hz, 1H), 3.62 (s, 3H), 3.60–3.57 (m, 2H), 3.54–3.46 (m, 4H), 2.65 (s, 3H), 2.39 (s, 3H), 1.68–1.64 (m, 3H). Its carbon NMR data are as follows: 13 C NMR (151MHz, CDCl3) δ 170.6, 166.1, 165.0, 163.8, 155.7, 154.7, 149.9, 142.4, 140.4, 136.8, 136.7, 136.4, 135.6, 132.8, 132.2, 130.9, 130.8, 130.5, 129.9 , 128.7, 125.9, 125.8, 114.8, 111.3, 102.6, 101.6, 70.5, 70.4, 70.0, 69.1, 55.8, 54.4, 52.1, 44.9, 44.2, 39.4, 39.1, 14.4, 13.1, 11.9. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 40 H 45 O7N7ClS + :802.2784; found: 802.2755; purity: 99.97%.

[0079] Synthesis of compound D21: Following the synthetic route of this embodiment and referring to the synthetic method of compound D1, compound D21 (46.7 mg, yield 52.5%) was obtained. Its 1H NMR spectrum data is as follows: 1 H NMR (600MHz, CDCl3) δ 7.60–7.52 (m, 2H), 7.44–7.40 (m, 2H), 7.35–7.32 (m, 2H), 7.20 (d, J =9.0Hz, 1H), 7.06 (d, J =2.4Hz, 1H), 7.01 (s, 1H), 6.97 (dd, J=9.0, 2.4 Hz, 1H), 6.20–6.17 (m, 1H), 5.49–5.45 (m, 2H), 4.90–4.88 (m, 1H), 4.74–4.71 (m, 1H), 3.87 (s, 3H), 3.85–3.83 (m, 3H), 3.73–3.70 (m, 9H), 3.69–3.66 (m, 6H), 3.65–3.59 (m, 3H), 3.56–3.51 (m, 3H), 3.48–3.44 (m, 1H), 2.68 (s, 3H), 2.44–2.42 (m, 3H), 1.72–1.69 (m, 3H). Its carbon NMR data are as follows: 13 C NMR (151MHz, CDCl3) δ 170.6, 166.2, 163.8, 162.3, 155.8, 154.7, 149.8, 142.8, 138.6, 136.9, 1 36.7, 136.7, 133.7, 132.1, 131.0, 130.8, 130.6, 129.9, 128.7, 128.7, 126 6, 124.9, 115.1, 111.3, 104.7, 102.7, 70.6, 70.5, 70.5, 70.3, 70.2, 70.1, 70.0, 69.9, 55.8, 54.3, 52.0, 45.0, 39.4, 39.1, 38.7, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 44 H 53 O9N7ClS + :890.3308; found: 890.3285; purity: 99.84%.

[0080] Synthesis of compound D22: Following the synthetic route of this embodiment, compound 7 is replaced with... Following the synthetic method for compound D1, compound D22 (69 mg, yield 93.2%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (600MHz, CDCl3) δ 7.42–7.38 (m, 2H), 7.35–7.32 (m, 2H), 7.21 (d, J =9.0Hz, 1H), 7.07 (d, J =2.4Hz, 1H), 6.95 (dd, J =9.0, 2.4Hz, 1H), 6.59 (s, 1H), 6.22 (s, 1H), 5.21 (s, 3H), 4.81 (t,J =6.8Hz, 1H), 4.02–3.93 (m, 2H), 3.92–3.87 (m, 2H), 3.85 (s, 3H), 3.78 (m, 1H), 3.75 (s, 3H), 3.73–3.69 (m, 1H), 3.57 (m, 2H), 2.67 (s, 3H), 2.40 (s, 3H), 1.75 (s, 2H), 1.68 (s, 3H). Its carbon NMR data are as follows: 13 C NMR (126MHz, CDCl3) δ 169.3, 165.9, 163.9, 163.3, 155.7, 154.7, 149.9, 142.4, 139.7, 136.9, 136.7, 136.7, 136.7, 132.8, 132.2, 131.1, 130.9, 130.5, 129.8, 128.7, 126.6, 126.1, 114.8, 111.4, 104.7, 102.5, 68.8, 61.8, 55.7, 54.4, 52.1, 51.8, 45.8, 45.0, 35.3, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 38 H 39 O5N7ClS + :740.2416; found: 740.2404; purity: 99.12%.

[0081] Synthesis of compound D27: Following the synthetic route of this embodiment, compound 7 is replaced with... Following the synthetic method for compound D1, compound D27 (63.6 mg, yield 83.9%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (600MHz, CDCl3) δ 7.43–7.39 (m, 2H), 7.35–7.32 (m, 2H), 7.19–7.16 (m, 1H), 7.05 (d, J =2.4Hz, 1H), 6.97–6.94 (m, 1H), 6.91 (s, 1H), 6.64–6.56 (m, 1H), 6.17 (d, J =9.6Hz, 1H), 5.43 (s, 2H), 4.94–4.90 (m, 1H), 4.88 (s, 1H), 4.85–4.80 (m, 1H), 4.65–4.58 (m, 1H), 4.32 (d, J=14.0Hz, 1H), 4.26–4.21 (m, 1H), 3.84 (s, 3H), 3.80 (s, 3H), 3.71–3.65 (m, 1H), 3.42 (dd, J =15.7, 6.1Hz, 1H), 3.34–3.26 (m, 1H), 2.67 (s, 3H), 2.40 (s, 3H), 2.15–2.08 (m, 2H), 2.06–1.99 (m, 2H), 1.68 (d, J =6.9Hz, 3H). Its carbon NMR data are: 13 C NMR (126MHz, CDCl3) δ168.9, 166.2, 163.9, 161.6, 155.9, 154.8, 149.7, 136.9, 136.8, 133.8, 132.1, 131.7, 131.7, 130.9, 130.8, 130.5, 129.9 ,129.8,128.7,128.7,126.5,124.9,115.5,111.3,104.5,102.6,55.7, 54.8, 54.3, 52.0, 46.8, 45.4, 45.0, 41.4, 32.2, 31.9, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 39 H 41 O5N7ClS + :754.2573; found: 754.2577; purity: 99.57%.

[0082] Synthesis of compound D32: Following the synthetic route of this embodiment, replace compound 7 in it. Following the synthetic method for compound D1, compound D32 (64.9 mg, yield 86.0%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (600MHz, CDCl3) δ 7.40 (d, J =8.2Hz, 2H), 7.33 (d, J =8.7Hz, 2H), 7.19 (d, J =9.0Hz, 1H), 7.05 (d, J =2.5Hz, 1H), 6.93 (dd, J =8.9, 2.5Hz, 2H), 6.55 (s, 1H), 6.20 (s, 1H), 5.15 (t, J =1.7Hz, 2H), 5.10 (d,J =1.9Hz, 1H), 4.63–4.59 (m, 1H), 4.39 (s, 2H), 4.09–4.03 (m, 1H), 3.84 (s, 3H), 3.76 (s, 3H), 3.57–3.52 (m, 1H), 3.37–3.32 (m, 1H), 3.12 (s, 2H), 2.67 (s, 3H), 2.40 (s, 3H), 1.88 (s, 4H), 1.67 (s, 3H). Its carbon NMR data are as follows: 13 C NMR (151MHz, CDCl3) δ 170.0, 166.0, 163.9, 162.9, 155.7, 154.7, 149.9, 142.5, 139.3, 136.8, 136.6, 136.6, 132.6, 132.1, 131.8, 131.0, 130.9, 130.5, 12 9.9, 128.7, 126.8, 125.9, 114.5, 111.3, 104.0, 102.5, 58.4, 55.8, 54.4, 52.1, 46.7, 44.8, 39.1, 31.9, 29.7, 29.5, 14.4, 13.1, 11.9. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 39 H 41 O5N7ClS + :754.2573; found: 754.2587; purity: 99.91%.

[0083] Example 3: Synthesis of compounds D23 to D26, D28 to D31, and D33 to D36 The synthesis route is as follows: .

[0084] Synthesis of compound D23 (When preparing compound D23, the Y in the structural formulas of compounds 13, 14, 16, and 17 is the same as the Y in the structural formula of compound D23, and the same principle applies to the preparation of the remaining compounds): Compound 6 (0.19 g, 1 mmol), EDCI (0.29 g, 1.5 mmol), HOBt (0.27 g, 2 mmol), and DIPEA (0.52 mL, 3 mmol) were dissolved together in DMF (5 mL) and then activated in an ice bath for 30 min. After activation, compound 15a (0.22 g, 1.2 mmol) was added to the reaction system, and the reaction mixture was allowed to react at room temperature overnight (12 h to ensure complete reaction). After the reaction was complete, the reaction solution was extracted with citric acid aqueous solution (5% by mass), saturated NaHCO3 aqueous solution, and saturated saline solution in a 1:1 volume ratio with EtOAc (i.e., a total of 3 extractions). The organic phase was dried and concentrated, and then separated by column chromatography (using PE and EtOAc in a 4:1 volume ratio as eluent) to obtain compound 11 (0.32 g, yield 88.7%).

[0085] Compound 11 (0.32 g, 0.89 mmol) was mixed with DCM (1 mL) and TFA (1 mL) and stirred at room temperature for 0.5 h. After the reaction was completed, the mixture was extracted three times with DCM and H2O in a volume ratio of 1:1. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a white oily substance, namely compound 12 (0.21 g, yield 92.4%).

[0086] Compound 13 (0.20 g, 0.68 mmol), EDCI (0.20 g, 1.02 mmol), HOBt (0.18 g, 1.36 mmol), DIPEA (0.36 mL, 2.04 mmol), and DMF (30 mL) were added sequentially to a round-bottom flask. The mixture was activated at 0 °C for 30 min, followed by the addition of compound 12 (0.21 g, 0.82 mmol). The mixture was stirred overnight at room temperature (12 h) and monitored by TLC (thin-layer chromatography). After the reaction was complete, the reaction solution was extracted with citric acid aqueous solution (5% by mass), saturated NaHCO3 aqueous solution, and saturated saline solution in a 1:1 volume ratio with EtOAc (i.e., three extractions). The organic phase was dried and concentrated, and then purified by column chromatography (using PE and EtOAc in a volume ratio of 8:1) to obtain compound 14 (0.29 g, yield 78.9%).

[0087] Compound 14 (0.29 g, 0.54 mmol) and DCM (2 mL) were mixed, and piperidine (0.4 mL, piperidine volume was 20% of DCM volume) was added. The mixture was stirred at room temperature for 2 h. The reaction solution was extracted three times with extract (DCM and H2O in a volume ratio of 1:1). The organic phase was dried, concentrated under reduced pressure, and purified by column chromatography (PE and EtOAc in a volume ratio of 4:1) to obtain compound 16 (0.15 g, yield 87.0%).

[0088] JQ1 (80 mg, 0.2 mmol), EDCI (57.51 g, 0.3 mmol), HOBt (54 mg, 0.4 mmol), and DIPEA (104 μL, 0.6 mmol) were dissolved in DMF (1 mL) and activated in an ice bath for 30 min. Then, compound 16 (0.06 g, 0.2 mmol) was added, and the mixture was stirred at room temperature overnight (12 h). After the reaction was complete, the reaction solution was extracted with citric acid aqueous solution (5% by mass), saturated NaHCO3 solution, and saturated saline solution in a 1:1 volume ratio with EtOAc (i.e., a total of 3 extractions). The organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure to obtain the crude product. Subsequently, the crude product was purified by column chromatography (using DCM and MeOH in a 20:1 volume ratio as eluent) to obtain compound 17 (0.12 g, yield 85.8%).

[0089] Compound 17 (0.12 g, 0.17 mmol) and compound B (44 mg, 0.204 mmol) were dissolved in DCM (1 mL). DABCO (4 mg, 0.034 mmol) was added to the reaction solution. The reaction system was stirred at room temperature for 0.5 h. The solvent was then removed by rotary evaporation to obtain the crude product. Subsequently, the crude product was separated by column chromatography (using DCM and MeOH in a volume ratio of 20:1 as eluent) to obtain compound D23 (0.12 g, yield 93.3%).

[0090] The proton NMR spectrum data of compound D23 are as follows: 1 H NMR (600MHz, CDCl3) δ 7.44–7.41 (m, 1H), 7.40–7.37 (m, 1H), 7.35–7.30 (m, 2H), 7.23–7.18 (m, 1H), 7.05 (dd, J=11.7, 2.4Hz, 1H), 6.96–6.92 (m, 1H), 6.59–6.53 (m, 1H), 6.23–6.18 (m, 1H), 5.20–5.18 (m, 1H), 5.1 8–5.16 (m, 1H), 4.81–4.76 (m, 1H), 4.62–4.48 (m, 2H), 4.36–4.28 (m, 1H), 4.14–4.05 (m, 1H), 3.88 (t, J =5.3Hz, 1H), 3.84 (d, J =5.4Hz, 3H), 3.81 (s, 2H), 3.78–3.76 (m, 2H), 3.74 (s, 1H), 3.72 (d, J =5.8Hz, 3H), 3.69–3.61 (m, 2H), 3.54 (d, J =24.4 Hz, 2H), 2.69–2.61 (m, 3H), 2.39 (s, 3H), 1.69–1.63 (m, 3H). The carbon NMR data for compound D23 are: 13 C NMR (126MHz, CDCl3) δ 172.0, 166.6, 166.2, 165.9, 163.3, 155.8, 154.8, 149.9, 149.8, 138.5, 136.7, 136.7, 136.6, 135.1, 132.8, 132.2, 130.9, 130.7, 130.5 , 129.9, 128.7, 127.2, 126.1, 114.9, 111.4, 104.7, 102.5, 55.8, 54.9, 54.3, 53.5, 52.2, 52.1, 49.6, 47.0, 45.0, 35.4, 14.4, 13.1, 11.8. The high-resolution mass spectrometry data of compound D23 are as follows (HRMS-ESI, m / z): [M+H] + calcd for C 40 H 42 O6N8ClS + :797.2631; found: 797.2595; purity: 98.45%.

[0091] Synthesis of compound 24: Following the synthetic method for compound D23, compound D24 (76.7 mg, yield 89.9%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (600MHz, CDCl3) δ 7.42–7.38 (m, 2H), 7.33 (m, 2H), 7.20 (d, J =9.0Hz, 1H), 7.05 (d,J =2.4Hz, 1H), 6.94 (dd, J =9.0, 2.5Hz, 1H), 6.63 (t, J =5.5Hz, 1H), 6.58–6.55 (m, 1H), 6.22–6.19 (m, 1H), 5.18 (m, 3H), 4.61 (m, 1H), 3.84 (s, 3H), 3.80–3.77 (m, 2H) ), 3.75 (m, 2H), 3.73 (s, 3H), 3.67 (s, 2H), 3.58–3.50 (m, 3H), 3.30 (m, 3H), 2.66 (s, 3H), 2.40 (s, 3H), 2.36 (t, J =7.7 Hz, 2H), 1.72–1.70 (m, 2H), 1.67 (s, 3H), 1.60–1.54 (m, 2H), 1.43–1.37 (m, 2H). Its carbon NMR data are as follows: 13 C NMR (151MHz, CDCl3) δ 171.7, 170.5, 165.9, 164.0, 163.3, 155.7, 154.8, 149.9, 142.6, 136.8, 136.7, 132.8, 132.2, 131.0, 130.9, 130.9, 130.5, 129.9, 128.8, 126.7, 126.0, 114.9, 111.4, 104.7, 102.5, 55.8, 54.6, 52.1, 45.4, 45.0, 43.7, 41.6, 39.5, 39.4, 33.1, 29.7, 29.3, 26.6, 24.7, 14.4, 13.1, 11.9. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 44 H 50 O6N8ClS + :853.3257; found: 853.3257; purity: 99.57%.

[0092] Synthesis of compound 25: Following the synthetic method for compound D23, compound D25 (78.8 mg, yield 92.1%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (600MHz, CDCl3) δ 7.40 (d, J =8.0Hz, 2H), 7.32 (m, 2H), 7.22 (s, 1H), 7.18 (d, J =8.9Hz, 1H), 7.04 (d, J=2.5Hz, 1H), 6.93 (m, 1H), 6.55 (s, 1H), 6.19 (s, 1H), 5.16 (d, J =6.4Hz, 3H), 4.64 (t, J =6.9Hz, 1H), 3.83 (m, 5H), 3.76 (m, 4H), 3.72 (d, J =1.3Hz, 3H), 3.71–3.66 (m, 2H), 3.58 (m, 5H), 3.48 (m, 2H), 3.38 (m, 1H), 2.70–2.65 (m, 2H), 2.63 (s, 3H), 2.39 (s, 3H), 1.66 (s, 3H). Its carbon NMR data are as follows: 13 C NMR (126MHz, CDCl3) δ 170.6, 169.9, 165.9, 163.9, 163.3, 155.7, 154.7, 149.9, 142.6, 139.3, 136.8, 136.7, 136.6, 132.8, 132.1, 131.0, 130.9, 130.9, 130.5, 129.9, 128.7, 126.6, 126.0, 114.8, 111.3, 104.8, 102.6, 69.6, 66.5, 55.8, 54.4, 53.5, 52.1, 45.5, 45.0, 41.7, 39.3, 39.1, 33.4, 29.7, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 43 H 48 O7N8ClS + :855.3050; found: 855.3055; purity: 99.31%.

[0093] Synthesis of compound D26: Following the synthetic method for compound D23, compound D26 (88.9 mg, yield 94.3%) was obtained. Its 1H NMR spectrum data are as follows: 1 H NMR (500MHz, CDCl3) δ 7.43 (s, 1H), 7.36 (d, J =8.0Hz, 2H), 7.27 (d, J =8.3Hz, 2H), 7.15 (d, J =8.9Hz, 1H), 7.00 (s, 1H), 6.89 (m, 1H), 6.49 (s, 1H), 6.14 (s, 1H), 5.13 (m, 3H), 4.63 (t, J=6.9Hz, 1H), 3.81–3.76 (m, 5H), 3.70–3.65 (m, 7H), 3.62 (m, 8H), 3.56 (m, 2H), 3.48 (m, 5H), 3.38 (m, 1H), 2.80 (s, 2H), 2.64 (t, J =6.5 Hz, 2H), 2.58 (s, 3H), 2.35 (s, 3H), 1.62 (s, 3H). Its carbon NMR data are: 13 C NMR (126MHz, CDCl3) δ 170.6, 170.0, 165.9, 163.8, 163.2, 155.7, 154.7, 149.8, 142.2, 138.3, 136.7 ,136.6,136.6,132.8,132.1,131.1,130.9,130.8,130.5,129.9,128.7,126.6 The high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H]. (Note: The numbers 126.0, 114.8, 111.3, 104.6, 102.5, 70.5, 70.5, 70.4, 70.2, 69.9, 67.3, 55.7, 54.3, 52.0, 50.4, 45.6, 44.9, 41.6, 39.4, 38.8, 33.5, 29.7, 14.4, 13.1, 11.7 are likely values ​​or values, but without further context, a precise translation is not possible.) + calcd for C 47 H 56 O9N8ClS + :943.3574; found: 943.3582; purity: 98.81%.

[0094] Synthesis of compound D28: Following the synthetic method of compound D23, replace compound 15a with 15b. ), to obtain compound D28, where Y is (77.7 mg, yield 95.8%). Its 1H NMR data are as follows: 1 H NMR (600MHz, CDCl3) δ 7.90 (s, 1H), 7.45 (d, J=8.5Hz, 1H), 7.40–7.36 (m, 1H), 7.34–7.31 (m, 1H), 7.29–7.26 (m, 1H), 7.20–7.13 (m, 2H), 7.06–7.02 (m, 1H) ), 6.99–6.89 (m, 2H), 6.16–6.14 (m, 1H), 5.44–5.39 (m, 2H), 4.89–4.86 (m, 1H), 4.81–4.69 (m, 2H), 4.68–4.6 2 (m, 1H), 4.30–4.11 (m, 4H), 3.85–3.83 (m, 3H), 3.79–3.77 (m, 3H), 3.73–3.66 (m, 1H), 3.18–3.08 (m, 1H), 2.82–2.75 (m, 1H), 2.63–2.58 (m, 3H), 2.41–2.38 (m, 3H), 2.05–1.95 (m, 2H), 1.79 (s, 3H), 1.68–1.65 (m, 2H). Its carbon NMR data are as follows: 13 C NMR (151MHz, CDCl3) δ 170.8, 166.9, 166.2, 163.7, 161.8, 155.9, 154.8, 150.0, 138.2, 137.0, 136.9, 136.8, 136.6, 136.1, 133.8, 132.3, 132.0, 131.1, 130.9, 130.1, 128.7, 126.6, 124.9, 115.1, 114.2, 111.4, 105.2, 55.8, 54.1, 53.9, 52.0, 46.6, 45.0, 44.0, 41.9, 41.4, 32.3, 31.2, 14.1, 13.1, 11.6. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 41 H 44 O6N8ClS + :811.2788; found: 811.2780; purity: 99.89%.

[0095] Synthesis of compound D29: Following the synthetic method of compound D23, replace compound 15a with compound 15b. ), to obtain compound D29, where Y is (48.7 mg, yield 56.2%). Its 1H NMR data are as follows: 1H NMR (600MHz, CDCl3) δ 7.41–7.35 (m, 2H), 7.34–7.30 (m, 2H), 7.18–7.13 (m, 1H), 7.00–6.97 (m, 1H), 6.94 (dd, J =9.1, 2.5Hz, 1H), 6.86 (d, J =7.6Hz, 1H), 6.82–6.74 (m, 1H), 6.65–6.57 (m, 1H), 6.16 (d, J =11.3Hz, 1H), 5.41 (d, J =5.1Hz, 2H), 4.86 (d, J =3.8Hz, 1H), 4.67–4.57 (m, 2H), 4.19–4.13 (m, 1H), 3.91–3.86 (m, 1H), 3.82 (s, 3H), 3.81–3 .77 (m, 3H), 3.68 (s, 1H), 3.59–3.53 (m, 1H), 3.36–3.24 (m, 3H), 3.17–3.10 (m, 1H), 2.70 (t, J =13.1Hz, 1H), 2.66–2.59 (m, 3H), 2.40–2.36 (m, 3H), 2.34–2.29 (m, 1H), 2.13–1.96 (m, 3H), 1.85 (s, 4H), 1.66 (s, 1H), 1.63 (d, J =12.4 Hz, 3H), 1.59–1.54 (m, 2H). Its carbon NMR data are: 13 C NMR (151MHz, CDCl3) δ 171.4, 170.5, 168.3, 166.2, 161.6, 155.7, 154.8, 149.9, 138.3, 136.9, 136 .8, 136.6, 133.8, 132.6, 132.1, 131.8, 131.7, 130.9, 130.4, 129.9, 128.7, 126.5, 124.9, 115.5, 114.1, 111.4, 104.5, 55.8, 54.5, 53.5, 52.0, 46.9, 45.1, 44.8, 41.0, 39.3, 32.9, 29.7, 29.4, 29.2, 26.7, 25.0, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 45 H 52 O6N8ClS +:867.3414; found: 867.3430; purity: 98.71%.

[0096] Synthesis of compound D30: Following the synthetic method of compound D23, replace compound 15a with compound 15b. ), to obtain compound D30, where Y is (55.4 mg, yield 63.7%). Its 1H NMR data are as follows: 1 H NMR (600MHz, CDCl3) δ 7.43–7.37 (m, 2H), 7.33–7.29 (m, 2H), 7.24–7.13 (m, 2H), 6.98–6.92 (m, 2H), 6.83 (d, J =27.4Hz, 1H), 6.17–6.13 (m, 1H), 5.45–5.34 (m, 2H), 4.89–4.83 (m, 1H), 4.73–4.62 (m, 2H), 4.18–4.1 0 (m, 1H), 3.94–3.85 (m, 2H), 3.83–3.80 (m, 3H), 3.79–3.77 (m, 3H), 3.76–3.72 (m, 1H), 3.70–3.66 (m, 1 The NMR spectra are as follows: 3.65–3.52 (m, 4H), 3.52–3.42 (m, 2H), 3.42–3.35 (m, 1H), 3.18–3.11 (m, 1H), 2.80–2.68 (m, 2H), 2.66–2.62 (m, 1H), 2.59–2.56 (m, 1H), 2.53 (s, 1H), 2.42–2.34 (m, 3H), 1.84 (s, 4H), 1.68–1.62 (m, 3H). The carbon NMR spectra are as follows: 13 C NMR (151MHz, CDCl3) δ 170.7, 169.7, 166.2, 163.9, 161.7, 155.7, 154.8, 149.9, 139.3, 138.2, 1 36.9, 136.8, 136.8, 136.6, 133.8, 132.1, 131.9, 130.9, 130.4, 129.9, 128 7, 126.5, 124.9, 115.2, 114.1, 111.3, 104.6, 69.6, 67.0, 55.8, 54.4, 52.5, 52.0, 46.9, 45.0, 44.9, 41.3, 39.3, 32.6, 31.5, 29.7, 14.4, 13.1, 11.7. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C44 H 50 O7N8ClS + :869.3206; found: 869.3222; purity: 97.36%.

[0097] Synthesis of compound D31: Following the synthetic method of compound D23, replace compound 15a with compound 15b. ), to obtain compound D31, where Y is (73.8 mg, yield 77.1%). Its 1H NMR data are as follows: 1 H NMR (600MHz, CDCl3) δ 7.40 (d, J =8.0Hz, 2H), 7.34–7.29 (m, 2H), 7.19–7.14 (m, 1H), 7.01 (d, J =2.3Hz, 1H), 6.97–6.91 (m, 2H), 6.90–6.84 (m, 1H), 6.15 (d, J =7.7Hz, 1H), 5.47–5.35 (m, 2H), 4.87 (s, 1H), 4.69–4.57 (m, 2H), 4.15–4.08 (m, 1 H), 3.94–3.88 (m, 1H), 3.83 (s, 3H), 3.79 (s, 3H), 3.77 (s, 1H), 3.71–3.63 (m, 8H) 3.63–3.56 (m, 2H), 3.55–3.35 (m, 4H), 3.15–3.05 (m, 1H), 2.74–2.65 (m, 2H), 2.65–2.61 (m, 1H), 2.58 (s, 3H), 2.45–2.33 (m, 3H), 2.00–1.90 (m, 6H), 1.66 (s, 3H). Its carbon NMR data are as follows: 13C NMR (151MHz, CDCl3) δ 170.7, 169.5, 166.2, 163.8, 161.6, 155.7, 154.8, 149.9, 139.3, 138.2, 136.9, 136.8, 136.7, 133.8, 132.1, 131.9, 131.0, 130.9, 130.6, 130.0, 128.7, 126.5, 1 24.9, 115.4, 114.1, 111.4, 104.6, 70.8, 70.6, 70.4, 70.4, 70.0, 67.5, 55.8, 54.3, 53.5, 52.0, 46.8, 45.0, 44.9, 41.0, 39.6, 32.0, 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 48 H 58 O9N8ClS + :957.3730; found: 957.3741; purity: 99.49%.

[0098] Synthesis of compound D33: Following the synthetic method of compound D23, replace compound 15a with compound 15c. ), to obtain compound D33, where Y is (73.7 mg, yield 90.9%). Its 1H NMR data are as follows: 1 H NMR (600MHz, CDCl3) δ 7.42 (dd, J =8.6, 2.7Hz, 2H), 7.34 (dd, J =8.7, 3.0Hz, 2H), 7.20–7.15 (m, 1H), 7.05 (d, J =2.5Hz, 1H), 6.98 (d, J =8.4Hz, 1H), 6.91 (dd, J =9.0, 2.4Hz, 1H), 6.40 (d, J =69.5Hz, 1H), 6.19 (d, J =9.7Hz, 1H), 5.72 (d, J =55.7Hz, 1H), 5.05 (d, J=6.0Hz, 2H), 4.90–4.80 (m, 1H), 4.51–4.42 (m, 1H), 4.24–4.09 (m, 2H), 4.07–3.91 (m, 2H), 3.82 (s, 5H), 3.76 (s, 1H), 3.73 (d, J =2.4Hz, 3H), 3.55–3.42 (m, 1H), 3.32–3.23 (m, 1H), 2.60 (d, J =42.3Hz, 3H), 2.40 (d, J =4.8Hz, 3H), 1.75 (s, 4H), 1.67 (d, J =3.1Hz, 3H). Its carbon NMR data are: 13 C NMR (151MHz, CDCl3) δ 172.2, 170.6, 167.5, 166.0, 162.7, 155.7, 154.7, 149.7, 137.0, 136.7, 136.7, 136.6, 136.6, 134.6, 132.6, 132.4, 132.2, 130.8, 130.1, 129.8, 128.8, 127.5, 126.9, 114.3, 114.1, 111.3, 104.0, 55.8, 55.0, 52.3, 52.0, 50.3, 49.9, 47.5, 47.1, 44.8, 31.4, 29.7, 14.4, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 41 H 44 O6N8ClS + :811.2788; found: 811.2787; purity: 98.98%.

[0099] Synthesis of compound D34: Following the synthetic method of compound D23, replace compound 15a with compound 15c. ), to obtain compound D34, where Y is (52.8 mg, yield 60.9%). Its 1H NMR data are as follows: 1 H NMR (600MHz, CDCl3) δ 7.44–7.36 (m, 2H), 7.35–7.28 (m, 2H), 7.15 (t, J =8.2Hz, 1H), 7.01 (d, J =6.7Hz, 2H), 6.90 (t, J=8.0Hz, 1H), 6.65–6.39 (m, 2H), 6.18–6.10 (m, 1H), 5.12 (d, J =6.9Hz, 2H), 5.05 (d, J =6.6Hz, 1H), 4.66–4.59 (m, 1H), 4.08–4.00 (m, 1H), 3.81 (d, J =7.3Hz, 3H), 3.72 (d, J =7.2Hz, 3H), 3.68 (d, J =7.7Hz, 1H), 3.60–3.52 (m, 1H), 3.35–3.26 (m, 2H), 3.24–3.13 (m, 2H), 2.91–2.82 (m, 1H), 2.81–2.72 (m, 1H), 2.65 (d, J =6.8Hz, 3H), 2.38 (d, J =6.8Hz, 3H), 2.11 (d, J =7.7Hz, 2H), 1.99–1.91 (m, 2H), 1.65 (d, J =7.1Hz, 3H), 1.62–1.57 (m, 2H), 1.52–1.48 (m, 2H), 1.41 (d, J =7.4Hz, 2H), 1.36 (d, J =7.6 Hz, 2H). Its carbon NMR data are: 13 C NMR (151MHz, CDCl3) δ 172.8, 170.5, 166.0, 164.0, 162.9, 155.7, 154.7, 149.9, 145.5, 139.3, 138 .1, 136.8, 136.6, 136.6, 132.6, 132.0, 131.7, 130.9, 130.5, 129.9, 128.8, 128.7, 126.7, 126.0, 114.5, 114.1, 111.3, 104.0, 55.8, 54.5, 52.1, 52.0, 46.6, 44.7, 39.3, 39.2, 36.5, 31.9, 30.2, 29.7, 26.3, 25.2, 14.2, 13.1, 11.8. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 45 H 52 O6N8ClS + :867.3414; found: 867.3427; purity: 97.97%.

[0100] Synthesis of compound D35: Following the synthetic method of compound D23, replace compound 15a with compound 15c. ), to obtain compound D35, where Y is (48.6 mg, yield 55.9%). Its 1H NMR data are as follows: 1 H NMR (600MHz, CDCl3) δ 7.45–7.34 (m, 2H), 7.30 (d, J =8.1Hz, 2H), 7.26–7.23 (m, 1H), 7.17–7.07 (m, 2H), 7.01 (d, J =2.4Hz, 1H), 6.90 (dd, J =9.0, 2.4Hz, 1H), 6.49 (s, 1H), 6.12 (d, J =5.3Hz, 1H), 5.09 (d, J =4.9Hz, 2H), 5.03–4.96 (m, 1H), 4.69–4.59 (m, 1H), 4.13–4.04 (m, 1H), 3.82 (s, 4H), 3.71 (s, 3H), 3.69–3.62 (m, 2H), 3.58–3.45 (m, 3H), 3.40–3.30 (m, 2H), 2.66 (s, 3H), 2.53–2.44 (m, 2H), 2.39 (s, 3H), 2.32–2.17 (m, 1H), 2.05–1.92 (m, 3H), 1.66 (s, 3H), 1.42 (s, 2H), 1.36 (s, 1H), 1.33 (s, 1H). Its carbon NMR data are as follows: 13 C NMR (151MHz, CDCl3) δ 171.1, 170.4, 166.0, 164.2, 162.9, 155.5, 154.7, 149.3, 142.0, 139.3, 1 37.1, 136.6, 136.2, 135.9, 132.6, 131.7, 131.4, 131.0, 130.7, 130.0, 128 0.8, 126.8, 125.9, 114.4, 114.1, 111.2, 104.0, 69.2, 66.9, 65.9, 55.8, 54.5, 52.0, 46.7, 44.7, 39.4, 37.1, 33.9, 32.0, 29.7, 29.7, 14.4, 13.2, 11.8. Its high-resolution mass spectrometry data are HRMS-ESI (m / z): [M+H] + calcd for C 44 H 50 O7N8ClS+ :869.3206; found: 869.3225; purity: 99.27%.

[0101] Synthesis of compound D36: Following the synthetic method of compound D23, replace compound 15a with compound 15c. ), to obtain compound D36, where Y is (66.1 mg, yield 69.1%). Its 1H NMR data are as follows: 1 H NMR (600MHz, CDCl3) δ 7.44–7.40 (m, 1H), 7.39 (d, J =8.2Hz, 2H), 7.30 (d, J =8.3Hz, 2H), 7.20–7.12 (m, 2H), 7.04–7.00 (m, 1H), 6.90 (dd, J =9.0, 2.5Hz, 1H), 6.52 (s, 1H), 6.16 (s, 1H), 5.12 (s, 2H), 5.04 (s, 1H), 4.68 (t, J =7.0Hz, 1H), 4.06–3.99 (m, 1H), 3.81 (s, 3H), 3.80–3.75 (m, 2H), 3.74 (d, J =1.7Hz, 1H), 3.72 (s, 3H), 3.69–3.61 (m, 9H), 3.61–3.55 (m, 2H), 3.53–3.44 (m, 3H), 3.42–3.36 (m, 1H), 3.26–2.89 (m, 2H), 2.63 (s, 5H), 2.54–2.50 (m, 2H), 2.38 (s, 3H), 1.97–1.88 (m, 2H), 1.65 (s, 3H). Its carbon NMR data are as follows: 13C NMR (151MHz, CDCl3) δ 171.0, 170.5, 166.0, 164.0, 162.8, 155.8, 154.7, 149.9, 142.6, 139.3, 136.9, 136.8, 136.7, 136.6, 132.6, 132.0, 131.8, 131.0, 130.6, 129.9, 128.7, 126.8, 1 25.9, 114.4, 111.3, 103.9, 102.5, 70.5, 70.4, 70.4, 70.2, 69.9, 67.4, 55.8, 55.3, 54.3, 52.1, 46.5, 44.8, 39.4, 38.7, 36.9, 31.9, 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 48 H 58 O9N8ClS + :957.3730; found: 957.3751; purity: 96.38%.

[0102] test: (1) High-content analysis and screening a. Construction and expression of dual-fluorescence screening models containing BD1 domain, BD2 domain, and BD1-BD2 dual domain. The genes encoding EGFP (green fluorescent protein) and mScarlet (red fluorescent protein) were fused with the active domains of the BRD4 protein (BD1 domain, BD2 domain, and BD1-BD2 dual domain, respectively) via a FLAG tag to construct recombinant plasmids Flag-BD1-mScarlet-P2A-EGFP, Flag-BD2-mScarlet-P2A-EGFP, and Flag-BD1-BD2-mScarlet-P2A-EGFP, respectively. These recombinant plasmids were then transfected into HEK-293T cells. Upon successful transfection, the cells expressed the 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. Because the fusion contains a P2A self-cleaving sequence, this sequence undergoes specific self-cleavage at the peptide bond between the glycine and proline residues at the C-terminus of the protein after expression. This self-cleavage results in the separation of EGFP from the mScarlet-tagged BD1, BD2, or BD1-BD2 domains, thus creating two independent fluorescent labels within the cell.

[0103] b. The compounds prepared in the above examples were serially diluted with DMEM high-glucose medium to prepare compound solutions with two concentration gradients of 1µM and 100nM.

[0104] HEK-293T cells in good condition after transfection with the recombinant plasmid were digested and counted at a concentration of 1×10⁻⁶. 4 Cells were evenly seeded at a density of 100 cells / well into 96-well plates and cultured overnight. Cell adhesion density reached 80% under an inverted microscope. The old culture medium was discarded, and compound solutions were added in triplicate for each concentration. An equal volume of PBS (phosphate-buffered saline) was added to the edge of each well to eliminate edge effects. The plates were incubated for 12 hours. After incubation, the plates were removed and gently shaken to ensure even cell distribution. The 96-well plates were then placed in a high-content analyzer, and automatic imaging and data acquisition were performed on each well according to a pre-programmed procedure.

[0105] In transfected HEK-293T cells, when the aforementioned compounds (i.e., degradative agents based on indole-based fused covalent warheads) are added, the effective compounds specifically degrade the target domains (BD1 domain, BD2 domain, or BD1-BD2 double domain) of the BRD4 protein. This leads to a synchronous decrease in the fluorescence of the covalently bound mScarlet, 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 aforementioned compounds in inducing target protein degradation can be objectively characterized. The smaller the mScarlet to EGFP ratio, the stronger the ability of the aforementioned compounds to degrade the BRD4 protein.

[0106] (2) Western blot assay Remove the culture dish, rinse with PBS, add RIPA lysis buffer (containing protease inhibitor), scrape off cells with a scraper, transfer to EP tubes, sonicate, centrifuge at 12000 rpm for 20 min at 4°C, and collect the supernatant to obtain the protein sample to be tested. Then, add 20 µL of PBS standard and 5 µL of the protein sample to each well of a 96-well plate (the blank control is 25 µL of PBS). Add 20 µL of BCA working solution to each well, mix well, and incubate at 37°C for 30 min. Cool, and measure the absorbance of each well at 562 nm using a microplate reader. Plot a standard curve with standard protein concentration and absorbance values ​​on the x and y axes, and calculate the protein content of the sample to be tested.

[0107] Mix the protein sample to be tested with 5× Loading Buffer at a volume ratio of 4:1, heat at 100℃ for 5 min to denature the protein, and place the denatured protein sample on ice for later use. Then set up the electrophoresis apparatus, add the pre-stained protein molecular weight standard, and then add the denatured protein sample, ensuring that the total volume of each well is consistent. First run the stacking gel at 80V (30 min), then run the separating gel at 120V (60 min) until the two-color Loading Buffer reaches the bottom of the gel.

[0108] PVDF (polyvinylidene fluoride) membranes were activated by immersion in methanol for 30 seconds and then equilibrated in buffer solution. The gel was then tightly adhered to the activated PVDF membrane, transfer buffer was added, and wet transfer (230 mA constant current, 120 min) was performed. After transfer, the membrane was stained with Ponceau S, and the distribution of visible protein bands on the membrane was observed to confirm successful transfer.

[0109] Block the membrane with 5% skim milk powder in TBST buffer (tris(hydroxymethyl)aminomethane) and incubate at room temperature for 1 hour. Then wash the membrane three times with TBST buffer, 5 minutes each time. Incubate the membrane with diluted primary antibody (target protein) overnight at 4°C. Remove the membrane and wash three times with TBST, 5 minutes each time. Add secondary antibody and incubate at room temperature for 1 hour. Wash three times with TBST buffer, 5 minutes each time.

[0110] Mix solutions A and B from the development kit in a 1:1 ratio to prepare the developing working solution, and transfer it to a light-protected container. Immerse the transfer membrane, which has undergone secondary antibody incubation, in the developing solution and incubate for 3 minutes in the dark. Subsequently, transfer the membrane to a chemiluminescence imaging system, optimize the exposure time based on the signal intensity, and acquire and save protein band images.

[0111] (3) Cell viability test (CCK-8 assay) Take cells in the logarithmic growth phase, digest and count them, and then sort them at a ratio of 4 × 10⁻⁶. 3 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured overnight. The old culture medium was removed, and fresh complete culture medium containing the test compound was added. Three replicates were made for each group, and a blank control and a negative control were also established. Cells were cultured for another 48 hours. After the reaction, 100 μL of 10% CCK-8 solution was added to each well, and the cells were incubated at 37°C in the dark for 1-2 hours (the specific time depends on the cell type). The absorbance was measured at 450 nm using a microplate reader. The relative cell viability was calculated by comparing the OD values ​​(absorbance at 450 nm) of the experimental group and the control group.

[0112] (4) Elution experiment MDA-MB-231 cells were cultured in 6-well plates to 80% confluence, and then incubated with complete medium containing 1 μM compound D29, 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 PBS at 37°C, 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.

[0113] The results are as follows: (1) Figure 3 This is a diagram showing the construction results of a high-content screening platform. Figure 3 As shown in (a) (where GAPDH represents meso-diaminopimelic acid dehydrogenase; the Flag-BD1-mScarlet recombinant plasmid is constructed by fusing the gene encoding mScarlet with BD1 via a FLAG tag), the successful construction and expression of the dual-fluorescence screening model containing the BD1 domain were confirmed by Western blotting; Figure 3As shown in (b), the dual-fluorescence screening model containing the BD1 domain was confirmed to be normally expressed through the high-content platform.

[0114] (2) Figure 4 The graph shows the activity test results of each compound in the above examples. Row min indicates that mScarlet / EGFP is 0.7, and row max indicates that mScarlet / EGFP is 1.0. Figure 4 The degradation effects of each compound on the BD1 domain, BD2 domain, and BD1-BD2 dual domain were demonstrated in HEK293T cells with a stable high-content screening model. The results showed that all compounds could degrade BRD4 protein.

[0115] (3) Figure 5 The graph shows the degradation effects of the four compounds with the highest degradation effects in the high-content analysis on endogenous BRD4 protein. Specifically, it shows the degradation effects of compounds D24, D25, D29 and D20 on BRD4 protein after treatment with MDA-MB-231 cells (triple-negative breast cancer cells) at concentrations of 0.1 μM and 1 μM for 12 h, indicating that each compound can degrade BRD4 protein in MDA-MB-231 cells.

[0116] (4) Figure 6 The graph shows the degradation activity results of compound D29, where (a) is the degradation effect of compound D29 on BET (BRD2, BRD3, BRD4, and BRDT, testis-specific bromine-containing domain proteins) family proteins, (b) is the semi-quantitative result of compound D29 on the degradation of BET family proteins, and (c) is the DC value of compound D29 on the degradation of BRD3 and BRD4 proteins. 50 (d) shows the time-dependent degradation results of compound D29 on BRD4 protein, (e) shows the semi-quantitative results of compound D29 on BRD4 protein degradation at different times, (f) shows the elution effect of compound D29, and (g) shows the semi-quantitative results of compound D29 on elution. The results indicate that compound D29 has a good degradation effect on both BRD3 and BRD4 proteins. When compound D29 degrades BRD3 protein, DC... 50 =20.9 nM, DC during the degradation of BRD4 protein 50 =20.3 nM. Compared to low-concentration compound D29, high-concentration compound D29 showed better degradation effects on BRD3 and BRD4 proteins. Furthermore, with increasing time, the degradation effect of compound D29 on BRD4 initially increased and then decreased, reaching its optimal effect at a concentration of 1 μM and a degradation time of 9 hours.

[0117] (5) To verify the degradation pathway of compound D29, compound D29 at a concentration of 1 μM was reacted with autophagy inhibitors CQ (chloroquine), BafA1 (bafloxacin), and four proteasome inhibitors 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). PYR41 (ethyl 4-[4-[(5-nitro-2-furanyl)methylene]-3,5-dioxo-1-pyrazolyl]benzoate), MG132 (N-[(benzyloxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucamide), and PS341 (bortezomib) were used to simultaneously treat MDA-MB-231 cells. High-content analysis was then performed to determine whether target protein degradation was competitively inhibited. The results are as follows: Figure 7 and Figure 8 As shown, ns indicates no significant difference. This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001. The result indicates that neither chloroquine inhibition of autophagosome acidification nor bafloxacin interference with lysosomal maturation effectively inhibited the degradation activity of compound D29. However, all four inhibitors of the ubiquitin-proteasome pathway significantly suppressed the degradation effect of compound D29 on BRD4 protein. This suggests that compound D29 mediates BRD4 protein degradation through the classic ubiquitin-proteasome pathway.

[0118] (6) The test results of the E3 ubiquitin ligase recruited by compound D29 are as follows: Figure 9 As shown, in stable cell lines with knockout of E3 ubiquitin ligases such as CRBN, VHL, DCAF11, and DCAF16, compound D29 can still effectively degrade BRD4 protein. This indicates that compound D29 depends on the ubiquitin-proteasome system and is independent of known E3 ubiquitin ligases such as CRBN, VHL, DCAF11, and DCAF16, suggesting a novel mechanism mediated by a novel E3 ubiquitin ligase.

[0119] Therefore, this invention constructs a series of degradative molecules targeting the BRD4 protein by introducing methyl acrylate onto an indole backbone and coupling it with JQ1. Compound D29 achieved optimal degradation of the BRD4 protein within 9 hours, with a degradation duration exceeding 24 hours. Its action depends on the ubiquitin-proteasome system and is independent of known E3 ubiquitin ligases such as CRBN, VHL, DCAF11, and DCAF16, suggesting a novel mechanism mediated by a novel E3 ubiquitin ligase. Transwell assays (cell migration assays) and scratch assays confirmed that compound D29 significantly inhibits the migration ability of MDA-MB-231 cells, indicating its potential as a cancer therapeutic.

[0120] 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 an indole-based fused covalent warhead, characterized in that, The structural formula of the degrading agent based on the indole group-fused covalent warhead is: R is a single bond or -NH-; For single bond, , , , , , , , , or ; -NH-, , or ; for or ;R 1 and R 2 Located at any connectable position on the benzene ring or pyridine ring, R 1 and R 2 Each can be independently -H, -F, -Cl, -Br, -CH3, -OCH3, -NO2, -CH2-O-Ph or -CN; Ph is phenyl; Indicates the connection site.

2. The degrading agent based on an indole-based fused covalent warhead according to claim 1, characterized in that, R is -NH-; for , , , , or ; It is -NH-.

3. The degrading agent based on an indole-based fused covalent warhead according to claim 1, characterized in that, R is -NH-; for , , or ; for , or .

4. The degrading agent based on an indole-based fused covalent warhead according to claim 1, characterized in that, R is a single bond. It is a single key. for , or .

5. The degrading agent based on an indole-based fused covalent warhead according to claim 1, characterized in that, R in 1 For -H, R in 2 For -H, -F, -Cl, -Br, -CH3, -OCH3, -NO2, -CH2-O-Ph or -CN; R in 1 -H; R in 2 It can be -H, -F, -Cl, -Br, -CH3, -OCH3, -NO2, -CH2-O-Ph or -CN.

6. The degrading agent based on an indole-based fused covalent warhead according to claim 1, characterized in that, for or R 1 and R 2 Each can be represented independently as -Cl or -OCH3.

7. A method for preparing a degrading agent based on an indole-based fused covalent warhead as described in any one of claims 1-6, characterized in that, Includes the following steps: After reacting compound A with compound B, the degrading agent based on the indole group-fused covalent warhead is obtained; The structural formula of compound A is: ； The structural formula of compound B is as follows: Boc represents tert-butyloxycarbonyl.

8. The preparation method according to claim 7, characterized in that, When R is -NH-, the preparation method of compound A includes the following steps: Will After reacting with compound C, compound A is obtained; The structural formula of compound C is: 。 9. The use of the indole-based fused covalent warhead degrader according to any one of claims 1-6 in the preparation of BRD4 protein degrader or BRD3 protein degrader.

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