Proteolytic chimeras targeting bromodomain-containing protein 4 and uses thereof

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a class of proteolytic chimeras targeting bromodomain protein 4 and their applications. These chimeras simultaneously possess structural fragments containing a bromodomain protein 4 ligand, a linker, and an E3 ligase ligand. They can recruit E3 ligases in cells to ubiquitinate bromodomain protein 4, followed by UPS-induced degradation of bromodomain protein 4. Experimental studies have demonstrated that these chimeras can target bromodomain protein 4 at low concentrations, inducing its ubiquitination and significantly reducing intracellular bromodomain protein 4 aggregation levels, thereby improving cell viability and reducing related cytotoxicity. This makes them highly suitable for treating diseases related to bromodomain protein 4.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to a class of proteolytic chimeras targeting bromodomain protein 4 and their applications. Background Technology

[0002] Bet proteins are a class of bromodomain (Brd) proteins containing two N-terminal bromodomains (BD) BD1 and BD2, an extra terminal domain (ET), and several conserved regions (A, B, and SEED regions). Each Brd4 protein contains four antiparallel α-helices (αA, αB, αC, and αZ), interconnected by two loops of different lengths (ZA and BC loops), forming a hydrophobic cavity that can accommodate electrically neutral acetylated lysine residues. This allows Bet proteins to bind to acetylated lysine residues at the tail of histones via their N-terminal bromodomains, altering chromatin structure and thus influencing various cellular biological processes. Recent studies have found that Brd4 proteins can affect physiological processes such as cell cycle, proliferation, and apoptosis, playing a crucial role in tumor cell invasion, metastasis, and malignant development. Hematologic malignancies such as acute myeloid leukemia (AML) and multiple myeloma (MM), as well as solid tumors such as breast cancer (BCa), glioblastoma (GBM), and renal cell carcinoma (RCC), are all associated with Brd4 dysfunction.

[0003] PROteolysis targeting chimera (PROTAC) is a bifunctional small molecule compound containing two different ligands: a ubiquitin ligase E3 ligand and a ligand that binds to a target protein in the cell. These two ligands are linked by a linker, forming a target protein ligand-linker-E3 compound. After entering the cell, the target protein (POI) ligand in the PROTAC molecule specifically binds to the corresponding target protein, while the other end recruits the E3 ligase, forming a POI-Linker-E3 ligase ternary complex. The E3 ligase mediates the ubiquitination of POI by the ubiquitin-conjugating enzyme E2. The ubiquitin-labeled POI is then recognized and degraded by the proteasome. Furthermore, PROTAC can be recycled multiple times within the cell. For example, Chinese patent application CN114573570A discloses a class of compounds targeting bromodomain protein 4. These compounds simultaneously contain α-synuclein ligands, linkers, and E3 ligase ligand fragments, enabling them to recruit intracellular E3 ligases to ubiquitinate α-synuclein, thereby achieving chemically induced degradation of α-synuclein via UPS. This significantly reduces intracellular α-synuclein aggregate levels and improves cell viability. However, such compounds are still under research and development, and the types available for clinical research and application remain relatively limited. Therefore, there is an urgent need to provide more proteolytic chimeras targeting bromodomain protein 4 for selection. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiency and inadequacy of existing proteolytic chimeras targeting bromodomain protein 4, and to provide a proteolytic chimera targeting bromodomain protein 4.

[0005] The purpose of this invention is to provide the application of the proteolytic chimera targeting bromodomain protein 4 in the preparation of drugs for treating diseases related to bromodomain protein 4.

[0006] Another object of the present invention is to provide a medicament for treating diseases related to bromodomain protein 4.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] A type of proteolytic chimera targeting bromodomain protein 4 has the structure of formula (I):

[0009]

[0010] Where X is -CO-(CH2) a -or -CO-(CH2-CH2-O)b -CH2-CH2-, where a is an integer from 9 to 13 and b is an integer from 1 to 5;

[0011] R is selected from any of the following structures:

[0012]

[0013] Preferably, X is -CO-(CH2). a -or -CO-(CH2-CH2-O) b -CH2-CH2-, where a is an integer from 9 to 13 and b is an integer from 1 to 3.

[0014] More preferably, X is -CO-(CH2). a -or -CO-(CH2-CH2-O) b -CH2-CH2-, a is 9 or 13, b is 2 or 3.

[0015] Specifically, the proteolytic chimera targeting bromodomain protein 4 has any of the following structures:

[0016]

[0017] Furthermore, the proteolytic chimera targeting bromine domain protein 4 also includes its pharmaceutically acceptable salts, solvates, or isomers.

[0018] The proteolytic chimera targeting bromodomain protein 4 provided by this invention possesses structural fragments containing a bromodomain protein 4 ligand, a linker, and an E3 ligase ligand. It can recruit E3 ligase in cells to ubiquitinate bromodomain protein 4, followed by UPS-induced degradation of bromodomain protein 4. Experimental studies have demonstrated that this proteolytic chimera can target bromodomain protein 4 at low concentrations, inducing its ubiquitination and significantly reducing intracellular bromodomain protein 4 aggregation levels, thereby improving cell viability and reducing related cytotoxicity. This makes it highly suitable for treating diseases related to bromodomain protein 4.

[0019] Therefore, the present invention also claims the use of the proteolytic chimera targeting bromodomain protein 4 in the preparation of medicaments for treating diseases related to bromodomain protein 4.

[0020] Preferably, the diseases associated with the bromodomain protein 4 include solid tumors such as acute myeloid leukemia, multiple myeloma, breast cancer, glioblastoma, and renal cell carcinoma.

[0021] In addition, the present invention also provides a medicament for treating diseases related to bromodomain protein 4, comprising the proteolytic chimera that targets bromodomain protein 4.

[0022] Furthermore, the drug also includes pharmaceutically acceptable excipients selected from one or more of diluents, lubricants, binders, disintegrants, surfactants, film-forming materials, coating materials, and capsule materials.

[0023] Furthermore, the dosage form of the drug is an injectable formulation, an oral formulation, a nebulized inhalation formulation, or a transdermal formulation.

[0024] The present invention has the following beneficial effects:

[0025] This invention provides a proteolytic chimera targeting bromodomain protein 4, containing structural fragments of a bromodomain protein 4 ligand, a linker, and an E3 ligase ligand. It can recruit E3 ligase in cells to ubiquitinate bromodomain protein 4, followed by UPS-induced degradation of bromodomain protein 4. Experimental studies have demonstrated that this proteolytic chimera can target bromodomain protein 4 at low concentrations, inducing its ubiquitination and significantly reducing intracellular bromodomain protein 4 aggregation levels, thereby improving cell viability and reducing related cytotoxicity. This makes it highly suitable for treating diseases related to bromodomain protein 4. Attached Figure Description

[0026] Figure 1 This is an immunoblot image and a statistical graph showing the results of the reduction in the total level of the proteolytic chimeric compound targeting bromodomain protein 4 on intracellular bromodomain protein 4 aggregates. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0028] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0029] Example 1: Preparation of intermediate compound 9 (E3 ligase VHL ligand)

[0030] The synthetic route for intermediate compound 9 is as follows:

[0031]

[0032] Specifically, the following steps are included:

[0033] Synthesis of S1 and Compound 3:

[0034] Compound 1 (3 mmol) and compound 2 (6 mmol) were dissolved in DMAC (8 mL), and a catalytic amount of palladium acetate (2 mg) and an equal amount of potassium acetate (6 mmol) were added. The mixture was heated to 150 °C and refluxed overnight with stirring. After the reaction was basically complete as monitored by TLC, water (25 mL) was added to quench the reaction. The mixture was extracted twice with an equal amount of DCM, and the organic phases were combined. The mixture was washed twice with water (50 mL) and once with saturated brine. The aqueous phase was discarded, and the mixture was dried over anhydrous magnesium sulfate. After filtration, the mixture was evaporated to dryness to obtain compound 3 as a brown solid with a yield of 45%.

[0035] Synthesis of S2 and Compound 4:

[0036] Compound 3 (1 mmol) was dissolved in MeOH (10 mL) and cooled to 0 °C. Cobalt chloride (1.5 mmol) was added under stirring, followed by sodium borohydride (5 mmol) in portions. The reaction was allowed to proceed for 1.5 hours. After the reaction was nearly complete as monitored by TLC, a small amount of dilute ammonia was slowly added to quench the reaction. The insoluble matter was filtered off, and the mixture was extracted three times with DCM. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, and then evaporated to dryness to obtain a dark brown oil. The oil was purified by column chromatography (DCM:MeOH = 20:1) to obtain compound 4, which was a yellow oil with a yield of 65%.

[0037] Synthesis of S3 and Compound 5:

[0038] Compound 4 (1 mmol) and an equal volume of Boc-Hyp-OH were dissolved in DMF (8 mL). Under stirring at room temperature, DIPEA (4 mmol) was added dropwise. After reacting for 10 minutes, HATU (1.1 mmol) was added, and the reaction was continued for 10 hours. When the reaction was basically complete as monitored by TLC, water (25 mL) was added to quench the reaction. The mixture was extracted twice with an equal volume of ethyl acetate. The organic phases were combined, washed twice with water (50 mL), and once with saturated brine. The aqueous phase was discarded, dried over anhydrous magnesium sulfate, filtered, and evaporated to dryness. The mixture was separated by column chromatography (DCM:MeOH = 20:1) to obtain compound 5 as a yellow solid with a yield of 75%.

[0039] Synthesis of S4 and Compound 6:

[0040] Compound 5 (1 mmol) was dissolved in a mixed solution of DCM:TFA = 1:1 (10 mL) and reacted at room temperature with stirring for 30 minutes. After the reaction was basically complete as monitored by TLC, DCM was added to the system in small amounts several times, and TFA in the solvent was removed under reduced pressure. This step did not require purification and yielded the product with the corresponding deprotected group, namely compound 6, which was a brown oil with a yield of 80%.

[0041] Synthesis of S5 and Compound 8:

[0042] Compound 6 (1 mmol) and an equal volume of compound 7 were dissolved in DMF (8 mL). Under stirring at room temperature, DIPEA (4 mmol) was added dropwise. After reacting for 10 minutes, HATU (1.1 mmol) was added, and the reaction was continued for 10 hours. When the reaction was basically complete as monitored by TLC, water (25 mL) was added to quench the reaction. The mixture was extracted twice with an equal volume of ethyl acetate. The organic phases were combined, washed twice with water (50 mL), and once with saturated brine. The aqueous phase was discarded, dried over anhydrous magnesium sulfate, filtered, and evaporated to dryness. The mixture was separated by column chromatography (DCM:MeOH = 15:1) to obtain compound 8 as a pale yellow solid with a yield of 70%.

[0043] Synthesis of S6 and Compound 9:

[0044] Compound 8 (1 mmol) was dissolved in a mixed solution of DCM:TFA = 1:1 (10 mL) and reacted at room temperature with stirring for 30 minutes. After the reaction was basically complete as monitored by TLC, DCM was added to the system in small amounts several times, and TFA in the solvent was removed under reduced pressure. This step did not require purification and yielded the deprotected product, compound 9, which was a brown oil with a yield of 85%.

[0045] Example 2: Preparation of intermediate compound 12 (E3 ligase CRBN ligand)

[0046] The synthetic route for intermediate compound 12 is as follows:

[0047]

[0048] Specifically, the following steps are included:

[0049] Compound 10 (500 mg, 1.82 mmol), compound 11, and potassium carbonate were dissolved in DMF (2 mL) at a stoichiometric ratio of 1:1.05:1.5 and stirred at room temperature for 2 hours. After confirming that the reaction was basically complete by TLC monitoring (PE:EA = 1:1 as the developing solvent), water (25 mL) was added to quench the reaction. The mixture was extracted twice with an equal volume of ethyl acetate. The organic phases were combined, washed twice with water (50 mL), and once with saturated brine. The aqueous phase was discarded, dried over anhydrous sodium sulfate, filtered, and the solvent was removed. The mixture was then separated by column chromatography (PE:EA = 3:1 / 1:1) to obtain compound 12 in 85% yield.

[0050] Example 3: Preparation of protein hydrolysis chimeric compound 17

[0051] The synthetic route for the protein hydrolysis chimeric compound 17 is as follows:

[0052]

[0053] Specifically, the following steps are included:

[0054] Synthesis of S1 and Compound 15:

[0055] Compound 14 (0.27 mmol), DCC (56 mg, 0.27 mmol), and DMAP (5 mg, 0.04 mmol) were dissolved in DCM (3 mL), and then RVX-208 (compound 13, 50 mg, 0.14 mmol) was added. The mixture was stirred at room temperature for 16 hours. After confirming that the reactants had basically reacted completely by TLC (developing solvent: DCM:MeOH = 20:1), DCM (25 mL) was added to the system for extraction. The mixture was washed twice with water (50 mL × 2) and once with saturated brine (50 mL). The aqueous phase was discarded, and the organic phase was dried over anhydrous sodium sulfate. After filtration, the solvent was removed, and the mixture was separated by column chromatography (DCM:MeOH = 50:1 / 40:1) to obtain compound 15 in 95% yield.

[0056] Synthesis of S2 and Compound 16:

[0057] Compound 15 was dissolved in a DCM:TFA = 1:1 (4 mL) mixed solution and stirred at room temperature for 2 hours. DCM was added to the system in small amounts several times, and TFA was continuously dried under vacuum to obtain crude compound 16 with a yield of 90%. It was used directly in the next step of the reaction without purification.

[0058] Synthesis of S3 and Compound 17:

[0059] Compound 12 (43 mg, 0.13 mmol), HATU (74 mg, 0.19 mmol), and DIEA (67 mg, 0.52 mmol) were dissolved in DMF (2 mL), and then compound 16 (0.13 mmol) was added. The mixture was stirred at room temperature for 16 hours. After confirming that the reactants were basically completely reacted by TLC (developing solvent: DCM:MeOH = 20:1), water (25 mL) was added to quench the reaction. The mixture was extracted twice with ethyl acetate (25 mL × 2), and the organic phases were combined. The mixture was washed twice with water (50 mL × 2) and once with saturated brine (50 mL). The aqueous phase was discarded and dried over anhydrous sodium sulfate. After filtration, the solvent was removed and the mixture was separated by column chromatography (DCM:MeOH = 50:1 / 30:1) to obtain compound 17 in 60% yield.

[0060] Compound 17 is a colorless oil. 1H NMR(600MHz,Chloroform-d)δ9.55(s,1H),7.77(s,2H),7.69(t,J=7.9Hz,1H),7.60(t,J=5.3Hz,1H),7.49 (d,J=7.3Hz,1H),7.18(d,J=8.4Hz,1H),6.82-6.78(m,1H),6.42(d,J=2.0Hz,1H),5.30(s,1H),5.02-4.98 (m,1H),4.67(s,2H),4.44-4.39(m,2H),4.02-3.98(m,2H),3.92(d,J=10.2Hz,6H),3.77(t,J=6.3Hz,2H), 3.62(dd,J=14.6,5.0Hz,6H),3.55(q,J=6.0,5.5Hz,2H),2.64(t,J=6.3Hz,2H),2.29(s,6H),2.15(s,1H). 13 C NMR(151MHz,Chloroform-d)δ171.74,168.85,161.45,161.34,158.36,153.72,136.90,133.58,131.59,119.51,117.91,117.18,9 8.15,70.31,70.25,69.89,69.44,67.96,66.44,63.72,56.27,55.73,49.26,39.00,34.93,31.43,22.61,16.31.HRMS(ESI):Calcd for C 42 H 46 N5O 14 + [M+H] + :844.30358,C 42 H 45 N5O 14 Na + [M+Na] + :866.28552,Found:844.30200,866.28369.

[0061] Example 4: Preparation of protein hydrolysis chimeric compound 21

[0062] The synthetic route for the protein hydrolysis chimeric compound 21 is as follows:

[0063]

[0064] Specifically, the following steps are included:

[0065] Synthesis of S1 and Compound 19:

[0066] Compound 18 (0.27 mmol), DCC (56 mg, 0.27 mmol), and DMAP (5 mg, 0.04 mmol) were dissolved in DCM (3 mL), and then RVX-208 (compound 13, 50 mg, 0.14 mmol) was added. The mixture was stirred at room temperature for 16 hours. After confirming that the reactants had basically reacted completely by TLC (evolving solvent: DCM:MeOH = 20:1), DCM (25 mL) was added to the system for extraction. The mixture was washed twice with water (50 mL × 2) and once with saturated brine (50 mL). The aqueous phase was discarded, and the organic phase was dried over anhydrous sodium sulfate. After filtration, the solvent was removed, and the mixture was separated by column chromatography (DCM:MeOH = 50:1 / 40:1) to obtain compound 19 in 95% yield.

[0067] Synthesis of S2 and Compound 20:

[0068] Compound 19 was dissolved in a DCM:TFA = 1:1 (4 mL) mixed solution and stirred at room temperature for 2 hours. DCM was added to the system in small amounts several times, and TFA was continuously dried under vacuum to obtain crude compound 20 with a yield of 90%. It was used directly in the next step of the reaction without purification.

[0069] Synthesis of S3 and Compound 21:

[0070] Compound 12 (43 mg, 0.13 mmol), HATU (74 mg, 0.19 mmol), and DIEA (67 mg, 0.52 mmol) were dissolved in DMF (2 mL), and then compound 20 (0.13 mmol) was added. The mixture was stirred at room temperature for 16 hours. After confirming that the reactants were basically completely reacted by TLC (developing solvent: DCM:MeOH = 20:1), water (25 mL) was added to quench the reaction. The mixture was extracted twice with ethyl acetate (25 mL × 2), and the organic phases were combined. The mixture was washed twice with water (50 mL × 2) and once with saturated brine (50 mL). The aqueous phase was discarded and dried over anhydrous sodium sulfate. After filtration, the solvent was removed and the mixture was separated by column chromatography (DCM:MeOH = 50:1 / 30:1) to obtain compound 21 in 60% yield.

[0071] Compound 21 is a colorless oil. 1H NMR(600MHz,Chloroform-d)δ9.39(s,1H),7.78(s,2H),7.75-7.71(m,1H),7.66(t,J=5.3Hz,1H),7.54(d,J=7.3Hz,1H),7 .20(d,J=8.4Hz,1H),6.85(s,1H),6.46(d,J=2.2Hz,1H),5.32(s,1H),4.97(dd,J=12.2,5.5Hz,1H),4.68(s,2H),4.46-4. 42(m,2H),4.07-4.02(m,2H),3.95(d,J=16.0Hz,7H),3.78(td,J=6.4,2.6Hz,2H),3.65(dd,J=11.6,4.7Hz,11H),3.60-3. 53(m,2H),2.95-2.87(m,1H),2.85-2.73(m,2H),2.66(t,J=6.4Hz,2H),2.34(s,6H),2.19-2.14(m,1H),1.41-1.20(m,4H). 13 CNMR(151MHz,Chloroform-d)δ168.59,167.00,166.68,165.81,165.17,161.39,158.56,136.95,133.63,131.72,128.05,119.48,104.85, 101.09,98.23,70.47,70.37,70.24,70.00,67.96,66.43,63.76,56.34,55.77,49.29,39.10,34.90,31.44,22.68,16.40.HRMS(ESI):Calcd for C 44 H 50 N5O 15 + [M+H] + :888.32979,C 44 H 49 N5O 15 Na + [M+Na] + :910.31174,Found:888.32806,910.31006.

[0072] Example 5: Preparation of protein hydrolysis chimeric compound 25

[0073] The synthetic route for the protein hydrolysis chimeric compound 25 is as follows:

[0074]

[0075] Specifically, the following steps are included:

[0076] Synthesis of S1 and Compound 23:

[0077] Compound 22 (0.27 mmol), DCC (56 mg, 0.27 mmol), and DMAP (5 mg, 0.04 mmol) were dissolved in DCM (3 mL), and then RVX-208 (compound 13, 50 mg, 0.14 mmol) was added. The mixture was stirred at room temperature for 16 hours. After confirming that the reactants had basically reacted completely by TLC (evolving solvent: DCM:MeOH = 20:1), DCM (25 mL) was added to the system for extraction. The mixture was washed twice with water (50 mL × 2) and once with saturated brine (50 mL). The aqueous phase was discarded, and the organic phase was dried over anhydrous sodium sulfate. After filtration, the solvent was removed, and the mixture was separated by column chromatography (DCM:MeOH = 50:1 / 40:1) to obtain compound 23 in 95% yield.

[0078] Synthesis of S2 and compound 24:

[0079] Compound 23 was dissolved in a DCM:TFA = 1:1 (4 mL) mixed solution and stirred at room temperature for 2 hours. DCM was added to the system in small amounts several times, and TFA was continuously dried to obtain crude compound 24 with a yield of 90%. It was used directly in the next step of the reaction without purification.

[0080] Synthesis of S3 and Compound 25:

[0081] Compound 12 (43 mg, 0.13 mmol), HATU (74 mg, 0.19 mmol), and DIEA (67 mg, 0.52 mmol) were dissolved in DMF (2 mL), and then compound 24 (0.13 mmol) was added. The mixture was stirred at room temperature for 16 hours. After confirming that the reactants had basically reacted completely by TLC (developing solvent: DCM:MeOH = 20:1), water (25 mL) was added to quench the reaction. The mixture was extracted twice with ethyl acetate (25 mL × 2), and the organic phases were combined. The mixture was washed twice with water (50 mL × 2) and once with saturated brine (50 mL). The aqueous phase was discarded and dried over anhydrous sodium sulfate. After filtration, the solvent was removed and the mixture was separated by column chromatography (DCM:MeOH = 50:1 / 30:1) to obtain compound 25 in 60% yield.

[0082] Compound 25 is a colorless oil. 1H NMR(500MHz,Chloroform-d)δ9.00(s,1H),7.83(t,J=6.7Hz,1H),7.76(dd,J=7.5,1.5Hz,1H) ,7.46(t,J=7.5Hz,1H),7.37(s,2H),7.13(dd,J=7.5,1.5Hz,1H),6.55(s,2H),5.50(t,J=7.0H z,1H),4.58(s,2H),4.42-4.29(m,4H),3.87(s,3H),3.81(s,3H),3.20-3.06(m,2H),2.61(t, J=7.1Hz,2H),2.31(t,J=7.1Hz,2H),2.27-2.07(m,8H),1.63-1.40(m,4H),1.34-1.16(m,9H). 13 C NMR(125MHz,Chloroform-d)δ173.88,173.17,170.41,168.84,167.18,167.00,161.91, 161.50,161.04,156.73,154.84,153.98,150.73,131.33,129.34,128.84,126.74,124. 81,117.63,116.05,109.69,103.64,95.37,68.39,67.83,64.05,56.47,55.78,52.27,3 9.17,34.41,31.74,29.52,29.37,28.54,27.37,25.43,25.30,16.18.HRMS(ESI):Calcd for C 45 H 52 N5O 12 + [M+H] + :854.35342,C 45 H 51 N5O 12 Na + [M+Na] + :876.34319,Found:854.35423,876.34428.

[0083] Example 6: Preparation of protein hydrolysis chimeric compound 29

[0084] The synthetic route for the protein hydrolysis chimeric compound 29 is as follows:

[0085]

[0086] Specifically, the following steps are included:

[0087] Synthesis of S1 and Compound 27:

[0088] Compound 26 (0.27 mmol), DCC (56 mg, 0.27 mmol), and DMAP (5 mg, 0.04 mmol) were dissolved in DCM (3 mL), and then RVX-208 (compound 13, 50 mg, 0.14 mmol) was added. The mixture was stirred at room temperature for 16 hours. After confirming that the reactants had basically reacted completely by TLC (developing solvent: DCM:MeOH = 20:1), DCM (25 mL) was added to the system for extraction. The mixture was washed twice with water (50 mL × 2) and once with saturated brine (50 mL). The aqueous phase was discarded, and the organic phase was dried over anhydrous sodium sulfate. After filtration, the solvent was removed, and the mixture was separated by column chromatography (DCM:MeOH = 50:1 / 40:1) to obtain compound 27 in 95% yield.

[0089] Synthesis of S2 and compound 28:

[0090] Compound 27 was dissolved in a DCM:TFA = 1:1 (4 mL) mixed solution and stirred at room temperature for 2 hours. DCM was added to the system in small amounts several times, and TFA was continuously dried to obtain crude compound 28 with a yield of 90%. It was used directly in the next step of the reaction without purification.

[0091] Synthesis of S3 and Compound 29:

[0092] Compound 12 (43 mg, 0.13 mmol), HATU (74 mg, 0.19 mmol), and DIEA (67 mg, 0.52 mmol) were dissolved in DMF (2 mL), and then compound 28 (0.13 mmol) was added. The mixture was stirred at room temperature for 16 hours. After confirming that the reactants were basically completely reacted by TLC (developing solvent: DCM:MeOH = 20:1), water (25 mL) was added to quench the reaction. The mixture was extracted twice with ethyl acetate (25 mL × 2), and the organic phases were combined. The mixture was washed twice with water (50 mL × 2) and once with saturated brine (50 mL). The aqueous phase was discarded and dried over anhydrous sodium sulfate. After filtration, the solvent was removed and the mixture was separated by column chromatography (DCM:MeOH = 50:1 / 30:1) to obtain compound 29 in 60% yield.

[0093] Compound 29 is a colorless oil. 1H NMR(500MHz,Chloroform-d)δ9.00(s,1H),7.80(t,J=6.7Hz,1H),7.70(dd,J=7.5,1.5Hz,1H) ,7.46(t,J=7.4Hz,1H),7.37(s,2H),7.13(dd,J=7.5,1.5Hz,1H),6.55(s,2H),5.50(t,J=7.0H z,1H),4.58(s,2H),4.42-4.29(m,4H),3.87(s,3H),3.81(s,3H),3.20-3.06(m,2H),2.61(t,J =7.1Hz,2H),2.31(t,J=7.1Hz,2H),2.26-2.07(m,8H),1.63-1.40(m,4H),1.33-1.16(m,15H). 13 C NMR(125MHz,Chloroform-d)δ173.83,172.70,170.41,168.84,167.18,167.00,162.11, 161.91,161.04,156.73,154.84,154.15,151.29,131.54,129.34,128.84,126.74,124. 81,117.63,115.12,109.24,103.65,95.37,68.16,67.83,64.07,56.47,55.55,52.27,3 9.18,34.50,32.21,29.63,29.36,29.13,28.54,27.23,25.18,16.18.HRMS(ESI):Calcd for C 49 H 60 N5O 12 + [M+H] + :910.41602,C 49 H 59 N5O 12 Na + [M+Na] + :932.40579,Found:910.41726,932.40618.

[0094] Example 7: Preparation of protein hydrolysis chimeric compound 33

[0095] The synthetic route for the protein hydrolysis chimeric compound 33 is as follows:

[0096]

[0097] Specifically, the following steps are included:

[0098] Synthesis of S1 and Compound 31:

[0099] Compound 30 (0.27 mmol), DCC (56 mg, 0.27 mmol), and DMAP (5 mg, 0.04 mmol) were dissolved in DCM (2 mL), and then RVX-208 (compound 13, 50 mg, 0.14 mmol) was added. The mixture was stirred at room temperature for 16 hours. After confirming that the reactants had basically reacted completely by TLC (developing solvent: DCM:MeOH = 20:1), DCM (25 mL) was added to the system for extraction. The mixture was washed twice with water (50 mL × 2) and once with saturated brine (50 mL). The aqueous phase was discarded, and the organic phase was dried over anhydrous sodium sulfate. After filtration, the solvent was removed, and the mixture was separated by column chromatography (DCM:MeOH = 120:1 / 80:1) to obtain compound 31 in 95% yield.

[0100] Synthesis of S2 and compound 32:

[0101] Compound 31 was dissolved in a DCM:TFA = 1:1 (6 mL) mixed solution and stirred at room temperature for 2 hours. DCM was added to the system in small amounts several times, and TFA was continuously dried to obtain crude compound 32 with a yield of 90%. It was used directly in the next reaction without purification.

[0102] Synthesis of S3 and compound 33:

[0103] Compound 32 (0.12 mmol), HATU (66 mg, 0.17 mmol), and DIEA (60 mg, 0.46 mmol) were dissolved in DMF (2 mL), and then compound 9 (50 mg, 0.12 mmol) was added. The mixture was stirred at room temperature for 16 hours. TLC monitoring (developing solvent: DCM:MeOH = 20:1) confirmed that the reactants had largely reacted. Water (25 mL) was added to quench the reaction, and the mixture was extracted twice with ethyl acetate (25 mL × 2). The organic phases were combined, washed twice with water (50 mL × 2), and once with saturated brine (50 mL). The aqueous phase was discarded, and the mixture was dried over anhydrous sodium sulfate. After filtration, the solvent was removed, and the mixture was separated by column chromatography (DCM:MeOH = 40:1 / 30:1) to obtain compound 33 in 60% yield.

[0104] Compound 33 is a colorless oil. 1H NMR(500MHz,Chloroform-d)δ7.48-7.41(m,1H),7.39-7.31(m,2H),6.55(s,1H),4.46-4.21(m,4H),3.87(s,1H),3.86- 3.72(m,3H),3.67-3.45(m,3H),2.69-2.50(m,2H),2.46(s,1H),2.25(s,3H),2.11(td,J=7.0,5.0Hz,1H),0.97(s,4H). 13 C NMR(125MHz,Chloroform-d)δ172.42,172.35,171.45,164.15,162.17,161.06,154.88,1 54.77,154.17,145.10,144.00,138.85,138.37,136.94,129.07,128.65,126.93,126.76 ,108.75,103.28,95.38,70.59,69.38,67.83,67.20,66.83,64.03,60.13,59.38,56.47, 55.55,54.01,43.38,37.84,36.11,35.89,35.15,26.81,17.03,16.18.HRMS(ESI):Calcd for C 50 H 63 N6O 12 S + [M+H] + :971.41464,C 50 H 62 N6O 12 SNa + [M+Na] + :993.40441,Found:971.41517,993.40529.

[0105] Example 8: Preparation of protein hydrolysis chimeric compound 37

[0106] The synthetic route for the protein hydrolysis chimeric compound 37 is as follows:

[0107]

[0108] Specifically, the following steps are included:

[0109] Synthesis of S1 and Compound 35:

[0110] Compound 34 (0.27 mmol), DCC (56 mg, 0.27 mmol), and DMAP (5 mg, 0.04 mmol) were dissolved in DCM (2 mL), and then RVX-208 (compound 13, 50 mg, 0.14 mmol) was added. The mixture was stirred at room temperature for 16 hours. After confirming that the reactants had basically reacted completely by TLC (developing solvent: DCM:MeOH = 20:1), DCM (25 mL) was added to the system for extraction. The mixture was washed twice with water (50 mL × 2) and once with saturated brine (50 mL). The aqueous phase was discarded, and the organic phase was dried over anhydrous sodium sulfate. After filtration, the solvent was removed, and the mixture was separated by column chromatography (DCM:MeOH = 120:1 / 80:1) to obtain compound 35 in 95% yield.

[0111] Synthesis of S2 and compound 36:

[0112] Compound 35 was dissolved in a DCM:TFA = 1:1 (6 mL) mixed solution and stirred at room temperature for 2 hours. DCM was added to the system in small amounts several times, and TFA was continuously dried to obtain crude compound 36 with a yield of 90%. It was used directly in the next reaction without purification.

[0113] Synthesis of S3 and compound 37:

[0114] Compound 36 (0.12 mmol), HATU (66 mg, 0.17 mmol), and DIEA (60 mg, 0.46 mmol) were dissolved in DMF (2 mL), and then compound 9 (50 mg, 0.12 mmol) was added. The mixture was stirred at room temperature for 16 hours. After confirming that the reactants had basically reacted completely by TLC (developing solvent: DCM:MeOH = 20:1), water (25 mL) was added to quench the reaction. The mixture was extracted twice with ethyl acetate (25 mL × 2), and the organic phases were combined. The mixture was washed twice with water (50 mL × 2) and once with saturated brine (50 mL). The aqueous phase was discarded and dried over anhydrous sodium sulfate. After filtration, the solvent was removed, and the mixture was separated by column chromatography (DCM:MeOH = 40:1 / 30:1) to obtain compound 37 in 60% yield.

[0115] Compound 37 is a colorless oil. 1H NMR(500MHz,Chloroform-d)δ7.48-7.41(m,1H),7.39-7.31(m,2H),6.55(s,1H),4.45-4.23(m,4H),3.87(s,1 H),3.86-3.45(m,9H),2.69-2.50(m,2H),2.46(s,1H),2.25(s,3H),2.11(td,J=7.0,5.0Hz,1H),0.97(s,4H). 13 C NMR(125MHz,Chloroform-d)δ172.45,171.65,171.45,164.15,162.24,161.06,154.77,15 4.53,154.17,144.76,143.92,138.83,138.34,136.94,129.50,128.65,126.93,126.76,10 8.75,103.67,95.37,70.65,70.43,69.38,67.83,67.20,66.83,64.03,60.13,59.38,56.4 7,55.55,54.01,43.38,37.87,36.11,35.89,35.15,26.81,17.03,16.21.HRMS(ESI):Calcd forC 52 H 67 N6O 13 S + [M+H] + :1015.44086,C 52 H 66 N6O 13 SNa + [M+Na] + :1037.43063,Found:1015.44124,1037.43135.

[0116] Example 9: Preparation of protein hydrolysis chimeric compound 41

[0117] The synthetic route for the protein hydrolysis chimeric compound 41 is as follows:

[0118]

[0119] Specifically, the following steps are included:

[0120] Synthesis of S1 and compound 39:

[0121] Compound 38 (0.27 mmol), DCC (56 mg, 0.27 mmol), and DMAP (5 mg, 0.04 mmol) were dissolved in DCM (2 mL), and then RVX-208 (compound 13, 50 mg, 0.14 mmol) was added. The mixture was stirred at room temperature for 16 hours. After confirming that the reactants had basically reacted completely by TLC (developing solvent: DCM:MeOH = 20:1), DCM (25 mL) was added to the system for extraction. The mixture was washed twice with water (50 mL × 2) and once with saturated brine (50 mL). The aqueous phase was discarded, and the organic phase was dried over anhydrous sodium sulfate. After filtration, the solvent was removed, and the mixture was separated by column chromatography (DCM:MeOH = 120:1 / 80:1) to obtain compound 39 in 95% yield.

[0122] Synthesis of S2 and Compound 40:

[0123] Compound 39 was dissolved in a DCM:TFA = 1:1 (6 mL) mixed solution and stirred at room temperature for 2 hours. DCM was added to the system in small amounts several times, and TFA was continuously dried under vacuum to obtain crude compound 40 with a yield of 90%. It was used directly in the next step of the reaction without purification.

[0124] Synthesis of S3 and Compound 41:

[0125] Compound 40 (0.12 mmol), HATU (66 mg, 0.17 mmol), and DIEA (60 mg, 0.46 mmol) were dissolved in DMF (2 mL), and then compound 9 (50 mg, 0.12 mmol) was added. The mixture was stirred at room temperature for 16 hours. After confirming that the reactants had basically reacted completely by TLC (developing solvent: DCM:MeOH = 20:1), water (25 mL) was added to quench the reaction. The mixture was extracted twice with ethyl acetate (25 mL × 2), and the organic phases were combined. The mixture was washed twice with water (50 mL × 2) and once with saturated brine (50 mL). The aqueous phase was discarded and dried over anhydrous sodium sulfate. After filtration, the solvent was removed, and the mixture was separated by column chromatography (DCM:MeOH = 40:1 / 30:1) to obtain compound 41 in 60% yield.

[0126] Compound 41 is a colorless oil. 1H NMR(600MHz,Chloroform-d)δ8.70(s,1H),7.85(s,2H),7.37(s,4H),7.29(s,1H),7.25(s,1H),6.90(s,1H),6.45(s,1H),4.82(d,J=9.0Hz,1H),4.76(t,J=8.0Hz,1H),4.61(dd,J=14.9,6.7Hz,1H),4.56(s,1H),4.47(dq,J=12.4,7.7,6.2Hz,2H),4.34(dt,J=12.2,3.9Hz,2H),4.05(s,2H),3.95(d,J=8.1Hz,6H),3.58(dd,J=11.3,3.2Hz,1H),2.53(s,4H),2.41(t,J=6.8Hz,2H),2.38(s,6H),2.22(dd,J=13.4,8.0Hz,2H),2.11(s,2H),2.07(s,1H),1.71-1.65(m,3H),1.41(q,J=6.8,6.0Hz,2H),1.35(s,3H),1.30(s,9H),1.23(d,J=7.1Hz,3H),1.18(d,J=6.7Hz,3H),0.95(s,10H),0.90(td,J=7.1,1.9Hz,1H). 13 CNMR(151MHz,Chloroform-d)δ173.83,173.47,170.99,150.32,138.13,131.65,129.54,129.50,128.12,98.22,69.99,69.85,63.30,58.85,57.22,56.88,56.06,55.83,43.27,37.07,35.90,35.70,34.49,33.93,31.44,30.20,29.70,29.48,29.14,29.05,28.70,26.47,25.86,25.61,25.03,24.94,16.44,16.03.HRMS(ESI):Calcd for C 53 H 69 N6O 10 S + [M+H] + :981.47904,C 53 H 68 N6O 10 SNa + [M+Na] +:1003.46098,Found:981.47711,1003.45892.

[0127] Example 10: Preparation of protein hydrolysis chimeric compound 45

[0128] The synthetic route for the protein hydrolysis chimeric compound 45 is as follows:

[0129]

[0130] Specifically, the following steps are included:

[0131] Synthesis of S1 and compound 43:

[0132] Compound 42 (0.27 mmol), DCC (56 mg, 0.27 mmol), and DMAP (5 mg, 0.04 mmol) were dissolved in DCM (2 mL), and then RVX-208 (compound 13, 50 mg, 0.14 mmol) was added. The mixture was stirred at room temperature for 16 hours. After confirming that the reactants had basically reacted completely by TLC (developing solvent: DCM:MeOH = 20:1), DCM (25 mL) was added to the system for extraction. The mixture was washed twice with water (50 mL × 2) and once with saturated brine (50 mL). The aqueous phase was discarded, and the organic phase was dried over anhydrous sodium sulfate. After filtration, the solvent was removed, and the mixture was separated by column chromatography (DCM:MeOH = 120:1 / 80:1) to obtain compound 43 in 95% yield.

[0133] Synthesis of S2 and compound 44:

[0134] Compound 43 was dissolved in a DCM:TFA = 1:1 (6 mL) mixed solution and stirred at room temperature for 2 hours. DCM was added to the system in small amounts several times, and TFA was continuously dried under vacuum to obtain crude compound 44 with a yield of 90%. It was used directly in the next reaction without purification.

[0135] Synthesis of S3 and Compound 45:

[0136] Compound 44 (0.12 mmol), HATU (66 mg, 0.17 mmol), and DIEA (60 mg, 0.46 mmol) were dissolved in DMF (2 mL), and then compound 9 (50 mg, 0.12 mmol) was added. The mixture was stirred at room temperature for 16 hours. After confirming that the reactants had basically reacted completely by TLC (developing solvent: DCM:MeOH = 20:1), water (25 mL) was added to quench the reaction. The mixture was extracted twice with ethyl acetate (25 mL × 2), and the organic phases were combined. The mixture was washed twice with water (50 mL × 2) and once with saturated brine (50 mL). The aqueous phase was discarded and dried over anhydrous sodium sulfate. After filtration, the solvent was removed, and the mixture was separated by column chromatography (DCM:MeOH = 40:1 / 30:1) to obtain compound 45 in 60% yield.

[0137] Compound 45 is a colorless oil. 1 H NMR(600MHz,Chloroform-d)δ8.69(s,1H),7.82(s,2H),7.38-7.28(m,6H),6.85(s,1H),6.47-6.39(m,2H),4.72 (t,J=8.0Hz,1H),4.68(d,J=9.0Hz,1H),4.59-4.52(m,2H),4.46-4.43(m,2H),4.33(dd,J=15.0,5.2Hz,1H),4.19 (d,J=11.2Hz,1H),4.05-4.02(m,2H),3.94(d,J=9.5Hz,6H),3.61(dd,J=11.1,3.1Hz,1H),2.51(s,4H),2.39(d,J =14.1Hz,9H),2.17(t,J=7.5Hz,4H),1.71-1.62(m,3H),1.52(d,J=7.2Hz,2H),1.39-1.17(m,30H),0.95(s,10H). 13C NMR(151MHz,Chloroform-d)δ173.81,171.81,170.98,161.25,150.35,138.12,131.71 ,131.63,129.47,128.24,128.07,104.84,98.18,69.99,63.39,58.75,57.32,56.83,5 6.18,55.76,43.22,36.59,35.94,35.43,34.25,30.20,29.70,29.36,29.32,29.28,29 .22,29.18,29.12,28.94,28.92,26.44,25.67,24.82,16.38,16.04.HRMS(ESI):Calcd for C 57 H 77 N6O 10 S + [M+H] + :1037.54164,C 57 H 76 N6O 10 SNa + [M+Na] + :1059.52358,Found:1037.53955,1059.52112.

[0138] Application Example: Determination of the intracellular degradation effect of a proteolytic chimera targeting bromodomain protein 4.

[0139] 1. Experimental materials:

[0140] Protein hydrolysate chimeric compound solution: The synthesized protein hydrolysate chimeric compounds 17, 21, 25, 29, 33, 37, 41, and 45 (purity verified by HPLC) were dissolved in DMSO at 1000 times the working concentration to prepare the corresponding stock solutions. They were stored for a short period at 4°C in the dark and diluted to the working concentration in the culture system before use.

[0141] The same method was used to prepare a solution of compound BRD4-1.

[0142]

[0143] Cell culture: HeLa cells (human cervical cancer cells) were seeded in DMEM complete medium (containing 10% fetal bovine serum, 100 U / mL penicillin and 0.1 mg / mL streptomycin), cultured at 37°C under a 5% CO2 atmosphere, and passaged according to standard methods.

[0144] 2. Experimental methods:

[0145] Protein level determination: Intracellular bromodomain protein 4 (BGP) levels were determined using Western blotting under various conditions. After drug treatment, cells were collected, added with SDS lysis buffer (containing a mixture of protease inhibitors), and lysed on ice for 20 min, followed by sonication. After complete lysis, the cells were centrifuged at 12000 RCF for 10 min at 4 °C. The supernatant was collected, and protein concentration was determined using the BCA method. 5× loading buffer was added, followed by denaturation at 4 °C for 10 min. The harvested protein samples were loaded onto SDS-PAGE gels, separated by electrophoresis using standard methods, transferred to PVDF membranes for immunoassay, and finally developed using chemiluminescence. ImageJ software was used to quantify the developed images. Using GAPDH as an internal control, the intracellular BGP levels of BGP in each group were calculated, and the protein degradation under corresponding conditions was assessed based on these values.

[0146] 3. Experimental Results

[0147] This invention evaluated the degradation effects of eight compounds targeting and degrading bromodomain protein 4 on intracellular bromodomain protein 4. The degradation rate (D) of intracellular bromodomain protein 4 after treatment with 10 μM compounds for 24 h was used as the indicator to evaluate the degradation effect of the compounds on bromodomain protein 4. D can be calculated using the following formula:

[0148]

[0149] The DMSO treatment group served as a negative control, and the protein background level refers to the expression level of bromodomain protein 4 in HeLa cells without any treatment (including PFF seeding, transfection, and compound treatment).

[0150] See the statistical chart of the immunoblot results obtained from the test. Figure 1 The calculated degradation rate D is listed in Table 1.

[0151] Table 1. Degradation effects of compounds on intracellular bromodomain protein 4

[0152] 17(PR-2) 49.63±5.72 21(PR-1) 56.45±0.76 25 35.51±4.77 29 28.08±0.72 33 43.13±3.70 37 28.67±0.28 41(PR-3) 73.58±2.57 45(PR-4) 54.69±2.62 BRD4-1 51.36±5.37

[0153] The results show that after treatment with 10 μM of the proteolytic chimera targeting bromodomain protein 4, the intracellular levels of bromodomain protein 4 aggregates decreased significantly. Among them, compound 41 showed the best degradation effect, with a degradation rate of 73.58 ± 2.57% of bromodomain protein 4 aggregates at a concentration of 10 μM.

[0154] In summary, this invention provides a class of proteolytic chimeric compounds targeting bromodomain protein 4, and the degradation effect of these compounds on intracellular bromodomain protein 4 was evaluated by Western blotting. The results consistently show that these compounds can effectively degrade intracellular bromodomain protein 4 at low concentrations (10 μM) and reduce its associated cytotoxicity.

[0155] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A class of proteolytic chimeras targeting bromodomain protein 4, characterized in that, The proteolytic chimera targeting bromodomain protein 4 has the structure of formula (I): （I） Where X is -CO-(CH2) a -or -CO-(CH2-CH2-O) b -CH2-CH2-, where a is an integer from 9 to 13 and b is an integer from 1 to 5; R is selected from any of the following structures: 、 。 2. The proteolytic chimera targeting bromine domain protein 4 according to claim 1, characterized in that, X is -CO-(CH2) a -or -CO-(CH2-CH2-O) b -CH2-CH2-, where a is an integer from 9 to 13 and b is an integer from 1 to 3.

3. The proteolytic chimera targeting bromine domain protein 4 according to claim 2, characterized in that, X is -CO-(CH2) a -or -CO-(CH2-CH2-O) b -CH2-CH2-, a is 9 or 13, b is 2 or 3.

4. The proteolytic chimera targeting bromodomain protein 4 according to claim 3, characterized in that, The proteolytic chimera targeting bromodomain protein 4 has any of the following structures: 。 5. The proteolytic chimera targeting bromodomain protein 4 according to any one of claims 1 to 4, characterized in that, It also includes its pharmaceutically acceptable salts.

6. The use of the proteolytic chimera targeting bromodomain protein 4 as described in any one of claims 1 to 5 in the preparation of a medicament for treating diseases related to bromodomain protein 4.

7. The application according to claim 6, characterized in that, The diseases associated with the bromodomain protein 4 include acute myeloid leukemia, multiple myeloma, breast cancer, glioblastoma, or renal cell carcinoma.

8. A drug for treating diseases related to bromodomain protein 4, characterized in that, The protein hydrolysis chimera includes any of the target bromine domain protein 4 as described in claims 1 to 5.

9. The drug according to claim 8, characterized in that, The drug also includes pharmaceutically acceptable excipients selected from one or more of diluents, lubricants, binders, disintegrants, surfactants, and film-forming materials.

10. The drug according to claim 8, characterized in that, The drug also includes pharmaceutically acceptable excipients selected from one or more of diluents, lubricants, binders, disintegrants, surfactants, and coating materials.

11. The drug according to claim 8, characterized in that, The drug also includes pharmaceutically acceptable excipients selected from one or more of diluents, lubricants, binders, disintegrants, surfactants, and capsule materials.

12. The drug according to claim 8, characterized in that, The dosage form of the drug is an injectable preparation, an oral preparation, a nebulized inhalation preparation, or a transdermal preparation.

Images