Proteolysis targeting chimera and preparation method thereof

Abstract

The invention discloses a proteolysis targeting chimera and a preparation method thereof, and relates to the technical field of medicine preparation, the key points of the technical scheme are as follows: the proteolysis targeting chimera comprises RD1, RD2, RD3, RD4, RD5, RD6, RD7, RD8, RD9, RD10, RD11, RD12, RD13 and RD14, and the structure of the proteolysis targeting chimera is composed of a general structure (warhead R1 of RNF4), a VHL ligand or CRBN ligand and a linker; the structure of warhead R1 of RNF4 is as follows: # imgabs0 #, the structure of VHL ligand is as follows: # imgabs1 #, and the structure of CRBN ligand is as follows: # imgabs2 #

Description

Technical Field

[0001] The present invention relates to the technical field of drug preparation, and more specifically, to a protein hydrolysis targeting chimera and a preparation method thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is a common malignant tumor with high metastasis rate and high mortality rate. The current drugs used in clinical treatment of liver cancer still cannot meet the needs of effectively alleviating the disease. Therefore, it is urgent to explore new therapeutic targets and mechanisms of action.

[0003] Proteolytic Targeting Chimera (PROTAC) is a bifunctional small molecule that has developed rapidly in recent years and has made significant progress in the treatment of various diseases. PROTACs are characterized by low dose and low toxicity. They do not interfere with protein function, but mediate the degradation of target proteins. This technology makes targets that were originally resistant or intolerant drug-drugable. PROTACs are composed of three components: a target protein ligand, an E3 ubiquitin ligase ligand, and a linker that connects the two parts. PROTACs promote the formation of a ternary complex between the target protein and the E3 ligase, thereby ubiquitin-tagging the target protein and subsequently degrading it by the ubiquitin proteasome system.

[0004] RNF4 belongs to the family of accessory chaperone proteins (STUb1) and is an E3 ligase that targets small ubiquitin-like modifiers (SUMO). SUMO is one of the ubiquitin-like proteins discovered in recent years. The protein modifies target proteins by forming covalent bonds with specific lysine residues on the target protein, a process similar to ubiquitination, commonly known as SUMOylation. It is an important post-translational modification of proteins in eukaryotes, regulating biological processes such as transcription factor activation and DNA damage repair. RNF4 consists of a RING domain and four tandem SUMO interaction motifs (SIMs). Its C-terminal RING domain exhibits enzymatic activity only in the form of dimers, while the N-terminal SIMs efficiently bind to long poly-SUMO chains. These motifs play a crucial role in mediating the ubiquitination of SUMOylated substrates. RNF4 has been shown to mediate ubiquitination of MDC1 and BRCA1, thereby playing a key role in homologous recombination repair. RNF4 is expressed at elevated levels in liver cancer cells, suggesting that it has an oncogenic role in the development of this malignancy. Furthermore, silencing of RNF4 has been shown to induce cell cycle arrest and apoptosis, thereby exerting a significant anti-tumor effect. Summary of the invention

[0005] The present invention aims to provide a proteolysis-targeted chimera and a method for preparing the same, and develops a selective RNF4 degrader RD6, which can effectively induce the degradation of RNF4, especially in liver cancer cell lines, including HEPG2, HCCLM6 and Huh7. In addition, the degrader RD6 exhibits significant anti-proliferative properties in liver cancer cell lines and shows promising in vivo efficacy in a mouse model transplanted with HCCLM6 cells. This provides a new chemical tool and new insights for exploring liver cancer and its treatment methods.

[0006] The above technical objectives of the present invention are achieved through the following technical solutions: a proteolysis targeting chimera, wherein the proteolysis targeting chimera includes RD1, RD2, RD3, RD4, RD5, RD6, RD7, RD8, RD9, RD10, RD11, RD12, RD13 and RD14,

[0007] The structure of the proteolysis targeting chimera consists of a common structure (the warhead R1 of RNF4), a VHL ligand or a CRBN ligand, and a linker;

[0008] The structure of the warhead R1 of the RNF4 is as follows:

[0009]

[0010] The VHL ligand structure is as follows:

[0011]

[0012] The CRBN ligand structure is as follows:

[0013]

[0014] The present invention further provides a method for preparing a proteolysis-targeted chimera: the synthetic routes of RD1 and RD3 are as follows:

[0015]

[0016] The present invention is further configured as follows: the synthetic route of RD2 is as follows:

[0017]

[0018] The present invention is further configured as follows: the synthetic route of RD4 is as follows:

[0019]

[0020] The present invention is further configured as follows: in the synthetic route of RD4, RD5-RD14 can be obtained by replacing the linker of RD4 in step a;

[0021] The linker of RD5 is

[0022] The linker of RD6 is

[0023] The linker of RD7 is

[0024] The linker of RD8 is

[0025] The linker of RD9 is

[0026] The linker of RD10 is

[0027] The linker of the RD11 is

[0028] The linker of RD12 is

[0029] The linker of RD13 is

[0030] The linker of RD14 is

[0031] The present invention is further configured as follows: the reagents and reaction conditions in the preparation method are as follows:

[0032] (a) K2CO3, acetone, reflux, 3d; (b) Chloroacetyl chloride, TEA, CH2Cl2, 0→room temperature, 1 hour; (c) TFA, CH2Cl2, room temperature, 1 hour; (d) Succinic anhydride, DMAP, TEA, CH2Cl2, room temperature, 2 hours; (e) HATU, DIPEA, DMF, room temperature, 2 hours.

[0033] The present invention further provides a pharmaceutical composition synthesized by the proteolysis targeting chimera.

[0034] The present invention further provides the use of the protein hydrolysis targeting chimera in preparing drugs for treating liver cancer.

[0035] In summary, the present invention has the following beneficial effects:

[0036] Through molecular docking studies of RNF4 inhibitor R1 and RNF4, a series of RNF4 degraders were synthesized to recruit VHL with R1. By improving the linker length and chemical properties in PROTAC, RD1-RD14 were successfully developed, among which RD6 was the most effective, which is a highly efficient and selective RNF4 degrader. RD6 showed strong anti-tumor efficacy and did not cause any changes in mouse body weight, indicating that it has no potential toxicity.

[0037] In summary, this invention revolves around RNF4 as a key therapeutic target for liver cancer, and ultimately achieves the rational design and synthesis of its effective degraders, providing new therapeutic strategies and insights for the treatment of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 a: Molecular docking analysis of R1 and RNF4; b: Structures of RD1-RD4 compounds; c: RD3 and RD4 can effectively induce the degradation of RNF4 in HepG2 cells (concentration 5 μM); d: Neither R1 nor the VHL ligand MDK-7526 induces the degradation of RNF4;

[0039] Figure 2 a: RD4 administration triggered time-dependent degradation of RNF4, reaching a peak after 6 hours of treatment; b: A 6-hour dose-response study of RD4 confirmed that RNF4 degradation depended on the concentration of RD4; c: RD4 triggered phosphorylation of histone H2AX; d: The 72-hour IC50 values ​​of RD4 in the three cell lines were precisely measured, 1.788 μM for HepG2, 0.4813 μM for HCCLM6, and 0.6642 μM for Huh7;

[0040] Figure 3 a: Antiproliferative effect of RD4-RD14; b: RNF4 degradation efficiency of RD4-RD14; c: Protac formed by replacing the flexible linker with a rigid linker; d: Biological activity of Protac with a rigid linker;

[0041] Figure 4 is the inhibitory effect of R1 on the growth of HepG2 cells;

[0042] Figure 5 The figure is the result of flow cytometry detection of cell apoptosis induced by compound RD4;

[0043] Figure 6 The IC50 results of RD5, RD6 and RD7 obtained by modifying compound RD4 on liver cancer cell lines are shown in the figure;

[0044] Figure 7This is the result of WB detection of RNF4 degradation activity of compounds RD5, RD6, and RD7 modified from compound RD4;

[0045] Figure 8 This is the result of WB detection of the DNA damage-inducing ability of compound RD6;

[0046] Fig. 9 This is the result diagram of the in vivo anti-tumor function verification of compound RD6. DETAILED DESCRIPTION

[0047] The following is combined with Figure 1-9 The present invention is described in further detail.

[0048] Example 1: Design and characterization of RNF4 degraders

[0049] A covalent RNF4 inhibitor R1 was selected as a targeting ligand for RNF4. Molecular docking analysis of R1 and RNF4 ( Figure 1 As shown in Figure a), the methyl group of the methoxy group in R1 is clearly exposed to the solvent environment, providing an ideal binding pocket for the development of potential PROTACs. Cereblon (CRBN) and Von hipel-lindau (VHL) were selected as alternative ligands for E3 ubiquitin ligases and synthesized together with the RNF4 inhibitor R1 to form RD1-RD4 compounds ( Figure 1 b), the ultimate goal is to target RNF4 for degradation. The results showed that RD4 has an anti-proliferative effect comparable to that of the RNF4 inhibitor R1. In addition, at a concentration of 5 μM, RD3 and RD4 can effectively induce the degradation of RNF4 in HepG2 cells. And the PROTAC degrader RD4, which uses VHL as an E3 ligase recruiter, showed better results than the PROTAC degrader using CRBN.

[0050] Example 2: Preparation of proteolysis-targeted chimeras

[0051] The proteolysis targeting chimera includes RD1, RD2, RD3, RD4, RD5, RD6, RD7, RD8, RD9, RD10, RD11, RD12, RD13 and RD14, and its structure is composed of a common structure (the warhead R1 of RNF4), a VHL ligand or a CRBN ligand, and a linker;

[0052] The structure of the RNF4 warhead R1 is as follows:

[0053]

[0054] The structure of VHL ligand is as follows:

[0055]

[0056] The CRBN ligand structure is as follows:

[0057]

[0058] The synthetic routes of RD1 and RD3 are as follows:

[0059]

[0060] The synthetic route of RD2 is as follows:

[0061]

[0062] The synthetic route of RD4 is as follows:

[0063]

[0064] In the synthetic route of RD4, replacing the linker of RD4 in step a can produce RD5-RD14;

[0065] The linker of RD5 is

[0066] The linker of RD6 is

[0067] The linker of RD7 is

[0068] The linker of RD8 is

[0069] The linker of RD9 is

[0070] The linker of RD10 is

[0071] The linker of the RD11 is

[0072] The linker of RD12 is

[0073] The linker of RD13 is

[0074] The linker of RD14 is

[0075] The reagents and reaction conditions in its preparation method are as follows:

[0076] (a) K2CO3, acetone, reflux, 3d; (b) Chloroacetyl chloride, TEA, CH2Cl2, 0→room temperature, 1 hour; (c) TFA, CH2Cl2, room temperature, 1 hour; (d) Succinic anhydride, DMAP, TEA, CH2Cl2, room temperature, 2 hours; (e) HATU, DIPEA, DMF, room temperature, 2 hours.

[0077] RD4 has an antiproliferative effect comparable to that of the RNF4 inhibitor R1, and the IC50 values ​​of R1 and RD4 were subsequently determined in HepG2 cells after 72 hours of treatment. The results showed that the IC50 values ​​of R1 and RD4 were 2.19 μM and 1.788 μM, respectively ( Figure 2 (d)

[0078] At a concentration of 5 μM, RD3 and RD4 could effectively induce the degradation of RNF4 in HepG2 cells ( Figure 1 (c).

[0079] Western blotting experiments confirmed that neither R1 nor the VHL ligand MDK-7526 induced the degradation of RNF4 ( Figure 1 (d)

[0080] RD4 administration triggered a time-dependent degradation of RNF4, reaching a peak after 6 h of treatment ( Figure 2 Therefore, we performed a 6-hour dose-response study using RD4 and the results confirmed that RNF4 degradation was indeed dependent on the concentration of RD4 ( Figure 2 (b). To further investigate the efficacy of RD4 in degrading RNF4 in different HCC cell types, we evaluated the degradation potential of RNF4 in HCC cell lines HepG2, HCCLM6, and Huh7 using western blotting. The results showed a clear dose-dependent correlation between RD4 and RNF4 degradation, and also indicated that RD4 also triggered the phosphorylation of histone H2AX ( Figure 2 (c). Compared with HepG2 cells, RD4 showed better degradation efficiency in HCCLM6 and Huh7 cells. Therefore, we precisely measured the 72-hour IC50 values ​​of RD4 in these three cell lines, and the results showed that HepG2 was 1.788μM, HCCLM6 was 0.4813μM, and Huh7 was 0.6642μM ( Figure 2 (d)

[0081] Example 3: Design of RNF4 degraders with multiple linkers

[0082] Initially, alkanes and polyethylene glycol were selected as linkers to modify the length of the linker of RD4, thereby synthesizing a series of novel RNF4 degraders with flexible linkers (Table 1). The antiproliferative efficacy and protein degradation ability of RD5-RD14 in the HepG2 cell line were evaluated using cell viability assays and western blots. It was found that RD5-RD7 continued to exhibit strong antiproliferative activity and effective degradation of RNF4. Moreover, the antiproliferative effect and RNF4 degradation efficiency of RD5-RD7 were comparable to each other, but significantly exceeded RD4 ( Figure 3 We hypothesized that the bioactivity of these degraders would be optimal when their linkers contained 2–6 atoms.

[0083] The flexible linkers in Table 1 were replaced with rigid linkers, including heterocyclic scaffolds (such as nitrogen pyrrole, piperazine or piperidine), benzene ring linkers and spiro rings, to form a series of PROTACs ( Figure 3 c). However, the compounds in this series did not show outstanding biological activity ( Figure 3 (d)

[0084] Table 1: Novel RNF4 degraders with flexible linkers

[0085]

[0086]

[0087] Example 4: Detection of compound RD4’s ability to degrade RNF4 and induce DNA damage by WB assay

[0088] After treating liver cancer cells HCCLM6, Huh7 and HepG2 with compound RD4 for 6 hours, cell lysates were collected for WB experiments. The experimental results showed that RD4 could degrade RNF4 in a concentration-dependent manner, and the detection of DNA damage marker γH2AX showed that RD4 could enhance the induction of DNA damage with the concentration gradient. Figure 2 As shown in c.

[0089] Specific steps:

[0090] WB protein sample preparation:

[0091] 1. Take 5x 10^ 5 Each well was inoculated in a six-well plate, and DMEM complete medium was added to make up the volume to 2 mL;

[0092] 2. After the cells have attached overnight, remove the supernatant medium and add 2 mL of compound diluted in a concentration gradient (dissolve the compound powder in DMSO to prepare a 20 mM stock solution, take 1 μL of the stock solution and add it to 2 mL of DMEM complete medium to dilute the concentration to 10 μM, and then dilute it in a gradient to 5 / 2.5 / 1.25 μM);

[0093] 3. Place in a CO2 incubator and incubate at 37°C for 6 hours.

[0094] 4. After the drug incubation time is over, remove the supernatant medium and add 1 mL of PBS to each well to wash the cells twice;

[0095] 5. Add 500 μL of 0.25% trypsin to each well to digest the cells, and incubate at 37°C in a CO2 incubator for 2 minutes;

[0096] 6. After the incubation, add 500 μL DMEM complete medium to each well for neutralization and digestion;

[0097] 7. Place the incubated cells in a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 mins;

[0098] 8. Add 1 mL of PBS to each tube to resuspend the cells, and centrifuge at 1000 rpm for 5 mins;

[0099] 9. Remove the RIPA lysis buffer and add protease inhibitors and phosphatase inhibitors for later use.

[0100] 10. Add about 200 μL of RIPA lysis buffer containing protease inhibitors to the digested cell pellet.

[0101] 11. After lysis on ice for 30 minutes, pipette up and down gently several times and transfer the cell lysate to a 1.5 mL EP tube.

[0102] 12. Centrifuge at 12000 rpm and 4°C for 15 minutes, transfer the supernatant to a new 1.5 mL EP tube, and store it in a -20°C refrigerator for later use.

[0103] Protein quantification:

[0104] The protein quantification method used in the present invention is the BCA method, and the BCA protein quantification kit produced by Shanghai Biyuntian Biotechnology Co., Ltd. is used. The specific experimental steps are as follows:

[0105] 1. Add 0, 1, 2, 4, 8, 12, 16, and 20 μl of the standard to the standard wells of a 96-well plate, and add standard diluent to make up to 20 μl, which is equivalent to the standard concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml respectively;

[0106] 2. Add 20 μL of sample to the sample wells of the 96-well plate;

[0107] 3. Add 200 μl BCA working solution to each well and place at 37°C for 30 minutes;

[0108] 4. Use a microplate reader to measure the absorbance of A562 or other wavelengths between 540-595 nm;

[0109] 5. Calculate the protein concentration of the sample based on the standard curve.

[0110] SDS-PAGE gel preparation

[0111] 1. Lower gel preparation: Take a 50mL centrifuge tube, use a pipette to add 4mL of lower gel solution and lower gel buffer solution and mix well;

[0112] 2. Add 80ul of coagulant APS;

[0113] 3. Pour into the 1.50mm plate to about 70% height;

[0114] 4. Use a pipetting gun to add anhydrous ethanol to make the upper edge flat and solidify (pour out the anhydrous ethanol and discard it after the remaining solution in the centrifuge tube solidifies);

[0115] 5. Top gel preparation: Take a plastic cup and add 1 mL of top gel solution and top gel buffer solution, mix well;

[0116] 6. Add accelerator APS 20uL;

[0117] 7. Use a dosing gun to fill the glass plate with the upper layer of glue;

[0118] 8. Add the comb (pay attention to the scale on the comb) and wait for it to solidify and set aside.

[0119] Electrophoresis: Add samples to the electrophoresis tank; run at 90V for 20 minutes until the samples run out of the concentrated gel, then change the voltage to 140V and run to the wire (15ug of protein sample each);

[0120] Transfer membrane: Take out the SDS-PAGE gel after electrophoresis from the electrophoresis tank and put it into primary water (the electrophoresis solution in the tank is recovered), take out the NC membrane and place it in the NC membrane buffer;

[0121] Place the membranes in a transfer apparatus in the order of +sponge-NC membrane-SDS-PAGE gel-sponge for transfer;

[0122] Dyeing: Dyeing with Ponceau red (Ponceau red is recycled after use), and washing in pure water after dyeing;

[0123] Cut the membrane: Cut the NC membrane stained with Ponceau red according to the requirements (TUBULIN: 55; RNF4: 35kd; γH2AX: 15kd), take out the required bands and put them in PBST to wash off the Ponceau red dye solution;

[0124] Blocking: Weigh 1 g skim milk powder, dissolve in 20 mL PBST (5%), block with milk, shake on a shaker at 40 rpm for 20 min, wash three times with PBST, shake on a shaker at 40 rpm for 7 min;

[0125] Primary antibody incubation: Incubate overnight at 4°C on a shaker at 40 rpm, recover the primary antibody, and wash three times with PBST on a shaker at 40 rpm for 7 min;

[0126] Secondary antibody incubation: incubate on a shaker at 40 rpm for 45 min, wash three times with PBST on a shaker at 40 rpm for 7 min (secondary antibody = 6 mL milk: 0.6 uL antibody: milk = 1:10000; GAPDH mouse antibody 6 mL, rabbit antibody 6 mL)

[0127] Exposure and development: After the ultrasensitive ECL developer is evenly incubated on the NC membrane, the developer is used to expose and detect the protein bands.

[0128] Example 5. IC of compound RD4 on various liver cancer cell lines 50

[0129] The RD4 IC was calculated by detecting the cell viability of three different liver cancer cell lines HCCLM6, Huh7, and HepG2 after being treated with compound RD4 for 72 hours. 50 They are 481.3nM, 644.2nM and 1.788μM respectively.

[0130] Compound Cell Level IC 50 Test, the specific experimental steps are as follows:

[0131] 1. Take 5000 hepatoma cells HCCLM6, Huh7, and HepG2 in the logarithmic growth phase and inoculate them into a 96-well plate. Add DMEM complete medium to make the volume 100 μL.

[0132] 2. After the cells have attached overnight, remove the supernatant medium and add 100 μL of the compound diluted in a concentration gradient (dissolve the compound powder in DMSO to prepare a 20 mM stock solution, take 1 μL of the stock solution and add it to 1 mL of DMEM complete medium to dilute the concentration to 20 μM, then dilute it in a gradient to 20 / 10 / 5 / 2.5 / 1.25 / 0.625 / 0.3125 / 0.15625 / 0.078125 μM);

[0133] 3. Place in a CO2 incubator and incubate at 37°C for 72 hours;

[0134] 4. After incubation, the cell viability was detected using the CellTiter-Glo Luminescent Cell Viability Assay Kit produced by Promega;

[0135] 5. Draw the IC50 curve based on the cell viability and calculate the IC50 value.

[0136] Example 6. Detection of cell apoptosis induced by compound RD4 by flow cytometry

[0137] After HepG2 cells were treated with compound RD4 for 6 hours, flow cytometry staining showed that RD4 could increase the proportion of HepG2 apoptotic cells, and the results proved that compound RD6 has the function of inducing apoptosis of liver cancer cells. Figure 5 shown.

[0138] Flow cytometry was used to detect compound-induced cell apoptosis. The specific experimental steps are as follows:

[0139] 1. Take 5x 10^ 5 Each well was inoculated in a six-well plate, and DMEM complete medium was added to make up the volume to 2 mL;

[0140] 2. After the cells have attached overnight, remove the supernatant medium and add 2 mL of compound diluted in a concentration gradient (dissolve the compound powder in DMSO to prepare a 20 mM stock solution, take 1 μL of the stock solution and add it to 2 mL of DMEM complete medium to dilute the concentration to 10 μM, and then dilute it in a gradient to 10 / 5 μM);

[0141] 3. Place in a CO2 incubator and incubate at 37°C for 6 hours.

[0142] 4. After the drug incubation time is over, remove the supernatant medium and add 1 mL of PBS to each well to wash the cells once;

[0143] 5. After digestion with EDTA-free trypsin, collect the cells by centrifugation at 1000 rpm and 4°C for 5 min.

[0144] 6. Wash the cells twice with pre-cooled PBS, centrifuging at 300g for 5 min at 4°C each time. Collect 1-5×10 5 cell.

[0145] 7. Aspirate and discard PBS, add 100 μL 1× Binding Buffer to resuspend the cells.

[0146] 8. Add 5 μL Annexin V-FITC and 10 μL PI Staining Solution and mix gently.

[0147] 9. Protect from light and incubate at room temperature for 15 minutes.

[0148] 10. Add 400 μL 1× Binding Buffer, mix well and place on ice, then detect using flow cytometer.

[0149] Example 7. IC of RD5, RD6 and RD7 obtained by modifying compound RD4 on liver cancer cell lines 50

[0150] The cell viability of the liver cancer cell line HepG2 treated with compounds RD5, RD6, and RD7 for 72 hours was detected and the IC values ​​of RD5, RD6, and RD7 were calculated. 50 The activity of modified RD5, RD6 and RD7 was significantly improved compared with the IC50 value of 1.788μM obtained by RD4 on HepG2 cells. Figure 6 shown.

[0151] Compound Cell Level IC 50 Test, the specific experimental steps are as follows:

[0152] 1. Take 5000 HepG2 liver cancer cells in the logarithmic growth phase and inoculate them into a 96-well plate. Add DMEM complete medium to make the volume 100 μL.

[0153] 2. After the cells have attached overnight, remove the supernatant medium and add 100 μL of the compound diluted in a concentration gradient (dissolve the compound powder in DMSO to prepare a 20 mM stock solution, take 1 μL of the stock solution and add it to 1 mL of DMEM complete medium to dilute the concentration to 20 μM, then dilute it in a gradient to 20 / 10 / 5 / 2.5 / 1.25 / 0.625 / 0.3125 / 0.15625 / 0.078125 μM);

[0154] 3. Place in a CO2 incubator and incubate at 37°C for 72 hours;

[0155] 4. After incubation, the cell viability was detected using the CellTiter-Glo Luminescent Cell Viability Assay Kit produced by Promega;

[0156] 5. Draw the IC50 curve based on the cell viability and calculate the IC50 value.

[0157] Example 8. WB detection of RNF4 degradation activity of compounds RD5, RD6 and RD7 obtained from compound RD4

[0158] After treating liver cancer cells HCCLM6, Huh7 and HepG2 with compounds RD4, RD5, RD6 and RD7 for 6 hours, the cell lysates were collected for WB experiments. The experimental results showed that RD4 could degrade RNF4 in a concentration-dependent manner, and compared with compound RD4, the modified compounds RD5, RD6 and RD7 had stronger RNF4 degradation activity, among which compound RD6 had the best RNF4 degradation activity. Figure 7 shown.

[0159] Western Blot (WB) was used to test the RNF4 degradation function of the compound. Specific steps:

[0160] WB protein sample preparation:

[0161] 1. Take 5x 10^ 5 Each well was inoculated in a six-well plate, and DMEM complete medium was added to make up the volume to 2 mL;

[0162] 2. After the cells have attached overnight, remove the supernatant medium and add 2 mL of compound diluted in a concentration gradient (dissolve the compound powder in DMSO to prepare a 20 mM stock solution, take 1 μL of the stock solution and add it to 4 mL of DMEM complete medium to dilute the concentration to 5 μM, and then dilute it in a gradient to 2.5 / 1.25 μM);

[0163] 3. Place in a CO2 incubator and incubate at 37°C for 6 hours.

[0164] 4. After the drug incubation time is over, remove the supernatant medium and add 1 mL of PBS to each well to wash the cells twice;

[0165] 5. Add 500 μL of 0.25% trypsin to each well to digest the cells, and incubate at 37°C in a CO2 incubator for 2 minutes;

[0166] 6. After the incubation, add 500 μL DMEM complete medium to each well for neutralization and digestion;

[0167] 7. Place the incubated cells in a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 mins;

[0168] 8. Add 1 mL of PBS to each tube to resuspend the cells, and centrifuge at 1000 rpm for 5 mins;

[0169] 9. Remove the RIPA lysis buffer and add protease inhibitors and phosphatase inhibitors for later use.

[0170] 10. Add about 200 μL of RIPA lysis buffer containing protease inhibitors to the digested cell pellet.

[0171] 11. After lysis on ice for 30 minutes, pipette up and down gently several times and transfer the cell lysate to a 1.5 mL EP tube.

[0172] 12. Centrifuge at 12000 rpm and 4°C for 15 minutes, transfer the supernatant to a new 1.5 mL EP tube, and store it in a -20°C refrigerator for later use.

[0173] Protein quantification:

[0174] The protein quantification method used in the present invention is the BCA method, and the BCA protein quantification kit produced by Shanghai Biyuntian Biotechnology Co., Ltd. is used. The specific experimental steps are as follows:

[0175] 1. Add 0, 1, 2, 4, 8, 12, 16, and 20 μl of the standard to the standard wells of a 96-well plate, and add standard diluent to make up to 20 μl, which is equivalent to the standard concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml respectively;

[0176] 2. Add 20 μL of sample to the sample wells of the 96-well plate;

[0177] 3. Add 200 μl BCA working solution to each well and place at 37°C for 30 minutes;

[0178] 4. Use a microplate reader to measure the absorbance of A562 or other wavelengths between 540-595 nm;

[0179] 5. Calculate the protein concentration of the sample based on the standard curve.

[0180] SDS-PAGE gel preparation

[0181] 1. Lower gel preparation: Take a 50mL centrifuge tube, use a pipette to add 4mL of lower gel solution and lower gel buffer solution and mix well;

[0182] 2. Add 80ul of coagulant APS;

[0183] 3. Pour into the 1.50mm plate to about 70% height;

[0184] 4. Use a pipetting gun to add anhydrous ethanol to make the upper edge flat and solidify (pour out the anhydrous ethanol and discard it after the remaining solution in the centrifuge tube solidifies);

[0185] 5. Top gel preparation: Take a plastic cup and add 1 mL of top gel solution and top gel buffer solution, mix well;

[0186] 6. Add accelerator APS 20uL;

[0187] 7. Use a dosing gun to fill the glass plate with the upper layer of glue;

[0188] 8. Add the comb (pay attention to the scale on the comb) and wait for it to solidify and set aside.

[0189] Electrophoresis: Add samples to the electrophoresis tank; run at 90V for 20 minutes until the samples run out of the concentrated gel, then change the voltage to 140V and run to the wire (15ug of protein sample each);

[0190] Transfer membrane: Take out the SDS-PAGE gel after electrophoresis from the electrophoresis tank and put it into primary water (the electrophoresis solution in the tank is recovered), take out the NC membrane and place it in the NC membrane buffer;

[0191] Place the membranes in a transfer apparatus in the order of +sponge-NC membrane-SDS-PAGE gel-sponge for transfer;

[0192] Dyeing: Dyeing with Ponceau red (Ponceau red is recycled after use), and washing in pure water after dyeing;

[0193] Cut the membrane: Cut the NC membrane stained with Ponceau red according to the requirements (TUBULIN: 55; RNF4: 35kd), take out the required bands and put them in PBST to wash off the Ponceau red dye solution;

[0194] Blocking: Weigh 1 g skim milk powder, dissolve in 20 mL PBST (5%), block with milk, shake on a shaker at 40 rpm for 20 min, wash three times with PBST, shake on a shaker at 40 rpm for 7 min;

[0195] Primary antibody incubation: Incubate overnight at 4°C on a shaker at 40 rpm, recover the primary antibody, and wash three times with PBST on a shaker at 40 rpm for 7 min;

[0196] Secondary antibody incubation: incubate on a shaker at 40 rpm for 45 min, wash three times with PBST on a shaker at 40 rpm for 7 min (secondary antibody = 6 mL milk: 0.6 uL antibody: milk = 1:10000; GAPDH mouse antibody 6 mL, rabbit antibody 6 mL)

[0197] Exposure and development: After the ultrasensitive ECL developer is evenly incubated on the NC membrane, the developer is used to expose and detect the protein bands.

[0198] Example 9. Detection of DNA damage induced by compound RD6 by WB

[0199] After treating HepG2 cells with compound RD6 for 6 hours, the cell lysate was collected for WB experiment. The experimental results showed that RD4 could upregulate the expression of DNA damage maker H2AX protein in a concentration-dependent manner, proving that compound RD6 could enhance its ability to induce DNA damage with increasing concentration. Figure 8 shown.

[0200] Western Blot (WB) was used to test the RNF4 degradation and DNA induction functions of the compounds. Specific steps:

[0201] WB protein sample preparation:

[0202] 1. Take 5x 10^ 5 Each well was inoculated in a six-well plate, and DMEM complete medium was added to make up the volume to 2 mL;

[0203] 2. After the cells have attached overnight, remove the supernatant medium and add 2 mL of compound diluted in a concentration gradient (dissolve the compound powder in DMSO to prepare a 20 mM stock solution, take 1 μL of the stock solution and add it to 4 mL of DMEM complete medium to dilute the concentration to 5 μM, and then dilute it in a gradient to 2.5 / 1.25 μM);

[0204] 3. Place in a CO2 incubator and incubate at 37°C for 6 hours.

[0205] 4. After the drug incubation time is over, remove the supernatant medium and add 1 mL of PBS to each well to wash the cells twice;

[0206] 5. Add 500 μL of 0.25% trypsin to each well to digest the cells, and incubate at 37°C in a CO2 incubator for 2 minutes;

[0207] 6. After the incubation, add 500 μL DMEM complete medium to each well for neutralization and digestion;

[0208] 7. Place the incubated cells in a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 mins;

[0209] 8. Add 1 mL of PBS to each tube to resuspend the cells, and centrifuge at 1000 rpm for 5 mins;

[0210] 9. Remove the RIPA lysis buffer and add protease inhibitors and phosphatase inhibitors for later use.

[0211] 10. Add about 200 μL of RIPA lysis buffer containing protease inhibitors to the digested cell pellet.

[0212] 11. After lysis on ice for 30 minutes, pipette up and down gently several times and transfer the cell lysate to a 1.5 mL EP tube.

[0213] 12. Centrifuge at 12000 rpm and 4°C for 15 minutes, transfer the supernatant to a new 1.5 mL EP tube, and store it in a -20°C refrigerator for later use.

[0214] Protein quantification:

[0215] The protein quantification method used in the present invention is the BCA method, and the BCA protein quantification kit produced by Shanghai Biyuntian Biotechnology Co., Ltd. is used. The specific experimental steps are as follows:

[0216] 1. Add 0, 1, 2, 4, 8, 12, 16, and 20 μl of the standard to the standard wells of a 96-well plate, and add standard diluent to make up to 20 μl, which is equivalent to the standard concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml respectively;

[0217] 2. Add 20 μL of sample to the sample wells of the 96-well plate;

[0218] 3. Add 200 μl BCA working solution to each well and place at 37°C for 30 minutes;

[0219] 4. Use a microplate reader to measure the absorbance of A562 or other wavelengths between 540-595 nm;

[0220] 5. Calculate the protein concentration of the sample based on the standard curve.

[0221] SDS-PAGE gel preparation

[0222] 1. Lower gel preparation: Take a 50mL centrifuge tube, use a pipette to add 4mL of lower gel solution and lower gel buffer solution and mix well;

[0223] 2. Add 80ul of coagulant APS;

[0224] 3. Pour into the 1.50mm plate to about 70% height;

[0225] 4. Use a pipetting gun to add anhydrous ethanol to make the upper edge flat and solidify (pour out the anhydrous ethanol and discard it after the remaining solution in the centrifuge tube solidifies);

[0226] 5. Top gel preparation: Take a plastic cup and add 1 mL of top gel solution and top gel buffer solution, mix well;

[0227] 6. Add accelerator APS 20uL;

[0228] 7. Use a dosing gun to fill the glass plate with the upper layer of glue;

[0229] 8. Add the comb (pay attention to the scale on the comb) and wait for it to solidify and set aside.

[0230] Electrophoresis: Add samples to the electrophoresis tank; run at 90V for 20 minutes until the samples run out of the concentrated gel, then change the voltage to 140V and run to the wire (15ug of protein sample each);

[0231] Transfer membrane: Take out the SDS-PAGE gel after electrophoresis from the electrophoresis tank and put it into primary water (the electrophoresis solution in the tank is recovered), take out the NC membrane and place it in the NC membrane buffer;

[0232] Place the membranes in a transfer apparatus in the order of +sponge-NC membrane-SDS-PAGE gel-sponge for transfer;

[0233] Dyeing: Dyeing with Ponceau red (Ponceau red is recycled after use), and washing in pure water after dyeing;

[0234] Cut the membrane: Cut the NC membrane stained with Ponceau red according to the requirements (TUBULIN: 55; γH2AX: 15kd), take out the required bands and put them in PBST to wash the Ponceau red dye solution;

[0235] Blocking: Weigh 1 g skim milk powder, dissolve in 20 mL PBST (5%), block with milk, shake on a shaker at 40 rpm for 20 min, wash three times with PBST, shake on a shaker at 40 rpm for 7 min;

[0236] Primary antibody incubation: Incubate overnight at 4°C on a shaker at 40 rpm, recover the primary antibody, and wash three times with PBST on a shaker at 40 rpm for 7 min;

[0237] Secondary antibody incubation: incubate on a shaker at 40 rpm for 45 min, wash three times with PBST on a shaker at 40 rpm for 7 min (secondary antibody = 6 mL milk: 0.6 uL antibody: milk = 1:10000; GAPDH mouse antibody 6 mL, rabbit antibody 6 mL)

[0238] Exposure and development: After the ultrasensitive ECL developer is evenly incubated on the NC membrane, the developer is used to expose and detect the protein bands.

[0239] Example 10. Verification of the anti-tumor function of compound RD6 in vivo

[0240] A Huh7 tumor xenograft model was constructed in wild-type nude mice, and compound RD6 (20 mg / kg or 5 mg / kg, intraperitoneal injection, once every two days) was used as a single drug treatment. After 3 weeks of administration, the tumor size of the RD6-treated group was significantly reduced compared with the control group. At the same time, there was no significant change in body weight in mice treated with compound RD6, indicating the safety of this treatment regimen. After the administration, we took out the tumor tissue and weighed it, and found that the tumor mass of the RD6-treated group was also significantly reduced compared with the control group. The results are shown in the figure below. Fig. 9 shown.

[0241] Validation of mouse subcutaneous liver cancer xenograft tumor model. The specific experimental steps are as follows:

[0242] Nude mice inoculated with liver cancer cells Huh7:

[0243] Materials and equipment preparation: 1mL syringe, Huh7 single cell suspension, cotton balls, alcohol.

[0244] 1. Use serum-free DMEM medium to resuspend the digested Huh7 cells into a single cell suspension (5x10^ 7 / mL) for later use;

[0245] 2. Draw an appropriate amount of Huh7 single cell suspension into the syringe and insert the needle (the needle cut is facing the scale, and the air in the needle should be expelled before injection);

[0246] 3. Take the nude mouse out of the cage, pinch one ear and turn it over, pinch the front and back limbs of the mouse with the ring finger and little finger, and disinfect the injection site with an alcohol cotton ball;

[0247] 4. Inject 100uL of single cell suspension into the subcutaneous area of ​​the mouse shoulder blade through the needle;

[0248] 5. Rotate out the needle and use a dry cotton ball to push the injection liquid from bottom to top in time to avoid leakage;

[0249] 6. Then, the drug was administered according to the experimental requirements, and the weight and tumor size of the mice were monitored every other day.

[0250] After the experiment, the mice were killed, and the tumor tissues were removed with scissors and forceps, and photographed and recorded.

[0251] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A proteolysis targeting chimera, characterized in that: The proteolytic targeting chimeras include RD1, RD2, RD3, RD4, RD5, RD6, RD7, RD8, RD9, RD10, RD11, RD12, RD13 and RD14, The structure of the proteolysis targeting chimera consists of a common structure (the warhead R1 of RNF4), a VHL ligand or a CRBN ligand, and a linker; The structure of the warhead R1 of the RNF4 is as follows: The VHL ligand structure is as follows: The CRBN ligand structure is as follows:

2. The method for preparing a proteolysis targeting chimera according to claim 1, characterized in that: The synthetic routes of described RD1 and RD3 are as follows:

3. The method for preparing a proteolysis targeting chimera according to claim 1, characterized in that: The synthetic route of the RD2 is as follows:

4. The method for preparing a proteolysis targeting chimera according to claim 1, characterized in that: The synthetic route of the RD4 is as follows:

5. A proteolysis-targeted chimera pharmaceutical composition and use according to claim 4, characterized in that: In the synthetic route of RD4, RD5-RD14 can be obtained by replacing the linker of RD4 in step a; The linker of RD5 is The linker of RD6 is The linker of RD7 is The linker of RD8 is The linker of RD9 is The linker of RD10 is The linker of the RD11 is The linker of RD12 is The linker of RD13 is The linker of RD14 is 6. A method for preparing a proteolysis-targeted chimera according to any one of claims 2 to 4, characterized in that: The reagents and reaction conditions in the preparation method are as follows: (a) K2CO3, acetone, reflux, 3d; (b) Chloroacetyl chloride, TEA, CH2Cl2, 0→room temperature, 1 hour; (c) TFA, CH2Cl2, room temperature, 1 hour; (d) Succinic anhydride, DMAP, TEA, CH2Cl2, room temperature, 2 hours; (e) HATU, DIPEA, DMF, room temperature, 2 hours.

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