BRD4 and p300 / CBP double-target PROTAC molecule as well as preparation method and application thereof

Abstract

The invention relates to BRD4 and p300 / CBP double-target PROTAC molecules as well as a preparation method and application thereof, and belongs to the technical field of medicinal chemistry. The compound has a general formula shown in the specification, wherein L is preferably selected from one of the group; and E is preferably selected from one of the group. The compound disclosed by the invention has remarkable anti-tumor activity on human prostatic cancer cell strains PC-3, DU145 and 22Rv1 in vitro, the IC50 values are all lower than 20nM, and particularly, the IC50 values are all lower than 10nM in the cells PC-3 and DU145. The compound I-c shows the optimal activity, the IC50 values of the compound I-c in PC-3 and DU145 cells are 2.80 nM and 6.62 nM respectively, and the IC50 values of the compound I-c are obviously superior to those of positive control drugs NEO2734, paclitaxel and ARV-771. As a novel BRD4 and p300 / CBP double-target PROTAC molecule, the compound disclosed by the invention has a definite action mechanism and excellent anti-tumor activity, and can be used as a candidate or lead compound for research and development of anti-tumor drugs; the synthesis method is simple and convenient, and has good popularization and application prospects.

Description

Technical Field

[0001] The present invention specifically relates to a novel class of BRD4 and p300 / CBP dual-target PROTAC molecules, a preparation method thereof, and an application thereof in the preparation of anti-tumor drugs, belonging to the technical field of medicinal chemistry. Background Art

[0002] BRD4 (bromodomain-containing protein 4) is a ubiquitously expressed transcriptional regulator belonging to the BET protein family with important biological functions. BRD4 regulates gene transcriptional elongation by recognizing and binding to acetylated lysine on chromatin, recruiting the transcription elongation factor P-TEFb, and promoting the phosphorylation of RNA polymerase II. BRD4 plays a key role in maintaining gene promoter activity and transcriptional activation of super-enhancer-associated genes, and is particularly crucial in cell cycle regulation. In addition, BRD4 is involved in DNA damage repair, activating the DNA damage response, and maintaining genomic stability. BRD4 is abnormally activated in a variety of malignant tumors and is closely associated with tumor occurrence and progression. BRD4 can bind to super-enhancer regions and activate multiple key tumor-driving genes, such as c-Myc and BCL-2, promoting tumor cell proliferation and the development of drug resistance. Therefore, BRD4 has become one of the hot targets for anti-tumor drug development.

[0003] p300 and CBP are two highly homologous histone acetyltransferases (HATs) that play a key role in chromatin remodeling and transcriptional regulation. They acetylate lysine residues in histone tails, leading to a relaxation of chromatin structure, thereby enhancing the binding of transcription factors to DNA and promoting gene transcriptional activation. During tumorigenesis and progression, p300 and CBP mediate the regulation of multiple cancer-related signaling pathways. They acetylate and activate multiple pro-proliferation transcription factors, including c-Myc and AP-1, thereby driving the expression of cell cycle-related genes and promoting tumor cell proliferation. Furthermore, p300 and CBP enhance the activity of anti-apoptotic transcription factors such as NF-κB, inhibiting apoptotic signaling pathways and maintaining the survival of tumor cells. Furthermore, p300 / CBP regulate the expression of various cytokines and chemokines, reshaping the tumor microenvironment and helping tumor cells evade immune surveillance, further promoting tumor progression. They are closely associated with multiple oncogenic signaling pathways (such as Wnt / β-catenin, Notch, and PI3K / Akt), playing a core regulatory role in the development, progression, and immune escape of malignant tumors. p300 and CBP have become key targets of interest in the new generation of anti-tumor drugs, particularly epigenetic targeted therapies.

[0004] PROTAC (Protein Degradation Targeting Chimera) is an emerging anti-tumor drug development technology that can selectively degrade specific oncogenic proteins. Unlike traditional small molecule inhibitors, PROTAC directly eliminates target proteins by recruiting the endogenous ubiquitin-proteasome system, eliminating pathogenic proteins at the source, which helps to overcome the common drug resistance problem of targeted drugs. Another major advantage of PROTAC is its "catalytic" mechanism of action: one PROTAC molecule can act repeatedly on multiple target protein molecules, so usually only a lower dose is required to achieve significant therapeutic effects, reducing potential toxicity and side effects. In addition, PROTAC can target "undruggable" proteins that are difficult for traditional inhibitors to act on, including proteins with inactive pockets or unclear functions, providing a new approach for the development of new anti-tumor drugs. At present, PROTAC has been widely used in the study of various tumor types and has shown excellent efficacy and selectivity. It is increasingly becoming a research and development hotspot and future direction in the field of tumor treatment.

[0005] BRD4 and p300 / CBP have a close regulatory relationship in various malignant tumors. They act together on super-enhancers to activate tumor-driving genes and promote tumor cell proliferation and survival. Therefore, the design and development of novel BRD4 and p300 / CBP dual-targeting PROTAC molecules is of great significance for the development of new anti-tumor drugs. Summary of the Invention

[0006] The first object of the present invention is to provide a PROTAC molecule that can simultaneously degrade BRD4 and p300 / CBP proteins, thereby achieving excellent degradation activity against BRD4 and p300 / CBP proteins.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned BRD4 and p300 / CBP dual-target PROTAC molecule.

[0008] Another object of the present invention is to provide the use of the above-mentioned BRD4 and p300 / CBP dual-target PROTAC molecule in the preparation of anti-tumor drugs.

[0009] To achieve the above objectives, the BRD4 and p300 / CBP dual-target PROTAC molecule of the present invention has the general structural formula:

[0010]

[0011] In the general formula I, L is selected from One of the following;

[0012] E is selected from One of them.

[0013] As a preferred solution, the BRD4 and p300 / CBP dual-target PROTAC molecule is a compound with the following structure:

[0014]

[0015] The preparation method of the compound of the present invention comprises the following steps:

[0016] 1. Synthesis of the BRD4 and p300 / CBP dual-target inhibitor M6

[0017]

[0018] (1) Preparation of Compound M2: Compound M1 and 2-(trifluoromethoxy)ethylamine are reacted in a polar organic solvent in the presence of an alkaline reagent under stirring at room temperature. After the reaction is completed, the reaction system is extracted, washed, dried, filtered, concentrated, and purified by column chromatography to obtain Compound M2. The polar organic solvent is selected from N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), N,N-diethylformamide (DEF), hexamethylphosphoramide (HMPA), acetonitrile (MeCN) or other solvents with similar solubility properties, and the alkaline reagent is selected from N,N-diisopropylethylamine, triethylamine, potassium tert-butoxide, potassium tert-butoxide, cesium carbonate, sodium carbonate and potassium carbonate.

[0019] (2) Preparation of Compound M3: Compound M2, zinc powder, and acetic acid were reacted in ethanol under stirring at room temperature. After the reaction, the reaction system was sequentially extracted, washed, dried, filtered, and concentrated. The resulting concentrate was separated and purified by column chromatography to obtain Compound M3.

[0020] (3) Preparation of Compound M4: Compound M3, 1-Boc-4-piperidinecarboxaldehyde, and sodium bisulfite were reacted in methanol solvent under heating and stirring. After the reaction, the reaction system was sequentially extracted, washed, dried, filtered, and concentrated. The resulting concentrate was separated and purified by column chromatography to obtain Compound M4.

[0021] (4) Preparation of Compound M5: Compound M4 and 1,3-dimethylpyridin-2(1H)-one-5-pinacol borate are reacted in a toluene solvent in the presence of an alkaline reagent and a palladium catalyst under heating and stirring. After the reaction is completed, the reaction system is sequentially extracted, washed, dried, filtered, and concentrated. The resulting concentrate is separated and purified by column chromatography to obtain Compound M5. The alkaline reagent is selected from one of potassium carbonate, sodium carbonate, sodium bicarbonate, cesium carbonate, and potassium phosphate, and the palladium catalyst is selected from one of tetrakis(triphenylphosphine)palladium, palladium acetate, bis(dibenzylideneacetone)palladium, and palladium dichloride.

[0022] (5) Preparation of Compound M6: Compound M5 was reacted in anhydrous dichloromethane in the presence of trifluoroacetic acid at room temperature with stirring. After completion of the reaction, the reaction system was sequentially extracted, washed, dried, filtered, and concentrated. The resulting concentrate was separated and purified by column chromatography to obtain Compound M6.

[0023] 2. Synthesis of BRD4 and p300 / CBP dual-targeting PROTAC molecules Ia–Id

[0024] (1) Synthesis of compounds 2a–2d: 4-Fluorothalidamide or 5-Fluorothalidamide and tert-butyl piperidine-4-carboxylate or tert-butyl 2-(piperidin-4-yl)acetate are reacted in a polar organic solvent in the presence of an alkaline reagent under heating and stirring. After the reaction is completed, the reaction system is sequentially extracted, washed, dried, filtered, and concentrated. The resulting concentrate is separated and purified by column chromatography to obtain compounds 2a–2d. The polar organic solvent is selected from one of DMF, DMSO, NMP, DEF, HMPA, and MeCN, or other solvents with similar solubility properties, and the alkaline reagent is selected from one of potassium tert-butoxide, N,N-diisopropylethylamine, triethylamine, cesium carbonate, potassium carbonate, and sodium carbonate.

[0025] (2) Synthesis of Compounds 3a–3d: Compounds 2a–2d were reacted separately in anhydrous dichloromethane in the presence of trifluoroacetic acid at room temperature with stirring. After completion of the reaction, the reaction system was sequentially extracted, washed, dried, filtered, and concentrated. The resulting concentrate was separated and purified by column chromatography to obtain Compounds 3a–3d.

[0026] (3) Preparation of compounds Ia–Id: Compounds 3a–3d were reacted with M6 and an amide coupling reagent, respectively, in a polar organic solvent in the presence of an alkaline reagent under stirring at room temperature. After the reaction, the reaction system is sequentially extracted, washed, dried, filtered, and concentrated, and the resulting concentrate is separated and purified by column chromatography to obtain compounds Ia–Id; wherein the amide coupling reagent is selected from one of O-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), O-(7-aza-1-hydroxybenzotriazole)-tetramethyluronium hexafluorophosphate (HATU), (1-phenyl-1H-benzotriazol-1-yl)dimethylsilylcarbonyl chloride (TBTU), carbonyldiimidazole (CDI), dicyclohexylcarbodiimide (DCC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI); and the alkaline reagent is selected from one of potassium tert-butoxide, N,N-diisopropylethylamine, and triethylamine.

[0027]

[0028] Advantages of the present invention:

[0029] A new class of PROTAC molecules synthesized in this invention can efficiently and selectively degrade BRD4 and p300 / CBP proteins simultaneously. Taking the representative compound Ic as an example, it can achieve complete degradation of BRD4 protein when treated with PC-3 cells at a concentration of 1.0nM for 12 hours; and complete degradation of p300 / CBP protein when treated at a concentration of 30nM for 24 hours. This series of PROTAC molecules showed significant anti-proliferative activity against human prostate cancer PC-3, DU145 and 22Rv1 cell lines in vitro, IC 50 The values ​​were all below 20 nM, especially in PC-3 and DU145 cells. 50 The values ​​were all below 10 nM. Among them, compound Ic had the strongest activity, with IC 50 The values ​​were 2.80nM and 6.62nM, respectively, significantly superior to the positive control drugs NEO2734, paclitaxel, and ARV-771. The PROTAC molecule described in this invention has dual degradation activities of BRD4 and p300 / CBP, a clear mechanism of action, and significant anti-tumor activity. It has the potential to be a candidate molecule for anti-tumor drugs. The synthetic route is simple and has good research and development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Figure 2 is a graph showing that compound Ic of the present invention promotes the efficient degradation of BRD4 in PC-3 tumor cells, wherein A shows the dose-dependent induction of BRD4 and c-Myc protein degradation after PC-3 cells were treated with different concentrations of compound Ic for 24 hours; B shows the time-dependent induction of BRD4 and c-Myc protein degradation after PC-3 cells were treated with 1.0 nM Ic for different time periods.

[0031] Figure 2 This is a graph showing that compound Ic of the present invention promotes the efficient degradation of p300 and CBP in PC-3 tumor cells.

[0032] Among them, A shows the dose-dependent induction of p300 and CBP protein degradation after PC-3 cells were treated with different concentrations of compound Ic for 24 hours; B shows the time-dependent induction of p300 and CBP protein degradation after PC-3 cells were treated with 30 nM concentration of compound Ic for different time periods.

[0033] Figure 3The graph shows that compound Ic of the present invention effectively induces degradation of BRD4 and CBP / p300 in DU145 and RM-1 cancer cells, wherein A is a Western blot analysis of BRD4 protein levels in DU145 cells after treatment with different concentrations of Ic for 12 hours; B is a Western blot analysis of p300 and CBP protein levels in DU145 cells after treatment with different concentrations of Ic for 24 hours; C is a Western blot analysis of BRD4 protein levels in RM-1 cells after treatment with different concentrations of Ic for 12 hours; D is a Western blot analysis of p300 and CBP protein levels in RM-1 cells after treatment with different concentrations of Ic for 24 hours. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is described in detail below with reference to the reaction scheme and examples.

[0035] Example 1 Synthesis of 5-bromo-2-nitro-N-(2-(trifluoromethoxy)ethyl)aniline (M2)

[0036]

[0037] 2-(Trifluoromethoxy)ethylamine hydrochloride (1.1 eq) was added to a 250 mL single-necked flask and dissolved in anhydrous DMSO. N,N-diisopropylethylamine (6.0 eq) was added dropwise and stirred at room temperature for 15 minutes. A solution of 4-bromo-2-fluoro-1-nitrobenzene (M1) (1.0 eq) in anhydrous DMSO was added and allowed to react at room temperature for 7-8 hours. After the reaction, the reaction system was added dropwise to ice water and stirred for 20 minutes. The precipitated solid was filtered and the filter cake was dried in a vacuum oven to obtain compound M2 as a yellow solid with a yield of 93%. 1 H NMR (400MHz, CDCl3) δ8.21 (s, 1H), 8.06 (d, 1H, J = 9.2Hz), 7.02 (d, 1H, J = 2.0Hz), 6.84(dd,1H,J1=2.0Hz,J2=9.2Hz), 4.24(t,2H,J=5.6Hz), 3.65(q,2H,J=5.6Hz). 13 C NMR (101MHz, CDCl3) δ145.09,131.83,131.61,128.37,121.52(q,J C-F =257.6Hz),119.65,116.05,64.97(q,J C-F =3.0Hz),41.71.HRMS(ESI)calcd for C9H9BrF3N2O3[M+H] + :328.9743,found:328.9735

[0038] Example 2 Synthesis of 5-bromo-N'-(2-(trifluoromethoxy)ethyl)phenyl-1,2-diamine (M3)

[0039]

[0040] M2 (1.1 eq), glacial acetic acid (3.0 eq), zinc powder (10.0 eq), and methanol solution were added sequentially to a 250 mL single-necked flask and stirred at room temperature for 4 hours. After completion of the reaction, the reaction system was filtered and the filtrate was concentrated in vacuo. The concentrate was adjusted to a pH of 7-8 with sodium bicarbonate solution and extracted with ethyl acetate. The ethyl acetate layers were combined and washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo. The concentrate was separated and purified on a silica gel column (petroleum ether:ethyl acetate = 4:1) to obtain compound M3 as a dark brown oil in an 89% yield. 1 H NMR (400MHz, CDCl3) δ6.85 (dd, 1H, J1 = 2.0Hz, J2 = 8.0Hz), 6.75 (d, 1H, J = 2.0Hz), 6 .63(d,1H,J=8.0Hz), 4.22(t,2H,J=5.2Hz), 4.43(t,2H,J=5.2Hz), 3.21(br,3H). 13 C NMR (101MHz, CDCl3) δ138.05,133.56,122.05,121.65(q,J C-F =255.5Hz),118.05,115.02,112.92,65.95(q,J C-F =3.0Hz),42.70.HRMS(ESI)calcd for C9H 11 BrF3N2O[M+H] + :299.0001,found:299.0006.

[0041] Example 3 Synthesis of tert-butyl 4-(6-bromo-1-(2-(trifluoromethoxy)ethyl)-1H-benzimidazol-2-yl)piperidine-1-carboxylate (M4)

[0042]

[0043] M3 (1.0 eq) was added to a 250 mL single-necked flask and dissolved in methanol. 1-Boc-piperidine-4-carboxaldehyde (1.1 eq) and sodium bisulfite (1.5 eq) were then added sequentially, followed by stirring at 60°C for 1.5 hours. After the reaction, the system was filtered, the filtrate was concentrated under vacuum, and the concentrate was extracted with ethyl acetate and distilled water. The ethyl acetate layers were combined, washed with distilled water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum. The concentrate was purified using a silica gel column (petroleum ether:ethyl acetate = 3:1) to obtain compound M4 as a white solid in an 82% yield. 1 H NMR (400MHz, DMSO-d6) δ7.90 (d, 1H, J = 2.0Hz), 7.53 (d, 1H, J = 8.4Hz), 7.32 (dd, 1H, J1 = 2.0Hz, J2 = 8.4Hz), 4.67 (t, 2H, J = 5.2Hz), 4. 39(t,2H,J=5.2Hz),4.06(d,2H,J=13.2Hz),3.25–3.17(m,1H),2.90(brs,2H),1.85–1.80(m,2H),1.75–1.64(m,2H),1.43(s,9H). 13 C NMR (101MHz, CDCl3) δ158.39,154.52,141.72,135.30,125.89,121.25(q,J C-F =257.6Hz),121.04,115.89,111.94,79.69,64.79(q,J C-F =3.0Hz),44.05,43.15,42.28,34.35,30.96,28.46.HRMS(ESI)calcdfor C 20 H 26 BrF3N3O3[M+H] + :492.1104,found:492.1102.

[0044] Example 4 Synthesis of tert-butyl 4-(6-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-(2-(trifluoromethoxy)ethyl)-1H-benzimidazol-2-yl)piperidine-1-carboxylate (M5)

[0045]

[0046] Tetrakis(triphenylphosphine)palladium (0.3 eq) was added to a 100 mL two-necked flask under nitrogen. A toluene solution of compound M4 (1.0 eq) was added, followed by a potassium carbonate solution (1.85 eq). Finally, a dehydrated ethanol solution of 1,3-dimethylpyridin-2(1H)-one-5-pinacol borate (1.1 eq) was added. The reaction was maintained at 90°C for 7 h. After completion of the reaction, the reaction system was extracted with ethyl acetate and distilled water. The ethyl acetate layers were combined, washed with distilled water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated in vacuo and purified on a silica gel column (petroleum ether:acetone = 3:1) to obtain compound M5 as a white solid in a 79% yield. 1 H NMR (400MHz, DMSO-d6) δ7.98 (d, 1H, J = 2.4Hz), 7.81 (dd, 1H, J1 = 1.2Hz, J2 = 2.8Hz), 7.76 (d,1H,J=1.6Hz),7.58(d,1H,J=8.4Hz),7.39(dd,1H,J1=1.6Hz,J2=8.4Hz),4.69(t,2H ,J=4.8Hz),4.44(t,2H,J=4.8Hz),4.07(d,2H,J=12.8Hz),3.54(s,3H),3.25–3.18(m,1 H),2.91(brs,2H),2.11(s,3H),1.84(d,2H,J=12.8Hz),1.76–1.66(m,2H),1.43(s,9H). 13 C NMR (101MHz, DMSO-d6) δ162.02,158.91,154.35,141.79,136.98,135.54,134.66,131.14,128.09,121.56(q,J C-F =254.5),120.17,119.28,118.63,107.57,79.14,67.16,41.96,37.75,33.36,31.24,28.57,21.52,17.63.HRMS(ESI)calcd forC 27 H 34 F3N4O4[M+H] + :535.2527,found:535.2529.

[0047] Example 5 Synthesis of 1,3-dimethyl-5-(2-(piperidin-4-yl)-1-(2-(trifluoromethoxy)ethyl)-1H-benzimidazol-6-yl)pyridin-2(1H)-one (M6)

[0048]

[0049] M5 (1.0 eq) was added to a 100 mL single-necked flask and dissolved in anhydrous dichloromethane. The mixture was stirred in an ice bath for 10 minutes. Trifluoroacetic acid was slowly added dropwise. After the addition, the ice bath was removed and the mixture was stirred at room temperature for 0.5 h. After the reaction, the reaction system was concentrated in vacuo to obtain M6, which was used directly in the next reaction without further purification as a white solid with an 84% yield. 1 HNMR (400MHz, DMSO-d6) δ8.03 (d, 1H, J = 2.6Hz), 7.83-7.82 (m, 2H), 7.60 (d, 1H, J = 8.0Hz), 7.40 (dd, 1H, J1 = 1.6Hz, J2 = 8.4Hz), 4 .71(t,2H,J=4.8Hz),4.45(t,2H,J=4.8Hz),3.54(s,3H),3.28-3.23(m,3H),2.91-2.85(m,2H),2.10(s,3H),2.04-1.84(m,4H). 13 C NMR (101MHz, DMSO-d6) δ162.04,158.69,141.79,136.97,135.57,134.70,131.11,128.05,121.54(q,J C-F =254.5Hz),120.09,119.30,118.61,107.62,67.25,67.22,44.23,41.97,37.76,32.31,29.67,17.62.HRMS(ESI)calcd for C 22 H 26 F3N4O2[M+H] + :435.2002,found:435.2003.

[0050] Example 6 Synthesis of tert-butyl 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperidine-4-carboxylate (2a)

[0051]

[0052] According to the above synthetic route II, tert-butyl 4-piperidinoate (1.1 eq) was added to a 100 mL round-bottom flask and dissolved in anhydrous DMF. N,N-diisopropylethylamine (2.0 eq) was added and stirred for 5 minutes. A solution of compound 1a (1.0 eq) in anhydrous DMF was added and stirred at 90°C for 4 hours. The reaction system was extracted with ethyl acetate and distilled water. The ethyl acetate layers were combined, washed with distilled water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo. The concentrate was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 3:2) to obtain compound 2a as a yellow solid with a yield of 71%. 1 H NMR (400MHz, DMSO-d6) δ11.12(s,1H),7.68(dd,1H,J1=7.2Hz,J2=8.4Hz),7.35–7.33(m,2H),5.11(dd,1H,J1=5.2Hz,J2=12.8Hz),3.64(d,2H ,J=12.4Hz),2.97–2.85(m,3H),2.63–2.54(m,2H),2.46–2.40(m,1H), 2.07–1.99(m,1H),1.93-1.88(m,2H),1.79-1.67(m,2H),1.42(s,9H). 13 C NMR (101 MHz, CDCl3) δ 13 C NMR(101MHz,DMSO-d6)δ167.26,166.50,163.62,162.67,161.93,146.00,130.77,129.34,118.97,11 2.51,110.67,75.68,47.14,46.82,44.34,37.50,27.93,27.19,26.66,23.41,17.93.HRMS(ESI)calcd for C 23 H 27 N3NaO6[M+Na] + :464.1792,found:464.1793.

[0053] Example 7 Synthesis of tert-butyl 2-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperidin-4-yl)acetate (2b)

[0054]

[0055] According to the method of Example 6, tert-butyl 2-(piperidin-4-yl)acetate was used to replace tert-butyl 4-piperidinane. Compound 2b was obtained as a yellow solid in a yield of 66%. 1H NMR (400MHz, CDCl3) δ8.28 (s, 1H), 7.57 (dd, 1H, J1 = 7.2Hz, J2 = 8.4Hz), 7.37 (d, 1H, J = 7.2Hz), 7.18 (d, 1H, J = 8.4Hz), 4.98 (dd, 1H, J1 = 5.2Hz, J2=12.4Hz),3.74–3.66(m,2H),2.96–2.66(m,6H),2.12–2.06(m,1H), 2.00–1.92(m,1H),1.87–1.83(m,3H),1.57–1.51(m,2H),1.47(s,9H). 13 C NMR (101 MHz, CDCl3) δ 13 C NMR (101MHz, CDCl3) δ178.77,178.04,175.24,172.28,171.52,155.16,141.01,138.83,129.13, 121.81,119.89,84.87,55.43,55.38,53.98,45.83,36.18,33.24,32.92,27.27.HRMS(ESI)calcd for C 24 H 29 N3NaO6[M+Na] + :478.1979,found:478.1979.

[0056] Example 8 Synthesis of tert-butyl 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)piperidine-4-carboxylate (2c)

[0057]

[0058] According to the method of Example 6, 1a was replaced by 1b to obtain compound 2c as a yellow solid in a yield of 67%. 1 H NMR (400MHz, CDCl3) δ8.39 (s, 1H), 7.68 (d, 1H, J = 8.4Hz), 7.29 (d, 1H, J = 2.0H z),7.06(dd,1H,J1=2.4Hz,J2=8.4Hz),4.97–4.92(m,1H),3.89(t,1H,J=4.0H z),3.86(t,1H,J=4.0Hz),3.11–3.04(m,2H),2.91–2.69(m,3H),2.52–2.45( m,1H),2.18–2.10(m,1H),2.03–1.99(m,2H),1.84–1.74(m,2H),1.46(s,9H). 13CNMR (101MHz, CDCl3) δ173.55,171.29,168.45,168.00,167.26,155.23,134.37,125.47,119. 00,118.03,108.79,80.75,49.12,47.39,41.52,31.45,28.07,27.43,22.74.HRMS(ESI)calcd for C 23 H 27 N3NaO6[M+Na] + :464.1792,found:464.1796.

[0059] Example 9 Synthesis of tert-butyl 2-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)piperidin-4-yl)acetate (2d)

[0060]

[0061] According to the method of Example 6, tert-butyl 2-(piperidin-4-yl)acetate was used to replace tert-butyl 4-piperidinanoate, and 1b was used to replace 1a to obtain compound 2d as a yellow solid in a yield of 64%. 1 H NMR (400MHz, CDCl3) δ8.16 (s, 1H), 7.67 (d, 1H, J = 8.8Hz), 7.28 (d, 1H, J = 2.0Hz), 7. 05(dd,1H,J1=2.4Hz,J2=8.8Hz),4.94(dd,1H,J1=5.2Hz,J2=12.4Hz),3.96(t,1H,J =4.0Hz),3.92(t,1H,J=4.0Hz),3.04–2.97(m,2H),2.91–2.72(m,3H),2.20–2.10( m,3H),2.07-2.00(m,1H),1.87–1.81(m,2H),1.46–1.45(s,9H),1.40–1.30(m,2H). 13 CNMR(101MHz, CDCl3)δ171.72,171.36,168.52,168.06,167.31,155.28,134.39,125.46,118.61, 117.81,108.65,80.61,49.10,48.03,42.09,32.99,31.45,31.13,28.15,22.75.HRMS(ESI)calcd for C 24 H 29 N3NaO6[M+Na] +:478.1949,found:478.1956.

[0062] Example 10 Synthesis of 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperidine-4-carboxylic acid (3a)

[0063]

[0064] Compound 2a (1.0 eq) was added to a 250 mL single-necked flask and dissolved in anhydrous dichloromethane. The mixture was stirred for 10 minutes under an ice bath. Then, a trifluoroacetic acid solution in anhydrous CH2Cl2 was slowly added dropwise. After the addition was complete, the ice bath was removed and the mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction system was concentrated in vacuo to obtain compound 3a. Compound 3a was used directly in the next step without further purification. HRMS (ESI) calculated for C 19 H 18 N3O6[MH] - :384.1201,found:384.1209.

[0065] Example 11 Synthesis of 2-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperidin-4-yl)acetic acid (3b)

[0066]

[0067] Following the method of Example 10, compound 2b was substituted for compound 2a to obtain compound 3b as a yellow solid in 89% yield. Compound 3b was used directly in the next reaction without further purification. HRMS (ESI) calculation for C 20 H 20 N3O6[MH] - :398.1358,found:398.1355.

[0068] Example 12 Synthesis of 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)piperidine-4-carboxylic acid (3c)

[0069]

[0070] According to the method of Example 10, compound 2c was used to replace 2a to obtain compound 3c as a yellow solid with a yield of 87%. 1HNMR(400MHz,DMSO-d6)δ12.32(s,1H),11.11(s,1H),7.66(d,1H,J=8.8Hz),7.34(d, 1H, J=2.4Hz), 7.25 (dd, 1H, J1=2.4Hz, J2=8.8Hz), 5.08 (dd, 1H, J1=5.6Hz, J2=13.2Hz ),3.98(dt,2H,J1=4.0Hz,J2=13.6Hz),3.12–3.05(m,2H),2.94–2.84(m,1H),2.62–2 .53(m,2H),2.06–1.99(m,1H),1.92–1.84(m,2H),1.65–1.55(m,2H).HRMS(ESI)calcd for C 19 H 18 N3O6[MH] - :384.1201,found:384.1191.

[0071] Example 13 Synthesis of 2-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)piperidin-4-yl)acetic acid (3d)

[0072]

[0073] According to the method of Example 10, compound 2d was used to replace 2a to obtain compound 3d as a yellow solid with a yield of 72%. 1 HNMR(400MHz,DMSO-d6)δ12.12(s,1H),11.10(s,1H),7.65(d,1H,J=8.8Hz),7.31 (d,1H,J=2.4Hz),7.23(dd,1H,J1=2.4Hz,J2=8.8Hz),5.07(dd,1H,J1=5.2Hz,J2=1 2.8Hz),4.06–4.02(m,2H),3.00–2.84(m,3H),2.62–2.54(m,2H),2.18(d,2H,J=7 .2Hz),2.05–1.93(m,1H),1.76(dd,2H,J1=3.6Hz,J2=13.2Hz),1.27–1.16(m,3H). 13C NMR(101MHz,DMSO-d6)δ173.92,173.31,170.61,168.10,167.44,155.38,134.51,125.47,118.09 ,117.88,108.24,60.24,49.19,47.73,32.82,31.45,31.10,22.66,21.24,14.56.HRMS(ESI)calcd forC 20 H 20 N3O6[MH] - :398.1358,found:398.1352.

[0074] Example 14 Synthesis of 4-[4-[4-[5-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-(2-(trifluoromethoxy)ethyl)-1H-benzimidazol-2-yl]piperidine-1-carbonyl]piperidin-1-yl]-2-[2,6-dioxopiperidin-3-yl]isoindoledione (Ia)

[0075]

[0076] Compound 3a (1.0 eq) and HATU (1.15 eq) were added sequentially to a 50 mL two-necked flask under nitrogen atmosphere. Dissolved in anhydrous DMF was added and stirred thoroughly. N,N-diisopropylethylamine (4.2 eq) was added and stirred for 0.5 hours. M6 in anhydrous DMF was then added dropwise. The reaction was allowed to react at room temperature for 1 hour. After completion of the reaction, the reaction system was extracted with ethyl acetate and distilled water. The ethyl acetate layers were combined, washed with distilled water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated in vacuo. The concentrate was separated and purified on a silica gel column (dichloromethane:methanol = 30:1-20:1) to obtain compound Ia as a yellow solid in a 54% yield. 1H NMR (400MHz, DMSO-d6) δ11.12(s,1H),7.98(d,1H,J=2.4Hz),7.81(d,1H,J=2.4Hz),7.78(d,1H,J=1.6Hz),7.71–7.66(m ,1H),7.60(d,1H,J=8.4Hz),7.41(dd,1H,J1=1.6Hz,J2=8.4Hz),7.36–7.32(m,2H),5.12(dd,1H,J1=5.6Hz,J2=12.8Hz), 4.73(t,2H,J=5.2Hz), 4.56(d,1H,J=12.8Hz), 4.47(t,2H,J=5.2Hz), 4.20(d,1H,J=13.2Hz), 4.09(d,2H,J=12.0Hz), 3. 55(s,3H),3.25–3.17(m,1H),3.05–2.85(m,4H),2.79–2.69(m,1H),2.63–2.56(m,2H),2.11(s,3H),2.06–1.66(m,10H). 13 C NMR(101MHz,DMSO-d6)δ172.54,172.28,170.33,168.07,167.42,162.00,158.78,15 5.29,141.62,136.95,135.44,134.68,134.50,131.25,128.09,125.51,121.55(q,J C-F =255.5Hz),120.24,119.16,118.57,118.09,117.98,108.28,107.64,67.16,49.78,47.20,45.12,42.0 1,41.55,37.78,37.34,33.53,32.30,31.61,31.32,28.14,27.91,27.07,21.85,17.65.HRMS(ESI)calcd forC 41 H 43 F3N7O7[M+H] + :802.3171,found:802.3170.

[0077] Example 15 Synthesis of 4-[4-[2-[4-[6-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-(2-(trifluoromethoxy)ethyl)-1H-benzimidazol-2-yl]piperidin-1-yl]-2-oxoethyl]piperidin-1-yl]-2-[2,6-dioxopiperidin-3-yl]isoindoledione (Ib)

[0078]

[0079] According to the method of Example 14, compound 3b was used to replace 3a to obtain compound Ib as a yellow solid with a yield of 43%. 1 HNMR(400MHz, DMSO-d6)δ11.10(s,1H),7.98(d,1H,J=2.4Hz),7.82–7.80(m,2H),7.70–7.65(m,1H),7.61(d,1H,J=8.4H z),7.43(d,1H,J=8.4Hz),7.36–7.31(m,2H),5.11(dd,1H,J1=5.2Hz,J2=12.8Hz),4.74(t,2H,J=5.2Hz),4.57(d,1H,J= 12.8Hz),4.45(t,2H,J=5.2Hz),4.11(d,1H,J=13.6Hz),3.70(d,2H,J=10.4Hz),3.54(s,3H),3.24–3.17(m,2H),2.93–2 .84(m,3H),2.77–2.69(m,1H),2.62–2.55(m,2H),2.40–2.33(m,2H),2.11(s,3H),1.93–1.60(m,8H),1.53–1.35(m,2H). 13 C NMR(101MHz,DMSO-d6)δ173.31,170.54,169.87,167.58,166.75,162.01,158.7 0,150.63,136.93,136.19,135.29,134.75,134.12,128.10,124.43,121.54(q,J C-F =255.5Hz),120.51,118.94,118.47,116.81,114.86,107.75,67.11,55.39,51.56,49.22,45.48,42 .10,41.34,39.11,37.78,33.46,32.73,32.25,31.97,31.44,31.19,22.54,17.65.HRMS(ESI)calcd for C 42 H 45 F3N7O7[M+H] + :816.3327,found:816.3327.

[0080] Example 16 Synthesis of 5-[4-[4-[6-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-(2-(trifluoromethoxy)ethyl)-1H-benzimidazol-2-yl]piperidine-1-carbonyl]piperidin-1-yl]-2-[2,6-dioxopiperidin-3-yl]isoindoledione (Ic)

[0081]

[0082] According to the method of Example 14, compound 3c was used to replace 3a to obtain compound Ic as a yellow solid in a yield of 44%. 1 HNMR (400MHz, DMSO-d6) δ11.10(s,1H),7.98(d,1H,J=2.8Hz),7.81(dd,1H,J1=1.6Hz,J2=2.4Hz),7.78(d,1H,J=1.6Hz),7.67(d,1H,J=8.4 Hz), 7.60 (d, 1H, J = 8.4Hz), 7.41 (dd, 1H, J1 = 1.6Hz, J2 = 8.4Hz), 7.35 (d, 1H, J = 2.0Hz), 7.26 (dd, 1H, J1 = 2.0Hz, J2 = 8.8Hz), 5.08 (dd, 1H, J1 = 5 .6Hz, J2=12.8Hz), 4.73(t,2H,J=5.2Hz), 4.54(d,1H,J=12.8Hz), 4.46(t,2H,J=5.2Hz), 4.20(d,1H,J=13.2Hz), 4.09(d,2H,J=12.8Hz),3. 54(s,3H),3.27–3.21(m,1H),3.17–3.03(m,3H),2.94–2.85(m,1H),2 .77–2.68(m,1H),2.62–2.54(m,2H),2.11(s,3H),2.06–1.60(m,10H). 13 C NMR(101MHz,DMSO-d6)δ173.32,172.56,170.61,168.09,167.44,162.02,158.82,15 5.29,141.66,136.97,135.50,134.66,134.52,131.20,128.09,125.49,121.56(q,J C-F=255.5Hz),120.23,119.24,118.62,118.06,118.02,108.27,107.61,67.17,49.20,47.21,45.13,4 1.99,41.56,37.77,37.34,33.54,32.31,31.45,31.33,28.13,27.90,22.66,17.63.HRMS(ESI)calcd for C 41 H 43 F3N7O7[M+H] + :802.3171,found:802.3163.

[0083] Example 17 Synthesis of 5-[4-[2-[4-[6-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-(2-(trifluoromethoxy)ethyl)-1H-benzimidazol-2-yl]piperidin-1-yl]-2-oxoethyl]piperidin-1-yl]-2-[2,6-dioxopiperidin-3-yl]isoindoledione (Id)

[0084]

[0085] According to the method of Example 14, compound 3d was used to replace 3a to obtain compound Id as a yellow solid in a yield of 57%. 1HNMR (400MHz, DMSO-d6) δ11.10 (s, 1H), 7.98 (d, 1H, J = 2.4Hz), 7.79 (dd, 1H, J1 = 1.6Hz, J2 = 2.8Hz), 7.77 (d, 1H, J = 1.6Hz), 7.65 (d, 1H, J = 8.4Hz), 7.5 8(d,1H,J=8.4Hz),7.39(dd,1H,J1=1.6Hz,J2=8.4Hz),7.33(d,1H,J=2.4Hz),7.25(dd,1H,J1=2.4Hz,J2=8.8Hz),5.07(dd,1H,J1=5.2Hz,J2=12.8H z), 4.71 (t, 2H, J = 5.2Hz), 4.54 (d, 1H, J = 12.8Hz), 4.45 (t, 2H, J = 4.8Hz), 4.08–4.02(m,3H),3.54(s,3H),3.30–3.26(m,1H),3.17–3.14(m,1H),3.0 0(t,2H,J=12.4Hz),2.93–2.84(m,1H),2.76–2.70(m,1H),2.61–2.54(m,2 H),2.40–2.28(m,2H),2.11(s,3H),2.03–1.99(m,3H),1.91–1.55(m,7H). 13 C NMR(101MHz,DMSO-d6)δ173.33,170.63,169.80,168.13,167.46,162.04,158.85,15 5.40,141.76,137.00,135.52,134.65,134.52,131.14,128.10,125.48,121.55(q,J C-F =255.5Hz),120.18,119.27,118.66,118.05,117.74,108.20,107.57,67.16,49.19,47.86,45.45,4 1.95,41.36,39.00,37.78,33.49,33.07,32.02,31.44,31.37,31.27,22.66,17.63.HRMS(ESI)calcd forC 42 H 45 F3N7O7[M+H] + :816.3327,found:816.3339.

[0086] The in vitro anti-tumor activity of the PROTAC molecules synthesized in the present invention and the degradation activity of BRD4 and p300 / CBP were evaluated.

[0087] 1. Evaluation of antitumor activity in vitro

[0088] 3×10 3 PC-3 cells were seeded into 96-well plates and incubated with different concentrations of PROTAC molecules Ia-Id, NEO2734, PTX, and ARV-771 at 37°C and 5% CO2 for 72 hours. Subsequently, 10 μL of CCK-8 reagent (TargetMol) was added to each well and incubated for another 2 hours. The absorbance was measured at a wavelength of 450 nm using a microplate reader (SpectraMax i3X, Molecular Devices, USA). Finally, the IC was calculated using GraphPad Prism software version 10.0. 50 value.

[0089] 2. Evaluation of PROTAC concentration-dependent induction of BRD4 and p300 / CBP degradation activity

[0090] In a 96-well plate, add 1 × 10 6 PC-3 cells / well were incubated with various concentrations of PROTAC Ic for 12 hours (for BRD4 detection) or 24 hours (for p300 / CBP detection). Cells were then washed twice with pre-chilled PBS and lysed in ice-cold RIPA buffer. The lysate was centrifuged to remove impurities, and protein concentration was determined using a BCA protein quantification kit (Beyotime, China, P0010). Equal amounts of cell extracts were separated by SDS-PAGE and transferred to nitrocellulose membranes (Millipore, USA, IPVH00010). The membranes were then incubated with the appropriate primary antibodies (BRD4, AB128874; p300, 54062S; CBP, 7389S) at 4°C overnight and then treated with HRP-conjugated secondary antibodies (ZSGB-BIO, China, ZB-2301) for 2 hours at room temperature. Finally, immunoblots were detected using an enhanced chemiluminescence system.

[0091] 3. Evaluation of PROTAC Time-Dependent Induction of BRD4, p300, and CBP Degradation Activity

[0092] In a 96-well plate, add 1 × 10 6PC-3 cells / well were incubated with 1 nM (for BRD4 detection) or 30 nM (for p300 / CBP detection) PROTAC molecule Ic for different times. Subsequently, the cells were washed twice with pre-chilled PBS and lysed in ice-cold RIPA buffer. After the lysate was centrifuged to remove impurities, the protein concentration was determined using a BCA protein quantification kit (Beyotime, China, P0010). Equal amounts of cell extracts were separated by SDS-PAGE and transferred to nitrocellulose membranes (Millipore, USA, IPVH00010). Subsequently, the membranes were incubated with appropriate primary antibodies overnight at 4°C and then treated with HRP-conjugated secondary antibodies (ZSGB-BIO, China, ZB-2301) at room temperature for 2 hours. Finally, immunoblots were detected using an enhanced chemiluminescence system.

[0093] 4. Experimental results

[0094] 4.1. In vitro antitumor activity evaluation results

[0095] Table 1 In vitro tumor activity evaluation results of target compounds

[0096]

[0097] a IC 50 Values ​​are expressed as mean ± SD

[0098] We evaluated the in vitro anti-tumor activity of four PROTAC molecules Ia-Id in three human prostate cancer cell lines (PC-3, DU145 and 22Rv1) and human normal prostate epithelial cells RWPE-1, and selected the BRD4-p300 / CBP dual-target small molecule inhibitor NEO2734, paclitaxel and BRD4 degrader ARV-771 as positive control drugs. According to the evaluation results in Table 1, PROTAC molecules Ia-Id showed excellent anti-tumor activity in all three cell lines, especially in PC-3 and DU145 cell lines, with IC 50 The values ​​were all below 10 nM. Among them, the most active compound Ic had an IC 50 The values ​​were 2.80nM and 6.62nM, respectively, which were significantly better than the positive control drugs NEO2734, paclitaxel and ARV-771. In PC-3 cells, the anti-tumor activity of compound Ic was 3.6 times, 2.4 times and 2 times that of NEO2734, paclitaxel and ARV-771, respectively, showing its excellent in vitro anti-tumor activity. In addition, in normal human prostate epithelial cells RWPE-1, compound Ic showed significantly lower cytotoxicity, IC 50The toxicity of paclitaxel was 4.38 μM, which was comparable to that of NEO2734 and ARV-771, while the toxicity of paclitaxel was significantly higher (IC 50 The selectivity index of compound Ic between PC-3 / RWPE-1 was over 1500, and the selectivity index between DU145 / RWPE-1 was over 600, indicating that compound Ic has good selectivity and a broad therapeutic window.

[0099] 4.2 Evaluation results of BRD4 and p300 / CBP degradation activity

[0100] We selected compound Ic with the strongest in vitro antitumor activity and evaluated its degradation activity against BRD4 and p300 / CBP proteins in PC-3 cells. Figure 1 The results in Figure A show that after treating PC-3 cells with different concentrations of compound Ic for 12 hours, the expression of BRD4 protein and c-Myc was significantly reduced. 50 The (half-maximal degradation concentration) value was less than 0.1 nM. At a concentration of 1.0 nM, BRD4 was almost completely degraded, demonstrating its excellent degradation activity. Figure 1 Middle B shows that after PC-3 cells were treated with 1.0 nM compound Ic for different time periods, the expression levels of BRD4 and c-Myc were significantly reduced, and BRD4 was almost completely degraded after 12 hours of treatment.

[0101] same, Figure 2 A shows that treatment of PC-3 cells with different concentrations of compound Ic for 24 hours can significantly reduce the expression of p300 and CBP proteins in a concentration-dependent manner, among which DC of p300 50 The value is 1.05nM, D max (maximum degradation rate) greater than 97%; DC for CBP protein 50 The value is 5.96nM, D max Greater than 96%. Figure 2 Figure B further shows that after treating PC-3 cells with 30nM compound Ic for different time periods, the expression levels of p300 and CBP were significantly reduced, and p300 / CBP was almost completely degraded after 24 hours of treatment. In addition, compound Ic was also able to effectively degrade BRD4 and CBP / p300 proteins in DU145 and RM-1 cancer cell lines ( Figure 3 These results indicate that compound Ic is an excellent dual-target degrader of BRD4 and p300 / CBP.

[0102] In summary, the BRD4 and p300 / CBP dual-target PROTAC molecules provided by the present invention can efficiently and simultaneously degrade BRD4 and p300 / CBP proteins and show excellent in vitro tumor activity, providing new compound molecules for the research and development of PROTAC molecules and anti-tumor drugs based on BRD4 and p300 / CBP dual targets, which can be used as further candidate or lead compounds for the development of anti-tumor therapeutic drugs.

Claims

1. A class of BRD4 and p300 / CBP dual-target PROTAC molecules, characterized by: It has the structure shown in general formula I: In the general formula I, L is selected from One of the following; E is selected from One of them.

2. A class of BRD4 and p300 / CBP dual-target PROTAC molecules according to claim 1, characterized in that One of the following compounds:

3. A method for preparing a BRD4 and p300 / CBP dual-target PROTAC molecule according to claim 1 or 2, characterized in that: This is achieved through the following steps:

1. Synthesis of Compound M6 (1) Preparation of Compound M2: Compound M1 and 2-(trifluoromethoxy)ethylamine are reacted in a polar organic solvent in the presence of an alkaline reagent under stirring at room temperature; after the reaction, the reaction system is extracted, washed, dried, filtered, concentrated, and purified by column chromatography to obtain Compound M2; wherein the polar organic solvent is selected from one of N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), N,N-diethylformamide (DEF), hexamethylphosphoramide (HMPA) and acetonitrile (MeCN); the alkaline reagent is selected from one of N,N-diisopropylethylamine, triethylamine, potassium tert-butoxide, potassium tert-butoxide, cesium carbonate, sodium carbonate and potassium carbonate; (2) Preparation of Compound M3: Compound M2, zinc powder, and acetic acid are stirred in ethanol at room temperature. After the reaction, the reaction system is sequentially extracted, washed, dried, filtered, and concentrated. The resulting concentrate is separated and purified by column chromatography to obtain Compound M3. (3) Preparation of Compound M4: Compound M3, 1-Boc-4-piperidinecarboxaldehyde, and sodium bisulfite are reacted in methanol solvent under heating and stirring; after the reaction, the reaction system is sequentially extracted, washed, dried, filtered, and concentrated, and the resulting concentrate is separated and purified by column chromatography to obtain Compound M4; (4) Preparation of Compound M5: Compound M4 and 1,3-dimethylpyridin-2(1H)-one-5-pinacol borate are reacted in a toluene solvent in the presence of an alkaline reagent and a palladium catalyst under heating conditions; after the reaction, the reaction system is sequentially extracted, washed, dried, filtered and concentrated, and the obtained concentrate is separated and purified by column chromatography to obtain Compound M5; wherein the alkaline reagent is selected from one of potassium carbonate, sodium carbonate, sodium bicarbonate, cesium carbonate and potassium phosphate, and the palladium catalyst is selected from one of tetrakis(triphenylphosphine)palladium, palladium acetate, bis(dibenzylideneacetone)palladium and palladium dichloride; (5) Preparation of Compound M6: Compound M5 was reacted in anhydrous dichloromethane solution in the presence of trifluoroacetic acid under stirring at room temperature. After the reaction, the reaction system was extracted, washed, dried, filtered, and concentrated in sequence. The resulting concentrate was separated and purified by column chromatography to obtain Compound M6.

2. Synthesis of BRD4 and p300 / CBP dual-targeting PROTAC molecules Ia–Id (1) Synthesis of compounds 2a–2d: 4-fluorothalidomide or 5-fluorothalidomide and tert-butyl piperidine-4-carboxylate or tert-butyl 2-(piperidin-4-yl)acetate are reacted in a polar organic solvent in the presence of an alkaline reagent under heating and stirring; after the reaction, the reaction system is sequentially extracted, washed, dried, filtered, and concentrated, and the resulting concentrate is separated and purified by column chromatography to obtain compounds 2a–2d; wherein the polar organic solvent is selected from one of DMF, DMSO, NMP, DEF, HMPA, and MeCN, and the alkaline reagent is selected from one of potassium tert-butoxide, N,N-diisopropylethylamine, triethylamine, cesium carbonate, potassium carbonate, and sodium carbonate; (2) Synthesis of compounds 3a–3d: Compounds 2a–2d were reacted in anhydrous dichloromethane solution in the presence of trifluoroacetic acid under stirring at room temperature. After the reaction, the reaction system was sequentially extracted, washed, dried, filtered, and concentrated. The resulting concentrate was separated and purified by column chromatography to obtain compounds 3a–3d. (3) Preparation of Compounds Ia–Id: Compounds 3a–3d are reacted with M6 and an amide coupling reagent, respectively, in a polar organic solvent in the presence of an alkaline reagent, and stirred at room temperature; after the reaction, the reaction system is sequentially extracted, washed, dried, filtered, and concentrated, and the resulting concentrate is separated and purified by column chromatography to obtain Compounds Ia–Id; wherein the amide coupling reagent is selected from O-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBT U), O-(7-aza-1-hydroxybenzotriazole)-tetramethyluronium hexafluorophosphate (HATU), (1-phenyl-1H-benzotriazole-1-yl)dimethylsilylcarbonyl chloride (TBTU), carbonyldiimidazole (CDI), dicyclohexylcarbodiimide (DCC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI); the alkaline reagent is selected from one of potassium tert-butoxide, N,N-diisopropylethylamine and triethylamine; 4. Use of a BRD4 and p300 / CBP dual-target PROTAC molecule according to any one of claims 1-2 in drug preparation, characterized in that: It is used as an active ingredient in the preparation of BRD4 and p300 / CBP dual-target PROTAC molecular drugs.

5. Use of a class of BRD4 and p300 / CBP dual-target PROTAC molecules in drug preparation according to claim 4, characterized in that: It is used as an active ingredient in the preparation of tumor treatment drugs.

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