PD-l1 protac compound, application and antitumor drug thereof

Abstract

The application provides a PD-L1 PROTACs compound and application and an antitumor drug thereof, and belongs to the field of tumor immunotherapy. The PD-L1 PROTACs compound has a structural general formula as shown in formula I. The application connects a PD-L1 small molecule inhibitor BMS202 and an E3 ubiquitin ligase ligand PG through a straight-chain alkane chain, a PEG chain and a nitrogen-containing heterocyclic alkane chain to synthesize a series of PD-L1 PROTACs compounds. The compound can effectively reduce the PD-L1 level in tumor cells, directly inhibit the combination of PD-1 and PD-L1, effectively enhance the expression of immune-related factors such as GzmB and Prf1 in the microenvironment of tumor tissues, and finally play an excellent antitumor effect.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy, specifically involving PD-L1 PROTACs compounds and their applications and anti-tumor drugs. Background Technology

[0002] The programmed death receptor-1 / programmed death ligand-1 (PD-1 / PD-L1) signaling pathway is a crucial component of tumor immunosuppression. It enhances tumor cell immune tolerance by inhibiting T lymphocyte activation, thereby achieving immune escape through tumor immunosuppression. Therefore, the PD-1 / PD-L1 immune checkpoint is an important drug development target in the field of tumor immunotherapy. In recent years, although several monoclonal antibody PD-1 / PD-L1 inhibitors, including nivolumab, pembrolizumab, and atezolizumab, have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of various solid tumors and hematological malignancies, related studies have shown that large molecule antibody drugs have limited ability to penetrate tumor tissue, and some tumors have low response rates to monoclonal antibodies. Furthermore, the immunogenicity of antibody drugs can induce the body to produce anti-antibodies, leading to loss of drug efficacy. Furthermore, the high cost, inconvenient storage and transportation, and limited administration methods of monoclonal antibodies also restrict the clinical application and development of PD-1 / PD-L1 antibody drugs. Compared with monoclonal antibodies, although small molecule inhibitors are more suitable for oral administration and can reduce target occupancy time by adjusting the drug's half-life, thereby avoiding serious immune-related adverse events, and possess advantages such as ease of transportation and storage, high stability, and excellent membrane permeability, the disclosed crystal structures of PD-1 / PD-L1 proteins show that the three main druggable cavity binding pockets at the PD-L1-PD-1 interaction interface have relatively shallow grooves and relatively flat structures, which greatly limits the development of PD-1 / PD-L1 small molecule inhibitors.

[0003] Protein-targeting chimeras (PROTACs) are a rising star in drug development. This technology utilizes bifunctional small molecules that can bind to both the target protein and E3 ubiquitination ligases, pulling the target protein to the vicinity of the ubiquitination E3 complex and inducing its ubiquitination and degradation, thereby achieving therapeutic effects. PROTACs offer a new pathway to overcome the major obstacles faced in the development of PD-1 / PD-L1 small molecule inhibitors because they can degrade "undruggable" targets without requiring extremely high affinity for the target protein. Furthermore, although PD-L1 protein is mainly expressed on tumor cell membranes, the process of PD-L1 protein moving from the cytoplasm to the cell membrane is a continuous self-renewal cycle. Therefore, the idea of ​​constructing PROTAC molecules based on PD-L1 small molecule inhibitors to induce the degradation of newly generated or circulating PD-L1 protein in the cytoplasm, thereby reducing the amount of PD-L1 protein ultimately transported to the cell membrane, is theoretically feasible.

[0004] Based on the numerous advantages of PROTACs, combined with the hydrophobicity of the binding pocket and its relatively flat structural characteristics in the PD-1 / PD-L1 interaction model, developing PROTACs based on PD-1 / PD-L1 proteins may be a more effective approach than simple small molecule inhibitors. Therefore, three small molecule-based PD-L1 PROTACs have been reported in recent years. Among them, Cheng et al. designed and synthesized 28 novel PD-L1 PROTACs based on resorcinol diphenyl ether structures, using PD-1 / PD-L1 small molecule inhibitors BMS-8, BMS-1198, and BMS-1233 as targets and pomalidomide as an E3 ubiquitination ligase ligand. The preferred compound was P22, which showed the best IC50 performance. 50 The value reached 39.2 nM, and flow cytometry and Western blot data showed that P22 could moderately reduce PD-L1 protein levels in a lysosome-dependent manner. Wang et al. utilized the ethylenediamine side chain in the structure of the PD-L1 small molecule inhibitor BMS37, and coupled it with pomalidomide through linkers of different chain lengths to design and synthesize 19 PD-L1 PROTAC molecules. Among them, the preferred compound 21a not only effectively induced the degradation of PD-L1 protein in various malignant cells in a proteasome-dependent manner, but also showed in vivo experiments that compound 21a (15 mg / kg) significantly reduced PD-L1 protein levels in MC-38 cancer cells, thereby promoting CD8+. +T cell invasion inhibits the growth of MC-38 in vivo. Furthermore, Y. Liu et al. combined four different ligands of VHL, CRBN, MDM2, and cIAPE3 ligases, as well as four PD-L1 ligands including BMS-37 and NP19, and obtained 21 novel PROTAC molecules by clicking the target protein ligand, linker, and E3 ligand. Screening revealed that PROTACs composed of the CRBNE3 ligase recruiter, BMS-37, and the C3 linker (BMS-37-C3) exhibited the highest PD-L1 degradation efficiency. Moreover, in a co-culture model, BMS-37-C3 significantly promoted the tumor-killing ability of T cells in a concentration-dependent manner between 0.3 and 1.0 μmol / L. In summary, although PD-L1 protein PROTACs have shown initial effectiveness, research and achievements in this field are still limited. Therefore, further exploration of combinations of PD-L1 PROTACs based on various PD-L1 small molecule inhibitors and different E3 ubiquitination ligands may be a further expansion of research in this field. Summary of the Invention

[0005] This invention provides PD-L1 PROTACs compounds, their applications, and anti-tumor drugs. A series of PD-L1 PROTACs compounds were synthesized by linking the PD-L1 small molecule inhibitor BMS202 and the E3 ubiquitination ligand PG with linkers of different types and lengths. These compounds effectively reduce the PD-L1 level in tumor cells, directly inhibit the binding of PD-1 to PD-L1, thereby effectively enhancing the expression of immune-related factors such as GzmB and Prf1 in the tumor tissue microenvironment, and ultimately exerting excellent anti-tumor effects.

[0006] The technical solution of the present invention is as follows:

[0007] This invention provides PD-L1 PROTACs compounds, the general structural formula of which is shown in Formula I:

[0008]

[0009] Linker is a straight-chain alkane chain and / or a PEG chain.

[0010] In one specific embodiment, the straight-chain hydrocarbon has the structural formula shown in formula (1):

[0011] Equation (1) is Where n1 is any integer from 1 to 11;

[0012] The structural formula of the PEG chain is shown in formula (2) or formula (3):

[0013] Equation (2) is Where n2 is any integer from 1 to 4;

[0014] Equation (3) is Where n3 is any integer from 1 to 4.

[0015] In one specific embodiment, the PD-L1 PROTACs compound has any one of the following structural formulas:

[0016]

[0017]

[0018] The present invention also provides the application of the PD-L1 PROTACs compounds in the preparation of antitumor drugs.

[0019] The present invention also provides an antitumor drug comprising the above-mentioned PD-L1 PROTACs compounds.

[0020] In one specific embodiment, the anti-tumor drug is a PD-L1 targeted anti-tumor drug.

[0021] In one specific embodiment, paclitaxel is also included.

[0022] In one specific embodiment, the mass ratio of paclitaxel to the PD-L1 PROTACs compound is 5-10:30-60. In one specific embodiment, the structural formula of the PD-L1 PROTACs compound is:

[0023]

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] This invention synthesizes a series of PD-L1 PROTACs molecules by linking the PD-L1 small molecule inhibitor BMS202 and the E3 ubiquitination ligand PG with linkers of different types and lengths. These PD-L1 PROTACs molecules can effectively reduce the PD-L1 level in tumor cells, directly inhibit the binding of PD-1 to PD-L1, thereby effectively enhancing the expression of immune-related factors such as GzmB and Prf1 in the tumor tissue microenvironment, and ultimately exerting excellent anti-tumor effects. Attached Figure Description

[0026] Figure 1 The results show the preliminary screening results for the degradation activity of A-series PD-L1 PROTACs molecules;

[0027] Figure 2The results show the preliminary screening results for the degradation activity of B-series PD-L1 PROTAC molecules;

[0028] Figure 3 The results show the preliminary screening results for the degradation activity of C-series PD-L1 PROTAC molecules;

[0029] Figure 4 Preliminary screening results of the degradation activity of PD-L1 PROTAC molecules in the D series;

[0030] Figure 5 The degradation effect of PA8 at gradient doses on PD-L1 is shown in the following figures: (A) WB graph of the degradation effect of PA8 at gradient doses on PD-L1 in 4T1 cells; (B) Statistical graph of the degradation effect of PA8 at gradient doses on PD-L1 in 4T1 cells; (C) Quantitative analysis of the degradation effect of PA8 on PD-L1 protein in 4T1 cells.

[0031] Figure 6 To show the PD-L1 degradation effect of the control molecule PA8B, (A) WB graph of the degradation effect of PA8B at gradient doses on PD-L1 in 4T1 cells; (B) Statistical graph of the degradation effect of PA8B at gradient doses on PD-L1 in 4T1 cells.

[0032] Figure 7 To investigate the time-dependent degradation of PD-L1 protein by PA8, (A) WB plot showing the degradation effect of 0.312 μM PA8 on 4T1 cells at different treatment durations; (B) Statistical graph showing the degradation effect of 0.312 μM PA8 on 4T1 cells at different treatment durations on PD-L1.

[0033] Figure 8 To investigate the degradation mechanism of PA8 on PD-L1, including (A) the effect of siCRBN knockdown on CRBN on the degradation effect of PA8 on PD-L1; and (B) the effect of MG132 on the degradation effect of PA8 on PD-L1.

[0034] Figure 9 To analyze the changes in PD-L1 levels in 4T1 cells after 24 hours of PA8 treatment by flow cytometry;

[0035] Figure 10 To analyze the changes in PD-L1 content on the cell membrane of 4T1 cells after 24 hours of PA8 treatment by immunofluorescence.

[0036] Figure 11 The results of in vitro antitumor activity studies of PA8;

[0037] Figure 12 For the in vitro safety study of PA8 molecules;

[0038] Figure 13 The in vivo antitumor activity of PA8 at different doses was measured, including (A) changes in body weight of mice during administration and (B) changes in tumor volume of mice during administration.

[0039] Figure 14 To illustrate the in vivo tumor-suppressing effect of PA8 at different dosages, (A) tumor stripping diagrams of different drug groups; (B) final tumor weight statistics of different drug groups;

[0040] Figure 15 To investigate the in vivo antitumor mechanism of PA8 molecules, the study included: (A) PD-L1 immunofluorescence staining analysis of some tumor tissue sections; (B) CD4 immunofluorescence staining analysis of some tumor tissue sections; (C) CD8 immunofluorescence staining analysis of some tumor tissue sections; and (D) qRT-PCR analysis of immune-related factors in tumor tissue.

[0041] Figure 16 The pharmacokinetic curves for PA8 via different routes of administration are shown. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0043] Example 1: Chemical Synthesis of PD-L1 PROTACs Molecules

[0044] This embodiment designed 31 PD-L1 PROTACs molecules in four series. Series A uses a straight-chain alkane as the linker, with amide bonds connecting it to BMS202 and PG at both ends, respectively. Series B uses polyethylene glycol as the linker, also with amide bonds connecting it to BMS202 and PG at both ends. Series C still uses a straight-chain alkane as the linker, but the linker to BMS202 is directly coupled with a carbon-nitrogen bond, while the linker to PG continues to use an amide bond. Series D uses a polyethylene glycol chain containing a triazole ring as the linker, with the linker to BMS202 coupled with a carbon-nitrogen bond and the linker to PG using amide condensation. Based on the specific structures of the four series of PD-L1 PROTACs mentioned above, the synthetic route of BMS202 was first analyzed. The proposed approach is to expose the amino group at the end of its aliphatic chain as a leaving group, and then use the carboxyl group or halogen at one end of the linker to perform amide condensation or carbon-nitrogen coupling. Next, the amino group at the other end of the linker undergoes deboocation and dehydration condensation with the carboxyl group at the PG end to form an amide bond, thus synthesizing series A, B, and C PROTACs. For the D series, 3-bromopropyne is first carbon-nitrogen coupled with the exposed free amino group of BMS202 to reserve the alkyne group as a leaving group. Then, the amino group at the end of the linker, containing an azide group and an amino group respectively, undergoes amide condensation with the carboxyl group at the PG end. Finally, coupling is achieved through an azide-alkyne cycloaddition reaction using the two reserved alkyne and azide groups respectively. The specific synthetic routes are as follows:

[0045] Synthesis Route 1:

[0046]

[0047] Reagents and reaction conditions: (a) HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate), DIPEA (N,N-diisopropylethylamine), DMF (dimethylformamide), rt (room temperature), .4h; (b) TFA (trifluoroacetic acid), DCM (dichloromethane), rt (room temperature), 1h; (c) HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate), DIPEA (N,N-diisopropylethylamine), DMF (dimethylformamide), rt (room temperature), .4h.

[0048] Synthesis Route 2:

[0049]

[0050] Reagents and reaction conditions: (a) K2CO3, DMF (dimethylformamide), rt (room temperature), .6h; (b) TFA (trifluoroacetic acid), DCM (dichloromethane), rt (room temperature), 1h; (c) HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate), DIPEA (N,N-diisopropylethylamine), DMF (dimethylformamide), rt (room temperature), .4h.

[0051] Synthesis Route 3:

[0052]

[0053] Reagents and reaction conditions: (a) K2CO3, DMF (dimethylformamide), rt (room temperature), overnight; (b) HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate), DIPEA (N,N-diisopropylethylamine), DMF (dimethylformamide), rt (room temperature), .4h; (c) CuSO4·5H2O, VcNa (sodium vitamin C), THF / H2O (tetrahydrofuran / water); rt (room temperature), .5h.

[0054] 1. Synthesis of intermediate compounds

[0055] Synthesis of intermediate compound S1

[0056]

[0057] Reagents and reaction conditions: (a) Toluene, Pd(OAc)2,t-Bu Xphos (2-di-tert-butylphosphino-2′,4′,6′-triisopropylbiphenyl), Cs2CO3, 80℃, 12h.

[0058] In a 50 mL round-bottom flask, 3-hydroxymethyl-2-methylbiphenyl (1982.6 mg, 10.0 mmol, 1 eq), 6-chloro-2-methoxy-pyridine-3-carboxaldehyde (1715.8 mg, 10.0 mmol, 1 eq), Cs₂CO₃ (4875.0 mg, 15.0 mmol, 1.5 eq), Pd(OAc)₂ (22.4 mg, 1.0 mmol, 0.1 eq), and t-Bu XPhos (42.4 mg, 1.0 mmol, 0.1 eq) were added sequentially. Anhydrous toluene was used as the reaction solvent. The mixture was heated under reflux in an oil bath at 80 °C for 12 h. Thin-layer chromatography (TLC) was used to monitor the reaction until complete. The reaction was quenched with water, and the mixture was extracted three times with EA (5 mL / extraction). The combined EA organic layers were backwashed twice with saturated brine, dried over an appropriate amount of anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product S1. After the crude product was completely dried, it was packed into a silica gel column with 200-300 mesh silica gel powder and purified by column chromatography under polar conditions of PE:EA = 20:1 to obtain a white powder product S1 with a yield of about 86.0%. 1 H NMR(400MHz, DMSO-d6)δ10.10(s,1H),8.04(dd,J=8.3,1.0Hz,1H),7.51–7.43(m,3H),7.40–7.35(m,1H),7.33–7.26(m,3H) ,7.21(d,J=7.5Hz,1H),6.60(d,J=8.4Hz,1H),5.56(s,2H),4.05(d,J=1.1Hz,3H),2.22(s,3H).HRMS(DART-TOF)calculated for C 22 H 21 NO3Na + [M+Na] + m / z 370.1521, found 370.1541.

[0059] Synthesis of intermediate compound S2:

[0060]

[0061] Reaction conditions: (a) AcOH (acetic acid), DCM (dichloromethane), rt (room temperature), 4h; (b) STAB (sodium triacetoxyborohydride), DCM (dichloromethane), 10h.

[0062] S1 (666.78 mg, 2.0 mmol, 1 eq) was dissolved in 20 mL of dichloromethane. Then, N-tert-butoxycarbonyl-1,2-ethylenediamine (384.50 mg, 2.4 mmol, 1.2 eq) and 1 mL of glacial acetic acid were added sequentially to the system. The mixture was stirred at room temperature for 4 h. Then, sodium triacetoxyborohydride (1271.64 mg, 2.4 mmol, 3 eq) was added to the reaction system, and the mixture was stirred at room temperature for another 10 h. The reaction was monitored by thin-layer chromatography (TLC) until complete. The reaction was quenched with water, extracted three times with DCM (5 mL / time), and the combined DCM organic layers were backwashed twice with saturated brine. The mixture was dried over an appropriate amount of anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude S2. After the crude product was completely dried, it was packed into a silica gel column with 200-300 mesh silica gel powder and purified by column chromatography under polar conditions of PE:EA = 15:1 to obtain a pale yellow oily liquid product S2 with a yield of about 78.0%. 1 H NMR(400MHz,Chloroform-d)δ7.42(td,J=7.7,6.6,2.9Hz,2H),7.37(d,J=7.4Hz,2H),7.34–7.26(m,3H),7.22–7.17(m,2H),6.33(d,J=7.8 Hz,1H),5.40(s,2H),3.96(s,3H),3.65(s,2H),3.22(q,J=5.8Hz,2H),2.68(s,2H),2.26(s,3H),1.44(s,9H).HRMS(DART-TOF)calculated for C 28 H 35 N3O4Na + [M+Na] + m / z500.2628, found 500.2633.

[0063] Synthesis of intermediate compound S3

[0064]

[0065] Reagents and reaction conditions: (a) TFA (trifluoroacetic acid), DCM (dichloromethane), rt (room temperature), 1h.

[0066] Intermediate S2 (954.52 mg, 2.0 mmol, 1 eq) was dissolved in 15 mL of dichloromethane, and then 1.5 mL (10%) of trifluoroacetic acid was added to the reaction solution. The mixture was stirred at room temperature for 1 h, and the reaction was monitored by TLC. After the reaction was complete, the reaction was quenched with water, and extracted three times with DCM (5 mL / time). The organic layers were combined, backwashed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude product S3. After complete drying, the crude product was packed into a silica gel column using a dry method with 200-300 mesh silica gel powder and purified by column chromatography under polar conditions of PE:EA = 5:1 to obtain a pale yellow solid product S3, with a yield of approximately 80.0%. 1 H NMR(400MHz,Chloroform-d)δ7.45–7.38(m,4H),7.37–7.33(m,1H),7.33–7.28(m,2H),7.26–7.21(m,2H),6.35(d,J=7.9Hz,1H) ,5.41(s,2H),3.98(s,3H),3.72(s,2H),3.45(t,J=5.7Hz,2H),2.83(t,J=5.6Hz,2H),2.27(s,3H).HRMS(DART-TOF)calculated for C 23 H 27 N3O2Na + [M+Na] + m / z400.2103, found 400.2112.

[0067] Synthesis of intermediate compound S4

[0068]

[0069] Reagents and reaction conditions: (a) K2CO3, DMF (dimethylformamide), rt (room temperature), overnight.

[0070] Intermediate S3 (377.49 mg, 1.0 mmol, 1 eq) was dissolved in 15 mL of DMF. Then, 3-bromopropyne (130.85 mg, 1.1 mmol, 1.1 eq) and potassium carbonate (276.42 mg, 2.0 mmol, 2 eq) were added sequentially to the reaction solution, and the mixture was stirred overnight at room temperature. The reaction was monitored by TLC. After the reaction was complete, the reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (5 mL each time). The organic layers were combined, backwashed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude product S4. After complete drying, the crude product was purified by column chromatography using 200-300 mesh silica gel powder at a polar condition of PE (petroleum ether):EA (ethyl acetate) = 1.5:1, yielding a pale yellow solid product S4, with a yield of approximately 72.0%. 1 H NMR(400MHz,Chloroform-d)δ7.46–7.39(m,5H),7.37–7.34(m,1H),7.33–7.29( m,2H),7.23(d,J=1.9Hz,1H),6.36(d,J=7.9Hz,1H),5.42(s,2H),4.00(d,J=3.7H z,1H),3.95(s,3H),3.57(s,2H),3.48(d,J=3.4Hz,1H),3.29(d,J=2.4Hz,2H),3. 11(q,J=7.3Hz,1H),2.28(s,3H),1.49–1.37(m,4H).HRMS(DART-TOF)calculated for C 26 H 29 N3O2Na + [M+Na] + m / z 438.2260, found 438.2212.

[0071] Synthesis of intermediate compound G1

[0072]

[0073] Reagents and reaction conditions: (a) C 35 H 24 Cl4FeP2Pd([1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex), K3PO4,1,4-Dioxane / H2O(1,4-dioxane / water), 110℃, 12h.

[0074] 2,6-bis(benzyloxy)-3-bromopyridine (1851.2 mg, 5.0 mmol, 1 eq), 4-hydroxyphenylboronic acid (1379.3 mg, 10.0 mmol, 2 eq), and potassium phosphate (2122.6 mg, 10.0 mmol, 2 eq) were weighed into a 100 mL round-bottom flask. 1,4-dioxane (24.0 mL) and water (6.0 mL) in a volume ratio of (6:1) were used as the reaction solvent. The mixture was first degassed by sonication for 10 minutes, and then [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloromethane dichloride complex (408.3 mg, 0.5 mmol, 0.1 eq) was added. The reaction flask was then evacuated and purged with nitrogen. The reaction mixture was stirred at 110°C for 16 hours under nitrogen protection. After the reaction was complete as monitored by TLC, the reaction solution was cooled to room temperature and filtered through diatomaceous earth. The filtrate was diluted with water, extracted three times with ethyl acetate, and the organic layer was washed with saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (200-300 mesh silica gel powder) to obtain product G1 (1552.9 mg, 81%). 1 HNMR(400MHz,DMSO-d6)δ9.81(s,1H),7.65(d,J=8.1Hz,1H),7.46–7.28(m,12H),6.82– 6.72(m,2H),6.51(d,J=8.1Hz,1H),5.37(d,J=13.4Hz,4H).HRMS(DART-TOF)calculated for C 25 H 21 NO3Na + [M+Na] + m / z 406.1521, found 406.1525.

[0075] Synthesis of intermediate compound G2

[0076]

[0077] Reagents and reaction conditions: (a) K2CO3, DMF (dimethylformamide), 80℃, 4h.

[0078] Intermediate G1 (766.9 mg, 2.0 mmol, 1 eq) was dissolved in 25 mL of DMF. Then, tert-butyl bromoacetate (780.2 mg, 4.0 mmol, 2 eq) and potassium carbonate (552.0 mg, 4.0 mmol, 2 eq) were added to the reaction solution, and the mixture was stirred at 80 °C for 4 h. The reaction was monitored by TLC. After completion, the reaction was quenched with water, extracted three times with EA (5 mL / time), and the organic layers were combined. The mixture was backwashed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude product G2. The crude product was purified by column chromatography (200-300 mesh silica gel powder) to obtain product G2 (796.1 mg, 80%). 1 H NMR(400MHz, DMSO-d6)δ7.70(dd,J=8.1,0.9Hz,1H),7.54–7.23(m,12H),6.99–6.88(m,2H),6.53(dd,J =8.1,0.9Hz,1H),5.38(d,J=12.9Hz,4H),4.66(s,2H),1.47–1.38(m,9H).HRMS(DART-TOF)calculated for C 31 H 31 NO5Na + [M+Na] + m / z 520.2202, found 520.2211.

[0079] Synthesis of intermediate compounds G3 and G4

[0080]

[0081] Reagents and reaction conditions: (a) Pd-C, H2, EtOH, rt (room temperature), 12h; (b) TFA (trifluoroacetic acid), DCM (dichloromethane), 2h.

[0082] Intermediate G2 (746.3 mg, 1.5 mmol, 1 eq) was weighed and dissolved in 20 mL of ethanol. Pd-C (79.8 mg, 0.75 mmol, 0.5 eq) was then added. The mixture was stirred at room temperature under a hydrogen atmosphere for 12 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was filtered through diatomaceous earth. The filtrate was diluted with water, extracted three times with ethyl acetate, and the organic layer was washed with saturated brine. The solution was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (200-300 mesh silica gel powder) to obtain product G3 (421.5 mg, 88%). 1H NMR (400MHz, DMSO-d6) δ10.81(s,1H),7.22–7.04(m,2H),6.92–6.77(m,2H),4.62(d,J=1.3Hz,2H),3.79(dd, J=11.5,4.9Hz,1H),2.74–2.40(m,2H),2.22–1.92(m,2H),1.43(d,J=1.4Hz,9H).HRMS(DART-TOF)calculated for C 17 H 21 NO5Na + [M+Na] + m / z 342.1420, found 342.1432.

[0083] The intermediate G3 (383.2 mg, 1.2 mmol, 1 eq) obtained above was dissolved in 20 mL of dichloromethane, and then 2 mL of trifluoroacetic acid (10%) was added to the reaction solution. The mixture was stirred at room temperature for 4 h, and the reaction was monitored by TLC. After the reaction was complete, the product G4 (284.3 mg, 90%) was concentrated to dryness under reduced pressure. 1H NMR (400MHz, DMSO-d6) δ10.79(s,1H),7.13(d,J=8.5Hz,2H),6.86(d,J=8.4Hz,2H),4.65(s,2H),3.79(dd,J=11.5,4.9Hz,1H),2.65(dd d,J=17.1,11.7,5.3Hz,1H),2.50–2.41(m,1H),2.16(qd,J=12.0,4.4Hz,1H),2.00(dq,J=13.6,4.9Hz,1H).HRMS(DART-TOF)calculated for C 13 H 13 NO5Na + [M+Na] + m / z 286.0794, found 286.0798.

[0084] Synthesis of intermediate compounds A1-A11, B1-B9

[0085]

[0086] Reagents and reaction conditions: (a) HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate), DIPEA (N,N-diisopropylethylamine), DMF (dimethylformamide), rt (room temperature), .4h.

[0087] General Synthetic Step I: The intermediate S3 (150 mg, 0.39 mmol, 1 eq) synthesized above was dissolved in 20 mL of DMF. Then, HATU (192.7 mg, 0.507 mmol, 1.3 eq) and DIPEA (151.2 mg, 1.17 mmol, 3 eq) were added to the reaction solution. The mixture was stirred at room temperature for 20–30 minutes. Then, Linker (1.1 eq) containing different lengths and compositions was weighed, dissolved in DMF, and slowly added dropwise to the reaction solution. The mixture was stirred magnetically at room temperature for 4 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction was quenched with water. The mixture was extracted three times with ethyl acetate (5 mL / time). The organic layers were combined, backwashed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (200–300 mesh silica gel powder) to obtain the target product.

[0088] Synthesis of intermediate compound A1:

[0089] ((2-((2-methoxy-6-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)pyridin-3-yl)methyl)amino)ethyl)amino)-2-oxoethyl)tert-butyl carbamate)

[0090] The synthesis method of A1 is the same as the general synthesis step I: that is, intermediate S3 and Boc-glycine are used as reaction raw materials, and intermediate A1 is obtained by amide condensation reaction. The product is a white solid powder with a yield of about 81%. 1 H NMR(400MHz,Chloroform-d)δ7.45–7.38(m,3H),7.37–7.33(m,1H),7.31(dt,J=6.6,1 .6Hz,3H),7.23(q,J=1.3Hz,2H),6.39(dd,J=8.0,1.4Hz,1H),5.45–5.37(m,2H),3.97( d,J=1.4Hz,3H),3.75–3.62(m,2H),3.56(dd,J=6.7,4.4Hz,2H),3.39(q,J=5.3Hz,2H), 2.27(d,J=1.3Hz,3H),2.04(d,J=1.3Hz,2H),1.45(s,9H).HRMS(DART-TOF)calculated for C 30 H 38 N4O5Na + [M+Na] + m / z 557.2842, found 557.2851.

[0091] The synthesis methods for intermediates A2-A11 follow the same logic.

[0092] Synthesis of intermediate compound B1:

[0093] (2-((2-methoxy-6-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)pyridin-3-yl)methyl)amino)ethyl)amino)-2-oxoethoxy)tert-butyl carbamate

[0094] The synthesis method of B1 is the same as the general synthesis step I: that is, intermediate S3 and (Boc-aminooxy)acetic acid are used as reaction raw materials, and intermediate B1 is obtained by amide condensation reaction. The product is a white solid powder with a yield of about 78%. 1 H NMR(400MHz,Chloroform-d)δ7.96(s,1H),7.42(td,J=7.8,7.2,4.4Hz,3H),7.37–7.34(m, 1H),7.32–7.29(m,3H),7.23(d,J=2.1Hz,2H),6.38(d,J=8.0Hz,1H),5.41(d,J=4.0Hz,2H) ,4.72(s,2H),4.53(d,J=6.3Hz,1H),3.96(s,3H),3.54(dd,J=6.9,4.0Hz,2H),3.40(q,J=5 .2Hz,2H),3.12(q,J=7.3Hz,3H),2.27(s,3H),1.47(s,9H).HRMS(DART-TOF)calculatedfor C 30 H 38 N4O6Na + [M+Na] + m / z 573.2791, found 573.2798.

[0095] The synthesis methods for intermediates B2-B9 follow the same logic.

[0096] Synthesis of intermediate compounds C1-C6

[0097]

[0098] Reagents and reaction conditions: (a) K2CO3, DMF (dimethylformamide), rt (room temperature), 6h.

[0099] General Synthesis Step II: The previously synthesized intermediate S3 (150 mg, 0.39 mmol, 1 eq) was dissolved in 20 mL of LDM. Then, linkers of different lengths and compositions (1.1 eq) were added to the reaction solution, followed by K2CO3 (161.46 mg, 1.17 mmol, 3 eq). The mixture was magnetically stirred at room temperature for 6 h, and the reaction was monitored by TLC. After the reaction was complete, the reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (5 mL each time). The organic layers were combined, backwashed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (200-300 mesh silica gel powder) to obtain the target product.

[0100] Synthesis of intermediate compound C1:

[0101] ((2-((2-methoxy-6-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)pyridin-3-yl)methyl)amino)ethyl)tert-butyl carbamate)

[0102] The synthesis method of C1 is the same as the general synthesis step II: using intermediate S3 and N-BOC-bromoethylamine as reaction raw materials, intermediate C1 is obtained by coupling bromine with primary amine. The product is a white solid powder with a yield of about 73%. 1 H NMR(400MHz,Chloroform-d)δ7.46–7.39(m,3H),7.38–7.35(m,2H),7.33–7.28(m,2H),7.26–7.20(m,2H),6.36(d,J=8.0Hz,1H),5.41(s,2H),3.97 (d,J=5.2Hz,3H),3.49(s,2H),3.30–3.14(m,2H),2.54–2.32(m,2H),2.27(s,3H),1.83(d,J=9.7Hz,4H),1.42(s,9H).HRMS(DART-TOF) calculated for C 30 H 40 N4O4Na + [M+Na] + m / z 543.3050, found 543.3050.

[0103] Similarly, the synthesis of intermediate compounds C2-C6 is similar to that of C1.

[0104] Synthesis of intermediate compounds D1-D4

[0105]

[0106] Reagents and reaction conditions: (a) HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate), DIPEA (N,N-diisopropylethylamine), DMF (dimethylformamide), rt (room temperature), .4h.

[0107] General Synthesis Step III: The intermediate G4 (160.00 mg, 0.61 mmol, 1 eq) synthesized above was dissolved in 20 mL of DMF. Then, HATU (300.38 mg, 0.79 mmol, 1.3 eq) and DIPEA (236.52 mg, 1.83 mmol, 3 eq) were added to the reaction solution. The mixture was stirred at room temperature for 20–30 minutes. Then, Linker (1.1 eq) containing different lengths and compositions was weighed, dissolved in DMF, and slowly added dropwise to the reaction solution. The mixture was stirred magnetically at room temperature for 4 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction was quenched with water, extracted three times with EA (5 mL / time), and the organic layers were combined. The organic layers were backwashed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (200–300 mesh silica gel powder) to obtain the target product.

[0108] Synthesis of intermediate compound D1:

[0109] N-(2-(2-azidoethoxy)ethoxy)ethyl)-2-(4-(2,6-dioxopiperidin-3-yl)phenoxy)acetamide

[0110] The synthesis method of D1 is the same as that of general synthesis step III: using intermediate G4 and 2-[2-(2-azidoethoxy)ethoxy]ethylamine as reactants, intermediate compound D1 is obtained by amide condensation reaction. The product is a white solid powder with a yield of about 73%. 1 H NMR(400MHz,Chloroform-d)δ8.17(s,1H),7.21–7.11(m,2H),6.98–6.86(m,2H) ),4.50(s,2H),3.74(dd,J=9.9,5.3Hz,1H),3.66(dd,J=5.6,4.6Hz,2H),3.63( p,J=1.1Hz,3H),3.60(dd,J=4.4,0.9Hz,1H),3.56(t,J=5.4Hz,2H),3.37(t,J=5.0Hz,2H),2.81–2.56(m,4H),2.35–2.17(m,2H).HRMS(DART-TOF) calculated for C 19 H 25 N5O6[M+H] +m / z420.4380, found 420.4478.

[0111] Similarly, the synthesis methods for intermediate compounds D2-D4 are similar to those for D1.

[0112] 2. Synthesis of final products PA0-PA11, PB1-PB9, and PC1-PC6

[0113]

[0114] Reagents and reaction conditions: (a) TFA (trifluoroacetic acid), DCM (dichloromethane), rt (room temperature), .2h; (b) HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate), DIPEA (N,N-diisopropylethylamine), DMF (dimethylformamide), rt (room temperature), .4h.

[0115] General Synthesis Step IV:

[0116] Weigh 0.1 mmol of the synthesized A, B, and C series intermediates into a 50 mL round-bottom flask. Using DCM as solvent, add an appropriate amount of TFA (final TFA concentration in the reaction solution is 5-10%) to the reaction system. Stir at room temperature for 40 min and monitor the reaction by TLC. After the reaction is complete, evaporate under reduced pressure to remove the solvent completely. The crude product obtained can be used directly in the next step of the reaction.

[0117] The crude product was dissolved in an appropriate amount of DMF, and then HATU (49.4 mg, 0.13 mmol, 1.3 eq) and DIPEA (38.7 mg, 0.3 mmol, 3 eq) were added to the system. The mixture was stirred at room temperature for 20–30 minutes. Then, intermediate G4 (28.9 mg, 0.11 mmol, 1.1 eq) was weighed, dissolved in DMF, and slowly added dropwise to the above reaction solution. The mixture was stirred magnetically at room temperature for 4 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction was quenched with water, and the mixture was extracted three times with EA (5 mL / time). The organic layers were combined, backwashed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (200–300 mesh silica gel powder) to obtain the target product. The structure of the target product was confirmed by NMR and mass spectrometry.

[0118] Synthesis of the final product PA0:

[0119] 2-(4-(2,6-dioxopiperidin-3-yl)phenoxy)-N-(2-methoxy-6-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)pyridin-3-yl)methyl)amino)ethyl)acetamide

[0120] Intermediate S3 (100.0 mg, 0.26 mmol, 1 eq) was weighed and dissolved in 20 mL of DMF. Then, HATU (129.2 mg, 0.34 mmol, 1.3 eq) and DIPEA (100.1 mg, 0.78 mmol, 3 eq) were added to the reaction solution. The mixture was stirred at room temperature for 20–30 minutes. Next, intermediate G4 (75.3 mg, 0.28 mmol, 1.1 eq) was weighed, dissolved in DMF, and slowly added dropwise to the above reaction solution. The mixture was then stirred magnetically at room temperature for 4 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction was quenched with water, and the mixture was extracted three times with EA (5 mL / extraction). The organic layers were combined, backwashed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (200–300 mesh silica gel powder) to obtain the target product PA0. 1 H NMR(400MHz,Chloroform-d)δ7.48–7.38(m,3H),7.38–7.28(m,3H),7.24(s,3H) ,7.13(d,J=7.7Hz,2H),6.95–6.88(m,2H),6.39(d,J=7.7Hz,1H),5.43(d,J=2.1 Hz,2H),4.49(s,2H),3.97(d,J=2.1Hz,3H),3.72(dd,J=9.7,5.3Hz,2H),3.57(d ,J=15.6Hz,2H),3.36(s,2H),3.16–3.08(m,1H),2.28(s,3H),1.47–1.33(m,4H). 13 C NMR (101MHz, CDCl3) δ173.43,172.42,170.30,162.64,160.38,157.35,142.90,141 .97,140.75,135.48,130.10,129.38,129.24,128.41,128.08,126.85,125.48,115 .05,108.08,101.96,77.28,77.26,67.18,67.09,53.66,47.23,46.50,45.79,44.2 3,39.47,38.63,31.07,28.41,26.35,16.31,8.63.HRMS(DART-TOF)calculatedfor C 36 H 38 N4O6Na + [M+Na] + m / z 645.2491, found 645.2498.

[0121] Synthesis of final product PA1:

[0122] 2-(4-(2,6-dioxopiperidin-3-yl)phenoxy)-N-(2-(2-methoxy-6-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)pyridin-3-yl)methyl)amino)ethyl)amino)-2-oxoethyl)acetamide

[0123] The synthesis method of PA1 is the same as the general synthesis step IV: first, intermediate A1 is debocized by TFA, and then its terminal primary amine is coupled to the carboxyl group of intermediate G4 by amide condensation to obtain the target compound PA1. The product is a grayish-white solid powder with a yield of about 80%. 1 H NMR(400MHz,Chloroform-d)δ7.45–7.38(m,3H),7.38–7.28(m,4H),7.23(d,J=6 .8Hz,2H),7.13(s,1H),7.11(s,1H),6.93–6.85(m,2H),6.33(d,J=8.0Hz,1H),5. 40(s,2H),4.48(s,2H),4.32(d,J=21.2Hz,2H),3.95(d,J=4.5Hz,3H),3.65(d,J= 6.8Hz,3H),3.50(d,J=11.4Hz,2H),3.33(s,2H),2.26(s,3H),1.29–1.19(m,4H). 13 C NMR (101MHz, CDCl3) δ173.28,172.33,170.17,168.28,162.78,160.43,156.80,142.91 ,141.98,140.39,135.43,134.56,130.63,130.12,129.50,129.38,129.12,128.45,128 .07,126.84,125.48,115.27,107.54,101.97,77.24,77.23,67.40,67.12,53.70,47.2 5,46.46,44.62,40.90,39.38,31.08,29.70,26.39,16.28.HRMS(DART-TOF)calculated forC 38 H 41 N5O7Na + [M+Na] + m / z 702.3006, found 702.3011.

[0124] Synthesis of the final product PA6

[0125]

[0126] The synthesis method of PA6 is the same as the general synthesis step IV: first, intermediate A6 is debocized by TFA, and then its terminal primary amine is coupled to the carboxyl group of intermediate G4 by amide condensation to obtain the target compound PA6. The product is a grayish-white solid powder with a yield of about 73%. 1 H NMR(400MHz,Chloroform-d)δ7.46–7.39(m,4H),7.38–7.34(m,1H),7.32–7.28(m,2H),7.24(d,J=2.1Hz,2 H),7.17(d,J=8.5Hz,2H),6.95–6.90(m,2H),6.39(d,J=8.0Hz,1H),5.42(s,2H),4.48(s,2H),3.97(s,3H), 3.74(dd,J=9.8,5.4Hz,1H),3.57(t,J=5.4Hz,2H),3.40(d,J=5.7Hz,2H),3.33(q,J=6.8Hz,2H),2.42(t,J =7.4Hz,2H),2.28(s,3H),2.04(s,1H),1.63(d,J=7.1Hz,2H),1.54(t,J=6.9Hz,2H),1.33(d,J=7.3Hz,9H). 13 C NMR (101MHz, CDCl3) δ175.90,173.70,172.83,168.10,162.30,160.14,156.67,142.85,141.96 ,139.32,135.59,134.50,130.88,130.03,129.38,129.36,128.39,128.07,126.83,125.45,115 .08,108.88,101.67,70.48,67.41,67.01,53.58,53.54,47.15,46.70,45.28,44.20,39.74,38 .91,33.00,31.06,29.27,28.88,26.56,25.07,16.30,16.27,8.65.HRMS(DART-TOF)calculated for C 43 H 51 N5O7[M+H] + m / z 750.3788, found 750.3791.

[0127] Synthesis of the final product PA7

[0128]

[0129] The synthesis method of PA7 is the same as the general synthesis step IV: first, intermediate A7 is debocized by TFA, and then its terminal primary amine is coupled to the carboxyl group of intermediate G4 by amide condensation to obtain the target compound PA7. The product is a grayish-white solid powder with a yield of about 72%. 1 H NMR(400MHz,Chloroform-d)δ8.55(s,1H),8.26(t,J=4.6Hz,1H),7.47–7.37(m,3H),7.38–7.33(m,1H),7.33–7.28(m,2H),7. 23(dd,J=7.5,5.8Hz,3H),7.16(d,J=8.6Hz,2H),6.97–6.88(m,2H),6.39(d,J=8.0Hz,1H),5.42(s,2H),4.47(s,2H),4.12(q,J =7.1Hz,1H),3.97(d,J=4.6Hz,3H),3.73(dd,J=9.7,5.3Hz,1H),3.56(dd,J=6.9,4.2Hz,2H),3.40(q,J=5.1Hz,2H),3.32(q,J= 6.8Hz,2H),2.42(t,J=7.4Hz,2H),2.28(s,3H),2.04(s,2H),1.62(q,J=7.1Hz,2H),1.52(q,J=6.9Hz,2H),1.41–1.20(m,10H). 13 C NMR (101MHz, CDCl3) δ192.36,176.07,173.62,172.76,168.07,162.29,160.14,156.69,142.85,14 1.96,139.33,135.59,134.49,130.86,130.03,129.54,129.36,128.07,126.84,125.45,115.08,10 8.91,101.67,77.53,77.41,77.21,76.89,70.50,67.46,66.96,53.58,53.54,47.14,46.68,45.97, 44.18,39.81,39.04,33.06,31.06,29.42,26.62,25.13,16.30,16.27.HRMS(DART-TOF)calculated for C 44 H 53 N5O7[M+H] + m / z 764.3945, found 764.3951.

[0130] Synthesis of the final product PA8

[0131]

[0132] The synthesis method of PA8 is the same as the general synthesis step IV: first, intermediate A8 is debocized by TFA, and then its terminal primary amine is coupled to the carboxyl group of intermediate G4 by amide condensation to obtain the target compound PA8. The product is a grayish-white solid powder with a yield of about 77%. 1 H NMR(400MHz,Chloroform-d)δ7.47–7.38(m,4H),7.38–7.33(m,1H),7.33–7.28(m,2H),7.26–7.22(m,2H ),7.20–7.13(m,2H),6.96–6.89(m,2H),6.39(d,J=8.0Hz,1H),5.42(s,2H),4.48(s,2H),3.97(s,3H),3 .73(dd,J=9.9,5.4Hz,1H),3.57(q,J=4.6,3.9Hz,2H),3.40(q,J=5.1Hz,2H),3.32(qd,J=6.9,3.2Hz,2H ),2.42(t,J=7.5Hz,2H),2.28(s,3H),1.62(d,J=7.9Hz,2H),1.52(t,J=7.1Hz,2H),1.40–1.20(m,14H). 13 CNMR(101MHz, CDCl3)δ192.39,176.39,173.20,172.33,167.95,162.37,160.16,157.81,157.44,15 6.73,142.90,141.98,139.26,135.55,134.53,130.71,130.08,129.38,128.40,128.08,126.85,125 .46,115.12,108.75,101.74,77.26,77.24,67.51,67.04,53.59,53.55,47.22,46.75,45.93,44.18 ,40.26,39.06,33.14,31.11,29.71,29.31,26.78,26.34,25.24,16.28.HRMS(DART-TOF)calculated for C 45 H 55 N5O7[M+H] + m / z 778.4101, found 778.4154.

[0133] The synthesis methods for the final products PA2-PA5 and PA9-PA11 can be deduced similarly.

[0134] Synthesis of final product PB1:

[0135] 2-(4-(2,6-dioxopiperidin-3-yl)phenoxy)-N-(2-((2-methoxy-6-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)pyridin-3-yl)methyl)amino)ethyl)amino)-2-oxoethoxy)acetamide

[0136] The synthesis method of PB1 is the same as the general synthesis step IV: that is, intermediate B1 is first debocized by TFA, and then its terminal primary amine is coupled to the carboxyl group of intermediate G4 by amide condensation to obtain the target compound PB1. The product is a white solid powder with a yield of about 82%. 1 H NMR(400MHz,Chloroform-d)δ8.63(s,1H),8.02(s,1H),7.87(s,1H),7.45–7.38(m,4H),7.38–7.33(m, 1H),7.33–7.28(m,2H),7.25–7.20(m,2H),7.13(d,J=8.3Hz,2H),6.95–6.86(m,2H),6.38(d,J=8.0Hz, 1H),5.41(s,2H),4.87(s,2H),4.53(d,J=8.1Hz,2H),4.27(s,1H),3.96(s,3H),3.72(dd,J=10.0,5.3H z,1H),3.50(q,J=4.8,3.9Hz,2H),3.40(t,J=5.9Hz,2H),2.96(s,2H),2.27(s,3H),2.26–2.18(m,4H). 13 C NMR(101MHz,DMSO)δ174.93,173.92,171.08,170.07,161.86,161.38,160.06,157.18,1 42.61,141.85,141.04,136.31,136.23,134.32,134.29,131.95,130.00,129.60,128.7 3,128.68,127.39,125.93,117.74,114.85,114.67,110.21,101.66,101.58,66.82,65. 11,53.80,46.98,44.80,43.68,31.87,26.49,19.15,16.38.HRMS(DART-TOF)calculated forC 38 H41 N5O8Na + [M+Na] + m / z 718.2955, found 718.2961.

[0137] The synthesis methods for the final products PB2-PB9 can be deduced similarly.

[0138] Synthesis of the final product PC1:

[0139] 2-(4-(2,6-dioxopiperidin-3-yl)phenoxy)-N-(2-((2-methoxy-6-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)pyridin-3-yl)methyl)amino)ethyl)acetamide

[0140] The synthesis method of PC1 is the same as the general synthesis step IV: first, intermediate C1 is debocized by TFA, and then its terminal primary amine is coupled to the carboxyl group of intermediate G4 by amide condensation to obtain the target compound PC1. The product is a white solid powder with a yield of about 82%. 1 H NMR(400MHz,Chloroform-d)δ7.52(t,J=9.1Hz,1H),7.44–7.40(m,3H),7.37–7.29(m ,3H),7.25–7.20(m,2H),7.13(s,1H),7.11(s,1H),6.92–6.86(m,2H),6.33(d,J=8.0 Hz,1H),5.40(s,2H),4.48(s,2H),3.95(d,J=4.5Hz,3H),3.72–3.61(m,4H),3.50(d, J=11.4Hz,2H),3.39–3.25(m,3H),2.83–2.52(m,2H),2.26(s,3H),1.30–1.07(m,4H). 13 C NMR(101MHz,DMSO)δ174.83,173.86,168.56,161.54,157.08,157.06,142.60,14 1.86,141.23,136.30,134.29,132.31,130.02,129.61,128.69,128.69,127.40, 125.93,114.98,114.80,101.56,99.98,99.98,67.46,66.73,64.03,53.78,46.9 8,45.57,38.21,31.86,26.44,21.47,21.45,16.37.HRMS(DART-TOF)calculated for C 38 H43 N5O6Na + [M+Na] + m / z688.3213, found 688.3221.

[0141] Similarly, the synthesis methods for the final products PC2-PC6 are similar to those for PC1.

[0142] Synthesis of final products PD1-PD4

[0143]

[0144] Reagents and reaction conditions: (a) CuSO4·5H2O, VcNa (sodium vitamin C), THF / H2O (tetrahydrofuran / water); rt (room temperature), .5h.

[0145] General synthetic step V: Weigh the previously synthesized D-series intermediate (0.10 mmol, 1.0 eq) into a 25 mL round-bottom flask, dissolve it in THF / H2O solvent (THF:H2O = 2:1), and add CuSO4·5H2O (0.15 mmol, 37.44 mg, 1.5 eq) to the reaction solution. After stirring at room temperature for 15 minutes, add intermediate compound S4 (0.15 mmol, 63.78 mg, 1.5 eq) to the reaction solution, and finally add sodium vitamins (0.2 mmol, 39.62 mg, 2.0 eq). Continue to stir magnetically at room temperature for 4 h. Monitor the reaction by TLC. After the reaction is complete, quench the reaction with water, extract three times with EA (5 mL / time), combine the organic layers, backwash the organic layer twice with saturated brine, dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (200-300 mesh silica gel powder) to obtain the target product, and the structure of the target product was confirmed by NMR and mass spectrometry.

[0146] Synthesis of the final product PD1:

[0147] 2-(4-(2,6-dioxopiperidin-3-yl)phenoxy)-N-(2-(2-(2-(2-methoxy-6-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)pyridin-3-yl)methyl)amino)ethyl)amino)methyl)1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl)ethyl)ethyl)acetamide

[0148] The synthesis of PD1 follows the same general synthetic step V: coupling the alkynyl end of intermediate D1 with the azide group of intermediate S4 via an azide-alkynyl cycloaddition reaction, ultimately yielding the target compound PD1. The product is a white solid powder with a yield of approximately 48%. ¹H NMR (400 MHz, Chloroform-d) δ. 8.06 (s, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.42–7.36 (m, 3H), 7.36–7.27 (m, 3H), 7.23–7.19 (m, 2H), 7.16 (d, J = 8.7 Hz, 1H), 7.09 (d, J = 8.2 Hz, 2H), 6.97–6.90 (m, 2H), 6.85 (d, J = 8.3 Hz, 2H), 6.37 (d, J = 8.0 Hz, 1H) ),5.37(s,2H),4.49(s,2H),4.07(s,2H),3.91(s,3H),3.83(d,J=9.7Hz,2H),3.68–3.56(m,5H),3.49(s,9H),3.37(t ,J=5.0Hz,1H),2.79–2.56(m,3H),2.32–2.25(m,1H),2.23(s,3H),2.15(d,J=13.8Hz,3H),1.30(d,J=34.5Hz,1H).13C NMR (101MHz, CDCl3) δ173.91,173.48,172.92,168.47,168.20,163.81,161.45,156.76,156.72,144 .69,142.90,141.91,135.21,134.57,130.94,130.17,129.60,129.52,129.36,128.47,128.08,126 .86,125.50,115.14,102.81,70.55,70.32,70.06,69.75,69.53,68.99,67.48,67.23,53.90,51.80 ,51.01,50.62,50.36,48.31,47.19,38.83,35.61,31.12,26.38,16.25.HRMS(DART-TOF)calculated forC45H54N8O8Na+[M+Na]+m / z 857.4065, found 857.4071.

[0149] Similarly, the synthetic routes for the final products PD2-PD4 are similar to those for PD1.

[0150] Example 2: Activity study of PD-L1 PROTACs molecules

[0151] I. Experimental Materials and Methods

[0152] 1. Cell lines and experimental animals

[0153] The cells used in this invention include mouse breast cancer cells 4T1 and human kidney epithelial cells 293T, both derived from the Cell Bank of the Chinese Academy of Sciences (https: / / www.cellbank.org.cn / ). 4T1 cells were cultured in RPMI 1640 medium containing 10% FBS and 1% penicillin-streptomycin Liquid (100×); 293T cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin Liquid.

[0154] The experimental animals used in this invention were female BALB / c mice, 6-8 weeks old, with an average weight of 18-20g. All mice were purchased from Beijing Huafukang Biotechnology Co., Ltd., and housed in the animal facility of the State Key Laboratory of Biotherapy (SPF grade) at Sichuan University, with housing conditions conducted in accordance with international standards.

[0155] 2. Experimental Methods

[0156] 2.1 Western Blot Experiment

[0157] Cell culture and drug administration: Following the aforementioned cell culture method, when the cell density in the culture dish reaches 80%–90%, cells are collected and resuspended in fresh culture medium. Appropriate cell concentrations are seeded into multiple 6cm culture dishes and cultured in a cell culture incubator for 24 hours until the cells are fully adhered. Then, different concentrations of the test compound solution prepared in advance with culture medium are added to the 6cm culture dishes and gently mixed. The dishes are then placed in the incubator for further culture.

[0158] Protein extraction: After the drug has been applied for a specific time, discard the culture medium and wash 2-3 times with pre-chilled sterile PBS. Add 100 μL of pre-chilled cell lysis buffer to a 6 cm culture dish, gently scrape adherent cells off the dish with a cell scraper, collect them in a 1.5 mL EP tube, and then place the tube on ice for lysis for 30 min, vortexing continuously to ensure complete cell lysis. After lysis for 30 min, centrifuge at 13000 rpm / min for 15 min at 4°C. After centrifugation, carefully aspirate the supernatant into a new EP tube using a pipette (be careful not to aspirate any precipitate).

[0159] Protein quantification: First, prepare protein standards of different concentrations according to the BCA protein quantification kit instructions. Then, add 10 μL / well of each concentration of protein standards to a 96-well plate. Next, add 10 μL / well of PBS (specific solution) and 200 μL / well of BCA protein quantification working solution to each well. Incubate the plate in a cell culture incubator for 30 min. After incubation, measure the absorbance of each well at 560 nm using a microplate reader. Finally, plot the BCA protein standard curve based on the absorbance values. Separately, add an appropriate amount of the extracted protein supernatant to a 96-well plate (10 μL / well). Follow the same steps as above. Finally, calculate the protein concentration based on the absorbance value and the BCA protein standard curve.

[0160] Protein denaturation: Based on the protein quantification results, the concentration of the extracted protein samples was adjusted to a uniform level using cell lysis buffer. Then, an appropriate volume of 5× loading buffer was added to bring the final concentration to 1×. The mixture was vortexed and heated in a 100℃ metal bath for 10 min to ensure complete protein denaturation. After heating, the samples were cooled to room temperature and centrifuged (1200 rpm, 5 min). Finally, they were stored at -20℃ for later use.

[0161] Gel preparation: Fix the gel preparation glass plate on the gel preparation rack. First, use a pipette to draw about 5 mL of separating gel into the gap between the two glass plates. Then, add an appropriate amount of isopropanol and press it flat. After the separating gel dries, use a paper towel to absorb all the isopropanol. Then, add about 1.5 mL of stacking gel and insert the comb (the preparation methods for separating gel and stacking gel are according to the kit instructions).

[0162] Sample loading and electrophoretic separation: Fix the prepared gel-containing glass plate onto the electrophoresis clamp and assemble it into the electrophoresis tank. Fill the tank with the pre-prepared electrophoresis buffer. Carefully remove the comb from the gel and gently blow air into the sample wells with a pipette to remove air bubbles and fill the wells with electrophoresis buffer. Then, add the previously prepared protein samples and protein markers to the sample wells, with a sample volume of approximately 10 μL per well. After sample loading, adjust the voltage to 80 V and electrophores at a constant voltage until the bromophenol blue and protein markers emerge from the stacking gel. Then, increase the voltage to 120 V and continue electrophoresis until the protein markers are completely separated. Stop the electrophoresis and remove the SDS-PAGE gel from the electrophoresis tank and place it in transfer buffer.

[0163] Transfer: Activate the PVDF membrane by immersing it in methanol for approximately 1 minute, and mark the lower left corner of the membrane with a ballpoint pen beforehand. Then, place the PVDF membrane in pre-cooled transfer buffer. Prepare several appropriately sized filter papers, pre-wetting them with pre-cooled transfer buffer. Then, construct a transfer "sandwich" structure in the following order: sponge (black) → filter paper (approximately 3 sheets) → gel → PVDF membrane → filter paper (approximately 3 sheets) → sponge (white). During the construction process, avoid air bubbles between the layers to prevent affecting the transfer effect. Next, insert the fixed "sandwich" clips into the transfer tank, place the transfer tank in the electrophoresis chamber, and then place the electrophoresis chamber in an ice-water bath. Adjust the voltage to 100V and begin transfer. Set the transfer time according to the molecular weight of the target protein; in this invention, the transfer time is generally 90 minutes.

[0164] Band blocking and primary antibody incubation: After transfer, the PVDF membrane was removed and washed three times with TBS / T buffer. It was then transferred to the prepared blocking buffer and placed on a shaker for 1 hour at room temperature. After blocking, the membrane was washed three times with TBS / T buffer for 10 minutes each time. Following washing, the PVDF membrane was cut to the appropriate position according to the molecular weight of the target protein (PD-L1, CRBN, GAPDH in this invention) and placed in an incubation box containing the corresponding antibody. The membrane was then incubated overnight at 4°C with gentle shaking.

[0165] Secondary antibody incubation and band exposure: The bands incubated with primary antibody overnight were washed three times with TBS / T buffer for 10 min each time. Then, secondary antibody solution corresponding to the primary antibody species was added to the antibody incubation chamber, and the chamber was placed on a shaker and incubated at room temperature for approximately 1.5 h. After incubation, the bands were stripped three times with TBS / T buffer for 10 min each time. Before exposure, the TBS / T buffer on the PVDF membrane was wiped dry, and the membrane was immersed in chemiluminescent solution before exposure on an e-blot exposure machine.

[0166] 2.2 MTT assay for cell proliferation inhibition

[0167] When the cell density in the cell culture dish reaches 70%–80%, collect cells according to the aforementioned cell passage method, add an appropriate amount of fresh culture medium and carefully resuspend the cells with a pipette. Add 20 μL of the cell suspension to a cell counting chamber, calculate the cell concentration using a cell counter, and then adjust the cell concentration with the culture medium. According to different cell growth rates, a certain number of cells are seeded into 96-well plates, with 100 μL of cell suspension seeded into each well, and the total number of cells is approximately 1000–1500. The edges of the wells are filled with sterile PBS, and the plates are placed in a cell culture incubator for 24 hours until the cells are completely adhered.

[0168] The test compound dissolved in DMSO was diluted to different concentration gradients using cell culture medium. The solution was added to the well plate at a volume of 100 μL per well in a biosafety cabinet. Three replicates were set for each drug concentration group. DMSO at a concentration of 0.1% was used as a control group.

[0169] After drug administration, the 96-well plate was placed in a cell culture incubator for 48 hours. The cell growth status of the drug administration group, positive control group, and blank group was observed under a regular inverted microscope. 20 μL of pre-prepared 5 mg / mL MTT solution was added to each well in a biosafety cabinet. The 96-well plate was then placed in a cell culture incubator for 4 hours in the dark.

[0170] After adding MTT and culturing for 4 hours, remove the 96-well plate, carefully discard the supernatant, gently remove any remaining liquid with a 1mL syringe, gently invert the plate, and then add 150μL of DMSO to each well to fully dissolve the crystals. Then, place the plate on a microplate reader and measure the absorbance of each well at 562nm. Calculate the average value of three replicates for each drug concentration. The relative cell growth inhibition rate of each target compound was calculated using the following formula: Cell growth inhibition rate = [1 - (A sample group - A blank group) / (A control group - A blank group)] × 100%. Then, GraphpadPrism 8.0 software was used to generate compound concentration-cell growth curves, and the IC50 of each target compound was calculated based on the results. 50 value.

[0171] 2.3 Immunofluorescence staining of cells

[0172] Preparation and addition of reagents to cell slides: a. First, disinfect the cell slides (14 mm in diameter) by immersing them in 75% alcohol. Then, allow the alcohol to evaporate naturally in a biosafety cabinet. After evaporation, place the slides in a 24-well plate. b. Seed the target cells into the 24-well plate according to the cell passage procedure and culture the cells in a cell culture incubator until they adhere (24 hours). c. Add the corresponding concentration of the test compound to the 24-well plate and continue incubation for another 24 hours.

[0173] Immunofluorescence staining: a. Remove the well plate, carefully aspirate the culture medium from the well plate, and wash the cell-covered slides three times with PBS for 3 minutes each time. b. Fix the cell-covered slides with 4% paraformaldehyde for 15 minutes, then wash the slides three times with PBS for 3 minutes each time. c. Punch the slides with 0.5% Triton X-100 (prepared with PBS) for 20 minutes. After punching, wash the slides three times with PBS for 3 minutes each time, then blot dry any remaining PBS with absorbent paper (punching permeates the cells, allowing the cells and nuclei to fully contact the antibody). d. Block the cells on the slides with 5% goat serum at room temperature for 1 hour to prevent non-specific antibody binding. e. Incubate with primary antibody, remove the blocking solution with absorbent paper, add a sufficient amount of primary antibody (diluted blocking solution) to each slide, cover with sealing film, and incubate overnight at 4°C in a dark box. f. Warm to room temperature for 30 min, remove the sealing film, place the slide in a 24-well plate, wash the slide three times with PBST for 3 minutes each time, blot off excess liquid with absorbent paper, add diluted fluorescent secondary antibody, incubate in a dark chamber at room temperature for 1 h, wash the slide three times with PBST for 3 minutes each time. g. Counterstain with DAPI to observe cell nuclei. h. Mounting and detection: Add DAPI-containing anti-fluorescence mounting medium to the working area of ​​the slide, then observe and acquire fluorescence images directly under a fluorescence microscope.

[0174] 2.4 Flow cytometry

[0175] 4T1 cells in logarithmic growth phase were seeded in 6-well plates and incubated. Once the cell density reached 70%–80%, the cells were treated with high and low concentrations of compound PA8 (0.312 μM) or PA8 (0.078 μM) for 24 hours, respectively. Cells were then collected, washed three times with PBS, blocked with 0.1% BSA for 30 minutes, and stained with FAM-labeled PD-L1 antibody (1:200 dilution) for 30 minutes. Finally, after washing three times with PBS, the number of FAM-positive cells was analyzed by flow cytometry.

[0176] 2.5 Co-culture of PBMC cells and tumor cells

[0177] Extraction of mouse peripheral blood PBMCs

[0178] This experiment used the PBMC cell extraction kit from Tianjin Haoyang Huake Biotechnology Co., Ltd., and the specific operating procedure is as follows:

[0179] a. Blood (3 mL) was collected from the abdominal aorta of BALB / c mice and added to TBD. TM In anticoagulant tubes specifically designed for cell separation.

[0180] b. Take anticoagulated blood and mix it with the sample diluent at a volume ratio of 1:1. Take another centrifuge tube, add an equal volume of separation solution to the diluted sample, and then carefully pipette the diluted blood sample onto the surface of the separation solution (be careful not to mix it with the separation solution). Centrifuge at 400-500g for 20-30 minutes.

[0181] c. After centrifugation, the liquid in the centrifuge tube will naturally separate into four layers. From top to bottom, these are the plasma layer, the ring-shaped milky white mononuclear cell layer, the clear separation fluid layer, and the red blood cell layer. Then, carefully use a pipette to aspirate the second layer of liquid into another 15 mL centrifuge tube, add 10 mL of washing solution to the resulting centrifuge tube, and mix the cells thoroughly.

[0182] d. Centrifuge the cells obtained in step c at 250g for 10min, and discard the supernatant. Resuspend the cells in 5mL of washing buffer, and centrifuge again at 250g for 10min. Repeat this operation 3 times.

[0183] e. After washing, discard the supernatant, resuspend the cells in 1 mL of RPMI 1640 medium containing fetal bovine serum, and culture them in an incubator according to the standard cell culture method until ready for use.

[0184] 2.6 PBMC cells / 4 T1 cells co-culture

[0185] a. Stimulate and activate the PBMC cells extracted in step (1) for 24 h with 50 ng / mL CD3 antibody, 50 ng / mL CD28 antibody and 10 ng / mL IL2.

[0186] b. The stimulated and activated PBMC cells were added to the pre-treated 4T1 cells, and the mixture was then co-cultured at 37°C and 5% CO2 for 12 h.

[0187] 2.7 Pharmacokinetic Experiments

[0188] The pharmacokinetic experiments in this invention were commissioned to Chengdu Xiapaison Pharmaceutical Technology Co., Ltd. Male SD rats aged 6-8 weeks, purchased from Chengdu Dashuo Experimental Animal Co., Ltd., were used as experimental animals. The test compound PA8 was administered intravenously (iv) and intraperitoneally (ip) at single doses of 3 mg / kg and 30 mg / kg, respectively. During the experiment, the solvent for PA8 was DMSO:EtOH:Cre EL:Saline (5:10:10:75, v / v / v / v), and the specific dosing regimen is shown in the table below:

[0189] Table 1. Dosing regimens for pharmacokinetic analysis in PA8 rats

[0190] iv 3 3 1.5 2 IP 3 30 3 10

[0191] Note: All animals in the treatment groups were fasted overnight (>12h) before administration, but water was allowed; they were allowed to eat 4h after administration.

[0192] Test drug preparation

[0193] a. Intravenous administration: Add 0.097 mL of DMSO to a glass bottle containing 3.22 mg of the test substance PA8, mix well, then add 0.193 mL of EtoH, vortex mix, then add 0.193 mL of Cre EL, vortex mix, and finally add 1.449 mL of Saline, sonicate and vortex mix to obtain a clear drug solution with a concentration of 1.5 mg / mL.

[0194] b. Intraperitoneal administration: Add 0.413 mL of DMSO to a glass bottle containing 27.5 mg of the test substance PA8, mix well, then add 0.825 mL of EtoH, vortex mix, then add 0.825 mL of Cre EL, vortex mix, and finally add 6.187 mL of Saline, sonicate and vortex mix to obtain a suspension with a concentration of 3 mg / mL.

[0195] Sample collection

[0196] a. Data collection time points: 5 min, 15 min, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after drug administration.

[0197] b. Collection method: Blood was collected from rats via the jugular vein, with each sample containing approximately 0.20 mL. After anticoagulation with heparin sodium (the blood sample was placed in an ice bath after collection), the plasma was separated by centrifugation at 6000g for 5 min at 4℃. The plasma was stored at -70℃ for testing.

[0198] Sample processing

[0199] a. Except for the blank plasma sample, add 250 μL of internal standard working solution to a 96-well plate containing 25 μL of standard curve plasma sample, quality control plasma sample, or test plasma sample; add 250 μL of methanol to the blank sample; b. Vortex all plasma samples for about 2 min; c. After vortexing the plasma samples, centrifuge at 3800 rpm for 15 min; d. Take 200 μL of the supernatant after centrifugation and inject it into LC-MS / MS for analysis.

[0200] Animal disposal

[0201] After the experiment, all animals were euthanized in accordance with the relevant SOPs of Chengdu Xiapaison Pharmaceutical Technology Co., Ltd.

[0202] Results Analysis

[0203] The plasma concentration of PA8 was determined by LC-MS / MS with gliclazide as an internal standard; the main pharmacokinetic parameters of the test sample were calculated using a Winnolin 8.3 non-compartmental model: AUC 0-t AUC 0-∞ MRT 0-∞ C max T max and T 1 / 2 The values ​​and their averages and standard deviations are also considered. For samples with concentrations below the lower limit of quantitation, when calculating pharmacokinetic parameters, the C0 value is considered. max Previously sampled samples were calculated as zero values, and upon reaching C... max Subsequent sampling points will be sampled using the Unquantifiable (BLQ) method.

[0204] 2.8 In vivo anti-tumor proliferation experiment

[0205] Establishment of a BALB / c mouse subcutaneous xenograft model of 4T1 breast cancer cells.

[0206] a. Cell preparation: After collecting 4T1 cells in the logarithmic growth phase in a clean bench, wash the cells three times with serum-free and antibiotic-free RPMI 1640 basal medium. Then, resuspend the cells in an appropriate volume of serum-free and antibiotic-free RPMI 1640 basal medium, count the cells, and adjust the cell density to 1×10⁶ cells / year using serum-free medium. 7 per mL.

[0207] b. Cell inoculation: First, the hair on the right rib area of ​​BALB / c mice that have been acclimatized for 5-7 days was shaved to expose the skin. Then, 100 μL / mouse of the above-mentioned density-adjusted 4T1 cell suspension was inoculated subcutaneously into the right rib area of ​​female BALB / c mice to establish a subcutaneous tumor-bearing model. The tumor was allowed to grow until it reached 100 mm². 3 After being placed in the left and right positions, the mice were randomly divided into groups of 6.

[0208] Preparation and administration of the test drug

[0209] a. Preparation of test drugs: Compound PA8 was prepared using DMSO:EtOH:Cre EL:Saline (5:10:10:75, v / v / v / v) as the solvent, with the solvent added in the order of DMSO→EtOH→Cre EL→Saline; the positive control paclitaxel was prepared using Tween-80:DMSO:PEG-400:Saline (5:5:40:50, v / v / v / v) as the solvent, with the solvent added in the order of DMSO→PEG-400→Tween-80→Saline.

[0210] b. Groups and dosing regimens:

[0211] Table 2. In vivo experimental groupings and dosing regimens

[0212] 1 PA8 30 Intraperitoneal injection (ip) 15 2 PA8 60 Intraperitoneal injection (ip) 15 3 PA8+Paclitaxel 30+10 Intraperitoneal injection (ip) 15 4 Paclitaxel 10 Intraperitoneal injection (ip) 15 5 Saline / Intraperitoneal injection (ip) 15

[0213] Note: The dosage volume for each group was 100 μL / animal. The PA8 group was administered once daily for 15 consecutive days, while the PA8+Paclitaxel and Paclitaxel groups were administered every other day (considering the toxicity of Paclitaxel).

[0214] Recording and processing of experimental data

[0215] The health status of the experimental mice was observed daily, including their food and water intake, mental state, and presence of diarrhea. The weight and tumor volume of the mice were measured every two days. The long axis (a, mm) and the short axis (b, mm) perpendicular to the long axis were measured and recorded using calipers. Tumor volume and tumor inhibition rate were calculated using the following formulas:

[0216] Tumor volume V = a × b 2 / 2

[0217] Tumor growth inhibition rate TGI = [1-(T n -T0) / (C n -C0)]×100%

[0218] Among them, T n T0 and T0 represent the average tumor volume of each group before and after drug administration, respectively; C n C0 and C0 represent the average tumor volume of the blank control group before and after drug administration, respectively.

[0219] All data processing was performed using GraphpadPrism 8 for statistics and calculations.

[0220] Treatment of laboratory animals

[0221] After 15 days of drug administration, blood was first collected from mice in each group via orbital blood sampling for subsequent hematologic analysis. Following blood collection, subcutaneous tumor tissue, as well as the heart, liver, spleen, lungs, and kidneys of each experimental group of mice, were dissected using animal anatomical tools. These tissues and organs were then soaked in paraformaldehyde solution for further tissue sectioning and analysis. Finally, the mouse carcasses were sent to the Sichuan University Waste Laboratory Animal Disposal Center for processing.

[0222] 2.9 Immunofluorescence staining and H&E staining of tissue sections

[0223] Take tumor tissue or major organs fixed with 4% paraformaldehyde and proceed as follows:

[0224] Tissue processing. a. Dehydration and clearing: Rinse the tissue with tap water overnight, then soak it in the following sequence: 75% EtOH for 1 hour, 85% EtOH for 1 hour, 95% EtOH three times for 1 hour each time, 100% EtOH three times for 1 hour each time, and xylene twice for 1 hour each time. b. Embedding: Soak the tissue in primary, secondary, and tertiary paraffin waxes for 20 minutes each time, and finally immerse the tissue in melted fresh paraffin. c. Sectioning and spreading: After trimming the embedded tissue block, cut it into 5μm thick paraffin strips using a microtome. Spread the paraffin strips in 50℃ warm water, then lift and fix them onto a clean glass slide. d. Drying: Place the lifted paraffin strips in a 65℃ oven and dry for 3 hours.

[0225] Immunofluorescence staining

[0226] The immunofluorescence staining was outsourced to Wuhan Saiweier Biotechnology Co., Ltd., and the main steps included: a. desodiuming of the sections; b. antigen retrieval; c. serum blocking; d. primary antibody incubation; e. secondary antibody incubation.

[0227] H&E staining

[0228] The H&E staining was also outsourced to Wuhan Saiweier Biotechnology Co., Ltd., and the main steps included: a. section preparation; b. hematoxylin staining and eosin staining; c. section fixation; d. mounting with neutral resin, followed by observation and photography under a microscope.

[0229] II. Results Analysis

[0230] 1. In vitro activity study of PD-L1 PROTACs molecules

[0231] 1.1 Screening for degradation activity of PD-L1 PROTACs

[0232] Given that several studies on PD-L1 inhibition have been conducted in 4T1 tumor cells, and further research reports that BMS202-based nanoparticles can exhibit similar effects to PD-L1 antibodies in 4T1 tumor-bearing models, we selected 4T1 cells as our experimental subject for screening the degradation activity of PD-L1 PROTACs. First, we treated adherent 4T1 cells with a gradient concentration of PD-L1 PROTACs (1.25–20 μM) for 24 h. Then, we extracted total protein from each treatment group by cell lysis. Finally, we performed Western blotting experiments on the pretreated total protein to investigate its degradation level of PD-L1 protein in 4T1 cells. The experimental results are as follows: Figures 1-4 As shown.

[0233] Depend on Figures 1-4 It was found that compounds PA6, PA7, and PA8 in the A series exhibited certain degradation effects on PD-L1 protein. Among them, PA8, composed of a 9-carbon straight-chain alkane linking BMS202 and PG, still showed excellent degradation effects at a concentration of 1.25 μM. In contrast, only PB4, PB5, and PB7 of the other series showed weak degradation effects at high concentrations. Therefore, we selected PA8 as the preferred molecule for further activity verification and mechanism exploration.

[0234] To pinpoint the optimal degradation concentration of PA8, the preferred molecule obtained from the above screening, we further explored its ability to degrade PD-L1 in 4T1 tumor cells by expanding the dosage concentration gradient. The results are as follows: Figure 5 As shown, PA8 exhibited degradation of PD-L1 protein in 4T1 cells at concentrations ranging from 0.078 to 2.5 μM. Its degradation of PD-L1 protein by DCs... 50 The concentration was 0.609 μM. Its degradation activity gradually increased with increasing drug concentration from 0.078 μM to 0.312 μM, reaching its maximum degradation efficiency (D0) at 0.312 μM. max =85%), but its activity against PD-L1 protein degradation gradually decreased when the dosage was increased from 0.312 μM onwards.

[0235] To verify whether replacing conventional CRBN-type E3 ubiquitination ligands with the newly reported E3 ubiquitination ligand structure PG is more effective, we designed and synthesized a control molecule, PA8B, with pomalidomide replacing PG, based on the structure of the aforementioned preferred compound PA8. We then investigated its degradation ability of PD-L1 protein in 4T1 tumor cells using gradient concentration administration. The results are as follows: Figure 6 As shown, the control molecule PA8B also has a certain ability to degrade PD-L1 protein in 4T1 tumor cells, but its optimal effective degradation concentration is higher than that of PA8 (0.312 μM), mainly concentrated in 5-10 μM. Therefore, it can be concluded that the preferred molecule in this study is PA8 with PG instead of pomalidomide at the E3 ubiquitination ligand end, which has a better degradation effect.

[0236] Furthermore, based on the screening results of the aforementioned study, we further investigated the time dependence of the preferred molecule PA8 on the degradation of PD-L1 protein in 4T1 cells at the optimal degradation concentration. The results are as follows: Figure 7As shown, significant degradation of PD-L1 protein was observed in 4T1 cells after treatment with 0.312 μM PA8 for 16 h, with maximum degradation observed at 24 h, and continued degradation capacity even after 48 h. This indicates that PA8 can rapidly degrade PD-L1 protein in 4T1 cells, and its degradation capacity is sustainable.

[0237] 1.2 Degradation mechanism and efficacy verification of PA8

[0238] To verify whether compound PA8 exerts its degradation effect on PD-L1 through a ubiquitin-protease system, we continued to use 4T1 tumor cells as the degradation target. We explored its degradation mechanism by knocking down CRBN expression with siCRBN and by using the protease inhibitor MG132 in combination with PA8. The experimental results are as follows: Figure 8 As shown.

[0239] Depend on Figure 8 As shown in Figure A, when siCRBN (25 nM) was used alone, CRBN protein was significantly degraded but had no effect on PD-L1 protein. When PA8 was used alone, the PD-L1 protein content in 4T1 cells was significantly reduced; however, when siCRBN and PA8 were used in combination, CRBN protein expression was still suppressed, while PD-L1 protein did not show degradation. Similarly, from... Figure 8 As shown in Figure B, when MG132 and PA8 are used in combination, the degradation effect of PA8 on PD-L1 also disappears. Therefore, it is not difficult to conclude that the degradation effect of PA8 on PD-L1 protein in 4T1 cells depends on the ubiquitin-protease system, which is a PD-L1 PROTAC that can simultaneously bind PD-L1 protein and E3 ligase.

[0240] To further verify the PD-L1 degradation effect of PA8, the preferred compound screened by Western blotting, flow cytometry and immunofluorescence analysis were used to analyze the degradation effect of PA8 on PD-L1 protein in 4T1 cells. Cells were treated with high and low concentrations of PA8 for 24 h, then collected, washed three times with PBS, blocked with 0.1% BSA for 30 min, and stained with FAM-labeled PD-L1 antibody (dilution 1:200) for 30 min. Finally, after washing three times with PBS, the number of FAM-positive cells was analyzed by flow cytometry. The experimental results are shown below. Figure 9As shown, both the high and low concentration groups had fewer FAM-positive cells than the control group, and the high concentration PA8 group had even fewer FAM-positive cells than the low concentration group. 4T1 cell smears were prepared, and after incubation with the optimal degradation concentration of PA8 (0.312 μM) for 24 hours, the changes in PD-L1 content on the cell membrane were analyzed using immunofluorescence staining with PD-L1 antibody. The experimental results are shown below. Figure 10 As shown, the PA8-treated group not only exhibited significantly weaker green fluorescence of PD-L1 on the cell membrane compared to the control group, but also showed relatively weaker green fluorescence of PD-L1 in the cell nucleus. Therefore, these results further demonstrate that compound PA8 is indeed a molecule that can effectively degrade PD-L1 protein in 4T1 cells. It can reduce not only the level of PD-L1 in the cell nucleus but also the level of PD-L1 located on the cell membrane. The reason for this effect may be that PA8 preferentially degrades the level of PD-L1 in the cell nucleus, leading to a reduction in the amount of PD-L1 transported from the cell nucleus to the cell membrane, and consequently, a relative reduction in the PD-L1 content at the cell membrane.

[0241] 1.3 Investigation of the in vitro antitumor activity of PA8

[0242] Since PD-L1 proteins themselves do not possess the ability to kill tumor cells, their possible pathway is to inhibit the PD-1 / PD-L1 signaling axis by degrading PD-L1 in tumor cells, thereby enhancing the killing effect of immune cells on tumor cells. Therefore, the adjuvant antitumor ability of PA8 was investigated using a co-culture model of PBMC cells and tumor cells. Specifically, PBMC cells were first isolated from the blood of 6-8 week old BALB / c mice and activated by stimulation with 50 ng / ml CD3 antibody, 50 ng / ml CD28 antibody, and 10 ng / ml IL2. The activated PBMC cells were then added to 4T1 cells pre-treated with 0.312 μM PA8 and co-cultured at 37°C and 5% CO2 for 12 h. Finally, the culture medium was collected, and the level of LDH released into the supernatant was detected using a lactate dehydrogenase (LDH) cytotoxicity assay kit (leagene) to assess and quantify the degree of cell damage. The experimental results are as follows: Figure 11As shown, in the control group without PBMC cells, the number of surviving 4T1 cells was slightly reduced in the BMS202 and PA8 treatment groups compared to the DMSO group, indicating that both have a certain degree of cytotoxicity. In the experimental groups co-cultured with PBMC cells and 4T1 cells, firstly, because PBMC cells themselves have the ability to kill tumor cells, the number of 4T1 cells in each treatment group was reduced compared to the experimental group without PBMC cells. More importantly, in the co-culture group, the 4T1 cell mortality rate in the PA8 treatment group was significantly increased compared to the DMSO and BMS202 treatment groups, reaching 71.2%. This suggests that the PD-L1 PROTAC molecule PA8 can significantly enhance the tumor-killing ability of PBMC cells.

[0243] 1.4 Preliminary in vitro safety study of PA8

[0244] The toxicity of the PD-L1 protacoder molecule PA8 and the PD-L1 inhibitor BMS202 within the PA8 structure to normal cells was assessed and compared using the MTT assay. Results are as follows: Figure 12 As shown, PA8 molecules affect the IC50 of 293T cells. 50 The value was 28.03 μM, and the IC50 of BMS202 against 293T cells was [value missing]. 50 The value was 21.47 μM. The above data indicates that both have relatively high safety. Moreover, the fact that PA8 is slightly higher than BMS202 may also confirm that the toxicity of BMS202 is further reduced after PROTACs than that of the original drug.

[0245] 2. In vivo activity study of PD-L1 PROTACs molecules

[0246] 2.1 Evaluation of the in vivo antitumor activity of PA8

[0247] In vitro experiments have fully demonstrated that the optimized PD-L1 protein molecule PA8 can effectively degrade the PD-L1 protein level in mouse breast cancer cells (4T1). Furthermore, co-culture experiments using PBMC cells and 4T1 cells have also confirmed that PA8 can effectively promote the killing effect of PBMC cells on tumor cells, exhibiting a certain in vitro anti-tumor effect. Based on this, the in vivo anti-tumor effect of PA8 was further explored. Firstly, a subcutaneous tumor-bearing model was established by inoculating 4T1 tumor cells into the right rib skin of BALB / c mice. The tumor volume was increased until it reached 100 mm². 3After treatment, tumor-bearing mice were randomly divided into 5 groups of 6 mice each. These included a saline group (100 μL, ip), a high-dose PA8 group (60 mg / kg, ip), a low-dose PA8 group (30 mg / kg, ip), and a positive control paclitaxel group (10 mg / kg, ip). In addition, considering the common clinical use of PD-L1 inhibitors in combination with paclitaxel for breast cancer treatment, a PA8 + paclitaxel combination therapy group (30 mg / kg + 10 mg / kg, ip) was also included. The dosage for each group was 100 μL per mouse. The PA8 group received the drug once daily for 15 consecutive days, while the PA8 + paclitaxel and paclitaxel groups received the drug every other day (considering the toxicity of paclitaxel), for a total of 15 days. During the treatment period, the health status of the mice was observed daily, including food and water intake, mental state, and the presence of diarrhea. The body weight and tumor volume of the mice were measured every two days.

[0248] Changes in body weight and tumor volume in mice during drug administration are as follows: Figure 13 As shown in Figure A, the mice in the paclitaxel group and the combination therapy group showed almost no increase in body weight, while the mice in both PA8 administration groups and the saline group showed a slight increase in body weight, indicating that PA8 has a certain degree of safety in vivo. The lack of significant weight gain in the two groups may be due to the inherent toxicity of paclitaxel. Regarding changes in tumor volume ( Figure 13 B) It can be seen that, except for the control group, all other treatment groups showed certain anti-tumor effects, and the order of anti-tumor activity was PA8+Paclitaxel group > PA8 high-dose group > Paclitaxel group > PA8 low-dose group. Among them, the tumor inhibition rate of PA8 high-dose group was 83.23%, the tumor inhibition rate of PA8 low-dose group was 75.04%, and the tumor inhibition rate of PA8+Paclitaxel group was 86.74%.

[0249] Fifteen days after administration, all experimental mice were euthanized by cervical dislocation. Subcutaneous tumor tissue was then removed from each group of mice using animal experimental surgical tools, and the tissue was weighed and photographed. Figure 14 As shown, tumor weight in all PA8-treated groups was significantly reduced compared to the control group. Specifically, the PA8 30 mg / kg group showed a 70.4% reduction in tumor weight, the PA8 60 mg / kg group showed a 74.5% reduction, and the group treated with PA8 30 mg / kg in combination with Paclitaxel 10 mg / kg showed an 83.2% reduction. Therefore, based on the above data, it is easy to conclude that the PD-L1 PROTAC molecule PA8 not only has significant anti-tumor effects when used alone, but also exhibits a more synergistic anti-tumor effect when used in combination with the conventional chemotherapy drug paclitaxel.

[0250] 2.2 Study on the in vivo antitumor mechanism of PA8

[0251] Tumor tissues from each group were further paraffin-embedded and sectioned. Immunofluorescence staining for PD-L1, CD4, and CD8 was performed on sections from the PA830 mg / kg and PA860 mg / kg groups, as well as the blank control group. Furthermore, total RNA was extracted from tumor tissues of the PA860 mg / kg and blank control groups, and the expression levels of immune-related genes were assessed using qRT-PCR. Figure 15 As shown in Figure A, the immunofluorescence intensity of PD-L1 in tumor tissue sections from both high- and low-dose PA8 groups was significantly weaker than that in the blank control group. Furthermore, the immunofluorescence of PD-L1 in the high-dose PA8 group was also slightly weaker than that in the low-dose group. This indicates that the selected preferred PD-L1 PROTAC molecule, PA8, not only effectively reduced PD-L1 levels in subcutaneous tumor-bearing models in in vivo experiments but also exhibited a certain dose-dependent effect. Figure 15 As shown in B and 15C, the immunofluorescence staining results of CD4 and CD8 in tumor tissue sections showed that the expression levels of CD4 and CD8 in both PA8-treated groups were higher than those in the control group, with the high-dose group showing higher levels than the low-dose group. Overall, PA8 showed a stronger effect on increasing CD8 levels than on CD4. However, as... Figure 15 As shown in Figure D, qRT-PCR experiments on total RNA from tumor tissues in the high-dose PA8 group and the blank control group revealed that the levels of immune-related factors GzmB, Prf1, and IFN-γ in the high-dose PA8 group were significantly higher than those in the blank control group. These experimental results indicate that: firstly, the PD-L1 PROTAC molecule PA8 can effectively reduce the PD-L1 protein level in tumor tissues; secondly, by effectively reducing the PD-L1 level in tumor tissues, it can effectively enhance the expression of related immune factors in the tumor tissue microenvironment, effectively "relaxing the brakes" on immune checkpoints and enhancing the body's own immune system's ability to kill tumor tissues.

[0252] 2.3 Preliminary in vivo safety evaluation of PA8

[0253] While studying the in vivo antitumor proliferation effect of PA8, a preliminary evaluation of its in vivo safety was also conducted. Firstly, no mice in any of the administration groups died during the administration period, and their weight, food and water intake, and mental status remained normal. Secondly, after administration, blood was collected from each group of mice via orbital sampling, and relevant blood phase analyses were performed. The final results of blood routine and blood biochemistry tests showed that all indicators in the PA8 administration groups were normal compared to the blank control group, and there were no significant differences between the groups. Furthermore, after administration, the mice in each group were dissected, and the heart, liver, spleen, lungs, and kidneys were dissected. These organs were further paraffin-embedded, sectioned, and stained with H&E. The internal organ structures of all groups of mice were normal, with no obvious lesions or congestion. In conclusion, the PD-L1 PROTAC molecule PA8 had no significant effect on the main health indicators of experimental mice, demonstrating good in vivo safety.

[0254] 3. Pharmacokinetics of PA8, a PD-L1 protacoder molecule

[0255] Based on the excellent degradation activity and antitumor effects of the PD-L1 protacoder PA8 in vitro and in vivo, we further explored its pharmacokinetics. Using 6-8 week old male SD rats as experimental animals, the test compound PA8 was administered intravenously (iv) and intraperitoneally (ip) at single doses of 3 mg / kg and 30 mg / kg, respectively. Blood samples (approximately 0.20 mL per sample) were collected from the jugular vein of the rats at 5 min, 15 min, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after administration. Finally, 200 μL of the pretreated supernatant was analyzed by LC-MS / MS to determine the plasma concentration of PA8 at each blood collection time point. The pharmacokinetic curves for each administration group are shown below. Figure 16 As shown, the plasma concentration of the PD-L1 protacoder PA8 decreased rapidly within 0.5 hours after intravenous administration, and fell below the lower limit of quantitation (LQ) of 2.00 ng / mL after 4 hours, indicating poor stability in plasma. However, with intraperitoneal administration, we detected that it reached its maximum plasma concentration in approximately 2 hours, and the plasma concentration remained above 60.00 ng / mL at 8 hours, demonstrating that intraperitoneal administration of PA8 is more conducive to maintaining its efficacy in the body for a longer period than intravenous injection.

[0256] Furthermore, based on the blood drug concentration measurements at the aforementioned time points, we calculated the main pharmacokinetic parameters of PA8 for intravenous and intraperitoneal administration using the Winnolin 8.3 non-compartmental model. Specifically, for PA8 administered intravenously, the elimination half-life T0 was calculated.1 / 2 The time curve area under the curve (AUC) is 0.952 ± 0.239 h. 0-inf The elimination half-life (T0) was 112 ± 32.3 h*ng / mL; when PA8 was administered intraperitoneally, the elimination half-life was T0. 1 / 2 The peak time was 5.30 ± 0.153 h, T. max The area under the time curve (AUC) is 2.0 ± 0.210 h. 0-inf The concentration was 1706±182 h*ng / mL; its bioavailability F=71.2±3.9%. These results indicate that it has good drug-like properties, laying a foundation for its further research.

[0257] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A PD-L1 PROTACs compound, characterized in that, Its general structural formula is: As shown: ； Mode ; The Linker structure is shown in equation (1): Equation (1) is , where n1 is any integer from 6 to 8.

2. The PD-L1 PROTACs compound according to claim 1, characterized in that, The structural formula of the PD-L1 PROTACs compound is as follows: .

3. The use of the PD-L1 PROTACs compound according to claim 1 or 2 in the preparation of antitumor drugs.

4. An antitumor drug, characterized in that, Including the PD-L1 PROTACs compound as described in claim 1 or 2.

5. An antitumor drug according to claim 4, characterized in that, The anti-tumor drug is a PD-L1 targeted anti-tumor drug.

6. The antitumor drug according to claim 4, characterized in that, It also includes paclitaxel.

7. The antitumor drug according to claim 6, characterized in that, The mass ratio of paclitaxel to PD-L1 PROTACs compounds is 5-10:30-60.

8. An antitumor drug according to claim 4, characterized in that, The structural formula of the PD-L1 PROTACs compound is as follows: 。

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