Molecular glue degrader and its application and drug

Abstract

The application discloses a molecular glue degrader and application and medicine thereof, and relates to the technical field of medicine development.The structural formula of the molecular glue degrader is as follows: the molecular glue degrader provided by the application can degrade BRD4 through an ubiquitin-proteasome pathway mediated by an E3 ubiquitin ligase CRBN, and has antitumor proliferation activity, anti-fibrosis activity, anti-inflammatory activity and osteoclast differentiation inhibition activity.The application enriches a molecular glue degrader molecular library and simultaneously realizes degradation of BRD4.

Description

Technical Field

[0001] This invention relates to the field of drug development technology, and in particular to a molecular adhesive degrader and its application in drugs. Background Technology

[0002] Targeted protein degradation (TPD) is a technology that has emerged in recent years and has fundamentally changed the logic of traditional drug development. Instead of relying on small molecules to inhibit the function of target proteins by occupying their active sites, it directly utilizes the cell's inherent protein clearance system, primarily the ubiquitin-proteasome system (UPS) and lysosomal pathway, to selectively and completely remove pathogenic proteins. This eliminates all biological activities of the pathogenic protein, including its enzymatic catalytic function, scaffolding role, non-enzymatic regulatory function, and potential toxic conformations. This "event-driven" pharmacological mechanism gives TPD several advantages that traditional inhibitors cannot match, such as catalytic action, the ability to target traditionally "undruggable" targets, overcoming drug resistance mutations, sustained efficacy, and potential reduction in off-target toxicity.

[0003] Molecular glue (MG), as a typical small molecule targeted protein degrader, has a similar working principle to protein degradation targeted chimeras (PROTAC), but has advantages such as small molecular weight, moderate lipid solubility, strong membrane permeability, high oral bioavailability, short synthetic route, and outstanding blood-brain barrier penetration ability. Therefore, molecular glue degraders are expected to become one of the important strategies for achieving precision treatment of diseases.

[0004] Bromodomain protein 4 (BRD4) belongs to the bromodomain and extra-terminal domain (BET) protein family and regulates various physiological processes such as cell proliferation and apoptosis, playing a crucial role in tumor cell proliferation and malignant progression. BRD4 contains two bromodomains, BD1 and BD2, which participate in the specific recognition of acetylated lysine residues. It functions as both a typical chromatin "reader" protein and a transcriptional coactivator, interacting directly or indirectly with various transcription factors to promote RNA polymerase II-mediated gene transcription. As a key regulator of cell proliferation, BRD4 originally plays a physiological role primarily in critical stages of growth and development and tissue damage repair. However, in the context of tumorigenesis, it is hijacked by cancer cells, becoming a key molecule promoting malignant transformation and progression. Studies have shown that abnormal activation of BRD4 is closely related to the occurrence and rapid progression of various malignant tumors, including prostate cancer, breast cancer, and lung cancer. Furthermore, BRD4 directly or indirectly participates in regulating intrinsic and extrinsic apoptosis pathways, and its abnormal activation often leads to enhanced tumor cell survival and apoptosis escape. The abnormally high expression of BRD4 and its central role in tumorigenesis and development make BRD4 a highly promising and valuable target for cancer treatment. However, current research on molecular gels targeting BRD4 is limited, and drug development faces significant challenges.

[0005] Therefore, exploring and developing novel molecular adhesive degraders, continuously enriching the molecular library of molecular adhesive degraders, and achieving the degradation of BRD4 to treat cancers related to BRD4 function are of great significance.

[0006] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the purpose of this invention is to provide a molecular adhesive degrader and its application in pharmaceuticals, aiming to enrich the molecular library of molecular adhesive degraders and achieve targeted degradation of BRD4.

[0008] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a molecular adhesive degrading agent, wherein the structural formula of the molecular adhesive degrading agent is: ; R1 is selected from one of the following groups: , , , , , , , , , , , , , and Boc represents tert-butyloxycarbonyl. R2 is selected from one of the following groups: Cl、 , , , , , , , , , , , , , , , , , and ; When R2 is Cl, R1 is not. or ; Indicates the connection site (hereinafter referred to as the connection site) These all represent connection sites, and their meanings will not be repeated when they appear again in the following text.

[0009] This invention targets BRD4 and chemically modifies its inhibitor (+)-JQ-1. By introducing a variety of dominant backbones into its chemical structure, the interaction between BRD4 and E3 ubiquitin ligase is enhanced, thereby achieving targeted degradation of BRD4. Specifically, based on the principle of molecular glue degrading agents enhancing protein-protein interaction (PPI), this invention modifies the structure of the BRD4 inhibitor by introducing dominant backbones (various nitrogen-containing heterocyclic structures) as cementing groups, thereby generating new protein interfaces and binding sites, enhancing the PPI between BRD4 and E3 ubiquitin ligase, and thus realizing the transformation of the BRD4 inhibitor into a BRD4 degrading agent.

[0010] Research has shown that the molecular glue degrader provided by this invention can degrade BRD4 via the ubiquitin-proteasome pathway mediated by the E3 ubiquitin ligase CRBN, and exhibits anti-tumor proliferation activity, anti-fibrotic activity, anti-inflammatory activity, and inhibitory activity against osteoclast differentiation. Therefore, this invention enriches the molecular glue degrader library while simultaneously achieving the degradation of BRD4.

[0011] Optionally, R1 is ; R2 is selected from one of the following groups: , , , , , , , , , , , , , , , , , and .

[0012] Optionally, R1 is selected from one of the following groups: , and ; R2 is Cl.

[0013] Optionally, R1 is selected from one of the following groups: , , , , , , , , , and ; R2 is .

[0014] In a second aspect, the present invention provides the application of the molecular glue degrader described above in the preparation of a drug that degrades bromine domain protein 4.

[0015] A third aspect of the present invention provides the use of the molecular gel degrader of the present invention as described above in the preparation of a medicament for treating cancer, wherein the cancer is breast cancer, lung cancer, liver cancer, glioma, gastric cancer, or colorectal cancer.

[0016] In a fourth aspect, the present invention provides the use of the molecular adhesive degrading agent described above in the preparation of an antifibrotic drug.

[0017] A fifth aspect of the present invention provides the use of the molecular glue degrading agent of the present invention as described above in the preparation of a medicament for treating osteoporosis.

[0018] In a sixth aspect, the present invention provides the use of the molecular adhesive degrading agent of the present invention as described above in the preparation of a medicament for treating periodontitis.

[0019] A seventh aspect of the present invention provides a medicament comprising the molecular glue degrading agent of the present invention as described above, and the medicament further comprising a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable additive.

[0020] Wherein, the pharmaceutically acceptable carrier includes at least one of the following: a flow aid, a diluent, a wetting agent, a suspending agent, a solvent, and an emulsifier; the pharmaceutically acceptable adjuvant includes at least one of the following: a preservative, an antioxidant, a softener, a colorant, a flavoring agent, a stabilizer, and an isotonic agent.

[0021] Beneficial effects: The molecular glue degrader provided by this invention can degrade BRD4 via the ubiquitin-proteasome pathway mediated by the E3 ubiquitin ligase CRBN, and exhibits anti-tumor proliferation activity, anti-fibrotic activity, anti-inflammatory activity, and inhibitory activity against osteoclast differentiation. This invention enriches the molecular glue degrader library while simultaneously achieving the degradation of BRD4. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the mechanism of action of molecular adhesive degraders.

[0023] Figure 2The graph shows the effect of compound B2 on the degradation of BRD4. (a) is a band diagram of BRD4 in MDA-MB-231 cells after treatment with different concentrations of compound B2 for 12 h, and (b) is a quantitative result of BRD4 level in MDA-MB-231 cells after treatment with different concentrations of compound B2 for 12 h.

[0024] Figure 3 The diagram shows the validation results of the degradation pathway of compound B2, where (a) is the validation result of the lysosomal pathway and (b) is the validation result of the proteasome pathway.

[0025] Figure 4 The results of the test for E3 ubiquitin ligase recruited by compound B2 are shown in the figure. (a) shows the ratio of red fluorescence to green fluorescence after HEK-293T cells carrying a dual-fluorescence reporter system knocked down CRBN; (b) shows the BRD4 level after HEK-293T cells knocked down CRBN.

[0026] Figure 5 This figure shows the results of compound B2 exhibiting antifibrotic activity in mouse embryonic fibroblasts.

[0027] Figure 6 The figures show the effects of compound B2 on the expression of different inflammatory factors and BRD4 in rat gingival tissue. Among them, (a) shows the effect on IL-1β expression, (b) shows the effect on TNF-α expression, (c) shows the effect on IL-10 expression, (d) shows the effect on iNOS expression, (e) shows the effect on CD206 expression, and (f) shows the effect on BRD4 expression.

[0028] Figure 7 Image showing the TRAP immunohistochemical results of a rat alveolar bone section.

[0029] Figure 8 The results show that compound B2 can inhibit osteoclast differentiation. Among them, (a) is the TRAP staining image of Raw264.7 osteoclasts in different treatment groups, (b) is the osteoclast count result of Raw264.7 in different treatment groups, (c) is the TRAP staining image of BMDM osteoclasts in different treatment groups, and (d) is the osteoclast count result of Raw264.7 in different treatment groups. Detailed Implementation

[0030] This invention provides a molecular adhesive degrader and its applications in pharmaceuticals. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

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

[0032] This invention is based on the principle of enhancing PPI (protein ligase) through molecular gel degrading agents. By introducing a dominant backbone as a gelling group, new protein interfaces and binding sites are generated, enhancing the PPI between BRD4 and E3 ubiquitin ligases, thereby converting the BRD4 inhibitor into a BRD4 degrading agent. The dominant backbone selected in this invention mainly includes various nitrogen-containing heterocyclic structures, which are coupled with different sites and reaction handles in the inhibitor (+)-JQ-1 structure. Based on the differences in the binding sites of the dominant backbone and the inhibitor, as well as the differences in the modification sites of the inhibitor structure, four types of compounds were designed and synthesized in the embodiments of this invention: Class A compounds (compounds A1 to A5) obtained by condensing the amino reaction handle of the inhibitor (introduced through a Suzuki coupling reaction) with the dominant backbone; Class B compounds (compounds B1 to B14) obtained by condensing the carboxylic acid reaction handle of the inhibitor (introduced through a Suzuki coupling reaction) with the dominant backbone; Class C compounds (compounds C1 to C3) obtained by coupling at the carboxylic acid end of the inhibitor; and Class D compounds (compounds D1 to D11) obtained by different modifications at both ends of the inhibitor. These compounds were identified as having BRD4 degradation activity using high-content screening and Western blotting techniques. Furthermore, studies revealed that these compounds, acting as molecular glue degraders, recruit the E3 ubiquitin ligase CRBN to degrade BRD4. A schematic diagram of the mechanism of action of the molecular glue degraders of this invention is shown below. Figure 1 As shown (where Ub represents ubiquitin), it induces the ubiquitination of the target protein by bringing the E3 ubiquitin ligase closer to the target protein, thereby enabling the target protein to be recognized and degraded by the proteasome.

[0033] The present invention will be further described below through specific embodiments.

[0034] The meanings of some symbols in the following embodiments and synthetic routes are as follows: SPhOS: 2-Dicyclohexylphosphine-2',6'-dimethoxybiphenyl; Pd(OAc)2: Palladium acetate; THF: Tetrahydrofuran; TFA: Trifluoroacetic acid; DCM: Dichloromethane; HBTU: O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate; HOBt: 1-Hydroxybenzotriazole; DIPEA: N,N-diisopropylethylamine; rt: room temperature; eq: equivalent; DMSO: Dimethyl sulfoxide; TRAP: Tartrate-resistant acid phosphatase; RANKL: ligand of nuclear factor κB receptor activator; M-CSF: Macrophage colony-stimulating factor; PBS: Phosphate Buffered Salt; DEPC: Diethyl pyrocarbonate.

[0035] Example 1: Synthesis of compounds A1 to A5 The structural formulas of compounds A1 to A5 are as follows: The specific structure of R2 is shown in Table 1.

[0036] Table 1. Specific structures of R2 in the structural formulas of compounds A1 to A5

[0037] The synthetic route for compound A1 is as follows: .

[0038] Synthesis of compound A1: Following the above synthetic route, compound JQ1-OMe (1.242 g, 3 mmol), compound s1 (0.957 g, 3 mmol), SPhOS (0.123 g, 0.3 mmol), KF (0.522 g, 9 mmol), Pd(OAc)2 (0.067 g, 0.3 mmol), THF (10 mL), and H2O (0.2 mL) were added sequentially to a pressure-resistant tube with a branched end. Nitrogen gas was purged using a long needle for 3 min, followed by three evacuations. The pressure-resistant tube was then sealed, and the reaction was carried out overnight at 80 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was complete, ethyl acetate (10 mL) was added, followed by washing with water (10 mL) and saturated saline solution (10 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified using a silica gel column in a methanol / DCM system to obtain compound s2. The characterization data of compound s2 are as follows: 1H NMR (600 MHz, CDCl3) δ 7.55 – 7.48 (m, 6H), 7.44 (d, J = 8.3 Hz, 2H), 6.72 (s, 1H), 4.64 (m, 1H), 3.78 (d, J = 1.1 Hz, 3H), 3.70 – 3.62 (m, 2H), 2.68 (s, 3H), 2.41 (s, 3H), 1.73 (s, 3H), 1.53 – 1.50 (m, 9H). 13 C NMR (151 MHz, CDCl3) δ 172.1, 164.7, 155.4, 152.6, 149.9, 142.7, 138.3, 136.5, 134.6, 131.9, 131.2, 130.8 , 130.5, 128.9, 127.6, 126.6, 118.7, 65.8, 53.7, 51.9, 36.7, 28.3, 15.2, 14.4, 13.1. HRMS.ESI(m / z): [M+H] + calcd forC 31 H 34 N5O4S + : 572.2326, found 572.2301. HPLC Purity: 95.42%. Among them, 1 1H NMR represents the hydrogen nuclear magnetic resonance spectrum. 13 C NMR represents carbon nuclear magnetic resonance spectroscopy, HRMS.ESI represents high-resolution mass spectrometry, HLPC represents high-performance liquid chromatography; calcdfor represents theoretical calculation results; found represents actual analytical results; Purity represents purity; and as mentioned below... 13 The meanings of C NMR, HRMS.ESI, HLPC, calcd for, found, and Purity are the same as those here.

[0039] Compound s2 (1.0 mmol) was placed in a flask, and DCM (2 mL) and TFA (2 mL) were added sequentially. The mixture was stirred at room temperature for 30 minutes, and the reaction progress was monitored by TLC. After the reaction was complete, the mixture was repeatedly concentrated under reduced pressure with DCM (10 mL × 3, i.e., concentrated 3 times, 10 mL each time), and purified by silica gel column chromatography using a methanol / DCM system to obtain compound s3. The characterization data of compound s3 are as follows: 1HNMR (600 MHz, CDCl3) δ 7.53 – 7.49 (m, 2H), 7.47 (d, J = 8.1 Hz, 2H), 7.40 (d, J = 8.5Hz, 2H), 6.73 (d, J = 8.5 Hz, 2H), 4.63 (m, 1H), 3.85 (s, 1H), 3.77 (s, 3H), 3.69 – 3.62 (m, 2H), 2.67 (s, 3H), 2.41 (s, 3H), 1.73 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 172.1, 164.7, 155.5, 149.8, 146.5, 143.2, 135.8, 131.8, 131.2, 130.9, 130 .3, 130.0, 128.8, 127.9, 126.0, 115.3, 53.7, 51.8, 36.7, 14.4, 13.0, 11.8. HRMS.ESI(m / z): [M+H] + calcd forC 26 H 26 N5O2S + : 472.1802, found 472.1807. HPLC Purity:96.79%.

[0040] In a round-bottom flask, compounds s3 (70.6 mg, 1.0 eq.) and s4 (33.8 mg, 1.2 eq.; i.e., 1-phenyl-1H-pyrazole-4-carboxylic acid) were dissolved sequentially in anhydrous DCM (5 mL). Under ice-water bath cooling, HBTU (68.2 mg, 1.2 eq.), HOBt (24.8 mg, 1.2 eq.), and DIPEA (78 μL, 3.0 eq.) were added sequentially. After reacting for ten minutes, the ice bath was removed, and the mixture was allowed to rise naturally to room temperature and stirred for 12 h. After the reaction was complete as determined by TLC, the reaction solution was washed and extracted sequentially with saturated citric acid aqueous solution (10 mL), saturated sodium carbonate aqueous solution (10 mL), and saturated saline solution (10 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain a white solid, compound A1. The characterization data of compound A1 are as follows: 1 H NMR (500 MHz, CDCl3) δ 8.80 (s, 1H), 8.72 (s, 1H), 8.23 ​​(s, 1H), 7.73 – 7.63 (m, 4H), 7.51 (s, 4H), 7.48 – 7.39 (m, 4H), 7.30 (t, J= 7.4 Hz, 1H), 4.69 – 4.62 (m, 1H), 3.72 (s, 3H), 3.69 – 3.58 (m, 2H), 2.66 (s, 3H), 2.42 (s, 3H), 1.74 (s, 3H). 13 C NMR (126 MHz, CDCl3) δ 172.2, 164.8, 161.0, 155.7, 150.2, 143.0, 140.4, 139.6, 138.2, 136.6, 135.8, 131.8, 131.4, 131.2, 130 .9, 129.7, 129.1, 129.0, 127.7, 127.5, 126.8, 120.8, 120.8, 119.5, 53.9, 52.0, 36.9, 14.6, 13.3, 11.9. HRMS.ESI (m / z): [M + H] + calcd forC 36 H 32 O3N7S + : 642.2282, found 642.2269. HPLC Purity:99.33%.

[0041] Synthesis of compound A2: Following the synthetic method for compound A1, compound s4 was replaced with compound s5 (i.e., 6-quinoxalocarboxylic acid), yielding a white solid, which is compound A2. The characterization data for compound A2 are as follows: 1 H NMR (500 MHz, CDCl3) δ 8.92 (q, J = 1.8 Hz, 2H), 8.62 (d, J = 2.0 Hz, 1H), 8.55 (s, 1H), 8.34 (dd, J = 8.8, 2.0 Hz, 1H), 8.22 (d, J = 8.7 Hz, 1H), 7.80 (d, J = 8.2 Hz, 2H), 7.61 (d, J = 8.3 Hz, 2H), 7.58 (d, J = 8.2Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 4.69 – 4.58 (m, 1H), 3.77 (s, 3H), 3.65 (t, J = 6.9 Hz, 2H), 2.67 (s, 3H), 2.43 (s, 3H), 1.75 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 172.3, 164.8, 155.6, 150.1, 146.6, 146.2, 144.5, 142.6, 142.4, 137.8, 137.0, 136.7, 136.3, 132.1, 131 .3, 131.0, 130.7, 130.6, 129.2, 128.8, 128.4, 127.9, 126.9, 120.8, 53.9, 52.1, 36.9, 14.6, 13.3, 12.0. HRMS.ESI (m / z): [M + H] + calcd for C 35 H 30 O3N7S + :628.2125,found628.2119. HPLC Purity:95.70%.

[0042] Synthesis of compound A3: Following the synthetic method for compound A1, compound s4 was replaced with compound s6 (i.e., 2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid), yielding a white solid, compound A3. The characterization data for compound A3 are as follows: 1 H NMR (500MHz, CDCl3) δ 11.76 (s, 1H), 9.81 (s, 1H), 7.86 (d, J = 7.8 Hz, 2H), 7.70 (d, J = 8.3 Hz, 1H), 7.62 – 7.31 (m, 7H), 7.15 – 7.05 (m, 1H), 6.94 (s, 1H), 6.75 (s, 1H), 4.62 (t, J = 7.1 Hz, 1H), 3.72 (d, J = 20.1 Hz, 3H), 3.67 – 3.57 (m, 2H), 2.63 (s, 3H), 2.39 (s, 3H), 1.71 (s, 3H). 13 C NMR (126 MHz, CDCl3) δ 172.2, 164.7, 163.3, 155.5, 150.2, 142.6, 137.9, 132.0, 131.3, 130.9, 129.2, 12 7.7, 126.9, 120.8, 119.2, 116.7, 53.8, 52.1, 36.8, 31.6, 29.8, 14.6, 13.3, 11.9. HRMS.ESI (m / z): [M + H] + calcd for C 36 H31 O4N6S + : 643.2122, found643.2122. HPLC Purity:99.03%.

[0043] Synthesis of compound A4: Following the synthetic method for compound A1, compound s4 was replaced with compound s7 (i.e., 2-methyl-6-quinolinecarboxylic acid), yielding a white solid, compound A4. The characterization data for compound A4 are as follows: 1 H NMR (500 MHz, CDCl3) δ 8.57 (s, 1H), 8.41 (d, J = 1.9 Hz, 1H), 8.18 (d, J = 8.5 Hz, 1H), 8.15 – 8.13 (m, 1H), 7.82 (dd, J =14.6, 8.4 Hz, 2H), 7.62 – 7.59 (m, 2H), 7.57 (d, J = 8.1 Hz, 2H), 7.52 (d, J = 8.1 Hz, 2H), 7.43 – 7.29 (m, 2H), 4.65 (dd, J = 7.9, 6.3 Hz, 1H), 3.77 (s, 3H), 3.72 – 3.59 (m, 2H), 2.78 (s, 3H), 2.67 (s, 3H), 2.43 (s, 3H), 1.75 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 172.0, 164.5, 159.3, 155.5, 150.4, 142.9, 139.6, 138.4, 136.6, 136.2, 135.3, 132.3, 131.5, 131.3, 129.2, 128.6, 12 8.4, 127.7, 127.1, 126.9, 126.7, 126.4, 123.7, 122.6, 121.1, 116.6, 111.8, 53.7, 52.2, 36.7, 21.9, 14.6, 13.3, 11.8. HRMS.ESI (m / z): [M + H] + calcd forC 37 H 33 O3N6S + : 641.23294, found 641.23205. HPLC Purity:95.03%.

[0044] Synthesis of compound A5: Following the synthetic method for compound A1, compound s4 was replaced with compound s8 (i.e., 2-oxoindoline-6-carboxylic acid), yielding a white solid, compound A5. The characterization data for compound A5 are as follows: 1 H NMR (500 MHz, CDCl3) δ9.14 (s, 1H), 8.67 (s, 1H), 7.78 – 7.66 (m, 4H), 7.56 – 7.42 (m, 6H), 6.81 (d, J = 7.9 Hz, 1H), 4.63 (t, J = 7.2 Hz, 1H), 3.73 (s, 3H), 3.66 – 3.55 (m, 2H), 3.39 (s, 2H), 2.64 (s, 3H), 2.39 (s, 3H), 1.70 (s, 3H). 13 C NMR (126 MHz, CDCl3) δ 177.7, 172.1, 166.2, 165.2, 155.5, 150.3, 146.2, 138.5, 135.7, 131.9, 131.4, 131 .2, 131.0, 129.2, 127.6, 126.8, 120.9, 109.6, 53.7, 52.1, 36.7, 14.6, 13.2, 11.8. HRMS.ESI (m / z): [M + H] + calcd forC 35 H 31 O4N6S + : 631.2104, found 631.2122. HPLC Purity:98.79%.

[0045] Example 2 Synthesis of compounds B1 to B14 The structural formulas of compounds B1 to B14 are as follows: The specific structure of R2 is shown in Table 2.

[0046] Table 2. Specific structures of R2 in the structural formulas of compounds B1 to B14

[0047] The synthetic route for compound B1 is as follows: .

[0048] Synthesis of compound B1: Following the above synthetic route and referring to the synthetic method of compound s3 in Example 1, compound s11 was synthesized using compound JQ1-OMe and compound s9 as starting materials. The characterization data of compound s11 are as follows: 1H NMR (400 MHz, CDCl3) δ8.18 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.5 Hz, 2H), 7.63 (d, J = 8.2 Hz, 2H), 7.56 (d, J = 8.2Hz, 2H), 6.29 (s, 1H), 4.68 (m, 1H), 3.79 (s, 3H), 3.71 – 3.64 (m, 2H), 2.75 (s, 3H), 2.43 (s, 3H), 1.75 (s, 3H). 13 C NMR (126 MHz, CDCl3) δ 172.0, 170.4, 164.7, 155.2, 150.1, 144.9, 142.1, 137.7, 131.7, 131.2, 131.0, 130.9, 130.7, 129.1, 129.1, 127.3, 127.1, 53.7, 52.0, 36.6, 14.4, 13.1, 11.7. HRMS.ESI (m / z): [M + H] + calcd for C 27 H 25 N4O4S + : 501.1591, found 501.1592. HPLC Purity:96.87%.

[0049] In a round-bottom flask, compound s11 (100 mg, 0.2 mmol, 1.0 eq.) was dissolved in DCM (2.0 mL). HBTU (91 mg, 0.24 mmol, 1.2 eq.), HOBt (33 mg, 0.24 mmol, 1.2 eq.), and DIPEA (77.5 mg, 0.6 mmol, 3.0 eq.) were added. After stirring at 0 °C for 30 min, compound s12 (65.8 mg, 0.4 mmol, 2.0 eq.) was added. The mixture was stirred at room temperature for 3 h. After the reaction was complete as determined by TLC, the reaction solution was diluted with DCM (20 mL). The solution was then washed and extracted successively with saturated citric acid aqueous solution (10 mL), saturated sodium carbonate aqueous solution (10 mL), and saturated saline solution (10 mL). The extract was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain a white solid, compound B1. The characterization data of compound B1 are as follows: 1 H NMR (600 MHz, CDCl3) δ 8.74 (s, 1H), 7.89 (d, J = 8.1 Hz, 2H), 7.59 (d, J=8.0 Hz, 2H), 7.58 – 7.49 (m, 4H), 7.41 – 7.34 (m, 1H), 4.83 (dt, J = 11.9, 5.6 Hz, 1H), 4.65 (dd, J = 7.8, 6.2 Hz, 1H), 3.78 (s, 3H), 3.70 – 3.61 (m, 2H), 2.87 – 2.74 (m, 2H), 2.68 (s, 3H), 2.42 (s, 3H), 2.20 – 2.07 (m, 2H), 1.73 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 172.26, 172.16, 167.2, 164.7, 155.5, 150.2, 143.7, 142.1, 132.5, 131.2, 130.8, 129.3, 127.9, 127.4, 127.3, 53.9, 52.1, 51.2, 36.8, 31.5, 25.1, 14.6, 13.3, 12.0. HRMS.ESI(m / z): [M +H] + calcd for C 31 H 31 O5N6S + : 611.2052, found 611.2071. HPLC Purity:98.79%.

[0050] Synthesis of compound B2: Following the synthetic method for compound B1, compound s12 was replaced with compound s13 (i.e., 5-aminoindol-2-one), yielding a white solid, compound B2. The characterization data for compound B2 are as follows: 1 H NMR (500 MHz, CDCl3) δ 8.74 (s, 1H), 8.60 (s, 1H), 7.95 (d, J = 7.8 Hz, 2H), 7.68 – 7.58 (m, 3H), 7.54 (q, J = 8.1 Hz, 4H), 7.38 – 7.30 (m, 1H), 6.77 (d, J = 8.2 Hz, 1H), 4.65 (t, J = 7.0 Hz, 1H), 3.75 (s, 3H), 3.63 (dq, J = 16.8, 9.2 Hz, 2H), 3.42 (s, 2H), 2.65 (s, 3H), 2.41 (s, 3H), 1.72 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 177.7, 172.2, 165.8, 164.8, 150.2, 143.3, 142.1, 139.5, 137.7, 134.1, 133.1, 132.0, 131.2, 131 .0, 130.8, 129.2, 128.1, 127.3, 126.0, 120.9, 118.6, 109.8, 53.9, 52.1, 36.8, 14.6, 13.2, 11.9. HRMS.ESI (m / z): [M + H] + calcd for C 35 H 31 O4N6S + :631.2113, found 631.2122. HPLC Purity:98.08%.

[0051] Synthesis of compound B3: Following the synthetic method for compound B1, compound s12 was replaced with compound s14 (i.e., 6-aminoindolone), yielding a white solid, compound B3. The characterization data for compound B3 are as follows: 1 H NMR (600 MHz, CDCl3) δ 8.76 (s, 1H), 8.63 (s, 1H), 7.95 (d, J = 7.9 Hz, 2H), 7.61 (d, J = 8.0 Hz, 2H), 7.58 – 7.52 (m, 4H), 7.50 (dd, J = 8.8, 2.0 Hz, 1H), 7.16 – 7.04 (m, 2H), 4.65 (t, J = 7.1 Hz, 1H), 3.75 (s, 3H), 3.69 – 3.59 (m, 2H), 3.39 (s, 2H), 2.65 (s, 3H), 2.41 (s, 3H), 1.71 (s, 3H). 13 C NMR (151MHz, CDCl3) δ 177.6, 172.2, 165.8, 164.9, 155.4, 154.3, 150.3, 143.4, 142.2, 138.2, 134.0, 131.3, 131.1, 130 .3, 129.3, 128.1, 127.4, 127.3, 125.3, 124.8, 114.0, 112.2, 53.9, 52.1, 36.7, 14.6, 13.3, 11.9. HRMS.ESI (m / z): [M + H] + calcd for C 35 H31 O4N6S + :631.2103, found 631.2122. HPLC Purity:99.96%.

[0052] Synthesis of compound B4: Following the synthetic method for compound B1, compound s12 was replaced with compound s15 (i.e., 2-oxoazacycloheptan-3-amine), yielding a white solid, compound B4. The characterization data for compound B4 are as follows: 1 H NMR (500 MHz, CDCl3) δ 7.88 – 7.84 (m, 2H), 7.74 – 7.67 (m, 3H), 7.62 (d, J = 8.4 Hz, 2H), 7.59 – 7.56 (m, 3H), 7.51 (d, J = 8.0 Hz, 2H), 7.33 – 7.28 (m, 3H), 4.71 – 4.63 (m, 1H), 4.64 – 4.56 (m, 1H), 4.23 – 4.16 (m, 1H), 3.73 (s, 1H), 3.67 – 3.61 (m, 4H), 3.60 – 3.54 (m, 2H), 3.14– 3.08 (m, 4H), 2.65 (s, 2H), 2.39 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 175.8, 171.8, 171.3, 165.9, 143.1, 142.2, 137.2, 133.2, 131.3, 129.1, 127.6, 127.2, 127 .2, 127.1, 124.7, 117.6, 60.8, 53.6, 52.6, 51.9, 42.1, 31.4, 28.7, 27.9, 14.2, 13.1, 11.5. HRMS.ESI(m / z):[M+H] + calcd for C 33 H 35 O4N6S + : 611.2435, found 611.2431. HPLC Purity:96.55%.

[0053] Synthesis of compound B5: Following the synthetic method for compound B1, compound s12 was replaced with compound s16 (i.e., 7-aminoquinoline), yielding a white solid, compound B5. The characterization data for compound B5 are as follows: 1H NMR (500 MHz, CDCl3) δ 8.90 –8.85 (m, 1H), 8.64 (s, 1H), 8.27 (s, 1H), 8.14 (t, J = 7.7 Hz, 2H), 8.02 (d, J = 7.9 Hz, 2H), 7.83 (d, J = 8.8 Hz, 1H), 7.68 (d, J = 7.9 Hz, 2H), 7.60 (d, J = 8.0 Hz, 2H), 7.56 (d, J = 8.0Hz, 2H), 7.34 (dd, J = 8.2, 4.3 Hz, 1H), 4.66 (t, J = 7.1 Hz, 1H), 3.77 (s, 3H), 3.64 (q, J =6.7 Hz, 2H), 2.68 (s, 3H), 2.43 (s, 3H), 1.74 (s, 3H). 13 C NMR (126 MHz, CDCl3) δ 172.3, 165.8, 164.7, 155.5, 150.9, 148.6, 143.9, 142.1, 139.6, 137.9, 136.2, 134.0, 132.2, 131.2, 130 .8, 128.8, 128.1, 127.6, 127.4, 125.7, 121.4, 120.4, 117.8, 114.2, 54.0, 52.1, 36.9, 14.6, 13.3, 12.0. HRMS.ESI (m / z): [M + H] + calcd for C 36 H 31 O3N6S + : 627.2173, found 627.2161. HPLC Purity:99.73%.

[0054] Synthesis of compound B6: Following the synthetic method for compound B1, compound s12 was replaced with compound s17 (i.e., 5-aminoindazole), yielding a pale yellow solid, compound B6. The characterization data for compound B6 are as follows: 1 H NMR (500 MHz, CDCl3) δ 8.22 (d, J = 29.1 Hz, 2H), 8.05 (s, 1H), 7.98 (d, J = 7.9 Hz, 2H), 7.68 (d, J= 8.0 Hz, 2H), 7.63 –7.54 (m, 4H), 7.52 (d, J = 8.8 Hz, 1H), 7.46 (d, J = 8.9 Hz, 1H), 4.67 (t, J = 7.0 Hz, 1H), 3.78 (s, 3H), 3.70 – 3.60 (m, 2H), 2.68 (s, 3H), 2.43 (s, 3H), 1.74 (s, 3H). 13 C NMR (126 MHz, CDCl3) δ 172.3, 164.7, 155.6, 143.6, 137.9, 135.2, 134.2, 131.6, 131.2, 130.8, 129.3, 127.9, 127.5, 127.4, 122.1, 112.5, 110.3, 54.0, 52.1, 36.9, 14.6, 13.3, 12.0. HRMS.ESI (m / z): [M + H] + calcd for C 34 H 30 O3N7S + : 616.2125, found 616.2115. HPLC Purity:99.13%.

[0055] Synthesis of compound B7: Following the synthetic method for compound B1, compound s12 was replaced with compound s18 (i.e., 1,3-benzothiazole-5-amine), yielding a pale yellow solid, compound B7. The characterization data for compound B7 are as follows: 1 H NMR (500 MHz, CDCl3) δ9.01 (s, 1H), 8.57 – 8.52 (m, 1H), 8.47 (d, J = 2.0 Hz, 1H), 8.00 (d, J = 8.1 Hz, 2H), 7.92 (d, J = 8.7 Hz, 1H), 7.88 (dd, J = 8.7, 2.0 Hz, 1H), 7.67 (d, J = 8.0 Hz, 2H), 7.60 (d, J = 8.2Hz, 2H), 7.56 (d, J = 8.1 Hz, 2H), 4.66 (dd, J = 8.0, 6.2 Hz, 1H), 3.77 (s, 3H), 3.63 (dt, J=8.9, 6.2 Hz, 2H), 2.66 (s, 3H), 2.43 (s, 3H), 1.74 (s, 3H). 13 C NMR (126 MHz, CDCl3) δ 172.2, 165.8, 164.6, 155.5, 155.2, 154.0, 150.1, 143.7, 142.1, 137.8, 137.1, 134.1, 132.2, 131.2, 130 .9, 130.8, 129.6, 129.2, 128.1, 127.5, 127.4, 122.1, 119.5, 115.1, 54.0, 52.1, 36.9, 14.6, 13.3, 12.0. HRMS.ESI (m / z): [M + H] + calcd for C 34 H 29 O3N6S2 + : 633.1737, found 633.1726. HPLC Purity:98.57%.

[0056] Synthesis of compound B8: Following the synthetic method for compound B1, compound s12 was replaced with compound s19 (i.e., 5-amino-3,4-dihydro-2(1H)-quinolinone), yielding a pale yellow solid, compound B8. The characterization data for compound B8 are as follows: 1 H NMR (500MHz, CDCl3) δ 8.03 (d, J = 8.3 Hz, 2H), 7.95 (d, J = 7.9 Hz, 2H), 7.69 (d, J = 7.9 Hz, 2H), 7.66 (s, 1H), 7.62 – 7.55 (m, 4H), 7.36 (d, J = 8.8 Hz, 1H), 4.66 (t, J = 7.2 Hz, 1H), 3.78 (s, 3H), 3.66 (t, J = 7.5 Hz, 2H), 2.99 (t, J = 7.5 Hz, 2H), 2.68 (s, 3H), 2.65 – 2.61 (m, 2H), 2.43 (s, 3H), 1.75 (s, 4H). 13C NMR (126 MHz, CDCl3) δ 172.3, 171.3, 155.6, 150.1, 142.2, 138.0, 134.1, 133.4, 132.3, 131.2, 129.3, 127.8, 12 7.6, 127.4, 124.7, 120.8, 119.8, 115.7, 54.0, 52.1, 36.9, 30.7, 25.7, 14.6, 13.3, 12.0. HRMS.ESI (m / z): [M + H] + calcd for C 36 H 33 O4N6S + : 645.2279, found 645.2269. HPLC Purity:99.95%.

[0057] Synthesis of compound B9: Following the synthetic method for compound B1, compound s12 was replaced with compound s20 (i.e., 2-amino-5-bromobenzothiazole), yielding a pale yellow solid, compound B9. The characterization data for compound B9 are as follows: 1 H NMR (600 MHz, CDCl3) δ 8.08 (d, J = 8.1 Hz, 2H), 7.67 (dd, J = 12.3, 8.2 Hz, 3H), 7.57 (q, J = 8.2 Hz, 4H), 7.36 (dd, J = 8.3, 1.8 Hz, 1H), 7.34 – 7.32 (m, 1H), 4.68 (dd, J = 8.0, 6.2 Hz, 1H), 3.78 (s, 3H), 3.68 (dd, J = 9.7, 7.0 Hz, 2H), 2.69 (s, 3H), 2.43 (s, 3H), 1.73 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 172.3, 165.9, 164.6, 161.3, 155.5, 150.1, 148.8, 145.2, 141.7, 138.2, 132.3, 131.2, 131.0, 130.8, 130 .8, 130.6, 129.3, 128.8, 127.9, 127.5, 127.3, 123.6, 122.6, 119.9, 54.0, 52.1, 36.9, 14.6, 13.3, 12.0. HRMS.ESI (m / z): [M + H] + calcd forC34 H 28 O3N6BrS2 + : 711.0842, found 711.0825. HPLC Purity:97.1%.

[0058] Synthesis of compound B10: Following the synthetic method for compound B1, compound s12 was replaced with compound s21 (i.e., 3-(aminomethyl)-4,6-dimethyl-1H-pyridin-2-one hydrochloride), yielding a pale yellow solid, compound B10. The characterization data for compound B10 are as follows: 1 H NMR (600 MHz, CDCl3) δ 12.33 (s, 1H), 7.90 (t, J = 5.7 Hz, 1H), 7.84 (d, J = 8.0 Hz, 2H), 7.59 (d, J = 8.0 Hz, 2H), 7.57 – 7.50 (m, 4H), 5.96 (s, 1H), 4.65 (dd, J = 8.0, 6.1 Hz, 1H), 4.57 (d, J = 5.7 Hz, 2H), 3.78 (s, 3H), 3.71 – 3.58 (m, 2H), 2.68 (s, 3H), 2.41 (s, 3H), 2.38 (s, 3H), 2.27 (s, 3H), 1.72 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 172.3, 166.5, 165.5, 164.6, 155.5, 150.8, 150.1, 143.0, 142.7, 142.2, 137.8, 134.0, 132.3, 131.2, 130.8 , 130.7, 129.2, 129.1, 127.7, 127.3, 127.2, 122.1, 110.2, 53.9, 52.0, 36.9, 19.8, 18.9, 14.6, 13.2, 12.0. HRMS.ESI (m / z): [M + H] + calcd for C 35 H 35 O4N6S + : 635.2435, found635.2425. HPLC Purity:96.0%.

[0059] Synthesis of compound B11: Following the synthetic method for compound B1, compound s12 was replaced with compound s22 (i.e., 7-chloro-4-(1-piperazinyl)quinoline), yielding a pale yellow solid, compound B11. The characterization data for compound B11 are as follows: 1 H NMR (500 MHz, CDCl3) δ 8.73 (d, J = 5.0 Hz, 1H), 8.06 (d, J = 2.1 Hz, 1H), 7.94 (d, J = 9.0 Hz, 1H), 7.69 –7.64 (m, 2H), 7.58 (d, J = 8.6 Hz, 2H), 7.55 (dd, J = 8.4, 2.9 Hz, 4H), 7.45 (dd, J = 9.0, 2.2Hz, 1H), 6.85 (d, J = 5.1 Hz, 1H), 4.65 (dd, J = 7.9, 6.3 Hz, 1H), 4.15 – 3.96 (m, 2H), 3.78 (s, 5H), 3.69 – 3.63 (m, 2H), 3.37 – 3.12 (m, 4H), 2.68 (s, 3H), 2.42 (s, 3H), 1.74 (s, 3H). 13 C NMR (126 MHz, CDCl3) δ 172.2, 170.4, 164.7, 156.6, 155.5, 151.8, 150.0, 142.2, 142.0, 137.8, 135.4, 134.7, 132.3, 131.2, 1 29.2, 129.0, 127.5, 127.3, 124.9, 121.8, 109.4, 53.9, 52.0, 36.9, 31.6, 29.8, 22.8, 14.6, 13.2, 12.0. HRMS.ESI (m / z): [M + H] + calcd forC 40 H 37 N7O3ClS + : 730.2362, found 730.2350. HPLC Purity:97.63%.

[0060] Synthesis of compound B12: Following the synthetic method for compound B1, compound s12 was replaced with compound s23 (i.e., 6-methyl-1H-pyrazolo[3,4-B]pyridine-3-amine), yielding a white solid, compound B12. The characterization data for compound B12 are as follows: 1H NMR (600 MHz, CDCl3) δ 8.21 – 8.15 (m, 2H), 7.84 (d, J = 8.3 Hz, 1H), 7.74 (d, J = 8.3 Hz, 1H), 7.70 – 7.59 (m, 5H), 7.55 (d, J = 8.1 Hz, 2H), 7.43 – 7.38 (m, 1H), 7.37 – 7.31 (m, 1H), 4.67 (dd, J = 7.7, 6.3 Hz, 1H), 3.78 (s, 3H), 3.67 (dd, J = 7.2, 4.2 Hz, 2H), 2.72 (s, 3H), 2.43 (s, 3H), 1.74 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 172.1, 170.5, 165.0, 164.9, 155.4, 150.4, 145.0, 142.3, 137.7, 131.7, 131.4, 131.4, 131.2, 131 .2, 130.8, 129.3, 127.5, 127.2, 126.7, 126.0, 117.7, 111.2, 53.8, 52.1, 36.7, 14.6, 13.3, 11.8. HRMS.ESI(m / z): [M+ H] + calcd for C 34 H 31 O3N8S + : 631.2234, found 631.2228. HPLC Purity:99.26%.

[0061] Synthesis of compound B13: Following the synthetic method for compound B1, compound s12 was replaced with compound s24 (i.e., 1,4,5,6-tetrahydropyrrolo-[3,4-C]-pyrazole dihydrochloride), yielding a white solid, compound B13. The characterization data for compound B13 are as follows: 1H NMR (600 MHz, CDCl3) δ 8.32 – 8.08 (m, 2H), 7.74 – 7.55 (m, 8H), 4.89 (s, 1H), 4.77 – 4.59 (m, 2H), 3.94 (s, 2H), 3.79 (dd, J = 3.8, 1.9 Hz, 3H), 3.69 – 3.64 (m, 2H), 2.70 (q, J = 2.4 Hz, 3H), 2.43 (d, J = 4.6 Hz, 3H), 1.80 – 1.71 (m, 3H). 13 C NMR (151 MHz, CDCl3) δ172.1, 170.5, 169.4, 167.8, 165.4, 164.4, 155.4, 155.3, 149.9, 141.8, 138.0, 137.7, 132.3, 131.1, 130.7, 1 30.2, 129.2, 129.1, 127.5, 127.3, 127.3, 127.2, 127.0, 126.9, 53.8, 52.2, 51.9, 50.7, 36.7, 14.5, 13.1, 11.8. HRMS.ESI (m / z): [M + H] + calcd for C 32 H 30 O3N7S + : 592.2125, found 592.2116. HPLC Purity:96.07%.

[0062] Synthesis of compound B14: Following the synthetic method for compound B1, compound s12 was replaced with compound s25 (i.e., 4,5,6,7-tetrahydro-3H-imidazo[4,5-C]pyridine dihydrochloride), yielding a white solid, compound B14. The characterization data for compound B14 are as follows: 1 H NMR (600 MHz, CDCl3) δ 7.8 (d, J = 58.4 Hz, 1H), 7.7 (d, J = 8.0 Hz, 1H), 7.6 – 7.6 (m, 1H), 7.6 – 7.5 (m, 6H), 4.7 (dd, J = 7.9, 6.2 Hz, 1H), 3.8 (d, J = 3.1 Hz, 3H), 3.7 – 3.6 (m, 2H), 3.1 (d, J = 22.4 Hz, 4H), 2.7 (s, 3H), 2.4 (s, 3H), 2.0 (q, J= 6.7 Hz, 1H), 1.8 (t, J =2.8 Hz, 3H), 1.6 (p, J = 7.7 Hz, 1H). 13 C NMR (151 MHz, CDCl3) δ 172.1, 164.6, 155.4, 149.9, 147.6, 146.9, 146.5, 144.5, 144.2, 144.0, 143.5, 141.8, 141.8, 140.9, 139.7, 139. 2, 138.7, 138.5, 137.5, 137.0, 132.1, 131.1, 130.7, 129.1, 53.8, 53.4, 51.9, 36.8, 26.0, 24.5, 14.5, 13.1, 11.9. HRMS.ESI (m / z): [M + H] + calcd for C 33 H 32 O3N7S + :606.2282, found 606.2270. HPLC Purity:99.7%.

[0063] Example 3 Synthesis of compounds C1 to C3 The structural formulas of compounds C1 to C3 are as follows: The specific structure of R1 is shown in Table 3.

[0064] Table 3. Specific structures of R1 in the structural formulas of compounds C1 to C3

[0065] The synthetic routes for compounds C1 to C3 are as follows: .

[0066] Synthesis of compound C1: Following the synthetic route of compound C1, compound JQ1-OMe (1.0 mmol, 1.0 eq) was dissolved in a mixed solvent (20 mL) of THF and H2O (THF to H2O volume ratio 3:1), and LiOH (4.0 mmol, 4.0 eq) was added. The mixture was stirred at room temperature for 4 h. After the reaction was complete, THF was removed by vacuum distillation. The remaining aqueous solution was acidified to pH 3 with hydrochloric acid (1M HCl), extracted with DCM, and the organic phase was washed with saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound JQ1-COOH, which could be used directly in the next step without further purification.

[0067] Following the synthetic method for compound B1, compound C1 was prepared using compound JQ1-COOH and compound s13 (i.e., 5-aminoindole-2-one) as starting materials. The characterization data for compound C1 are as follows: 1 H NMR (500 MHz, CDCl3) δ 9.46 (d, J =63.9 Hz, 1H), 8.94 (d, J = 43.2 Hz, 1H), 7.48 (d, J = 23.4 Hz, 1H), 7.40 (d, J = 7.8 Hz, 2H), 7.31 (dd, J = 8.2, 3.9 Hz, 3H), 6.72 (dd, J = 22.8, 8.3 Hz, 1H), 4.74 (dd, J = 9.2, 5.4 Hz, 1H), 3.94 – 3.78 (m, 1H), 3.59 – 3.46 (m, 1H), 3.42 (d, J = 12.9 Hz, 2H), 2.68 (s, 3H), 2.41 (s, 3H), 1.68 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 177.8, 177.7, 168.8, 164.4, 155.8, 150.2, 139.4, 138.9, 137.1, 133.6, 131.3, 131.1, 130.6, 130.1, 128.9, 125.8, 119.8, 117.5, 109.7, 54.7, 40.1, 36.6, 14.5, 13.3, 12.0. HRMS.ESI (m / z): [M + H] + calcd forC 27 H 24 ClN6O2S + : 531.1364, found 531.1350. HPLC Purity:95.9%.

[0068] Synthesis of compound C2: Following the synthetic method of compound B1, compound s27 was obtained from compound JQ1-COOH and compound s26 (methyl 4-aminomethylbenzoate); following the synthetic method of compound JQ1-COOH, compound s27 was demethylated to obtain compound s28; following the synthetic method of compound B1, compound s28 and compound s13 (5-aminoindol-2-one) were used as starting materials to obtain compound C2. The characterization data of compound C2 are as follows: 1H NMR (600 MHz, CDCl3) δ 10.40 (s, 1H), 8.33 (d, J = 21.4 Hz, 1H), 8.05 (d, J = 48.2 Hz, 1H), 7.84 (s, 1H), 7.57 – 7.45 (m, 2H), 7.40 (d, J = 8.1 Hz, 2H), 7.31 (d, J = 7.8 Hz, 2H), 7.23 (d, J = 8.0 Hz, 2H), 5.03 (d, J = 15.3 Hz, 1H), 4.89 – 4.75 (m, 1H), 3.91 (dt, J = 40.8, 15.4 Hz, 2H), 3.36 (d, J = 14.7 Hz, 1H), 3.10 (d, J =21.3 Hz, 1H), 3.04 – 2.85 (m, 2H), 2.65 (d, J = 28.3 Hz, 3H), 2.41 (s, 3H), 1.66 (d, J = 16.7 Hz, 3H). 13 C NMR (151 MHz, CDCl3) δ 178.3, 170.3, 164.8, 164.4, 155.6, 150.0, 142.4, 140.1, 137.0, 136.6, 132.9, 132.8, 132.1, 131.3, 13 1.1, 130.7, 130.0, 128.9, 128.5, 127.1, 126.2, 117.8, 111.2, 42.9, 38.9, 36.5, 29.8, 14.5, 13.3, 12.0. HRMS.ESI(m / z): [M+ H] + calcd for C 35 H 31 O4N6S + : 631.2104, found 631.2122. HPLC Purity:98.79%.

[0069] Synthesis of compound C3: Following the synthetic method of compound B1, compound S30 was obtained from compound JQ1-COOH and compound S29 (tert-butyl 4-aminobutyric acid ester) as starting materials; following the synthetic method of compound S3, compound S30 was de-tert-butyl esterified by TFA to obtain compound S31; following the synthetic method of compound B1, compound S31 and compound S13 (5-aminoindol-2-one) were used as starting materials to obtain compound C3. The characterization data of compound C3 are as follows: 1 H NMR (600 MHz, CDCl3) δ 9.12 (s, 1H), 8.31 (d, J = 23.6 Hz, 1H), 7.59 (s, 1H), 7.41 (s, 2H), 7.33 (d, J = 8.3 Hz, 2H), 6.73 (d, J = 8.5 Hz, 1H), 4.68 (d, J = 6.5 Hz, 1H), 3.70 – 3.51 (m, 2H), 3.45 (s, 2H), 3.35 (d, J = 19.5 Hz, 1H), 3.25 (s, 1H), 2.68 (s, 3H), 2.42 (s, 3H), 2.33 (s, 1H), 1.97 (s, 2H), 1.86 (s, 2H), 1.68 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 177.4, 171.6, 171.2, 164.6, 155.8, 150.3, 138.5, 137.3, 136.4, 131.5, 13 1.2, 130.0, 129.0, 117.6, 109.7, 54.6, 38.4, 36.6, 34.1, 14.6, 13.3, 12.0. HRMS.ESI (m / z): [M + H] + calcd for C 35 H 29 O3N7ClS + : 662.1747, found 662.1751. HPLC Purity:97.98%.

[0070] Example 4 Synthesis of compounds D1 to D11 The structural formulas of compounds D1 to D11 are as follows: The specific structure of R1 is shown in Table 4.

[0071] Table 4. Specific structures of R1 in the structural formulas of compounds D1 to D11

[0072] The synthetic routes for compounds D1 and D2 are as follows: .

[0073] Synthesis of compound D1: Following the synthetic method of compound JQ1-COOH and the above route, using compound B2 as a starting material, a white solid, namely compound D1, was obtained. The characterization data of compound D1 are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 10.44 (s, 1H), 10.28 (s, 1H), 8.14 (dd, J = 8.5, 2.0 Hz, 2H), 8.05 – 7.85 (m, 5H), 7.77 (s, 1H), 7.62 (d, J = 8.0Hz, 3H), 6.88 (dd, J = 8.3, 1.8 Hz, 1H), 4.59 – 4.55 (m, 1H), 4.09 (dd, J = 7.2, 1.8 Hz, 1H), 2.69 (d, J = 1.9 Hz, 3H), 2.50 (s, 3H), 2.05 (d, J = 1.9 Hz, 2H), 1.76 (s, 3H), 1.60 –1.50 (m, 1H). 13 C NMR (126 MHz, DMSO-d6) δ 176.40, 174.51, 172.55, 170.39, 164.66, 163.67, 155.15, 149.86, 141.84, 140.90, 139.83, 137.48, 134.27, 133.14, 132.04, 130. 73, 130.09, 129.98, 129.09, 128.37, 126.85, 126.67, 125.96, 120.06, 117.89, 108.84, 59.80, 29.05, 20.79, 14.11, 12.74, 11.31. LC-MS (Liquid Chromatography-Mass Spectrometry): [M + H] + calcd forC 34 H 29 N6O4S + : 617.20, found 617.2. HPLC Purity:97.11%.

[0074] Synthesis of compound D2: Following the synthetic method for compound B1 and the above synthetic route, using compound D1 and compound 32 (morpholine) as starting materials, a white solid, compound D2, was obtained. The characterization data for compound D2 are as follows: 1H NMR (600 MHz, MeOD) δ 8.04– 8.00 (m, 2H), 7.86 (dt, J = 8.4, 1.0 Hz, 1H), 7.83 – 7.80 (m, 2H), 7.77 – 7.74 (m, 2H), 7.63 (d, J = 2.0 Hz, 1H), 7.57 (d, J = 8.3 Hz, 2H), 6.89 (d, J = 8.4 Hz, 1H), 4.74 (t, J = 6.9Hz, 1H), 3.82 – 3.77 (m, 4H), 3.72 – 3.69 (m, 2H), 3.67 – 3.61 (m, 4H), 3.55 (s, 2H), 2.71 (s, 3H), 2.45 (s, 3H), 1.74 (s, 3H). 13 C NMR (151 MHz, MeOD) δ 169.6, 142.1, 137.7, 132.0, 127.9, 126.9, 126.8, 126.7, 125.7, 121.1, 118.6, 117.3, 110.1, 10 9.2, 66.3, 53.4, 42.0, 34.5, 30.4, 29.4, 29.2, 29.1, 28.9, 22.3, 17.0, 13.03, 13.00, 11.6, 10.2. HRMS.ESI(m / z):[M+H] + calcd for C 38 H 36 N7O4S + : 686.2544, found 686.2549. HPLC Purity:95.29%.

[0075] Synthesis of compound D3: Following the synthetic method for compound D2, using compound D1 and compound s33 (N-tert-butyloxycarbonylpiperazine) as starting materials, a white solid, namely compound D3, was obtained. The characterization data for compound D3 are as follows: 1 H NMR (600 MHz, MeOD) δ 8.04 – 7.98 (m, 2H), 7.82 – 7.78 (m, 2H), 7.76 – 7.71 (m, 2H), 7.64 – 7.60 (m, 1H), 7.56 (d, J =8.1 Hz, 2H), 7.49 (dd, J = 8.4, 2.1 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 4.73 (t,J = 6.9 Hz, 1H), 3.81 – 3.72 (m, 2H), 3.69 – 3.59 (m, 6H), 3.54 (s, 2H), 3.47 (d, J = 16.9 Hz, 2H), 2.70 (s, 3H), 2.45 (s, 3H), 1.73 (s, 3H), 1.49 (s, 9H). 13 C NMR (151 MHz, MeOD) δ 178.4, 178.4, 169.6, 166.8, 165.6, 155.9, 154.9, 150.7, 142.1, 137.7, 134.1, 133.1, 132.0, 131.7, 130.94, 130.91, 129.1, 127.9, 126 .9, 126.8, 126.2, 121.0, 118.7, 118.5, 109.2, 80.3, 54.0, 45.2, 41.4, 34.7, 29.4, 29.4, 29.3, 29.2, 29.1, 29.0, 27.3, 22.3, 13.0, 11.6. HRMS.ESI (m / z): [M + H] + calcd for C 43 H 45 N8O5S + : 785.3228, found 785.3227. HPLC Purity:97.46%.

[0076] Synthesis of compound D4: Following the synthetic method for compound D2, using compound D1 and compound s34 (N-tert-butoxycarbonyl-1,2-ethylenediamine) as starting materials, a white solid, namely compound D4, was obtained. The characterization data for compound D4 are as follows: 1 H NMR (600 MHz, MeOD) δ 8.01 (dd, J = 8.2, 4.2 Hz, 2H), 7.86 (d, J = 8.3 Hz, 1H), 7.82 – 7.79 (m, 2H), 7.76 – 7.73 (m, 2H), 7.64 – 7.61 (m, 1H), 7.58 (t, J = 5.7 Hz, 2H), 6.89 (d, J = 8.3 Hz, 1H), 4.67 (dd, J =8.7, 5.4 Hz, 1H), 3.72 (d, J = 2.4 Hz, 2H), 3.55 (s, 2H), 3.36 (d, J= 6.4 Hz, 2H), 3.24 (d, J =6.0 Hz, 2H), 2.71 (d, J = 3.7 Hz, 3H), 2.45 (s, 3H), 1.73 (s, 3H), 1.42 (s, 9H). 13 C NMR (151MHz, MeOD) δ 178.51, 171.70, 166.83, 165.76, 155.70, 150.79, 143.08, 142.08, 141.48, 137.61 ,134.08,133.13,132.01,130.92,130.90,129.13,128.14,127.93,126.90,126.78 , 126.75, 125.74, 121.05, 118.55, 110.14, 109.25, 78.79, 67.47, 53.72, 39.29, 37 .34, 30.42, 29.38, 29.31, 29.04, 27.39, 26.72, 25.10, 13.08, 13.05, 11.57, 10.26. HRMS.ESI (m / z): [M + H] + calcd for C 41 H 43 N8O5S + : 759.3072, found 759.3080. HPLC Purity:95.97%.

[0077] Synthesis of compound D5: Following the synthetic method for compound D2, using compound D1 and compound s35 (ammonia water) as raw materials, a white solid, namely compound D5, was obtained. The characterization data for compound D5 are as follows: 1 H NMR (600 MHz, MeOD) δ 8.06 – 8.00 (m, 2H), 7.86 – 7.80 (m, 2H), 7.79 – 7.75 (m, 2H), 7.63 (d, J = 3.4 Hz, 1H), 7.62 – 7.58 (m, 2H), 7.50 (d, J = 2.3 Hz, 1H), 6.79 (d, J = 8.8 Hz, 1H), 4.63 (t, J = 7.1 Hz, 1H), 3.77 (s, 1H), 3.54 (d, J = 7.2 Hz, 2H), 3.35 (s, 1H), 2.72 (s, 3H), 2.50 – 2.45 (m, 3H), 1.76 (s, 3H).13 CNMR (126 MHz, DMSO) δ 176.4, 163.6, 155.4, 149.8, 141.9, 140.9, 137.6, 134.3, 132.1, 130.7, 130.1, 129.1, 128.4, 126.9, 126.7, 125.5, 120.1, 117.9, 108.9, 36.1, 29.1, 22.2, 14.2, 12.8, 11.4. HRMS.ESI (m / z): [M + H] + calcd for C 34 H 30 N7O3S + : 616.2125, found616.2119. HPLC Purity:96.73%.

[0078] Synthesis of compound D6: Following the synthetic method for compound D2, using compound D1 and compound s36 (2-[2-(2-aminoethoxy)ethoxy]ethanol) as starting materials, a white solid, namely compound D6, was obtained. The characterization data for compound D6 are as follows: 1 H NMR (600 MHz, MeOD) δ 8.01 (d, J = 8.0 Hz, 2H), 7.80 (d, J = 8.2 Hz, 2H), 7.77 – 7.72 (m, 2H), 7.63 (s, 1H), 7.58 (d, J = 8.0 Hz, 2H), 7.49 (dd, J = 8.4, 2.1 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 4.67 (dd, J = 8.8, 5.3 Hz, 1H), 3.68 – 3.66 (m, 5H), 3.59 – 3.56 (m, 2H), 3.55 (s, 2H), 3.48 (td, J = 6.4, 3.0 Hz, 2H), 3.35 (s, 5H), 2.71 (s, 3H), 2.45 (s, 3H), 1.73 (s, 3H). 13C NMR (151 MHz, MeOD) δ 178.5, 175.7, 172.3, 165.6, 155.7, 150.8, 142.1, 137.6, 134.1, 133.1, 132.0, 129.1, 127.9, 126.9, 126.8, 121.1, 1 18.6, 109.3, 72.3, 72.3, 70.1, 70.0, 69.97, 69.3, 60.8, 54.5, 53.8, 42.6, 39.1, 37.4, 29.4, 17.9, 13.1, 11.6, 10.3. LC-MS: [M + H] + calcdfor C 40 H 42 N7O6S + : 748.2, found 748.2. HPLC Purity:97.19%.

[0079] Synthesis of compound D7: Following the synthetic method for compound D2, using compound D1 and compound s37 (valine amide hydrochloride) as starting materials, a white solid, namely compound D7, was obtained. The characterization data for compound D7 are as follows: 1 H NMR (500 MHz, MeOD) δ 8.07 – 8.00 (m, 2H), 7.82 (dd, J = 8.4, 1.5 Hz, 2H), 7.77 (t, J = 8.7 Hz, 2H), 7.62 (dd, J = 9.9, 4.2 Hz, 2H), 7.50 (dd, J = 8.2, 2.1 Hz, 1H), 7.43 (d, J = 2.1 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 5.34 (dd, J = 5.5, 4.2 Hz, 1H), 4.72 – 4.65 (m, 1H), 4.34 (d, J = 5.7 Hz, 1H), 3.71 (s, 1H), 3.57 (s, 1H), 3.56 – 3.48 (m, 2H), 2.72 (s, 3H), 2.47 (s, 3H), 1.74 (d, J = 6.6 Hz, 3H), 1.08 –0.98 (m, 6H). 13C NMR (126 MHz, DMSO) δ 176.5, 173.3, 171.1, 170.0, 169.8, 166.5, 164.7, 163.8, 162.4, 155.4, 155. 3, 153.5, 149.9, 141.9, 140.9, 139.9, 137.6, 134.3, 133.2, 132.1, 130.8, 130 .1, 129.2, 128.4, 126.8, 125.6, 122.8, 120.1, 118.3, 118.0, 115.7, 111.0, 10 8.9, 104.0, 99.1, 54.1, 48.7, 46.2, 31.6, 29.5, 19.5, 18.8, 14.2, 12.8, 11.4. HRMS.ESI (m / z): [M + H] + calcd for C 39 H 39 N8O4S + : 715.2809, found 715.2788. HPLC Purity:96.95%.

[0080] Synthesis of compound D8: Following the synthetic method for compound D2, using compound D1 and compound s38 (tryptophan hydrochloride) as starting materials, a white solid, namely compound D8, was obtained. The characterization data for compound D8 are as follows: 11H NMR (500 MHz, MeOD) δ 8.02 (d, J = 8.0 Hz, 2H), 7.82 (d, J = 8.3 Hz, 1H), 7.78 (d, J = 8.5 Hz, 1H), 7.72 (d, J = 8.3 Hz, 1H), 7.67 (d, J = 7.9 Hz, 1H), 7.65 – 7.58 (m, 3H), 7.54 (d, J = 8.2 Hz, 1H), 7.48 (d, J = 7.3 Hz, 2H), 7.45 – 7.39 (m, 2H), 7.32 (dd, J = 15.6, 8.0 Hz, 1H), 7.23 (dd, J = 8.5, 2.1 Hz, 1H), 7.18 (s, 1H), 7.18 – 7.11 (m, 1H), 7.11 – 7.05 (m, 1H), 7.05 – 6.96 (m, 1H), 6.90 (d, J = 8.3 Hz, 1H), 4.60 – 4.56 (m, 1H), 3.56 (s, 2H), 2.70 (s, 3H), 2.44 (s, 3H), 2.27 (t, J = 7.4 Hz, 1H), 2.19 (s, 1H), 2.07 – 1.98 (m, 2H), 1.70 (d, J = 2.1 Hz, 3H), 1.60 (s, 2H). 13 C NMR (126 MHz, DMSO) δ 176.2, 173.4, 169.5, 164.5, 163.5, 155.2, 149.7, 141.7, 140.6, 139.7, 137.3, 136.0, 135.9, 134.1, 133.0, 131.9, 131.8, 130.5, 130.0, 129.8, 129.1, 128.9, 128.2, 127.3, 126.6, 126.5, 125.8, 125.6, 123.5, 123.3, 120.7, 119.9, 118.3, 118.10, 117.7, 111.2, 110.5, 108.7, 97.0, 73.9, 54.8, 48.5, 35.9, 28.9, 28.6, 14.0, 12.6, 11.2. HRMS.ESI (m / z): [M + H] + calcd for C 45 H 40 N9O4S + : 802.2918, found 802.2906. HPLC Purity: 95.29%.

[0081] Synthesis of compound D9: Following the synthetic method for compound D2, using compound D1 and compound s39 (tyrosine amide hydrochloride) as starting materials, a white solid, namely compound D9, was obtained. The characterization data for compound D9 are as follows: 1 H NMR (500 MHz, MeOD) δ 7.99 (d, J = 8.1 Hz, 2H), 7.83 – 7.77 (m, 3H), 7.74 (dd, J = 8.5, 4.1 Hz, 3H), 7.61 – 7.51 (m, 3H), 7.46 (d, J = 8.0 Hz, 3H), 7.18 (d, J = 2.3 Hz, 1H), 7.11 – 7.05 (m, 2H), 6.98 (d, J = 8.4Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 6.69 (d, J = 8.5 Hz, 2H), 6.59 (d, J = 8.5 Hz, 1H), 3.53 (s, 2H), 3.45 (s, 2H), 3.17 (s, 2H), 2.68 (d, J = 7.2 Hz, 3H), 2.42 (s, 3H), 2.00 (s, 2H), 1.69 (d, J = 2.1 Hz, 3H). 13 C NMR (126 MHz, DMSO) δ 176.4, 173.6, 173.3, 171.8, 169.6, 164.7, 163.6, 156.4, 155.8, 155.4, 155.3, 149 .9, 141.9, 140.8, 139.8, 134.2, 133.1, 132.0, 132.0, 1307, 130.4, 130.1, 130.0, 12 9.2, 129.1, 128.5, 128.4, 128.1, 126.7, 126.6, 126.0, 125.6, 123.2, 122.7, 120.1, 117.9, 115.6, 115.3, 114.9, 108.8, 46.2, 36.1, 31.6, 29.5, 18.8, 14.2, 12.7, 11.3. HRMS.ESI (m / z): [M + H] + calcd for C 43 H 39 N8O5S + : 779.2759, found 779.2750. HPLC Purity:96.55%.

[0082] Synthesis of compound D10: Following the synthetic method for compound D2, using compound D1 and compound s40 (leucine amide hydrochloride) as starting materials, a white solid, namely compound D10, was obtained. The characterization data for compound D10 are as follows:1 H NMR (500 MHz, MeOD) δ 8.08 – 7.96 (m, 3H), 7.91 – 7.81 (m, 3H), 7.80 – 7.73 (m, 3H), 7.60 (dt, J = 13.9, 8.7 Hz, 4H), 7.51 (d, J = 2.1 Hz, 1H), 7.50 – 7.33 (m, 1H), 7.23 (dd, J = 8.5, 2.2 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 4.72 – 4.66 (m, 1H), 4.58 (s, 1H), 4.46 (dt, J = 10.8, 5.4 Hz, 1H), 3.57 (s, 2H), 3.48 (d, J = 7.3 Hz, 1H), 2.72 (d, J = 1.9 Hz, 3H), 2.46 (d, J = 3.0 Hz, 3H), 2.03 (d, J = 6.2 Hz, 1H), 1.74 (d, J = 8.1 Hz, 4H), 1.02 – 0.94 (m, 6H). 13 C NMR (151 MHz, MeOD) δ176.6, 171.6, 166.9, 165.8, 155.8, 150.9, 137.6, 130.9, 12 9.2, 129.1, 127.9, 126.9, 126.8, 121.9, 118.6, 109.3, 53.9, 53.8, 51.8 ,51.6,40.7,40.4,31.7,30.5,30.4,29.5,29.4,29.4,29.4,29.3,29.2 ,29.1,28.9,26.7,24.7,24.6,22.4,22.3,22.2,20.4,20.2,13.1,11.6. HRMS.ESI (m / z): [M + H] + calcd for C 40 H 41 N8O4S + :729.2966,found729.2952. HPLC Purity:95.37%.

[0083] Synthesis of compound D11: Following the synthetic method for compound D2, using compound D1 and compound s41 (phenylalanine hydrochloride) as raw materials, a white solid, namely compound D11, was obtained. The characterization data for compound D11 are as follows: 1H NMR (500 MHz, MeOD) δ 8.06 – 8.01 (m, 2H), 7.98 (s, 2H), 7.87 – 7.71 (m, 5H), 7.63 (s, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.52 (dd, J = 15.7, 8.2 Hz, 3H), 7.37 – 7.24 (m, 4H), 7.25 – 7.16 (m, 3H), 6.90 (d, J = 8.4Hz, 1H), 4.68 (dd, J = 10.2, 5.1 Hz, 1H), 4.59 (dd, J = 13.5, 6.5 Hz, 1H), 3.57 (s, 1H), 3.48 (s, 2H), 2.99 (s, 3H), 2.86 (s, 4H), 2.71 (d, J = 4.8 Hz, 2H), 2.45 (s, 2H). 13 C NMR (126MHz, DMSO) δ 176.4, 174.5, 170.4, 164.6, 155.3, 149.8, 142.9, 138.4, 138.1, 135.5, 130.1, 130.0, 129.7, 129.5, 129.2, 129.1, 128.5, 128.4, 128.1 , 127.7, 126.7, 126.2, 125.5, 125.3, 123.6, 122.7, 120.0, 118.7, 117.7, 110.3, 108.8, 33.8, 31.3, 29.0, 24.5, 22.1, 14.1, 12.7, 11.3. HRMS.ESI (m / z): [M + H] + calcd for C 43 H 39 N8O4S + :763.2809,found763.2794. HPLC Purity:96.42%.

[0084] The compounds prepared in the above embodiments were tested: (1) High-content screening experiment A recombinant plasmid was constructed by fusing red fluorescent protein (mScarlet) with the BRD4 protein domains (tandem BD1-BD2, N44-E460 of human BRD4 (UniProt: O60885)) and using green fluorescent protein (EGFP) as an internal control. The recombinant plasmid was then transfected into HEK-293T cells. Well-transfected HEK-293T cells were digested and counted at a concentration of 1 × 10⁻⁶ cells. 4Cells were evenly seeded at a density of 100 cells / well into 96-well plates and cultured overnight. Cell adhesion density was observed to be 70%-80% under an inverted microscope. The test compound was diluted with DMEM high-glucose medium to prepare solutions of different concentrations. The old medium was discarded, and the compound solution was added, with three replicates for each concentration. An equal volume of PBS buffer was added to the edge of each well to eliminate edge effects. The plates were incubated for 12 hours, and after incubation, the plates were removed and gently shaken to ensure uniform 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-set program.

[0085] In transfected HEK-293T cells, the addition of the aforementioned compounds resulted in the effective degradation of the target domain (BD1-BD2 double domain) of BRD4, leading to a synchronous decrease in the fluorescence of the red fluorescent protein bound to it, while the green fluorescence remained stable. Therefore, by quantitatively analyzing the change in the red to green fluorescence ratio using a high-content imaging system, the efficacy of the aforementioned compounds in inducing target protein degradation can be objectively characterized. The smaller the red to green fluorescence ratio, the stronger the ability of the aforementioned compounds to degrade BRD4 protein.

[0086] (2) Western blot experiment Wash the cell plate with PBS, add RIPA lysis buffer (containing protease inhibitors), scrape off cells and transfer to EP tubes for lysis (8 cycles at 20% power, 2 seconds each). Centrifuge at 12000 rpm for 20 min at 4 °C, and transfer the supernatant to a new EP tube. Take a fixed volume of the supernatant, add 5× Loading Buffer, mix well, and heat at 95 °C for 8 min before use or storage at -80 °C. Perform protein quantification and prepare protein electrophoresis gels of different concentrations as needed.

[0087] Install the electrophoresis apparatus, add electrophoresis buffer until it is level with the top, remove the comb to expose the sample wells, and add markers (protein molecular weight standards) and protein samples sequentially. Set the electrophoresis conditions (70 V, 30 min) and run the gel. Once the buffer is level, separate the gel under voltage conditions (120 V, 60 min). Stop the process when the two-color loading buffer reaches the bottom. Immerse a suitably sized PVDF (polyvinylidene fluoride) membrane in methanol for 1 min, rinse with transfer buffer, and then cover the gel block after electrophoresis. Remove excess gel, transfer the membrane to an electrotransfer clamp to remove air bubbles, clamp it tightly, and place it in the electrotransfer tank. Transfer the membrane at a constant current of 230 mA under ice bath conditions, setting the electrotransfer time according to the protein molecular weight.

[0088] After electroporation, the membrane was placed in 5% skim milk and slowly swirled at room temperature for 1 hour to block. After removal, it was rinsed three times with 1×TBST buffer for 20 minutes each time. The membrane containing the target protein was excised and labeled, and then incubated with primary antibody overnight at 4°C. After removal, it was rinsed three times with 1×TBST buffer for 20 minutes each time, and then incubated with secondary antibody at room temperature for 1 hour. After removal, it was rinsed three times with 1×TBST buffer for 20 minutes each time. In a light-protected container, equal volumes of solutions A and B from the development kit were mixed to prepare the developing solution. The membrane was immersed and incubated for 3 minutes, then developed and analyzed.

[0089] (3) Effect of CRBN on the degradation of BRD4 by compound B2 A CRBN knockdown cell model was established by constructing an sgRNA plasmid targeting the CRBN gene (denoted as sgCRBN) and transfecting it into HEK-293T cells and HEK-293T cells carrying a dual-fluorescent reporter system using CRISPR / Cas9 gene editing technology. Subsequently, the CRBN-knockdown cells were treated with compounds B2 and DMSO, respectively. High-content screening (HCS) and Western blotting analysis were then used to evaluate the ability of compound B2 to mediate BRD4 degradation under conditions lacking the E3 ubiquitin ligase CRBN, thereby verifying whether its BRD4 degradation activity depends on the CRBN-mediated ubiquitin-proteasome pathway.

[0090] In addition, a plasmid targeting the CRBN gene (denoted as sgControl, which is a meaningless sequence) was constructed and transfected into HEK-293T cells and HEK-293T cells carrying a dual-fluorescent reporter system using CRISPR / Cas9 gene editing technology to establish a control cell model (i.e., CRBN was not knocked down). The control cell models were then treated with compounds B2 and DMSO, respectively, and tested using the same method described above.

[0091] (4) Test of antifibrotic activity of compound B2 in mouse embryonic fibroblasts NIH-3T3 cells were seeded and cultured in 6-well plates. When cell confluence reached approximately 70%-80%, serum-containing medium was added for acclimatization. Subsequently, cells were stimulated with transforming growth factor-β1 (TGF-β1, 5 ng / mL) to induce a fibrotic phenotype, while different concentrations of compound B2 were added for 24 h of intervention. Simultaneously, cells were stimulated with TGF-β1 (5 ng / mL) alone for 24 h as a control. After treatment, Western blotting was used to detect the protein levels of fibrosis-related proteins.

[0092] (5) q-PCR detection of inflammatory factor expression in rat gingival tissue After modeling, the rats were administered the drug every two days at the modeling site for 14 days. The low-dose and high-dose groups received 20 μL of the corresponding concentration of compound B2 (5 μM and 50 μM, respectively) at the modeling site, designated as the PD+B2 (5 μM) group and the PD+B2 (50 μM) group, respectively. The negative control group received an equal volume of hydrogel, designated as the PD group. The positive control group received 0.1% minocycline, designated as the PD+minocycline (0.1%) group. The control group (i.e., normal rats) received an equal volume of hydrogel. On day 14, the rats were sacrificed, and the maxillae were harvested, and the gingival tissue was dissected. In a fume hood, 1 mL of total RNA extraction reagent (Trizol) was added to each tissue sample, and the tissue was ground and lysed at room temperature for 5 min. 200 µL of chloroform was added to an EP tube, and the tube was vigorously shaken until it turned pink, then allowed to stand for 3 min. Subsequently, the tubes were centrifuged at 12000 g for 15 min at 4 ℃. At this point, the RNA is in the upper aqueous phase. Carefully aspirate 400 µL of the aqueous phase into a new EP tube, add 400 µL of isopropanol, invert and mix well, and let stand for 10 min to precipitate the RNA. Centrifuge at 12000 g for 15 min at 4 ℃, discard the supernatant, and retain the precipitate. Add 500 µL of 75% ethanol to wash the precipitate, centrifuge at 7500 g for 5 min at 4 ℃, and repeat this step twice. Discard the supernatant and dry in a fume hood for 2 min. Add 25 µL of DEPC to dissolve the RNA, and incubate in a 55 ℃ water bath for 10 min. Measure the RNA concentration using a NanoDrop 2000 instrument. Store the RNA at -80 ℃ or use it for the next experiment. Perform reverse transcription to obtain cDNA according to the kit instructions. Then perform qRT-PCR experiments. Prepare the following reaction system (Table 5) according to the Novizan Bio SYBR qPCR MasterMix kit instructions and add it to the qPCR plate, protecting it from light.

[0093] Table 5. Reaction System

[0094] After adding reagents to the qPCR plate, seal the plate with sealing film and briefly centrifuge to mix. Place the plate in a real-time quantitative PCR instrument, and follow the reaction program shown in Table 6.

[0095] Table 6. Reaction Procedure

[0096] After the reaction is complete, copy the data and perform the result calculation. The calculation formula is as follows: Ct = Target gene Ct - Internal reference gene Ct, Ct= Ct (control group) - Ct (experimental group), R=2- Ct Ct represents the cycle threshold, and R represents the relative expression level.

[0097] Histological morphological observation of periodontitis: After the rats were sacrificed, the left maxilla was removed, fixed with 4% paraformaldehyde, embedded in paraffin, prepared into slides, and then observed using TRAP staining.

[0098] (6) Experiment on the inhibition of osteoclast differentiation by compound B2 For RAW264.7 cells: RAW264.7 cells were cultured in DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin-dextrose antibody). Cells in the logarithmic growth phase were collected and divided into a control group, an OC (osteoclast differentiation) group, and an OC+B2 group. The treatments for each group were as follows: Control group: Logarithmic growth phase cells were resuspended in MEM medium (containing 10% fetal bovine serum and 1% penicillin and antibiotics) and seeded at a density of 20,000 cells / well in 24-well plates for 8 days, with the medium changed every two days; OC group: Logarithmic growth phase cells were resuspended in MEM medium (containing 10% fetal bovine serum and 1% penicillin and antibiotics), seeded at a density of 20,000 cells / well in 24-well plates, and 100 ng / mL RANKL was added. The cells were cultured for 8 days, with the medium changed every two days.

[0099] OC+B2 group: Logarithmic growth phase cells were resuspended in MEM medium (containing 10% fetal bovine serum and 1% penicillin and antibiotics) and seeded at a density of 20,000 cells / well in 24-well plates. 100 ng / mL of RANKL and compound B2 (25 nM) were added and cultured for 8 days, with the medium changed every two days.

[0100] For BMDM cells: 6-8 week old C57BL / 6JGpt mice were sacrificed and disinfected by immersion in 75% ethanol for 15 min. Under aseptic conditions, the femurs and tibias were separated, and after removing attached muscle tissue, they were immersed in 10% penicillin / streptomycin phosphate buffer for 5 min. The epiphyses were cut off, and the bone marrow cavity was repeatedly flushed with MEM complete culture medium to collect the bone marrow cell suspension. The cell suspension was gently pipetted to mix and filtered through a 70 μm cell sieve to remove bone fragments and tissue clumps. The cells were then collected by centrifugation, and the supernatant was discarded. The obtained bone marrow cells were seeded in culture dishes and divided into a control group, an OC group, and an OC+B2 group. The treatment for each group was as follows: Control group: The obtained bone marrow cells were cultured in α-MEM complete medium containing 10% fetal bovine serum and 1% penicillin and streptomycin, and 50 ng / mL M-CSF was added. The culture was carried out for 8 days, and the medium was changed every two days. OC group: The obtained bone marrow cells were cultured in α-MEM complete medium containing 10% fetal bovine serum and 1% penicillin and streptomycin, and 50 ng / mL M-CSF and 100 ng / mL RANKL were added. The culture was carried out for 8 days, and the medium was changed every two days. OC+B2 group: The obtained bone marrow cells were cultured in α-MEM complete medium containing 10% fetal bovine serum and 1% penicillin and streptomycin, and 50 ng / mL M-CSF, 100 ng / mL RANKL and compound B2 (25 nM) were added. The culture was carried out for 8 days, and the medium was changed every two days.

[0101] (7) Cell viability assay After cell digestion and counting, 3000–5000 cells per well were seeded in 96-well plates and incubated overnight. The compound was serially diluted with complete culture medium to prepare gradient concentrations, and the medium was replaced with each well of the 96-well plate, with three auxiliary wells for each concentration. PBS was added to the side wells of the plate, and the plates were incubated for 48 h. 10 μL of CCK-8 reagent was added to each well of the 96-well plate, vortexed, and incubated for 2 h. After incubation, the absorbance (OD) at 450 nm was measured using a microplate reader, and cell viability was calculated.

[0102] II. The test results are as follows: (1) The activity screening results of compounds based on the high content screening platform are shown in Table 7.

[0103] Table 7. Degradation activity of compounds on BRD4

[0104] The degradation activity A represents a red fluorescence to green fluorescence ratio of 0.8-0.9 at a compound concentration of 100 nM (the ratio is 1 when the compound cannot degrade BRD4); the degradation activity B represents a red fluorescence to green fluorescence ratio of 0.7-0.8 at a compound concentration of 100 nM; and the degradation activity C represents a red fluorescence to green fluorescence ratio of 0.6-0.7 at a compound concentration of 100 nM.

[0105] It is evident that all compounds in the above embodiments can degrade BRD4.

[0106] (2) The degradation activity results of compound B2 are as follows: Figure 2 As shown, compound B2 can effectively degrade BRD4 protein, and the higher the concentration of compound B2, the better the degradation effect.

[0107] (3) To verify the degradation pathway of compound B2, compound B2 at a concentration of 0.1 μM was reacted with autophagy inhibitors CQ (chloroquine), Baf (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 (4-[4-[( [5-nitro-2-furanyl]methylene]-3,5-dioxo-1-pyrazolyl]ethyl 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. Then, high-content analysis was performed to determine whether target protein degradation was competitively inhibited. The results were as follows: Figure 3 As shown, ns indicates no significant difference; This indicates that p < 0.01; The result indicates that neither chloroquine inhibition of autophagosome acidification nor bafloxacin interference with lysosomal maturation effectively inhibited the degradation activity of compound B2. However, all four inhibitors of the ubiquitin-proteasome pathway significantly suppressed the degradation effect of compound B2 on BRD4. This suggests that compound B2 mediates BRD4 degradation through the classic ubiquitin-proteasome pathway.

[0108] (4) To verify the effect of CRBN on the degradation of BRD4 by compound B2, high-content screening experiments were performed by knocking down CRBN in HEK-293T cells. The results showed that... Figure 4 As shown, ns indicates no significant difference; Indicated by p < 0.0001. The results showed that knocking down CRBN increased the red to green fluorescence ratio (mScarlet / EGFP), indicating a decrease in the BRD4 degradation activity of compound B2. Furthermore, Western blotting analysis of BRD4 levels further confirmed that knocking down CRBN reduced the compound's BRD4 degradation activity. These results indicate that compound B2 recruits CRBN as the E3 ubiquitin ligase.

[0109] (5) The results of the antifibrotic activity of compound B2 in mouse embryonic fibroblasts are as follows: Figure 5 As shown, compound B2 can effectively reduce the expression of fibronectin, type I collagen and α-smooth muscle actin, indicating that compound B2 has anti-fibrotic activity.

[0110] (6) The effects of different doses of compound B2 on the expression of inflammatory factors and BRD4 in rat gingival tissue are as follows: Figure 6 As shown, # indicates that compared with the control group, P < 0.05; ## indicates that compared with the control group, P < 0.01; #### indicates that compared with the control group, P < 0.0001. This indicates that compared to the PD group, p < 0.01. This indicates that compared to the PD group, p < 0.001. The result indicates that, compared with the PD group, p < 0.0001. This suggests that compound B2 can reduce the level of gingival inflammation in rats with periodontitis.

[0111] The results of TRAP immunohistochemistry of rat alveolar bone sections are as follows: Figure 7 As shown, both the compound B2 treatment group and the positive control group reduced osteoclast differentiation, with the high-dose group showing the least osteoclast differentiation. These results indicate that compound B2 can inhibit osteoclast differentiation in rats with periodontitis.

[0112] (7) Compound 2 can inhibit osteoclast differentiation as shown in the following results. Figure 8 As shown, OC indicates osteoclast induction. #### indicates that P < 0.0001 compared to the control group.

[0113] (8) The results of the antiproliferative activity of compound B2 against different cancer cells are shown in Table 8. It can be seen that compound B2 has antiproliferative activity against breast cancer, lung cancer, liver cancer, glioma and colorectal cancer.

[0114] Table 8. Results of the antiproliferative activity of compound B2 against different cancer cells.

[0115] in, a IC represents compound B2 50 The value is the average of three repeated experiments, and the data were obtained from a 48-hour CCK8 experiment.

[0116] In summary, this invention provides a molecular glue degrader and its applications in pharmaceuticals. This invention targets BRD4 and chemically modifies its inhibitor (+)-JQ-1. By introducing a diverse range of dominant backbones into its chemical structure, the interaction between BRD4 and E3 ubiquitin ligase is enhanced, achieving targeted degradation of BRD4. Specifically, based on the principle of enhanced PPI by molecular glue degraders, this invention structurally modifies the BRD4 inhibitor, introducing dominant backbones (various nitrogen-containing heterocyclic structures) as binding groups, thereby generating new protein interfaces and binding sites, enhancing the PPI between BRD4 and E3 ubiquitin ligase, thus converting the BRD4 inhibitor into a BRD4 degrader. The molecular glue degrader provided by this invention can degrade BRD4 via the ubiquitin-proteasome pathway mediated by the E3 ubiquitin ligase CRBN, and exhibits antitumor proliferation activity, antifibrotic activity, anti-inflammatory activity, and inhibitory activity against osteoclast differentiation. This invention enriches the molecular glue degrader library while simultaneously achieving BRD4 degradation.

[0117] 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 molecular adhesive degrading agent, characterized in that, The structural formula of the molecular adhesive degrader is: ； R1 is selected from one of the following groups: , , , , , , , , , , , , , and Boc represents tert-butyloxycarbonyl. R2 is selected from one of the following groups: Cl、 , , , , , , , , , , , , , , , , , and ; When R2 is Cl, R1 is not. or ; Indicates the connection site.

2. The molecular adhesive degrading agent according to claim 1, characterized in that, R1 is ; R2 is selected from one of the following groups: , , , , , , , , , , , , , , , , , and .

3. The molecular adhesive degrading agent according to claim 1, characterized in that, R1 is selected from one of the following groups: , and ; R2 is Cl.

4. The molecular adhesive degrading agent according to claim 1, characterized in that, R1 is selected from one of the following groups: , , , , , , , , , and ; R2 is .

5. The use of the molecular gel degrading agent according to any one of claims 1-4 in the preparation of a drug that degrades bromine domain protein 4.

6. The use of the molecular gel degrading agent according to any one of claims 1-4 in the preparation of a medicament for treating cancer, wherein the cancer is breast cancer, lung cancer, liver cancer, glioma, gastric cancer, or colorectal cancer.

7. The use of the molecular glue degrading agent according to any one of claims 1-4 in the preparation of antifibrotic drugs.

8. The use of the molecular glue degrading agent according to any one of claims 1-4 in the preparation of a medicament for treating osteoporosis.

9. The use of the molecular adhesive degrading agent according to any one of claims 1-4 in the preparation of a medicament for treating periodontitis.

10. A drug, characterized in that, The drug comprises the molecular glue degrading agent according to any one of claims 1-4, and the drug further comprises a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable additive.

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