A compound targeting degradation of p300 / cbp protein and synthesis method and application thereof

Abstract

The application discloses a compound for targeted degradation of p300 / CBP protein and a synthesis method and application thereof, and relates to the technical field of medicinal chemistry.The application first provides a method for constructing proteolysis targeting chimera and other bifunctional molecules by using a SuFEx reaction; the application uses a fluoro sulfonate and a sulfonyl fluoride analog of a compound CPI644 as a p300 / CBP protein ligand, and connects, through a SuFEx reaction, an E3 ubiquitin ligase ligand 5-OH and 4-OH thalidomide, which are previously introduced with different lengths of alkane as a connecting arm, to prepare two series of 12 PROTACs molecules; the PROTACs molecules designed in the application have the ability to degrade p300 / CBP protein, and the PROTACs molecules can degrade p300 / CBP protein in a concentration-dependent manner in human breast cancer cells; the PROTACs molecules show good antitumor effects, and have a wide application prospect in the diagnosis and treatment of tumors.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a compound that targets and degrades p300 / CBP protein, its synthesis method, and its applications. Background Technology

[0002] Currently, p300 / CBP is an important target in disease research, and the design and discovery of high-quality small molecule inhibitors targeting p300 / CBP is also a hot topic among researchers. However, due to potential problems with the specificity, selectivity, and safety of small molecule inhibitors targeting p300 / CBP, protein degradation-targeting chimeras (PROTACs) targeting p300 / CBP have attracted great interest. Novel p300 / CBP PROTAC degraders show promise as a potential cancer therapy targeting p300 / CBP.

[0003] Unlike traditional small molecule inhibitors, PROTACs degrade non-drug-forming proteins. Furthermore, due to their event-driven nature, PROTACs offer advantages such as reduced dosage, improved drug resistance, and reduced off-target toxicity. PROTACs are bifunctional molecules, with one end linked to a ligand for E3 ubiquitin ligase and the other end linked to a ligand for the target protein, connected by a linker arm of appropriate length. The PROTAC molecule, E3 ubiquitin ligase, and target protein form a ternary complex. This complex allows the polyubiquitin chain to bind to the substrate protein, ultimately leading to protein degradation via UPS (Underlying Protein-Stage Compression).

[0004] It should be noted that the linker arm between these two ligands plays a very important role. If the linker arm is too short, the two ligands will mutually hinder their binding to the target protein or ubiquitinase, respectively; if the linker arm is too long, the PROTACs molecule will have difficulty bringing the E3 ubiquitin ligase closer to the target protein, thus preventing ubiquitination from occurring.

[0005] The SuFEx reaction, proposed by Sharpless et al., is a novel "click chemistry" approach based on the reaction of arylsulfonyl fluorides or aryl fluorosulfonates with silyl ethers or amino compounds. The SuFEx reaction offers several potential advantages, including: mild reaction conditions and ease of operation; strong substrate tolerance and good biocompatibility; significantly higher chemical stability of its substrates (sulfur(VI) fluorides) compared to the more common S-Cl, facilitating the preparation and storage of raw materials; and the formation of "sulfonamide" or "sulfate" linkages when arylsulfonyl fluorides or aryl fluorosulfonates react with amino compounds, which are advantageous structural members in pharmaceuticals and may introduce novel properties to PROTAC molecules.

[0006] Therefore, based on the above research background, the inventors believe that how to design a new PROTAC molecule based on the SuFEx reaction and construct a synthetic method for PROTAC molecules are technical problems that urgently need to be solved by those skilled in the art.

[0007] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a compound for targeted degradation of p300 / CBP protein, its synthesis method, and its application, thereby resolving the issues raised in the background section.

[0009] A compound that targets and degrades p300 / CBP protein, with the general structural formula as follows: Figure 4 Equations (A) and (B) are shown in the figure.

[0010] Preferred compounds that target the degradation of p300 / CBP protein have the following structural formulas: Figure 5 One of formulas I 1-I 6 and II 1-II 6.

[0011] A method for synthesizing a compound that targets and degrades p300 / CBP protein as described above, with the reaction route detailed below. Figure 3 and Figure 4 .

[0012] Preferably, the method for synthesizing compounds that target the degradation of p300 / CBP protein includes the following steps: Mixing 2-fluoro-3-bromonitrobenzene (Formula 1) and (R)-3-aminobutyric acid (Formula 2) and reacting them with a substitution reaction yields intermediate 3. Under the conditions of iron powder and acetic acid, the nitro group on intermediate 3 is reduced to an amino group and then cyclized to obtain intermediate 4; Intermediate 4 was subjected to Suzuki-Miyaura cross-coupling with boron reagent 5 to obtain olefin 6; Alkene 6 is Heck-coupled with the bromophenylsulfonyl fluoride shown in Formula 7 to obtain sulfonyl fluoride IV. Intermediate 4 was Heck-coupled with styrene 8 to obtain compound 9; Compound 9 was acylated with thioyl fluoride gas under alkaline conditions to give fluorosulfonate III; Fluorosulfonate III and sulfonyl fluoride IV were reacted with thalidomide containing linking arms of different alkane lengths via SuFEx reactions to obtain the target compounds shown in formulas (A) and (B).

[0013] The use of a compound as described above that targets and degrades p300 / CBP protein in the preparation of a drug for degrading p300 / CBP protein.

[0014] The use of a compound as described above that targets and degrades p300 / CBP protein in the preparation of a drug for cancer treatment.

[0015] Preferably, compounds that target and degrade p300 / CBP proteins are used as active ingredients in the preparation of tumor therapeutic drugs.

[0016] Preferably, the dosage form of the drug is an oral preparation, a mucosal delivery preparation, or an injection.

[0017] It is important to note that p300 / CBP proteins play a crucial role in tumorigenesis and development. They promote tumor cell growth and survival by regulating key processes such as gene transcription, cell cycle, and apoptosis. Overexpression or abnormal activity of p300 / CBP is a characteristic of many cancers, making them potential targets for cancer therapy.

[0018] The present invention provides a compound for targeted degradation of p300 / CBP protein, its synthesis method, and its application, which have the following beneficial effects: 1. This invention provides for the first time a method for constructing protein hydrolysis-targeting chimeras and other bifunctional molecules using the SuFEx reaction; this invention uses the fluorosulfonate and sulfonyl fluoride analog of compound CPI644 as p300 / CBP protein ligands, and connects them with E3 ubiquitin ligase ligands 5-OH and 4-OH thalidomide with alkane linkages of different lengths as linking arms via the SuFEx reaction to prepare two series of 12 PROTAC molecules; 2. The PROTACs molecules designed in this invention have the ability to degrade p300 / CBP protein, and the PROTACs molecules can degrade p300 / CBP protein in a concentration-dependent manner in human breast cancer cells; these PROTACs molecules have shown good anti-tumor effects and have broad application prospects in the diagnosis and treatment of tumors. Attached image description: Figure 1 This is a predicted diagram of the binding of compound CPI644 to the bromine domain (PDB: 5DBM) of the p300 / CBP protein; Figure 2 Design route for the synthesis of PROTACs based on the SuFEx reaction of compound CPI644 fluorosulfonate III and sulfonyl fluoride IV; Figure 3 Synthetic routes for fluorosulfonate III and sulfonyl fluoride IV; Figure 4 Synthetic route diagram for target PROTAC molecules; Figure 5 The structural formulas of 12 target PROTAC molecules; Figure 6 The degradation results of p300 protein by PROTAC molecules; Figure 7 The mechanism by which compounds I-4 and II-4 induce p300 protein degradation. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] To address the aforementioned technical problems, this invention provides a compound for targeted degradation of p300 / CBP protein, its synthesis method, and its application, thereby resolving the issues raised in the background section.

[0021] I. Design Concept of the Invention Compared to p300 / CBP HAT inhibitors, the development of p300 / CBP bromine domain inhibitors has progressed more rapidly, with various structural types of p300 / CBP bromine domain inhibitors reported to date. Among them, Genentech's compound CPI644 exhibits good affinity for the CBP bromine domain. The relevant research data for compound CPI644 are as follows: ITC (Isothermal titration calorimetry) experiments show K d = 0.084 uM; NanoBRETCellular Assays indicate that CPI 644 is equivalent to CBP's EC50. 50 = 0.53 uM, for EP300 EC 50 = 0.36 uM (Reference: S. Ghosh, A. Taylor, M. Chin, et al., J. Biol. Chem. 291 (2016) 13014-13027). These results suggest that compound CPI644 can serve as a ligand targeting the p300 / CBP bromine domain.

[0022] Docking experiment of compound CPI644 molecule with p300 / CBP protein bromine domain (PDB: 5DBM) (see details) Figure 1The results indicate that CPI644 binds to the acetyllysine recognition site of the p300 / CBP protein: its benzodiazepine core forms a key hydrogen bond with ASN1168 and a π-π stack with TRP1125. It should be noted that the sulfonamide group at the end of CPI644 points towards the solvent region. These results suggest that the sulfonamide at the end of CPI644 can be rationally coupled with E3 ubiquitin ligase ligands to prepare corresponding PROTAC molecules.

[0023] Based on the docking results of CPI644 molecules with the bromine domain molecules of p300 / CBP proteins, we designed and modified the sulfonamide groups at the end of CPI644 to fluorosulfonate III and sulfonyl fluoride IV (see details). Figure 2 The fluorosulfonate group in compound III and the sulfonyl fluoride group in compound IV can both be extended by the SuFEx reaction, that is, reacting with an amino-containing E3 ubiquitin ligase fragment to generate PROTACs molecules linked with ammonium sulfate or sulfonamide, respectively.

[0024] II. Specific synthetic methods for compounds that target and degrade p300 / CBP protein The synthetic methods of fluorosulfonate III and sulfonyl fluoride IV are as follows: Figure 3 As shown. A substitution reaction was carried out using commercially available starting material 1 (2-fluoro-3-bromonitrobenzene) and 2 ((R)-3-aminobutyric acid) to obtain intermediate 3. Then, under the conditions of iron powder and acetic acid, the nitro group was reduced to an amino group, and the intermediate 4 was obtained through self-cyclization. Intermediate 4 was Suzuki-Miyaura cross-coupled with boron reagent 5 to obtain olefin 6 (olefin 6 was prepared according to the synthetic route in the literature: J. Biol. Chem. 2016, 291, (25), 13014), and then Heck-coupled with bromophenylsulfonyl fluoride 7 to obtain sulfonyl fluoride IV. Intermediate 4 was Heck-coupled with styrene 8 to obtain 9, and then acylated with sulfuryl fluoride gas under alkaline conditions to obtain fluorosulfonate III.

[0025] After obtaining fluorosulfonate III and sulfonyl fluoride IV, fluorosulfonate III and sulfonyl fluoride IV were respectively subjected to SuFEx reactions with thalidomide fragments (which are known compounds) containing linking arms of different alkane lengths (see details). Figure 4 ), and 12 target PROTAC molecules were prepared; (Table 1).

[0026] Table 1. Synthetic information of target PROTACs molecules 4 n = 1 I-1 (79%) II-1 (76%) 4 n = 2 I-3 (88%) II-3 (78%) 4 n = 3 I-5 (72%) II-5 (81%) 5 n = 1 I-2 (82%) II-2 (72%) 5 n = 2 I-4 (85%) II-4 (84%) 5 n = 3 I-6 (78%) II-6 (82%) Reaction conditions: Fluorosulfonate III or sulfonyl fluoride IV (1.0 equiv.), thalidomide with linker arms of varying lengths (2.0 equiv.), HOBt (5 mol%), DIPEA (1.0 equiv.), and TMDS (2.0 equiv.) were reacted in anhydrous DMSO.

[0027] III. Preparation Examples of Specific Key Compounds Example 1: Synthesis of Compound 5 Compound 4 (100 mg, 0.39 mmol, 1.0 eq), boron reagent 5 (98 μL, 0.57 mmol, 1.5 eq), PdCl2(dppf) (27 mg, 0.04 mmol, 0.1 eq), Cs2CO3 (260 mg, 0.80 mmol, 2.0 eq), dioxane (1 mL), and H2O (0.3 mL) were added to a microwave reaction tube. The system was bubbled with nitrogen for 5 min to create an anaerobic environment. The temperature was set to 100 °C, and the microwave reaction was carried out for 45 min. After cooling to room temperature, the reaction was detected by TLC to confirm its completion. The product was extracted from the aqueous phase into the organic phase using ethyl acetate (3 × 30 mL). The aqueous and organic phases were monitored separately by TLC to ensure that all the product had entered the organic phase and that there was no product left in the aqueous phase. The organic phases were then combined, washed with saturated brine to absorb excess water, and thoroughly dried with anhydrous sodium sulfate. The anhydrous sodium sulfate was filtered off, and the organic phase was concentrated under reduced pressure using a rotary evaporator. The mixture was then separated by silica gel column chromatography (DCM:MeOH = 10:1) to give compound 5 as a brown solid (72 mg, 80%). 1H NMR (400 MHz, DMSO-d6) d 8.92 (s, 1H), 7.32 (dd, J = 7.9, 1.2 Hz, 1H), 7.17 (dd, J = 6.6, 1.2 Hz, 1H), 7.00 (dd, J =7.9, 6.6 Hz, 1H), 6.86 (dd, J = 16.3, 10.8 Hz, 1H), 6.26 (d, J = 8.5 Hz, 1H), 5.62 (dd, J = 16.2, 2.4 Hz, 1H), 5.44 (dd, J = 10.8, 2.4 Hz, 1H), 4.09 (m,1H), 2.68 (dd, J = 16.4, 6.4 Hz, 1H), 2.47- 2.39 (m, 1H), 1.29 (d, J = 6.6Hz, 3H). ESI-MS: m / z calcd. for C 12 H 15 N2O＋ [M+H] ＋ 203.1, found 203.0. Example 2: Synthesis of Compound IV Dissolve 5 (50 mg, 0.24 mmol, 1.0 eq) and 7 (88 mg, 0.37 mmol, 1.5 eq) in DMF (10 mL). Add TEA (0.6 mL, 4.80 mmol, 20.0 eq) and tris(o-methylphenyl)phosphine (15 mg, 0.05 mmol, 0.2 eq) to the system. After purging with nitrogen three times, add Pd(OAc)2 (11 mg, 0.05 mmol, 0.2 eq), and purge with nitrogen three more times. Heat to 150 °C and react overnight. The reaction is then monitored by TLC until complete. The reaction solution was diluted with water, and the product was extracted from the aqueous phase into the organic phase with ethyl acetate (3 × 30 mL). The aqueous and organic phases were monitored by TLC to ensure that all the product entered the organic phase and that there was no product left in the aqueous phase. The organic phases were then combined, washed with saturated brine to absorb excess water, dried thoroughly with anhydrous sodium sulfate, and filtered off the anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure using a rotary evaporator and then separated by silica gel column chromatography (PE:EA = 1:1) to give compound IV as a brown solid (62 mg, 47%). 1 H NMR (400 MHz, cdcl3) δ 8.09(ddd, J = 14.3, 8.1, 1.3 Hz, 1H), 8.01 (s, 1H), 7.86 (d, J = 8.0 Hz, 2H),7.81 – 7.66 (m, 1H), 7.56 – 7.40 (m, 1H), 7.37 – 7.19 (m, 3H), 5.30 (s, 1H), 4.18 (s, 1H), 2.66 (ddd, J = 17.5, 13.3, 4.7 Hz, 1H), 2.42 (dt, J = 13.3, 8.0Hz, 1H), 1.40 (dd, J = 6.3, 1.2 Hz, 3H). ESI-MS: m / z calcd. for C 18 H 18 FN2O3S ＋ [M+H] ＋ 361.1, found 361.1. Example 3: Synthesis of Compound 9 Dissolve 4 (50 mg, 0.2 mmol, 1.0 eq) and 8 (35 mg, 0.3 mmol, 1.5 eq) in DMF (10 mL). Add TEA (277 μL, 2.0 mmol, 10.0 eq) and tris(o-methylphenyl)phosphine (12 mg, 0.04 mmol, 0.2 eq) to the system. After purging with nitrogen three times, add Pd(OAc)2 (4 mg, 0.04 mmol, 0.2 eq), and purge with nitrogen three more times. Heat to 150 °C and react overnight. The reaction is then monitored by TLC until complete. The reaction solution was diluted with water, and the product was extracted from the aqueous phase into the organic phase with ethyl acetate (3 × 30 mL). The aqueous and organic phases were monitored by TLC to ensure that all the product entered the organic phase and that there was no product left in the aqueous phase. The organic phases were then combined, washed with saturated brine to absorb excess water, dried thoroughly with anhydrous sodium sulfate, and filtered off the anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure using a rotary evaporator and then separated by silica gel column chromatography (DCM:MeOH = 10:1) to give compound 9 as a yellow solid (30 mg, 51%). 1 H NMR (400 MHz, DMSO-d6) δ 8.99 (s,1H), 8.78 (s, 1H), 7.49 (dd, J = 8.0, 1.3 Hz, 1H), 7.36 – 7.21 (m, 3H), 7.21– 7.08 (m, 4H), 6.79 (dd, J 1.29 (d, J = 6.6 Hz, 3H). ESI-MS: m / z calcd. for C 18 H 19 N2O2 ＋ [M+H] ＋ 295.1, found 295.2. Example 4: Synthesis of Compound III Dissolve 9 (30 mg, 0.1 mmol, 1.0 eq) in DCM (5 mL), add TEA (41 μL, 0.3 mmol, 3.0 eq), and replace SO2F2 gas. React for 2 h. The reaction is stopped by TLC. Dilute the reaction solution with water, and extract the product from the aqueous phase to the organic phase with ethyl acetate (3 × 30 mL). Monitor the aqueous and organic phases separately by TLC to ensure that all product has entered the organic phase and that there is no product in the aqueous phase. Combine the organic phases, wash with saturated brine to absorb excess water, dry thoroughly with anhydrous sodium sulfate, filter out the anhydrous sodium sulfate, and concentrate the organic phase under reduced pressure using a rotary evaporator. No further purification is required; proceed directly to the next step.

[0028] Example 5: General method for synthesizing target PROTACs Compound III or IV (0.1 mmol, 1.0 eq), thalidomide with linker arms of varying lengths (0.1 mmol, 1.0 eq), and HOBT (1.0 mg, 0.005 mmol, 0.05 eq), TMDS (35 μL, 0.2 mmol, 2.0 eq), and DIPEA (17 μL, 0.1 mmol, 1.0 eq) were added to the system. The reaction was allowed to proceed at room temperature for 24 h after the addition was complete. The reaction was then monitored by TLC until the reaction was complete. The reaction solution was diluted with water, and the product was extracted from the aqueous phase into the organic phase using ethyl acetate (3 × 30 mL). TLC was performed on both the aqueous and organic phases separately to ensure that all product had entered the organic phase and that no product remained in the aqueous phase. The organic phases were then combined, washed with saturated brine to absorb excess water, dried thoroughly with anhydrous sodium sulfate, and filtered to remove the anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure using a rotary evaporator and then separated using silica gel plates.

[0029] I-1: Pale yellow powder, 57.5 mg, 79%. 1H NMR (400 MHz, DMSO) δ 11.16 (s, 1H),9.54 (s, 1H), 8.82 (t, J = 5.6 Hz, 1H), 8.31 (t, J = 5.8 Hz, 1H), 8.10 – 8.01(m, 1H), 7.78 (dd, J = 8.5, 7.3 Hz, 1H), 7.59 (d, J = 16.1 Hz, 1H), 7.49 (d,J = 7.3 Hz, 1H), 7.44 – 7.32 (m, 6H), 7.27 (d, J = 16.1 Hz, 1H), 6.96 – 6.79(m, 2H), 5.14 (dd, J = 12.9, 5.4 Hz, 2H), 4.81 (s, 2H), 4.05 (dq, J = 11.6,6.6 Hz, 2H), 3.34 (q, J = 6.4 Hz, 2H), 3.25 (q, J = 6.3 Hz, 3H), 2.91 (ddd, J= 17.4, 13.9, 5.5 Hz, 1H), 2.66 – 2.53 (m, 2H), 2.52 – 2.41 (m, 2H), 2.15(dd, J = 13.0, 7.2 Hz, 1H), 2.10 – 2.01 (m, 1H), 2.00 (s, 1H), 1.28 (d, J =6.1 Hz, 3H), 1.19 (t, J = 7.1 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 173.34,172.32, 172.26, 170.90, 170.42, 167.81, 167.25, 165.90, 155.60, 147.67,137.91, 137.43, 133.52, 132.85, 131.33, 131.04, 129.92, 129.12, 127.48,127.34, 127.27, 126.89, 125.13, 124.13, 123.01, 122.81, 122.51, 122.27,122.11, 121.38, 120.85, 119.63, 117.20, 116.53, 116.25, 67.96, 60.29, 56.23,56.13, 49.29, 43.14, 40.73, 38.86, 31.45, 23.76, 23.68, 22.51, 21.28, 14.59.ESI-MS: m / z calcd. for C 35 H 35 N6O 10 S ＋ [M+H] ＋ 731.2, found 731.2. I-2: Pale yellow powder, 60.8 mg, 82%. 1H NMR (400 MHz, DMSO) δ 11.15 (s, 1H),9.52 (s, 1H), 8.82 (t, J = 5.6 Hz, 1H), 8.45 (t, J = 5.7 Hz, 1H), 8.08 – 8.01(m, 1H), 7.91 – 7.83 (m, 1H), 7.58 (d, J = 16.1 Hz, 1H), 7.50 – 7.41 (m, 2H),7.41 – 7.35 (m, 5H), 7.28 (t, J = 14.8 Hz, 2H), 6.95 – 6.86 (m, 2H), 6.86 –6.79 (m, 1H), 5.18 – 5.07 (m, 2H), 4.72 (s, 2H), 4.04 (q, J = 7.1 Hz, 2H),3.32 (t, J = 6.1 Hz, 3H), 3.25 (t, J = 6.1 Hz, 2H), 3.23 – 3.15 (m, 1H), 2.97– 2.84 (m, 1H), 2.65 – 2.52 (m, 2H), 2.52 – 2.38 (m, 2H), 2.19 – 2.02 (m,2H), 2.00 (s, 2H), 1.26 (t, J = 6.9 Hz, 4H), 1.18 (t, J = 7.1 Hz, 3H). 13C NMR(101 MHz, DMSO) δ 173.31, 172.27, 172.21, 171.96, 170.87, 170.44, 167.67,167.28, 167.23, 163.48, 163.44, 155.33, 147.67, 137.91, 137.38, 134.20,132.85, 131.33, 131.01, 129.91, 129.37, 129.11, 127.49, 127.33, 127.26,126.87, 125.83, 124.78, 124.06, 122.99, 122.80, 122.49, 122.25, 122.08,121.34, 121.26, 119.61, 116.25, 110.08, 67.75, 60.28, 56.20, 56.10, 49.48,43.33, 43.08, 40.73, 38.95, 38.77, 31.45, 24.14, 23.67, 23.08, 22.55, 21.60,21.27, 14.59.ESI-MS: m / z calcd. for C 35 H 35 N6O 10 S ＋ [M+H] ＋ 731.2, found 731.2. I-3: Yellow powder, 66.8 mg, 88%. 1H NMR (600 MHz, DMSO) δ 11.11 (s, 1H), 9.49(s, 1H), 8.53 (t, J = 5.5 Hz, 1H), 8.04 – 7.95 (m, 2H), 7.85 – 7.77 (m, 1H),7.57 – 7.46 (m, 2H), 7.43 – 7.31 (m, 5H), 7.30 – 7.21 (m, 1H), 6.94 – 6.89(m, 1H), 6.89 – 6.82 (m, 1H), 5.15 – 5.08 (m, 1H), 4.76 (d, J = 5.1 Hz, 2H),4.03 (qd, J = 6.7, 5.3 Hz, 1H), 3.18 – 3.07 (m, 4H), 2.93 – 2.84 (m, 1H),2.69 (d, J = 1.3 Hz, 4H), 2.63 – 2.56 (m, 1H), 2.53 (d, J = 16.0 Hz, 8H),2.43 (dd, J = 13.0, 5.0 Hz, 1H), 2.21 – 2.10 (m, 1H), 2.06 – 1.97 (m, 2H),1.53 – 1.48 (m, 2H), 1.48 (d, J = 7.9 Hz, 2H), 1.37 – 1.28 (m, 2H), 1.24 (dd,J = 14.5, 5.6 Hz, 6H), 1.17 (td, J = 7.1, 1.3 Hz, 1H), 0.90 – 0.83 (m, 1H). 13C NMR (101 MHz, DMSO) δ 173.48, 172.61, 170.46, 167.42, 167.28, 166.06,155.51, 147.71, 137.88, 137.58, 133.43, 132.75, 131.20, 130.02, 129.20,127.32, 127.26, 123.07, 122.98, 122.53, 122.18, 121.47, 120.81, 117.13,116.60, 82.28, 67.96, 56.22, 49.27, 43.72, 40.63, 40.59, 38.36, 34.84, 31.39,27.15, 26.87, 26.54, 23.62, 22.46. ESI-MS: m / z calcd. for C 37 H 39 N6O 10 S ＋ [M+H] ＋ 759.2, found 759.2. I-4: Pale yellow powder, 64.5 mg, 85%. 11H NMR (400 MHz, DMSO) δ 11.15 (s, 1H), 9.52 (s, 1H), 8.30 (q, J = 5.1 Hz, 1H), 8.06 – 7.99 (m, 1H), 7.88 (d, J = 8.3 Hz, 1H), 7.57 (d, J = 16.1 Hz, 1H), 7.45 (d, J = 2.3 Hz, 1H), 7.43 – 7.35 (m, 4H), 7.35 – 7.22 (m, 2H), 6.95 – 6.71 (m, 2H), 5.18 – 5.05 (m, 2H), 4.72 (d, J = 2.8 Hz, 2H), 4.10 – 3.99 (m, 1H), 3.13 (dt, J = 6.7, 3.6 Hz, 5H), 2.90 (ddd, J = 17.0, 14.0, 5.4 Hz, 1H), 2.59 (dd, J = 15.5, 11.9 Hz, 3H), 2.51 – 2.36 (m, 2H), 2.19 – 1.98 (m, 3H), 1.56 – 1.46 (m, 4H), 1.26 (t, J = 6.0 Hz, 4H), 1.23 – 1.15 (m, 1H). 13 13C NMR (101 MHz, DMSO) δ 173.30, 172.18, 170.44, 167.30, 167.24, 167.17, 163.57, 147.84, 137.89, 134.20, 132.86, 131.30, 129.96, 129.08, 127.37, 127.22, 127.15, 125.81, 124.01, 123.00, 122.39, 122.07, 121.32, 109.97, 67.83, 60.27, 56.09, 49.48, 43.79, 40.74, 38.34, 31.45, 27.02, 26.69, 23.69, 22.54, 21.28, 14.60. ESI-MS: m / z calcd. for C 37 H 39 N6O 10 S ＋ [M+H] ＋ 759.2,found 759.2. I-5: Yellow powder, 6.1 mg, 72%. 1H NMR (600 MHz, DMSO) δ 11.13 (s, 1H), 9.51(s, 1H), 8.56 (q, J = 4.0 Hz, 1H), 8.06 – 8.00 (m, 2H), 7.84 – 7.77 (m, 1H),7.57 (d, J = 16.1 Hz, 1H), 7.49 (d, J = 7.3 Hz, 1H), 7.45 – 7.31 (m, 5H),7.26 (d, J = 16.1 Hz, 1H), 6.90 – 6.84 (m, 2H), 5.76 (s, 1H), 5.16 – 5.09 (m,1H), 5.09 – 5.05 (m, 1H), 4.78 (s, 2H), 4.16 (s, 1H), 4.05 (tt, J = 7.3, 3.6Hz, 1H), 3.18 (d, J = 3.1 Hz, 3H), 3.16 – 3.11 (m, 2H), 3.11 – 3.02 (m, 2H),2.90 (ddd, J = 17.0, 13.9, 5.4 Hz, 1H), 2.64 – 2.51 (m, 3H), 2.47 – 2.38 (m,1H), 2.14 (dd, J = 12.9, 7.2 Hz, 1H), 2.04 (dtd, J = 12.5, 4.9, 2.0 Hz, 1H),1.55 – 1.37 (m, 4H), 1.25 (dd, J = 14.7, 8.9 Hz, 7H). 13C NMR (151 MHz, DMSO)δ 173.25, 172.16, 171.72, 170.35, 167.21, 167.11, 165.97, 155.54, 147.77,137.87, 137.37, 133.50, 132.80, 131.25, 129.93, 129.04, 127.44, 127.26,127.16, 127.05, 122.95, 122.89, 122.37, 122.06, 121.23, 120.81, 117.25,116.47, 68.06, 56.01, 55.38, 55.35, 49.28, 49.05, 43.98, 43.77, 40.73, 40.50, 38.72, 31.41, 29.44, 29.37, 26.34, 26.31, 26.15, 23.63, 22.47 . ESI-MS: m / zcalcd. for C 39 H 43 N6O 10 S ＋ [M+H] ＋ 787.2, found 787.3. I-6: Yellow powder, 60.7 mg, 78%. 1H NMR (400 MHz, DMSO) δ 9.53 (s, 1H), 8.31(t, J = 5.8 Hz, 1H), 8.04 (dd, J = 6.1, 3.3 Hz, 1H), 7.87 (dd, J = 8.3, 2.2Hz, 1H), 7.58 (d, J = 16.1 Hz, 1H), 7.44 (d, J = 2.4 Hz, 1H), 7.43 – 7.30 (m,6H), 7.30 – 7.22 (m, 2H), 6.93 – 6.84 (m, 2H), 5.14 (dt, J = 12.8, 3.4 Hz,1H), 5.08 (s, 1H), 4.73 (s, 2H), 4.04 (ddt, J = 14.2, 7.1, 4.0 Hz, 1H), 3.10(t, J = 6.9 Hz, 4H), 2.90 (tt, J = 13.6, 5.4 Hz, 2H), 2.65 – 2.54 (m, 2H),2.44 (dd, J = 13.1, 4.9 Hz, 2H), 2.19 – 1.95 (m, 4H), 1.54 – 1.34 (m, 6H),1.26 (d, J = 5.8 Hz, 5H), 0.85 (s, 1H). 13 C NMR (151 MHz, DMSO) δ 173.23,171.97, 171.70, 170.37, 167.27, 167.20, 167.05, 163.57, 159.19, 158.94,158.69, 158.44, 147.78, 134.17, 133.02, 131.19, 130.20, 129.13, 127.23,127.20, 127.18, 125.74, 123.99, 123.26, 122.92, 122.44, 122.14, 121.87,121.37, 116.72, 114.80, 109.86, 67.83, 60.22, 56.23, 55.34, 49.47, 43.98,40.58, 40.47, 38.74, 31.41, 29.42, 29.35, 26.35, 26.15, 23.25, 22.88, 22.52,14.52. ESI-MS: m / z calcd. for C 39 H 43 N6O10 S ＋ [M+H] ＋ 787.2, found 787.3. II-1: Yellow powder, 53.9 mg, 76%. 1 H NMR (600 MHz, DMSO) δ 11.12 (s, 1H), 9.49 (s, 1H), 8.08 (d, J = 7.9 Hz, 1H), 8.04 (dt, J = 6.1, 3.1 Hz, 1H), 7.98 (t, J = 6.0 Hz, 1H), 7.85 (dd, J = 8.0, 1.4 Hz, 1H), 7.79 – 7.71 (m, 2H), 7.64 (t, J = 7.6 Hz, 1H), 7.50 – 7.43 (m, 3H), 7.41 (dd, J = 6.4, 3.0 Hz, 1H), 7.35 (d, J = 8.6 Hz, 1H), 6.89 (q, J = 4.2 Hz, 2H), 5.18 – 5.01 (m, 2H), 4.71 (d, J = 1.6 Hz, 2H), 4.04 (q, J = 7.0 Hz, 1H), 3.20 (q, J = 6.6 Hz, 2H), 3.01 – 2.78 (m, 3H), 2.47 – 2.38 (m, 2H), 2.13 (dd, J = 13.0, 7.2 Hz, 1H), 2.03 (dtd, J = 12.8, 5.3, 2.2 Hz, 1H), 1.37 – 1.29 (m, 1H), 1.24 (dd, J = 13.5, 4.9 Hz, 5H). 13 C NMR (101 MHz, DMSO) δ 173.28, 172.16, 170.85, 170.38, 167.64, 167.23, 165.88, 155.57, 137.41, 133.54, 126.16, 123.29, 122.20, 120.89, 117.26, 116.52, 67.97, 60.27, 56.05, 55.42, 49.29, 49.09, 48.97, 40.92, 31.45, 23.70, 22.51, 21.27, 14.59. ESI-MS: m / z calcd. for C 35 H 35 N6O9S＋ [M+H] ＋ 715.2, found 715.2. II-2: Yellow powder, 51.1 mg, 72%. 1 H NMR (600 MHz, DMSO) δ 11.12 (s, 1H), 9.49 (s, 1H), 8.22 (t, J = 5.9 Hz, 1H), 8.09 (dd, J = 8.0, 1.2 Hz, 1H), 7.99 (t, J = 6.0 Hz, 1H), 7.90 – 7.82 (m, 2H), 7.75 (d, J = 15.8 Hz, 1H), 7.69 – 7.57 (m, 1H), 7.50 – 7.40 (m, 4H), 7.40 – 7.32 (m, 1H), 6.93 – 6.86 (m, 2H), 5.15 – 5.08 (m, 2H), 4.64 (s, 2H), 4.11 (q, J = 5.2 Hz, 2H), 4.03 (q, J = 7.1 Hz, 2H), 3.19 (dd, J = 17.0, 5.9 Hz, 6H), 2.95 – 2.90 (m, 2H), 2.90 – 2.85 (m, 1H), 2.64 – 2.59 (m, 1H), 2.59 – 2.50 (m, 4H), 2.43 (dd, J = 12.9, 5.0 Hz, 1H), 2.13 (dd, J = 13.0, 7.2 Hz, 1H), 2.05 (dtd, J = 13.0, 5.4, 2.4 Hz, 1H), 1.99 (s, 2H), 1.25 (d, J = 6.2 Hz, 3H), 1.18 (t, J = 7.1 Hz, 2H). 13C NMR(151 MHz, DMSO) δ 173.24, 172.13, 170.82, 170.37, 167.49, 167.23, 167.18,163.35, 138.00, 137.55, 136.58, 134.17, 133.01, 132.73, 129.85, 129.13,129.07, 128.94, 128.57, 127.83, 126.12, 125.76, 124.04, 123.26, 122.18,121.24, 121.12, 109.98, 67.69, 60.22, 56.00, 55.37, 49.46, 49.07, 42.00,40.70, 40.51, 38.97, 31.42, 23.65, 22.52, 21.22, 14.55. ESI-MS: m / z calcd.for C 35 H 35 N6O9S ＋ [M+H] ＋ 715.2, found 715.3. II-3: Yellow powder, 57.9 mg, 78%. 1H NMR (600 MHz, DMSO) δ 11.11 (s, 1H),9.49 (s, 1H), 8.09 (dd, J = 8.0, 1.3 Hz, 1H), 7.92 (t, J = 5.8 Hz, 1H), 7.87– 7.80 (m, 2H), 7.79 (dd, J = 7.2, 1.4 Hz, 1H), 7.76 (s, 1H), 7.67 – 7.62 (m,1H), 7.51 – 7.43 (m, 3H), 7.42 (dd, J = 7.2, 2.3 Hz, 1H), 7.36 (d, J = 8.6Hz, 1H), 6.93 – 6.86 (m, 2H), 5.16 – 5.05 (m, 2H), 4.74 (s, 2H), 4.04 (q, J =6.6 Hz, 1H), 3.06 (q, J = 6.4 Hz, 2H), 2.89 (ddd, J = 17.1, 13.9, 5.5 Hz,1H), 2.82 (q, J = 6.3 Hz, 2H), 2.60 (ddd, J = 17.0, 4.2, 2.5 Hz, 1H), 2.47 –2.41 (m, 1H), 2.13 (dd, J = 13.0, 7.2 Hz, 1H), 2.03 (dtd, J = 12.8, 5.2, 2.3Hz, 1H), 1.39 (dd, J = 6.3, 3.1 Hz, 3H), 1.26 – 1.24 (m, 3H). 13 C NMR (151MHz, DMSO) δ 173.24, 172.13, 170.35, 167.20, 167.14, 165.95, 155.53, 137.99,137.40, 133.50, 132.87, 129.00, 128.78, 128.47, 127.79, 126.27, 123.23,122.17, 121.13, 120.85, 117.26, 116.51, 68.06, 56.01, 49.27, 42.48, 40.89,40.71, 38.27, 31.42, 27.05, 26.63, 23.65, 22.46. ESI-MS: m / z calcd. forC 37 H 39 N6O9S ＋ [M+H]＋ 743.2, found 743.2. II-4: Yellow powder, 62.1 mg, 84%. 1 H NMR (600 MHz, DMSO) δ 11.12 (s, 1H), 9.50 (s, 1H), 8.17 (t, J = 5.9 Hz, 1H), 8.10 (dd, J = 7.9, 1.2 Hz, 1H), 7.88–7.85 (m, 2H), 7.82 (t, J = 5.8 Hz, 1H), 7.78 (d, J = 15.8 Hz, 1H), 7.65 (td, J = 7.6, 1.4 Hz, 1H), 7.51 – 7.40 (m, 4H), 7.37 (dd, J = 8.3, 2.3 Hz, 1H), 6.94 – 6.87 (m, 2H), 5.15 – 5.08 (m, 2H), 4.68 (s, 2H), 4.14 – 4.02 (m, 1H), 3.18 (d, J = 5.0 Hz, 1H), 3.05 (q, J = 6.3 Hz, 2H), 2.83 (q, J = 6.3 Hz, 2H), 2.52 – 2.41 (m, 7H), 2.14 (dd, J = 12.9, 7.2 Hz, 1H), 2.10 – 2.00 (m, 1H), 1.43 – 1.33 (m, 4H), 1.26 (d, J = 6.2 Hz, 4H). 13 C NMR (101 MHz, DMSO) δ 172.31, 170.85, 170.19, 169.67, 167.20, 167.03, 161.43, 138.09, 136.53, 133.91, 133.40, 131.42, 130.37, 128.63, 128.54, 128.31, 127.87, 127.53, 127.38, 126.20, 125.54, 124.06, 124.02, 122.82, 121.65, 108.38, 67.77, 52.50, 49.98, 43.97, 43.52, 40.15, 29.91, 26.43, 26.33, 24.73, 20.89. ESI-MS: m / z calcd. for C 37 H 39 N6O9S ＋[M+H] ＋ 743.2, found 743.2. II-5: Yellow powder, 62.4 mg, 81%. 1 H NMR (600 MHz, DMSO) δ 11.12 (s, 1H), 9.49 (s, 1H), 8.10 (dd, J = 7.8, 1.2 Hz, 1H), 7.92 – 7.84 (m, 2H), 7.84 – 7.75 (m, 3H), 7.68 – 7.62 (m, 1H), 7.51 – 7.40 (m, 4H), 7.38 (d, J = 8.6 Hz, 1H), 6.94 – 6.86 (m, 2H), 5.15 – 5.10 (m, 1H), 5.10 – 5.07 (m, 1H), 4.75 (s, 2H), 4.04 (q, J = 6.2 Hz, 1H), 3.07 (p, J = 6.3 Hz, 2H), 2.94 – 2.84 (m, 1H), 2.81 (q, J = 6.7 Hz, 2H), 2.48 – 2.40 (m, 1H), 2.13 (dd, J = 12.9, 7.3 Hz, 1H), 2.04 (dtd, J = 13.0, 5.4, 2.4 Hz, 1H), 1.33 (dt, J = 14.7, 7.1 Hz, 5H), 1.28 – 1.21 (m, 6H), 1.21 – 1.17 (m, 2H), 1.17 – 1.14 (m, 1H), 1.14 – 1.08 (m, 2H). 13C NMR (151 MHz, DMSO) δ 173.24, 172.13, 170.34, 167.20, 167.06,165.97, 155.51, 137.97, 137.38, 136.54, 133.50, 132.86, 132.77, 129.90,129.06, 128.74, 128.44, 127.77, 126.28, 123.23, 122.17, 121.14, 120.83,117.28, 116.51, 68.08, 56.03, 49.27, 42.67, 40.90, 40.71, 40.52, 38.70,31.41, 29.53, 29.34, 26.25, 26.11, 23.65, 22.46. ESI-MS: m / z calcd. forC 39 H 43 N6O9S ＋ [M+H] ＋ 771.2, found 771.1. II-6: Yellow powder, 63.2 mg, 82%. 1H NMR (600 MHz, DMSO) δ 11.11 (s, 1H),9.49 (s, 1H), 8.15 (t, J = 5.8 Hz, 1H), 8.10 (dd, J = 8.0, 1.2 Hz, 1H), 7.86(dt, J = 6.6, 2.4 Hz, 2H), 7.85 – 7.75 (m, 2H), 7.68 – 7.62 (m, 1H), 7.51 –7.31 (m, 5H), 6.94 – 6.86 (m, 2H), 5.15 – 5.07 (m, 2H), 4.69 (s, 2H), 4.04(q, J = 6.6 Hz, 1H), 3.06 (dq, J = 13.2, 6.6 Hz, 2H), 2.93 – 2.84 (m, 2H),2.80 (q, J = 6.6 Hz, 2H), 2.69 (s, 1H), 2.63 – 2.57 (m, 1H), 2.43 (dd, J =12.9, 5.0 Hz, 1H), 2.13 (dd, J = 13.0, 7.3 Hz, 1H), 2.05 (dtd, J = 12.7, 5.3,2.2 Hz, 1H), 1.42 – 1.33 (m, 3H), 1.31 (dd, J = 13.3, 6.1 Hz, 3H), 1.26 –1.18 (m, 8H), 1.17 (d, J = 8.3 Hz, 1H), 1.11 (dt, J = 14.4, 8.6 Hz, 3H), 0.85(t, J = 6.8 Hz, 1H). 13C NMR (151 MHz, DMSO) δ 173.24, 172.12, 170.37, 167.27,167.20, 167.00, 163.57, 137.97, 136.54, 134.17, 132.86, 132.77, 129.90,129.06, 128.74, 128.44, 127.77, 126.28, 125.76, 123.98, 123.23, 122.17,121.38, 121.14, 109.86, 67.83, 56.03, 49.46, 42.69, 40.90, 40.72, 40.52,38.71, 32.01, 31.42, 30.30, 29.90, 29.52, 29.45, 29.40, 26.31, 26.13, 23.65,22.56, 22.52, 14.42. ESI-MS: m / z calcd. for C 39 H 43 N6O9S ＋ [M+H] ＋ 771.2, found 771.4. IV. Pharmacological assays for the activity of PROTACs molecules The obtained target compound was used to perform p300 protein degradation experiments in human breast cancer cells MDA-MB-468. The Western blotting procedure included: discarding the culture medium from the plate and washing once with PBS. 100 μL of pre-prepared 2% SDS solution was added to each well, ensuring the solution evenly covered all cells. The mixture was continuously stirred with a pipette until the cells at the bottom were completely digested and a viscous liquid was formed. This liquid was then transferred to a 1.5 mL EP tube and heated in a 100°C metal bath for 15 min. The viscous liquid in the EP tube became clear and transparent, which was the cell protein solution. The protein concentration of each sample was detected using a BCA kit. 1 / 4 volume of 5× loading buffer was added to the protein solution and mixed thoroughly. The samples were added to SDS-PAGE wells (total protein 40 μg / well). The electrophoresis apparatus was set to a constant voltage of 80 V. After the samples left the stacking gel, protein separation was completed using a constant voltage current of 150 V. The protein samples on the separating gel were transferred to a PVDF membrane. The electrophoresis apparatus was set to a constant current of 400 mA, and the transfer time was 60 min. The PVDF membrane was placed in TBST solution containing 5% BSA and blocked at room temperature for 1 h. After blocking, the membrane was washed three times with TBST for 5 min each time. The PVDF band containing the target protein was completely immersed in the primary antibody solution, ensuring full contact, and incubated overnight with gentle shaking at 4°C. The PVDF membrane was washed five times with TBST for 6 min each time. After washing, the PVDF band was completely immersed in the primary antibody solution, ensuring full contact, and incubated for 2 h with gentle shaking at room temperature. After washing, the membrane was washed five times with TBST for 6 min each time. The PVDF band was uniformly covered with ECL chemiluminescence buffer, and the protein bands were detected using a Bio-Rad gel imaging system.

[0030] Western blotting results show (see details) Figure 6 The 12 PROTAC molecules designed and synthesized in this invention can all degrade p300 protein in a concentration-dependent manner in MDA-MB-468 cells. Among them, I-4 and II-4 showed better protein degradation ability in their respective series, with their DC... 50 The values ​​are 0.18 uM and 1.04 uM, respectively.

[0031] Next, taking I-4 and II-4 as examples, a mechanism verification experiment using competitive inhibition confirmed that I-4 and II-4 induce the degradation of p300 protein through the ubiquitin-proteasome pathway (see details). Figure 7First, I-4 and II-4 significantly degraded p300 protein. Next, MDA-MB-468 cells were pretreated with the proteasome inhibitor MG132, followed by treatment with compounds I-4 and II-4. After treatment, the degradation of p300 activity by both compounds was significantly reduced, suggesting that the degradation of the target protein by PROTACs occurs via the proteasome pathway. Then, MDA-MB-468 cells were pretreated with the CRBN ligand thalidomide and the p300 protein ligand CPI644, respectively, before the addition of compounds I-4 and II-4. After treatment, the degradation of p300 activity by both compounds was again significantly reduced, suggesting that the degradation of the target protein by PROTACs occurs through the formation of a p300-PROTACs-CRBN ternary complex.

[0032] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A compound that targets and degrades p300 / CBP protein, characterized in that, The general structural formulas are shown in equations (A) and (B): .

2. The compound for targeted degradation of p300 / CBP protein according to claim 1, characterized in that, The structural formula is one of formulas I1-I6 and II1-II6: 。 3. A method for synthesizing a compound for targeted degradation of p300 / CBP protein as described in claim 1 or 2, characterized in that, The synthetic reaction route is as follows: ； 。 4. The use of the compound as described in claim 1 for targeting and degrading p300 / CBP protein in the preparation of a drug for degrading p300 / CBP protein.

5. The use of a compound as described in claim 1 that targets and degrades p300 / CBP protein in the preparation of a medicament for cancer treatment.

6. The use of the compound according to claim 5, which targets and degrades p300 / CBP protein, in the preparation of a drug for tumor treatment, characterized in that, Application of compounds that target and degrade p300 / CBP protein as active ingredients in the preparation of tumor therapeutic drugs.

7. The use of the compound according to claim 6, which targets and degrades p300 / CBP protein, in the preparation of a drug for tumor treatment, characterized in that, The dosage form of the drug is oral preparation, mucosal delivery preparation or injection.

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