PD-L1 protein degradation targeting chimera as well as preparation method and application thereof

Abstract

The invention provides a PD-L1 protein degradation targeting chimera as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The protein degradation targeting chimera of PD-L1 provided by the invention is a PROTAC molecule based on an isoindoline structure, and the molecular weight of a warhead part of the PROTAC molecule is only 382.5070. The PD-L1 protein degradation targeting chimera provided by the invention has high PD-L1 protein degradation activity, and has excellent tumor treatment potential. In addition, the preparation method of the PD-L1 protein degradation targeting chimera is short in synthetic route, low in cost and easy to realize industrial production.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a PD-L1 protein degradation targeting chimera, its preparation method, and its application. Background Technology

[0002] Immune checkpoint (IC) pathways negatively regulate T cell activation pathways to prevent excessive autoimmunity and maintain immune homeostasis. However, the expression of ICs and their ligands in the tumor microenvironment is often upregulated, becoming a substantial barrier to initiating an effective anti-tumor immune response. ICIs aim to restore anti-tumor immune responses by blocking co-inhibitory signaling pathways. The most widely used targets are cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), programmed cell death receptor-1 (PD-1 or CD279), and programmed cell death ligand-1 (PD-L1 or CD274 or B7 homolog 1). Among these, PD-L1 is overexpressed on the surface of malignant tumor cells, and its binding mediates tumor immune escape, which is an important reason for cancer treatment failure. Therefore, ICIs targeting PD-1 / PD-L1 have significant clinical value. PD-L1 inhibitors have shown clinical efficacy in the treatment of many cancers. Currently approved PD-L1 antibody drugs (such as atezolizumab and durvalumab) have shown good therapeutic effects in the treatment of melanoma, non-small cell lung cancer, and urothelial carcinoma.

[0003] However, PD-L1 monoclonal antibody drugs have drawbacks such as long half-life, poor oral bioavailability and tissue penetration, adverse immune reactions, high cost, and difficult storage. Currently, there are no small molecule inhibitors targeting PD-L1 available on the market. Therefore, in recent years, a large amount of basic research has been dedicated to developing small molecule inhibitors targeting PD-L1. However, small molecule inhibitors using the "site-driven" mechanism still suffer from problems such as high oral effective doses, short half-lives, increased toxicity and side effects, and drug resistance. Many studies have shown that protein degradation has better anti-cancer activity than protein regulation. Proteolysis-targeting chimera (PROTAC) molecules are heterobifunctional molecules that exert their effects by having ligands at both ends simultaneously bind to the target protein and E3 ubiquitin ligase. This drives the ubiquitination of exposed lysine residues on the target protein by the E3 ubiquitin ligase complex. After polyubiquitination, the target protein is recognized and degraded by the proteasome. PROTAC molecules degrade the target protein through an event-driven mechanism (MOA), achieving transient binding and elimination of the target protein, and the molecules are reusable. This eliminates the need for high concentrations of drugs to tightly bind to the active site of the target protein. Therefore, PROTAC-based PD-L1 degraders have unique advantages such as reduced toxicity, overcoming drug resistance, expanding the therapeutic window, and eliminating the entire protein.

[0004] Current limited research is based on a series of PD-L1 inhibitors developed by BMS, whose protein targeting warheads alone have a molecular weight exceeding 420. The synthesis of the entire PROTAC molecule involves more steps, resulting in a larger molecular weight. Only a few molecules exhibit good in vitro PD-L1 degradation activity. For example, Cheng et al. developed a novel PROTAC molecule based on the PD-L1 inhibitor BMS-1198 (molecular weight 638.1450), which reduced PD-L1 expression in MDA-MB-231 cells by 21% and 35% in vitro at 1 mM and 10 mM, respectively. Wang et al. developed a PD-L1 PROTAC degradation molecule based on a derivative of BMS-37 (molecular weight 448.5630), which reduced PD-L1 expression in MC38 cells by approximately 30% in vitro at a concentration of 5 μM. Liu et al. reported a PROTAC molecule based on BMS-37 (molecular weight 448.5630), BMS-37-C3. In in vitro experiments, 1 μM BMS-37-C3 reduced PD-L1 expression on the surface of A375 cells to 38.7%; Li et al. disclosed a PROTAC molecule based on BMS-202 (molecular weight 419.5250), and in in vitro experiments, PA8 (molecular weight 777.9630) achieved the maximum degradation efficiency of PD-L1 protein in 4T1 cells at 0.312 μM (D max=85%); Wang et al. disclosed a PROTAC molecule based on BMS1001 (molecular weight 594.6640), in which 5 μM concentration of CL-F-B1 (933.0310) reduced the expression of PD-L1 on the surface of MC38 cells by 67.55%.

[0005] Therefore, there is an urgent need for compounds with relatively small molecular weights and simple synthesis to enrich the relevant compound library, further explore structure-activity relationships, and lay the foundation for finding PROTAC-based PD-L1 degraders with better activity and lower cost. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a PD-L1 protein degradation targeting chimera, its preparation method, and its application. The PD-L1 protein degradation targeting chimera provided by this invention has a small molecular weight and high PD-L1 protein degradation activity.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a PD-L1 protein degradation-targeting chimera with the structure shown in Formula I: Formula I.

[0008] This invention provides a method for preparing the above-mentioned PD-L1 protein degradation-targeting chimera, comprising the following steps: In the presence of a condensing agent, a compound having the structure shown in Formula P-C7 undergoes a first condensation reaction with a compound having the structure shown in Formula P-C4 to obtain a protein degradation-targeting chimera of PD-L1 having the structure shown in Formula I. Formula P-C7; Formula P-C4.

[0009] Preferably, the molar ratio of the compound having the structure shown in formula P-C7 to the compound having the structure shown in formula P-C4 is 1 to 1.1:1.

[0010] Preferably, the condensing agent is HOBt and EDC; The temperature of the first condensation reaction is 20~30℃, and the time is 10~15 h.

[0011] Preferably, the method for preparing the compound having the structure shown in formula P-C7 includes the following steps: In the presence of an organic base and a catalyst, a compound having the structure shown in formula P-C1 undergoes a Suzuki coupling reaction with a compound having the structure shown in formula P-C2 to obtain a compound having the structure shown in formula P-C3. The compound having the structure shown in formula P-C3 undergoes a deprotection reaction to obtain the compound having the structure shown in formula P-C5. In the presence of a condensing agent, a compound having the structure shown in formula P-C5 undergoes a second condensation reaction with Boc-L-proline to obtain a compound having the structure shown in formula P-C6. The compound having the structure shown in formula P-C6 undergoes a deprotection reaction to obtain the compound having the structure shown in formula P-C7. Formula P-C1; Formula P-C2; Formula P-C3; Formula P-C5; Formula P-C6.

[0012] Preferably, the organic base comprises potassium acetate, and the catalyst comprises a Pd(II) catalyst; The Suzuki coupling reaction was carried out at a temperature of 98-105°C for 8-12 hours.

[0013] Preferably, the method for preparing the compound having the structure shown in formula P-C4 includes the following steps: Glutaric anhydride reacts with pomalidomide in a third condensation reaction to give a compound having the structure shown in formula P-C4.

[0014] Preferably, the molar ratio of glutaric anhydride to pomalidomide is 15~30:1; The third condensation reaction is carried out at a temperature of 65-90°C for 18-25 hours.

[0015] This invention provides the application of the above-mentioned protein degradation targeting chimera in the preparation of PD-L1 protein degrading agents.

[0016] This invention provides the application of the above-mentioned PD-L1 protein degradation targeting chimera in the preparation of antitumor drugs.

[0017] This invention provides a PD-L1 protein degradation targeting chimera with the structure shown in Formula I (denoted as P-C8). The PD-L1 protein degradation targeting chimera provided by this invention is a PROTAC molecule based on an isoindoline structure, with a warhead molecular weight of only 382.5070. The PD-L1 protein degradation targeting chimera provided by this invention exhibits high PD-L1 protein degradation activity and possesses excellent potential for tumor therapy.

[0018] This invention provides a method for preparing the above-mentioned PD-L1 protein degradation targeting chimera. The preparation method provided by this invention has a short synthetic route, low cost, and is easy to realize industrial production. Attached Figure Description

[0019] Figure 1 Synthetic route for P-C8, the protein degradation-targeting chimera of PD-L1; Figure 2 The hydrogen NMR spectrum of P-C8; Figure 3 The hydrogen NMR spectrum of P-C8; Figure 4 Western blot (WB) results of PD-L1 protein degradation by different concentrations of P-C8 over 48 hours; Figure 5 This is a grayscale analysis chart of the WB bands. Detailed Implementation

[0020] This invention provides a PD-L1 protein degradation-targeting chimera with the structure shown in Formula I: Formula I.

[0021] This invention provides a method for preparing the above-mentioned PD-L1 protein degradation-targeting chimera, comprising the following steps: In the presence of a condensing agent, a compound having the structure shown in Formula P-C7 undergoes a first condensation reaction with a compound having the structure shown in Formula P-C4 to obtain a protein degradation-targeting chimera of PD-L1 having the structure shown in Formula I. Formula P-C7; Formula P-C4.

[0022] In this invention, the method for preparing a compound having the structure shown in formula P-C7 preferably includes the following steps: In the presence of an organic base and a catalyst, a compound having the structure shown in formula P-C1 undergoes a Suzuki coupling reaction with a compound having the structure shown in formula P-C2 to obtain a compound having the structure shown in formula P-C3. The compound having the structure shown in formula P-C3 undergoes a deprotection reaction to obtain the compound having the structure shown in formula P-C5. In the presence of a condensing agent, a compound having the structure shown in formula P-C5 undergoes a second condensation reaction with Boc-L-proline to obtain a compound having the structure shown in formula P-C6. The compound having the structure shown in formula P-C6 undergoes a deprotection reaction to obtain the compound having the structure shown in formula P-C7. Formula P-C1; Formula P-C2; Formula P-C3; Formula P-C5; Formula P-C6.

[0023] In this invention, in the presence of an organic base and a catalyst, a compound having the structure shown in formula P-C1 undergoes a Suzuki coupling reaction with a compound having the structure shown in formula P-C2 to obtain a compound having the structure shown in formula P-C3. In this invention, the molar ratio of the compound having the structure shown in formula P-C1 to the compound having the structure shown in formula P-C2 is preferably 1.05 to 1.25:1, more preferably 1.2:1; the organic base preferably includes potassium acetate, and the molar ratio of the organic base to the compound having the structure shown in formula P-C2 is preferably 3.5 to 4.5:1, more preferably 4:1; the catalyst preferably includes a Pd(II) catalyst, more preferably a tetratetraphenylphosphine palladium, palladium dichloride and 1,1'-bis(diphenylphosphine)ferrocene palladium(II) dichloromethane complex, further preferably a 1,1'-bis(diphenylphosphine)ferrocene palladium(II) dichloromethane complex, and the molar ratio of the compound having the structure shown in formula PC to the catalyst is preferably 1:0.05 to 0.2, more preferably 1:0.1.

[0024] In this invention, the organic solvent used in the Suzuki coupling reaction is preferably dioxane; the Suzuki coupling reaction is preferably carried out under nitrogen protection; the temperature of the Suzuki coupling reaction is preferably 98~105℃, more preferably 100℃; and the time is preferably 8~12 h, more preferably 10 h.

[0025] Following the Suzuki coupling reaction, the present invention preferably performs post-treatment on the obtained Suzuki coupling reaction solution, the post-treatment preferably including the following steps: The Suzuki coupling reaction solution was concentrated under reduced pressure, extracted, dried, filtered, and purified by column chromatography.

[0026] In this invention, the extraction method is preferably to use dichloromethane to redissolve the reaction product after vacuum concentration, followed by water extraction; the extraction is preferably performed three times, and the dichloromethane layers are combined after extraction. The drying method is preferably anhydrous Na2SO4 drying; the eluent for column chromatography purification is preferably petroleum ether (PE) and ethyl acetate (EA), with a preferred volume ratio of PE to ethyl acetate of 15:1.

[0027] In this invention, a compound having the structure shown in formula P-C3 undergoes a deprotection reaction to obtain a compound having the structure shown in formula P-C5. In this invention, the deprotection reagent used in the deprotection reaction is preferably trifluoroacetic acid; the deprotection reaction is preferably carried out in an organic solvent, preferably dichloromethane. In this invention, the temperature of the deprotection reaction is preferably room temperature, and the time is preferably 35-70 min, more preferably 40-50 min.

[0028] Following the Boc deprotection reaction, the present invention preferably performs post-treatment on the resulting Boc deprotection reaction solution, which preferably includes the following steps: The pH of the Boc-protected reaction solution was adjusted to 8, and extraction and vacuum concentration were performed.

[0029] In this invention, the alkaline reagent used to adjust the pH value is preferably a saturated sodium bicarbonate solution; the extractant used for extraction is preferably dichloromethane.

[0030] In the presence of a condensing agent, this invention involves a second condensation reaction between a compound having the structure shown in formula P-C5 and Boc-L-proline to obtain a compound having the structure shown in formula P-C6. Prior to the reaction, the pH of the compound having the structure shown in formula P-C5 is preferably adjusted to 8-10, more preferably 8-9, and the reagent used for pH adjustment is preferably N-methylmorpholine. The molar ratio of the compound having the structure shown in formula P-C5 to Boc-L-proline is preferably 1:1.1-1.3. The condensing agent preferably includes HOBt and EDC, the molar ratio of HOBt to EDC is preferably 1-1.1:1, more preferably 1:1, and the molar ratio of HOBt to Boc-L-proline is preferably 1.08-1.25:1.

[0031] In this invention, the organic solvent used in the second condensation reaction is preferably tetrahydrofuran; the temperature of the second condensation reaction is preferably room temperature, and the time is preferably overnight.

[0032] After the second condensation reaction, the present invention preferably performs post-treatment on the obtained second condensation reaction solution, the post-treatment preferably including the following steps: The second condensation reaction solution was filtered, concentrated under reduced pressure, washed, dried, and purified by column chromatography.

[0033] In this invention, the washing method is preferably as follows: the product concentrated under reduced pressure is dissolved in ethyl acetate, and then washed three times each with saturated NaHCO3 solution, saturated NaCl solution, saturated KHSO4 solution, saturated NaCl solution, saturated NaHCO3 solution, and saturated NaCl solution. In this invention, the drying is preferably performed with anhydrous Na2SO4; the eluent for column chromatography purification is preferably petroleum ether (PE) and ethyl acetate (EA), and the volume ratio of petroleum ether to ethyl acetate is preferably 2:1.

[0034] In this invention, a compound having the structure shown in formula P-C6 undergoes a deprotection reaction to obtain a compound having the structure shown in formula P-C7. In this invention, the deprotection reagent used in the deprotection reaction is preferably trifluoroacetic acid; the deprotection reaction is preferably carried out in an organic solvent, preferably dichloromethane. In this invention, the temperature of the deprotection reaction is preferably room temperature, and the time is preferably 35-70 min, more preferably 40-50 min.

[0035] After the Boc deprotection reaction, the present invention preferably dissolves the resulting Boc deprotection reaction solution in ethyl acetate and adds hydrochloric acid to precipitate a solid.

[0036] In this invention, the method for preparing the compound having the structure shown in formula P-C4 preferably includes the following steps: Glutaric anhydride reacts with pomalidomide in a third condensation reaction to give a compound having the structure shown in formula P-C4.

[0037] In this invention, the molar ratio of glutaric anhydride to pomalidomide is preferably 15-30:1, more preferably 16.7:1; the temperature of the third condensation reaction is preferably 65-90°C, more preferably 80-90°C; and the time is preferably 18-25 h, more preferably 20 h.

[0038] Following the third condensation reaction, the present invention preferably performs post-processing on the obtained third condensation reaction product, the post-processing preferably including the following steps: The product of the third condensation reaction was cooled and dissolved in ethyl acetate, followed by extraction and column chromatography purification.

[0039] In this invention, the reagent used for extraction is preferably water; the eluent used for column chromatography purification is preferably dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is preferably 20:1.

[0040] In this invention, a compound having the structure shown in formula P-C7 and a compound having the structure shown in formula P-C4 undergo a first condensation reaction in the presence of a condensing agent to obtain a protein degradation-targeting chimera of PD-L1 having the structure shown in formula I. In this invention, the molar ratio of the compound having the structure shown in formula P-C7 to the compound having the structure shown in formula P-C4 is preferably 1 to 1.1:1, more preferably 1:1. Before the first condensation reaction, the pH value of the compound having the structure shown in formula P-C7 is preferably adjusted to 8 to 10, more preferably 8 to 9. The reagent used to adjust the pH value is preferably N-methylmorpholine. In this invention, the condensing agent is preferably HOBt and EDC, the molar ratio of HOBt to EDC is preferably 1 to 1.1:1, more preferably 1:1, and the molar ratio of HOBt to the compound having the structure shown in formula P-C4 is preferably 1.18 to 1.22:1, more preferably 1.21:1. In this invention, the organic solvent used in the first condensation reaction is preferably tetrahydrofuran; the temperature of the first condensation reaction is preferably 20~30°C, more preferably room temperature, and the time is preferably 10~15 h, more preferably overnight.

[0041] Following the first condensation reaction, the present invention preferably performs post-processing on the obtained first condensation reaction product, the post-processing preferably including the following steps: The first condensation reaction product was purified by filtration, vacuum concentration, extraction, and column chromatography.

[0042] In this invention, the reagents used for extraction are preferably EA and H2O; the eluents used for column chromatography separation are preferably dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is preferably 25:1.

[0043] In this invention, the synthetic route of the PD-L1 protein degradation-targeting chimeric compound (P-C8) is as follows: Figure 1 As shown, Figure 1 In the following samples, (a) AcOK, Pd(II), dioxane, 100℃; (b) AcOK, Pd(II), dioxane, 100℃; (c) AcOK, Pd(II), dioxane, 100℃; (d) TFA, DCM, rt; (e) Boc-L-Pro, EDC, HOBt, THF, rt; (f) TFA, DCM, rt; (g) glutaric anhydride, 80℃; (h) EDC, HOBt, THF, rt.

[0044] This invention uses phenylboronic acid, 1,3-dibromo-2-methylbenzene, 5-bromoisoindoline-2-tert-butyl carbonate, and bis(pinacol)diboron as raw materials, and obtains compounds P-C1, P-C2, and P-C3 sequentially via Suzuki coupling. P-C3 undergoes debonding of Boc protection via trifluoroacetic acid (TFA) to generate P-C5, which then condenses with Boc-L-proline and debonds the Boc protecting group to yield P-C7. This invention also uses pomalidomide and glutaric anhydride as raw materials, undergoing a condensation reaction to obtain P-C4, which is then condensed with P-C7 to produce the target compound P-C8.

[0045] This invention provides the application of the above-mentioned PD-L1 protein degradation targeting chimera in the preparation of PD-L1 protein degrading agents.

[0046] This invention provides the application of the above-mentioned PD-L1 protein degradation-targeting chimera in the preparation of antitumor drugs. In this invention, the tumor is preferably one or more of lung cancer, liver cancer, colorectal cancer, breast cancer, and melanoma.

[0047] The following detailed description, in conjunction with embodiments, illustrates the PD-L1 protein degradation targeting chimera provided by the present invention, its preparation method, and its application. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0048] Example 1: Synthesis of P-C8, a protein degradation-targeting chimera of PD-L1 according to Figure 1 The route for synthesizing P-C8 is as follows: (1) Synthesis of compound P-C1 Phenylated boric acid (1.00 g, 8.20 mmol) was dissolved in 15 mL of dioxane, and 1,3-dibromo-2-methylbenzene (4.10 g, 16.4 mmol) and potassium acetate (3.20 g, 32.7 mmol) were added. The system was protected with nitrogen and heated to 100 °C before adding Pd(II) catalyst (334 mg, 0.409 mmol). The reaction was carried out at 100 °C for 8 h. TLC monitoring showed the disappearance of the reactants (PE). The reaction solution was cooled and concentrated under reduced pressure to remove dioxane. The solution was reconstituted with dichloromethane and extracted three times with water. The dichloromethane layers were combined, dried over anhydrous Na₂SO₄, and filtered. Column chromatography purification (PE, R) was performed. f = 0.6) yielded a colorless liquid P-Cl (1.29 g, yield 64.1%).

[0049] (2) Synthesis of compound P-C2 5-Bromoisoindoline-2-tert-butyl carbonate (1.20 g, 4.04 mmol) and bis(pinacol)diboron (1.13 g, 4.45 mmol) were dissolved in 12 mL of dioxane, and potassium acetate (1.58 g, 16.1 mmol) was added. The system was protected with nitrogen and heated to 100 °C, then Pd(II) (165 mg, 0.202 mmol) catalyst was added, and the reaction was allowed to proceed for 18 h. TLC monitoring showed the disappearance of the reactants (PE:EA = 5:1). The reaction solution was cooled and concentrated under reduced pressure to remove dioxane. The solution was reconstituted with dichloromethane and extracted three times with water. The dichloromethane layers were combined, dried over anhydrous Na₂SO₄, and filtered. Column chromatography purification was performed (PE:EA = 15:1, R…). f = 0.2) to give white solid P-C2 (1.02 g, yield 73.4%).

[0050] (3) Synthesis of compound P-C3 Compounds P-C1 (674 mg, 2.73 mmol) and P-C2 (785 mg, 2.28 mmol) were dissolved in 12 mL of dioxane, and potassium acetate (892 mg, 9.10 mmol) was added. The system was protected with nitrogen and heated to 100 °C, then Pd(II) (186 mg, 0.228 mmol) catalyst was added, and the reaction was allowed to proceed for 10 h. TLC monitoring showed that the reactants had largely disappeared (PE:EA = 15:1). The reaction solution was cooled and concentrated under reduced pressure to remove dioxane. The solution was redissolved in dichloromethane and extracted three times with water. The dichloromethane layers were combined, dried over anhydrous Na₂SO₄, and filtered. Column chromatography purification was performed (PE:EA = 15:1, R…). f = 0.3) to give white solid P-C3 (492 mg, yield 56.2%).

[0051] (4) Synthesis of compound P-C4 Glutaric anhydride (5.6 g, 49.1 mmol) was heated to 90 °C to melt, and pomalidomide (800 mg, 2.94 mmol) was added. The mixture was stirred at 90 °C for 20 h until no obvious yellow solid was observed. TLC monitoring showed that the reaction starter disappeared (DCM: MeOH = 10: 1). The reaction solution was cooled and dissolved in ethyl acetate, repeatedly extracted with water several times, and purified by column chromatography (DCM: MeOH = 20: 1, R...). f = 0.2), to give white solid P-C4 (210 mg, yield 37.0%).

[0052] (5) Synthesis of compound P-C7 P-C3 (332 mg, 0.862 mmol) was dissolved in 4 mL of dichloromethane, and 1 mL of trifluoroacetic acid was added. The mixture was stirred at room temperature for 40 min. TLC monitoring showed the disappearance of the starting material. The pH was adjusted to 8 by adding saturated sodium bicarbonate solution, followed by extraction with dichloromethane. The organic phases were combined, and the solvent was removed by concentration under reduced pressure to obtain a yellow oily liquid, P-C5. This was directly used for the next condensation step without purification.

[0053] Add 2 mL of N-methylmorpholine to P-C5 (123 mg, 0.431 mmol) to adjust the pH to 8, and stir at room temperature for 20 min to obtain solution A. Dissolve Boc-L-Pro (102 mg, 0.474 mmol) in 4 mL of tetrahydrofuran, add HOBt (110 mg, 0.516 mmol), stir in an ice bath for 10 min, and add EDC (80 mg, 0.52 mmol) to obtain solution B. Pour solution A into solution B and stir overnight at room temperature. TLC plate monitoring showed that the reaction starting material disappeared (PE:EA = 1:1). Filter the reaction solution to remove insoluble impurities, concentrate the filtrate under reduced pressure, dissolve the residue with EA, and wash three times each with saturated NaHCO3 solution, saturated NaCl solution, saturated KHSO4 solution, saturated NaCl solution, saturated NaHCO3 solution, and saturated NaCl solution. Combine the organic layers, dry with anhydrous Na2SO4, filter, and purify by column chromatography (PE:EA = 2:1, R...). f = 0.2) to obtain white solid P-C6 (151 mg), TLC comparison was consistent with the standard. Boc was removed by TFA in the same steps as above, the resulting oily substance was dissolved in a small amount of ethyl acetate, 1 N hydrochloric acid was added dropwise to the solution, allowed to stand, and filtered to obtain white solid P-C7 (68 mg, total yield 23.6%).

[0054] (6) Synthesis of compound P-C8 Add 2 mL of N-methylmorpholine to P-C7 (55 mg, 0.14 mmol) to adjust the pH to 8, and stir at room temperature for 20 min to obtain solution A. Dissolve P-C4 (56 mg, 0.14 mmol) in 4 mL of tetrahydrofuran, add HOBt (37 mg, 0.17 mmol), stir in an ice bath for 10 min, and add EDC (27 mg, 0.17 mmol) to obtain solution B. Pour solution A into solution B and stir overnight at room temperature. TLC monitoring showed that the reaction starter disappeared (DCM: MeOH = 10:1). Filter to remove insoluble impurities, concentrate the filtrate under reduced pressure, and extract with EA / H2O. Combine the organic phases, concentrate under reduced pressure, and then separate and purify on a large plate (DCM: MeOH = 25: 1, R f = 0.2) to give white solid P-C8 (28 mg, yield 25%).

[0055] The proton NMR spectrum of P-C8 is as follows: Figure 2 As shown. 1 H NMR (300 MHz, DMSO- d 6 ) δ 11.14 (s,1H), 9.71 (s, 1H), 8.40 (m, 1H), 7.83 (t, J = 7.9 Hz, 1H), 7.66 – 7.15 (m,12H), 5.26 – 4.53 (m, 6H), 3.50 (m, 3H), 2.99 – 2.76 (m, 2H), 2.63 (m, 2H),2.44 – 2.25 (m, 3H), 2.10 – 1.98 (m, 5H), 1.96 – 1.72 (m, 4H). The proton NMR spectrum of P-C8 is as follows: Figure 3 As shown, C 44 H 41 N5O7[M+H] + The calculated value is 752.3084, and the measured value is 752.3058.

[0056] Evaluation of PD-L1 protein degradation activity in test cases The level of PD-L1 expressed in cells after treatment with the test compound was detected by Western blotting (WB) to evaluate the ability of the test compound to degrade PD-L1 protein in vitro.

[0057] 1. Protein extraction and quantification MC38 cells were seeded into 6-well plates, with 2 mL of cell suspension added to each well, at a cell density of 1 × 10⁻⁶ cells / well. 5 Cells / well. Incubate at 37℃, 5% CO2 for 12–24 h until cell adhesion is achieved. Treat with different concentrations of the test compound, and include a blank control. After 48 h, remove the 6-well plate, aspirate the culture medium, wash with 1 mL of pre-chilled PBS buffer, add 80 μL LRIPA lysis buffer (containing a phosphatase inhibitor), scrape cells to one side of the well with a spatula, and transfer the cell debris and lysis buffer mixture to an EP tube using a pipette. Centrifuge at 13000 rpm for 10 min. Store the supernatant at -20℃.

[0058] Protein quantification was performed using a BCA kit. In a 96-well plate, 200 μL of freshly prepared BCA mixture and 10 μL of standard protein solution or protein lysis buffer were added to each well. The plate was incubated at 37°C for 30 min, then transferred to room temperature and incubated for 20 min. The absorbance at 562 nm was read using a multi-mode microplate reader, and the loading volume was calculated based on a loading rate of 20 μg per well.

[0059] 2. SDS-PAGE gel electrophoresis Heat the protein samples at 100°C for 5 min, cool them, and then mix them with buffer at a ratio of protein sample:buffer solution of 4:1. Remove the pre-made gel and place it into a mold. Add each protein sample and 10 μL of marker to the sample wells, place the mold in the electrophoresis tank, add electrophoresis buffer, and electrophoresis at 140 V for about 50 min.

[0060] 3. Transfer and sealing Preparation of transfer buffer: Take 50 mL of rapid transfer buffer (20×) + 850 mL of distilled water + 100 mL of anhydrous ethanol.

[0061] Pre-soak the PVDF membrane in methanol and the sponge in transfer buffer, ensuring no air bubbles. Remove the gel and cut it according to the target protein molecular weight. Place the sponge pad, gel, transfer membrane, and sponge pad from bottom to top on the transfer clamp, removing air bubbles to create a sandwich structure. Place the prepared transfer structure in the electrophoresis tank and transfer at a constant current of 400 mA for 1 h. Remove the PVDF membrane, trim any excess, and mark it. Block with 5% skim milk in TBST solution at room temperature for 1 h (shaking at 20 rpm).

[0062] 4. Immunohistochemical staining The blocked membrane was washed three times with TBST (shaking at 37 rpm for 5 min each time), and incubated overnight at 4°C with primary antibody diluted 1:5000. After washing three times with TBST (shaking at 37 rpm for 8 min each time), secondary antibody diluted 1:2000 was added, and the membrane was incubated at room temperature for 1 h. The membrane was then washed three times with TBST (shaking at 37 rpm for 8 min each time). ECL developing solution was prepared (equal volumes of the two liquids were mixed), and the membrane was exposed and photographed. ImageJ was used to analyze the grayscale values ​​of the bands.

[0063] Western blot (WB) results of PD-L1 protein degradation by different concentrations of P-C8 after 48 hours are as follows: Figure 4 As shown. By Figure 4 It can be seen that the expression level of PD-L1 in MC38 cells under P-C8 treatment was significantly lower than that in the control group, and this expression was concentration-dependent.

[0064] The grayscale value analysis chart of the WB bands is shown below. Figure 5 As shown, by Figure 5It can be seen that a P-C8 concentration of 5 μM can reduce PD-L1 protein expression by 38%, and 25 μM can reduce it by 79%. This demonstrates that P-C8 has good activity in degrading PD-L1 protein.

[0065] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A PD-L1 protein degradation-targeting chimera, characterized in that, It has the structure shown in Equation I: Equation I.

2. The method for preparing the PD-L1 protein degradation-targeting chimera according to claim 1, characterized in that, Includes the following steps: In the presence of a condensing agent, a compound having the structure shown in Formula P-C7 undergoes a first condensation reaction with a compound having the structure shown in Formula P-C4 to obtain a protein degradation-targeting chimera of PD-L1 having the structure shown in Formula I. Formula P-C7; Formula P-C4.

3. The preparation method according to claim 2, characterized in that, The molar ratio of the compound having the structure shown in formula P-C7 to the compound having the structure shown in formula P-C4 is 1 to 1.1:

1.

4. The preparation method according to claim 2, characterized in that, The condensing agents are HOBt and EDC; The temperature of the first condensation reaction is 20~30℃, and the time is 10~15 h.

5. The preparation method according to claim 2, characterized in that, The method for preparing the compound having the structure shown in formula P-C7 includes the following steps: In the presence of an organic base and a catalyst, a compound having the structure shown in formula P-C1 undergoes a Suzuki coupling reaction with a compound having the structure shown in formula P-C2 to obtain a compound having the structure shown in formula P-C3. The compound having the structure shown in formula P-C3 undergoes a deprotection reaction to obtain the compound having the structure shown in formula P-C5. In the presence of a condensing agent, a compound having the structure shown in formula P-C5 undergoes a second condensation reaction with Boc-L-proline to obtain a compound having the structure shown in formula P-C6. The compound having the structure shown in formula P-C6 undergoes a deprotection reaction to obtain the compound having the structure shown in formula P-C7. Formula P-C1; Formula P-C2; Formula P-C3; Formula P-C5; Formula P-C6.

6. The preparation method according to claim 5, characterized in that, The organic base includes potassium acetate, and the catalyst includes a Pd(II) catalyst; The Suzuki coupling reaction was carried out at a temperature of 98-105°C for 8-12 hours.

7. The preparation method according to claim 2, characterized in that, The method for preparing the compound having the structure shown in formula P-C4 includes the following steps: Glutaric anhydride reacts with pomalidomide in a third condensation reaction to give a compound having the structure shown in formula P-C4.

8. The preparation method according to claim 7, characterized in that, The molar ratio of glutaric anhydride to pomalidomide is 15~30:1; The third condensation reaction is carried out at a temperature of 65-90°C for 18-25 hours.

9. The application of the PD-L1 protein degradation targeting chimera according to claim 1 or the PD-L1 protein degradation targeting chimera prepared by any one of claims 2 to 8 in the preparation of PD-L1 protein degrading agents.

10. The use of the PD-L1 protein degradation targeting chimera according to claim 1 or the PD-L1 protein degradation targeting chimera prepared by any one of claims 2 to 8 in the preparation of antitumor drugs.

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