BRD4 and p300 / CBP double-target degradation agent as well as preparation method and application thereof

Abstract

The invention relates to a BRD4 and p300 / CBP double-target degradation agent as well as a preparation method and application thereof, and belongs to the technical field of medicinal chemistry. The compound has the following general formula: BRD4 and p300 / CBP protein can be efficiently degraded at the same time in a concentration-dependent and time-dependent manner, the compound shows remarkable anti-tumor activity on a PC-3 cell line in vitro, and IC50 is lower than 30 nM. The overall activity is superior to that of positive control drugs NEO2734, paclitaxel and ARV-771, and the anti-proliferation effect is remarkably superior to that of existing control drugs. The compound can be used as a candidate or lead compound for the development of antitumor drugs, is simple and convenient in synthesis method, and has good development and application prospects.

Description

Technical Field

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

[0002] BRD4, also known as bromodomain-containing protein 4, is a member of the BET protein family. It is widely present in various biological tissues and plays a key role in biological regulation. It specifically recognizes and binds to acetylated lysine sites in chromatin, thereby recruiting the transcription elongation factor P-TEFb. This process promotes the phosphorylation of RNA polymerase II, effectively regulating the elongation phase of gene transcription. BRD4 plays a crucial role in maintaining gene promoter activity and regulating the transcriptional activation of genes associated with super-enhancers, particularly in the fine regulation of the cell cycle. Furthermore, BRD4 participates in DNA damage repair mechanisms, activating related responses to ensure genomic stability. Research has shown that aberrant BRD4 activation is closely associated with the progression of various malignancies. By binding to super-enhancers, BRD4 activates tumor-related genes such as c-Myc and BCL-2, thereby promoting rapid tumor cell proliferation and enhancing drug resistance. Given its importance in tumorigenesis and progression, BRD4 has become a hot topic in the development of anti-cancer drugs.

[0003] p300 and CBP are highly homologous histone acetyltransferases that are widely involved in multiple transcriptional regulatory pathways and play important biological roles. By acetylating histones, p300 and CBP relax chromatin structure, thereby enhancing the binding of transcription factors and promoting gene expression. Furthermore, p300 and CBP regulate the function of multiple transcription factors, such as c-Myc and AP-1, thereby activating the expression of genes associated with cell proliferation, significantly promoting tumor cell proliferation. p300 and CBP also inhibit apoptotic signaling by enhancing the activity of anti-apoptotic transcription factors such as NF-κB, thereby maintaining tumor cell survival. Furthermore, p300 and CBP play an important role in regulating the expression of cytokines and chemokines. p300 and CBP can also enhance the interaction between tumor cells and surrounding immune cells. By upregulating the expression of tumor-promoting cytokines and inhibiting the production of anti-tumor cytokines, p300 and CBP lead to the formation of an immunosuppressive microenvironment. This altered immunosuppressive microenvironment not only facilitates the survival and proliferation of tumor cells but also effectively evades the host immune system, allowing tumor cells to persist and spread in the body for extended periods of time. Because p300 and CBP play a key role in the development of malignant tumors, they have become important targets for the development of new anti-tumor drugs.

[0004] PROTAC (Proteolysis Targeting Chimera) is an emerging and promising anti-tumor drug development technology that can selectively recruit the endogenous ubiquitin-proteasome system to target and degrade specific oncogenic proteins, thereby achieving functional elimination rather than inhibition. Unlike traditional small molecule inhibitors that only block protein function, PROTAC completely eliminates pathogenic proteins by inducing ubiquitination of target proteins and guiding them into the proteasome degradation pathway, significantly reducing the risk of drug resistance caused by target mutations or feedback activation. PROTAC has many advantages: first, it can target "undruggable" proteins that are difficult for traditional drugs to act on, such as transcription factors and scaffold proteins that have no catalytic activity or obvious binding pockets; second, as a "catalytic" molecule, PROTAC can be recycled in the cell, and one molecule can repeatedly induce the degradation of multiple target proteins. Therefore, a low dose is often required to achieve significant drug efficacy, reducing the risk of systemic toxicity and adverse reactions; third, the complete degradation of the target protein can more lastingly inhibit the oncogenic signaling pathway and enhance the anti-tumor efficacy. At present, multiple PROTAC molecules targeting proteins such as AR, ER, BCL-xL, p300 / CBP have entered the clinical stage, showing good safety and preliminary efficacy, pushing this technology to become a research hotspot and industry focus in the field of precision medicine and 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 the proliferation and survival of tumor cells. Therefore, the design and development of novel BRD4 and p300 / CBP dual-targeting PROTAC degraders 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 class of compounds that can simultaneously degrade BRD4 and p300 / CBP proteins, thereby achieving excellent degradation activity on BRD4 and p300 / CBP proteins.

[0007] Another object of the present invention is to provide a method for preparing the aforementioned BRD4 and p300 / CBP dual-target degrader. Yet another object of the present invention is to provide the use of the aforementioned BRD4 and p300 / CBP dual-target degrader in the preparation of an anti-tumor drug.

[0008] To achieve the above objectives, the general structural formula of the BRD4 and p300 / CBP dual-target degrader involved in the present invention is:

[0009]

[0010] In the general formula I, L is selected from

[0011] One of the following;

[0012] E is selected from One of them.

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

[0014]

[0015]

[0016] The preparation method of the compound of the present invention comprises the following steps:

[0017] 1. Synthesis of Compound M6

[0018]

[0019] (1) Preparation of Compound M2: Compound M1 and 2-(trifluoromethoxy)ethylamine are reacted in dimethyl sulfoxide (DMSO) in the presence of an alkaline reagent under stirring at room temperature. 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 M2. 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.

[0020] (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.

[0021] (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.

[0022] (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 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.

[0023] (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.

[0024] 2. Synthesis of BRD4 and p300 / CBP Dual-Target Degrader I

[0025]

[0026] (1) Preparation of compounds 2a–2j and 4a–4h: 4-hydroxythalidomide or 5-hydroxythalidomide is reacted with tert-butyl bromoalkanoate or terminal dibromoalkane 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, and the resulting concentrate is separated and purified by column chromatography to obtain compounds 2a–2j and 4a–4h. The polar organic solvent is selected from N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), N,N-diethylformamide (DEF), hexamethylphosphoric triamide (HMPA) and acetonitrile (MeCN) or other solvents with similar solubility properties, and the alkaline reagent is selected from sodium bicarbonate, potassium bicarbonate, sodium carbonate and potassium carbonate.

[0027] (2) Preparation of compounds 3a–3j: Compounds 2a–2j were reacted separately in anhydrous dichloromethane in the presence of trifluoroacetic acid with 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 compounds 3a–3j.

[0028] (3) Preparation of Compounds Ia–Ij: Compounds 3a–3j were reacted with M6 and an amide coupling reagent, respectively, in DMF in the presence of an alkaline reagent under stirring at room temperature. 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 Ia–Ij. 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-benzotriazole-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.

[0029] (4) Preparation of Compounds Ik–Ir: Compounds 4a–4h were reacted with M6 and an amide coupling reagent, respectively, in DMF in the presence of an alkaline reagent, under stirring at room temperature. 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 Ik–Ir. The amide coupling reagent was selected from one of HBTU, HATU, TBTU, DCC, and EDCI; and the alkaline reagent was selected from one of potassium tert-butoxide, N,N-diisopropylethylamine, and triethylamine.

[0030] Advantages of the present invention:

[0031] The compounds of the present invention can simultaneously degrade BRD4 and p300 / CBP proteins in a highly efficient, concentration-dependent, and time-dependent manner. For example, the representative compound Ig, when treated with PC-3 cells at a concentration of 1.0 nM for 12 hours, completely degraded BRD4 protein; and when treated with a concentration of 30 nM for 24 hours, completely degraded p300 / CBP protein. The compounds of the present invention exhibited significant antitumor activity against human prostate cancer PC-3 cell lines in vitro, with IC 50 The IC values ​​of compounds Ia, Ib, Ie, Ig, Ii, Im, In, Ip, Iq, and Ir were all less than 30 nM. 50 The values ​​were all below 10 nM, and the overall activity was better than that of the positive control drugs NEO2734, paclitaxel and ARV-771. In particular, compound Ig had an IC 50The value was 1.98 nM, demonstrating significantly superior antiproliferative activity to the positive control drug. The compounds of this invention represent a novel class of dual-target degraders for BRD4 and p300 / CBP with a well-defined mechanism of action, providing an important foundation for the development of this class of targeted degraders. These compounds have excellent druggability and can serve as candidate or lead compounds for further development of anti-tumor drugs. Their simple synthesis facilitates large-scale application. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Figure 3 is a graph showing that the compound Ig of the present invention promotes the efficient degradation of BRD4 in PC-3 tumor cells, wherein A shows the dose-dependent induction of BRD4 protein degradation after PC-3 cells are treated with different concentrations of compound Ig for 24 hours; B shows the time-dependent induction of BRD4 protein degradation after PC-3 cells are treated with 10.0 nM compound Ig for different time periods.

[0033] Figure 2 The graph shows that the compound Ig of the present invention promotes the efficient degradation of p300 and CBP in PC-3 tumor cells, wherein A shows the dose-dependent induction of p300 and CBP protein degradation after PC-3 cells are treated with different concentrations of compound Ig for 24 hours; B shows the time-dependent induction of p300 and CBP protein degradation after PC-3 cells are treated with 30 nM concentration of compound Ig for different time periods. 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 vacuum dried to obtain compound M2 as a yellow solid in a 93% yield. 1H 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 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 oily liquid with a yield of 89%. 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 100 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 separated and purified on 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 in 84% yield. 1 H NMR (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 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)oxy)acetate (2a)

[0051]

[0052] To a 100 mL single-necked flask, 4-fluorothalidomide (1.0 eq), tert-butyl 2-bromoacetate (1.1 eq), sodium bicarbonate (1.2 eq), and sodium iodide (0.3 eq) were added, followed by anhydrous DMF, and the mixture was stirred at 70°C for 72 hours. 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 afford compound 2a as a white solid in 84% yield. 1 H NMR (400MHz, CDCl3) δ8.46 (s, 1H), 7.67 (dd, 1H, J1 = 7.2Hz, J2 = 8.4Hz), 7.50 (d, 1H, J = 7.2Hz), 7.10 (d, 1H, J = 8 .4Hz), 5.00 (dd, 1H, J1=5.2Hz, J2=12.0Hz), 4.79 (s, 2H), 2.91–2.75 (m, 3H), 2.18–2.11 (m, 1H), 1.48 (s, 9H). 13 C NMR (101MHz, CDCl3) δ171.31,168.17,166.93,166.87,165.50,155.48,136.31,133.8 9,119.73,117.54,116.89,83.13,66.51,49.17,31.37,28.04,22.56.HRMS(ESI)calcd for C 19 H 24 N3O7[M+NH4] + :406.1609,found:406.1597.

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

[0054]

[0055] According to the method of Example 6, tert-butyl 4-bromobutyrate was used to replace tert-butyl 2-bromoacetate to obtain compound 2b as a white solid with a yield of 66%. 1H NMR (400MHz, CDCl3) δ8.32 (s, 1H), 7.68 (dd, 1H, J1 = 7.6Hz, J2 = 8.4Hz), 7.46 (dd, 1H, J1 = 0.8Hz, J2 = 7.2Hz), 7.25 (d, 1H, J = 8.4Hz), 4. 97(dd,1H,J1=5.2Hz,J2=12.0Hz),4.24.(t,2H,J=6.0Hz),2.96–2.70(m,3H),2.51(t,2H,J=7.2Hz),2.18–2.10(m,3H),1.45(s,9H). 13 C NMR (101MHz, CDCl3) δ172.40,171.04,168.14,167.06,165.68,156.48,136.59,133.79,1 19.02,117.18,115.93,80.60,68.27,49.09,31.42,28.12,24.34,22.64.HRMS(ESI)calcd for C 21 H 24 N2NaO7[M+Na] + :439.1476,found:439.1473.

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

[0057]

[0058] According to the method of Example 6, tert-butyl 6-bromovalerate was used to replace tert-butyl 2-bromoacetate to obtain compound 2c as a white solid with a yield of 62%. 1 H NMR (400MHz, CDCl3) δ8.29(s,1H),7.67(dd,1H,J1=7.2Hz,J2=8.4Hz),7.45(dd,1H,J1=0.8Hz,J2=7.2Hz),7.21(d,1H,J=8.4Hz),4.96(dd,1H,J1=5.2H z, J2=12.4Hz), 4.19. (t, 2H, J=6.4Hz), 2.91-2.69 (m, 3H), 2.33 (t, 2H, J=7. 2Hz),2.16–2.10(m,1H),1.96–1.89(m,2H),1.85–1.77(m,2H),1.45(s,9H). 13C NMR (101MHz, CDCl3) δ172.75,171.18,168.23,167.08,165.65,156.55,136.51,133.81,118.93, 117.15,115.81,80.29,69.00,49.08,34.96,31.39,28.30,28.12,22.62,21.43.HRMS(ESI)calcd for C 22 H 26 N2NaO7[M+Na] + :453.1632,found:453.1627.

[0059] Example 9 Synthesis of tert-butyl 7-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)oxy)heptanoate (2d)

[0060]

[0061] According to the method of Example 6, tert-butyl 7-bromoheptanoate was used to replace tert-butyl 2-bromoacetate to obtain compound 2d as a white solid with a yield of 60%. 1 H NMR (400MHz, CDCl3) δ8.29 (s, 1H), 7.67 (t, 1H, J = 7.2Hz), 7.44 (d, 1H, J = 5.6Hz), 7.21 (d, 1H, J = 8.4Hz), 4.96 (dd, 1H, J1 = 5.2Hz, J2 = 12.0Hz), 4.17 (t, 2H ,J=6.8Hz),2.91–2.71(m,3H),2.23(t,2H,J=6.8Hz),2.14–2.10(m,1H),1. 92–1.85(m,2H),1.75–1.61(m,2H),1.55–1.51(m,2H),1.47–1.37(m,11H). 13 C NMR (101MHz, CDCl3) δ173.15,171.04,168.14,167.08,165.66,156.70,136.46,133.83,118.98,117.16,1 15.73,80.01,69.36,49.10,35.47,31.40,28.74,28.70,28.13,25.52,24.93,22.63.HRMS(ESI)calcdfor C 24 H 30 N2NaO7[M+Na] + :481.1945,found:481.1958.

[0062] Example 10 Synthesis of tert-butyl 9-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)nonanoate (2e)

[0063]

[0064] According to the method of Example 6, tert-butyl 9-bromononanoate was used to replace tert-butyl 2-bromoacetate to obtain compound 2e as a white solid with a yield of 61%. 1 H NMR (400MHz, CDCl3) δ8.35 (s, 1H), 7.67 (dd, 1H, J1 = 7.2Hz, J2 = 8.4Hz), 7.44 (d, 1H ,J=7.2Hz),7.21(d,1H,J=8.4Hz),4.97(dd,1H,J1=5.2Hz,J2=12.4Hz),4.17(t,2 H,J=6.8Hz),2.91–2.69(m,3H),2.21(t,2H,J=7.6Hz),2.16–2.10(m,1H),1.91–1 .84(m,2H),1.61–1.54(m,2H),1.52–1.46(m,2H),1.44(s,9H),1.39–1.28(m,6H). 13 C NMR (101MHz, CDCl3) δ173.36,171.14,168.20,167.12,165.71,156.72,136.49,133.81,118.93,117.10,115.6 9,79.97,69.44,49.08,35.60,31.40,29.17,29.10,29.00,28.86,28.13,25.76,25.06,22.64.HRMS(ESI)calcd for C 26 H 34 N2NaO7[M+Na] + :509.2258,found:509.2255.

[0065] Example 11 Synthesis of tert-butyl 10-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)decanoate (2f)

[0066]

[0067] According to the method of Example 6, tert-butyl 10-bromodecanoate was used to replace tert-butyl 2-bromoacetate to obtain compound 2f as a white solid with a yield of 64%. 1H NMR (400MHz, CDCl3) δ8.32 (s, 1H), 7.67 (dd, 1H, J1 = 7.6Hz, J2 = 8.4Hz), 7.44 (d, 1H ,J=7.2Hz),7.22(d,1H,J=8.4Hz),4.97(dd,1H,J1=5.2Hz,J2=12.0Hz),4.17(t,2 H,J=6.8Hz),2.91–2.69(m,3H),2.20(t,2H,J=7.6Hz),2.16–2.10(m,1H),1.91–1 .84(m,2H),1.61–1.54(m,2H),1.52–1.46(m,2H),1.44(s,9H),1.38–1.24(m,8H). 13 C NMR (101MHz, CDCl3) δ173.43,171.16,168.21,167.13,165.71,156.72,136.49,133.80,118.89,117.08,115.6 8,79.97,69.43,49.07,35.62,31.41,29.32,29.21,29.08,28.87,28.13,25.76,25.11,22.64.HRMS(ESI)calcd for C 27 H 36 N2NaO7[M+Na] + :523.2415,found:523.2412.

[0068] Example 12 tert-Butyl 11-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)undecanoate (2 g)

[0069]

[0070] According to the method of Example 6, tert-butyl 11-bromodecaneate was used to replace tert-butyl 2-bromoacetate to obtain 2 g of compound as a white solid. The yield was 63%. 1H NMR (400MHz, CDCl3) δ8.40 (s, 1H), 7.67 (dd, 1H, J1 = 7.2Hz, J2 = 8.4Hz), 7.44 (d, 1H ,J=7.2Hz),7.22(d,1H,J=8.4Hz),4.97(dd,1H,J1=5.2Hz,J2=12.0Hz),4.17(t,2H ,J=6.8Hz),2.91–2.70(m,3H),2.20(t,2H,J=7.6Hz),2.14–2.10(m,1H),1.91–1. 84(m,2H),1.59–1.53(m,2H),1.51–1.48(m,2H),1.44(s,9H),1.36–1.26(m,10H). 13 C NMR (101MHz, CDCl3) δ173.41,171.18,168.23,167.13,165.71,156.73,136.48,133.80,118.91,117.08,115.67,79.9 4,69.46,49.07,35.62,31.41,29.44,29.37,29.27,29.25,29.08,28.89,28.13,25.80,25.10,22.63.HRMS(ESI)calcd for C 28 H 38 N2NaO7[M+Na] + :537.2571,found:537.2574.

[0071] Example 13 Synthesis of tert-butyl 7-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)oxy)heptanoate (2h)

[0072]

[0073] According to synthetic route II and the method of Example 6, compound 2h was obtained as a colorless oil in a yield of 47% by replacing 1a with 1b and replacing 2-bromoacetate with tert-butyl 7-bromoheptanoate. 1H NMR (400MHz, DMSO-d6) δ11.14(s,1H),7.83(d,1H,J=8.4Hz),7.42(d,1H,J=2.4 Hz), 7.34 (dd, 1H, J1 = 2.4Hz, J2 = 8.4Hz), 5.13 (dd, 1H, J1 = 5.6Hz, J2 = 13.2Hz), 4. 16(t,2H,J=6.4Hz),2.95–2.85(m,1H),2.63–2.55(m,2H),2.19(t,2H,J=7.2Hz ),2.09–1.92(m,1H),1.78-1.71(m,2H),1.55–1.47(m,2H),1.46–1.23(m,13H). 13 C NMR(101MHz,DMSO-d6)δ173.26,172.74,170.41,167.36,167.29,164.55,134.41,125.77,123.32,121.1 6,109.28,79.82,69.17,49.42,35.13,31.43,28.66,28.51,28.22,25.50,24.98,22.55.HRMS(ESI)calcd for C 24 H 30 N2NaO7[M+Na] + :481.1945,found:481.1944.

[0074] Example 14 Synthesis of tert-butyl 8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)oxy)octanoate (2i)

[0075]

[0076] According to synthetic route II and the method of Example 6, compound 2i was obtained as a colorless oil with a yield of 41%, by replacing 1a with 1b and replacing 2-bromoacetate with tert-butyl 8-bromooctanoate. 1H NMR (400MHz, DMSO-d6) δ11.13(s,1H),7.83(d,1H,J=8.4Hz),7.42(d,1H,J=2.0 Hz), 7.34 (dd, 1H, J1 = 2.4Hz, J2 = 8.4Hz), 5.13 (dd, 1H, J1 = 5.6Hz, J2 = 13.2Hz), 4. 17(t,2H,J=6.8Hz),2.94–2.85(m,1H),2.62–2.54(m,2H),2.18(t,2H,J=7.2Hz ),2.09–2.02(m,1H),1.78–1.71(m,2H),1.54–1.47(m,2H),1.42–1.22(m,15H). 13 C NMR(101MHz,DMSO-d6)δ173.26,172.77,170.42,167.37,167.29,164.56,134.42,125.77,123.32,12 1.18,109.28,79.80,69.21,49.41,35.19,31.43,28.78,28.23,25.66,25.00,22.54.HRMS(ESI)calcd for C 25 H 32 N2NaO7[M+Na] + :495.2102,found:495.2108.

[0077] Example 15 Synthesis of tert-butyl 9-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)oxy)nonanoate (2j)

[0078]

[0079] According to synthetic route II and the method of Example 6, compound 2j was obtained as a colorless oil with a yield of 43%, by replacing 1a with 1b and replacing 2-bromoacetate with tert-butyl 9-bromononanoate. 1H NMR (400MHz, DMSO-d6) δ11.14(s,1H),7.83(d,1H,J=8.4Hz),7.42(d,1H,J=2.4 Hz), 7.34 (dd, 1H, J1 = 2.4Hz, J2 = 8.4Hz), 5.13 (dd, 1H, J1 = 5.6Hz, J2 = 13.2Hz), 4. 16(t,2H,J=6.8Hz),2.94–2.85(m,1H),2.62–2.55(m,2H),2.17(t,2H,J=7.2Hz ),2.09–1.99(m,1H),1.78–1.71(m,2H),1.54–1.46(m,2H),1.42–1.22(m,17H). 13 C NMR(101MHz,DMSO-d6)δ173.25,172.77,170.41,167.36,167.29,164.57,134.42,125.77,123.31,121.17,1 09.27,79.79,69.25,49.41,35.22,31.43,29.09,29.01,28.79,28.22,25.76,25.06,22.55.HRMS(ESI)calcd for C 26 H 34 N2NaO7[M+Na] + :509.2258,found:509.2260.

[0080] Example 16 Synthesis of 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)oxy)acetic acid (3a)

[0081]

[0082] 2a (1.0 eq) was added to a 250 mL single-necked bottle, dissolved in anhydrous dichloromethane, stirred for 10 minutes under ice bath conditions, and then a solution of trifluoroacetic acid in anhydrous CH2Cl2 was slowly added dropwise. After the addition was completed, the mixture was slowly warmed to room temperature and the stirring reaction was continued for 0.5 hours. After the reaction was completed, the reaction system was concentrated under reduced pressure to obtain compound 3a as a white solid with a yield of 95%. Compound 3a did not require further purification and was used directly in the next reaction. HRMS (ESI) calculated for C 15 H 11 N2O7[MH] - :331.0572,found:359.0883.

[0083] Example 17 Synthesis of 4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)oxy)butanoic acid (3b)

[0084]

[0085] According to the method of Example 16, 2a was replaced by 2b to obtain compound 3b as a white solid in a yield of 95%. 1 H NMR (400MHz, DMSO-d6) δ12.21(s,1H),11.13(s,1H),7.81(dd,1H,J1=7.6Hz,J2=8.0Hz),7.52(d,1H,J=8.8Hz),7.45(d,1H,J=7.2Hz),5. 09(dd,1H,J1=5.2Hz,J2=12.8Hz),4.23(t,2H,J=6.4Hz),2.94–2.85(m,1H),2.62–2.54(m,2H),2.46(t,2H,J=7.6Hz),2.08-1.95(m,3H). 13 CNMR(101MHz,DMSO-d6)δ174.53,173.30,170.45,167.31,165.82,156.27,137.55,133. 69,120.23,116.76,115.77,68.28,49.20,31.42,30.19,24.48,22.46.HRMS(ESI)calcd for C 17 H 15 N2O7[MH] - :359.0885,found:359.0883.

[0086] Example 18 Synthesis of 6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)oxy)hexanoic acid (3c)

[0087]

[0088] Following the method of Example 16, 2c was substituted for 2a to obtain compound 3c as a white solid in a yield of 92%. HRMS (ESI) calculated for C 18 H 17 N2O7[MH] - :373.1041,found:373.1044.

[0089] Example 19 Synthesis of 7-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)oxy)nonanoic acid (3d)

[0090]

[0091] According to the method of Example 16, 2a was replaced by 2d to obtain compound 3d as a white solid in a yield of 92%. HRMS (ESI) calculated for C 20 H 21 N2O7[MH] - :401.1354,found:401.1357.

[0092] Example 20 Synthesis of 9-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)oxy)nonanoic acid (3f)

[0093]

[0094] According to the method of Example 16, 2e was used to replace 2a to obtain compound 3e as a white solid in a yield of 95%. 1 H NMR (400MHz, DMSO-d6) δ11.13 (s, 1H), 7.81 (dd, 1H, J1=7.2Hz, J2=8.4Hz), 7.51 (d,1H,J=8.8Hz),7.44(d,1H,J=7.2Hz),5.09(dd,1H,J1=5.2Hz,J2=12.8Hz),4 .20(t,2H,J=6.4Hz),2.93–2.84(m,1H),2.62–2.54(m,2H),2.19(t,2H,J=7.6H z),2.10–2.01(m,1H),1.79–1.72(m,2H),1.51–1.41(m,4H),1.38–1.04(m,6H). 13 C NMR(101MHz,DMSO-d6)δ175.01,173.29,170.46,167.34,165.79,156.48,137.51,133.70,120.22,116.6 4,115.59,69.23,49.19,34.13,31.43,29.17,29.03,28.97,28.86,25.72,24.95,22.47.HRMS(ESI)calcd for C 22 H 25 N2O7[MH] - :429.1667,found:429.1663.

[0095] Example 21 Synthesis of 10-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)decanoic acid (3f)

[0096] According to the method of Example 16, 2a was replaced by 2f to obtain compound 3f as a white solid in a yield of 94%. 1 H NMR (400MHz, DMSO-d6) δ12.00(s,1H),11.13(s,1H),7.81(dd,1H, J1=7.6Hz, J2=8. 0Hz), 7.51 (d, 1H, J = 8.8Hz), 7.44 (d, 1H, J = 7.2Hz), 5.09 (dd, 1H, J1 = 5.6Hz, J2 = 12. 8Hz),4.19(t,2H,J=6.4Hz),2.94–2.84(m,1H),2.62–2.53(m,2H),2.19(t,2H,J=7 .6Hz),2.07–2.01(m,1H),1.79–1.72(m,2H),1.50–1.42(m,4H),1.33–1.07(m,8H). 13 C NMR(101MHz,DMSO-d6)δ175.01,173.29,170.46,167.33,165.79,156.48,137.50,133.70,120.21,116.65,1 15.58,69.23,49.18,34.12,31.43,29.32,29.15,29.09,29.01,28.88,25.73,24.97,22.47.HRMS(ESI)calcd for C 23 H 28 N2NaO7[M+Na] + :467.1789,found:467.1786.

[0097] Example 22 11-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)undecanoic acid (3 g)

[0098]

[0099] According to the method of Example 16, 2g was used to replace 2a to obtain compound 3g as a white solid with a yield of 95%. 1H NMR(400MHz,DMSO-d6)δ12.00(s,1H),11.13(s,1H),7.82-7.78(m,1H),7.51(d ,1H,J=8.4Hz),7.44(d,1H,J=7.2Hz),5.09(dd,1H,J1=5.2Hz,J2=12.8Hz),4.1 9(t,2H,J=6.0Hz),2.94–2.85(m,1H),2.62–2.54(m,2H),2.19(t,2H,J=7.6Hz) ,2.05–2.02(m,1H),1.77–1.73(m,2H),1.57–1.46(m,4H),1.33–1.06(m,10H). 13 C NMR (101MHz, DMSO-d6) δ175.02,173.29,170.45,167.34,165.78,156.48,137.50,133.70,120.21,116.64,115. 58,69.23,49.18,34.13,31.43,29.41,29.33,29.21,29.13,29.03,28.88,25.75,24.97,22.47.HRMS(ESI)calcd for C 24 H 29 N2O7[MH] - :457.1980,found:457.1977.

[0100] Example 23 Synthesis of 7-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)oxy)heptanoic acid (3h)

[0101]

[0102] According to the method of Example 16, 2h was used to replace 2a to obtain compound 3h as a white solid in a yield of 94%. 1H NMR (400MHz, DMSO-d6) δ12.01 (s, 1H), 11.13 (s, 1H), 7.83 (d, 1H, J = 8.4Hz), 7. 42(d,1H,J=2.0Hz),7.35(dd,1H,J1=2.4Hz,J2=8.4Hz),5.13(dd,1H,J1=5.2H z,J2=12.8Hz),4.17(t,2H,J=6.8Hz),2.93–2.85(m,1H),2.62–2.55(m,2H),2 .22(t,2H,J=7.6Hz),2.09–2.03(m,1H),1.79–1.72(m,2H),1.56–1.23(m,6H). 13 C NMR(101MHz,DMSO-d6)δ174.96,173.26,170.43,167.37,167.30,164.56,134.41,125.77,123 .32,121.18,109.30,69.20,49.42,34.05,31.43,28.68,25.56,24.87,22.54.HRMS(ESI)calcd for C 20 H 22 N2NaO7[M+Na] + :425.1319,found:425.1323.

[0103] Example 24 Synthesis of tert-butyl 8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)oxy)octanoate (3i)

[0104]

[0105] According to the method of Example 16, 2a was replaced by 2i to obtain compound 3i as a white solid in 90% yield. 1 H NMR (400MHz, DMSO-d6) δ12.00 (s, 1H), 11.14 (s, 1H), 7.83 (d, 1H, J = 8.0Hz), 7. 42(d,1H,J=2.4Hz),7.35(dd,1H,J1=2.0Hz,J2=8.4Hz),5.13(dd,1H,J1=5.6H z,J2=13.2Hz),4.16(t,2H,J=6.8Hz),2.95–2.85(m,1H),2.63–2.53(m,2H),2 .21(t,2H,J=7.6Hz),2.09–2.03(m,1H),1.78–1.72(m,2H),1.54–1.23(m,8H). 13C NMR(101MHz,DMSO-d6)δ175.00,173.27,170.43,167.37,167.30,164.57,134.41,125.77,123.31,12 1.18,109.28,69.24,49.41,34.09,31.43,28.94,28.86,28.78,25.69,24.90,22.54.HRMS(ESI)calcd for C 21 H 24 N2NaO7[M+Na] + :439.1476,found:439.1481.

[0106] Example 25 Synthesis of tert-butyl 9-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)oxy)nonanoate (3j)

[0107]

[0108] According to the method of Example 16, 2a was replaced by 2j to obtain compound 3j as a white solid in a yield of 96%. 1 H NMR (400MHz, DMSO-d6) δ11.99(s,1H),11.13(s,1H),7.83(d,1H,J=8.4Hz),7. 42(d,1H,J=2.4Hz),7.34(dd,1H,J1=2.4Hz,J2=8.4Hz),5.12(dd,1H,J1=5.6Hz ,J2=13.2Hz),4.17(t,2H,J=6.8Hz),2.94–2.85(m,1H),2.62–2.54(m,2H),2. 19(t,2H,J=7.6Hz),2.09–1.99(m,1H),1.78–1.71(m,2H),1.53–1.16(m,10H). 13 C NMR(101MHz,DMSO-d6)δ174.99,173.27,170.43,167.37,167.30,164.57,134.42,125.78,123.31,121.1 9,109.28,69.26,49.41,34.12,31.42,29.16,29.04,28.97,28.82,25.78,24.95,22.54.HRMS(ESI)calcd forC 22 H 26 N2NaO7[M+Na] + :453.1632,found:453.1630.

[0109] Example 26 Synthesis of 4-(3-bromopropoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (4a)

[0110]

[0111] According to Synthesis Route II, compound 1b (1.0 eq), 1,3-dibromopropane (5.0 eq), sodium bicarbonate (2.0 eq), and sodium iodide (0.3 eq) were added sequentially to a 150 mL single-necked flask. Anhydrous DMF was added and the mixture was stirred at 70°C for 72 hours. 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 afford compound 4a as a white solid in 40% yield. 1 H NMR (400MHz, DMSO-d6) δ11.19(s,1H),7.89(dd,1H,J1=7.2Hz,J2=8.4Hz),7.61(d,1H,J=8.4Hz),7.53(d,1H,J=7.2Hz),5.15(dd,1H,J1=5. 6Hz, J2=12.8Hz), 4.37(t,2H,J=6.0Hz), 3.79(t,2H,J=6.8Hz), 2.99–2.89(m,1H), 2.68–2.55(m,2H), 2.39–2.33(m,2H), 2.18–2.06(m,1H). 13 C NMR(101MHz,DMSO-d6)δ173.29,170.44,167.28,165.82,156.06,137.62,133.68,1 20.29,116.91,116.00,66.97,49.22,32.07,31.42,31.34,22.46.HRMS(ESI)calcd for C 16 H 15 BrN2NaO5[M+Na] + :417.0057,found:417.0048.

[0112] Example 27 Synthesis of 4-(4-bromobutoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (4b)

[0113]

[0114] According to the method of Example 26, 1,4-dibromobutane was used instead of 1,3-dibromopropane to obtain compound 4b as a white solid with a yield of 44%. 1 H NMR (400MHz, DMSO-d6) δ11.18(s,1H),7.87(dd,1H,J1=7.2Hz,J2=8.4Hz),7.56(d,1H,J=8.4Hz),7.50(d,1H,J=7.2Hz),5.14(dd,1H,J1=5. 6Hz, J2=12.8Hz), 4.30(t,2H,J=6.4Hz), 3.72(t,2H,J=6.8Hz), 2.98–2.89(m,1H), 2.67–2.58(m,2H), 2.17–2.05(m,3H), 1.98–1.91(m,2H). 13 C NMR (101MHz, DMSO-d6) δ173.29,170.45,167.32,165.83,156.28,137.53,133.67,120. 19,116.71,115.72,68.49,49.19,35.38,31.42,29.50,27.57,22.47.HRMS(ESI)calcd for C 17 H 17 BrN2NaO5[M+Na] + :431.0213,found:431.0215.

[0115] Example 28 Synthesis of 4-(5-bromopentyloxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (4c)

[0116]

[0117] According to the method of Example 26, 1,5-dibromopentane was used instead of 1,3-dibromopropane to obtain compound 4c as a white solid with a yield of 45%. 1H NMR (400MHz, DMSO-d6) δ11.18 (s, 1H), 7.87 (dd, 1H, J1=7.2Hz, J2=8.4Hz), 7.58 (d,1H,J=8.4Hz),7.50(d,1H,J=7.2Hz),5.14(dd,1H,J1=5.6Hz,J2=12.8Hz),4 .28(t,2H,J=6.4Hz),3.62(t,2H,J=6.8Hz),2.99–2.90(m,1H),2.67–2.58(m,2 H),2.15–2.06(m,1H),1.99-1.91(m,2H),1.89–1.81(m,2H),1.68–1.60(m,2H). 13 C NMR(101MHz,DMSO-d6)δ173.29,170.46,167.33,165.78,156.40,137.51,133.69,120.25, 116.67,115.65,69.12,49.18,35.53,32.29,31.43,27.97,24.59,22.47.HRMS(ESI)calcd for C 18 H 19 BrN2NaO5[M+Na] + :445.0370,found:445.0344.

[0118] Example 29 Synthesis of 4-(6-bromohexyloxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (4d)

[0119]

[0120] According to the method of Example 26, 1,6-dibromohexane was used instead of 1,3-dibromopropane to obtain compound 4d as a white solid with a yield of 40%. 1 H NMR (400MHz, DMSO-d6) δ11.13(s,1H),7.81(dd,1H,J1=7.6Hz,J2=8.8Hz),7.51(d,1H,J=8.4Hz),7.44(d,1H,J=7.2Hz),5.09(dd,1H,J1=5.6Hz,J2=1 2.8Hz),4.20(t,2H,J=6.4Hz),3.54(t,2H,J=6.8Hz),2.94–2.85(m,1H),2 .63–2.53(m,2H),2.06–2.01(m,1H),1.86–1.74(m,4H),1.53–1.43(m,4H). 13C NMR(101MHz,DMSO-d6)δ173.28,170.45,167.33,165.80,156.44,137.51,133.70,120.23,116 .68,115.62,69.13,49.19,35.57,32.62,31.43,28.68,27.63,24.90,22.47.HRMS(ESI)calcd for C 19 H 21 BrN2NaO5[M+Na] + :459.0526,found:459.0521.

[0121] Example 30 Synthesis of 4-(7-bromoheptyloxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (4e)

[0122]

[0123] According to the method of Example 26, 1,7-dibromoheptane was used instead of 1,3-dibromopropane to obtain compound 4e as a white solid with a yield of 41%. 1 H NMR (400MHz, DMSO-d6) δ11.13 (s, 1H), 7.81 (t, 1H, J = 8.0Hz), 7.52 (d, 1H, J = 8.8Hz), 7.44 (d, 1H, J = 7.2Hz), 5.09 (dd, 1H, J1 = 5.6Hz, J2 = 12.8Hz) ,4.21(t,2H,J=6.4Hz),3.54(t,2H,J=6.8Hz),2.93–2.84(m,1H),2.62 –2.53(m,2H),2.07–1.99(m,1H),1.84–1.72(m,4H),1.50–1.36(m,6H). 13 C NMR(101MHz,DMSO-d6)δ173.29,170.45,167.33,165.79,156.45,137.50,133.70,120.21,116.65 ,115.60,69.18,49.18,35.68,32.64,31.43,28.75,28.19,27.92,25.62,22.47.HRMS(ESI)calcd for C 20 H 23 BrN2NaO5[M+Na] + :473.0683,found:473.0683.

[0124] Example 31 Synthesis of 4-(8-bromooctyloxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (4f)

[0125]

[0126] According to the method of Example 26, 1,8-dibromooctane was used instead of 1,3-dibromopropane to obtain compound 4f as a white solid with a yield of 47%. 1 H NMR (400MHz, DMSO-d6) δ11.13(s,1H),7.81(dd,1H,J1=7.2Hz,J2=8.4Hz),7.52(d,1H,J=8.4Hz),7.44(d,1H,J=7.2Hz),5.08(dd,1H,J1=5.2Hz,J2=1 2.8Hz),4.20(t,2H,J=6.4Hz),3.53(t,2H,J=6.8Hz),2.93–2.84(m,1H),2 .63–2.51(m,2H),2.06–1.98(m,1H),1.83–1.72(m,4H),1.49–1.23(m,8H). 13 C NMR(101MHz,DMSO-d6)δ173.27,170.44,167.33,165.78,156.48,137.50,133.70,120.22,116.65,11 5.59,69.23,49.19,35.68,32.68,31.43,28.98,28.82,28.52,27.94,25.68,22.48.HRMS(ESI)calcd forC 21 H 25 BrN2NaO5[M+Na] + :487.0839,found:487.0841.

[0127] Example 32 Synthesis of 4-(9-bromononyloxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (4 g)

[0128]

[0129] According to the method of Example 26, 1,9-dibromononane was used instead of 1,3-dibromopropane to obtain 4 g of compound as a white solid with a yield of 52%. 1H NMR (400MHz, DMSO-d6) δ11.12(s,1H),7.81(dd,1H,J1=7.2Hz,J2=8.4Hz),7.51(d,1H,J=8.4Hz),7.44(d,1H,J=7.2Hz),5.08(dd,1H,J1=5.6Hz,J2=12 .8Hz),4.20(t,2H,J=6.4Hz),3.52(t,2H,J=6.8Hz),2.93–2.84(m,1H),2. 62–2.53(m,2H),2.06–1.99(m,1H),1.82–1.72(m,4H),1.49–1.23(m,10H). 13 C NMR(101MHz,DMSO-d6)δ173.27,170.44,167.33,165.78,156.48,137.50,133.71,120.22,116.66,115.5 9,69.23,49.19,35.69,32.71,31.43,29.25,29.00,28.84,28.47,27.96,25.68,22.47.HRMS(ESI)calcd forC 22 H 27 BrN2NaO5[M+Na] + :501.0996,found:501.1000.

[0130] Example 33 Synthesis of 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-oxoethoxy]-2-[2,6-dioxopiperidin-3-yl]isoindole-1,3-dione (Ia)

[0131]

[0132] Compound 3a (1.0 eq) and HATU (1.15 eq) were added sequentially to a 50 mL two-necked flask under nitrogen. Dissolved in anhydrous DMF solution was added and stirred until uniform. N,N-diisopropylethylamine (4.2 eq) was added and stirred for half an hour. M6 (1.0 eq) in anhydrous DMF was then added dropwise. The mixture was stirred 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 was concentrated in vacuo. The concentrate was purified on a silica gel column (dichloromethane:methanol = 30:1-20:1) to obtain compound Ia as a yellow solid in a 39% yield. 1H NMR (400MHz, DMSO-d6) δ11.15(d,1H,J=5.2Hz),7.99(d,1H,J=2.8Hz),7.82–7.78(m,3H),7.60(d,1 H,J=8.4Hz),7.47(d,1H,J=7.2Hz),7.42–7.37(m,2H),5.34–5.21(m,2H),5.12(dd,1H,J1=5.2Hz,J 2=12.8Hz),4.73(t,2H,J=5.2Hz),4.48-4.41(m,3H),3.96(d,1H,J=13.2Hz),3.55(s,3H),3.30–3. 17(m,2H),2.95–2.81(m,2H),2.62–2.54(m,2H),2.12(s,3H),2.07–1.89(m,4H),1.80–1.70(m,1H). 13 C NMR(101MHz,DMSO-d6)δ173.31,170.45,167.31,165.77,165.27,162.04,158.7 5,156.22,141.74,136.99,135.55,134.68,133.55,131.19,128.11,121.57(q,J C-F =255.5Hz),120.73,120.22,119.28,118.64,116.56,115.89,107.61,67.19,66.57,49.2 2,44.27,41.99,41.61,37.78,33.27,31.58,31.42,30.98,22.47,17.64.HRMS(ESI)calcd for C 37 H 36 F3N6O8[M+H] + :749.2541,found:749.2547.

[0133] Example 34 Synthesis of 4-[4-[4-[6-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-[2-(trifluoromethoxy)ethyl]-1H-benzimidazol-2-yl]piperidin-1-yl]-4-oxobutoxy]-2-[2,6-dioxopiperidin-3-yl]isoindole-1,3-dione (Ib)

[0134]

[0135] According to the method of Example 33, 3a was replaced by 3b to obtain compound Ib as a yellow solid in a yield of 43%. 1H NMR (400MHz, DMSO-d6) δ11.14(d,1H,J=12.0Hz),7.98(d,1H,J=2.4Hz),7.85–7.81(m,3H),7.57(d,2H,J=8.4Hz),7.46(d ,1H,J=7.2Hz),7.39(dd,1H,J1=1.6Hz,J2=8.4Hz),5.09(dd,1H,J1=5.2Hz,J2=12.8Hz),4.70(t,2H,J=5.2Hz),4.53(d,1H ,J=12.8Hz),4.45(t,2H,J=5.2Hz),4.28(t,2H,J=5.6Hz),4.05(d,1H,J=12.8Hz),3.54(s,3H),3.29–3.16(m,2H),2.92– 2.83(m,1H),2.78–2.72(m,1H),2.63–2.51(m,4H),2.11(s,3H),2.04–2.01(m,3H),1.91–1.79(m,3H),1.71–1.62(m,1H). 13 C NMR(101MHz,DMSO-d6)δ173.28,170.47,170.28,167.34,165.89,162.02,158.83,15 6.36,141.82,137.55,136.97,135.56,134.65,133.70,131.13,128.09,121.55(q,J C-F =255.5Hz),120.29,120.15,119.30,118.64,116.78,115.71,107.56,68.69,67.18,49.20,45.2 1,41.96,41.42,37.77,33.44,31.83,31.41,31.21,28.78,24.89,22.45,17.64.HRMS(ESI)calcd for C 39 H 40 F3N6O8[M+H] + :777.2854,found:777.2857.

[0136] Example 35 Synthesis of 4-[5-[4-[6-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-[2-(trifluoromethoxy)ethyl]-1H-benzimidazol-2-yl]piperidin-1-yl]-4-oxobutoxy]-2-[2,6-dioxopiperidin-3-yl]isoindole-1,3-dione (Ic)

[0137] become

[0138]

[0139] According to the method of Example 33, 3a was replaced by 3c to obtain compound Ic as a yellow solid in 38% yield. 1 H NMR (400MHz, DMSO-d6) δ11.14(s,1H),7.98(d,1H,J=2.8Hz),7.84–7.77(m,3H),7.57(d,1H,J=8.0Hz),7.54(d,1H,J =8.8Hz),7.45(d,1H,J=7.2Hz),7.39(d,1H,J=8.8Hz),5.10–5.04(m,1H),4.70(t,2H,J=5.2Hz),4.51(d,1H,J=13.2H z),4.45(t,2H,J=4.8Hz),4.26(s,2H,J=6.0Hz),4.04(d,1H,J=13.2Hz),3.54(s,3H),3.32–3.25(m,1H),3.20–3.13 (m,1H),2.88–2.80(m,1H),2.71–2.67(m,1H),2.58–2.44(m,4H),2.11(s,3H),2.01–1.99(m,1H),1.87–1.63(m,8H). 13 C NMR(101MHz,DMSO-d6)δ173.25,170.81,170.46,167.33,165.84,162.02,158.87,15 6.45,141.74,137.51,136.98,135.54,134.66,133.70,131.15,128.09,121.55(q,J C-F =256.5Hz),120.19,119.26,118.63,116.65,115.60,107.58,69.23,67.19,49.20,45.22,41.98 ,41.32,37.76,33.48,32.50,31.94,31.39,31.22,28.48,22.48,21.97,17.64.HRMS(ESI)calcd for C 40 H 42 F3N6O8[M+H] + :791.3011,found:791.3014.

[0140] Example 36 Synthesis of 4-[(7-[4-(6-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-[2-(trifluoromethoxy)ethyl]-1H-benzimidazol-2-yl]piperidin-1-yl]-7-oxoheptyl)oxy]-2-[2,6-dioxopiperidin-3-yl]isoindole-1,3-dione (Id)

[0141]

[0142] According to the method of Example 33, 3a was replaced by 3d to obtain compound Id as a yellow solid in a yield of 37%. 1 H NMR (400MHz, DMSO-d6) δ11.14(s,1H),7.97(d,1H,J=2.4Hz),7.82–7.76(m,3H),7.57(d,1H,J=8.0Hz),7.52(d,1H,J=8.4Hz),7.44(d,1H,J=7.2 Hz), 7.39 (dd, 1H, J1 = 2.0Hz, J2 = 8.4Hz), 5.09 (dd, 1H, J1 = 5.2Hz, J2 = 12.8Hz), 4.71 (t, 2H, J = 5.2Hz), 4.52 (d, 1H, J = 12.8Hz), 4.45 (t, 2H, J = 5.2H z), 4.21 (t, 2H, J = 6.4Hz), 4.02 (d, 1H, J = 13.6Hz), 3.54 (s, 3H), 3.32–3. 26(m,1H),3.20–3.14(m,1H),2.93–2.84(m,1H),2.74–2.68(m,1H),2.6 0–2.53(m,2H),2.44–2.32(m,2H),2.11(s,3H),2.07–1.99(m,1H),1.95 –1.74(m,5H),1.71–1.62(m,1H),1.60–1.47(m,4H),1.43–1.34(m,2H). 13 C NMR(101MHz,DMSO-d6)δ173.29,170.90,170.47,167.34,165.82,162.01,158.87,15 6.48,141.83,137.49,136.98,135.55,134.65,133.71,131.11,128.08,121.56(q,J C-F=255.5Hz),120.22,120.14,119.29,118.63,116.68,115.59,107.55,69.20,67.17,49.19,45.30,41.94,4 1.34,37.77,33.48,32.65,31.94,31.42,31.25,28.89,28.77,25.58,25.30,22.47,17.64.HRMS(ESI)calcd forC 42 H 46 F3N6O8[M+H] + :819.3324,found:819.3325.

[0143] Example 37 Synthesis of 4-[(9-[4-(6-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-[2-(trifluoromethoxy)ethyl]-1H-benzimidazol-2-yl]piperidin-1-yl]-9-oxononyl)oxy]-2-[2,6-dioxopiperidin-3-yl]isoindole-1,3-dione (Ie)

[0144]

[0145] According to the method of Example 33, 3a was replaced by 3e to obtain compound Ie as a yellow solid in a yield of 39%. 1H NMR(400MHz,DMSO-d6)δ11.14(s,1H),7.98(d,1H,J=2.4Hz),7.82–7.77(m,3H),7.57(d,1H,J=8.4Hz),7.51(d,1H,J=8.4Hz),7.44(d,1H,J=7.2Hz),7.38(dd,1H,J1=1.6Hz,J2=8.4Hz),5.09(dd,1H,J1=5.2Hz,J2=12.8Hz),4.70(t,2H,J=5.2Hz),4.51(d,1H,J=12.8Hz),4.45(t,2H,J=5.2Hz),4.20(t,2H,J=6.4Hz),4.01(d,1H,J=12.8Hz),3.54(s,3H),3.31–3.26(m,1H),3.20–3.13(m,1H),2.93–2.84(m,1H),2.74–2.67(m,1H),2.61–2.54(m,2H),2.42–2.26(m,2H),2.11(s,3H),2.06–2.00(m,1H),1.92–1.83(m,2H),1.80–1.73(m,3H),1.70–1.60(m,1H),1.55–1.42(m,4H),1.34–1.26(m,6H). 13 C NMR(101MHz,DMSO-d6)δ173.30,170.97,170.48,167.35,165.80,162.03,158.87,156.48,141.82,137.49,136.99,135.54,134.64,133.69,131.11,128.09,121.55(q,J C-F =254.5Hz),120.19,120.14,119.29,118.66,116.64,115.57,107.55,69.25,67.17,49.19,45.32,41.95,41.32,37.78,33.48,32.83,31.94,31.42,31.25,29.34,29.26,29.12,28.88,25.75,25.43,22.47,17.64.HRMS(ESI)calcd for C 44 H 48 F3N6O8[M-H] - :845.3491,found:845.3495.

[0146] Example 38 Synthesis of 4-[(10-[4-(6-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-[2-(trifluoromethoxy)ethyl]-1H-benzimidazol-2-yl]piperidin-1-yl]-10-oxodecanyl)oxy]-2-[2,6-dioxopiperidin-3-yl]isoindole-1,3-dione (If)

[0147]

[0148] According to the method of Example 33, 3a was replaced by 3f to obtain compound If as a yellow solid in a yield of 45%. 1 H NMR (400MHz, DMSO-d6) δ11.12(s,1H),7.97(d,1H,J=2.8Hz),7.82–7.76(m,3H),7.57(d,1H,J=8.4Hz),7.50(d,1H,J=8.4Hz),7.43(d,1H,J= 6.8Hz), 7.39 (d, 1H, J = 8.4Hz), 5.09 (dd, 1H, J1 = 4.8Hz, J2 = 12.8Hz), 4.71 (d, 2H, J = 5.2Hz), 4.51 (d, 1H, J = 12.8Hz), 4.45 (t, 2H, J = 5.2Hz), 4. 19(t,2H,J=6.0Hz),4.00(d,1H,J=13.2Hz),3.54(s,3H),3.29–3.26(m,1H),3.20–3.13(m,1H),2.93–2.83(m,1H),2.73–2.67(m,1H),2.61– 2.56(m,2H),2.39–2.27(m,2H),2.11(s,3H),2.05–2.01(m,1H),1.91– 1.72(m,5H),1.66–1.62(m,1H),1.53–1.43(m,4H),1.35–1.22(m,8H). 13 C NMR(101MHz,DMSO-d6)δ173.29,170.96,170.47,167.34,165.79,162.02,158.83,15 6.48,141.62,137.47,136.96,135.50,134.66,133.70,131.19,128.09,121.55(q,J C-F=254.5Hz),120.19,119.21,118.60,116.65,115.56,107.58,69.23,67.16,49.19,45.31,41.98,41.32,37.77,33 .48,32.81,31.94,31.43,31.22,29.39,29.31,29.29,29.11,28.88,25.74,25.44,22.48,17.64.HRMS(ESI)calcd forC 45 H 52 F3N6O8[M+H] + :861.3794,found:861.3794.

[0149] Example 39 Synthesis of 4-[(11-[4-(6-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-[2-(trifluoromethoxy)ethyl]-1H-benzimidazol-2-yl]piperidin-1-yl]-11-oxoundecyl)oxy]-2-[2,6-dioxopiperidin-3-yl]isoindole-1,3-dione (1 g)

[0150]

[0151] According to the method of Example 33, 3a was replaced by 3g to obtain compound Ig as a yellow solid with a yield of 40%. 1H NMR(400MHz,DMSO-d6)δ11.14(s,1H),7.97(d,1H,J=2.4Hz),7.82–7.76(m,3H),7.57(d,1H,J=8.4Hz),7.50(d,1H,J=8.4Hz),7.43(d,1H,J=7.2Hz),7.38(dd,1H,J1=1.6Hz,J2=8.8Hz),5.09(dd,1H,J1=5.6Hz,J2=12.8Hz),4.70(t,2H,J=5.2Hz),4.51(d,1H,J=12.8Hz),4.45(t,2H,J=4.8Hz),4.18(t,2H,J=6.4Hz),4.01(d,1H,J=12.8Hz),3.54(s,3H),3.31–3.24(m,1H),3.20–3.13(m,1H),2.93–2.84(m,1H),2.76–2.67(m,1H),2.62–2.52(m,2H),2.39–2.27(m,2H),2.11(s,3H),2.06–1.99(m,1H),1.92–1.60(m,6H),1.53–1.42(m,4H),1.36–1.23(m,10H). 13 C NMR(101MHz,DMSO-d6)δ173.28,170.94,170.46,167.33,165.78,162.01,158.85,156.47,141.81,137.46,136.96,135.54,134.63,133.69,131.11,128.08,121.55(q,J C-F =254.5Hz),120.16,120.13,119.28,118.63,116.64,115.55,107.54,69.22,67.18,49.19,45.31,41.95,41.31,37.76,33.48,32.82,31.95,31.43,31.21,29.45,29.42,29.38,29.33,29.16,28.88,25.75,25.44,22.48,17.63.HRMS(ESI)calcd for C 46 H 52 F3N6O8[M-H] - :873.3804,found:873.3807.

[0152] Example 40 Synthesis of 5-[(7-[4-(6-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-[2-(trifluoromethoxy)ethyl]-1H-benzimidazol-2-yl]piperidin-1-yl]-7-oxoheptyl)oxy]-2-[2,6-dioxopiperidin-3-yl]isoindole-1,3-dione (Ih)

[0153]

[0154] According to the method of Example 33, 3a was replaced by 3h to obtain compound Ih as a yellow solid in a yield of 44%. 1 H NMR (400MHz, DMSO-d6) δ11.14(s,1H),7.98(d,1H,J=2.4Hz),7.84–7.77(m,3H),7.57(d,1H,J=8.4Hz),7.43(d,1H,J=2.4Hz),7.39(d,1H,J =8.4Hz),7.36(dd,1H,J1=2.4Hz,J2=8.4Hz),5.12(dd,1H,J1=5.2Hz,J2=12.8Hz),4.71(t,2H,J=4.8Hz),4.52(d,1H,J=12.8Hz),4.45(t,2 H,J=4.8Hz),4.19(t,2H,J=6.0Hz),4.02(d,1H,J=12.8Hz),3.54(s,3H),3.30–3.23(m,1H),3.20–3.14(m,1H),2.94–2.85(m,1H),2.74–2. 67(m,1H),2.61–2.54(m,2H),2.42–2.31(m,2H),2.11(s,3H),2.07–1 .99(m,1H),1.91–1.75(m,5H),1.69–1.52(m,3H),1.48–1.36(m,4H). 13 C NMR(101MHz,DMSO-d6)δ173.29,170.91,170.45,167.38,167.31,164.57,162.02,15 8.84,141.54,136.97,135.47,134.69,134.41,128.09,125.77,123.30,121.54(q,J C-F=255.5Hz),121.19,120.25,119.18,118.59,109.28,107.61,69.24,67.17,49.40,45.30,41.99,41.33 ,37.78,33.47,32.71,31.93,31.42,31.22,28.97,28.77,25.72,25.34,22.54,17.65.HRMS(ESI)calcd for C 42 H 46 F3N6O8[M+H] + :819.3324,found:819.3331.

[0155] Example 41 Synthesis of 5-[(8-[4-(6-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-[2-(trifluoromethoxy)ethyl]-1H-benzimidazol-2-yl]piperidin-1-yl]-8-oxooctyl)oxy]-2-[2,6-dioxopiperidin-3-yl]isoindole-1,3-dione (Ii)

[0156]

[0157] According to the method of Example 33, 3a was replaced by 3i to obtain compound Ii as a yellow solid with a yield of 32%. 1 H NMR (400MHz, DMSO-d6) δ11.13(s,1H),7.99(d,1H,J=2.4Hz),7.83-7.80(m,3H),7.60(d,1H,J=8.4Hz),7.42(d,2H,J=2.4Hz), 7.34(dd,1H,J1=2.4Hz,J2=8.4Hz),5.12(dd,1H,J1=5.6Hz,J2=12.8Hz),4.75(s,2H),4.54(d,1H,J=13.6Hz),4.47(t,2H,J=5 .2Hz),4.18(t,2H,J=6.4Hz),4.03(d,1H,J=13.6Hz),3.54(s,3H),3.30-3.14(m,2H),2.94-2.84(m,1H),2.73-2.67(m,1H),2 .62-2.53(m,2H),2.32-2.29(m,2H),2.11(s,3H),2.06-2.01(m,1H),1.96-1.73(m,5H),1.68-1.50(m,3H),1.46-1.29(m,6H); 13C NMR(101MHz,DMSO-d6)δ173.27,170.94,170.43,167.37,167.30,164.57 ,162.02,158.74,136.93,135.32,134.74,134.41,132.33,132.03,131. 93,129.13,129.02,128.11,125.75,123.30,122.81,121.18,120.46,12 0.28,118.96,118.49,109.27,107.72,69.27,68.98,67.13,67.10,56.2 9,49.41,45.29,42.09,41.31,41.25,40.59,40.38,40.17,39.96,39.75 ,39.55,39.34,37.77,35.89,33.45,32.77,32.59,31.88,31.43,31.15, 30.06,29.23,29.19,29.03,28.95,28.82,27.56,25.85,25.77,25.35,2 2.95,22.54,20.36,19.74,19.10,17.64,14.63,11.72.HRMS(ESI)calcd for C 43 H 48 F3N6O8[M+H] + :833.3481,found:833.3473.

[0158] Example 42 Synthesis of 5-[(9-[4-(6-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-[2-(trifluoromethoxy)ethyl]-1H-benzimidazol-2-yl]piperidin-1-yl]-9-oxononyl)oxy]-2-[2,6-dioxopiperidin-3-yl]isoindole-1,3-dione (Ij)

[0159]

[0160] According to the method of Example 33, 3a was replaced by 3j to obtain compound Ij as a yellow solid in 60% yield. 1H NMR(400MHz,DMSO-d6)δ11.13(s,1H),7.97(d,1H,J=2.8Hz),7.83–7.77(m,3H),7.58(d,1H,J=8.4Hz),7.41–7.38(m,2H),7.34(dd,1H,J1=2.4Hz,J2=8.4Hz),5.13(dd,1H,J1=5.2Hz,J2=12.8Hz),4.71(t,2H,J=5.2Hz),4.54(d,1H,J=12.8Hz),4.46(t,2H,J=4.8Hz),4.17(t,2H,J=6.4Hz),4.01(d,1H,J=12.8Hz),3.54(s,3H),3.31–3.27(m,1H),3.18–3.14(m,1H),2.95–2.85(m,1H),2.75–2.68(m,1H),2.62–2.55(m,2H),2.42–2.27(m,2H),2.11(s,3H),2.07–2.01(m,1H),1.95–1.70(m,5H),1.68–1.61(m,1H),1.55–1.51(m,2H),1.46–1.41(m,2H),1.37–1.35(m,6H). 13 C NMR(101MHz,DMSO-d6)δ173.27,170.94,170.43,167.36,167.29,164.55,162.02,158.82,141.57,136.94,135.48,134.64,134.38,131.20,128.09,125.73,123.27,121.54(q,J C-F =255.5Hz),121.11,120.22,119.18,118.61,109.24,107.57,69.26,67.15,49.42,45.31,41.99,41.32,37.76,33.48,32.80,31.92,31.43,31.22,29.32,29.24,29.13,28.84,25.80,25.41,22.54,17.62.HRMS(ESI)calcd forC 44 H 48 F3N6O8[M-H] - :845.3491,found:845.3494.

[0161] Example 43 Synthesis of 4-(3-(4-(6-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-(2-trifluoromethoxyethyl)-1H-benzimidazol-2-yl)piperidin-1-yl)propoxy)-2-(2,6-dioxopiperidin-3-yl)isophthalic acid-1,3-dione (Ik)

[0162]

[0163] M6 (1.0 eq) was first added to a 100 mL single-necked flask and dissolved in anhydrous DMF. Compound 4a (1.0 eq), EDCI, and sodium bicarbonate (4.0 eq) were then added and stirred at 80°C for 72 hours. 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 using a silica gel column (dichloromethane:methanol = 20:1-10:1) to obtain compound Ik as a yellow solid in a 32% yield. 1 H NMR (400MHz, DMSO-d6) δ11.14(s,1H),7.98(d,1H,J=2.4Hz),7.85–7.80(m,2H),7.75(d,1H, J=1.6Hz),7.60–7.55(m,2H),7.46(d,1H,J=7.2Hz),7.38(dd,1H,J1=1.6Hz,J2=8.4Hz),5.1 0(dd,1H,J1=5.6Hz,J2=12.8Hz),4.66(t,2H,J=5.2Hz),4.43(t,2H,J=5.2Hz),4.29(t,2H,J =6.4Hz),3.54(s,3H),3.03–2.84(m,4H),2.61–2.53(m,4H),2.11(s,3H),2.09–1.81(m,9H). 13 C NMR(101MHz,DMSO-d6)δ173.30,170.48,167.34,165.84,162.01,159.47,156.4 8,141.89,137.53,136.99,135.56,134.62,133.70,130.97,128.07,121.56(q,J C-F=254.5Hz),120.27,120.04,119.27,118.67,116.72,115.66,107.45,67.65,67.18,54.8 5,53.64,49.20,41.98,37.76,33.83,31.55,31.43,26.51,22.47,17.64.HRMS(ESI)calcd for C 38 H 40 F3N6O7[M+H] + :749.2905,found:749.2907.

[0164] Example 44 Synthesis of 4-(4-(4-(6-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-(2-trifluoromethoxyethyl)-1H-benzimidazol-2-yl)piperidin-1-yl)butoxy)-2-(2,6-dioxopiperidin-3-yl)isophthalic acid-1,3-dione (I1)

[0165]

[0166] According to the method of Example 43, 4a was replaced by 4b to obtain compound I1 as a yellow solid in a yield of 36%. 1 H NMR (400MHz, DMSO-d6) δ11.14(s,1H),8.00(d,1H,J=2.4Hz),7.87–7.81(m,2H),7.78(d,1H,J=2 .0Hz),7.60(d,1H,J=8.4Hz),7.56(d,1H,J=8.8Hz),7.47(d,1H,J=7.2Hz),7.40(dd,1H,J1=1.6 Hz, J2=8.4Hz), 5.10 (dd, 1H, J1=5.6Hz, J2=13.2Hz), 4.67 (t, 2H, J=5.2Hz), 4.44 (t, 2H, J=4.8Hz ), 4.27 (t, 2H, J = 6.4Hz), 3.54 (s, 3H), 3.37–2.84 (m, 4H), 2.64–2.50 (m, 4H), 2.11–1.67 (m, 14H). 13 C NMR(101MHz,DMSO-d6)δ173.30,170.47,167.34,165.88,162.02,159.04,156.4 2,141.83,137.57,136.99,135.57,134.66,133.69,131.05,128.06,121.55(q,J C-F=255.5Hz),120.28,120.08,119.30,118.64,116.68,115.67,107.51,70.24,69.09,67.1 9,57.42,53.01,49.19,42.00,37.77,31.42,30.78,26.58,22.47,17.64.HRMS(ESI)calcd for C 39 H 42 F3N6O7[M+H] + :763.3062,found:763.3063.

[0167] Example 45 Synthesis of 4-((5-(4-(6-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-(2-trifluoromethoxyethyl)-1H-benzimidazol-2-yl)piperidin-1-yl)pentyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isophthalic acid-1,3-dione (Im)

[0168]

[0169] According to the method of Example 43, 4a was replaced by 4c to obtain compound Im as a yellow solid in 33% yield. 1 H NMR (400MHz, DMSO-d6) δ11.14(s,1H),7.99(d,1H,J=2.4Hz),7.85–7.81(m,2H),7.78(d,1H,J=1 .6Hz),7.60(d,1H,J=8.4Hz),7.54(d,1H,J=8.4Hz),7.46(d,1H,J=7.2Hz),7.40(dd,1H,J1=1.6 Hz, J2=8.4Hz), 5.09 (dd, 1H, J1=5.6Hz, J2=12.8Hz), 4.68 (t, 2H, J=5.2Hz), 4.44 (t, 2H, J=5.2Hz ), 4.24 (t, 2H, J = 6.4Hz), 3.54 (s, 3H), 2.92–2.83 (m, 4H), 2.59–2.52 (m, 4H), 2.11–1.49 (m, 16H). 13 C NMR(101MHz,DMSO-d6)δ173.30,170.48,167.33,165.85,162.02,156.44,14 1.79,137.55,136.97,135.57,134.68,133.69,131.13,128.07,121.55(q,J C-F=254.5Hz),120.24,120.13,119.33,118.59,116.65,115.66,107.55,69.12,67.17,5 5.40,52.71,49.19,42.02,37.77,31.41,28.57,23.51,22.48,17.64.HRMS(ESI)calcd for C 40 H 44 F3N6O7[M+H] + :777.3218,found:777.3218.

[0170] Example 46 Synthesis of 4-((6-(4-(6-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-(2-trifluoromethoxyethyl)-1H-benzimidazol-2-yl)piperidin-1-yl)hexyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isophthalic acid-1,3-dione (In)

[0171]

[0172] According to the method of Example 43, 4a was replaced by 4d to obtain compound In as a yellow solid in a yield of 35%. 1 H NMR (400MHz, DMSO-d6) δ11.12(s,1H),7.98(d,1H,J=2.4Hz),7.84–7.79(m,2H),7.76(d,1H,J=2 .0Hz),7.59(d,1H,J=8.4Hz),7.53(d,1H,J=8.8Hz),7.45(d,1H,J=7.2Hz),7.39(dd,1H,J1=2.0 Hz, J2=8.4Hz), 5.09 (dd, 1H, J1=5.6Hz, J2=13.2Hz), 4.67 (t, 2H, J=5.2Hz), 4.43 (t, 2H, J=5.2Hz ), 4.22 (t, 2H, J = 6.4Hz), 3.54 (s, 3H), 3.17–2.84 (m, 4H), 2.62–2.41 (m, 4H), 2.27–1.33 (m, 18H). 13 C NMR(101MHz,DMSO-d6)δ173.27,170.44,167.33,165.81,162.01,159.11,156.4 9,141.87,137.51,136.96,135.58,134.62,133.72,131.06,128.08,121.56(q,J C-F=255.5Hz),120.26,120.09,119.30,118.64,116.70,115.61,107.48,69.23,67.17,58.10,53.2 0,49.21,42.00,37.74,31.43,30.92,28.81,26.90,26.22,25.62,22.49,17.62.HRMS(ESI)calcd for C 41 H 46 F3N6O7[M+H] + :791.3375,found:791.3348.

[0173] Example 47 Synthesis of 4-((7-(4-(6-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-(2-trifluoromethoxyethyl)-1H-benzimidazol-2-yl)piperidin-1-yl)heptyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isophthalic acid-1,3-dione (Io)

[0174]

[0175] According to the method of Example 43, 4a was replaced by 4e to obtain compound Io as a yellow solid in a yield of 25%. 1 H MR(400MHz,DMSO-d6)δ11.14(s,1H),8.00(d,1H,J=2.8Hz),7.85–7.79(m,3H),7.61(d,1H ,J=8.4Hz),7.53(d,1H,J=8.4Hz),7.45(d,1H,J=7.2Hz),7.41(dd,1H,J1=2.0Hz,J2=8.4H z),5.09(dd,1H,J1=5.6Hz,J2=12.8Hz),4.70(t,2H,J=5.2Hz),4.45(t,2H,J=4.8Hz),4.2 2(t,2H,J=6.4Hz),3.54(s,3H),2.99–2.84(m,4H),2.61–2.46(m,4H),2.33–1.33(m,20H). 13 CNMR(101MHz,DMSO-d6)δ173.31,170.48,167.34,165.82,162.03,156.47,141.7 3,137.53,136.95,136.30,135.61,134.71,133.69,131.22,128.07,121.54(q,J C-F=255.5Hz),120.23,120.17,119.37,118.57,116.65,115.61,107.61,69.25,67.1 9,49.20,42.04,37.76,31.43,28.75,26.77,25.64,22.48,17.62.HRMS(ESI)calcd for C 42 H 48 F3N6O7[M+H] + :805.3531,found:805.3520.

[0176] Example 48 Synthesis of 4-((8-(4-(6-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-(2-trifluoromethoxyethyl)-1H-benzimidazol-2-yl)piperidin-1-yl)octyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isophthalic acid-1,3-dione (Ip)

[0177]

[0178] According to the method of Example 43, 4a was replaced by 4f to obtain compound Ip as a yellow solid in a yield of 34%. 1 H NMR(400MHz,DMSO-d6)δ10.47(s,1H),8.03(d,1H,J=2.8Hz),7.87–7.76(m,3H),7.62(d,1 H,J=8.8Hz),7.48(d,1H,J=8.8Hz),7.40(d,1H,J=7.2Hz),7.22(brs,1H),4.74(t,2H,J=5. 2Hz),4.55(dd,1H,J1=4.4Hz,J2=10.8Hz),4.48(t,2H,J=5.2Hz),4.20(t,2H,J=6.4Hz),3 .55(s,3H),3.11–3.04(m,4H),2.44–1.99(m,12H),1.82–1.75(m,4H),1.53–1.23(m,10H). 13 CNMR(101MHz,DMSO-d6)δ173.12,170.40,167.81,166.30,162.03,157.85,156.2 2,141.71,136.98,136.95,135.61,134.71,134.23,131.26,128.08,121.53(q,J C-F=254.5Hz),120.20,119.65,119.40,118.58,117.22,115.30,107.62,69.15,67.16,52.15,51.6 9,42.04,37.76,30.93,28.96,28.94,28.90,26.62,25.71,23.96,17.62,17.59.HRMS(ESI)calcd for C 43 H 50 F3N6O7[M+H] + :819.3688,found:819.3689.

[0179] Example 49 Synthesis of 4-((9-(4-(6-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-(2-trifluoromethoxyethyl)-1H-benzimidazol-2-yl)piperidin-1-yl)nonyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isophthalic acid-1,3-dione (1q)

[0180]

[0181] According to the method of Example 43, 4a was replaced by 4g to obtain compound Iq as a yellow solid with a yield of 35%. 1 H NMR (400MHz, DMSO-d6) δ11.15(s,1H),7.99(d,1H,J=2.8Hz),7.83–7.78(m,3H),7.60(d,1H,J=8.4H z),7.52(d,1H,J=8.4Hz),7.44(d,1H,J=7.2Hz),7.40(dd,1H,J1=1.6Hz,J2=8.4Hz),5.09(dd,1H,J1 =5.6Hz, J2=12.8Hz), 4.68(t,2H,J=5.2Hz), 4.44(t,2H,J=4.8Hz), 4.21(t,2H,J=6.0Hz), 3.54(s,3 H),3.17-2.73(m,4H),2.73–2.53(m,4H),2.11–1.84(m,9H),1.80–1.73(m,2H),1.64–1.22(m,13H). 13 C NMR(101MHz,DMSO-d6)δ173.30,170.47,167.33,165.80,162.01,158.62,156.4 8,141.80,137.50,136.95,135.57,134.66,133.70,131.13,128.07,121.54(q,JC-F =254.5Hz),120.19,120.13,119.33,118.60,116.64,115.58,107.53,69.24,67.15,57.56,52.58,49.19,42.01,3 7.76,32.72,31.43,30.10,29.34,29.20,29.10,28.88,27.05,25.75,25.43,22.48,21.56,17.63.HRMS(ESI)calcd for C 44 H 52 F3N6O7[M+H] + :833.3845,found:833.3845.

[0182] Example 50 Synthesis of 4-((10-(4-(6-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-(2-trifluoromethoxyethyl)-1H-benzimidazol-2-yl)piperidin-1-yl)decyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isophthalic acid-1,3-dione (Ir)

[0183]

[0184] According to the method of Example 43, 4a was replaced by 4h to obtain compound Ir as a yellow solid in a yield of 29%. 1 H NMR (400MHz, DMSO-d6) δ11.14(s,1H),8.00(d,1H,J=2.8Hz),7.83-7.77(m,3H),7.60(d,1H,J=8.4Hz),7.52( d,1H,J=8.4Hz),7.45(d,1H,J=7.2Hz),7.40(dd,1H,J1=1.6Hz,J2=8.4Hz),5.09(dd,1H,J1=5.2Hz,J2=12.8Hz ),4.69(t,2H,J=5.2Hz),4.45(t,2H,J=5.2Hz),4.20(t,2H,J=6.4Hz),3.54(s,3H),3.22–2.85(m,4H),2.80- 2.57(m,4H),2.12-1.91(m,8H),1.80–1.72(m,2H),1.67–1.58(m,2H),1.50–1.43(m,2H),1.36–1.17(m,12H). 13C NMR(101MHz,DMSO-d6)δ173.30,170.46,167.33,165.80,162.02,158.34,156.4 7,141.76,137.50,136.95,135.58,134.68,133.69,131.17,128.07,121.54(q,J C-F =254.5Hz),120.20,120.15,119.35,118.59,116.63,115.58,107.56,69.24,67.16,57.17,52.17,49.19,42.02,37.7 6,31.43,31.15,29.50,29.39,29.32,29.19,29.14,28.88,26.96,25.76,24.91,22.48,21.57,17.63.HRMS(ESI)calcd for C 45 H 54 F3N6O7[M+H] + :847.4001,found:847.3991.

[0185] The compounds synthesized in the present invention were evaluated for their in vitro anti-tumor activity and BRD4 and p300 / CBP degradation activities.

[0186] 1. Evaluation of antitumor activity in vitro

[0187] 3×10 3 PC-3 cells were seeded into 96-well plates and incubated with different concentrations of PROTAC molecules Ia-Ir, 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.

[0188] 2. Evaluation of compound concentration-dependent induction of BRD4 and p300 / CBP degradation activity

[0189] In a 96-well plate, add 1 × 10 6PC-3 cells were incubated with various concentrations of PROTAC Ig for 12 hours (for BRD4 detection) or 24 hours (for p300 / CBP detection) using 1000 PC-3 cells / well. 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.

[0190] 3. Evaluation of compound time-dependent induction of BRD4, p300, and CBP degradation activity

[0191] In a 96-well plate, add 1 × 10 6 PC-3 cells / well were incubated with 1 nM (for BRD4 detection) or 30 nM (for p300 / CBP detection) PROTAC molecule Ig for different times. Subsequently, the cells were washed twice with pre-chilled PBS and lysed in ice-cold RIPA buffer. After centrifugation 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 the 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.

[0192] 4. Experimental results

[0193] 4.1. In vitro antitumor activity evaluation results

[0194] Table 1 In vitro tumor activity evaluation results of target compounds

[0195]

[0196] a IC 50 Values ​​are expressed as mean ± SD

[0197] bNot detected

[0198] We evaluated the in vitro antitumor activity of the synthesized Ia-Ir compounds in human prostate cancer cell line PC-3, using the BRD4 and p300 / CBP dual-target small molecule inhibitor NEO2734, paclitaxel, and the BRD4 degrader ARV-771 as positive control drugs. According to the evaluation results in Table 1, compounds Ia-Ir exhibited excellent antitumor activity in PC-3 cells, with most compounds having IC 50 The value is less than 10nM. Among them, the IC of the most active compound Ig 50 The value was 1.96nM, which was significantly better than the positive control drugs NEO2734, paclitaxel and ARV-771, and was 5.0 times, 4.5 times and 2.5 times that of NEO2734, paclitaxel and ARV-771, respectively, demonstrating its excellent in vitro anti-tumor activity.

[0199] Table 2 In vitro tumor activity evaluation results of target compound Ig

[0200]

[0201] a IC 50 Values ​​are expressed as mean ± SD

[0202] In addition, compound Ig has excellent in vitro antitumor activity in three cell lines: DU145, 22Rv1, and RM-1 (Table 2). In particular, in DU145 and RM-1 cell lines, compound Ig's antitumor activity is significantly better than that of the positive control drugs NEO2734, paclitaxel, and ARV-771. In normal human prostate epithelial cells RWPE-1, compound Ig exhibits significantly lower cytotoxicity, IC 50 was 3.89 μM, which was comparable to NEO2734 and ARV-771, while paclitaxel showed obvious toxicity (IC 50 The selectivity index of compound Ig between PC-3 / RWPE-1 was over 1500, and the selectivity index between DU145 / RWPE-1 was over 600, indicating the good selectivity and broad therapeutic window of compound Ig.

[0203] 4.2 Evaluation results of BRD4 and p300 / CBP degradation activity

[0204] We selected compound Ig with the strongest in vitro antitumor activity and evaluated its degradation activity against BRD4, p300, and CBP proteins in PC-3 cells. Figure 1The results in Figure A show that after treating PC-3 cells with different concentrations of compound Ig for 12 hours, the expression of BRD4 protein and c-Myc was significantly reduced. 50 (half-maximal degradation concentration) value reached 0.27nM, D max The maximum degradation rate was greater than 92%. 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 10.0 nM PROTAC molecule Ig 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.

[0205] same, Figure 2 A shows that treatment of PC-3 cells with different concentrations of compound Ig for 24 hours can significantly reduce the expression of p300 and CBP proteins in a concentration-dependent manner, among which the expression of DC for p300 is significantly decreased. 50 The value is 1.26nM, D max Greater than 83%; DC for CBP 50 The value is 10.50nM, D max Greater than 82%. Figure 2 Experimental results in Figure B further showed that treatment of PC-3 cells with 30 nM compound Ic for various time periods significantly reduced the expression of p300 and CBP, and almost completely degraded p300 / CBP after 24 hours of treatment. These results demonstrate that compound Ig is an excellent dual-target degrader of BRD4 and p300 / CBP.

[0206] In summary, the BRD4 and p300 / CBP dual-target degrader provided by the present invention can efficiently degrade BRD4 and p300 / CBP proteins simultaneously and exhibit excellent in vitro tumor activity, which can provide new compound entities and research directions for the development of degraders or anti-tumor drugs based on the dual targets of BRD4 and p300 / CBP.

Claims

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

3. A method for preparing a class of BRD4 and p300 / CBP dual-target degraders 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 dimethyl sulfoxide (DMSO) 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 Compound M2; wherein 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 reacted in an ethanol solvent under stirring 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 conditions. 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 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-target degrader I (1) Preparation of compounds 2a–2j and 4a–4h: 4-hydroxythalidomide or 5-hydroxythalidomide is reacted with tert-butyl bromoalkanoate or terminal dibromoalkane 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–2j and 4a–4h; wherein the polar organic solvent is selected from one of N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), N,N-diethylformamide (DEF), hexamethylphosphoric triamide (HMPA) and acetonitrile (MeCN); and the alkaline reagent is selected from one of sodium bicarbonate, potassium bicarbonate, sodium carbonate and potassium carbonate; (2) Preparation of compounds 3a–3j: Compounds 2a–2j were 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 compounds 3a–3j. (3) Preparation of Compounds Ia–Ij: Compounds 3a–3j were reacted with M6 and an amide coupling reagent, respectively, in DMF in the presence of an alkaline reagent under stirring at room temperature; after the reaction, the reaction system was sequentially extracted, washed, dried, filtered, and concentrated, and the resulting concentrate was separated and purified by column chromatography to obtain Compounds Ia–Ij; wherein the amide coupling reagent was selected from O-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) , 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) Preparation of compounds Ik–Ir: Compounds 4a–4h were reacted with M6 and an amide coupling reagent, respectively, in DMF in the presence of an alkaline reagent under stirring at room temperature; after the reaction, the reaction system was sequentially extracted, washed, dried, filtered, and concentrated, and the resulting concentrate was separated and purified by column chromatography to obtain compounds Ik–Ir; wherein the amide coupling reagent was selected from one of HBTU, HATU, TBTU, DCC, and EDCI; and the alkaline reagent was selected from one of potassium tert-butoxide, N,N-diisopropylethylamine, and triethylamine.

4. Use of a dual-target degrader of BRD4 and p300 / CBP as claimed in 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 degrader drugs.

5. Use of a dual-target degrader of BRD4 and p300 / CBP 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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