Preparation method and application of p65 protein degradation agent

Abstract

The invention relates to the technical field of biological medicine, in particular to a preparation method and application of a p65 protein degradation agent, the structural formula of the compound is as shown in formula I: # imgabs0 # experiment results show that the compound (formula I) provided by the invention can significantly down-regulate the p65 protein level and presents a dose-dependent effect. The HOOK effect observed under the condition of high concentration prompts that the formula I possibly forms a ternary complex with p65 and E3 ubiquitin ligase CRBN to mediate degradation of p65, and important evidence is provided for the action mechanism of the compound. In addition, the compound (formula I) can effectively reduce gene expression of key inflammatory factors (such as IL-1beta, IL-6, TNF-alpha, iNOS and MCP-1) in LPS-induced RAW 264.7 macrophages, and shows potential application value in the aspects of regulating inflammatory response and improving lipid metabolism.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular to a preparation method and application of a p65 protein degrader. Background Art

[0002] p65 (RelA) is one of the core members of the NF-κB (nuclear factor κB) transcription factor family and plays a key role in regulating biological processes such as inflammation, immunity, cell survival, and metabolism. The heterodimer formed by p65 and p50 is the main active form of the classical NF-κB pathway. The activation of p65 depends on the stimulation of the IKK complex by upstream signals (such as TNFα, IL1β, LPS, etc.), which then leads to the phosphorylation, ubiquitination, and degradation of IκBα, releasing the p65 / p50 dimer and promoting its nuclear translocation. In the nucleus, p65 binds to the κB site in the promoter or enhancer region of target genes through its N-terminal Rel homology domain (RHD), driving gene transcription.

[0003] By directly or indirectly regulating the expression of a variety of inflammation-related genes, as a core regulator of the inflammatory response, p65 amplifies the inflammatory signal by inducing the expression of pro-inflammatory cytokines (such as IL1β, IL6, TNFα) and chemokines (such as MCP1). In addition, the abnormal activation of p65 is closely related to the occurrence and development of various diseases, such as chronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease; metabolic diseases such as non-alcoholic steatohepatitis (NASH) and type 2 diabetes, as well as cancers.

[0004] Currently, the intervention strategies for p65 mainly include small molecule inhibitors and gene silencing technologies, but both have certain limitations. In terms of small molecule inhibitors, existing drugs (such as BAY11-7082, IKKβ inhibitors, etc.) mainly target upstream kinases of the NF-κB pathway (such as IKKβ), rather than directly acting on p65. For example, IKKβ, as a key kinase in the classical NF-κB pathway, its inhibition will block the phosphorylation and degradation of IκBα, thereby indirectly inhibiting the nuclear translocation of p65. However, IKKβ is widely involved in the signal regulation of various immune cells, and its non-specific inhibition may cause systemic immunosuppression and significantly increase the risk of infection. In addition, studies have shown that the NF-κB signaling pathway has a complex cross-regulation network with other key pathways such as Wnt and Nrf2, further exacerbating the off-target effect. More importantly, small molecule inhibitors can only block the activation process of p65 (such as inhibiting its phosphorylation or nuclear translocation), but cannot clear the already activated p65 protein. Therefore, in some diseases (such as chronic inflammation or fibrosis), simply inhibiting the activity of p65 may be difficult to reverse the existing pathological damage, and strategies such as protein knockout, degradation, or silencing need to be combined to completely interrupt the inflammatory signal transduction.

[0005] In the field of gene silencing technology, siRNA or nucleic acid aptamers need to be delivered to target cells with the aid of lipid nanoparticles or viral vectors. However, siRNA is easily degraded by lysosomal enzymes during intracellular transport and requires a combination of escape strategies (such as pH-sensitive liposomes) to improve its cytoplasmic release efficiency. In addition, delivery vectors may activate pattern recognition receptors such as TLR3 / 7, triggering non-specific inflammatory responses and limiting their clinical applications.

[0006] Therefore, targeted regulation of p65 protein homeostasis has become an important strategy for intervening in related pathological processes. In recent years, the emerging targeted protein degradation technology has achieved specific clearance of disease-related proteins by leveraging the intracellular natural degradation systems (such as the ubiquitin-proteasome system or the lysosomal pathway). Among numerous TPD strategies, the proteolysis-targeting chimera (PROTAC) technology has stood out with its mature R & D system. Its core mechanism relies on heterobifunctional small molecules that bind to the target protein (such as p65) at one end and recruit E3 ubiquitin ligase at the other end. By forming a ternary complex of "target protein - PROTAC - E3 ligase", it induces polyubiquitination labeling of the target protein, which is ultimately recognized and degraded by the proteasome. Different from the "occupation-driven" mode of traditional small molecule inhibitors, PROTAC follows an "event-driven" mechanism and still has high degradation ability even for targets lacking active pockets or with low affinity (such as "undruggable" targets like transcription factors and scaffold proteins). Currently, multiple PROTAC molecules globally have entered the clinical research stage, covering multiple fields such as solid tumors, hematological malignancies, and immune metabolic diseases, providing a new idea for breaking through the bottleneck of traditional drug development. Summary of the Invention

[0007] The object of the present invention is to solve the drawbacks existing in the prior art and to propose a preparation method and application of a p65 protein degrader.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A p65 protein degrader, the structure of the degrader is shown in Formula I:

[0010]

[0011] A preparation method of a p65 protein degrader, the synthetic reaction structural formula of the degrader of Compound Formula I is shown as follows:

[0012]

[0013] Its preparation method includes the following steps:

[0014] A certain amount of bromoacetyl bromide (2.13 mL, 4.94 g, 24.6 mmol) was added to a 50 mL anhydrous tetrahydrofuran (THF) suspension containing pomalidomide (2.23 g, 8.2 mmol). The mixture was stirred under reflux for 16 h (TLC monitoring: petroleum ether / ethyl acetate = 1:1, Rf ≈ 0.3); after the reaction was completed, the reaction mixture was concentrated, and after adding diethyl ether, pale yellow solid 1a (2.75 g, yield 85.5%) was directly precipitated;

[0015] Sodium azide (988.0 mg, 15.2 mmol) was slowly added to a 50 mL acetone suspension of compound 1a (3.00 g, 7.6 mmol). The mixture was stirred under reflux overnight under nitrogen protection; after cooling when the reaction was detected to be complete by TLC, diethyl ether was added to the reaction solution to precipitate the product, and yellow solid compound 1b (3.29 g, yield 86.5%) was obtained;

[0016] Under an ice bath and nitrogen protection, triethylene glycol (9.82 g, 65.4 mmol) was added to a 150 mL anhydrous THF suspension of potassium tert-butoxide (tBuOK, 3.82 g, 33.0 mmol). The mixture was stirred vigorously for 30 min, and then propargyl bromide (3.64 mL, 80% toluene solution, 32.7 mmol) was added dropwise. After the addition was completed, the mixture was stirred at room temperature for another 18 h; after the reaction was completed (TLC monitoring: petroleum ether / ethyl acetate = 1:1, Rf ≈ 0.1), the reaction mixture was diluted with THF and filtered through diatomaceous earth; after the filtrate was concentrated, it was purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1:1), and pale yellow liquid compound 3a (4.75 g, yield 76.5%) was obtained;

[0017] At -10 °C and under nitrogen protection, DIAD (1.80 g, 9.0 mmol) was slowly added dropwise to a 20 mL anhydrous THF solution containing Diphyllin (1.14 g, 3.0 mmol), compound 3a (2.25 g, 15.0 mmol) and triphenylphosphine (2.36 g, 9.0 mmol); after the addition was completed, the mixture was stirred at room temperature for 16 h; after the reaction was completed (TLC monitoring: petroleum ether / ethyl acetate = 1:2, Rf ≈ 0.15), the reaction mixture was concentrated and purified by column chromatography (petroleum ether / ethyl acetate = 1:3), and white solid compound 2a (1.47 g, yield 89.1%) was obtained;

[0018] Compound 2a and compound 1b were mixed and reacted to prepare compound I.

[0019] The present invention also provides the use of a p65 protein degrader prepared by the above preparation method in regulating inflammation-related diseases.

[0020] By adopting the above technical solutions, the present invention successfully designed and synthesized a compound (Formula I) with p65 degradation activity, and systematically verified its mechanism of action and biological functions. Through Western blot experiments, the present invention confirmed that the compound (Formula I) could significantly down-regulate the p65 protein level in a dose-dependent manner in LO2 cells. As the concentration of the compound increased, the degradation efficiency of p65 gradually increased, but a typical HOOK effect (i.e., a decrease in degradation efficiency) was observed under high-concentration conditions, which was consistent with the characteristics of PROTAC molecules forming ternary complexes. The appearance of the HOOK effect indicated that the compound (Formula I) mediated the polyubiquitination and proteasome-dependent degradation of p65 by forming a "p65-PROTAC-CRBN" ternary complex with p65 and CRBN (E3 ubiquitin ligase). This result not only verified the p65 degradation activity of the compound (Formula I), but also provided direct evidence for its mechanism of action.

[0021] In the LPS-induced RAW 264.7 macrophage model of the present invention, its regulatory effect on pro-inflammatory factors was detected. The results showed that the compound (Formula I) could significantly inhibit the expression of a variety of key inflammatory factors (such as IL1β, IL6, TNFα, MCP1, etc.). This result was highly correlated with the degradation level of p65, further confirming that the compound (Formula I) blocked the NF-κB signaling pathway by degrading p65, thereby achieving transcriptional regulation of inflammatory factors.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention applied PROTAC to the targeted degradation of p65, breaking through the limitations of traditional small molecule inhibitors in p65 regulation. As a transcription factor, p65 lacks a pharmacophore pocket that can be bound by traditional small molecules and belongs to a typical "undruggable" target. The PROTAC technology forms a "p65-PROTAC-E3 ligase" ternary complex through a heterobifunctional small molecule (binding to p65 at one end and recruiting E3 ubiquitin ligase at the other end), uses intermolecular forces to stabilize weak binding ligands, induces polyubiquitination labeling of p65, and is finally recognized and degraded by the proteasome. This mechanism not only overcomes the deficiency of traditional inhibitors in targeting p65, but also realizes the effective degradation of p65 protein, rather than simply inhibiting its activity. By degrading p65, the present invention can significantly inhibit the expression of its downstream inflammatory factors, thereby blocking the cascade amplification of inflammatory signals. Description of the Drawings

[0024] Figure 1 Schematic diagram of the (A) cytotoxicity of Formula I at different concentrations of the present invention to LO2 and its (B) effect on the p65 protein level;

[0025] Figure 2 Schematic diagram of the effect of Formula I (20 μM) of the present invention on the gene expression of key inflammatory factors (IL1β, IL6, TNFα, iNOS, MCP1) in LPS-induced RAW 264.7 macrophages;

[0026] Figure 3 High-resolution mass spectrum of Formula I of the present invention;

[0027] Figure 4 1H nuclear magnetic resonance spectrum of Formula I of the present invention (400 MHz, measured in DMSO-d6);

[0028] Figure 5 13C nuclear magnetic resonance spectrum of Formula I of the present invention (101 MHz, measured in DMSO-d6). Detailed implementation manners

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, so that those skilled in the art can better understand the advantages and features of the present invention, and thus make a clearer definition of the protection scope of the present invention. The embodiments described in the present invention are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0030] Example 1

[0031] A p65 protein degrader, the structure of the degrader is shown in Formula I:

[0032]

[0033] A preparation method of a p65 protein degrader, the synthesis reaction structural formula of the degrader of Compound Formula I is shown as follows:

[0034]

[0035] Its preparation method includes the following steps:

[0036] A certain amount of bromoacetyl bromide (2.13 mL, 4.94 g, 24.6 mmol) was added to a 50 mL anhydrous tetrahydrofuran (THF) suspension containing pomalidomide (2.23 g, 8.2 mmol), and the mixture was stirred under reflux for 16 h (TLC monitoring: petroleum ether / ethyl acetate = 1:1, Rf ≈ 0.3); after the reaction was completed, the reaction mixture was concentrated, and after adding ether, a pale yellow solid 1a (2.75 g, yield 85.5%) was directly precipitated. Compound 1a: 1HNMR (400 MHz, DMSO-d6) δ 11.16 (s, 1H), 10.26 (s, 1H), 8.46 (dd, J = 8.4, 0.7 Hz, 1H), 7.87 (dd, J = 8.4, 7.4 Hz, 1H), 7.68 (dd, J = 7.4, 0.7 Hz, 1H), 5.17 (dd, J = 12.8, 5.4 Hz, 1H), 4.33 (s, 2H), 2.90 (ddd, J = 16.9, 13.8, 5.4 Hz, 1H), 2.61 (m, 2H), 2.09 (m, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 172.76, 169.77, 167.50, 166.58, 165.72, 136.28, 135.64, 131.57, 126.08, 119.10, 117.57, 48.98, 30.94, 29.94, 21.97. ESI-MS: m / z 396, 394 [M+H] + 。

[0037] Sodium azide (988.0 mg, 15.2 mmol) was slowly added to a 50 mL acetone suspension of compound 1a (3.00 g, 7.6 mmol). The mixture was stirred under nitrogen protection at reflux overnight. After cooling when the reaction was complete by TLC detection, ether was added to the reaction solution to precipitate the product, and yellow solid compound 1b (3.29 g, yield 86.5%) was obtained. Compound 1b: 1 HNMR (400 MHz, DMSO-d6) δ 11.16 (s, 1H), 10.12 (s, 1H), 8.52 (dd, J = 8.5, 0.8 Hz, 1H), 7.87 (dd, J = 8.5, 7.4 Hz, 1H), 7.66 (dd, J = 7.4, 0.8 Hz, 1H), 5.17 (dd, J = 12.8, 5.4 Hz, 1H), 4.33 (s, 2H), 2.92 (m, 1H), 2.59 (m, 2H), 2.07 (m, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 172.76, 169.76, 167.69, 167.09, 166.60, 136.35, 135.61, 131.51, 125.91, 118.90, 117.31, 51.88, 48.98, 30.95, 21.97. ESI-MS: m / z 357 [M+H] + 。

[0038] Under an ice bath and nitrogen protection, triethylene glycol (9.82 g, 65.4 mmol) was added to a 150 mL anhydrous THF suspension of potassium tert-butoxide (tBuOK, 3.82 g, 33.0 mmol). Stir vigorously for 30 min. Subsequently, propargyl bromide (3.64 mL, 80% toluene solution, 32.7 mmol) was added dropwise. After the addition was complete, stirring was continued at room temperature for 18 h. After the reaction was completed (TLC monitoring: petroleum ether / ethyl acetate = 1:1, Rf ≈ 0.1), the reaction mixture was diluted with THF and filtered through diatomaceous earth. The filtrate was concentrated and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1:1) to obtain a pale yellow liquid compound 3a (4.75 g, yield 76.5%). Compound 3a: 1 HNMR(400MHz,Chloroform-d)δ4.27(d,J=2.4Hz,2H),3.77(m,10H),3.67(m,2H),2.53(t,J=2.4Hz,1H). 13 C NMR(101MHz,Chloroform-d)δ79.56,74.79,72.65,70.62,70.33,70.28,69.08,61.68,58.42。

[0039] At -10 °C and under nitrogen protection, DIAD (1.80 g, 9.0 mmol) was slowly added dropwise to a 20 mL anhydrous THF solution containing Diphyllin (1.14 g, 3.0 mmol), compound 3a (2.25 g, 15.0 mmol), and triphenylphosphine (2.36 g, 9.0 mmol). After the addition was complete, the mixture was stirred at room temperature for 16 h. After the reaction was completed (TLC monitoring: petroleum ether / ethyl acetate = 1:2, Rf ≈ 0.15), the reaction mixture was concentrated and purified by column chromatography (petroleum ether / ethyl acetate = 1:3) to obtain a white solid compound 2a (1.47 g, yield 89.1%). Compound 2a: 1 H NMR(400MHz,Methanol-d4)δ7.67(s,1H),7.02(s,1H),6.94(d,J=7.8Hz,1H),6.75(d,J=1.7Hz,1H),6.74(dd,J=7.8,1.7Hz,1H),6.04(dd,J=7.1,1.2Hz,2H),5.55(s,2H),4.38(m,2H),4.14(d,J=2.4Hz,2H),3.91(m,2H),3.75(m,2H),3.72(s,3H),3.67(m,2H),3.63(s,3H),2.81(t,J=2.4Hz,1H).ESI-MS:m / z 551[M+H]+ 。

[0040] Mix compound 2a and compound 1b for reaction to obtain compound I. Compound I: 1 H NMR(400MHz,DMSO-d6)δ11.19(s,1H),10.19(s,1H),8.35(d,J=8.4Hz,1H),8.14(s,1H),7.82(t,J=7.9Hz,1H),7.66(d,J=7.3Hz,1H),7.63(s,1H),7.04(d,J=7.9Hz,1H),6.97(s,1H),6.89(d,J=1.6Hz,1H),6.77(dd,J=7.9,1.6Hz,1H),6.13(s,2H),5.58(s,2H),5.54(s,2H),5.16(dd,J=12.8,5.4Hz,1H),4.56(s,2H),4.37(dd,J=5.4,3.1Hz,2H),3.94(s,3H),3.83(m,2H),3.67(m,2H),3.66(s,3H),3.57(qd,J=5.0,2.7Hz,6H),2.91(ddd,J=16.7,13.7,5.4Hz,1H),2.59(m,2H),2.08(m,1H). 13 C NMR(101MHz,DMSO-d6)δ172.88,169.88,169.19,167.18,166.62,165.69,151.30,149.96,146.96,146.87,146.41,144.03,136.19,135.35,133.40,131.71,129.57,128.35,126.80,126.06,125.73,123.67,119.21,118.90,117.99,110.90,108.01,105.52,101.17,100.75,71.87,70.06,69.96,69.78,69.68,68.99,66.54,63.47,59.85,55.64,55.24,52.29,49.01,30.99,22.02.HR-ESI-MS:m / z907.3088[M+H] + ,calcd for C 45 H 43 N6O 15 ,907.2786。

[0041] Example 2

[0042] Degradation effect of the compound (Formula I) of the present invention on p65 protein

[0043] Cell culture and drug treatment: LO2 cells in good growth state were seeded in 96-well plates (5×10 3 cells per well), and cultured until adherent under the conditions of 37 °C and 5% CO2. Subsequently, the compound (Formula I) was diluted to different concentrations (0, 10, 20, 30, 40, and 50 μM) with complete medium, and 100 μL of the drug-containing culture solution was added to each well and treated for 24 hours.

[0044] Cell viability assay (MTT method): After the compound (Formula I) was treated for 24 hours, 10 μL of MTT solution was added to each well and cultured for another 4 hours. After removing the culture solution, 150 μL of DMSO was added to dissolve the formazan crystals, and shaken on a shaker for 30 minutes. The absorbance value at a wavelength of 490 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the relative cell viability was calculated based on the blank control group.

[0045] As Figure 1 shown in A, in the concentration range of 0 to 50 μM, compound I had almost no obvious effect on the growth of LO2 cells, indicating that within this concentration range, the compound did not have a toxic effect on the cells.

[0046] Western blot analysis: Cell protein extraction: After LO2 cells were treated with 0, 5, 10, 20 μM D-P for 24 hours, the culture medium was discarded, and the cells were washed and digested with PBS. After centrifuging to collect the cells, protease inhibitors were added for lysis, and the cells were scraped off after standing on ice for 5 - 10 minutes. The supernatant was taken after centrifuging at 12,000 rpm for 15 minutes. The protein concentration was measured according to the kit instructions. Electrophoresis and membrane transfer: A 10% SDS-PAGE gel was prepared, and after loading the samples, electrophoresis separation was carried out successively at 80 V and 120 V. When transferring the membrane, a "black gel-white membrane" sequence was used to assemble the sandwich structure, and the membrane was transferred at a constant current of 260 mA for 70 minutes. Antibody incubation and development: After membrane transfer, the PVDF membrane was blocked, primary antibody (overnight at 4 °C) and secondary antibody (incubated at room temperature for 2 hours) were added. After each incubation, the membrane was washed 3 times with TBST, 10 minutes each time. Finally, the developing solution was prepared and developed according to the instructions.

[0047] The results of Western blot are as Figure 1As shown in Figure B, compound (Formula I) can significantly down-regulate the p65 protein level in LO2 cells in a dose-dependent manner. As the concentration increases, the degradation efficiency of p65 gradually increases, but a HOOK effect (decrease in degradation efficiency) is observed at high concentrations (such as 50 μM). This phenomenon suggests that compound (Formula I) mediates the polyubiquitination and proteasome-dependent degradation of p65 by forming a "p65-PROTAC-CRBN" ternary complex with p65 and CRBN. This result not only verifies the p65 degradation activity of compound (Formula I), but also provides direct evidence for its mechanism of action.

[0048] Example 3

[0049] Effect of the compound (Formula I) of the present invention on the expression of inflammatory factors in LPS-induced RAW 264.7 macrophages

[0050] Cell culture and drug treatment: RAW 264.7 cells were cultured in HG-DMEM (containing 20% FBS and 2‰ PS) until the density reached 90%, seeded in 24-well plates at 160,000 cells / well, and after the cells adhered for 12 hours, they were grouped and treated: compound (Formula I, 20 μM) or positive control BAY 11-7082 (5 μM) was added for pretreatment for 12 hours; then LPS (final concentration 100 ng / mL) was added for stimulation for 12 hours.

[0051] RNA extraction and reverse transcription: 300 μL of TRIzol reagent was added to each well to lyse the cells, transferred to an EP tube, and after adding chloroform for layering, centrifuged (12,000 rpm, 15 minutes). The upper aqueous phase was aspirated, isopropanol was added to precipitate RNA, and placed at -20 °C for 2 hours or overnight. After centrifugation, the precipitate was washed with 70% ethanol, dried and dissolved in RNAase-free water, and the RNA concentration was measured. 1 μg of RNA was taken for reverse transcription reaction (37 °C for 15 minutes, inactivated at 85 °C for 15 seconds), and diluted to 200 μL for standby.

[0052] qPCR amplification: 4 μL of cDNA template was taken, and the forward and reverse primer mixture (5 μM, 1 μL) and SYBR Green qPCR Master Mix (5 μL) were added to prepare a 10 μL reaction system. The amplification conditions were: pre-denaturation at 95 °C for 5 minutes, denaturation at 95 °C for 30 seconds, annealing at 60 °C for 30 seconds, and cycling 40 times.

[0053] The qPCR results are as Figure 2It was shown that the compound (Formula I) significantly inhibited the gene expression of multiple key inflammatory factors (such as IL-1β, IL-6, TNF-α, iNOS, MCP-1) induced by LPS. This result was highly correlated with the degradation level of p65, further confirming that the compound (Formula I) blocked the NF-κB signaling pathway by degrading p65, thereby achieving transcriptional regulation of inflammatory factors.

[0054] In summary, the compound (Formula I) provided by the present invention can effectively degrade the p65 protein and significantly inhibit the expression of inflammatory factors induced by LPS. Its mechanism of action may involve forming a ternary complex with p65 and CRBN, mediating the polyubiquitination and proteasome-dependent degradation of p65. These findings provide an important theoretical basis and experimental evidence for the development of anti-inflammatory drugs.

[0055] The descriptions and practices disclosed in the present invention are easy to think about and understand for those of ordinary skill in the art. Without departing from the principle of the present invention, several improvements and refinements can also be made. Therefore, the modifications or improvements made without deviating from the spirit of the present invention should also be regarded as within the protection scope of the present invention.

Claims

1. A p65 protein degradation agent, characterized in that: The structure of the degradation agent is shown in Formula I:

2. The method for preparing a p65 protein degradation agent according to claim 1, characterized in that: The synthetic reaction formula of the compound formula I of the degradation agent is as follows: The preparation method thereof comprises the following steps: A certain amount of bromoacetyl bromide was added to a 50 mL anhydrous tetrahydrofuran suspension containing pomalidomide, and the mixture was stirred under reflux for 16 h. After the reaction was completed, the reaction mixture was concentrated, and ether was added to directly precipitate a light yellow solid 1a. Sodium azide was slowly added to a 50 mL acetone suspension of compound 1a, and the mixture was stirred under reflux conditions overnight under nitrogen protection; after TLC detection, the reaction mixture was cooled and ether was added to the reaction solution to precipitate the product, thereby obtaining a yellow solid compound 1b; In an ice bath and under nitrogen protection, triethylene glycol was added to a 150 mL anhydrous THF suspension of potassium tert-butoxide; the mixture was stirred vigorously for 30 min, and then propargyl bromide was added dropwise. After the addition was complete, stirring was continued for 18 h at room temperature; after the reaction was completed, the reaction mixture was diluted with THF and filtered with diatomaceous earth; the filtrate was concentrated and purified by column chromatography to obtain a light yellow liquid compound 3a; At -10 °C, under nitrogen protection, DIAD was slowly added dropwise to a 20 mL anhydrous THF solution containing diphyllin, compound 3a and triphenylphosphine; After the addition was completed, the mixture was stirred at room temperature for 16 h. After the reaction was completed, the reaction mixture was concentrated and purified by column chromatography to obtain a white solid compound 2a. Compound 2a and compound 1b are mixed and reacted to obtain compound I.

3. Use of a p65 protein degrader prepared by the preparation method of claim 2 in regulating inflammation-related diseases.

Images