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

Abstract

The application discloses a degrading agent based on an indole group fused covalent warhead, and a preparation method and application thereof, relates to the technical field of drug development, and the structural formula of the degrading agent is as follows: R is a single bond or -NH-; is a single bond,,,,,,,,, or ; is -NH-,,, or ; is or ; R 1 and R 2 Each is independently -H, -F, -Cl, -Br, -CH3, -OCH3, -NO2, -CH2-O-Ph or -CN; Ph is a phenyl group; represents a connection site. The application uses an indole skeleton and an acrylic ester to construct an E3 ubiquitin ligase ligand, and then connects the BRD4 protein inhibitor JQ1 through a linker, so that a series of degrading agents based on an indole group fused covalent warhead are obtained, E3 ubiquitin ligase ligand library and protein degradation targeting chimera molecule library are enriched, and the targeted degradation of BRD4 protein is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug development, and particularly relates to a degrader based on an indole group fused covalent warhead and a preparation method and application thereof. BACKGROUND

[0002] The generation and development of most diseases are related to abnormal expression or aggregation of proteins. In view of this pathological mechanism, the traditional drug development idea is to develop various small molecule or protein inhibitors to occupy and block the active sites of target proteins and inhibit the functional activity of the proteins. However, more than 80% of proteins in the human proteome lack a druggable active site, and it is difficult to target them by traditional small molecule inhibitors.

[0003] Targeted protein degradation technology (TPD) is a technology developed in recent years, which utilizes the main endogenous pathways of protein and organelle degradation in eukaryotic cells: lysosomal pathway and ubiquitin-proteasome system (UPS) pathway, and realizes functional intervention by specifically inducing degradation of target proteins, and shows great potential in various disease treatment fields, especially in tumor treatment, intervention of neurodegenerative diseases and regulation of metabolic disorders, and has important application value, providing a new solution for “undruggable” targets that are difficult to deal with by traditional small molecule inhibitors and gene interference technology.

[0004] PROTACs based on UPS are an important research direction in the field of TPD, such as Figure 1 as shown in the formula (Ub represents ubiquitin and ATP represents adenine nucleotide triphosphate), which contains three key components: an E3 ubiquitin ligase binding ligand, a target protein (POI) specific ligand (POI ligand) and a linker connecting the two. This kind of molecule forms a ternary complex by simultaneously binding E3 ubiquitin ligase and target protein, and under the synergistic action of E1 ubiquitin activating enzyme, E2 ubiquitin binding enzyme and E3 ubiquitin ligase, it promotes E3 ubiquitin ligase to label ubiquitin molecules on the target protein. The target protein modified by multiple rounds of ubiquitination is then recognized and degraded by the proteasome, while the protein degradation targeting chimera molecule can be recycled in this process. Due to the catalytic nature of this mechanism, even if the protein degradation targeting chimera has a low affinity for the target protein or is bound to a non-active site, it can still effectively induce degradation of the target protein under substoichiometric conditions.

[0005] In recent years, significant progress has been made in the field of protein degradation targeting chimera, successfully achieving the degradation of various proteins, including endosome proteins, protein kinases, transcription regulators, and some proteins that are traditionally considered "undruggable", and at least a dozen protein degradation targeting chimeras have entered clinical trials. Despite the rapid development of protein degradation targeting chimera technology, the limited number and types of available E3 ubiquitin ligases and their ligands remain a key problem that needs to be addressed in current protein degradation targeting chimera technology. Although the human genome encodes hundreds of E3 ubiquitin ligases, to date, most developed protein degradation targeting chimera molecules still mainly rely on the two E3 ubiquitin ligases of CRBN or VHL. The recent emergence of drug resistance of CRBN or VHL-based protein degradation targeting chimeras highlights the urgent need to develop other E3 ubiquitin ligases and their ligands to fully utilize the capabilities of protein degradation targeting chimeras. In addition, the specific expression of E3 ubiquitin ligases in cells and tissues provides a theoretical basis for the development of protein degradation targeting chimera molecules with higher target selectivity.

[0006] Bromodomain-containing protein 4 (BRD4) is a member of the bromodomain and extraterminal domain (BET) protein family, which can recognize acetylated histones and localize to the promoter or enhancer region of the target gene, initiating and maintaining the expression of tumor-related genes. BRD4 protein is closely related to the regulation of various transcription factors and chromatin modification, involved in DNA damage repair and maintenance of telomere function, thereby maintaining the survival of tumor cells.

[0007] Therefore, it is of great significance to explore and develop new E3 ubiquitin ligases and their ligands to overcome the limitations of existing technologies, continuously enrich the protein degradation targeting chimera molecule library, achieve the degradation of target proteins such as BRD4 protein, and treat cancers related to the function of target proteins.

[0008] Therefore, the prior art still needs to be improved and developed. SUMMARY

[0009] Based on the shortcomings of the prior art described above, the purpose of the present application is to provide a degradation agent based on an indole group fused covalent warhead and its preparation method and application, aiming to enrich the protein degradation targeting chimera molecule library and achieve targeted degradation of BRD4 protein.

[0010] The technical scheme of the present application is as follows:

[0011] In a first aspect of the present application, a degradation agent based on an indole group fused covalent warhead is provided, wherein the structure of the degradation agent based on an indole group fused covalent warhead is:

[0012] ;

[0013] R is a single bond or -NH-;

[0014] (i.e. linker 1) is a single bond, , , , , , , , , or ;

[0015] (i.e. linker 2) is -NH-, , or ;

[0016] is or ; R 1 and R 2 are located at any connectable position on the benzene ring or the 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;

[0017] represents a connection site (if no special description is made, the connection site represented by in the structural formula below means a connection site, and the meaning thereof will not be described again when appears again below).

[0018] The present application has found, through research, that the covalent warhead of an electrophilic group introduced in the backbone structure can form a stable covalent bond with a specific amino acid residue in the E3 ubiquitin ligase or substrate receptor subunit, thereby enhancing the binding capacity and degradation efficiency of the E3 ubiquitin ligase ligand, and targeting the E3 ubiquitin ligase lacking a traditional small molecule binding pocket to expand the range of targetable E3 ubiquitin ligases. In addition, the present application takes BRD4 protein as a phenotypic research model. First, based on the property that methyl acrylate can specifically covalently bind to the cysteine (Cys) residue (HS, sulfhydryl) in the protein (as shown in Figure 2 ), it is combined on the indole skeleton to develop a new E3 ubiquitin ligase ligand, and then the E3 ubiquitin ligase ligand (obtained by introducing the covalent warhead acrylate into the indole skeleton) is coupled with the BRD4 protein inhibitor JQ1 with good targeting to construct a degradation agent based on the BRD4 protein target.

[0019] The degradation agent based on the indole group fusion covalent warhead provided by the application can recruit an E3 ubiquitin ligase (which is different from the known E3 ubiquitin ligases such as CRBN, VHL, DCAF11 and DCAF16) and a target protein (BRD4 protein) to form a ternary complex, induce ubiquitination of the BRD4 protein, and finally realize degradation after the ubiquitinated BRD4 protein is recognized by a proteasome. The degradation agent based on the indole group fusion covalent warhead can also recruit an E3 ubiquitin ligase (which is different from the known E3 ubiquitin ligases such as CRBN, VHL, DCAF11 and DCAF16) and BRD3 (bromodomain-containing protein 3) to form a ternary complex, induce ubiquitination of the BRD3 protein, and finally realize degradation. The E3 ubiquitin ligase ligand in the degradation agent based on the indole group fusion covalent warhead has strong binding capacity with the E3 ubiquitin ligase, thereby improving the degradation efficiency of the BRD4 protein and the BRD3 protein. In addition, the degradation agent based on the indole group fusion covalent warhead exhibits significant anti-proliferation activity in various tumor cell lines, can down-regulate downstream signal pathway proteins of c-Myc (cell myeloma virus cancer gene homolog), and can significantly inhibit the migration ability of MDA-MB-231 cells (triple-negative breast cancer cells), which indicates the potential of the degradation agent based on the indole group fusion covalent warhead as a cancer treatment drug.

[0020] The degradation agent based on the indole group fusion covalent warhead provided by the application not only realizes targeted degradation of the BRD4 protein, but also realizes targeted degradation of the BRD3 protein, can recruit a kind of E3 ubiquitin ligase that has not been reported, and enriches the E3 ubiquitin ligand library and the protein degradation targeted chimera molecule library.

[0021] Optionally, R is -NH-; is , , , , or ; is -NH-.

[0022] Optionally, R is -NH-; is , , or ;

[0023] is , or .

[0024] Optionally, R is a single bond, is a single bond, For , or .

[0025] Optionally, R in R is -H, 1 -H, R in R is -H, -F, -Cl, -Br, -CH3, -OCH3, -NO2, -CH2-O-Ph or -CN; 2 -H,

[0026] R in R is -H; 1 -H, R in R is -H, -F, -Cl, -Br, -CH3, -OCH3, -NO2, -CH2-O-Ph or -CN. 2 -H,

[0027] Optionally, or , R 1 and R 2 are each independently -Cl or -OCH3. In a second aspect of the present application, a preparation method of the degradation agent based on the indole group fusion covalent warhead according to the present application is provided, and the preparation method comprises the following steps:

[0028] After the compound A is reacted with the compound B, the degradation agent based on the indole group fusion covalent warhead is obtained.

[0029] The structural formula of the compound A is as follows:

[0030]

[0031] ;

[0032] The structural formula of the compound B is as follows: , and Boc represents tert-butyloxy carbonyl.

[0033] In the present application, the N-allylation reaction of the compound A and the compound B is performed to obtain the degradation agent based on the indole group fusion covalent warhead.

[0034] Optionally, when R is -NH-, the preparation method of the compound A comprises the following steps:

[0035] After the compound D is reacted with the compound C, the compound A is obtained. The structural formula of the compound C is as follows:

[0036] .

[0037] ​​Specifically, the preparation method of the compound C comprises the following steps:

[0038] The compound E is added into dichloromethane and trifluoroacetic acid, and after reaction, the compound C is obtained;

[0039] The compound E has the following structural formula: .

[0040] R in the structural formula of the compound A and the compound C above, , , R 1 and R 2 are respectively same as R , , R 1 and R 2 in the structural formula of the degradation agent based on the indole group fusion covalent warhead in the above.

[0041] In a third aspect, the present application provides application of the degradation agent based on the indole group fusion covalent warhead in the present application in the above in preparation of a BRD4 protein degradation agent or a BRD3 protein degradation agent.

[0042] Beneficial effects: the present application uses an indole skeleton and an acrylic ester to construct an E3 ubiquitin ligase ligand, and then connects the BRD4 protein inhibitor JQ1 through a linker, so that a series of degradation agents based on the indole group fusion covalent warhead are obtained. The degradation agent based on the indole group fusion covalent warhead can target BRD3 protein and BRD4 protein and efficiently degrade BRD3 protein and BRD4 protein. Therefore, the present application not only realizes the targeted degradation of BRD4 protein, but also realizes the degradation of BRD3 protein while enriching the E3 ubiquitin ligase ligand library and the protein degradation targeting chimera molecule library. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 It is a schematic diagram of the mechanism of the protein degradation targeting chimera degrading target proteins in the prior art.

[0044] Figure 2 It is a schematic diagram of the mechanism of the degradation agent based on the indole group fusion covalent warhead degrading BRD4 protein in the present application.

[0045] Figure 3 It is a result graph of construction and expression of a high-content screening platform, wherein (a) is a result graph of protein immunoblotting of a double fluorescence screening model, and (b) is a fluorescence graph of the double fluorescence screening model.

[0046] Figure 4 It is a result graph of activity test of each compound in different concentrations (100 nM, 1 µM) in the examples.

[0047] Figure 5 Figure 4 is a graph of degradation effect of the top four compounds in the high content analysis results on endogenous BRD4 protein.

[0048] Figure 6 Figure 5 is a graph of degradation activity results of compound D29, wherein (a) is a graph of degradation effect of compound D29 on BET family proteins, (b) is a semi-quantitative graph of degradation of BET family proteins by compound D29, (c) is a DC 50 Figure 5 is a graph of degradation activity results of compound D29, wherein (a) is a graph of degradation effect of compound D29 on BET family proteins, (b) is a semi-quantitative graph of degradation of BET family proteins by compound D29, (c) is a DC

[0049] Figure 7 Figure 6 is a graph of degradation mechanism verification results of compound D29, wherein (a) is a graph of degradation mechanism results of compound D29 under high content analysis microscope, (b) is a semi-quantitative graph of degradation mechanism verification of compound D29 under high content analysis microscope.

[0050] Figure 8 Figure 7 is a graph of results of lysosome pathway inhibition experiment and proteasome pathway inhibition experiment of compound D29, wherein (a) is a graph of results of lysosome pathway inhibition experiment of compound D29, (b) is a semi-quantitative graph of results of lysosome pathway inhibition experiment of compound D29, (c) is a graph of results of proteasome pathway inhibition experiment of compound D29, (d) is a semi-quantitative graph of results of proteasome pathway inhibition experiment of compound D29.

[0051] Figure 9 Figure 8 is a graph of test results of E3 ubiquitin ligase recruited by compound D29. DETAILED DESCRIPTION

[0052] The present application provides a degrader based on an indole group fused covalent warhead and a preparation method and application thereof. In order to make the purpose, technical scheme and effect of the present application more clear and definite, 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.

[0053] 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.

[0054] If the description of "first", "second" and the like is involved in the embodiments of the present application, the description of "first", "second" and the like is only for the purpose of description, and cannot be understood as indicating or implying the relative importance of the indicated technical features or implicitly indicating the number of the indicated technical features.

[0055] The present application is further illustrated by the following specific examples.

[0056] The meanings of some symbols in the following examples and synthetic routes are as follows:

[0057] DIPEA: N,N-diisopropylethylamine;

[0058] EDCl: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride;

[0059] HOBt: 1-hydroxybenzotriazole;

[0060] DMF: N,N-dimethylformamide;

[0061] r.t.: room temperature;

[0062] e.q.: equivalent;

[0063] TFA: trifluoroacetic acid;

[0064] DCM: dichloromethane;

[0065] MeOH: methanol;

[0066] DABCO: 1,4-diazabicyclo[2.2.2]octane;

[0067] EtOAc: ethyl acetate;

[0068] PE: petroleum ether;

[0069] JQ1: the structural formula is .

[0070] In the following examples, the structural formulae of compounds D1 to D16 are as follows: wherein, The specific structures of the indole skeleton part are shown in Table 1 and Table 2.

[0071] Table 1, the specific structure of the indole skeleton part in the structural formulae of compounds D1 to D10

[0072]

[0073] Table 2, the specific structure of the indole skeleton part in the structural formulae of compounds D11 to D16

[0074]

[0075] The structural formula of compound D17 to compound D21 is:

[0076] ; wherein the specific structure of X is shown in Table 3.

[0077] Table 3, the specific structure of X in the structural formula of compound D17 to compound D21

[0078]

[0079] The structural formula of compound D22 to compound D36 is: , wherein, and The specific structure is shown in Table 4, Table 5 and Table 6.

[0080] Table 4, the specific structure of and in the structural formula of compound D22 to compound D26

[0081]

[0082] Table 5, the specific structure of and in the structural formula of compound D27 to compound D31

[0083]

[0084] Table 6, the specific structure of and in the structural formula of compound D32 to compound D36

[0085]

[0086] Example 1 synthesis of compound D1 to compound D16

[0087] The synthesis route is as follows:

[0088] .

[0089] In this synthesis route, the specific structure of in the structural formula of final product compound D1 to compound D16 is shown in Table 1 and Table 2.

[0090] In the process of preparing compound D1 to compound D16, the specific structure of in the structural formula of compound 1, compound 3, compound 4, compound 5 in the above synthesis route is respectively in the structural formula of corresponding prepared compound D1 to compound D16.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.

[0091] Synthesis of compound D1:

[0092] 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%).

[0093] 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%).

[0094] 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), then the reaction system was activated in ice bath for 30 min, and then JQ1 (80 mg, 0.2 mmol) was added, and the reaction was stirred at room temperature overnight (i.e. 12 h). After the reaction was completed, the reaction solution was extracted with aqueous citric acid solution (5% by mass fraction of citric acid), saturated NaHCO3 aqueous solution and saturated brine as extraction liquid (i.e. 3 times of extraction), and the organic phase was dried over anhydrous sodium sulfate and rotary evaporated under reduced pressure to obtain the crude product. Then column chromatography (eluent: DCM and MeOH in a volume ratio of 20:1) was used for purification to obtain compound 5 (0.10 g, yield 84.7%).

[0095] 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), and then DABCO (4 mg, 0.034 mmol) was added to the reaction solution, and the reaction system was stirred at room temperature for 0.5 h, and then the solvent was removed by rotary evaporation to obtain the crude product, which was then separated by column chromatography (eluent: DCM and MeOH in a volume ratio of 20:1) to obtain compound D1 (107 mg, yield 90.2%).

[0096] The nuclear magnetic resonance hydrogen spectrum data of compound D1 are 1 H NMR (600 MHz, CDCl3) δ 7.96-7.90 (m, 1H), 7.88-7.75 (m, 1H), 7.59 (d, J =8.0 Hz, 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).

[0097] The nuclear magnetic resonance carbon spectrum data of compound D1 are 13C NMR (151 MHz, CDC13) 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, 129.8, 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.

[0098] HRMS-ESI (m / z) for C + calcd for C 36 H 37 O4N7ClS + : 698.2311; found: 698.2309; purity: 99.32% (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).

[0099] Synthesis of compound D2:

[0100] Referring to the synthesis method of compound D1, compound D2 (63.7 mg, yield 89.3%) was obtained. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (600 MHz, CDC13) 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 = 8.3 Hz, 1H), 6.78-6.70 (m, 1H), 6.14 (s, 1H), 5.51-5.41 (m, 2H), 4.87 (d, J = 2.2 Hz, 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 nuclear magnetic resonance carbon spectrum data is 13C NMR (151 MHz, CDC13) 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 is HRMS-ESI (m / z): [M+H] + calcd for C 36 H 36 O4N7ClFS + : 716.2217; found: 716.2211; purity: 99.55%.

[0101] Synthesis of compound D3:

[0102] Referring to the synthesis method of compound D1, compound D3 (38.4 mg, 52.5%) was obtained. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (400 MHz, CDC13) δ 7.97-7.87 (m, 1H), 7.69-7.57 (m, 1H), 7.36 (d, J = 8.0 Hz, 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 = 2.2 Hz, 3H), 3.56-3.36 (m, 6H), 2.63 (d, J = 2.4 Hz, 3H), 2.36 (d, J = 2.3 Hz, 3H), 1.81-1.74 (m, 2H), 1.60 (s, 3H). Its nuclear magnetic resonance carbon spectrum data is 13C NMR (101 MHz, CDC13) 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 is HRMS-ESI (m / z): [M+H] + calcd for C 36 H 36 O4N7Cl2S + : 732.1921; found: 732.1925; purity: 99.34%.

[0103] Synthesis of compound D4:

[0104] Referring to the synthesis method of compound D1, compound D4 (65.3 mg, yield 84.3%) was obtained. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (400 MHz, CDC13) δ 8.09-7.95 (m, 1H), 7.80-7.66 (m, 1H), 7.39 (d, J = 8.3 Hz, 2H), 7.34-7.27 (m, 3H), 7.22 (d, J = 8.3 Hz, 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 nuclear magnetic resonance carbon spectrum data is 13C NMR (101 MHz, CDC13) 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 is HRMS-ESI (m / z): [M+H] + calcd for C 36 H 36 O4N7BrClS + : 776.1416; found: 776.1417; purity: 99.77%.

[0105] Synthesis of compound D5:

[0106] Referring to the synthesis method of compound D1, compound D5 (44 mg, yield 61.4%) was obtained. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (600 MHz, CDC13) δ 7.85-7.75 (m, 1H), 7.39 (d, J = 7.8 Hz, 3H), 7.30 (d, J = 7.6 Hz, 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 nuclear magnetic resonance carbon spectrum data is 13CNMR (151 MHz, CDC13) 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 spectrum data is HRMS-ESI (m / z): [M+H] + calcd for C 36 H 36 O4N7ClFS + : 716.2217; found: 716.2213; purity: 99.65%.

[0107] Synthesis of compound D6:

[0108] Referring to the synthesis method of compound D1, compound D6 (63.6 mg, yield 82.0%) was obtained. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (400 MHz, CDC13) δ 7.89-7.74 (m, 1H), 7.65-7.49 (m, 2H), 7.27 (d, J = 8.3 Hz, 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 nuclear magnetic resonance carbon spectrum data is 13C NMR (101 MHz, CDC13) 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 is HRMS-ESI (m / z): [M+H] + calcd for C 36 H 36 O4N7BrClS + : 776.1416; found: 776.1416; purity: 99.91%.

[0109] Synthesis of compound D7:

[0110] Referring to the synthesis method of compound D1, compound D7 (43.5 mg, yield 61.1%) was obtained. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (400 MHz, CDC13) 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 = 8.5 Hz, 1H), 7.01-6.93 (m, 1H), 6.11 (s, 1H), 5.44 (s, 2H), 4.83 (d, J = 5.1 Hz, 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 nuclear magnetic resonance carbon spectrum data is 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%.

[0111] Synthesis of compound D8:

[0112] 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 (151 MHz, CDC13) 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 spectrum data was HRMS-ESI (m / z): [M+H] + calcd for C 37 H 39 O5N7ClS + : 728.2416; found: 728.2434; purity: 99.15%.

[0113] Synthesis of compound D9:

[0114] Referring to the synthesis method of compound D1, compound D9 (44.9 mg, yield 60.5%) was obtained. Its nuclear magnetic resonance hydrogen spectrum data was 1 H NMR (600 MHz, CDC13) δ 8.56-8.48 (m, 1H), 8.21-8.08 (m, 2H), 7.48 (t, J = 6.5 Hz, 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 nuclear magnetic resonance carbon spectrum data was 13C NMR (151 MHz, CDC13) 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 is HRMS-ESI (m / z): [M+H] + calcd for C 36 H 36 O6N8ClS + : 743.2162; found: 743.2154; purity: 99.33%.

[0115] Synthesis of compound D10:

[0116] Referring to the synthesis method of compound D1, compound D10 (60.5 mg, yield 75.2%) was obtained. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (400 MHz, CDC13) 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 = 3.7 Hz, 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 = 3.7 Hz, 3H), 2.29 (d, J = 3.8 Hz, 3H), 1.71-1.60 (m, 2H), 1.54 (d, J = 3.6 Hz, 3H). Its nuclear magnetic resonance carbon spectrum data is 13C NMR (101 MHz, CDC13) δ 171.5, 166.2, 164.2, 162.2, 155.7, 153.8, 150.0, 137.4, 137.0, 136.9, 136.4, 135.6, 133.8, 132.2, 132.0, 131.1, 131.0, 130.6, 129.8, 128.8, 128.5, 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 spectrum data is HRMS-ESI (m / z): [M+H] + calcd for C 43 H 43 O5N7ClS + : 804.2729; found: 804.2733; purity: 96.22%.

[0117] Synthesis of compound D11:

[0118] Referring to the synthesis method of compound D1, compound D11 (63.5 mg, yield 88.8%) was obtained. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (600 MHz, CDC13) δ 7.78-7.70 (m, 1H), 7.51-7.48 (m, 1H), 7.43-7.35 (m, 3H), 7.30 (d, J = 8.0 Hz, 2H), 7.07 (s, 1H), 6.92 (dd, J = 9.8, 2.2 Hz, 1H), 6.89-6.84 (m, 1H), 6.15 (s, 1H), 5.42 (s, 2H), 4.88 (s, 1H), 4.67 (d, J = 7.0 Hz, 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 nuclear magnetic resonance carbon spectrum data is 13C NMR (151 MHz, CDC13) 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 spectrum data is HRMS-ESI (m / z): [M+H] + calcd for C 36 H 36 O4N7ClFS + : 716.2217; found: 716.2210; purity: 99.53%.

[0119] Synthesis of compound D12:

[0120] Referring to the synthesis method of compound D1, compound D12 (72.2 mg, yield 93.0%) was obtained. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (400 MHz, CDC13) δ 7.93-7.83 (m, 1H), 7.74-7.63 (m, 1H), 7.41-7.34 (m, 4H), 7.27 (d, J = 8.7 Hz, 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 nuclear magnetic resonance carbon spectrum data is 13C NMR (101 MHz, CDC13) 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 is HRMS-ESI (m / z): [M+H] + calcd for C 36 H 36 O4N7BrClS + : 776.1416; found: 776.1420; purity: 98.78%.

[0121] Synthesis of compound D13:

[0122] Referring to the synthesis method of compound D1, compound D13 (63.4 mg, yield 87.6%) was obtained. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (400 MHz, CDC13) δ 8.11 - 8.00 (m, 1H), 7.68 - 7.50 (m, 3H), 7.37 (d, J = 8.2 Hz, 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 nuclear magnetic resonance carbon spectrum data is 13C NMR (101 MHz, CDC13) 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 is HRMS-ESI (m / z): [M+H] + calcd for C 37 H 36 O4N8ClS + : 723.2263; found: 723.2260; purity: 97.10%.

[0123] Synthesis of compound D14:

[0124] Referring to the synthesis method of compound D1, compound D14 (41.9 mg, yield 54.7%) was obtained. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (600 MHz, CDC13) δ 7.98-7.88 (m, 1H), 7.47 (d, J = 8.1 Hz, 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 nuclear magnetic resonance carbon spectrum data is 13C NMR (151 MHz, CDC13) 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 spectrum data is HRMS-ESI (m / z): [M+H] + calcd for C 36 H 35 O4N7Cl3S + : 766.1531; found: 766.1528; purity: 99.89%.

[0125] Synthesis of compound D15:

[0126] Referring to the synthesis method of compound D1, compound D15 (71.2 mg, yield 93.9%) was obtained. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (600 MHz, CDC13) 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 = 2.8 Hz, 1H), 6.98 (s, 1H), 6.65 (s, 1H), 6.12 (d, J = 2.0 Hz, 1H), 5.44 (d, J = 2.2 Hz, 2H), 4.83 (d, J = 2.1 Hz, 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 nuclear magnetic resonance carbon spectrum data is 13C NMR (151 MHz, CDC13) 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 spectrum data is HRMS-ESI (m / z): [M+H] + calcd for C 38 H 41 O6N7ClS + : 758.2522; found: 758.2521; purity: 99.57%.

[0127] Synthesis of compound D16:

[0128] Referring to the synthesis method of compound D1, compound D16 (37.5 mg, yield 53.6%) was obtained. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (600 MHz, CDC13) δ 8.47 (d, J = 5.4 Hz, 1H), 8.19 (d, J = 7.6 Hz, 1H), 7.69 (d, J = 7.3 Hz, 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 = 5.7 Hz, 1H), 5.59-5.41 (m, 2H), 5.10 (d, J = 5.5 Hz, 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 nuclear magnetic resonance carbon spectrum data is 13C NMR (151 MHz, CDCI3) d 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, 1 1.9. Its high resolution mass spectrum data is HRMS-ESI (m / z): [M+H] + calcd for C 35 H 36 O4N8ClS + : 699.2263; found: 699.2258; purity: 99.91%.

[0129] Synthesis of compound D17 to compound D21, compound D22, compound D27 and compound D32

[0130] The synthesis route is:

[0131] ;

[0132] In the process of preparing compound D17 to compound D21, the specific structure of X in the structural formula of compound 7, compound 8, compound 9 and compound 10 in the above synthesis route is the same as the specific structure of X in the structural formula of the corresponding prepared compound D17 to compound D21. For example, X in the structural formula of compound D17 is , then the X in the structural formula of the corresponding compound 7, compound 8, compound 9 and compound 10 used for preparing compound D17 is also . The preparation of the remaining compounds is similar.

[0133] Synthesis of compound D17:

[0134] According to the synthesis route of this embodiment, compound D17 (61.2 mg, yield 85.7%) is obtained by referring to the synthesis method of compound D1. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (600 MHz, CDCI3) d 7.65 (m, 1 H), 7.52 (t, J = 6.0 Hz, 1 H), 7.34 (dd, J = 8.6, 2.0 Hz, 2H), 7.27 (s, 1 H), 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%.

[0135] Synthesis of compound D18:

[0136] 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 spectrum data is 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%.

[0137] Synthesis of compound D19:

[0138] 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%.

[0139] Synthesis of compound D20:

[0140] 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, 2.4 Hz, 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 = 2.8 Hz, 2H), 3.78 (s, 3H), 3.76 (s, 2H), 3.73 - 3.70 (m, 1H), 3.68 (d, J = 1.8 Hz, 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 nuclear magnetic resonance spectrum data is 13 C NMR (151 MHz, CDC13) 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 spectrum data is HRMS-ESI (m / z): [M+H] + calcd for C 40 H 45 O7N7ClS + : 802.2784; found: 802.2755; purity: 99.97%.

[0141] Synthesis of compound D21:

[0142] According to the synthetic route of this example, referring to the synthesis method of compound D1, compound D21 (46.7 mg, yield 52.5%) was obtained. Its hydrogen nuclear magnetic resonance spectrum data is 1 H NMR (600 MHz, CDC13) δ 7.60 - 7.52 (m, 2H), 7.44 - 7.40 (m, 2H), 7.35 - 7.32 (m, 2H), 7.20 (d, J = 9.0 Hz, 1H), 7.06 (d, J = 2.4 Hz, 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 nuclear magnetic resonance spectrum data is 13 C NMR (151 MHz, CDC13) δ 170.6, 166.2, 163.8, 162.3, 155.8, 154.7, 149.8, 142.8, 138.6, 136.9, 136.7, 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 spectrum data is HRMS-ESI (m / z): [M+H] + calcd for C 44 H 53 O9N7ClS + : 890.3308; found: 890.3285; purity: 99.84%.

[0143] Synthesis of compound D22:

[0144] According to the synthetic route of this example, compound 7 in the route was replaced by , and compound D22 (69 mg, yield 93.2%) was obtained according to the synthetic method of compound D1. Its hydrogen nuclear magnetic resonance spectrum data is 1 H NMR (600 MHz, CDC13) δ 7.42-7.38 (m, 2H), 7.35-7.32 (m, 2H), 7.21 (d, J = 9.0 Hz, 1H), 7.07 (d, J = 2.4 Hz, 1H), 6.95 (dd, J= 9.0, 2.4 Hz, 1H), 6.59 (s, 1H), 6.22 (s, 1H), 5.21 (s, 3H), 4.81 (t, J = 6.8 Hz, 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 nuclear magnetic resonance spectrum data is 13 C NMR (126 MHz, CDC13) 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 spectrum data is HRMS-ESI (m / z): [M+H] + calcd for C 38 H 39 O5N7ClS + : 740.2416; found: 740.2404; purity: 99.12%.

[0145] Synthesis of compound D27:

[0146] According to the synthetic route of this example, compound 7 in which is replaced by , compound D27 (63.6 mg, yield 83.9%) was obtained according to the synthetic method of compound D1. Its hydrogen nuclear magnetic resonance spectrum data is 1 H NMR (600 MHz, CDC13) δ 7.43 - 7.39 (m, 2H), 7.35 - 7.32 (m, 2H), 7.19 - 7.16 (m, 1H), 7.05 (d, J = 2.4 Hz, 1H), 6.97 - 6.94 (m, 1H), 6.91 (s, 1H), 6.64 - 6.56 (m, 1H), 6.17 (d, J= 9.6 Hz, 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 = 14.0 Hz, 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, 6.1 Hz, 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 = 6.9 Hz, 3H). Its carbon nuclear magnetic resonance spectrum data is 13 C NMR (126 MHz, CDC13) δ 168.9, 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 spectrum data is HRMS-ESI (m / z): [M+H] + calcd for C 39 H 41 O5N7ClS + : 754.2573; found: 754.2577; purity: 99.57%.

[0147] Synthesis of compound D32:

[0148] According to the synthetic route of this example, compound 7 in which is replaced Compound D32 (64.9 mg, yield 86.0%) was obtained according to the synthetic method of compound D1. Its hydrogen nuclear magnetic resonance spectrum data is 1 H NMR (600 MHz, CDC13) δ 7.40 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 8.7 Hz, 2H), 7.19 (d, J = 9.0 Hz, 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%.

[0149] Example 3: Synthesis of compounds D23 to D26, D28 to D31, and D33 to D36

[0150] The synthesis route is as follows:

[0151] .

[0152] 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):

[0153] 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 in DMF (5 mL) and 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 (i.e. 12 h, to ensure that the reaction was complete). After the reaction was complete, the reaction liquid was extracted with an aqueous citric acid solution (5% by mass), a saturated NaHCO3 aqueous solution and saturated brine as the extraction liquid (i.e. 3 extractions in total) at a volume ratio of 1:1 with EtOAc. The organic phase was dried, concentrated, and then separated by column chromatography (eluent: PE and EtOAc at a volume ratio of 4:1) to obtain compound 11 (0.32 g, yield 88.7%).

[0154] Compound 11 (0.32 g, 0.89 mmol) was mixed with DCM (1 mL) and TFA (1 mL), stirred at room temperature for 0.5 h, and after the reaction was complete, extracted 3 times with DCM and H2O at a volume ratio of 1:1, and the organic phase was collected, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain white oil, i.e. compound 12 (0.21 g, yield 92.4%).

[0155] 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 sequentially added to a round-bottom flask, activated at 0°C for 30 min, and then compound 12 (0.21 g, 0.82 mmol) was added, and stirred at room temperature overnight (i.e. 12 h), and monitored by TLC (thin layer chromatography). After the reaction was complete, the reaction liquid was extracted with an aqueous citric acid solution (5% by mass), a saturated NaHCO3 aqueous solution and saturated brine as the extraction liquid (i.e. 3 extractions in total) at a volume ratio of 1:1 with EtOAc. The organic phase was dried, concentrated, and then purified by column chromatography (eluent: PE and EtOAc at a volume ratio of 8:1) to obtain compound 14 (0.29 g, yield 78.9%).

[0156] 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%).

[0157] 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%).

[0158] 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%).

[0159] 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%.

[0160] Synthesis of compound 24:

[0161] 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%.

[0162] Synthesis of compound 25:

[0163] 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= 8.9 Hz, 1 H), 7.04 (d, J = 8.9 Hz, 1 H), 6.93 (m, 1 H), 6.55 (s, 1 H), 6.19 (s, 1 H), 5.16 (d, J = 2.5 Hz, 1 H), 4.64 (t, J = 6.4 Hz, 3 H), 4.34 (d, J = 6.4 Hz, 3 H), 3.82 (m, 5 H), 3.76 (m, 4 H), 3.72 (d, J = 6.9 Hz, 1 H), 3.68 (m, 1 H), 3.64 (m, 1 H), 3.58 (m, 5 H), 3.48 (m, 2 H), 3.38 (m, 1 H), 2.70 - 2.65 (m, 2 H), 2.63 (s, 3 H), 2.39 (s, 3 H), 1.66 (s, 3 H). Its carbon nuclear magnetic resonance spectrum data is J = 2.5 Hz, 1 H), 6.93 (m, 1 H), 6.55 (s, 1 H), 6.19 (s, 1 H), 5.16 (d, J = 2.5 Hz, 1 H), 4.64 (t, J = 6.4 Hz, 3 H), 4.34 (d, J = 6.4 Hz, 3 H), 3.82 (m, 5 H), 3.76 (m, 4 H), 3.72 (d, J = 6.9 Hz, 1 H), 3.83 (m, 5 H), 3.76 (m, 4 H), 3.72 (d, J = 1.3 Hz, 3 H), 3.71 - 3.66 (m, 2 H), 3.58 (m, 5 H), 3.48 (m, 2 H), 3.38 (m, 1 H), 2.70 - 2.65 (m, 2 H), 2.63 (s, 3 H), 2.39 (s, 3 H), 1.66 (s, 3 H). Its carbon nuclear magnetic resonance spectrum data is 13 C NMR (126 MHz, CDC13) 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 spectrum data is HRMS-ESI (m / z): [M+H] + calcd for C 43 H 48 O7N8ClS + : 855.3050; found: 855.3055; purity: 99.31%.

[0164] Synthesis of compound D26:

[0165] Referring to the synthesis method of compound D23, compound D26 (88.9 mg, yield 94.3%) was obtained. Its hydrogen nuclear magnetic resonance spectrum data is 1 H NMR (500 MHz, CDC13) δ 7.43 (s, 1 H), 7.36 (d, J = 8.0 Hz, 2 H), 7.27 (d, J = 8.3 Hz, 2 H), 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.) + calcd for C 47 H 56 O9N8ClS + :943.3574; found: 943.3582; purity: 98.81%.

[0166] Synthesis of compound D28:

[0167] 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 HNMR (600MHz, CDCl3) δ 7.90 (s, 1H), 7.45 (d, J= 8.5 Hz, 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), 4.68-4.62 (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 nuclear magnetic resonance spectrum data is 13 C NMR (151 MHz, CDC13) 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 spectrum data is HRMS-ESI (m / z): [M+H] + calcd for C 41 H 44 O6N8ClS + : 811.2788; found: 811.2780; purity: 99.89%.

[0168] Synthesis of compound D29:

[0169] Referring to the synthesis method of compound D23, compound 15a therein is replaced by compound 15b ( ), to obtain compound D29, wherein Y is (48.7 mg, yield 56.2%). Its hydrogen nuclear magnetic resonance spectrum data is 1H NMR (600 MHz, CDC13) 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, 2.5 Hz, 1H), 6.86 (d, J = 7.6 Hz, 1H), 6.82 - 6.74 (m, 1H), 6.65 - 6.57 (m, 1H), 6.16 (d, J = 11.3 Hz, 1H), 5.41 (d, J = 5.1 Hz, 2H), 4.86 (d, J = 3.8 Hz, 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 = 13.1 Hz, 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 nuclear magnetic resonance spectrum data is 13 C NMR (151 MHz, CDC13) 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 spectrum data is HRMS-ESI (m / z): [M+H] + calcd for C 45 H 52 O6N8ClS +: 867.3414; found: 867.3430; purity: 98.71%.

[0170] Synthesis of compound D30:

[0171] Referring to the synthesis method of compound D23, compound 15a therein is replaced by compound 15b ( ), to obtain compound D30, wherein Y is (55.4 mg, yield 63.7%). Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (600 MHz, 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 = 27.4 Hz, 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), 4.18-4.10 (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, 1H), 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). Its nuclear magnetic resonance carbon spectrum data is 13 C NMR (151 MHz, CDCl3) δ 170.7, 169.7, 166.2, 163.9, 161.7, 155.7, 154.8, 149.9, 139.3, 138.2, 136.9, 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 spectrum data is HRMS-ESI (m / z): [M+H] +Caled for C 44 H 50 O7N8ClS + : 869.3206; found: 869.3222; purity: 97.36%.

[0172] Synthesis of compound D31:

[0173] Referring to the synthesis method of compound D23, compound 15a therein was replaced by compound 15b ( ), to obtain compound D31, wherein Y is (73.8 mg, yield 77.1%). Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (600 MHz, CDC13) δ 7.40 (d, J = 8.0 Hz, 2H), 7.34 - 7.29 (m, 2H), 7.19 - 7.14 (m, 1H), 7.01 (d, J = 2.3 Hz, 1H), 6.97 - 6.91 (m, 2H), 6.90 - 6.84 (m, 1H), 6.15 (d, J = 7.7 Hz, 1H), 5.47 - 5.35 (m, 2H), 4.87 (s, 1H), 4.69 - 4.57 (m, 2H), 4.15 - 4.08 (m, 1H), 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 nuclear magnetic resonance carbon spectrum data is 13C NMR (151 MHz, CDC13) 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, 124.9, 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 spectrum data was HRMS-ESI (m / z): [M+H] + calcd for C 48 H 58 O9N8ClS + : 957.3730; found: 957.3741; purity: 99.49%.

[0174] Synthesis of compound D33:

[0175] Referring to the synthesis method of compound D23, compound 15a therein was replaced by compound 15c ( ), to obtain compound D33, wherein Y is (73.7 mg, yield 90.9%). Its nuclear magnetic resonance hydrogen spectrum data was 1 H NMR (600 MHz, CDC13) δ 7.42 (dd, J = 8.6, 2.7 Hz, 2H), 7.34 (dd, J = 8.7, 3.0 Hz, 2H), 7.20-7.15 (m, 1H), 7.05 (d, J = 2.5 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 6.91 (dd, J = 9.0, 2.4 Hz, 1H), 6.40 (d, J = 69.5 Hz, 1H), 6.19 (d, J = 9.7 Hz, 1H), 5.72 (d, J = 55.7 Hz, 1H), 5.05 (d, J= 6.0 Hz, 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 = 2.4 Hz, 3H), 3.55-3.42 (m, 1H), 3.32-3.23 (m, 1H), 2.60 (d, J = 42.3 Hz, 3H), 2.40 (d, J = 4.8 Hz, 3H), 1.75 (s, 4H), 1.67 (d, J = 3.1 Hz, 3H). Its carbon nuclear magnetic resonance spectrum data is 13 C NMR (151 MHz, CDC13) 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 spectrum data is HRMS-ESI (m / z): [M+H] + calcd for C 41 H 44 O6N8ClS + : 811.2788; found: 811.2787; purity: 98.98%.

[0176] Synthesis of compound D34:

[0177] Referring to the synthesis method of compound D23, compound 15a therein is replaced by compound 15c ( ), to obtain compound D34, wherein Y is (52.8 mg, yield 60.9%). Its hydrogen nuclear magnetic resonance spectrum data is 1 H NMR (600 MHz, CDC13) δ 7.44-7.36 (m, 2H), 7.35-7.28 (m, 2H), 7.15 (t, J = 8.2 Hz, 1H), 7.01 (d, J = 6.7 Hz, 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%.

[0178] Synthesis of compound D35:

[0179] Referring to the synthesis method of compound D23, compound 15a therein is replaced by compound 15c ( ), to obtain compound D35, wherein Y is (48.6 mg, yield 55.9%). Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (600 MHz, 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 nuclear magnetic resonance carbon spectrum data is 13 C NMR (151 MHz, CDCl3) δ 171.1, 170.4, 166.0, 164.2, 162.9, 155.5, 154.7, 149.3, 142.0, 139.3, 137.1, 136.6, 136.2, 135.9, 132.6, 131.7, 131.4, 131.0, 130.7, 130.0, 128.8, 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 spectrum data is HRMS-ESI (m / z): [M+H] + calcd for C 44 H50 O7N8ClS + : 869.3206; found: 869.3225; purity: 99.27%.

[0180] Synthesis of compound D36:

[0181] Referring to the synthesis method of compound D23, compound 15a therein was replaced by compound 15c ( ), to obtain compound D36, wherein Y is (66.1 mg, yield 69.1%). Its nuclear magnetic resonance hydrogen spectrum data are 1 H NMR (600 MHz, CDC13) δ 7.44-7.40 (m, 1H), 7.39 (d, J = 8.2 Hz, 2H), 7.30 (d, J = 8.3 Hz, 2H), 7.20-7.12 (m, 2H), 7.04-7.00 (m, 1H), 6.90 (dd, J = 9.0, 2.5 Hz, 1H), 6.52 (s, 1H), 6.16 (s, 1H), 5.12 (s, 2H), 5.04 (s, 1H), 4.68 (t, J = 7.0 Hz, 1H), 4.06-3.99 (m, 1H), 3.81 (s, 3H), 3.80-3.75 (m, 2H), 3.74 (d, J = 1.7 Hz, 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 nuclear magnetic resonance carbon spectrum data are 13C NMR (151 MHz, CDC13) 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, 125.9, 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 spectrum data is HRMS-ESI (m / z): [M+H] + calcd for C 48 H 58 O9N8ClS + : 957.3730; found: 957.3751; purity: 96.38%.

[0182] Test:

[0183] (1) High content analysis screening

[0184] a. Construction and expression of a dual fluorescence screening model containing BD1 domain, BD2 domain and BD1-BD2 double domain

[0185] The coding genes of EGFP (green fluorescent protein) and mScarlet (red fluorescent protein) were fused with the active domains (BD1 domain, BD2 domain and BD1-BD2 double domain) of BRD4 protein through FLAG tag to construct Flag-BD1-mScarlet-P2A-EGFP recombinant plasmid, Flag-BD2-mScarlet-P2A-EGFP recombinant plasmid and Flag-BD1-BD2-mScarlet-P2A-EGFP recombinant plasmid, respectively. Subsequently, the recombinant plasmids were transfected into HEK-293T cells. After successful transfection, Flag-BD1-mScarlet-P2A-EGFP double fluorescent protein fusion, Flag-BD2-mScarlet-P2A-EGFP double fluorescent protein fusion and Flag-BD1-BD2-mScarlet-P2A-EGFP double fluorescent protein fusion were expressed in the cells. Since the fusion contains P2A self-cleavage sequence, this sequence will specifically self-cleave at the peptide bond between the C-terminal glycine and proline after protein expression. The result of self-cleavage will cause the separation of EGFP and mScarlet labeled BD1 domain, BD2 domain or BD1-BD2 domain, thereby forming two independent fluorescent labels in the cells.

[0186] b. The compound prepared in the above example was gradiently diluted with DMEM high glucose medium to prepare compound solutions with two concentration gradients of 1 µM and 100 nM.

[0187] The well-transfected HEK-293T cells were digested and counted, and 1×10 4 The cells were inoculated into a 96-well plate at a density of 1×10

[0188] Transfected HEK-293T cells, when adding the above-mentioned compound (i.e. the degradation agent based on the fusion of the covalent warhead of the indole group), the effective compound can specifically degrade the target domain of the BRD4 protein (BD1 domain, BD2 domain or BD1-BD2 double domain), resulting in synchronous reduction of 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 above-mentioned compound in inducing target protein degradation can be objectively characterized. The smaller the ratio of mScarlet to EGFP, the stronger the ability of the above-mentioned compound to degrade BRD4 protein.

[0189] (2) Western blot test

[0190] Take out the culture dish, rinse with PBS, add RIPA lysis buffer (containing protease inhibitors), scrape the cells with a spatula, transfer to an EP tube, sonicate, centrifuge at 12000 rpm for 20 min at 4°C, take the supernatant, and obtain the protein sample to be tested. Then, take 20 μL of PBS standard and 5 μL of the protein sample to be tested and add them to a 96-well plate (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 down, and measure the absorbance of each well at 562 nm wavelength using a microplate reader. Plot the standard curve with the standard protein concentration and absorbance value as the horizontal and vertical coordinates, and calculate the protein content of the sample to be tested.

[0191] Mix the protein sample to be tested with 5x Loading Buffer (loading buffer) at a volume ratio of 4:1, heat at 100°C for 5 min to denature the protein, and obtain the denatured protein sample to be tested, which is placed on ice for standby. Then install the electrophoresis device, add the pre-stained protein molecular weight marker, then add the denatured protein sample to be tested, and ensure that the total volume of each sample well is consistent. First run the concentrated gel at 80V (30 min), then run the separation gel at 120V (60 min), until the double-color Loading Buffer reaches the bottom of the gel.

[0192] Soak the PVDF (polyvinylidene fluoride) membrane in methanol for 30 s for activation treatment, and balance in buffer for standby. Then tightly attach the gel to the activated PVDF membrane, add the transfer buffer, and use wet transfer method (230 mA constant current, 120 min) for membrane transfer. After completing the transfer operation, use the Ponceau staining solution to stain the transfer membrane, and confirm the successful transfer by observing the distribution of protein bands on the membrane.

[0193] Block the membrane with 5% skim milk powder in TBST buffer (Tris-Hydroxymethyl Amino Methane Buffer) for 1 h at room temperature. Then wash the membrane with TBST buffer for 3 times, 5 min each time. Incubate the membrane with the diluted primary antibody (for the target protein) at 4°C overnight. Take out the membrane and wash with TBST for 3 times, 5 min each time. Add the secondary antibody and incubate at room temperature for 1 h. Wash with TBST buffer for 3 times, 5 min each time.

[0194] Mix the A and B in the developing kit at a ratio of 1:1 to prepare the developing working solution and transfer it to a light-proof container. Immerse the transfer film after completing the secondary antibody incubation in the developing solution and incubate for 3 min in the dark. Then transfer the membrane to the chemiluminescence imaging system, optimize the exposure time according to the signal intensity, and collect and save the protein band image.

[0195] (3) Cell activity test (CCK-8 method)

[0196] Take the logarithmic phase cells, digest and count, and inoculate 4x10 3 cells / well in a 96-well plate and incubate overnight. Remove the old culture medium and add fresh complete culture medium containing the test compound, 3 replicates per group, and set up blank control and negative control groups at the same time, and continue to culture for 48 h. After the reaction is completed, add 100 μL of 10% CCK-8 solution to each well, incubate at 37°C for 1-2 h (the specific time is determined according to the cell type) in the dark. Determine the absorbance at 450 nm wavelength with a microplate reader, and calculate the relative survival rate of cells by comparing the OD values (i.e. the absorbance of the solution at 450 nm wavelength) of the experimental and control groups.

[0197] (4) Elution experiment

[0198] 6-well plate culture MDA-MB-231 cells to 80% confluence, add complete culture medium containing 1 μM compound D29, and set 0.1% DMSO (methyl sulfoxide) as control, incubate at 37°C, 5% CO2 for 12 h. After 12 h, wash gently with 37°C PBS for 2 times, and change the drug-free culture medium. Collect cells at 0 h, 6 h, 12 h, 24 h, 36 h, and 48 h after elution for 6 time points for Western blot test.

[0199] The results are as follows:

[0200] (1) Figure 3 The results are as follows: Figure 3As 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 3 As shown in (b), the dual-fluorescence screening model containing the BD1 domain was confirmed to be normally expressed through the high-content platform.

[0201] (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.

[0202] (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.

[0203] (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 with low concentration of compound D29, high concentration of compound D29 has better degradation effect on BRD3 protein and BRD4 protein. In addition, with the increase of time, the degradation rate of compound D29 on BRD4 first increases and then decreases, and when the concentration of compound D29 is 1 μM, the degradation time is 9 h, and the optimal degradation effect is reached.

[0204] (5) In order to verify the degradation pathway of compound D29, MDA-MB-231 cells were treated with 1 μM of compound D29 and autophagy inhibitors CQ (chloroquine), BafA1 (bafilomycin) and four proteasome inhibitors 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), PYR41 (4-[4-[(5-nitro-2-furanyl)methylene]-3, 5-dioxo-1-pyrazolidinyl]benzoic acid ethyl ester), MG132 (N-[(benzyloxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide) and PS341 (bortezomib) respectively, and then whether the degradation of target protein was competitively inhibited was analyzed by high content, and the results are shown in Figure 7 and Figure 8 ns indicates no significant difference, p < 0.05, p < 0.01, p < 0.001, p < 0.0001. The results show that whether autophagosome acidification is inhibited by chloroquine or lysosome maturation is interfered by bafilomycin, the degradation activity of compound D29 cannot be effectively hindered. However, the degradation effect of compound D29 on BRD4 protein can be obviously inhibited by the four ubiquitin-proteasome pathway inhibitors. Therefore, it can be explained that compound D29 mediates the degradation of BRD4 protein through the classical ubiquitin-proteasome pathway.

[0205] (6) The test results of E3 ubiquitin ligase recruited by compound D29 are shown in Figure 9 It can be seen that in the stable cell lines in which CRBN, VHL, DCAF11 and DCAF16 E3 ubiquitin ligases are knocked out, compound D29 can still effectively degrade BRD4 protein, indicating that compound D29 depends on the ubiquitin-proteasome system and is independent of known CRBN, VHL, DCAF11 and DCAF16 E3 ubiquitin ligases, suggesting a new mechanism mediated by a new type of E3 ubiquitin ligase.

[0206] Therefore, the present application constructs a series of degradation agent molecules based on BRD4 protein target by introducing methyl acrylate on the indole skeleton and coupling it with JQ1, wherein compound D29 achieves the best degradation of BRD4 protein within 9 hours, and the degradation duration is more than 24h, its effect depends on the ubiquitin-proteasome system and is independent of known E3 ubiquitin ligases such as CRBN, VHL, DCAF11 and DCAF16, suggesting a new mechanism mediated by a new E3 ubiquitin ligase. It is proved by Transwell experiment (cell migration experiment) and scratch experiment that compound D29 can significantly inhibit the migration ability of MDA-MB-231 cells, which indicates its potential as a cancer treatment drug.

[0207] It should be understood that the application of the present application is not limited to the above examples, and can be improved or changed according to the above description for those of ordinary skill in the art, 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 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 ; When R and When all are single bonds, for , 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 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.

5. 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.

6. A method for preparing a degrading agent based on an indole-based fused covalent warhead as described in any one of claims 1-5, 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.

7. The preparation method according to claim 6, 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: 。 8. The use of the indole-based fused covalent warhead degrader according to any one of claims 1-5 in the preparation of BRD4 protein degrader or BRD3 protein degrader.

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