Heterobifunctional compound for targeted degradation of BRD4 based on HSP70, preparation, and use

Abstract

Provided in the present invention are a heterobifunctional compound for targeted degradation of BRD4 based on HSP70, preparation, and use, belonging to the technical fields of pharmaceutical synthesis and chemical engineering. In the present application, an HSP70 inhibitor is selected as a ligand moiety A that binds to HSP70, a BRD4 inhibitor is selected as a ligand moiety B that binds to BRD4, and the ligand A of HSP70 and the ligand B of BRD4 are linked by means of a linking chain, Linker, to obtain a series of bifunctional degradation agents that can degrade BRD4 to different degrees. The obtained degradation agents have the characteristics of high selectivity and strong activity, and can overcome the disadvantages of BRD4 inhibitors, such as poor selectivity for tumor tissues and strong toxic and side effects.

Description

Heterobifunctional compounds based on HSP70-targeted degradation of BRD4, their preparation and application Technical Field

[0001] This invention belongs to the field of pharmaceutical synthesis and chemical technology, specifically involving a class of heterobifunctional compounds based on HSP70-targeted degradation of BRD4, and discloses specific preparation methods and related applications. Background Technology

[0002] Molecular chaperones, also known as companion proteins, are key regulators of protein homeostasis. They are primarily composed of the heat shock protein (HSP) family and its cochaperones. Molecular chaperones mainly participate in protein folding, maturation, and protein degradation mediated by the ubiquitin-proteasome system, ultimately regulating cell proliferation and apoptosis. Dysfunction of the molecular chaperone system is highly correlated with the occurrence and development of diseases such as cancer, autoimmune diseases, inflammation, infectious diseases, and neurodegenerative diseases, making them potential targets for drug development. The heat shock protein (HSP) family mainly includes HSP60, HSP70, HSP90, and HSP100. HSP70 guides protein unfolding, breakdown, refolding, or degradation; HSP90 acts in the late folding stage, recognizing partially folded proteins and assisting their maturation or degradation via the proteasome; both HSP70 and HSP90 can recognize misfolded proteins and guide their degradation via the ubiquitin-proteasome system (UPS). However, compared to HSP90, HSP70 contains more substrate proteins, including kinases (such as AKT), transcription factors, steroid hormone receptors, and E3 ubiquitin ligases. HSP70 not only assists in the folding of many substrate proteins and mutant oncogenic proteins, but also guides the degradation of its substrate proteins by the proteasome system. In tumor tissues, the HSP70 complex is highly activated compared to normal tissues, and small molecule compounds binding to HSP70 exhibit unique tumor selectivity.

[0003] BRD4, or Bromodomain Protein 4, is a member of the bromodomain and C-terminal extradomain (BET) protein family. This family consists of BRD2, BRD3, BRD4, and BRDT, all of which bind to acetylated lysine residues on histone tails and other proteins. As epigenetic markers, acetylated lysine residues, once bound to BET family proteins, lead to the recruitment of RNA polymerases and other transcription-related proteins to these sites, thereby regulating transcription initiation and elongation. BRD4 proteins are associated with large protein complexes regulating gene transcription, including mediators, PAFc, and superelongation complexes. The kinase activity of BRD4 can directly phosphorylate and activate RNA polymerase II, thereby regulating gene transcriptional expression. Many human diseases are closely related to BRD4 proteins, such as tumors, autoimmune or inflammatory diseases, and viral infections. Inhibitors and degraders targeting BRD4 have significant application value in anticancer and anti-inflammatory drugs and other fields, and have long been a focus of attention for major pharmaceutical companies and research institutions.

[0004] Although BRD4 inhibitors have shown good efficacy against various disease types, especially hematologic malignancies, they still have some limitations. Because these drugs reversibly bind to target proteins based on occupation-driven pharmacology, high doses are often required to achieve therapeutic effects. Clinical trials have shown dose-limiting toxicities such as thrombocytopenia, fatigue, diarrhea, vomiting, anemia, and hyperbilirubinemia. Furthermore, pan-BET inhibitors, due to their lack of selectivity for multiple bromine domains, can lead to off-target effects and serious safety issues.

[0005] The continuous development of PROTAC technology has provided new ideas for the application of BRD4 inhibitors. BRD4 ligand, E3 ubiquitin ligase ligand, and intermediate linker chain constitute the basic structure of PROTAC-based BRD4 degraders. These degraders can be divided into two categories based on the type of E3 ubiquitin ligase: CRBN-based BRD4 degraders and VHL-based BRD4 degraders. Although these two types of degraders based on PROTAC technology have good efficacy, some problems exist. For example, both are non-tissue-specific E3 ligases, which may lead to systemic off-target effects and toxic side effects; both are non-essential proteins for tumor growth, and small molecule PROTACs based on them are prone to drug resistance. Understanding new E3 enzymes and developing corresponding PROTACs is of great significance, but also faces significant challenges.

[0006] PROTAC technology and molecular glue both belong to the targeted protein degradation (TPD) technology. Compared with PROTAC, molecular glue has a smaller molecular weight and is more like the small molecule drugs currently used. However, the discovery of molecular glue is more accidental, and only a small number of protein-protein interactions mediated by molecular glue have been reported so far.

[0007] To address the aforementioned issues, Ranok Biotech has developed a novel targeted protein degradation technology: chaperone-mediated protein degradation (CHAMP). Compared to other targeted protein degradation pathways, CHAMPs offer several advantages. For instance, they utilize HSP90 / HSP70 to recruit E3 ubiquitin ligases for ubiquitination and degradation, potentially helping to overcome resistance caused by E3 ubiquitin ligase mutations in PROTAC. Furthermore, their selective accumulation in tumor tissues promises improved safety. Therefore, CHAMPs show great promise as a novel targeted protein degradation technology for drug development. The company has previously developed a heterobifunctional small molecule that simultaneously targets BRD4 and HSP90 using the properties of HSP90, and their selective protein degrader for BRD4 developed using CHAMP technology has already entered Phase II clinical trials.

[0008] However, no heterobifunctional small molecule capable of simultaneously targeting BRD4 and HSP70 has yet been discovered in current technologies. Based on the CHAMPs design concept and the function of HSP70 in ubiquitinizing and degrading substrate proteins, the design and synthesis of an HSP70-BRD4 bifunctional molecule could potentially provide new chemical tools and drug discovery strategies for the field of molecular targeted therapy. Summary of the Invention

[0009] The purpose of this invention is to provide a heterobifunctional compound based on HSP70-targeted degradation of BRD4. When a BRD4 inhibitor and an HSP70 ligand form a compound through a linker chain, the HSP70 ligand can be used to target tumor cells, thereby significantly improving the inhibitory effect of the BRD4 inhibitor on the target protein in tumor cells, and thus solving the problems of high toxicity and low selectivity of existing BRD4 inhibitors.

[0010] The specific technical solution of this invention is as follows: a class of heterobifunctional compounds based on HSP70-targeted degradation of BRD4 or their pharmaceutically acceptable salts, wherein the heterobifunctional compound comprises a ligand portion A that binds to HSP70, a ligand portion B that binds to BRD4, and a linker connecting the two, and its structure is as follows:

[0011] A-Linker-B.

[0012] Preferably, the structure of the heterobifunctional compound is shown in formula (I):

[0013] Furthermore, the linker includes: carbon-carbon single bonds, carbon-carbon double bonds, carbon-carbon triple bonds, aryl bonds, heteroaryl bonds, cycloalkyl bonds, cycloalkenyl bonds, heterocyclic bonds, fused bicyclic bonds, fused heterobicyclic bonds, spirobicyclic bonds, spiroheterocyclic bonds, carbon-nitrogen bonds, carbon-sulfur bonds, carbon-phosphorus bonds, nitrogen-nitrogen bonds, carbonyl (-C(=O)-), C1-6 alkylene groups, amide bonds, ether bonds, disulfide bonds, piperazine bonds, biphenyl diamine bonds, phenylpiperazine bonds, carboxylic acid ester bonds, sulfonate bonds, carbamate bonds, and combinations thereof.

[0014] As a preferred option, the Linker's structure is as follows:

[0015] Where n is any natural number between 2 and 12, and m is any natural number between 1 and 3.

[0016] Preferably, the heterobifunctional compounds based on HSP70-targeted degradation of BRD4 include compounds with structures shown in formulas AB-01 to 19:

[0017] In one embodiment, this application relates to a pharmaceutical composition comprising the aforementioned heterobifunctional compound based on HSP70-targeted degradation of BRD4 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0018] Furthermore, the pharmaceutical composition may be formulated as an injectable fluid, aerosol, cream, gel, pill, capsule, syrup, transdermal patch, or excipient.

[0019] Furthermore, the pharmaceutical composition also includes other therapeutic agents. The pharmaceutical composition disclosed in this application can be used in combination with other therapeutic agents, and can be formulated into the same dosage form or into separate dosage forms with other therapeutic agents. The other therapeutic agents are tumor-targeting drugs, tumor chemotherapy drugs, tumor immunotherapy drugs, and tumor drug conjugates.

[0020] The aforementioned small molecule drug conjugates or their pharmaceutically acceptable salts, or pharmaceutical compositions, can be used to prepare drugs for the prevention or treatment of tumors. These tumors include, but are not limited to, gastric cancer, ovarian cancer, cervical cancer, esophageal cancer, lymphoma, glioma, colon cancer, colorectal cancer, prostate cancer, liver cancer, non-small cell lung cancer, breast cancer, epithelial cell carcinoma, multiple myeloma, and leukemia.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] 1. In this application, the HSP70 inhibitor Apoptozole is selected as the ligand that binds to HSP70, and the BRD4 inhibitor (+)-JQ-1 is selected as the ligand that binds to BRD4. The two are linked by a linker chain to obtain a series of bifunctional degraders that can degrade BRD4 to different degrees.

[0023] 2. The HSP70-BRD4 heterobifunctional compounds provided in this application can degrade BRD4 protein in leukemia MV-4-11 cells to varying degrees. Among them, compound AB-13 has the best degradation activity, with a degradation rate of 90% when 30 nM is administered.

[0024] 3. This application proposes a novel method for targeting protein degradation, providing new chemical tools and drug discovery strategies for the field of molecular targeted therapy. Detailed Implementation

[0025] The technical solution of the present invention will be further described below with reference to the accompanying drawings, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention that do not depart from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention.

[0026] I. Synthesis of Hsp70 ligand (4-((2-(3,5-bis(trifluoromethyl)phenyl)-4,5-bis(4-methoxyphenyl)-1H-imidazol-1-yl)methyl)benzoic acid):

[0027] 4-Aminomethylbenzoic acid (19.8 mmol), 3,5-bis(trifluoromethyl)benzaldehyde (25.8 mmol), 4,4'-dimethoxyphenol ester (25.8 mmol), and ammonium acetate (119 mmol) were added to a two-necked round-bottom flask. A reflux condenser was installed on the main neck of the flask, and a rubber stopper was placed on the side neck. The flask was then protected with nitrogen. Glacial acetic acid (60 mL) was added through the rubber stopper on the side neck using a syringe. The mixture was heated to 100 °C and stirred for 12 h. After cooling, the reaction mixture was transferred to a separating funnel, and 200 mL of ethyl acetate was added. The mixture was washed three times each with 100 mL of distilled water and saturated sodium bicarbonate solution. After standing, a solid precipitated. The solid was filtered through a Buchner funnel and dried to give compound a (6.53 g, 53% yield) as a white solid. 1H NMR (300MHz, DMSO) δ12.94(s,1H),8.19(s,2H),8.12(s,1H),7.80(d,J=8.2Hz,2H),7.43(d,J=8.9Hz,2H),7.2 9(d,J=8.7Hz,2H),7.02(dd,J=8.6,2.2Hz,4H),6.84(d,J=8.9Hz,2H),5.26(s,2H),3.78(s,3H),3.71(s,3H).

[0028] II. Synthesis of Hsp70 binding ligands and linker chains

[0029] 1) Synthesis of Compound 1:

[0030] Step 1: Synthesis of tert-butyl (2-(4-((2-(3,5-bis(trifluoromethyl)phenyl)-4,5-bis(4-methoxyphenyl)-1H-imidazol-1-yl)methyl)benzamido)ethyl)carbamate:

[0031] Compound a (0.479 mmol) was dissolved in anhydrous dichloromethane (DCM, 10 mL), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 0.958 mmol), 1-hydroxybenzotriazole (HOBT, 0.958 mmol), and anhydrous triethylamine (Et3N, 1.912 mmol) were added. The mixture was stirred at room temperature for 30 min, and then N-tert-butoxycarbonyl-1,2-ethylenediamine (0.719 mmol) was added, and the mixture was stirred at room temperature for 3 h. The reaction solution was washed once with 30 mL of distilled water and once with saturated sodium chloride solution. The organic phase was concentrated under reduced pressure and purified by silica gel chromatography (DCM / MeOH = 150:1 elution) to give intermediate compound b (297 mg, yield 81%) as a white solid.

[0032] 1 H NMR (300MHz, DMSO) δ8.40(t,J=5.5Hz,1H),8.23(s,2H),8.13(s,1H),7.72(d,J=8.0Hz,2H),7.45-7.41(m,2H),7.30-7.26(m,2H),7.03-6.96( m,4H),6.89(d,J=5.7Hz,1H),6.86-6.82(m,2H),5.26(s,2H),3.78(s,3 H),3.71(s,3H),3.24(q,J=6.2Hz,2H),3.10-3.03(m,2H),1.34(s,9H).

[0033] Step 2: Synthesis of Compound 1

[0034] Add 5 mL of hydrogen chloride-ethyl acetate solution (2 mol / L) to compound b (0.387 mmol), stir at room temperature for 1 h, concentrate the mixture under reduced pressure to give the hydrochloride salt of compound 1 (268 mg, yield 99%) as a pale yellow solid, which does not require further purification for the next step.

[0035] 2) Synthesis of compound 2:

[0036] The synthesis method is the same as that of compound 1, except that N-tert-butoxycarbonyl-1,3-propanediamine is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine.

[0037] Intermediate compound c (297 mg, yield 79%) was obtained as a white solid. 1 H NMR(300MHz,DMSO)δ8.37(t,J=5.6Hz,1H),8.23(s,2H),8.13(s,1H),7.72( d,J=8.0Hz,2H),7.47-7.40(m,2H),7.34-7.26(m,2H),7.06-6.95(m,4H),6 .84(d,J=8.8Hz,2H),6.79(s,1H),5.26(s,2H),3.79(s,3H),3.72(s,3H),3 .21(q,J=6.6Hz,2H),2.94(d,J=6.4Hz,2H),1.65-1.53(m,2H),1.36(s,9H).

[0038] The hydrochloride salt of compound 2 was obtained (264 mg, 77% yield) as a pale yellow solid, which did not require further purification for the next step.

[0039] 3) Synthesis of compound 3:

[0040] The synthesis method is the same as that of compound 1, except that N-tert-butoxycarbonyl-1,4-butanediamine is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine.

[0041] Intermediate compound d (286 mg, yield 75%) was obtained as a white solid. 1H NMR (300MHz, DMSO) δ8.42(t,J=5.6Hz,1H),8.22(d,J=1.7Hz,2H),8.14(s,1 H),7.76-7.68(m,2H),7.46-7.39(m,2H),7.32-7.25(m,2H),7.04-6.94(m,4 H),6.83(dd,J=7.4,5.3Hz,3H),5.25(s,2H),3.78(s,3H),3.71(s,3H),3.19 (d,J=6.2Hz,2H),2.90(d,J=6.5Hz,2H),1.41(d,J=5.8Hz,4H),1.35(s,9H).

[0042] The hydrochloride salt of compound 3 was obtained (256 mg, yield 74%) as a pale yellow solid, which did not require further purification for the next step.

[0043] 4) Synthesis of compound 4:

[0044] The synthesis method is the same as that of compound 1, except that N-tert-butoxycarbonyl-1,2-ethylenediamine is replaced with N-(5-aminopentyl)carbamate tert-butyl ester.

[0045] Intermediate compound e (302 mg, yield 78%) was obtained as a white solid. 1 H NMR(300MHz,DMSO)δ8.37(s,1H),8.23(s,2H),8.13(s,1H),7.72(d,J=7.9Hz,2H) ,7.47-7.40(m,2H),7.29(d,J=8.2Hz,2H),6.99(dd,J=17.6,8.2Hz,4H),6.87-6. 81(m,2H),6.75(s,1H),5.25(s,2H),3.79(s,3H),3.72(s,3H),3.19(d,J=6.4Hz, 2H),2.88(d,J=6.4Hz,2H),1.47(t,J=7.3Hz,2H),1.34(s,9H),1.28-1.14(m,4H).

[0046] The hydrochloride salt of compound 4 was obtained (271 mg, 97% yield) as a pale yellow solid, which did not require further purification for the next step.

[0047] 5) Synthesis of compound 5:

[0048] The synthesis method is the same as that of compound 1, except that N-tert-butoxycarbonyl-1,6-hexanediamine is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine.

[0049] Intermediate compound f (308 mg, yield 78%) was obtained as a white solid. 1 H NMR(300MHz,DMSO)δ8.38(s,1H),8.22(s,2H),8.13(s,1H),7.71(d,J=7.9Hz,2 H),7.43(d,J=8.4Hz,2H),7.29(d,J=8.2Hz,2H),6.99(dd,J=17.0,8.1Hz,4H),6 .84(d,J=8.5Hz,2H),6.77(s,1H),5.25(s,2H),3.78(s,3H),3.71(s,3H),3.19( d,J=6.6Hz,2H),2.87(d,J=6.7Hz,2H),1.46(s,2H),1.35(s,12H),1.24(s,4H).

[0050] The hydrochloride salt of compound 5 was obtained (279 mg, 99% yield) as a pale yellow solid, which did not require further purification for the next step.

[0051] 6) Synthesis of compound 6:

[0052] The synthesis method is the same as that of compound 1, except that compound N-tert-butoxycarbonyl-1,7-diaminoheptane is replaced with compound 1-tert-butoxycarbonyl-1,2-ethylenediamine.

[0053] The intermediate compound g (293 mg, yield 73%) was obtained as a white solid. 1 H NMR (300MHz, DMSO) δ8.37(t,J=5.6Hz,1H),8.22(s,2H),8.12(s,1H),7.71(d,J=8.0H z,2H),7.47-7.39(m,2H),7.29(d,J=8.2Hz,2H),6.99(dd,J=17.0,8.1Hz,4H),6.84(d d,J=9.1,2.8Hz,2H),6.74(s,1H),5.25(s,2H),3.78(s,3H),3.71(s,3H),3.19(d,J=6 .5Hz,2H),2.87(d,J=6.4Hz,2H),1.46(s,2H),1.36(s,2H),1.35(s,9H),1.24(s,6H).

[0054] The hydrochloride salt of compound 6 was obtained (263 mg, 98% yield) as a pale yellow solid, which did not require further purification for the next step.

[0055] 7) Synthesis of compound 7:

[0056] The synthesis method is the same as that of compound 1, except that compound 1-tert-butoxycarbonyl-1,8-diaminooctane is used instead of compound N-tert-butoxycarbonyl-1,2-ethylenediamine.

[0057] Intermediate compound h (303 mg, yield 75%) was obtained as a white solid. 1 H NMR(300MHz,DMSO)δ8.36(d,J=6.2Hz,1H),8.22(s,2H),8.12(s,1H),7.71(d,J=7 .9Hz,2H),7.47-7.39(m,2H),7.29(d,J=8.5Hz,2H),7.05-6.94(m,4H),6.87-6.80 (m,2H),6.74(s,1H),5.25(s,2H),3.78(s,3H),3.71(s,3H),3.19(d,J=6.6Hz,2H) ,2.87(q,J=6.5Hz,2H),1.46(s,2H),1.35(s,9H),1.30(s,2H),1.27-1.19(m,8H).

[0058] The hydrochloride salt of compound 7 was obtained (278 mg, 99% yield) as a pale yellow solid, which did not require further purification for the next step.

[0059] 8) Synthesis of compound 8:

[0060] The synthesis method is the same as that of compound 1, except that compound 1-tert-butoxycarbonyl-1,9-diaminononane is used instead of compound N-tert-butoxycarbonyl-1,2-ethylenediamine.

[0061] Intermediate compound i (328 mg, yield 80%) was obtained as a white solid. 1H NMR (300MHz, DMSO) δ8.38(t,J=5.7Hz,1H),8.22(s,2H),8.13(s,1H),7.72(d,J=7.9H z,2H),7.47-7.39(m,2H),7.34-7.27(m,2H),7.00(dd,J=16.2,8.3Hz,4H),6.84(d,J =8.8Hz,2H),6.77(d,J=5.7Hz,1H),5.25(s,2H),3.79(s,3H),3.72(s,3H),3.19(q,J =6.5Hz,2H),2.87(q,J=6.5Hz,2H),1.47(s,2H),1.36(s,9H),1.24(d,J=7.1Hz,12H).

[0062] The hydrochloride salt of compound 8 was obtained (293 mg, 96% yield) as a pale yellow solid, which did not require further purification for the next step.

[0063] 9) Synthesis of compound 9:

[0064] The synthesis method is the same as that of compound 1, except that compound 1-tert-butoxycarbonyl-1,10-diaminodecane is used instead of compound N-tert-butoxycarbonyl-1,2-ethylenediamine.

[0065] Intermediate compound j (312 mg, yield 72%) was obtained as a white solid. 1 H NMR (300MHz, DMSO) δ8.38(t,J=5.5Hz,1H),8.21(s,2H),8.13(s,1H),7.71(d,J=8.1 Hz,2H),7.43(d,J=8.8Hz,2H),7.29(d,J=8.5Hz,2H),6.99(dd,J=16.7,8.3Hz,4H),6 .84(d,J=8.7Hz,2H),6.75(s,1H),5.24(s,2H),3.78(s,3H),3.71(s,3H),3.19(d,J= 6.5Hz,2H),2.86(q,J=6.5Hz,2H),1.46(s,2H),1.35(s,9H),1.23(d,J=7.5Hz,14H).

[0066] The hydrochloride salt of compound 9 was obtained (276 mg, 97% yield) as a pale yellow solid, which did not require further purification for the next step.

[0067] 10) Synthesis of compound 10:

[0068] The synthesis method is the same as that of compound 1, except that N-tert-butoxycarbonyl-1,2-ethylenediamine is replaced with (11-aminoundecyl)carbamate tert-butyl ester.

[0069] Intermediate compound k (317 mg, 70% yield) was obtained as a white solid. 1 H NMR(300MHz,DMSO)δ8.37(d,J=6.2Hz,1H),8.21(s,2H),8.13(s,1H),7.71(d,J=8.0 Hz,2H),7.43(d,J=8.4Hz,2H),7.30(d,J=8.2Hz,2H),7.00(dd,J=16.6,8.1Hz,4H),6 .84(d,J=8.5Hz,2H),6.76(s,1H),5.25(s,2H),3.78(s,3H),3.72(s,3H),3.19(d,J= 6.6Hz,2H),2.87(d,J=6.5Hz,2H),1.46(s,2H),1.36(s,9H),1.23(d,J=7.5Hz,16H).

[0070] The hydrochloride salt of compound 10 was obtained (280 mg, 96% yield) as a pale yellow solid, which did not require further purification for the next step.

[0071] 11) Synthesis of compound 11:

[0072] The synthesis method is the same as that of compound 1, except that compound 1-tert-butoxycarbonyl-1,12-diaminododecane is used instead of compound N-tert-butoxycarbonyl-1,2-ethylenediamine.

[0073] Intermediate compound 1 (335 mg, yield 77%) was obtained as a white solid. 1 H NMR (300MHz, DMSO) δ8.37(t,J=5.5Hz,1H),8.21(s,2H),8.12(s,1H),7.71(d,J=8.0Hz, 2H),7.43(d,J=8.5Hz,2H),7.29(d,J=8.3Hz,2H),6.99(dd,J=16.2,8.2Hz,4H),6.84(d, J=8.5Hz,2H),6.74(s,1H),5.24(s,2H),3.78(s,3H),3.71(s,3H),3.19(q,J=6.4Hz,2H ),2.86(q,J=6.6Hz,2H),1.46(s,2H),1.35(s,9H),1.31(s,2H),1.23(d,J=8.7Hz,16H).

[0074] The hydrochloride salt of compound 11 was obtained (303 mg, 98% yield) as a pale yellow solid, which did not require further purification for the next step.

[0075] 12) Synthesis of compound 12:

[0076] The synthesis method is the same as that of compound 1, except that compound N-tert-butoxycarbonyl-1,2-ethylenediamine is replaced with compound [2-(2-aminoethoxy)ethyl]carbamate tert-butyl ester.

[0077] Intermediate compound m (303 mg, yield 78%) was obtained as a white solid. 1 H NMR(300MHz,DMSO)δ8.44(t,J=5.5Hz,1H),8.23(d,J=1.7Hz,2H),8.13(s,1H),7 .75-7.70(m,2H),7.45-7.40(m,2H),7.31-7.26(m,2H),7.04-6.95(m,4H),6.86- 6.81(m,2H),6.75(d,J=5.8Hz,1H),5.26(s,2H),3.78(s,3H),3.71(s,3H),3.46( d, J=5.5Hz, 2H), 3.37 (dd, J=6.8, 5.1Hz, 4H), 3.05 (q, J=5.9Hz, 2H), 1.32 (s, 9H).

[0078] The hydrochloride salt of compound 12 was obtained (263 mg, 95% yield) as a pale yellow solid, which did not require further purification for the next step.

[0079] 13) Synthesis of compound 13:

[0080] The synthesis method is the same as that of compound 1, except that compound N-tert-butoxycarbonyl-1,2-ethylenediamine is replaced with compound 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate tert-butyl ester.

[0081] Intermediate compound n (336 mg, yield 82%) was obtained as a white solid. 1H NMR(300MHz,DMSO)δ8.49(t,J=5.5Hz,1H),8.22(d,J=1.7Hz,2H),8.14(s,1H) ,7.73(d,J=8.3Hz,2H),7.45-7.40(m,2H),7.32-7.27(m,2H),7.04-6.96(m,4 H),6.86-6.82(m,2H),6.78(t,J=5.8Hz,1H),5.25(s,2H),3.78(s,3H),3.71( s,3H),3.48(d,J=5.5Hz,6H),3.35(s,4H),3.03(q,J=6.0Hz,2H),1.34(s,9H).

[0082] The hydrochloride salt of compound 13 was obtained (299 mg, 97% yield) as a pale yellow solid, which did not require further purification for the next step.

[0083] 14) Synthesis of compound 14:

[0084] The synthesis method is the same as that of compound 1, except that N-tert-butyloxycarbonyl-1,2-ethylenediamine is replaced by compound (2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate tert-butyl ester.

[0085] Intermediate compound O (327 mg, yield 76%) was obtained as a white solid. 1 H NMR (300MHz, DMSO) δ8.51-8.44(m,1H),8.22(s,2H),8.13(s,1H),7.73(d,J= 8.0Hz,2H),7.46-7.40(m,2H),7.32-7.26(m,2H),7.00(dd,J=11.7,8.3Hz,4 H),6.88-6.81(m,2H),6.76(s,1H),5.25(s,2H),3.78(s,3H),3.71(s,3H),3 .46(s,4H),3.35(dd,J=7.3,4.4Hz,8H),3.03(q,J=6.0Hz,2H),1.35(s,9H).

[0086] The hydrochloride salt of compound 14 was obtained (296 mg, 98% yield) as a pale yellow solid, which did not require further purification for the next step.

[0087] 15) Synthesis of compound 15:

[0088] The synthesis method is the same as that of compound 1, except that N-tert-butoxycarbonyl-1,2-ethylenediamine is replaced with compound ((trans-4-(aminomethyl)cyclohexyl)methyl)carbamate tert-butyl ester.

[0089] Intermediate compound p (264 mg, yield 65%) was obtained as a white solid. 1 H NMR (300MHz, DMSO) δ8.36 (s, 1H), 8.22 (s, 2H), 8.13 (s, 1H), 7.72 (d, J = 7.9Hz, 2H), 7.4 4(d,J=8.9Hz,2H),7.30(d,J=8.2Hz,2H),7.00(dd,J=19.5,8.1Hz,4H),6.84(d,J=8.6 Hz,2H),6.78(s,1H),5.25(s,2H),3.79(s,3H),3.72(s,3H),3.05(s,2H),2.75(t,J=6 .0Hz,2H),1.75-1.62(m,4H),1.37(s,9H),1.32-1.19(m,2H),0.82(q,J=11.3Hz,4H).

[0090] The hydrochloride salt of compound 15 was obtained (228 mg, 94% yield) as a pale yellow solid, which did not require further purification for the next step.

[0091] 16) Synthesis of compound 16:

[0092] The synthesis method is the same as that of compound 1, except that N-tert-butyloxycarbonyl-1,2-ethylenediamine is replaced with piperazine-1-carboxylic acid tert-butyl ester.

[0093] The intermediate compound q (305 mg, yield 80%) was obtained as a transparent oil. 1 H NMR (300MHz, DMSO) δ8.20(s,2H),8.11(s,1H),7.43(d,J=8.3Hz,2H),7.29(dd,J=18.0,8.0Hz,4H),7.04(d,J=8.1Hz,2H),6.9 6(d,J=7.7Hz,2H),6.84(d,J=8.3Hz,2H),5.25(s,2H),3.79(s,3H),3.71(s,3H),3.52(s,4H),3.20-2.99(m,4H),1.40(s,9H).

[0094] The hydrochloride salt of compound 16 was obtained (269 mg, 97% yield) as a pale yellow solid, which did not require further purification for the next step.

[0095] 17) Synthesis of compound 17:

[0096] The synthesis method is the same as that of compound 1, except that compound N-tert-butoxycarbonyl-1,2-ethylenediamine is replaced with compound (4-[4-(2,2-dimethylpropionyl)piperazin-1-yl]phenyl)amine.

[0097] Intermediate compound r (292 mg, yield 69%) was obtained as a light brown solid. 1 H NMR (300MHz, DMSO) δ10.01 (s, 1H), 8.25 (s, 2H), 8.15 (s, 1H), 7.84 (d, J = 8. 0Hz,2H),7.60(s,2H),7.48-7.40(m,2H),7.31(d,J=8.3Hz,2H),7.03(d,J =8.2Hz,4H),6.97-6.89(m,2H),6.88-6.81(m,2H),5.29(s,2H),3.79(s,3 H), 3.72 (s, 3H), 3.44 (d, J = 5.9Hz, 4H), 3.04 (t, J = 5.0Hz, 4H), 1.42 (s, 9H).

[0098] The hydrochloride salt of compound 17 was obtained (251 mg, 93% yield) as a brown solid, which did not require further purification for the next step.

[0099] 18) Synthesis of compound 18:

[0100] The synthesis method is the same as that of compound 1, except that N-tert-butyloxycarbonyl-1,2-ethylenediamine is replaced with (4'-amino-[1,1'-biphenyl]-4-yl)carbamate tert-butyl ester.

[0101] Intermediate compound S (256 mg, yield 60%) was obtained as a brown solid. 1H NMR(300MHz,DMSO)δ10.26(s,1H),9.44(s,1H),8.27(s,2H),8.16(s,1H),7. 89(d,J=7.9Hz,2H),7.82(d,J=8.3Hz,2H),7.61(d,J=8.5Hz,2H),7.55(d,J= 4.8Hz,4H),7.45(d,J=8.7Hz,2H),7.35-7.30(m,2H),7.09-7.02(m,4H),6.8 6(dd,J=9.2,2.5Hz,2H),5.31(s,2H),3.80(s,3H),3.72(s,3H),1.49(s,9H).

[0102] The hydrochloride salt of compound 18 was obtained (220 mg, 93% yield) as a dark brown solid, which did not require further purification for the next step.

[0103] 19) Synthesis of compound 19:

[0104] Step 1: Preparation of benzyl piperazine-1-carboxylate:

[0105] The preparation method is the same as that of compound 1, except that compound N-tert-butoxycarbonyl-1,2-ethylenediamine is replaced with compound 4-(4-aminobenzyl)piperazine-1-carboxylic acid benzyl ester.

[0106] The intermediate compound t (286 mg, yield 64%) was obtained as a white solid. 1 H NMR (300MHz, DMSO) δ10.19(s,1H),8.87(s,1H),8.25(s,2H),8.15(s,1H),7.85(d,J=8.0Hz,2H),7.69(d,J=8.0Hz,2H),7.48-7.40(m,2H), 7.38-7.23(m,9H),7.09-7.00(m,4H),6.84(d,J=8.9Hz,2H),5.30(s, 2H),5.06(s,2H),3.79(s,3H),3.71(s,3H),3.43(s,6H),2.33(s,4H).

[0107] Step 2: Preparation of 4-((2-(3,5-bis(trifluoromethyl)phenyl)-4,5-bis(4-methoxyphenyl)-1H-imidazol-1-yl)methyl)-N-(4-(piperazin-1-ylmethyl)phenyl)benzamide:

[0108] Compound t was dissolved in methanol (5 mL), and 10% Pd / C (containing 55% water) was added. The mixture was stirred overnight at room temperature under hydrogen atmosphere. The mixture was filtered through diatomaceous earth, and the filtrate was concentrated and purified by silica gel chromatography (DCM / MeOH = 40:1 elution) to give compound 19 (196 mg, yield 80%) as a clear oil.

[0109] 1 H NMR (300MHz, DMSO) δ10.17(s,1H),8.26(s,2H),8.15(s,1H),7.85(d,J=7.9Hz ,2H),7.68(d,J=8.0Hz,2H),7.45(d,J=8.4Hz,2H),7.32(d,J=8.1Hz,2H),7.25 (d,J=8.1Hz,2H),7.04(dd,J=8.5,3.0Hz,4H),6.85(d,J=8.4Hz,2H),5.30(s,2 H),3.79(s,3H),3.72(s,3H),3.42(s,2H),2.82(t,J=5.0Hz,4H),2.36(s,4H).

[0110] In the following examples, the BRD4 ligand used in the preparation of bimolecular compounds AB-01 to 19 is a compound obtained by carboxylating the BRD4 inhibitor (+)-JQ-1, with the following structure:

[0111] Example 1: Preparation of bimolecular compound AB-01

[0112] (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetic acid (0.382 mmol) was dissolved in anhydrous dichloromethane (10 mL), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.764 mmol), 1-hydroxybenzotriazole (0.764 mmol), and anhydrous triethylamine (1.528 mmol) were added. The mixture was stirred at room temperature for 30 minutes, and then the hydrochloride of compound 1 (0.382 mmol) was added. The mixture was stirred at room temperature for 6 hours. The reaction solution was washed once with 30 mL of distilled water and once with saturated sodium chloride aqueous solution. The organic phase was concentrated under reduced pressure and purified by silica gel chromatography (DCM / MeOH = 60:1 elution) to obtain compound AB-01 (246 mg, yield 62%) as a white solid.

[0113] 1H NMR (300MHz, DMSO) δ8.47(d,J=4.5Hz,1H),8.38(d,J=6.0Hz,1H),8.23(s,2H),8.13(s,1H),7.7 2(d,J=8.0Hz,2H),7.42(t,J=7.1Hz,6H),7.28(d,J=8.4Hz,2H),7.00(dd,J=10.4,8.4Hz,4H),6. 86-6.80(m,2H),5.26(s,2H),4.50(t,J=7.1Hz,1H),3.74(d,J=19.2Hz,6H),3.32-3.27(m,2H),3 .23(dd,J=6.9,3.4Hz,2H),2.56(s,3H),2.38(s,3H),1.57(s,3H).MS(ESI)m / z=1051.2970[M+H] +

[0114] Example 2: Preparation of bimolecular compound AB-02

[0115] The preparation method of AB-02 is the same as that of AB-01 in Example 1. The compound AB-02 (217 mg, yield 65%) was synthesized as a white solid.

[0116] 1 H NMR (300MHz, DMSO) δ8.41(s,1H),8.26(s,1H),8.23(s,2H),8.12(s,1H),7.73(d,J=8.0Hz,2H ),7.46-7.38(m,6H),7.29(d,J=8.2Hz,2H),7.00(dd,J=11.0,8.3Hz,4H),6.84(d,J=8.5Hz,2H ),5.26(s,2H),4.51(t,J=7.1Hz,1H),3.78(s,3H),3.72(s,3H),3.28(d,J=6.7Hz,2H),3.25- 3.18(m,2H),3.13(d,J=5.5Hz,2H),2.58(s,3H),2.39(s,3H),1.74-1.64(m,2H),1.59(s,3H).

[0117] MS(ESI)m / z = 1065.3115[M+H] +

[0118] Example 3: Preparation of bimolecular compound AB-03

[0119] The preparation method of AB-03 is the same as that of AB-01 in Example 1. The compound AB-03 (192 mg, yield 54%) was synthesized as a white solid.

[0120] 1 H NMR (300MHz, DMSO) δ8.44(t,J=5.5Hz,1H),8.23(d,J=1.7Hz,2H),8.22(s,1H),8.14(s,1H),7.73(d,J=8.2Hz,2H), 7.43(ddd,J=10.6,5.4,2.8Hz,6H),7.32-7.27(m,2H),7.03-6.95(m,4H),6.86-6.81(m,2H),5.26(s,2H),4.49(dd ,J=8.3,5.9Hz,1H),3.77(s,3H),3.71(s,3H),3.27(d,J=15.0Hz,2H),3.23-3.17(m,2H),3.15(d,J=8.5Hz,1H),3. 09(dd,J=11.6,5.6Hz,1H),2.58(s,3H),2.38(s,3H),1.60-1.57(m,3H),1.50(s,4H).MS(ESI)m / z=1079.3265[M+H] +

[0121] Example 4: Preparation of bimolecular compound AB-04

[0122] The preparation method of AB-04 is the same as that of AB-01 in Example 1. The compound AB-04 (191 mg, yield 49%) was synthesized as a white solid.

[0123] 1H NMR (300MHz, DMSO) δ8.39(d,J=6.0Hz,1H),8.23(s,2H),8.19(s,1H),8.13(s,1H),7.74(d,J=7.9Hz,2H),7.51-7. 39(m,6H),7.33-7.26(m,2H),7.00(dd,J=15.2,8.3Hz,4H),6.88-6.80(m,2H),5.26(s,2H),4.50(dd,J=8.1,6.0H z,1H),3.78(s,3H),3.72(s,3H),3.40(s,1H),3.22(q,J=6.9,5.9Hz,3H),3.10(dt,J=19.7,6.5Hz,2H),2.59(s,3 H),2.40(s,3H),1.61(s,3H),1.48(dd,J=14.4,7.3Hz,4H),1.32(d,J=11.1Hz,2H).MS(ESI)m / z=1093.3424[M+H] +

[0124] Example 5: Preparation of bimolecular compound AB-05

[0125] The preparation method of AB-05 is the same as that of AB-01 in Example 1. The compound AB-05 (151 mg, yield 39%) was synthesized as a white solid.

[0126] 1 H NMR (300MHz, DMSO) δ8.40(t,J=5.7Hz,1H),8.22(s,2H),8.18(s,1H),8.12(s,1H),7.72(d,J= 8.0Hz,2H),7.43(d,J=9.3Hz,6H),7.29(d,J=8.3Hz,2H),6.99(dd,J=15.9,8.2Hz,4H),6.83(d ,J=8.7Hz,2H),5.25(s,2H),4.50(t,J=7.0Hz,1H),3.77(s,3H),3.71(s,3H),3.19(d,J=6.9Hz ,4H),3.08(d,J=7.9Hz,2H),2.58(s,3H),2.37(s,3H),1.59(s,3H),1.45(s,4H),1.29(s,4H).

[0127] MS(ESI)m / z = 1107.3570 [M+H] +

[0128] Example 6: Preparation of bimolecular compound AB-06

[0129] The preparation method of AB-06 is the same as that of AB-01 in Example 1. The compound AB-06 (177 mg, yield 46%) was synthesized as a white solid.

[0130] 1 H NMR (300MHz, DMSO) δ8.38(d,J=6.1Hz,1H),8.22(s,2H),8.16(s,1H),8.12(s,1H),7.72(d,J =7.9Hz,2H),7.51-7.39(m,6H),7.30(d,J=8.2Hz,2H),6.99(dd,J=15.9,8.1Hz,4H),6.88-6 .80(m,2H),5.25(s,2H),4.50(t,J=7.1Hz,1H),3.78(s,3H),3.72(s,3H),3.21(t,J=7.3Hz, 4H),3.09(s,2H),2.58(s,3H),2.39(s,3H),1.61(s,3H),1.45(s,4H),1.27(d,J=6.8Hz,6H).

[0131] MS(ESI)m / z=1121.3719[M+H] +

[0132] Example 7: Preparation of bimolecular compound AB-07

[0133] The preparation method of AB-07 is the same as that of AB-01 in Example 1. The compound AB-07 (210 mg, yield 55%) was synthesized as a white solid.

[0134] 1H NMR (300MHz, DMSO) δ8.39(d,J=5.7Hz,1H),8.22(s,2H),8.16(t,J=5.4Hz,1H),8.13(s,1H),7.72(d,J=7. 9Hz,2H),7.50-7.40(m,6H),7.30(d,J=8.2Hz,2H),7.00(dd,J=15.6,8.1Hz,4H),6.88-6.81(m,2H),5.25 (s,2H),4.50(dd,J=8.0,5.9Hz,1H),3.78(s,3H),3.72(s,3H),3.25-3.15(m,4H),3.08(d,J=7.8Hz,2H), 2.59(s,3H),2.40(s,3H),1.62(s,3H),1.44(s,4H),1.25(d,J=8.0Hz,8H).MS(ESI)m / z=1135.3866[M+H] +

[0135] Example 8: Preparation of bimolecular compound AB-08

[0136] The preparation method of AB-08 is the same as that of AB-01 in Example 1. The compound AB-08 (194 mg, yield 51%) was synthesized as a white solid.

[0137] 1 H NMR (300MHz, DMSO) δ8.38(t,J=5.3Hz,1H),8.22(s,2H),8.18(d,J=5.4Hz,1H),8.13(s,1H),7.72(d,J= 7.9Hz,2H),7.51-7.39(m,6H),7.30(d,J=8.2Hz,2H),7.00(dd,J=14.8,8.2Hz,4H),6.89-6.78(m,2H),5 .25(s,2H),4.50(dd,J=8.1,5.8Hz,1H),3.78(s,3H),3.72(s,3H),3.15(ddt,J=33.8,12.1,7.5Hz,6H) ,2.59(s,3H),2.39(s,3H),1.61(s,3H),1.44(s,4H),1.29-1.20(m,10H).MS(ESI)m / z=1149.4059[M+H] +

[0138] Example 9: Preparation of bimolecular compound AB-09

[0139] The preparation method of AB-09 is the same as that of AB-01 in Example 1. The compound AB-09 (178 mg, yield 47%) was synthesized as a white solid.

[0140] 1 H NMR (300MHz, DMSO) δ8.38(t,J=5.6Hz,1H),8.22-8.20(m,2H),8.17(t,J=5.6Hz,1H),8.12(s,1H),7.71(d,J=8.1Hz,2H),7.48-7.40(m,6H),7. 32-7.27(m,2H),7.05-6.95(m,4H),6.86-6.80(m,2H),5.24(s,2H),4. 49(dd,J=8.3,5.8Hz,1H),3.78(s,3H),3.71(s,3H),3.24-3.02(m,6H).

[0141] MS(ESI)m / z=1163.4211[M+H] +

[0142] Example 10: Preparation of bimolecular compound AB-10

[0143] The preparation method of AB-10 is the same as that of AB-01 in Example 1. The compound AB-10 (131 mg, yield 35%) was synthesized as a white solid.

[0144] 1 H NMR (300MHz, DMSO) δ8.38(t,J=5.7Hz,1H),8.22(s,2H),8.17(d,J=5.5Hz,1H),8.12(s,1H),7.72(d,J=8.0Hz,2H) ,7.43(dd,J=8.8,6.2Hz,6H),7.30(d,J=8.2Hz,2H),7.00(dd,J=14.8,8.1Hz,4H),6.84(dd,J=9.3,2.5Hz,2H),5. 25(s,2H),4.50(dd,J=8.2,5.7Hz,1H),3.78(s,3H),3.72(s,3H),3.21(dt,J=16.9,6.1Hz,4H),3.09(dd,J=17.9, 6.5Hz,2H),2.59(s,3H),2.39(s,3H),1.62(s,3H),1.44(s,2H),1.29-1.19(m,16H).MS(ESI)m / z=1177.4352[M+H] +

[0145] Example 11: Preparation of bimolecular compound AB-11

[0146] The preparation method of AB-11 is the same as that of AB-01 in Example 1. The compound AB-11 (123 mg, yield 33%) was synthesized as a white solid.

[0147] 1 H NMR (300MHz, DMSO) δ8.38(t,J=5.7Hz,1H),8.22(s,2H),8.17(t,J=5.6Hz,1H),8.12(s,1H),7.72(d,J=7.9Hz, 2H),7.43(dd,J=9.0,6.1Hz,6H),7.30(d,J=8.3Hz,2H),7.00(dd,J=14.8,8.1Hz,4H),6.84(d,J=8.5Hz,2H),5. 25(s,2H),4.50(dd,J=8.4,5.7Hz,1H),3.78(s,3H),3.72(s,3H),3.21(dt,J=18.1,6.2Hz,4H),3.14-2.99(m, 2H),2.59(s,3H),2.40(s,3H),1.62(s,3H),1.44(s,4H),1.23(d,J=8.2Hz,16H).MS(ESI)m / z=1191.4497[M+H] +

[0148] Example 12: Preparation of bimolecular compound AB-12

[0149] The preparation method of AB-12 is the same as that of AB-01 in Example 1. The compound AB-12 (114 mg, yield 28%) was synthesized as a white solid.

[0150] 1H NMR (300MHz, DMSO) δ8.47(t,J=5.4Hz,1H),8.30(t,J=5.5Hz,1H),8.22(s,2H),8.12(s,1H),7.75(d,J=8.3Hz, 2H),7.48-7.43(m,3H),7.42-7.38(m,3H),7.30-7.26(m,2H),7.02-6.94(m,4H),6.85-6.81(m,2H),5.24(s,2H ),4.49(dd,J=8.1,6.0Hz,1H),3.77(s,3H),3.71(s,3H),3.52(d,J=5.7Hz,2H),3.47(t,J=5.7Hz,2H),3.41(t, J=5.5Hz,2H),3.29(s,1H),3.27-3.21(m,2H),3.20-3.14(m,1H),2.57(s,3H),2.39-2.36(m,3H),1.59(s,3H).

[0151] MS(ESI)m / z = 1095.3225 [M+H] +

[0152] Example 13: Preparation of bimolecular compound AB-13

[0153] The preparation method of AB-13 is the same as that of AB-01 in Example 1. The compound AB-13 (217 mg, yield 56%) was synthesized as a white solid.

[0154] 1 H NMR (300MHz, DMSO) δ8.50(t,J=5.5Hz,1H),8.29(t,J=5.6Hz,1H),8.22(s,2H),8.13(s,1H),7.74(d,J=8. 3Hz,2H),7.48-7.39(m,6H),7.31-7.26(m,2H),7.03-6.96(m,4H),6.86-6.81(m,2H),5.25(s,2H),4.49(d d,J=7.9,6.2Hz,1H),3.77(s,3H),3.71(s,3H),3.51(d,J=8.4Hz,6H),3.47-3.36(m,4H),3.28-3.17(m,4H ),3.08(dd,J=7.3,4.3Hz,2H),2.58(s,3H),2.41-2.37(m,3H),1.60(s,3H).MS(ESI)m / z=1139.3475[M+H] +

[0155] Example 14: Preparation of bimolecular compound AB-14

[0156] The preparation method of AB-14 is the same as that of AB-01 in Example 1. The compound AB-14 (182 mg, yield 48%) was synthesized as a white solid.

[0157] 1 H NMR (300MHz, DMSO) δ8.45(t,J=5.6Hz,1H),8.26(s,1H),8.22(s,2H),8.11(s,1H),7.73(d,J=8.2Hz,2H),7 .48-7.40(m,6H),7.31-7.26(m,2H),7.03-6.96(m,4H),6.85-6.80(m,2H),5.25(s,2H),4.50(dd,J=7.9,6. 2Hz,1H),3.78(s,3H),3.71(s,3H),3.51(d,J=1.5Hz,8H),3.48(d,J=5.7Hz,2H),3.43(t,J=5.8Hz,2H),3.3 7(d,J=5.7Hz,2H),3.29-3.19(m,4H),2.58(s,3H),2.39(s,3H),1.61(s,3H).MS(ESI)m / z=1183.3741[M+H] +

[0158] Example 15: Preparation of the bimolecular compound AB-15

[0159] The preparation method of AB-15 is the same as that of AB-01 in Example 1. The synthesized compound AB-15 (77 mg, yield 20%) is a white solid.

[0160] 1H NMR (300MHz, DMSO) δ8.39(s,1H),8.22(s,2H),8.15(s,1H),8.12(s,1H),7.74(d,J=7.9Hz,2H),7.48-7.38(m, 6H),7.30(d,J=8.3Hz,2H),6.99(dd,J=17.1,8.1Hz,4H),6.88-6.80(m,2H),5.25(s,2H),4.50(dd,J=8.7,5.3 Hz,1H),3.78(s,3H),3.72(s,3H),3.26(d,J=8.8Hz,1H),3.16(d,J=5.0Hz,1H),3.08(s,3H),2.93-2.84(m,1H ),2.59(s,3H),2.39(s,3H),1.75(s,4H),1.59(s,3H),1.43(s,2H),0.88(s,4H).MS(ESI)m / z=1133.3788[M+H] +

[0161] Example 16: Preparation of the bimolecular compound AB-16

[0162] The preparation method of AB-16 is the same as that of AB-01 in Example 1. Compound AB-16 (207 mg, yield 52%) was synthesized as a white solid.

[0163] 1 H NMR (300MHz, DMSO) δ8.22 (s, 2H), 8.13 (s, 1H), 7.51-7.41 (m, 8H), 7.31 (d, J = 8. 1Hz,4H),7.04(d,J=8.6Hz,2H),6.97(d,J=7.8Hz,2H),6.87-6.81(m,2H),5.26 (s,2H),4.58(s,1H),3.79(s,3H),3.71(s,3H),3.61-3.40(m,6H),3.17(d,J=5 .1Hz,2H),2.59(s,4H),2.41(s,4H),1.63(s,3H).MS(ESI)m / z=1077.3113[M+H] +

[0164] Example 17: Preparation of the bimolecular compound AB-17

[0165] The preparation method of AB-17 is the same as that of AB-01 in Example 1. The compound AB-17 (157 mg, yield 41%) was synthesized as a white solid.

[0166] 1 H NMR (300MHz, DMSO) δ10.04(s,1H),8.26(s,2H),8.16(s,1H),7.85(d,J=7.9Hz,2H),7.62(d,J=8.5Hz,2H),7.46 (q,J=7.3,5.6Hz,6H),7.32(d,J=8.2Hz,2H),7.01(dd,J=19.8,8.3Hz,6H),6.85(d,J=8.9Hz,2H),5.30(s,2H),4 .60(t,J=6.6Hz,1H),3.84(s,2H),3.79(s,3H),3.72(s,3H),3.64(d,J=6.6Hz,2H),3.49(d,J=6.2Hz,1H),3.44( d,J=6.5Hz,1H),3.22(s,2H),3.09(s,2H),2.60(s,3H),2.42(s,3H),1.63(s,3H).MS(ESI)m / z=1168.3507[M+H] +

[0167] Example 18: Preparation of the bimolecular compound AB-18

[0168] The preparation method of AB-18 is the same as that of AB-01 in Example 1. The compound AB-18 (118 mg, yield 35%) was synthesized as a light brown solid.

[0169] 1 H NMR (300MHz, DMSO) δ10.45(s,1H),10.30(s,1H),8.27(s,2H),8.16(s,1H),7.87(dd,J=16.1,8.1H z,4H),7.74(d,J=8.4Hz,2H),7.65(d,J=8.3Hz,4H),7.46(dd,J=9.1,2.0Hz,6H),7.36-7.29(m,2H ),7.12-7.02(m,4H),6.86(dd,J=9.2,2.6Hz,2H),5.32(s,2H),3.80(s,3H),3.73(s,3H),3.54(d, J=7.0Hz,1H),3.12(td,J=7.2,4.7Hz,1H),2.62(s,3H),2.43(s,3H).MS(ESI)m / z=1174.3209[M+H] +

[0170] Example 19: Preparation of the bimolecular compound AB-19

[0171] The preparation method of AB-19 is the same as that of AB-01 in Example 1. The synthesized compound AB-19 (90 mg, yield 32%) is a grayish-white solid.

[0172] 1 H NMR (300MHz, DMSO) δ10.18(s,1H),8.26(s,2H),8.14(s,1H),7.86(d,J=7.9Hz,2H),7.71(d,J=8.1Hz,2H),7.5 5-7.39(m,6H),7.30(t,J=8.7Hz,4H),7.04(dd,J=8.4,3.9Hz,4H),6.84(d,J=8.8Hz,2H),5.30(s,2H),4.57(t ,J=6.6Hz,1H),3.79(s,3H),3.72(s,3H),3.65(s,2H),3.57(d,J=7.1Hz,1H),3.48(s,3H),3.41(d,J=6.3Hz,1 H),2.59(s,3H),2.45(s,1H),2.40(s,3H),2.31(d,J=15.6Hz,2H),1.62(s,3H).MS(ESI)m / z=1182.3721[M+H] +

[0173] Related performance tests

[0174] 1. Western blotting assay to detect the degradation activity of compounds.

[0175] Cell lines and cell culture reagents

[0176] The MV-4-11 cell line was purchased from the Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All cell lines used were cultured in RPMI 1640 medium containing 10% fetal bovine serum. 1% (v / v) of a 100X penicillin-streptomycin stock solution (15140-122, Gibco ThermoFisher, USA) was added to the medium before use. All cells were tested for mycoplasma contamination and cultured in a 37°C incubator with 5% CO2 and saturated humidity (Thermo Fisher Scientific). Cells adhered to the culture vessel and cancer cells were passaged every 1-2 days.

[0177] Experimental Methods: MV-4-11 human myeloid monocytic leukemia cells in logarithmic growth phase were prepared into a cell suspension and seeded into 6-well cells. After overnight culture, when the cell density reached approximately 80%, the test compound was added to the desired final concentration. Simultaneously, an equal volume of DMSO was added to the blank control group. Cell status was observed and recorded under a microscope after a period of further culture. Cells were digested with 1 mL of trypsin, centrifuged at 3000 rpm for 3 minutes, washed three times with PBS, and lysed with an appropriate amount of RIPA lysis buffer (containing 1% volume of a protease inhibitor mixture (P1005, Beyotime, CHN) and 1% volume of PMSF working solution (174 μg / mL, i.e., 1 mM) (MKR692, MyBios, cience, CHN)). The cells were gently pipetted and mixed thoroughly. After lysis on ice for 40 minutes, the cells were centrifuged at 12000 rpm for 15 minutes at 4°C, and the supernatant was collected. Quantify protein according to the BCA quantitative kit standard operating procedure. Adjust the loading volume according to the protein concentration to ensure the same total protein loading for all samples. Add 5× Loading Buffer to the remaining protein sample, mix well, boil at 100℃ for 10 minutes, and store at -20℃. Prepare stacking and separating gels separately. Add an appropriate amount of marker (26616, ThermoFisher, USA) to the edge of the sample wells. Load 30 μg of protein per well. For SDS-PAGE electrophoresis, first electrophore at a constant voltage of 60V until the band at the top of the marker separates and leaves the stacking gel. Then increase the voltage to 120V until the leading edge of the bromophenol blue band reaches the bottom of the separating gel. Finally, transfer the membrane at a constant current of 300mA for 1.5 hours.

[0178] Transfer procedure: Porous pad (negative electrode) - sponge pad - filter paper - adhesive - membrane - filter paper - sponge pad - porous pad (positive electrode). All materials must be cooled beforehand. The PVDF membrane size must exceed the cut adhesive but not the size of the black and white board to avoid short circuits. All layers in the module must be free of air bubbles. After transfer, remove the PVDF membrane, cut the required strips, block with 5% skim milk at room temperature for one hour, incubate with primary antibody (diluted according to instructions) at 4°C overnight, remove the primary antibody, and wash three times with 1×TBST for 10 minutes each time. Secondary antibody (1:20000 dilution) was incubated at room temperature for 0.5 hours, followed by washing three times with 1×TBST for 10 minutes each time. After washing, the membrane was transferred to a fully automated chemiluminescence imaging analysis system (Tanon5200, Tanon, CHN) for detection. The system was pre-cooled for 30 minutes. Equal volumes of solution A and solution B of the detection solution Tanon™ High-sig ECL Western Blotting Substrate (180-5001, Tanon, CHN) were mixed. The strip was immersed in the mixture and then placed on the detection plate for imaging.

[0179] The degradation activity of compounds AB-01 to 19 against BRD4 protein is shown in Table 1:

[0180] Table 1. Degradation rate of BRD4 protein by different compounds at 30 nM in MV-4-11 cells

[0181] As shown in Table 1, compounds AB-12, AB-13, and AB-14 exhibited significant degradation effects on BRD4 protein in MV-4-11 cells. Among them, compound AB-13 achieved a degradation rate of 90% when administered at 30 nM.

[0182] 2. Cell proliferation assay (CCK-8 assay)

[0183] Logarithmic growth phase MV-4-11 cells were harvested, digested with trypsin for 2–3 minutes, centrifuged to discard the digestion solution, washed twice with PBS, and resuspended in fresh serum-containing medium. Cells were seeded at a density of 3000 cells per well, approximately 100 μL per well. Cells were then cultured at 37°C in a 5% CO2 incubator until complete adherence. A gradient of drug concentrations was then added, with three replicates for each concentration, and control wells were included. Cells were cultured for another 72 hours. 10 μL of CCK8 solution was added to each well, and the cells were incubated at 37°C for 30 minutes. The absorbance at 450 nm was measured using a microplate reader, and the inhibitory rate of the compound on tumor cells was calculated. Finally, data analysis was performed using a GraphPad Prism 9.5 to calculate the IC50.

[0184] The antiproliferative activity of compounds AB-01-19 in MV-4-11 cells is shown in Table 2:

[0185] Table 2. Antiproliferative activity of different compounds in MV-4-11 cells

[0186] As can be seen from Table 2, compounds AB-12, AB-13, and AB-14 exhibited good anti-proliferative activity in MV-4-11 cells, with IC50 values ​​less than 100 nM.

[0187] In summary, the Hsp70-BRD4 bimolecular degrader provided by this invention has significant degradation activity against BRD4 protein and is an effective BRD4 protein degrader. This application proposes a novel method for targeted protein degradation, providing new chemical tools and drug discovery strategies for the field of molecular targeted therapy.

[0188] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A heterobifunctional compound or a pharmaceutically acceptable salt thereof that targets and degrades BRD4 based on HSP70, characterized in that, The heterobifunctional compound comprises a ligand A that binds to HSP70, a ligand B that binds to BRD4, and a linker connecting the two, with the following structure: A-Linker-B.

2. The heterobifunctional compound based on HSP70-targeted degradation of BRD4 as described in claim 1, characterized in that, The structure of the heterobifunctional compound is shown in formula (I):

3. The heterobifunctional compound based on HSP70-targeted degradation of BRD4 as described in claim 1, characterized in that, Linker can be a carbon-carbon single bond, carbon-carbon double bond, carbon-carbon triple bond, aryl bond, heteroaryl bond, cycloalkyl bond, cycloalkenyl bond, heterocyclic bond, fused bicyclic bond, fused heterocyclic bond, spirobicyclic bond, spiroheterocyclic bond, carbon-nitrogen bond, carbon-sulfur bond, carbon-phosphorus bond, nitrogen-nitrogen bond, carbonyl (-C(=O)-), C1-6 alkylene group, amide bond, ether bond, disulfide bond, piperazine bond, biphenyl diamine bond, phenylpiperazine bond, carboxylic acid ester bond, sulfonate bond, carbamate bond, or a combination thereof.

4. The heterobifunctional compound based on HSP70-targeted degradation of BRD4 as described in claim 3, characterized in that, The structure of Linker is as follows: Where n is any natural number between 2 and 12, and m is any natural number between 1 and 3.

5. The heterobifunctional compound based on HSP70-targeted degradation of BRD4 as described in claim 2, characterized in that, The heterobifunctional compounds include compounds with structures shown in formulas AB-01 to 19 below:

6. A pharmaceutical composition, characterized in that, It comprises any one of the heterobifunctional compounds based on HSP70 targeting the degradation of BRD4 according to any one of claims 1-5, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, wherein The pharmaceutical composition is formulated as an injectable fluid, aerosol, cream, gel, pill, capsule, syrup, transdermal patch, or excipient.

8. The pharmaceutical composition of claim 6, wherein It also includes other therapeutic agents, such as chemotherapy drugs, targeted drugs, immunotherapy drugs, and drug-drug conjugates.

9. The use of any small molecule drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1-5, or the pharmaceutical composition as described in claims 6-8, in the preparation of a drug for the prevention or treatment of tumors.

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